WO2023178169A2 - Compositions and methods for treating the pathophysiology of severe viral infection - Google Patents

Compositions and methods for treating the pathophysiology of severe viral infection Download PDF

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WO2023178169A2
WO2023178169A2 PCT/US2023/064419 US2023064419W WO2023178169A2 WO 2023178169 A2 WO2023178169 A2 WO 2023178169A2 US 2023064419 W US2023064419 W US 2023064419W WO 2023178169 A2 WO2023178169 A2 WO 2023178169A2
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Jian Qing XU
Robert Mandell
Beverly W. Lubit
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Anemoi Biotech Holdings, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • Viruses depend on contact with a compatible host cell for replication. Viral replication begins with attachment of a virus to a host cell, which is followed by entry of the viral genome into the host where it begins to direct the synthesis of viral proteins and nucleic acid molecules. During replication, the nucleic acid and protein capsid structures are synthesized separately and then assembled within the host cell by packaging viral genomes into protein coats.
  • the two primary patterns of infection are acute infections and persistent infections. In acute infections, some viruses rapidly kill the cell while producing a burst of new infectious particles (cytopathic viruses), while others infect cells and actively produce infectious particle without causing immediate host cell death (noncytopathic viruses). In persistent infections (e.g., latent infections, slow, abortive and transforming infections), some viruses infect, but neither kill the cell nor produce any viral progeny. Replicated viruses remain inert unless they attach to the surface of another compatible host cell.
  • the host immune response to viral infection [0006]
  • the human immune system is a complex arrangement of cells and molecules that maintain immune homeostasis to preserve the integrity of the organism by elimination of all elements judged to be dangerous.
  • Responses in the immune system may generally be divided into two arms, referred to as “innate immunity” and “adaptive immunity.”
  • the two arms of immunity do not operate independently of each other, but rather work together to elicit effective immune responses.
  • the innate arm of the immune system is a nonspecific fast response to pathogens that is predominantly responsible for an initial inflammatory response via a number of soluble factors, including the complement system and the chemokine/cytokine system; and a number of specialized cell types, including mast cells, macrophages, dendritic cells (DCs), and natural killer cells (NKs).
  • Complement activation [0008]
  • the complement system is a system of soluble pattern recognition receptors (PRRs) and effector molecules that detect and destroy microorganisms.
  • complement is activated through the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP).
  • CP classical pathway
  • LP lectin pathway
  • AP alternative pathway
  • PRMs soluble pattern recognition molecules
  • PAMP pathogen-associated molecular pattern
  • DAMP damage-associated molecular pattern
  • complement component C1 which comprises a recognition protein (C1q) associated with proteases (C1r and C1s) either recognizes a microbial surface directly or binds to antibodies already bound to a pathogen.
  • C1q ligands include antigen- antibody complexes, molecular patterns on certain bacteria, viruses, parasites, and mycoplasma, C-reactive protein (CRP) in complex with exposed phosphocholine residues on bacteria; pentraxin ⁇ 3 (PTX ⁇ 3), serum amyloid P component, ⁇ amyloid fibrils, as well as tissue damage elements such as DNA and mitochondrial membranes [Bajik, G., et al. EMBO J.
  • C1r autoactivates and subsequently cleaves C1s, which may then cleave C4 into the fragments C4a and C4b.
  • the nascent C4b can be covalently bound to the activator via an exposed internal thioester leading to irreversible tagging of the activator.
  • C2 binds activator ⁇ bound C4b and is cleaved by C1s to generate the active serine protease C2a bound to C4b resulting in the CP C3 convertase C4b2a [Id., citing Muller ⁇ Eberhard, HJ et al, J Exp Med (1967) 125: 359–3801967].
  • the C3 convertase cleaves C3 into the anaphylatoxin C3a and the major opsonin of the complement system, C3b, which like C4b, becomes covalently coupled to the activator through its exposed thioester [Id., citing Law SK, Dodds AW. Protein Sci (1997) 6: 263–274).
  • the lectin pathway is initiated by soluble carbohydrate-binding proteins – mannose-binding lectin (MBL) and the ficolins—that bind to particular carbohydrate structures on microbial surfaces.
  • MBL-associated serine proteases MASPs
  • Activation of the lectin pathway is initiated by the collectins MBL and CL ⁇ LK or one of three ficolins.
  • MBL and CL ⁇ LK harbor Ca2+ ⁇ dependent carbohydrate ⁇ recognition domains (CRDs) and collagen ⁇ like regions through which they trimerize.
  • trimers oligomerize in larger complexes, allowing high ⁇ avidity binding (K D ⁇ 10 ⁇ 9 M) based on multiple low ⁇ affinity interactions of their CRDs (K D ⁇ 10 ⁇ 3 M) [Id., citing Kawasaki et al. J Biochem (1983) 94: 937–947; Degn SE, Thiel S. Scand J Immunol (2013) 78: 181–193].
  • Ficolins are structurally similar to collectins, but instead of C ⁇ type lectin domains they possess fibrinogen (FBG) ⁇ like domains for PAMP recognition [Id., citing Matsushita M Ficolins in complement activation. Mol Immunol (2013) 55: 22–26].
  • Ficolins recognize motifs containing acetylated groups, including non ⁇ sugars such as N ⁇ acetyl ⁇ glycine, N ⁇ acetyl ⁇ cysteine, and acetylcholine. Besides conferring avidity, the oligomerization of collectins and ficolins allows these PRMs to discriminate not only specific monosaccharides or acetylated groups but also specific patterns of sugars and acetyl groups characteristic to pathogens.
  • the LP PRMs form complexes with MBL ⁇ associated serine proteases (MASPs), which are always present as dimers.
  • MASPs MBL ⁇ associated serine proteases
  • MASP ⁇ 1 and MASP ⁇ 2 are structural and functional homologs of C1r and C1s from the CP, but there are important differences between PRM–protease complexes from the two pathways.
  • the C1 complex has a defined stoichiometry (a hexamer of the heterotrimeric C1q subunit in complex with a C1r2s2 tetramer)
  • the LP PRMs are polydisperse oligomers of trimers.
  • a tetramer is the most abundant oligomer and this carries only a single MASP ⁇ 1 or MASP ⁇ 2 dimer, but the more rare, larger oligomers may simultaneously carry both dimers [Id., citing Dahl MR, et al.
  • MASP ⁇ 1 in complex with an activator ⁇ bound PRM autoactivates and cleaves MASP ⁇ 2 as well as C2, whereas activated MASP ⁇ 2 cleaves C4 and C2 resulting in the same C3 convertase as in the CP, that is, C4b2a [Id., citing Matsushita, M et al. J Immunol (2000) 165: 2637–2642; Rossi, V. et al.
  • the alternative pathway can be initiated by spontaneous hydrolysis and activation of complement component C3, which can then bind directly to microbial surfaces. Activation through the CP and LP results in deposition of C3b on the activator, which recruits factor B (FB) in the first step of the AP. The resulting proconvertase C3bB is subsequently cleaved by factor D (FD), generating the AP C3 convertase C3bBb (Id., citing Fearon, DT et al.
  • the three pathways converge at the step whereby enzymatic activity of a C3 convertase is generated.
  • Cleavage of C3 is the critical step in complement activation and leads directly or indirectly to all the effector activities of the complement system.
  • the C3 convertase is bound covalently to the pathogen surface, where it cleaves C3 to generate large amounts of C3b, the main effector molecule of the complement system, and C3a, a small peptide that binds to specific receptors and helps induce inflammation.
  • the terminal pathway (TP) of complement is initiated when a threshold density of C3b molecules on an activator has been reached.
  • the C3 convertases can recruit another C3b molecule to form C3bBb3b [Id., citing Medicus, RG et al. J Exp Med (1976) 144: 1076– 1093] and C4b2a3b [Id., citing Takata, Y et al. J Exp Med (1987) 165: 1494–1507], the AP and CP C5 convertases, respectively. Through cleavage of C5, they generate the potent chemoattractant C5a and C5b.
  • MAC lytic membrane attack complex
  • the anaphylatoxins C3a and C5a released when the convertases cleave C3 and C5, exert their biological functions upon binding to seven ⁇ transmembrane domain (7TM) receptors in the membranes of host cells.
  • 7TM seven ⁇ transmembrane domain
  • C3aR and C5aR1 CD88
  • GPCR G protein ⁇ coupled receptors
  • C5aR2 previously known as C5L2
  • C5aR2 is structurally similar to C5aR1 but does not couple to heterotrimeric G proteins [Id., citing Li, R. et al. FASEB J (2013) 27: 855–864, 2013].
  • C5aR2 was first considered as a decoy receptor, limiting the availability of the C5a and C5adesArg ligands to C5aR1. Decoy receptors do not undergo ligand ⁇ induced internalization but are rather continuously recycled between the cell membrane and the intracellular compartments, thereby removing their extracellular ligand [Id., citing Weber, M. et al. Mol Biol Cell (2004) 15: 2492–2508]. Thus, it has been suggested that C5aR2 may reduce the cellular responses to pro ⁇ inflammatory molecules and thereby actively regulate inflammatory processes [Id., citing Rittirsch, D. et al. Nat Med (2008) 14: 551–557].
  • C3b receptor 1 is a large CCP module ⁇ based glycoprotein expressed on almost all peripheral blood cells except NK and T cells [Id., citing Fearon, DT. J Exp Med (1980) 152: 20–30; Tedder, TF et al, J Immunol (1983) 130: 1668–1673].
  • CR1 binds C3b and C4b with high affinity and iC3b and C3d with a lower affinity [Id., citing Reynes, M. et al J Immunol (1985)135: 2687–2694].
  • CR1 on erythrocytes may bind C3b ⁇ containing immune complexes as part of removal processes, whereas on phagocytic cells it promotes C3b/C4b ⁇ coated particle uptake.
  • CR1 also plays an important role in the germinal centers of lymph nodes where it is found on follicular dendritic cells (FDCs) capturing complement ⁇ opsonized antigens that serve to stimulate B cells [Id., citing Heesters, BA et al. (2013) Nat Rev Immunol 14: 495–504].
  • FDCs follicular dendritic cells
  • CR2 binds C3b, iC3b, and C3d with the same affinity in agreement with the crystal structure of the CR2 ⁇ C3d complex revealing recognition of a surface patch on the TE domain accessible in all three ligands but concealed in C3 prior to cleavage [Id.].
  • CR3 and CR4 are integrin ⁇ type heterodimeric receptors (CD11b/CD18 and CD11c/CD18) having distinct ⁇ chains, ⁇ M and ⁇ X, respectively, but sharing a common ⁇ 2 ⁇ chain. Both are phagocytic receptors expressed on myeloid leukocytes and NK cells and share iC3b as ligand [Id., citing Metlay, JP et al. (1990) J Exp Med 171: 1753–1771; Ross, GD (2000) Crit Rev Immunol 20: 197–222]. However, structural studies indicate that the receptors bind to different epitopes of iC3b.
  • CR3 was shown to recognize the TE domain of iC3b [Id., citing Bajic, G. et al. (2013) Proc Natl Acad Sci USA 110: 16426–16431], whereas CR4 binds quite far from this in the C3c moiety of iC3b [Id., citing Chen, X. et al. (2012) Proc Natl Acad Sci USA 109: 4586–4591].
  • CR3 and CR2 may bind simultaneously to the iC3b TE domain [Id., citing Bajic, G.
  • CRIg CRIg
  • Kupffer cells an immunoglobulin ⁇ type receptor expressed on liver ⁇ resident macrophages
  • CRIg The binding of CRIg to C3b selectively inhibits the interaction of C3 and C5 with the AP, but not with the CP convertases.
  • the complement system also influences adaptive immunity. For example, opsonization of pathogens (meaning the coating of the surface of a pathogen that makes it more easily ingested by phagocytes) by complement facilitates their uptake by phagocytic APCs that express complement receptors, which enhances presentation of pathogen antigens to T cells. B cells express receptors for complement proteins that enhance their responses to complement-coated antigens.
  • complement fragments also can act to influence cytokine production by APCs, thereby influencing the direction and extent of the subsequent adaptive immune response.
  • Complement fragments can be generated by other means besides the three canonical activation routes.
  • the cross-talk with the coagulation system has regained attention due to studies indicating that thrombin, coagulation factors XIa, Xa, and IXa, and plasmin effectively cleave C3 and C5 and generate C3a and C5a [Bajik, G. et al. EMBO J. (2015) 34(22): 2735-57, citing Huber ⁇ Lang, M. et al. (2006) Nat Med 12: 682–687; Amara U. et al.
  • C3 can also be produced intracellularly by CD4+ T cells. This C3 is processed by the T ⁇ cell lysosomal protease cathepsin L, yielding biologically active C3a and C3b [Id., citing Liszewski, MK et al. (2013) Immunity 39: 1143–1157]. Tonic intracellular C3a generation is required for homeostatic T ⁇ cell survival, whereas shuttling of this intracellular C3 activation system to the cell surface upon T ⁇ cell stimulation additionally induces autocrine proinflammatory cytokine production.
  • C3aR activation via intrinsic generation of C3a appears to be an integral part of human Th1 immunity [Id., citing Ghannam, A. et al, (2014) Mol Immunol 58: 98–107].
  • Thrombin slowly cleaves C5 and generates C5a, but under conditions with normal convertase activity, this is possibly not a physiologically significant reaction.
  • Clotting ⁇ induced production of thrombin instead leads to cleavage of C5 or C5b in the CUB domain.
  • C5a can be released from such CUB ⁇ digested C5 by the conventional C5 convertases, and the combined action of thrombin and convertases appears to enhance the efficiency of the lytic pathway [Id., citing Krisinger, MJ et al.
  • MASP ⁇ 1 has been reported to activate coagulation [Id., citing Takahashi, K. et al. (2011) Immunobiology 216: 96–102; La Bonte, LR et al. (2012) J Immunol 188: 885–891] and to initiate endothelial cell signaling via cleavage of protease ⁇ activated receptor 4 [Id., citing Megyeri, M. et al. (2009) J Immunol 183: 3409–3416]. [00021] The complement system has been implicated as a contributor to the observed tissue damage that occurs in such severe virus infections as influenza A virus H1Ni [Wang, R. et al.
  • the anaphylatoxin C5a is one of the most potent inflammatory peptides. [Id. citing Marc, MM, et al. Am. J. Respir. Cell Mol. Biol. (2004) 31: 216-19]. It is a strong chemoattractant for neutrophils and monocytes and activates these cells to generate oxidative bursts with release of reactive oxygen species (ROS), especially O 2 and H 2 O 2 . [Id., citing Guo, RF, Ward, PA. Annu. Rev. Immunol. (2005) 23: 821-52]. C5a mediates neutrophil attraction, aggregation, activation and subsequent pulmonary endothelial damage.
  • ROS reactive oxygen species
  • C5a activates macrophages and endothelial cells and promotes vascular leakage and the release of Neutrophil Extracellular Traps (NETs).
  • NETs are primarily composed of DNA from neutrophils, which bind pathogens with anti-microbial proteins, and increase the permeability of the alveolar-capillary barrier by cleaving endothelial actin cytoskeleton, E-cadherin and VE-cadherin.
  • Saffarzadeh M. et al. PLoS One (2012) 7: e32366
  • the antimicrobial peptide LL-37 in NET structures is cytotoxic and pro-apoptotic toward endothelial and epithelial cells [Id., citing Aarbiou, J. et al. Inflamm. Res.
  • C5a is also a potent chemoattractant for T cells [Id., citing Nataf, S., e al., J. Immunol. (1999) 162: 4018-23; Tsuji, RF et al. J. Immunol. (2000) 165: 1588-98], B cells [Id., citing Ottonello, L, et al. J. Immunol.
  • DCs can then take up antigen and are primed for T cell help. [Id., citing Kim, AH, et al. J. Immunol. (2004) 173: 2524-29].
  • C5a induces upregulation of CD11b /CD18 expression on neutrophils. [Id., citing Guo, RF, Ward, PA. Annu. Rev. Imunol. (2005) 23: 821-52].
  • IL-8 levels have been found to correlate with neutrophil numbers and the degree of lung dysfunction. [Id., citing Williams, TJ, Jose, PJ. Novartis Found Symp. (2001) 234: 136-41].
  • C5a directly activates endothelial cells to upregulate adhesion molecules, such as P-selectin, and C5a and TNF- ⁇ cooperate to enhance upregulation of intracellular adhesion molecule 1 and E-selectin [Id., citing Ward, PA. Ann. NY Acad. Sci. (1996) 796: 104-112].
  • Strategies of innate immunity that defend against intracellular pathogens [00024] Viruses are obligate intracellular pathogens – they must invade host cells to replicate.
  • innate immunity includes soluble defenses, such as antimicrobial peptides (e.g., defensins, athelicidins, and histatins) and phagocytic cells (macrophages, neutrophils and dendritic cells) that can engulf and destroy pathogens before they become intracellular. Macrophages and neutrophils constitutively express cell-surface receptors that stimulate the phagocytosis and intracellular killing of microbes bound to them, although some also signal through other pathways to trigger other responses, e.g., cytokine production.
  • antimicrobial peptides e.g., defensins, athelicidins, and histatins
  • phagocytic cells macrophagocytic cells
  • Macrophages and neutrophils constitutively express cell-surface receptors that stimulate the phagocytosis and intracellular killing of microbes bound to them, although some also signal through other pathways to trigger other responses, e.g.,
  • phagocytic receptors include several members of the C-type lectin-like family (e.g., Dectin-1, and the mannose receptor (MR); scavenger receptors that recognize various anionic polymers and acetylated low density lipoproteins; and complement receptors and Fc receptors that bind to complement coated microbes or to antibodies bound to the surface of microbes that facilitate phagocytosis.
  • C-type lectin-like family e.g., Dectin-1, and the mannose receptor (MR)
  • scavenger receptors that recognize various anionic polymers and acetylated low density lipoproteins
  • complement receptors and Fc receptors that bind to complement coated microbes or to antibodies bound to the surface of microbes that facilitate phagocytosis.
  • the nucleic acid sensing toll like receptors – TLR3, TLR-7, TLR-8 and TLR-9 are endosomal nucleotide sensors involved in the recognition of viruses.
  • TLR-3 is expressed by macrophages, conventional dendritic cells, and intestinal epithelial cells; it recognizes double-stranded RNA which is a replicative intermediate of many types of viruses.
  • TLR-7 and TLR-9 are expressed by plasmacytoid dendritic cells, B cells and eosinophils; TLR-8 is expressed primarily by monocytes and macrophages.
  • TLR-7 and TLR-8 are activated by single-stranded RNA.
  • the virus genome for example of orthomyxoviruses (such as influenza) and flaviviruses (such as West Nile virus) consist of single stranded RNA.
  • TLR-7 When extracellular particles of these viruses are endocytosed by macrophages or dendritic cells, they are uncoated in the acidic environment of endosomes and lysosomes, exposing the ssRNA genome for recognition by TLR-7.
  • TLR-8 is physiologically most similar to TLR7, recognizes viral ssRNA, and is predominantly expressed in monocytes. [Petrasek, J. et al., Advances in Clin. Chem. (2013) 59: 255-201].
  • TLR-9 recognizes unmethylated CpG nucleotides; in the genomes of bacteria and many viruses, CpG dinucleotides remain unmethylated. [Janeway’s Immunology, 9th Ed.
  • Macrophages and neutrophils secrete lipid mediators of inflammation – prostaglandins, leukotrienes, and platelet-activating factor (PAF) – which are rapidly produced by enzymatic pathways that degrade membrane phospholipids.
  • PAF platelet-activating factor
  • Signaling by mammalian TLRs in various cell types induces a diverse range of intracellular responses that together result in the production of inflammatory cytokines, chemotactic factors, antimicrobial peptides, and the antiviral cytokines interferon ⁇ and interferon ⁇ .
  • Viral RNAs produced within a cell are sensed by RIG-1 like receptors, which bind to viral RNA using an RNA helicase-like domain in their carboxy terminal, which has a DexH tetrapeptide amino acid motif and is a subgroup of DEAD-box family proteins.
  • the RLR proteins also contain two amino terminal CARD domains that interact with adaptor proteins and activate signaling to produce type 1 interferons when viral RNAs are bound.
  • RIG- 1 discriminates between host and viral RNA by sensing differences at the 5’ end of single stranded RNA transcripts – most RNA viruses do not replicate in the nucleus where addition of a 7-methylguanosine to the 5’triphosphate (called capping) occurs, and their RNA genomes do not undergo this modification.
  • RIG-1 senses the unmodified 5’-triphosphate end of the ssRNA viral genome.
  • MDA-5 (melanoma differentiation-associated 5, also called hellicard, is similar in structure to RIG-1, but it senses dsRNA.
  • the RLR family member LGP2 (encoded by DHX58) retains a helicase domain but lacks CARD domains.
  • RIG-1 and MDA-5 are in the cytoplasm in an auto-inhibited configuration that is stabilized by interactions between the CARD and helicase domains.
  • Sensing of viral RNAs activates signaling by RIG-1 and MDA-5, which leads to type 1 interferon production.
  • viral RNA associates with the helicase domains of RIG-1 or MDA-5, freeing the two CARD domains for other interactions.
  • the more amino- proximal portion of the two CARD domains can then recruit E3 ligases, including TRIM25 and RIPLET, which initiate K63-linked polyubiquitin scaffolds, which appear to help RIG-1 and MDA-5 interact with a downstream adaptor protein called mitochondrial antiviral signaling protein (MACVS).
  • MAVS is attached to the outer mitochondrial membrane and contains a CARD domain that may bind RIG-1 and MDA-5. This aggregation of CARD domains may initiate aggregation of MAVs, which propagates signals by recruiting various TRAF family E3 ubiquitin ligases, including TRAF2, TRAF3, TRAF5, and TRAF6.
  • NK cells Natural killer cells
  • ILCs cytotoxic population of innate lymphoid cells
  • Virus-infected cells can become susceptible to being killed by NK cells by a variety of mechanisms. First, some viruses inhibit all protein synthesis in their host cells; synthesis of MHC class I proteins would be blocked in infected cells, which would make them correspondingly less able to inhibit NK cells through their MHC-specific receptors, and they would become more susceptible to being killed. Second, many viruses can selectively prevent the export of MHC class I molecules to the cell surface, or induce their degradation once there. Virally infected cells can still be killed by NK cells even if the cells do not downregulate MHC, provided that ligands for activating receptors are induced.
  • NK cells that target ligands for the activating receptors on NK cells can thwart NK cell recognition and killing of virus-infected cells.
  • NK cells also express receptors that more directly sense the presence of infection or other perturb ations in a cell.
  • Activating receptors include the natural cytotoxicity receptors (NCRs) NKp30, NKp44, and NKp46, which are immunoglobulin-like receptors, and the C-type lectin-like family members LY49H and NKG2D. Recognition by NKG2D acts as a generalized ‘danger’ signal to the immune system.
  • NKG2D is expressed by various T cells, including all human CD8 T cells, ⁇ T cells, activated murine CD8 T cells and invariant NKT cells. In these cells recognition of NKG2D ligands provides a potent co-stimulatory signal that enhances their effector functions.
  • the conventional NK (cNK) cell pool consists of a circulating compartment and a tissue-resident compartment in the gut intraepithelial layer and lamina intestinal layer.
  • cNK cells are able to sense pathogens, oncogenesis and tissue damage signals. Activation and turnover of cNK cells rely on the overall signal input of activating signals, inhibitory signals, and exogenous cytokine signals, which further leads to the alteration of specific transcription factors and a group of pro-apoptotic proteins and ultimately determines the fate of cNK cells. [Id., citing Viant C, et al. J Exp Med. (2017) 214:491–510]. Upon activation, cNK cells exert their cytotoxicity function by releasing the pore forming cytolytic protein–perforin and the cytotoxic protein–granzyme.
  • cNK cells also utilize tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) pathways and antibody-dependent cellular cytotoxicity (ADCC) (Id., citing Caligiuri MA. Blood. (2008) 112:461–9).
  • TNF tumor necrosis factor
  • TRAIL apoptosis-inducing ligand
  • ADCC antibody-dependent cellular cytotoxicity
  • cNK cells possess strong cytokine production ability, including TNF, IFN- ⁇ , and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Id., citing Souza-Fonseca-Guimaraes F, et al. J Biol Chem. (2013) 288:10715–21).
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • cNK education posits that the threshold of activation of cNKs throughout their development is modulated by adjusting the expression level of their activating receptors and inhibitory receptors (“education”).
  • cNK cell arming meaning the downregulation of inhibitory receptors that could upregulate the threshold of activation
  • cNK cell licensing meaning the scenario where activating receptors are downregulated to endow cNK cells with increased receptivity to activating signals
  • ILCs Innate lymphoid cells (ILCs) are the innate counterparts of T lymphocytes. They lack adaptive antigen receptors generated by the recombination of genetic elements. [Vivier, E. et al. Cell (2018) 174: 1054-66, citing Spits, et al. Nat. Rev. Immunol. (2013) 13: 145-49; Eberl, G et al.
  • ILCs express interleukin-7 receptor ⁇ (CD127). ILCs may be activated by signals from other cells around them upon exposure to foreign antigens (including microbes), rather than by being directly activated by foreign antigens. Some ILCs express TLRs that recognize microbes, and the cells may be directly activated by the PAMPs of microbes. However, there have been some reports showing that ILCs express various kinds of receptors for cytokines, danger signals, neuropeptides and lipid mediators that are more dominant than TLRs.
  • ILCs are generally thought to be tissue-resident cells that differentiate into mature effector cells in tissues, and show minimal movement between organs. Instead, they have functional plasticity that enables them to respond promptly to microenvironmental changes, thereby precluding any need for differentiation and/or migration of new ILC subsets adapted to a new environment.
  • transdifferentiation has been shown between ILC1s and ILC3s [Id., citing Bernink JH, et al. Immunity (2015) 43:146–160, Bernink JH, et al. Nat Immunol (2013) 14:221–229], between ILC1s and ILC2 [Id., citing Bal SM, et al.
  • Group 1 ILCs currently are divided into 3 different subtypes, according to their expression of cytokines and transcription factors: group 1 ILCs (ILC1s), group 2 ILCs (ILC2s), and group 3 ILCs (ILC3s).
  • ILC1s are defined as ILCs that express T box-expressed in T cells (T-bet) and produce interferon (IFN- ⁇ ); they include conventional natural killer cells (cNK) and are considered to be involved in anti-viral immunity, like Th1 cells.
  • ICL2s are defined as ILCs that express GATA-binding protein 3 and produce such cytokines as IL-4, IL-5, IL-9, and IL-13, as well as the epidermal growth factor, amphiregulin; like Th2 cells, they are considered to be involved in anti-helminth immunity.
  • ILC3s are defined as ILCs that express retinoic acid receptor-related orphan receptor- ⁇ t and produce cytokines, such as IL-17A, IL-22 and GM-CSF; they include both natural cytotoxicity receptor (NCR)- ILC3s and NCR+ ILC3s, and are considered to be involved in antibacterial immunity, like Th17 cells.
  • NCR natural cytotoxicity receptor
  • ILC3s are the predominant population in mucosal tissues, including the lung and gut, whereas the proportion of ILC2s is a little higher in the skin compared to mucosal tissues [Id., citing Bal SM, et al. Nat Immunol (2016) 17:636–645).
  • the proportion of the ILC subsets is influenced by age; although ILC3s are the predominant population in the fetal human lung, their proportion decreases while the proportions of ILC1s and ILC2s increase with age in the adult human lung. [Id., citing Bal SM, et al. Nat Immunol (2016) 17:636–645]. There is substantial heterogeneity in each subset of ILCs. Moreover ILCs show different phenotypes depending on the organ. (Id., citing Ricardo- Gonzalez RR, et al. (2016) 19:1093–1099).
  • ILC2s from different organs share canonical markers such as GATA3 and IL-7R, expression of IL-33R, IL-25R, and IL- 18R1 differs depending on the organ.
  • ILCregs regulatory ILCs
  • ILCregs produce regulatory cytokines such as IL-10 and/or TGF ⁇ , but they do not express FOXP3, the canonical transcription factor of Tregs. It remains controversial wither ILCregs represents an independent effector subset, or just a temporary state of ILCs. [00039] There is increasing evidence to suggest that like T helper cell subsets, ILC subsets also display a certain degree of plasticity, which enables them to adjust to their microenvironment. Thus, ILC subsets can change their phenotype and functional capacities.
  • ILC2s from different organs share canonical markers such as GATA3 and IL-7R
  • expression of IL-33R, IL-25R, and IL-18R1 differs depending on the organ.
  • Ricardo-Gonzalez RR, et al. Tissue signals imprint ILC2 identity with anticipatory function. Nat Immunol (2016) 19:1093–1099].
  • This requires accessible polarizing signals in the tissue in which conversion occurs, together with the expression of cognate cytokine receptors and key transcription factors in the responding ILCs.
  • Vivier, E. et al. Cell (2018) 174: 1054-66 [Vivier, E. et al. Cell (2018) 174: 1054-66].
  • IL12 is a major activator of ILC1s and promotes their secretion of IFN- ⁇ . [Orimo, K. et al. Allergy Asthma Immunol. Res. (2020) 12 (3): 381-98, citing Bernink JH, et al. Nat Immunol (2013) 14:221–229].
  • the major physiological producers of IL12 are APCs, such as dendritic cells and macrophages.
  • INF- ⁇ produced by ILC1s in response to DC-derived IL-12 during viral infection suppresses early viral growth, suggesting that the IL-12-ILC1 axis may be involved in anti-viral immunity.
  • ILC2s citing Bal SM, et al. Nat Immunol (2016) 17:636–645; Silver JS, et al. Nat Immunol (2016) 17:626–635; Ohne Y, et al. Nat Immunol (2016) 17:646–655
  • ILC3s citing Bernink JH, et al. Immunity (2015) 43:146–160] into INF- ⁇ producing ILC1s, a mechanism that may be involved in immune responses to viral infections and in the pathophysiology of COPD.
  • IL-15 activates ILC1s to produce IFN- ⁇ .
  • IL-15 is known to be produced by APCs, a subset of thymic epithelial cells, and by stromal cells. In the airways, human bronchial epithelial cells produce IL-15 in response to respiratory syncytial virus infection [Id., citing Zdrenghea MT, et al. Eur Respir J 2012;39:712–720].
  • IL-15–positive cells have been reported to be increased inpatients with sarcoidosis, tuberculosis or chronic bronchitis compared to asthmatic patients and healthy subjects, [Id., citing Muro S, et al. Allergy Clin Immunol (2001) 108:970–975] suggesting the involvement of IL-15 in the pathophysiology of these diseases.
  • IL-18 [00042] IL-18 also activates ILC2s and ILC3s to produce their signature cytokines, (Id., citing 12, 16) suggesting that IL-18 may be a pan-activator of ILCs. Furthermore, IL-18 and IL-12 together promote conversion of ILC2s to ILC1s.
  • IL-18 is produced by APCs such as macrophages and DCs.
  • APCs such as macrophages and DCs.
  • IL-18 was shown to be released from human bronchial epithelial cells upon human rhinovirus infection [Id., citing Briend E, et al. Respir Res 2017;18:159] and Alternaria extract stimulation [Id., citing Murai H, et al. Biochem Biophys Res Commun (2015) 464:969–974] in vitro.
  • cigarette smoke exposure induced IL-18 production by alveolar macrophages in the mouse lungs.
  • ILC2s IL-25 activates ILC2s and promotes type 2 cytokine production.
  • Various kinds of immune cells such as macrophages, eosinophils and T cells, have been shown to produce IL-25.
  • tuft cells including intestinal tuft cells, brush cells in the lower airways and solitary chemosensory cells (SCCs) in nasopharyngeal tissue—have attracted broad attention as major sources of IL- 25.
  • SCCs single chemosensory cells
  • intestinal tuft cells produce IL-25 after sensing microbial metabolites through succinate receptors or taste receptors during protozoan and helminth infections, which results in activation of ILC2s and promotion of an anti-helminth response.
  • SCCs in the human upper respiratory tract Id., citing Kohanski MA, et al. J Allergy Clin Immunol (2018) 142:460– 469.e7] and brush cells in the murine lower respiratory tract [Id., citing Bankova LG, et al.
  • IL-33 Unlike other cytokines that are newly synthesized upon stimulation and secreted via the endoplasmic reticulum/Golgi pathway, IL-33 is constitutively expressed in cells at the mucosal barrier and released from the nucleus in active form in response to tissue damage. [Id., citing Cayrol C, Girard JP. Immunol Rev (2016) 281:154–168]. It is believed to be one of the “alarmins” that gather components of the repair response to the sites of injury. However, several studies suggest that IL-33 may be actively secreted from live cells, including bronchial epithelial cells [Id., citing Hristova M, et al.
  • IL-33 is recognized as one of the major activators of ILC2s that induce production of type 2 cytokines.
  • IL-33 is released from alveolar epithelial cells in response to tissue damage caused by fungi such as Alternaria and Aspergillus and viruses such as respiratory syncytial virus (RSV) and rhinovirus (RV).
  • fungi such as Alternaria and Aspergillus
  • viruses such as respiratory syncytial virus (RSV) and rhinovirus (RV).
  • RSV respiratory syncytial virus
  • RV rhinovirus
  • IL-33 is released from bronchial epithelial cells located more centrally, [Id., citing Cayrol C, Girard JP. Immunol Rev (2016) 281:154–168] similar to IL-25 and thymic stromal lymphopoietin (TSLP).
  • TSLP thymic stromal lymphopoietin
  • IL-33 may play a major role in the developing phase of acquired immunity and that epithelial damage may induce more severe allergic airway inflammation during infancy than during adulthood through the IL-33–ILC2s axis.
  • stromal cells [Id., citing Dahlgren MW, et al.
  • TSLP Thymic stromal lymphopoietin
  • TSLP was shown to induce corticosteroid resistance in murine ILC2s through activation of an intracellular signaling molecule, signal transducer and activator of transcription 5.
  • TSLP is produced by various kinds of cells including DCs, vascular endothelial cells, macrophages and mast cells.
  • DCs DCs
  • vascular endothelial cells vascular endothelial cells
  • macrophages macrophages
  • mast cells In the airways, similar to IL-25 and IL-33, TSLP is produced mainly by airway epithelial cells in response to exposure to bacteria, fungi and viruses. [Id., citing Varricchi G, et al. Front Immunol 2018;9:1595].
  • IL-27 is generally produced by DCs and macrophages. In mice, IL-27 suppresses the proliferation and cytokine production of ILC2 cells in vitro, [Id., citing Moro K, et al. Nat Immunol (2016) 17:76–86, Duerr CU, et al.
  • IFNs are classified as type 1 ( ⁇ / ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ), type 2 ( ⁇ ) or type 3 ( ⁇ ), depending on the type of receptor through which they signal.
  • the type I IFNs are the largest group and include IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ and IFN- ⁇ .
  • IFN- ⁇ family of 12 closely related human genes and IFN- ⁇ , the product of a single gene, are best understood; less well studied are IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ and IFN- ⁇ , which may offer equal or superior biological activities compared to IFN ⁇ / ⁇ with less adverse effects.
  • Type I IFNs combat viral infection both directly by inhibiting viral replication in infected cells and indirectly by stimulating the adaptive immune system [Zhou, Z. et al. J. Virology (2007) 81 (14): 7749-58, citing Biron, C. A. (1994) Curr. Opin.
  • IFN- ⁇ - receptors IFN- ⁇ - receptors
  • IFNARs IFN- ⁇ - receptors
  • JK Janus kinase
  • STAT activator of transcription
  • IFN- ⁇ exhibits increased anti-influenza activity in cultured cells compared to INF- ⁇ 2, albeit less than IFN- ⁇ 1a [Id., citing Skorvanoa, L. et al. Acta Virol.2015] 59: 413-17].
  • IFN- ⁇ was first identified in human keratinocytes and then in dendritic cells and monocytes [Id., citing Nardelli, B. et al. J. Immunol. (2002) 169: 4822-30]. While it is primarily viewed as a keratinocyte-specific IFN dedicated to skin immune responses, it can also induce antiviral responses in other human cell types. [Id., citing LaFleur, DW, et al.
  • IFN ⁇ is induced earlier than IFN- ⁇ in cultured cells in response to virus infection [Id., citing Honda, K., et al. Immunity (2006) 25: 349-60]; many virus-encoded proteins interfere with the production of IFN through various mechanisms [Id., citing Garda- Sastre, A. Cell Host Microbe (2017) 22: 176-84].
  • the NS1 proteins of some IAVs are capable of inhibiting the 3’ end processing of cellular pre-mRNAs by binding to cleavage and polyadenylation specific factor (CPSF30) and accordingly blocking the production of mature mRNAs, including those of IFN- ⁇ and IFN- ⁇ [Id., citing Krug, RM. Curr. Opin. Virol. (2015) 12: 106] [00053] IFN- ⁇ . IFN- ⁇ was first described in 2004. It consists of 192 amino acids and shares about 30% homology with IFN- ⁇ and IFN- ⁇ in humans [Li, S-F et al. Cell Physiol. Biochem. (2016) 51: 2377-96, citing Zwarthoff, EC et al.
  • IFN- ⁇ is constitutively expressed in the lung, brain, skin, small intestine, rectum, jejunum, and reproductive tissues; it shows substantial expression in the uterus, cervix, vagina, and ovarian tissue [Id., citing Zwarthoff, EC et al. Nucleic Acids Res. (1985) 13: 791- 804; Demers, A. et al. J. Leukoc. Biol. (2014) 96: 1101-7].
  • IFN- ⁇ also exerts its biological activity by stimulating immune mediators and activating the JAK-STAT signal pathways in vitro and in vivo [Id., citing Zwarthoff, EC et al. Nucleic Acids Res. (1985) 13: 791-804].
  • a recombinant vaccinia virus co-expressing HIV gag or pol genes and murine IFN- ⁇ (VV-HIV- IFN- ⁇ ) inhibits growth of VV in L929 murine cell lines and increases upregulation of activation markers (CD69 and CD86) and antiviral protein expression [Id., citing Day, SL et al. J. Immunol. (2008) 180: 7158-66].
  • IFN- ⁇ has a higher binding affinity for IFNAR1 [Id., citing Stifter, SA et al. J. Biol. Chem. (2016) 293: 3168-79]. [00056] There are differences between IFN- ⁇ and other IFNs. First, the antiviral, natural killer cell-cytotoxicity activity and antiproliferative activities of IFN- ⁇ are weaker than IFN- ⁇ and IFN- ⁇ [Id., citing Peng, FW et al. Protein Expr. Purific. (2007) 53: 356-62].
  • IFN- ⁇ exhibits antiviral activity against cells derived from species that have near relatives and are expected to be homologous cells [Id., citing Guo, Y. et al. Gene (2015) 558: 25-30; Yang, L. et al. J. Interferon Cytokine Res. (2013) 33: 760-68].
  • IFN- ⁇ differs from IFN- ⁇ in macrophages by inducing an antiviral state mediated by different factors [Id., citing Li, SF et al. Intl Immunopharmacol. (2017) 52: 253-60].
  • IFN- ⁇ mediates more genes and upregulates genes more than IFN- ⁇ in the type I IFN signaling pathway, whereas IFN- ⁇ induces more genes in the TNF- ⁇ pathway and more ROS generation and phagocyte activation than IFN- ⁇ , to block HIV replication [Id., citing Li, SF et al.
  • IFN- ⁇ IFN- ⁇ genes have been identified in humans as well as in other animal groups including feline, porcine, equine, rabbit, cattle, and serotine bat, but not noted in canines or mice [Id., citing Zhou, H. et al. Int. J. Mol. Sci. (2014) 15: 21045-68]. Treatment with IFN- ⁇ is suggested to be effective for patients who are resistant to IFN- ⁇ , because the antigenic structure of IFN- ⁇ is distantly related to IFN- ⁇ , ⁇ , ⁇ , with no crossreaction with antibodies against these other IFNs [Id., citing Adolf, GR. Mult. Scler.
  • FeIFN- ⁇ Recombinant FeIFN- ⁇ is approved for treatment of FLV and FIV infections in some countries.
  • FIFN- ⁇ has 13 subtypes that have high similarity (95% to 99%) at the nucleic acid and amino acid level. All of them contain an N-terminal secretory signal sequence at position 1 to 23. Lengths of mature FeIFN- ⁇ subtype polypeptides are 173 aa (except FeIFN- ⁇ 2 and FeIFN- ⁇ 4 which have 180 aa).
  • the mature amino acid sequence of FeIFN- ⁇ has six additional amino acids at the carboxyl-end and an N-glycosylation recognition site that differs from other mammalian subtypes [Id., citing Yang, LM et al. J.
  • IFN- ⁇ has antiviral activities similar to other types I IFNs. However, unlike IFN- ⁇ , it has cross-species activity to some extent. This activity indicates that cells have a tendency to be insensitive to IFN- ⁇ from distantly related species [Id., citing Guo, Y. et al. Gene (2015) 558: 25-30]. IFN- ⁇ is involved in the nonspecific response based on increased expression of several acute phase proteins and MHC I molecules; upregulation of the phagocytic activities of whole blood cells, macrophages and NK cell activities; and decreased concurrent viral excretion [Id., citing Domenech, A. et al. Vet Immunol. Immunopathol. (2011) 143: 301-6; Gil, S.
  • IL-6 plasma levels decrease and proviral load increase in FIV-cats treated with rFeIFN- ⁇ by a subcutaneous licensed protocol.
  • IL-6 mRNA expression decreases in an oral group.
  • Viremia and other cytokines do not change with therapy [Id., citing Leal, RO et al. Res. Vet Sci. (2015) 99: 87-95].
  • Cytotoxic effects e.g. apoptosis, necrosis, and early senescence
  • human IFN ⁇ gene lipofection induced by catiolic lipid-mediated interferon-beta gene transfer to human tumor cells were reported to show the same or a superior effect to that of high doses of the exogenously applied recombinant IFN ⁇ protein [Id., citing Villaverde, MS, et al., Cancer Gene Ther.
  • IFN- ⁇ lipofection and expression is equal to or more effective than rFIFN- ⁇ protein at suppressing cell growth by inducing ROS generation, mitochondrial potential disruption and calcium uptake [Id., citing Villaverde, MS et al. Cytokine 84: 47-55].
  • IFN- ⁇ is mainly expressed in the uterus [Li, S-f et al. Cell Physiol. Biochem. (2016) 51: 2377-96], although it is also detected in other tissues, such as Peyer’s patch, ovary, liver and peritoneal macrophages, although at low levels.
  • IFNK gene On the basis of the identification of a mutant IFNK gene in human lung epithelial A549 cells and subsequent demonstration that wild-type IFN- ⁇ , but not the mutant, failed to contain IAV in cultured human cells, they pinned down chromodomain helicase DNA binding protein 6 (CHD6) as the major effector molecule mediating the anti-influenza activity of IFN- ⁇ . Compared to its induction by IFN- ⁇ , CHD-6 was less induced by IFN- ⁇ and IFN- ⁇ [collectively IFN- ⁇ / ⁇ ] and dispensible for IFN- ⁇ / ⁇ -mediated inhibition of IAV replication. They also identified the upstream signaling required by IFN- ⁇ to stimulate CHD6 expression.
  • CH6 chromodomain helicase DNA binding protein 6
  • IFN- ⁇ Unlike IFN- ⁇ / ⁇ , which transduce antiviral signal preferentially through IFNAR1, IFN- ⁇ required the engagement of both IFNAR1 and IFNAR2. [00061] The binding by IFN- ⁇ to the individual IFNARs is weaker than the binding of IFN- ⁇ / ⁇ [Id., citing Harris, BD et al. J. Biol. Chem. (2016) 293: 16057-068], suggesting different modes of action between IFN- ⁇ / ⁇ and IFN- ⁇ . IFN- ⁇ also was distinct in that it induced CHD6 through a p38-cFos axis, rather than the canonical JAK-STAT pathway.
  • IFN ⁇ / ⁇ therefore use multiple signaling pathways to activate a diverse array of ISGs, exerting profound effects on both virus and cells, while IFN- ⁇ instead exhibited a selective use of downstream signaling, resulting in a relatively narrower spectrum of downstream targets, among which some effector genes are preferentially stimulated, as seen for CHD6.
  • Such focused strategy underlying the observed dominance of a single effector molecule in the antiviral activity of IFN- ⁇ -CHD6 for influenza as shown here, and Sp100 for human papillomavirus (HPV) as shown in a previous study [Id., citing Habiger, C. et al. J. Virol.
  • IFN- ⁇ seems more closely related to the IFN- ⁇ , IFN- ⁇ , and IFN- ⁇ cluster than to IFN- ⁇ .
  • the IFN- ⁇ family comprises a large number of members with diversity that is greater than other multigene porcine or horse IFN families such as IFN- ⁇ and IFN- ⁇ [Id., citing Zhao, X. et al. J. Interferon Cytokine Res.
  • IFN- ⁇ s are probably highly glycosylated, as they display one or two potential N-glycosylation sites in OvIFN- ⁇ and the porcine IFN. All IFN- ⁇ s have two other cysteine residues, Cys77 and Cys128, except for IFN- ⁇ 2 and IFN- ⁇ 7, which have an additional COOH-terminal cysteine residue, Cys166. IFN- ⁇ s exhibit antiviral and immunomodulatory activity through typical type I IFN signaling, with lower antiviral activities than IFN- ⁇ [Id., citing Sang, Y. et al. Physiol. Genomics (2010) 42: 248-58]. However, the differential affinity of IFN- ⁇ for certain hosts could influence their biological activities.
  • IFN- ⁇ which is involved in maternal recognition of pregnancy, shares about 75% identity with IFN- ⁇ and has 172 aa with two disulfide bridges (1-99, 29-139) and an amino terminal proline. Not every IFN- ⁇ is glycosylated. For instance, ovine IFN- ⁇ lacks glycosylation, while bovine IFN- ⁇ is N-glycosylated at ASN78 and caprine IFN- ⁇ is a mixture of nonglycosylated and glycosylated forms.
  • IFN- ⁇ has 85% sequence identity to IFN- ⁇ in ruminants [Id., citing DeCarlo, CA et al. Lab Invest. (2010) 90: 1482-91].
  • IFN- ⁇ possesses antiviral activity and antiproliferative effects.
  • IFN- ⁇ has a receptor binding domain at the C-terminus and a biologically active site at the N-terminus [Id., citing Pontzer, CH et al. J. Interferon Res. (1994) 14: 133-41].
  • IFN- ⁇ is suggested to have comparable antiviral activity effects as IFN- ⁇ from the same species.
  • IFN- ⁇ also stimulates some interleukin expression and secretion such as IL-6 and IL-8.
  • the mechanism involved in inducing cytokine secretion is dependent on STAT3 rather than STAT1 signaling [Id., citing Tanikawa, N. et al. J. Interferon Cytokine Res. (2017) 37: 456-66].
  • IFN- ⁇ displays > 30 times less toxicity than IFN- ⁇ [45, 88]. This difference in cytotoxicity is illustrated by the differential selectivity of individual N-termini towards receptors and the differential degree of receptor avidity [Id., citing Pontzer, CH et al. J. Interferon Res. (1994) 14: 133-41]. [00065] IFN- ⁇ . A seemingly secreted glycoprotein, IFN- ⁇ is composed of 182 aa residues with a signal peptide of 21 amino acids at the N-terminal end and an N-linked glycosylation site at amino acid residue 68. IFN- ⁇ lacks an internal transmembrane domain.
  • IFN- ⁇ shares high nucleotide homology with IFN- ⁇ and IFN- ⁇ at residues 45-60, 105-115, and 135-165, corresponding to the N-terminal half of the AB loop, the C helix, and the DE loop, together with helices D and E [Id., citing Oritani, K.
  • IFN- ⁇ shares activities with other type I IFNs such as IFN- ⁇ : It induces the surface expression of MHC class I, enhances CTL activities, and inhibits growth of lymphohematopoietic cell lines as strongly as IFN- ⁇ .
  • This kind of IFN has relatively higher antiviral activity than IFN- ⁇ [Id., citing Kawamoto, S. et al. Exp. Hematol. (2004) 32: 797- 805].
  • IFN- ⁇ Signals induced by IFN- ⁇ are similar but distinct in contrasted to signals of other type I IFNs.
  • IFN regulatory factor-1 IRF-1
  • IRF-1-deficient fibroblasts IRF-1-deficient fibroblasts
  • IFN- ⁇ does not inhibit colony production of myeloid and erythroid progenitors while IFN- ⁇ is known to suppress lymphohematopoiesis [Id., citing Kawamoto, S. et al. Exp. Hematol. (2004) 32: 797-805].
  • IFN- ⁇ suppresses the proliferation of megakaryocyte progenitors without influencing megakaryocyte differentiation, although higher concentrations are required.
  • a possible explanation is that IFN- ⁇ induces lower expression of Daxx and weaker phosphorylation of Tyk2 and Crk than IFN- ⁇ [Id., citing Ishida, N. et al. Exp. Hematol.
  • IFN- ⁇ Formation of IFN- ⁇ is distinct from other known IFNs. IFN- ⁇ is constitutively produced by mature T lymphocytes in the spleen and thymus and by bronchial epithelial and salivary duct cells in healthy mice [Id., citing Oritani, K. et al. Blood (2003) 101: 178-85] (V) Unlike IFN- ⁇ / ⁇ , IFN- ⁇ gene expression in lymph nodes is unchanged with lipopolysaccharide injection or herpes simplex virus infection [Id., citing Oritani, K. et al. Blood (2003) 101: 178-85]. [00067] Type II IFN- ⁇ .
  • IFN- ⁇ is the sole type II interferon; the dominating biological role of IFN- ⁇ seems to be stimulation of the adaptive immune system, primarily activation of T cells [ Zhou, Z. et al. J. Virology (2007) 81 (14): 7749-58., citing Biron, C. A. (1994). Curr. Opin. Immunol.6:530-538, Muller, U., et al. (1994) Science 264:1918-1921]. [00068] Type III IFNs.
  • Type III interferons are the products of three IFN- ⁇ genes, IL- 28A, IL-28B, and IL-29, which bind a heterodimeric IFN- ⁇ receptor composed on a unique IL- 28R ⁇ subunit and the ⁇ subunit of the IL-10 receptor.
  • the type III IFNR has a more restricted tissue distribution pattern.
  • the IL10R2 chain is ubiquitously expressed in all tissues and cells, the expression of the IFN ⁇ R1 varies widely between different organs and at the cellular level is restricted to epithelial cells. [Zhou, P.
  • Type I interferons are inducible and are synthesized by many cell types after infection by diverse viruses. Almost all types of cells can produce IFN- ⁇ and IFN- ⁇ in response to activation of several innate sensors.
  • type I interferons are induced by RIG-1 and MDA-5 (the sensors of cytoplasmic viral RNA) downstream of MAVs, and by signaling from cGAS (the sensor of cytoplasmic DNA) downstream of STING.
  • Plasmacytoid dendritic cells also called interferon-producing cells (IPCs) or natural interferon-producing cells, make abundant type I interferons, which may result from the efficient coupling of viral recognition by TLRs to the pathways of interferon production.
  • pDCs express a subset of TLRs that includes TLR-7 and TLR-9, which are endosomal sensors of viral RNA and of the nonmethylated CpG residues present in the genomes of many DNA viruses.
  • pDCs express CXCR3, a receptor for chemokines CXCL9, CXCL10, and CXCR11, which are produced by T cells, which allows pDCs to migrate from the blood into lymph nodes in which there is an ongoing inflammatory response to a pathogen.
  • Interferons help defend against viral infections in several ways. IFN- ⁇ induces cells to make IFN- ⁇ , thus amplifying the interferon response. Interferons act to induce a state of resistance to viral replication in all cells.
  • IFN- ⁇ and IFN- ⁇ bind to a common cell surface receptor, the interferon-a receptor (IFNAR), which uses the JAK and STAT pathways.
  • IFNAR uses the kinases Tyk2 and Jak1 to activate the factors STAT1 and STAT2, which can interact with IRF9 and form a complex called ISGF4, which binds to the promoters of many interferon stimulated genes (ISGs). [Janeway’s Immunology, 9th Ed.
  • ISG encodes the enzyme oligoadenylate synthetase, which polymerizes ATP into 2’-5’ linked oligomers, which activate an endoribonuclase that then degrades viral RNA.
  • a second protein induced by IFN- ⁇ and IFN- ⁇ is protein kinase R (PKR), a dsRNA-dependent protein kinase, which phosphorylates the ⁇ subunit of eukaryotic initiation factor 2 (eIF2 ⁇ ), thus suppressing protein translation and contributing to the inhibitor of viral replication.
  • PLR protein kinase R
  • eIF2 ⁇ eukaryotic initiation factor 2
  • IFIT1 and IFIT2 are GTPases belong to the dynamin protein family; how they interfere with virus replication is not understood. [Janeway’s Immunology, 9th Ed. (2017) Garland Science, New York, pp 122-125] [00072]
  • the interferon-induced protein with tetratricoid repeats (IFIT) family contains four human and three mouse proteins that function in restraining the translation of viral RNA into proteins. IFIT1 and IFIT2 can suppress the translation of normal capped mRNAs by binding to subunits of the eukaryotic initiation factor 3 (eIF3) complex, which prevents eIF3 from interacting with eIF2 to form the 43S pre-initiation complex.
  • eIF3 eukaryotic initiation factor 3
  • IFIT1 vesicular stomatitis virus
  • IFIT1 also suppresses translation of viral RNA that lacks a normal host modification of the 5’ cap.
  • Many viruses e.g., West Nile virus, and SARS coronavirus, have acquired a 2’e-O- methyltransferase (MTase) that produce cap-1 or cap-2 on their viral transcripts, thus evading restriction by IFIT1.
  • MTase 2’e-O- methyltransferase
  • IFITM interferon-induced transmembrane protein
  • IFITM2 interferes with membrane fusion in late endosomes, and so restricts the influenza A virus, which undergoes fusion there.
  • Interferons stimulate production of the chemokines CXCL9, CXCL10, and CXCL11, which recruit lymphocytes to sites of infection, and increases expression of MHC class I molecules on all types of cells.
  • Type 1 and 2 IFNs have been shown to suppress type 2 cytokine production by ILC2s, both in vitro and in vivo.
  • IFN- ⁇ and - ⁇ are macrophages and DCs.
  • IFN- ⁇ is produced by activated Th1 cells and ILC1s, including NK cells, which are activated mainly through TLRs.
  • NK cells which are activated mainly through TLRs.
  • ILC2s activated ILC2s.
  • Lipid inflammatory mediators Although lipids are primarily involved in the formation of cell membranes of organs, various reports have shown that bioactive lipids or lipid mediators also play crucial roles in immune responses and the maintenance of homeostasis. Cysteinyl leukotrienes (CysLTs) as well as prostaglandin (PG) D2 are products of arachidonic acid and were known to be major pro-inflammatory lipid mediators of allergic disorders from early days.
  • Cysteinyl leukotrienes Cysteinyl leukotrienes
  • PG prostaglandin
  • Mast cells activated by immunoglobulin (Ig) E-crosslinking are the major source of PGD2 in terms of quantity, but other leukocytes, including eosinophils, Th2 cells, DCs and cytokine-activated ILC2s, [Id., citing Maric J, et al. J Allergy Clin Immunol (2019) 143:2202–2214.e5] also produce PGD2. Since human ILC2s are identified as lineage-negative cells expressing chemoattractant receptor-homologous molecules on Th2 cells (CRTH2), [Id., citing Mjösberg JM, et al.
  • CRTH2 chemoattractant receptor-homologous molecules
  • CysLTs are generally produced by leukocytes such as eosinophils, mast cells, macrophages and basophils. CysLTs act directly on ILC2s to enhance their ability to produce type 2 cytokines, both in vivo and in vitro.
  • Neuropeptides are peptides that are expressed in the nervous system and exhibit physiological activity. They are present not only in the central nervous system, but also in the nervous system of peripheral tissues such as the lungs, and they function as signal transmitters between cells. Among several neuropeptides known to act on ILC2s, vasoactive intestinal peptide (VIP) was the first one shown to modulate ILC2 activation. VIP belongs to the glucagon/secretin family and is highly expressed in intestinal neurons, where it coordinates pancreatic secretion with smooth muscle relaxation in response to feeding.
  • VIP vasoactive intestinal peptide
  • Both lung and intestinal ILC2s express VIP receptors, including VIP receptor type 1 and type 2, and VIP simulation induces IL-5 production by the cells.
  • the IL-5 produced in turn activates sensory neurons to produce VIP [Id., citing Talbot S, et al. Neuron (2015) 87:341–354], which may exacerbate allergic airway inflammation.
  • Lung ILC2s also express receptors for another neuropeptide, called neuromedin U (NMU), whereas ILC1s and ILC3s do not. NMU is thought to directly activate lung ILC2s to proliferate and produce type 2 cytokines. [Id., citing Wallrapp A, et al. Nature (2017) 549:351–356].
  • Calcitonin gene-related peptide is a calcitonin gene product, like the thyroid hormone calcitonin and it is involved in the regulation of blood calcium levels.
  • CGRP Calcitonin gene-related peptide
  • PNECs pulmonary neuroendocrine cells
  • ILC2s are localized in close proximity to PNECs and that CGRP enhances type 2 cytokine production by lung ILC2s in the presence of IL-33 or IL-25, [Id., citing Sui P, et al. Science (2016) 360:eaan8546] suggesting that interaction between PNECs and ILC2s may be involved in allergic airway inflammation.
  • Both lung and intestinal ILC2s express the ⁇ 2-adrenergic receptor ( ⁇ 2-AR), which is a receptor for epinephrine released by sympathetic nerve stimulation.
  • ⁇ 2-AR ⁇ 2-adrenergic receptor
  • Sex Steroids Sex Steroids
  • ILC2s peripheral tissues
  • Androgen receptors are expressed on lung ILC2s [Id., citing Cephus JY, et al. Cell Reports (2017) 21:2487–2499] as well as ILC2 progenitors (ILC2Ps) in bone marrow (BM), [Id., citing Laffont S, et al. J Exp Med 2017;214:1581–1592], whereas estrogen receptors are expressed on lung ILC2s and uterine ILC2s [Id., citing Bartemes K, et al.
  • ILC3s IL-23 is a major activator of ILC3s that induces production of inflammatory cytokines such as IL-17 and IL-22. IL-23 also induces conversion of ILC1s to ILC3s in conjunction with IL-1 ⁇ and retinoic acid, [Id., citing Bernink JH, et al. Immunity (2015) 43:146–160] and ILC2s to ILC3s in conjunction with IL-1 ⁇ and TGF- ⁇ . [Id., citing Bernink JH, et al. Nat Immunol (2019) 20:992–1003, Golebski K, et al.
  • IL-23 is generally produced by DCs and macrophages.
  • IL-1 ⁇ [00080] IL-1 ⁇ is a major activator of ILC3s that induces IL-17A production. [Id., citing Kim HY, et al. Nat Med (2014) 20:54–61] While IL-1 ⁇ is a potent activator of ILC2s that induce type 2 cytokine production, [Id., citing Bal SM, et al. Nat Immunol (2016) 17:636–645] it also induces conversion of ILC2s to ILC1Ss together with IL-12, [Id., citing Bal SM, et al.
  • IL-1 ⁇ is produced by DCs in response to exposure to chitin and IL- 33 [Id., citing Arae, K. et al. Sci. Rep. (2016) 8: 11721] and by nasal epithelial cells exposed to Staphylococcus aureus or Pseudomonas aeruginosa.
  • Vitamins [00081] Retinoic acid (RA)–which is a metabolite of vitamin A (Vit A)–and vitamin D (Vit D) is known to regulate ILCs. [Id., citing Morita H, et al. J Allergy Clin Immunol (2019) 143:2190–2201.e9, Seehus CR, et al. Nat Commun (2017) 8:1900, Bernink JH, et al. Immunity (2015) 43:146–160, Golebski K, et al. Nat Commun (2019) 10:2162, Konya V, et al.
  • RA is synthesized from a Vit A metabolite, retinal, by cells having enzymes such as retinaldehyde dehydrogenase (ALDH)1A1, ALDH1A2 and ALDH1A3.
  • ADH retinaldehyde dehydrogenase
  • RA is generally synthesized by CD103+ DCs, intestinal epithelial cells and lamina intestinal stromal cells in the gut that express ALDHs.
  • bronchial epithelial cells express ALDHs in response to IL-13 stimulation [Id., citing Morita H, et al.
  • Vit D can be absorbed by oral intake, but it is synthesized mainly in the skin upon exposure to ultraviolet light from the sun.
  • RA enhances activation of ILC3s by IL-1 ⁇ and IL-23 to increase production of IL-22, and it also induces conversion of ILC1s to ILC3s in conjunction with IL-1 ⁇ and IL-23.
  • RA inhibits development of ILC2s from ILC2Ps in mouse BM69 and induces conversion of ILC2s to IL-10–producing ILCregs in both humans and mice.
  • ILC2Ps ILC2Ps
  • IL-10 ILCregs
  • RA inhibits development of ILC2s from ILC2Ps in mouse BM69 and induces conversion of ILC2s to IL-10–producing ILCregs in both humans and mice.
  • cytokines such as IL-22, IL-17F and GM-CSF
  • ILC3s are emerging as key orchestrators and regulators of adaptive immune responses, either through indirect modulation of bystander cells that subsequently modulate the adaptive immune response or directly via both soluble mediators and cell contact-dependent interactions with adaptive lymphocytes. [Domingues, RG, Hepworth, MR. Front. Immunol. (2020) 11:116].
  • ILC3s In addition to their function as tissue-resident cytokine producing cells, ILC3s have the capacity to participate in multiple cellular circuits through direct cell-cell modulation of T cell responses, as well as the release of soluble mediators that augment adaptive immune function and development. For example, ILC3s can control the magnitude and quality of the CD4+ T cell response via antigen presentation in the context of MHC class II. At steady state, ILC3s lack co-stimulatory molecule expression and appear to limit CD4+ T cell responses; however, this interaction may be altered in inflammatory scenarios via upregulation of costimulatory molecules such as CD4- CD80 and CD86, which favor the promotion of a T cell response.
  • costimulatory molecules such as CD4- CD80 and CD86
  • ILCs act to modulate the survival of recirculating memory CD4+ T cells via interactions via OX40L and CD30L.
  • ILC3 regulation of T follicular helper (TFH) cell responses has consequences for the priming of germinal center B cells and the induction of T dependent IgA responses toward colon-dwelling commensal microbes.
  • ILC3s also can modulate adaptive immune cells through the production of regulatory cytokines and growth factors. For example ILC3 directly support B cell responses in the spleen through provision of critical growth factors such as BAFF/APRIL. Similarly they modulate the magnitude of the T cell response within the intestinal tract through production of soluble mediators.
  • ILC3-derived IL-22 induces epithelial serum amyloid A (SAA) protein, which subsequently promotes local Th17 responses and acts to limit colonization with segmented filamentous bacteria (SGF) via induction of antimicrobial peptides.
  • SAA epithelial serum amyloid A
  • SGF segmented filamentous bacteria
  • ILC3 facilitate the establishment of a regulatory and tolerogenic environment in the gut by promoting Treg responses.
  • ILC subsets are a potent source of IL-2 in the small intestine which provide survival signals for Tregs. [Domingues, RG, Hepworth, MR. Front. Immunol. (2020) 11:116]. [00083] While most pathogens can overcome innate immune responses, the adaptive immune response is required to eliminate them and to prevent subsequent reinfection.
  • the adaptive arm involves a specific, delayed and longer-lasting response by various types of cells that create long-term immunological memory against a specific antigen. It can be further subdivided into cellular and humoral branches, the former largely mediated by T cells and the latter by B cells. This arm further encompasses cell lineage members of the adaptive arm that have effector functions in the innate arm, thereby bridging the gap between the innate and adaptive immune response. [00085] Generally speaking, these immune responses are initiated by an encounter between an individual and a foreign substance, e.g., an infectious microorganism.
  • the infected individual rapidly responds with both a humoral immune response with the production of antibody molecules specific for the antigenic determinants/epitopes of the immunogen, and a cell mediated immune response with the expansion and differentiation of antigen-specific regulatory and effector T-lymphocytes, including cells that produce cytokines and killer T cells, capable of lysing infected cells.
  • Primary immunization with a given microorganism evokes antibodies and T cells that are specific for the antigenic determinants/epitopes found on that microorganism; these usually fail to recognize or recognize only poorly antigenic determinants expressed by unrelated microbes [Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed.
  • Nonconventional lymphocytes bearing a phenotype that is rare in the blood, spleen, or lymph nodes of undiseased individuals are encountered at high frequency in different localizations, e.g., alpha/beta TCR+CD4-CD8- cells in the bone marrow and gut epithelium, particular invariant gamma/delta TCR+CD4-CD8 alpha+CD8 beta- and gamma/delta TCR+CD4-CD8 alpha-CD8 beta- T cells in various epithelia, or CD5+ B cells in the peritoneum.
  • the antigen receptor repertoire is different in each localization.
  • gamma/delta TCR gene products dominant in each site, and the proportion of cells expressing transgenic and endogenous alpha/beta TCR and immunoglobulin gene products follows a gradient, with a maximum of endogenous gene expression in the peritoneum, intermediate values in other peripheral lymphoid organs (spleen, lymph nodes), and minimum values in thymus and bone marrow.
  • Forbidden T cells that bear self-superantigen-reactive V beta gene products are physiologically detected among alpha/beta TCR+CD4-CD8- lymphocytes of the bone marrow, as well as in the gut.
  • self-peptide-specific gamma/delta T cells are present among intestinal intraepithelial lymphocytes, and CD5+ B cells produce low-affinity cross- reactive autoantibodies in a physiological fashion. It appears that, in contrast to the bulk of T and B lymphocytes, certain gamma/delta and alpha/beta T cells found in the periphery, as well as most CD5+ B cells, do not depend on the thymus or bone marrow for their development, respectively, but arise from different, nonconventional lineages.
  • local induction or selection processes may be important in the diversification of peripheral lymphocyte compartments. Selection may be exerted by local antigens, antigen-presenting cells whose function varies in each anatomical localization, cytokines, and cell-matrix interactions, thus leading to the expansion and maintenance of some clones, whereas others are diluted out or deleted.
  • ECM extracellular matrix
  • ECM electrospray mediated endoderms
  • the ECM helps hold the cells and compartments together and provides an organized lattice or scaffold within which cells can migrate and interact with one another.
  • cells in a compartment can be held in place by direct cell-cell adhesions.
  • such compartments include four major types, a connective tissue (CT) compartment, an epithelial tissue (ET) compartment, a muscle tissue (MT) compartment and a nervous tissue (NT) compartment, which are derived from three embryonic germ layers: ectoderm, mesoderm and endoderm.
  • CT connective tissue
  • ET epithelial tissue
  • MT muscle tissue
  • NT nervous tissue
  • the NT and portions of the ET compartments are differentiated from the ectoderm; the CT, MT and certain portions of the ET compartments are derived from the mesoderm; and further portions of the ET compartment are derived from the endoderm.
  • HSC hematopoietic stem cells
  • HSCs Hematopoietic stem cells
  • anatomical sites the yolk sac, the aorta-gonadmesonephros region, the placenta and the fetal liver
  • HSC precursors Once HSC precursors have been specified from mesoderm, they have to mature into functional HSCs and undergo self-renewing divisions to generate a pool of HSCs.
  • developing HSCs migrate through various embryonic niches, which provide signals for their establishment and the conservation of their self-renewal ability. [Mikkola, HKA, Orkin, SH. Development (2006) 133: 3733-44].
  • B & T lymphocytes have to receive contact-dependent activation signals from immobile cells in situ, and exert the majority of their functions via direct intercellular interactions. Lymphocytes are influenced in their behavior by local antigens and metabolites and are embedded in a complex network of interactions with neighboring accessory cells (e.g., B cells and macrophages. ECM proteins continuously interact with signal-transducing receptors on lymphoid cells. [Kroemer, G. et al. Adv. Immunol. (1993) 53: 157-216]. [00091] Lymphocytes located outside of the thymus and bone marrow are considered as peripheral cells.
  • Peripheral lymphocytes are contained in the classic lymphoid organs (spleen, lymph nodes, tonsils and Peyer’s patches), the epidermis, the mucosae of the gastrointestinal, respiratory, and female reproductive tracts, and in mesoderm derivatives (e.g., the pleuroperitoneal cavity. Lymphocytes in the lung interstitium are as numerous as those of the circulating blood pool. [Kroemer, G. et al. Adv. Immunol. (1993) 53: 157-216, citing Pabst, R. (1992) Immunology Today 13: 119-22]. [00092] T cells are extremely heterogeneous in specificity, activation requirements, life span, and functional properties.
  • T cells produce a nearly infinite antigen receptor repertoire via somatic diversification processes, including gene rearrangements and somatic mutation. They can also be classified into subpopulations that differ in the expression of classes of the T cell receptor (TCR; ⁇ / ⁇ or ⁇ / ⁇ heterodimers) and CD antigens, in the activation state, or in functional terms.
  • TCR T cell receptor
  • CD4 and CD8 are found on mutually exclusive ⁇ / ⁇ T lymphocyte subsets in the periphery.
  • CD4+ ⁇ / ⁇ T cells are predominantly of the helper phenotype, whereas CD8 (usually a heterodimer composed of CD8 ⁇ and CD8 ⁇ is mainly expressed on cytotoxic and suppressor T cells.
  • CD4- T lymphocytes can effect cytotoxicity and suppression, and a more stringent correlation exists between CD4/CD8 expression and MHC gene products expressed by target or antigen presenting cells (APC).
  • CD4+ T cells interact with cells expressing MHC class II; whereas CD8+ T cells are class I restricted [Id., citing Moller, G. (Ed ) Immunol. Rev. (1989) 109: 5-153, Parnes, JR. Adv. Immunol.44: 265-311; Bierer, BE et al. (1989) Annu. Rev. Immunol. (1989) 7: 579-99; Auffray, C. et al. Trends Biotechnol.
  • PBLs Peripheral blood lymphocytes
  • B cells T cells and natural killer cells.
  • T lymphocytes the distribution of B cells follows a nonrandom pattern.
  • IgA-bearing lymphocytes are highly represented in mucosa-associated lymphoid structures (e.g., lamina intestinal and Peyer’s patches), the nonkeratinizing external surfaces of the body (gut and exocrine glands, including the lactating mammary gland, urogenital epithelia and upper respiratory tract) attract predominantly IgA-secreting plasma cells.
  • mucosa-associated lymphoid structures e.g., lamina limbaloid structures
  • the nonkeratinizing external surfaces of the body gut and exocrine glands, including the lactating mammary gland, urogenital epithelia and upper respiratory tract
  • IgA secreting cells are infrequent and most plasma cells secrete IgM or IgG.
  • lymphoid follicle is a compartment of primarily B cells, which represents a unique microenvironment.
  • VH gene families are also inhomogeneous [Kroemer, G. et al. Adv. Immunol. (1993) 53: 157-216, citing, Freitas, A.A. et al. Int. Immunol. (1989) 1: 342-54].
  • B cells may be divided into two classes according to the expression of CD5, a signal-transducing receptor [Id., citing Alberola-Ila, J. et al. J. Immunol.
  • B1 cells represent the CD5+ (Ly-1+) subset, and have the phenotype IgM high IgD low -Mac-1 (CD11b/CD18)+CD45 low FceR-IL-5R+.
  • B2 “ conventional” cells have a similar phenotype except that they lack CD5 (Id. citing Hayakawa, K., et al. J. Exp. Med. (1983) 157: 202-15; Wetzel, GD. Eur. J. Immunol.
  • CD5+ B cells are endowed with the capacity of self-renewal, i.e., they may expand in the absence of any cell input from IgM- precursors, unlike conventional B cells [Id., citing Immunberg, LA, et al.
  • Memory T cells [00096] The vast majority of human memory T cells reside in tissue sites, including lymphoid tissues, intestines, lungs and skin. By the end of puberty, lymphoid tissues, mucosal sites and the skin are populated predominantly by memory T cells, which persist throughout adult life and represent the most abundant lymphocyte population throughout the body.
  • Memory T cells in humans are classically distinguished by the phenotype CD45RO+CD45RA-, and comprise heterogeneous populations of memory T cell subsets.
  • Na ⁇ ve T cells uniformly express CCR7, reflecting their predominant residence in lymphoid tissue.
  • Memory T cells are subdivided into CD45RA-CCR7+ central memory T (TCM) cells, which traffic to lymphoid tissues, and CD45RA-CCR7- effector memory T (TEM) cells, which can migrate to multiple peripheral tissue sites.
  • TCM central memory T
  • TEM CD45RA-CCR7- effector memory T
  • both T CM and T EM cell subsets produce effector cytokines in response to viruses, antigens and other stimuli [Id., citing Wang A, et al. Sci Transl Med. (2012) 4:149ra12030-33; Pedron B, et al. Pediatr Res. (2011) 69:106–111; Champagne P, et al. Nature. (2001) 410:106–111; Ellefsen K, et al. Eur J Immunol. (2002) 32:3756–3764], although TCM cells exhibit a higher proliferative capacity. (Id. citing Wang A, et al. Sci Transl Med. (2012) 4:149ra120, Fearon DT, et al.
  • T SCM Tmemory stem
  • TRM tissue resident memory T
  • Mouse CD4+T RM cells can be generated in the lungs from adoptive transfer or activated (effector) T cells [Id., citing Teijaro JR, et al. J Immunol. (2011) 187:5510–5514] or following respiratory virus infection [Id., citing Turner, DL, et al. Mucosal Immunol. (2014) 7 (3): 501- 510], and are distinguished from splenic and circulating memory T cells by their upregulation of the early activation marker CD69, their tissue-specific retention in niches of the lung [Id., citing Turner, DL, et al. Mucosal Immunol.
  • CD4+ TRM cells generated following infection have been identified in multiple mouse tissues, including skin [Id., citing Clark RA, et al. Sci Transl Med.
  • memory CD4+ T cells predominate throughout the body and persist as CCR7+ or CCR7- subsets localized to lymphoid tissues and mucosal sites, respectively, whereas memory CD8+ T cells persist as mainly CCR7- subsets in all sites, with low numbers of CD8 TCM cells in lymphoid tissues and negligible numbers of these cells in other sites [Id., citing Sathaliyawala T, et al. Immunity (2013) 38:187–197]. Most memory T cells in human mucosal, lymphoid and peripheral tissue sites such as skin express the putative T RM cell marker CD69 [Id., citing Goronzy JJ, Weyand CM. Nat Immunol.
  • memory T cells in the small intestine and colon express the gut- homing receptor CCR9 [Id., citing Kunkel EJ, et al. J Exp Med. (2000) 192:761–768] and the integrin ⁇ 4 ⁇ 7 [Id., citing Agace WW. Trends Immunol. (2008) 29:514–522], and memory T cells in the lungs upregulate CCR6 expression [Id., citing Purwar R, et al. PLoS One. (2011) 6:e16245].
  • mucosal sites such as lung and intestines.
  • lung dendritic cells induce migration of protective T cells to the gastrointestinal tract.
  • TRM can be multifunctional and also exhibit qualitative functional differences.
  • a substantial fraction of human lung T RM cells produce multiple pro- inflammatory cytokines [Id., citing Purwar R, et al. PLoS One. 2011;6:e16245], and human intestinal TRM cells are also multifunctional [Id. citing Sathaliyawala T, et al. Immunity. 2013;38:187–197].
  • Other functions appear to be confined to specific subsets and/or tissue sites.
  • IL-17 is produced by a subset of CD4+ T RM cells in mucosal sites, particularly in intestines in healthy individuals [Id., citing Sathaliyawala T, et al. Immunity (2013) 38:187– 197], by CCR6+ memory T cells in peripheral blood [Id., citing Singh SP, et al. J Immunol. (2008) 180:214–221, Wan Q, et al. J Exp Med. (2011) 208:1875–1887], and by a subset of CD161+ T cells in inflamed tissue, such as the skin of patients with psoriasis [Id., citing Cosmi L, et al.
  • TRM cells in tissue sites can adopt multiple or distinct functional attributes, which may also depend on tissue-specific inflammation.
  • human memory T cells exhibit cross-reactivity to antigenic epitopes not previously encountered, which may be due to intrinsic properties of TCR recognition [Id., citing Sewell AK. Nat Rev Immunol.2012;12:669–677] and to the range and breadth of human antigenic experience.
  • CD4+ and CD8+ T cells specific for unique epitopes of avian influenza strain H5N1 were detected in healthy individuals that were not exposed to H5N1 infection assessed by serology [Id., citing Lee LY, et al. J Clin Invest. (2008) 118 (10): 3478-90; Roti M, et al. J Immunol. (2008) 180:1758–1768].
  • HIV- specific memory T cells have been identified in HIV-negative individuals [Id., citing Su, LF et al. Immunity (2013) 38: 373-83].
  • Virus-specific memory T cells also show cross-reactivity to alloantigens, autoantigens and unrelated pathogens [Id., citing D'Orsogna LJ, et al. Transpl Immunol. (2010) 23:149–155, Wucherpfennig KW. Mol Immunol. (2004) 40:1009–1017]: EBV-specific human memory T cells generated in HLA-B8 individuals exhibit allogeneic cross-reactivity to HLA-B44 [Id., citing Burrows SR, et al. J Exp Med.
  • T cells recognize epitopes from unrelated microbial pathogens [Id., citing Su LF, et al. Immunity. (2013) 38:373–383]. Furthermore, T cells specific for the autoantigen myelin basic protein (MBP) recognized multiple epitopes from viral and bacterial pathogens [Id., citing Wucherpfennig KW. Mol Immunol. (2004) 40:1009–1017, Wucherpfennig KW, Strominger JL. Cell. (1995) 80:695–705].
  • MBP myelin basic protein
  • This cross- reactivity may enable memory T cells to mediate protection without initial disease — a phenomenon known as heterologous immunity [Id., citing Welsh RM, Selin LK. Nat Rev Immunol. (2002) 2:417–426]. Heterologous immunity has been demonstrated in humans where EBV infection expanded clones of influenza virus-specific T cells [Id., citing Clute SC, et al. J Clin Invest.2005;115:3602–3612]. [000103] Analysis of human samples has revealed that influenza-specific TRM can be found in substantial numbers in lung tissue, highlighting their role in natural infection.
  • CD8+ T RM had a diverse T cell receptor (TCR) repertoire, high proliferative capacities, and were polyfunctional [Muruganandah, V., et al. (2016). Front. Immunol., 9, 1574. doi:10.3389/fimmu.2018.01574].
  • Influenza infection history suggests a greater level of protection against re-infections is likely due to the accumulation of CD8+ TRM in the lungs.
  • the natural immune response to influenza A virus infection in a rhesus monkey model demonstrated that a large portion of influenza-specific CD8+ T cells generated in the lungs were phenotypically confirmed as CD69+CD103+ TRM.
  • CD8+ T cells are effectors when an antigen is present and become memory when the antigen is eliminated.
  • a memory T cell population comprises multiple subtypes of cells, distributed in diverse anatomic compartments and possibly recirculating among them.
  • the memory CD8+ T cell response to most viruses is diverse in phenotype and function and undergoes dynamic changes during its development and maintenance in vivo. This heterogeneity is related to the nature of the infecting virus, its cellular tropism, the anatomic location of the infection, and the location of the CD8+ T cells.
  • Mucosal immune system While the mucosal surfaces of the body have a protective barrier of mucus, they are highly vulnerable to infection and possess a complex array of innate and adaptive mechanisms of immunity. The adaptive immune system of the mucosa-associated lymphoid tissues differs from that of the rest of the peripheral lymphoid system in several respects.
  • T cells The types and distribution of T cells differ, with significantly greater numbers of ⁇ : ⁇ T cells in the gut mucosa compared with peripheral lymph nodes and blood.
  • the major antibody type secreted across the epithelial cells lining mucosal surfaces is secretory polymeric IgA.
  • IgA The major antibody type secreted across the epithelial cells lining mucosal surfaces.
  • the mucosal immune system protects internal mucosal surfaces, such as the linings of the gut, respiratory tract and urogenital tracts, which are the site of entry for virtually all pathogens and other antigens.
  • the mucosa-associated lymphoid tissues lining the gut are known as gut-associated lymphoid tissue or GALT.
  • the other principal sites within the gut mucosal immune system for the induction of immune responses are the Peyer's patches of the small intestine, the appendix, and solitary lymphoid follicles of the large intestine and rectum.
  • Peyer's patches are an important site for the induction of immune responses in the small intestine and have a distinctive structure, forming domelike structures extending into the lumen of the intestine.
  • the overlying layer of follicle-associated epithelium of the Peyer's patches contains specialized epithelial cells (microfold cells or M cells) that have microfolds on their luminal surface, instead of the microvilli present on the absorptive epithelial cells of the intestine. They are much less prominent than the absorptive gut epithelial cells, known as enterocytes, and form a membrane overlying the lymphoid tissue within the Peyer's patch.
  • M cells lack a thick surface glycocalyx and do not secrete mucus, they are adapted to interact directly with molecules and particles within the lumen of the gut. M cells take up molecules and particles from the gut lumen by endocytosis or phagocytosis. This material is then transported through the interior of the cell in vesicles to the basal cell membrane, where it is released into the extracellular space by transcytosis. At their basal surface, the cell membrane of M cells is extensively folded around underlying lymphocytes and antigen-presenting cells, which take up the transported material released from the M cells and process it for antigen presentation. [Immunobiology: The Immune System in Health and disease. Janeway, CA et al Eds., 5 th Ed.
  • lymphoid tissue in which induction of immune responses occurs within the mucosal immune system, small foci of lymphocytes and plasma cells, which are scattered widely throughout the lamina intestinal of the gut wall, represent the effector cells of the gut mucosal immune system. As naive lymphocytes, these cells emerge from the primary lymphoid organs of bone marrow and thymus to enter the inductive lymphoid tissue of the mucosal immune system via the bloodstream. They may encounter foreign antigens presented within the organized lymphoid tissue of the mucosal immune system and become activated to effector status.
  • the activated lymphocytes traffic via the lymphatics draining the intestines pass through mesenteric lymph nodes, and eventually wind up in the thoracic duct, from which they circulate in the blood throughout the entire body. They reenter the mucosal tissues from the small blood vessels lining the gut wall and other sites of MALT, such as the respiratory or reproductive mucosa, and the lactating breast; these small vessels express the mucosal adressin MAdCAM-1. In this way, an immune response that may be started by foreign antigens presented in a limited number of Peyer's patches is disseminated throughout the mucosa of the body.
  • lymphocyte trafficking is distinct from and parallel to that of lymphocytes in the rest of the peripheral lymphoid system.
  • Immunobiology The Immune System in Health and disease. Janeway, CA et al Eds., 5 th Ed. (2001) Garland Publishing, New York, Ch.10, p.482-493].
  • the distinctiveness of the mucosal immune system from the rest of the peripheral lymphoid system is further underlined by the different lymphocyte repertoires in the different compartments.
  • the T cells of the gut can be divided into two types. One type bears the conventional ⁇ : ⁇ T-cell receptors in conjunction with either CD4 or CD8, and participates in conventional T-cell responses to foreign antigens.
  • the second class is made up of T cells with unusual surface phenotypes such as TCR ⁇ : ⁇ and CD8 ⁇ : ⁇ TCR ⁇ : ⁇ .
  • the receptors of these T cells do not bind to the normal MHC:peptide ligands. Instead, they bind to a number of different ligands, including MHC class IB molecules.
  • These highly specialized T cells are abundant in the epithelium of the gut and have a restricted repertoire of T-cell receptor specificities. Unlike conventional T cells, many of these cells do not undergo positive and negative selection in the thymus, and express receptors with sequences that have undergone no or minimal divergence from their germline-encoded sequences. These cells may be classified in phylogenetic terms as being at the interface between innate and adaptive immunity.
  • T cells bearing a ⁇ : ⁇ receptor are especially abundant in the gut mucosa compared with other lymphoid tissues.
  • T-cell receptor that uses the V ⁇ 1 gene segment, carries an activating C-type lectin NK receptor, NKG2D.
  • NKG2D binds to two MHC-like molecules—MIC-A and MIC-B—that are expressed on intestinal epithelial cells in response to cellular injury and stress.
  • the injured cells may then be recognized and killed by the subset of ⁇ : ⁇ T cells.
  • the V ⁇ 1-containing receptor on these T cells may also play a part in allowing them to survey tissues for injured cells.
  • Some human T cells expressing this receptor bind to CD1c, one of the isotypes of the CD1 family of MHC class I-like molecules. This protein, which shows increased expression on activated monocytes and dendritic cells, presents endogenous lipid and glycolipid antigens to some types of T cell.
  • CD1c one of the isotypes of the CD1 family of MHC class I-like molecules.
  • This protein which shows increased expression on activated monocytes and dendritic cells, presents endogenous lipid and glycolipid antigens to some types of T cell.
  • these T cells secrete IFN- ⁇ , which may have an important role in polarizing the response of conventional T cells bearing ⁇ : ⁇ receptors toward a TH1 response.
  • Respiratory syncytial virus (RSV), one of many ( ⁇ 200) viruses known as a common cold virus, predominately affects infants and leads to long ⁇ term lung disease.
  • RSV Respiratory syncytial virus
  • ⁇ 200 viruses known as a common cold virus
  • the contribution of ⁇ T cells to RSV infection has been tested in mice infected with RSV with or without immunization with a live vaccine vector expressing RSV F protein.
  • V ⁇ 4+ ⁇ T cells were enhanced in the lungs and produced IFN ⁇ , RANTES, IL ⁇ 10, IL ⁇ 4 and IL ⁇ 5 in a time ⁇ dependent manner after challenge of sensitized mice.
  • ⁇ T cells Depletion of ⁇ T cells reduced lung inflammation and disease severity and slightly increased peak viral replication without compromising viral clearance during secondary challenge in vaccinated mice. [Id., citing Dodd J, et al. J Immunol (2009) 182:1174–81].
  • ⁇ T cells are the main producers of IL ⁇ 17A.
  • Exogenous IL ⁇ 17A administration decreases inflammation in RSV ⁇ infected neonates, whereas neutralization of IL ⁇ 17A increases lung inflammation and airway mucus in RSV ⁇ infected adults.
  • RSV disease severity is in part mediated by a lack of IL ⁇ 17A+ ⁇ T cells in the lungs of neonates.
  • RSV infection elevates Th1 cytokine- and suppresses Th2 cytokine-expression in lung ⁇ T cells.
  • Ovalbumin (OVA) challenge induces a large influx of ⁇ T cells into the lungs.
  • OVA ⁇ induced infiltration and activation of ⁇ T cells were inhibited, suggesting that RSV protected against subsequent OVA ⁇ induced allergic responses by inhibiting Th2 ⁇ type ⁇ T cells.
  • V ⁇ 9V ⁇ 2 T cells that are activated in vitro by aminobisphosphonate pamidronate efficiently kill influenza virus-infected lung alveolar epithelial cells and inhibit virus replication in a cell ⁇ to ⁇ cell contact manner.
  • the cytotoxic activity of V ⁇ 9V ⁇ 2 T cells requires NKG2D activation and involves perforin/granzyme B, TRAIL and FasL. [Id., citing Li H, et al. Cell Mol Immunol (2013) 10:159–64, Tu W, et al.
  • Immunoglobulin A (sIgA) is the predominant antibody isotype in the mucosal immune system. Normally serum IgA shows a monomeric structure, while mucosal IgA is polymeric.
  • Secretory IgA (sIgA) comprises two monomeric IgAs, secretory component (SC) and J chain. There are also trimeric sIgA, tetrameric sIgA, and larger polymeric IgA in the upper respiratory tract of healthy humans. Among them, tetrameric IgA has broad neutralizing activity against influenza viruses [Li, Y. et al. Biomed. Res.
  • GALT gut ⁇ associated lymphoid tissue
  • PPs Peyer's patches
  • ILFs isolated lymphoid follicles
  • MNs mesenteric lymph nodes
  • GALT contains at least 80% plasma cells (PCs) and 90% sIgA of the body [Id., citing Brandtzaeg P., et al. (1999) 171(1):45–87]
  • NALT nasopharynx-associated lymphoid tissues
  • BALT bronchus-associated lymphoid tissues
  • Dendritic cells exist in the subepithelial dome (SED) beneath the follicle-associated epithelium (FAE) of the Peyer’s patches; mucosal antigens are captured by the underlying DCs which extend their dendrites [Id., citing Farache J., et al. Immunity. (2013) 38(3):581–595] or through the transcytosis of M cells [Id., citing Mabbott N. A., et al. Mucosal Immunology. (2013) 6(4):666– 677].
  • SED subepithelial dome
  • FAE follicle-associated epithelium
  • T cells and B cells Upon antigen presentation by DCs, T cells and B cells are activated and IgA class switch recombination (CSR) is mediated in the mucosal B cells.
  • CSR IgA class switch recombination
  • T cells participate in this process by either a T-dependent (TD) mechanism or a T-independent (TI) mechanism.
  • TD T-dependent
  • TI T-independent
  • the T-dependent mechanism requires interaction between CD40 on the surface of B cells and its ligand CD40L derived from T cells, resulting in high-affinity antigen-specific IgA production to neutralize pathogens [Id., citing Pabst O. Nature Reviews Immunology (2012) 12(12):821–832].
  • T follicular helper Tfh
  • Foxp3 +Treg T follicular helper
  • Th17 cells are involved in promoting the IgA response in the intestine by the release of various cytokines, such as IL ⁇ 4, IL ⁇ 5, IL ⁇ 6, IL ⁇ 10, IL ⁇ 13, IL-17A, and IL-21, to further promote the CSR to IgA [Id., citing Cao A. T., et al. Mucosal Immunology. (2015) 8(5):1072–1082].
  • the T-independent (TI) mechanism produces commensal-reactive IgA through innate immune cells such as innate lymphoid cells (ILCs) and plasmacytoid dendritic cells (pDC) [Id., citing Boyaka P. N. J. Immunology. (2017) 199(1):9– 16, Pabst O. Nature Reviews Immunology (2012) 12(12):821–832, Kubinak J. L., et al. Cell Host & Microbe. (2015) 17(2):153–163].
  • ILCs innate lymphoid cells
  • pDC plasmacytoid dendritic cells
  • BAFF B-cell activating factor of the TNF family
  • APRIL A proliferation-inducing ligand
  • sIgA is concentrated in the mucus out layer [Rogier E., et al. Pathogens. (2014) 3(2):390–403], noncovalently cross-linking microorganisms, and promoting the microorganisms to clump together in situ. Peristaltic bowel movements then help remove the bacterial clumps. These processes of agglutination, entrapment, and clearance together are called immune exclusion [Id., citing Mantis N. J., et al. Mucosal Immunology. (2011) 4(6):603–611]. [000115] sIgAs also have more extensive protective functions.
  • sIgA coating and the steric hindrance help block microbial adhesins to interact with the epithelium
  • sIgA can also inhibit specifically pathogens by direct recognition of receptor-binding domains such as reovirus type 1 Lang (T1L) [Id., citing Helander A., et al. J. Virology. (2003) 77(14):7964– 7977].
  • T1L reovirus type 1 Lang
  • the advanced glycosylated IgA heavy chain and SC serve as competitive inhibitors of the pathogen adhesion process [Id., citing Mantis N. J., et al. Mucosal Immunology. 2011;4(6):603–611].
  • Blocking pathogens from interacting with epithelial cells is not the exclusive mechanism by which sIgA exerts its protective function.
  • sIgA may have direct effects on impacting the bacterial viability or changing pathogenicity.
  • sIgA can interact with flagella to inhibit Salmonella bacterial motility [Id., citing Forbes S. J., et al. Infection and Immunity. (2008) 76(9):4137–4144], as well as to protect from cholera toxin- induced fluid accumulation in a ligated intestinal loop model [Lycke N., et al. Scandinavian Journal of Immunology. 1987;25(4):413–419].
  • CbpA choline binding protein A
  • a surface protein of Streptococcus pneumoniae [Id., citing Corthesy B. Frontiers in Immunology. (2013) 4:p.185]; the galactose residues of free SC were shown to neutralize Clostridium difficile toxin A and enteropathogenic E. coli intimin [Id., citing Perrier C., et al. Journal of Biological Chemistry. (2006) 281(20):14280–14287].
  • Fc ⁇ RI CD89
  • the most important IgA host receptor is widely expressed in cell types including neutrophils, eosinophils, monocytes, and macrophages.
  • sIgA also interacts with the polymeric immunoglobulin Fc receptor pIgR, transferrin receptor (Tfr/CD71), asialoglycoprotein receptor (ASGPR), Fc ⁇ / ⁇ R (also known as CD351), FcRL4 (an immunoregulatory receptor specifically expressed by memory B cells localized in sub-epithelial regions of lymphoid tissues; [Jourdan, M. et al., PLoS One (2017) doi.org/10.1371/journal.pone.0179793], and dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN; CD209) [Id., citing Mkaddem S.
  • DC-SIGN dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin
  • IAV Escape from Host Immune Surveillance [000117] To establish a successful infection, IAVs have evolved multiple strategies to circumvent host immunity. For example, it is well known that IAV infection triggers robust production of IFNs that induce the expression of numerous antiviral molecules or ISGs.
  • IFNs have a strong antiviral activity, they cannot fully control IAV infection due to the virus-mediated suppression of IFNs signaling.
  • Hemagglutinin of IAVs has been shown to facilitate IFNAR ubiquitination and degradation, reducing the levels of IFNAR, and thus suppressing the expression of IFN- stimulated antiviral proteins [Chen, X. et al., Frontiers Immunol. (2016) 9: 320, citing Xia C, et al. J Virol (2015) 90(5):2403–17].
  • H9- A and H9-B may provide a novel mechanism for H9N2 virus to counteract humoral immunity [Id., citing Peacock T, et al. Sci Rep (2016) 6:18745].
  • a study has shown that the escape of H5N1 from vaccine-mediated immunity is caused by the addition of N-glycosylation sites on the globular head of HA [Id., citing Hervé PL, et al. Virology (2015) 486(8):134–45].
  • antibody response against NA of IAV cannot inhibit viral infection, but restrain its diffusion, thus lowering the severity of influenza.
  • IAVs employ NA protein to block the recognition of HA by natural cytotoxicity receptors, NKp46, and NKp44 receptors and evade the NKp46-mediated elimination, leading to minimized clearance of infected cells by NK cells [Id., citing Baron Y, et al. J Infect Dis (2014) 210(3):410]. [000119]
  • Nonstructural protein-1 of IAVs is the most important IFN antagonist protein, acting on multiple targets and suppressing the host IFN response.
  • Viral RNA invading the host cell causes RIG-I ubiquitination by a RING-finger E3 ubiquitin ligase named as TRIM25, which is essential for RIG-I signaling pathway to trigger host antiviral innate immunity [Id., citing Gack MU, et al. Proc Natl Acad Sci U S A (2008) 105(43):16743].
  • TRIM25 RING-finger E3 ubiquitin ligase
  • NS1 protein can inhibit the TRIM25-mediated RIG-I ubiquitination, thereby blocking RIG-I activation [Id., citing Gack MU, et al Cell Host Microbe (2009) 5(5):439–49].
  • NS1 has an inhibitory effect on protein kinase RNA-activated (also known as protein kinase R, PKR), but the effect relies on the induced expression of vault RNAs (a kind of small non-coding RNA with approximately 100 bases). They are initially described as fornix RNP complex components [Id., citing Kedersha NL, Rome LH. J Cell Biol (1986) 103(3):699–709]. Through NS1 protein, influenza virus induces the expression of vault RNA that inhibits the activation of PKR and the production of IFNs and ultimately promotes the replication of the virus.
  • PKR protein kinase R
  • NS1 protein can block the phosphorylation of these kinases and eventually destroy the NF- ⁇ B complex predominating in nucleus as well as the expression of downstream genes [Id., citing remindle A, et al. J Virol (2012) 86(18):10211–7, Gao S, et al Cell Microbiol (2012) 14(12):1849]. Also, through the JAK-STAT pathway, NS1 protein can block IFN-mediated downstream signaling pathway and weaken the antiviral effect mediated by the downstream effector molecules induced by IFNs.
  • IKK I ⁇ B kinases
  • NS1 acts mainly by lowering the phosphorylation levels of STAT1, STAT2, and STAT3, preventing STAT2 from entering into the nucleus to bind to the DNA sequence of ISGs promoter region, leading to reduced expression of ISGs [Id., citing Jia D, et al. PLoS One (2010) 5(11):e13927].
  • NS1 is not only involved in host innate immunity, but also affects adaptive immunity via modulating the maturation and the capacity of DCs to induce T cell responses [Id., citing Fernandez-Sesma A, et al. J Virol (2006) 80(13):6295–304].
  • influenza virus NS1 can bind to cellular double-stranded DNA (dsDNA), counteract the recruitment of RNA polymerase II (Pol II) to DNA, and finally block the transcription of IFNs and ISGs [Id., citing Anastasina M, et al. Biochim Biophys Acta (2016) 1859(11):1440–8].
  • dsDNA double-stranded DNA
  • Pol II RNA polymerase II
  • IFNs and ISGs Id., citing Anastasina M, et al. Biochim Biophys Acta (2016) 1859(11):1440–8].
  • the Antagonism of Other IAV Proteins [000121] Studies have found that PB1-F2 protein has a mitochondrial positioning signal, via interacting with MAVS, to counteract RLR-mediated activation of IFN signaling pathway (Id., citing Varga ZT, et al. PLoS Pathog (2011) 7(6):e1002067).
  • PB2 protein a member of the viral polymerase complex
  • IFN antagonism citing Iwai A, et al. J Biol Chem (2010) 285(42):32064–74
  • PB2 interacts with the MAVS to evade the host IFN antiviral response, which is similar to the action mode of PB1-F2 protein [Id., citing Grimm D, et al. Proc Natl Acad Sci U S A (2007) 104(16):6806– 11].
  • Viral M2 protein may inhibit the activation of TLR pathway and the generation of IFNs via blocking host autophagy [Id., citing Münz C. et al. Cell Host Microbe (2014) 15(2):130–1, Beale R, et al. Cell Host Microbe (2014) 15(2):239–47].
  • Wound healing refers to the processes by which the body repairs trauma to any of its tissues, especially those caused by physical means and with interruption of continuity.
  • a wound-healing response can be viewed as comprising four separate phases, comprising: 1) an initial phase post injury involving hemostasis; 2) a second phase involving inflammation; 3) a third phase involving granulation and proliferation; and 4) a fourth phase involving remodeling and maturation.
  • the culmination of the wound-healing response results in the replacement of normal tissue structures with fibroblastic mediated scar tissue. Processes involved in the wound healing response, however, can go awry and produce an exuberance of fibroblastic proliferation, which can result in tissue damage, including hypertrophic scarring (a widened or unsightly scar that does not extend the original boundaries of the wound).
  • FIG. 1 shows an illustrative representation of the classical coagulation cascades. It is generally recognized that these systems function together and interact in vivo.
  • the intrinsic coagulation pathway is initiated when blood contacts any surface except normal endothelial and blood cells.
  • This pathway also known as the contact activation pathway, begins with formation of the primary complex on collagen by high-molecular weight kininogen (HMWK), prekallikrein, and coagulation factor (Factor) XII (Hageman factor).
  • HMWK high-molecular weight kininogen
  • Factor coagulation factor
  • Prekallikrein is converted to kallikrein and Factor XII becomes Factor XI la.
  • Factor Xlla converts Factor XI into Factor Xla.
  • Factor Xla activates Factor IX, which, with its co-factor FVIIIa form the tenase complex, which activates Factor X to Factor Xa.
  • tissue factor pathway also known as the tissue factor pathway
  • tissue factor pathway tissue factor
  • proconvertin enzyme coagulation factor VII
  • Factor VII is activated to Factor VIla by different proteases, including thrombin (Factor lla), Factors Xa, IXa, Xlla and the Factor Vlla-TF complex itself.
  • the Factor Vlla-TF complex activates Factors IX and X.
  • Factor Xa tissue factor pathway inhibitor
  • Factor Xa and its cofactor Va form the prothrombinase complex which activates the conversion of prothrombin to thrombin.
  • Thrombin then activates other components of the coagulation cascade, including Factors V and VIII (which activates Factor XI, which, in turn, activates Factor IX), and activates and releases Factor VIII from being bound to von Willebrand Factor (vWF).
  • Factors VIla and IXa together form the "tenase" complex, which activates Factor X, and so the cycle continues.
  • FIG.2 shows an illustration of the cell-surface based model of coagulation in vivo (Monroe Arterioscler Thromb Vase Biol. 2002; 22:1381 -1389).
  • tissue factor which is present on the subendothelium, tissues not normally exposed to blood, activated monocytes and endothelium when activated by inflammation.
  • Factors VII and Vila bind to tissue factor and adjacent collagen.
  • the factor Vila- tissue factor complex activates factor X and IX.
  • Factor Xa activates factor V, forming a prothrombinase complex (factor Xa, Va and calcium) on the tissue factor expressing cell.
  • coagulation is amplified as platelets adhere to the site of injury in the blood vessel.
  • Thrombin is activated by platelet adherence and then acts to fully activate platelets, enhance their adhesion and to release factor V from the platelet alpha granules.
  • Thrombin on the surface of activated platelets activates factors V, VIII and XI, with subsequent activation of factor IX.
  • the tenase complex (factors IXa, VIIa and calcium) now is present on platelets where factor Xa can be produced and can generate another prothrombinase complex on the platelet so that there can be large-scale production of thrombin.
  • Propagation the third step, and is a combination of activation of the prothrombinase complexes that allow large amounts of thrombin to be generated from prothrombin. More platelets can be recruited, as well as activation of fibrin polymers and factor XIII.
  • Proteases of the coagulation cascade have been shown to activate protease activated receptors (PARs), a family of G-protein-coupled receptors. [Moretti, S. et al.
  • PARs have been cloned from the mammalian genome. PARS are activated by proteolytic cleavage of their N-terminal extracellular domain, which releases a new amino terminus sequence, which then acts as a tethered ligand to bind intramolecularly the receptor on its second extracellular loop. [Cirino, G. & Vergnolle, N. Curr. Opin. Pharmacol. (2006) 6: 428-34]. Thrombin activates PAR1, PAR3, and PAR4, whereas trypsin and mast cell tryptase activate PAR2. [Schmidlin, F. et al. Am. J. Crit. Care Med.
  • neutrophil elastase and cathepsin G are able to cleave the N-terminal domain of PARs downstream from the activating protease cleavage site, thereby preventing activation of the receptor [Cirino, G. & Vergnolle, N. Curr. Opin. Pharmacol. (2006) 6: 428-34]., citing Dulon, S. et al. Am. J. Respir. Cell Mol. Biol. (2003) 28: 339-46].
  • This disarming mechanism could be used by pathogens to avoid PAR-driven host immune responses; studies have shown that lung (Pseudomonas aeruginosa) [Id., citing Dulon, S.
  • PAR1 has multiple roles on many cell types. On endothelial cells, it can have barrier protective or barrier disruptive roles, depending on the agonist and co-receptors present on the cell. [Arachiche, A. et al. J. Biol. Chem. (2013) 288 (45): 32553-62, citing Riewald, M., & Ruf, W. J. Biol. Chem.
  • PAR4 is the primary signaling receptor on platelets of many species. In human platelets, PAR1 and PAR4 have both overlapping and unique signaling functions. [Id., citing Holinstat, M. et al. J. Biol. Chem. (2006) 281: 26665- 74; Voss, B. et al. Mol. Pharmacol.
  • PAR4 interacts with PAR1 to enhance PAR4 activation [Arachiche, A. et al. J. Biol. Chem. (2013) 288 (45): 32553-62, citing Nieman, MT. Biochemistry (2008) 47: 13279-86; Leger, A. et al. Circulation (2006) 113: 1244-54].
  • the activation of PAR1 and PAR4 requires the receptors to be cleaved by thrombin.
  • PAR1 which has a hirudin-like sequence that binds tightly to thrombin exosite I [Id., citing Liu, L. et al. J. Biol. Chem.
  • PAR4 does not bind to exosite I and is a poor thrombin substrate when it is expressed on cells alone [Id., citing Nieman, MT. Biochemistry (2008) 47: 13279-86; Bah, A. et al. Proc. Nat. Acad. Sci. USA (2007) 104: 11603-8].
  • the inefficient activation of PAR4 is overcome by co-expression of PAR1 on human platelets and PAR3 on mouse platelets [Id., citing Leger, AJ et al. Circulation (2006) 113: 1244-54; Kahn, M. et al. Nature (1998) 394: 690-94].
  • the proposed model for PAR1 or PAR3 enhancing PAR4 activation is that after cleavage of PAR1 or PAR3, thrombin remains bound to the hirudin-like sequence via exosite I and cleaves an adjacent PAR4.
  • the exosite I interaction likely holds thrombin in the protease conformation for efficient cleavage of PAR4 [Id., citing Nieman, MT. Biochemistry (2008) 47: 13279-86; Leger, A. et al. Circulation (2006) 113: 1244-54; Nakanishi-Matsui, M. et al. Nature (2000) 404: 609-13].
  • PAR1 or PAR3 co-expression with PAR4 is a 10-fold reduction in the EC50 of thrombin activation of PAR4 [Id., citing Nieman, MT. Biochemistry (2008) 47: 13279-86; Leger, A. et al. Circulation (2006) 113: 1244-54; Nakanishi-Matsui, M. et al. Nature (2000) 404: 609-13].
  • PAR1 and PAR4 In addition to PAR1 and PAR4 having important independent roles in platelet signaling, the two receptors act synergistically by PAR1 enhancing PAR4 activation. It was demonstrated that PAR1 and PAR4 require allosteric changes induced via receptor cleavage by ⁇ -thrombin to mediate heterodimer formation.
  • P2Y12 has been linked to important aspects of platelet activation, including enhancement of dense granule secretion, recruitment of additional platelets to the site of vascular injury, and enhancement of the efficacy of other pro-coagulant agonists.
  • ⁇ -arrestin 1 and ⁇ -arrestin 2 are well-known negative regulators of G-protein- coupled receptor (GPCR) signaling. Upon GPCR activation, ⁇ -arrestins translocate to the cell membrane and bind to the agonist-occupied receptors. This uncouples these receptors from G proteins and promotes their internalization, thus causing desensitization.
  • ⁇ -arrestins also regulate transcription. They function as scaffold proteins that interact with several cytoplasmic proteins and link GPCRs to intracellular signaling pathways such as MAPK cascades. In response to activation of certain GPCRs, ⁇ - arrestins translocate from the cytoplasm to the nucleus and associate with transcription cofactors, such as p300 and cAMP response element binding protein (CREB) at the promoters of target genes to promote transcription. They also interact with regulators of transcription factors, such as I ⁇ B ⁇ and MDM2 in the cytoplasm, regulating the NF- ⁇ B and p53 transcription factors, respectively [Ma, L., & Pei, G. J. Cell Sci. (2007) 120: 213-18].
  • transcription cofactors such as p300 and cAMP response element binding protein (CREB)
  • PARs act as “sensors” of extracellular protease gradients.
  • serine proteases such as thrombin, trypsin, and mast cell tryptase
  • PARs act as “sensors” of extracellular protease gradients.
  • PARs protease-activated receptors
  • certain microbial proteases can also activate mammalian PARs.
  • Activated PARs couple to signaling cascades that affect, among others, coagulation and inflammatory responses. [Id., citing Cirino, G. & Vergnolle, N. Curr. Opin. Pharmacol. (2006) 6: 428-34].
  • the role of PARs in inflammation is complex, as individual PARs have both proinflammatory and protective roles in the airway [Id., citing Cocks, TM et a.
  • PAR1 has been identified as a major endogenous mediator of lung inflammation and fibrosis in a model of bleomycin-induced lung injury. [Cirino, G. & Vergnolle, N. Curr. Opin. Pharmacol. (2006) 6: 428-34, citing Howell, DC et al. Am. J. Pathol. (2005) 166: 1353-65].
  • Fibrin has been shown to contribute to the innate immune response to bacterial infections by increasing the expression of inflammatory mediators. [Id., citing Degen, JL et al. J. Thromb. Haaemost. (2007) 5 (1): 24-31; Esmon, CT et al. J. Thromb. Haemost. (2011) 1: 182-88].
  • PAR-1 expression is increased in endothelial cells after Dengue and CMV viral infection.
  • Huerta-Zepeda A. et al. Thromb. Haemost. (2008) 99 (5)L 936-43; [Popovic, M. et al.
  • hMPV-infected mice treated with the PAR1 agonist TFLLR-NH2 showed increased mortality, when compared to infected mice which were left untreated.
  • Thrombin generation was shown to occur downstream of PAR1 activation in infected mice via exposure to tissue factor as part of the inflammatory response; thrombin inhibition by argatroban reduced the pathogenicity of the infection with no additive effect to that induced by PAR1 inhibition.
  • the inflammatory phase begins during the hemostasis phase. Thrombocytes, as well as recruited white blood cells, release numerous factors to ramp up the healing process.
  • PDGF-A, B platelet-derived growth factor
  • TGF- ⁇ transforming growth factor beta
  • the processes of inflammation, collagen degradation and collagenogenesis, myoblastic creation from transformed fibroblasts, growth of new blood vessels, and reepithelialization are mediated by a host of cytokines and growth factors.
  • the interleukins strongly influence the inflammatory process.
  • Vascular endothelial growth factor (VEGF) and other factors enhance blood vessel formation, and some have multiple roles, such as fibroblast growth factor (FGF)-2, which affects not only the process of angiogenesis but also that of reepithelialization.
  • FGF fibroblast growth factor
  • Vasoactive amines such as histamine and serotonin, are released from dense bodies found in thrombocytes.
  • PDGF is chemotactic for fibroblasts and, along with TGF- ⁇ , is a potent modulator of fibroblastic mitosis, leading to prolific collagen fibril construction in later phases.
  • Fibrinogen is cleaved into fibrin, and the framework for completion of the coagulation process is formed. Fibrin provides the structural support for cellular constituents of inflammation. This process starts immediately after the insult and may continue for a few days.
  • PMNs polymorphonuclear leukocytes
  • monocytes/macrophages a polymorphonuclear leukocytes
  • PMNs engorge the wound.
  • TGF- ⁇ facilitates PMN migration from surrounding blood vessels, from which they extrude themselves from these vessels. These cells cleanse the wound, clearing it of debris.
  • the PMNs attain their maximal numbers in 24-48 hours and commence their departure by hour 72.
  • chemotactic agents are released, including FGF-2, TGF- ⁇ and TGF-a, PDGF-A,B, and plasma-activated complements C3a and C5a (anaphylactic toxins). They are sequestered by macrophages or interred within the scab or eschar (Id.; Habif. Dermatologic surgical procedures. Clinic Dermatology: A Color Guide to Diagnosis and Therapy.3rd ed.1996.809-810). [000147] As the process continues, monocytes also exude from surrounding blood vessels. Once they leave the vessel, these are termed macrophages.
  • the macrophages continue the cleansing process, manufacture various growth factors during days 3-4, and orchestrate the multiplication of endothelial cells with the sprouting of new blood vessels, the duplication of smooth muscle cells, and the creation of the milieu created by the fibroblast.
  • Many factors influencing the wound healing process are secreted by macrophages, including TGFs, cytokines and interleukins , tumor necrosis factor (TNF), and PDGF.
  • TGFs tumor necrosis factor
  • PDGF tumor necrosis factor
  • procollagen is the precursor of all collagen types and is transformed within the cell's rough endoplasmic reticulum, where proline and lysine are hydroxylated. Disulfide bonds are established, allowing 3 tropocollagen strands to form a triple left-handed triple helix, termed procollagen.
  • GAGs glycosaminoglycans
  • fibronectin produced by fibroblasts.
  • GAGs include heparin sulfate, hyaluronic acid, chondroitin sulfate, and keratin sulfate.
  • Proteoglycans are GAGs that are bonded covalently to a protein core and contribute to matrix deposition.
  • Angiogenesis results from parent vessel offshoots.
  • vascular endothelial growth factor means the formation of primitive vascular structures via the differentiation of endothelial precursor cells (EPCs), which is distinct from angiogenesis- also can be seen.
  • FGF-2 Basic FGF
  • EPCs endothelial precursor cells
  • Re-epithelization occurs with the migration of cells from the periphery of the wound and accessory or adjoining tissues. This process commences with the spreading of cells within 24 hours. Division of peripheral cells occurs in hours 48-72, resulting in a thin epithelial cell layer, which bridges the wound.
  • Epidermal growth factors are believed to play a key role in this aspect of wound healing.
  • This succession of subphases can last up to 4 weeks in the clean and uncontaminated wound.
  • the wound undergoes constant alterations, known as remodeling, which can last for years after the initial injury occurred.
  • Collagen is degraded and deposited in an equilibrium-producing fashion, resulting in no change in the amount of collagen present in the wound.
  • the collagen deposition in normal wound healing reaches a peak by the third week after the wound is created. Contraction of the wound is an ongoing process resulting in part from the proliferation of specialized fibroblasts termed myofibroblasts, which provide mechanical support and integrity to the tissue after initial injury.
  • Wound contraction occurs to a greater extent with secondary healing (i.e., healing by second intention, which describes a wound left open and allowed to close by reepithelialization and contraction by myofibroblasts) than with primary healing (i.e., healing by first intention, which describes a wound closed by approximation of wound margins or by placement of a graft or flap, or wounds created and closed in the operating room, unlike via reepithelialization and contraction by myofibroblasts).
  • Maximal tensile strength (the greatest longitudinal stress a substance can bear without tearing apart) of the wound is achieved by the 12th week, and the ultimate resultant scar has only 80% of the tensile strength of the original skin that it has replaced.
  • Platelets and the immunopathology of viral infections [000156] Platelets form a functional link between wound healing, coagulation and immune responses. [000157] Platelets are now recognized as major inflammatory cells with key roles in the innate and adaptive response. [Hottz, ED, et al. Frontiers in Medicine (2016) 5: 121]. Activated platelets have key thromboinflammatory functions linking coagulation to immune responses in various infections, including viral infections.
  • platelets Besides their central role in hemostasis, platelets modulate inflammatory reactions and immune responses by direct interaction with leukocytes and endothelial cells and via release of soluble inflammatory mediators that enhance recruitment of leukocytes and trigger their activation [Assinger, A. Frontiers Immunology (2014): 00649, citing Weyrich, AS, et al. J. Thromb. Haemost. (2003) 1: 1897-905; von Hundelshausen, P. & Weber, C. Cir. Res. (2007) 100: 27-40; Semple, JW et al. Nat. Rev. Immunol. (2011) 11: 264-74; Karshovska, E. et al. Thromb. Haemost. (2013) 110: 894-902].
  • Platelets express surface receptors, such as lectins, integrins, and toll-like receptors (TLR), allowing them to directly interact with several pathogens.
  • Platelets also express Fc receptors by which they can recognize immunocomplexes. [Id., citing Anderson, CL et al. Semin. Thromb. Hemost. (1995) 21: 1-9].
  • Platelets are produced in the bone marrow by megakaryocytes, which develop from hematopoietic stem cells. Megakaryocytes first undergo linage commitment, followed by endomitosis resulting in polyploidy [Id., citing Patel, SR et al. J. Clin. Invest.
  • megakaryocytes After endoplasmic maturation, megakaryocytes form proplatelets, which bud off thousands of platelets and microparticles into the blood stream [Id., citing Kuter, DJ. Br. J. Haematol. (2014) 165: 248-58]. Megakaryopoiesis is triggered by a variety of cytokines (e.g., GM-CSF, IL-3, IL-6, IL-11, FGF4, and SDF-1), with thrombopoietin (TPO) being the most important. TPO not only triggers megakaryocyte development but also plays a role in maintaining stem cells [Id., citing de Graaf, CA & Metcalf, D.
  • cytokines e.g., GM-CSF, IL-3, IL-6, IL-11, FGF4, and SDF-1
  • TPO thrombopoietin
  • Viruses can modulate platelet production at various steps of development. Some viruses (e.g., simian immunodeficiency virus (SIV), which triggers TPO production via up- regulation of tumor growth factor (TGF) ⁇ [Id., citing Metcalf Pate, KA, et al. J. Acquir. Immune Defic. Syndr. (2014) 65: 510-6]; human herpes virus 6, which can interfere with TPO- inducible megakaryocytic colony formation [Id., citing Isomura, H. et al. J. Gen. Virol.
  • SIV simian immunodeficiency virus
  • TGF tumor growth factor
  • HIV human immunodeficiency virus
  • CMV cytomegalovirus
  • HCV cytomegalovirus
  • Viral infection of megakaryocytes can increase apoptosis and decreases the maturation and ploidy of megakaryocytes. Moreover, virus-infected megakaryocytes have been shown to express less surface c-Mpl, which is the receptor for TPO [Id., citing Gibellini, D. e al. World J. Virol. (2013) 2: 91-101]. [000164] Platelet–virus interaction can occur via a variety of platelet receptors.
  • platelets exhibit several pattern recognition receptors (PRR) including those from the toll-like receptor, NOD-like receptor, and C-type lectin receptor family and are first-line sentinels in detecting and responding to pathogens in the vasculature.
  • PRR pattern recognition receptors
  • G-protein-coupled receptors While traditional platelet activation by G-protein-coupled receptors is usually rapid, platelet PRR activation in responses to infectious and immune stimuli can be delayed and sustained, lasting hours after initial aggregation and secretion [Id., citing Vieira-de-Abreu, A. et al. Semin. Immunopathol. (2012) 34: 5-30; Shashkin, PN, et al.
  • PRR from different classes have been shown to be expressed and functional in platelets, including those from C-type lectin receptors (CLR) and toll-like receptors (TLRs) families [Id., citing Middleton, EA et al. Physiol. Rev. (2016) 96: 1211-59; Cognasse, F. et al. Immunol. Cell Biol. (2005) 83: 196098; D’Atri, LP & Schattner, M. Front. Biosci. (Landmark Ed. ) 2017] 22: 1867-83].
  • CLR C-type lectin receptors
  • TLRs toll-like receptors
  • CLRs are a large family of surface proteins containing at least one carbohydrate- binding domain which are specialized in the recognition of bacterial, fungi, or viral glycans [Id., citing Shiokawa, M. et al. Curr. Opinion. Microbiol. (2017) 40: 123-30; Marakalala, MJ & Ndlovu, H. PLoS Pathog. (2017) 13: e1006333.10.1371/journal.ppat.1006333].
  • Some viruses can exploit certain CLR for viral attachment and entry in host cells, including in platelets [Id., citing Turville, SG et al. Nat. Immunol. (2002) 3: 975-83; Chaipan, C.
  • TLRs a family of transmembrane cellular sensors, are the best described class of PRR in platelets. While virtually all TLRs (1–10 in human) are detected at some level (mRNA or protein) in platelets [Id., citing Vieira-de-Abreu, A. et al. Semin. Immunopathol. (2012) 34; 5-10; Middleton, EA et al. Physiol. Rev. (2016) 96: 1211-59; Semple, JW, et al. Nat. Rev. Immunol. (2011) 11: 264-74; Cognasse, F. et al., Immunol. Cell Biol.
  • NLR NOD-like receptor
  • I and melanoma differentiation-associated gene 5 which are highly specialized in viral RNA recognition, are expressed in human megakaryocytes in response to type I interferon (IFN- ⁇ and - ⁇ ) [Id., citing Lutteke, N. et al. Virology (2010) 405: 70-80], but their expression in platelets, as well the ability of platelets to express other IFN-stimulated genes (ISGs) and to perform IFN-induced antiviral response are not known.
  • IFN- ⁇ and - ⁇ type I interferon
  • megakaryocytes express PRR and cytokine receptors, and there is evidence that TLR agonists or cytokine engagement affects megakaryocytic maturation and thrombopoiesis [Id., citing D’Atri, LP et al. J. Thromb. Haemost. (2015) 13: 839-50; Negrotto, S. et al. J. Thromb Haemost. (2011) 9: 277-85; Beaulieu, LM et al., Arterioscler. Thromb. Vasc. Biol. (2014) 34: 552-64; Beaulieu, LM et al., Blood (2011) 117: 5963-74].
  • Megakaryocytes and megakaryocytic cell lines respond to viral infections or viral PAMPs by secreting high levels of ⁇ and ⁇ IFN [Id., citing D’Atri, LP et al. J. Thromb. Haemost. (2015) 13: 839-50; Lutteke, N. et al. Virology (2010) 405: 70-80; Negrotto, S. et al. J. Thromb. Haemost. (2011) 9: 2477-85; Rivadeneyra, L. et al., Thromb. Haemost. (2015) 114: 982-93; Pozner, RG, et al. PLoS Pathog.
  • Platelet-virus interactions are mainly mediated by integrins, surface lectins, and TLRs.
  • integrins integrins
  • surface lectins integrins
  • TLRs integrins
  • EMCV Encephalomyocarditis virus
  • Rotavirus utilizes the collagen receptor GPIa/IIa to bind to platelets [Id., citing Flaujac, C. et al. Cell Mol. Life Sci. (2010) 67: 545-56; Coulson, BS et al. Proc. Nat. Acad. Sci.
  • GPIIb/IIIa fibrinogen receptor
  • GPIIb/IIIa does not seem to be the unique receptor for platelet–adenovirus interaction, as inhibition of GPIIb/IIIa does not alter platelet internalization of adenoviruses [Id., citing Gupalo, E. et al. Platelets (2013) 24: 383-91].
  • GPIIb/IIIa is the most abundant platelet integrin and displays bi-directional signaling functions.
  • GPIIb/IIIa affinity Inside-out signaling, which is positively regulated by various platelet agonists, is mediated by intracellular protein–protein interactions and biochemical reactions that regulate GPIIb/IIIa affinity [Id., citing Coller, BS & Shattil, SJ. Blood (2008): 112: 3011- 25].
  • These intracellular processes triggering GPIIb/IIIa activation are complex and include recruitment of talin, which separates the GPIIb and the GPIIIa subunits, and kindlins, which are involved in GPIIb/IIIa activation [Id., citing Moser, M. et al. Nat. Med. (2008) 14: 325- 30] independent of talin recruitment [Id., citing Kahner, BN et al.
  • G protein subunit G ⁇ 13 directly binds to the cytoplasmic domain of GPIIIa and promotes ligand binding to GPIIb/IIIa [Id., citing Gong, H. et al. Science (2010) 327: 340-4].
  • GPIIb/IIIa GPIIb/IIIa
  • Outside-in signaling via receptor binding promotes actin polymerization and platelet spreading [Id., citing Coller, BS & Shattil, SJ. Blood (2008) 112: 3011-25] and can thereby enhance virus attachment to endothelial cells but also promote platelet clearance.
  • lectin receptors such as C-type lectin domain family 2 (CLEC-2) and cell-specific intercellular adhesion molecule-3-grapping non-integrin (DC-SIGN) [Id., citing Chaipan, C. et al. J. Virol. (2006) 80: 8951-60].
  • Platelets and/or megakaryocytes can further interact with HIV envelope proteins via C—X–C chemokine receptor type 4 (CXCR4) or via chemokine (C–C motif) ligand (CCL) 3 (MIP-1 ⁇ ) and 5 (RANTES) [Id., citing Flaujac, C. et al. Cell Mol. Life Sci. (2010) 67: 545-56].
  • CXCR4 C–X–C chemokine receptor type 4
  • MIP-1 ⁇ chemokine (C–C motif) ligand
  • RANTES chemokine receptor type 4
  • HIV-1 changes its co-receptor usage from CCR5 to CXCR4 only; this receptor change represents a switch to non-CD4-dependent platelet activation at late stages of disease.
  • platelets express the Coxsackie virus-specific Coxsackie- adenovirus receptor (CAR) [Id., citing Othman, M. et al. Blood (2007) 109: 2832-9] and HCV interacts with platelets via collagen receptor GPVI [Id., citing Zahn, A. et al. J. Gen. Virol. (2006) 87: 2243-51]. [000175] These direct interactions often result in platelet activation and adhesion of activated platelets to leukocytes. Platelet binding to neutrophils triggers phagocytosis of platelets [Id., citing Koupenova, M. et al. Blood (2014) 124: 791-802; Maugeri, N.
  • CAR Coxsackie virus-specific Coxsackie- adenovirus receptor
  • cytokines such as interleukin 6 (IL-6), in target cells [Id., citing Bouwman, J. et al. Eur. J. Clin. Invest. (2002) 32: 759-66].
  • Platelets can be activated by these cytokines, leading to platelet–leukocyte interactions, which foster leukocyte and endothelial activation, further amplifying platelet activation and enhancing their clearance by splenic macrophages or Kupffer cells in the liver [Id., citing Nunez, D. et al. Eur. J. Immunol. (1987) 17: 515-20].
  • Monocytes that encounter dengue virus for example, start generating platelet activating factor (PAF) [Id., citing Zapata, JC et al. PLoS Negl. Trop. Dis. (2014) 8: e2858], which is a lipid mediator that triggers platelet activation. This leads to enhanced apoptosis of platelets and accelerates platelet clearance in secondary dengue infection [Id., citing Alonzo, MT et al. J. Infect. Dis. (2012) 205: 1321-9]. [000178] Several virus infections activate the coagulation cascade via induction of tissue factor (TF) expression in target cells.
  • TF tissue factor
  • thrombin by the activated coagulation cascade causes platelet activation and subsequent clearance via protease activating receptor (PAR) signaling [Id., citing Antoniak, S. & Mackman, N. Blood (2014) 123: 2605-13].
  • PARs on platelets, endothelial cells, and leukocytes are important modulators during viral infections, which modulate innate immune responses and exert positive and negative effects on TLR- dependent responses [Id., citing Antoniak, S. & Mackman, N. Blood (2014) 123: 2605-13].
  • Platelets also recognize viral particles coated with immunoglobulins via their Fc ⁇ RII receptor, which results in Fc receptor-mediated platelet activation, aggregation, and platelet clearance [Id., citing Anderson, CL et al. Semin. Thromb. Hemost. (1995) 21: 1-9; Cox, D. et al. J. Thromb. Haemost. (2011) 9: 1097-107].
  • Fc ⁇ RII-mediated platelet activation depends on IgG and GPIIb/IIIa engagement and involves ADP and thromboxane A2 (TxA2) feedback mechanisms to cause platelet aggregation, which is further enhanced by CXCL4 [Id., citing Arman, M.
  • thrombocytopenia refers to a drop in platelet count caused by either decreased platelet production or increased platelet destruction. It is associated with an increased bleeding risk.
  • Viral infection can either enhance platelet activation resulting in pro-thrombotic events, or diminish platelet responses leading to bleeding complications.
  • Activation of the coagulation cascade has been observed in various virus infections, including HIV, dengue, and Ebola virus infection [Id., citing Antoniak, S. & Mackman, N. Blood (2014) 123: 2605-13].
  • thrombocytopenia is a common result of viral infections and associated with an increased bleeding risk.
  • VHF outbreaks lead to the deaths of thousands of people every year and are caused by different enveloped RNA viruses, which include Arenaviridae (e.g., Lassa virus), Bunyavirideae (e.g., hantavirus), Filioviridae (e.g., Marburg and Ebola virus), and Flaviviridae (e.g., dengue virus).
  • Arenaviridae e.g., Lassa virus
  • Bunyavirideae e.g., hantavirus
  • Filioviridae e.g., Marburg and Ebola virus
  • Flaviviridae e.g., dengue virus.
  • Thrombocytopenia in response to viral infections is often multifactorial. Rapidly induced thrombocytopenia in response to viral infections is mediated via enhanced platelet destruction.
  • HIV results in impaired survival of bone marrow megakaryocytes and their precursors.
  • HIV also decreases the number and activity of human progenitor cells and decreases megakaryocyte maturation and ploidy.
  • HIV surface glycoprotein gp120 leads to increased megakaryocyte apoptosis in vitro due to increased TGF ⁇ and down-regulation of the proliferation-inducing ligand tumor necrosis factor ligand superfamily member 13 (TNFSF13).
  • TGFSF13 proliferation-inducing ligand tumor necrosis factor ligand superfamily member 13
  • gp120 interacts with CD4, which is expressed by immature megakaryocytes, which also express CCR5, and leads to their infection [Id., citing Louache, F. et al. Blood (1991) 78: 1697-1805].
  • HIV infection of megakaryocytes can lead to reduced TPO receptor (c-Mpl) expression.
  • platelet production is impaired by suppression of megakaryopoiesis via infection of hematopoietic progenitor cells or indirectly via altered cytokine levels in the bone marrow due to impaired stromal cell function [Id., citing Zapata, JC et al. PLoS Negl. Trop Dis. (2014) 8: e2858]. Platelets from patients with dengue infection present signs of activation, mitochondrial dysfunction, and enhanced apoptosis, which may contribute to the genesis of thrombocytopenia [Id., citing Hottz, ED et al. J. Thromb. Haemost (2013) 11: 951-62; Hottz, ED et al. J. Immunol.
  • NS-1 Dengue virus-induced anti-non-structural protein-1 (NS-1) induces complement- mediated lysis of platelets and thereby further accelerates thrombocytopenia [Id., citing Sun, DS et al. J. Thromb. Haemost. (2007) 5: 2291-9].
  • NS-1 can also activate endothelial cells and leads to increased vascular permeability and further platelet activation [Hottz, ED et al. Blood (2013) 122: 3405-14].
  • Dengue virus-infected patients show increased levels of E-selectin on their endothelial cell surface, which promotes adhesion and clearance of platelets [Id., citing Hottz, ED et al. Blood (2013) 122: 3405-14; Chuansumrit, A. & Chaiyaratana, W. Thromb. Res. (2014) 133: 10-16] as well as enhanced activation of the coagulation cascade [Id., citing Nascimento, EJ et al. Crit. Rev. Immunol. (2014) 34: 227-40].
  • LCMV lymphocytic choriomeningitis virus
  • Junin virus results in thrombocytopenia and decreased agonist-induced platelet responses in mice [Id., citing Iannacone, M. et al. Proc. Natl Acad. Sci. USA (2008) 105: 629-34; Pozner, RG, et al. PLoS Pathog. (2010) 6: e1000847].
  • platelet depletion in LMCV- infected mice results in lethal hemorrhagic anemia [Id., citing Iannacone, M. et al. Proc. Natl Acad. Sci. USA (2008) 105: 629-34].
  • IFN interferon
  • IFN ⁇ / ⁇ receptor signaling represents an important paracrine repressor of megakaryopoiesis, which directly inhibits TPO-induced signaling through induction of suppressor of cytokine signaling 1 (SOCS-1) [Id., citing Wang, Q. et al. Blood (2000) 96: 2093-9], induction of 2′5′- oligoadenylate synthetase (OAS) [Id., citing Iannacone, M. et al. Proc. Natl Acad. Sci.
  • SOCS-1 suppressor of cytokine signaling 1
  • OFS 2′5′- oligoadenylate synthetase
  • Viruses also possess enzymes, which can modulate platelet functions.
  • Influenza virus exhibits neuraminidase (sialidase), which hydrolyses the terminal sialic acid residues from host cell receptors and thereby decreases the life span of platelets by targeting platelets for rapid clearance in the liver and spleen [Id., citing Sorensen, AL et al. Blood (2009) 114: 1645-54].
  • neuraminidase sialidase
  • mycoviral neuraminidase has been shown to reduce platelet life span by cleaving sialic acid in the platelet membrane [Id., citing Terada, H. et al. Blood (1966) 28: 213-28].
  • Vascular permeability [000193] The molecular and genetic events that regulate vascular permeability are not fully understood. [000194] Vascular permeability by any measure is dramatically increased in acute and chronic inflammation, cancer, and wound healing. This hyperpermeability is mediated by acute or chronic exposure to vascular permeabilizing agents, particularly vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A).
  • vascular permeability factor/vascular endothelial growth factor particularly vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A).
  • VPF/VEGF vascular endothelial growth factor
  • VEGF-A vascular endothelial growth factor
  • Three distinctly different types of vascular permeability can be distinguished, based on the different types of microvessels involved, the composition of the extravasate, the anatomic pathways by which molecules of different size cross-vascular endothelium, the time course over which permeability is measured; and the
  • Basal vascular permeability (BVP) of normal tissues the basal vascular permeability (AVH) that occurs in response to a single, brief exposure to VEGF-A or other vascular permeabilizing agents, and the chronic vascular hyperpermeability (CVH) that characterizes pathological angiogenesis.
  • BVP basal vascular permeability
  • ABV acute vascular hyperpermeability
  • CVH chronic vascular hyperpermeability
  • BVP Basal vascular permeability
  • Nagy, et al state that the primary function of several major organs (heart, lungs, kidneys) and of larger blood vessels (arteries, arterioles, veins, venules) is to supply the capillaries, and thus the tissues, with nutrients and to clear waste products.
  • the molecules exchanged consist largely of gases (O 2 and CO2), water, small molecules such as salts and sugars, and only small amounts of plasma proteins. The process is driven largely by diffusion.
  • the extent of BVP varies considerably in different normal tissues and is subject to substantial change in response to changes in hydrostatic pressure, opening of closed vessels, surface area available for exchange, blood flow, etc.
  • Water and lipophilic solutes are able to diffuse through endothelial cells; they also pass readily through inter-endothelial cell junctions and through endothelial fenestrae.
  • Fenestrae are greatly thinned (70–150-nm diameter) zones of microvascular endothelium that can be induced by VEGF-A [60]. They are found in small numbers in many types of vascular endothelium and are especially numerous in specialized vascular beds that supply tissues that secrete protein hormones. They are induced in other types of vascular endothelium by VEGF-A [Id., citing Roberts, WG, Palade, GE. J.
  • Acute vascular hyperpermeability A rapid increase in vascular permeability occurs when the microvasculature is exposed acutely to any of a number of vascular permeabilizing factors, e.g., VEGF-A, histamine, serotonin, PAF, etc. Single exposure to any of these permeability factors results in a rapid but self-limited (complete by 20–30 min) influx of plasma into the tissues.
  • vascular permeabilizing factors e.g., VEGF-A, histamine, serotonin, PAF, etc.
  • the fluid passing from the circulation into normal tissues under basal conditions is a plasma filtrate, i.e., a fluid consisting largely of water and small solutes but containing very little plasma protein.
  • the fluid that extravasates in AVH is rich in plasma proteins, approaching the levels found in plasma, and is referred to as an exudate.
  • plasma proteins that extravasate are fibrinogen and various members of the blood clotting cascade.
  • Fibrin forms a gel that traps water and other solutes, restraining their clearance by lymphatics or capillaries and resulting in tissue swelling (edema). Fibrin in tissues has other functions that are discussed below. However, as long as the permeability stimulus is not continuous, the deposited fibrin is rapidly degraded without further consequences.
  • AVH also differs from BVP in that the vascular leakage takes place not from capillaries but from post-capillary venules, highly specific vessels just downstream of capillaries [Id., citing Majno, G. et al. J. Biophys. Biochem. Cytol. (1961) 11: 607-26; Majno, G. et al. J. Cell Biol. (1969) 42: 647-672]. Whereas capillaries have a flattened endothelium, venules are lined by a much taller, cuboidal endothelium.
  • VVO vesiculo-vacuolar organelle
  • VVOs are grape-like clusters comprised of hundreds of uncoated, cytoplasmic vesicles and vacuoles that together form an organelle that traverses venular endothelial cytoplasm from lumen to albumen. VVOs often extend to inter-endothelial cell interfaces and their individual vesicles (unlike caveolae) commonly open to the inter-endothelial cell cleft.
  • the vesicles and vacuoles comprising VVOs vary in size from those the size of caveolae to vacuoles with volumes as much as 10-fold larger [Id., citing Feng, D. et al. Microcirculation (1999) 6: 23- 44]. These vesicles and vacuoles are linked to each other and to the luminal and abluminal plasma membranes by stomata that are normally closed by thin diaphragms that appear similar to those found in caveolae. Whether VVOs somehow take the place of caveolae in caveolin-1 null mice and thereby contribute to the increased permeability observed in these animals needs to be investigated.
  • vascular permeability inducing agents cause the diaphragms interconnecting vesicles and vacuoles to open, thereby providing a transcellular pathway for plasma and plasma-protein extravasation.
  • the underlying mechanism could be mechanical, as was the endothelial cell contraction mechanism originally postulated by Majno [Majno, G. et al. J. Cell Biol. (1969) 42: 647-72]. If so, the actin–myosin contractions induced by permeability factors would act to pull apart the diaphragms linking adjacent VVO vesicles and vacuoles, resulting in a transcellular rather than an inter-endothelial cell (paracellular) route for plasma extravasation.
  • Chronic vascular hyperpermeability (CVH)
  • acute exposure to VEGF-A results in immediate but self-limited hyperpermeability of normal venules
  • chronic exposure results in profound changes in venular structure and function that lead to the chronic hyperpermeability of pathological angiogenesis as found in tumors, healing wounds, and chronic inflammatory diseases such as rheumatoid arthritis, psoriasis, cellular immunity, etc.
  • chronic inflammatory diseases such as rheumatoid arthritis, psoriasis, cellular immunity, etc.
  • the fluid that extravasates is an exudate that approaches the overall composition of plasma.
  • fluid leakage in CVH does not take place from any type of normal blood vessel. Instead, whether in tumors or wounds, the blood vessels that leak are newly formed, highly abnormal angiogenic blood vessels; these are primarily mother vessels (MV), and also, to a lesser extent, glomeruloid microvascular proliferations (GMP) that form from MV [Id., citing Nagy JA, et al. Lab Invest. (2006) 86: 767-80; Pettersson, A. et al. Lab Invest. (2000) 80: 99-115; Sundberg, C. et al. Am. J. Pathol.
  • MVs Mother Vessels
  • MVs are greatly enlarged sinusoids that arise from preexisting normal venules by a process that involves pericyte detachment, vascular basal lamina degradation, and a 4–5-fold increase in lumen size that is accompanied by extensive endothelial cell thinning.
  • Poiseuille’s law indicates that blood flow is proportional to the fourth power of the vascular radius.
  • MV exhibit sluggish blood flow because of their hyperpermeability to plasma which results in a striking increase in hematocrit.
  • tissue factor to trigger the clotting system and deposit fibrin [Id., citing Dvorak, HF et al. Science (1981) 212: 923-24; Van De Water, L. et al. Cancer Res. (1985) 45: 5521-25].
  • Tissue factor is expressed on many tumor cells as well as host interstitial cells and is induced in endothelial cells by VEGF-A [Id., citing Dvorak, HF, Rickles, FR (2005) Hemostasis and thrombosis in cancer.
  • fibrin In addition to its fluid trapping properties, fibrin also has a number of other properties when it persists over time as in tumors and healing wounds. It provides a pro-angiogenic provisional stroma that induces and is later replaced by the ingrowth of new blood vessels and fibroblasts and the laying down of mature fibro-vascular stroma [Id., citing Dvorak, HF. Am. J. Pathol. (2003) 162: 1747-57; Dvorak, HF, et al. J. Immunol.
  • Fibrin interacts with integrins expressed by multiple cell types and so supports the migration of tumor cells as well as host mesenchymal cells (endothelial cells, pericytes, fibroblasts) and inflammatory cells (neutrophils, monocytes). Fibrin also sequesters growth factors, protecting them from degradation, and induces the expression of proangiogenic molecules such as IL-8 and tissue factor. Fragment E, a fibrin breakdown product, is directly pro-angiogenic [Id., citing Dvorak H (2007) Tumor blood vessels. Aird, W.
  • MV develop from normal venules by a process that involves extensive vascular enlargement with consequent endothelial cell thinning
  • processes that are thought to be facilitated, at least in part, by a transfer of VVO membranes to the plasma membrane, MV, as well as the GMP that derive from MV contain many fewer and less complex VVOs than normal venular endothelium.
  • the path length for molecular extravasation is greatly shortened as tracers such as ferritin need to pass through only a few, often only one or two, vesicles or vacuoles to reach the ablumen [Id., citing Nagy, JA, et al. Lab Invest. (2006) 86 (8): 767-80].
  • Macromolecules also extravasate through fenestrae that are present in both MV and GMP [Id., citing Feng, D. et al. Microcirculation (1999) 6: 23-44]. Pores of the type that have been described in AVH have also been found in the endothelial cells of blood vessels supplying tumors [Id., citing Majno, G. et al. J. Cell Biol. (1969) 42: 647-72; Freng, D. et al. Microvascular Research (2000) 59: 24-37; Roberts, WG, Palade, GE. J. Cell Sci. (1995) 109 (6): 2369-79].
  • Virus infection induces increased permeability of the lung microvasculature
  • IAV influenza A virus
  • vasculotide activates the cell-cell adhesion protein TEK [Id., citing Van Slyke, P. et al. Tissue Eng. Part A (2009) 15: 1269-8049] and had been shown to maintain barrier function of human microvascular endothelial cells treated in vitro with lipopolysaccharide and to improve alveolar-capillary barrier function and survival in mice during experimental endotoxemia [Id., citing David, S. et al.
  • ANGPTL4 angiopoietin-like 4
  • PR8 angiopoietin-1
  • ANGPTL4 interacts with integrin ⁇ 5 ⁇ 1, VE-cadherin, and claudin-5 to weaken cell-cell interactions and loosen the epithelial barrier [Id., citing Huang, RL, et al. Blood (2011) 118: 3990-4002].
  • Sphingosine 1-phosphate is a physiological lipid signaling intermediate involved in both immunoregulation and epithelial barrier function [Id., citing Chi H. Trends Pharmacol Sci (2011) 32: 16–24, Natarajan, V. et al. (2013) Am J Respir Cell Mol Biol 49: 6– 17].
  • AAL(R) The S1P analog 2-amino-4-(4-heptyloxyphenyl)-2-methylbutanol [AAL(R)] showed some success in protecting mice from infection with either 2009 pandemic or mouse-adapted H1N1 strains [Id., citing Walsh, KB, et al. Proc Natl Acad Sci USA (2011) 108: 12018–12023]. Mice treated with AAL(R) 1 h after infection showed improvements in lung damage and survival accompanied by reduction in proinflammatory cytokine production, including type I interferons and macrophage chemokines compared with vehicle-treated controls. [Id., citing Walsh, KB et el. Proc Natl Acad Sci USA (2011) 108: 12018–12023].
  • AAL(R) is phosphorylated in vivo by sphingosine kinase 2 to AFD(R) ([(2R)-2-amino-4-(4- heptoxyphenyl)-2-methylbutyl] dihydrogen phosphate), which stimulates S1P receptors (S1PR) 1, 3, 4, and 5 [Id., citing Jary, E., et al. Mol. Pharmacol. (2010) 78: 685-92; Oldstone, MBA, et al. Virology (2013) 4435: 92-101].
  • S1P is a regulator of endothelial cell adhesion [Id., citing Natarajan, V. et al. Am J Respir Cell Mol Biol (2013) 49: 6–17; Rosen, H. et al. Trends Immunol. (2007) 28: 102-107]. It is therefore possible that mortality and cytokine storm are both effects of the permeabilized alveolar-capillary barrier function, rather than the cytokine storm being directly causative of mortality. [Id.] Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) [000210] ALI and its more severe form, ARDS, are syndromes of acute respiratory failure that result from acute pulmonary edema and inflammation.
  • ALI acute lung injury
  • ARDS acute respiratory distress syndrome
  • ALI/ARDS is a cause of acute respiratory failure that develops in patients of all ages from a variety of clinical disorders, including sepsis (pulmonary and nonpulmonary), pneumonia (bacterial, viral, and fungal), aspiration of gastric and oropharyngeal contents, major trauma, and several other clinical disorders, including severe acute pancreatitis, drug over dose, and blood products [Ware, L. and Matthay, M., N Engl J Med, (2000) 342:1334-1349,]. Most patients require assisted ventilation with positive pressure. The primary physiologic abnormalities are severe arterial hypoxemia as well as a marked increase in minute ventilation secondary to a sharp increase in pulmonary dead space fraction.
  • ALI/ARDS Patients with ALI/ARDS develop protein-rich pulmonary edema resulting from exudation of fluid into the interstitial and airspace compartments of the lung secondary to increased permeability of the barrier. Additional pathologic changes indicate that the mechanisms involved in lung edema are complex and that edema is only one of the pathophysiologic events in ALI/ARDS.
  • One physiologic consequence is a significant decrease in lung compliance that results in an increased work of breathing [Nuckton T. et al., N Engl J Med. (2002) 346:1281-1286,], one of the reasons why assisted ventilation is required to support most patients.
  • MV ventilator-associated lung injury
  • HVT high tidal volumes
  • MK2 mechanical ventilation
  • ACE levels increase in the brochoalveolar fluid of individuals with ARDS [Id., citing Idell, S. et al., Chest (1987) 91: 52-56]; ACE is thought to influence both vascular permeability and the air-vessel interface, as well as maintain pneumocyte viability. Furthermore, treatment of rats with ALI using ACE antagonists delays the onset of ARDS. [Id., citing Raiden, S., et al. J. Pharmacol. Exp. Ther. (2002) 303: 45-51].
  • ACE2 As for ACE2, ACE2 has been shown to have an opposing function to ACE and protects against lung injury. [Id., citing Imai, Y. et al. Nature (2005) 436 (7047): 112-116].
  • the protective effect of ACE2 seems to result partially from the conversion of angiotensin II by ACE2 to angiotensin1-7, thereby reducing angiotensin II binding to the cell membrane receptors AT1aR (angiotensin II type 1a receptor) and AT2R (angiotensin II type 2 receptor). It is believed that angiotensin II binding to AT1aR will stimulate lung injury, whereas binding to AT2R reduces lung injury.
  • Kuba et al proposed that binding of SARS-CoV to ACE2 downregulates ACE2, thus leaving angiotensin II unmodified, allowing it to continue to bind to the AT1aR to aggravate the lung injury and produce lung edema. [Kuba, K. et al., Nat. Med. (2005) 11: 875-79]. [000214] Age-dependent differences in host defense and the pulmonary renin-angiotensin system may be responsible for observed differences in epidemiology of ARDS between children and adults.
  • Viral mediated ALI The underlying pathophysiology of virally mediated ALI is not well understood, and it is likely that there are unique signature mechanisms to each viral strain that converge onto a common end pathway resulting in diffuse alveolar damage (DAD). It remains to be seen whether epithelial injury is the primary lesion or is coincident to endothelial injury. Most community-acquired respiratory viral pneumonias are inhaled and bind to receptors in the upper respiratory tract.
  • DAD diffuse alveolar damage
  • the viruses initially infect the respiratory epithelium, it is possible that this is merely a portal of entry, and the important steps in alveolar damage are mediated primarily by endothelial injury resulting in elaboration of cytokines and chemokines and recruitment of both innate and adaptive immune cells.
  • the specific cytokine profiles vary by viral pathogen and may be driven by macrophages, epithelial cells, endothelial cells, or some combination of crosstalk. If lung injury is not primarily mediated by viral infection, but rather is a result of the inflammatory host response, then viral clearance may not be central to the resolution of lung injury. [Hendrickson, CM, Matthay, MA Semin. Respir. Crit. Care Med. (2013) 34: 475-86].
  • DFA direct fluorescence assays
  • PCR-based assays PCR-based assays
  • viral culture PCR-based assays
  • RSV respiratory syncytial virus
  • adenovirus adenovirus
  • metapneumovirus adenovirus
  • rhinovirus enterovirus
  • coronavirus cytomegalovirus
  • CMV cytomegalovirus
  • Cytomegalovirus [000217]
  • CMV Cytomegalovirus
  • the virion of CMV a member of the herpesvirus family; contains not only DNA, but also four species of mRNA.
  • CMV is a common pathogen in severely immunosuppressed populations (especially organ transplant recipients), but was previously thought to be an uncommon pathogen in nosocomial pneumonia.
  • Seroprevalence for CMV in adults ranges from 50 to 90%.
  • CMV has been detected in nonimmunocompromised adults with critical illness and is thought to be reactivation of latent infection. It is associated with longer intensive care unit (ICU) and hospital stays, longer duration of mechanical ventilation, and increased rates of nosocomial infections. CMV infection is associated with increased interleukin (IL)-6 and IL-8 in vitro and in vivo, and these cytokines have been associated with ALI and ARDS.
  • IL interleukin
  • IL-8 interleukin-6 and IL-8 in vitro and in vivo
  • cytokines have been associated with ALI and ARDS.
  • VAP ventilator-associated pneumonia
  • Hantavirus are enveloped viruses with a negative sense, single-stranded RNA genome that belong to the family Bunyaviridae [Saggioro, FP, et al. J. Infectious Disease (2007) 195 (10): 1541-49, citing Simmons, JH, Riley, LK. Comp. Med. (2002) 52: 97-110]. Distinct hantavirus types are distributed throughout the world and are associated with different primary rodent reservoirs [Id., citing Simmons, JH, Riley, LK. Comp. Med. (2002) 52: 97- 110; Lednicky, J.A. Arch. Pathol. Lab Med.
  • HPS is an acute respiratory illness, first identified in Four Corners, southwestern United States, in 1993, characterized by a capillary-leak syndrome in the lungs and clinically presenting as an adult respiratory distress syndrome [Id., citing Centers for Disease Control and Prevention. Outbreak of acute illness—southeastern United States, 1993, MMWR Morb Mortal Wkly Rep, 1993, vol.42 (pg.421-4); Nolte, KB, et al. Hum. Pathol. (1995) 26: 110-20; Zaki, SR, et al. Am. J. Pathol. (1995) 146: 552-79].
  • Influenza viruses belong to the family Orthomyoviridae, and are enveloped negative-sense RNA viruses with segmented genomes. There are three antigenically distinct subtypes, A, B, and C, which circulate globally among human populations.
  • Influenza A viruses are subdivided based on antigenic characterization of the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). There are 16 HA subtypes and nine NA subtypes. The clinical response to influenza infection ranges from mild disease to severe pneumonia, and it remains unclear whether the inflammatory response to infection is protective or pathogenic. Infection rarely induces symptoms of lower respiratory tract infections or severe lung injury. Influenza virus infection has been a global health concern since the 1918 Spanish flu pandemic. Three influenza pandemics occurred in the 20th century. The pandemics of 1918, 1957, and 1968 were caused by different antigenic subtypes of influenza A: H1N1, H2N2, and H3N2, respectively.
  • H1N1 pandemic A virus originated from the triple- reassortment of swine influenza (H1) virus circulating in North American pigs.
  • H1N1 pandemic A virus
  • Nucleic acid sequencing showed that the HA, nucleoprotein (NP), and nonstructural protein (NS) gene segments were from the classical swine viruses; PB1 gene segment from human seasonal H3N2 influenza viruses; and PB2 and PA genes from avian influenza viruses.
  • NA and M gene segments were genetically different from previously isolated human pathogens and found to originate from Eurasian swine influenza strains.
  • H1N1-infected patients were children or adults aged ⁇ 60 years; most recovered uneventfully, and the overall mortality was not higher than that of seasonal influenza.
  • Risk factors for more severe infection by pandemic H1N1 include extremes of age, underlying medical illness, obesity, and pregnancy.
  • IFN signaling results in the transcription and translation of cell death-inducing ISGs.
  • infected cells in which the internal activation of antiviral ISGs is not sufficient to restrict viral replication are sacrificed to prevent the release of infectious progeny virions to limit viral spreading.
  • TRAIL Tumor necrosis factor-related apoptosis-inducing ligand
  • TRAIL signaling outcomes can differ largely depending on its delivered form (membrane-bound versus soluble), the availability of DRs on the target cell membrane, alternate intracellular pathways that might be activated, and the pathogen itself, which might exploit TRAIL-induced pathways for its own survival and replication.
  • TRAIL signaling is often part of an IFN-driven overshooting inflammatory reaction that promotes unspecific tissue injury and thus disease severity by increasing functional and structural changes in infected and non-infected cells.
  • Macrophage-expressed IFN- ⁇ contributes to apoptotic alveolar epithelial cell injury in severe influenza virus pneumonia.
  • the relationship between TRAIL induction and IAV strain-specific pathogenicity also translates to the highly pathogenic avian H5N1 IAV, causing severe pneumonia in mice as well as in humans [Id., citing Uiprasertkul M, et al. Emerg Infect Dis (2007) 13(5):708–12, Szretter KJ, et al. J Virol (2007) 81(6):2736–44].
  • TRAIL signaling upon IAV infection has also been implicated in the induction of necroptosis in fibroblasts, DCs, and lung epithelial cells [Id., citing Rodrigue-Gervais IG, et al. Cell Host Microbe (2014) 15(1):23–35, Nogusa S, et al. Cell Host Microbe (2016) 20(1):13–24, Hartmann B, et al. J Immunol (2015) 194(1 Suppl):127.2–127.2].
  • TRAIL-induced and AMPK-mediated downregulation of the Na,K-ATPase a major driver of vertical ion and fluid transport from the alveolar airspace toward the interstitium, resulted in a reduced capacity of IAV-infected mice to clear excessive fluid from the alveoli.
  • TRAIL signaling contributes to intensive edema formation, a hallmark of disease in virus-induced ARDS [Id., citing Matthay MA, et al. J Clin Invest (2012) 122(8):2731–40].
  • TRAIL has also been shown to be upregulated on NK, DC, and on CD4+ and CD8+ T cells after IAV infection [Id., citing Ishikawa E, et al. J Virol (2005) 79(12):7658–63].
  • Bacterial superinfection after viral injury [000226] Severe viral infections of the respiratory tract often are followed by outgrowth of colonizing Gram-positive bacteria that aggravates the course of illness.
  • type I IFNs activate the histone methyltransferase Setdb2, which in turn represses the Cxcl1 promoter and thus impairs neutrophil recruitment and bacterial clearance.
  • type I IFN production decreases CCL2 production, thus inhibiting macrophage recruitment, which as well has been reported to have detrimental effects on bacterial clearance and disease progression in bacterial superinfection after viral insult in vivo [Id., citing Nakamura S, et al. J Clin Invest (2011) 121(9):3657–65).
  • type I IFNs also impair ⁇ T cell function and IL-17 release, which was shown to increase susceptibility to S.
  • Respiratory Syncytial Virus [000228] Human respiratory syncytial virus (RSV) is an enveloped, nonsegmented negative-strand RNA virus of family Paramyxoviridae. It is the most complex member of the family in terms of the number of genes and proteins. Respiratory syncytial virus is an important cause of respiratory tract infections especially in children worldwide; it is the major cause of lower respiratory tract illness in young children. [Falsey, AR et al., J. Clin. Microbiol. (2002) 40(3): 817-20, citing Hall, C. B. N. Engl. J. Med. (2001) 334:1917-1928].
  • soluble TRAIL released from leukocytes was elevated in the bronchoalveolar lavage fluid of patients with RSV-associated respiratory failure, suggesting that similar to IAV, TRAIL contributes to RSV-induced epithelial injury and disease progression [Id., citing Bem RA, Bos AP, Wösten-van Asperen RM, Bruijn M, Lutter R, Sprick MR, et al. Potential role of soluble TRAIL in epithelial injury in children with severe RSV infection. Am J Respir Cell Mol Biol (2010) 42(6):697–705].
  • SARS coronavirus is an enveloped RNA virus that replicates with transcription of discontinuous nested messenger RNA (mRNA).
  • mRNA messenger RNA
  • the reservoir for the virus is thought to be civet cats, a nocturnal mammal considered a delicacy in southern China.
  • Horseshoe bats may also be a reservoir.
  • the incubation period is 2 to 7 days before symptom onset, and peak viral shedding in respiratory secretions occurs relatively late, between 6 and 11 days.
  • the virus is spread through respiratory secretion shedding and via contact with fomites. Airborne transmission, particularly on international flights, contributed to superspreader outbreak phenomenon.
  • SARS-CoV infection drives cells into apoptosis by a PKR-driven but eIF2 ⁇ -independent pathway [Id., citing Krähling V, et al. J Virol (2009) 83(5):2298–309], which might—similarly as seen in IAV infection—suggest a PKR-induced and autocrine/paracrine executed activation of apoptosis. [000234] Also MERS-CoV, which causes pneumonia and respiratory failure, has been demonstrated to induce profound cell death within 24 h of infection, irrespective of viral titers produced by the infected cells.
  • type I IFN expression is strongly reduced in MERS- CoV in comparison to seasonal human CoV in in vitro infection models, including human monocyte-derived macrophages, Calu-3, and human lung fibroblasts [Id., citing Lau, SK et al. J. Gen. Virol. (2013) 94 (Pt.12): 2679-90; Zhou, J. et al. J. Infect. Dis. (2014) 209 (9): 1331- 42], which might also dampen downstream TRAIL induction. Therefore, the exact mechanism by which MERS-CoV promotes cell death remains to be investigated.
  • Zika virus is an arbovirus, which can be transmitted to humans by Aedes mosquitoes as well as by sexual interactions.
  • ZIKV is closely related to some important human pathogens, such as dengue virus (DENV), yellow fever virus (YFV), west nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) [Yang, C. et al. Virologica Sinica (2019) 34: 168-174, citing Wang Q, et al. J. Virol. (2017) 91: e01049-1].
  • DECV dengue virus
  • YFV yellow fever virus
  • WNV west nile virus
  • JEV Japanese encephalitis virus
  • TBEV tick-borne encephalitis virus
  • IUI Intrauterine infection or inflammation
  • chorioamnionitis is responsible for ⁇ 40% of preterm labor cases [Cappeltti, M. et al. Front. Immunol. (2020) 11: 649; citing Kim, CJ et al. Am. J. Obstat. Gynecol. (2015) 213: S29-S52] ).
  • Prematurity which affects nearly 10% of pregnancies world-wide, is the most significant cause of perinatal mortality or morbidity [Id., citing Iams, JD, et al.
  • Acute chorioamnionitis which is characterized by neutrophilic infiltration and inflammation at the maternal fetal interface is a relatively common complication of pregnancy and can have devastating consequences including preterm labor, maternal infections, fetal infection/inflammation, fetal lung, brain, and gastrointestinal tract injury. [000238] Most commonly, acute chorioamnionitis is a result of ascending infection with relatively low-virulence organisms such as the Ureaplasma species. In a minority of cases, microorganisms can also invade the placenta by the hematogenous route, and the profile of organisms is different compared to the ascending route.
  • Microorganisms that invade the placenta by the hematogenous route include Listeria monocytogenes [Id., citing Gellin, BG et al. Am. J. Epidemiol. (1991) 133: 392-401], Zika virus [Id., citing Msorekar, IU, Diamond MS. N. Engl. J. Med. (2016) 375: 481-84; Tabata, T. et al., Cell Host Microbe (2016) 20: 155- 66], Treponema pallidum [Id., citing Arora, N. et al. Cell Host Microbe (2017) 21 : 561-67]; Cytomegalovirus [Pereira, L. Anu. Rev.
  • Viral infections can prime or accentuate bacterial infection- mediated preterm labor and the intensity of inflammatory response at the maternal-fetal interface [Id., citing Cardenas, I. et al. Am. J. Reprod. Immunol. (2011) 65: 110-7; Aracicot, K. et al. J. Immunol. (2013) 191: 934-41; Kwon, JY, et al., am. J. Reprod. Immunol. (2014) 71: 387-90; Racicot, K. et al., Am. J. Reprod. Immunol. (2016) 75: 451-60; Racicot, K. et al. J. Imunol. (2017) 198: 3029-32].
  • IFN type I Interferon
  • IFNAR IFN receptor
  • AXL is a TAM family tyrosine kinase that was initially described as the entry receptor for Zika. [Chen, J. et al. Nature Microbiollogy (2016) 3: 302-309].
  • astrocytes It is highly expressed in astrocytes, radial glia cells, and endothelial cells in human brain.
  • Astrocytes play an important role during infection of neurotropic flativiruses, and type I IFN signaling in primary astrocytes has been reported to play an important role in restricting Zikv infection.
  • the susceptibility of astrocytes to divergent WNV strains is a key determinant for viral propagation and fitness. [Id., citing Hussmann, KL, et al. J. Gen.
  • astrocytes can express pro-inflammatory cytokines and chemokines during the infection of tick-borne encephalitis virus. [Id., citing Palus, M. et al. J. Gen. Virol. (2014) 95: 2411-26]. That astrocytes are the primary target of Zika in the brain during peripheral infection in newborn immunocompetent mice has been suggested. [Id., citing van den Pol, AN, J. Neurosci. (2017) 37: 2161-75].
  • AXL is unlikely to function as an entry receptor for Zika, and may instead promote Zika infection in human astrocytes by antagonizing type I interferon signaling.
  • the acute lung injury caused by the virus must be repaired to regain lung function; any dysregulation in this wound healing process leads to fibrosis.
  • the repair of lung tissue after infection or mechanical trauma normally occurs in a controlled series of events beginning with damage signals sent from infected cells, which recruit inflammatory cells, which then induce secretion of growth factors, which activates basement membrane repair and finally leads to the replacement of injured tissue. Under normal circumstances, the wound healing response is downregulated once the injury is repaired.
  • Acute kidney injury (AKI) and coronavirus infection [000241]
  • a previous study on SARS-CoV infection showed that the virus RNA is effectively detected in urine 10 days after the onset of symptoms, and the excretion gradually decreased until day 21 [Martinez-Rojas, M.A. et al. Am. J. Physiol. Renal Physiol. (2020) 318 (6): F1454-62, citing Peiris, JSM et al. Lancet (2003) 361L 1767-72].
  • Autopsies of SARS- CoV-confirmed patients demonstrated the virus presence in tubular epithelial cells by immunohistochemistry and in situ hybridization [Id., citing Ding, Y. et al. J. Pathol.
  • a small fraction of plasma proteins is filtered in the renal glomeruli, and most of them are effectively reabsorbed in the proximal tubule, so that basically no proteins appear in normal urine.
  • the glomerular filtration barrier depends on adequate function of its three components: endothelial cells, the glomerular basement membrane, and podocytes [Id., citing Cara-Fuentes, G. et al. Pediatr. Nephrol. (2016) 31: 2179-89].
  • Podocytes are known to be particularly sensitive to RAAS homeostasis, with angiotensin-1–7 being the most abundant product, probably due to the specific expression of ACE2 in this region [Id., citing Velez, JCQ, et al. Am. J.
  • Physiol. Renal Physiol. (2007) 293: F398-F407 If a pathological process increases glomerular levels of angiotensin II, podocytes acquire a dysfunctional phenotype mediated by cellular responses to this octapeptide due to shear stress and resulting in single nephron hyperfiltration.
  • This phenotype involves Ca2+ signaling, cytoskeleton restructure, and nephrin internalization, which finally is manifested by proteinuria [Id., citing Konigshausen, E. et al. Sci. Rep. (2016) 6: 39513; Srivastava, T. et al. Am. J. Physiol. Renal Physiol.
  • AKI might occur as a result of intrarenal inflammation, increased vascular permeability, and volume depletion, which is translated in the findings of autopsies of erythrocyte aggregates obstructing the lumen of capillaries without platelet or fibrinoid material.
  • an imbalance in components of the RAAS can contribute to kidney injury by changing renal hemodynamics, altering tubular handling of electrolytes with a higher metabolic demand, and inducing proinflammatory phenotypes in both epithelial and immune cells.
  • Trefoil factor family of peptides [000247]
  • the trefoil factor family (TFF) is a family of peptides with a three-loop trefoil domain that have a close association with mucins and are mainly synthesized and secreted by mucin secreting epithelial cells lining the gastrointestinal tract. [Wong, WM, et al. Gut (1999) 44(6): 890-95].
  • TFFs trefoil peptides or trefoil factors
  • the first trefoil peptide discovered was pS2/TFF1 or breast cancer estrogen inducible gene— discovered during a search for estrogen induced mRNAs from the mammary carcinoma cell line MCF7 in 1982. [Wong, WM, et al. Gut (1999) 44(6): 890-95, citing Masiakowski, P. et al. Nucleic Acids Res. (1982) 10: 7895-7903].
  • TFF2 previously spasmolytic polypeptide, SP
  • ITF/TFF3 intestinal trefoil factor
  • ITF/TFF3 intestinal trefoil factor
  • the trefoil domain is characterized by a sequence of amino acid residues, in which 6 cysteines are linked by 3 disulphide bonds to form the “trefoil” disulphide loop structure or the clover-like shaped structure [Aamann, L. et al. World J. Gastroenterol. (2014) 20 (12): 3223-30, citing Thim. L.
  • TFF1 and TFF3 only contain one trefoil domain but have a seventh free cysteine, which is essential for the formation of dimers [Id., citing Thim, L. et al. Biochemistry (1995) 34: 4757-64].
  • TFF2 contains two trefoil domains; in the amphibian Xenopus, there are molecules containing multiple trefoil domains [Wong, WM, et al. Gut (1999) 44(6): 890-95, citing Gmachl, M. et al. FEBS Lett. (1990) 260: 145-48; Hauser, F. and Hoffman, W. J. Biol. Chem. (1991) 266: 21306-09; Hauser, F. et al., J. Biol. Chem. (1992) 267: 14451-55; Hoffman, W., Hauser, F. Trends Biochem. Sci. (1993) 18: 239-43].
  • TFF1 and TFF2 are major expression sites for TFF1 and TFF2 in vivo.
  • TFF3 it is the intestine
  • TFF peptides also appear in saliva, gastric juice, urine, blood and breast milk [Id., citing Klellev, S. Cell Mol. Life Sci. (2009) 66: 1350-69; Roa, B and Tortolero, S. Bratisl. Med. J. (2016) 117: 332-39].
  • TFF peptides are ectopically expressed after wounding, in inflammatory diseases, and in various tumors. [Id, citing Klellev, S. Cell Mol. Life Sci. (2009) 66: 1350-69]. They are also secreted in an endocrine manner, for example, in the immune and central nervous system [Id., citing Klellev, S. Cell Mol. Life Sci. (2009) 66: 1350-69; Hoffmann, W, J. Med. Chem. (2009) 52: 6505-10]. TFF interactions [000251] TFF signaling through canonical receptor-lligand interactions has long been debated.
  • Chemokine receptor types 4 and 7 have been reported to mediate TFF2 and TFF3-induced chemotaxis. [Id., citing Klellev, S. Cell Mol. Life Sci. (2009) 66: 1350-69, Otto, W. and Thim, L. Cell Mol. Life Sci. (2005) 62: 2939-46].
  • Typical activated cascades include ERK1/2, JNK, Akt, and NF- ⁇ B. [Id., citing Klellev, S. Cell Mol. Life Sci. (2009) 66: 1350-69, Deckow, J. et al. Ophthalmol. Vis. Sci. (2016) 5;7: 56-65].
  • TFF2 triggers ERK1/2 signaling via CXCR4 [Id., citing Hoffmann, W, J. Med. Chem. (2009) 52: 6505-10], which might be linked to the recruitment of immune cells [Id., citing Deckow, J. et al. Ophthalmol. Vis. Sci. (2016) 5;7: 56-65].
  • TFF3 also interacts with CXCR4 and CXCR7 expressed on ocular surface tissues, where cell migration is induced via an ERK1/2-independent signaling pathway. [Id., citing Deckow, J. et al. Ophthalmol. Vis. Sci. (2016) 5;7: 56-65].
  • TFF2 and TFF3 activate PAR4 and PAR2 respectively [Id., citing Roa, B and Tortolero, S. Bratisl. Med. J. (2016) 117: 332-39, 9].
  • PAR knockdown abolishes the mucosal healing effect of TFF2 [Id., citing Zhang, Y. et al. Cell Mol. Lif Sci. (2011) 68: 3771-80].
  • TFF3 activates PAR2, but not PAR1, as shown in cytosolic CA2+ activity measurements in HT-29 cells, causing downregulation of proinflammatory cytokines and upregulation of defensing expression [Id., citing Roa, B and Tortolero, S. Bratisl. Med. J.
  • TFF2 binds noncovalently to integrin ⁇ 1, as determined by affinity chromatography. [Id., citing Hoffmann, W. Intl. J. Oncol. (2015) 47: 806-16; Otto, W. and Thim, L. Cell Mol. Life Sci. (2005) 62: 2939-46] This is of interest because integrins play an important role in cell migration, which is enhanced by TFF peptides.
  • TFF1 is produced mainly in the stomach, in superficial cells of the body and antral mucosa. TFF2 is abundant in the mucous neck cells in the body and in antral glands of the stomach [Wong, WM, et al.
  • TFF3 is expressed throughout the intestine and is abundant in salivary glands. It has been shown that both TFF1 and TFF2 are expressed by mucous neck cells in the corpus of the stomach [Id., citing Hanby, AM et al. J. Pathol.
  • TFF1 was first identified in human breast carcinoma cell lines by virtue of its regulation by estrogen [Id., citing Masiakowski, P. et al. Nucleic Acids Res. (1982) 10: 7895- 7903; May, FEB, Westley, BR. Cancer Res. (1986) 46: 6034-40]; TFF1 mRNA can be detected in 68% of breast tumors.
  • TFF1 has since been found to be expressed in a variety of other carcinomas including those of the stomach, pancreas, lung, endometrium, ovary (particularly mucinous carcinomas), prostate, bladder, cervix, and pancreas, and in medullary carcinoma of the thyroid and mucinous carcinoma of the skin.
  • TFF1 has since been found to be expressed in a variety of other carcinomas including those of the stomach, pancreas, lung, endometrium, ovary (particularly mucinous carcinomas), prostate, bladder, cervix, and pancreas, and in medullary carcinoma of the thyroid and mucinous carcinoma of the skin.
  • TFF3 expression is also induced in breast carcinomas in a hormone dependent manner. [Id., citing Poulsom, R. et al. J. Pathol. (1997) 183: 30-38]. TFF2 is not expressed in breast carcinomas. Id., citing Tomasetto, C. et al. EMBO J. (1990) 9: 407-14].
  • Trefoil factors are expressed in a wide variety of ulcerative conditions of the gastrointestinal tract, from Barrett’s esophagus [Id., citing Hanby, AM et al. J. Pathol. (1994) 173: 213-19] to gastric [Id., citing Levi, S. et al. Eur. J. Gastroenterol. Hepatol. (1993) 5 (suppl. 3): S39-S43; Alison, MR et al. J. Pathol. (1995) 175: 405-14] and duodenal ulcers [Id., citing Hanby, AM et al. J. Pathol.
  • hTFF1, hTFF2 and hTFF3, and rTFF3 have all been produced in Escherichia coli or yeast. All three mammalian trefoil factors are motogens, namely able to promote cell migration without promoting cell division, and are all upregulated at sites of mucosal injury and stimulate the repair process. Thus they participate in mucosal repair by stimulating the migration of surviving cells from the edge of the damaged region over the denuded area, a process called epithelial restitution, and essential for the repair of both minor and more extensive lesions.
  • TFF2 and TFF3 act as motogens when cell monolayers are wounded, in a transforming growth factor beta (TGF- ⁇ ) independent manner.
  • TGF- ⁇ transforming growth factor beta
  • TFF epidermal growth factor
  • TGF- ⁇ TGF- ⁇ receptors on the basolateral side of the epithelium.
  • trefoil factor family 2 (hTFF2), a secretory peptide with a molecular mass of 14,284 Da, which comprises 106 amino acid residues, is a Ca-independent, pH-resistant lectin that binds with high affinity to ⁇ -GlcNAc- capped O-glycans on MUC6 [Morozov, V. et al. Molecules (2016) 23 (5): 1151, citing Hanisch, F-G, et al. J. Biol. Chem. (2014) 289: 27363-75].
  • hTFF2 can reverse the growth inhibitory effect of porcine stomach mucin (PSM), which expresses antibiotic glycans similar to human MUC6 [Id.]. Many viruses have also exploited lectins for their own benefits during infection.
  • PSM porcine stomach mucin
  • dengue viruses utilize a c-type lectin, dendritic cell-specific ICAM- 3-grabbing non-integrins (DC-SIGN), on dendritic cells to enter the cells and replicate [Id., citing Tassaneetrithep, B. et al. J. Exp. Med. (2003) 197: 823-29].
  • HIV-1 mainly employs DC- SIGN on dendritic cells to promote the transfer to CD4+ T lymphocytes and galectin-1 to facilitate attachment to CD4+ T cells [Id., citing St-Pierre, C. et al. J. Virol.
  • Viruses themselves can encode lectins on their surface for cell targeting, among them noroviruses, rotaviruses, and influenza viruses.
  • the “leucine-rich repeat and immunoglobulin-like domain-containing NoGo” or “LINGO” family of proteins has been described in the nervous system where they play a role in axonal regeneration, neuronal survival, oligodendrocyte differentiation, and myelination.
  • LINGO-1 is characterized as a negative regulator of neuronal survival, axonal regeneration, and oligodendrocyte precursor cell (OPC) differentiation into mature myelinating oligodendrocytes.
  • OPC oligodendrocyte precursor cell
  • LINGO2 is highly expressed in patients with advanced gastric cancer and is involved in cell motility, tumorigenic ability and angiogenesis. [Id., citing Jo, JH et al. Int. J. Mol. Sci. (2019) 20 (3): 555]. LINGO activation usually occurs via homotypic or heterotypic interaction with other membrane proteins, such as the Nogo-A receptor and p75 neurotrophin receptor interaction with LINGO1 and EGF interaction with LINGO3. [Id., citing Zullo, K. et al. J. Immunol. (2019) 202: 192; Cobreet, L. et al. Br. J. Pharmacol. (2015) 172 (3): 841-56].
  • TFF2 and TFF3 have been described as natural ligands for the LINGO receptor interacting protein. [Id., citing Belle, NM et al. Nat. Commun. (2019) 10(1): 4408; Zullo, K. et al. J. Immunol. (2019) 202: 192]. TFF2 interaction with LINGO3 triggers ERK signaling, mediating tissue repair at the mucosal interface. [Id., citing Zullo, K. et al. J. Immunol. (2019) 202: 192].
  • LINGO3 -/- mice display a phenotype that resembles TFF2 deficiency, such as impairment of mucosal regeneration and accumulation of immune cells secreting inflammatory cytokines even without stimuli.
  • TFF3 interaction with LINGO2 disrupts LINGO2/EGFR interactions, enhancing activation of the EGFR pathway; thus this framework mediates epithelial repair.
  • a LINGO2-TFF3 interaction is supported by immunoprecipitation and colocalization studies at the IEC cell surface. [Id., citing Belle, NM, et al. Nat. Commun. (2019) 10(1): 4408].
  • LINGO2 and LINGO3 have been implicated as the receptors of TFF3 and TFF2, respectively [Rossi, HL et al. Am. J. Resp. Cell & Molec. Biol. (2022) 66 (3): 252-258, citing Belle, NM, et al. Nat. Commun. (2019) 10 (1): 4408; Zullo, KM. et al. Scand. J. Gastroenterol. (2021) 56: 791-805].
  • TFF3 binding to LINGO2 removes it from EGFR, allowing EGFR signaling to occur [Id., citing Belle, NM, et al. Nat. Commun.
  • TFF1 [000262] In a TFF1 knockout mouse model all mice showed loss of expression of gastric mucus and showed notably elongated pits, occupying the whole mucosa. Epithelial cells exhibited severe hyperplasia and high grade dysplasia, adenomas develop in the antrum of the stomach, and some of these progress into simply invasive carcinomas.
  • TFF1 may be a specific tumor suppressor gene for the stomach.
  • TFF1 may be a specific tumor suppressor gene for the stomach.
  • the dimeric form of TFF1 which forms dimers via Cys58, is more potent than the monomeric form in preventing indomethacin induced gastric damage and is also a stronger stimulant of the rate of migration of cells at the leading edge of wounded monolayers, suggesting that the Cys58 residue may play an important role in the biological function of TFF1.
  • TFF2 [000263] Within the context of mucosal repair, TFF2 is known for its involvement in mucosal repair, protection and proliferation, especially within both digestive and respiratory systems [Ghanemi, A. et al. Animals (2020) 10: 1646]. TFF2 is an important component and a stabilizer of the gastric mucus with the property of binding to the mucin MUC6 [Id., citing Heuer, F. et al. Int. J. Mol. Sci. (2019) 20: 5871], and is involved in tissue remodeling [Id., citing Royce, SG et al.
  • TFF2-deficient mice show increased susceptibility to NSAID injury. [Id., citing Farrell, JJ et al. J. Clin. Investig. (2002) 109: 193- 204]. [000264] Several studies suggest that upregulation of TFF may initiate the healing and repairing process that counteracts the inflammation-induced damage and some have described a potential anti-inflammatory effect. For example, TFF2 has been shown to be overexpressed (or upregulated) following inflammatory conditions [Id., citing Hoffmann, W. Int. J. Oncol.
  • TFF2 TFF2
  • IL-4 and IL-13 induce TFF2 [Id., citing Nikolaidis, NM et al. Am. J. Respir. Cell Mol. Biol. (2003) 29: 458-64]
  • hypoxia leads to upregulated TFF expression.
  • TFF2 treatment also has been reported to reduce fibrosis (subepithelial collagen deposition) in a murine model of chronic allergic airways disease [Id., citing Royce, SG et al. A. J. Respir. Cell Mol. Biol. (2013) 48: 135-44].
  • TFF2 mRNA levels increase within 30 minutes after mucosal injury induced by a cryoprobe on the serosal surface of the rat stomach.
  • TFF2 increases cell migration in in vitro models of cell wounding.
  • TFF3 also plays an important role in the repair and healing of the gastrointestinal tract. There is impaired mucosal healing and death from extensive colitis in TFF3 knockout mice after oral administration of dextran sulfate sodium, an agent that only causes mild to severe epithelial injury in wild type mice. [Id., citing Mashimo, H.
  • TFF3 has been implicated in mucosal protection and repair processes, stimulating cell migration via chemotaxis [Id. citing Chwieralski, CE et al. Am. J. Respir. Cell Mol Biol. (2004) 31: 527-37]. Because TFF peptides are secreted with mucin and because the amount of mucus is increased in lung diseases, it was hypothesized that the amount of TFF should also be increased in pulmonary diseases.
  • TFF3 was the most abundant of the peptides, followed by TFF1 and TFF2.
  • a correlation between TFF1 and TFF3 concentrations and FEV1 measurements was found, i.e., individuals with low FEV1 tended to have higher concentrations of TFF1 and TFF3 in the BAL fluid.
  • Goblet cell metaplasia and hyperplasia, together with periibronchiolar inflammation and fibrosis is characteristic in the pathobiology of COPD [Id., citing Jeffery, PK. Thorax (1998) 53: 129-36], and this appeared to be preceded by an increase in TFF production.
  • TFF secretion also could be a consequence of the increased amounts of mucin secretion in combination with the presence of several inflammatory cytokines. Inappropriate mucus production due to metaplasia in the bronchi has several detrimental effects, including a reduction in bronchiolar antiprotase, leading to epithelial destruction.
  • the exact stimulus for the goblet cell to increase expression of TFF is not known, but epithelial damage could be a mediator, since TFFs have been shown to be involved in protection and regeneration in other tissues. [Id., citing National Institute for Health and Care Excellence.
  • the mucous cells adjacent to the UACL express TFF1, with TFF1 co-packaged with mucous granules.
  • Neuroendocrine cells adjacent to the UACL in Crohn’s disease also express TFF1.
  • the function of these cell lineages is very different. Mucus is released into the lumen where it has lubricating and protective functions, whereas endocrine secretions release various regulatory peptides into the local circulation that act via paracrine or autocrine mechanisms to produce manifold effects on the gut.
  • TFF1 tissue necrosis factor 1
  • two different lineages suggests that it may have a dual mechanism of action—one is through the interaction with the overlying mucus layer and the other through an action on putative trefoil receptors on the basolateral surface of the epithelial cells.
  • An animal model of UACL in which UACL was induced in the distal esophagus by esophago-jejunal anastomosis has been reported. [Id., citing Hanby, AM et al. Am. J. Pathol. (1997) 151: 1819-24].
  • TFF3 mediates epithelial chloride transport after application to the basolateral surface of monolayers of colorectal carcinoma cells or of rat jejunum [Id., citing Chinery, R., Cox, HM. Br. J. Pharmacol. (1995) 115: 77-80] Furthermore, TFF3 seemed to bind a protein present in membrane preparations of colorectal epithelial cells, accompanied by phosphorylation of tyrosine, and the phosphorylation occurs within 10 seconds—suggestive of a receptor mediated response. [Id., citing Chinery, R., Cox, HM.
  • TFF3 also has been shown to decrease extracellular signal related protein kinase (ERK) activity; ERK is a member of the mitogen activated protein kinases (MAPK) family. [Id., citing Kanai, M. et al. Proc. Nat. Acad. Sci. USA (1998) 95: 178-82].
  • ERK extracellular signal related protein kinase
  • MAPK mitogen activated protein kinases
  • TFF3 The close association of TFF3 with EGFR has been shown by a synergistic action of TFF3 on electrogenic chloride transport [Id., citing Chinery, R., Cox, HM, Br. J. Pharmacol. (1995) 115: 77-80] cell migration and cytoprotection.[Id., citing Chinery, R., Playford, RJ. Clin. Sci. (1995) 88: 401-403].
  • the trefoil factor family molecules were identified as small, reparative cytokines (6-18 kDa) that promote rapid movement of epithelia over denuded basement membrane [Taupin, D., Podolsky, DK. Nat. Rev. Mol. Cell Biol.
  • TFF2 Does TFF2 play a role in modulating immune responses in vivo? [000274] The TFFs act to promote the speed of gastric epithelial restitution in vivo after acute and modest gastric injury. [Xue, L. et al. Gut (2010) 59 (9): 1184-91]. TFF2 activates calcium-dependent signaling and ERK1/2 phosphorylation via the CXCR4 receptor. [Xue, L. et al. J. Biol. Chem. (2011) 286 (44): 38375-82]. TFF2 is upregulated in Helicobacter spp- infected gastric tissues of both humans and mice.
  • TFF2 may play a protective role by modulating levels of gastric IFN-gamma in the development of H. pylori-associated premalignancy of the distal stomach.
  • TFF2 modulates Ca2+ and AKT signaling in lymphoblastic Jurkat cells; these effects appear to be mediated through the CXCR4 receptor.
  • TFF2 expression is not limited to the gastrointestinal tract; it is also present in macrophages and lymphocytes.
  • TFF2 may function in part as an anti-inflammatory cytokine, showing some similarity in this respect to IL10.
  • Kurt-Jones EA et al. Infect. Immun. (2007) 75: 471-80
  • TFF2 -/- mice show a hyper-inflammatory phenotype.
  • TFF2 -/- mice exhibit increased IFN ⁇ levels and increased mucosal CD4+ T cells.
  • TFF2 transcription is regulated by p53 and KLF4 transcription factors through an AP-1 site.
  • AP-1 binding sequences are promoter/enhancer elements that play a role in gene induction in mammalian cells. [Zhou, H. et al. DNA Res. (2005) 12: 139-50].
  • TFF2 induces cell apoptosis and inhibits cell migration in part by downregulating TFF2 expression through an AP-1 site.
  • TFF2 (a) promotes type 2 lung immunopathology induced by allergen or IL-13; (b) controls the extent of lung injury caused by migratory infectious stage larvae (L3); and (c) modulates early TH2 development. [Wills-Kkarp, M. et al. J. Exp. Med. (2011) 209 (3): 607-22].
  • Hookworms damage alveolar architecture during their circuitous migration from skin to GI tract; Nippostrongylus brasiliensis (N.b.) larvae enter the lung within 24h and cause petechial hemorrhage for 3 days before parasites egress to infest the GI tract [Id., citing Hung, LY et al. Proc. Nat. Acad. Sci. USA (2013) 110 (1): 282-87; Herbert, DR et al., J. Exp. Med. (20009) 206 (13): 2947-57].
  • Macrophages (M ⁇ ) reside in virtually every organ, where tissue-specific cues instruct unique transcriptional signatures in resident and emigrant M ⁇ to facilitate host defense, immunoregulation, and tissue homeostasis [Hung, L-Y, et al. Mucosal Immunol. (2019) 12 (1): 64-76, citing Lavin, Y. et al. Cell (2014) 159 (6): 1312-26].
  • Lung M ⁇ alveolar macrophages (AM) and interstitial macrophages (IM) have been long recognized as immunosuppressive cells that mediate efferocytosis (meaning the clearance of apoptotic cells by phagocytes), and phagocytic clearance of inhaled particles and pathogens.
  • Communication between AM and alveolar epithelia enforces an immunosuppressive lung environment [Id., citing Westphalen, K. et al. Nature (2014) 159 (6): 1312-26], but whether such crosstalk regulates epithelial proliferation remained obscure.
  • Alveolar macrophage (AM)-derived TGF- ⁇ promotes Foxp3+Treg expansion [Id., citing Soroosh, P.
  • TGF- ⁇ promotes fibrosis and restricts epithelial cell proliferation [Id., citing Siegel, PM, Massague, J. Nat. Rev. Cancer (2003) 3 (11): 807-21] [000281] Mouse strains deficient for Tff1, Tff2, or Tff3 possess constitutive defects in gastrointestinal (GI) barrier function [Hung, L-Y, et al. Mucosal Immunol. (2019) 12 (1): 64- 76, Aamann, L. et al. World J. Gastroenterol. (2014) 20 (12): 3223-30’ Taupin, DR, et al., Proc. Nat.
  • TFF2 can be produced by gastric epithelia, peritoneal macrophages and splenic T lymphocytes [Id., citing Dubeykovskaya, Z. et al. Nature Commun. (2016) 7: 10517], how this molecule regulates epithelial repair is understudied.
  • TFF2 deficiency in all of its potential cellular sources was shown to exacerbate lung damage caused by the murine hookworm Nippostrongylus brasiliensis (N.b.), and data has shown that both bone marrow (BM)-derived and non-BM-derived TFF2 drives repair.
  • BM bone marrow
  • TFF2 conditionally deficient mice on a C57BL/6 background were generated by expressing an internal ribosomal entry site-fluorescent tandem dimer (td)-Tomato cassette (IRES-TdTomato) immediately following the stop codon in exon 4 of Tff2, with two loxP sites flanking exons 2 and 3’UTR.
  • td internal ribosomal entry site-fluorescent tandem dimer
  • TFF2-tdTomato flox mice (designated “Tre-Tom”) had no gross abnormalities and were born at the expected female:male ratios. Site-specific recombination and germ-line transmission were confirmed by Southern blot. As expected, Tff2 mRNA was constitutively expressed in a small population of Td-tomato+ gastric epithelial cells sorted from the stomach tissue of naive Tre-tom mice, which was consistent with Tff2 expression in the stomach [Id., citing Farrell, JJ et al. J. Clin. Invst. (2002) 109 (2): 193-204].
  • Tff2 expression in the pulmonary compartment 300 ⁇ m sections of agarose-filled lung tissue were prepared using established methods [Id., citing van Rijt, LS, et al. J. Exp. Med. (2005) 201(6): 981-91] and subjected to confocal imaging under steady-state and infectious conditions. Whereas baseline Td-tomato expression was sparse but punctate within the distal lung compartment under steady-state conditions, large, focal aggregates of Td-Tomato+ cells were seen within the alveolar compartment at d4 following hookworm infection. These data indicated that hookworm infection induced an increase of TFF2 expression occurred within large cells in the alveoli.
  • CD11cCreTFF2flox mice were generated through intercross between Tretom and CD11cCre strains to test whether myeloid-derived TFF2 was biologically important [Id., citing van Rijt, LS, et al. J. Exp. Med. (2005) 201 (6): 981-91].
  • Tff2 transcripts were higher in lung epithelia sorted from CD11cCreTFF2flox mice than epithelia in CD11cCre controls, suggesting a compensatory Tff2 upregulation in epithelial cells due to deletion in the myeloid compartment.
  • CD11cCreTFF2flox mice had a reduced percentage of BrdU+ cells within alveolar type 2 cell populations (CD45 ⁇ EpCAM+ pro-SPC+) when compared to CD11cCre controls at day 9 post-infection.
  • Oxygen saturation (SpO2) levels were significantly lower in CD11cCreTFF2flox mice at d9 post-infection, implying a defect in lung function rebound.
  • Acute cytotoxicity of bleomycin causes severe alveolar cell damage, transient weight loss, pulmonary inflammation, and chronic collagen accumulation [Id., citing Hogan, BL et al., Cell Stem Cell (2014) 15(2): 123-38].
  • Bleomycin treatment transiently increased BAL TFF2 levels in both mouse strains, whereas CD11cCre controls underwent a 3-fold increase at d9; this early induction was reduced by myeloid-specific TFF2 deficiency.
  • CD11cCreTFF2flox mice produced higher TFF2 levels than CD11cCre controls by d16, suggestive of compensatory production from a non-myeloid source at later stages when fibrosis occurs.
  • CD11cCreTFF2flox animals experienced a transient, but significantly greater, weight loss than CD11cCre controls during the acute injury phase, but subsequently recovered in the latter phase.
  • the total protein levels in BAL fluid were higher in CD11cCre TFF2flox; also, the CD45 ⁇ EpCAM+ BrdU+ pro-SPC+ population of lung epithelia were significantly reduced in percentage from CD11cCreTFF2flox mice compared to CD11cCre mice, concordant with significantly reduced mRNA expression levels for Spc (encoding surfactant protein C) and Cc10 (encoding Clara cell 10kDa protein) at d9.
  • M ⁇ -epithelial repair assay utilized primary mouse airway epithelial cells cultured under bi-phasic conditions (i.e. air-liquid interface) in trans-well inserts with bone marrow M ⁇ (BMM ⁇ ) attached to the basolateral surface directly underneath.
  • the two cell populations were separated across a semi-permeable barrier (10 ⁇ m thick with pore size 0.4 ⁇ m) and evaluated following a pipette-mediated scratch wound to the epithelial monolayer.
  • Tracheal epithelia were initially used due to the high trans-epithelial cell resistance (TER) levels (generally >1000 ⁇ ).
  • TER trans-epithelial cell resistance
  • M ⁇ cultured alone lacked electrical resistance, but epithelia cultured alone maintained TER levels over 1000 ⁇ .
  • M ⁇ significantly accelerated the TER rebound following pipette- mediated scratch because M ⁇ -epithelia co-cultures resulted in 100% restoration of baseline TER levels by d3, further increasing to 130% by d4 post-scratch.
  • rTFF2 In the absence of M ⁇ , rTFF2 treatment increased proliferation only 2-fold. Application of rTFF2 alone (without M ⁇ ) significantly accelerated TER rebound over the scratched, mock-treated cultures. Moreover, WT alveolar macrophages (AM) significantly accelerated TER restoration, but TFF2 ⁇ / ⁇ AM were less able to promote repair, whereas lack of TFF2 within the epithelia impaired TER rebound irrespective of the AM genotype. [000289] To address whether macrophages also promoted recovery of epithelia population relevant to the distal lung compartment, primary alveolar type 2 (AT2) cells were used instead of MTEC.
  • AT2 primary alveolar type 2
  • WT M ⁇ also induced greater EdU incorporation (EdU is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis) and TER recovery than TFF2 ⁇ / ⁇ M ⁇ .
  • EdU is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis
  • TFF2 ⁇ / ⁇ M ⁇ M ⁇ induced recovery of epithelial barrier function and also epithelial proliferation through a TFF2-dependent mechanism.
  • RNA-sequencing was completed on WT or TFF2 ⁇ / ⁇ BMM ⁇ recovered from MERA at d4 and compared to reads obtained from na ⁇ ve BMM ⁇ exposed to quiescent MTEC.
  • TFF2 mRNA transcripts were not different between WT M ⁇ exposed to intact or damaged epithelia, but WT M ⁇ exposed to damaged MTEC were found to up-regulate canonical M2 genes such as: Mgl2, Socs2, and Arg1.
  • WT M ⁇ exposed to damaged MTEC were found to up-regulate canonical M2 genes such as: Mgl2, Socs2, and Arg1.
  • comparison of RNA transcripts between WT and TFF2 ⁇ / ⁇ M ⁇ that were both exposed to damaged MTEC revealed that Camkk2b expression was significantly (>2-fold) under-represented in TFF2 ⁇ / ⁇ M ⁇ . Because Camkk2b is implicated in the calcium dependent pathway for Wnt glycoprotein expression [Id., citing Qu, F., et al. Front. Biosci. (Landmark Ed) (2013) 18: 493-503; Wang, Q. et al.
  • M ⁇ -derived TFF2 may promote epithelial repair, at least partially, via regulating Wnt expression.
  • cDNA-derived from different M ⁇ populations recovered from d4 of MERA were screened against a panel of 84 different Wnt pathway genes. Wnt4a and Wnt16 were 2-fold increased in WT M ⁇ exposed to damaged epithelia vs. WT M ⁇ exposed to non-scratched, quiescent epithelia.
  • Wnt4a and Wnt16 were both 2–3-fold reduced in Tff2 ⁇ / ⁇ M ⁇ exposed to injured epithelia compared to WT M ⁇ exposed to injured epithelia.
  • Inoculation of WT MERA cultures with anti-Wnt4 Ab abrogated the TER rebound compared to control IgG treatment.
  • Real-time PCR quantification of Wnt4 and Wnt16 mRNA transcript levels in CD103+ DC, IM, and AM populations FACS-sorted from lung tissues of WT and Tff2 ⁇ / ⁇ mice at 4d post N.b confirmed that TFF2 deficiency impaired Wnt4 expression in alveolar macrophages, but did not alter mRNA expression for Arg1, Retnla and Nos2.
  • CD11cCre and CD11cCreTFF2flox mice were administered either an rWnt4/Wnt16/R-spondin1 cocktail (1 ⁇ g) or saline control (vehicle).
  • rWnt4/Wnt16/R-spondin treatment caused a 2-fold increase in the percentage of EpCAM+BrdU+pro-Spc+ cell populations recovered from the distal lung compartment of the CD11cCreTFF2flox mice. No increased BrdU incorporation was noted in R-spondin only treated animals.
  • CXCR4/SDF-1 signaling [000293]
  • the chemokine receptor CXCR4 is a member of a family of seven-span transmembrane G-protein coupled chemokine receptors. It is ubiquitously expressed and evolutionarily conserved. Its ligand, stromal cell derived factor 1 (SDF-1) called CXCL12, is likewise highly conserved.
  • CXCR4 is intensively studied in different autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, and autoimmune disorders of the central nervous system, such as multiple sclerosis, for its involvement in leukocyte chemotaxis.
  • rheumatoid arthritis systemic lupus erythematosus
  • autoimmune disorders of the central nervous system such as multiple sclerosis, for its involvement in leukocyte chemotaxis.
  • the CXCL12/CXCR-4 axis comprising the chemokine receptor CXCR4 and its ligand CXCL12 (also known as stromal cell-derived factor 1, or SDF-1), plays a crucial role in the homing of stem and progenitor cells in the bone marrow and controls their mobilization into peripheral blood and tissues in homeostatic conditions, as well as after tissue injury or stress. Upregulation of CXCL12 in hypoxic conditions with subsequent mobilization of CXCR4- positive stem and progenitor cells [Id., citing Ceradini, DJ et al. Nature Medf.
  • CXCL12 also binds a second chemokine receptor (CXCR7 or RDC1) with an even 100-fold higher affinity compared with CXCR4.
  • CXCR7 has been implicated in cell survival and adhesion [Id., citing Burns, JM et al J. Exp. Med. (2006) 203: 2201-13], and can mediate CXCL12-directed T cell chemotaxis independently from CXCR4 [Id., citing Balabanian, K. et al. J. Biol. Chem. (2005) 280: 35760-66; Kumar, R. et al Cell Immunol. (2012).272: 230-41).
  • CXCL12 scavenging function of CXCR7 can also positively influence CXCR4-mediated migration by preventing the downregulation of CXCR4 surface expression and function through excessive CXCL12 concentrations [Id., citing Sanchez-Alcaniz et al. Neuron (2011) 69: 77-90].
  • CXCR7 is able to mediate CXCL12-induced MAPK activation independently from CXCR4 (Id., citing Wang, Y. et al. Neuron (2011) 69: 61-76).
  • CXCR7 does not bind to or induce the activation of heterotrimeric G-proteins as typical in classical GPCR signaling, but depends on ligand-induced ⁇ -arrestin recruitment [Id., citing Rajagopal, S. et al. Proc. Ntl. Acad. Sci. USA (2010) 107: 628-32].
  • Neutrophils [000295] The CXCL12/CXCR4 axis maintains neutrophil homeostasis primarily by regulation of neutrophil release from the bone marrow in a cell-autonomous fashion [Id., citing Eash, KJ et al. Blood (2009) 113: 4711-19].
  • Senescent neutrophils in the periphery expressing high levels of CXCR4 home back to the bone marrow to be cleared [Id., citing Martin, C. et al. Immunity (2003) 19: 583-93].
  • activated neutrophils downregulate CXCR4 expression putatively postponing their clearance (Bruhl, H. et al. Eur. J. Immunol. (2003) 33: 3028-37; Martin, C. et al. Immunity (2003) 19: 583-93].
  • Lymphocytes [000296] CXCR4 on lymphocytes plays an essential role during B-cell development [Id., citing Nagasawa, T. et al.
  • CXCL12 is able to trigger B-cell chemotaxis in vitro through CXCR4 [Id., citing (Klasen, C. et al. J. Immunol. (2014) 192: 5273-84). Platelets [000297] CXCR4 expression (mRNA, protein) was reported on platelets [Wang, JF et al. Blood (1998) 92: 756-64; Kowalska, MA et al. Br. J. Haematol.
  • CXCL12 is a weak platelet agonist, however still amplifying platelet activation, adhesion and chemokine release triggered by low doses of primary platelet agonists, such as adenosine diphosphate (ADP) and thrombin, or arterial flow conditions [Id., citing (Kowalska, MA et al. Br. J. Haematol. (1999) 104: 220-29; Gear, AR et al. Blood (2001) 97: 937-45). Furthermore, CXCL12 gradients was shown to induce platelet migration and transmigration in vitro involving PI3K signaling [Id., citing Kraemer, BF et al. J. Mol. Med.
  • the described invention proposes a biofunctional fusion protein containing a biologically active immunomodulatory component operatively linked to a biologically active anti-viral component.
  • the biologically active immunomodulatory component reduces exacerbated inflammatory responses in the host in response to severe virus infection.
  • the biologically active anti-viral component reduces viral load.
  • the described invention provides a method for reducing damaging effects of a severe respiratory virus infection in a susceptible subject comprising a pharmaceutically acceptable carrier and a recombinant bifunctional fusion protein comprising a recombinant biologically active immunomodulatory component operatively linked to a recombinant biologically active anti-viral component, the recombinant immunomodulatory component comprising a recombinant human trefoil factor 1 (hTFF1); a recombinant human trefoil factor 2 (hTFF2), or a recombinant human trefoil factor 3 (hTFF3), wherein the pharmaceutical composition is cytoprotective.
  • hTFF1 human trefoil factor 1
  • hTFF2 a recombinant human trefoil factor 2
  • hTFF3 recombinant human trefoil factor 3
  • the recombinant bifunctional fusion protein comprises the recombinant human trefoil factor 1 (hTFF1) or a biologically active fragment or variant thereof, the biologically active hTFF2 molecule or a biologically active fragment or variant thereof, or the recombinant human trefoil factor 3 (hTFF3) or a biologically active fragment or variant thereof joined by its C-terminal end to a linker sequence, which is joined to an N-terminal end of a biologically active recombinant human interferon molecule, fragment or variant thereof.
  • hTFF1 human trefoil factor 1
  • hTFF2 biologically active fragment or variant thereof
  • hTFF3 recombinant human trefoil factor 3
  • the C-terminal end of the recombinant interferon molecule, fragment or variant sequence is further joined to a recombinant Fc derived antibody domain comprising a constant region of a human immunoglobulin heavy chain.
  • the recombinant interferon molecule is a recombinant type I interferon or biologically active fragment thereof selected from IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ and IFN- ⁇ .
  • the recombinant IFN is a human interferon.
  • the recombinant hTFF2 molecule is a human protein of SEQ ID NO: 52 (NCBI Ref NP_005414).
  • the recombinant hTFF1 molecule is a protein of SEQ ID NO:# __
  • the recombinant hTFF3 molecule is a protein of SEQ ID NO:# __ .
  • the recombinant interferon molecule is a human IFN- ⁇ of SEQ ID NO: 53 (NCBI Ref NP_000596.2); or the recombinant interferon molecule is a human IFN- ⁇ of SEQ ID NO: 54 (NCBI Ref NM_020124.3); or the interferon is a recombinant human IFN- ⁇ of SEQ ID NO: 55 (NCBI Ref.002177.3).
  • the recombinant interferon is a recombinant human interferon- ⁇ as disclosed by Chon, TW and Bixler, S, J. Interferon & Cytokine Res. (2010) 30 (7): 477-85: [000300] MAFVLSLLMALVLVSYGPGGSLGCDLSQNHVLVGRKNLRLLDEMRR LSPHFCLQDRKDFALPQEMVEGGQLQEAQAISVLHEMLQQSFNLFHTEHSSAAWDT TLLEPCRTGLHQQLDNLDACLGQVMGEEDSALGRTGPTLALKRYFQGIHVLKEKGY SDCAWETVRLEIMRSFSSLISLQERLRMMDGDLSSP (SEQ ID NO: 34) [000301] According to some embodiments, the Fc-derived antibody domain is a human protein of SEQ ID NO: 56 (NCBI Ref 4CDH_A) According to some embodiments, the amino acid sequence of the recombinant fusion protein is SEQ ID NO: 56
  • the sequences are codon-optimized to improve gene expression.
  • the recombinant fusion protein is produced in CHO cells.
  • the method further comprises encapsulating the recombinant fusion protein into particles.
  • the recombinant TFF2-IFN, TFF1-IFN or TFF-3 IFN fusion protein is encapsulated within the polymer matrix of a plurality of microparticles.
  • the respiratory virus is a respiratory syncytial virus (RSV), an Ebola virus, a cytomegalovirus, a Hanta virus, an influenza virus, a coronavirus, a Zika virus, A West Nile virus, a dengue virus, a Japanese encephalitis virus, a tick-borne encephalitis virus, a yellow fever virus, a rhinovirus, an adenovirus, a herpes virus, an Epstein Barr virus, a measles virus, a mumps virus, a rotavirus, a cocksackie virus, a norovirus, or an encephalomyocarditis virus (EMCV).
  • RSV respiratory syncytial virus
  • Ebola virus Ebola virus
  • a cytomegalovirus a Hanta virus
  • influenza virus a coronavirus
  • a Zika virus Zika virus
  • a West Nile virus a dengue virus
  • a Japanese encephalitis virus a tick
  • the method stimulates repair of a mucosal injury, modulates an immune response, or both.
  • the administering occurs parenterally, by inhalation, or by insufflation.
  • the susceptible patient includes a very young subject, an elderly subject, a subject who is ill; an immunocompromised subject, a subject with long term health conditions, a subject who is obese, or a subject that is physically weak due to malnutrition or dehydration.
  • the damaging effects of the severe respiratory virus infection include one or more of: primary viral pneumonia; superimposed bacterial pneumonia; disruption or injury to alveolar epithelium, endothelium or both; acute lung injury (ALI); acute respiratory distress syndrome (ARDS); symptoms of shock; excessive complement activation; a pathological increase in vascular permeability; endothelial activation, loss of barrier function and consequent microvascular leak; thrombotic complications; kidney damage; or elevated concentrations of one or more inflammatory mediators in plasma (hypercytokinemia), compared to a normal healthy subject.
  • symptoms of shock include low blood pressure, lightheadedness, shortness of breath, and rash.
  • the thrombotic complications include one or more of formation of pulmonary microthrombi, acute pulmonary embolism, deep-vein thrombosis, ischemic stroke, myocardial infarction, or systemic arterial embolism.
  • the inflammatory mediator is one or more of interferon ⁇ , interferon ⁇ , interferon- ⁇ , interferon- ⁇ , complement, prostaglandin D2, vasoactive intestinal peptide (VIP), nterleukin-1-beta (IL-1 ⁇ ), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 (IL-12), IL-17, tumor necrosis factor-alpha (TNF- ⁇ ).
  • the method may stimulate repair of a mucosal injury, modulates an immune response, or both.
  • repair of a mucosal injury comprises epithelial proliferation; or repair of a mucosal injury restores an epithelial barrier, an endothelial barrier or both; or the immune response comprises recruitment of innate and adaptive immune cells.
  • the innate immune cells comprise macrophages, dendritic cells (DCs), innate lymphoid cells (ILCs), and natural killer cells (NKs); or the adaptive immune cells include ⁇ T cells, ⁇ T cells, and B cells.
  • the method comprises aerosolizing the composition in a form selected from a dry powder, a suspension or a solution and administering the aerosolized composition to the respiratory system.
  • the administering of the aerosolized composition to the respiratory system occurs parenterally, by inhalation, or by insufflation.
  • the composition is a solution.
  • the administering to the respiratory system is by an inhalation delivery device or a solid particulate therapeutic aerosol generator.
  • the solid particulate aerosol generator is an insufflator.
  • the inhalation delivery device is a nebulizer, a metered-dose inhaler, or a dry powder inhaler (DPI).
  • the respirable particles range in size from about 1 to 10 microns, inclusive; or the particles for nasal administration (insufflation), range in size from 10-500 ⁇ M, inclusive.
  • the susceptible patient includes a very young subject, an elderly subject, a subject who is ill; an immunocompromised subject, a subject with long term health conditions, a subject who is obese, or a subject that is physically weak due to malnutrition or dehydration.
  • the pharmaceutical composition further comprises a supportive therapy or an additional therapeutic agent selected from one or more of an immunomodulatory agent, an analgesic agent, an anti-inflammatory agent, an anti-infective agent, an anti-malarial agent, an anti-viral agent or an anti-fibrotic agent.
  • the immunomodulatory agent can be used as a monotherapy or in combination with the supportive therapy, the analgesic agent, the anti-inflammatory agent, the anti-infective agent, the anti-malarial agent, the anti-viral agent or the anti-fibrotic agent.
  • the immunomodulatory agent is selected from the group consisting of methotrexate; a glucocorticoid, cyclosporine, tacrolimus and sirolimus; a recombinant interferon selected from IFN- ⁇ ; IFN- ⁇ -2b, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ and IFN- ⁇ ; a recombinant IL-2 receptor inhibitor; a PDE4 inhibitor; a hyperimmune globulin prepared from a donor with high titers of a desired antibody; a TNF ⁇ inhibitor/antagonist; an IL-1 ⁇ inhibitor; a chimeric IL-1Ra; an IL-6 inhibitor; an IL-12/ IL-23 inhibitor selected from ustekinumab, briakinumab; an IL-23 inhibitor selected from guselkumab, tildrakizumab; a compound that targets T
  • the glucocorticoid is a corticosteroid selected from prednisone, dexamethasone, azathioprine, mycophenolate, mycophenolate mofetil, and combinations thereof; or the recombinant IL-2 inhibitor is denileukin diftitox; or the PDE4 inhibitor is cilomilast; or the TNF ⁇ inhibitor/antagonist is selected from the group consisting of etanercept; adalimumab; infliximab, certolizumab pegol, or golimumab; or the IL-1 ⁇ inhibitor is selected from rilonacept; canakinumab; and Anakinra; or the IL-6 inhibitor is selected from tocilizumab, siltuximab, sarilumab, olokizumab, and sirukumab; or the compound that targets TLR4 signaling is selected from (ethyl 4-(4
  • a physiologic or supraphysiological dose of the recombinant interferon selected from recombinant IFN- ⁇ ; IFN- ⁇ -2b, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ and IFN- ⁇ or a PEGylated form thereof boosts immune defenses of the subject.
  • the analgesic agent is selected from the group consisting of codeine, hydrocodone, oxycodone, methadone, hydromorphone, morphine, and fentanyl.
  • the anti-inflammatory agent is selected from aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac nabumetone, naproxen, nintedanib, oxaprozin, pirfenidone, piroxicam, salsalate, sarilumab (Kevzara®) sulindac, tolmetin, and combinations thereof.
  • the anti-infective agent is amoxicillin, doxycycline, demeclocycline; eravacycline, minocycline, ormadacycline, tetracycline, cephalexin, defotaxime, cetazidime, cefuroxime, ceftaroline; ciprofloxacin, levofloxacin, moxifloxacin, clindamycin, lincomycin, metronidazole, azithromycin; clarithromycin, erythromycin, sulfamethoxazle and trimethoprim; sulfasalazine, amoxicillin and clavulanate; vancomycin, dalbavancin, oritavancin, telavancin, gentamycin, tobramycin, amikacin, imipenem and cilastatin, meropenem, doripenem, or ertapenem.
  • the anti-malarial agent is selected from quinine, quinidine, chloroquine, hydroxychloroquine, amodiaquine, mefloquine, halofantrine, lumefantrine, piperaquine, and tafenoquine; an antifolate compound selected from pyrimethamine, proguanil, chlorproguanil, trimethoprim; an artemisinin compound selected from artemisinin, dihydroartemisinin, artemether, artesunate; and atovaquone.
  • the anti-viral agent is selected from acyclovir, gancidovir, foscarnet; ribavirin; amantadine, azidodeoxythymidine /zidovudine), nevirapine, a tetrahydroimidazobenzodiazepinone (TIBO) compound; efavirenz; remdecivir, lopinavir/ritonavir, umifenovir, favipiravir, ivermectin, and delavirdine.
  • the anti-fibrotic agent is selected from nintedanib, pirfenidone, and combinations thereof.
  • the anti-viral agent is an agent that inhibits viral entry and decreases viral load.
  • the anti-viral agent that inhibits or blocks viral entry is a synthetic peptide selected from the group consisting of: NP-1, SEQ ID NO: 18; NP-2; SEQ ID NO: 19; NP-3; SEQ ID NO: 20; NP-4; SEQ ID NO: 21; CP-1; SEQ ID NO: 22; CP-2; SEQ ID NO: 23; HR2P; SEQ ID NO: 25; OC43-HR2, SEQ ID NO: 26; EK1, SEQ ID NO: 27; EK1P, SEQ ID NO: 28; EK1C, SEQ ID NO: 29.
  • the anti-viral agent that inhibits or blocks viral entry is a dipeptidyl peptidase 4 (DPP4) inhibitor; an ACE2 inhibitor; a transmembrane serine protease TMRSS2 inhibitor; a cathepsin B inhibitor, a cathepsin L inhibitor or a cathepsin B/ L inhibitor.
  • DPP4 dipeptidyl peptidase 4
  • TMRSS2 transmembrane serine protease
  • TMRSS2 transmembrane serine protease
  • cathepsin B inhibitor a cathepsin L inhibitor or a cathepsin B/ L inhibitor.
  • the supportive therapy is therapeutic apheresis comprising a virion removing step.
  • the therapeutic apheresis reduces viral load.
  • the described invention provides a method for reducing progression of symptoms of a severe respiratory virus infection in a susceptible human subject, comprising administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant bifunctional fusion protein comprising a recombinant biologically active immunomodulatory component operatively linked to a recombinant biologically active anti-viral component.
  • the recombinant biologically active immunomodulatory component comprises a recombinant trefoil factor 1 (hTFF1), a recombinant human trefoil factor 2 (hTFF2), or a recombinant human trefoil factor 3 (hTFF3) and a vehicle, wherein the method recues symptoms of the severe virus infection.
  • hTFF1 recombinant trefoil factor 1
  • hTFF2 recombinant human trefoil factor 2
  • hTFF3 recombinant human trefoil factor 3
  • the recombinant bifunctional fusion protein comprises the recombinant biologically active hTFF1 molecule, fragment or variant thereof; the recombinant biologically active hTFF2 molecule, fragment or variant thereof, or the recombinant biologically active hTFF3 molecule, fragment or variant thereof joined by its C- terminal end to a linker sequence, which is joined to an N-terminal end of a recombinant biologically active recombinant human interferon molecule, fragment or variant.
  • the C-terminal end of the recombinant interferon molecule, fragment or variant sequence is further joined to a recombinant Fc derived antibody domain comprising a recombinant constant region of a human immunoglobulin heavy chain.
  • the recombinant interferon molecule is a type I interferon or biologically active fragment thereof selected from recombinant IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ and IFN- ⁇ .
  • the recombinant IFN is a human interferon.
  • the recombinant hTFF1 molecule is a recombinant human protein of SEQ ID NO: (NCBI Reference Sequence: NM_003225.3).
  • the hTFF2 molecule is a recombinant human protein of SEQ ID NO: 52 (NCBI Ref NP_005414).
  • the recombinant hTFF3 molecule is a human protein of SEQ ID NO:38__( NCBI Reference Sequence: NM_003226.4).
  • the recombinant interferon molecule is a human IFN- ⁇ of SEQ ID NO: 53 (NCBI Ref NP_000596.2); or the recombinant interferon molecule is a human IFN- ⁇ of SEQ ID NO: 54 (NCBI Ref NM_020124.3); or the recombinant interferon is a human IFN- ⁇ of SEQ ID NO: 55 (NCBI Ref. NM_002177.3).
  • the recombinant interferon is a human interferon- ⁇ as disclosed by Chon, TW and Bixler, S, J. Interferon & Cytokine Res.
  • the Fc-derived antibody domain is a recombinant human protein of SEQ ID NO: 56 (NCBI Ref 4CDH_A)
  • the amino acid sequence of the recombinant fusion protein is SEQ ID NO: 35.
  • the sequences are codon-optimized to improve gene expression.
  • the recombinant fusion protein is produced in CHO cells.
  • the method further comprises encapsulating the recombinant fusion protein into particles.
  • the recombinant TFF2-IFN fusion protein is encapsulated within the polymer matrix of a plurality of microparticles.
  • the respiratory virus is a respiratory syncytial virus (RSV), an Ebola virus, a cytomegalovirus, a Hanta virus, an influenza virus, a coronavirus, a Zika virus, a West Nile virus, a dengue virus, a Japanese encephalitis virus, a tick-borne encephalitis virus, a yellow fever virus, a rhinovirus, an adenovirus, a herpes virus, an Epstein Barr virus, a measles virus, a mumps virus, a rotavirus, a cocksackie virus, a norovirus, or an encephalomyocarditis virus (EMCV).
  • RSV respiratory syncytial virus
  • Ebola virus Ebola virus
  • a cytomegalovirus a Hanta virus
  • an influenza virus a coronavirus
  • a Zika virus a West Nile virus
  • a dengue virus a Japanese encephalitis virus
  • symptoms of the severe respiratory virus infection include one or more of: primary viral pneumonia; superimposed bacterial pneumonia; disruption or injury to alveolar epithelium, endothelium or both; acute lung injury (ALI); acute respiratory distress syndrome (ARDS); symptoms of shock; excessive complement activation; a pathological increase in vascular permeability; endothelial activation, loss of barrier function and consequent microvascular leak; thrombotic complications; kidney damage; or elevated concentrations of one or more inflammatory mediators in plasma (hypercytokinemia), compared to a normal healthy subject.
  • symptoms of shock include low blood pressure, lightheadedness, shortness of breath, and rash.
  • the thrombotic complications include one or more of formation of pulmonary microthrombi, acute pulmonary embolism, deep-vein thrombosis, ischemic stroke, myocardial infarction, or systemic arterial embolism.
  • the inflammatory mediator is one or more of interferon ⁇ , interferon ⁇ , interferon- ⁇ , interferon- ⁇ , complement, prostaglandin D2, vasoactive intestinal peptide (VIP), nterleukin-1-beta (IL-1 ⁇ ), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 (IL-12), IL-17, tumor necrosis factor-alpha (TNF- ⁇ ).
  • the method stimulates repair of a mucosal injury, modulates an immune response, or both.
  • repair of a mucosal injury comprises epithelial proliferation; or repair of a mucosal injury restores an epithelial barrier, an endothelial barrier or both; or the immune response comprises recruitment of innate and adaptive immune cells.
  • the innate immune cells comprise macrophages, dendritic cells (DCs), innate lymphoid cells (ILCs), and natural killer cells (NKs)
  • the adaptive immune cells include ⁇ T cells, ⁇ T cells, and B cells.
  • the susceptible patient includes a very young subject, an elderly subject, a subject who is ill; an immunocompromised subject, a subject with long term health conditions, a subject who is obese, or a subject that is physically weak due to malnutrition or dehydration.
  • the method comprises aerosolizing the composition in a form selected from a dry powder, a suspension or a solution and administering the aerosolized composition to the respiratory system.
  • the administering of the aerosolized composition to the respiratory system occurs parenterally, by inhalation, or by insufflation.
  • the composition is a solution. [000330] .
  • the administering is by an inhalation delivery device or a solid particulate therapeutic aerosol generator.
  • the solid particulate aerosol generator is an insufflator.
  • the inhalation delivery device is a nebulizer, a metered-dose inhaler, or a dry powder inhaler (DPI).
  • respirable particles range in size from about 1 to 10 microns, inclusive; or particles for nasal administration (insufflation), range in size from 10-500 ⁇ M, inclusive.
  • the pharmaceutical composition further comprises a supportive therapy or an additional therapeutic agent selected from one or more of an immunomodulatory agent, an analgesic agent, an anti-inflammatory agent, an anti-infective agent, an anti-malarial agent, an anti-viral agent or an anti-fibrotic agent.
  • the immunomodulatory agent can be used as a monotherapy or in combination with the supportive therapy, the analgesic agent, the anti-inflammatory agent, the anti-infective agent, the anti-malarial agent, the anti-viral agent or the anti-fibrotic agent.
  • the immunomodulatory agent is selected from the group consisting of methotrexate; a glucocorticoid; cyclosporine, tacrolimus and sirolimus; recombinant interferon selected from IFN- ⁇ , IFN- ⁇ -2b, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , , IFN- ⁇ , IFN- ⁇ and IFN- ⁇ ; a recombinant IL-2 receptor inhibitor; a PDE4 inhibitor; a hyperimmune globulin prepared from a donor with high titers of a desired antibody; a TNF ⁇ inhibitor/antagonist; an IL-1 ⁇ inhibitor; a chimeric IL-1Ra; or an IL-6 inhibitor; an IL-12/ IL- 23 inhibitor selected from ustekinumab or briakinumab, an IL-23 inhibitor selected from guselkumab, or tildrakizumab; or a
  • the glucocorticoid is a corticosteroid selected from prednisone, dexamethasone, azathioprine, mycophenolate, mycophenolate mofetil,, and combinations thereof;
  • the recombinant IL-2 inhibitor is denileukin diftitox;
  • the PDE4 inhibitor is cilomilast;
  • the TNF ⁇ inhibitor/antagonist is selected from the group consisting of etanercept; adalimumab; infliximab, certolizumab pegol, or golimumab;
  • the IL-1 ⁇ inhibitor is selected from rilonacept; canakinumab; and Anakinra;
  • the IL-6 inhibitor is selected from tocilizumab, siltuximab, sarilumab, olokizumab, and sirukumab;
  • the compound that targets TLR4 signaling is selected from (ethyl 4-(4’-
  • a physiologic or supraphysiological dose of the recombinant interferon selected from IFN- ⁇ ; IFN- ⁇ -2b, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , , IFN- ⁇ , IFN- ⁇ and IFN- ⁇ boosts the subject’s defenses.
  • the analgesic agent is selected from the group consisting of codeine, hydrocodone, oxycodone, methadone, hydromorphone, morphine, and fentanyl.
  • the anti-inflammatory agent is selected from aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac nabumetone, naproxen, nintedanib, oxaprozin, pirfenidone, piroxicam, salsalate, sarilumab (Kevzara®) sulindac, tolmetin, and combinations thereof.
  • the anti-infective agent is amoxicillin, doxycycline, demeclocycline; eravacycline, minocycline, ormadacycline, tetracycline, cephalexin, defotaxime, cetazidime, cefuroxime, ceftaroline; ciprofloxacin, levofloxacin, moxifloxacin, clindamycin, lincomycin, metronidazole, azithromycin; clarithromycin, erythromycin, sulfamethoxazle and trimethoprim; sulfasalazine, amoxicillin and clavulanate; vancomycin, dalbavancin, oritavancin, telavancin, gentamycin, tobramycin, amikacin, imipenem and cilastatin, meropenem, doripenem, or ertapenem.
  • the anti-malarial agent is selected from quinine, quinidine, chloroquine, hydroxycloroquine, amodiaquine, mefloquine, halofantrine, lumefantrine, piperaquine, and tafenoquine; an antifolate compound, selected from pyrimethamine, proguanil, chlorproguanil, trimethoprim; cepharanthine/selamectin/mefloquine hydrochloride; an artemisinin compound selected from artemisinin, dihydroartemisinin, artemether, artesunate; and atovaquone.
  • the anti-viral agent is selected from acyclovir, gancidovir, foscarnet; ribavirin; amantadine, azidodeoxythymidine /zidovudine), nevirapine, a tetrahydroimidazobenzodiazepinone (TIBO) compound; efavirenz; remdecivir, lopinavir/ritonavir, umifenovir, favipiravir, ivermectin, and delavirdine.
  • the anti-fibrotic agent is selected from nintedanib, pirfenidone, and combinations thereof.
  • the antiviral agent inhibits viral entry and decreases viral load.
  • the anti-viral agent that blocks viral entry is a synthetic peptide selected from the group consisting of: NP-1, SEQ ID NO: 18; NP- 2; SEQ ID NO: 19; NP-3; SEQ ID NO: 20; NP-4; SEQ ID NO: 21; CP-1; SEQ ID NO: 22; CP- 2; SEQ ID NO: 23; HR2P; SEQ ID NO: 25; OC43-HR2, SEQ ID NO: 26; EK1, SEQ ID NO: 27; EK1P, SEQ ID NO: 28; and EK1C, SEQ ID NO: 29.
  • the anti-viral agent that inhibits viral entry is a dipeptidyl peptidase 4 (DPP4) inhibitor; an ACE2 inhibitor; a transmembrane serine protease TMRSS2 inhibitor; a cathepsin B inhibitor, a cathepsin L inhibitor or a cathepsin B/ L inhibitor.
  • DPP4 dipeptidyl peptidase 4
  • TMRSS2 transmembrane serine protease
  • TMRSS2 transmembrane serine protease
  • cathepsin B inhibitor a cathepsin L inhibitor or a cathepsin B/ L inhibitor.
  • the supportive therapy is therapeutic apheresis comprising a virion removing step.
  • the therapeutic apheresis reduces viral load.
  • FIG. 1 is a schematic of the contact activation (intrinsic) and the tissue factor (extrinsic) coagulation pathways.
  • FIG. 2 is a schematic depicting a three stage cell-surface based model of coagulation, comprising initiation, priming, and propagation. Taken from Monroe et al. Arterioscler Thromb Vase Biol. (2002) 22:1381 -1389.
  • FIG.3 is a schematic showing the principal stages in complement activation by the classical, lectin, and alternative pathways. [Taken from Molecular Biology of the Cell, 4 th Ed., Bruce Alberts, et al. ed. Garland Science, New York (2002), Fig. 25-41].
  • FIG 4A is a schematic of the challenge model of influenza virus.
  • FIG.4B is a graph of body weight change vs. days post infection for mice infected with H7N9 influenza A virus, H9N2 influenza A virus, and a PBS control.
  • FIG.4C is a plot of percent survival vs.
  • FIG 5A is a Log 10 (p Value) vs. Log 2 (H7N9/H9N2 plot showing genes that are significantly differentially expressed in the lung tissues of mice after infection with H7N9 and H9N2 influenza viruses.
  • FIG.5B is a plot of expression level of TFF2 vs days post infection for mice infected with H7N9 and H9N2.
  • FIG. 5C is a plot of TFF2 mRNA relative value versus time for mice infected with H7N9 and H9N2. Fluorescence quantitative PCR was used to verify the expression of TFF2 in lung tissues.
  • FIG.5D is a Western Blot showing expression of TFF2 at different time points after infection with H7N9 and H9N2.
  • FIG.6A shows vector construction maps of TFF2 eukaryotic expression vectors pSV1.0 (left), pSV1.0-TFF2 (middle), pSV1.0-TFF2-6xHis ("6xHis" disclosed as SEQ ID NO: 33) (right).
  • FIG.6B shows in vitro expression of TFF2 in cell lines and secreted into the cell supernatant.
  • FIG. 7A is a schematic of the mouse model to show the protective effect of TFF2 protein treatment on influenza virus H7N9, H9N2, PR8 challenged mice.
  • FIG.7B top is a plot of percent survival versus days post infection for (B) mice infected with H7N9, mice infected with H7N9 treated with mTFF2 supernatant; and mice infected with H7N9s treated with hTFF2 supernatant.
  • FIG. 7A is a schematic of the mouse model to show the protective effect of TFF2 protein treatment on influenza virus H7N9, H9N2, PR8 challenged mice.
  • FIG.7B top is a plot of percent survival versus days post infection for (B) mice infected with H7N9, mice infected with H7N9 treated with mTFF2 supernatant; and mice infected with H7N9s treated with hTFF2 supernatant.
  • FIG. 7B (bottom) is a plot of body weight change vs. days post infection for mice infected with H7N9, and mice infected with H7N9 treated with mTFF2.
  • TFF2 increased the survival rate of H7N9 infected mice and reduced weight loss, compared to the control.
  • FIG. 7C (top) is a plot of body weight change vs. days post infection of mice infected with H9N2 and mice treated with H9N2 + mTFF2 supernatant.
  • FIG.7C (bottom) is a plot of body weight change vs. days post infection for wild type and TFF2 KO mice.
  • TFF2 protein reduces the weight loss caused by H9N2 infection.
  • FIG.7D (top) is a plot of percent survival vs. days post infection of mice infected with PR8 and mice infected with PR8 treated with mTFF2 supernatant. TFF2 protein also protects PR8 infected mice and reduces the lethality and weight loss caused by PR8 infection.
  • FIG.8A shows the effect of TFF2 on H7N9 influenza virus replication in mice.
  • FIG.8B shows the effect of TFF2 on H7N9 and N9N2 replication in vitro lung epithelial cell line A549.
  • FIG.8C shows the effect of TFF2 addition on the microstructure of lung tissue in mice infected with H7N9 at 0 days, 1 day, 3 days, and 7 days post-infection.
  • FIG.8D shows the effects of mTFF2 on the expression of inflammatory factors: tumor necrosis factor alpha (TNF- ⁇ ) and interleukin 6 (IL-6) at 0 days, 1 day, 3 days and 7 days post-infection.
  • FIG.8E shows the effect of TFF2 on lung tissue microstructure in an acute lung inflammation model induced by lipopolysaccharide (LPS): left, control; middle, LPS; right, LPS + mTFF-His.
  • LPS lipopolysaccharide
  • FIG.9A shows a 10% SDS gel stained with coomassie blue with samples from days 0, 3, 4, 7 and 10 after transfection of epiCHO cells with a TFF2-IFN ⁇ -Fc construct and expression of the TFF2-IFN ⁇ Fc in transfected CHO cell supernatants.
  • FIG. 9B shows a Western blot; TFF2-IFN ⁇ -Fc was detected by anti-Fc antibodies (primary antibody: mouse anti-human Fc monoclonal; secondary antibody: HRP-conjugated goat anti-mouse polyclonal).
  • FIG. 10A shows a Western blot of resolved epiCHO cell lysates after transfection with a TFF2- IFN ⁇ -Fc construct.
  • TFF2-IFN ⁇ -Fc was detected by anti-Fc antibodies (primary antibody: mouse anti-human Fc monoclonal; secondary antibody: HRP- conjugated goat anti-mouse polyclonal).
  • the anti-Fc intensity was maximum at day 5, consistent with the maximum viable cell number in culture.
  • the Fc positive species in the supernatants confirms secretion.
  • 10B shows a Western blot of resolved epiCHO cell lysates after transfection with a GAPDH construct probed with rabbit anti-human anti-GAPDH antibodies (secondary antibody HRP-conjugated goat-anti-rabbit polyclonal antibodies).
  • the anti- GAPDH antibody Western blot shows a prominent species at ⁇ 40 kD present in both transfected and control cell supernatants at days 0, 3, 5, 7 and 10, indicating that similar amounts of control and transfected cellular proteins are present in resolved samples at each time point. Note: less material is apparent at day 10 likely due to cellular degradation.
  • FIG.11A shows a 10% SDS gel stained with coomassie blue containing elution fractions 1 through 9 from Protein A affinity chromatography of the expressed fusion product.
  • the expected species has a molecular weight of about 70 kDa. Smaller Fc reactive species indicate proteolysis, which will be addressed in subsequent runs.
  • FIG. 11B shows a Western blot of the resolved elution fractions 1 through 9 from Protein A affinity chromatography of the expressed fusion product detected by anti-Fc antibodies (primary antibody: mouse anti-human Fc monoclonal; secondary antibody: HRP- conjugated goat anti-mouse polyclonal), indicating a purification efficiency of less than 30.0% for this pilot run.
  • primary antibody mouse anti-human Fc monoclonal
  • secondary antibody HRP- conjugated goat anti-mouse polyclonal
  • active ingredient ("AI”, “active pharmaceutical ingredient”, “API”, or “bulk active”) or “active agent” are used interchangeably to refer to the substance in a drug that is pharmaceutically active.
  • additional active ingredient refers to an agent, other than the recombinant fusion protein of the described composition, that exerts a pharmacological, or any other beneficial activity.
  • administer means to give or to apply.
  • administering includes in vivo administration, as well as administration directly to tissue ex vivo.
  • the administering occurs nasally, by insufflation, intratracheally, orally, parenterally, intravenously, or intraperitoneally.
  • alveolar type II cells refers to the progenitors for alveolar type I cells. Alveolar type I cells cover 95 percent of the alveolar surface; they comprise the major gas exchange surface of the alveolus and are integral to the maintenance of the permeability barrier function of the alveolar membrane. AT2 cells are the only pulmonary cells that synthesize, store, and secrete all components of pulmonary surfactant important to regulate surface tension, preventing atelectasis and maintaining alveolar fluid balance within the alveolus.
  • amino acid residue or “amino acid” or “residue” are used interchangeably to refer to an amino acid that is incorporated into a protein, a polypeptide, or a peptide, including, but not limited to, a naturally occurring amino acid and known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.
  • the amino acids may be L- or D-amino acids.
  • An amino acid may be replaced by a synthetic amino acid, which is altered so as to increase the half-life of the peptide, increase the potency of the peptide, or increase the bioavailability of the peptide.
  • the single letter designation for amino acids is used predominately herein.
  • A is alanine
  • C cysteine
  • D is aspartic acid
  • E is glutamic acid
  • F is phenylalanine
  • G is glycine
  • H histidine
  • I is isoleucine
  • K is lysine
  • L leucine
  • M methionine
  • N is asparagine
  • P proline
  • Q is glutamine
  • R arginine
  • S is serine
  • T is threonine
  • V valine
  • W is tryptophan
  • Y tyrosine.
  • [000361] The following represents groups of amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
  • ACE2 angiotensin-converting enzyme 2
  • ACE2 refers to a type 1 integral membrane glycoprotein [Tikellils, C. and Thomas MC.
  • the extracellular domain of ACE2 enzyme contains a single catalytic metallopeptidase unit that shares 42% sequence identity and 61% sequence similarity with the catalytic domain of ACE [[Id., citing Donoghue, M. et al. Cir. Res. (2000) 87 (5): E1-E9].
  • ACE2 activity is not antagonized by conventional ACE inhibitors [Id., citing Rice, GI et al. Biochemical J. (2004) 383 (1): 45-51].
  • the major substrate for ACE2 appears to be (Ang II) [Id., citing Donoghue, M.
  • ACE2 is able to cleave the C-terminal amino acid from angiotensin I, vasoactive bradykinin, des-Arg- kallidin (also known as des-Arg10 Lys-bradykinin), Apelin-13 and Apelin-36 [Id., citing Kuba, K. et al.
  • the noncatalytic C-terminal domain of ACE2 shows 48% sequence identity with collectrin [Id., citing Zhang, H. et al. J. Biol. Chem. (2001) 276 (20): 17132-39], a protein shown to have an important role in neutral amino acid reabsorption from the intestine and the kidney [Id., citing Kowalczuk, S. et al. The FASEB J. (2008) 22 (8): 2880-87]; .
  • the removed amino acid then becomes available for reabsorption.
  • the cytoplasmic tail of ACE2 also contains calmodulin-binding sites [Id., citing DW Lambert, et al. FEBS Letters (2008) 582 (2): 385-90] which may influence shedding of its catalytic ectodomain.
  • ACE2 has also been associated with integrin function, independent of its angiotensinase activity.
  • the term “antibody” includes, by way of example, both naturally occurring and non-naturally occurring antibodies. Specifically, the term “antibody” includes polyclonal antibodies and monoclonal antibodies, and fragments thereof.
  • antibody includes chimeric antibodies and wholly synthetic antibodies, and fragments thereof.
  • Antibodies are serum proteins the molecules of which possess small areas of their surface that are complementary to small chemical groupings on their targets. These complementary regions (referred to as the antibody combining sites or antigen binding sites) of which there are at least two per antibody molecule, and in some types of antibody molecules ten, eight, or in some species as many as 12, may react with their corresponding complementary region on the antigen (the antigenic determinant or epitope) to link several molecules of multivalent antigen together to form a lattice.
  • the basic structural unit of a whole antibody molecule consists of four polypeptide chains, two identical light (L) chains (each containing about 220 amino acids) and two identical heavy (H) chains (each usually containing about 440 amino acids).
  • the two heavy chains and two light chains are held together by a combination of noncovalent and covalent (disulfide) bonds.
  • the molecule is composed of two identical halves, each with an identical antigen-binding site composed of the N-terminal region of a light chain and the N- terminal region of a heavy chain. Both light and heavy chains usually cooperate to form the antigen binding surface.
  • An antibody may be an oligoclonal antibody, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a multi-specific antibody, a bi-specific antibody, a catalytic antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an anti-idiotypic antibody, and an antibody that can be labeled in soluble or bound form, as well as fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences provided by known techniques.
  • Monoclonal antibodies can be generated by fusing mouse spleen cells from an immunized donor with a mouse myeloma cell line to yield established mouse hybridoma clones that grow in selective media.
  • a hybridoma cell is an immortalized hybrid cell resulting from the in vitro fusion of an antibody-secreting B cell with a myeloma cell.
  • In vitro immunization which refers to primary activation of antigen-specific B cells in culture, is another well-established means of producing mouse monoclonal antibodies.
  • Diverse libraries of immunoglobulin heavy (VH) and light (V ⁇ and V ⁇ ) chain variable genes from peripheral blood lymphocytes also can be amplified by polymerase chain reaction (PCR) amplification.
  • Genes encoding single polypeptide chains in which the heavy and light chain variable domains are linked by a polypeptide spacer can be made by randomly combining heavy and light chain V-genes using PCR.
  • a combinatorial library then can be cloned for display on the surface of filamentous bacteriophage by fusion to a minor coat protein at the tip of the phage.
  • the technique of guided selection is based on human immunoglobulin V gene shuffling with rodent immunoglobulin V genes.
  • the method entails (i) shuffling a repertoire of human ⁇ light chains with the heavy chain variable region (VH) domain of a mouse monoclonal antibody reactive with an antigen of interest; (ii) selecting half-human Fabs on that antigen (iii) using the selected ⁇ light chain genes as “docking domains” for a library of human heavy chains in a second shuffle to isolate clone Fab fragments having human light chain genes; (v) transfecting mouse myeloma cells by electroporation with mammalian cell expression vectors containing the genes; and (vi) expressing the V genes of the Fab reactive with the antigen as a complete IgG1, ⁇ antibody molecule in the mouse myeloma.
  • VH heavy chain variable region
  • An antibody may be from any species.
  • the term antibody also includes binding fragments of the antibodies of the invention. Binding fragments of an antibody can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Exemplary fragments include Fv, Fab, Fab', single stranded antibody (svFC), dimeric variable region (Diabody) and di-sulphide stabilized variable region (dsFv).
  • Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases. For example, computerized comparison methods can be used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function.
  • antibody construct refers to a polypeptide comprising one or more of the antigen-binding portions of the invention linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen-binding portions.
  • linker polypeptides are well known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123).
  • An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art.
  • Antibody portions, such as Fab and F(ab')2 fragments can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies.
  • antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques.
  • the term “antigen” and its various grammatical forms refers to any substance that can stimulate the production of antibodies and can combine specifically with them.
  • the term “antigenic determinant” or “epitope” as used herein refers to an antigenic site on a molecule.
  • the term “attenuate” as used herein refers to render less virulent, to weaken or reduce in force, intensity, effect or quantity.
  • binding specificity as used herein involves both binding to a specific partner and not binding to other molecules.
  • Functionally important binding may occur at a range of affinities from low to high, and design elements may suppress undesired cross- interactions. Post-translational modifications also can alter the chemistry and structure of interactions. “Promiscuous binding” may involve degrees of structural plasticity, which may result in different subsets of residues being important for binding to different partners. “Relative binding specificity” is a characteristic whereby in a biochemical system a molecule interacts with its targets or partners differentially, thereby impacting them distinctively depending on the identity of individual targets or partners. [000372]
  • bioavailable and its other grammatical forms as used herein refers to the ability of a substance to be absorbed and sued by the body.
  • biocompatible refers to a material that is generally non-toxic to the recipient and does not possess any significant untoward effects to the subject and, further, that any metabolites or degradation products of the material are non-toxic to the subject. Typically a substance that is “biocompatible” causes no clinically relevant tissue irritation, injury, toxic reaction, or immunological reaction to living tissue.
  • biodegradable refers to a material that will erode to soluble species or that will degrade under physiologic conditions to smaller units or chemical species that are, themselves, non-toxic.
  • CARD domain refers to the family subclass of the caspase recruitment domain.
  • the NLR proteins utilize the CARD for binding to downstream signaling molecules through CARD-CARD interactions in order to ultimately initiate the innate immune and inflammatory responses [Id., citing Inohara N., Nu ⁇ ez G. Nat. Rev. Immunol. (2003) 3, 371–382 ; Park, HH, et al. Annu. Rev. Immunol. (2007) 25, 561–586].
  • carrier as used herein describes a material that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the active compound of the composition of the described invention. Carriers must be of sufficiently high purity and of sufficiently low toxicity to render them suitable for administration to the mammal being treated.
  • the carrier can be inert, or it can possess pharmaceutical benefits, cosmetic benefits or both.
  • excipient “carrier”, or “vehicle” are used interchangeably to refer to carrier materials suitable for formulation and administration of pharmaceutically acceptable compositions described herein. Carriers and vehicles useful herein include any such materials know in the art which are nontoxic and do not interact with other components.
  • the carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide for the desired bulk, consistency, etc., when combined with an active agent and other components of a given composition.
  • class switch recombination or “CSR” as used herein refers to antibody class switching, which occurs in mature B cells in response to antigen stimulation and costimulatory signals.
  • switch a unique type of intrachromosomal deletional recombination within special G-rich tandem repeated DNA sequences [called switch, or S, regions located upstream of each of the heavy chain constant (CH) region genes, except C ⁇ ].
  • the recombination is initiated by the B cell–specific activation-induced cytidine deaminase (AID), which deaminates cytosines in both the donor and acceptor S regions.
  • AID activity converts several dC bases to dU bases in each S region, and the dU bases are then excised by the uracil DNA glycosylase UNG; the resulting basic sites are nicked by apurinic/apyrimidinic endonuclease (APE).
  • APE apurinic/apyrimidinic endonuclease
  • AID attacks both strands of transcriptionally active S regions, but how transcription promotes AID targeting is not entirely clear. Mismatch repair proteins are then involved in converting the resulting single-strand DNA breaks to double-strand breaks with DNA ends appropriate for end-joining recombination. Proteins required for the subsequent S- S recombination include DNA-PK, ATM, Mre11-Rad50-Nbs1, ⁇ H2AX, 53BP1, Mdc1, and XRCC4-ligase IV. B cells undergo antibody, or Ig, class switching in vivo after immunization or infection or upon appropriate activation in culture.
  • CD40L CD154
  • TLR4 Toll-like receptor 4
  • LPS lipopolysaccharide
  • Naive B cells have the potential to switch to any isotype, and cytokines secreted by T cells and other cells direct the isotype switch.
  • the predominant mechanism for regulating isotype specificity was by regulation of transcription through S regions, and only transcriptionally active S regions undergo CSR. [Tavnezer, J. et al., Annu Rev. Immunnol. (2008) 26: 261-92].
  • costimulatory molecule refers to molecules that are displayed on the cell surface that have a role in enhancing the activation of a T cell that is already being stimulated through its TCR.
  • HLA proteins which present foreign antigen to the T cell receptor, require costimulatory proteins which bind to complementary receptors on the T cell’s surface to result in enhanced activation of the T cell.
  • co- stimulatory molecules refers to highly active immunomodulatory proteins that play a critical role in the development and maintenance of an adaptive immune response (Kaufman and Wolchok eds., General Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)).
  • TCR cognate T cell receptor
  • cytokine refers to small soluble protein substances secreted by cells which have a variety of effects on other cells.
  • Cytokines mediate many important physiological functions including growth, development, wound healing, and the immune response. They act by binding to their cell-specific receptors located in the cell membrane, which allows a distinct signal transduction cascade to start in the cell, which eventually will lead to biochemical and phenotypic changes in target cells. Generally, cytokines act locally.
  • type I cytokines which encompass many of the interleukins, as well as several hematopoietic growth factors
  • type II cytokines including the interferons and interleukin-10
  • TNF tumor necrosis factor
  • IL-1 immunoglobulin super-family members
  • chemokines a family of molecules that play a critical role in a wide variety of immune and inflammatory functions.
  • the same cytokine can have different effects on a cell depending on the state of the cell. Cytokines often regulate the expression of, and trigger cascades of, other cytokines.
  • DPI dry powder inhaler
  • the term “dry powder inhaler” or “DPI” as used herein refers to a device similar to a metered-dose inhaler, but where the drug is in powder form. The patient exhales out a full breath, places the lips around the mouthpiece, then quickly breathes in the powder. Dry powder inhalers do not require the timing and coordination that are necessary with MDIs. DPIs contain an active drug mixed with an excipient containing much larger particles (e.g., lactose) to which the drug attaches. During aerosolization, the active drug is stripped from the carrier and inhaled while the carrier particles impact on the mouth and throat and are ingested.
  • DC dendritic cells
  • MHC major histocompatibility complex
  • CD11c CD11c
  • DCs express many other molecules which allow their classification into various subtypes.
  • DC and macrophages have distinct characteristics [Id., citing Delamarre L, Science (2005) 307:1630–4; Geissmann F, et al. Science (2010) 327:656– 61; van Montfoort N, et al. Proc Natl Acad Sci USA. (2009) 106:6730–5] and ontogeny, so that, currently, little doubt remains that they belong to distinct lineages [Id., citing Haniffa M, et al. (2013) 120:1–49; Hashimoto D, et al. Immunity (2013) 38:792–804; Hettinger J, et al. Nat Immunol.
  • DC are found in two different functional states, “mature” and “immature”.
  • DC maturation is triggered by tissue homeostasis disturbances, detected by the recognition of pathogen- associated molecular patterns (PAMP) or damage-associated molecular patterns (DAMPs) [Id., citing Hemmi H, et al.
  • Mature DCs express higher levels of the chemokine receptor, CCR7 [Id., citing Förster R, et al. Cell (1999) 99:23– 33; Ohl L, et al. Immunity (2004) 21:279–88; Sallusto F, et al. Eur J Immunol. (1998) 28:2760– 9; Steinman RM. The control of immunity and tolerance by dendritic cell. Pathol Biol. (2003) 51:59–60] and secrete cytokines essential for T-cell activation [Id., citing Reis e Sousa C.
  • DCs When interacting with CD4+ T cells, DCs may induce their differentiation into different T helper (Th) subsets [Id., citing Iwasaki A, Medzhitov R. Nat Immunol. (2015) 16:343–353] such as Th1 [Amsen D, et al. Cell (2004) 117:515–26; Constant S, et al. J Exp Med (1995) 182:1591–6; Hosken NA, et al. J Exp Med. (1995) 182:1579–84; Kadowaki N. Allergol Int. (2007) 56:193–9; Maekawa Y, et al. Immunity (2003) 19:549–59; Pulendran B, et al.
  • Th1 T helper subsets
  • T cell differentiation in each subtype is a complex phenomenon, that can be influenced by the cytokines in the DC tissue of origin [Id., citing Rescigno M. Dendritic cell-epithelial cell crosstalk in the gut. Immunol Rev. (2014) 260:118–28], their maturation state [Id., citing Reis e Sousa C. Nature Rev Immunol.
  • DCs present a unique characteristic: the ability to perform cross-presentation [Id., citing Coulon P-G, et al. J Immunol. (2016) 197:517–32; Delamarre L, Mellman I. Semin Immunol. (2011) 23:2–11; Jung S, et al. Immunity (2002) 17:211–20; Segura E, Amigorena S. Adv Immunol. (2015) 127:1–31; Segura E, Villadangos JA. Curr Opin Immunol.
  • MHC-I class I MHC molecules
  • This feature allows DC to trigger responses against intracellular antigens from other cell types, thus providing means for the system to deal with threats that avoid professional APC [Id., citing Coulon P-G, et al. J Immunol. (2016) 197:517–32, Bevan MJ. Cross-priming for a secondary cytotoxic response to minor H antigens with H-2 congenic cells which do not cross-react in the cytotoxic assay. J Exp Med. (1976) 143:1283–8, Sánchez-Paulete AR, et al. Ann Oncol. (2017) 28:xii74.
  • Immature DC are poor inducers of na ⁇ ve lymphocyte effector responses, since they have low surface expression of co-stimulatory molecules, low expression of chemokine receptors, and do not release immunostimulatory cytokines [Id., citing Trombetta ES, Mellman I. Annu Rev Immunol. (2005) 23:975–1028, Steinman RM, Swanson J. J Exp Med. (1995) 182:283–8].
  • Tregs [Id., citing Saito M, et al. J Exper Med. (2011) 208:235–49, Sela U, et al. PLoS ONE (2016) 11:e0146412).
  • These “tolerogenic DC” express less co-stimulatory molecules and proinflammatory cytokines, but upregulate the expression of inhibitory molecules (like PD-L1 and CTLA-4), secrete anti-inflammatory cytokines (IL-10, for example) [Id., citing Manicassamy S, Pulendran B. Immunol Rev. (2011) 241:206–27, Grohmann U, et al. Nat Immunol.
  • immature DC can be harmful to the body. It is known that DCs that are unable to induce lymphocyte effector responses may contribute to the immune system's failure to fight infections [Id., citing Campanelli AP, et al. J Infect Dis. (2006) 193:1313–22, Montagnoli C, et al. J Immunol. (2002) 169:6298–308] or tumors [Id., citing Baleeiro RB, et al. Cancer Immunol Immunother (2008) 57:1335–45; Almand B, et al. Clin Cancer Res.
  • D value or “mass division diameter” as used herein, refer to the diameter which, when all particles in a sample are arranged in order of ascending mass, divides the sample's mass into specified percentages. The percentage mass below the diameter of interest is the number expressed after the "D".
  • the D10 diameter is the diameter at which 10% of a sample's mass is comprised of smaller particles
  • the D50 is the diameter at which 50% of a sample's mass is comprised of smaller particles.
  • the D50 is also known as the "mass median diameter” as it divides the sample equally by mass. While D-values are based on a division of the mass of a sample by diameter, the actual mass of the particles or the sample does not need to be known.
  • a relative mass is sufficient as D-values are concerned only with a ratio of masses. This allows optical measurement systems to be used without any need for sample weighing.
  • DAD diffuse alveolar damage
  • DAD diffuse alveolar damage
  • Delayed release may or may not involve gradual release of drug over an extended time, and thus may or may not be “sustained release.”
  • the term “dispersion”, as used herein, refers to a two-phase system, in which one phase is distributed as droplets in the second, or continuous phase. In these systems, the dispersed phase frequently is referred to as the discontinuous or internal phase, and the continuous phase is called the external phase and comprises a continuous process medium.
  • the particle size is 0.5 ⁇ m. In colloidal dispersions, size of the dispersed particle is in the range of approximately 1 nm to 0.5 ⁇ m.
  • a molecular dispersion is a dispersion in which the dispersed phase consists of individual molecules; if the molecules are less than colloidal size, the result is a true solution.
  • drug load refers to the amount of drug encapsulated and to be released by a particle.
  • ECOG performance status scale refers to a scale used to assess how a patient's disease is progressing, assess how the disease affects the daily living abilities of the patient, and determine appropriate treatment and prognosis.
  • encapsulated and microencapsulated are used herein to refer generally to a bioactive agent that is incorporated into any sort of long-acting formulation or technology regardless of shape or design; therefore, an “encapsulated” or “microencapsulated” bioactive agent may include bioactive agents that are incorporated into a particle or a microparticle.
  • entrapped as used herein means to catch, hold, capture, enmesh, entangle, ensnare, snare, or trap.
  • fusion protein refers to a peptide, polypeptide or protein constructed by combining multiple protein domains or polypeptides for the purpose of creating a single peptide or protein with functional properties derived from each of the original proteins or polypeptides. Creation of a fusion protein may be accomplished by operatively ligating or linking two different nucleotides sequences that encode each protein domain or polypeptide via recombinant DNA technology, thereby creating a new polynucleotide sequences that codes for the desired fusion peptide or protein. Alternatively, a fusion peptide may be created by chemically joining the desired protein domains.
  • GALTs refers to gut-associated lymphoid tissues, which are part of the mucosa-associated lymphoid tissues (MALTs).
  • MALTs mucosa-associated lymphoid tissues
  • the histological components of GALTs mainly includes Peyer’s patches, crypt patches, isolated lymphoid follicles (ILFs) appendix and mesenteric lymph nodes (mLNs).
  • IVFs isolated lymphoid follicles
  • MNLNs mesenteric lymph nodes
  • GALTs include microfold (M) cells, which are capable of transferring antigens but not processing or presenting them [Id., citing Mabbott, NA et al. Microfold (M) cells: important immunosurveillance posts in the intestinal epithelium, Mucosal Immunol. (2013) 6:666].
  • Conventional lymphocytes such as helper T cells (Th cells) (Id., citing Dunkley, M., Husband, A.
  • phagocytes including dendritic cells (Id., citing Coombes, JL., et al.
  • a functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF ⁇ -and-retinoid acid-dependent mechanism.
  • J. Exp. Med. (2007) 204: 1757-64; Siddiqui, K., Powrie, F. CD103+ GALT DCs promote Foxp3+ regulatory T cells. Mucosal Immunol.
  • lymphoid cells Id., citing Pearson, C. et al. Lymphoid microenvironments and innate lymphoid cells in the gut. Trends Immunol. (2012) 33: 289-96; Wojno, EDT; Artis, D. Innate lymphoid cells: balancing immunity, inflammation and tissue repair in the intestine. Cell Host Microbe (2012) 12: 445-57).
  • ILCs innate lymphoid cells
  • GATA-3 and “GATA binding protein 3” are used interchangeably to refer to a member of the GATA family of conserved zinc-finger transcription factors, several of which are involved in hematopoiesis. GATA-3 is highly expressed in T cells and a wide variety of other tissues, including the CNS and fetal liver. In T cells, Gata3 acts at multiple stages of thymocyte differentiation.
  • Gata3 is also necessary for single-positive CD4 thymocyte development as well as for Th1-Th2 lineage commitment [Ting, CN et al., Nature. (1996) 384(6608):474-8; Zhang, DH et al., J Biol Chem. (1997) 272(34):21597-603; Zheng W, Flavell RA. Cell. (1997) 89(4):587-96; Zhang, DH et al., J Immunol.
  • GATA3 acts either as a transcriptional activator or repressor through direct action at many critical loci encoding cytokines, cytokine receptors, signaling molecules as well as transcription factors that are involved in the regulation of T(h)1 and T(h)2 differentiation [Jenner, RG et al., Proc Natl Acad Sci U S A. (2009) 106(42):17876-81].
  • Th2 lineage specific cytokine gene such as IL5
  • Th1 lineage specific genes IL-12 receptor ⁇ 2 and STAT4 as well as neutralizing RUNX3 function through protein-protein interaction.
  • Mice lacking Gata3 produce IFN-gamma rather than Th2 cytokines (IL5 and IL13) in response to infection [Zhu, J et al., Nat Immunol. (2004)5(11):1157-65]. It acts in mutual opposition to the transcription factor T-bet, as T-bet promotes whereas GATA3 represses Fut7 transcription [Hwang, ES et al., Science. (2005) 21;307(5708):430-3].
  • Tbx21 acts with Tbx21 to regulate cell lineage-specific expression of lymphocyte homing receptors and cytokine in both Th1 and Th2 lymphocyte subsets [Chen, GY et al., Proc Natl Acad Sci U S A. (2006) 103(45):16894-9].
  • Enforced expression of Gata3 during T cell development induced CD4(+)CD8(+) double-positive (DP) T cell lymphoma [Nawijn, MC et al., J Immunol. (2001)167(2):724-32a; Nawijn, MC et al., J Immunol. (2001)167(2):715-23].
  • Gata3 is essential for the expression of the cytokines IL-4, IL-5 and IL-13 that mediate allergic inflammation. Gata3 overexpression causes enhanced allergen-induced airway inflammation and airway remodeling, including subepithelial fibrosis, and smooth muscle cell hyperplasia [Kiwamoto, T et al., Am J Respir Crit Care Med. (2006) 174(2):142-51]. It additionally has a critical function in regulatory T cells and immune tolerance since deletion of Gata3 specifically in regulatory T cells led to a spontaneous inflammatory disorder in mice [Wang, Y et al., Immunity (2011) 35(3):337-48].
  • IL-4R ⁇ refers to the cytokine-binding receptor chain for IL-4.
  • the terms “immune response” and “immune-mediated” are used interchangeably herein to refer to any functional expression of a subject’s immune system, against either foreign or self-antigens, whether the consequences of these reactions are beneficial or harmful to the subject.
  • the term “immune system” as used herein refers to a complex network of cells, tissues, organs, and the substances they make that helps the body fight infections and other diseases.
  • the immune system includes white blood cells and organs and tissues of the lymph system, such as the thymus, spleen, tonsils, lymph nodes, lymph vessels, and bone marrow.
  • immunocompromised refers to having a weakened immune system and a reduced ability to fight infections and other diseases. Immunocompromised subjects include patients receiving long-term (>3 months) or high-dose (>0.5 mg/kg/day) steroids or other immunosuppressant drugs, solid-organ transplant recipients, patients with a solid tumor requiring chemotherapy in the last 5 years or with a hematological malignancy whatever the time since diagnosis and who received treatments, and patients with primary immune deficiency.
  • immunosuppressant drugs include patients receiving long-term (>3 months) or high-dose (>0.5 mg/kg/day) steroids or other immunosuppressant drugs, solid-organ transplant recipients, patients with a solid tumor requiring chemotherapy in the last 5 years or with a hematological malignancy whatever the time since diagnosis and who received treatments, and patients with primary immune deficiency.
  • immunosuppressant drugs include patients receiving long-term (>3 months) or high-dose (>0.5 mg/kg/day) steroids or other immunosuppressant drugs, solid-
  • immunomodulatory refers to a substance, agent, or cell that is capable of augmenting or diminishing immune responses directly or indirectly, e.g., by expressing chemokines, cytokines and other mediators of immune responses.
  • the term “immunostimulatory amount” refers to an amount of an immunogenic composition that stimulates an immune response by a measurable amount, for example, as measured by ELISPOT assay (cellular immune response), ICS (intracellular cytokine staining assay) and major histocompatibility complex (MHC) tetramer assay.
  • ELISPOT assay cellular immune response
  • ICS intracellular cytokine staining assay
  • MHC major histocompatibility complex
  • inflammation refers to the physiologic process by which vascularized tissues respond to injury. See, e.g., FUNDAMENTAL IMMUNOLOGY, 4th Ed., William E. Paul, ed. Lippincott-Raven Publishers, Philadelphia (1999) at 1051-1053, incorporated herein by reference.
  • inflammatory mediators e.g
  • acute inflammation refers to the rapid, short-lived (minutes to days), relatively uniform response to acute injury characterized by accumulations of fluid, plasma proteins, and neutrophilic leukocytes.
  • injurious agents that cause acute inflammation include, but are not limited to, pathogens (e.g., bacteria, viruses, parasites), foreign bodies from exogenous (e.g. asbestos) or endogenous (e.g., urate crystals, immune complexes), sources, and physical (e.g., burns) or chemical (e.g., caustics) agents.
  • chronic inflammation refers to inflammation that is of longer duration and which has a vague and indefinite termination. Chronic inflammation takes over when acute inflammation persists, either through incomplete clearance of the initial inflammatory agent or as a result of multiple acute events occurring in the same location. Chronic inflammation, which includes the influx of lymphocytes and macrophages and fibroblast growth, may result in tissue scarring at sites of prolonged or repeated inflammatory activity.
  • inflammatory mediators or “inflammatory cytokines” as used herein refers to the molecular mediators of the inflammatory process. These soluble, diffusible molecules act both locally at the site of tissue damage and infection and at more distant sites.
  • inflammatory mediators are activated by the inflammatory process, while others are synthesized and/or released from cellular sources in response to acute inflammation or by other soluble inflammatory mediators.
  • inflammatory mediators of the inflammatory response include, but are not limited to, plasma proteases, complement, kinins, clotting and fibrinolytic proteins, lipid mediators, prostaglandins, leukotrienes, platelet-activating factor (PAF), peptides and amines, including, but not limited to, histamine, serotonin, and neuropeptides, and proinflammatory cytokines, including, but not limited to, interleukin-1-beta (IL-1 ⁇ ), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor- alpha (TNF- ⁇ ), interferon-gamma (IF- ⁇ ), and interleukin-12 (IL-12).
  • IL-1 ⁇ interleukin-1-beta
  • IL-4
  • IL-1 IL-6
  • TNF- ⁇ pro-inflammatory mediators
  • IL-1 IL-6
  • TNF- ⁇ pro-inflammatory mediators
  • IL-1 IL-6
  • TNF- ⁇ pro-inflammatory mediators
  • IL-1 IL-6
  • TNF- ⁇ pro-inflammatory mediators
  • IL-1 IL-6
  • TNF- ⁇ hepatocytes in an acute phase response to synthesize acute-phase proteins that activate complement.
  • IL-1 refers to the act of drawing in a medicated vapor with the breath.
  • inhalation delivery device refers to a machine/apparatus or component that produces small droplets or an aerosol from a liquid or dry powder aerosol formulation and is used for administration through the mouth in order to achieve pulmonary administration of a drug, e.g., in solution, powder, and the like.
  • inhalation delivery device examples include, but are not limited to, a nebulizer, a metered-dose inhaler, and a dry powder inhaler (DPI).
  • inhalation delivery device examples include, but are not limited to, a nebulizer, a metered-dose inhaler, and a dry powder inhaler (DPI).
  • DPI dry powder inhaler
  • Inhibition may include a reduction or decrease of the amount, rate, action function, or process of a substance by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%.
  • the term “injury” as used herein refers to damage or harm to a structure or function of the body caused by an outside agent or force, which may be physical or chemical.
  • insufflation refers to the act of delivering air, a gas, or a powder under pressure to a cavity or chamber of the body.
  • nasal insufflation relates to the act of delivering air, a gas, or a powder under pressure through the nose.
  • IL interleukin
  • the term “interleukin (IL)” as used herein refers to a cytokine secreted by, and acting on, leukocytes. Interleukins regulate cell growth, differentiation, and motility, and stimulates immune responses, such as inflammation.
  • interleukins examples include, interleukin-1 (IL-1), interleukin-1 ⁇ (IL-1 ⁇ ), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-12 (IL-12).
  • isolated is used herein to refer to material, such as, but not limited to, a nucleic acid, peptide, polypeptide, or protein, which is: (1) substantially or essentially free from components that normally accompany or interact with it as found in its naturally occurring environment.
  • substantially free or “essentially free” are used herein to refer to considerably or significantly free of, or more than about 95% free of, or more than about 99% free of such components.
  • the isolated material optionally comprises material not found with the material in its natural environment; or (2) if the material is in its natural environment, the material has been synthetically (non-naturally) altered by deliberate human intervention to a composition and/or placed at a location in the cell (e.g., genome or subcellular organelle) not native to a material found in that environment.
  • the term “lectin” as used herein refers to a class of proteins that bind specifically to certain sugars and so cause agglutination of particular cell types.
  • LINGO refers to the “leucine-rich repeat and immunoglobulin-like domain-containing NoGo” family of proteins.
  • lymphocyte refers to a small white blood cell formed in lymphatic tissue throughout the body and in normal adults making up about 22-28% of the total number of leukocytes in the circulating blood that plays a large role in defending the body against disease.
  • lymphocytes are specialized in that they are committed to respond to a limited set of structurally related antigens. This commitment, which exists before the first contact of the immune system with a given antigen, is expressed by the presence on the lymphocyte’s surface membrane of receptors specific for determinants (epitopes) on the antigen. Each lymphocyte possesses a population of receptors, all of which have identical combining sites.
  • lymphocytes differs from another clone in the structure of the combining region of its receptors and thus differs in the epitopes that it can recognize. Lymphocytes differ from each other not only in the specificity of their receptors, but also in their functions.
  • B-cells B-lymphocytes
  • T-cells T-lymphocytes
  • B-lymphocytes B-lymphocytes
  • B-lymphocytes are derived from hematopoietic cells of the bone marrow. A mature B-cell can be activated with an antigen that expresses epitopes that are recognized by its cell surface.
  • the activation process may be direct, dependent on cross-linkage of membrane Ig molecules by the antigen (cross-linkage-dependent B-cell activation), or indirect, via interaction with a helper T-cell, in a process referred to as cognate help.
  • a helper T-cell in a process referred to as cognate help.
  • receptor cross-linkage stimuli and cognate help synergize to yield more vigorous B- cell responses.
  • Cross-linkage dependent B-cell activation requires that the antigen express multiple copies of the epitope complementary to the binding site of the cell surface receptors because each B-cell expresses Ig molecules with identical variable regions. Such a requirement is fulfilled by other antigens with repetitive epitopes, such as capsular polysaccharides of microorganisms or viral envelope proteins. Cross-linkage-dependent B-cell activation is a major protective immune response mounted against these microbes. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4 th Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)).
  • Cognate help allows B-cells to mount responses against antigens that cannot cross-link receptors and, at the same time, provides costimulatory signals that rescue B cells from inactivation when they are stimulated by weak cross-linkage events.
  • Cognate help is dependent on the binding of antigen by the B-cell’s membrane immunoglobulin (Ig), the endocytosis of the antigen, and its fragmentation into peptides within the endosomal/lysosomal compartment of the cell. Some of the resultant peptides are loaded into a groove in a specialized set of cell surface proteins known as class II major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the resultant class II/peptide complexes are expressed on the cell surface and act as ligands for the antigen-specific receptors of a set of T-cells designated as CD4+ T- cells.
  • the CD4+ T-cells bear receptors on their surface specific for the B-cell’s class II/peptide complex.
  • B-cell activation depends not only on the binding of the T cell through its T cell receptor (TCR), but this interaction also allows an activation ligand on the T-cell (CD40 ligand) to bind to its receptor on the B-cell (CD40) signaling B-cell activation.
  • T helper cells secrete several cytokines that regulate the growth and differentiation of the stimulated B- cell by binding to cytokine receptors on the B cell.
  • CD40 ligand is transiently expressed on activated CD4+ T helper cells, and it binds to CD40 on the antigen-specific B cells, thereby transducing a second costimulatory signal.
  • the latter signal is essential for B cell growth and differentiation and for the generation of memory B cells by preventing apoptosis of germinal center B cells that have encountered antigen. Hyperexpression of the CD40 ligand in both B and T cells is implicated in the pathogenic autoantibody production in human SLE patients.
  • T-lymphocytes [000426] T-lymphocytes derive from precursors in hematopoietic tissue, undergo differentiation in the thymus, and are then seeded to peripheral lymphoid tissue and to the recirculating pool of lymphocytes. T-lymphocytes or T cells mediate a wide range of immunologic functions.
  • T cells differ from B cells in their mechanism of antigen recognition. Immunoglobulin, the B cell’s receptor, binds to individual epitopes on soluble molecules or on particulate surfaces.
  • B-cell receptors see epitopes expressed on the surface of native molecules. Antibody and B-cell receptors evolved to bind to and to protect against microorganisms in extracellular fluids. In contrast, T cells recognize antigens on the surface of other cells and mediate their functions by interacting with, and altering, the behavior of these antigen- presenting cells (APCs).
  • APCs antigen-presenting cells
  • APCs display three types of protein molecules on their surface that have a role in activating a T cell to become an effector cell: (1) MHC proteins, which present foreign antigen to the T cell receptor; (2) costimulatory proteins which bind to complementary receptors on the T cell surface; and (3) cell-cell adhesion molecules, which enable a T cell to bind to the antigen-presenting cell (APC) for long enough to become activated.
  • MHC proteins which present foreign antigen to the T cell receptor
  • costimulatory proteins which bind to complementary receptors on the T cell surface
  • cell-cell adhesion molecules which enable a T cell to bind to the antigen-presenting cell (APC) for long enough to become activated.
  • APC antigen-presenting cell
  • T cells express T cell receptors (TCR) consisting of ⁇ and ⁇ chains.
  • TCR T cell receptors
  • a small group of T cells express receptors made of ⁇ and ⁇ chains.
  • CD4+ T cells are two important sublineages: those that express the coreceptor molecule CD4 (CD4+ T cells); and those that express CD8 (CD8+ T cells). These cells differ in how they recognize antigen and in their effector and regulatory functions.
  • CD4+ T cells are the major regulatory cells of the immune system. Their regulatory function depends both on the expression of their cell-surface molecules, such as CD40 ligand whose expression is induced when the T cells are activated, and the wide array of cytokines they secrete when activated.
  • T cells also mediate important effector functions, some of which are determined by the patterns of cytokines they secrete.
  • the cytokines can be directly toxic to target cells and can mobilize potent inflammatory mechanisms.
  • T cells particularly CD8+ T cells can develop into cytotoxic T- lymphocytes (CTLs) capable of efficiently lysing target cells that express antigens recognized by the CTLs.
  • CTLs cytotoxic T- lymphocytes
  • T cell receptors recognize a complex consisting of a peptide derived by proteolysis of the antigen bound to a specialized groove of a class II or class I MHC protein.
  • the CD4+ T cells recognize only peptide/class II complexes while the CD8+ T cells recognize peptide/class I complexes.
  • the TCR’s ligand i.e., the peptide/MHC protein complex
  • APCs antigen-presenting cells
  • class II MHC molecules bind peptides derived from proteins that have been taken up by the APC through an endocytic process. These peptide-loaded class II molecules are then expressed on the surface of the cell, where they are available to be bound by CD4+ T cells with TCRs capable of recognizing the expressed cell surface complex.
  • CD4+ T cells are specialized to react with antigens derived from extracellular sources.
  • class I MHC molecules are mainly loaded with peptides derived from internally synthesized proteins, such as viral proteins. These peptides are produced from cytosolic proteins by proteolysis by the proteosome and are translocated into the rough endoplasmic reticulum. Such peptides, generally nine amino acids in length, are bound into the class I MHC molecules and are brought to the cell surface, where they can be recognized by CD8+ T cells expressing appropriate receptors.
  • T cell system particularly CD8+ T cells, the ability to detect cells expressing proteins that are different from, or produced in much larger amounts than, those of cells of the remainder of the organism (e.g., vial antigens) or mutant antigens (such as active oncogene products), even if these proteins in their intact form are neither expressed on the cell surface nor secreted.
  • vial antigens e.g., vial antigens
  • mutant antigens such as active oncogene products
  • Helper T cells are T cells that stimulate B cells to make antibody responses to proteins and other T cell-dependent antigens.
  • T cell-dependent antigens are immunogens in which individual epitopes appear only once or a limited number of times such that they are unable to cross-link the membrane immunoglobulin (Ig) of B cells or do so inefficiently.
  • B cells bind the antigen through their membrane Ig, and the complex undergoes endocytosis. Within the endosomal and lysosomal compartments, the antigen is fragmented into peptides by proteolytic enzymes and one or more of the generated peptides are loaded into class II MHC molecules, which traffic through this vesicular compartment.
  • the resulting peptide/class II MHC complex is then exported to the B-cell surface membrane.
  • T cells with receptors specific for the peptide/class II molecular complex recognize this complex on the B-cell surface.
  • B-cell activation depends both on the binding of the T cell through its TCR and on the interaction of the T-cell CD40 ligand (CD40L) with CD40 on the B cell. T cells do not constitutively express CD40L.
  • CD40L expression is induced as a result of an interaction with an APC that expresses both a cognate antigen recognized by the TCR of the T cell and CD80 or CD86.
  • CD80/CD86 is generally expressed by activated, but not resting, B cells so that the helper interaction involving an activated B cell and a T cell can lead to efficient antibody production.
  • the initial induction of CD40L on T cells is dependent on their recognition of antigen on the surface of APCs that constitutively express CD80/86, such as dendritic cells.
  • Such activated helper T cells can then efficiently interact with and help B cells.
  • CD4+ T cells tend to differentiate into cells that principally secrete the cytokines IL-4, IL-5, IL-6, and IL-10 (T H2 cells) or into cells that mainly produce IL-2, IFN- ⁇ , and lymphotoxin (T H1 cells).
  • T H2 cells are very effective in helping B-cells develop into antibody-producing cells
  • TH1 cells are effective inducers of cellular immune responses, involving enhancement of microbicidal activity of monocytes and macrophages, and consequent increased efficiency in lysing microorganisms in intracellular vesicular compartments.
  • T cells with the phenotype of T H2 cells are efficient helper cells
  • TH1 cells also have the capacity to be helpers.
  • T cells involved in Induction of Cellular Immunity T cells also may act to enhance the capacity of monocytes and macrophages to destroy intracellular microorganisms.
  • helper T cells enhances several mechanisms through which mononuclear phagocytes destroy intracellular bacteria and parasitism including the generation of nitric oxide and induction of tumor necrosis factor (TNF) production.
  • the TH1 cells are effective in enhancing the microbicidal action because they produce IFN- ⁇ .
  • two of the major cytokines produced by TH2 cells, IL-4 and IL-10 block these activities.
  • T cell anergy a tolerance mechanism in which the T cells are intrinsically functionally inactivated following an antigen encounter (Scwartz, R. H., “T cell anergy,” Annu. Rev. Immunol., 21: 305-334 (2003)) are important mechanisms that contribute to the downregulation of the immune response.
  • a third mechanism is provided by active suppression of activated T cells by suppressor or regulatory CD4+ T (Treg) cells.
  • CD4+ Tregs that constitutively express the IL-2 receptor alpha (IL-2R ⁇ ) chain are a naturally occurring T cell subset that are anergic and suppressive.
  • IL-2R ⁇ IL-2 receptor alpha
  • CD4+ CD25+ are a naturally occurring T cell subset that are anergic and suppressive.
  • Depletion of CD4 + CD25 + Tregs results in systemic autoimmune disease in mice. Furthermore, transfer of these Tregs prevents development of autoimmune disease.
  • Human CD4 + CD25 + Tregs are generated in the thymus and are characterized by the ability to suppress proliferation of responder T cells through a cell-cell contact-dependent mechanism, the inability to produce IL-2, and the anergic phenotype in vitro.
  • Human CD4 + CD25 + T cells can be split into suppressive (CD25 high ) and nonsuppressive (CD25 low ) cells, according to the level of CD25 expression.
  • a member of the forkhead family of transcription factors, FOXP3 has been shown to be expressed in murine and human CD4 + CD25 + Tregs and appears to be a master gene controlling CD4 + CD25 + Treg development. (Battaglia, M.
  • CTL Cytotoxic T Lymphocytes
  • CTL-mediated lysis appears to be a major mechanism for the destruction of virally infected cells.
  • macrophage refers to a mononuclear, actively phagocytic cell arising from monocyte stem cells in the bone marrow. These cells are widely distributed in the body and vary in morphology and motility.
  • Phagocytic activity is typically mediated by serum recognition factors, including certain immunoglobulins and components of the complement system, but also may be nonspecific. Macrophages also are involved in both the production of antibodies and in cell-mediated immune responses, particularly in presenting antigens to lymphocytes. They secrete a variety of immunoregulatory molecules.
  • MHC Major Histocompatibility Complex
  • HLA HLA
  • MHCs are encoded by a large gene group and can be organized into three subgroups- class I, class II, and class III.
  • HLA Human leukocyte antigen
  • H-2 human leukocyte antigen
  • Both species have three main MHC class I genes, which are called HLA- A, HLA-B, and HLA-C in humans, and H2-K, H2-D and H2-L in the mouse. These encode the ⁇ chain of the respective MHC class I proteins.
  • the other subunit of an MHC class I molecule is ⁇ 2-microglobulin.
  • the class II region includes the genes for the ⁇ and ⁇ chains (designated A and B) of the MHC class II molecules HLA-DR, HLA-DP, and HLA-DQ in humans. Also in the MHC class II region are the genes for the TAP1:TAP2 peptide transporter, the PSMB (or LMP) genes that encode proteasome subunits, the genes encoding the DM ⁇ and BM ⁇ chains (DMA and DMB), the genes encoding the ⁇ and ⁇ chains of the DO molecule (DOA and DOB, respectively), and the gene encoding tapasin (TAPBP).
  • the class II genes encode various other proteins with functions in immunity.
  • the DMA and DMB genes encoding the subunits of the HLA-DM molecule that catalyzes peptide binding to MHC class II molecules are related to the MHC class II genes, as are the DOA and DOB genes that encode the subunits of the regulatory HLA-DO molecule.
  • DOA and DOB genes that encode the subunits of the regulatory HLA-DO molecule.
  • group 1 which includes CD1a, b, and c
  • group 2 which consists of CD1d.
  • Group 1 CD1s can present antigens to a wide variety of T cells, whereas CD1d presents antigens mostly to NKT cells. (Brutkiewicz. “CD1d Ligands: The Good, the Bad, and the Ugly.” The Journal of Immunology (2006) 177 (2) 769-775). While CD1d structurally resembles MHC Class I molecules, it traffics through the endosome of the exogenous antigen presentation pathway.
  • CD1d presents lipid antigens, and requires the presence of particular mechanisms to induce uptake of these molecules by APCs and subsequent loading onto CD1d molecules.
  • Lipid transfer protein such as apolipoprotein E and fatty acid amide hydrolase (FAAH) have been shown to enhance the presentation of certain antigens by CD1d.
  • Loading efficiency can be enhanced by specific proteins, such as saposins and microsomal triglyceride transfer protein , present in the endosomal and lysosomal compartments of cells by promoting lipid antigen exchange. Similar to MHC antigens, lipid antigens can also be processed by lysosomal enzymes to yield active compounds, as demonstrated in the case of CD1d for synthetic antigens, microbial antigens, and self-antigens. [Giradi and Zajonc (2012).
  • MHC Class I-like molecules are nonclassical MHC type molecules; while including Cd1d CD1a, CD1b, CD1c, CD1e, and MR1 are also expressed on APCs and can activate various subsets of T cells. [Kumar and Delovitch (2014) “Different subsets of natural killer T cells may vary in their roles in health and disease.” Immunology 142: 321-336]. Other non-classical histocompatibility molecules include MR1, which activate MAIT cells.
  • MMAD mass median aerodynamic diameter
  • matrix refers to a three-dimensional network of fibers that contains voids (or “pores”) where the woven fibers intersect. The structural parameters of the pores, including the pore size, porosity, pore interconnectivity/ tortuosity and surface area, affect how substances (e.g., fluid, solutes) move in and out of the matrix.
  • tuning of the drug delivery system comprises controlling the porosity and rate of pore formation.
  • the polymer is porous.
  • the polymer is nonporous.
  • the polymer comprises a channel forming agent or porogen, e.g., CaCl 2 .
  • the term “metered-dose inhaler”, “MDI”, or “puffer” as used herein refers to a pressurized, hand-held device that uses propellants to deliver a specific amount of medicine (“metered dose”) to the lungs of a patient.
  • propellant refers to a material that is used to expel a substance usually by gas pressure through a convergent, divergent nozzle.
  • the pressure may be from a compressed gas, or a gas produced by a chemical reaction.
  • the exhaust material may be a gas, liquid, plasma, or, before the chemical reaction, a solid, liquid or gel.
  • Propellants used in pressurized metered dose inhalers are liquefied gases, traditionally chlorofluorocarbons (CFCs) and increasingly hydrofluoroalkanes (HFAs).
  • Suitable propellants include, for example, a chlorofluorocarbon (CFC), such as trichlorofluoromethane (also referred to as propellant 11), dichlorodifluoromethane (also referred to as propellant 12), and 1,2-dichloro-1,1,2,2-tetrafluoroethane (also referred to as propellant 114), a hydrochlorofluorocarbon, a hydrofluorocarbon (HFC), such as 1,1,1,2- tetrafluoroethane (also referred to as propellant 134a, HFC-134a, or HFA-134a) and 1,1,1,2,3,3,3-heptafluoropropane (also referred to as propellant 227, HFC-227, or HFA-227), carbon dioxide, dimethyl ether, butane, propane, or mixtures thereof.
  • CFC chlorofluorocarbon
  • trichlorofluoromethane also referred to as propellant 11
  • the propellant includes a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or mixtures thereof.
  • a hydrofluorocarbon is used as the propellant.
  • HFC-227 and/or HFC-134a are used as the propellant.
  • the particles may be of any order release kinetics, including zero order release, first order release, second order release, delayed release, sustained release, immediate release, etc., and any combination thereof.
  • the particles may include, in addition to therapeutic agent(s), any of those materials routinely used in the art of pharmacy and medicine, including, but not limited to, erodible, nonerodible, biodegradable, or nonbiodegradable material or combinations thereof.
  • the particles may be microcapsules that contain the therapeutic agent in a solution or in a semi-solid state.
  • the particles may be of virtually any shape.
  • microparticulate composition refers to a composition comprising a microparticulate formulation and a pharmaceutically acceptable carrier, where the microparticulate formulation comprises a therapeutic agent and a plurality of microparticles.
  • microparticulate formulation refers to a composition comprising a microparticulate formulation and a pharmaceutically acceptable carrier, where the microparticulate formulation comprises a therapeutic agent and a plurality of microparticles.
  • minimum effective concentration “minimum effective dose”, or “MEC” are used interchangeably to refer to the minimum concentration of the recombinant fusion protein of the present disclosure required to produce a desired pharmacological effect in most patients.
  • mitogen refers to a substance that stimulates mitosis.
  • modulate as used herein means to regulate, alter, adapt, or adjust to a certain measure or proportion.
  • molecule refers to a chemical unit composed of one or more atoms, whereby the atoms of the molecule are held together by chemical bonds.
  • monomer refers to a molecule that may bind chemically to other molecules to form a polymer.
  • trimer refers to a substance that enhances cell migratory processes (motogenic effect), show anti-apoptotic effects and are inflammatory modulators.
  • morphogen refers to a substance that governs the movement and development of cells during morphogenesis by forming a concentration gradient in the developing tissue.
  • MALT lymphoid tissue
  • MALT mucosa-associated lymphoid tissue
  • GALT GALT
  • NALT NALT
  • BALT BALT
  • MAIT mucosa-associated invariant T cells
  • mucosal epithelia refers to mucus-coated epithelia lining the body’s internal cavities that connect with the outside (e.g., the gut, airways, and vaginal tract).
  • mucosal mast cells refers to specialized mast cells present in mucosa. They produce little histamine but large amounts of prostaglandins and leukotrienes.
  • mutation refers to a change of the DNA sequence within a gene or chromosome of an organism resulting in the creation of a new character or trait not found in the parental type, or the process by which such a change occurs in a chromosome, either through an alteration in the nucleotide sequence of the DNA coding for a gene or through a change in the physical arrangement of a chromosome.
  • Three mechanisms of mutation include substitution (exchange of one base pair for another), addition (the insertion of one or more bases into a sequence), and deletion (loss of one or more base pairs).
  • myeloid as used herein means of or pertaining to bone marrow.
  • Granulocytes and monocytes are differentiated descendants from common progenitors derived from hematopoietic stem cells in the bone marrow. Commitment to either lineage of myeloid cells is controlled by distinct transcription factors followed by terminal differentiation in response to specific colony-stimulating factors and release into the circulation. Upon pathogen invasion, myeloid cells are rapidly recruited into local tissues via various chemokine receptors, where they are activated for phagocytosis as well as secretion of inflammatory cytokines, thereby playing major roles in innate immunity. [Kawamoto, H., Minato, N. Intl J. Biochem. Cell Biol. (2004) 36 (8): 1374-9].
  • nanoparticle refers to a particle whose extension in all three dimensions lies between 1 and 1000 nanometers. Because of their small size, nanoparticles have a very large surface area to volume ratio when compared to bulk material, which enables nanoparticles to possess unexpected optical, physical and chemical properties that are not found in bulk samples of the same material. They can be classified into different classes based on their properties, shapes or sizes. The different groups include fullerenes, metal NPs, ceramic NPs, and polymeric NPs. Fullerenes and carbon nanotubes (CNTs) represent two major classes of carbon-based NPs. Fullerenes contain nanomaterial that are made of globular hollow cage such as allotropic forms of carbon.
  • NF ⁇ B refers to a proinflammatory transcription factor that switches on multiple inflammatory genes, including cytokines, chemokines, proteases, and inhibitors of apoptosis, resulting in amplification of the inflammatory response [Barnes, PJ, (2016) Pharmacol. Rev. 68: 788-815].
  • the molecular pathways involved in NF- ⁇ B activation include several kinases.
  • IKK inhibitor of ⁇ B kinase
  • IKK- ⁇ and IKK- ⁇ a regulatory subunit IKK- ⁇ (or NF ⁇ B essential modulator [Id., citing Hayden, MS and Ghosh, S (2012) Genes Dev. 26: 203-234].
  • the IKK complex phosphorylates Nf- ⁇ B- bound I ⁇ Bs, targeting them for degradation by the proteasome and thereby releasing NF- ⁇ B dimers that are composed of p65 and p50 subunits, which translocate to the nucleus where they bind to ⁇ B recognition sites in the promoter regions of inflammatory and immune genes, resulting in their transcriptional activation.
  • This response depends mainly on the catalytic subunit IKK- ⁇ (also known as IKK2), which carries out I ⁇ B phosphorylation.
  • the noncanonical (alternative) pathway involves the upstream kinase NF- ⁇ B-inducing kinase (NIK) that phosphorylates IKK- ⁇ homodimers and releases RelB and processes p100 to p52 in response to certain members of the TNF family, such as lymphotoxin- ⁇ [Id., citing Sun, SC. (2012) Immunol. Rev.246: 125-140].
  • This pathway switches on different gene sets and may mediate different immune functions from the canonical pathway.
  • Dominant-negative IKK- ⁇ inhibits most of the proinflammatory functions of NF- ⁇ B, whereas inhibiting IKK- ⁇ has a role only in response to limited stimuli and in certain cells, such as B-lymphocytes.
  • the noncanonical pathway is involved in development of the immune system and in adaptive immune responses.
  • the coactivator molecule CD40 which is expressed on antigen-presenting cells, such as dendritic cells and macrophages, activates the noncanonical pathway when it interacts with CD40L expressed on lymphocytes [Id., citing Lombardi, V et al. (2010) Int. Arch. Allergy Immunol.151: 179-89].
  • NK natural killer cells
  • NK cells secrete cytokines such as IFN ⁇ and TNF ⁇ , which act on other immune cells, like macrophages and dendritic cells, to enhance the immune response.
  • cytokines such as IFN ⁇ and TNF ⁇
  • Activating receptors on the NK cell surface recognize molecules expressed on the surface of cancer cells and infected cells and switch on the NK cell.
  • Inhibitory receptors act as a check on NK cell killing.
  • Most normal healthy cells express MHCI receptors, which mark them as “self.”
  • Inhibitory receptors on the surface of the NK cell recognize cognate MHCI, which switches off the NK cell, preventing it from killing.
  • NK cell releases cytotoxic granules containing perforin and granzymes, which leads to lysis of the target cell.
  • Natural killer reactivity including cytokine secretion and cytotoxicity, is controlled by a balance of several germ-line encoded inhibitory and activating receptors such as killer immunoglobulin-like receptors (KIRs) and natural cytotoxicity receptors (NCRs).
  • KIRs killer immunoglobulin-like receptors
  • NCRs natural cytotoxicity receptors
  • the presence of the MHC Class I molecule on target cells serves as one such inhibitory ligand for MHC Class I-specific receptors, the Killer cell Immunoglobulin-like Receptor (KIR), on NK cells.
  • nebulizer refers to a device used to administer liquid medication in the form of a mist inhaled into the lungs. Nebulizers, which actively aerosolize a liquid formulation and operate continuously once loaded, require either compressed air or an electrical supply.
  • Exemplary nebulizers include, a vibrating mesh nebulizer, a jet nebulizer (also known as an atomizer) and an ultrasonic wave nebulizer.
  • Exemplary vibrating mesh nebulizers include, but are not limited to, Respironics i-Neb, Omron MicroAir, Beurer Nebulizer IHSO and Aerogen Aeroneb.
  • Respironics i-Neb, Omron MicroAir, Beurer Nebulizer IHSO and Aerogen Aeroneb Acorn-I, Acorn-II, AquaTower, AVA-NEB, Cirrhus, Dart, DeVilbiss 646, Downdraft, Fan Jet, MB-5, Misty Neb, Salter Labs 8900, Sidestream, Updraft-II, and Whisper Jet are examples of a jet nebulizer.
  • Exemplary ultrasonic nebulizers include, but are not limited to, an Omron NE-U17 nebulizer and a Beurer Nebulizer IH30.
  • the term “neutrophils” or “polymorphonuclear neutrophils (PMNs)” as used herein refers to the most abundant type of white blood cells in mammals, which form an essential part of the innate immune system. They form part of the polymorphonuclear cell family (PMNs) together with basophils and eosinophils. Neutrophils are normally found in the blood stream.
  • neutrophils are one of the first-responders of inflammatory cells to migrate toward the site of inflammation. They migrate through the blood vessels, then through interstitial tissue, following chemical signals such as interleukin- 8 (IL-8) and C5a in a process called chemotaxis, the directed motion of a motile cell or part along a chemical concentration gradient toward environmental conditions it deems attractive and/or away from surroundings it finds repellent.
  • IL-8 interleukin- 8
  • C5a chemical signals
  • chemotaxis the directed motion of a motile cell or part along a chemical concentration gradient toward environmental conditions it deems attractive and/or away from surroundings it finds repellent.
  • NLRs Nucleotide-binding Oligomerization Domain (NOD)-like receptors
  • Subfamilies of NLRs can be distinguished based on the other protein domains they contain.
  • the NOD subfamily has an amino-terminal caspase recruitment domain (CARD), which is structurally related to the T1R death domain in MyD88, and can dimerize with CARD domains on other proteins to induce signaling.
  • CARD caspase recruitment domain
  • NOD proteins recognize fragments of bacterial cell wall peptidoglycans, although it is not known if they do so through direct binding or through accessory proteins.
  • NOD1 senses ⁇ -glutamyl diaminopimelic acid (iE-DAP), a breakdown product of peptidoglycans of Gram negative and some Gram positive bacteria, whereas NOD2 recognizes muramyl dipeptide (MDP), which is present in the peptidoglycans of most bacteria. Id. Other members of the NOD family, including NLRX1 and NLRC5, have been identified, but their function is less well understood.
  • iE-DAP ⁇ -glutamyl diaminopimelic acid
  • MDP muramyl dipeptide
  • NOD1 or NOD2 When NOD1 or NOD2 recognizes its ligand, it recruits the CARD-containing serine-threonine kinase RIP2 (also known as RICK and RIPK2), which associates with the E3 ligases cIAP1, CIAP2, and XIAP, whose activity generates a polyubiquitin scaffold, which recruits TAK1 and IKK and results in activation of NF ⁇ B.
  • NF ⁇ B then induces the expression of genes for inflammatory cytokines and for enzymes involved in the production of NO.
  • Macrophages and dendritic cells express both TLRs and NOD1 and NOD2, and are activated by both pathways.
  • NOD1 may also function as a systemic activator of innate immunity.
  • NOD2 is strongly expressed in the Paneth cells of the gut where it regulates the expression of potent anti-microbial peptides such as the ⁇ - and ⁇ - defensins.
  • NLRX1 and NLRC5 have been identified, but their function is less well understood.
  • the NLRP family another subfamily of NLR proteins, has a pyrin domain in place of the CARD domain at their amino termini. Humans have 14 NLR proteins containing pyrin domains, of which NLRP3 (also known as NAPL3 or cryopyrin) is the best characterized.
  • NLRP3 resides in an inactive form in the cytoplasm, where its leucine rich repeat (LRR) domains are thought to bind the head-shock chaperone protein HSP90 and the co-chaperone SGT1.
  • LRR leucine rich repeat
  • NRLP3 signaling is induced by reduced intracellular potassium, the generation of reactive oxygen species, or the disruption of lysosomes by particulate or crystalline matter. For example, death of nearby cells can release ATP into the extracellular space, which would activate the purinergic receptor P2X7, which is a potassium channel, and allow potassium ion efflux.
  • a model proposed for ROS-induced NLRP3 activation involves intermediate oxidation of sensor proteins collectively called thioredoxin (TRX).
  • TRX proteins are bound to thioredoxin-interacting protein (TXNIP). Oxidation of TRX by ROS causes dissociation of TXNIP from TRX. The free TXNIP may then displace HSP90 and SGT1 from NLRP3, again causing its activation. In both cases, NLRP3 activation involves aggregation of multiple monomers via their leucine-rich repeat (LRR) and NOD domains to induce signaling. Phagocytosis of particulate matter (e.g. the adjuvant alum), may lead to the rupture of lysosomes and release of the active protease cathepsin B, which can activate NLRP3.
  • LRR leucine-rich repeat
  • NLR signaling leads to the generation of pro- inflammatory cytokines and to cell death through formation of an inflammasome, a multiprotein complex. Activation of the inflammasome proceeds in several stages.
  • NLRP molecules triggers autocleavage of procaspase I, which releases active caspase 1 - Aggregation of LRR domains of several NLRP3 molecules, or other NLRP molecules by a specific trigger or recognition event, which induces the pyrin domains of NLRP3 to interact with pyrin domains of ASC (also called PYCARD), an adaptor protein composed of an amino terminal pyrin domain and a carboxy terminal CARD domain, which further drives the formation of a polymeric ASC filament, with the pyrin domains in the center and the CARD domains facing outward; the CARD domains then interact with CARD domains of the inactive protease pro-caspase 1, initiating its CARD-dependent polymerization into discrete caspase 1 filaments.
  • ASC also called PYCARD
  • Active caspase 1 then carries out ATP-dependent proteolytic processing of proinflammatory cytokines, particularly 1L-1 ⁇ and IL-18, into their active forms, and induces a form of cell death (pyroptosis) associated with inflammation because of the release of these pro-inflammatory cytokines upon cell rupture.
  • proinflammatory cytokines particularly 1L-1 ⁇ and IL-18
  • pyroptosis cell death
  • a priming step which can result from TLR signaling, must first occur in which cells inducer and translate the mRNAs that encode the pro-forms of IL-1, IL-18 or other cytokines for inflammasome activation to produce inflammatory cytokines.
  • the TLR-3 agonist poly I:C can be used experimentally to prime cells for triggering of the inflammasome. Janeway’s Immunobiology. 9th ed., GS, Garland Science, Taylor & Francis Group, 2017, at 100].
  • Inflammasome activation also can involve proteins of the PYHIN family, which have an H inversion (HIN) domain in place of an LRR domain. There are four PYIN proteins in humans. Id. At 100.
  • a noncanonical inflammasome (caspase I-independent) pathway uses the protease caspase 11, which therefore is both a sensor and an effector molecule, to detect intracellular LPS.
  • the term “operatively linked” as used herein refers to a linkage in which two or more protein domains or peptides are ligated or combined via recombinant DNA technology or chemical reaction such that each protein domain or polypeptide of the resulting fusion peptide retains its original function.
  • the term “oxygen saturation” (SpO2) as used herein refers to a measurement of how much oxygen the blood is carrying as a percentage of the maximum it could carry. For a healthy individual, the normal SpO 2 should be between 96% to 99%.
  • parenteral refers to introduction into the body by means other than through the digestive tract, for example, without limitation, by way of an injection (i.e., administration by injection), including, for example, subcutaneously (i.e., an injection beneath the skin), intramuscularly (i.e., an injection into a muscle), intravenously (i.e., an injection into a vein), or infusion techniques.
  • injection i.e., administration by injection
  • particles refers to refers to an extremely small constituent (e.g., nanoparticles, microparticles, or in some instances larger) in or on which is contained the composition as described herein.
  • pathogenesis refers to the development of a disease and the chain of events leading to that disease and its sequelae.
  • pathological refers to indicative of or caused by disease.
  • pathophysiology and its various grammatical forms as used herein refers to derangement of function in an individual or organ due to a disease.
  • PRRs pattern recognition receptors
  • PRRs pathogen associated molecular patterns
  • TLR toll-like receptors
  • NLR nucleotide oligomerization receptors
  • CLR C-type leptin receptors
  • RIG-1 like receptors RLR
  • the subunits are linked by peptide bonds, except where noted.
  • FDA considers any polymer composed of 40 or fewer amino acids to be a peptide.
  • the peptides, polypeptides or proteins described herein may be chemically synthesized or recombinantly expressed.
  • Synthetic polypeptides prepared using the well-known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can include natural and unnatural amino acids.
  • Amino acids used for peptide synthesis may be standard Boc (N- ⁇ -amino protected N- ⁇ -t-butyloxycarbonyl) amino acid resin with the standard deprotecting, neutralization, coupling and wash protocols of the original solid phase procedure of Merrifield (1963, J. Am. Chem. Soc.
  • Solid phase peptide synthesis may be accomplished by techniques familiar to those in the art and provided, for example, in Stewart and Young, 1984, Solid Phase Synthesis, Second Edition, Pierce Chemical Co., Rockford, Ill.; Fields and Noble, 1990, Int. J. Pept. Protein Res. 35:161-214, or using automated synthesizers.
  • the polypeptides useful in the described invention may comprise D-amino acids (which are resistant to L-amino acid- specific proteases in vivo), a combination of D- and L-amino acids, and various "designer" amino acids (e.g., ⁇ -methyl amino acids, C- ⁇ -methyl amino acids, and N- ⁇ -methyl amino acids, etc.) to convey special properties.
  • Synthetic amino acids include ornithine for lysine, and norleucine for leucine or isoleucine.
  • the polypeptides can have peptidomimetic bonds, such as ester bonds, to prepare peptides with novel properties.
  • a peptide may be generated that incorporates a reduced peptide bond, i.e., R 1 -CH 2 -NH-R 2 , where R 1 and R 2 are amino acid residues or sequences.
  • a reduced peptide bond may be introduced as a dipeptide subunit.
  • Such a polypeptide would be resistant to protease activity, and would possess an extended half-live in vivo.
  • amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • the essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids.
  • polypeptide polypeptide
  • peptide protein
  • proteins also are inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.
  • polypeptides may not be entirely linear.
  • polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslational events, including natural processing event and events brought about by human manipulation which do not occur naturally.
  • Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well.
  • the peptide is of any length or size.
  • a peptidomimetic typically arises from modification of an existing peptide in order to alter the molecule's properties.
  • pharmaceutical composition is used herein to refer to a composition that is employed to prevent, reduce in intensity, cure or otherwise treat a target condition or disease.
  • polymer refers to a large molecule, or macromolecule, composed of many repeated subunits.
  • polymer matrix refers to a matrix formed by polymers.
  • potentiate and its other grammatical forms as used herein means to increase the power, effect, or potency, of; to enhance, to augment the activity of.
  • the term “priming” as used herein refers to the process whereby T cells and B cell precursors encounter the antigen for which they are specific.
  • the term “unprimed cells” also referred to as virgin, na ⁇ ve, or inexperienced cells) as used herein refers to T cells and B cells that have generated an antigen receptor (TCR for T cells, BCR for B cells) of a particular specificity, but have never encountered the antigen.
  • TCR antigen receptor
  • Priming involves several steps: antigen uptake, processing, and cell surface expression bound to class II MHC molecules by an antigen presenting cell, recirculation and antigen-specific trapping of helper T cell precursors in lymphoid tissue, and T cell proliferation and differentiation.
  • Helper T cells express CD4, but not all CD4 T cells are helper cells. Id. The signals required for clonal expansion of helper T cells differ from those required by other CD4 T cells.
  • the critical antigen-presenting cell for helper T cell priming appears to be a macrophage; and the critical second signal for helper T cell growth is the macrophage product interleukin 1 (IL-1). Id. If the primed T cells and/or B cells receive a second, co-stimulatory signal, they become activated T cells or B cells. [000495]
  • the term “progression” as used herein refers to the course of a disease as it becomes worse or spreads in the body.
  • the term “(human) peripheral blood mononuclear cells” or “hPBMCs” refers to lymphocytes (B cells, T cells and NK cells), monocytes and dendritic cells.
  • recombinant refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
  • the term “recombinant” and grammatical variations thereof are meant to relate to or denote an organism, protein, or genetic material formed by or using recombined DNA comprising DNA pieces from different sources or from different parts of the same source.
  • the term “recombinant DNA” means a DNA molecule formed through recombination methods to splice fragments of DNA from a different source or from different parts of the same source.
  • two or more different sources of DNA are cleaved using restriction enzymes and joined together using ligases.
  • the term “recombinant protein” or “recombinant domains” and grammatical variations thereof means a protein molecule formed through recombination methods originating from spliced fragments of DNA from a different source or from different parts of the same source.
  • the term “recombinant microbe” or “recombinant bacteria” and grammatical variations thereof mean a microbe/bacteria that comprises one or more recombinant DNA/protein molecules.
  • the term “reduce” and its various grammatical forms as used herein refers to a diminution, a decrease, an attenuation or abatement of a degree, intensity, extent, size, amount, density or number.
  • release and its various grammatical forms, refers to dissolution of an active drug component and diffusion of the dissolved or solubilized species by a combination of the following processes: (1) hydration of a matrix, (2) diffusion of a solution into the matrix; (3) dissolution of the drug; and (4) diffusion of the dissolved drug out of the matrix.
  • RAAS Renin-Angiotensin-aldosterone System
  • RAS renin-angiotensin system
  • renin angiotensin II
  • aldosterone angiotensin II
  • ACE Angiotensin converting enzyme
  • Ang II elicits its effects by activating two receptors: type 1 angiotensin II (AT1) receptor and type 2 angiotensin II (AT2) receptor [Ingraham, NE, et al. Eur. Respir. J. (2020); DOI: 10.1183/13993003.00912-2020, citing Balakumar, P. & Jagadeesh, G. Cell Signal (2014) 26: 2147-60].
  • AT1 receptor type 1 angiotensin II receptor
  • AT2 angiotensin II receptor
  • AT2 receptor stimulation causes vasodilation, decreased platelet aggregation, and the promotion of insulin action.
  • the expression of AT2 receptor is low in healthy adults [Id., citing Dandona, P. et al. J. Hum. Hypertens. (2007) 21: 20-27].
  • Ang II's effects in adults are modulated and balanced indirectly by angiotensin II converting enzyme (ACE2), which converts Ang II into lung-protective Angiotensin-(1–7) (Ang- [1–7]), similar to effects seen from AT2 receptor stimulation [Id., citing Ghazi, L. & Grawz, P. F1000Research 2017; 6: F1000, Faculty Rev-1297. doi:10.12688/f1000research.9692.1; Warner, FJ et al. Cell Mol. Life Sci. (2004) 61: 2704-13].
  • the term “restore” and its various grammatical forms as used herein refers to bringing back to a former or normal condition, to recover or renew.
  • lymphoid tissues refers to sites where lymphocytes interact with each other and nonlymphoid cells to generate immune responses to antigens. These include the spleen, lymph nodes, and mucosa-associated lymphoid tissues (MALT).
  • Sequelae and its various grammatical forms as used herein means a pathological condition resulting from a prior disease, injury or attack.
  • shock as used herein refers to a critical condition brought on by a sudden drop in blood flow through the body, where the circulatory system fails to maintain adequate blood flow, sharply curtailing the delivery of oxygen and nutrients to vital organs.
  • the term “sign” as used herein refers to a healthcare provider’s evidence of disease.
  • the term “splice variant” as used herein refers to a recombinant DNA molecule derived from cutting and resealing of DNA from different sources that can result in an altered protein-coding sequence from the wild-type sequence.
  • the term “stability” and its other grammatical forms as used herein with respect to a pharmaceutical product refers to the capability of a particular formulation to remain within its physical, chemical, microbiological, therapeutic and toxicological specifications. Stabilizers may be used to help an active pharmaceutical ingredient (API) maintain the desirable properties of the product until it is consumed by the patient.
  • API active pharmaceutical ingredient
  • the term “susceptible subject” as used herein refers to an individual vulnerable to developing infection when their body is invaded by an infectious agent.
  • individuals vulnerable to developing a serious lung infection include, without limitation, the very young, the elderly, those who are ill; those who are receiving immunosuppressants; those with long term health conditions; those that are obese; and those who are physically weak, e.g., due to malnutrition or dehydration.
  • sustained release also referred to as "extended release” is used herein in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant blood levels of a drug over an extended time period.
  • sustained release biodegradable polymers include polyesters, polyester polyethylene glycol copolymers, polyamino-derived biopolymers, polyanhydrides, hydrogels, polyorthoesters, polyphosphazenes, SAIB, photopolymerizable biopolymers, protein polymers, collagen, polysaccharides, chitosans, and alginates.
  • symptom refers to a patient’s subjective evidence of disease.
  • T cell exhaustion refers to a state of T cell dysfunction that arises during many chronic infections and cancer. It is defined by poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Modulating pathways overexpressed in exhaustion — for example, by targeting programmed cell death protein 1 (PD1) and cytotoxic T lymphocyte antigen 4 (CTLA4) — can reverse this dysfunctional state and reinvigorate immune responses [Wherry EJ and Kurachi, M. Nature (2015) 15: 486-99, citing Wherry EJ. Nat. Immunol. (2011) 131:492–499; Schietinger A, Greenberg PD. Trends Immunol.
  • PD1 programmed cell death protein 1
  • CTL4 cytotoxic T lymphocyte antigen 4
  • inhibitory receptors include the inhibitory pathways mediated by PD1 in response to binding of PD1 ligand 1 (PDL1) and/or PDL2. [Id., citing Okazaki T, et al., Nature Immunol. (2013) 14:1212–1218, Odorizzi PM, Wherry EJ. J.
  • Exhausted T cells can co- express PD1 together with lymphocyte activation gene 3 protein (LAG3), 2B4 (also known as CD244), CD160, T cell immunoglobulin domain and mucin domain-containing protein 3 (TIM3; also known as HAVCR2), CTLA4 and many other inhibitory receptors [Id., citing Blackburn SD, et al. Nat. Immunol. (2009) 10:29–37].
  • LAG3 lymphocyte activation gene 3 protein
  • 2B4 also known as CD244
  • CD160 T cell immunoglobulin domain and mucin domain-containing protein 3
  • CTLA4 tumor necrosis factor 4
  • inhibitory receptors such as PD1 might regulate T cell function in several ways [Id., citing Schietinger A, Greenberg PD. Trends Immunol. (2014) 35:51–60; Odorizzi PM, Wherry EJ. J. Immunol.
  • ectodomain competition which refers to inhibitory receptors sequestering target receptors or ligands and/or preventing the optimal formation of microclusters and lipid rafts (for example, CTLA4);
  • intracellular mediators which can cause local and transient intracellular attenuation of positive signals from activating receptors such as the TCR and co-stimulatory receptors [Id., citing Parry RV, et al. Molec. Cell. Biol. (2005) 25:9543–9553; Yokosuka T, et al. J. Exp. Med. (2012) 209:1201–1217; Clayton KL, et al. J.
  • Co-stimulatory receptors also are involved in T cell exhaustion [Id., citing Odorizzi PM, Wherry EJ. J. Immunol. (2012) 188:2957–2965]. For example, desensitization of co-stimulatory pathway signaling through the loss of adaptor molecules can serve as a mechanism of T cell dysfunction during chronic infection.
  • TNFR tumor necrosis factor receptor
  • TRAF1 tumor necrosis factor receptor-associated factor 1
  • Adoptive transfer of CD8+ T cells expressing TRAF1 enhanced control of chronic LCMV infection compared with transfer of TRAF1- deficient CD8+ T cells, which indicates a crucial role for TRAF1-dependent co-stimulatory pathways in this setting [Id., citing Wang C, et al. J. Exp. Med. (2012) 209:77–91].
  • 4-1BB also known as CD137 and TNFRSF9
  • 4-1BB is a TNFR family member and positive co-stimulatory molecule that is expressed on activated T cells.
  • Combining PD1 blockade and treatment with an agonistic antibody to 4-1BB dramatically improved exhausted T cell function and viral control [Id, citing Vezys V, et al. J. Immunol. (2011) 187:1634–1642].
  • Soluble molecules are a second class of signals that regulate T cell exhaustion; these include immunosuppressive cytokines such as IL-10 and transforming growth factor- ⁇ (TGF ⁇ ) and inflammatory cytokines, such as type I interferons (IFNs) and IL-6.
  • immunosuppressive cytokines such as IL-10 and transforming growth factor- ⁇ (TGF ⁇ )
  • inflammatory cytokines such as type I interferons (IFNs) and IL-6.
  • IFNs type I interferons
  • IL-6 type I interferons
  • Eomesodermin facilitates the cooperative maintenance of the pool of antiviral CD8+ T cells during chronic viral infection.
  • T-bet is reduced in virus-specific CD8+ T cells; this reduction correlates with T cell dysfunction.
  • Eomes mRNA expression is up-regulated in exhausted CD8+ T cells during chronic infection.
  • T follicular helper (Tfh) cells refers to a distinct subset of CD4+ T lymphocytes, specialized in B cell help and in regulation of antibody responses. They develop within secondary lymphoid organs (SLO) and can be identified based on their unique surface phenotype, cytokine secretion profile, and signature transcription factor. They support B cells to produce high-affinity antibodies toward antigens, in order to develop a robust humoral immune response and are crucial for the generation of B cell memory. They are essential for infectious disease control and optimal antibody responses after vaccination.
  • SLO secondary lymphoid organs
  • Th1 cells refers to a lineage of CD4+ effector T cells that promotes cell-mediated immune responses and is required for host defense against intracellular viral and bacterial pathogens. Th1 cells secrete IFN-gamma, IL-2, IL-10, and TNF-alpha/beta.
  • IL-12 and IFN- ⁇ make naive CD4+ T cells highly express T-bet and STAT4 and differentiate to Th1 cells.
  • Th2 cells refers to a lineage of CD4+ effector T cells that secrete IL-4, IL-5, IL-9, IL-13, and IL-17E/IL-25. These cells are required for humoral or antibody-mediated immunity and play an important role in coordinating the immune response to large extracellular pathogens.
  • IL-4 make naive CD4+ T cells highly express STAT6 and GATA3 and differentiate to Th2 cells.
  • Th17 cells refers to a CD4+ T-cell subset characterized by production of interleukin-17 (IL-17).
  • IL-17 is a highly inflammatory cytokine with robust effects on stromal cells in many tissues, resulting in production of inflammatory cytokines and recruitment of leukocytes, especially neutrophils, thus creating a link between innate and adaptive immunity.
  • Th17 cell development is ROR ⁇ t.
  • thrombosis refers to the formation of a blood clot (thrombus) within a blood vessel, which prevents blood from flowing normally through the circulatory system.
  • a blood clot thrombus
  • endothelial infection with influenza virus has been shown to increase the adhesion of human platelets to primary human lung microvascular endothelial cells via fibronectin, contributing to mortality from acute lung injury.
  • a blood clot that forms in the veins (a venous thromboembolism) can cause deep vein thrombosis and pulmonary embolisms.
  • Deep vein thrombosis occurs when a blood clot forms in a major vein, usually in the leg, which stops blood from flowing easily through the vein, which can lead to swelling, discoloration and pain.
  • Patients with DVT are at risk for developing post-thrombotic syndrome (PTS), which can involve chronic leg swelling, calf pain calf heaviness/fatigue, skin discoloration and/or venous ulcers.
  • PTS post-thrombotic syndrome
  • PE pulmonary embolism
  • PE is a blood clot that has traveled to the lungs. It often starts as a DVT where a piece of the clot breaks off and is carried to the lungs.
  • TRM tissue-resident memory T cell
  • TLR tumor necrosis factor-related apoptosis-inducing ligand, a member of the TNF cytokine family expressed on the cell surface of some cells, e.g., NK cells, that induces cell death in target cells by ligation of the “death” receptors DR4 and DR5.
  • TAM21 refers to tripartite motif-containing 21, a cytosolic Fc receptor and E3 ligase that is activated by IgG and can ubiquitinate viral proteins after an antibody coated virus enters the cytoplasm.
  • TAM25 refers to an E3 ubiquitin ligase involved in signaling by RIG-1 and MDA-5 for the activation of MAVs.
  • variants are used herein to refer to nucleotide or polypeptide sequences with substantial identity to a reference nucleotide or polypeptide sequence.
  • sequences may be the result of changes, either naturally or by design, in sequence or structure. Natural changes may arise during the course of normal replication or duplication in nature of the particular nucleic acid sequence. Designed changes may be specifically designed and introduced into the sequence for specific purposes. Such specific changes may be made in vitro using a variety of mutagenesis techniques. Such sequence variants generated specifically may be referred to as “mutants” or “derivatives” of the original sequence. A skilled artisan likewise can produce polypeptide variants having single or multiple amino acid substitutions, deletions, additions or replacements, but biologically equivalent to the wild type sequence.
  • variants may include inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non- conservative amino acids; (b) variants in which one or more amino acids are added; (c) variants in which at least one amino acid includes a substituent group; (d) variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at conserved or non-conserved positions; and (d) variants in which a target protein is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the target protein, for example, an epitope for an antibody.
  • a target protein is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the target protein, for example, an epitope for an antibody.
  • vascular permeability means the net amount of a solute, typically a macromolecule, that has crossed a vascular bed and accumulated in the interstitium in response to a vascular permeabilizing agent or at a site of pathological angiogenesis.
  • the term “viral load” as used herein refers to a measurement of the amount of a virus in an organism, typically in the bloodstream, usually stated in virus particles per milliliter.
  • wild-type refers to the most common phenotype of an organism, strain, gene, protein, nucleic acid, or characteristic as it occurs in nature.
  • wild-type and “naturally occurring” are used interchangeably.
  • WNT proteins refers to a group of secreted lipid- modified signaling proteins that activate various pathways in different types of cells. These pathways can be classified as canonical and non-canonical Wnt pathways [Qu, F. et al.
  • Wnt1 and WNT3A Some members of the Wnt family, such as Wnt1 and WNT3A, interact with Frizzled (FZD) receptors and their co- receptors, low-density lipoprotein receptor-related receptor 5/6 (LRP5/6), activating Dishevelled (DSH) family proteins, and leading to a change in the amount of nuclear ⁇ -catenin [Id., cigin Komiya, Y., Habas, R.
  • Frizzled Frizzled receptors
  • LRP5/6 low-density lipoprotein receptor-related receptor 5/6
  • DSH Dishevelled
  • DSH protects ⁇ -catenin from proteolytic degradation triggered by another complex comprising axin, glycogen synthase kinase-3 and the adenomatous polyposis coli protein.
  • the blockade of ⁇ -catenin destruction raises the levels of cytoplasmic ⁇ -catenin, thus increasing the likelihood of some ⁇ -catenin translocating to the nucleus.
  • T-cell factor TNF
  • lymphoid enhancer-binding factor transcription factors promotes specific gene expression [ id., citing Macdonald, BT, et al. Cell (2007) 131: 1204.e1-1204.e2].
  • the canonical and non-canonical pathways differ in terms of the specific ligands that activate each pathway, and whether or not ⁇ -catenin is activated.
  • the canonical pathway employs ⁇ -catenin to transmit signals, while the non- canonical pathway utilizes calcium/calmodulin-dependent kinase II (CaMKII) or Rho to exert its effects.
  • Wnt4, WNT5A, and Wnt11 activate the planar cell polarity (PCP) and the Wnt/Ca2+/CaMKII pathways.
  • WNT3A has been regarded as an activator of the canonical Wnt signaling pathway.
  • AHACs primary adult human articular chondrocytes
  • WNT3A resulted in the activation of the non-canonical Wnt/Ca2+/CaMKII pathway [Id., citing Nalesso, G. et al., J. Cell Biol. (2011) 193: 551-54].
  • the PCP pathway is involved in the regulation of cytoskeletal structure, while the Wnt/Ca2+/CaMKII pathway regulates intracellular Ca2+ levels.
  • Ligand binding causes an increase in intracellular Ca2+, which in turn activates CaMKII.
  • CaMKII activates transforming growth factor (TGF)- ⁇ -activated kinase and Nemo-like kinase, which can interfere with TCF/ ⁇ -catenin signaling in the canonical pathway [Id., citing Semenov, MV et al. Cell (2007) 131: 1378.e1-1378.e2].
  • TGF transforming growth factor
  • the described invention provides a method for reducing damaging effects of a severe virus infection in a subject comprising administering a pharmaceutical composition comprising a vehicle/carrier and a recombinant bifunctional fusion protein comprising a recombinant biologically active immunomodulatory component operatively linked to a recombinant biologically active antiviral component.
  • the recombinant immunomodulatory component comprises a recombinant human trefoil factor 1, a sequence variant or a splice- variant thereof.
  • the recombinant immunomodulatory component comprises a recombinant human trefoil factor 2, a sequence variant, or a splice- variant thereof. According to some embodiments, the recombinant immunomodulatory component comprises a recombinant human trefoil factor 2, a sequence variant or a splice- variant thereof.
  • the recombinant fusion protein comprises a recombinant biologically active human TFF1 molecule, fragment or variant thereof, a recombinant human TFF2 molecule, fragment or variant thereof, or a recombinant human TFF3 molecule, fragment or variant thereof joined by its C-terminal end to a linker sequence, which is joined to an N-terminal end of a recombinant biologically active interferon molecule, fragment or variant.
  • the linker can be from 6-20 amino acids in length, inclusive.
  • the recombinant interferon component is pegylated.
  • the C-terminal end of the recombinant interferon molecule, fragment or variant sequence is further joined to a recombinant Fc derived antibody domain comprising a constant region of a human immunoglobulin heavy chain.
  • the Fc component can: increase stability and aggregation resistance of the recombinant fusion protein; . extend serum half-life of the recombinant fusion protein; enhance Fc-mediated effector functions of the recombinant fusion protein; or all of the above.
  • activity of the recombinant TFF1, TFF2 or TFF3 component of the recombinant fusion protein may be assayed in an animal model of inflammation.
  • activity of the IFN component of the recombinant fusion protein may be assayed by showing inhibition of virus replication, activation of APCs, notably DCs [See, e.g., Kolumam, et al. J. Exp. Med. (2005) 202 (5): 637-50, citing Lucas, T. et al. J. Immunol. (1998) 161: 1947-56; Biron, CA Immunity (2001) 14: 661-64; Le Bon, A. et al. Nat.
  • the recombinant interferon molecule is a recombinant type I interferon or biologically active fragment thereof selected from IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ and IFN- ⁇ .
  • the recombinant IFN is a human interferon.
  • the recombinant interferon molecule is a recombinant human IFN- ⁇ of SEQ ID NO: 57 (NCBI Ref NM_000605.4)._According to some embodiments, the recombinant interferon molecule is a recombinant human IFN- ⁇ of SEQ ID NO: 54 (NCBI Ref NM_020124.3). According to some embodiments, the recombinant interferon is a recombinant human IFN- ⁇ of SEQ ID NO: 55 (NCBI Ref. NM 0021773).
  • the recombinant interferon is a recombinant human interferon- ⁇ as disclosed by Chon, TW and Bixler, S, J. Interferon & Cytokine Res. (2010) 30 (7): 477-85: [000535] MAFVLSLLMALVLVSYGPGGSLGCDLSQNHVLVGRKNLRLLDEMRR LSPHFCLQDRKDFALPQEMVEGGQLQEAQAISVLHEMLQQSFNLFHTEHSSAAWDT TLLEPCRTGLHQQLDNLDACLGQVMGEEDSALGRTGPTLALKRYFQGIHVLKEKGY SDCAWETVRLEIMRSFSSLISLQERLRMMDGDLSSP (SEQ ID NO: 34) [000536] According to some embodiments, the recombinant Fc-derived antibody domain is a recombinant human protein of SEQ ID NO: 56 (NCBI Ref: 4CDH_A).
  • the amino acid sequence of the recombinant hTFF1-IFN ⁇ -IgG1 fusion protein is SEQ ID NO: 66: RRKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK
  • Green Homo sapiens TFF1 ORF w/o Leader
  • Blue linker sequence
  • Gold homo sapiens interferon kappa w/o Leader
  • Black Homo sapiens human IgG1 Fc domain.
  • the amino acid sequence of the recombinant hTFF2-IFN ⁇ -IgG1 Fc fusion protein is SEQ ID NO: 35: THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
  • Green Homo sapiens TFF2 ORF w/o Leader
  • Blue linker sequence
  • Gold homo sapiens interferon kappa w/o Leader
  • Black Homo sapiens human IgG1 Fc domain.
  • the amino acid sequence of the recombinant hTFF3-IFN ⁇ -IgG1 fusion protein is SEQ ID NO: 67: RKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK
  • Green Homo sapiens TFF3 ORF w/o Leader
  • Blue linker sequence
  • Gold homo sapiens interferon kappa w/o Leader
  • Black Homo sapiens human IgG1 Fc domain.
  • the sequences are codon-optimized to improve gene expression.
  • the recombinant fusion protein is produced in CHO cells.
  • the severe virus infection is a severe respiratory virus infection.
  • the respiratory virus is a cytomegalovirus.
  • the respiratory virus is Hanta virus.
  • the respiratory virus is an influenza virus.
  • the respiratory virus is a MERS virus.
  • the respiratory virus is a respiratory syncytial virus.
  • the respiratory virus is a SARS-CoV virus.
  • the respiratory virus is Zika virus.
  • the respiratory virus is a West Nile virus. According to some embodiments, the respiratory virus is a dengue virus. According to some embodiments, the respiratory virus is a Japanese encephalitis virus. According to some embodiments, the respiratory virus is a tick-borne encephalitis virus. According to some embodiments, the respiratory virus is a yellow fever virus. According to some embodiments, the respiratory virus is a rhinovirus. According to some embodiments, the respiratory virus is an adenovirus. According to some embodiments, the respiratory virus is a herpes virus. According to some embodiments, the respiratory virus is an Epstein Barr virus. According to some embodiments, the respiratory virus is a measles virus. According to some embodiments, the respiratory virus is a mumps virus.
  • the respiratory virus is a rotavirus.
  • the respiratory virus is a cocksackie virus.
  • the respiratory virus is a norovirus.
  • the respiratory virus is an encephalomyocarditis virus (EMCV).
  • EMCV encephalomyocarditis virus
  • the composition of the described invention is cytoprotective, meaning it protects cells from damaging effects of a severe virus infection.
  • the composition is cytoprotective to one or more of gastrointestinal tissue, lung tissue, heart tissue, kidney tissue, brain tissue or vascular tissue
  • the composition of the described invention is cytoprotective to mucosal tissue.
  • the mucosal tissue is lung tissue.
  • the mucosal tissue is gastrointestinal tissue.
  • the damaging effects of a severe virus infection comprise primary viral pneumonia.
  • the damaging effects of a severe virus infection comprise superimposed bacterial pneumonia.
  • the damaging effects of a severe virus infection comprise disruption or injury to alveolar epithelium, endothelium or both.
  • the damaging effects of a severe virus infection comprise acute lung injury (ALI).
  • the damaging effects of a severe virus infection comprise acute respiratory distress syndrome (ARDS).
  • the damaging effects of a severe virus infection comprise symptoms of shock, including low blood pressure, lightheadedness, shortness of breath, and rash.
  • the damaging effects of a severe virus infection comprise excessive complement activation.
  • the effects comprise a pathological increase in vascular permeability.
  • the damaging effects of a severe virus infection comprise thrombotic complications.
  • the thrombotic complications include one or more of formation of pulmonary microthrombi, acute pulmonary embolism, deep-vein thrombosis, ischemic stroke, myocardial infarction, or systemic arterial embolism.
  • the damaging effects of a severe virus infection comprise kidney damage.
  • the damaging effects of a severe virus infection comprise elevated concentrations of one or more inflammatory mediator in plasma (hypercytokinemia), compared to a normal healthy subject.
  • the inflammatory mediator is one or more of complement, prostaglandin D2, vasoactive intestinal peptide (VIP), interleukin-1-beta (IL-1 ⁇ ), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 (IL-12), IL-17, or tumor necrosis factor-alpha (TNF- ⁇ ).
  • VIP vasoactive intestinal peptide
  • IL-1 ⁇ interleukin-1-beta
  • IL-6 interleukin-6
  • IL-8 interleukin-8
  • IL-12 interleukin-12
  • IL-17 tumor necrosis factor-alpha
  • TNF- ⁇ tumor necrosis factor-alpha
  • the composition stimulates repair of a mucosal injury.
  • the repair comprises epithelial proliferation.
  • the repair restores an epithelial barrier, an endothelial barrier or both.
  • the composition modulates an immune response.
  • the immune response comprises recruitment of innate and adaptive immune cells.
  • the innate immune cells comprise macrophages, dendritic cells (DCs), innate lymphoid cells (ILCs), and natural killer cells (NKs).
  • the adaptive immune cells include ⁇ T cells, ⁇ T cells, and B cells.
  • the damaging effects comprise one or more of endothelial activation, a loss of barrier function and consequent microvascular leak.
  • endothelial activation refers to changes to the endothelium under the stimulation of agents that allow it to participate in the inflammatory response. [Hunt, B.J., K.M.
  • the prothrombotic effects of endothelial cell activation include loss of the surface anticoagulant molecules thrombomodulin and heparan sulphate; reduced fibrinolytic potential due to enhanced plasminogen activator inhibitor type 1 release; loss of the platelet antiaggregatory effects of ecto-ADPases and prostacyclin; and production of platelet activating factor, nitric oxide, and expression of tissue factor.
  • Cytokines are synthesized, including interleukin [Id., citing Pober, JS, et al.
  • endothelial cell activation Two stages of endothelial cell activation exist [Id., citing Bach, FH et al. Nature Medicine (1995) 1: 869-73]; the first, endothelial cell stimulation or endothelial cell activation type I, does not require de novo protein synthesis or gene upregulation and occurs rapidly. Effects include the retraction of endothelial cells, expression of P selectin, and release of von Willebrand factor. The second response, endothelial cell activation type II, requires time for the stimulating agent to cause an effect via gene transcription and protein synthesis.
  • the genes involved are those for adhesion molecules, cytokines, and tissue factor.
  • Endothelial cell activation is a graded rather than an all or nothing response—for example, changes in endothelial cell integrity range from simple increases in local permeability to major endothelial cell contraction, exposing large areas of subendothelium. Activation may occur locally, as in transplant rejection, [Id., citing Bach, FH et al. Nature Medicine (1995) 1: 869-73] or systemically, as in septicemia and the systemic inflammatory response.
  • the described invention relates to all routes of administration including intramuscular, subcutaneous, sublingual, intravenous, intraperitoneal, intranasal, intratracheal, topical, intradermal, intramucosal, intracavernous, intrarectal, into a sinus, gastrointestinal, intraductal, intrathecal, intraventricular, intrapulmonary, into an abscess, intraarticular, subpericardial, into an axilla, into the pleural space, intradermal, intrabuccal, transmucosal, transdermal, via inhalation, via insufflation, via nebulizer, and via subcutaneous injection.
  • the administering is parenterally.
  • the administering occurs by inhalation.
  • the administering is by insufflation.
  • Respiratory Delivery The respiratory tract originates from the nose and terminates deep in the lung at the alveolar sac. The upper respiratory tract is composed of the nose, pharynx and nasal cavity, whereas the lower tract is composed of the larynx, bronchi and alveoli. On the basis of functional zones, the respiratory system can be divided into two regions, the conducting airway and the respiratory region. [Thakur, AK et al. Ch. 22 in Targeting Chronic Inflammatory Lung Diseases Using Advanced Drug Delivery Systems (2020) pp.475- 491].
  • the conducting airway includes the nasal cavity, sinuses, nasopharynx, oropharynx, larynx, trachea, bronchi and bronchioles.
  • the main physiological functions of this airway are filtering and conditioning of the air.
  • the respiratory airway includes the bronchioles, terminal bronchioles, alveolar ducts and alveolar sacs. Around 300 million alveoli are present in the lungs.
  • the entire pulmonary region is lined by a continuous layer of pulmonary epithelial cells, which performs such functions as acting as a barrier and protection of the region by secreting mucus, surfactant proteins and antimicrobial peptides, repairing and regenerating epithelial cells; and modulating the response of smooth muscle and inflammatory cells.
  • Goblet cells are the mucus-secreting cells that secrete mucus glycoproteins responsible for trapping and removing particles.
  • Some approaches to enhance the residence time of a delivery system in the lungs include molecular engineering, and use of penetration enhancers, enzyme inhibitors, and bioadhesive polymers.
  • Particle deposition in the lungs is governed by size, shape and density of the particles.
  • the principle mechanisms guiding inhaled particle deposition in the respiratory airway include sedimentation due to gravity, Brownian diffusion, and impaction by inertial force (meaning the condition of being pressed closely together and firmly fixed). Larger particles (>10 ⁇ m) are retained in the oropharyngeal region and larynx due to impaction.
  • Particles between 2 ⁇ m and 10 ⁇ m normally are deposited in the tracheobronchial region. Particles of 0.5 ⁇ m -2 ⁇ m normally are deposited in the alveoli and small conducting airways due to gravitational sedimentation. Particles ⁇ 0.5 ⁇ m generally are not deposited and are expelled in exhalation [Id., citing Moreno-Sastre, M. et al. J. Antimicrob. Chemother. (2015) 70: 2945-55; Yu, CP and Taulbee, DB. Inhaled Part. (1975) 4 (Pt.1): 35-47].
  • Impaction is most effective when air and particle velocities are higher than in the peripheral region of the lungs.
  • particle deposition is mainly guided by diffusion and sedimentation due to smaller velocities and hence longer residence times [Id., citing Hoffmann, W. J. Aerosol Sci. (2011) 42: 693- 724].
  • Numerous natural polymers, synthetic polymers, and copolymers have been studied as carriers for pulmonary drug delivery [Vinjamuri, BP et al.
  • Chitosan which is obtained by partial deactylation of chitin, is a copolymer linked by ⁇ -1,4 of D- glucosamine and N-acetylglucosamine carrying a cationic charge, thereby adhering to negatively charged surfaces and chelating metal ions; it is biocompatible, biodegradable, mucoadhesive, antibacterial and nontoxic, although poorly water soluble [Id., citing Yamamoto, H. et al. J. Controlled Release (2005) 102: 373-81].
  • Commonly used synthetic polymers include polylactic acid (PLA), polyethylene glycol (PEG), polyvinyl alcohol, and acrylyic acid derivatives.
  • PLGA Polylactic-co-glycolic acid
  • PGA polyglycolic acid
  • PLGA particles degrade by bulk erosion, undergoing hydrolysis to PLA and PGA.
  • PLGA and gelatin-based nanoparticles have been used for pulmonary protein and DNA delivery [Id., citing Menon, JU et al, Acta Biomater. (2014) 10: 2643-52].
  • the carrier is a controlled release carrier.
  • controlled release is intended to refer to any drug-containing formulation in which the manner and profile of drug release from the formulation are controlled.
  • the controlled release of the pharmaceutical formulation is mediated by changes in temperature.
  • the controlled release of the pharmaceutical formulation is mediated by changes in pH.
  • the carrier is a delayed release carrier.
  • the delayed release carrier comprises a biodegradable polymer.
  • the biodegradable polymer is a synthetic polymer.
  • the biodegradable polymer is a naturally occurring polymer.
  • the carrier is a sustained release carrier.
  • the sustained-release carrier comprises a biodegradable polymer.
  • the biodegradable polymer is a synthetic polymer.
  • the biodegradable polymer is a naturally occurring polymer.
  • the carrier is a short-term release carrier.
  • short-term release means that the carrier is constructed and arranged to deliver therapeutic levels of the biologically active fusion protein for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours.
  • the short term release carrier delivers therapeutic levels of the biologically active fusion protein for about 1, 2, 3, or 4 days.
  • the carrier is a long-term release carrier.
  • the term “long-term” release means that the carrier is constructed and arranged to deliver therapeutic levels of the recombinant fusion protein for at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days.
  • the long-term-release carrier comprises a biodegradable polymer.
  • the biodegradable polymer is a synthetic polymer.
  • the recombinant fusion protein of the present disclosure can be covalently attached to polyethylene glycol (PEG) polymer chains.
  • the recombinant fusion protein of the present disclosure is stapled with hydrocarbons to generate hydrocarbon-stapled fusion proteins that are capable of forming stable alpha-helical structure (Schafmeister, C. et al., J. Am. Chem. Soc., 2000, 122, 5891- 5892, incorporated herein by reference in its entirety).
  • the carrier comprises particles.
  • the recombinant fusion protein of the present invention is encapsulated or entrapped into microspheres, nanocapsules, liposomes, or microemulsions, or comprises d-amino acids in order to increase stability, to lengthen delivery, or to alter activity of the recombinant fusion protein.
  • these techniques can lengthen the stability and release simultaneously by hours to days, or delay the uptake of the drug by nearby cells.
  • the composition is formulated as a solution.
  • a carbohydrate e.g., a polyol (e.g., mannitol, sorbitol, or xylitol) trehalose, or lactose may be used as an excipient to protect the protein from denaturation/degradation upon lyophilization, spray drying and reconstitution and to improve aerosol performance.
  • a polyol e.g., mannitol, sorbitol, or xylitol
  • lactose may be used as an excipient to protect the protein from denaturation/degradation upon lyophilization, spray drying and reconstitution and to improve aerosol performance.
  • compositions for use according to the described invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit can be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the pharmaceutical composition is packaged in an inhalation device, including, for example, but not limited to a nebulizer, a metered-dose inhaler (MDI), and a dry powder inhaler (DPI).
  • the pharmaceutical composition is a liquid for aerosolized delivery using a nebulizer.
  • the flow-rate of the pharmaceutical composition is at least 0.3 ml/min, and the pharmaceutical composition is delivered as 2 mm particles, with distribution into deepest alveoli.
  • the therapeutic amount of the pharmaceutical composition is administered via an inhalation device.
  • the inhalation device that can be used for administering the pharmaceutical composition include, but are not limited to, a nebulizer, a metered-dose inhaler (MDI), a dry powder inhaler (DPI), and a dry powder nebulizer.
  • the dry powder is produced by a spray drying process.
  • the dry powder is produced by micronization.
  • the dry powder comprises microparticles with Mass Median Aerodynamic Diameter (MMAD) of 1 to 5 microns.
  • MMAD Mass Median Aerodynamic Diameter
  • Particle characteristics play a role in performance of polymeric particulate systems, which is dependent on the amount of drug loaded into the particle. For example, if the load percent is low, drug diffusion from a particulate delivery system comprising large particle size may be slow compared to that from small particles or a colloidal dispersion, because in the latter, the drug has a shorter path to cover for diffusion to take place. If the load percent is high, however, then the larger particles have a faster diffusion rate than small particles due to the amount of drug loaded into the microparticle available space.
  • MMAD Mass Median Aerodynamic Diameter
  • Nanoparticles are solid particles ranging in size from 1 nm to 1000 nm Some potential advantages of nanoparticles for pulmonary delivery include sustained release, drug targeting, dose reduction, and improved patient compliance. Prolonged residence time of nanoparticles in pulmonary regions may be achieved through the use of mucoadhesive polymeric materials.
  • Microparticles are solid particles ranging in size from 1 ⁇ m to 1000 ⁇ m.
  • the pharmaceutical composition further comprises at least one additional therapeutic agent.
  • the additional therapeutic agent is selected from a supportive therapy, an immunomodulatory agent, an analgesic agent, an anti-inflammatory agent, an anti-infective agent, an anti-malarial agent, an anti-viral agent or an anti-fibrotic agent.
  • the supportive therapy is therapeutic apheresis comprising a virion removing step.
  • the therapeutic apheresis reduces viral load.
  • each of these agents can be used alone as a monotherapy or in combination with a second agent, e.g., an immunomodulatory agent with a second immunomodulatory agent, e.g., methotrexate; an immunomodulatory agent with an analgesic agent; an immunomodulatory agent with an anti-inflammatory agent; an immunomodulatory agent with an anti-infective agent; an immunomodulatory agent with an anti-malarial agent; an immunomodulatory agent with an anti-viral agent; an immunomodulatory agent with an antifibrotic agent; or an immunomodulatory agent with a supportive therapy.
  • a second agent e.g., an immunomodulatory agent with a second immunomodulatory agent, e.g., methotrexate; an immunomodulatory agent with an analgesic agent; an immunomodulatory agent with an anti-inflammatory agent; an immunomodulatory agent with an
  • cytokine synthesis drugs that inhibit cytokine synthesis
  • drugs that inhibit cytokine synthesis e.g., glucocorticoids, cyclosporine A, tacrolimus, myophnolate- helper lymphocyte (Th2)-selective inhibitors
  • humanized blocking antibodies to cytokines or their receptors soluble receptors that mop up secreted cytokines, low molecular weight receptor antagonists, and drugs that block the signal transduction pathways activated by cytokines.
  • Cyclosporine, tacrolimus and sirolimus are immunosuppressive medications that inhibit T cell activation through a series of calcium-dependent signal events involved in cytokine gene transcription.
  • Cyclosporine inhibits the activation of helper T cells. Tacrolimus also interferes with T cell receptor-dependent cell activation. CSA and tacrolimus inhibit IL- 2, IL-3, Il-4, IFN- ⁇ , GM-CSF, and TNF ⁇ production. Transcriptional factors nuclear factor of activated T cells (NF-AT), NF- ⁇ B, and PU-box are inhibited by tacrolimus. T cell receptor mediated apoptosis of lymphocytes and thymocytes is augmented by tacrolimus.
  • Sirolimus is a macrocyclic lactone produced by Streptomyces hygroscopicis that inhibits T lymphocyte activation and proliferation that occurs in response to antigenic and cytokine stimulation; it binds intracellularly to the immunophilin, FK binding protein-12, which becomes an immunosuppressive complex, which in turn binds to and inhibits activation of mammalian regulatory kinase (target of rapamycin, mTOR). This inhibition suppresses cytokine-driven T cell proliferation, inhibiting the progression from the G1 to S phases of the cell cycle. [Nelson, RP, and Ballow, M. J. Allergy Clin. Immunol. (2003) 111 (2): S720- 732].
  • the immunomodulatory agent is a recombinant interferon, e.g., one or more of IFN- ⁇ ; IFN- ⁇ -2b, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , or IFN- ⁇ .
  • the recombinant interferon is in a PEGylated form.
  • the immunomodulatory agent is a recombinant IL-2 receptor inhibitor (e.g., denileukin diftitox).
  • the immunomodulatory agent is a phosphodiesterase 4 (PDE4) inhibitor, e.g., cilomilast, a second generation PDE4 inhibitor with anti-inflammatory effects that target bronchoconstriction, mucus hypersecretion and airway remodeling.
  • PDE4 phosphodiesterase 4
  • the immunomodulatory agent is a hyperimmune globulin prepared from a donor with high titers of a desired antibody.
  • the immunomodulatory agent targets pro- inflammatory cytokines.
  • the immunomodulatory agent is a TNF ⁇ inhibitor/antagonist [e.g., etanercept; adalimumab; infliximab, certolizumab pegol, golimumab].
  • the immunomodulatory agent is an IL-1 ⁇ inhibitor [e.g., rilonacept; canakinumab; Anakinra].
  • the immunomodulatory agent is chimeric IL- 1Ra. This molecule is a fusion of the N-terminal peptide of IL-1 ⁇ and IL-1Ra, resulting in inactive IL-1Ra. Because the IL-1 ⁇ N-terminal peptide contains several protease sites clustered around the caspase-1 site, local proteases at sites of inflammation can cleave chimeric IL-1Ra and activate IL-1Ra. [Rider, P. et al. J. Immunol.
  • the immunomodulatory agent is an IL-6 inhibitor [e.g., tocilizumab, siltuximab, sarilumab, olokizumab, or sirukumab].
  • the immunomodulatory agent is an IL-12/ IL- 23 inhibitor (e.g., ustekinumab, briakinumab), or an IL23 inhibitor (e.g., guselkumab, tildrakizumab).
  • the immunomodulatory agent targets cytokine signaling pathways, e.g., compounds targeting TLR-4 signaling, e.g., enamionone E121 (ethyl 4-(4’-chlorophenyl) amino-6 methyl-2-oxocytlohex-3-en-1-aote), an aniline enaminone); JODI 18b and 19, [Szollosi, DE et al. J. Pharmacy & Pharmacol.
  • cytokine signaling pathways e.g., compounds targeting TLR-4 signaling, e.g., enamionone E121 (ethyl 4-(4’-chlorophenyl) amino-6 methyl-2-oxocytlohex-3-en-1-aote), an aniline enaminone); JODI 18b and 19, [Szollosi, DE et al. J. Pharmacy & Pharmacol.
  • TAK-242 (resatorvid, Takeda), TLR-C34, a 2-acetamidopyranoside that inhibits TLR4 signaling [Olusayo, A., et al. J. Applied Toxicol (2019) 39(4). Doi: 10/1002/jat.3771]; C35 [Neal, MD. Et al. PLoS One (2013) 8 (6): e65779].
  • the immunomodulatory agent is a p38 MAPK inhibitor, e.g., SB203580, 4-(4’-Fluorophenyl)-2-(4’-methylsulfinylphenyl)-5- (4’-pyridyl)- imidazole, a pyridinyl imidazole inhibitor used to elucidate the roles of p38 mitogen-activated protein (MAP) kinase [Cuenda, A. et al. FEBS Lett.
  • MAP mitogen-activated protein
  • SB203580 inhibits also the phosphorylation and activation of protein kinase B (PKB, also known as Akt) [Lali, F.V. et al. J. Biol. Chem. (2000) 275 (10): 7395-402].
  • PPKB protein kinase B
  • SB239063 [trans-4-[4-(4-Fluorophenyl)- 5-(2-methoxy-4-pyrimidinyl)-1H-imidazol-1-yl]cyclohexanol; Barone. Et al J. Pharmacol. Exp.
  • the immunomodulatory agent targets Janus kinase signaling (e.g., tofacitinub, baricitinib, or upadacitinib).
  • the immunomodulatory agent targets cell adhesion molecules to reduce leukocyte recruitment, e.g., molecules that are ⁇ 4 integrin inhibitors [e.g., vedolizumab, natalizumab]. [Szollosi, DE et al. J. Pharmacy & Pharmacol. (2016) 70: 18-26]. [000587] According to some embodiments, the immunomodulatory agent is a recombinant anti-inflammatory cytokine, e.g., IL-4, IL-10, IL-11.
  • Immunomodulation also includes therapies that boost an individual’s defenses by providing physiologic or supraphysiologic doses of exogenous cytokines, e.g., to treat viral infections.
  • a parameter for measuring activation state of lymphocytes is cytokine release profile.
  • ELISPOT enzyme linked immunospot
  • ELISPOT is a technique that was developed for the detection of secreted proteins, such as cytokines and growth factors. It is performed using a PVDF or nitrocellulose membrane 96- well plate pre-coated with an antibody specific to the secreted protein. Cells are added to the plate and attach to the coated membrane. Cells are then stimulated and the secreted protein binds to the antibody.
  • a detection antibody is added that binds specifically to the bound protein.
  • the resulting antibody complex can be detected either through enzymatic action to produce a colored substrate or with fluorescent tags.
  • the membrane can be analyzed by manually counting the spots or with an automated reader designed for this purpose. Each secreting cell appears as a spot of color or fluorescence.
  • another parameter for measuring activation of lymphocytes is by quantifying cellular subset differentiation.
  • the differentiation of CD45+/CD3+ T-lymphocytes to CD45+/CD3+/CD4+ helper T-lymphocytes, CD45+/CD3+/ CD8+ cytotoxic T-lymphocytes, and CD45+/CD3+/CD25+ activated T- lymphocytes can be quantified by flow cytometry analysis.
  • the immunomodulatory agent is a corticosteroid.
  • the corticosteroid is selected from prednisone, dexamethasone, azathioprine, mycophenolate, and mycophenolate mofetil.
  • analgesic agent refers to an agent producing diminished sensation to pain without loss of consciousness.
  • the analgesic agent is selected from codeine, hydrocodone, oxycodone, methadone, hydromorphone, morphine, fentanyl, and combinations thereof.
  • An “anti-inflammatory agent” is a substance that reduces inflammation (redness, swelling, and pain) in the body by inhibiting inflammatory mediators in the body that cause inflammation.
  • the anti-inflammatory agent is selected from aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac nabumetone, naproxen, nintedanib, oxaprozin, pirfenidone, piroxicam, salsalate, sarilumab (Kevzara®) sulindac, tolmetin, and combinations thereof.
  • anti-viral agent as used herein means any of a group of chemical substances having the capacity to inhibit the replication of or to destroy viruses used chiefly in the treatment of viral diseases.
  • the antiviral agent inhibits viral entry, thereby decreasing viral load.
  • the anti-viral agent is selected from acyclovir, gancidovir, foscarnet; ribavirin; amantadine, azidodeoxythymidine /zidovudine), nevirapine, a tetrahydroimidazobenzodiazepinone (TIBO) compound; efavirenz; remdecivir, lopinavir/ritonavir, umifenovir, favipiravir, ivermectin, and delavirdine.
  • the agent that blocks viral entry is a peptide inhibitor.
  • the peptide inhibitor is a fusion peptide.
  • the S glycoprotein is a type 1 transmembrane glycoprotein common to all HCoVs.
  • the S proteins consist of two subunits S1 and S2.
  • the S1 subunit binds the cellular receptor through its receptor binding domain (RBD), followed by conformational changes in the S2 subunit, which allows the fusion peptide to insert into the host target cell membrane.
  • RBD receptor binding domain
  • the heptad repeat 1 (HR1) region in the SR2 unit forms a homotrimeric assembly, which exposes three highly conserved grooves on the surface that bind heptad repeat 2 (HR2).
  • This six-helix bundle (6-HB) core structure formed during the fusion process helps bring the viral and cellular membranes into close proximity for viral fusion and entry.
  • the HR region in the S2 subunit is conserved among various HCoVs and plays a pivotal role in HCoV infections by forming the 6-HB that mediates viral fusion.
  • the mode of interaction between HR1 and HR2 is conserved among CoVs such that certain residues in the HR1 helices interact with certain residues in the HR2 helices.
  • the fusion peptide is a synthetic peptide derived from HR1 and HR2 regions of SARS-CoV spike protein as described in Liu, S. et al. Lancet (2004) 363: 938-47, and as shown in Table 1 below. [000598] Table 1 [000599] As described in Liu, S. et al. Lancet (2004) 363: 938-47, the HR1 and H2 sequences tend to form a coiled-coil structure, and the amino acid sequences of peptides derived from the HR1 and HR2 regions of SARS-CoV spike protein are similar to those from the HIV-1 gp41 HR1 and HR2 regions.
  • a fusion peptide of Table 1 may be used as a lead in designing more potent anti-SARS-CoV peptides.
  • HR1P and HR2P spanning residues 998-1039 in the HR1 domain [ANKFNQALGAMQTGFTTTNEAFQKVQDAVNNNAQALSKLASE, SEQ ID NO: 24] and 1251-1286 in the HR2P domain [SLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKEL, SEQ ID NO: 25], respectively, from MERS-CoV can form a stable six-helix bundle fusion core structure.
  • HR2P can effectively inhibit MERS-CoV replication and its spike protein-mediated cell–cell fusion.
  • Introduction of hydrophilic residues into HR2P results in significant improvement of its stability, solubility and antiviral activity.
  • the HR2P analogues can be further developed into effective viral fusion inhibitors.
  • the viral entry inhibitor is peptide OC43- HR2P [LAEADANVVAQIKVLASNTADFGEQIADLANNFANAIL, SEQ ID NO: 26] as described in Xia, et al., Sci. Advisor (2019) 5: eaav4580.
  • the viral entry inhibitor is EK1 [SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL, SEQ ID NO: 27] as described in Xia, et al., Sci. Advisor (2019) 5: eaav4580.
  • EK1 is a modified derivative of OC43-HR2P.
  • cholesterol (Chol) and palmitic acid (Palm) were covalently attached to the C-terminus of EK1 sequence under the help of a flexible polyethylene glycol (PEG) spacer, and the corresponding lipopeptides EK1C and EK1P were constructed.
  • the viral entry inhibitor is EK1P [SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL, SEQ ID NO: 28] or a PEG-based spacer-containing derivative [PEG4-C (Palm) thereof as disclosed by Xia, S. et al. Cell Res.
  • the viral entry inhibitor is EK1C [SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL, SEQ ID NO: 29] or a PEG-based spacer-containing derivative [PEG4-C (Chol)] thereof as disclosed by Xia, S. et al. Cell Res. (2020) 30(4): 343-55. [000604] As described by Xia et al. Cell Res.
  • EK1C cholesteryl EK1 with multiple linkers were constructed, where the glycine/serine-based linker, i.e., GSG, or PEG- based spacer was employed between EK1 and the cholesterol moiety.
  • GSG glycine/serine-based linker
  • PEG- based spacer PEG- based spacer
  • the agent that blocks viral entry is a dipeptidyl peptidase 4 (DPP4) inhibitor.
  • Drugs in the DPP-4 inhibitor class that are approved for use to treat type 2 diabetes include sitagliptin, saxagliptin, linagliptin, and alogliptin.
  • the agent that blocks viral entry is an ACE2 inhibitor.
  • ACE2 angiotensin-converting enzyme 2
  • peptide inhibitors of the enzyme were developed by selection of constrained peptide libraries displayed on phage [McKee, DL et al., Pharmacol. Res. (2020) 157: 104859, citing Huang, L. et al. J. Biol. Chem.
  • DX600 the most potent inhibitor of which, termed DX600, with the amino acid sequence of Ac-GDYSHCSPLRYYPWWKCTYPDPEGGG-NH2 [SEQ ID NO: 31] had a Ki of 2.8 nm and an IC50 of 10.1 ⁇ M.
  • DX600 is a potent ACE2 inhibitor specific for only human ACE2 [Id., citing Pedersen, KB et al. Am J. Physiol. Regul. Integr. Comp. Physiol. (2011) 301(5): R1293-99; Ye, M. et aql. Hypertension (2012) 60(3): 730-40].
  • the phytochemical nicotianamine (CAS number: 34441 ⁇ 14-0), a metal chelator ubiquitously present in higher plants [Id., citing Takahashi, M. et al., Plant Cell (2003) 15(6): 1263-80], was identified in high concentrations in soybean, and was shown as a potent inhibitor of human ACE2 with an IC50 of 84 nM [Id., citing Takahashi, S. et al. Biomed Res. (2015) 36(3): 219- 224]. Because dietary phytochemicals as naturally occurring compounds display a wide safety profile and less pharmacological side effects [Id., citing Naujokat, C. & McKee, DL. Curr. Med. Chem.
  • nicotianamine constitutes a candidate drug for ACE2 inhibition and thus blockade of SARS-CoV-2 cell entry.
  • a clinical-grade soluble recombinant human ACE2 protein hrsACE2
  • hrsACE2 a clinical-grade soluble recombinant human ACE2 protein
  • the anti-viral agent is a protease inhibitor that inhibits a host cell protease to block viral entry.
  • the anti- viral agent is a serine protease TMPRSS2 inhibitor, e.g., camostat (FOY-305), [N,N- dimethylcarbamoylmethyl 4-(4-guanidinobenzoyloxy)-phenylacetate] methanesulfate and camostat mesilate (FoipanTM), alternatively termed camostat mesylate, (NI-03), (CAS number: 59721 ⁇ 28-7); or Nafamostat mesilate (BuipelTM), (6-amidino-2-naphthyl-4-guanidino benzoate-dimethanesulfonate) (FUT-175), (CAS number: 81525 ⁇ 10-2).
  • camostat e.g., camostat (FOY-305), [N,N- dimethylcarbamoylmethyl 4-(4-guanidinobenzoyloxy)-phenylacetate] methanesulfate and camostat mes
  • SARS-CoV can use the endosomal cysteine proteases cathepsin B and L (CatB/L) [Id., citing Simmons et al., 2005) and the transmembrane serine protease TMPRSS2 [Id., citing Glowacka, I. et al. J. Virol. (2011) 85: 4122-34, Matsuyama, S. et al. J. Virol. (2010) 84: 12658-664, Shulla, K. et al. J. Virol. (2011) 85: 873-82] for S protein priming in cell lines, and inhibition of both proteases is required for robust blockade of viral entry [Id., citing Kawase, M et al.
  • TMPRSS2 activity is essential for viral spread and pathogenesis in the infected host whereas CatB/L activity is dispensable [Id., citing Iwata-Yoshikawa, N. et al. J. Virol. (2019) 93: 10.1128/JVL01815-18, Shirato, K. et al. Virology (2016) 91: 10.1128/JVL01387-16, Shirato, K. et al. Virology (2016) 517: 9-15, Zhou, P et al. Antiviral Res. (2015) 116: 76-84].
  • the protease inhibitor is cysteine protease inhibitor K11777, ((2S)-N-[(1E,3S)-1- (benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2- ⁇ €-4-methylpiperazine-1-carbonyl]amino ⁇ -3- phenylpropanamide, or a P3 derivative thereof which inhibits SARS-CoV and Ebola virus entry.
  • the anti-viral agent comprises sera from a convalescent patient, e.g., a coronavirus patient, such as a SARSCoV, a MERS, or a COVID- 19 patient.
  • a convalescent patient e.g., a coronavirus patient, such as a SARSCoV, a MERS, or a COVID- 19 patient.
  • anti-malarial agent refers to a substance used for treatment of clinical Plasmodium falciparum malaria
  • the anti-malarial agent is selected from an aryl aminoalcohol compound selected from quinine, quinidine, chloroquine, amodiaquine, mefloquine, halofantrine, lumefantrine, piperaquine, and tafenoquine; an antifolate compound, selected from pyrimethamine, proguanil, chlorproguanil, trimethoprim; an artemisinin compound selected from artemisinin, dihydroartemisinin, artemether, artesunate; and atovaquone.
  • Chloroquine phosphate inhibits terminal phosphorylation of ACE2, and hydroxychloroquine elevates the pH in endosomes which are involved in virus cell entry [McKee, DL, et al. Pharmacol. Res. (2020) 157: 104859, citing 44, 45].
  • cepharanthine an anti-inflammatory alkaloid from Stephania cepharantha Hayata
  • selamectin an avermectin isolated from Streptomyces avermitilis and used as an anti-helminthic and parasiticide drug in veterinary medicine
  • mefloquine hydrochloride Lifloquine hydrochloride
  • anti-malarial agent refers to a substance used for treatment of clinical Plasmodium falciparum malaria.
  • the anti-malarial agent is an aryl aminoalcohol compound selected from quinine, quinidine, chloroquine, amodiaquine, mefloquine, halofantrine, lumefantrine, piperaquine, and tafenoquine.
  • the anti-malarial agent is an antifolate compound selected from pyrimethamine, proguanil, chlorproguanil, and trimethoprim.
  • the anti-malarial agent is an artemisinin compound selected from artemisinin, dihydroartemisinin, artemether, artesunate; and atovaquone.
  • Chloroquine phosphate inhibits terminal phosphorylation of ACE2, and hydroxychloroquine elevates the pH in endosomes which are involved in virus cell entry [McKee, DL, et al. Pharmacol. Res. (2020) 157: 104859, citing Vincent, MJ et al. (2005) Virol. J.2: 69; Bari, MAA. Pharmacol. Res. Perspect. (2017) 5(1): e0029344, 45].
  • cepharanthine an anti- inflammatory alkaloid from Stephania cepharantha Hayata
  • selamectin an avermectin isolated from Streptomyces avermitilis and used as an anti- helminthic and parasiticide drug in veterinary medicine
  • mefloquine hydrochloride Lifloquine hydrochloride
  • anti-infective agent means any of a group of chemical substances having the capacity to inhibit the growth of, or to destroy microorganisms, used chiefly in the treatment of infectious diseases.
  • the anti- infective agent is selected from amoxicillin, doxycycline, demeclocycline; eravacycline, minocycline, ormadacycline, tetracycline, cephalexin, defotaxime, cetazidime, cefuroxime, ceftaroline; ciprofloxacin, levofloxacin, moxifloxacin clindamycin, lincomycin, metronidazole, azithromycin; clarithromycin, erythromycin, sulfamethoxazle and trimethoprim; sulfasalazine, amoxicillin and clavulanate; vancomycin, dalbavancin, oritavancin,
  • anti-fibrotic agent refers to a substance that inhibits or reduces tissue scarring
  • the anti-fibrotic agent is selected from nintedanib, pirfenidone, and combinations thereof.
  • the described invention provides a method for reducing progression of symptoms of a severe virus infection in a subject comprising administering a pharmaceutical composition comprising a vehicle/carrier, a bifunctional fusion protein comprising a biologically active immunomodulatory component operatively linked to.
  • a biologically active antiviral component wherein the immunomodulatory component comprising a recombinant trefoil factor 2, a sequence variant, or a splice variant thereof.
  • the severe virus infection is a severe respiratory virus infection.
  • the respiratory virus is a cytomegalovirus.
  • the respiratory virus is Hanta virus.
  • the respiratory virus is an influenza virus.
  • the respiratory virus is a MERS virus.
  • the respiratory virus is a respiratory syncytial virus.
  • the respiratory virus is a SARS-CoV virus.
  • the respiratory virus is a Zika virus.
  • the respiratory virus is a West Nile virus.
  • the respiratory virus is a dengue virus.
  • the respiratory virus is a Japanese encephalitis virus.
  • the respiratory virus is a tick-borne encephalitis virus.
  • the respiratory virus is a yellow fever virus.
  • the respiratory virus is a rhinovirus.
  • the respiratory virus is an adenovirus.
  • the respiratory virus is a herpes virus.
  • the respiratory virus is an Epstein Barr virus.
  • the respiratory virus is a measles virus.
  • the respiratory virus is a mumps virus.
  • the respiratory virus is a rotavirus.
  • the respiratory virus is a Cocksackie virus.
  • the respiratory virus is a norovirus.
  • the respiratory virus is an encephalomyocarditis virus (EMCV).
  • EMCV encephalomyocarditis virus
  • the symptoms of a severe virus infection comprise primary viral pneumonia.
  • the symptoms of a severe virus infection comprise superimposed bacterial pneumonia.
  • symptoms of a severe virus infection comprise disruption or injury to alveolar epithelium, endothelium or both.
  • the symptoms of a severe virus infection comprise acute lung injury (ALI).
  • the symptoms of a severe virus infection comprise acute respiratory distress syndrome (ARDS).
  • the symptoms of a severe virus infection comprise symptoms of shock, including low blood pressure, lightheadedness, shortness of breath, and rash.
  • the symptoms of a severe virus infection comprise excessive complement activation.
  • the symptoms comprise a pathological increase in vascular permeability.
  • the symptoms of a severe virus infection comprise thrombotic complications.
  • the thrombotic complications include one or more of formation of pulmonary microthrombi, acute pulmonary embolism, deep-vein thrombosis, ischemic stroke, myocardial infarction, or systemic arterial embolism.
  • the symptoms of a severe virus infection comprise kidney damage.
  • the symptoms of a severe virus infection comprise elevated concentrations of one or more inflammatory mediator in plasma (hypercytokinemia), compared to a normal healthy subject.
  • the inflammatory mediator is one or more of interferon ⁇ , interferon ⁇ , interferon- ⁇ , interferon- ⁇ , interferon- ⁇ -1b, complement, prostaglandin D2, vasoactive intestinal peptide (VIP), interleukin-1-beta (IL-1 ⁇ ), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 (IL-12), IL-17, tumor necrosis factor-alpha (TNF- ⁇ ).
  • VIP vasoactive intestinal peptide
  • IL-1 ⁇ interleukin-6
  • IL-8 interleukin-8
  • IL-12 interleukin-12
  • IL-17 tumor necrosis factor-alpha
  • TNF- ⁇ tumor necrosis factor-alpha
  • the composition stimulates repair of a mucosal injury.
  • the repair comprises epithelial proliferation.
  • the repair restores an epithelial barrier, an endothelial barrier or both.
  • the composition modulates an immune response.
  • the immune response comprises recruitment of innate and adaptive immune cells.
  • the innate immune cells comprise macrophages, dendritic cells (DCs), innate lymphoid cells (ILCs), and natural killer cells (NKs).
  • the adaptive immune cells include ⁇ T cells, ⁇ T cells, and B cells.
  • the step of administering occurs nasally, intratracheally, orally, parenterally, topically, or by inhalation.
  • the administering is parenterally (by injection).
  • the administering occurs by inhalation. According to some embodiments, the administering is by insufflation.
  • the pharmaceutical composition further comprises at least one additional therapeutic agent or a supportive therapy.
  • the additional therapeutic agent is selected from one or more of an immunomodulatory agent, an analgesic agent, an anti-inflammatory agent, an anti-infective agent, an anti-malarial agent, an anti-viral agent or an anti-fibrotic agent.
  • each of these agents can be used alone as a monotherapy or in combination with a second agent, e.g., an immunomodulatory agent with a second immunomodulatory agent, e.g., methotrexate; an immunomodulatory agent with an analgesic agent, an immunomodulatory agent with an anti-inflammatory agent, an immunomodulatory agent with an anti-infective agent, an immunomodulatory gent with an anti-malarial agent, an immunomodulatory agent with an antiviral, an immunomodulatory agent with an antifibrotic agent; an immunomodulatory agent with supportive therapy.
  • a second agent e.g., an immunomodulatory agent with a second immunomodulatory agent, e.g., methotrexate
  • an immunomodulatory agent with an analgesic agent an immunomodulatory agent with an anti-inflammatory agent
  • an immunomodulatory agent with an anti-infective agent an immunomodulatory gent with an anti-malarial agent
  • an immunomodulatory agent with an antiviral an immunomodulatory agent with an antifibrotic agent
  • cytokine synthesis drugs that inhibit cytokine synthesis
  • drugs that inhibit cytokine synthesis e.g., glucocorticoids, cyclosporine A, tacrolimus, myophnolate- helper lymphocyte (Th2)-selective inhibitors
  • humanized blocking antibodies to cytokines or their receptors soluble receptors that mop up secreted cytokines, low molecular weight receptor antagonists, and drugs that block the signal transduction pathways activated by cytokines.
  • the immunomodulatory agent is a corticosteroid selected from prednisone, azathioprine, dexamethasone, mycophenolate, mycophenolate mofetil, and combinations thereof.
  • cyclosporine, tacrolimus and sirolimus are immunosuppressive medications that inhibit T cell activation through a series of calcium- dependent signal events involved in cytokine gene transcription. Cyclosporine inhibits the activation of helper T cells. Tacrolimus also interferes with T cell receptor-dependent cell activation. CSA and tacrolimus inhibit IL-2, IL-3, Il-4, IFN- ⁇ , GM-CSF, and TNF- ⁇ production. Transcriptional factors nuclear factor of activated T cells (NF-AT), NF- ⁇ B, and PU-box are inhibited by tacrolimus.
  • NF-AT nuclear factor of activated T cells
  • T cell receptor mediated apoptosis of lymphocytes and thymocytes is augmented by tacrolimus.
  • Sirolimus rapamycin
  • rapamycin is a macrocyclic lactone produced by Streptomyces hygroscopicis that inhibits T lymphocyte activation and proliferation that occurs in response to antigenic and cytokine stimulation; it binds intracellularly to the immunophilin, FK binding protein-12, which becomes an immunosuppressive complex, which in turn binds to and inhibits activation of mammalian regulatory kinase (target of rapamycin, mTOR). This inhibition suppresses cytokine-driven T cell proliferation, inhibiting the progression from the G1 to S phases of the cell cycle.
  • the immunomodulatory agent is a recombinant interferon, e.g., TFN- ⁇ ; IFN- ⁇ -2b, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ and IFN- ⁇ .
  • the recombinant interferon is in a PEGylated form.
  • the immunomodulatory agent is a recombinant IL-2 receptor inhibitor (e.g., denileukin diftitox).
  • the immunomodulatory agent is a PDE4 inhibitor, e.g., cilomilast, a second generation PDE4 inhibitor with anti-inflammatory effects that target bronchoconstriction, mucus hypersecretion and airway remodeling.
  • a PDE4 inhibitor e.g., cilomilast
  • the immunomodulatory agent is a hyperimmune globulin prepared from a donor with high titers of a desired antibody.
  • the immunomodulatory agent targets pro- inflammatory cytokines.
  • the immunomodulatory agent is a TNF ⁇ inhibitor/antagonist [e.g., etanercept; adalimumab; infliximab, certolizumab pegol, or golimumab].
  • the immunomodulatory agent is an IL-1 ⁇ inhibitor [e.g., rilonacept; canakinumab; or Anakinra].
  • the immunomodulatory agent is chimeric IL- 1Ra. This molecule is a fusion of the N-terminal peptide of IL-1 ⁇ and IL-1Ra, resulting in inactive IL-1Ra. Because the IL-1 ⁇ N-terminal peptide contains several protease sites clustered around the caspase-1 site, local proteases at sites of inflammation can cleave chimeric IL-1Ra and turn IL-1Ra active. [Rider, P. et al. J.
  • the immunomodulatory agent is an IL-6 inhibitor (e.g., tocilizumab, siltuximab, sarilumab, olokizumab, or sirukumab).
  • the immunomodulatory agent is an IL-12/ IL- 23 inhibitor (e.g., ustekinumab, briakinumab) an IL23 inhibitor (e.g., guselkumab, or tildrakizumab).
  • the immunomodulatory agent targets cytokine signaling pathways, e.g., compounds targeting TLR4 signaling, e.g., enamionone E121 (ethyl 4-(4’-chlorophenyl) amino-6 methyl-2-oxocytlohex-3-en-1-aote), an aniline enaminone); JODI 18b and 19, [Szollosi, DE et al. J. Pharmacy & Pharmacol.
  • cytokine signaling pathways e.g., compounds targeting TLR4 signaling, e.g., enamionone E121 (ethyl 4-(4’-chlorophenyl) amino-6 methyl-2-oxocytlohex-3-en-1-aote), an aniline enaminone); JODI 18b and 19, [Szollosi, DE et al. J. Pharmacy & Pharmacol.
  • TAK-242 (resatorvid, Takeda), TLR-C34, a 2-acetamidopyranoside that inhibits TLR4 signaling [Olusayo, A., et al. J. Applied Toxicol (2019) 39(4). Doi: 10/1002/jat.3771]; or C35 [Neal, MD. Et al. PLoS One (2013) 8 (6): e65779].
  • the immunomodulatory agent is a p38 MAPK inhibitor, e.g., SB203580, 4-(4’-fluorophenyl)-2-(4’-methylsulfinylphenyl)-5- (4’-pyridyl)- imidazole, a pyridinyl imidazole inhibitor used to elucidate the roles of p38 mitogen-activated protein (MAP) kinase [Cuenda, A. et al. FEBS Lett. (1995) 364: 229-33]; SB203580 inhibits also the phosphorylation and activation of protein kinase B (PKB, also known as Akt [Lali, F.V.
  • PKA protein kinase B
  • the immunomodulatory agent targets Janus kinase signaling (e.g., tofacitinub, baricitinib, or upadacitinib).
  • the immunomodulatory agent targets cell adhesion molecules to reduce leukocyte recruitment, e.g., molecules that are ⁇ 4 integrin inhibitors, e.g., vedolizumab, or natalizumab. [Szollosi, DE et al. J. Pharmacy & Pharmacol. (2016) 70: 18-26].
  • the immunomodulatory agent is a recombinant anti-inflammatory cytokine, e.g., IL-4, IL-10,or IL-11.
  • Immunomodulation also includes therapies that boost an individual’s defenses by providing physiologic or supraphysiologic doses of exogenous cytokines, e.g., to treat viral infections.
  • a parameter for measuring activation state of lymphocytes is cytokine release profile.
  • ELISPOT enzyme linked immunospot, is a technique that was developed for the detection of secreted proteins, such as cytokines and growth factors.
  • Another parameter for measuring activation of lymphocytes is by quantifying cellular subset differentiation.
  • the differentiation of CD45+/CD3+ T-lymphocytes to CD45+/CD3+/CD4+ helper T-lymphocytes, CD45+/CD3+/ CD8+ cytotoxic T-lymphocytes, and CD45+/CD3+/CD25+ activated T- lymphocytes can be quantified by flow cytometry analysis.
  • the immunomodulatory agent is a corticosteroid.
  • the corticosteroid is selected from prednisone, azathioprine, dexamethasone, mycophenolate, mycophenolate mofetil, and combinations thereof.
  • analgesic agent refers to an agent producing diminished sensation to pain without loss of consciousness.
  • the analgesic agent is selected from codeine, hydrocodone, oxycodone, methadone, hydromorphone, morphine, fentanyl, and combinations thereof.
  • An “anti-inflammatory agent” is a substance that reduces inflammation (redness, swelling, and pain) in the body by inhibiting inflammatory mediators in the body that cause inflammation.
  • the anti-inflammatory agent is selected from aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac nabumetone, naproxen, nintedanib, oxaprozin, pirfenidone, piroxicam, salsalate, sarilumab (Kevzara®) sulindac, tolmetin, and combinations thereof.
  • anti-viral agent as used herein means any of a group of chemical substances having the capacity to inhibit the replication of or to destroy viruses used chiefly in the treatment of viral diseases.
  • the anti-viral agent is selected from acyclovir, gancidovir, foscarnet; ribavirin; amantadine, azidodeoxythymidine /zidovudine), nevirapine, a tetrahydroimidazobenzodiazepinone (TIBO) compound; efavirenz; remdecivir, lopinavir/ritonavir (KaletraTM), umifenovir (ArbidolTM), favipiravir (AviganTM) and delavirdine.
  • the anti-viral agent is an agent that inhibits virus entry and thereby reduces viral load.
  • the agent that blocks viral entry is a peptide inhibitor.
  • the peptide inhibitor is a fusion protein.
  • the S glycoprotein is a type 1 transmembrane glycoprotein common to all HCoVs.
  • the S proteins consist of two subunits S1 and S2.
  • the S1 subunit binds the cellular receptor through its receptor binding domain (RBD), followed by conformational changes in the S2 subunit, which allows the fusion peptide to insert into the host target cell membrane.
  • the heptad repeat 1 (HR1) region in the SR2 unit forms a homotrimeric assembly, which exposes three highly conserved grooves on the surface that bind heptad repeat 2 (HR2).
  • This six-helix bundle (6-HB) core structure formed during the fusion process helps bring the viral and cellular membranes into close proximity for viral fusion and entry.
  • the HR region in the S2 subunit is conserved among various HCoVs and plays a pivotal role in HCoV infections by forming the 6-HB that mediates viral fusion.
  • the mode of interaction between HR1 and HR2 is conserved among CoVs such that certain residues in the HR1 helices interact with certain residues in the HR2 helices.
  • the fusion peptide is a synthetic peptide derived from HR1 and HR2 regions of SARS-CoV spike protein as described in Liu, S. et al. Lancet (2004) 363: 938-47, and as shown in Table 2 below.
  • Table 2 [000649] As described in Liu, S. et al. Lancet (2004) 363: 938-47, the HR1 and H2 sequences tend to form a coiled-coil structure, and the amino acid sequences of peptides derived from the HR1 and HR2 regions of SARS-CoV spike protein are similar to those from the HIV-1 gp41 HR1 and HR2 regions.
  • a fusion peptide of Table 2 may be used as a lead in designing more potent anti-SARS-CoV peptides.
  • HR1P and HR2P spanning residues 998-1039 in the HR1 domain [ANKFNQALGAMQTGFTTTNEAFQKVQDAVNNNAQALSKLASE, SEQ ID NO: 24] and 1251-1286 in the HR2P domain [SLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKEL, SEQ ID NO: 25], respectively, from MERS-CoV can form a stable six-helix bundle fusion core structure.
  • HR2P can effectively inhibit MERS-CoV replication and its spike protein-mediated cell–cell fusion.
  • Introduction of hydrophilic residues into HR2P results in significant improvement of its stability, solubility and antiviral activity.
  • the HR2P analogues can be further developed into effective viral fusion inhibitors.
  • the viral entry inhibitor is peptide OC43- HR2P [LAEADANVVAQIKVLASNTADFGEQIADLANNFANAIL, SEQ ID NO: 26] as described in Xia, et al., Sci. Advisor (2019) 5: eaav4580.
  • the viral entry inhibitor is EK1 [SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL, SEQ ID NO: 27] as described in Xia, et al., Sci. Advisor (2019) 5: eaav4580.
  • EK1 is a modified derivative of OC43-HR2P.
  • cholesterol (Chol) and palmitic acid (Palm) were covalently attached to the C-terminus of EK1 sequence under the help of a flexible polyethylene glycol (PEG) spacer, and the corresponding lipopeptides EK1C and EK1P were constructed.
  • the viral entry inhibitor is EK1P [SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL, SEQ ID NO: 28] or a PEG-based spacer-containing derivative [PEG4-C (Palm) thereof as disclosed by Xia, S. et al. Cell Res.
  • the viral entry inhibitor is EK1C [SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL, SEQ ID NO: 29] or a PEG-based spacer-containing derivative [PEG4-C (Chol)] thereof as disclosed by Xia, S. et al. Cell Res. (2020) 30(4): 343-55. [000654] As described by Xia et al. Cell Res.
  • EK1C cholesteryl EK1 with multiple linkers were constructed, where the glycine/serine-based linker, i.e., GSG, or PEG- based spacer was employed between EK1 and the cholesterol moiety.
  • GSG glycine/serine-based linker
  • PEG- based spacer PEG- based spacer
  • the agent that blocks viral entry is a dipeptidyl peptidase 4 (DPP4) inhibitor.
  • Drugs in the DPP-4 inhibitor class that are approved for use to treat type 2 diabetes include sitagliptin, saxagliptin, linagliptin, and alogliptin.
  • the agent that blocks viral entry is an ACE2 inhibitor.
  • ACE2 angiotensin-converting enzyme 2
  • peptide inhibitors of the enzyme were developed by selection of constrained peptide libraries displayed on phage [McKee, DL et al., Pharmacol. Res. (2020) 157: 104859, citing Huang, L. et al. J. Biol. Chem.
  • DX600 the most potent inhibitor of which, termed DX600, with the amino acid sequence of Ac-GDYSHCSPLRYYPWWKCTYPDPEGGG-NH2 [SEQ ID NO: 31] had a Ki of 2.8 nm and an IC50 of 10.1 ⁇ M.
  • DX600 is a potent ACE2 inhibitor specific for only human ACE2 [Id., citing Pedersen, KB et al. Am J. Physiol. Regul. Integr. Comp. Physiol. (2011) 301(5): R1293-99; Ye, M. et aql. Hypertension (2012) 60(3): 730-40].
  • the phytochemical nicotianamine (CAS number: 34441 ⁇ 14-0), a metal chelator ubiquitously present in higher plants [Id., citing Takahashi, M. et al. Plant Cell (2003) 15(6): 1263-80], was identified in high concentrations in soybean, and was shown as a potent inhibitor of human ACE2 with an IC50 of 84 nM [Id., citing Takahashi, S. et al. Biomed Res. (2015) 36(3): 219- 224]. Because dietary phytochemicals as naturally occurring compounds display a wide safety profile and less pharmacological side effects [Id., citing Naujokat, C. & McKee, DL. Curr. Med. Chem.
  • nicotianamine constitutes a candidate drug for ACE2 inhibition and thus blockade of SARS-CoV-2 cell entry.
  • a clinical-grade soluble recombinant human ACE2 protein hrsACE2
  • hrsACE2 a clinical-grade soluble recombinant human ACE2 protein
  • the anti-viral agent is a protease inhibitor that inhibits a host cell protease to block viral entry.
  • the anti- viral agent is a serine protease TMPRSS2 inhibitor, e.g., camostat (FOY-305), [N,N- dimethylcarbamoylmethyl 4-(4-guanidinobenzoyloxy)-phenylacetate] methanesulfate and camostat mesilate (FoipanTM), alternatively termed camostat mesylate, (NI-03), (CAS number: 59721 ⁇ 28-7); or Nafamostat mesilate (BuipelTM), (6-amidino-2-naphthyl-4-guanidino benzoate-dimethanesulfonate) (FUT-175), (CAS number: 81525 ⁇ 10-2).
  • camostat e.g., camostat (FOY-305), [N,N- dimethylcarbamoylmethyl 4-(4-guanidinobenzoyloxy)-phenylacetate] methanesulfate and camostat mes
  • SARS-CoV can use the endosomal cysteine proteases cathepsin B and L (CatB/L) [Id., citing Simmons et al., 2005) and the transmembrane serine protease TMPRSS2 [Id., citing Glowacka, I. et al. J. Virol. (2011) 85: 4122-34, Matsuyama, S. et al. J. Virol. (2010) 84: 12658-664, Shulla, K. et al. J. Virol. (2011) 85: 873-82] for S protein priming in cell lines, and inhibition of both proteases is required for robust blockade of viral entry [Id., citing Kawase, M et al.
  • TMPRSS2 activity is essential for viral spread and pathogenesis in the infected host whereas CatB/L activity is dispensable [Id., citing Iwata-Yoshikawa, N. et al. J. Virol. (2019) 93: 10.1128/JVL01815-18, Shirato, K. et al. Virology (2016) 91: 10.1128/JVL01387-16, Shirato, K. et al. Virology (2016) 517: 9-15, Zhou, P et al. Antiviral Res. (2015) 116: 76-84].
  • the protease inhibitor is cysteine protease inhibitor K11777, ((2S)-N-[(1E,3S)-1- (benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2- ⁇ €-4-methylpiperazine-1-carbonyl]amino ⁇ -3- phenylpropanamide, or a P3 derivative thereof which inhibits SARS-CoV and Ebola virus entry.
  • the anti-viral agent comprises sera from a convalescent patient, e.g., a coronavirus patient, e.g., a SARSCoV, a MERs, or a COVID-19 patient.
  • a convalescent patient e.g., a coronavirus patient, e.g., a SARSCoV, a MERs, or a COVID-19 patient.
  • anti-malarial agent refers to a substance used for treatment of clinical Plasmodium falciparum malaria
  • the anti-malarial agent is selected from an aryl aminoalcohol compound selected from quinine, quinidine, chloroquine, hydroxychloroquine, amodiaquine, mefloquine, halofantrine, lumefantrine, piperaquine, and tafenoquine; an antifolate compound, selected from pyrimethamine, proguanil, chlorproguanil, trimethoprim; an artemisinin compound selected from artemisinin, dihydroartemisinin, artemether, artesunate; and atovaquone.
  • Chloroquine phosphate inhibits terminal phosphorylation of ACE2, and hydroxychloroquine elevates the pH in endosomes which are involved in virus cell entry [McKee, DL, et al. Pharmacol. Res. (2020) 157: 104859, citing Vincent, MJ et al. (2005) Virol. J.2: 69; Bari, MAA. Pharmacol. Res. Perspect. (2017) 5(1): e00293].
  • cepharanthine an anti- inflammatory alkaloid from Stephania cepharantha Hayata
  • selamectin an avermectin isolated from Streptomyces avermitilis and used as an anti- helminthic and parasiticide drug in veterinary medicine
  • mefloquine hydrochloride Lifloquine hydrochloride
  • anti-infective agent means any of a group of chemical substances having the capacity to inhibit the growth of, or to destroy microorganisms, used chiefly in the treatment of infectious diseases.
  • the anti- infective agent is selected from amoxicillin, doxycycline, demeclocycline; eravacycline, minocycline, ormadacycline, tetracycline, cephalexin, defotaxime, cetazidime, cefuroxime, ceftaroline; ciprofloxacin, levofloxacin, moxifloxacin clindamycin, lincomycin, metronidazole, azithromycin; clarithromycin, erythromycin, sulfamethoxazle and trimethoprim; sulfasalazine, amoxicillin and clavulanate; vancomycin, dalbavancin, oritavancin,
  • anti-fibrotic agent refers to a substance that inhibits or reduces tissue scarring
  • the anti-fibrotic agent is selected from nintedanib, pirfenidone, and combinations thereof.
  • the supportive therapy is therapeutic apheresis comprising a virion removing step.
  • the therapeutic apheresis reduces viral burden.
  • Therapeutic apheresis is an extracorporeal treatment that can separate blood components (plasma and/or cellular components) from the patient's blood for the treatment of conditions in which a pathogenic substance in the blood is causing morbidity.
  • the therapeutic apheresis is consistent with then current guidelines of the American Society for Apheresis (ASFA). See, e.g., Szczepiorkowski ZM, Bandarenko N, Kim HC, et al. Apheresis Applications Committee of the American Society for Apheresis Guidelines on the use of therapeutic apheresis in clinical practice-evidence-based approach from the Apheresis Applications Committee of the American Society for Apheresis. J Clin Apher.2007;22:106–175; Szczepiorkowski M, Winters J, Bandarenko N, et al.

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Abstract

According to one aspect the described invention provides a method for reducing damaging effects of a severe virus infection in a susceptible subject comprising administering a pharmaceutical composition comprising a vehicle and a recombinant bifunctional fusion protein comprising a recombinant biologically active immunomodulatory component operatively linked to a recombinant biologically active anti-viral component, the recombinant immunomodulatory component comprising a recombinant biologically active human trefoil protein molecule, fragment or variant and the recombinant anti-viral component comprising a recombinant biologically active human interferon molecule, fragment or variant, wherein the method rescues symptoms of the severe virus infection.

Description

COMPOSITIONS AND METHODS FOR TREATING THE PATHOPHYSIOLOGY OF SEVERE VIRAL INFECTION CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of priority to U.S. provisional application 63/320,119 (filed March 15, 2022) and to U.S. provisional application 63/371,303 (filed August 12, 2022). The disclosure of each of these applications is incorporated herein by reference in entirety. SEQUENCE LISTING [0002] The instant application contains a Sequence Listing which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing was created on March 14, 2023, is named 130189-00520_SL.xml and is 121,160 bytes in size. FIELD OF THE INVENTION [0003] The described invention relates to compositions and methods for treating pathophysiology caused by severe viral infection. BACKGROUND OF THE INVENTION Viral infections – general principles [0004] Viruses depend on contact with a compatible host cell for replication. Viral replication begins with attachment of a virus to a host cell, which is followed by entry of the viral genome into the host where it begins to direct the synthesis of viral proteins and nucleic acid molecules. During replication, the nucleic acid and protein capsid structures are synthesized separately and then assembled within the host cell by packaging viral genomes into protein coats. The completed viral progeny exit the host cell, sometimes acquiring a piece of host cell membrane as a viral envelope. The two primary patterns of infection are acute infections and persistent infections. In acute infections, some viruses rapidly kill the cell while producing a burst of new infectious particles (cytopathic viruses), while others infect cells and actively produce infectious particle without causing immediate host cell death (noncytopathic viruses). In persistent infections (e.g., latent infections, slow, abortive and transforming infections), some viruses infect, but neither kill the cell nor produce any viral progeny. Replicated viruses remain inert unless they attach to the surface of another compatible host cell. [Principles of Virology Flint, SJ, Enquist LWQ, Krug, RM, Racaniello, VR, Skalka, AM, Eds. (2000) ASM Press, Washington, DC, Chapter 15, pp.519-551] [0005] To initiate an infection in an individual host, sufficient virus must be available to initiate infection, the cells at the site of infection must be susceptible and permissive for the virus, and the local host antiviral defense systems must be absent or at least initially ineffective. A severe virus infection attacks the host on multiple fronts. Some host defenses may be overcome passively by an overwhelming inoculum of virus. Many viruses have evolved active mechanisms for bypassing or disarming host defenses. The host immune response to viral infection [0006] The human immune system is a complex arrangement of cells and molecules that maintain immune homeostasis to preserve the integrity of the organism by elimination of all elements judged to be dangerous. Responses in the immune system may generally be divided into two arms, referred to as “innate immunity” and “adaptive immunity.” The two arms of immunity do not operate independently of each other, but rather work together to elicit effective immune responses. Innate immune response [0007] The innate arm of the immune system is a nonspecific fast response to pathogens that is predominantly responsible for an initial inflammatory response via a number of soluble factors, including the complement system and the chemokine/cytokine system; and a number of specialized cell types, including mast cells, macrophages, dendritic cells (DCs), and natural killer cells (NKs). Complement activation [0008] The complement system is a system of soluble pattern recognition receptors (PRRs) and effector molecules that detect and destroy microorganisms. In the presence of pathogens or of antibody bound to pathogens, soluble plasma proteins that in the absence of infection circulate in an inactive form becomes activated, so that particular complement proteins interact with each other to form the pathways of complement activation, which are initiated in different ways. As shown schematically in FIG.3, complement is activated through the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). In the classical and lectin pathways, binding of soluble pattern recognition molecules (PRMs) to a pathogen-associated molecular pattern (PAMP) or a damage-associated molecular pattern (DAMP) (the activator) activates zymogen proteases in complex with the PRMs. The classical pathway is initiated when complement component C1, which comprises a recognition protein (C1q) associated with proteases (C1r and C1s) either recognizes a microbial surface directly or binds to antibodies already bound to a pathogen. Exemplary C1q ligands include antigen- antibody complexes, molecular patterns on certain bacteria, viruses, parasites, and mycoplasma, C-reactive protein (CRP) in complex with exposed phosphocholine residues on bacteria; pentraxin‐3 (PTX‐3), serum amyloid P component, β‐amyloid fibrils, as well as tissue damage elements such as DNA and mitochondrial membranes [Bajik, G., et al. EMBO J. (2015) 34 (22) 2735-57, citing Kang, YH et al. Adv Exp Med Biol (2009) 653: 117–128], and DAMPS such as DNA, histones, and annexins A2 and A5 exposed by apoptotic cells [Id., citing Martin, M. et al. J Biol Chem (2012) 287: 33733–33744]. The proteins SCARF1 and LAIR have been implicated as immunomodulatory receptors for C1q-opsonized apoptotic cells. [Id., citing Son, M. et al. Proc Natl Acad Sci USA (2012) 109: E3160–E3167; Ramirez-Ortiz et al, Nat Immunol (2013) 14: 917–926]. Following C1q‐ligand binding, C1r autoactivates and subsequently cleaves C1s, which may then cleave C4 into the fragments C4a and C4b. The nascent C4b can be covalently bound to the activator via an exposed internal thioester leading to irreversible tagging of the activator. C2 binds activator‐bound C4b and is cleaved by C1s to generate the active serine protease C2a bound to C4b resulting in the CP C3 convertase C4b2a [Id., citing Muller‐Eberhard, HJ et al, J Exp Med (1967) 125: 359–3801967]. The C3 convertase cleaves C3 into the anaphylatoxin C3a and the major opsonin of the complement system, C3b, which like C4b, becomes covalently coupled to the activator through its exposed thioester [Id., citing Law SK, Dodds AW. Protein Sci (1997) 6: 263–274). [0009] The lectin pathway is initiated by soluble carbohydrate-binding proteins – mannose-binding lectin (MBL) and the ficolins—that bind to particular carbohydrate structures on microbial surfaces. MBL-associated serine proteases (MASPs), which associate with the recognition proteins, then trigger cleavage of complement proteins and activation of the pathway. [00010] Activation of the lectin pathway (LP) is initiated by the collectins MBL and CL‐ LK or one of three ficolins. MBL and CL‐LK harbor Ca2+‐dependent carbohydrate‐ recognition domains (CRDs) and collagen‐like regions through which they trimerize. Such trimers oligomerize in larger complexes, allowing high‐avidity binding (K D ≈ 10−9 M) based on multiple low‐affinity interactions of their CRDs (K D ≈ 10−3 M) [Id., citing Kawasaki et al. J Biochem (1983) 94: 937–947; Degn SE, Thiel S. Scand J Immunol (2013) 78: 181–193]. Ficolins are structurally similar to collectins, but instead of C‐type lectin domains they possess fibrinogen (FBG)‐like domains for PAMP recognition [Id., citing Matsushita M Ficolins in complement activation. Mol Immunol (2013) 55: 22–26]. Ficolins recognize motifs containing acetylated groups, including non‐sugars such as N‐acetyl‐glycine, N‐acetyl‐cysteine, and acetylcholine. Besides conferring avidity, the oligomerization of collectins and ficolins allows these PRMs to discriminate not only specific monosaccharides or acetylated groups but also specific patterns of sugars and acetyl groups characteristic to pathogens. The LP PRMs form complexes with MBL‐associated serine proteases (MASPs), which are always present as dimers. MASP‐1 and MASP‐2 are structural and functional homologs of C1r and C1s from the CP, but there are important differences between PRM–protease complexes from the two pathways. Whereas the C1 complex has a defined stoichiometry (a hexamer of the heterotrimeric C1q subunit in complex with a C1r2s2 tetramer), the LP PRMs are polydisperse oligomers of trimers. For MBL, a tetramer is the most abundant oligomer and this carries only a single MASP‐1 or MASP‐2 dimer, but the more rare, larger oligomers may simultaneously carry both dimers [Id., citing Dahl MR, et al. Immunity (2001) 15: 127–135; Teillet, F. et al, J Immunol (2005) 174: 2870–2877; Degn, SE et al J Immunol (2013) 191: 1334–1345]. MASP‐ 1 in complex with an activator‐bound PRM autoactivates and cleaves MASP‐2 as well as C2, whereas activated MASP‐2 cleaves C4 and C2 resulting in the same C3 convertase as in the CP, that is, C4b2a [Id., citing Matsushita, M et al. J Immunol (2000) 165: 2637–2642; Rossi, V. et al. J Biol Chem (2001) 276: 40880–40887; Chen CB, Wallis R J Biol Chem (2004)279: 26058–26065]. [00011] The alternative pathway (AP) can be initiated by spontaneous hydrolysis and activation of complement component C3, which can then bind directly to microbial surfaces. Activation through the CP and LP results in deposition of C3b on the activator, which recruits factor B (FB) in the first step of the AP. The resulting proconvertase C3bB is subsequently cleaved by factor D (FD), generating the AP C3 convertase C3bBb (Id., citing Fearon, DT et al. J Exp Med (1973) 138: 1305–1313], which is functionally homologous to the CP C3 convertase C4b2a. A positive feedback amplification loop is initiated as multiple copies of C3b are deposited on the activator leading to further assembly of the AP C3 convertase. Regardless of the initiating pathway, up to 90% of the deposited C3b molecules are generated through the AP [Id., citing Harboe, M. et al, Clin Exp Immunol (2004) 138: 439–446; Harboe, M. et al. Mol Immunol 47: 373–380]. This amplification is rapidly terminated on host cells by various regulators, but proceeds on pathogens and altered host tissues lacking such regulators. [00012] The three pathways converge at the step whereby enzymatic activity of a C3 convertase is generated. Cleavage of C3 is the critical step in complement activation and leads directly or indirectly to all the effector activities of the complement system. The C3 convertase is bound covalently to the pathogen surface, where it cleaves C3 to generate large amounts of C3b, the main effector molecule of the complement system, and C3a, a small peptide that binds to specific receptors and helps induce inflammation. [00013] The terminal pathway (TP) of complement is initiated when a threshold density of C3b molecules on an activator has been reached. The C3 convertases can recruit another C3b molecule to form C3bBb3b [Id., citing Medicus, RG et al. J Exp Med (1976) 144: 1076– 1093] and C4b2a3b [Id., citing Takata, Y et al. J Exp Med (1987) 165: 1494–1507], the AP and CP C5 convertases, respectively. Through cleavage of C5, they generate the potent chemoattractant C5a and C5b. The latter forms the lytic membrane attack complex (MAC, also called C5b‐9) together with C6, C7, C8, and multiple C9 molecules in membranes of pathogens lacking a protective cell wall like Gram‐negative bacteria [Id., citing Laursen, NS et al. Curr Mol Med (2012) 12: 1083–1097; Berends, ET et al. FEMS Microbiol Rev (2014) 38: 1146– 1171]. [00014] All three pathways have the final outcome of killing the pathogen, either directly or by facilitating its phagocytosis, and inducing inflammatory responses that help to fight infection. [00015] The anaphylatoxins C3a and C5a, released when the convertases cleave C3 and C5, exert their biological functions upon binding to seven‐transmembrane domain (7TM) receptors in the membranes of host cells. Two of these receptors, C3aR and C5aR1 (CD88), are G protein‐coupled receptors (GPCR), whereas the third, C5aR2 (previously known as C5L2), is structurally similar to C5aR1 but does not couple to heterotrimeric G proteins [Id., citing Li, R. et al. FASEB J (2013) 27: 855–864, 2013]. C5aR2 was first considered as a decoy receptor, limiting the availability of the C5a and C5adesArg ligands to C5aR1. Decoy receptors do not undergo ligand‐induced internalization but are rather continuously recycled between the cell membrane and the intracellular compartments, thereby removing their extracellular ligand [Id., citing Weber, M. et al. Mol Biol Cell (2004) 15: 2492–2508]. Thus, it has been suggested that C5aR2 may reduce the cellular responses to pro‐inflammatory molecules and thereby actively regulate inflammatory processes [Id., citing Rittirsch, D. et al. Nat Med (2008) 14: 551–557]. Additionally, some studies report concerted action of C5aR1 and C5aR2 in adipocyte metabolism and immunity as well as formation of C5aR1/C5aR2 heterocomplexes [Id., citing Bamberg, CE et al. Adv Exp Med Biol (2010) 632: 117–142; Poursharifi, P. et al. Mol Cell Endocrinol (2014) 382: 325–333]. [00016] Signaling through C3aR and C5aR1 triggers chemotaxis, oxidative burst, histamine release, and leukotriene and interleukin synthesis [Id., citing Klos, A. et al, Mol Immunol (2009) 46: 2753–2766]. [00017] There are five known C3b receptors on the surface of cells, especially immune cells. Complement receptor 1 (CR1, CD35) is a large CCP module‐based glycoprotein expressed on almost all peripheral blood cells except NK and T cells [Id., citing Fearon, DT. J Exp Med (1980) 152: 20–30; Tedder, TF et al, J Immunol (1983) 130: 1668–1673]. CR1 binds C3b and C4b with high affinity and iC3b and C3d with a lower affinity [Id., citing Reynes, M. et al J Immunol (1985)135: 2687–2694]. CR1 on erythrocytes may bind C3b‐containing immune complexes as part of removal processes, whereas on phagocytic cells it promotes C3b/C4b‐coated particle uptake. CR1 also plays an important role in the germinal centers of lymph nodes where it is found on follicular dendritic cells (FDCs) capturing complement‐ opsonized antigens that serve to stimulate B cells [Id., citing Heesters, BA et al. (2013) Nat Rev Immunol 14: 495–504]. CR2 (CD21), also possessing a CCP architecture, is primarily present on B cells and FDCs. It is important in trapping of C3‐opsonized antigens by FDCs in the germinal centers and stimulating B cells for affinity maturation, isotype switching, and memory [Id., citing Fang, Y. et al. (1998) J Immunol 160: 5273–5279; Carroll, MC (2000) Adv Immunol 74: 61–88]. CR2 binds C3b, iC3b, and C3d with the same affinity in agreement with the crystal structure of the CR2‐C3d complex revealing recognition of a surface patch on the TE domain accessible in all three ligands but concealed in C3 prior to cleavage [Id.]. [00018] CR3 and CR4 are integrin‐type heterodimeric receptors (CD11b/CD18 and CD11c/CD18) having distinct α‐chains, αM and αX, respectively, but sharing a common β2‐ chain. Both are phagocytic receptors expressed on myeloid leukocytes and NK cells and share iC3b as ligand [Id., citing Metlay, JP et al. (1990) J Exp Med 171: 1753–1771; Ross, GD (2000) Crit Rev Immunol 20: 197–222]. However, structural studies indicate that the receptors bind to different epitopes of iC3b. CR3 was shown to recognize the TE domain of iC3b [Id., citing Bajic, G. et al. (2013) Proc Natl Acad Sci USA 110: 16426–16431], whereas CR4 binds quite far from this in the C3c moiety of iC3b [Id., citing Chen, X. et al. (2012) Proc Natl Acad Sci USA 109: 4586–4591]. CR3 and CR2 may bind simultaneously to the iC3b TE domain [Id., citing Bajic, G. et al (2013) Proc Natl Acad Sci USA 110: 16426–16431], and since CR3 is expressed on subcapsular sinus macrophages (SSMs), it is plausible that complement‐bearing immune complexes could be conveyed from CR3‐positive SSMs to CR2‐positive naïve B cells within lymph nodes [Id., citing Phan, TG et al. (2007) Nat Immunol 8: 992–1000; Bajic, G. et al. (2013) Proc Natl Acad Sci USA 110: 16426–16431; Heesters, BA et al. (2014) Nat Rev Immunol 14: 495–504]. SSM are poorly endocytic, and appear to retain ICs on their surface during the IC shuttling from the sinus‐lining to the follicular side [Id., citing Phan, TG et al. (2009) Nat Immunol 10: 786–793]. The fifth C3b receptor is CRIg (VSIG4), an immunoglobulin‐type receptor expressed on liver‐resident macrophages (Kupffer cells), which plays an important role in the clearance of pathogens from the circulation through interaction with surface‐bound C3b and iC3b opsonins [Id., citing Helmy, KY et al. (2006) Cell 124: 915– 927]. The binding of CRIg to C3b selectively inhibits the interaction of C3 and C5 with the AP, but not with the CP convertases. [00019] Besides acting in innate immunity, the complement system also influences adaptive immunity. For example, opsonization of pathogens (meaning the coating of the surface of a pathogen that makes it more easily ingested by phagocytes) by complement facilitates their uptake by phagocytic APCs that express complement receptors, which enhances presentation of pathogen antigens to T cells. B cells express receptors for complement proteins that enhance their responses to complement-coated antigens. Several complement fragments also can act to influence cytokine production by APCs, thereby influencing the direction and extent of the subsequent adaptive immune response. [Janeway’s Immunology, 9th Ed. (2017) Garland Science, New York, Chapter 2, 49-51]. [00020] Complement fragments can be generated by other means besides the three canonical activation routes. The cross-talk with the coagulation system has regained attention due to studies indicating that thrombin, coagulation factors XIa, Xa, and IXa, and plasmin effectively cleave C3 and C5 and generate C3a and C5a [Bajik, G. et al. EMBO J. (2015) 34(22): 2735-57, citing Huber‐Lang, M. et al. (2006) Nat Med 12: 682–687; Amara U. et al. (2010) J Immunol 185: 5628–5636; Berends, ET et al. (2014) FEMS Microbiol Rev 38: 1146– 1171]. C3 can also be produced intracellularly by CD4+ T cells. This C3 is processed by the T‐cell lysosomal protease cathepsin L, yielding biologically active C3a and C3b [Id., citing Liszewski, MK et al. (2013) Immunity 39: 1143–1157]. Tonic intracellular C3a generation is required for homeostatic T‐cell survival, whereas shuttling of this intracellular C3 activation system to the cell surface upon T‐cell stimulation additionally induces autocrine proinflammatory cytokine production. Thus, C3aR activation via intrinsic generation of C3a appears to be an integral part of human Th1 immunity [Id., citing Ghannam, A. et al, (2014) Mol Immunol 58: 98–107]. Thrombin slowly cleaves C5 and generates C5a, but under conditions with normal convertase activity, this is possibly not a physiologically significant reaction. Clotting‐induced production of thrombin instead leads to cleavage of C5 or C5b in the CUB domain. C5a can be released from such CUB‐digested C5 by the conventional C5 convertases, and the combined action of thrombin and convertases appears to enhance the efficiency of the lytic pathway [Id., citing Krisinger, MJ et al. (2012) Blood 120: 1717–1725]. Conversely, MASP‐1 has been reported to activate coagulation [Id., citing Takahashi, K. et al. (2011) Immunobiology 216: 96–102; La Bonte, LR et al. (2012) J Immunol 188: 885–891] and to initiate endothelial cell signaling via cleavage of protease‐activated receptor 4 [Id., citing Megyeri, M. et al. (2009) J Immunol 183: 3409–3416]. [00021] The complement system has been implicated as a contributor to the observed tissue damage that occurs in such severe virus infections as influenza A virus H1Ni [Wang, R. et al. Emerging Microbes and Infections (2015) 4: e28., citing Garcia, CC. et al. PLoS One (2013) 8: e64443; Berdal, JE et al., J. Infect. (2011) 63: 308-16], H5N1 [Id., citing Sun, S. et al. Am. J. Respir. Cell Mol. Biol. (2013) 49: 221-230], H7N9 [Id., citing Sun, S. et al. Cllin. Infect. Dis. (2014) 60: 586-95], SARS-CoV [Id., citing Huang, KJ, et al. J. Med. Virol. (2005) 75: 185-94]; and MERS-CoV [Id., citing Zhou, J. et al. J. Infect. Dis. (2014) 209: 1331-1442]. Studies suggest the synthesis of complement components by human alveolar macrophages and synthesis and secretion of complement components by pulmonary alveolar type II epithelial cells. [Id., citing Ackerman, SK et al., Immunology (1978) 35: 369-72; Coi, FS, et al., Clin. Immunol. Immunopathol. (1983) 27: 153-59; Strunk, RC et al., J. Clin. Invest. (1988) 81: 1419-146]. [00022] Among the complement activation products, the anaphylatoxin C5a is one of the most potent inflammatory peptides. [Id. citing Marc, MM, et al. Am. J. Respir. Cell Mol. Biol. (2004) 31: 216-19]. It is a strong chemoattractant for neutrophils and monocytes and activates these cells to generate oxidative bursts with release of reactive oxygen species (ROS), especially O2 and H2O2. [Id., citing Guo, RF, Ward, PA. Annu. Rev. Immunol. (2005) 23: 821-52]. C5a mediates neutrophil attraction, aggregation, activation and subsequent pulmonary endothelial damage. [Id., citing Stevens, JH, Raffom. TA. Postgrad. Med. J. (1984) 60: 505- 513; Tate, RM, Repine, JE. Am. Rev. Respir. Dis. (1983) 128: 802-806; Craddock, PR et al., J. Clin. Invest. (1977) 60: 260-64; Sacks, T. et al. J. Clin. Invest. (1978) 61: 1161-67]. C5a activates macrophages and endothelial cells and promotes vascular leakage and the release of Neutrophil Extracellular Traps (NETs). [Id., citing Guo, RF, Ward, PA. Annu. Rev. Immunol; (2005) 23: 821-52]. NETs are primarily composed of DNA from neutrophils, which bind pathogens with anti-microbial proteins, and increase the permeability of the alveolar-capillary barrier by cleaving endothelial actin cytoskeleton, E-cadherin and VE-cadherin. [Id., citing Saffarzadeh, M. et al. PLoS One (2012) 7: e32366]. The antimicrobial peptide LL-37 in NET structures is cytotoxic and pro-apoptotic toward endothelial and epithelial cells [Id., citing Aarbiou, J. et al. Inflamm. Res. (2006) 55: 119-127]. NETs also induce the release of proinflammatory cytokines. [Id., citing Saffarzadeh, M. et al. PLoS One (2012) 7: e32366]. [00023] C5a is also a potent chemoattractant for T cells [Id., citing Nataf, S., e al., J. Immunol. (1999) 162: 4018-23; Tsuji, RF et al. J. Immunol. (2000) 165: 1588-98], B cells [Id., citing Ottonello, L, et al. J. Immunol. (1999) 162: 6510-17], and DCs [Id., citing Morelli, A., et al., Immunology (1996) 89: 126-34; Sozzani, S. et al., J. Immunol. (1995) 155: 3292- 95; Mrowietz, U. et al. Exp. Dermatol. (2001) 10: 238-45; Yang, D. et al. J. Immunol. (2000) 165: 2694-2702], which release cytokines, such as TNF-α, IL-1β, IL-6, and IL-8 [Id., citing Hopken, U., et al. Eur. J. Immunol. (1996) 26: 1103-09; Strieter, RM et al. Am. J. Pathol. (1992) 141: 397-407]. DCs can then take up antigen and are primed for T cell help. [Id., citing Kim, AH, et al. J. Immunol. (2004) 173: 2524-29]. The process of leukocyte adhesion to endothelial cells is the first critical step in neutrophil migration into an area of inflammation. C5a induces upregulation of CD11b /CD18 expression on neutrophils. [Id., citing Guo, RF, Ward, PA. Annu. Rev. Imunol. (2005) 23: 821-52]. IL-8 levels have been found to correlate with neutrophil numbers and the degree of lung dysfunction. [Id., citing Williams, TJ, Jose, PJ. Novartis Found Symp. (2001) 234: 136-41]. C5a directly activates endothelial cells to upregulate adhesion molecules, such as P-selectin, and C5a and TNF-α cooperate to enhance upregulation of intracellular adhesion molecule 1 and E-selectin [Id., citing Ward, PA. Ann. NY Acad. Sci. (1996) 796: 104-112]. Strategies of innate immunity that defend against intracellular pathogens [00024] Viruses are obligate intracellular pathogens – they must invade host cells to replicate. Two strategies of innate immunity defend against intracellular pathogens. [00025] One is to destroy pathogens before they infect cells. To this end, innate immunity includes soluble defenses, such as antimicrobial peptides (e.g., defensins, athelicidins, and histatins) and phagocytic cells (macrophages, neutrophils and dendritic cells) that can engulf and destroy pathogens before they become intracellular. Macrophages and neutrophils constitutively express cell-surface receptors that stimulate the phagocytosis and intracellular killing of microbes bound to them, although some also signal through other pathways to trigger other responses, e.g., cytokine production. These phagocytic receptors include several members of the C-type lectin-like family (e.g., Dectin-1, and the mannose receptor (MR); scavenger receptors that recognize various anionic polymers and acetylated low density lipoproteins; and complement receptors and Fc receptors that bind to complement coated microbes or to antibodies bound to the surface of microbes that facilitate phagocytosis. [00026] The nucleic acid sensing toll like receptors – TLR3, TLR-7, TLR-8 and TLR-9, are endosomal nucleotide sensors involved in the recognition of viruses. TLR-3 is expressed by macrophages, conventional dendritic cells, and intestinal epithelial cells; it recognizes double-stranded RNA which is a replicative intermediate of many types of viruses. TLR-7 and TLR-9 are expressed by plasmacytoid dendritic cells, B cells and eosinophils; TLR-8 is expressed primarily by monocytes and macrophages. TLR-7 and TLR-8 are activated by single-stranded RNA. The virus genome for example of orthomyxoviruses (such as influenza) and flaviviruses (such as West Nile virus) consist of single stranded RNA. When extracellular particles of these viruses are endocytosed by macrophages or dendritic cells, they are uncoated in the acidic environment of endosomes and lysosomes, exposing the ssRNA genome for recognition by TLR-7. TLR-8 is physiologically most similar to TLR7, recognizes viral ssRNA, and is predominantly expressed in monocytes. [Petrasek, J. et al., Advances in Clin. Chem. (2013) 59: 255-201]. TLR-9 recognizes unmethylated CpG nucleotides; in the genomes of bacteria and many viruses, CpG dinucleotides remain unmethylated. [Janeway’s Immunology, 9th Ed. (2017) Garland Science, New York, at 91] [00027] Macrophages and neutrophils secrete lipid mediators of inflammation – prostaglandins, leukotrienes, and platelet-activating factor (PAF) – which are rapidly produced by enzymatic pathways that degrade membrane phospholipids. Signaling by mammalian TLRs in various cell types induces a diverse range of intracellular responses that together result in the production of inflammatory cytokines, chemotactic factors, antimicrobial peptides, and the antiviral cytokines interferon α and interferon β. [Janeway’s Immunology, 9th Ed. (2017) Garland Science, New York, at 92] [00028] Viral RNAs produced within a cell are sensed by RIG-1 like receptors, which bind to viral RNA using an RNA helicase-like domain in their carboxy terminal, which has a DexH tetrapeptide amino acid motif and is a subgroup of DEAD-box family proteins. The RLR proteins also contain two amino terminal CARD domains that interact with adaptor proteins and activate signaling to produce type 1 interferons when viral RNAs are bound. RIG- 1 discriminates between host and viral RNA by sensing differences at the 5’ end of single stranded RNA transcripts – most RNA viruses do not replicate in the nucleus where addition of a 7-methylguanosine to the 5’triphosphate (called capping) occurs, and their RNA genomes do not undergo this modification. RIG-1 senses the unmodified 5’-triphosphate end of the ssRNA viral genome. MDA-5 (melanoma differentiation-associated 5, also called hellicard, is similar in structure to RIG-1, but it senses dsRNA. The RLR family member LGP2 (encoded by DHX58) retains a helicase domain but lacks CARD domains. It appears to cooperate with RIG-1 and MDA-5 in the recognition of viral RNA. Before infection by viruses, RIG-1 and MDA-5 are in the cytoplasm in an auto-inhibited configuration that is stabilized by interactions between the CARD and helicase domains. [Id.] [00029] Sensing of viral RNAs activates signaling by RIG-1 and MDA-5, which leads to type 1 interferon production. Upon infection, viral RNA associates with the helicase domains of RIG-1 or MDA-5, freeing the two CARD domains for other interactions. The more amino- proximal portion of the two CARD domains can then recruit E3 ligases, including TRIM25 and RIPLET, which initiate K63-linked polyubiquitin scaffolds, which appear to help RIG-1 and MDA-5 interact with a downstream adaptor protein called mitochondrial antiviral signaling protein (MACVS). MAVS is attached to the outer mitochondrial membrane and contains a CARD domain that may bind RIG-1 and MDA-5. This aggregation of CARD domains may initiate aggregation of MAVs, which propagates signals by recruiting various TRAF family E3 ubiquitin ligases, including TRAF2, TRAF3, TRAF5, and TRAF6. Their further production of K63-linked polyubiquitin leads to activation of TBK1 and IRF3 and production of type 1 interferons as described for TLR-3 signaling, and to activation of Nf-κB. [Janeway’s Immunology, 9th Ed. (2017) Garland Science, New York, at 102-103]. [00030] Alternatively, the innate immune system can recognize and kill cells infected by some pathogens. Natural killer cells (NK cells), the only cytotoxic population of innate lymphoid cells (ILCs) [Jiao, Y. et al., Front. Immunol. (2020) 11: 282] are instrumental in keeping certain viral infections in check before cytotoxic T cells of the adaptive immune response become functional. Virus-infected cells can become susceptible to being killed by NK cells by a variety of mechanisms. First, some viruses inhibit all protein synthesis in their host cells; synthesis of MHC class I proteins would be blocked in infected cells, which would make them correspondingly less able to inhibit NK cells through their MHC-specific receptors, and they would become more susceptible to being killed. Second, many viruses can selectively prevent the export of MHC class I molecules to the cell surface, or induce their degradation once there. Virally infected cells can still be killed by NK cells even if the cells do not downregulate MHC, provided that ligands for activating receptors are induced. Viruses that target ligands for the activating receptors on NK cells can thwart NK cell recognition and killing of virus-infected cells. NK cells also express receptors that more directly sense the presence of infection or other perturb ations in a cell. Activating receptors include the natural cytotoxicity receptors (NCRs) NKp30, NKp44, and NKp46, which are immunoglobulin-like receptors, and the C-type lectin-like family members LY49H and NKG2D. Recognition by NKG2D acts as a generalized ‘danger’ signal to the immune system. Besides being expressed by a subset of NK cells, NKG2D is expressed by various T cells, including all human CD8 T cells, γδ T cells, activated murine CD8 T cells and invariant NKT cells. In these cells recognition of NKG2D ligands provides a potent co-stimulatory signal that enhances their effector functions. [Janeway’s Immunology, 9th Ed. (2017) Garland Science, New York, pp125-130] [00031] The conventional NK (cNK) cell pool consists of a circulating compartment and a tissue-resident compartment in the gut intraepithelial layer and lamina propria layer. [Jiao, Y. et al., Front. Immunol. (2020) 11: 282] cNK cells are able to sense pathogens, oncogenesis and tissue damage signals. Activation and turnover of cNK cells rely on the overall signal input of activating signals, inhibitory signals, and exogenous cytokine signals, which further leads to the alteration of specific transcription factors and a group of pro-apoptotic proteins and ultimately determines the fate of cNK cells. [Id., citing Viant C, et al. J Exp Med. (2017) 214:491–510]. Upon activation, cNK cells exert their cytotoxicity function by releasing the pore forming cytolytic protein–perforin and the cytotoxic protein–granzyme. cNK cells also utilize tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) pathways and antibody-dependent cellular cytotoxicity (ADCC) (Id., citing Caligiuri MA. Blood. (2008) 112:461–9). At the same time, cNK cells possess strong cytokine production ability, including TNF, IFN-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Id., citing Souza-Fonseca-Guimaraes F, et al. J Biol Chem. (2013) 288:10715–21). [00032] The theory of cNK education posits that the threshold of activation of cNKs throughout their development is modulated by adjusting the expression level of their activating receptors and inhibitory receptors (“education”). The processes of cNK cell arming (meaning the downregulation of inhibitory receptors that could upregulate the threshold of activation) and cNK cell licensing (meaning the scenario where activating receptors are downregulated to endow cNK cells with increased receptivity to activating signals) ensure the appropriate activation strategy, namely, to limit self-reaction of cNK cells that do not recognize self MHC class I molecules by inhibitory receptors. Generally, educated cNK cells, marked by the elevated expression of the activating receptor DNAM-1, exhibit higher reactivity to missing- self targets with increased degranulation and cytokine production capability [Id., citing Enqvist M, et al. J Immunol. (2015) 194:4518–27]. It has been hypothesized that the gut may be one of the centers for cNK cells to obtain normal function and acquire education. Gain of cytotoxic function of cNK cells is dependent on the priming step by commensal bacteria in a dendritic cell dependent manner [Id., citing Ganal SC, et al. Immunity. (2012) 37:171–86] and commensal lactic acid bacteria are a key regulator in the cross-talk between cNK cells. Lactic acid bacteria activate immature dendritic cells in the gut to produce key cytokines, including IL-12 and IL-15, and to favor the activation and proliferation of cNK cells [Id., citing Rizzello V, et al. BioMed Res Int. (2011) 2011:473097]. ILCs [00033] Innate lymphoid cells (ILCs) are the innate counterparts of T lymphocytes. They lack adaptive antigen receptors generated by the recombination of genetic elements. [Vivier, E. et al. Cell (2018) 174: 1054-66, citing Spits, et al. Nat. Rev. Immunol. (2013) 13: 145-49; Eberl, G et al. Science (2015) 348: aaa6566; Artis, D. and Spits, H. Nature (2015) 517: 293-301]. All ILCs express interleukin-7 receptorα (CD127). ILCs may be activated by signals from other cells around them upon exposure to foreign antigens (including microbes), rather than by being directly activated by foreign antigens. Some ILCs express TLRs that recognize microbes, and the cells may be directly activated by the PAMPs of microbes. However, there have been some reports showing that ILCs express various kinds of receptors for cytokines, danger signals, neuropeptides and lipid mediators that are more dominant than TLRs. [Id.] [00034] ILCs are generally thought to be tissue-resident cells that differentiate into mature effector cells in tissues, and show minimal movement between organs. Instead, they have functional plasticity that enables them to respond promptly to microenvironmental changes, thereby precluding any need for differentiation and/or migration of new ILC subsets adapted to a new environment. For example, transdifferentiation has been shown between ILC1s and ILC3s [Id., citing Bernink JH, et al. Immunity (2015) 43:146–160, Bernink JH, et al. Nat Immunol (2013) 14:221–229], between ILC1s and ILC2 [Id., citing Bal SM, et al. Nat Immunol (2016) 17:636–645; Silver JS, et al. Nat Immunol 2016;17:626–635; Ohne Y, et al. Nat Immunol (2016) 17:646–655], and between ILC2s and ILC3s [Id. citing Bernink JH, et al. Nat Immunol (2019) 20:992–1003, Golebski K, et al. Nat Commun (2019) 10:2162].Group 1 ILCs currently are divided into 3 different subtypes, according to their expression of cytokines and transcription factors: group 1 ILCs (ILC1s), group 2 ILCs (ILC2s), and group 3 ILCs (ILC3s). [00035] ILC1s are defined as ILCs that express T box-expressed in T cells (T-bet) and produce interferon (IFN-γ); they include conventional natural killer cells (cNK) and are considered to be involved in anti-viral immunity, like Th1 cells. [Orimo, K. et al., Allergy Asthma Immunol. Res. (2020) 19(3): 381-98] [00036] ICL2s are defined as ILCs that express GATA-binding protein 3 and produce such cytokines as IL-4, IL-5, IL-9, and IL-13, as well as the epidermal growth factor, amphiregulin; like Th2 cells, they are considered to be involved in anti-helminth immunity. [Id.] [00037] ILC3s are defined as ILCs that express retinoic acid receptor-related orphan receptor-γt and produce cytokines, such as IL-17A, IL-22 and GM-CSF; they include both natural cytotoxicity receptor (NCR)- ILC3s and NCR+ ILC3s, and are considered to be involved in antibacterial immunity, like Th17 cells. [Id.] In humans, ILC3s are the predominant population in mucosal tissues, including the lung and gut, whereas the proportion of ILC2s is a little higher in the skin compared to mucosal tissues [Id., citing Bal SM, et al. Nat Immunol (2016) 17:636–645). The proportion of the ILC subsets is influenced by age; although ILC3s are the predominant population in the fetal human lung, their proportion decreases while the proportions of ILC1s and ILC2s increase with age in the adult human lung. [Id., citing Bal SM, et al. Nat Immunol (2016) 17:636–645]. There is substantial heterogeneity in each subset of ILCs. Moreover ILCs show different phenotypes depending on the organ. (Id., citing Ricardo- Gonzalez RR, et al. (2018) 19:1093–1099). For example, although ILC2s from different organs share canonical markers such as GATA3 and IL-7R, expression of IL-33R, IL-25R, and IL- 18R1 differs depending on the organ. [Id., citing Ricardo-Gonzalez RR, et al. Nat Immunol (2018) 19:1093–1099]. [00038] Another ILC subset, called regulatory ILCs (ILCregs), which resemble regulatory T cells (Tregs) and have regulatory functions, has been reported. [Id., citing Morita H, et al. Allergy Clin Immunol (2019) 143:2190–2201.e9; Wang S, et al. Cell (2017) 171:201– 216.e18; Seehus CR, et al. Nat Commun (2017) 8:1900]. ILCregs produce regulatory cytokines such as IL-10 and/or TGFβ, but they do not express FOXP3, the canonical transcription factor of Tregs. It remains controversial wither ILCregs represents an independent effector subset, or just a temporary state of ILCs. [00039] There is increasing evidence to suggest that like T helper cell subsets, ILC subsets also display a certain degree of plasticity, which enables them to adjust to their microenvironment. Thus, ILC subsets can change their phenotype and functional capacities. For example, although ILC2s from different organs share canonical markers such as GATA3 and IL-7R, expression of IL-33R, IL-25R, and IL-18R1 differs depending on the organ. [Id., citing Ricardo-Gonzalez RR, et al. Tissue signals imprint ILC2 identity with anticipatory function. Nat Immunol (2018) 19:1093–1099]. This requires accessible polarizing signals in the tissue in which conversion occurs, together with the expression of cognate cytokine receptors and key transcription factors in the responding ILCs. [Vivier, E. et al. Cell (2018) 174: 1054-66]. Airway ILCs ILC1s-- IL-12 [00040] IL12 is a major activator of ILC1s and promotes their secretion of IFN-γ. [Orimo, K. et al. Allergy Asthma Immunol. Res. (2020) 12 (3): 381-98, citing Bernink JH, et al. Nat Immunol (2013) 14:221–229]. The major physiological producers of IL12 are APCs, such as dendritic cells and macrophages. In the mouse lung, INF-γ produced by ILC1s in response to DC-derived IL-12 during viral infection suppresses early viral growth, suggesting that the IL-12-ILC1 axis may be involved in anti-viral immunity. [Id., citing Weizman OE, et al. Cell (2017) 171:795–808.e12]. Furthermore, IL-12 mediates the transdifferentiation of ILC2s [Id., citing Bal SM, et al. Nat Immunol (2016) 17:636–645; Silver JS, et al. Nat Immunol (2016) 17:626–635; Ohne Y, et al. Nat Immunol (2016) 17:646–655] and ILC3s [Id., citing Bernink JH, et al. Immunity (2015) 43:146–160] into INF-γ producing ILC1s, a mechanism that may be involved in immune responses to viral infections and in the pathophysiology of COPD. IL-15 [00041] Like IL-12, IL-15 activates ILC1s to produce IFN-γ. IL-15 is known to be produced by APCs, a subset of thymic epithelial cells, and by stromal cells. In the airways, human bronchial epithelial cells produce IL-15 in response to respiratory syncytial virus infection [Id., citing Zdrenghea MT, et al. Eur Respir J 2012;39:712–720]. In human airway diseases, IL-15–positive cells have been reported to be increased inpatients with sarcoidosis, tuberculosis or chronic bronchitis compared to asthmatic patients and healthy subjects, [Id., citing Muro S, et al. Allergy Clin Immunol (2001) 108:970–975] suggesting the involvement of IL-15 in the pathophysiology of these diseases. IL-18 [00042] IL-18 also activates ILC2s and ILC3s to produce their signature cytokines, (Id., citing 12, 16) suggesting that IL-18 may be a pan-activator of ILCs. Furthermore, IL-18 and IL-12 together promote conversion of ILC2s to ILC1s. [Id., citing Silver JS, et al. Nat Immunol (2016) 17:626–635]. IL-18 is produced by APCs such as macrophages and DCs. In regard to the airways, IL-18 was shown to be released from human bronchial epithelial cells upon human rhinovirus infection [Id., citing Briend E, et al. Respir Res 2017;18:159] and Alternaria extract stimulation [Id., citing Murai H, et al. Biochem Biophys Res Commun (2015) 464:969–974] in vitro. In addition, cigarette smoke exposure induced IL-18 production by alveolar macrophages in the mouse lungs. [Id., citing Kang MJ, et al. J Immunol (2007) 178:1948– 1959]. In humans, the levels of IL-18 in bronchoalveolar lavage fluids (BALFs) were significantly higher in patients with COPD than in healthy subjects, and even higher in patients with acute exacerbations of COPD. [Id., citing Wang H, et al. Inflammation (2018) 41:1321– 1333]. In addition, the expression of IL-18 in lung epithelial cells was significantly increased in patients with severe COPD compared to healthy individuals who never smoked. [Id., citing Briend E, et al. Respir Res 2017;18:159]. These findings suggest that IL-18 may be involved in the pathophysiology of COPD. ILC2s IL-25 [00043] IL-25 activates ILC2s and promotes type 2 cytokine production. Various kinds of immune cells, such as macrophages, eosinophils and T cells, have been shown to produce IL-25. Recently, bottle-shaped epithelial-lineage cells expressing taste receptors, named tuft cells—including intestinal tuft cells, brush cells in the lower airways and solitary chemosensory cells (SCCs) in nasopharyngeal tissue—have attracted broad attention as major sources of IL- 25. [Id., citing Schneider C, et al. Nat Rev Immunol (2019) 19:584–593]. In mice, intestinal tuft cells produce IL-25 after sensing microbial metabolites through succinate receptors or taste receptors during protozoan and helminth infections, which results in activation of ILC2s and promotion of an anti-helminth response. [Id., citing Schneider C, et al. Nat Rev Immunol (2019) 19:584–593]. Similarly, recent findings have suggested that SCCs in the human upper respiratory tract [Id., citing Kohanski MA, et al. J Allergy Clin Immunol (2018) 142:460– 469.e7] and brush cells in the murine lower respiratory tract [Id., citing Bankova LG, et al. Sci Immunol (2018) 3:eaat9453] are major producers of IL-25 in the airways. Besides allergic disorders, the concentration of IL-25 and the number of ILC2s were increased in BALF from patients with idiopathic pulmonary fibrosis and in the lungs of mice with helminth–induced lung fibrosis compared to controls, [Id., citing Hams E, et al. Proc Natl Acad Sci U S A (2014) 111:367–372] suggesting possible involvement of the IL-25–ILC2 axis in lung fibrosis as well. IL-33 [00044] Unlike other cytokines that are newly synthesized upon stimulation and secreted via the endoplasmic reticulum/Golgi pathway, IL-33 is constitutively expressed in cells at the mucosal barrier and released from the nucleus in active form in response to tissue damage. [Id., citing Cayrol C, Girard JP. Immunol Rev (2018) 281:154–168]. It is believed to be one of the “alarmins” that gather components of the repair response to the sites of injury. However, several studies suggest that IL-33 may be actively secreted from live cells, including bronchial epithelial cells [Id., citing Hristova M, et al. J Allergy Clin Immunol (2016) 137:1545– 1556.e11] and fibroblasts, even in the absence of necrosis. Although the mechanisms of IL-33 secretion are not fully understood, adenosine triphosphate-induced purinoceptor-dependent activation of epithelial nicotinamide adenine dinucleotide phosphate oxidase, i.e., dual oxidase 1, may be involved. [Id., citing Hristova M, et al. J Allergy Clin Immunol (2016) 137:1545– 1556.e11]. IL-33 is recognized as one of the major activators of ILC2s that induce production of type 2 cytokines. In mice, IL-33 is released from alveolar epithelial cells in response to tissue damage caused by fungi such as Alternaria and Aspergillus and viruses such as respiratory syncytial virus (RSV) and rhinovirus (RV). [Id., citing Cayrol C, Girard JP. Immunol Rev (2018) 281:154–168]. Meanwhile, in humans, IL-33 is released from bronchial epithelial cells located more centrally, [Id., citing Cayrol C, Girard JP. Immunol Rev (2018) 281:154–168] similar to IL-25 and thymic stromal lymphopoietin (TSLP). [00045] The expression of IL-33 in the lungs peaks during infancy, and declines with age. The number of ILC2s in the lungs also peaks in infancy. [Id., citing de Kleer IM, et al. Immunity (2016) 45:1285–1298]. These findings suggest that IL-33 may play a major role in the developing phase of acquired immunity and that epithelial damage may induce more severe allergic airway inflammation during infancy than during adulthood through the IL-33–ILC2s axis. In addition to epithelial cells, stromal cells, [Id., citing Dahlgren MW, et al. Immunity (2019) 50:707–722.e6] endothelial cells, fibroblasts [Id., citing Cayrol C, Girard JP. Immunol Rev 2018;281:154–168] and platelets [Id., citing Takeda T, et al. J Allergy Clin Immunol (2016) 138:1395–1403.e6] may produce IL-33. Thymic stromal lymphopoietin (TSLP) [00046] Like other epithelial–derived cytokines such as IL-33 and IL-25, TSLP is recognized as a major activator of ILC2s that induces production of type 2 cytokines. However, unlike other epithelial-derived cytokines, TSLP was shown to induce corticosteroid resistance in murine ILC2s through activation of an intracellular signaling molecule, signal transducer and activator of transcription 5. [Id., citing Kabata H, , et al. Nat Commun 2013;4:2675]. TSLP is produced by various kinds of cells including DCs, vascular endothelial cells, macrophages and mast cells. In the airways, similar to IL-25 and IL-33, TSLP is produced mainly by airway epithelial cells in response to exposure to bacteria, fungi and viruses. [Id., citing Varricchi G, et al. Front Immunol 2018;9:1595]. Adventitial stromal cells localize with ILC2s in adventitial niches around the lung bronchi and large vessels, and support ILC2s through constitutive expression of TSLP and IL-33. [Id., citing Dahlgren MW, et al. Immunity (2019) 50:707– 722.e6]. IL-27 [00047] IL-27 is generally produced by DCs and macrophages. In mice, IL-27 suppresses the proliferation and cytokine production of ILC2 cells in vitro, [Id., citing Moro K, et al. Nat Immunol (2016) 17:76–86, Duerr CU, et al. Nat Immunol (2016)17:65–75] and it also suppresses Alternaria-induced eosinophilic airway inflammation by regulating ILC2 activation in vivo. [Id., citing Moro K, et al. Nat Immunol (2016) 17:76–86]. Interferons [00048] IFNs are classified as type 1 (α/β, κ, ε, ω, δ, τ), type 2 (γ) or type 3 (λ), depending on the type of receptor through which they signal. [00049] The type I IFNs are the largest group and include IFN-α, IFN-β, IFN-ε, IFN-ω, IFN-κ, IFN-δ, IFN-τ and IFN-ζ. Li, S-f et al. Cellular Physiol. & Biochem. (2018) 51: 2377- 96]. The IFN-α family of 12 closely related human genes and IFN-β, the product of a single gene, are best understood; less well studied are IFN-κ, IFN-ε, IFN-ω, IFN-τ and IFN-ζ,, which may offer equal or superior biological activities compared to IFN α/β with less adverse effects. [00050] Type I IFNs combat viral infection both directly by inhibiting viral replication in infected cells and indirectly by stimulating the adaptive immune system [Zhou, Z. et al. J. Virology (2007) 81 (14): 7749-58, citing Biron, C. A. (1994) Curr. Opin. Immunol.6:530-538, Ida-Hosonuma, M., et al. (2005) J. Virol.79:4460-4469, Sen, G. C., and P. Lengyel. (1992). J. Biol. Chem.267:5017-5020, Stark, G. R., et al. (1998) Annu. Rev. Biochem.67:227-264]. [00051] The engagement of type I IFNs and their cell surface receptors (IFN-α- receptors, or IFNARs) activates Janus kinase (JAK)-signal transducer and activator of transcription (STAT)-signaling, which promotes the transcription of a large array of IFN-stimulated genes (ISGs) to exert antiviral activities [He, Y. et a., Sci. Signal.13(2020) eaaz3381]. Although all type I IFNs can bind to IFNARs, their antiviral activities appear to be distinct. For example, IFN-ω exhibits increased anti-influenza activity in cultured cells compared to INF-α2, albeit less than IFN-β1a [Id., citing Skorvanoa, L. et al. Acta Virol.2015] 59: 413-17]. IFN-κ was first identified in human keratinocytes and then in dendritic cells and monocytes [Id., citing Nardelli, B. et al. J. Immunol. (2002) 169: 4822-30]. While it is primarily viewed as a keratinocyte-specific IFN dedicated to skin immune responses, it can also induce antiviral responses in other human cell types. [Id., citing LaFleur, DW, et al. J. Biol. Chem. (2001) 276: 39765-71]. [00052] IFNβ is induced earlier than IFN-α in cultured cells in response to virus infection [Id., citing Honda, K., et al. Immunity (2006) 25: 349-60]; many virus-encoded proteins interfere with the production of IFN through various mechanisms [Id., citing Garda- Sastre, A. Cell Host Microbe (2017) 22: 176-84]. For example, the NS1 proteins of some IAVs are capable of inhibiting the 3’ end processing of cellular pre-mRNAs by binding to cleavage and polyadenylation specific factor (CPSF30) and accordingly blocking the production of mature mRNAs, including those of IFN-α and IFN-β [Id., citing Krug, RM. Curr. Opin. Virol. (2015) 12: 106] [00053] IFN-ε. IFN-ε was first described in 2004. It consists of 192 amino acids and shares about 30% homology with IFN-α and IFN-β in humans [Li, S-F et al. Cell Physiol. Biochem. (2018) 51: 2377-96, citing Zwarthoff, EC et al. Nucleic Acids Res. (1985) 13: 791- 804]. Unlike IFN-α/β, IFN-ε is constitutively expressed in the lung, brain, skin, small intestine, rectum, jejunum, and reproductive tissues; it shows substantial expression in the uterus, cervix, vagina, and ovarian tissue [Id., citing Zwarthoff, EC et al. Nucleic Acids Res. (1985) 13: 791- 804; Demers, A. et al. J. Leukoc. Biol. (2014) 96: 1101-7]. [00054] Studies demonstrate that IFN-ε is positively modulated by hormones, seminal plasma, and TNF-α stimulation and expression correlates negatively with progesterone levels [Id., citing Zwarthoff, EC et al. Nucleic Acids Res. (1985) 13: 791-804; Fung, KY, et al. Science (2013) 339: 1088-928]. Two stable stem-loop structures (loops 1 and 2) were identified in the 5′-untranslated region of IFN-ε mRNA, and they markedly suppresses IFN-ε mRNA expression. However, only loop 1 is essential for enhancing mRNA expression unless the loop structure is disrupted [Id., citing Matsumiya, T. et al. J. Immunol. (2013) 191: 1907- 15]. The molecular transporter and chaperone Importin9 [IPO9], which binds to the IFN-ε 5’- untranslated region (UTR) stem-loop structures, affects IFN-ε constitutive expression. Expression levels of IFN-ε decrease following IPO9 overexpression and increase in response to IPO9 silencing [Id., citing Matsumiya, T. et al. J. Immunol. (2013) 191: 1907-15]. These findings suggest that the IPO9, with the participation of stem-loop structure 1, serves as a negative, specific, post-transcriptional modulator of IFN-ε mRNA. [00055] It has been demonstrated that IFN-ε also exerts its biological activity by stimulating immune mediators and activating the JAK-STAT signal pathways in vitro and in vivo [Id., citing Zwarthoff, EC et al. Nucleic Acids Res. (1985) 13: 791-804]. For example, a recombinant vaccinia virus co-expressing HIV gag or pol genes and murine IFN-ε (VV-HIV- IFN-ε) inhibits growth of VV in L929 murine cell lines and increases upregulation of activation markers (CD69 and CD86) and antiviral protein expression [Id., citing Day, SL et al. J. Immunol. (2008) 180: 7158-66]. Between IFNAR1 and IFNAR2, IFN-ε has a higher binding affinity for IFNAR1 [Id., citing Stifter, SA et al. J. Biol. Chem. (2018) 293: 3168-79]. [00056] There are differences between IFN-ε and other IFNs. First, the antiviral, natural killer cell-cytotoxicity activity and antiproliferative activities of IFN-ε are weaker than IFN-α and IFN-β [Id., citing Peng, FW et al. Protein Expr. Purific. (2007) 53: 356-62]. Second, some studies showed that IFN-ε exhibits antiviral activity against cells derived from species that have near relatives and are expected to be homologous cells [Id., citing Guo, Y. et al. Gene (2015) 558: 25-30; Yang, L. et al. J. Interferon Cytokine Res. (2013) 33: 760-68]. Third, IFN-ε differs from IFN-α in macrophages by inducing an antiviral state mediated by different factors [Id., citing Li, SF et al. Intl Immunopharmacol. (2017) 52: 253-60]. For example, studies have revealed that numbers of genes or expression levels induced by type I IFN, IL-6, and TNF pathways in response to IFN-α and IFN-ε are not identical, in spite of some overlap among IL- 1α, IL-1RA, IL-4, VEGF, and GCSF [Id., citing Li, SF et al. Intl Immunopharmacol. (2017) 52: 253-60]. IFN-α mediates more genes and upregulates genes more than IFN-ε in the type I IFN signaling pathway, whereas IFN-ε induces more genes in the TNF-α pathway and more ROS generation and phagocyte activation than IFN-α, to block HIV replication [Id., citing Li, SF et al. Intl Immunopharmacol. (2017) 52: 253-60]. [00057] IFN-ω. IFN-ω genes have been identified in humans as well as in other animal groups including feline, porcine, equine, rabbit, cattle, and serotine bat, but not noted in canines or mice [Id., citing Zhou, H. et al. Int. J. Mol. Sci. (2014) 15: 21045-68]. Treatment with IFN- ω is suggested to be effective for patients who are resistant to IFN-α, because the antigenic structure of IFN-ω is distantly related to IFN-α, β, λ, with no crossreaction with antibodies against these other IFNs [Id., citing Adolf, GR. Mult. Scler. (1995) S44-47]. Recombinant FeIFN-ω is approved for treatment of FLV and FIV infections in some countries. FIFN-ω has 13 subtypes that have high similarity (95% to 99%) at the nucleic acid and amino acid level. All of them contain an N-terminal secretory signal sequence at position 1 to 23. Lengths of mature FeIFN-ω subtype polypeptides are 173 aa (except FeIFN-ω2 and FeIFN-ω4 which have 180 aa). The mature amino acid sequence of FeIFN-ω has six additional amino acids at the carboxyl-end and an N-glycosylation recognition site that differs from other mammalian subtypes [Id., citing Yang, LM et al. J. Interferon Cytokine Res. (2007) 27: 119-27]. In addition, seven prolines are conserved among these subtypes, four at positions 4, 26, 39, and 117 of mature proteins, similar to other mammalian IFN-ω proteins at positions 4, 26, 39, and 116, respectively [Id., citing Yang, LM et al. J. Interferon Cytokine Res. (2007) 27: 119-27; Roberts, RM et al. Prog. Nucleic Acid Res. Mol. Biol. (1997) 56: 287-325]. The cysteines at positions 1, 29, 100, and 140 of the mature proteins correspond to IFN-ω at positions 1, 29, 99, and 139. [00058] IFN-ω has antiviral activities similar to other types I IFNs. However, unlike IFN-α, it has cross-species activity to some extent. This activity indicates that cells have a tendency to be insensitive to IFN-ω from distantly related species [Id., citing Guo, Y. et al. Gene (2015) 558: 25-30]. IFN-ω is involved in the nonspecific response based on increased expression of several acute phase proteins and MHC I molecules; upregulation of the phagocytic activities of whole blood cells, macrophages and NK cell activities; and decreased concurrent viral excretion [Id., citing Domenech, A. et al. Vet Immunol. Immunopathol. (2011) 143: 301-6; Gil, S. et al. Res. Vet Sci (2014) 96: 79-85; DeMari, K. et al. J. Vet Intern. Med. (2004) 18: 477-82; Leal, RO et al. J. Small Anim. Pract. (2014) 55: 39-45]. Different therapy protocols might also contribute to the distinct expression of innate immunity cytokines following IFN-ω treatment. For example, IL-6 plasma levels decrease and proviral load increase in FIV-cats treated with rFeIFN-ω by a subcutaneous licensed protocol. IL-6 mRNA expression decreases in an oral group. Viremia and other cytokines (IL-1, IL-4, IL-10, IL- 12p40, IFN-γ and TNF-α) do not change with therapy [Id., citing Leal, RO et al. Res. Vet Sci. (2015) 99: 87-95]. Cytotoxic effects (e.g. apoptosis, necrosis, and early senescence) of human IFNβ gene lipofection induced by catiolic lipid-mediated interferon-beta gene transfer to human tumor cells were reported to show the same or a superior effect to that of high doses of the exogenously applied recombinant IFNβ protein [Id., citing Villaverde, MS, et al., Cancer Gene Ther. (2012) 19: 420-30]. Based on these findings, the researchers also found that fIFN- ω lipofection and expression is equal to or more effective than rFIFN-ω protein at suppressing cell growth by inducing ROS generation, mitochondrial potential disruption and calcium uptake [Id., citing Villaverde, MS et al. Cytokine 84: 47-55]. [00059] IFN-κ. IFN-κ is mainly expressed in the uterus [Li, S-f et al. Cell Physiol. Biochem. (2018) 51: 2377-96], although it is also detected in other tissues, such as Peyer’s patch, ovary, liver and peritoneal macrophages, although at low levels. [00060] He et al. reported a study in which they analyzed the expression of genes encoding different type I IFNs during infection of epidemic-causing H7N9 virus and an H9N2 virus in a mouse model [He, Y. et al. Science Signaling (2020) 13 (626) DOI: 10.1126/scisignal.aaz3381]. They identified IFNK, the gene that encodes IFN-κ, as the most differentially expressed type I IFN gene in the early phase of infection, being the only one increased after H9N2 infection, but decreased after H7N9 infection. They then used cultured human cells to study the action of IFN-κ against IAV infection. On the basis of the identification of a mutant IFNK gene in human lung epithelial A549 cells and subsequent demonstration that wild-type IFN-κ, but not the mutant, failed to contain IAV in cultured human cells, they pinned down chromodomain helicase DNA binding protein 6 (CHD6) as the major effector molecule mediating the anti-influenza activity of IFN-κ. Compared to its induction by IFN-κ, CHD-6 was less induced by IFN-α and IFN-β [collectively IFN-α/β] and dispensible for IFN-α/β-mediated inhibition of IAV replication. They also identified the upstream signaling required by IFN-κ to stimulate CHD6 expression. Unlike IFN-α/β, which transduce antiviral signal preferentially through IFNAR1, IFN-κ required the engagement of both IFNAR1 and IFNAR2. [00061] The binding by IFN-κ to the individual IFNARs is weaker than the binding of IFN-α/β [Id., citing Harris, BD et al. J. Biol. Chem. (2018) 293: 16057-068], suggesting different modes of action between IFN-α/β and IFN-κ. IFN-κ also was distinct in that it induced CHD6 through a p38-cFos axis, rather than the canonical JAK-STAT pathway. IFNα/β therefore use multiple signaling pathways to activate a diverse array of ISGs, exerting profound effects on both virus and cells, while IFN-κ instead exhibited a selective use of downstream signaling, resulting in a relatively narrower spectrum of downstream targets, among which some effector genes are preferentially stimulated, as seen for CHD6. Such focused strategy, underlying the observed dominance of a single effector molecule in the antiviral activity of IFN-κ-CHD6 for influenza as shown here, and Sp100 for human papillomavirus (HPV) as shown in a previous study [Id., citing Habiger, C. et al. J. Virol. (2016) 90: 694-704] may bestow a benefit on the host by constraining responding cells from overreacting. Last, they showed that preapplication of IFN-κ protected mice against lethal IAV infection with H7N9. [00062] IFN-δ. IFN-δ seems more closely related to the IFN-α, IFN-τ, and IFN-ω cluster than to IFN-β. [Li, S-f et al. Cell Physiol. Biochem. (2018) 51: 2377-96]. The IFN-δ family comprises a large number of members with diversity that is greater than other multigene porcine or horse IFN families such as IFN-α and IFN-ω [Id., citing Zhao, X. et al. J. Interferon Cytokine Res. (2012) 32: 378-85]. Some IFN-δs are probably highly glycosylated, as they display one or two potential N-glycosylation sites in OvIFN-δ and the porcine IFN. All IFN- δs have two other cysteine residues, Cys77 and Cys128, except for IFN-δ2 and IFN-δ7, which have an additional COOH-terminal cysteine residue, Cys166. IFN-δs exhibit antiviral and immunomodulatory activity through typical type I IFN signaling, with lower antiviral activities than IFN-α [Id., citing Sang, Y. et al. Physiol. Genomics (2010) 42: 248-58]. However, the differential affinity of IFN-δ for certain hosts could influence their biological activities. [00063] IFN-τ. IFN-τ, which is involved in maternal recognition of pregnancy, shares about 75% identity with IFN-ω and has 172 aa with two disulfide bridges (1-99, 29-139) and an amino terminal proline. Not every IFN-τ is glycosylated. For instance, ovine IFN-τ lacks glycosylation, while bovine IFN-τ is N-glycosylated at ASN78 and caprine IFN-τ is a mixture of nonglycosylated and glycosylated forms. Although secretion is specific to ruminant mammals (e.g., sheep, cows, oxen, goats, gazelle, giraffe and deer), all have several variants of IFN-τ, except for giraffes, and 7 putative analogs of IFN-τ are identified in humans [Id., citing Roberts, RM. Cytokine Growth Factor Rev. (2007) 18: 403-8; Whaley, AE et al. J. Biol. Chem. (1994) 269: 10864-8; Duc-Goiran, P. et al. Proc. Nat. Acad. Sci. USA (1985) 82: 5010- 4; Leaman, DW and Roberts, RM. J. Interferon Res. (1992) 12: 1-11]. Studies show that human trophoblast IFN in placental trophoblast cells has 85% sequence identity to IFN-τ in ruminants [Id., citing DeCarlo, CA et al. Lab Invest. (2010) 90: 1482-91]. [00064] Similar to IFN-α and IFN-β, IFN-τ possesses antiviral activity and antiproliferative effects. IFN-τ has a receptor binding domain at the C-terminus and a biologically active site at the N-terminus [Id., citing Pontzer, CH et al. J. Interferon Res. (1994) 14: 133-41]. IFN-τ is suggested to have comparable antiviral activity effects as IFN-α from the same species. It has high species specificity and some biological activities are remarkably decreased when administered to another species [Id., citing Ealy, AD et al. Biol. Reprod. (1998) 58: 566-73; Ealy, AD et al. Endocrinol. (2001) 142: 2906-15]. IFN-τ also stimulates some interleukin expression and secretion such as IL-6 and IL-8. However, the mechanism involved in inducing cytokine secretion is dependent on STAT3 rather than STAT1 signaling [Id., citing Tanikawa, N. et al. J. Interferon Cytokine Res. (2017) 37: 456-66]. IFN-τ displays > 30 times less toxicity than IFN-α [45, 88]. This difference in cytotoxicity is illustrated by the differential selectivity of individual N-termini towards receptors and the differential degree of receptor avidity [Id., citing Pontzer, CH et al. J. Interferon Res. (1994) 14: 133-41]. [00065] IFN-ζ. A seemingly secreted glycoprotein, IFN-ζ is composed of 182 aa residues with a signal peptide of 21 amino acids at the N-terminal end and an N-linked glycosylation site at amino acid residue 68. IFN-ζ lacks an internal transmembrane domain. It appears to have an IFN-like globular structure of five long α-helices and one short helix in the middle of a loop connecting helices B and C, with possible disulfide bonds between residues 52 and 157 and between residues 80 and 130 [Id., citing Oritani, K. et al. Cytokine Growth Factor Rev. (2001) 12: 337-48]. IFN-ζ shares high nucleotide homology with IFN-α and IFN- β at residues 45-60, 105-115, and 135-165, corresponding to the N-terminal half of the AB loop, the C helix, and the DE loop, together with helices D and E [Id., citing Oritani, K. et al. Cytokine Growth Factor Rev. (2001) 12: 337-48]. [00066] IFN-ζ shares activities with other type I IFNs such as IFN-α: It induces the surface expression of MHC class I, enhances CTL activities, and inhibits growth of lymphohematopoietic cell lines as strongly as IFN-α. This kind of IFN has relatively higher antiviral activity than IFN-α [Id., citing Kawamoto, S. et al. Exp. Hematol. (2004) 32: 797- 805]. Apart from their antiviral effects, other differences exist among them: (I) Signals induced by IFN-ζ are similar but distinct in contrasted to signals of other type I IFNs. Studies revealed that IFN regulatory factor-1 (IRF-1) dependency for antiviral activities is distinct between IFN- ζ and IFN-α. A higher concentration of IFN-ζ is needed compared to IFN-α for antiviral activity and transcription of proteins in IRF-1-deficient fibroblasts [Id., citing Kawamoto, S. et al. J. Virol. (2003) 77: 9622-31] (II) Some common adverse effects such as myelosuppression and fever are not observed in mice treated with IFN-ζ compared to IFN-α [Id., citing Kawamoto, S. et al. Exp. Hematol. (2004) 32: 797-805] (III) IFN-ζ does not inhibit colony production of myeloid and erythroid progenitors while IFN-α is known to suppress lymphohematopoiesis [Id., citing Kawamoto, S. et al. Exp. Hematol. (2004) 32: 797-805]. In addition, IFN-ζ suppresses the proliferation of megakaryocyte progenitors without influencing megakaryocyte differentiation, although higher concentrations are required. A possible explanation is that IFN- ζ induces lower expression of Daxx and weaker phosphorylation of Tyk2 and Crk than IFN-α [Id., citing Ishida, N. et al. Exp. Hematol. (2005) 33: 495-503] (IV) Formation of IFN-ζ is distinct from other known IFNs. IFN-ζ is constitutively produced by mature T lymphocytes in the spleen and thymus and by bronchial epithelial and salivary duct cells in healthy mice [Id., citing Oritani, K. et al. Blood (2003) 101: 178-85] (V) Unlike IFN-α/β, IFN-ζ gene expression in lymph nodes is unchanged with lipopolysaccharide injection or herpes simplex virus infection [Id., citing Oritani, K. et al. Blood (2003) 101: 178-85]. [00067] Type II IFN-γ. IFN-γ is the sole type II interferon; the dominating biological role of IFN-γ seems to be stimulation of the adaptive immune system, primarily activation of T cells [ Zhou, Z. et al. J. Virology (2007) 81 (14): 7749-58., citing Biron, C. A. (1994). Curr. Opin. Immunol.6:530-538, Muller, U., et al. (1994) Science 264:1918-1921]. [00068] Type III IFNs. Type III interferons are the products of three IFN-γ genes, IL- 28A, IL-28B, and IL-29, which bind a heterodimeric IFN-λ receptor composed on a unique IL- 28Rα subunit and the β subunit of the IL-10 receptor. Unlike the ubiquitously expressed type I IFNR complex, the type III IFNR has a more restricted tissue distribution pattern. Although the IL10R2 chain is ubiquitously expressed in all tissues and cells, the expression of the IFNλR1 varies widely between different organs and at the cellular level is restricted to epithelial cells. [Zhou, P. et al., PLoS One (2011) 6(9): e15385, citing Witte K, Witte E, et al. Cytokine Growth Factor Rev. (2010) 21:237–251, Witte K, et al. Genes Immun. (2009) 10:702–14; Sommereyns C, et al. PLoS Pathog. (2008) 4:e1000017; Donnelly RP, et al. J Leukoc Biol. (2004) 76:314–21. [00069] Type I interferons are inducible and are synthesized by many cell types after infection by diverse viruses. Almost all types of cells can produce IFN-α and IFN-β in response to activation of several innate sensors. For example, type I interferons are induced by RIG-1 and MDA-5 (the sensors of cytoplasmic viral RNA) downstream of MAVs, and by signaling from cGAS (the sensor of cytoplasmic DNA) downstream of STING. Plasmacytoid dendritic cells (pDCs), also called interferon-producing cells (IPCs) or natural interferon-producing cells, make abundant type I interferons, which may result from the efficient coupling of viral recognition by TLRs to the pathways of interferon production. pDCs express a subset of TLRs that includes TLR-7 and TLR-9, which are endosomal sensors of viral RNA and of the nonmethylated CpG residues present in the genomes of many DNA viruses. pDCs express CXCR3, a receptor for chemokines CXCL9, CXCL10, and CXCR11, which are produced by T cells, which allows pDCs to migrate from the blood into lymph nodes in which there is an ongoing inflammatory response to a pathogen. [Janeway’s Immunology, 9th Ed. (2017) Garland Science, New York, pp 122-125] [00070] Interferons help defend against viral infections in several ways. IFN-β induces cells to make IFN-α, thus amplifying the interferon response. Interferons act to induce a state of resistance to viral replication in all cells. IFN-α and IFN-β bind to a common cell surface receptor, the interferon-a receptor (IFNAR), which uses the JAK and STAT pathways. IFNAR uses the kinases Tyk2 and Jak1 to activate the factors STAT1 and STAT2, which can interact with IRF9 and form a complex called ISGF4, which binds to the promoters of many interferon stimulated genes (ISGs). [Janeway’s Immunology, 9th Ed. (2017) Garland Science, New York, pp 122-125] [00071] One ISG encodes the enzyme oligoadenylate synthetase, which polymerizes ATP into 2’-5’ linked oligomers, which activate an endoribonuclase that then degrades viral RNA. A second protein induced by IFN-α and IFN-β is protein kinase R (PKR), a dsRNA- dependent protein kinase, which phosphorylates the α subunit of eukaryotic initiation factor 2 (eIF2α), thus suppressing protein translation and contributing to the inhibitor of viral replication. Mx (myxoma resistant) proteins also are induced by type I interferons. Mx1 and Mx2 are GTPases belong to the dynamin protein family; how they interfere with virus replication is not understood. [Janeway’s Immunology, 9th Ed. (2017) Garland Science, New York, pp 122-125] [00072] The interferon-induced protein with tetratricoid repeats (IFIT) family contains four human and three mouse proteins that function in restraining the translation of viral RNA into proteins. IFIT1 and IFIT2 can suppress the translation of normal capped mRNAs by binding to subunits of the eukaryotic initiation factor 3 (eIF3) complex, which prevents eIF3 from interacting with eIF2 to form the 43S pre-initiation complex. Mice lacking IFIT1 or IFIT2 show increased susceptibility to infection by certain viruses, e.g., vesicular stomatitis virus. IFIT1 also suppresses translation of viral RNA that lacks a normal host modification of the 5’ cap. Many viruses, e.g., West Nile virus, and SARS coronavirus, have acquired a 2’e-O- methyltransferase (MTase) that produce cap-1 or cap-2 on their viral transcripts, thus evading restriction by IFIT1. [Janeway’s Immunology, 9th Ed. (2017) Garland Science, New York, at 122-125] [00073] Members of the interferon-induced transmembrane protein (IFITM) family are strongly induced by type I interferons. There are four functional IFITM genes in humans and in mice; these encode protein that have two transmembrane domains and are localized to various vesicular compartments of the cell. IFITM protein act to inhibit, or restrict, viruses at early steps of infection. IFITM1 appears to interfere with the fusion of viral membranes with the membrane of the lysosome, which is required for introducing some viral genomes into the cytoplasm. Viruses that must undergo this fusion event in lysosomes, e.g., Ebola virus, are restricted by IFITM1. IFITM2 interferes with membrane fusion in late endosomes, and so restricts the influenza A virus, which undergoes fusion there. [Janeway’s Immunology, 9th Ed. (2017) Garland Science, New York, at 122-125] [00074] Interferons stimulate production of the chemokines CXCL9, CXCL10, and CXCL11, which recruit lymphocytes to sites of infection, and increases expression of MHC class I molecules on all types of cells. [Janeway’s Immunology, 9th Ed. (2017) Garland Science, New York, at 122-125] [00075] Type 1 and 2 IFNs have been shown to suppress type 2 cytokine production by ILC2s, both in vitro and in vivo. [Id., citing Moro K, et al. Nat Immunol (2016) 17:76–86, Duerr CU, et al. Nat Immunol (2016) 17:65–75]. The major producers of IFN-α and -β are macrophages and DCs. IFN-γ is produced by activated Th1 cells and ILC1s, including NK cells, which are activated mainly through TLRs. In mice, the deficiency of type 1 IFN during influenza virus and helminth infections results in severe or prolonged eosinophilic airway inflammation mediated by activated ILC2s. In humans, dozens of reports have shown impaired production of type 1 and 3 IFNs by cultured primary bronchial epithelial cells, BAL cells, peripheral blood mononuclear cells (PBMCs) and plasmacytoid DCs in response to infection with viruses such as RSV and rhinovirus (RV) in patients with asthma compared to healthy individuals. [Id., citing Edwards MR, et al. J Allergy Clin Immunol (2017) 140:909–920]. Therefore, dysregulation of ILC2 activity by type 1 and 3 IFNs during viral infection in asthmatic patients may result in the development and exacerbation of allergic airway inflammation. Lipid inflammatory mediators [00076] Although lipids are primarily involved in the formation of cell membranes of organs, various reports have shown that bioactive lipids or lipid mediators also play crucial roles in immune responses and the maintenance of homeostasis. Cysteinyl leukotrienes (CysLTs) as well as prostaglandin (PG) D2 are products of arachidonic acid and were known to be major pro-inflammatory lipid mediators of allergic disorders from early days. Mast cells activated by immunoglobulin (Ig) E-crosslinking are the major source of PGD2 in terms of quantity, but other leukocytes, including eosinophils, Th2 cells, DCs and cytokine-activated ILC2s, [Id., citing Maric J, et al. J Allergy Clin Immunol (2019) 143:2202–2214.e5] also produce PGD2. Since human ILC2s are identified as lineage-negative cells expressing chemoattractant receptor-homologous molecules on Th2 cells (CRTH2), [Id., citing Mjösberg JM, et al. Nat Immunol (2011) 12:1055–1062] which is the PGD2 receptor, PGD2 influences ILC2s in a variety of ways, including their migration [Id., citing Winkler C, et al. J Allergy Clin Immunol (2019) 144:61–69.e7] and production of IL-13. [Id., citing Doherty TA, Broide DH. J Allergy Clin Immunol (2018) 141:1587–1589]. CysLTs are generally produced by leukocytes such as eosinophils, mast cells, macrophages and basophils. CysLTs act directly on ILC2s to enhance their ability to produce type 2 cytokines, both in vivo and in vitro. [Id., citing Doherty TA, Broide DH. J Allergy Clin Immunol (2018) 141:1587–1589]. There are some lipid molecules that inhibit ILC2 activation. PGI2, PGE2 and lipoxin A4—also products of arachidonic acid—suppress ILC2s' cytokine production and proliferation, in vitro and in vivo. [Id., citing Doherty TA, Broide DH. J Allergy Clin Immunol (2018) 141:1587–1589]. LTE4 and PGD2 reportedly induce Th2 cytokines, including IL-4, synergistically in purified human peripheral blood ILC2s. [Id., citing Salimi M, et al. J Allergy Clin Immunol (2017) 140:1090– 1100.e11]. Neuropeptides [00077] Neuropeptides are peptides that are expressed in the nervous system and exhibit physiological activity. They are present not only in the central nervous system, but also in the nervous system of peripheral tissues such as the lungs, and they function as signal transmitters between cells. Among several neuropeptides known to act on ILC2s, vasoactive intestinal peptide (VIP) was the first one shown to modulate ILC2 activation. VIP belongs to the glucagon/secretin family and is highly expressed in intestinal neurons, where it coordinates pancreatic secretion with smooth muscle relaxation in response to feeding. Both lung and intestinal ILC2s express VIP receptors, including VIP receptor type 1 and type 2, and VIP simulation induces IL-5 production by the cells. The IL-5 produced in turn activates sensory neurons to produce VIP [Id., citing Talbot S, et al. Neuron (2015) 87:341–354], which may exacerbate allergic airway inflammation. Lung ILC2s also express receptors for another neuropeptide, called neuromedin U (NMU), whereas ILC1s and ILC3s do not. NMU is thought to directly activate lung ILC2s to proliferate and produce type 2 cytokines. [Id., citing Wallrapp A, et al. Nature (2017) 549:351–356]. Calcitonin gene-related peptide (CGRP) is a calcitonin gene product, like the thyroid hormone calcitonin and it is involved in the regulation of blood calcium levels. CGRP is widely distributed in the central and peripheral nervous systems; It was also produced by non-neuronal cells in the airways—called pulmonary neuroendocrine cells (PNECs)─after OVA challenge in an OVA-sensitized mouse model. [Id., citing Sui P, et al. Science (2018) 360:eaan8546]. It has been reported that ILC2s are localized in close proximity to PNECs and that CGRP enhances type 2 cytokine production by lung ILC2s in the presence of IL-33 or IL-25, [Id., citing Sui P, et al. Science (2018) 360:eaan8546] suggesting that interaction between PNECs and ILC2s may be involved in allergic airway inflammation. Besides the neuropeptides that induce activation of ILC2s, there is also a neuropeptide that regulates activation of ILC2s. Both lung and intestinal ILC2s express the β2-adrenergic receptor (β2-AR), which is a receptor for epinephrine released by sympathetic nerve stimulation. Treatment with a β2-AR agonist, salmeterol, suppressed proliferation and type 2 cytokine production by ILC2s in an IL-33-induced airway inflammation model. (Id., citing Moriyama S, et al. Science (2018) 359:1056–1061). These findings suggest that β2-AR agonists used as therapeutic agents for asthma may work not only as a bronchodilator, but also as a suppressor of type 2 inflammation induced by ILC2s. Sex Steroids [00078] Sex steroids, such as estrogen and androgen, are steroid hormones that are produced mainly by the reproductive organs and modulate reproductive functions. In addition to their effects on the reproductive organs, sex steroids have recently been shown to have effects on immune cells, including ILC2s in peripheral tissues. Androgen receptors are expressed on lung ILC2s [Id., citing Cephus JY, et al. Cell Reports (2017) 21:2487–2499] as well as ILC2 progenitors (ILC2Ps) in bone marrow (BM), [Id., citing Laffont S, et al. J Exp Med 2017;214:1581–1592], whereas estrogen receptors are expressed on lung ILC2s and uterine ILC2s [Id., citing Bartemes K, et al. J Immunol (2018) 200:229–236], but not on ILC2Ps in BM. [Id., citing Laffont S, et al. J Exp Med (2017) 214:1581–1592]. These findings indicate that androgens may influence both the development of ILC2s in BM and the activation of ILC2s in peripheral tissues, whereas estrogens may influence mainly ILC2s in peripheral tissues. Androgens and estrogens are thought to exert opposite effects on ILC2s. Androgen signaling inhibits differentiation of ILC2Ps into ILC2s [Id., citing Laffont S, et al. J Exp Med (2017) 214:1581–1592] and also activation of ILC2s. (Id., citing Cephus JY, et al. Cell Reports (2017) 21:2487–2499, Laffont S, et al. J Exp Med (2017) 214:1581–1592). In contrast, estrogen has been suggested to have supportive effects on ILC2s. [Id., citing Bartemes K, et al. J Immunol (2018) 200:229–236]. Indeed, the numbers of lung ILC2s [Id., citing Laffont S, et al. J Exp Med (2017) 214:1581–1592] and BM ILC2Ps are significantly lower in adult male mice than in adult female mice in the steady state. [Id., citing Laffont S, et al. J Exp Med (2017) 214:1581–1592]. ILC3s IL-23 [00079] IL-23 is a major activator of ILC3s that induces production of inflammatory cytokines such as IL-17 and IL-22. IL-23 also induces conversion of ILC1s to ILC3s in conjunction with IL-1β and retinoic acid, [Id., citing Bernink JH, et al. Immunity (2015) 43:146–160] and ILC2s to ILC3s in conjunction with IL-1β and TGF-β. [Id., citing Bernink JH, et al. Nat Immunol (2019) 20:992–1003, Golebski K, et al. Nat Commun (2019) 10:2162]. IL-23 is generally produced by DCs and macrophages. IL-1β [00080] IL-1β is a major activator of ILC3s that induces IL-17A production. [Id., citing Kim HY, et al. Nat Med (2014) 20:54–61] While IL-1β is a potent activator of ILC2s that induce type 2 cytokine production, [Id., citing Bal SM, et al. Nat Immunol (2016) 17:636–645] it also induces conversion of ILC2s to ILC1Ss together with IL-12, [Id., citing Bal SM, et al. Nat Immunol (2016) 17:636–645, Ohne Y, et al. Nat Immunol (2016) 17:646–655] and to ILC3s together with IL-23 and TGF-β. [Id., citing Golebski K, et al. Nat Commun (2019) 10:2162]. In the airways, IL-1β is produced by DCs in response to exposure to chitin and IL- 33 [Id., citing Arae, K. et al. Sci. Rep. (2018) 8: 11721] and by nasal epithelial cells exposed to Staphylococcus aureus or Pseudomonas aeruginosa. [Id., citing Golebski K, et al. Nat Commun (2019) 10:2162]. Vitamins [00081] Retinoic acid (RA)–which is a metabolite of vitamin A (Vit A)–and vitamin D (Vit D) is known to regulate ILCs. [Id., citing Morita H, et al. J Allergy Clin Immunol (2019) 143:2190–2201.e9, Seehus CR, et al. Nat Commun (2017) 8:1900, Bernink JH, et al. Immunity (2015) 43:146–160, Golebski K, et al. Nat Commun (2019) 10:2162, Konya V, et al. J Allergy Clin Immunol (2018) 141:279–292]. RA is synthesized from a Vit A metabolite, retinal, by cells having enzymes such as retinaldehyde dehydrogenase (ALDH)1A1, ALDH1A2 and ALDH1A3. RA is generally synthesized by CD103+ DCs, intestinal epithelial cells and lamina propria stromal cells in the gut that express ALDHs. In the airways, bronchial epithelial cells express ALDHs in response to IL-13 stimulation [Id., citing Morita H, et al. J Allergy Clin Immunol (2019) 143:2190–2201.e9], suggesting that these cells could be the source of RA during allergic airway inflammation. Vit D can be absorbed by oral intake, but it is synthesized mainly in the skin upon exposure to ultraviolet light from the sun. RA enhances activation of ILC3s by IL-1β and IL-23 to increase production of IL-22, and it also induces conversion of ILC1s to ILC3s in conjunction with IL-1β and IL-23. [Id., citing Bernink JH, et al. Immunity (2015) 43:146–160]. In addition, RA inhibits development of ILC2s from ILC2Ps in mouse BM69 and induces conversion of ILC2s to IL-10–producing ILCregs in both humans and mice. [Id., citing Morita H, et al. J Allergy Clin Immunol (2019) 143:2190–2201.e9, Seehus CR, et al. Nat Commun (2017) 8:1900]. In contrast to the positive effects of RA on ILC3s, Vit D suppresses production of cytokines such as IL-22, IL-17F and GM-CSF by ILC3s by down- regulating the IL-23/IL-23R pathway, [Id., citing Konya V, et al. J Allergy Clin Immunol (2018) 141:279–292] and it also prevents IL-1β-, IL-23- and TGF-β-induced conversion of ILC2s to ILC3s. [Id., citing Golebski K, et al. Nat Commun (2019) 10:2162]. [00082] ILC3s are emerging as key orchestrators and regulators of adaptive immune responses, either through indirect modulation of bystander cells that subsequently modulate the adaptive immune response or directly via both soluble mediators and cell contact-dependent interactions with adaptive lymphocytes. [Domingues, RG, Hepworth, MR. Front. Immunol. (2020) 11:116]. In addition to their function as tissue-resident cytokine producing cells, ILC3s have the capacity to participate in multiple cellular circuits through direct cell-cell modulation of T cell responses, as well as the release of soluble mediators that augment adaptive immune function and development. For example, ILC3s can control the magnitude and quality of the CD4+ T cell response via antigen presentation in the context of MHC class II. At steady state, ILC3s lack co-stimulatory molecule expression and appear to limit CD4+ T cell responses; however, this interaction may be altered in inflammatory scenarios via upregulation of costimulatory molecules such as CD4- CD80 and CD86, which favor the promotion of a T cell response. Further, ILCs act to modulate the survival of recirculating memory CD4+ T cells via interactions via OX40L and CD30L. In addition, ILC3 regulation of T follicular helper (TFH) cell responses has consequences for the priming of germinal center B cells and the induction of T dependent IgA responses toward colon-dwelling commensal microbes. ILC3s also can modulate adaptive immune cells through the production of regulatory cytokines and growth factors. For example ILC3 directly support B cell responses in the spleen through provision of critical growth factors such as BAFF/APRIL. Similarly they modulate the magnitude of the T cell response within the intestinal tract through production of soluble mediators. For example, ILC3-derived IL-22 induces epithelial serum amyloid A (SAA) protein, which subsequently promotes local Th17 responses and acts to limit colonization with segmented filamentous bacteria (SGF) via induction of antimicrobial peptides. In addition, ILC3 facilitate the establishment of a regulatory and tolerogenic environment in the gut by promoting Treg responses. Finally ILC subsets are a potent source of IL-2 in the small intestine which provide survival signals for Tregs. [Domingues, RG, Hepworth, MR. Front. Immunol. (2020) 11:116]. [00083] While most pathogens can overcome innate immune responses, the adaptive immune response is required to eliminate them and to prevent subsequent reinfection. Adaptive immune response [00084] The adaptive arm involves a specific, delayed and longer-lasting response by various types of cells that create long-term immunological memory against a specific antigen. It can be further subdivided into cellular and humoral branches, the former largely mediated by T cells and the latter by B cells. This arm further encompasses cell lineage members of the adaptive arm that have effector functions in the innate arm, thereby bridging the gap between the innate and adaptive immune response. [00085] Generally speaking, these immune responses are initiated by an encounter between an individual and a foreign substance, e.g., an infectious microorganism. The infected individual rapidly responds with both a humoral immune response with the production of antibody molecules specific for the antigenic determinants/epitopes of the immunogen, and a cell mediated immune response with the expansion and differentiation of antigen-specific regulatory and effector T-lymphocytes, including cells that produce cytokines and killer T cells, capable of lysing infected cells. Primary immunization with a given microorganism evokes antibodies and T cells that are specific for the antigenic determinants/epitopes found on that microorganism; these usually fail to recognize or recognize only poorly antigenic determinants expressed by unrelated microbes [Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia, (1999), at p.102]. [00086] As a consequence of this initial response, the immunized individual develops a state of immunologic memory. If the same or a closely related microorganism is encountered again, a secondary response ensues. This secondary response generally consists of an antibody response that is more rapid, greater in magnitude and composed of antibodies that bind to the antigen with greater affinity and that are more effective in clearing the microbe from the body, and a similarly enhanced and often more effective T-cell response. However, immune responses against infectious agents do not always lead to elimination of the pathogen [Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia, (1999), at p.102]. Compartmentalization of the immune system [00087] The periphery of the immune system--as opposed to the central lymphoid organs--contains inhomogeneously distributed B and T cells whose phenotype, repertoire, developmental origin, and function are highly divergent. Nonconventional lymphocytes bearing a phenotype that is rare in the blood, spleen, or lymph nodes of undiseased individuals are encountered at high frequency in different localizations, e.g., alpha/beta TCR+CD4-CD8- cells in the bone marrow and gut epithelium, particular invariant gamma/delta TCR+CD4-CD8 alpha+CD8 beta- and gamma/delta TCR+CD4-CD8 alpha-CD8 beta- T cells in various epithelia, or CD5+ B cells in the peritoneum. The antigen receptor repertoire is different in each localization. Thus, different gamma/delta TCR gene products dominant in each site, and the proportion of cells expressing transgenic and endogenous alpha/beta TCR and immunoglobulin gene products follows a gradient, with a maximum of endogenous gene expression in the peritoneum, intermediate values in other peripheral lymphoid organs (spleen, lymph nodes), and minimum values in thymus and bone marrow. Forbidden T cells that bear self-superantigen-reactive V beta gene products are physiologically detected among alpha/beta TCR+CD4-CD8- lymphocytes of the bone marrow, as well as in the gut. Violating previous ideas on self-tolerance preservation, self-peptide-specific gamma/delta T cells are present among intestinal intraepithelial lymphocytes, and CD5+ B cells produce low-affinity cross- reactive autoantibodies in a physiological fashion. It appears that, in contrast to the bulk of T and B lymphocytes, certain gamma/delta and alpha/beta T cells found in the periphery, as well as most CD5+ B cells, do not depend on the thymus or bone marrow for their development, respectively, but arise from different, nonconventional lineages. In addition to divergent lineages that are targeted to different organs guided by a spatiotemporal sequence of tissue- specific homing receptors, local induction or selection processes may be important in the diversification of peripheral lymphocyte compartments. Selection may be exerted by local antigens, antigen-presenting cells whose function varies in each anatomical localization, cytokines, and cell-matrix interactions, thus leading to the expansion and maintenance of some clones, whereas others are diluted out or deleted. [00088] In multicellular organisms, cells that are specialized to perform common functions are usually organized into cooperative assemblies embedded in a complex network of secreted extracellular macromolecules, the extracellular matrix (ECM), to form specialized tissue compartments. Individual cells in such tissue compartments are in contact with ECM macromolecules. The ECM helps hold the cells and compartments together and provides an organized lattice or scaffold within which cells can migrate and interact with one another. In many cases, cells in a compartment can be held in place by direct cell-cell adhesions. In vertebrates, such compartments include four major types, a connective tissue (CT) compartment, an epithelial tissue (ET) compartment, a muscle tissue (MT) compartment and a nervous tissue (NT) compartment, which are derived from three embryonic germ layers: ectoderm, mesoderm and endoderm. The NT and portions of the ET compartments are differentiated from the ectoderm; the CT, MT and certain portions of the ET compartments are derived from the mesoderm; and further portions of the ET compartment are derived from the endoderm. [00089] The lifelong production of blood cells depends on hematopoietic stem cells (HSC) and their ability to self-renew and to differentiate into all blood lineages. Hematopoietic stem cells (HSCs) develop during embryogenesis in a complex process that involves multiple anatomical sites (the yolk sac, the aorta-gonadmesonephros region, the placenta and the fetal liver), Once HSC precursors have been specified from mesoderm, they have to mature into functional HSCs and undergo self-renewing divisions to generate a pool of HSCs. During this process, developing HSCs migrate through various embryonic niches, which provide signals for their establishment and the conservation of their self-renewal ability. [Mikkola, HKA, Orkin, SH. Development (2006) 133: 3733-44]. [00090] B & T lymphocytes have to receive contact-dependent activation signals from immobile cells in situ, and exert the majority of their functions via direct intercellular interactions. Lymphocytes are influenced in their behavior by local antigens and metabolites and are embedded in a complex network of interactions with neighboring accessory cells (e.g., B cells and macrophages. ECM proteins continuously interact with signal-transducing receptors on lymphoid cells. [Kroemer, G. et al. Adv. Immunol. (1993) 53: 157-216]. [00091] Lymphocytes located outside of the thymus and bone marrow are considered as peripheral cells. Peripheral lymphocytes are contained in the classic lymphoid organs (spleen, lymph nodes, tonsils and Peyer’s patches), the epidermis, the mucosae of the gastrointestinal, respiratory, and female reproductive tracts, and in mesoderm derivatives (e.g., the pleuroperitoneal cavity. Lymphocytes in the lung interstitium are as numerous as those of the circulating blood pool. [Kroemer, G. et al. Adv. Immunol. (1993) 53: 157-216, citing Pabst, R. (1992) Immunology Today 13: 119-22]. [00092] T cells are extremely heterogeneous in specificity, activation requirements, life span, and functional properties. T cells produce a nearly infinite antigen receptor repertoire via somatic diversification processes, including gene rearrangements and somatic mutation. They can also be classified into subpopulations that differ in the expression of classes of the T cell receptor (TCR; α/β or γ/δ heterodimers) and CD antigens, in the activation state, or in functional terms. For example, the differentiation antigens CD4 and CD8 are found on mutually exclusive α/β T lymphocyte subsets in the periphery. CD4+ α/β T cells are predominantly of the helper phenotype, whereas CD8 (usually a heterodimer composed of CD8α and CD8β is mainly expressed on cytotoxic and suppressor T cells. This functional distinction is not absolute, because some CD4- T lymphocytes can effect cytotoxicity and suppression, and a more stringent correlation exists between CD4/CD8 expression and MHC gene products expressed by target or antigen presenting cells (APC). CD4+ T cells interact with cells expressing MHC class II; whereas CD8+ T cells are class I restricted [Id., citing Moller, G. (Ed ) Immunol. Rev. (1989) 109: 5-153, Parnes, JR. Adv. Immunol.44: 265-311; Bierer, BE et al. (1989) Annu. Rev. Immunol. (1989) 7: 579-99; Auffray, C. et al. Trends Biotechnol. (1991) 9: 124-30). A majority of the γ/δ do not express either CD4 or CD8; however a significant fraction displays CD8 and exerts a suppressor or cytotoxic function [Id., citing Bandeira, A. et al. Proc. Natl Acad. Sci. USA (1991) 88: 43-47]. A minor population that expresses CD4 exhibits a helper phenotype (Id., citing 11). T cells may differ in their activation state, which may or may not be reflected by the expression of activation markers. [Id. citing Crabtree, GR. Science (1989) 243: 355-361]. [00093] The best characterized lymphocyte populations in humans are those contained in the peripheral blood. Peripheral blood lymphocytes (PBLs), which are mature lymphocytes that circulate in the blood rather than being localized to organs, include B cells, T cells and natural killer cells. [Chiu, Po-Llin, et al. Scientific Reports (2019) 9: article 8145]. [00094] By analogy to T lymphocytes, the distribution of B cells follows a nonrandom pattern. Surface IgA-bearing lymphocytes are highly represented in mucosa-associated lymphoid structures (e.g., lamina propria and Peyer’s patches), the nonkeratinizing external surfaces of the body (gut and exocrine glands, including the lactating mammary gland, urogenital epithelia and upper respiratory tract) attract predominantly IgA-secreting plasma cells. In nonmucosal sites (peripheral lymph nodes, spleen and skin), IgA secreting cells are infrequent and most plasma cells secrete IgM or IgG. Similarly, distinct differentiation and activation stages of B lymphocytes are discontinuously distributed in different zones of lymphoid follicles (a lymphoid follicle is a compartment of primarily B cells, which represents a unique microenvironment). The expression of different VH gene families is also inhomogeneous [Kroemer, G. et al. Adv. Immunol. (1993) 53: 157-216, citing, Freitas, A.A. et al. Int. Immunol. (1989) 1: 342-54]. B cells may be divided into two classes according to the expression of CD5, a signal-transducing receptor [Id., citing Alberola-Ila, J. et al. J. Immunol. (1992) 148: 1287-93] that interacts with the B cell surface marker CD72/Lub-2 [Id., citing Van de Velde, H. et al. Nature (London) (1992) 148: 1287-93]. B1 cells represent the CD5+ (Ly-1+) subset, and have the phenotype IgMhighIgDlow-Mac-1 (CD11b/CD18)+CD45lowFceR-IL-5R+. B2 “ conventional” cells have a similar phenotype except that they lack CD5 (Id. citing Hayakawa, K., et al. J. Exp. Med. (1983) 157: 202-15; Wetzel, GD. Eur. J. Immunol. (1989) 19: 1701-08; Herzenberg, LA, et al. Immunol. Rev. (1986) 93: 81-109; Waldschmidt, TJ et al. Int. Immunol. (1991) 3: 305-315; Marcos, MAR, et al. Scand. J. Immunol. (1991) 34: 129-35; Kasaian, MT et al. J. Immunol. (1992) 148: 2690- 2702]. CD5+ B cells are endowed with the capacity of self-renewal, i.e., they may expand in the absence of any cell input from IgM- precursors, unlike conventional B cells [Id., citing Herzenberg, LA, et al. Immunol. Rev. (1986) 93: 81-109, Hayakawa, K. et al. Eur. J. Immunol. (1986) 4: 243-52; Forster, I., Rajewsky, K. Eur. J. Immunol. (1987) 17: 521-28]. [00095] A large proportion of mature lymphocytes continuously traffic from the bloodstream into lymphoid organs and tissue, then to the collecting efferent lymphatics, and eventually back to the bloodstream. Lymphocyte migration follows a nonrandom pattern. Naïve T cells migrate into lymph nodes, whereas memory T cells traffic preferentially into nonlymphoid tissue [Id., citing Mackay, CF (1991) Immunol. Today (1991) 12: 189-92; Pober, JS, Cotran, RS Adv. Immunol. (1991) 50: 261-302; Dustin, ML, Springer, TA Annu. Rev. Immunol.9: 27-66; Oppenheimer-Marks, N. et al. J. Immunol. (1990) 145: 140-48]. Memory T cells [00096] The vast majority of human memory T cells reside in tissue sites, including lymphoid tissues, intestines, lungs and skin. By the end of puberty, lymphoid tissues, mucosal sites and the skin are populated predominantly by memory T cells, which persist throughout adult life and represent the most abundant lymphocyte population throughout the body. [00097] Memory T cells in humans are classically distinguished by the phenotype CD45RO+CD45RA-, and comprise heterogeneous populations of memory T cell subsets. [Farber, DL, et al. Nat. Rev. Immunol. (2014) 14(1): 24-35] Naïve T cells uniformly express CCR7, reflecting their predominant residence in lymphoid tissue. Memory T cells are subdivided into CD45RA-CCR7+ central memory T (TCM) cells, which traffic to lymphoid tissues, and CD45RA-CCR7- effector memory T (TEM) cells, which can migrate to multiple peripheral tissue sites. Functionally, both TCM and TEM cell subsets produce effector cytokines in response to viruses, antigens and other stimuli [Id., citing Wang A, et al. Sci Transl Med. (2012) 4:149ra12030-33; Pedron B, et al. Pediatr Res. (2011) 69:106–111; Champagne P, et al. Nature. (2001) 410:106–111; Ellefsen K, et al. Eur J Immunol. (2002) 32:3756–3764], although TCM cells exhibit a higher proliferative capacity. (Id. citing Wang A, et al. Sci Transl Med. (2012) 4:149ra120, Fearon DT, et al. Immunol Rev.2006;211:104–118). A new subset, Tmemory stem (TSCM) cells, which resemble naïve T cells in that they are CD45RA+CD45RO- and express high levels of the co-stimulatory receptors CD27 and CD28, IL-7 receptor α chain (IL7Rα), CD62L and CCR7, have high proliferative capacity and are both self-renewing and multipotent in that they can further differentiate into other subsets, including TCM and TEM cells [Id. citing Gattinoni L, et al. Nat Med. (2011) 17:1290–1297, Gattinoni L, et al. Clin Cancer Res. (2010) 16:4695–4701]. A progressive differentiation pathway based on signal strength and/or extent of activation, places naïve (TN), TSCM, TCM and TEM cells in a differentiation hierarchy, serving as precursors for effector T cells [Id. citing Gattinoni L, et al. Nat Rev Cancer. (2012) 12:671–684; Klebanoff CA, et al. Immunol Rev. (2006) 211:214–224; Lanzavecchia A, Sallusto F. Nat Rev Immunol. (2002) 2:982–987]. [00098] In mice, tissue resident memory T (TRM) cells are a non-circulating subset that resides in peripheral tissue sites and, in some cases, elicits rapid in situ protective responses. Mouse CD4+TRM cells can be generated in the lungs from adoptive transfer or activated (effector) T cells [Id., citing Teijaro JR, et al. J Immunol. (2011) 187:5510–5514] or following respiratory virus infection [Id., citing Turner, DL, et al. Mucosal Immunol. (2014) 7 (3): 501- 510], and are distinguished from splenic and circulating memory T cells by their upregulation of the early activation marker CD69, their tissue-specific retention in niches of the lung [Id., citing Turner, DL, et al. Mucosal Immunol. (2014) 7 (3): 501-510] and their enhanced ability to mediate protection to influenza virus infection compared to circulating memory CD4+ T cells [Id. citing Teijaro JR, et al. J Immunol. (2011) 187:5510–5514]. An analogous non- circulating CD4+ TRM cell subset has been identified in the bone marrow of mice following systemic virus infection that exhibits enhanced helper functions. [Id., citing Herndler- Brandstetter D, et al. J Immunol. (2011) 186:6965–6971]. CD8+ TRM cells generated following infection have been identified in multiple mouse tissues, including skin [Id., citing Clark RA, et al. Sci Transl Med. (2012) 4:117ra117; Liu L, et al. Nat Med. (2010) 16:224–227], vaginal mucosa [Id., citing Mackay LK, et al. Proc Natl Acad Sci U S A. (2012) 109:7037–7042, Shin H, Iwasaki A. Nature. (2012) 491:463–467], intestine [Id., citing Klonowski KD, et al. Immunity. (2004) 20:551–562, Masopust D, et al. J Exp Med. (2010) 207:553–564, Masopust D, et al. J Immunol. (2006) 176:2079–2083], lungs [Id., citing Turner, DL, et al. Mucosal Immunol. (2014) 7 (3): 501-510, Anderson KG, et al. J Immunol. (2012) 189:2702–2706] and brain [Id., citing Wakim LM, et al. Proc Natl Acad Sci U S A. (2010) 107:17872–17879]. They are distinguished from splenic and circulating memory CD8+ T cells by their increased expression of CD69 and by expression of the epithelial cell binding integrin αEβ7 (also known as CD103 [Id, citing Mueller SN, et al. Annu Rev Immunol. (2013) 31:137–161, Mackay LK, et al. Proc Natl Acad Sci U S A. (2012) 109:7037–7042, Casey KA, et al. J Immunol. (2012) 188:4866–4875; Masopust D, Picker LJ. J Immunol. (2012) 188:5811–5817; Gebhardt T, Mackay LK. Front Immunol. (2012) 3:340]. [00099] In humans, memory CD4+ T cells predominate throughout the body and persist as CCR7+ or CCR7- subsets localized to lymphoid tissues and mucosal sites, respectively, whereas memory CD8+ T cells persist as mainly CCR7- subsets in all sites, with low numbers of CD8 TCM cells in lymphoid tissues and negligible numbers of these cells in other sites [Id., citing Sathaliyawala T, et al. Immunity (2013) 38:187–197]. Most memory T cells in human mucosal, lymphoid and peripheral tissue sites such as skin express the putative TRM cell marker CD69 [Id., citing Goronzy JJ, Weyand CM. Nat Immunol. (2013) 14:428–436; Nikolich- Zugich J, Rudd BD. Curr Opin Immunol. (2010) 22:535–540; Clark RA, et al. J Immunol. (2006) 176:4431–4439, Mueller SN, et al. Annu Rev Immunol. (2013) 31:137–161, Casey KA, et al. J Immunol. (2012) 188:4866–4875], whereas circulating blood memory T cells uniformly lack CD69 expression. [Id., citing Sathaliyawala T, et al. Immunity (2013) 38:187–197]. [000100] Human TRM cells also exhibit tissue-specific properties, suggesting in situ influences. For example, memory T cells in the small intestine and colon express the gut- homing receptor CCR9 [Id., citing Kunkel EJ, et al. J Exp Med. (2000) 192:761–768] and the integrin α4β7 [Id., citing Agace WW. Trends Immunol. (2008) 29:514–522], and memory T cells in the lungs upregulate CCR6 expression [Id., citing Purwar R, et al. PLoS One. (2011) 6:e16245]. There is also evidence for crosstalk between mucosal sites, such as lung and intestines. For example lung dendritic cells induce migration of protective T cells to the gastrointestinal tract. [Id. citing Ruane D, et al. J Exp Med. (2013) 210:1871–1888]. [000101] There is evidence that TRM can be multifunctional and also exhibit qualitative functional differences. A substantial fraction of human lung TRM cells produce multiple pro- inflammatory cytokines [Id., citing Purwar R, et al. PLoS One. 2011;6:e16245], and human intestinal TRM cells are also multifunctional [Id. citing Sathaliyawala T, et al. Immunity. 2013;38:187–197]. Other functions appear to be confined to specific subsets and/or tissue sites. For example, IL-17 is produced by a subset of CD4+ TRM cells in mucosal sites, particularly in intestines in healthy individuals [Id., citing Sathaliyawala T, et al. Immunity (2013) 38:187– 197], by CCR6+ memory T cells in peripheral blood [Id., citing Singh SP, et al. J Immunol. (2008) 180:214–221, Wan Q, et al. J Exp Med. (2011) 208:1875–1887], and by a subset of CD161+ T cells in inflamed tissue, such as the skin of patients with psoriasis [Id., citing Cosmi L, et al. J Exp Med.2008;205:1903–1916]. Thus, while predominant memory T cell functions, such as IFNγ production, are broadly distributed among multiple memory T cell subsets and tissues, TRM cells in tissue sites can adopt multiple or distinct functional attributes, which may also depend on tissue-specific inflammation. [000102] Despite their specificity, human memory T cells exhibit cross-reactivity to antigenic epitopes not previously encountered, which may be due to intrinsic properties of TCR recognition [Id., citing Sewell AK. Nat Rev Immunol.2012;12:669–677] and to the range and breadth of human antigenic experience. Memory CD4+ and CD8+ T cells specific for unique epitopes of avian influenza strain H5N1 were detected in healthy individuals that were not exposed to H5N1 infection assessed by serology [Id., citing Lee LY, et al. J Clin Invest. (2008) 118 (10): 3478-90; Roti M, et al. J Immunol. (2008) 180:1758–1768]. In addition, HIV- specific memory T cells have been identified in HIV-negative individuals [Id., citing Su, LF et al. Immunity (2013) 38: 373-83]. Virus-specific memory T cells also show cross-reactivity to alloantigens, autoantigens and unrelated pathogens [Id., citing D'Orsogna LJ, et al. Transpl Immunol. (2010) 23:149–155, Wucherpfennig KW. Mol Immunol. (2004) 40:1009–1017]: EBV-specific human memory T cells generated in HLA-B8 individuals exhibit allogeneic cross-reactivity to HLA-B44 [Id., citing Burrows SR, et al. J Exp Med. (1994) 179:1155–1161], and influenza virus- and HIV-specific memory CD4+ T cells recognize epitopes from unrelated microbial pathogens [Id., citing Su LF, et al. Immunity. (2013) 38:373–383]. Furthermore, T cells specific for the autoantigen myelin basic protein (MBP) recognized multiple epitopes from viral and bacterial pathogens [Id., citing Wucherpfennig KW. Mol Immunol. (2004) 40:1009–1017, Wucherpfennig KW, Strominger JL. Cell. (1995) 80:695–705]. This cross- reactivity may enable memory T cells to mediate protection without initial disease — a phenomenon known as heterologous immunity [Id., citing Welsh RM, Selin LK. Nat Rev Immunol. (2002) 2:417–426]. Heterologous immunity has been demonstrated in humans where EBV infection expanded clones of influenza virus-specific T cells [Id., citing Clute SC, et al. J Clin Invest.2005;115:3602–3612]. [000103] Analysis of human samples has revealed that influenza-specific TRM can be found in substantial numbers in lung tissue, highlighting their role in natural infection. Despite expressing low levels of granzyme B and CD107a, these CD8+ TRM had a diverse T cell receptor (TCR) repertoire, high proliferative capacities, and were polyfunctional [Muruganandah, V., et al. (2018). Front. Immunol., 9, 1574. doi:10.3389/fimmu.2018.01574]. Influenza infection history suggests a greater level of protection against re-infections is likely due to the accumulation of CD8+ TRM in the lungs. Furthermore, the natural immune response to influenza A virus infection in a rhesus monkey model demonstrated that a large portion of influenza-specific CD8+ T cells generated in the lungs were phenotypically confirmed as CD69+CD103+ TRM. Unlike lung parenchymal TRM, airway CD8+ TRM are poorly cytolytic and participate in early viral replication control by producing a rapid and robust IFN-γ response. Bystander CD8+ TRM may also take part in the early immune response to infection through antigen non-specific, NKG2D-mediated immunity. The generation of functional TRM that protect against heterosubtypic influenza infection appear to be dependent on signals from CD4+ T cells. [Muruganandah, V., et al. (2018). Frontiers in Immunology, 9, 1574. doi:10.3389/fimmu.2018.01574]. [000104] According to the paradigm of a typical CD8+ T cell response to acute viruses, CD8+ T cells are effectors when an antigen is present and become memory when the antigen is eliminated. However, it has become apparent that in viral infections, a memory T cell population comprises multiple subtypes of cells, distributed in diverse anatomic compartments and possibly recirculating among them. The memory CD8+ T cell response to most viruses is diverse in phenotype and function and undergoes dynamic changes during its development and maintenance in vivo. This heterogeneity is related to the nature of the infecting virus, its cellular tropism, the anatomic location of the infection, and the location of the CD8+ T cells. In resolved acute infections, the presence of memory CD8+ T cells at the site of the original virus entry and replication is crucial for a rapid response to a secondary infection. In latent infections, the presence of memory CD8+ T cells at sites of virus persistence is important for immune surveillance of virus reactivation. [Racanelli, V. et al., Rev. Med. Virol. (2011) 21 (6): 347-357]. Mucosal immune system [000105] While the mucosal surfaces of the body have a protective barrier of mucus, they are highly vulnerable to infection and possess a complex array of innate and adaptive mechanisms of immunity. The adaptive immune system of the mucosa-associated lymphoid tissues differs from that of the rest of the peripheral lymphoid system in several respects. The types and distribution of T cells differ, with significantly greater numbers of γ:δ T cells in the gut mucosa compared with peripheral lymph nodes and blood. The major antibody type secreted across the epithelial cells lining mucosal surfaces is secretory polymeric IgA. [Immunobiology: The Immune System in Health and disease. Janeway, CA et al Eds., 5th Ed. (2001), Garland Publishing, New York, Ch.10, p.482-493]. [000106] The mucosal immune system protects internal mucosal surfaces, such as the linings of the gut, respiratory tract and urogenital tracts, which are the site of entry for virtually all pathogens and other antigens. The mucosa-associated lymphoid tissues lining the gut are known as gut-associated lymphoid tissue or GALT. The tonsils and adenoids, which form a ring, known as Waldeyer's ring, at the back of the mouth at the entrance of the gut and airways, represent large aggregates of mucosal lymphoid tissue, which often become extremely enlarged in childhood because of recurrent infections. The other principal sites within the gut mucosal immune system for the induction of immune responses are the Peyer's patches of the small intestine, the appendix, and solitary lymphoid follicles of the large intestine and rectum. Peyer's patches are an important site for the induction of immune responses in the small intestine and have a distinctive structure, forming domelike structures extending into the lumen of the intestine. The overlying layer of follicle-associated epithelium of the Peyer's patches contains specialized epithelial cells (microfold cells or M cells) that have microfolds on their luminal surface, instead of the microvilli present on the absorptive epithelial cells of the intestine. They are much less prominent than the absorptive gut epithelial cells, known as enterocytes, and form a membrane overlying the lymphoid tissue within the Peyer's patch. Since M cells lack a thick surface glycocalyx and do not secrete mucus, they are adapted to interact directly with molecules and particles within the lumen of the gut. M cells take up molecules and particles from the gut lumen by endocytosis or phagocytosis. This material is then transported through the interior of the cell in vesicles to the basal cell membrane, where it is released into the extracellular space by transcytosis. At their basal surface, the cell membrane of M cells is extensively folded around underlying lymphocytes and antigen-presenting cells, which take up the transported material released from the M cells and process it for antigen presentation. [Immunobiology: The Immune System in Health and disease. Janeway, CA et al Eds., 5th Ed. (2001) Garland Publishing, New York, Ch.10, p.482-493]. [000107] In addition to the organized lymphoid tissue in which induction of immune responses occurs within the mucosal immune system, small foci of lymphocytes and plasma cells, which are scattered widely throughout the lamina propria of the gut wall, represent the effector cells of the gut mucosal immune system. As naive lymphocytes, these cells emerge from the primary lymphoid organs of bone marrow and thymus to enter the inductive lymphoid tissue of the mucosal immune system via the bloodstream. They may encounter foreign antigens presented within the organized lymphoid tissue of the mucosal immune system and become activated to effector status. From these sites, the activated lymphocytes traffic via the lymphatics draining the intestines, pass through mesenteric lymph nodes, and eventually wind up in the thoracic duct, from which they circulate in the blood throughout the entire body. They reenter the mucosal tissues from the small blood vessels lining the gut wall and other sites of MALT, such as the respiratory or reproductive mucosa, and the lactating breast; these small vessels express the mucosal adressin MAdCAM-1. In this way, an immune response that may be started by foreign antigens presented in a limited number of Peyer's patches is disseminated throughout the mucosa of the body. This pathway of lymphocyte trafficking is distinct from and parallel to that of lymphocytes in the rest of the peripheral lymphoid system. [Immunobiology: The Immune System in Health and disease. Janeway, CA et al Eds., 5th Ed. (2001) Garland Publishing, New York, Ch.10, p.482-493]. [000108] The distinctiveness of the mucosal immune system from the rest of the peripheral lymphoid system is further underlined by the different lymphocyte repertoires in the different compartments. The T cells of the gut can be divided into two types. One type bears the conventional α:β T-cell receptors in conjunction with either CD4 or CD8, and participates in conventional T-cell responses to foreign antigens. The second class is made up of T cells with unusual surface phenotypes such as TCRγ:δ and CD8α:α TCRα:β. The receptors of these T cells do not bind to the normal MHC:peptide ligands. Instead, they bind to a number of different ligands, including MHC class IB molecules. These highly specialized T cells are abundant in the epithelium of the gut and have a restricted repertoire of T-cell receptor specificities. Unlike conventional T cells, many of these cells do not undergo positive and negative selection in the thymus, and express receptors with sequences that have undergone no or minimal divergence from their germline-encoded sequences. These cells may be classified in phylogenetic terms as being at the interface between innate and adaptive immunity. [Immunobiology: The Immune System in Health and disease. Janeway, CA et al Eds., 5th Ed. (2001) Garland Publishing, New York, Ch.10, p.482-493]. [000109] T cells bearing a γ:δ receptor are especially abundant in the gut mucosa compared with other lymphoid tissues. One subset of these γ:δ T cells in humans, which expresses a T-cell receptor that uses the Vδ1 gene segment, carries an activating C-type lectin NK receptor, NKG2D. NKG2D binds to two MHC-like molecules—MIC-A and MIC-B—that are expressed on intestinal epithelial cells in response to cellular injury and stress. The injured cells may then be recognized and killed by the subset of γ:δ T cells. The Vδ1-containing receptor on these T cells may also play a part in allowing them to survey tissues for injured cells. Some human T cells expressing this receptor bind to CD1c, one of the isotypes of the CD1 family of MHC class I-like molecules. This protein, which shows increased expression on activated monocytes and dendritic cells, presents endogenous lipid and glycolipid antigens to some types of T cell. In response to antigen presentation by CD1c, these T cells secrete IFN- γ, which may have an important role in polarizing the response of conventional T cells bearing α:β receptors toward a TH1 response. This is closely analogous, although opposite in effect, to the polarization toward TH2 cells induced by secretion of IL-4 by NK 1.1+ T cells (NK1+ T cells) responding to CD1d. [Immunobiology: The Immune System in Health and disease. Janeway, CA et al Eds., 5th Ed. (2001) Garland Publishing, New York, Ch.10, p.482-493]. Involvement of γδ T cells in viral lung infections [000110] Lung‐resident γδ T cells play critical roles in anti‐viral immune responses and are involved in virus‐induced lung inflammation and injury. Respiratory syncytial virus (RSV), one of many (~ 200) viruses known as a common cold virus, predominately affects infants and leads to long‐term lung disease. [Cheng, M., and Hu, Shilian. Immunology (2017) 151: 375- 84]. The contribution of γδ T cells to RSV infection has been tested in mice infected with RSV with or without immunization with a live vaccine vector expressing RSV F protein. Vγ4+ γδ T cells were enhanced in the lungs and produced IFN‐γ, RANTES, IL‐10, IL‐4 and IL‐5 in a time‐dependent manner after challenge of sensitized mice. Depletion of γδ T cells reduced lung inflammation and disease severity and slightly increased peak viral replication without compromising viral clearance during secondary challenge in vaccinated mice. [Id., citing Dodd J, et al. J Immunol (2009) 182:1174–81]. Using a neonatal mouse model of RSV, it was found that neonates failed to develop IL‐17A responses of the type observed in adult mice. In adults, γδ T cells are the main producers of IL‐17A. Exogenous IL‐17A administration decreases inflammation in RSV‐infected neonates, whereas neutralization of IL‐17A increases lung inflammation and airway mucus in RSV‐infected adults. Hence, RSV disease severity is in part mediated by a lack of IL‐17A+ γδ T cells in the lungs of neonates. [Id., citing Huang H, et al. Immunol Cell Biol (2015) 93:126–35]. Additionally, RSV infection elevates Th1 cytokine- and suppresses Th2 cytokine-expression in lung γδ T cells. Ovalbumin (OVA) challenge induces a large influx of γδ T cells into the lungs. When mice were previously infected with RSV, the OVA‐induced infiltration and activation of γδ T cells were inhibited, suggesting that RSV protected against subsequent OVA‐induced allergic responses by inhibiting Th2‐type γδ T cells. [Id., citing Zhang L, et al. J Med Virol (2013) 85:149–56] [000111] During influenza virus infection, RORγt‐positive αβ and γδ T cells, as well as innate lymphoid cells, express enhanced IL‐22 as early as 2 days post‐infection. Although IL‐ 22 plays no role in the control of influenza A virus replication, IL‐22 is beneficial during sublethal influenza A virus infection but not lethal influenza A virus infection, which limits lung inflammation and injury after a secondary challenge with S. pneumoniae. [Id., citing Ivanov S, et al. J Virol (2013) 87:6911–24] Type I interferon induction during influenza virus infection increases susceptibility to secondary S. pneumoniae infection by negative regulation of γδ T cells with decreased IL‐17 production. [Id., citing Li W, et al. J Virol (2012) 86:12304– 12]. Human Vγ9Vδ2 T cells that are activated in vitro by aminobisphosphonate pamidronate efficiently kill influenza virus-infected lung alveolar epithelial cells and inhibit virus replication in a cell‐to‐cell contact manner. The cytotoxic activity of Vγ9Vδ2 T cells requires NKG2D activation and involves perforin/granzyme B, TRAIL and FasL. [Id., citing Li H, et al. Cell Mol Immunol (2013) 10:159–64, Tu W, et al. J Exp Med (2011) 208:1511–22] Secretory IgA (sIgA) [000112] Immunoglobulin A (IgA) is the predominant antibody isotype in the mucosal immune system. Normally serum IgA shows a monomeric structure, while mucosal IgA is polymeric. Secretory IgA (sIgA) comprises two monomeric IgAs, secretory component (SC) and J chain. There are also trimeric sIgA, tetrameric sIgA, and larger polymeric IgA in the upper respiratory tract of healthy humans. Among them, tetrameric IgA has broad neutralizing activity against influenza viruses [Li, Y. et al. Biomed. Res. Intl (2020) 2020: 2032057]. [000113] The classical sIgA inductive sites are gut‐associated lymphoid tissue (GALT) including Peyer's patches (PPs), isolated lymphoid follicles (ILFs), and mesenteric lymph nodes (MLNs). GALT contains at least 80% plasma cells (PCs) and 90% sIgA of the body [Id., citing Brandtzaeg P., et al. (1999) 171(1):45–87] The nasopharynx-associated lymphoid tissues (NALT) and the bronchus-associated lymphoid tissues (BALT) are also mucosal immune inductive sites [Id., citing Brandtzaeg P. Vaccine. (2007) 25(30): 5467–5484]. Dendritic cells exist in the subepithelial dome (SED) beneath the follicle-associated epithelium (FAE) of the Peyer’s patches; mucosal antigens are captured by the underlying DCs which extend their dendrites [Id., citing Farache J., et al. Immunity. (2013) 38(3):581–595] or through the transcytosis of M cells [Id., citing Mabbott N. A., et al. Mucosal Immunology. (2013) 6(4):666– 677]. Upon antigen presentation by DCs, T cells and B cells are activated and IgA class switch recombination (CSR) is mediated in the mucosal B cells. T cells participate in this process by either a T-dependent (TD) mechanism or a T-independent (TI) mechanism. The T-dependent mechanism requires interaction between CD40 on the surface of B cells and its ligand CD40L derived from T cells, resulting in high-affinity antigen-specific IgA production to neutralize pathogens [Id., citing Pabst O. Nature Reviews Immunology (2012) 12(12):821–832]. T follicular helper (Tfh), Foxp3 +Treg, and Th17 cells are involved in promoting the IgA response in the intestine by the release of various cytokines, such as IL‐4, IL‐5, IL‐6, IL‐10, IL‐13, IL-17A, and IL-21, to further promote the CSR to IgA [Id., citing Cao A. T., et al. Mucosal Immunology. (2015) 8(5):1072–1082]. The T-independent (TI) mechanism produces commensal-reactive IgA through innate immune cells such as innate lymphoid cells (ILCs) and plasmacytoid dendritic cells (pDC) [Id., citing Boyaka P. N. J. Immunology. (2017) 199(1):9– 16, Pabst O. Nature Reviews Immunology (2012) 12(12):821–832, Kubinak J. L., et al. Cell Host & Microbe. (2015) 17(2):153–163]. In the TI pathway, BAFF (B-cell activating factor of the TNF family) and APRIL (A proliferation-inducing ligand), two members of the TNF family, are responsible for stimulating CSR to IgG or IgA in humans [Id., citing Lycke N., et al. Scandinavian Journal of Immunology. (1987) 25(4):413–419]. [000114] Traditionally, IgA is thought of as a noninflammatory antibody at mucosal sites. Due to its polymeric structure and the oligosaccharide side chains of SC [Id., citing Phalipon A., et al. Immunity. (2002) 17(1):107–115], sIgA is concentrated in the mucus out layer [Rogier E., et al. Pathogens. (2014) 3(2):390–403], noncovalently cross-linking microorganisms, and promoting the microorganisms to clump together in situ. Peristaltic bowel movements then help remove the bacterial clumps. These processes of agglutination, entrapment, and clearance together are called immune exclusion [Id., citing Mantis N. J., et al. Mucosal Immunology. (2011) 4(6):603–611]. [000115] sIgAs also have more extensive protective functions. Firstly, sIgA coating and the steric hindrance help block microbial adhesins to interact with the epithelium, sIgA can also inhibit specifically pathogens by direct recognition of receptor-binding domains such as reovirus type 1 Lang (T1L) [Id., citing Helander A., et al. J. Virology. (2003) 77(14):7964– 7977]. The advanced glycosylated IgA heavy chain and SC serve as competitive inhibitors of the pathogen adhesion process [Id., citing Mantis N. J., et al. Mucosal Immunology. 2011;4(6):603–611]. Blocking pathogens from interacting with epithelial cells is not the exclusive mechanism by which sIgA exerts its protective function. In addition, sIgA may have direct effects on impacting the bacterial viability or changing pathogenicity. For example, sIgA can interact with flagella to inhibit Salmonella bacterial motility [Id., citing Forbes S. J., et al. Infection and Immunity. (2008) 76(9):4137–4144], as well as to protect from cholera toxin- induced fluid accumulation in a ligated intestinal loop model [Lycke N., et al. Scandinavian Journal of Immunology. 1987;25(4):413–419]. SC has been shown to interact with choline binding protein A (CbpA), a surface protein of Streptococcus pneumoniae, [Id., citing Corthesy B. Frontiers in Immunology. (2013) 4:p.185]; the galactose residues of free SC were shown to neutralize Clostridium difficile toxin A and enteropathogenic E. coli intimin [Id., citing Perrier C., et al. Journal of Biological Chemistry. (2006) 281(20):14280–14287]. [000116] FcαRI (CD89), the most important IgA host receptor, is widely expressed in cell types including neutrophils, eosinophils, monocytes, and macrophages. [Id., citing Otten M. A., van Egmond M. Immunology Letters. (2004) 92(1-2):23–31]. It mediates several biological effects such as antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, antigen presentation and release of cytokines, superoxide generation, calcium mobilization, and degranulation [Id., citing Monteiro R. C., Van De Winkel J. G. J. Annual Review of Immunology. (2003) 21(1):177–204]. Although sIgA is not able to activate phagocytosis by neutrophils or Kupffer cells, sIgA can initiate respiratory burst activity by neutrophils [Id., citing van Egmond M., et al. Nature Medicine. (2000) 6(6):680–685]. This process is dependent on the expression of the integrin co-receptor Mac‐1 (CD11b/CD18), suggesting that sIgA needs this integrin co-receptor to bind or activate FcαRI [Id., citing van Spriel A. B., et al. Blood. (2001) 97(8):2478–2486]. Besides FcαRI, sIgA also interacts with the polymeric immunoglobulin Fc receptor pIgR, transferrin receptor (Tfr/CD71), asialoglycoprotein receptor (ASGPR), Fcα/μR (also known as CD351), FcRL4 (an immunoregulatory receptor specifically expressed by memory B cells localized in sub-epithelial regions of lymphoid tissues; [Jourdan, M. et al., PLoS One (2017) doi.org/10.1371/journal.pone.0179793], and dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN; CD209) [Id., citing Mkaddem S. B., et al. Fc Receptors. Vol.382. Berlin, Germany: Springer; 2014. IgA, IgA receptors, and their anti-inflammatory properties; pp.221–235.]. Interactions between dengue virus E protein and DC-SIGN are essential for dengue virus infection of dendritic cells. [Lozach, PY, et al. J. Biol. Chem. (2005) 280 (25): 23698-708]. sIgA immune complexes can reverse transport back into the lamina propria via the transferrin receptor on epithelial cells [Li, Y. et al., BioMed. Res. Intl. (2020) 2020: 2032057, citing Matysiak-Budnik T., et al. Journal of Experimental Medicine. (2008) 205(1):143–154.] or via interaction with dectin-1 through M cells [Id., citing Rochereau N., et al. PLoS Biology. (2013) 11(9) doi: 10.1371/journal.pbio.1001658.e1001658]. IAV Escape from Host Immune Surveillance [000117] To establish a successful infection, IAVs have evolved multiple strategies to circumvent host immunity. For example, it is well known that IAV infection triggers robust production of IFNs that induce the expression of numerous antiviral molecules or ISGs. Although IFNs have a strong antiviral activity, they cannot fully control IAV infection due to the virus-mediated suppression of IFNs signaling. [000118] Hemagglutinin of IAVs has been shown to facilitate IFNAR ubiquitination and degradation, reducing the levels of IFNAR, and thus suppressing the expression of IFN- stimulated antiviral proteins [Chen, X. et al., Frontiers Immunol. (2018) 9: 320, citing Xia C, et al. J Virol (2015) 90(5):2403–17]. It has been reported that two discrete antigenic sites, H9- A and H9-B, may provide a novel mechanism for H9N2 virus to counteract humoral immunity [Id., citing Peacock T, et al. Sci Rep (2016) 6:18745]. In addition, a study has shown that the escape of H5N1 from vaccine-mediated immunity is caused by the addition of N-glycosylation sites on the globular head of HA [Id., citing Hervé PL, et al. Virology (2015) 486(8):134–45]. In contrast, antibody response against NA of IAV cannot inhibit viral infection, but restrain its diffusion, thus lowering the severity of influenza. IAVs employ NA protein to block the recognition of HA by natural cytotoxicity receptors, NKp46, and NKp44 receptors and evade the NKp46-mediated elimination, leading to minimized clearance of infected cells by NK cells [Id., citing Baron Y, et al. J Infect Dis (2014) 210(3):410]. [000119] Nonstructural protein-1 of IAVs is the most important IFN antagonist protein, acting on multiple targets and suppressing the host IFN response. Viral RNA invading the host cell causes RIG-I ubiquitination by a RING-finger E3 ubiquitin ligase named as TRIM25, which is essential for RIG-I signaling pathway to trigger host antiviral innate immunity [Id., citing Gack MU, et al. Proc Natl Acad Sci U S A (2008) 105(43):16743]. However, NS1 protein can inhibit the TRIM25-mediated RIG-I ubiquitination, thereby blocking RIG-I activation [Id., citing Gack MU, et al Cell Host Microbe (2009) 5(5):439–49]. Moreover, NS1 has an inhibitory effect on protein kinase RNA-activated (also known as protein kinase R, PKR), but the effect relies on the induced expression of vault RNAs (a kind of small non-coding RNA with approximately 100 bases). They are initially described as fornix RNP complex components [Id., citing Kedersha NL, Rome LH. J Cell Biol (1986) 103(3):699–709]. Through NS1 protein, influenza virus induces the expression of vault RNA that inhibits the activation of PKR and the production of IFNs and ultimately promotes the replication of the virus. In a recent reverse genetic investigation, it was found that after interfering with NS1, the phosphorylation level of PKR dramatically increased, which was attenuated by forced expression of vault RNAs [Id., citing Li F, et al. Nucleic Acids Res (2015) 43(21):10321–37]. These data indicate that IAV has evolved a critical mechanism by which NS1-mediated PKR inhibition is mediated by upregulation of the host factor vault RNAs that inactivates PKR and blocks the production of downstream effector molecules of IFNs. [000120] In addition, studies have shown that through the interaction with IκB kinases (IKK) α and β, two important kinases in NF-κB pathway, NS1 protein can block the phosphorylation of these kinases and eventually destroy the NF-κB complex predominating in nucleus as well as the expression of downstream genes [Id., citing Rückle A, et al. J Virol (2012) 86(18):10211–7, Gao S, et al Cell Microbiol (2012) 14(12):1849]. Also, through the JAK-STAT pathway, NS1 protein can block IFN-mediated downstream signaling pathway and weaken the antiviral effect mediated by the downstream effector molecules induced by IFNs. Specifically, NS1 acts mainly by lowering the phosphorylation levels of STAT1, STAT2, and STAT3, preventing STAT2 from entering into the nucleus to bind to the DNA sequence of ISGs promoter region, leading to reduced expression of ISGs [Id., citing Jia D, et al. PLoS One (2010) 5(11):e13927]. NS1 is not only involved in host innate immunity, but also affects adaptive immunity via modulating the maturation and the capacity of DCs to induce T cell responses [Id., citing Fernandez-Sesma A, et al. J Virol (2006) 80(13):6295–304]. Evidence also indicates that influenza virus NS1 can bind to cellular double-stranded DNA (dsDNA), counteract the recruitment of RNA polymerase II (Pol II) to DNA, and finally block the transcription of IFNs and ISGs [Id., citing Anastasina M, et al. Biochim Biophys Acta (2016) 1859(11):1440–8]. The Antagonism of Other IAV Proteins [000121] Studies have found that PB1-F2 protein has a mitochondrial positioning signal, via interacting with MAVS, to counteract RLR-mediated activation of IFN signaling pathway (Id., citing Varga ZT, et al. PLoS Pathog (2011) 7(6):e1002067). Investigation of the interaction between the virus and host by systematic biology analysis has revealed that PB2 protein, a member of the viral polymerase complex, also plays roles in IFN antagonism [Id., citing Iwai A, et al. J Biol Chem (2010) 285(42):32064–74]. Furthermore, PB2 interacts with the MAVS to evade the host IFN antiviral response, which is similar to the action mode of PB1-F2 protein [Id., citing Grimm D, et al. Proc Natl Acad Sci U S A (2007) 104(16):6806– 11]. Viral M2 protein may inhibit the activation of TLR pathway and the generation of IFNs via blocking host autophagy [Id., citing Münz C. et al. Cell Host Microbe (2014) 15(2):130–1, Beale R, et al. Cell Host Microbe (2014) 15(2):239–47]. Wound healing [000122] The term "wound healing" refers to the processes by which the body repairs trauma to any of its tissues, especially those caused by physical means and with interruption of continuity. [000123] A wound-healing response can be viewed as comprising four separate phases, comprising: 1) an initial phase post injury involving hemostasis; 2) a second phase involving inflammation; 3) a third phase involving granulation and proliferation; and 4) a fourth phase involving remodeling and maturation. The culmination of the wound-healing response results in the replacement of normal tissue structures with fibroblastic mediated scar tissue. Processes involved in the wound healing response, however, can go awry and produce an exuberance of fibroblastic proliferation, which can result in tissue damage, including hypertrophic scarring (a widened or unsightly scar that does not extend the original boundaries of the wound). Initial Phase - Hemostasis [000124] An initial injury results in an outflow of blood and lymphatic fluid. This is also the process during which the initial reparative blood clot is created. Both the intrinsic coagulation pathways, so called because all of the components are intrinsic to plasma, and the extrinsic coagulation pathways are activated. The intrinsic and extrinsic systems converge to activate the final common pathways causing fibrin formation. FIG. 1 shows an illustrative representation of the classical coagulation cascades. It is generally recognized that these systems function together and interact in vivo. [000125] The intrinsic coagulation pathway is initiated when blood contacts any surface except normal endothelial and blood cells. This pathway, also known as the contact activation pathway, begins with formation of the primary complex on collagen by high-molecular weight kininogen (HMWK), prekallikrein, and coagulation factor (Factor) XII (Hageman factor). Prekallikrein is converted to kallikrein and Factor XII becomes Factor XI la. Factor Xlla converts Factor XI into Factor Xla. Factor Xla activates Factor IX, which, with its co-factor FVIIIa form the tenase complex, which activates Factor X to Factor Xa. [000126] The extrinsic coagulation pathway, also known as the tissue factor pathway, generates a thrombin burst and is initiated when tissue thromboplastin activates Factor VII. Upon vessel injury, tissue factor (TF), a nonenzymatic lipoprotein cofactor that greatly increases the proteolytic efficiency of Factor VIla, is exposed to the blood and enzyme coagulation factor VII (proconvertin) circulating in the blood. Once bound to TF, Factor VII is activated to Factor VIla by different proteases, including thrombin (Factor lla), Factors Xa, IXa, Xlla and the Factor Vlla-TF complex itself. The Factor Vlla-TF complex activates Factors IX and X. The activation of Factor Xa by the Factor Vlla-TF complex almost immediately is inhibited by tissue factor pathway inhibitor (TFPI). Factor Xa and its cofactor Va form the prothrombinase complex which activates the conversion of prothrombin to thrombin. [000127] Thrombin then activates other components of the coagulation cascade, including Factors V and VIII (which activates Factor XI, which, in turn, activates Factor IX), and activates and releases Factor VIII from being bound to von Willebrand Factor (vWF). Factors VIla and IXa together form the "tenase" complex, which activates Factor X, and so the cycle continues. [000128] As currently understood, coagulation in vivo is a 3-step process centered on cell surfaces. FIG.2 shows an illustration of the cell-surface based model of coagulation in vivo (Monroe Arterioscler Thromb Vase Biol. 2002; 22:1381 -1389).In the first step, coagulation begins primarily by initiation with tissue factor, which is present on the subendothelium, tissues not normally exposed to blood, activated monocytes and endothelium when activated by inflammation. Factors VII and Vila bind to tissue factor and adjacent collagen. The factor Vila- tissue factor complex activates factor X and IX. Factor Xa activates factor V, forming a prothrombinase complex (factor Xa, Va and calcium) on the tissue factor expressing cell. In the second step, coagulation is amplified as platelets adhere to the site of injury in the blood vessel. Thrombin is activated by platelet adherence and then acts to fully activate platelets, enhance their adhesion and to release factor V from the platelet alpha granules. Thrombin on the surface of activated platelets activates factors V, VIII and XI, with subsequent activation of factor IX. The tenase complex (factors IXa, VIIa and calcium) now is present on platelets where factor Xa can be produced and can generate another prothrombinase complex on the platelet so that there can be large-scale production of thrombin. Propagation, the third step, and is a combination of activation of the prothrombinase complexes that allow large amounts of thrombin to be generated from prothrombin. More platelets can be recruited, as well as activation of fibrin polymers and factor XIII. [000129] Proteases of the coagulation cascade have been shown to activate protease activated receptors (PARs), a family of G-protein-coupled receptors. [Moretti, S. et al. Mucosal Immunol. (2008) 1(2): 156-68]. Four PARs have been cloned from the mammalian genome. PARS are activated by proteolytic cleavage of their N-terminal extracellular domain, which releases a new amino terminus sequence, which then acts as a tethered ligand to bind intramolecularly the receptor on its second extracellular loop. [Cirino, G. & Vergnolle, N. Curr. Opin. Pharmacol. (2006) 6: 428-34]. Thrombin activates PAR1, PAR3, and PAR4, whereas trypsin and mast cell tryptase activate PAR2. [Schmidlin, F. et al. Am. J. Crit. Care Med. (2001) 164: 1276-81] Once the tethered ligand has bound the receptor, G proteins coupled to the receptor are activated and orchestrate intracellular signaling. [Cirino, G. & Vergnolle, N. Curr. Opin. Pharmacol. (2006) 6: 428-34]. Activation of PARs by proteases is irreversible; once activated, they are internalized, and new pools of receptors have to be addressed to the plasma membrane to observe PAR signaling again. [000130] It has been demonstrated that neutrophil elastase and cathepsin G are able to cleave the N-terminal domain of PARs downstream from the activating protease cleavage site, thereby preventing activation of the receptor [Cirino, G. & Vergnolle, N. Curr. Opin. Pharmacol. (2006) 6: 428-34]., citing Dulon, S. et al. Am. J. Respir. Cell Mol. Biol. (2003) 28: 339-46]. This disarming mechanism could be used by pathogens to avoid PAR-driven host immune responses; studies have shown that lung (Pseudomonas aeruginosa) [Id., citing Dulon, S. et al. Am. J. Respir. Cell Mo. Biol. (2005) 32: 411-19] and mouth (Teponema denticola) [Id., citing Holzhausen, M. et al. Am. J. Pathol (2006) 168: 1189-99] pathogens can disarm PAR2. [000131] PAR1 has multiple roles on many cell types. On endothelial cells, it can have barrier protective or barrier disruptive roles, depending on the agonist and co-receptors present on the cell. [Arachiche, A. et al. J. Biol. Chem. (2013) 288 (45): 32553-62, citing Riewald, M., & Ruf, W. J. Biol. Chem. (2005) 280: 19808-14; Russo, A. et al. Proc. Natl. Acad. Sci. USA (2009) 106: 6393-97; Soh, U. & Trejo, J. Proc. Natl Acad. Sci USA (211) 108: E1372- 80; Bae, JS, et al. Blood (2007) 110: 3909-16]. PAR4 is the primary signaling receptor on platelets of many species. In human platelets, PAR1 and PAR4 have both overlapping and unique signaling functions. [Id., citing Holinstat, M. et al. J. Biol. Chem. (2006) 281: 26665- 74; Voss, B. et al. Mol. Pharmacol. (2007) 71: 1399-1406]. For example, PAR4 activation produces a prolonged signal that is required for stable clot formation [Id., citing Kahn, ML et al. J. Clin. Invest. (1999) 103: 879-87; Covic, L. et al. Biochemistry (2000) 39: 5458-67; Covic, L. et al. Thromb. Haemost. (2002) 87: 722-27; Mazharian, A. et al. J. Biol. Chem. (2007) 282: 5478-87]. [000132] Transactivation of PARs by intermolecular binding between two different PARs has been demonstrated. [Cirino, G. & Vergnolle, N. Curr. Opin. Pharmacol. (2006) 6: 428-34, citing Chen, J., et al. J. Biol. Chem. (1994) 269: 16041-45; O’Brien, PJ et al. J. Biol. Chem. (2000) 275: 13502-9]. Cleaved PAR1 tethered ligand can bind and activate an adjacent uncleaved PAR2. This mechanism of activation was shown to be less efficient than intramolecular binding of the tethered ligand. In platelets, PAR3 acts as a cofactor for PAR4 activation by thrombin: PAR3 recognizes and binds thrombin, which then activates adjacent PAR4. [Id., citing Nakanishi-Matsui, M. et al. Nature (2000) 404: 609-13]. [000133] On human platelets, PAR4 interacts with PAR1 to enhance PAR4 activation [Arachiche, A. et al. J. Biol. Chem. (2013) 288 (45): 32553-62, citing Nieman, MT. Biochemistry (2008) 47: 13279-86; Leger, A. et al. Circulation (2006) 113: 1244-54]. The activation of PAR1 and PAR4 requires the receptors to be cleaved by thrombin. PAR1). PAR1, which has a hirudin-like sequence that binds tightly to thrombin exosite I [Id., citing Liu, L. et al. J. Biol. Chem. (1991) 266: 16977-80] is an excellent thrombin substrate [Id., citing Nieman, MT & Schmaier, AH (2007) Biochemistry 46: 8603-10; Jacques, SL et al. J. Biol. Chem. (2000) 275: 40671-78]. Based on biochemical and structural data from other tight exosite I binders, the PAR1 hirudin-like sequence likely induces thrombin into the protease conformation [Id., citing Kamath, P. et al. J. Biol. Chem. (2010) 285: 28651-58; Huntington, JA. Biochim. Biohys. Acta. (2012) 1824: 246-52]. In contrast, PAR4 does not bind to exosite I and is a poor thrombin substrate when it is expressed on cells alone [Id., citing Nieman, MT. Biochemistry (2008) 47: 13279-86; Bah, A. et al. Proc. Nat. Acad. Sci. USA (2007) 104: 11603-8]. The inefficient activation of PAR4 is overcome by co-expression of PAR1 on human platelets and PAR3 on mouse platelets [Id., citing Leger, AJ et al. Circulation (2006) 113: 1244-54; Kahn, M. et al. Nature (1998) 394: 690-94]. The proposed model for PAR1 or PAR3 enhancing PAR4 activation is that after cleavage of PAR1 or PAR3, thrombin remains bound to the hirudin-like sequence via exosite I and cleaves an adjacent PAR4. In addition to recruiting thrombin to the surface of cells, the exosite I interaction likely holds thrombin in the protease conformation for efficient cleavage of PAR4 [Id., citing Nieman, MT. Biochemistry (2008) 47: 13279-86; Leger, A. et al. Circulation (2006) 113: 1244-54; Nakanishi-Matsui, M. et al. Nature (2000) 404: 609-13]. The functional significance of PAR1 or PAR3 co-expression with PAR4 is a 10-fold reduction in the EC50 of thrombin activation of PAR4 [Id., citing Nieman, MT. Biochemistry (2008) 47: 13279-86; Leger, A. et al. Circulation (2006) 113: 1244-54; Nakanishi-Matsui, M. et al. Nature (2000) 404: 609-13]. [000134] In addition to PAR1 and PAR4 having important independent roles in platelet signaling, the two receptors act synergistically by PAR1 enhancing PAR4 activation. It was demonstrated that PAR1 and PAR4 require allosteric changes induced via receptor cleavage by α-thrombin to mediate heterodimer formation. [Arachiche, A. et al. J. Biol. Chem. (2013) 288 (45): 32553-62]. PAR1-PAR4 heterodimers were not detected when unstimulated; however, when the cells were stimulated with 10 nm α-thrombin, a strong interaction between PAR1 and PAR4 was detected by bioluminescence resonance energy transfer. In contrast, activating the receptors without cleavage using PAR1 and PAR4 agonist peptides (TFLLRN (SEQ ID NO: 14) and AYPGKF (SEQ ID NO: 16), respectively) did not enhance heterodimer formation. Preventing PAR1 or PAR4 cleavage with point mutations or hirugen also prevented the induction of heterodimers. Mutations in PAR1 or PAR4 at the heterodimer interface likewise prevented PAR1-assisted cleavage of PAR4. [000135] It has been reported that pro- and anti-angiogenic proteins are separated in distinct subpopulations of alpha-granules in platelets and megakaryocytes that can undergo differential release in vitro [Italiano, Jr. JE et al. Blood (2008) 111 (3): 1227-33]. The treatment of human platelets with a selective PAR4 agonist (AYPGKF-NH2 (SEQ ID NO: 16)) resulted in release of endostatin-containing alpha granules, but not VEGF-containing alpha granules, while a selective PAR1 agonist (TFLLR-NH2 (SEQ ID NO: 17)) liberated VEGF, but not endostatin-containing granules. [000136] In addition to PARs, platelets express two purinergic GPCRs, P2Y1 and P2Y12, that are activated by dense granule release of ADP. [Smith, TH et al. J. Biol. Chem. (2017) 292 (33): 13867-78, citing Leon, C. et al. Arterioscler. Thromb. Vasc. Biol. (2003) 23: 1941- 47; Jin, J. et al. J. Biol. Chem.273: 2030-34]. P2Y12 has been linked to important aspects of platelet activation, including enhancement of dense granule secretion, recruitment of additional platelets to the site of vascular injury, and enhancement of the efficacy of other pro-coagulant agonists. [Id., citing Cattaneo, M. J. Thromb. Haemost. (2015) 13: S10-S16; Savi, et al. Proc. Natl. Acad. Sci. USA (2006) 103: 11069-74]. Using bioluminescence resonance energy transfer, immunofluorescence microscopy and co-immunoprecipitation in cells expressing receptors exogenously and endogenously, it has been demonstrated that PAR4 and P2Y12 specifically interact and form dimers expressed at the platelet surface, and that activation of PAR4 but not of P2Y12 drives internalization of the PAR4-P2Y12 heterodimer. [Id.] Activated PAR4 internalization was required for recruitment of β-arrestin to endocytic vesicles, which was dependent on co-expression of P2Y12. Stimulation of the Par4-P2Y12 heterodimer promoted β-arrestin and Akt co-localization to intracellular vesicles. [Id.] Moreover, activated PAR4-P2Y12 internalization was required for sustained Akt activation, recruitment to endosomes and AKT signaling. [Id.] [000137] β-arrestin 1 and β-arrestin 2 are well-known negative regulators of G-protein- coupled receptor (GPCR) signaling. Upon GPCR activation, β-arrestins translocate to the cell membrane and bind to the agonist-occupied receptors. This uncouples these receptors from G proteins and promotes their internalization, thus causing desensitization. However, accumulating evidence indicates that β-arrestins also regulate transcription. They function as scaffold proteins that interact with several cytoplasmic proteins and link GPCRs to intracellular signaling pathways such as MAPK cascades. In response to activation of certain GPCRs, β- arrestins translocate from the cytoplasm to the nucleus and associate with transcription cofactors, such as p300 and cAMP response element binding protein (CREB) at the promoters of target genes to promote transcription. They also interact with regulators of transcription factors, such as IκBα and MDM2 in the cytoplasm, regulating the NF-κB and p53 transcription factors, respectively [Ma, L., & Pei, G. J. Cell Sci. (2007) 120: 213-18]. [000138] Through their unique ability to sense serine proteases, such as thrombin, trypsin, and mast cell tryptase, PARs act as “sensors” of extracellular protease gradients.[Moretti, S. et al. Mucosal Immunity (2008) 1: 156-68] Id., citing Coughlin, SR. Nature (2000) 407: 258- 64; Ossovskaya, VS & Bunnett, NW. Physiol. Rev. (2004) (84) 579-621]. During inflammation, host- and microbial-derived proteases trigger the activation of protease-activated receptors (PARs). However, certain microbial proteases can also activate mammalian PARs. [Id., citing Kauffman, HF & van der Heide, S. Curr. Allergy Asthma Rep. (2003) 3: 430-37] Activated PARs couple to signaling cascades that affect, among others, coagulation and inflammatory responses. [Id., citing Cirino, G. & Vergnolle, N. Curr. Opin. Pharmacol. (2006) 6: 428-34]. The role of PARs in inflammation is complex, as individual PARs have both proinflammatory and protective roles in the airway [Id., citing Cocks, TM et a. Nature Z(1999) 398: 156-60], the gastrointestinal tract [Id., citing Ossovskaya, VS & Bunnett, NW. Physiol. Rev. (2004) 84: 579-621; Fiorucci, S. et al. Proc. Natl. Acad. Sci. USA (2001) 98: 13936-41; Vergnolle, N. e al. Trends Pharmacol. Sci. (2001) 22: 146-52; Fiorucci, S. & Distrutti, E. Trends Pharmacol. Sci. (2002) 23: 153-55; Coughlin, SR & Camerer, E. J. Clin. Invest. (2003) 111: 25-27], and the brain [Id., citing Noorbakhsh, F. et al. J. Exp. Med. (2006) 203: 425-35] depending on disease context and cellular type. [000139] For example, PAR1 has been identified as a major endogenous mediator of lung inflammation and fibrosis in a model of bleomycin-induced lung injury. [Cirino, G. & Vergnolle, N. Curr. Opin. Pharmacol. (2006) 6: 428-34, citing Howell, DC et al. Am. J. Pathol. (2005) 166: 1353-65]. Several studies have shown protective effects of pharmacological activation of PAR2 in vivo in LPS induced inflammation [Id., citing Moffatt, JD et al. Am. J. Respir. Cell Mol. Biol. (2002) 26: 680-84] or airway allergy models [Id., citing De Campo, BA & Henry, PJ. Br. J. Pharmacol. (2005) 144: 1100-8]. Other studies have demonstrated that PAR2- deficient mice are protected against ovalbumin-induced airway allergic immune response. [Id.]. In isolated human bronchi, activation of both PAR1 and PAR2 causes a moderate contractile response that, at least in part, is mediated by direct activation of smooth muscle cell receptors. [Schmidlin, F. et al. Am. J. Crit. Care Med. (2001) 164: 1276-81]. [000140] Uncontrolled tissue factor-dependent activation of coagulation during infection leads to disseminated intravascular coagulation. [Antoniak, S. et al. J. Clin. Investig.123(3): 1310-1322, citing Geisbert, TW et al. J. Infect. Dis. (2003) 188 (11): 1618-29 (Ebola); Taylor, FB et al. Circ. Shock (1991) 33: 127-34 (E. coli); Ruf, W. Trends Immunol. (2004) 25 (9): 461-6413, 24-26 (Ebola)]. Fibrin has been shown to contribute to the innate immune response to bacterial infections by increasing the expression of inflammatory mediators. [Id., citing Degen, JL et al. J. Thromb. Haaemost. (2007) 5 (1): 24-31; Esmon, CT et al. J. Thromb. Haemost. (2011) 1: 182-88]. PAR-1 expression is increased in endothelial cells after Dengue and CMV viral infection. [Antoniak, S. et al. J. Clin. Investig. 123(3): 1310-1322., citing Huerta-Zepeda, A. et al. Thromb. Haemost. (2008) 99 (5)L 936-43; [Popovic, M. et al. J. Thromb Thrombolysis. (2010) 30 (2): 164-71]. Studies with cultured endothelial cells have found that tissue factor, thrombin, PAR-1, and PAR-2 contribute to the infectivity of the DNA virus herpes simplex virus type 1, [Id., citing Sutherland, MR, et al. J. Thromb. Haemost. (2007) 5(5): 1055-61; Sutherland, MR et al. Blood (2012) 119 (15): 3638-45]. [000141] When an antiviral response was induced in macrophages and mice with the double stranded RNA mimetic polyinosinic:polycytidylic acid (poly I:C), activation of PAR1 enhanced poly I:C induction of IFNβ and interferon (IFN)-γ inducible protein CXCL10 expression in the murine macrophage cell line RAW264.7, bone marrow-derived mouse macrophages and mouse spenocytes. Human interferon-inducible protein 10 (IP-10 or CXCL10) is a chemokine of the CXC family [Gotsch, F. et al. J. Matern. Fetal Neonatal Med. (2007) 20 (11): 777-92]. Members of this chemokine family have pro-inflammatory properties and act as modulators of angiogenesis in conditions such as wound healing, ischemia and neoplasia. [000142] Next, poly I:C was used to induce a type I IFN innate immune response in the spleen and plasma of wild type (WT) and PAR1-/- mice. The results showed that poly I:C treated PAR1-/- mice and WT mice given the thrombin inhibitor dabigatran etexilate exhibited significantly less IFNβ and CXCL10 expression in the spleen and plasma than WT mice. These studies were interpreted to suggest that thrombin activation of PAR-1 contributes to the antiviral innate immune response in mice. [Antoniak, S. et al. J. Innate Immun. (2017) 9: 181- 92]. [000143] In vitro, the PAR1 antagonist RWJ-56110 has been shown to reduce the replication of respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) in A549 cells. [Le, VB et al. Br. J. Pharmacol. (2018) 175: 388-403]. In agreement with these results, RWJ-56110-treated mice were protected against RSV and hMPV infections, as indicated by less weight loss and mortality. This protective effect in mice correlated with decreased lung viral replication and inflammation. In contrast, hMPV-infected mice treated with the PAR1 agonist TFLLR-NH2 (SEQ ID NO: 17) showed increased mortality, when compared to infected mice which were left untreated. Thrombin generation was shown to occur downstream of PAR1 activation in infected mice via exposure to tissue factor as part of the inflammatory response; thrombin inhibition by argatroban reduced the pathogenicity of the infection with no additive effect to that induced by PAR1 inhibition. [000144] The inflammatory phase (see below) begins during the hemostasis phase. Thrombocytes, as well as recruited white blood cells, release numerous factors to ramp up the healing process. Alpha-granules liberate platelet-derived growth factor (PDGF-A, B), platelet factor IV, and transforming growth factor beta (TGF-β). The processes of inflammation, collagen degradation and collagenogenesis, myoblastic creation from transformed fibroblasts, growth of new blood vessels, and reepithelialization are mediated by a host of cytokines and growth factors. The interleukins strongly influence the inflammatory process. Vascular endothelial growth factor (VEGF) and other factors enhance blood vessel formation, and some have multiple roles, such as fibroblast growth factor (FGF)-2, which affects not only the process of angiogenesis but also that of reepithelialization. Vasoactive amines, such as histamine and serotonin, are released from dense bodies found in thrombocytes. PDGF is chemotactic for fibroblasts and, along with TGF-β, is a potent modulator of fibroblastic mitosis, leading to prolific collagen fibril construction in later phases. Fibrinogen is cleaved into fibrin, and the framework for completion of the coagulation process is formed. Fibrin provides the structural support for cellular constituents of inflammation. This process starts immediately after the insult and may continue for a few days. Second Phase: Inflammation [000145] The early component of the inflammatory phase is predominated by the influx of the polymorphonuclear leukocytes (PMNs) and the later component of the inflammatory phase is predominated by monocytes/macrophages. [000146] Within the first 6-8 hours, PMNs engorge the wound. TGF-β facilitates PMN migration from surrounding blood vessels, from which they extrude themselves from these vessels. These cells cleanse the wound, clearing it of debris. The PMNs attain their maximal numbers in 24-48 hours and commence their departure by hour 72. Other chemotactic agents are released, including FGF-2, TGF-β and TGF-a, PDGF-A,B, and plasma-activated complements C3a and C5a (anaphylactic toxins). They are sequestered by macrophages or interred within the scab or eschar (Id.; Habif. Dermatologic surgical procedures. Clinic Dermatology: A Color Guide to Diagnosis and Therapy.3rd ed.1996.809-810). [000147] As the process continues, monocytes also exude from surrounding blood vessels. Once they leave the vessel, these are termed macrophages. The macrophages continue the cleansing process, manufacture various growth factors during days 3-4, and orchestrate the multiplication of endothelial cells with the sprouting of new blood vessels, the duplication of smooth muscle cells, and the creation of the milieu created by the fibroblast. Many factors influencing the wound healing process are secreted by macrophages, including TGFs, cytokines and interleukins , tumor necrosis factor (TNF), and PDGF. Third Phase: Granulation and Proliferation [000148] The granulation and proliferation phase consists of an overall and ongoing process, comprising subphases termed the "fibroplasia, matrix deposition, angiogenesis and re- epithelialization" subphases (Cho & Lo. Dermatol Clin.1998 Jan; 16(1 ): 25-47). [000149] By days 5-7, fibroblasts have migrated into the wound, laying down new collagen of subtypes I and III. Early in normal wound healing, type III collagen predominates but is later replaced by type I collagen. [000150] Tropocollagen is the precursor of all collagen types and is transformed within the cell's rough endoplasmic reticulum, where proline and lysine are hydroxylated. Disulfide bonds are established, allowing 3 tropocollagen strands to form a triple left-handed triple helix, termed procollagen. As the procollagen is secreted into the extracellular space, peptidases in the cell membrane cleave terminal peptide chains, creating true collagen fibrils. [000151] The wound is suffused with glycosaminoglycans (GAGs) and fibronectin produced by fibroblasts. These GAGs include heparin sulfate, hyaluronic acid, chondroitin sulfate, and keratin sulfate. Proteoglycans are GAGs that are bonded covalently to a protein core and contribute to matrix deposition. [000152] Angiogenesis results from parent vessel offshoots. The formation of new vasculature requires extracellular matrix and basement membrane degradation followed by migration, mitosis, and maturation of endothelial cells. Basic FGF (FGF-2) and vascular endothelial growth factor are believed to modulate angiogenesis. Vasculogenesis (meaning the formation of primitive vascular structures via the differentiation of endothelial precursor cells (EPCs), which is distinct from angiogenesis- also can be seen. [000153] Re-epithelization occurs with the migration of cells from the periphery of the wound and accessory or adjoining tissues. This process commences with the spreading of cells within 24 hours. Division of peripheral cells occurs in hours 48-72, resulting in a thin epithelial cell layer, which bridges the wound. Epidermal growth factors are believed to play a key role in this aspect of wound healing. [000154] This succession of subphases can last up to 4 weeks in the clean and uncontaminated wound. Fourth Phase: Remodeling and Maturation [000155] After the third week, the wound undergoes constant alterations, known as remodeling, which can last for years after the initial injury occurred. Collagen is degraded and deposited in an equilibrium-producing fashion, resulting in no change in the amount of collagen present in the wound. The collagen deposition in normal wound healing reaches a peak by the third week after the wound is created. Contraction of the wound is an ongoing process resulting in part from the proliferation of specialized fibroblasts termed myofibroblasts, which provide mechanical support and integrity to the tissue after initial injury. Wound contraction occurs to a greater extent with secondary healing (i.e., healing by second intention, which describes a wound left open and allowed to close by reepithelialization and contraction by myofibroblasts) than with primary healing (i.e., healing by first intention, which describes a wound closed by approximation of wound margins or by placement of a graft or flap, or wounds created and closed in the operating room, unlike via reepithelialization and contraction by myofibroblasts). Maximal tensile strength (the greatest longitudinal stress a substance can bear without tearing apart) of the wound is achieved by the 12th week, and the ultimate resultant scar has only 80% of the tensile strength of the original skin that it has replaced. At the end of tissue repair, the reconstructed ECM takes over the mechanical load and myofibroblasts disappear by massive apoptosis (Tomasek et al. Nat Rev Mol Cell Biol.2002 May; 3(5): 349-63). Platelets and the immunopathology of viral infections [000156] Platelets form a functional link between wound healing, coagulation and immune responses. [000157] Platelets are now recognized as major inflammatory cells with key roles in the innate and adaptive response. [Hottz, ED, et al. Frontiers in Medicine (2018) 5: 121]. Activated platelets have key thromboinflammatory functions linking coagulation to immune responses in various infections, including viral infections. Besides their central role in hemostasis, platelets modulate inflammatory reactions and immune responses by direct interaction with leukocytes and endothelial cells and via release of soluble inflammatory mediators that enhance recruitment of leukocytes and trigger their activation [Assinger, A. Frontiers Immunology (2014): 00649, citing Weyrich, AS, et al. J. Thromb. Haemost. (2003) 1: 1897-905; von Hundelshausen, P. & Weber, C. Cir. Res. (2007) 100: 27-40; Semple, JW et al. Nat. Rev. Immunol. (2011) 11: 264-74; Karshovska, E. et al. Thromb. Haemost. (2013) 110: 894-902]. [000158] Platelets express surface receptors, such as lectins, integrins, and toll-like receptors (TLR), allowing them to directly interact with several pathogens. [000159] Platelets also express Fc receptors by which they can recognize immunocomplexes. [Id., citing Anderson, CL et al. Semin. Thromb. Hemost. (1995) 21: 1-9]. [000160] Platelets are produced in the bone marrow by megakaryocytes, which develop from hematopoietic stem cells. Megakaryocytes first undergo linage commitment, followed by endomitosis resulting in polyploidy [Id., citing Patel, SR et al. J. Clin. Invest. (2005) 115: 3348- 54]. After endoplasmic maturation, megakaryocytes form proplatelets, which bud off thousands of platelets and microparticles into the blood stream [Id., citing Kuter, DJ. Br. J. Haematol. (2014) 165: 248-58]. Megakaryopoiesis is triggered by a variety of cytokines (e.g., GM-CSF, IL-3, IL-6, IL-11, FGF4, and SDF-1), with thrombopoietin (TPO) being the most important. TPO not only triggers megakaryocyte development but also plays a role in maintaining stem cells [Id., citing de Graaf, CA & Metcalf, D. Cell Cycle (2011) 10: 1582-9]. [000161] Viruses can modulate platelet production at various steps of development. Some viruses (e.g., simian immunodeficiency virus (SIV), which triggers TPO production via up- regulation of tumor growth factor (TGF) β [Id., citing Metcalf Pate, KA, et al. J. Acquir. Immune Defic. Syndr. (2014) 65: 510-6]; human herpes virus 6, which can interfere with TPO- inducible megakaryocytic colony formation [Id., citing Isomura, H. et al. J. Gen. Virol. (2000) 81: 663-73]; human herpes virus 7, which impairs the survival and differentiation of megakaryocytes [Id., citing Gonelli, A. et al. Haematologica (2002) 87: 1223-5], are able to influence the cytokine profile of the host, resulting in altered TPO production in the liver. [000162] Some viruses also directly interfere with TPO production by destruction of liver tissue as shown for hepatitis C virus (HCV) [Id., citing Afdhal, N. et al. J. Hepatol. (2008) 48: 1000-7]. The resulting drop in TPO production results in delayed megakaryocyte development and a decrease in platelet production. Other viruses infect bone marrow stromal cells and hematopoietic stem cells, resulting in altered cytokine production and decreased number of progenitor cells, thereby disturbing hematopoiesis [Id., citing Kolb-Maurer, A. & Goebel, W. EMS Microbiol. Lett. (2003) 226: 203-7]. [000163] For example, human immunodeficiency virus (HIV), cytomegalovirus (CMV), and HCV are known to replicate in megakaryocytes [Id., citing Chelucci, C. et al. Blood (1998) 91: 1225-34; Lil, X. et al. J:. Viral Hepat. (1999) 6: 107-14; Crapnell, K. et al. Blood (2000) 95: 487-93] and many more viruses can interact with megakaryocytes modulating their proliferation and function [Id., citing Li, X. et al. J. Viral Hepat. (1999) 6: 107-14; Flaujac, C. et al. Cell Mol. Life Sci. (2010) 67: 545-56; Yang, M. et al. Hematology (2005) 10: 101-5; Lutteke, N. et al. Virology (2010) 405: 70-80; Passos, AM et al. Acta Haematol. (2010) 124: 13-18; Carballal, G. et al. J. Infect. Dis. (1981) 143: 7-14]. Viral infection of megakaryocytes can increase apoptosis and decreases the maturation and ploidy of megakaryocytes. Moreover, virus-infected megakaryocytes have been shown to express less surface c-Mpl, which is the receptor for TPO [Id., citing Gibellini, D. e al. World J. Virol. (2013) 2: 91-101]. [000164] Platelet–virus interaction can occur via a variety of platelet receptors. Recent studies have demonstrated that platelets exhibit several pattern recognition receptors (PRR) including those from the toll-like receptor, NOD-like receptor, and C-type lectin receptor family and are first-line sentinels in detecting and responding to pathogens in the vasculature. [Hottz, ED, et al. Frontiers in Medicine (2018) 5: 121]. While traditional platelet activation by G-protein-coupled receptors is usually rapid, platelet PRR activation in responses to infectious and immune stimuli can be delayed and sustained, lasting hours after initial aggregation and secretion [Id., citing Vieira-de-Abreu, A. et al. Semin. Immunopathol. (2012) 34: 5-30; Shashkin, PN, et al. J. Immunol. (2008) 181: 3495-502]. PRR from different classes have been shown to be expressed and functional in platelets, including those from C-type lectin receptors (CLR) and toll-like receptors (TLRs) families [Id., citing Middleton, EA et al. Physiol. Rev. (2016) 96: 1211-59; Cognasse, F. et al. Immunol. Cell Biol. (2005) 83: 196098; D’Atri, LP & Schattner, M. Front. Biosci. (Landmark Ed. ) 2017] 22: 1867-83]. [000165] CLRs are a large family of surface proteins containing at least one carbohydrate- binding domain which are specialized in the recognition of bacterial, fungi, or viral glycans [Id., citing Shiokawa, M. et al. Curr. Opinion. Microbiol. (2017) 40: 123-30; Marakalala, MJ & Ndlovu, H. PLoS Pathog. (2017) 13: e1006333.10.1371/journal.ppat.1006333]. Some viruses can exploit certain CLR for viral attachment and entry in host cells, including in platelets [Id., citing Turville, SG et al. Nat. Immunol. (2002) 3: 975-83; Chaipan, C. et al. J. Virol. (2006) 80: 8951-60]. [000166] The TLRs, a family of transmembrane cellular sensors, are the best described class of PRR in platelets. While virtually all TLRs (1–10 in human) are detected at some level (mRNA or protein) in platelets [Id., citing Vieira-de-Abreu, A. et al. Semin. Immunopathol. (2012) 34; 5-10; Middleton, EA et al. Physiol. Rev. (2016) 96: 1211-59; Semple, JW, et al. Nat. Rev. Immunol. (2011) 11: 264-74; Cognasse, F. et al., Immunol. Cell Biol. (2005) 83: 196-98; Cognasse, F. et al., Front. Immunol. (2015) 6: 83; Koupenova, M. et al. Arterioscler. Thromb. Vasc. Bio. (2015) 35: 1030-7; Rowley, JW et al. Blood (2011) 118 (14): e101- 11.10.1182/blood-2011-030339705], most functional characterization of TLR-mediated platelet responses was reported for those specialized in bacterial molecules, especially regarding platelet TLR-4 activation by lipopolysaccharide (LPS) from Gram-negative bacteria [Id., citing Shashkin, PN et al. J. Immunol. (2008) 181: 3495-502; Brown, GT & McIntyre, TM, J. Immunol. (2011) 186: 5489-96; Aslam, R. et al., Blood (2006) 107(2): 637-41; Andonegui, G. et al. Blood (2005) 106: 2417-23; Stahl, AL et al. Blood (2006) 108: 167-76; Clark, SR et al. Nat. Med. (2007) 13: 463-69]. The expression and functionality of endosomal TLR-3, -7, and -9, related to recognition and response to viral genome in nucleated cells, have also been reported in platelets [Id., citing Thon, JN, et al. J. Cell Biol. (2012) 198: 561-74; Koupenova, M. et al., Blood (2014) 124: 791-802; D’Atri, LP et al., J. Thromb. Haemost. (2015) 13: 839-50]. [000167] Two major cytoplasmic PRRs from the NOD-like receptor (NLR) family were reported to be expressed and functional in platelets: the nucleotide-binding domain leucine rich repeat containing pyrin 3 (NLRP3), a major sensor for the activation of inflammasome that recognizes various bacterial, viral, and tissue damage signals; and the nucleotide-binding oligomerization domain 2 that recognizes the bacterial cell wall peptidoglycan component muramyl dipeptide [Id., citing Hottz, ED, et al., Blood (2013) 122: 3405-14; Zhang, S. et al. Circulation (2015) 131: 1160-70; Hottz, ED, et al. Mediators Inflamm. (2015) 22015: 12-15]. Other intracellular sensors including retinoic acid-inducible gene I and melanoma differentiation-associated gene 5, which are highly specialized in viral RNA recognition, are expressed in human megakaryocytes in response to type I interferon (IFN-α and -β) [Id., citing Lutteke, N. et al. Virology (2010) 405: 70-80], but their expression in platelets, as well the ability of platelets to express other IFN-stimulated genes (ISGs) and to perform IFN-induced antiviral response are not known. [000168] In addition, megakaryocytes express PRR and cytokine receptors, and there is evidence that TLR agonists or cytokine engagement affects megakaryocytic maturation and thrombopoiesis [Id., citing D’Atri, LP et al. J. Thromb. Haemost. (2015) 13: 839-50; Negrotto, S. et al. J. Thromb Haemost. (2011) 9: 277-85; Beaulieu, LM et al., Arterioscler. Thromb. Vasc. Biol. (2014) 34: 552-64; Beaulieu, LM et al., Blood (2011) 117: 5963-74]. Megakaryocytes and megakaryocytic cell lines respond to viral infections or viral PAMPs by secreting high levels of α and β IFN [Id., citing D’Atri, LP et al. J. Thromb. Haemost. (2015) 13: 839-50; Lutteke, N. et al. Virology (2010) 405: 70-80; Negrotto, S. et al. J. Thromb. Haemost. (2011) 9: 2477-85; Rivadeneyra, L. et al., Thromb. Haemost. (2015) 114: 982-93; Pozner, RG, et al. PLoS Pathog. (2010) 6: e1000847.10.1371/journal.ppat.10000847], which reduce platelet production in vitro through an autocrine IFNAR signaling [Id., citing D’Atri, LP et al. J. Thromb. Haemost. (2015) 13: 839-50; Rivadeneyra, L. et al. Thromb. Haemost. (2015) 114: 982-93; Pozner, RG et al. PLoS Pathog. (2010) 6: e10000847.10.1371/journal.ppat.10000847; Yamane, A. et al. Blood (2008) 112: 542-50]. [000169] Platelet-virus interactions are mainly mediated by integrins, surface lectins, and TLRs. [Assinger, A. Frontiers Immunology (2014): 00649]. For example, platelets can bind CMV via TLR2, which triggers platelet activation and degranulation and results in enhanced platelet interaction with neutrophils [, citing Assinger, A. et al. Arterioscler. Thromb. Vasc. Bio. (2014) 34: 801-9]. Encephalomyocarditis virus (EMCV) interacts with platelet TLR7, which also leads to platelet degranulation and direct platelet–neutrophil interactions [Id., citing Koupenova, M. et al. Blood (2014) 124: 791-802]. Platelet fragments subsequently get phagocytized by neutrophils, thereby contributing to the drop in platelet count [Id., citing Koupenova, M. et al. Blood (2014) 124: 791-802]. [000170] Rotavirus utilizes the collagen receptor GPIa/IIa to bind to platelets [Id., citing Flaujac, C. et al. Cell Mol. Life Sci. (2010) 67: 545-56; Coulson, BS et al. Proc. Nat. Acad. Sci. USA (19997) 94: 5389-94], while hantavirus and adenoviruses interact with platelets via the fibrinogen receptor GPIIb/IIIa [Id., citing Mackow, ER & Gavrilovskaya, IN. Curr. Top. Microbiol. Immuno. (2001) 256: 91-115] However, GPIIb/IIIa does not seem to be the unique receptor for platelet–adenovirus interaction, as inhibition of GPIIb/IIIa does not alter platelet internalization of adenoviruses [Id., citing Gupalo, E. et al. Platelets (2013) 24: 383-91]. In addition, hantavirus-infected endothelial cells can bind to quiescent platelets via GPIIb/IIIa [Id., citing Gavrilovskaya, IN et al. J. Virol. (2010) 84: 4832-9], which results in platelet activation and clearance but might also influence vascular permeability [Id., citing Goeijenbier, M. et al. J. Med. Virol. (2012) 84: 1680-96]. [000171] GPIIb/IIIa is the most abundant platelet integrin and displays bi-directional signaling functions. Inside-out signaling, which is positively regulated by various platelet agonists, is mediated by intracellular protein–protein interactions and biochemical reactions that regulate GPIIb/IIIa affinity [Id., citing Coller, BS & Shattil, SJ. Blood (2008): 112: 3011- 25]. These intracellular processes triggering GPIIb/IIIa activation are complex and include recruitment of talin, which separates the GPIIb and the GPIIIa subunits, and kindlins, which are involved in GPIIb/IIIa activation [Id., citing Moser, M. et al. Nat. Med. (2008) 14: 325- 30] independent of talin recruitment [Id., citing Kahner, BN et al. PLoS One (2012) 7: e34056]. Further, G protein subunit Gα13 directly binds to the cytoplasmic domain of GPIIIa and promotes ligand binding to GPIIb/IIIa [Id., citing Gong, H. et al. Science (2010) 327: 340-4]. [000172] Outside-in signaling via receptor binding promotes actin polymerization and platelet spreading [Id., citing Coller, BS & Shattil, SJ. Blood (2008) 112: 3011-25] and can thereby enhance virus attachment to endothelial cells but also promote platelet clearance. [000173] Epstein–Barr virus (EBV) interaction with platelets occurs via complement receptor 2 (CR2) [Id., citing Nunez, D. et al. Eur. J. Immuno. (1987) 17: 515-20]. HIV and dengue virus activate platelets by binding to lectin receptors such as C-type lectin domain family 2 (CLEC-2) and cell-specific intercellular adhesion molecule-3-grapping non-integrin (DC-SIGN) [Id., citing Chaipan, C. et al. J. Virol. (2006) 80: 8951-60]. Platelets and/or megakaryocytes can further interact with HIV envelope proteins via C–X–C chemokine receptor type 4 (CXCR4) or via chemokine (C–C motif) ligand (CCL) 3 (MIP-1α) and 5 (RANTES) [Id., citing Flaujac, C. et al. Cell Mol. Life Sci. (2010) 67: 545-56]. After many years of infection, HIV-1 changes its co-receptor usage from CCR5 to CXCR4 only; this receptor change represents a switch to non-CD4-dependent platelet activation at late stages of disease. [000174] In addition, platelets express the Coxsackie virus-specific Coxsackie- adenovirus receptor (CAR) [Id., citing Othman, M. et al. Blood (2007) 109: 2832-9] and HCV interacts with platelets via collagen receptor GPVI [Id., citing Zahn, A. et al. J. Gen. Virol. (2006) 87: 2243-51]. [000175] These direct interactions often result in platelet activation and adhesion of activated platelets to leukocytes. Platelet binding to neutrophils triggers phagocytosis of platelets [Id., citing Koupenova, M. et al. Blood (2014) 124: 791-802; Maugeri, N. et al. Platelets (2014) 25: 224-5] and platelet activation itself promotes platelet clearance in spleen and liver [Id., citing Grozovsky, R. et al. Curr. Opin. Hematol. (2010) 17: 585-9]. [000176] Host defense mechanisms in response to viral infections can also lead to platelet activation. For example, many viral infections lead to systemic inflammation, which in turn triggers platelet activation and decreases platelet life span [Id., citing Badimon, L. et al. Circulation (1992) 86: 11174-85]. Among others, influenza virus, rhinovirus, and CMV infection result in up-regulation of cytokines, such as interleukin 6 (IL-6), in target cells [Id., citing Bouwman, J. et al. Eur. J. Clin. Invest. (2002) 32: 759-66]. [000177] Platelets can be activated by these cytokines, leading to platelet–leukocyte interactions, which foster leukocyte and endothelial activation, further amplifying platelet activation and enhancing their clearance by splenic macrophages or Kupffer cells in the liver [Id., citing Nunez, D. et al. Eur. J. Immunol. (1987) 17: 515-20]. Monocytes that encounter dengue virus, for example, start generating platelet activating factor (PAF) [Id., citing Zapata, JC et al. PLoS Negl. Trop. Dis. (2014) 8: e2858], which is a lipid mediator that triggers platelet activation. This leads to enhanced apoptosis of platelets and accelerates platelet clearance in secondary dengue infection [Id., citing Alonzo, MT et al. J. Infect. Dis. (2012) 205: 1321-9]. [000178] Several virus infections activate the coagulation cascade via induction of tissue factor (TF) expression in target cells. Generation of thrombin by the activated coagulation cascade causes platelet activation and subsequent clearance via protease activating receptor (PAR) signaling [Id., citing Antoniak, S. & Mackman, N. Blood (2014) 123: 2605-13]. PARs on platelets, endothelial cells, and leukocytes are important modulators during viral infections, which modulate innate immune responses and exert positive and negative effects on TLR- dependent responses [Id., citing Antoniak, S. & Mackman, N. Blood (2014) 123: 2605-13]. [000179] Platelets also recognize viral particles coated with immunoglobulins via their FcγRII receptor, which results in Fc receptor-mediated platelet activation, aggregation, and platelet clearance [Id., citing Anderson, CL et al. Semin. Thromb. Hemost. (1995) 21: 1-9; Cox, D. et al. J. Thromb. Haemost. (2011) 9: 1097-107]. FcγRII-mediated platelet activation depends on IgG and GPIIb/IIIa engagement and involves ADP and thromboxane A2 (TxA2) feedback mechanisms to cause platelet aggregation, which is further enhanced by CXCL4 [Id., citing Arman, M. et al., Blood (2014) 123: 3166-74]. [000180] Furthermore, B-lymphocyte production of antibodies against some viruses has been shown to interfere with platelet survival. These antibodies, which usually target surface glycoproteins of viruses, show a cross-reactivity with platelet surface integrins such as GPIIb/IIIa or GPIb-IX-V [Id., citing Goeijenbier, M. et al. J. Med. Virol. (2012) 84: 1680-96]. This so called idiopathic thrombocytopenic purpura (ITP) or platelet autoantibody-induced thrombocytopenia has been described for HCV, HIV, CMV, EBV, hantavirus, varicella zoster virus, herpes viruses, and severe acute respiratory syndrome coronavirus [Id., citing Goeijenbier, M. et al. J. Med. Virol. (2012) 84: 1680-96]. [000181] The term “thrombocytopenia” as used herein refers to a drop in platelet count caused by either decreased platelet production or increased platelet destruction. It is associated with an increased bleeding risk. [000182] Viral infection can either enhance platelet activation resulting in pro-thrombotic events, or diminish platelet responses leading to bleeding complications. [000183] Activation of the coagulation cascade has been observed in various virus infections, including HIV, dengue, and Ebola virus infection [Id., citing Antoniak, S. & Mackman, N. Blood (2014) 123: 2605-13]. Changes in the activation of the coagulation cascade and modulation of platelet count and function, which are also observed during viral infections, lead to an increased risk of disseminated vascular coagulation (DIC), deep vein thrombosis (DVT) thrombosis, and hemorrhage in infected patients [Id., citing Goeijenbier, M. et al. J. Med. Virol. (2012) 84: 1680-96]. [000184] Thrombocytopenia is a common result of viral infections and associated with an increased bleeding risk. Approximately 10% of HIV positive patients and up to 60% of patients with acquired immunodeficiency syndrome (AIDS) suffer from thrombocytopenia, which can lead to severe bleeding in these patients [Id., citing Gibellini, D. et al. World J. Virol. (2013) 2: 91-101]. In many viral infections, platelet function and aggregation in response to different agonists are diminished [Id., citing Flaujac, C. et al. Cell Mol. Life Sci. (2010) 67: 545-56], causing bleeding complications in viral hemorrhagic fevers (VHF) [Id., citing Zapata, JC, et al. PLoS Negl. Trop. Dis. (2014) 8: e2858]. VHF outbreaks lead to the deaths of thousands of people every year and are caused by different enveloped RNA viruses, which include Arenaviridae (e.g., Lassa virus), Bunyavirideae (e.g., hantavirus), Filioviridae (e.g., Marburg and Ebola virus), and Flaviviridae (e.g., dengue virus). [000185] Thrombocytopenia in response to viral infections is often multifactorial. Rapidly induced thrombocytopenia in response to viral infections is mediated via enhanced platelet destruction. [000186] For example, HIV results in impaired survival of bone marrow megakaryocytes and their precursors. HIV also decreases the number and activity of human progenitor cells and decreases megakaryocyte maturation and ploidy. HIV surface glycoprotein gp120 leads to increased megakaryocyte apoptosis in vitro due to increased TGFβ and down-regulation of the proliferation-inducing ligand tumor necrosis factor ligand superfamily member 13 (TNFSF13). Further, gp120 interacts with CD4, which is expressed by immature megakaryocytes, which also express CCR5, and leads to their infection [Id., citing Louache, F. et al. Blood (1991) 78: 1697-1805]. Furthermore, HIV infection of megakaryocytes can lead to reduced TPO receptor (c-Mpl) expression. [000187] In dengue virus infection, platelet production is impaired by suppression of megakaryopoiesis via infection of hematopoietic progenitor cells or indirectly via altered cytokine levels in the bone marrow due to impaired stromal cell function [Id., citing Zapata, JC et al. PLoS Negl. Trop Dis. (2014) 8: e2858]. Platelets from patients with dengue infection present signs of activation, mitochondrial dysfunction, and enhanced apoptosis, which may contribute to the genesis of thrombocytopenia [Id., citing Hottz, ED et al. J. Thromb. Haemost (2013) 11: 951-62; Hottz, ED et al. J. Immunol. (2014) 193: 1864-72]. Further, enhanced destruction of platelets occurs due to cross-reaction of platelets with anti-dengue virus antibodies. Dengue virus-induced anti-non-structural protein-1 (NS-1) induces complement- mediated lysis of platelets and thereby further accelerates thrombocytopenia [Id., citing Sun, DS et al. J. Thromb. Haemost. (2007) 5: 2291-9]. NS-1 can also activate endothelial cells and leads to increased vascular permeability and further platelet activation [Hottz, ED et al. Blood (2013) 122: 3405-14]. Dengue virus-infected patients show increased levels of E-selectin on their endothelial cell surface, which promotes adhesion and clearance of platelets [Id., citing Hottz, ED et al. Blood (2013) 122: 3405-14; Chuansumrit, A. & Chaiyaratana, W. Thromb. Res. (2014) 133: 10-16] as well as enhanced activation of the coagulation cascade [Id., citing Nascimento, EJ et al. Crit. Rev. Immunol. (2014) 34: 227-40]. [000188] Arenavirus infection by either lymphocytic choriomeningitis virus (LCMV) or Junin virus results in thrombocytopenia and decreased agonist-induced platelet responses in mice [Id., citing Iannacone, M. et al. Proc. Natl Acad. Sci. USA (2008) 105: 629-34; Pozner, RG, et al. PLoS Pathog. (2010) 6: e1000847]. As a consequence, platelet depletion in LMCV- infected mice results in lethal hemorrhagic anemia [Id., citing Iannacone, M. et al. Proc. Natl Acad. Sci. USA (2008) 105: 629-34]. This effect is caused by diminished platelet responses, rather than solely a drop in platelet count. The underlying mechanism of altered platelet production and reduced platelet reactivity was found to rely on virus-induced production of interferon (IFN) α/β [Id., citing Iannacone, M. et al. Proc. Natl Acad. Sci. USA (2008) 105: 629-34, Pozner, RG, et al. PLoS Pathog. (2010) 6: e1000847]. Junin virus mainly infects CD34+ cells not megakaryocytes but impairs proplatelet formation and platelet release via IFNα/β receptor signaling [Id., citing Pozner, RG, et al. PLoS Pathog. (2010) 6: e1000847]. IFNα/β receptor signaling represents an important paracrine repressor of megakaryopoiesis, which directly inhibits TPO-induced signaling through induction of suppressor of cytokine signaling 1 (SOCS-1) [Id., citing Wang, Q. et al. Blood (2000) 96: 2093-9], induction of 2′5′- oligoadenylate synthetase (OAS) [Id., citing Iannacone, M. et al. Proc. Natl Acad. Sci. USA (2008) 105: 629-34], and decease of nuclear factor erythroid 2 (NF-E2) expression [Id., citing Pozner, RG, et al. PLoS Pathog. (2010) 6: e1000847]. [000189] Hantavirus can directly interact with and activate platelets via GPIIb/IIIa [Id., citing Mackow, ER & Gavrilovskaya, IN, Curr. Top. Microbiol. Immunol. (2001) 256: 91- 115] and infection of megakaryocytes with hantavirus induces the up-regulation of human leukocyte antigen (HLA) class 1 molecules on the megakaryocyte surface, which leads to cytotoxic T-lymphocyte-mediated destruction of megakaryocytes [Id., citing Lutteke, N. et al. Virology (2010) 405: 70-80]. [000190] Viruses also possess enzymes, which can modulate platelet functions. For example, Influenza virus exhibits neuraminidase (sialidase), which hydrolyses the terminal sialic acid residues from host cell receptors and thereby decreases the life span of platelets by targeting platelets for rapid clearance in the liver and spleen [Id., citing Sorensen, AL et al. Blood (2009) 114: 1645-54]. As another example, mycoviral neuraminidase has been shown to reduce platelet life span by cleaving sialic acid in the platelet membrane [Id., citing Terada, H. et al. Blood (1966) 28: 213-28]. Besides the effect on platelet live span, neuroaminidase further alters megakaryocyte ploidy as well as morphology and size of platelets [Id., citing Stenberg, PE et al. J. Cell Physiol. (1991) 147: 7-16]. Newcastle virus can directly disrupt platelet cell membrane, resulting in platelet lysis [Id., citing Turpie, AG et al. Lab Invest. (1973) 28: 575-83]. Human parvovirus 19 cannot reproduce in megakaryocytes, but triggers a drop in platelet count via platelet activation [Id., citing Srivastava, A. et al. Blood (1990) 76: 1997-2004]. [000191] In line with the observation that platelet dysfunction increases the risk of hemorrhages and therefore mortality, it has been demonstrated that pharmacological inhibition of either platelets or the coagulation cascade increases the mortality of H1N1-infected mice [Id., citing Antoniak, S. & Mackman, N. Blood (2014) 123: 2605-13]. Thus, aspirin treatment, which inhibits platelet activation via inhibition of cyclooxygenase and subsequent thromboxane A2 production, has been hypothesized to have worsened the incidence and severity of the influenza pandemics in the 1910s [Id., citing Starko, JM. Clin. Infect. Dis. (2009) 49: 1405-10]. [000192] Altered platelet gene expression and functional responses in patients infected with SARS-CoV-2 have recently been reported. Manne, BK et al. Blood (2020) doi.org/10.1182/blood.2020007214. The receptor for SARS-CoV-2 binding, ACE2, was not detected by mRNA or protein in platelets. mRNA from the SARSCoV-2 N1 gene was detected in platelets from 2/25 COVID-19 patients, suggesting platelets may take-up SARS-COV-2 mRNA independent of ACE2. Resting platelets from COVID-19 patients had increased P- selectin expression basally and upon activation. Circulating platelet-neutrophil, monocyte and T cell aggregates were all significantly elevated in COVID-19 patients compared to healthy donors. Vascular permeability [000193] The molecular and genetic events that regulate vascular permeability are not fully understood. [000194] Vascular permeability by any measure is dramatically increased in acute and chronic inflammation, cancer, and wound healing. This hyperpermeability is mediated by acute or chronic exposure to vascular permeabilizing agents, particularly vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A). Three distinctly different types of vascular permeability can be distinguished, based on the different types of microvessels involved, the composition of the extravasate, the anatomic pathways by which molecules of different size cross-vascular endothelium, the time course over which permeability is measured; and the animals and vascular beds that are being investigated. These are the basal vascular permeability (BVP) of normal tissues, the acute vascular hyperpermeability (AVH) that occurs in response to a single, brief exposure to VEGF-A or other vascular permeabilizing agents, and the chronic vascular hyperpermeability (CVH) that characterizes pathological angiogenesis. [Nagy, JA, et al. Angiogenesis (2008) 11(2): 1009- 119] Basal vascular permeability (BVP) [000195] Molecular exchange in normal tissues takes place primarily in capillaries. Indeed, Nagy, et al state that the primary function of several major organs (heart, lungs, kidneys) and of larger blood vessels (arteries, arterioles, veins, venules) is to supply the capillaries, and thus the tissues, with nutrients and to clear waste products. [Nagy, JA, et al. Angiogenesis (2008) 11(2): 1009-119]. The molecules exchanged consist largely of gases (O2 and CO2), water, small molecules such as salts and sugars, and only small amounts of plasma proteins. The process is driven largely by diffusion. The extent of BVP varies considerably in different normal tissues and is subject to substantial change in response to changes in hydrostatic pressure, opening of closed vessels, surface area available for exchange, blood flow, etc. [000196] Water and lipophilic solutes (e.g., gases such as O2 and CO2) are able to diffuse through endothelial cells; they also pass readily through inter-endothelial cell junctions and through endothelial fenestrae. Fenestrae are greatly thinned (70–150-nm diameter) zones of microvascular endothelium that can be induced by VEGF-A [60]. They are found in small numbers in many types of vascular endothelium and are especially numerous in specialized vascular beds that supply tissues that secrete protein hormones. They are induced in other types of vascular endothelium by VEGF-A [Id., citing Roberts, WG, Palade, GE. J. Cell Sci. (1995) 108 (Pt.6): 2369-2379]. Fenestrae are closed by a thin diaphragm, similar structurally to the diaphragms closing the stomata found in caveolae and vesiculo-vacuolar organelles (VVOs) [Id., citing Dvorak, A. (2007) Electron microscopic-facilitated understanding of endothelial cell biology: contributions established during the 1950s and 1960s. Aird, W. Cambridge University Press, New York; Stan R (2007) Endothelial structures involved in vascular permeability. Aird, W. Cambridge University Press, New York]. Small lipophilic molecules can also dissolve in endothelial cell membranes and so pass from the vascular lumen to the interstitium. [000197] However, none of these routes provided a satisfactory explanation for the passage of large molecules. Acute vascular hyperpermeability (AVH) [000198] A rapid increase in vascular permeability occurs when the microvasculature is exposed acutely to any of a number of vascular permeabilizing factors, e.g., VEGF-A, histamine, serotonin, PAF, etc. Single exposure to any of these permeability factors results in a rapid but self-limited (complete by 20–30 min) influx of plasma into the tissues. [000199] Not only is the quantity of extravasated fluid greatly increased above that found in BVP but its composition is greatly changed. As already noted, the fluid passing from the circulation into normal tissues under basal conditions is a plasma filtrate, i.e., a fluid consisting largely of water and small solutes but containing very little plasma protein. However, the fluid that extravasates in AVH is rich in plasma proteins, approaching the levels found in plasma, and is referred to as an exudate. Among the plasma proteins that extravasate are fibrinogen and various members of the blood clotting cascade. When these come into contact with tissue factor, a protein that is normally expressed by many interstitial cells, the clotting system is activated and the exudate clots to deposit fibrin [Id., citing Dvorak, HF, et al. Science (1981) 212: 923- 24; Van De Water, L. et al. Cancer Res. (1985) 45: 5521-25]. Fibrin forms a gel that traps water and other solutes, restraining their clearance by lymphatics or capillaries and resulting in tissue swelling (edema). Fibrin in tissues has other functions that are discussed below. However, as long as the permeability stimulus is not continuous, the deposited fibrin is rapidly degraded without further consequences. [000200] AVH also differs from BVP in that the vascular leakage takes place not from capillaries but from post-capillary venules, highly specific vessels just downstream of capillaries [Id., citing Majno, G. et al. J. Biophys. Biochem. Cytol. (1961) 11: 607-26; Majno, G. et al. J. Cell Biol. (1969) 42: 647-672]. Whereas capillaries have a flattened endothelium, venules are lined by a much taller, cuboidal endothelium. Majno also proposed a mechanism of protein leakage, namely that histamine and other vascular permeabilizing agents induced endothelial cells to contract and pull apart to form intercellular (paracellular) gaps of sufficient size to permit plasma-protein extravasation. [000201] The vesiculo-vacuolar organelle (VVO) in venular endothelium offers an alternative, trans-endothelial cell route for plasma extravasation in response to permeability factors [Id., citing Kohn, S. et al. Lab Invest. (1992) 67: 596-607; Dvorak, AM et al. J. Leuk. Biol. (1996) 59: 100-115; Feng, D. et al. Microvascular Res. (2000) 59: 24-37; Feng, D. et al. J. Exp. Med. (1996) 183: 1981-86; Feng, D. et al. J. Physiol. (1997) 504 (Pt. 3): 747-61]. VVOs are grape-like clusters comprised of hundreds of uncoated, cytoplasmic vesicles and vacuoles that together form an organelle that traverses venular endothelial cytoplasm from lumen to albumen. VVOs often extend to inter-endothelial cell interfaces and their individual vesicles (unlike caveolae) commonly open to the inter-endothelial cell cleft. The vesicles and vacuoles comprising VVOs vary in size from those the size of caveolae to vacuoles with volumes as much as 10-fold larger [Id., citing Feng, D. et al. Microcirculation (1999) 6: 23- 44]. These vesicles and vacuoles are linked to each other and to the luminal and abluminal plasma membranes by stomata that are normally closed by thin diaphragms that appear similar to those found in caveolae. Whether VVOs somehow take the place of caveolae in caveolin-1 null mice and thereby contribute to the increased permeability observed in these animals needs to be investigated. It seems that vascular permeability inducing agents cause the diaphragms interconnecting vesicles and vacuoles to open, thereby providing a transcellular pathway for plasma and plasma-protein extravasation. The underlying mechanism could be mechanical, as was the endothelial cell contraction mechanism originally postulated by Majno [Majno, G. et al. J. Cell Biol. (1969) 42: 647-72]. If so, the actin–myosin contractions induced by permeability factors would act to pull apart the diaphragms linking adjacent VVO vesicles and vacuoles, resulting in a transcellular rather than an inter-endothelial cell (paracellular) route for plasma extravasation. Three-dimensional (3D) reconstructions at the electron microscopic level have demonstrated that many of the openings induced in venular endothelium by permeability factors are in fact trans-endothelial cell pores [Id., citing Feng, D. et al. J. Physiol. (1997) 504 (Pt.3): 747-61; Feng, D. et al. Microcirculation (1999) 6: 23-44; Neal, CR, Michel, CC. J. Physiol. (1995) 488 (Pt.2): 427-37]. Chronic vascular hyperpermeability (CVH) [000202] Whereas acute exposure to VEGF-A results in immediate but self-limited hyperpermeability of normal venules, chronic exposure results in profound changes in venular structure and function that lead to the chronic hyperpermeability of pathological angiogenesis as found in tumors, healing wounds, and chronic inflammatory diseases such as rheumatoid arthritis, psoriasis, cellular immunity, etc. [Id., citing Dvorak H (2007) Tumor blood vessels. Aird, W. Cambridge University Press, New York; Dvorak, HF, Am. J. Pathol. (2003) 162: 1747-57; Nagy, JA et al. Cancer Res. (1995) 55: 360-68]. As in AVH, the fluid that extravasates is an exudate that approaches the overall composition of plasma. [000203] In contrast to BVP and AVH, fluid leakage in CVH does not take place from any type of normal blood vessel. Instead, whether in tumors or wounds, the blood vessels that leak are newly formed, highly abnormal angiogenic blood vessels; these are primarily mother vessels (MV), and also, to a lesser extent, glomeruloid microvascular proliferations (GMP) that form from MV [Id., citing Nagy JA, et al. Lab Invest. (2006) 86: 767-80; Pettersson, A. et al. Lab Invest. (2000) 80: 99-115; Sundberg, C. et al. Am. J. Pathol. (2001) 158: 1145-60; Brown, LF, et al. EXS (1997) 79: 233; Ren, G. et al. J, Histochem. Cytochem. (2002) 50: 71-79]. Mother Vessels (MVs) are greatly enlarged sinusoids that arise from preexisting normal venules by a process that involves pericyte detachment, vascular basal lamina degradation, and a 4–5-fold increase in lumen size that is accompanied by extensive endothelial cell thinning. Poiseuille’s law indicates that blood flow is proportional to the fourth power of the vascular radius. Nonetheless, MV exhibit sluggish blood flow because of their hyperpermeability to plasma which results in a striking increase in hematocrit. [000204] The protein-rich exudates in CVH interact with tissue factor to trigger the clotting system and deposit fibrin [Id., citing Dvorak, HF et al. Science (1981) 212: 923-24; Van De Water, L. et al. Cancer Res. (1985) 45: 5521-25]. Tissue factor is expressed on many tumor cells as well as host interstitial cells and is induced in endothelial cells by VEGF-A [Id., citing Dvorak, HF, Rickles, FR (2005) Hemostasis and thrombosis in cancer. In: Colman RW, Hirsh J, Marder VJ, Clowes AW, George JN (eds) Homeostasis and thrombosis. Lippincott Williams & Wilkins, Philadelphia]. In addition to its fluid trapping properties, fibrin also has a number of other properties when it persists over time as in tumors and healing wounds. It provides a pro-angiogenic provisional stroma that induces and is later replaced by the ingrowth of new blood vessels and fibroblasts and the laying down of mature fibro-vascular stroma [Id., citing Dvorak, HF. Am. J. Pathol. (2003) 162: 1747-57; Dvorak, HF, et al. J. Immunol. (1979) 122: 166-74, Dvorak, HF et al. J. Natl Cancer Inst. (1979) 62: 1459-7259]. Fibrin interacts with integrins expressed by multiple cell types and so supports the migration of tumor cells as well as host mesenchymal cells (endothelial cells, pericytes, fibroblasts) and inflammatory cells (neutrophils, monocytes). Fibrin also sequesters growth factors, protecting them from degradation, and induces the expression of proangiogenic molecules such as IL-8 and tissue factor. Fragment E, a fibrin breakdown product, is directly pro-angiogenic [Id., citing Dvorak H (2007) Tumor blood vessels. Aird, W. Cambridge University Press, New York; Dvorak, HR, Rickles, FR. (2005) Hemostasis and thrombosis in cancer. In: Colman RW, Hirsh J, Marder VJ, Clowes AW, George JN (eds) Homeostasis and thrombosis. Lippincott Williams & Wilkins, Philadelphia]). [000205] Macromolecules such as ferritin extravasate from MV and glomeruloid microvascular proliferations (GMP) largely by a transcellular route [Id., citing Nagy, JA, et al. Lab Invest. (2006) 86 (8): 767-80]. Because MV develop from normal venules by a process that involves extensive vascular enlargement with consequent endothelial cell thinning, processes that are thought to be facilitated, at least in part, by a transfer of VVO membranes to the plasma membrane, MV, as well as the GMP that derive from MV, contain many fewer and less complex VVOs than normal venular endothelium. However, the path length for molecular extravasation is greatly shortened as tracers such as ferritin need to pass through only a few, often only one or two, vesicles or vacuoles to reach the ablumen [Id., citing Nagy, JA, et al. Lab Invest. (2006) 86 (8): 767-80]. Macromolecules also extravasate through fenestrae that are present in both MV and GMP [Id., citing Feng, D. et al. Microcirculation (1999) 6: 23-44]. Pores of the type that have been described in AVH have also been found in the endothelial cells of blood vessels supplying tumors [Id., citing Majno, G. et al. J. Cell Biol. (1969) 42: 647-72; Freng, D. et al. Microvascular Research (2000) 59: 24-37; Roberts, WG, Palade, GE. J. Cell Sci. (1995) 109 (6): 2369-79]. While such openings have often been called intercellular, careful 3D reconstructions of serial electron microscopic sections have shown that many pores induced by vascular permeabilizing agents are in fact transcellular pores that pass through endothelial cell cytoplasm [Id., citing Feng, D. et al. J. Physiol. (1997) 504 (3): 747-61; Neal, CR, Michel, CC. J. Physiol. (1995) 488 (2): 427-37]. Virus infection induces increased permeability of the lung microvasculature [000206] A longitudinal study of epithelial barrier breakdown in mice infected with virulent PR8 or less virulent x31 strains of influenza A virus (IAV) showed continued inflammation in survivors even 30 days after infection, by which time acute markers of infection, including viral presence, had resolved. [Gregory, DJ and Kobzik, L., Am. J. Physiol. Lung Cell Mol. Physiol. (2015) 309(10): L1041-L1046, citing Sanders, CJ et al. Am J Physiol Lung Cell Mol Physiol (2013) 304: L481–L488]. Others have also reported sustained lung damage, with increased barrier permeability to radiolabeled albumin even 3 weeks after nonlethal infection. [Id., citing Gotts, JE. Am. J. Physiol. Lung Cell Mol. Physiol. (307): L395- L406]. The more commonly used measure of total protein in bronchoalveolar lavage fluid returned to normal within about 3 wk, indicating that improved protein clearance compensates for the sustained permeability [Id., citing Gotts, JE. Am. J. Physiol. Lung Cell Mol. Physiol. (307): L395-L406]. This is reflected in the slow recovery of lung capacity measured by plethysmography [Id., citing (Julander, JG, et al. Antiviral Res. (2011) 92: 228-36). [000207] While a possible beneficial effect of this sustained permeability is to allow accumulation of mediators such as plasma gelsolin, which protects against postinfluenza bacterial pneumonia by enhancing macrophage function [Id., citing Yang, Z. et al. Am. J. Physiol. Lung Cell Mol. Physiol.309: L11-L16], earlier interventions to preserve the alveolar- capillary barrier during the acute phase of primary influenza infection (or other insults) may have beneficial effects. Daily administration of a synthetic angiopoietin-1 mimic improves survival of mice infected with PR8 or x31 [Id., citing Sugiyama, MG et al. Sci Rep (2015) 5: 11030, 2015]. The mimic, called vasculotide, activates the cell-cell adhesion protein TEK [Id., citing Van Slyke, P. et al. Tissue Eng. Part A (2009) 15: 1269-8049] and had been shown to maintain barrier function of human microvascular endothelial cells treated in vitro with lipopolysaccharide and to improve alveolar-capillary barrier function and survival in mice during experimental endotoxemia [Id., citing David, S. et al. Am J Physiol Lung Cell Mol Physiol 300: L851–L862, 2011]. [000208] Similarly, a monoclonal antibody used to neutralize angiopoietin-like 4 (ANGPTL4) improved both barrier function and infiltration following experiential infection with PR8 [Id., citing Li, L. Cell Rep (2015) 10: 654–63]. In contrast to angiopoietin-1, ANGPTL4 interacts with integrin α5β1, VE-cadherin, and claudin-5 to weaken cell-cell interactions and loosen the epithelial barrier [Id., citing Huang, RL, et al. Blood (2011) 118: 3990-4002]. Its expression is elevated in both experimentally infected mouse lungs and human postmortem lung tissue, suggesting a maladaptive response [Id., citing Li, L. Cell Rep. (2015) 10: 654-63]. [000209] Sphingosine 1-phosphate (S1P) is a physiological lipid signaling intermediate involved in both immunoregulation and epithelial barrier function [Id., citing Chi H. Trends Pharmacol Sci (2011) 32: 16–24, Natarajan, V. et al. (2013) Am J Respir Cell Mol Biol 49: 6– 17]. The S1P analog 2-amino-4-(4-heptyloxyphenyl)-2-methylbutanol [AAL(R)] showed some success in protecting mice from infection with either 2009 pandemic or mouse-adapted H1N1 strains [Id., citing Walsh, KB, et al. Proc Natl Acad Sci USA (2011) 108: 12018–12023]. Mice treated with AAL(R) 1 h after infection showed improvements in lung damage and survival accompanied by reduction in proinflammatory cytokine production, including type I interferons and macrophage chemokines compared with vehicle-treated controls. [Id., citing Walsh, KB et el. Proc Natl Acad Sci USA (2011) 108: 12018–12023]. AAL(R) is phosphorylated in vivo by sphingosine kinase 2 to AFD(R) ([(2R)-2-amino-4-(4- heptoxyphenyl)-2-methylbutyl] dihydrogen phosphate), which stimulates S1P receptors (S1PR) 1, 3, 4, and 5 [Id., citing Jary, E., et al. Mol. Pharmacol. (2010) 78: 685-92; Oldstone, MBA, et al. Virology (2013) 4435: 92-101]. A more selective S1PR1 agonist, CYM5442, also reduced cytokine production by acting on endothelial cells rather than lymphocytes [Id., citing Teijaro, JR, et al. Cell (2011) 146: 980-991]. Given that excessive cytokine production is correlated with poor prognosis [Id., citing Cheng, XW, et al. Respir. Physiol. Neurobiol. (2011) 175: 185-187; Liu, Q. et al. Cell Mol. Immunol. (2015) doi:10.1038/cmi.2015.74; Oldstone, MBA, et al. Virology (2013) 435: 92-101; Peiris JSM, et al. Curr Opin Immunol (2010) 22: 475–481, 2010], it has been suggested that pathology is mediated by an endothelial cell-driven cytokine storm, which is suppressed by S1P analogs [Id., citing Oldstone, MBA, et al. Virology (2013) 435: 92-101]. However, suppression of the cytokine response failed to match the therapeutic benefit of S1P analogs or angiopoietin (ant)agonists [Id., citing Salomon, R. et al. Proc Natl Acad Sci USA 104: 12479–12481, 2007]. In addition to regulating immune cell activity and recruitment, S1P is a regulator of endothelial cell adhesion [Id., citing Natarajan, V. et al. Am J Respir Cell Mol Biol (2013) 49: 6–17; Rosen, H. et al. Trends Immunol. (2007) 28: 102-107]. It is therefore possible that mortality and cytokine storm are both effects of the permeabilized alveolar-capillary barrier function, rather than the cytokine storm being directly causative of mortality. [Id.] Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) [000210] ALI and its more severe form, ARDS, are syndromes of acute respiratory failure that result from acute pulmonary edema and inflammation. ALI/ARDS is a cause of acute respiratory failure that develops in patients of all ages from a variety of clinical disorders, including sepsis (pulmonary and nonpulmonary), pneumonia (bacterial, viral, and fungal), aspiration of gastric and oropharyngeal contents, major trauma, and several other clinical disorders, including severe acute pancreatitis, drug over dose, and blood products [Ware, L. and Matthay, M., N Engl J Med, (2000) 342:1334-1349,]. Most patients require assisted ventilation with positive pressure. The primary physiologic abnormalities are severe arterial hypoxemia as well as a marked increase in minute ventilation secondary to a sharp increase in pulmonary dead space fraction. Patients with ALI/ARDS develop protein-rich pulmonary edema resulting from exudation of fluid into the interstitial and airspace compartments of the lung secondary to increased permeability of the barrier. Additional pathologic changes indicate that the mechanisms involved in lung edema are complex and that edema is only one of the pathophysiologic events in ALI/ARDS. One physiologic consequence is a significant decrease in lung compliance that results in an increased work of breathing [Nuckton T. et al., N Engl J Med. (2002) 346:1281-1286,], one of the reasons why assisted ventilation is required to support most patients. [000211] It was suggested that mechanical ventilation (MV), a mainstay treatment for ALI, potentially contributes to and worsens permeability by exacting mechanical stress on various components of the respiratory system causing ventilator-associated lung injury (VALI) [Fan, E. et al., JAMA (2005) 294:2889-2896; MacIntyre N., Ches (2005), 128:561 S-567,]. A 2000 trial demonstrated a significant improvement in survival in patients ventilated with low (LVT) compared to high tidal volumes (HVT) [The Acute Respiratory Distress Syndrome N. Ventilation with Lower Tidal Volumes as Compared with Traditional Tidal Volumes for Acute Lung Injury and the Acute Respiratory Distress Syndrome. N Engl J Med. (2000) 342:1301- 1308,]. Other than ventilating at lower tidal volumes, which presumably imparts lower mechanical stress, there is little mechanistic understanding of the pathophysiology and no directed therapies for VALI. [000212] It has been suggested that the high tidal volumes (HVT) of mechanical ventilation (MV) results in phosphorylation of p38 MAP kinase, activation of MK2, and phosphorylation of HSPB1, a process that causes actin to disassociate from HSPB1 and polymerize to form stress fibers, which ultimately leads to paracellular gaps and increased vascular permeability. Furthermore, it was shown that inhibiting p38 MAP kinase or its downstream effector MK2 prevents the phosphorylation of HSPB1 and protects from increased vascular permeability by abrogating actin stress fiber formation and cytoskeletal rearrangement, suggesting that targeted inhibition of MK2 could be a potential therapeutic strategy for the treatment of acute lung injury [Damarla, M. et al., PLoS ONE (2009) 4(2): E4600]. The renin-angiotensin system in acute lung injury [000213] An increasing body of evidence supports the role of an activated renin- angiotensin system (RAS) in acute lung injury. [Nicholls, J., Peiris, M., Nature Medicine (2005) 11 (8): 821-22]. ACE levels increase in the brochoalveolar fluid of individuals with ARDS [Id., citing Idell, S. et al., Chest (1987) 91: 52-56]; ACE is thought to influence both vascular permeability and the air-vessel interface, as well as maintain pneumocyte viability. Furthermore, treatment of rats with ALI using ACE antagonists delays the onset of ARDS. [Id., citing Raiden, S., et al. J. Pharmacol. Exp. Ther. (2002) 303: 45-51]. As for ACE2, ACE2 has been shown to have an opposing function to ACE and protects against lung injury. [Id., citing Imai, Y. et al. Nature (2005) 436 (7047): 112-116]. The protective effect of ACE2 seems to result partially from the conversion of angiotensin II by ACE2 to angiotensin1-7, thereby reducing angiotensin II binding to the cell membrane receptors AT1aR (angiotensin II type 1a receptor) and AT2R (angiotensin II type 2 receptor). It is believed that angiotensin II binding to AT1aR will stimulate lung injury, whereas binding to AT2R reduces lung injury. Kuba et al proposed that binding of SARS-CoV to ACE2 downregulates ACE2, thus leaving angiotensin II unmodified, allowing it to continue to bind to the AT1aR to aggravate the lung injury and produce lung edema. [Kuba, K. et al., Nat. Med. (2005) 11: 875-79]. [000214] Age-dependent differences in host defense and the pulmonary renin-angiotensin system may be responsible for observed differences in epidemiology of ARDS between children and adults. In particular, higher levels of markers involved in the neutrophil response (e.g., levels of myeloperoxidase, interleukin (IL-6), IL-10 and p-selectin) were seen with increasing age, whereas ICAM-1 was higher in neonates. While preclinical studies had suggested that age-dependent differences in the RAS system are responsible for observed differences in epidemiology of ARDS between children and adults, age was not associated with changes in the pulmonary renin-angiotensin system (RAS); indeed, no differences in activity of ACE and ACE2 were seen. [Schouten, LR et al. Ann. Intensive Care (2019) 9: 55]. Viral mediated ALI [000215] The underlying pathophysiology of virally mediated ALI is not well understood, and it is likely that there are unique signature mechanisms to each viral strain that converge onto a common end pathway resulting in diffuse alveolar damage (DAD). It remains to be seen whether epithelial injury is the primary lesion or is coincident to endothelial injury. Most community-acquired respiratory viral pneumonias are inhaled and bind to receptors in the upper respiratory tract. Although the viruses initially infect the respiratory epithelium, it is possible that this is merely a portal of entry, and the important steps in alveolar damage are mediated primarily by endothelial injury resulting in elaboration of cytokines and chemokines and recruitment of both innate and adaptive immune cells. The specific cytokine profiles vary by viral pathogen and may be driven by macrophages, epithelial cells, endothelial cells, or some combination of crosstalk. If lung injury is not primarily mediated by viral infection, but rather is a result of the inflammatory host response, then viral clearance may not be central to the resolution of lung injury. [Hendrickson, CM, Matthay, MA Semin. Respir. Crit. Care Med. (2013) 34: 475-86]. [000216] In clinical practice, viral infections are diagnosed using direct fluorescence assays (DFA), PCR-based assays, and viral culture. These methods only assess the most well- known human pathogens, including, without limitation, influenza A and B, parainfluenza, respiratory syncytial virus (RSV), adenovirus, metapneumovirus, rhinovirus, enterovirus, coronavirus, and cytomegalovirus (CMV). Research laboratories have the ability to detect many more viruses with nucleic acid amplification methods, although the clinical significance of these viruses is not well understood. [Hendrickson, CM, Matthay, MA Semin. Respir. Crit. Care Med. (2013) 34: 475-86]. Cytomegalovirus (CMV) [000217] For example, the virion of CMV, a member of the herpesvirus family; contains not only DNA, but also four species of mRNA. [Huang, E-S, Johnson, RA, Nature Medicine (2000) 6: 863-64]. CMV is a common pathogen in severely immunosuppressed populations (especially organ transplant recipients), but was previously thought to be an uncommon pathogen in nosocomial pneumonia. With new diagnostic tools, the importance of detection, prevention, and treatment of CMV pneumonia in a broader population of critically ill patients is being reconsidered. Seroprevalence for CMV in adults ranges from 50 to 90%. CMV has been detected in nonimmunocompromised adults with critical illness and is thought to be reactivation of latent infection. It is associated with longer intensive care unit (ICU) and hospital stays, longer duration of mechanical ventilation, and increased rates of nosocomial infections. CMV infection is associated with increased interleukin (IL)-6 and IL-8 in vitro and in vivo, and these cytokines have been associated with ALI and ARDS. [Id., citing Limaye, AP, Boeckh, M. Rev. Med. Virol. (2010) 20(g): 372-79] Autopsy studies suggest that CMV may be an important pathogen in ventilator-associated pneumonia (VAP). [Id., citing Chiche, L. et al. Curr. Opin. Infect. Dis. (2011) 24 (2): 152-56]. Hantavirus [000218] Hantaviruses are enveloped viruses with a negative sense, single-stranded RNA genome that belong to the family Bunyaviridae [Saggioro, FP, et al. J. Infectious Disease (2007) 195 (10): 1541-49, citing Simmons, JH, Riley, LK. Comp. Med. (2002) 52: 97-110]. Distinct hantavirus types are distributed throughout the world and are associated with different primary rodent reservoirs [Id., citing Simmons, JH, Riley, LK. Comp. Med. (2002) 52: 97- 110; Lednicky, J.A. Arch. Pathol. Lab Med. (2003) 127: 30-35; Peters, CH, Khan, AS. Clin. Infect. Dis. (2002) 34: 1224-31; Johnson, KM. Curr. Top. Microbiol. Immunol. (2001) 256: 1-14]. The spectrum of clinical symptoms caused by hantavirus infections in humans varies from subclinical presentation to pulmonary involvement progressing with shock (hantavirus pulmonary syndrome [HPS]) or severe hemorrhagic fever with renal involvement (hemorrhagic fever with renal syndrome) [Id., citing Zaki, SR, Peters, CJ. Viral hemorrhagic fevers. In: Connor DH, Chandler FW, Schwartz DA, Manz HJ, Lack EE, eds. Pathology of infectious diseases.1st ed., Stamford: Appleton & Lange, 1997, vol.1 (pg.347-64; Duchin, JS, et al. N. Engl. J. Med. (1994) 330: 949-55; Lee, HW, et al. J. Infect. Dis. (1998) 137: 298- 308; Centers for Disease Control and Prevention. Outbreak of acute illness—southwestern United States, 1993, MMWR Morb Mortal Wkly Rep, 1993, vol. 42 (pg. 421-4)]. HPS is an acute respiratory illness, first identified in Four Corners, southwestern United States, in 1993, characterized by a capillary-leak syndrome in the lungs and clinically presenting as an adult respiratory distress syndrome [Id., citing Centers for Disease Control and Prevention. Outbreak of acute illness—southwestern United States, 1993, MMWR Morb Mortal Wkly Rep, 1993, vol.42 (pg.421-4); Nolte, KB, et al. Hum. Pathol. (1995) 26: 110-20; Zaki, SR, et al. Am. J. Pathol. (1995) 146: 552-79]. it is well known that several species of the hantavirus genus have wide distribution in the Americas, causing HPS with similar clinical manifestations [Id., citing Johnson, AM et al. J. Med. Virol. (1999) 59: 527-35; Moreli, ML et al., Mem. Inst. Oswaldo Cruz (2004) 99: 633-38]. Inflammatory cytokines produced locally after activation of lymphomononuclear cells in the lungs and systemic production of TNFα, IL-10, IL-4, and IFNγ by activated mononuclear cells have been suggested to play a role in the pathogenesis of HPS [Id., citing Mori, M. et al. J. Infect. Dis. (1999) 179: 295-302; Borges, AA. Et al Microbes Infect. (2006) 8: 2324-30]. SNV virions and antigen were identified in endothelial cells in the lungs and other organs in fatal cases of HPS [Id., citing Nolte, KB et al. Hum. Pathol. (1995) 26: 110-20; Zaki, SR et al. Am. J. Pathol. (1995) 146: 552-79]. Influenza [000219] Influenza viruses belong to the family Orthomyoviridae, and are enveloped negative-sense RNA viruses with segmented genomes. There are three antigenically distinct subtypes, A, B, and C, which circulate globally among human populations. Influenza A viruses are subdivided based on antigenic characterization of the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). There are 16 HA subtypes and nine NA subtypes. The clinical response to influenza infection ranges from mild disease to severe pneumonia, and it remains unclear whether the inflammatory response to infection is protective or pathogenic. Infection rarely induces symptoms of lower respiratory tract infections or severe lung injury. Influenza virus infection has been a global health concern since the 1918 Spanish flu pandemic. Three influenza pandemics occurred in the 20th century. The pandemics of 1918, 1957, and 1968 were caused by different antigenic subtypes of influenza A: H1N1, H2N2, and H3N2, respectively. In March 2009 a novel influenza virus emerged in Mexico and the United States and quickly spread worldwide. The pandemic A (H1N1) virus originated from the triple- reassortment of swine influenza (H1) virus circulating in North American pigs. [Huang, E-S, Johnson, RA, Nature Medicine (2000) 6: 863-64]] Nucleic acid sequencing showed that the HA, nucleoprotein (NP), and nonstructural protein (NS) gene segments were from the classical swine viruses; PB1 gene segment from human seasonal H3N2 influenza viruses; and PB2 and PA genes from avian influenza viruses. NA and M gene segments were genetically different from previously isolated human pathogens and found to originate from Eurasian swine influenza strains. Studies in ferrets and mice showed that, compared with seasonal H1N1, intranasal inoculation with pandemic H1N1 causes a higher morbidity, higher viral titers in lung tissue, and viral shedding in the gastrointestinal tract, suggesting a more invasive pathogen. [Id., citing Maines T R, et al. Science (2009) 325(5939):484–487; Munster V J, et al. Science. (2009) 325(5939):481–4830; Itoh Y, Shinya K, Kiso M. et al. In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses. Nature.2009;460(7258):1021– 1025] [000220] The majority of H1N1-infected patients were children or adults aged < 60 years; most recovered uneventfully, and the overall mortality was not higher than that of seasonal influenza. Risk factors for more severe infection by pandemic H1N1 include extremes of age, underlying medical illness, obesity, and pregnancy. [Id., citing Loouie, JK et al. N. Eng. J. Med. (2010) 362 (1): 27-35; Centers for Disease Control and Prevention (CDC), MMWR Morb. Mortal. Wkly Rep. (2009) 58 (38): 1071-74; Jamieson, DJ et al. Lancet (2009) 374 (9688): 451-58; Jain, S. et al. N. Eng. J. Med. (2009) 361 (20): 1935-44]. However, some previously healthy patients without comorbidities developed rapidly progressive pneumonia, ARDS, multiorgan failure, and death, with ARDS reported to be the prominent cause of death. Although epidemiological data from the H1N1 pandemic suggested that obese patients are at higher risk for ALI and have elevated levels of various inflammatory cytokines, the cytokine profiles from sera collected from ARDSNet participants showed that obese patients with ALI induced by a variety of mechanisms had lower levels of proinflammatory cytokines IL-6 and IL-8. [Id., citing Stapleton, RD et al. Chest (2010) 138 (3): 568-77]. This finding suggested that the description of serum cytokines profiles varies by disease state, and it is unlikely that cytokine profiles are conserved among mechanisms of ALI from viral infections. This study did find elevated levels of surfactant protein D (SP-D) and von Willebrand factor (vWF), markers for endothelial injury, in obese patients with ALI. [Hendrickson, CM, Matthay, MA Semin. Respir. Crit. Care Med. (2013) 34: 475-86] Three distinct pulmonary histological patterns have been described in autopsy studies of patients infected with pandemic H1N1; DAD, necrotizing bronchiolitis, and DAD with intense pulmonary hemorrhage. [Id., citing Mauad, T. et al. Am. J. Respir. Crit. Care Med. (2010) 181 (1): 72-79] Several case series found a significant proportion of patients with severe H1N1 infection had secondary bacterial pneumonia with Streptococcus, Staphylococcus, and Haemophilus species, and a few cases with multiple bacterial pathogens isolated. [Id., citing Cheng, VC et al. J. Infect. (2009) 59(5): 366-70; Centers for Disease Control and Prevention (CDC) MMWR Morb. Mortal. Wkly Rep. (2009) 58 (27): 749-52]. These autopsy studies show H1N1 results in a nonspecific final pathological pattern and may predispose to superimposed bacterial infections. A cohort study by Rice et al [Id., citing Rice, TW et al. Crit. Care Med. (2012) 40 (5): 1487-98] found that bacterial coinfection was common in patients infected with pandemic H1N1 admitted to ARDSNet ICUs in North America. Thirty percent of patients in this study had evidence of bacterial coinfection, and of the patients with bacterial infections, 11% had Staphylococcus aureus in blood or respiratory cultures and 8% had Streptococcus pneumoniae. Human studies from serum, BAL fluid, and autopsy specimens have not shed much light on the molecular mechanisms involved in H1N1-induced ALI. [Hendrickson, CM, Matthay, MA Semin. Respir. Crit. Care Med. (2013) 34: 475-86] [000221] In addition to antiviral, immunomodulatory and pro-inflammatory ISGs, IFN signaling results in the transcription and translation of cell death-inducing ISGs. In the context of viral infection, infected cells in which the internal activation of antiviral ISGs is not sufficient to restrict viral replication are sacrificed to prevent the release of infectious progeny virions to limit viral spreading. However, especially in the lung, the disruption of the alveolar epithelial barrier by cell death of infected cells, and also noninfected bystander cells induced by factors such as TRAIL, significantly contributes to worsened disease outcome. [Peteranderl, C., Herold, S. Front. Immunol. (2017) :8: 313]. [000222] Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the TNF cytokine family expressed on the cell surface of some cells (such as NK cells), which induces cell death in target cells by ligation of the death receptors DR4 and DR5, has been reported to be inducible by both type I and type III IFNs. Its signaling outcomes can differ largely depending on its delivered form (membrane-bound versus soluble), the availability of DRs on the target cell membrane, alternate intracellular pathways that might be activated, and the pathogen itself, which might exploit TRAIL-induced pathways for its own survival and replication. In acute respiratory infection, TRAIL signaling is often part of an IFN-driven overshooting inflammatory reaction that promotes unspecific tissue injury and thus disease severity by increasing functional and structural changes in infected and non-infected cells. [Id.] [000223] Davidson et al. [Id., citing Davidson S, et al. Nat Commun (2014) 5:3864] demonstrated that type I IFN application to IAV-infected mice increased morbidity and lung injury, which could be attributed to both DR5 and TRAIL upregulation inducing epithelial cell apoptosis. It was reported that the IAV strain used in these studies, A/PR8 (H1N1), which is highly pathogenic for mice, induced an approximately 800-fold induction in macrophage TRAIL expression, whereas the lower pathogenic virus A/X-31 (H3N2) only stimulated TRAIL by a factor of eight. [Id., citing Högner K, , et al. Macrophage-expressed IFN-β contributes to apoptotic alveolar epithelial cell injury in severe influenza virus pneumonia. PLoS Pathog (2013) 9(2):e1003188]. The relationship between TRAIL induction and IAV strain-specific pathogenicity also translates to the highly pathogenic avian H5N1 IAV, causing severe pneumonia in mice as well as in humans [Id., citing Uiprasertkul M, et al. Emerg Infect Dis (2007) 13(5):708–12, Szretter KJ, et al. J Virol (2007) 81(6):2736–44]. Moreover, human infection with both the highly pathogenic H5N1 as well as the pandemic 1918 H1N1 IAV strains are characterized by a massive influx of mononuclear phagocytes into the alveoli, which is correlated with extensive alveolar epithelial cell apoptosis [Id., citing Cheung CY, et al. Lancet (2002) 360(9348):1831–7, Perrone LA, et al. PLoS Pathog (2008) 4(8):e1000115]. Additionally, macrophages gained from bronchoalveolar lavages of patients presenting with ARDS caused by the pandemic H1N1/2009 virus strain showed high surface expression and release of TRAIL [Id., citing Högner K, et al. PLoS Pathog (2013) 9(2):e1003188]. Another report demonstrated that in highly pathogenic avian influenza, in addition to macrophages also the alveolar epithelium might be involved in causing elevated levels of TRAIL in the alveolar space [Id., citing Hui KP, et al. Sci Rep (2016) 6:28593]. Besides its role in apoptosis, TRAIL signaling upon IAV infection has also been implicated in the induction of necroptosis in fibroblasts, DCs, and lung epithelial cells [Id., citing Rodrigue-Gervais IG, et al. Cell Host Microbe (2014) 15(1):23–35, Nogusa S, et al. Cell Host Microbe (2016) 20(1):13–24, Hartmann B, et al. J Immunol (2015) 194(1 Suppl):127.2–127.2]. [000224] In IAV infection, DR5 expression is elevated on infected alveolar epithelial cells, but not in non-infected cells in vivo, which might impact on TRAIL susceptibility to apoptosis induction [Id., citing Högner K, et al. PLoS Pathog (2013) 9(2):e1003188]. However, both infected as well as neighboring bystander cells were found to be targeted for apoptosis induction by macrophage-released TRAIL. [000225] The TRAIL-induced and AMPK-mediated downregulation of the Na,K-ATPase, a major driver of vertical ion and fluid transport from the alveolar airspace toward the interstitium, resulted in a reduced capacity of IAV-infected mice to clear excessive fluid from the alveoli. Thus, TRAIL signaling contributes to intensive edema formation, a hallmark of disease in virus-induced ARDS [Id., citing Matthay MA, et al. J Clin Invest (2012) 122(8):2731–40]. The effect of TRAIL on Na,K-ATPase expression was induced independently of cell death pathways elicited by caspases, as treatment of cells and mice with a specific caspase-3 inhibitor diminished apoptosis in alveolar epithelial cells but still allowed for the reduction of the Na,K-ATPase [Id., citing Peteranderl C, et al. J Clin Invest (2016) 126(4):1566–80. Conclusively, treatment of IAV-infected mice with neutralizing antibodies directed against TRAIL or the abrogation of recruitment of TRAIL+ bone marrow-derived macrophages inhibited apoptosis of both non-infected and bystander cells. Thus, lung leakage due to loss of alveolar barrier function was reduced, whereas alveolar fluid clearance capacity was enhanced, resulting in reduced edema, improved survival, and outcome upon IAV challenge in vivo. However, TRAIL has also been shown to be upregulated on NK, DC, and on CD4+ and CD8+ T cells after IAV infection [Id., citing Ishikawa E, et al. J Virol (2005) 79(12):7658–63]. Bacterial superinfection after viral injury [000226] Severe viral infections of the respiratory tract often are followed by outgrowth of colonizing Gram-positive bacteria that aggravates the course of illness. This is well documented for IAV, where “super” infections with Streptococcus pneumoniae and Staphylococcus aureus are the most frequent and increase viral pneumonia-associated morbidity and mortality [Id., citing Rynda-Apple A, et al. Infect Immun (2015) 83(10):3764– 70]. Virus-induced elevation of the type I IFN response levels might promote secondary bacterial outgrowth by several mechanisms [[Id., citing McNab F, et al. Nat Rev Immunol (2015) 15(2):87–103]. It has been repeatedly demonstrated that lack of type I IFN signaling results in better bacterial clearance and increased survival rates in IAV- and S. pneumoniae- superinfected mice [Id., citing Shahangian A, et al. J Clin Invest (2009) 119(7):1910–20; Nakamura S, et al. J Clin Invest (2011) 121(9):3657–65; Li W, et al. J Virol (2012) 86(22):12304–12]. [000227] Bacterial clearance from the lung has been reported to rely on sufficient phagocyte generation, recruitment, and survival. Type I IFN has been demonstrated to cause apoptosis in bone marrow-derived granulocytes, affecting the numbers of recruited neutrophils [Id., citing Navarini AA, et al. Proc Natl Acad Sci USA (2006) 103(42):15535–9], and to impair expression of the cytokines CXCL1 (or KC) and CXCL2 (or MIP-2), thus inhibiting neutrophil recruitment to the lungs with severe effects on survival of superinfected mice [Id., citing Shahangian A, et al. J Clin Invest (2009) 119(7):1910–20]. A report by Schliehe et al. [Id., citing Schliehe C, et al. Nat Immunol (2015) 16(1):67–74] demonstrated that type I IFNs activate the histone methyltransferase Setdb2, which in turn represses the Cxcl1 promoter and thus impairs neutrophil recruitment and bacterial clearance. Moreover, type I IFN production decreases CCL2 production, thus inhibiting macrophage recruitment, which as well has been reported to have detrimental effects on bacterial clearance and disease progression in bacterial superinfection after viral insult in vivo [Id., citing Nakamura S, et al. J Clin Invest (2011) 121(9):3657–65). In addition, type I IFNs also impair γδ T cell function and IL-17 release, which was shown to increase susceptibility to S. pneumoniae superinfection after IAV challenge [Id., citing Li W, et al. J Virol (2012) 86(22):12304–12]. Also in S. aureus pneumonia, a robust type I IFN response is correlated to excessive morbidity and tissue injury [Id., citing Parker D, et al. PLoS Pathog (2014) 10(2):e1003951]. In a model of polyI:C, S. aureus (methicillin-resistant strain, MRSA) superinfection, polyI:C treatment prior to bacterial infection enhanced type I IFN levels and decreased bacterial clearance and survival [Id., citing Tian X, et al. PLoS One (2012) 7(9):e41879]. Furthermore, late type I IFN induction was reported to render mice more susceptible to secondary bacterial pneumonia in a model of IAV– MRSA superinfection. [Shepardson KM, et al. MBio (2016) 7(3):e506–16]. Using the IAV– S. pneumoniae superinfection mouse model, it was shown that IAV-induced TRAIL has a detrimental effect on overall mortality [Id., citing Ellis GT, et al. EMBO Rep (2015) 16(9):1203–18]; TRAIL-induced epithelial injury enhanced bacterial outgrowth of S. pneumoniae—administered at day 5 after IAV infection—markedly. Importantly, administration of anti-TRAIL neutralizing antibodies enhanced bacterial control by the host organism. Respiratory Syncytial Virus (RSV) [000228] Human respiratory syncytial virus (RSV) is an enveloped, nonsegmented negative-strand RNA virus of family Paramyxoviridae. It is the most complex member of the family in terms of the number of genes and proteins. Respiratory syncytial virus is an important cause of respiratory tract infections especially in children worldwide; it is the major cause of lower respiratory tract illness in young children. [Falsey, AR et al., J. Clin. Microbiol. (2002) 40(3): 817-20, citing Hall, C. B. N. Engl. J. Med. (2001) 334:1917-1928]. In recent years it has been recognized that RSV infection may be severe in certain adult populations, including the elderly, persons with cardiopulmonary disease, and the immunocompromised [Id., citing Englund, J. A.et al. Ann. Intern. Med. (1988) 109:203-208; Falsey, A. R., et al. J. Am. Geriatr. Soc. (1992) 40:115-119; Sorvillo, F. J., et al. J. Infect. (1984) 9:252-256; Walsh, E. E., et al. Am. J. Respir. Crit. Care Med. (1999) 160:791-795]. [000229] Generally, there seem to be virus-elicited anti-apoptotic mechanisms active in the lung epithelium, as RSV-infected primary human airway cells show a minimal cytopathic effect [Peteranderl, C. and Herold, S. Front. Immunol. (2017) 8: 313, citing Zhang L, et al. J Virol (2002) 76(11):5654–66]. However, several cell lines including small airway cells, primary tracheal-bronchial cells, and A549 and HEp-2 showed increased expression of TRAIL and its ligands DR4 and DR5 in an in vitro RSV infection model [Id., citing Kotelkin A, et al. J Virol (2003) 77(17):9156–72]. Moreover, soluble TRAIL released from leukocytes was elevated in the bronchoalveolar lavage fluid of patients with RSV-associated respiratory failure, suggesting that similar to IAV, TRAIL contributes to RSV-induced epithelial injury and disease progression [Id., citing Bem RA, Bos AP, Wösten-van Asperen RM, Bruijn M, Lutter R, Sprick MR, et al. Potential role of soluble TRAIL in epithelial injury in children with severe RSV infection. Am J Respir Cell Mol Biol (2010) 42(6):697–705]. SARSCoV [000230] The SARS coronavirus (SARS CoV) is an enveloped RNA virus that replicates with transcription of discontinuous nested messenger RNA (mRNA). The reservoir for the virus is thought to be civet cats, a nocturnal mammal considered a delicacy in southern China. Horseshoe bats may also be a reservoir. The incubation period is 2 to 7 days before symptom onset, and peak viral shedding in respiratory secretions occurs relatively late, between 6 and 11 days. The virus is spread through respiratory secretion shedding and via contact with fomites. Airborne transmission, particularly on international flights, contributed to superspreader outbreak phenomenon. Several epidemiological studies using logistic regression showed that older age and underlying comorbid conditions (diabetes, chronic obstructive pulmonary disease, hepatitis B infection, cancer, and cardiac disease) were associated with worse outcomes including ICU admission, mechanical ventilation, and death. [Huang, E-S, Johnson, RA, Nature Medicine (2000) 6: 863-64], citing Peiris, JS, et al. Lancet (2003) 361 (9366): 1319- 25; Booth CM, et al. JAMA (2003) 289 (21): 2801-97]. [000231] Postmortem pathology studies and various in vitro and in vivo model systems of SARS CoV infection suggest that the virus enters through the respiratory tract and binds ACE2 in the alveolar epithelium. Infection is followed by serological evidence of increased ACE activity and decreased ACE2 activity. The signaling pathways that are activated by binding the SARS CoV to ACE2 and the subsequent downstream cytokine elaboration appear to share common features with other mechanisms of ALI and result in a pathological phenotype indistinguishable from other mechanisms of lung injury. [Hendrickson, CM, Matthay, MA Semin. Respir. Crit. Care Med. (2013) 34: 475-86] [000232] Animal models of ALI that are rescued by administration of intravenous or intraperitoneal agents to restore the balance of ACE and ACE2 activity suggest that the vascular endothelium is also involved in lung injury and that therapy may be delivered systemically rather than to the alveolar epithelium, the site of initial viral binding and infection. The epidemiological features of persons at high risk of severe infection and complications do not suggest a particular host defect. While the important downstream targets upregulated by ACE and downregulated by ACE2 are not yet well understood, and the relative contributions of endothelial and epithelial processes to the development of lung injury and the molecular pathways that link initial binding of the virus to alveolar cells with the development of diffuse alveolar damage (DAD) are under active investigation, it was suggested that the pathways involved in lung injury induced by SARS CoV infection may be common to other mechanisms of injury, including LPS, acid aspiration, and cecal ligation and perforation, and therefore may provide the framework for developing a more universal therapy for lung injury mediated by other infectious and noninfectious processes. [Hendrickson, CM, Matthay, MA Semin. Respir. Crit. Care Med. (2013) 34: 475-86]. [000233] In CoV respiratory tract infection, TRAIL levels, but less so FasL, have been reported to be markedly elevated Peteranderl, C. and Herold, S. Front. Immunol. (2017) 8: 313., citing Law HKW, et al. BMC Immunol (2009) 10:35; Marfè G, et al. PLoS One (2011) 6(11):e27313]. For SARS-CoV that presents with a severe damage to both the upper and lower respiratory tract, DCs respond with a strong induction of TRAIL production, which was suggested to correlate to increased cellular lung infiltrations present in SARS-CoV patients [Id., citing Law HKW, et al. BMC Immunol (2009) 10:35]. SARS-CoV infection drives cells into apoptosis by a PKR-driven but eIF2α-independent pathway [Id., citing Krähling V, et al. J Virol (2009) 83(5):2298–309], which might—similarly as seen in IAV infection—suggest a PKR-induced and autocrine/paracrine executed activation of apoptosis. [000234] Also MERS-CoV, which causes pneumonia and respiratory failure, has been demonstrated to induce profound cell death within 24 h of infection, irrespective of viral titers produced by the infected cells. However, type I IFN expression is strongly reduced in MERS- CoV in comparison to seasonal human CoV in in vitro infection models, including human monocyte-derived macrophages, Calu-3, and human lung fibroblasts [Id., citing Lau, SK et al. J. Gen. Virol. (2013) 94 (Pt.12): 2679-90; Zhou, J. et al. J. Infect. Dis. (2014) 209 (9): 1331- 42], which might also dampen downstream TRAIL induction. Therefore, the exact mechanism by which MERS-CoV promotes cell death remains to be investigated. Zika virus [000235] Zika virus (ZIKV) is an arbovirus, which can be transmitted to humans by Aedes mosquitoes as well as by sexual interactions. As a member of the Flaviviridae family of positive strand RNA, ZIKV is closely related to some important human pathogens, such as dengue virus (DENV), yellow fever virus (YFV), west nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) [Yang, C. et al. Virologica Sinica (2019) 34: 168-174, citing Wang Q, et al. J. Virol. (2017) 91: e01049-1]. [000236] The Zika virus outbreak in South America in 2016 was associated with fetal birth defects, including neonatal microencephaly. [000237] Intrauterine infection or inflammation (IUI), also known as chorioamnionitis, is responsible for ~40% of preterm labor cases [Cappelletti, M. et al. Front. Immunol. (2020) 11: 649; citing Kim, CJ et al. Am. J. Obstat. Gynecol. (2015) 213: S29-S52] ). Prematurity, which affects nearly 10% of pregnancies world-wide, is the most significant cause of perinatal mortality or morbidity [Id., citing Iams, JD, et al. Lancet (2008) 371: 164-75]. Acute chorioamnionitis, which is characterized by neutrophilic infiltration and inflammation at the maternal fetal interface is a relatively common complication of pregnancy and can have devastating consequences including preterm labor, maternal infections, fetal infection/inflammation, fetal lung, brain, and gastrointestinal tract injury. [000238] Most commonly, acute chorioamnionitis is a result of ascending infection with relatively low-virulence organisms such as the Ureaplasma species. In a minority of cases, microorganisms can also invade the placenta by the hematogenous route, and the profile of organisms is different compared to the ascending route. Microorganisms that invade the placenta by the hematogenous route include Listeria monocytogenes [Id., citing Gellin, BG et al. Am. J. Epidemiol. (1991) 133: 392-401], Zika virus [Id., citing Msorekar, IU, Diamond MS. N. Engl. J. Med. (2016) 375: 481-84; Tabata, T. et al., Cell Host Microbe (2016) 20: 155- 66], Treponema pallidum [Id., citing Arora, N. et al. Cell Host Microbe (2017) 21 : 561-67]; Cytomegalovirus [Pereira, L. Anu. Rev. Virol. (2018) 5: 273-99], Plasmodium species [Sharma, L., Shukla, G. Front. Med. (20017) 4: 117], and microorganisms causing toxoplasmosis, syphilis, varicella-zoster, parvovirus B19, Rubella, and Herpes infections (TORCH) [Stegmann, BJ, Carely, JC. Curr. Womens Health Rep. (2002) 2: 253-8]. These organisms gain access through the maternal circulation to the intervillous space, from where they invade the villi and fetal circulation. [Cappelletti, M. et al. Front. Immunol. (2020) 11: 649]. Viral infections, such as influenza virus, can prime or accentuate bacterial infection- mediated preterm labor and the intensity of inflammatory response at the maternal-fetal interface [Id., citing Cardenas, I. et al. Am. J. Reprod. Immunol. (2011) 65: 110-7; Aracicot, K. et al. J. Immunol. (2013) 191: 934-41; Kwon, JY, et al., am. J. Reprod. Immunol. (2014) 71: 387-90; Racicot, K. et al., Am. J. Reprod. Immunol. (2016) 75: 451-60; Racicot, K. et al. J. Imunol. (2017) 198: 3029-32]. Specifically, pathogen/pathogen-associated molecular pattern-driven activation of type I Interferon (IFN)/IFN receptor (IFNAR) was sufficient to prime for systemic and uterine proinflammatory chemokine and cytokine production and induction of preterm birth in mice [Id., citing Cappelletti, M. et al. JCI Insight (2017) 2: e91288]. [000239] The Zika outbreak also was associated with atypical Guillain-Barre syndrome in adults. AXL is a TAM family tyrosine kinase that was initially described as the entry receptor for Zika. [Chen, J. et al. Nature Microbiollogy (2018) 3: 302-309]. It is highly expressed in astrocytes, radial glia cells, and endothelial cells in human brain. [Id., citing Nowakowski, T. et al. Cell Stem Cell (2016) 18: 591-96]. Astrocytes play an important role during infection of neurotropic flativiruses, and type I IFN signaling in primary astrocytes has been reported to play an important role in restricting Zikv infection. [Id., citing Lindqvist, R. et al. J. Neuroinflamm. (2016) 13: 277]. The susceptibility of astrocytes to divergent WNV strains is a key determinant for viral propagation and fitness. [Id., citing Hussmann, KL, et al. J. Gen. Virol. (92014) 95: 1991-2003; Hussman, KLL. J. Virol. (2013) 87: 2814-22]. In addition, primary human astrocytes can express pro-inflammatory cytokines and chemokines during the infection of tick-borne encephalitis virus. [Id., citing Palus, M. et al. J. Gen. Virol. (2014) 95: 2411-26]. That astrocytes are the primary target of Zika in the brain during peripheral infection in newborn immunocompetent mice has been suggested. [Id., citing van den Pol, AN, J. Neurosci. (2017) 37: 2161-75]. Meanwhile, Chen et al reported that in astrocytes cultured in vitro, AXL knockout did not block the entry of Zika. Instead, the presence of AXL attenuated the Zika virus-induced activation of type I interferon signaling genes, including several type 1 interferons and IFN-stimulating genes. Knocking out type I IFN receptor α chain (IFNAR1) restored the vulnerability of AXL knockout astrocytes to Zika infection. Further experiments suggested that AXL regulates the expression of SOCS1, a known type 1 IFN signaling suppressor, in a STAT1/STAT2 dependent manner. Collectively, these results demonstrate that AXL is unlikely to function as an entry receptor for Zika, and may instead promote Zika infection in human astrocytes by antagonizing type I interferon signaling. [000240] After a viral infection, the acute lung injury caused by the virus must be repaired to regain lung function; any dysregulation in this wound healing process leads to fibrosis. The repair of lung tissue after infection or mechanical trauma normally occurs in a controlled series of events beginning with damage signals sent from infected cells, which recruit inflammatory cells, which then induce secretion of growth factors, which activates basement membrane repair and finally leads to the replacement of injured tissue. Under normal circumstances, the wound healing response is downregulated once the injury is repaired. However, when either the infectious burden overwhelms the system or there is persistent damage (e.g., with hepatitis C virus infection), the wound healing response can become dysregulated, resulting in scarring and fibrosis. When fibrosis occurs, it leads to reduced lung function, resulting in a low quality of life and often death. Traditionally, corticosteroids are used for the treatment of acute respiratory distress syndrome (ARDS) and pulmonary fibrosis. However, during a viral infection, these interventions dampen the immune response and often result in worsened disease. Acute kidney injury (AKI) and coronavirus infection [000241] A previous study on SARS-CoV infection showed that the virus RNA is effectively detected in urine 10 days after the onset of symptoms, and the excretion gradually decreased until day 21 [Martinez-Rojas, M.A. et al. Am. J. Physiol. Renal Physiol. (2020) 318 (6): F1454-62, citing Peiris, JSM et al. Lancet (2003) 361L 1767-72]. Autopsies of SARS- CoV-confirmed patients demonstrated the virus presence in tubular epithelial cells by immunohistochemistry and in situ hybridization [Id., citing Ding, Y. et al. J. Pathol. (2004) 203: 622-30]. In addition, 35% of heart specimens from SARS-CoV-infected patients revealed the coexistence of viral RNA and reduced ACE2 protein expression [Id., citing Oudit, GY et al. Eur. J. Clin. Invest. (2009) 39: 618-25]. A retrospective study during the SARS-CoV outbreak found that only 6% of SARS-CoV-infected patients exhibited AKI [Id., citing Chu, KH, et al. Kidney Int. (2005) 67: 698-705]. However, since almost 92% of patients with SARS with AKI, died AKI was a fatal complication of SARS. This study also evaluated whether active replication of SARS-CoV existed in the tubular cells of postmortem patients infected with SARS-CoV by analyzing the presence of viral particles using electron transmission microscopy. The authors found that SARS-CoV was not detectable in any of the analyzed samples and suggested that renal impairment was likely related to multi-organ failure, and suggested that AKI in patients with SARS-CoV could be the result of cytokine release syndrome (CRS) rather than active viral replication in the kidney. [Id., citing Tisoncik, JR et al. Microbiol. Mol. Biol. Rev. (2012) 76: 16-32]. [000242] In contrast, recent studies have reported that the human kidney is a specific target for SARS-CoV-2 infection [Id., citing Diao, B. et al. (2020) medRxiv: 2020.03.04.2003112010.1101/2020.03.04.20031120; Farkash, EA, et al. J. Am. Soc. Nephrol. (2020) 31: ASN 2020040432; Pan, XW, et al. Intensive Care Med. (2020) 2-4. doi:10.1007/s00134-020-06026-1; Su, H et al. Kidney Int. doi:10.1016/j.kint.2020.04.003]. Diao et al. examined viral nucleocapsid protein in the kidney of postmortem patients and found that SARS-CoV-2 antigens accumulated in renal epithelial tubules, suggesting that SARS- CoV-2 infects the human kidney directly, which leads to kidney dysfunction and contributes to viral spreading in the body. [Diao, B. et al. medRxiv: 2020.03.04.20031120 10.1101/2020.03.04.20031120]. One explanation offered for the difference between the higher renal tropism of SARS-CoV-2 versus SARS-CoV is the increased affinity of SARS-CoV-2 for ACE2, allowing greater viral load in several organs, and especially into the kidney, which may act as viral reservoir [Id., citing Perico, L. et al. Nephron doi:10.1159/000507305]. An additional study of 26 autopsies found virus particles characteristic of SARS-CoV-2 in the proximal tubular epithelium and podocytes by electronic microscopy, foot process effacement and occasional vacuolation and detachment of podocytes from the glomerular basement membrane [Id., citing Su, H. et al. doi:10.1016/j.kint.2020.04.003]. [000243] Without being limited by theory, these findings, along with the consensual physiological role of ACE2 in the kidneys, raise the possibility of a complex multifactorial pathophysiology explaining kidney abnormalities in COVID-19, involving a direct cytopathic effect of the virus, a local disruption in Renin Angiotensin Aldosterone System (RAAS) homeostasis, and a systemic inflammatory response to infection. [000244] The most frequent finding of kidney dysfunction in patients with COVID-19 is mild to moderate proteinuria [Id., citing Cheng, Y. et al. Kidney Int. (2020) 97: 8829-38]. A small fraction of plasma proteins is filtered in the renal glomeruli, and most of them are effectively reabsorbed in the proximal tubule, so that basically no proteins appear in normal urine. The glomerular filtration barrier depends on adequate function of its three components: endothelial cells, the glomerular basement membrane, and podocytes [Id., citing Cara-Fuentes, G. et al. Pediatr. Nephrol. (2016) 31: 2179-89]. Podocytes are known to be particularly sensitive to RAAS homeostasis, with angiotensin-1–7 being the most abundant product, probably due to the specific expression of ACE2 in this region [Id., citing Velez, JCQ, et al. Am. J. Physiol. Renal Physiol. (2007) 293: F398-F407]. If a pathological process increases glomerular levels of angiotensin II, podocytes acquire a dysfunctional phenotype mediated by cellular responses to this octapeptide due to shear stress and resulting in single nephron hyperfiltration. This phenotype involves Ca2+ signaling, cytoskeleton restructure, and nephrin internalization, which finally is manifested by proteinuria [Id., citing Konigshausen, E. et al. Sci. Rep. (2016) 6: 39513; Srivastava, T. et al. Am. J. Physiol. Renal Physiol. (2018) 314: F22-F34]. The actual tropism of SARS-CoV-2 to podocytes has been recently determined; without being limited by theory, it has been hypothesized that proteinuria is a partial consequence of direct podocyte infection with potential RAAS alterations, which together would affect the glomerular filtration barrier and result in increased filtration of plasmatic proteins. [000245] The incidence of AKI in SARS-CoV-2-infected patients has been variable, and it has been found predominantly in critically ill patients [Id., citing Wang, L. et al. Am. J. Nephrol. (2020) 51: 343-48; Yang, X. et al. Lancet Respir. Med. (2020) 8: 475-81]. It has been reported that patients in the intensive care unit have higher levels of IL-1β, IL-8, interferon-γ, and TNF-α, among other cytokines, compared with noncritically ill patients [Id., citing Huang, C. et al. Lancet (2020) 395: 497-506]. This suggest a potential role of CRS, also named as “cytokine storm,” comparable with sepsis-associated AKI, where the uncontrolled systemic inflammatory response leads to kidney dysfunction. The occurrence of CRS in COVID-19 has been documented since the first reports of the disease [Id., citing Huang, C. et al. Lancet (20230) 395: 497-506; Wu, C. et al. JAMA Intern. Med. doi:10.1001/jamainternmed.2020.0994]. In patients with CRS, AKI might occur as a result of intrarenal inflammation, increased vascular permeability, and volume depletion, which is translated in the findings of autopsies of erythrocyte aggregates obstructing the lumen of capillaries without platelet or fibrinoid material. [000246] It is well known that an imbalance in components of the RAAS can contribute to kidney injury by changing renal hemodynamics, altering tubular handling of electrolytes with a higher metabolic demand, and inducing proinflammatory phenotypes in both epithelial and immune cells. This imbalance could contribute to the renal dysfunction observed in severe patients with COVID-19, which could also be accompanied by a decrease in ACE2 activity [Id., citing Briet, M., Schiffrin, EL. Nat. Rev. Nephrol. (2010) 6: 261-73; Mattson, DL, Nat. Rev. Nephrol. (2019) 15: 290-300; Rodriguez-Romo, R. et al. Kidney Int. (2016) 89: 363-73]. Sepsis-associated AKI is believed to be multifactorial, involving the kidney inflammatory response, microcirculatory dysfunction, and metabolic reprogramming with mitochondrial injury Id., citing Peerapornratana, S. et al. Kidney Int. (2019) 96: 1083-99]. These mechanisms are compatible with the current understanding of SARS-CoV-2 infection and biology, supporting the prevailing hypothesis that COVID-19-associated AKI takes place in a severe disease scenario with a complex pathophysiological network. Trefoil factor family of peptides. [000247] The trefoil factor family (TFF) is a family of peptides with a three-loop trefoil domain that have a close association with mucins and are mainly synthesized and secreted by mucin secreting epithelial cells lining the gastrointestinal tract. [Wong, WM, et al. Gut (1999) 44(6): 890-95]. They are highly conserved during evolution and are heat, acid and enzyme resistant. While their abundant expression in distinct patterns in the normal physiological state and ectopic expression in various ulcerative conditions suggests an important role in mucosal defense and repair, the physiologically relevant functions of TFFs are not clear, and how TFFs work is not fully understood. [Aamann, L. et al. World J. Gastroenterol. (2014) 20 (12): 3223- 30]. [000248] In humans, three trefoil peptides or trefoil factors (TFF) are known. The first trefoil peptide discovered was pS2/TFF1 or breast cancer estrogen inducible gene— discovered during a search for estrogen induced mRNAs from the mammary carcinoma cell line MCF7 in 1982. [Wong, WM, et al. Gut (1999) 44(6): 890-95, citing Masiakowski, P. et al. Nucleic Acids Res. (1982) 10: 7895-7903]. In the same year, TFF2 (formerly spasmolytic polypeptide, SP) was purified and extracted from porcine pancreas during the preparation of porcine insulin. [Id., citing Jorgensen, KD, et al. Regul. Pept. (1982) 3: 231-43; Jorgensen, KH et al. Regul. Pept. (1982) 3: 207-19; Thim, L. et al. Regul. Pept. (1982) 3: 221-30] Several years later, it was noticed that these peptides share a common novel sequence motif [Id., citing Baker, ME Biochem. J. (1988) 253: 307-09; Thim, L. (1988) Biochem. J.253: 309] later named the trefoil domain [Id., citing Thim, L. FEBS Lett. (1989) 250: 85-90] or P-domain [Id., citing Hoffman, W. J. Biol. Chem. (1988) 263: 7686-90]. The third mammalian protein in the family, ITF/TFF3 (previously called intestinal trefoil factor, ITF or hP1.B) was later discovered as a rat cDNA sequence in 1991 [Id., citing Suemori, S. et al. Proc. Natl Acad Sci. USA (1991) 88: 11017- 21] and the human cDNA sequence was reported in 1993. [Id., citing Hauser, F. et al. Proc. Nat. Acad. Sci. USA (1993) 90: 6961-65; Podolsky, DK et al. J. Biol. Chem. (1993) 268: 6694-6702; Podolsky, DK, et al. J. Biol. Chem. (1993) 268: 12230]. These peptides are resistant to thermal and enzymatic digestion, largely because of the structure of the TFF domain, which is encoded by shuffled modules highly conserved during evolution from amphibians to mammals. [000249] The trefoil domain is characterized by a sequence of amino acid residues, in which 6 cysteines are linked by 3 disulphide bonds to form the “trefoil” disulphide loop structure or the clover-like shaped structure [Aamann, L. et al. World J. Gastroenterol. (2014) 20 (12): 3223-30, citing Thim. L. FEBS Lett. (1989) 250: 85-90]. The resistance of the peptides to proteolytic digestion, acids and thermal degradation seems to be caused by this compact trefoil structure of the peptides [Id., citing Thim, L. et al. Biochem. (1995) 34: 4757- 64; Jorgensen, KH, et al. Regul. Pept. (1982) 3: 207-19]. TFF1 and TFF3 only contain one trefoil domain but have a seventh free cysteine, which is essential for the formation of dimers [Id., citing Thim, L. et al. Biochemistry (1995) 34: 4757-64]. TFF2 contains two trefoil domains; in the amphibian Xenopus, there are molecules containing multiple trefoil domains [Wong, WM, et al. Gut (1999) 44(6): 890-95, citing Gmachl, M. et al. FEBS Lett. (1990) 260: 145-48; Hauser, F. and Hoffman, W. J. Biol. Chem. (1991) 266: 21306-09; Hauser, F. et al., J. Biol. Chem. (1992) 267: 14451-55; Hoffman, W., Hauser, F. Trends Biochem. Sci. (1993) 18: 239-43]. [000250] The major expression site for TFF1 and TFF2 in vivo is the stomach, and for TFF3 it is the intestine [Emidio, NB et al. Trends in Biochemical Sci. (2019) 44 (5): 387-390, citing Klellev, S. Cell Mol. Life Sci. (2009) 66: 1350-69]. TFF peptides also appear in saliva, gastric juice, urine, blood and breast milk [Id., citing Klellev, S. Cell Mol. Life Sci. (2009) 66: 1350-69; Roa, B and Tortolero, S. Bratisl. Med. J. (2016) 117: 332-39]. Pathologically, TFF peptides are ectopically expressed after wounding, in inflammatory diseases, and in various tumors. [Id, citing Klellev, S. Cell Mol. Life Sci. (2009) 66: 1350-69]. They are also secreted in an endocrine manner, for example, in the immune and central nervous system [Id., citing Klellev, S. Cell Mol. Life Sci. (2009) 66: 1350-69; Hoffmann, W, J. Med. Chem. (2009) 52: 6505-10]. TFF interactions [000251] TFF signaling through canonical receptor-lligand interactions has long been debated. Chemokine receptor types 4 and 7 (CXCR4 and CXCR7) have been reported to mediate TFF2 and TFF3-induced chemotaxis. [Id., citing Klellev, S. Cell Mol. Life Sci. (2009) 66: 1350-69, Otto, W. and Thim, L. Cell Mol. Life Sci. (2005) 62: 2939-46]. Typical activated cascades include ERK1/2, JNK, Akt, and NF-κB. [Id., citing Klellev, S. Cell Mol. Life Sci. (2009) 66: 1350-69, Deckow, J. et al. Ophthalmol. Vis. Sci. (2016) 5;7: 56-65]. For example, it has been reported that TFF2 triggers ERK1/2 signaling via CXCR4 [Id., citing Hoffmann, W, J. Med. Chem. (2009) 52: 6505-10], which might be linked to the recruitment of immune cells [Id., citing Deckow, J. et al. Ophthalmol. Vis. Sci. (2016) 5;7: 56-65]. TFF3 also interacts with CXCR4 and CXCR7 expressed on ocular surface tissues, where cell migration is induced via an ERK1/2-independent signaling pathway. [Id., citing Deckow, J. et al. Ophthalmol. Vis. Sci. (2016) 5;7: 56-65]. [000252] It has been reported that TFF2 and TFF3 activate PAR4 and PAR2 respectively [Id., citing Roa, B and Tortolero, S. Bratisl. Med. J. (2016) 117: 332-39, 9]. PAR knockdown abolishes the mucosal healing effect of TFF2 [Id., citing Zhang, Y. et al. Cell Mol. Lif Sci. (2011) 68: 3771-80]. TFF3 activates PAR2, but not PAR1, as shown in cytosolic CA2+ activity measurements in HT-29 cells, causing downregulation of proinflammatory cytokines and upregulation of defensing expression [Id., citing Roa, B and Tortolero, S. Bratisl. Med. J. (2016) 117: 332-39]. [000253] Porcine TFF2 binds noncovalently to integrin β1, as determined by affinity chromatography. [Id., citing Hoffmann, W. Intl. J. Oncol. (2015) 47: 806-16; Otto, W. and Thim, L. Cell Mol. Life Sci. (2005) 62: 2939-46] This is of interest because integrins play an important role in cell migration, which is enhanced by TFF peptides. [000254] TFF1 is produced mainly in the stomach, in superficial cells of the body and antral mucosa. TFF2 is abundant in the mucous neck cells in the body and in antral glands of the stomach [Wong, WM, et al. Gut (1999) 44(6): 890-95., citing Tomasetto, C. et al. EMBO J. (1990) 9: 407-14; Hanby, AM, et al. Gastroenterology (1993) 105: 1110-16] and the acini and distal ducts of Brunner’s gland in the duodenum. TFF3 is expressed throughout the intestine and is abundant in salivary glands. It has been shown that both TFF1 and TFF2 are expressed by mucous neck cells in the corpus of the stomach [Id., citing Hanby, AM et al. J. Pathol. (1999) 187: 331-337] in which the secretory phenotype is remarkably similar to that of a reparative lineage in the gut—the ulcer associated cell lineage (UACL) [Id., citing Wright, NA, et al. Nature (1990) 3423: 82-85]. [000255] TFF1 was first identified in human breast carcinoma cell lines by virtue of its regulation by estrogen [Id., citing Masiakowski, P. et al. Nucleic Acids Res. (1982) 10: 7895- 7903; May, FEB, Westley, BR. Cancer Res. (1986) 46: 6034-40]; TFF1 mRNA can be detected in 68% of breast tumors. [Id., citing Henry, JA et al. Br. J. Cancer (1991) 63: 615-22] TFF1 has since been found to be expressed in a variety of other carcinomas including those of the stomach, pancreas, lung, endometrium, ovary (particularly mucinous carcinomas), prostate, bladder, cervix, and pancreas, and in medullary carcinoma of the thyroid and mucinous carcinoma of the skin. [Id., citing Bonkoff, H. et al. Human Pathol. (1995) 26: 824-28; Henry, JA, et al. Br. J. Cancer (1989) 61: 32-38; Cappelletti, V. et al., Eur. J. Cancer (1992) 28: 1315- 18; Foekens, JA, et al. Cancer Res. (1990) 50: 3832-37; Pallud, C. et al. Histopathol. (1993) 23: 249-56; Rio, MC, et al. Proc. Nat. Acad. Sci. USA (1987) 84: 9243-47; Skilton, RA, et al. Br. J. Cancer (1989) 60: 168-75; Poulsom, R. et al. J. Pthol. (1997) 183: 30-38; Machado, JC, et al. Eur. J. Cancer Prev. (1996) 5: 169-79; Henry, JA et al. Br. J. Cancer (1991) 64: 677-82; Luqmani, Y. et al. Intl. J. Cancer (1989) 44: 806-12; Muller, W., Borchard, F. J. Pathol. (19939) 171: 263-69; Machado, JC, et al. Eur. J. Cancer (1996) 32: 1585-90; Theisinger, B. et al. Eur. J. Cancer (1991) 27: 770-73; Taupin, D. et al. Lab Invest. (1996) 75: 25-32; Higashiyama, M. et al. Eur. J. Cancer (1994) 30: 792-97; Higashiyama, M., et al. Anticancer Res. (1996) 16: 2351-56; Welter, C. et al. Lab Invest. (1992) 66: 187-92; Collier, JD et al., J. Gastroenterol. Hepatol. (1995) 10: 296-300; Wang, DG et al. J. Pathol. (1998) 184: 408-13]. In breast carcinoma, expression is significantly associated with estrogen receptor status, responsiveness to hormone therapy and favorable prognosis. [Id., citing Bonkoff, H. et al. Human Pathol. (1995) 26: 824-28; Henry, JA, et al. Br. J. Cancer (1989) 61: 32-38; Cappelletti, V. et al., Eur. J. Cancer (1992) 28: 1315-18; Foekens, JA, et al. Cancer Res. (1990) 50: 3832-37; Pallud, C. et al. Histopathol. (1993) 23: 249-56; Rio, MC, et al. Proc. Nat. Acad. Sci. USA (1987) 84: 9243-47]. TFF3 expression is also induced in breast carcinomas in a hormone dependent manner. [Id., citing Poulsom, R. et al. J. Pathol. (1997) 183: 30-38]. TFF2 is not expressed in breast carcinomas. Id., citing Tomasetto, C. et al. EMBO J. (1990) 9: 407-14]. [000256] Trefoil factors are expressed in a wide variety of ulcerative conditions of the gastrointestinal tract, from Barrett’s esophagus [Id., citing Hanby, AM et al. J. Pathol. (1994) 173: 213-19] to gastric [Id., citing Levi, S. et al. Eur. J. Gastroenterol. Hepatol. (1993) 5 (suppl. 3): S39-S43; Alison, MR et al. J. Pathol. (1995) 175: 405-14] and duodenal ulcers [Id., citing Hanby, AM et al. J. Pathol. (1993) 169: 355-360], and also in the small and large intestine in Crohn’s disease [Id., citing Wright, NA et al. Gastroenterology (1993) 104: 12-20; Weight, NA et al. J. Pathol. (19990) 1162: 279-84; Rio, MC et al. Gastroenterology (1991) 100: 375- 79], which suggests their importance as molecules involved in the repair of gastrointestinal mucosa. [Id., citing Hanby, AM, Wright, NA. J. Pathol. (1993) 171: 3-4] [000257] Recombinant hTFF1, hTFF2 and hTFF3, and rTFF3 have all been produced in Escherichia coli or yeast. All three mammalian trefoil factors are motogens, namely able to promote cell migration without promoting cell division, and are all upregulated at sites of mucosal injury and stimulate the repair process. Thus they participate in mucosal repair by stimulating the migration of surviving cells from the edge of the damaged region over the denuded area, a process called epithelial restitution, and essential for the repair of both minor and more extensive lesions. They are also morphogens: murine mammary carcinoma cell lines transfected with human TFF1 show an enhanced dispersed growth pattern in three dimensional collagen gels. [Id., citing Williams, R. et al. J. Cell Sci. (1996) 109: 63-71] TFF2 and TFF3 act as motogens when cell monolayers are wounded, in a transforming growth factor beta (TGF-β) independent manner. [Id., citing Dignass, A. et al. J. Clin. Invest. (1994) 94: 376-83; Playford, RJ et al. Gstroenterology (1995) 108: 108-16]. The established gut repair peptides epidermal growth factor (EGF) and TGF-α are mitogens and also act as motogens, but here the effect is mediated through TGF-β receptors on the basolateral side of the epithelium. [Id., citing Levi, S. et al. Eur. J. Gastroenterol. Hepatol. (1993) 5 (suppl.3): S39-S43]. [000258] TFF peptides are expressed by mucus producing cells throughout the normal gastrointestinal tract in a site specific manner; there is some evidence to suggest that trefoil factors might be involved in mucus polymerization. There is a strong association between the expression of trefoil factors and mucins, and both contribute to mucosal defense. [000259] A 2018 report provided evidence that human trefoil factor family 2 (hTFF2), a secretory peptide with a molecular mass of 14,284 Da, which comprises 106 amino acid residues, is a Ca-independent, pH-resistant lectin that binds with high affinity to α-GlcNAc- capped O-glycans on MUC6 [Morozov, V. et al. Molecules (2018) 23 (5): 1151, citing Hanisch, F-G, et al. J. Biol. Chem. (2014) 289: 27363-75]. It was suggested that this feature of hTFF2 suggests that it may block the growth inhibitory potential of antibiotic glycans upon binding to them, and it therefore could be regarded as a probiotic factor in Helicobacter pylori growth control. In line with these considerations, it has been shown that hTFF2 can reverse the growth inhibitory effect of porcine stomach mucin (PSM), which expresses antibiotic glycans similar to human MUC6 [Id.]. Many viruses have also exploited lectins for their own benefits during infection. As an example, dengue viruses utilize a c-type lectin, dendritic cell-specific ICAM- 3-grabbing non-integrins (DC-SIGN), on dendritic cells to enter the cells and replicate [Id., citing Tassaneetrithep, B. et al. J. Exp. Med. (2003) 197: 823-29]. HIV-1 mainly employs DC- SIGN on dendritic cells to promote the transfer to CD4+ T lymphocytes and galectin-1 to facilitate attachment to CD4+ T cells [Id., citing St-Pierre, C. et al. J. Virol. (2011) 85: 11742- 51; Geijtenbeek, TB e al., Cell (2000) 100: 587-97]. Viruses themselves can encode lectins on their surface for cell targeting, among them noroviruses, rotaviruses, and influenza viruses. [Id.] [000260] The “leucine-rich repeat and immunoglobulin-like domain-containing NoGo” or “LINGO” family of proteins has been described in the nervous system where they play a role in axonal regeneration, neuronal survival, oligodendrocyte differentiation, and myelination. LINGO-1 is characterized as a negative regulator of neuronal survival, axonal regeneration, and oligodendrocyte precursor cell (OPC) differentiation into mature myelinating oligodendrocytes. Three LINGO-1 homologs named LINGO-2, LINGO-3 and LINGO-4 also have been described and can form hetocomplexes with LINGO 1. [Guillemain, A. et al. FASEB J. (2020) 34 (10): 13641-53]. LINGO also exists in the gastrointestinal tract, but its function there is poorly characterized. [Emidio, NB et al. ACS Pharmacology & Translational Sci. (2020) 3: 583-97]. LINGO2 is highly expressed in patients with advanced gastric cancer and is involved in cell motility, tumorigenic ability and angiogenesis. [Id., citing Jo, JH et al. Int. J. Mol. Sci. (2019) 20 (3): 555]. LINGO activation usually occurs via homotypic or heterotypic interaction with other membrane proteins, such as the Nogo-A receptor and p75 neurotrophin receptor interaction with LINGO1 and EGF interaction with LINGO3. [Id., citing Zullo, K. et al. J. Immunol. (2019) 202: 192; Cobreet, L. et al. Br. J. Pharmacol. (2015) 172 (3): 841-56]. TFF2 and TFF3 have been described as natural ligands for the LINGO receptor interacting protein. [Id., citing Belle, NM et al. Nat. Commun. (2019) 10(1): 4408; Zullo, K. et al. J. Immunol. (2019) 202: 192]. TFF2 interaction with LINGO3 triggers ERK signaling, mediating tissue repair at the mucosal interface. [Id., citing Zullo, K. et al. J. Immunol. (2019) 202: 192]. LINGO3-/- mice display a phenotype that resembles TFF2 deficiency, such as impairment of mucosal regeneration and accumulation of immune cells secreting inflammatory cytokines even without stimuli. [Id., citing Zullo, K. et al. J. Immunol. (2019) 202: 192]. TFF3 interaction with LINGO2 disrupts LINGO2/EGFR interactions, enhancing activation of the EGFR pathway; thus this framework mediates epithelial repair. A LINGO2-TFF3 interaction is supported by immunoprecipitation and colocalization studies at the IEC cell surface. [Id., citing Belle, NM, et al. Nat. Commun. (2019) 10(1): 4408]. [000261] LINGO2 and LINGO3 have been implicated as the receptors of TFF3 and TFF2, respectively [Rossi, HL et al. Am. J. Resp. Cell & Molec. Biol. (2022) 66 (3): 252-258, citing Belle, NM, et al. Nat. Commun. (2019) 10 (1): 4408; Zullo, KM. et al. Scand. J. Gastroenterol. (2021) 56: 791-805]. In the gastrointestinal tract, TFF3 binding to LINGO2 removes it from EGFR, allowing EGFR signaling to occur [Id., citing Belle, NM, et al. Nat. Commun. (2019) 10 (1): 4408] LINGO3 is required for exogenous TFF2 to speed airway epithelial repair in culture; both are located in the nasal polyp epithelium [Id., citing Zullo, KM. et al. Scand. J. Gastroenterol. (2021) 56: 791-805]. TFF1 [000262] In a TFF1 knockout mouse model all mice showed loss of expression of gastric mucus and showed notably elongated pits, occupying the whole mucosa. Epithelial cells exhibited severe hyperplasia and high grade dysplasia, adenomas develop in the antrum of the stomach, and some of these progress into frankly invasive carcinomas. It was therefore proposed that TFF1 may be a specific tumor suppressor gene for the stomach. [Wong, WM, et al. Gut (1999) 44(6): 890-95, citing Lebefvre, O. et al. Science (1996) 274: 259-62] It has been shown that the dimeric form of TFF1, which forms dimers via Cys58, is more potent than the monomeric form in preventing indomethacin induced gastric damage and is also a stronger stimulant of the rate of migration of cells at the leading edge of wounded monolayers, suggesting that the Cys58 residue may play an important role in the biological function of TFF1. [Marchbank, T. et al. J. Pathol. (1998) 185: 153-58; Chadwick, MP, et al. Biochem. (1997) 327: 117-23]. TFF2 [000263] Within the context of mucosal repair, TFF2 is known for its involvement in mucosal repair, protection and proliferation, especially within both digestive and respiratory systems [Ghanemi, A. et al. Animals (2020) 10: 1646]. TFF2 is an important component and a stabilizer of the gastric mucus with the property of binding to the mucin MUC6 [Id., citing Heuer, F. et al. Int. J. Mol. Sci. (2019) 20: 5871], and is involved in tissue remodeling [Id., citing Royce, SG et al. Am. J. Respir. Cell Mol. Biol. (2013) 48: 135-44, Royce, SG et al. J. Asthma (2011) 48: 653-9]. Studies have reported that TFF2-deficient mice show increased susceptibility to NSAID injury. [Id., citing Farrell, JJ et al. J. Clin. Investig. (2002) 109: 193- 204]. [000264] Several studies suggest that upregulation of TFF may initiate the healing and repairing process that counteracts the inflammation-induced damage and some have described a potential anti-inflammatory effect. For example, TFF2 has been shown to be overexpressed (or upregulated) following inflammatory conditions [Id., citing Hoffmann, W. Int. J. Oncol. (2015) 47: 806-16], such as in asthma [Id., citing Royce, SG et al. Am. J. Respir. Cell Mol. Biol. (2013) 48: 135-44], gastrointestinal ulcerative disease [Id., citing Ortiz-Masia, D. et al. FASEB J. (2010) 24z: 136-45], and allergic airway inflammation [Id., citing Nikolaidis, NM et al. Am. J. Respir. Cell Mol. Biol. (2003) 29: 458-64]. A recombinant human TFF2 was shown to reduce colitis inflammation in a rat model and to increase the colonic epithelial repair rate [Id., citing Tran, CP et al. Gut (1999) 44: 636-42]. In the lung, IL-4 and IL-13 induce TFF2 [Id., citing Nikolaidis, NM et al. Am. J. Respir. Cell Mol. Biol. (2003) 29: 458-64], and hypoxia leads to upregulated TFF expression. [Id., citing Hernandez, C. et al. Br. J. Pharmacol. (2009) 156: 262-72]. TFF2 treatment also has been reported to reduce fibrosis (subepithelial collagen deposition) in a murine model of chronic allergic airways disease [Id., citing Royce, SG et al. A. J. Respir. Cell Mol. Biol. (2013) 48: 135-44]. [000265] TFF2 mRNA levels increase within 30 minutes after mucosal injury induced by a cryoprobe on the serosal surface of the rat stomach. [Wong, WM, et al. Gut (1999) 44(6): 890-95., citing Alison, MR et al. J. Pathol. (1995) 175: 405-14]. TFF2 increases cell migration in in vitro models of cell wounding. [Id., citing Dignass, A. et al. J. Clin. Invest. (1994) 94: 376-83; Playford, RJ et al. Gastroenterology (1995) 108: 108-116] and it also acts as a cytoprotective agent in rats treated with indomethacin. [Id., citing Playford, RJ et al. Gastroenterology (1995) 108: 108-16; Babyatsky, MW et al. Gastroenterol. (1996) 110: 489- 97]. TFF3 [000266] TFF3 also plays an important role in the repair and healing of the gastrointestinal tract. There is impaired mucosal healing and death from extensive colitis in TFF3 knockout mice after oral administration of dextran sulfate sodium, an agent that only causes mild to severe epithelial injury in wild type mice. [Id., citing Mashimo, H. et al. Science (1996) 274: 262-65] Repletion of TFF3 deficient mice by luminal supply of recombinant TFF3 resulted in restoration of normal healing and enhanced epithelial migration after acetic acid induced mucosal injury. Although the expression of the two other gastrointestinal trefoil factors seemed to be normal, the TFF3 deficient mice still had poor epithelial regeneration after injury, indicating that in this model, TFF1 and TFF2 cannot compensate for this deficiency. Involvement of TFFs in tissue regeneration, proliferation and protection in human lung diseases [000267] Immunohistochemical studies have localized TFF1 and TFF3 to goblet cells, Clara cells, and submucosal glands with TFF3 being most abundant. [Vilby, N-E et al. Peptides (2015) 90-95, citing dos Santos Silva, E. et al. J. Pathol. (2000) 190: 133-42; Wiede, A. et al. Am. J. Respir. Crit. Care Med. (1999) 159: 1330-5]. In one study, TFF2 mRNA has been detected in the lungs of asthma patents but not in healthy individuals. [Id., citing Kuperman, DA et al. J. Allergy Clin. Immunol. (2005116: 305-11]. TFF3 has been implicated in mucosal protection and repair processes, stimulating cell migration via chemotaxis [Id. citing Chwieralski, CE et al. Am. J. Respir. Cell Mol Biol. (2004) 31: 527-37]. Because TFF peptides are secreted with mucin and because the amount of mucus is increased in lung diseases, it was hypothesized that the amount of TFF should also be increased in pulmonary diseases. [000268] Results of a study of 92 patients undergoing diagnostic bronchoscopy for radiological findings in the form of tumor like infiltrates, atelectasis, bronchiecktasiae and abscess and symptoms such as hemoptysis, prolonged cough and recurring pneumonia of which 27 patients were diagnosed with COPD based on the results of their pulmonary function test (FEV1/FVC < 70%), and 39 individuals were diagnosed with pulmonary malignancies. BAL fluid was collected for measurement of acellular components and standardization of BAL [Id., citing Haslam, PL and Baughman RP, Eur. Resp J. (1999) 14: 245-8]. In all patients, TFF3 was the most abundant of the peptides, followed by TFF1 and TFF2. A correlation between TFF1 and TFF3 concentrations and FEV1 measurements was found, i.e., individuals with low FEV1 tended to have higher concentrations of TFF1 and TFF3 in the BAL fluid. Goblet cell metaplasia and hyperplasia, together with periibronchiolar inflammation and fibrosis is characteristic in the pathobiology of COPD [Id., citing Jeffery, PK. Thorax (1998) 53: 129-36], and this appeared to be preceded by an increase in TFF production. It was hypothesized that the increased TFF secretion also could be a consequence of the increased amounts of mucin secretion in combination with the presence of several inflammatory cytokines. Inappropriate mucus production due to metaplasia in the bronchi has several detrimental effects, including a reduction in bronchiolar antiprotase, leading to epithelial destruction. [Id., citing Jeffery, PK. Thorax (1998) 53: 129-36]. The exact stimulus for the goblet cell to increase expression of TFF is not known, but epithelial damage could be a mediator, since TFFs have been shown to be involved in protection and regeneration in other tissues. [Id., citing National Institute for Health and Care Excellence. CG101 Chronic obstructive pulmonary disease. NICE guideline www,guidance.nice.org.uk; 2013]. Since the cause of COPD development is extensive and persistent toxic damage, this seemed to result in an increased production and hypersecretion of the peptides. In addition, increased levels of TFF1 and TFF2 in patients with malignant lesions in the lungs was observed. The Ulcer Associated Cell Lineage (UACL) [000269] The importance of the trefoil factors in mucosal repair and healing are illustrated in the ulcer associated cell lineage (UACL). All trefoil factors are expressed in the UACL developing in humans in response to chronic ulceration. [Id., citing Hauser, F. et al. Proc. Nat. Acad. Sci. USA (1993)90: 6961-65; Wright, NA, et al. Nature (1990) 343: 82-85; Wright, NA et al. J. Pathol. (1990) 162: 279-84; Hanby, AM, Wright NA (1993) J. Pathol.171: 3-4]. In situ hybridization showed TFF2 mRNA in the acini and lower duct cells, whereas TFF1 mRNA and protein were localized in large amounts in the upper duct and all surface cells. TFF3 is expressed throughout the UACL with some glands showing stronger expression than others. Hauser, F. et al. Proc. Nat. Acad. Sci. USA (1993) 90: 6961-65]. The mucous cells adjacent to the UACL express TFF1, with TFF1 co-packaged with mucous granules. Neuroendocrine cells adjacent to the UACL in Crohn’s disease also express TFF1. [Id., citing Wright, NA et al. Gastroenterology (1993) 104: 12-20] The function of these cell lineages is very different. Mucus is released into the lumen where it has lubricating and protective functions, whereas endocrine secretions release various regulatory peptides into the local circulation that act via paracrine or autocrine mechanisms to produce manifold effects on the gut. Without being limited by theory, the expression of TFF1 by two different lineages suggests that it may have a dual mechanism of action—one is through the interaction with the overlying mucus layer and the other through an action on putative trefoil receptors on the basolateral surface of the epithelial cells. An animal model of UACL in which UACL was induced in the distal esophagus by esophago-jejunal anastomosis has been reported. [Id., citing Hanby, AM et al. Am. J. Pathol. (1997) 151: 1819-24]. [000270] The protective effect of subcutaneous TFF2 was reported seen at infused doses as small as 25 μg/kg/h, substantially less than that needed if given orally (1–15 mg/rat). [Id., citing Babyatsky, MW et al. Gastroenterology (1996) 110: 489-97], which suggested the presence of basolateral receptors or of transporting proteins which mediate the action of TFF2. [Id., citing Playford, RJ, et al. Gastroenterology (1995) 108: 108-116] Receptors for TFF2 were proposed in epithelial membranes that activate adenlyate cyclase [Id., citing Frandsen, EK, et al. Regul. Pepti. (1986) 16: 291-97]. [000271] TFF3 mediates epithelial chloride transport after application to the basolateral surface of monolayers of colorectal carcinoma cells or of rat jejunum [Id., citing Chinery, R., Cox, HM. Br. J. Pharmacol. (1995) 115: 77-80] Furthermore, TFF3 seemed to bind a protein present in membrane preparations of colorectal epithelial cells, accompanied by phosphorylation of tyrosine, and the phosphorylation occurs within 10 seconds—suggestive of a receptor mediated response. [Id., citing Chinery, R., Cox, HM. Peptides (1995) 16: 749-55]. Autoradiographic study of frozen rat gastrointestinal tissue using 125I-labelled rTFF3 showed the presence of a high density of specific 125I-rTFF3 binding sites in the gastric, colonic and jejunal mucosal glands [Id., citing Chinery, R., Cox, HM. Peptides (1995) 16: 749-55]. [000272] It had been hypothesized that trefoil peptides may act closely with EGFR, APC, the E-cadherin–catenin complex and the MAPK family On the molecular level, it has been shown that recombinant rTFF3 causes tyrosine phosphorylation of β-catenin and the EGF receptor (EGFR) in the HT29 colonic carcinoma cell line: tyrosine phosphorylation of β- catenin was associated with reduced membranous E-cadherin expression, with consequent perturbation of intercellular adhesion, and promotion of cell motility [Id., citing Liu, D. et al. Lab Invest. (1997) 77: 557-63] Moreover, it has been shown, in the HT29 cell line, which harbors an adenomatous polyposis coli (APC) mutation but has a normal E-cadherin–catenin complex, that rTFF3 leads to downregulation of E-cadherin, decreased cell–cell and cell– substratum adhesion, downregulation of expression of APC and α- and β-catenin, translocation of APC from the cytoplasm to the nucleus, and the induction of apoptotic changes. [Id., citing Efstathiou, JA, et al. Proc. Natl. Acad. Sci. USA (1998) 95: 3122-27]. TFF3 also has been shown to decrease extracellular signal related protein kinase (ERK) activity; ERK is a member of the mitogen activated protein kinases (MAPK) family. [Id., citing Kanai, M. et al. Proc. Nat. Acad. Sci. USA (1998) 95: 178-82]. The effect of TFF3 on ERK activity was blocked by a tyrosine phosphatase inhibitor, indicating that abrogation of the MAPK pathway by TFF3 is mediated by tyrosine phosphatase or a dual specific phosphatase. The close association of TFF3 with EGFR has been shown by a synergistic action of TFF3 on electrogenic chloride transport [Id., citing Chinery, R., Cox, HM, Br. J. Pharmacol. (1995) 115: 77-80] cell migration and cytoprotection.[Id., citing Chinery, R., Playford, RJ. Clin. Sci. (1995) 88: 401-403]. [000273] More generally, the trefoil factor family molecules were identified as small, reparative cytokines (6-18 kDa) that promote rapid movement of epithelia over denuded basement membrane [Taupin, D., Podolsky, DK. Nat. Rev. Mol. Cell Biol. (2003) 4 (9): 721- 32]. Does TFF2 play a role in modulating immune responses in vivo? [000274] The TFFs act to promote the speed of gastric epithelial restitution in vivo after acute and modest gastric injury. [Xue, L. et al. Gut (2010) 59 (9): 1184-91]. TFF2 activates calcium-dependent signaling and ERK1/2 phosphorylation via the CXCR4 receptor. [Xue, L. et al. J. Biol. Chem. (2011) 286 (44): 38375-82]. TFF2 is upregulated in Helicobacter spp- infected gastric tissues of both humans and mice. Data showed that TFF2 may play a protective role by modulating levels of gastric IFN-gamma in the development of H. pylori-associated premalignancy of the distal stomach. [Fox, JG et al. Am. J. Pathol. (2007) 171 (5): 1520-28; TFF2 modulates Ca2+ and AKT signaling in lymphoblastic Jurkat cells; these effects appear to be mediated through the CXCR4 receptor. [Dubeykovskaya, Z. et al. J. Niol. Chem. (2009) 284 (6)L 3650-62]. [000275] TFF2 expression is not limited to the gastrointestinal tract; it is also present in macrophages and lymphocytes. [Fox, JG et al. Am. J. Pathol. (2007) 171 (5): 1520-28, citing Cook, GA et al. FEBS Lett. (1999) 456: 155-59]. It has been hypothesized that TFF2 may function in part as an anti-inflammatory cytokine, showing some similarity in this respect to IL10. [Id., citing Kurt-Jones, EA et al. Infect. Immun. (2007) 75: 471-80] [000276] Wang, et al. developed a TFF2/spasmolytic polypeptide-deficient mouse model. [Id., citing Farrell, JJ et al. J. Clin. Invest. (2002) 109: 193-204]. These TFF2-/- mice show a hyper-inflammatory phenotype. In response to long term H. felis infection, TFF2-/- mice exhibit increased IFNγ levels and increased mucosal CD4+ T cells. [000277] TFF2 transcription is regulated by p53 and KLF4 transcription factors through an AP-1 site. [Tu, SP et al. Am. J. Physiol. Gastrointestinal Liver Physiol. (2009) 297 (2): G385-96]; AP-1 binding sequences are promoter/enhancer elements that play a role in gene induction in mammalian cells. [Zhou, H. et al. DNA Res. (2005) 12: 139-50]. P53 induces cell apoptosis and inhibits cell migration in part by downregulating TFF2 expression through an AP-1 site. [Tu, SP et al. Am. J. Physiol. Gastrointestinal Liver Physiol. (2009) 297 (2): G385-96] [000278] Results have shown that TFF2 (a) promotes type 2 lung immunopathology induced by allergen or IL-13; (b) controls the extent of lung injury caused by migratory infectious stage larvae (L3); and (c) modulates early TH2 development. [Wills-Kkarp, M. et al. J. Exp. Med. (2011) 209 (3): 607-22]. This mechanism involves rapid, coordinated IL-33 production from lung epithelia, alveolar macrophages, and inflammatory DCs. IL-33 induction required CXCR4, but occurred independently of the TLR adaptor molecule MyD88. Collectively, these experiments demonstrated a role for TFF2 in the regulation of IL-33 release at mucosal surfaces and the development of type 2 immune responses. [Id.] Macrophage-induced recovery of epithelial barrier function and epithelial proliferation through TFF2-dependent mechanisms [000279] Using a hookworm lung injury model in mice, Hung et al tested whether lung myeloid macrophages utilize a TFF2/Wnt axis as a mechanism that drives epithelial proliferation following lung injury. [Hung, L-Y et al., Mucosal Immunol. (2019) 12 (1): 64- 76]. Hookworms damage alveolar architecture during their circuitous migration from skin to GI tract; Nippostrongylus brasiliensis (N.b.) larvae enter the lung within 24h and cause petechial hemorrhage for 3 days before parasites egress to infest the GI tract [Id., citing Hung, LY et al. Proc. Nat. Acad. Sci. USA (2013) 110 (1): 282-87; Herbert, DR et al., J. Exp. Med. (20009) 206 (13): 2947-57]. IL-4Rα-driven M2 cells suppress pulmonary IL-17 responses during N.b. infection [Id., citing Chen, F. et al. Nat .Med. (2012) 18 (2): 260-66]. [000280] Macrophages (Mφ) reside in virtually every organ, where tissue-specific cues instruct unique transcriptional signatures in resident and emigrant Mφ to facilitate host defense, immunoregulation, and tissue homeostasis [Hung, L-Y, et al. Mucosal Immunol. (2019) 12 (1): 64-76, citing Lavin, Y. et al. Cell (2014) 159 (6): 1312-26]. Lung Mφ (alveolar macrophages (AM) and interstitial macrophages (IM)) have been long recognized as immunosuppressive cells that mediate efferocytosis (meaning the clearance of apoptotic cells by phagocytes), and phagocytic clearance of inhaled particles and pathogens. Communication between AM and alveolar epithelia enforces an immunosuppressive lung environment [Id., citing Westphalen, K. et al. Nature (2014) 159 (6): 1312-26], but whether such crosstalk regulates epithelial proliferation remained obscure. Alveolar macrophage (AM)-derived TGF-β promotes Foxp3+Treg expansion [Id., citing Soroosh, P. et al., J. Exp. Med. (2013) 210 (4): 775-88], but TGF-β promotes fibrosis and restricts epithelial cell proliferation [Id., citing Siegel, PM, Massague, J. Nat. Rev. Cancer (2003) 3 (11): 807-21] [000281] Mouse strains deficient for Tff1, Tff2, or Tff3 possess constitutive defects in gastrointestinal (GI) barrier function [Hung, L-Y, et al. Mucosal Immunol. (2019) 12 (1): 64- 76, Aamann, L. et al. World J. Gastroenterol. (2014) 20 (12): 3223-30’ Taupin, DR, et al., Proc. Nat. Acad. Sci. USA (2000) 97 (2): 799-804; McBerry, C. et al., J. Immunol. (2012) 189 (6): 3078-84], but the critical source(s) and function(s) for TFF’s in non-GI tissues are still unclear. Even though TFF2 can be produced by gastric epithelia, peritoneal macrophages and splenic T lymphocytes [Id., citing Dubeykovskaya, Z. et al. Nature Commun. (2016) 7: 10517], how this molecule regulates epithelial repair is understudied. TFF2 deficiency in all of its potential cellular sources was shown to exacerbate lung damage caused by the murine hookworm Nippostrongylus brasiliensis (N.b.), and data has shown that both bone marrow (BM)-derived and non-BM-derived TFF2 drives repair. [Id., citing Wills-Karp, M. et al., J. Exp. Med. (2012) 209(3): 607-22; Hung, LY et al. Am. J. Pathol. (2018) 188 (5): 1161-70]. Moreover, the demonstration that myeloid cells influenced by Type 2 immune responses (i.e. IL-4Rα signaling) regenerate lung following partial pneumonectomy [Id., citing Lerchner, AJ et al., Cell Stem Cell (2017) 21 (1): 120-134], prompted speculation that Mφ could repair damaged lung tissue by producing TFF2. [000282] To address whether myeloid-derived TFF2 was involved in lung repair, TFF2 conditionally deficient mice on a C57BL/6 background were generated by expressing an internal ribosomal entry site-fluorescent tandem dimer (td)-Tomato cassette (IRES-TdTomato) immediately following the stop codon in exon 4 of Tff2, with two loxP sites flanking exons 2 and 3’UTR. TFF2-tdTomato flox mice (designated “Tre-Tom”) had no gross abnormalities and were born at the expected female:male ratios. Site-specific recombination and germ-line transmission were confirmed by Southern blot. As expected, Tff2 mRNA was constitutively expressed in a small population of Td-tomato+ gastric epithelial cells sorted from the stomach tissue of naive Tre-tom mice, which was consistent with Tff2 expression in the stomach [Id., citing Farrell, JJ et al. J. Clin. Invst. (2002) 109 (2): 193-204]. To evaluate Tff2 expression in the pulmonary compartment, 300μm sections of agarose-filled lung tissue were prepared using established methods [Id., citing van Rijt, LS, et al. J. Exp. Med. (2005) 201(6): 981-91] and subjected to confocal imaging under steady-state and infectious conditions. Whereas baseline Td-tomato expression was sparse but punctate within the distal lung compartment under steady-state conditions, large, focal aggregates of Td-Tomato+ cells were seen within the alveolar compartment at d4 following hookworm infection. These data indicated that hookworm infection induced an increase of TFF2 expression occurred within large cells in the alveoli. Based on their morphology and location, it was hypothesized that some of these large TFF2 positive cells were of a hematopoietic lineage. [000283] CD11cCreTFF2flox mice were generated through intercross between Tretom and CD11cCre strains to test whether myeloid-derived TFF2 was biologically important [Id., citing van Rijt, LS, et al. J. Exp. Med. (2005) 201 (6): 981-91]. Real-time PCR was used to compare Tff2 mRNA transcript levels between TFF2 deficient, CD11cCreTFF2flox and CD11cCre controls in 4 distinct FACS-sorted lung populations: lung epithelia (CD45−EpCAM+), alveolar macrophages (AM) (CD64+, CD11b−, CD11c+), interstitial macrophages (IM) (CD64+. CD11b+, CD11c−), and CD103+DC (CD64−, CD103+, CD11c+). Results showed that AM underwent infection-induced Tff2 expression by d4 post-infection. TFF2 was also evident in CD103 DC and interstitial macrophages and expression was lost in CD11cCreTFF2flox mice. Tff2 transcripts were higher in lung epithelia sorted from CD11cCreTFF2flox mice than epithelia in CD11cCre controls, suggesting a compensatory Tff2 upregulation in epithelial cells due to deletion in the myeloid compartment. [000284] CD11cCreTFF2flox mice had a reduced percentage of BrdU+ cells within alveolar type 2 cell populations (CD45− EpCAM+ pro-SPC+) when compared to CD11cCre controls at day 9 post-infection. Oxygen saturation (SpO2) levels were significantly lower in CD11cCreTFF2flox mice at d9 post-infection, implying a defect in lung function rebound. Concerning the initial injury caused by larvae, the BAL RBC numbers were increased at day 3, reflective of more severe injury in CD11cCreTFF2flox mice compared to CD11cCre controls. The alveolar space within infected CD11cCreTFF2flox mice was markedly larger than CD11cCre controls evidenced by areas of enlarged airspace suggesting that resolution of hookworm injury was abnormal in CD11cCreTFF2flox mice. [000285] Next, the bleomycin model of lung injury was used to determine whether myeloid-derived TFF2 was important for lung repair in a non-infectious context lacking excess Type 2 cytokine production [Id., citing Wilson, MS et al. J. Exp. Med. (2010) 207(3): 535-52]. Acute cytotoxicity of bleomycin causes severe alveolar cell damage, transient weight loss, pulmonary inflammation, and chronic collagen accumulation [Id., citing Hogan, BL et al., Cell Stem Cell (2014) 15(2): 123-38]. Bleomycin treatment transiently increased BAL TFF2 levels in both mouse strains, whereas CD11cCre controls underwent a 3-fold increase at d9; this early induction was reduced by myeloid-specific TFF2 deficiency. On the other hand, CD11cCreTFF2flox mice produced higher TFF2 levels than CD11cCre controls by d16, suggestive of compensatory production from a non-myeloid source at later stages when fibrosis occurs. CD11cCreTFF2flox animals experienced a transient, but significantly greater, weight loss than CD11cCre controls during the acute injury phase, but subsequently recovered in the latter phase. At day 9, the total protein levels in BAL fluid were higher in CD11cCre TFF2flox; also, the CD45− EpCAM+ BrdU+ pro-SPC+ population of lung epithelia were significantly reduced in percentage from CD11cCreTFF2flox mice compared to CD11cCre mice, concordant with significantly reduced mRNA expression levels for Spc (encoding surfactant protein C) and Cc10 (encoding Clara cell 10kDa protein) at d9. Taken together, these data demonstrate that lack of myeloid-derived TFF2 reduced epithelial proliferation during recovery from bleomycin injury. [000286] To determine whether macrophage (Mφ)-driven epithelial proliferation following injury was a direct or indirect process, a co-culture system was developed to isolate the Mφ-epithelial cell interaction in vitro. This system, referred to as the macrophage-epithelial repair assay (MERA) utilized primary mouse airway epithelial cells cultured under bi-phasic conditions (i.e. air-liquid interface) in trans-well inserts with bone marrow Mφ (BMMφ) attached to the basolateral surface directly underneath. The two cell populations were separated across a semi-permeable barrier (10 μm thick with pore size 0.4 μm) and evaluated following a pipette-mediated scratch wound to the epithelial monolayer. Tracheal epithelia were initially used due to the high trans-epithelial cell resistance (TER) levels (generally >1000Ω). As expected, Mφ cultured alone lacked electrical resistance, but epithelia cultured alone maintained TER levels over 1000Ω. [000287] Upon injury, Mφ significantly accelerated the TER rebound following pipette- mediated scratch, because Mφ-epithelia co-cultures resulted in 100% restoration of baseline TER levels by d3, further increasing to 130% by d4 post-scratch. However, in the absence of Mφ, the epithelia only recovered 65% of baseline TER levels by d4 post-injury. The ability of Mφ to promote TER rebound did not require rIL-4 or rIL-13 administration, nor were any defects in TER recovery observed using IL-4Rα−/− Mφ. In contrast, using TFF2 deficient Mφ reduced the re-epithelialization rate and resulted in only 45-50% TER restoration by d4 post- wounding. Detectable levels of TFF2 protein were found in the mouse tracheal epithelial cell (MTEC) supernatant lacking Mφ, but the presence of Mφ increased supernatant TFF2 levels 2-fold. [000288] To determine whether epithelial barrier restoration and proliferation were functionally linked, BrdU incorporation was assessed on MTEC harvested on d4 and gated on EpCAM+, junctional adhesion molecule-1 (JAM-1+) cells to ensure evaluation of mature epithelial cells devoid of macrophages. Of the live EpCAM+ cells recovered from the apical side of the transwell insert, only low levels of epithelial proliferation occurred in cultures lacking Mφ, but the BrdU+ epithelial population increased 7-fold with WT Mφ. TFF2−/− Mφ promoted a moderate increase in proliferation over cultures lacking Mφ, but IL-4Rα−/− Mφ stimulated an 8-fold increase in BrdU+ epithelia over cultures lacking Mφ. In the absence of Mφ, rTFF2 treatment increased proliferation only 2-fold. Application of rTFF2 alone (without Mφ) significantly accelerated TER rebound over the scratched, mock-treated cultures. Moreover, WT alveolar macrophages (AM) significantly accelerated TER restoration, but TFF2−/− AM were less able to promote repair, whereas lack of TFF2 within the epithelia impaired TER rebound irrespective of the AM genotype. [000289] To address whether macrophages also promoted recovery of epithelia population relevant to the distal lung compartment, primary alveolar type 2 (AT2) cells were used instead of MTEC. In this setting, WT Mφ also induced greater EdU incorporation (EdU is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis) and TER recovery than TFF2−/− Mφ. Taken together, Mφ induced recovery of epithelial barrier function and also epithelial proliferation through a TFF2-dependent mechanism. [000290] To further understand the mechanism acting downstream of TFF2 expression in myeloid cells, RNA-sequencing was completed on WT or TFF2−/− BMMφ recovered from MERA at d4 and compared to reads obtained from naïve BMMφ exposed to quiescent MTEC. TFF2 mRNA transcripts were not different between WT Mφ exposed to intact or damaged epithelia, but WT Mφ exposed to damaged MTEC were found to up-regulate canonical M2 genes such as: Mgl2, Socs2, and Arg1. However, comparison of RNA transcripts between WT and TFF2−/− Mφ that were both exposed to damaged MTEC revealed that Camkk2b expression was significantly (>2-fold) under-represented in TFF2−/− Mφ. Because Camkk2b is implicated in the calcium dependent pathway for Wnt glycoprotein expression [Id., citing Qu, F., et al. Front. Biosci. (Landmark Ed) (2013) 18: 493-503; Wang, Q. et al. PLoS One (2010) 5 (5): e10456], which in turn regulates multiple aspects of epithelial cell biology, it was hypothesized that Mφ-derived TFF2 may promote epithelial repair, at least partially, via regulating Wnt expression. [000291] To test this hypothesis, cDNA-derived from different Mφ populations recovered from d4 of MERA were screened against a panel of 84 different Wnt pathway genes. Wnt4a and Wnt16 were 2-fold increased in WT Mφ exposed to damaged epithelia vs. WT Mφ exposed to non-scratched, quiescent epithelia. Wnt4a and Wnt16 were both 2–3-fold reduced in Tff2−/− Mφ exposed to injured epithelia compared to WT Mφ exposed to injured epithelia. Inoculation of WT MERA cultures with anti-Wnt4 Ab abrogated the TER rebound compared to control IgG treatment. Real-time PCR quantification of Wnt4 and Wnt16 mRNA transcript levels in CD103+ DC, IM, and AM populations FACS-sorted from lung tissues of WT and Tff2−/− mice at 4d post N.b confirmed that TFF2 deficiency impaired Wnt4 expression in alveolar macrophages, but did not alter mRNA expression for Arg1, Retnla and Nos2. [000292] To confirm that non-canonical Wnt signaling functioned in the same pathway as TFF2 in the context of N.b.-induced lung injury, CD11cCre and CD11cCreTFF2flox mice were administered either an rWnt4/Wnt16/R-spondin1 cocktail (1μg) or saline control (vehicle). At d4 post-infection, rWnt4/Wnt16/R-spondin treatment caused a 2-fold increase in the percentage of EpCAM+BrdU+pro-Spc+ cell populations recovered from the distal lung compartment of the CD11cCreTFF2flox mice. No increased BrdU incorporation was noted in R-spondin only treated animals. These data were interpreted as implying that Wnt4/16 act downstream of TFF2 as part of Mφ−driven repair processes that restore damaged epithelia. CXCR4/SDF-1 signaling [000293] The chemokine receptor CXCR4 is a member of a family of seven-span transmembrane G-protein coupled chemokine receptors. It is ubiquitously expressed and evolutionarily conserved. Its ligand, stromal cell derived factor 1 (SDF-1) called CXCL12, is likewise highly conserved. CXCR4 is intensively studied in different autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, and autoimmune disorders of the central nervous system, such as multiple sclerosis, for its involvement in leukocyte chemotaxis. [Doring, Y. et al. Frontiers Physiology (2014): doi:10.3389/fphys.2014.00212, citing Debnath, B. et al. Theranostics (2013) 3: 47-75; Domanska, UM et al. Eur. J. Cancer (2013) 49: 219- 30]. The CXCL12/CXCR-4 axis, comprising the chemokine receptor CXCR4 and its ligand CXCL12 (also known as stromal cell-derived factor 1, or SDF-1), plays a crucial role in the homing of stem and progenitor cells in the bone marrow and controls their mobilization into peripheral blood and tissues in homeostatic conditions, as well as after tissue injury or stress. Upregulation of CXCL12 in hypoxic conditions with subsequent mobilization of CXCR4- positive stem and progenitor cells [Id., citing Ceradini, DJ et al. Nature Medf. (2004) 10: 858- 64] has promoted researchers to explore the role and therapeutic value of progenitor cells and the CXCR12/CXCR4 axis in diverse models of ischemic injury, including in heart, kidney, lung and brain. [000294] CXCL12 also binds a second chemokine receptor (CXCR7 or RDC1) with an even 100-fold higher affinity compared with CXCR4. [Id., citing Balabanian, K. et al. J. Biol. Chem. (2005) 280: 35760-66; Burns, JM et al J. Exp. Med. (2006) 203: 2201-13] CXCR7 has been implicated in cell survival and adhesion [Id., citing Burns, JM et al J. Exp. Med. (2006) 203: 2201-13], and can mediate CXCL12-directed T cell chemotaxis independently from CXCR4 [Id., citing Balabanian, K. et al. J. Biol. Chem. (2005) 280: 35760-66; Kumar, R. et al Cell Immunol. (2012).272: 230-41). Binding of the chemokine ligands CXCL12 and CXCL11 (also called I-TAC) to CXCR7 enhances continuous CXCR7 internalization and delivery of the chemokine ligands to the lysosomes for degradation [Id., citing Luker, KE et al. Oncogene (2010) 29: 4599-4610; Naumann, U. et al. PLoS One (2010) 5: e9175]. Such CXCR7-mediated regulation of available CXCL12 concentrations has associated CXCR7 with a function as decoy receptor, reducing acute CXCL12/CXCR4 signaling [Luker, KE et al. Oncogene (2010) 29: 4599-4610]. Furthermore, study of a CXCR7 agonist recently suggested downregulation of CXCR4 protein levels by CXCR7 signaling as another negative regulatory mechanism of CXCR7 toward the CXCL12/CXCR4 axis [Id., citing Uto-Konomi, A. et al. Biochem. Biophys. Res. Commun. (2013) 431: 722-76]. Also, heterodimerization of CXCR7 with CXCR4 interferes with CXCR4-induced Gαi protein-mediated signaling and favors β- arrestin-linked signaling [Id., citing Levoye, A.et al., Blood (2009) 113: 6085-93; Decaillot, FM et al. J. Biol. Chem. (2011) 286: 32188-97]. On the other hand, the CXCL12 scavenging function of CXCR7 can also positively influence CXCR4-mediated migration by preventing the downregulation of CXCR4 surface expression and function through excessive CXCL12 concentrations [Id., citing Sanchez-Alcaniz et al. Neuron (2011) 69: 77-90]. In addition to its modulatory effect on CXCL12/CXCR4 signaling, CXCR7 is able to mediate CXCL12-induced MAPK activation independently from CXCR4 (Id., citing Wang, Y. et al. Neuron (2011) 69: 61-76). Although the precise signaling mechanisms downstream of CXCR7 is not understood, CXCR7 does not bind to or induce the activation of heterotrimeric G-proteins as typical in classical GPCR signaling, but depends on ligand-induced β-arrestin recruitment [Id., citing Rajagopal, S. et al. Proc. Ntl. Acad. Sci. USA (2010) 107: 628-32]. Neutrophils [000295] The CXCL12/CXCR4 axis maintains neutrophil homeostasis primarily by regulation of neutrophil release from the bone marrow in a cell-autonomous fashion [Id., citing Eash, KJ et al. Blood (2009) 113: 4711-19]. Senescent neutrophils in the periphery expressing high levels of CXCR4 home back to the bone marrow to be cleared [Id., citing Martin, C. et al. Immunity (2003) 19: 583-93]. In contrast, activated neutrophils downregulate CXCR4 expression putatively postponing their clearance (Bruhl, H. et al. Eur. J. Immunol. (2003) 33: 3028-37; Martin, C. et al. Immunity (2003) 19: 583-93]. Lymphocytes [000296] CXCR4 on lymphocytes plays an essential role during B-cell development [Id., citing Nagasawa, T. et al. Nature (1996) 382: 635-38] and T-cell homeostasis (Id., citing Bleul, CC et al. J. Exp. Med. (1996) 184: 1101-9; Zou, YR. et al. Nature (1998) 393: 595-99). Furthermore, the CXCL12/CXCR4 axis drives chemotaxis or fugetaxis of T-cells in various pathophysiological settings [Id., citing (Poznansky, MC et al. Nat. Med. (2000) 6: 543-48; Dunussi-Joannopoulos, K. et al. Blood (2002) 100: 1551-58; Fernandis, AZ et al. J. Biol. Chem. (2003) 278: 9536-43; Okabe, S. et al. Blood (2005) 105: 474-80; Zhang, D. et al., Arthritis Rheum. (2005) 52: 38839-49. Similarly, CXCL12 is able to trigger B-cell chemotaxis in vitro through CXCR4 [Id., citing (Klasen, C. et al. J. Immunol. (2014) 192: 5273-84). Platelets [000297] CXCR4 expression (mRNA, protein) was reported on platelets [Wang, JF et al. Blood (1998) 92: 756-64; Kowalska, MA et al. Br. J. Haematol. (1999) 104: 220-29]. Addition of CXCL12 to platelets from healthy donors induced platelet aggregation, which could be inhibited by blocking CXCR4. The latter implies an atherogenic, pro-thrombotic, and plaque- destabilizing role for the CXCL12/CXCR4 axis in vivo [Id., citing Falk, E. et al. Circulation (1995) 92: 657-71; Abi-Younes, S. et al. Cir. Res. (2000) 86: 131-38]. In contrast, others report CXCL12 to be a weak platelet agonist, however still amplifying platelet activation, adhesion and chemokine release triggered by low doses of primary platelet agonists, such as adenosine diphosphate (ADP) and thrombin, or arterial flow conditions [Id., citing (Kowalska, MA et al. Br. J. Haematol. (1999) 104: 220-29; Gear, AR et al. Blood (2001) 97: 937-45). Furthermore, CXCL12 gradients was shown to induce platelet migration and transmigration in vitro involving PI3K signaling [Id., citing Kraemer, BF et al. J. Mol. Med. (2010) 88: 1277- 88]. In addition, CXCL12 was shown to trigger CXCR4 internalization and cyclophilin A- dependent CXCR7 externalization on (mouse and human) platelets, resulting in prolonged platelet survival. Mice lacking the cytosolic chaperone cyclophilin A showed less CXCL12- induced rescue of platelets from activation-induced apoptosis through CXCR7 engagement. Hence, it has been hypothesized that differential regulation of CXCR4/CXCR7 surface expression on platelets upon CXCL12 exposure at sites of platelet activation/accumulation may orchestrate platelet survival, subsequently impacting platelet-mediated physiological mechanisms [Id., citing Chatterjee, M. et al. FASEB J.(2014) 28. Doi:10.1096/fj.14-249730]. [000298] The described invention proposes a biofunctional fusion protein containing a biologically active immunomodulatory component operatively linked to a biologically active anti-viral component. The biologically active immunomodulatory component reduces exacerbated inflammatory responses in the host in response to severe virus infection. The biologically active anti-viral component reduces viral load. .SUMMARY OF THE INVENTION [000299] According to one aspect, the described invention provides a method for reducing damaging effects of a severe respiratory virus infection in a susceptible subject comprising a pharmaceutically acceptable carrier and a recombinant bifunctional fusion protein comprising a recombinant biologically active immunomodulatory component operatively linked to a recombinant biologically active anti-viral component, the recombinant immunomodulatory component comprising a recombinant human trefoil factor 1 (hTFF1); a recombinant human trefoil factor 2 (hTFF2), or a recombinant human trefoil factor 3 (hTFF3), wherein the pharmaceutical composition is cytoprotective. According to some embodiments, the recombinant bifunctional fusion protein comprises the recombinant human trefoil factor 1 (hTFF1) or a biologically active fragment or variant thereof, the biologically active hTFF2 molecule or a biologically active fragment or variant thereof, or the recombinant human trefoil factor 3 (hTFF3) or a biologically active fragment or variant thereof joined by its C-terminal end to a linker sequence, which is joined to an N-terminal end of a biologically active recombinant human interferon molecule, fragment or variant thereof. According to some embodiments, the C-terminal end of the recombinant interferon molecule, fragment or variant sequence is further joined to a recombinant Fc derived antibody domain comprising a constant region of a human immunoglobulin heavy chain. According to some embodiments, the recombinant interferon molecule is a recombinant type I interferon or biologically active fragment thereof selected from IFN-α, IFN-β, IFN-ε, IFN-ω, IFN-κ, IFN-δ, IFN-τ and IFN-ζ. According to some embodiments the recombinant IFN is a human interferon. According to some embodiments, the recombinant hTFF2 molecule is a human protein of SEQ ID NO: 52 (NCBI Ref NP_005414). According to some embodiments, the recombinant hTFF1 molecule is a protein of SEQ ID NO:# __ According to some embodiments, the recombinant hTFF3 molecule is a protein of SEQ ID NO:# __ .According to some embodiments, the recombinant interferon molecule is a human IFN-α of SEQ ID NO: 53 (NCBI Ref NP_000596.2); or the recombinant interferon molecule is a human IFN-κ of SEQ ID NO: 54 (NCBI Ref NM_020124.3); or the interferon is a recombinant human IFN-ω of SEQ ID NO: 55 (NCBI Ref.002177.3). According to some embodiments, the recombinant interferon is a recombinant human interferon-τ as disclosed by Chon, TW and Bixler, S, J. Interferon & Cytokine Res. (2010) 30 (7): 477-85: [000300] MAFVLSLLMALVLVSYGPGGSLGCDLSQNHVLVGRKNLRLLDEMRR LSPHFCLQDRKDFALPQEMVEGGQLQEAQAISVLHEMLQQSFNLFHTEHSSAAWDT TLLEPCRTGLHQQLDNLDACLGQVMGEEDSALGRTGPTLALKRYFQGIHVLKEKGY SDCAWETVRLEIMRSFSSLISLQERLRMMDGDLSSP (SEQ ID NO: 34) [000301] According to some embodiments, the Fc-derived antibody domain is a human protein of SEQ ID NO: 56 (NCBI Ref 4CDH_A) According to some embodiments, the amino acid sequence of the recombinant fusion protein is SEQ ID NO: 35. According to some embodiments, the sequences are codon-optimized to improve gene expression. According to some embodiments, the recombinant fusion protein is produced in CHO cells. .According to some embodiments, the method further comprises encapsulating the recombinant fusion protein into particles. According to some embodiments, the recombinant TFF2-IFN, TFF1-IFN or TFF-3 IFN fusion protein is encapsulated within the polymer matrix of a plurality of microparticles. [000302] According to some embodiments, the respiratory virus is a respiratory syncytial virus (RSV), an Ebola virus, a cytomegalovirus, a Hanta virus, an influenza virus, a coronavirus, a Zika virus, A West Nile virus, a dengue virus, a Japanese encephalitis virus, a tick-borne encephalitis virus, a yellow fever virus, a rhinovirus, an adenovirus, a herpes virus, an Epstein Barr virus, a measles virus, a mumps virus, a rotavirus, a cocksackie virus, a norovirus, or an encephalomyocarditis virus (EMCV). According to some embodiments, the method stimulates repair of a mucosal injury, modulates an immune response, or both. According to some embodiments, the administering occurs parenterally, by inhalation, or by insufflation. According to some embodiments, the susceptible patient includes a very young subject, an elderly subject, a subject who is ill; an immunocompromised subject, a subject with long term health conditions, a subject who is obese, or a subject that is physically weak due to malnutrition or dehydration. [000303] According to some embodiments, the damaging effects of the severe respiratory virus infection include one or more of: primary viral pneumonia; superimposed bacterial pneumonia; disruption or injury to alveolar epithelium, endothelium or both; acute lung injury (ALI); acute respiratory distress syndrome (ARDS); symptoms of shock; excessive complement activation; a pathological increase in vascular permeability; endothelial activation, loss of barrier function and consequent microvascular leak; thrombotic complications; kidney damage; or elevated concentrations of one or more inflammatory mediators in plasma (hypercytokinemia), compared to a normal healthy subject. According to some embodiments, symptoms of shock include low blood pressure, lightheadedness, shortness of breath, and rash. According to some embodiments, the thrombotic complications include one or more of formation of pulmonary microthrombi, acute pulmonary embolism, deep-vein thrombosis, ischemic stroke, myocardial infarction, or systemic arterial embolism. According to some embodiments,; the inflammatory mediator is one or more of interferon α, interferon β, interferon-κ, interferon-γ, complement, prostaglandin D2, vasoactive intestinal peptide (VIP), nterleukin-1-beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 (IL-12), IL-17, tumor necrosis factor-alpha (TNF-α). [000304] According to some embodiments, the method may stimulate repair of a mucosal injury, modulates an immune response, or both. According to some embodiments, repair of a mucosal injury comprises epithelial proliferation; or repair of a mucosal injury restores an epithelial barrier, an endothelial barrier or both; or the immune response comprises recruitment of innate and adaptive immune cells. According to some embodiments, the innate immune cells comprise macrophages, dendritic cells (DCs), innate lymphoid cells (ILCs), and natural killer cells (NKs); or the adaptive immune cells include αβ T cells, γδT cells, and B cells. [000305] According to some embodiments, the method comprises aerosolizing the composition in a form selected from a dry powder, a suspension or a solution and administering the aerosolized composition to the respiratory system. According to some embodiments, the administering of the aerosolized composition to the respiratory system occurs parenterally, by inhalation, or by insufflation. According to some embodiments, the composition is a solution. [000306] According to some embodiments, the administering to the respiratory system is by an inhalation delivery device or a solid particulate therapeutic aerosol generator. According to some embodiments, the solid particulate aerosol generator is an insufflator. According to some embodiments, the inhalation delivery device is a nebulizer, a metered-dose inhaler, or a dry powder inhaler (DPI). According to some embodiments, the respirable particles range in size from about 1 to 10 microns, inclusive; or the particles for nasal administration (insufflation), range in size from 10-500 µM, inclusive. [000307] According to some embodiments, the susceptible patient includes a very young subject, an elderly subject, a subject who is ill; an immunocompromised subject, a subject with long term health conditions, a subject who is obese, or a subject that is physically weak due to malnutrition or dehydration. [000308] According to some embodiments, the pharmaceutical composition further comprises a supportive therapy or an additional therapeutic agent selected from one or more of an immunomodulatory agent, an analgesic agent, an anti-inflammatory agent, an anti-infective agent, an anti-malarial agent, an anti-viral agent or an anti-fibrotic agent. According to some embodiments, the immunomodulatory agent can be used as a monotherapy or in combination with the supportive therapy, the analgesic agent, the anti-inflammatory agent, the anti-infective agent, the anti-malarial agent, the anti-viral agent or the anti-fibrotic agent. [000309] According to some embodiments, the immunomodulatory agent is selected from the group consisting of methotrexate; a glucocorticoid, cyclosporine, tacrolimus and sirolimus; a recombinant interferon selected from IFN-α; IFN-α-2b, IFN-β, IFN-γ, IFN-κ, IFN-ω, IFN-δ, IFN-τ and IFN-ζ; a recombinant IL-2 receptor inhibitor; a PDE4 inhibitor; a hyperimmune globulin prepared from a donor with high titers of a desired antibody; a TNFα inhibitor/antagonist; an IL-1β inhibitor; a chimeric IL-1Ra; an IL-6 inhibitor; an IL-12/ IL-23 inhibitor selected from ustekinumab, briakinumab; an IL-23 inhibitor selected from guselkumab, tildrakizumab; a compound that targets TLR4 signaling; a p38 MAPK inhibitor, a compound that targets Janus kinase signaling; a compound that targets cell adhesion molecules to reduce leukocyte recruitment; and a recombinant anti-inflammatory cytokine. [000310] According to some embodiments, the glucocorticoid is a corticosteroid selected from prednisone, dexamethasone, azathioprine, mycophenolate, mycophenolate mofetil, and combinations thereof; or the recombinant IL-2 inhibitor is denileukin diftitox; or the PDE4 inhibitor is cilomilast; or the TNFα inhibitor/antagonist is selected from the group consisting of etanercept; adalimumab; infliximab, certolizumab pegol, or golimumab; or the IL-1β inhibitor is selected from rilonacept; canakinumab; and Anakinra; or the IL-6 inhibitor is selected from tocilizumab, siltuximab, sarilumab, olokizumab, and sirukumab; or the compound that targets TLR4 signaling is selected from (ethyl 4-(4’-chlorophenyl) amino-6 methyl-2-oxocyclohex-3-en-1-aote (enamionone E121),t; JODI 18b; JODI 19, resatorvid, TLR-C34; and C35; the p38 MAPK inhibitor is selected from the group consisting of 4-(4’- fluorophenyl)-2-(4’-methylsulfinylphenyl)-5- (4’-pyridyl)-imidazole (SB203580), trans-4-[4- (4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)-1H-imidazol-1-yl]cyclohexanol (SB239063), and 4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1- ol (RWJ 67657); or the compound that targets Janus kinase signaling is tofacitinub, baricitinib, or upadacitinib; or the compound that targets a cell adhesion molecule to reduce leukocyte recruitment is an α4 integrin inhibitor selected from vedolizumab and natalizumab; or the recombinant anti-inflammatory cytokine is IL-4, IL-10, or IL-11; or the interferon is in a PEGylated form. [000311] According to some embodiments, a physiologic or supraphysiological dose of the recombinant interferon selected from recombinant IFN-α; IFN-α-2b, IFN-β, IFN-γ, IFN-κ, IFN-ω, IFN-δ, IFN-τ and IFN-ζ or a PEGylated form thereof boosts immune defenses of the subject. [000312] According to some embodiments, the analgesic agent is selected from the group consisting of codeine, hydrocodone, oxycodone, methadone, hydromorphone, morphine, and fentanyl. [000313] According to some embodiments, the anti-inflammatory agent is selected from aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac nabumetone, naproxen, nintedanib, oxaprozin, pirfenidone, piroxicam, salsalate, sarilumab (Kevzara®) sulindac, tolmetin, and combinations thereof. [000314] According to some embodiments, the anti-infective agent is amoxicillin, doxycycline, demeclocycline; eravacycline, minocycline, ormadacycline, tetracycline, cephalexin, defotaxime, cetazidime, cefuroxime, ceftaroline; ciprofloxacin, levofloxacin, moxifloxacin, clindamycin, lincomycin, metronidazole, azithromycin; clarithromycin, erythromycin, sulfamethoxazle and trimethoprim; sulfasalazine, amoxicillin and clavulanate; vancomycin, dalbavancin, oritavancin, telavancin, gentamycin, tobramycin, amikacin, imipenem and cilastatin, meropenem, doripenem, or ertapenem. [000315] According to some embodiments, the anti-malarial agent is selected from quinine, quinidine, chloroquine, hydroxychloroquine, amodiaquine, mefloquine, halofantrine, lumefantrine, piperaquine, and tafenoquine; an antifolate compound selected from pyrimethamine, proguanil, chlorproguanil, trimethoprim; an artemisinin compound selected from artemisinin, dihydroartemisinin, artemether, artesunate; and atovaquone. [000316] According to some embodiments, the anti-viral agent is selected from acyclovir, gancidovir, foscarnet; ribavirin; amantadine, azidodeoxythymidine /zidovudine), nevirapine, a tetrahydroimidazobenzodiazepinone (TIBO) compound; efavirenz; remdecivir, lopinavir/ritonavir, umifenovir, favipiravir, ivermectin, and delavirdine. [000317] According to some embodiments, the anti-fibrotic agent is selected from nintedanib, pirfenidone, and combinations thereof. [000318] According to some embodiments, the anti-viral agent is an agent that inhibits viral entry and decreases viral load. [000319] According to some embodiments, the anti-viral agent that inhibits or blocks viral entry is a synthetic peptide selected from the group consisting of: NP-1, SEQ ID NO: 18; NP-2; SEQ ID NO: 19; NP-3; SEQ ID NO: 20; NP-4; SEQ ID NO: 21; CP-1; SEQ ID NO: 22; CP-2; SEQ ID NO: 23; HR2P; SEQ ID NO: 25; OC43-HR2, SEQ ID NO: 26; EK1, SEQ ID NO: 27; EK1P, SEQ ID NO: 28; EK1C, SEQ ID NO: 29. According to some embodiments, the anti-viral agent that inhibits or blocks viral entry is a dipeptidyl peptidase 4 (DPP4) inhibitor; an ACE2 inhibitor; a transmembrane serine protease TMRSS2 inhibitor; a cathepsin B inhibitor, a cathepsin L inhibitor or a cathepsin B/ L inhibitor. [000320] According to some embodiments, the supportive therapy is therapeutic apheresis comprising a virion removing step. According to some embodiments, the therapeutic apheresis reduces viral load. [000321] According to another aspect, the described invention provides a method for reducing progression of symptoms of a severe respiratory virus infection in a susceptible human subject, comprising administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant bifunctional fusion protein comprising a recombinant biologically active immunomodulatory component operatively linked to a recombinant biologically active anti-viral component. According to some embodiments the recombinant biologically active immunomodulatory component comprises a recombinant trefoil factor 1 (hTFF1), a recombinant human trefoil factor 2 (hTFF2), or a recombinant human trefoil factor 3 (hTFF3) and a vehicle, wherein the method recues symptoms of the severe virus infection. According to some embodiments, the recombinant bifunctional fusion protein comprises the recombinant biologically active hTFF1 molecule, fragment or variant thereof; the recombinant biologically active hTFF2 molecule, fragment or variant thereof, or the recombinant biologically active hTFF3 molecule, fragment or variant thereof joined by its C- terminal end to a linker sequence, which is joined to an N-terminal end of a recombinant biologically active recombinant human interferon molecule, fragment or variant. According to some embodiments, the C-terminal end of the recombinant interferon molecule, fragment or variant sequence is further joined to a recombinant Fc derived antibody domain comprising a recombinant constant region of a human immunoglobulin heavy chain. According to some embodiments, the recombinant interferon molecule is a type I interferon or biologically active fragment thereof selected from recombinant IFN-α, IFN-β, IFN-ε, IFN-ω, IFN-κ, IFN-δ, IFN- τ and IFN-ζ. According to some embodiments, the recombinant IFN is a human interferon. According to some embodiments, the recombinant hTFF1 molecule is a recombinant human protein of SEQ ID NO: (NCBI Reference Sequence: NM_003225.3). According to some embodiments, the hTFF2 molecule is a recombinant human protein of SEQ ID NO: 52 (NCBI Ref NP_005414). According to some embodiments, the recombinant hTFF3 molecule is a human protein of SEQ ID NO:38__( NCBI Reference Sequence: NM_003226.4). According to some embodiments, the recombinant interferon molecule is a human IFN-α of SEQ ID NO: 53 (NCBI Ref NP_000596.2); or the recombinant interferon molecule is a human IFN-κ of SEQ ID NO: 54 (NCBI Ref NM_020124.3); or the recombinant interferon is a human IFN-ω of SEQ ID NO: 55 (NCBI Ref. NM_002177.3). According to some embodiments, the recombinant interferon is a human interferon-τ as disclosed by Chon, TW and Bixler, S, J. Interferon & Cytokine Res. (2010) 30 (7): 477-85: [000322] MAFVLSLLMALVLVSYGPGGSLGCDLSQNHVLVGRKNLRLLDEMRR LSPHFCLQDRKDFALPQEMVEGGQLQEAQAISVLHEMLQQSFNLFHTEHSSAAWDT TLLEPCRTGLHQQLDNLDACLGQVMGEEDSALGRTGPTLALKRYFQGIHVLKEKGY SDCAWETVRLEIMRSFSSLISLQERLRMMDGDLSSP (SEQ ID NO: 34) [000323] According to some embodiments, the Fc-derived antibody domain is a recombinant human protein of SEQ ID NO: 56 (NCBI Ref 4CDH_A) According to some embodiments, the amino acid sequence of the recombinant fusion protein is SEQ ID NO: 35. According to some embodiments, the sequences are codon-optimized to improve gene expression. According to some embodiments, the recombinant fusion protein is produced in CHO cells..According to some embodiments, the method further comprises encapsulating the recombinant fusion protein into particles. According to some embodiments, the recombinant TFF2-IFN fusion protein is encapsulated within the polymer matrix of a plurality of microparticles. [000324] According to some embodiments, the respiratory virus is a respiratory syncytial virus (RSV), an Ebola virus, a cytomegalovirus, a Hanta virus, an influenza virus, a coronavirus, a Zika virus, a West Nile virus, a dengue virus, a Japanese encephalitis virus, a tick-borne encephalitis virus, a yellow fever virus, a rhinovirus, an adenovirus, a herpes virus, an Epstein Barr virus, a measles virus, a mumps virus, a rotavirus, a cocksackie virus, a norovirus, or an encephalomyocarditis virus (EMCV). [000325] According to some embodiments, symptoms of the severe respiratory virus infection include one or more of: primary viral pneumonia; superimposed bacterial pneumonia; disruption or injury to alveolar epithelium, endothelium or both; acute lung injury (ALI); acute respiratory distress syndrome (ARDS); symptoms of shock; excessive complement activation; a pathological increase in vascular permeability; endothelial activation, loss of barrier function and consequent microvascular leak; thrombotic complications; kidney damage; or elevated concentrations of one or more inflammatory mediators in plasma (hypercytokinemia), compared to a normal healthy subject. [000326] According to some embodiments, symptoms of shock include low blood pressure, lightheadedness, shortness of breath, and rash. According to some embodiments, the thrombotic complications include one or more of formation of pulmonary microthrombi, acute pulmonary embolism, deep-vein thrombosis, ischemic stroke, myocardial infarction, or systemic arterial embolism. According to some embodiments the inflammatory mediator is one or more of interferon α, interferon β, interferon-κ, interferon-γ, complement, prostaglandin D2, vasoactive intestinal peptide (VIP), nterleukin-1-beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 (IL-12), IL-17, tumor necrosis factor-alpha (TNF-α). [000327] According to some embodiments, the method stimulates repair of a mucosal injury, modulates an immune response, or both. According to some embodiments, repair of a mucosal injury comprises epithelial proliferation; or repair of a mucosal injury restores an epithelial barrier, an endothelial barrier or both; or the immune response comprises recruitment of innate and adaptive immune cells. According to some embodiments, the innate immune cells comprise macrophages, dendritic cells (DCs), innate lymphoid cells (ILCs), and natural killer cells (NKs) According to some embodiments, the adaptive immune cells include αβ T cells, γδT cells, and B cells. [000328] According to some embodiments, the susceptible patient includes a very young subject, an elderly subject, a subject who is ill; an immunocompromised subject, a subject with long term health conditions, a subject who is obese, or a subject that is physically weak due to malnutrition or dehydration. [000329] According to some embodiments, the method comprises aerosolizing the composition in a form selected from a dry powder, a suspension or a solution and administering the aerosolized composition to the respiratory system. According to some embodiments, the administering of the aerosolized composition to the respiratory system occurs parenterally, by inhalation, or by insufflation. According to some embodiments, the composition is a solution. [000330] . According to some embodiments, the administering is by an inhalation delivery device or a solid particulate therapeutic aerosol generator. According to some embodiments, the solid particulate aerosol generator is an insufflator. According to some embodiments, the inhalation delivery device is a nebulizer, a metered-dose inhaler, or a dry powder inhaler (DPI). According to some embodiments, respirable particles range in size from about 1 to 10 microns, inclusive; or particles for nasal administration (insufflation), range in size from 10-500 µM, inclusive. [000331] According to some embodiments, the pharmaceutical composition further comprises a supportive therapy or an additional therapeutic agent selected from one or more of an immunomodulatory agent, an analgesic agent, an anti-inflammatory agent, an anti-infective agent, an anti-malarial agent, an anti-viral agent or an anti-fibrotic agent. According to some embodiments, the immunomodulatory agent can be used as a monotherapy or in combination with the supportive therapy, the analgesic agent, the anti-inflammatory agent, the anti-infective agent, the anti-malarial agent, the anti-viral agent or the anti-fibrotic agent. [000332] According to some embodiments, the immunomodulatory agent is selected from the group consisting of methotrexate; a glucocorticoid; cyclosporine, tacrolimus and sirolimus; recombinant interferon selected from IFN-α, IFN-α-2b, IFN-β, IFN-γ, IFN-κ, IFN- ω, , IFN-δ, IFN-τ and IFN-ζ; a recombinant IL-2 receptor inhibitor; a PDE4 inhibitor; a hyperimmune globulin prepared from a donor with high titers of a desired antibody; a TNFα inhibitor/antagonist; an IL-1β inhibitor; a chimeric IL-1Ra; or an IL-6 inhibitor; an IL-12/ IL- 23 inhibitor selected from ustekinumab or briakinumab, an IL-23 inhibitor selected from guselkumab, or tildrakizumab; or a compound that targets TLR4 signaling; a p38 MAPK inhibitor, a compound that targets Janus kinase signaling selected from the group consisting of tofacitinub, baricitinib, and upadacitinib; a compound that targets cell adhesion molecules to reduce leukocyte recruitment; and a recombinant anti-inflammatory cytokine selected from the group consisting of IL-4, IL-10, and IL-11. [000333] According to some embodiments, the glucocorticoid is a corticosteroid selected from prednisone, dexamethasone, azathioprine, mycophenolate, mycophenolate mofetil,, and combinations thereof; the recombinant IL-2 inhibitor is denileukin diftitox; the PDE4 inhibitor is cilomilast; the TNFα inhibitor/antagonist is selected from the group consisting of etanercept; adalimumab; infliximab, certolizumab pegol, or golimumab; the IL-1β inhibitor is selected from rilonacept; canakinumab; and Anakinra; the IL-6 inhibitor is selected from tocilizumab, siltuximab, sarilumab, olokizumab, and sirukumab; the compound that targets TLR4 signaling is selected from (ethyl 4-(4’-chlorophenyl) amino-6 methyl-2-oxocytlohex-3-en-1-aote (enamionone E121), JODI 18b; JODI 19, resatorvid, TLR-C34 and C35; the p38 MAP inhibitor is selected from the group consisting of 4-(4’-fluorophenyl)-2-(4’-methylsulfinylphenyl)-5- (4’-pyridyl)-imidazole (SB203580), trans-4-[4-(4-fluorophenyl)-5-(2-methoxy-4- pyrimidinyl)-1H-imidazol-1-yl]cyclohexanol (SB239063); and 4-[4-(4-fluorophenyl)-1-(3- phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol (RWJ 67657); the compound that targets Janus kinase signaling is tofacitinub, baricitinib, or upadacitinib; the compound that targets a cell adhesion molecule to reduce leukocyte recruitment is an α4 integrin inhibitor selected from vedolizumab and natalizumab; the recombinant anti-inflammatory cytokine is IL-4, IL-10, or IL-11; or the recombinant interferon is in a PEGylated form. [000334] According to some embodiments, a physiologic or supraphysiological dose of the recombinant interferon selected from IFN-α; IFN-α-2b, IFN-β, IFN-γ, IFN-κ, IFN-ω, , IFN- δ, IFN-τ and IFN-ζ boosts the subject’s defenses. [000335] According to some embodiments, the analgesic agent is selected from the group consisting of codeine, hydrocodone, oxycodone, methadone, hydromorphone, morphine, and fentanyl. [000336] According to some embodiments, the anti-inflammatory agent is selected from aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac nabumetone, naproxen, nintedanib, oxaprozin, pirfenidone, piroxicam, salsalate, sarilumab (Kevzara®) sulindac, tolmetin, and combinations thereof. [000337] According to some embodiments, the anti-infective agent is amoxicillin, doxycycline, demeclocycline; eravacycline, minocycline, ormadacycline, tetracycline, cephalexin, defotaxime, cetazidime, cefuroxime, ceftaroline; ciprofloxacin, levofloxacin, moxifloxacin, clindamycin, lincomycin, metronidazole, azithromycin; clarithromycin, erythromycin, sulfamethoxazle and trimethoprim; sulfasalazine, amoxicillin and clavulanate; vancomycin, dalbavancin, oritavancin, telavancin, gentamycin, tobramycin, amikacin, imipenem and cilastatin, meropenem, doripenem, or ertapenem. [000338] According to some embodiments, the anti-malarial agent is selected from quinine, quinidine, chloroquine, hydroxycloroquine, amodiaquine, mefloquine, halofantrine, lumefantrine, piperaquine, and tafenoquine; an antifolate compound, selected from pyrimethamine, proguanil, chlorproguanil, trimethoprim; cepharanthine/selamectin/mefloquine hydrochloride; an artemisinin compound selected from artemisinin, dihydroartemisinin, artemether, artesunate; and atovaquone. [000339] According to some embodiments, the anti-viral agent is selected from acyclovir, gancidovir, foscarnet; ribavirin; amantadine, azidodeoxythymidine /zidovudine), nevirapine, a tetrahydroimidazobenzodiazepinone (TIBO) compound; efavirenz; remdecivir, lopinavir/ritonavir, umifenovir, favipiravir, ivermectin, and delavirdine. [000340] According to some embodiments, the anti-fibrotic agent is selected from nintedanib, pirfenidone, and combinations thereof. [000341] According to some embodiments, the antiviral agent inhibits viral entry and decreases viral load. According to some embodiments, the anti-viral agent that blocks viral entry is a synthetic peptide selected from the group consisting of: NP-1, SEQ ID NO: 18; NP- 2; SEQ ID NO: 19; NP-3; SEQ ID NO: 20; NP-4; SEQ ID NO: 21; CP-1; SEQ ID NO: 22; CP- 2; SEQ ID NO: 23; HR2P; SEQ ID NO: 25; OC43-HR2, SEQ ID NO: 26; EK1, SEQ ID NO: 27; EK1P, SEQ ID NO: 28; and EK1C, SEQ ID NO: 29. According to some embodiments, the anti-viral agent that inhibits viral entry is a dipeptidyl peptidase 4 (DPP4) inhibitor; an ACE2 inhibitor; a transmembrane serine protease TMRSS2 inhibitor; a cathepsin B inhibitor, a cathepsin L inhibitor or a cathepsin B/ L inhibitor. [000342] According to some embodiments, the supportive therapy is therapeutic apheresis comprising a virion removing step. According to some embodiments, the therapeutic apheresis reduces viral load. BRIEF DESCRIPTION OF DRAWINGS [000343] FIG. 1 is a schematic of the contact activation (intrinsic) and the tissue factor (extrinsic) coagulation pathways. [000344] FIG. 2 is a schematic depicting a three stage cell-surface based model of coagulation, comprising initiation, priming, and propagation. Taken from Monroe et al. Arterioscler Thromb Vase Biol. (2002) 22:1381 -1389. [000345] FIG.3 is a schematic showing the principal stages in complement activation by the classical, lectin, and alternative pathways. [Taken from Molecular Biology of the Cell, 4th Ed., Bruce Alberts, et al. ed. Garland Science, New York (2002), Fig. 25-41]. In all three pathways, the reactions of complement activation usually take place on the surface of an invading pathogen. C2-C9 and factors B and D are the reacting components of the complement system; various other components regulate the system. Early components are shown based on position relative to the cleavage of C3 block (above and below) accordingly. [000346] FIG 4A is a schematic of the challenge model of influenza virus. FIG.4B is a graph of body weight change vs. days post infection for mice infected with H7N9 influenza A virus, H9N2 influenza A virus, and a PBS control. FIG.4C is a plot of percent survival vs. days post infection for mice infected with H7N9 influenza A virus, H9N2 influenza A virus, and a PBS control. [000347] FIG 5A is a Log10(p Value) vs. Log2 (H7N9/H9N2 plot showing genes that are significantly differentially expressed in the lung tissues of mice after infection with H7N9 and H9N2 influenza viruses. FIG.5B is a plot of expression level of TFF2 vs days post infection for mice infected with H7N9 and H9N2. FIG. 5C is a plot of TFF2 mRNA relative value versus time for mice infected with H7N9 and H9N2. Fluorescence quantitative PCR was used to verify the expression of TFF2 in lung tissues. FIG.5D is a Western Blot showing expression of TFF2 at different time points after infection with H7N9 and H9N2. [000348] FIG.6A shows vector construction maps of TFF2 eukaryotic expression vectors pSV1.0 (left), pSV1.0-TFF2 (middle), pSV1.0-TFF2-6xHis ("6xHis" disclosed as SEQ ID NO: 33) (right). FIG.6B shows in vitro expression of TFF2 in cell lines and secreted into the cell supernatant. FIG. 6C shows result of polyacrylamide gel electrophoresis (PAGE) verifying purification of TFF2-6xHis protein ("6xHis" disclosed as SEQ ID NO: 33) at varying concentrations of imidazole. [000349] FIG. 7A is a schematic of the mouse model to show the protective effect of TFF2 protein treatment on influenza virus H7N9, H9N2, PR8 challenged mice. FIG.7B (top) is a plot of percent survival versus days post infection for (B) mice infected with H7N9, mice infected with H7N9 treated with mTFF2 supernatant; and mice infected with H7N9s treated with hTFF2 supernatant. FIG. 7B (bottom) is a plot of body weight change vs. days post infection for mice infected with H7N9, and mice infected with H7N9 treated with mTFF2. TFF2 increased the survival rate of H7N9 infected mice and reduced weight loss, compared to the control. FIG. 7C (top) is a plot of body weight change vs. days post infection of mice infected with H9N2 and mice treated with H9N2 + mTFF2 supernatant. FIG.7C (bottom) is a plot of body weight change vs. days post infection for wild type and TFF2 KO mice. TFF2 protein reduces the weight loss caused by H9N2 infection. The weight loss is aggravated and the weight recovery time is prolonged in TFF2 gene knockout mice. FIG.7D (top) is a plot of percent survival vs. days post infection of mice infected with PR8 and mice infected with PR8 treated with mTFF2 supernatant. TFF2 protein also protects PR8 infected mice and reduces the lethality and weight loss caused by PR8 infection. [000350] FIG.8A shows the effect of TFF2 on H7N9 influenza virus replication in mice. FIG.8B shows the effect of TFF2 on H7N9 and N9N2 replication in vitro lung epithelial cell line A549. FIG.8C shows the effect of TFF2 addition on the microstructure of lung tissue in mice infected with H7N9 at 0 days, 1 day, 3 days, and 7 days post-infection. FIG.8D shows the effects of mTFF2 on the expression of inflammatory factors: tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) at 0 days, 1 day, 3 days and 7 days post-infection. FIG.8E shows the effect of TFF2 on lung tissue microstructure in an acute lung inflammation model induced by lipopolysaccharide (LPS): left, control; middle, LPS; right, LPS + mTFF-His. FIG. 8F shows the effect of TFF on expression of inflammatory factors TNF-α (left) and IL-6 (right). [000351] FIG.9A shows a 10% SDS gel stained with coomassie blue with samples from days 0, 3, 4, 7 and 10 after transfection of epiCHO cells with a TFF2-IFNκ-Fc construct and expression of the TFF2-IFNκFc in transfected CHO cell supernatants. FIG. 9B shows a Western blot; TFF2-IFNκ-Fc was detected by anti-Fc antibodies (primary antibody: mouse anti-human Fc monoclonal; secondary antibody: HRP-conjugated goat anti-mouse polyclonal). [000352] FIG. 10A shows a Western blot of resolved epiCHO cell lysates after transfection with a TFF2- IFNκ-Fc construct. TFF2-IFNκ-Fc was detected by anti-Fc antibodies (primary antibody: mouse anti-human Fc monoclonal; secondary antibody: HRP- conjugated goat anti-mouse polyclonal). The anti-Fc intensity was maximum at day 5, consistent with the maximum viable cell number in culture. The Fc positive species in the supernatants confirms secretion. [000353] FIG. 10B shows a Western blot of resolved epiCHO cell lysates after transfection with a GAPDH construct probed with rabbit anti-human anti-GAPDH antibodies (secondary antibody HRP-conjugated goat-anti-rabbit polyclonal antibodies). The anti- GAPDH antibody Western blot shows a prominent species at ~ 40 kD present in both transfected and control cell supernatants at days 0, 3, 5, 7 and 10, indicating that similar amounts of control and transfected cellular proteins are present in resolved samples at each time point. Note: less material is apparent at day 10 likely due to cellular degradation. [000354] FIG.11A shows a 10% SDS gel stained with coomassie blue containing elution fractions 1 through 9 from Protein A affinity chromatography of the expressed fusion product. The expected species has a molecular weight of about 70 kDa. Smaller Fc reactive species indicate proteolysis, which will be addressed in subsequent runs. [000355] FIG. 11B shows a Western blot of the resolved elution fractions 1 through 9 from Protein A affinity chromatography of the expressed fusion product detected by anti-Fc antibodies (primary antibody: mouse anti-human Fc monoclonal; secondary antibody: HRP- conjugated goat anti-mouse polyclonal), indicating a purification efficiency of less than 30.0% for this pilot run. DETAILED DESCRIPTION OF THE INVENTION Glossary [000356] The terms "active ingredient" ("AI", "active pharmaceutical ingredient", "API", or "bulk active") or “active agent” are used interchangeably to refer to the substance in a drug that is pharmaceutically active. As used herein, the phrase "additional active ingredient" refers to an agent, other than the recombinant fusion protein of the described composition, that exerts a pharmacological, or any other beneficial activity. [000357] The term "administer" as used herein means to give or to apply. The term "administering" as used herein includes in vivo administration, as well as administration directly to tissue ex vivo. According to some embodiments, the administering occurs nasally, by insufflation, intratracheally, orally, parenterally, intravenously, or intraperitoneally. [000358] The term “alveolar type II cells (AT2 cells) as used herein refers to the progenitors for alveolar type I cells. Alveolar type I cells cover 95 percent of the alveolar surface; they comprise the major gas exchange surface of the alveolus and are integral to the maintenance of the permeability barrier function of the alveolar membrane. AT2 cells are the only pulmonary cells that synthesize, store, and secrete all components of pulmonary surfactant important to regulate surface tension, preventing atelectasis and maintaining alveolar fluid balance within the alveolus. [000359] The terms “amino acid residue” or “amino acid” or “residue” are used interchangeably to refer to an amino acid that is incorporated into a protein, a polypeptide, or a peptide, including, but not limited to, a naturally occurring amino acid and known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids. The amino acids may be L- or D-amino acids. An amino acid may be replaced by a synthetic amino acid, which is altered so as to increase the half-life of the peptide, increase the potency of the peptide, or increase the bioavailability of the peptide. [000360] The single letter designation for amino acids is used predominately herein. As is well known by one of skill in the art, such single letter designations are as follows: A is alanine; C is cysteine; D is aspartic acid; E is glutamic acid; F is phenylalanine; G is glycine; H is histidine; I is isoleucine; K is lysine; L is leucine; M is methionine; N is asparagine; P is proline; Q is glutamine; R is arginine; S is serine; T is threonine; V is valine; W is tryptophan; and Y is tyrosine. [000361] The following represents groups of amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). [000362] The term “angiotensin-converting enzyme 2” or “ACE2” as used herein refers to a type 1 integral membrane glycoprotein [Tikellils, C. and Thomas MC. Intl J. Peptides (2012) 256294, citing Tipnis, SR et al. J. Biol. Chem. (2000) 275 (43): 33238-43] that is expressed and active in most tissues. The highest expression of ACE2 is observed in the kidney, the endothelium, the lungs, and in the heart [Id., citing Donoghue, M. et al. Cir. Res. (2000) 87 (5): E1-E9, Tipnis, SR et al. J. Biol. Chem. (2000) 275 (43): 33238-43]. The extracellular domain of ACE2 enzyme contains a single catalytic metallopeptidase unit that shares 42% sequence identity and 61% sequence similarity with the catalytic domain of ACE [[Id., citing Donoghue, M. et al. Cir. Res. (2000) 87 (5): E1-E9]. However, unlike ACE, it functions as a carboxypeptidase, rather than a dipeptidase, and ACE2 activity is not antagonized by conventional ACE inhibitors [Id., citing Rice, GI et al. Biochemical J. (2004) 383 (1): 45-51]. The major substrate for ACE2 appears to be (Ang II) [Id., citing Donoghue, M. et al. Circulation Res. (2000) 87 (5): E1-E9; turner, AJ and Hooper NM, Trends in Pharmacological Sci. (2002) 23 (4): 177-83; Rice, GI et al. Biochemical J. (2004) 383 (1): 45-51], although other peptides may also be degraded by ACE2, albeit at lower affinity. For example, ACE2 is able to cleave the C-terminal amino acid from angiotensin I, vasoactive bradykinin, des-Arg- kallidin (also known as des-Arg10 Lys-bradykinin), Apelin-13 and Apelin-36 [Id., citing Kuba, K. et al. Circulation Res. (2007) 101 (4): e32-e42] as well as other possible targets [Id., citing Vickers, C. et al. J. Biol. Chem. (2002) 277 (17): 14838-43]. The noncatalytic C-terminal domain of ACE2 shows 48% sequence identity with collectrin [Id., citing Zhang, H. et al. J. Biol. Chem. (2001) 276 (20): 17132-39], a protein shown to have an important role in neutral amino acid reabsorption from the intestine and the kidney [Id., citing Kowalczuk, S. et al. The FASEB J. (2008) 22 (8): 2880-87]; . the removed amino acid then becomes available for reabsorption. The cytoplasmic tail of ACE2 also contains calmodulin-binding sites [Id., citing DW Lambert, et al. FEBS Letters (2008) 582 (2): 385-90] which may influence shedding of its catalytic ectodomain. In addition, ACE2 has also been associated with integrin function, independent of its angiotensinase activity. [000363] As used herein, the term "antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies. Specifically, the term "antibody" includes polyclonal antibodies and monoclonal antibodies, and fragments thereof. Furthermore, the term "antibody" includes chimeric antibodies and wholly synthetic antibodies, and fragments thereof. [000364] Antibodies are serum proteins the molecules of which possess small areas of their surface that are complementary to small chemical groupings on their targets. These complementary regions (referred to as the antibody combining sites or antigen binding sites) of which there are at least two per antibody molecule, and in some types of antibody molecules ten, eight, or in some species as many as 12, may react with their corresponding complementary region on the antigen (the antigenic determinant or epitope) to link several molecules of multivalent antigen together to form a lattice. [000365] The basic structural unit of a whole antibody molecule consists of four polypeptide chains, two identical light (L) chains (each containing about 220 amino acids) and two identical heavy (H) chains (each usually containing about 440 amino acids). The two heavy chains and two light chains are held together by a combination of noncovalent and covalent (disulfide) bonds. The molecule is composed of two identical halves, each with an identical antigen-binding site composed of the N-terminal region of a light chain and the N- terminal region of a heavy chain. Both light and heavy chains usually cooperate to form the antigen binding surface. [000366] The principle of complementarity, which often is compared to the fitting of a key in a lock, involves relatively weak binding forces (hydrophobic and hydrogen bonds, van der Waals forces, and ionic interactions), which are able to act effectively only when the two reacting molecules can approach very closely to each other and indeed so closely that the projecting constituent atoms or groups of atoms of one molecule can fit into complementary depressions or recesses in the other. Antigen-antibody interactions show a high degree of specificity, which is manifest at many levels. Brought down to the molecular level, specificity means that the combining sites of antibodies to an antigen have a complementarity not at all similar to the antigenic determinants of an unrelated antigen. Whenever antigenic determinants of two different antigens have some structural similarity, some degree of fitting of one determinant into the combining site of some antibodies to the other may occur, and that this phenomenon gives rise to cross-reactions. Cross reactions are of major importance in understanding the complementarity or specificity of antigen-antibody reactions. Immunological specificity or complementarity makes possible the detection of small amounts of impurities/contaminations among antigens. [000367] An antibody may be an oligoclonal antibody, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a multi-specific antibody, a bi-specific antibody, a catalytic antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an anti-idiotypic antibody, and an antibody that can be labeled in soluble or bound form, as well as fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences provided by known techniques. Monoclonal antibodies (mAbs) can be generated by fusing mouse spleen cells from an immunized donor with a mouse myeloma cell line to yield established mouse hybridoma clones that grow in selective media. A hybridoma cell is an immortalized hybrid cell resulting from the in vitro fusion of an antibody-secreting B cell with a myeloma cell. In vitro immunization, which refers to primary activation of antigen-specific B cells in culture, is another well-established means of producing mouse monoclonal antibodies. Diverse libraries of immunoglobulin heavy (VH) and light (Vκ and Vλ) chain variable genes from peripheral blood lymphocytes also can be amplified by polymerase chain reaction (PCR) amplification. Genes encoding single polypeptide chains in which the heavy and light chain variable domains are linked by a polypeptide spacer (single chain Fv or scFv) can be made by randomly combining heavy and light chain V-genes using PCR. A combinatorial library then can be cloned for display on the surface of filamentous bacteriophage by fusion to a minor coat protein at the tip of the phage. The technique of guided selection is based on human immunoglobulin V gene shuffling with rodent immunoglobulin V genes. The method entails (i) shuffling a repertoire of human λ light chains with the heavy chain variable region (VH) domain of a mouse monoclonal antibody reactive with an antigen of interest; (ii) selecting half-human Fabs on that antigen (iii) using the selected λ light chain genes as “docking domains” for a library of human heavy chains in a second shuffle to isolate clone Fab fragments having human light chain genes; (v) transfecting mouse myeloma cells by electroporation with mammalian cell expression vectors containing the genes; and (vi) expressing the V genes of the Fab reactive with the antigen as a complete IgG1, λ antibody molecule in the mouse myeloma. An antibody may be from any species. The term antibody also includes binding fragments of the antibodies of the invention. Binding fragments of an antibody can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Exemplary fragments include Fv, Fab, Fab', single stranded antibody (svFC), dimeric variable region (Diabody) and di-sulphide stabilized variable region (dsFv). Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases. For example, computerized comparison methods can be used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. See, for example, Bowie et al. Science 253:164 (1991), which is incorporated by reference in its entirety. An antibody other than a "bispecific" or "bifunctional" antibody is understood to have each of its binding sites identical. [000368] The term “antibody construct” as used herein refers to a polypeptide comprising one or more of the antigen-binding portions of the invention linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen-binding portions. Such linker polypeptides are well known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123). An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art. Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques. [000369] The term “antigen” and its various grammatical forms refers to any substance that can stimulate the production of antibodies and can combine specifically with them. The term “antigenic determinant” or “epitope” as used herein refers to an antigenic site on a molecule. [000370] The term "attenuate" as used herein refers to render less virulent, to weaken or reduce in force, intensity, effect or quantity. [000371] The term “binding specificity” as used herein involves both binding to a specific partner and not binding to other molecules. Functionally important binding may occur at a range of affinities from low to high, and design elements may suppress undesired cross- interactions. Post-translational modifications also can alter the chemistry and structure of interactions. “Promiscuous binding” may involve degrees of structural plasticity, which may result in different subsets of residues being important for binding to different partners. “Relative binding specificity” is a characteristic whereby in a biochemical system a molecule interacts with its targets or partners differentially, thereby impacting them distinctively depending on the identity of individual targets or partners. [000372] The term “bioavailable” and its other grammatical forms as used herein refers to the ability of a substance to be absorbed and sued by the body. [000373] The term "biocompatible" as used herein refers to a material that is generally non-toxic to the recipient and does not possess any significant untoward effects to the subject and, further, that any metabolites or degradation products of the material are non-toxic to the subject. Typically a substance that is "biocompatible" causes no clinically relevant tissue irritation, injury, toxic reaction, or immunological reaction to living tissue. [000374] The term "biodegradable" as used herein refers to a material that will erode to soluble species or that will degrade under physiologic conditions to smaller units or chemical species that are, themselves, non-toxic. [000375] The term “CARD domain” as used herein refers to the family subclass of the caspase recruitment domain. The formation of apoptotic and inflammatory multiprotein complexes together with defined signaling episodes in innate immunity heavily relies on members of the death domain family and particularly on the family subclass of the caspase recruitment domain (CARD). [Palacios-Rodriguez, Y. et al., Polypeptide Modulators of Caspase Recruitment Domain (CARD)-Card-mediated protein-protein interactions. J. Biol. Chem. (2011) 286 (52): 44457-66, citing Varfolomeev, E. et al. Cell (2007) 131: 669-81] The interaction between the CARD of Apaf-1 (apoptotic protease-activating factor) and the CARD of procaspase-9 (PC9) in the mitochondria-mediated apoptotic intrinsic pathway is essential for the recruitment of PC9 into the apoptosome and its subsequent activation [Id., citing Acehan, D., et al. Mol. Cell (2002) 9: 423-32]. On the other hand, proteins like those of the NOD-like receptor (NLR) family (in particular NOD-1, NOD-2, and NLRP-1) act as intracellular scrutiny devices and signaling initiators to face microbial aggressions [Id., citing Proell, M. et al. PLoS One (2008) 3: e2119]. The NLR proteins utilize the CARD for binding to downstream signaling molecules through CARD-CARD interactions in order to ultimately initiate the innate immune and inflammatory responses [Id., citing Inohara N., Nuñez G. Nat. Rev. Immunol. (2003) 3, 371–382 ; Park, HH, et al. Annu. Rev. Immunol. (2007) 25, 561–586]. [000376] The term “carrier” as used herein describes a material that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the active compound of the composition of the described invention. Carriers must be of sufficiently high purity and of sufficiently low toxicity to render them suitable for administration to the mammal being treated. The carrier can be inert, or it can possess pharmaceutical benefits, cosmetic benefits or both. The terms "excipient", "carrier", or "vehicle" are used interchangeably to refer to carrier materials suitable for formulation and administration of pharmaceutically acceptable compositions described herein. Carriers and vehicles useful herein include any such materials know in the art which are nontoxic and do not interact with other components. The carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide for the desired bulk, consistency, etc., when combined with an active agent and other components of a given composition. [000377] The term “class switch recombination” or “CSR” as used herein refers to antibody class switching, which occurs in mature B cells in response to antigen stimulation and costimulatory signals. It occurs by a unique type of intrachromosomal deletional recombination within special G-rich tandem repeated DNA sequences [called switch, or S, regions located upstream of each of the heavy chain constant (CH) region genes, except Cδ]. The recombination is initiated by the B cell–specific activation-induced cytidine deaminase (AID), which deaminates cytosines in both the donor and acceptor S regions. AID activity converts several dC bases to dU bases in each S region, and the dU bases are then excised by the uracil DNA glycosylase UNG; the resulting basic sites are nicked by apurinic/apyrimidinic endonuclease (APE). AID attacks both strands of transcriptionally active S regions, but how transcription promotes AID targeting is not entirely clear. Mismatch repair proteins are then involved in converting the resulting single-strand DNA breaks to double-strand breaks with DNA ends appropriate for end-joining recombination. Proteins required for the subsequent S- S recombination include DNA-PK, ATM, Mre11-Rad50-Nbs1, γH2AX, 53BP1, Mdc1, and XRCC4-ligase IV. B cells undergo antibody, or Ig, class switching in vivo after immunization or infection or upon appropriate activation in culture. Engagement of the CD40 receptor on B cells by CD154 (CD40L) or, specifically for mouse B cells, the Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS), provides crucial signaling for CSR. AID expression is induced in mouse splenic B cells activated to switch in culture, and also in vivo, with especially high levels detected in germinal center (GC) B cells, which are undergoing SHM and probably CSR. CSR requires cell proliferation, appearing to require a minimum of two complete rounds of cell division for IgG and IgA CSR and perhaps additional rounds for IgE CSR. This requirement appears to be at least partly due to the requirements for induction of AID expression. Naive B cells have the potential to switch to any isotype, and cytokines secreted by T cells and other cells direct the isotype switch. At the time of publication, the predominant mechanism for regulating isotype specificity was by regulation of transcription through S regions, and only transcriptionally active S regions undergo CSR. [Tavnezer, J. et al., Annu Rev. Immunnol. (2008) 26: 261-92]. [000378] The term “costimulatory molecule” as used herein refers to molecules that are displayed on the cell surface that have a role in enhancing the activation of a T cell that is already being stimulated through its TCR. For example, HLA proteins, which present foreign antigen to the T cell receptor, require costimulatory proteins which bind to complementary receptors on the T cell’s surface to result in enhanced activation of the T cell. The term “co- stimulatory molecules” as used herein refers to highly active immunomodulatory proteins that play a critical role in the development and maintenance of an adaptive immune response (Kaufman and Wolchok eds., General Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)). The two signal hypothesis of T cell response involves the interaction between an antigen bound to an HLA molecule and with its cognate T cell receptor (TCR), and an interaction of a co-stimulatory molecule and its ligand. Specialized APCs, which are carriers of a co-stimulatory second signal, are able to activate T cell responses following binding of the HLA molecule with TCR. By contrast, somatic tissues do not express the second signal and thereby induce T cell unresponsiveness (Id.). Many of the co-stimulatory molecules involved in the two-signal model can be blocked by co-inhibitory molecules that are expressed by normal tissue (Id.). In fact, many types of interacting immunomodulatory molecules expressed on a wide variety of tissues may exert both stimulatory and inhibitory functions depending on the immunologic context (Id.). [000379] The term "cytokine" as used herein refers to small soluble protein substances secreted by cells which have a variety of effects on other cells. Cytokines mediate many important physiological functions including growth, development, wound healing, and the immune response. They act by binding to their cell-specific receptors located in the cell membrane, which allows a distinct signal transduction cascade to start in the cell, which eventually will lead to biochemical and phenotypic changes in target cells. Generally, cytokines act locally. They include type I cytokines, which encompass many of the interleukins, as well as several hematopoietic growth factors; type II cytokines, including the interferons and interleukin-10; tumor necrosis factor ("TNF")-related molecules, including TNFα and lymphotoxin; immunoglobulin super-family members, including interleukin 1 ("IL-1"); and the chemokines, a family of molecules that play a critical role in a wide variety of immune and inflammatory functions. The same cytokine can have different effects on a cell depending on the state of the cell. Cytokines often regulate the expression of, and trigger cascades of, other cytokines. [000380] The term “cytoprotective” as used herein refers to protecting cells from damage. [000381] The term “dry powder inhaler” or “DPI” as used herein refers to a device similar to a metered-dose inhaler, but where the drug is in powder form. The patient exhales out a full breath, places the lips around the mouthpiece, then quickly breathes in the powder. Dry powder inhalers do not require the timing and coordination that are necessary with MDIs. DPIs contain an active drug mixed with an excipient containing much larger particles (e.g., lactose) to which the drug attaches. During aerosolization, the active drug is stripped from the carrier and inhaled while the carrier particles impact on the mouth and throat and are ingested. DPIs synchronize drug delivery with inhalation. [000382] The term “dendritic cells (DC)” as used herein refers to professional antigen presenting cells, which induce naïve T cell activation and effector differentiation. [Patente, TA, et al., Frontiers Immunol. (2019) doi.org/10.3389/fimmu.2018.03176]. Human DC are identified by their high expression of major histocompatibility complex (MHC) class II molecules (MHC-II) and of CD11c, both of which are found on other cells, like lymphocytes, monocytes and macrophages [Id., citing Carlens J, et al. J Immunol. (2009) 183:5600–7; Drutman SB, et al. J Immunol. (2012) 188:3603–10; Hochweller K, Set al. Eur J Immunol. (2008) 38:2776–83; Huleatt JW, Lefrançois L. J Immunol. (1995) 154:5684–93; Rubtsov AV, et al. Blood (2011) 118:1305–15; Probst HC, et al. Clin Exp Immunol. (2005) 141:398–404; Vermaelen K, Pauwels R. Cytometry (2004) 61A:170–7]. DCs express many other molecules which allow their classification into various subtypes. Although some of the DC subtypes were originally described as macrophages, DC and macrophages have distinct characteristics [Id., citing Delamarre L, Science (2005) 307:1630–4; Geissmann F, et al. Science (2010) 327:656– 61; van Montfoort N, et al. Proc Natl Acad Sci USA. (2009) 106:6730–5] and ontogeny, so that, currently, little doubt remains that they belong to distinct lineages [Id., citing Haniffa M, et al. (2013) 120:1–49; Hashimoto D, et al. Immunity (2013) 38:792–804; Hettinger J, et al. Nat Immunol. (2013) 14:821–30; McGovern N, et al. Immunity (2014) 41:465–77; Naik SH, et al. Nature (2013) 496:229–32; Schulz C, et al. Science (2012) 336:86–90; Schraml BU, et al. Cell (2013) 154:843–58; Wang J, et al. Mol Med Rep. (2017) 16:6787–93; Yona S, et al. Immunity (2013) 38:79–91]. DC are found in two different functional states, “mature” and “immature”. These are distinguished by many features, but the ability to activate antigen- specific naïve T cells in secondary lymphoid organs is the hallmark of mature DC [Id., citing Hawiger D, Inaba K, et al. J Exp Med. (2001) 194:769–79; Steinman RM, et al. Ann NY Acad Sci. (2003) 987:15–25; Worbs T, et al. Nat Rev Immunol. (2017) 17:30–48]. DC maturation is triggered by tissue homeostasis disturbances, detected by the recognition of pathogen- associated molecular patterns (PAMP) or damage-associated molecular patterns (DAMPs) [Id., citing Hemmi H, et al. Chem Immunol Aller. (2005) 86:120–135, Cerboni S, et al. Adv Immunol. (2013) 120:211–237]. Maturation turns on metabolic, cellular, and gene transcription programs allowing DCs to migrate from peripheral tissues to T-dependent areas in secondary lymphoid organs, where T lymphocyte-activating antigen presentation may occur [Id., citing Alvarez D, et al. Immunity (2008) 29:325–42; Dong H, Bullock TNJ. Front Immunol. (2014) 5:24; Friedl P, Gunzer M. Trends Immunol. (2001) 22:187–91; Henderson RA, et al. J Immunol. (1997) 159:635–43; Randolph GJ, et al. Nature Rev Immunol. (2005) 5:617–28 Imai Y, et al. Histol Histopathol. (1998) 13:469–510]. During maturation, DC lose adhesive structures, reorganize the cytoskeleton and increase their motility [Id., citing Winzler C, et al. J Exp Med. (1997) 185:317–28). DC maturation also leads to a decrease in their endocytic activity but increased expression of MHC-II and co-stimulatory molecules [Id., citing Reis e Sousa C. Nature Rev Immunol. (2006) 6:476–83; Steinman RM. Annu Rev Immunol. (2012) 30:1–22; Trombetta ES, Mellman I. Annu Rev Immunol. (2005) 23:975–1028]. Mature DCs express higher levels of the chemokine receptor, CCR7 [Id., citing Förster R, et al. Cell (1999) 99:23– 33; Ohl L, et al. Immunity (2004) 21:279–88; Sallusto F, et al. Eur J Immunol. (1998) 28:2760– 9; Steinman RM. The control of immunity and tolerance by dendritic cell. Pathol Biol. (2003) 51:59–60] and secrete cytokines essential for T-cell activation [Id., citing Reis e Sousa C. Nature Rev Immunol. (2006) 6:476–83, Caux C, et al. J Exp Med. (1994) 180:1263–72; Jensen SS, Gad M. J Inflamm (Lond) (2010) 7:37; Tan JKH, O'Neill HC. J Leukocyte Biol. (2005) 78:319–324; Iwasaki A, Medzhitov R. Nat Immunol. (2015) 16:343–353]. Thus, the interaction between mature DCs and antigen-specific T cells is the trigger of antigen-specific immune responses [Id., citing Luft T,. Blood (2006) 107:4763–9, Jonuleit H. Arch Dermatol Res. (1996) 289:1–8]. When interacting with CD4+ T cells, DCs may induce their differentiation into different T helper (Th) subsets [Id., citing Iwasaki A, Medzhitov R. Nat Immunol. (2015) 16:343–353] such as Th1 [Amsen D, et al. Cell (2004) 117:515–26; Constant S, et al. J Exp Med (1995) 182:1591–6; Hosken NA, et al. J Exp Med. (1995) 182:1579–84; Kadowaki N. Allergol Int. (2007) 56:193–9; Maekawa Y, et al. Immunity (2003) 19:549–59; Pulendran B, et al. Proc Natl Acad Sci USA. (1999) 96:1036–41, Th2 [Id., citing Constant S, et al. J Exp Med (1995) 182:1591–6, Hosken NA, et al. J Exp Med. (1995) 182:1579–84, Jenkins SJ, P. et al. J Immunol. (2007) 179:3515–23, Soumelis V, et al. Nat Immunol. (2002) 3:673–680], Th17 [Id., citing Bailey SL, Nat Immunol. (2007) 8:172–80; Iezzi G, et al. Proc Natl Acad Sci USA. (2009) 106:876–81; Huang G, et al. Cell Mol Immunol. (2012) 9:287–95], or other CD4+ T cell subtypes [Id., citing Levings MK, et al. Blood (2005) 105:1162–9]. T cell differentiation in each subtype is a complex phenomenon, that can be influenced by the cytokines in the DC tissue of origin [Id., citing Rescigno M. Dendritic cell-epithelial cell crosstalk in the gut. Immunol Rev. (2014) 260:118–28], their maturation state [Id., citing Reis e Sousa C. Nature Rev Immunol. (2006) 6:476–83] and cause of tissue imbalance [Id., citing Vega-Ramos J, et al. Curr Opin Pharmacol. (2014) 17:64–70]. DCs present a unique characteristic: the ability to perform cross-presentation [Id., citing Coulon P-G, et al. J Immunol. (2016) 197:517–32; Delamarre L, Mellman I. Semin Immunol. (2011) 23:2–11; Jung S, et al. Immunity (2002) 17:211–20; Segura E, Amigorena S. Adv Immunol. (2015) 127:1–31; Segura E, Villadangos JA. Curr Opin Immunol. (2009) 21:105–110], defined as the presentation, in the context of class I MHC molecules (MHC-I), of antigens captured from the extracellular milieu. This feature allows DC to trigger responses against intracellular antigens from other cell types, thus providing means for the system to deal with threats that avoid professional APC [Id., citing Coulon P-G, et al. J Immunol. (2016) 197:517–32, Bevan MJ. Cross-priming for a secondary cytotoxic response to minor H antigens with H-2 congenic cells which do not cross-react in the cytotoxic assay. J Exp Med. (1976) 143:1283–8, Sánchez-Paulete AR, et al. Ann Oncol. (2017) 28:xii74. doi: 10.1093/annonc/mdx727] and, even, to prime CD8+ lymphocytes in the absence of CD4+ T cells [Id., citing McCoy KD, et al. J Exp Med. (1999) 189:1157–62, Young JW, Steinman RM. J Exp Med. (1990) 171:1315–32]. Cross-presentation is involved also in the induction of tolerance to intracellular self-antigens that are not expressed by APC and, then, called, cross-tolerance [Kurts C, et al. J Exp Med. (1997) 186:239–45, Rock KL, Shen L. Immunol Rev. (2005) 207:166–83]. [000383] Before receiving maturation stimuli, DC are said to be in an “immature state.” Immature DC are poor inducers of naïve lymphocyte effector responses, since they have low surface expression of co-stimulatory molecules, low expression of chemokine receptors, and do not release immunostimulatory cytokines [Id., citing Trombetta ES, Mellman I. Annu Rev Immunol. (2005) 23:975–1028, Steinman RM, Swanson J. J Exp Med. (1995) 182:283–8]. These “immature” cells, though, are very efficient in antigen capture due to their high endocytic capacity, via receptor-mediated endocytosis, including lectin- [Id., citing Geijtenbeek TB, et al. Cell (2000) 100:575–585; Sallusto F, et al. J Exp Med. (1995) 182:389–400; Valladeau J, et al. Cell Immunol. (1994) 159:323–30; Medzhitov R, et al. Nature (1997) 388:394–7; Muzio M, et al. J Immunol. (2000) 164:5998–6004], FC- and complement receptors [Id., citing Muzio M, et al. J Immunol. (2000) 164:5998–6004) and macropinocytosis (Id., citing Sallusto F, et al. J Exp Med. (1995) 182:389–400). Thus, immature DCs act not only as sentinels against invading pathogens [Id., citing Worbs T, et al. Nat Rev Immunol. (2017) 17:30–48, Wilson NS, et al. Blood (2004) 103:2187–95], but also as tissue scavengers, capturing apoptotic and necrotic cells [Id., citing Albert ML, et al. Nature (1998) 392:86–9). [000384] This latter feature confers to immature DC an essential role in the induction and maintenance of immune tolerance [Id., citing Steinman RM, et al. Ann NY Acad Sci. (2003) 987:15–25, Castellano G, et al. Mol Immunol. (2004) 41:133–40; Deluce-Kakwata-Nkor N, et al. Transfus Clin Biol. (2018) 25:90–5; Liu J, Cao X. J Autoimmun. (2015) 63:1–12; Shiokawa A, et al. Immunology (2017) 152:52–64]. Apoptotic cells that arise in consequence of natural tissue turnover [Id., citing Huang FP, et al. J Exp Med. (2000) 191:435–44, Steinman RM, et al. J Exp Med. (2000) 191:411–416] are internalized by DCs but do not induce their maturation [Id., citing Steinman RM, et al. Ann NY Acad Sci. (2003) 987:15–25, Liu K, et al. J Exp Med. (2002) 196:1091–1097; Stuart LM, et al. J Immunol. (2002) 168:1627–35; Wallet MA, et al. J Exper Med. (2008) 205:219–32]. Thus, their antigens are presented to T cells without the activating co-stimulatory signals that a mature DC would deliver, resulting in T cell apoptosis [Id., citing Kurts C, et al. J Exp Med. (1997) 186:239–45, Hong J, et al. Chin Med J. (2013) 126:2139–44], anergy [Id., citing Manicassamy S, Pulendran B. Immunol Rev. (2011) 241:206–27, Zhu H-C, et al. Cell Immunol. (2012) 274:12–8] or development into Tregs [Id., citing Saito M, et al. J Exper Med. (2011) 208:235–49, Sela U, et al. PLoS ONE (2016) 11:e0146412). [000385] These “tolerogenic DC” express less co-stimulatory molecules and proinflammatory cytokines, but upregulate the expression of inhibitory molecules (like PD-L1 and CTLA-4), secrete anti-inflammatory cytokines (IL-10, for example) [Id., citing Manicassamy S, Pulendran B. Immunol Rev. (2011) 241:206–27, Grohmann U, et al. Nat Immunol. (2002) 3:1097–101; Morelli AE, Thomson AW. Nature Rev Immunol. (2007) 7:610–21; Sakaguchi S, et al. Nat Rev Immunol. (2010) 10:490–500] and are essential to prevent responses against healthy tissues [Id., citing Hawiger D. J Exp Med. (2001) 194:769– 79, Steinman RM, et al. Ann NY Acad Sci. (2003) 987:15–25, Idoyaga J, et al. J Clin Invest. (2013) 123:844–54; Mahnke K, et al. Blood (2003) 101:4862–9; Yates SF, et al. J Immunol (2007) 179:967–76; Yogev N, et al. Immunity (2012) 37:264–75]. [000386] However, in some contexts, immature DC can be harmful to the body. It is known that DCs that are unable to induce lymphocyte effector responses may contribute to the immune system's failure to fight infections [Id., citing Campanelli AP, et al. J Infect Dis. (2006) 193:1313–22, Montagnoli C, et al. J Immunol. (2002) 169:6298–308] or tumors [Id., citing Baleeiro RB, et al. Cancer Immunol Immunother (2008) 57:1335–45; Almand B, et al. Clin Cancer Res. (2000) 6:1755–66; Bella SD, et al. Br J Cancer (2003) 89:1463–72; Dunn GP, et al. Immunity (2004) 21:137–48; Johnson DJ, Ohashi PS. Anna NY Acad Sci. (2013) 1284:46– 51; Vicari AP, et al. Semin Cancer Biol. (2002) 12:33–42]. In these situations, DC, even after recognition of pathogens or other changes in microenvironment, fail to increase the co- stimulatory molecules required to activate T cells, thus allowing the disease to “escape” immune control. [000387] The terms “D value” or “mass division diameter” as used herein, refer to the diameter which, when all particles in a sample are arranged in order of ascending mass, divides the sample's mass into specified percentages. The percentage mass below the diameter of interest is the number expressed after the "D". For example, the D10 diameter is the diameter at which 10% of a sample's mass is comprised of smaller particles, and the D50 is the diameter at which 50% of a sample's mass is comprised of smaller particles. The D50 is also known as the "mass median diameter" as it divides the sample equally by mass. While D-values are based on a division of the mass of a sample by diameter, the actual mass of the particles or the sample does not need to be known. A relative mass is sufficient as D-values are concerned only with a ratio of masses. This allows optical measurement systems to be used without any need for sample weighing. [000388] From the diameter values obtained for each particle a relative mass can be assigned according to the following relationship: Mass of a sphere= π/6 d3 ρ [000389] Assuming that ρ is constant for all particles and cancelling all constants from the equation: Relative mass= d3 each particle's diameter is therefore cubed to give its relative mass. These values can be summed to calculate the total relative mass of the sample measured. The values may then be arranged in ascending order and added iteratively until the total reaches 10%, 50% or 90% of the total relative mass of the sample. The corresponding D value for each of these is the diameter of the last particle added to reach the required mass percentage. [000390] The term “DAD” as used herein refers to diffuse alveolar damage (DAD), which is manifested by injury to alveolar lining and endothelial cells, pulmonary edema, hyaline membrane formation and later by proliferative changes involving alveolar and bronchiolar lining cells and interstitial cells (Katzenstein, AL et al. Am J Pathol (1976) 85:209). [000391] The term "delayed release" is used herein in its conventional sense to refer to a drug formulation in which there is a time delay between administration of the formulation and the release of the drug therefrom. "Delayed release" may or may not involve gradual release of drug over an extended time, and thus may or may not be "sustained release.” [000392] The term “dispersion”, as used herein, refers to a two-phase system, in which one phase is distributed as droplets in the second, or continuous phase. In these systems, the dispersed phase frequently is referred to as the discontinuous or internal phase, and the continuous phase is called the external phase and comprises a continuous process medium. For example, in course dispersions, the particle size is 0.5 µm. In colloidal dispersions, size of the dispersed particle is in the range of approximately 1 nm to 0.5 µm. A molecular dispersion is a dispersion in which the dispersed phase consists of individual molecules; if the molecules are less than colloidal size, the result is a true solution. [000393] The term “drug load” as used herein refers to the amount of drug encapsulated and to be released by a particle. [000394] The term “ECOG performance status scale” as used herein refers to a scale used to assess how a patient's disease is progressing, assess how the disease affects the daily living abilities of the patient, and determine appropriate treatment and prognosis. The terms “encapsulated” and “microencapsulated” are used herein to refer generally to a bioactive agent that is incorporated into any sort of long-acting formulation or technology regardless of shape or design; therefore, an “encapsulated” or “microencapsulated” bioactive agent may include bioactive agents that are incorporated into a particle or a microparticle. [000395] The term “entrapped” as used herein means to catch, hold, capture, enmesh, entangle, ensnare, snare, or trap. [000396] The term “fusion protein” as used herein refers to a peptide, polypeptide or protein constructed by combining multiple protein domains or polypeptides for the purpose of creating a single peptide or protein with functional properties derived from each of the original proteins or polypeptides. Creation of a fusion protein may be accomplished by operatively ligating or linking two different nucleotides sequences that encode each protein domain or polypeptide via recombinant DNA technology, thereby creating a new polynucleotide sequences that codes for the desired fusion peptide or protein. Alternatively, a fusion peptide may be created by chemically joining the desired protein domains. [000397] The term “GALTs” as used herein refers to gut-associated lymphoid tissues, which are part of the mucosa-associated lymphoid tissues (MALTs). The histological components of GALTs mainly includes Peyer’s patches, crypt patches, isolated lymphoid follicles (ILFs) appendix and mesenteric lymph nodes (mLNs). [Jiao, Y. et al., Crosstalk between gut microbiota and innate immunity and its implication in autoimmune disease. Front. Immunol. (2020) 11: 282; citing Brandtzaeg, P. et al. Terminology: nomenclature of mucosa- associated lymphoid tissue. Mucosal Immunol. (2008) 1: 31; Mowat, AM. Anatomical basis of tolerance and immunity to intestinal antigens. Nat. Rev. Immunol. (2003) 3: 331-41] constituent cells of GALTs include microfold (M) cells, which are capable of transferring antigens but not processing or presenting them [Id., citing Mabbott, NA et al. Microfold (M) cells: important immunosurveillance posts in the intestinal epithelium, Mucosal Immunol. (2013) 6:666]. Conventional lymphocytes, such as helper T cells (Th cells) (Id., citing Dunkley, M., Husband, A. Distribution and functional characteristics of antigen-specific helper T cells arising after Peyer’s patch immunization. J. Immunol. (1987) 61: 475; Kiyono, H. e al. Murine Peyer’s patch T cell clones. Characterization of antigen-specific helper T cells for immunoglobulin A responses. J. Exp. Med. (1982) 156: 1115-30]; Tregs (Id., citing Coombes, JL., et al. A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGFβ-and-retinoid acid-dependent mechanism. J. Exp. Med. (2007) 204: 1757-64; Siddiqui, K., Powrie, F. CD103+ GALT DCs promote Foxp3+ regulatory T cells. Mucosal Immunol. (2008) 1 (Suppl. 1): 534-8), cytotoxic T lymphocytes (Id. citing Nelson, DL et al. Cytotoxic effector cell function in organized gut-associated lymphoid tissue (GALT). Cell Immunol. (1976) 22: 65-75), IgA producing B cells (Id., citing Mora, JR et al. Generation of gut-homing IgA-secreting B cells by intestinal dendritic cells. Science (2006) 314: 1157-60), phagocytes, including dendritic cells (Id., citing Coombes, JL., et al. A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGFβ-and-retinoid acid-dependent mechanism. J. Exp. Med. (2007) 204: 1757-64; Siddiqui, K., Powrie, F. CD103+ GALT DCs promote Foxp3+ regulatory T cells. Mucosal Immunol. (2008) 1 (Suppl.1): 534-8), macrophages, and other nonconventional lymphocytes, such as innate lymphoid cells (ILCs) (Id., citing Pearson, C. et al. Lymphoid microenvironments and innate lymphoid cells in the gut. Trends Immunol. (2012) 33: 289-96; Wojno, EDT; Artis, D. Innate lymphoid cells: balancing immunity, inflammation and tissue repair in the intestine. Cell Host Microbe (2012) 12: 445-57). The gut microbiota shapes the structural development of GALTs and primes its immune response to initiate host defense and to maintain tolerance against commensal bacteria via PRR-PAMP recognition and epigenetic modulators like short-chain fatty acids (SCFAs). [Id.] [000398] The terms “GATA-3” and “GATA binding protein 3” are used interchangeably to refer to a member of the GATA family of conserved zinc-finger transcription factors, several of which are involved in hematopoiesis. GATA-3 is highly expressed in T cells and a wide variety of other tissues, including the CNS and fetal liver. In T cells, Gata3 acts at multiple stages of thymocyte differentiation. It is indispensable for early thymic progenitor differentiation [Hosoya, T. et al., J Exp Med.2009206(13):2987-3000] and for thymocytes to pass through beta selection and T cell commitment. Gata3 is also necessary for single-positive CD4 thymocyte development as well as for Th1-Th2 lineage commitment [Ting, CN et al., Nature. (1996) 384(6608):474-8; Zhang, DH et al., J Biol Chem. (1997) 272(34):21597-603; Zheng W, Flavell RA. Cell. (1997) 89(4):587-96; Zhang, DH et al., J Immunol. (1998) 161(8):3817-21; Pai, SY et al. Immunity (2003) 19(6):863-753]. As master regulator of Th2 lineage commitment, GATA3 acts either as a transcriptional activator or repressor through direct action at many critical loci encoding cytokines, cytokine receptors, signaling molecules as well as transcription factors that are involved in the regulation of T(h)1 and T(h)2 differentiation [Jenner, RG et al., Proc Natl Acad Sci U S A. (2009) 106(42):17876-81]. For example, it regulates the expression of Th2 lineage specific cytokine gene such as IL5 and represses the Th1 lineage specific genes IL-12 receptor β2 and STAT4 as well as neutralizing RUNX3 function through protein-protein interaction. Mice lacking Gata3 produce IFN-gamma rather than Th2 cytokines (IL5 and IL13) in response to infection [Zhu, J et al., Nat Immunol. (2004)5(11):1157-65]. It acts in mutual opposition to the transcription factor T-bet, as T-bet promotes whereas GATA3 represses Fut7 transcription [Hwang, ES et al., Science. (2005) 21;307(5708):430-3]. It also acts with Tbx21 to regulate cell lineage-specific expression of lymphocyte homing receptors and cytokine in both Th1 and Th2 lymphocyte subsets [Chen, GY et al., Proc Natl Acad Sci U S A. (2006) 103(45):16894-9]. Enforced expression of Gata3 during T cell development induced CD4(+)CD8(+) double-positive (DP) T cell lymphoma [Nawijn, MC et al., J Immunol. (2001)167(2):724-32a; Nawijn, MC et al., J Immunol. (2001)167(2):715-23]. [000399] Gata3 is essential for the expression of the cytokines IL-4, IL-5 and IL-13 that mediate allergic inflammation. Gata3 overexpression causes enhanced allergen-induced airway inflammation and airway remodeling, including subepithelial fibrosis, and smooth muscle cell hyperplasia [Kiwamoto, T et al., Am J Respir Crit Care Med. (2006) 174(2):142-51]. It additionally has a critical function in regulatory T cells and immune tolerance since deletion of Gata3 specifically in regulatory T cells led to a spontaneous inflammatory disorder in mice [Wang, Y et al., Immunity (2011) 35(3):337-48]. [000400] The term “IL-4Rα” as used herein refers to the cytokine-binding receptor chain for IL-4. [000401] The terms “immune response” and “immune-mediated” are used interchangeably herein to refer to any functional expression of a subject’s immune system, against either foreign or self-antigens, whether the consequences of these reactions are beneficial or harmful to the subject. [000402] The term “immune system” as used herein refers to a complex network of cells, tissues, organs, and the substances they make that helps the body fight infections and other diseases. The immune system includes white blood cells and organs and tissues of the lymph system, such as the thymus, spleen, tonsils, lymph nodes, lymph vessels, and bone marrow. [000403] The term “immunocompromised” as used herein refers to having a weakened immune system and a reduced ability to fight infections and other diseases. Immunocompromised subjects include patients receiving long-term (>3 months) or high-dose (>0.5 mg/kg/day) steroids or other immunosuppressant drugs, solid-organ transplant recipients, patients with a solid tumor requiring chemotherapy in the last 5 years or with a hematological malignancy whatever the time since diagnosis and who received treatments, and patients with primary immune deficiency. [000404] The term “immunogen” and its various grammatical forms as used herein refers to a substance that elicits an immune response. [000405] The terms “immunomodulatory”, “immune modulator”, “immunomodulatory,” and “immune modulatory” are used interchangeably herein to refer to a substance, agent, or cell that is capable of augmenting or diminishing immune responses directly or indirectly, e.g., by expressing chemokines, cytokines and other mediators of immune responses. [000406] As used herein, the term “immunostimulatory amount” refers to an amount of an immunogenic composition that stimulates an immune response by a measurable amount, for example, as measured by ELISPOT assay (cellular immune response), ICS (intracellular cytokine staining assay) and major histocompatibility complex (MHC) tetramer assay. [000407] As used herein the term “immunosuppressive amount” refers to an amount of an immunosuppressive composition that suppresses an immune response, for example, as measured by ELISPOT assay (cellular immune response), ICS (intracellular cytokine staining assay) and major histocompatibility complex (MHC) tetramer assay. [000408] The term “inflammation” as used herein refers to the physiologic process by which vascularized tissues respond to injury. See, e.g., FUNDAMENTAL IMMUNOLOGY, 4th Ed., William E. Paul, ed. Lippincott-Raven Publishers, Philadelphia (1999) at 1051-1053, incorporated herein by reference. During the inflammatory process, cells involved in detoxification and repair are mobilized to the compromised site by inflammatory mediators. Inflammation is often characterized by a strong infiltration of leukocytes at the site of inflammation, particularly neutrophils (polymorphonuclear cells). These cells promote tissue damage by releasing toxic substances at the vascular wall or in uninjured tissue. Traditionally, inflammation has been divided into acute and chronic responses. [000409] The term “acute inflammation” as used herein refers to the rapid, short-lived (minutes to days), relatively uniform response to acute injury characterized by accumulations of fluid, plasma proteins, and neutrophilic leukocytes. Examples of injurious agents that cause acute inflammation include, but are not limited to, pathogens (e.g., bacteria, viruses, parasites), foreign bodies from exogenous (e.g. asbestos) or endogenous (e.g., urate crystals, immune complexes), sources, and physical (e.g., burns) or chemical (e.g., caustics) agents. [000410] The term “chronic inflammation” as used herein refers to inflammation that is of longer duration and which has a vague and indefinite termination. Chronic inflammation takes over when acute inflammation persists, either through incomplete clearance of the initial inflammatory agent or as a result of multiple acute events occurring in the same location. Chronic inflammation, which includes the influx of lymphocytes and macrophages and fibroblast growth, may result in tissue scarring at sites of prolonged or repeated inflammatory activity. [000411] The term “inflammatory mediators” or “inflammatory cytokines” as used herein refers to the molecular mediators of the inflammatory process. These soluble, diffusible molecules act both locally at the site of tissue damage and infection and at more distant sites. Some inflammatory mediators are activated by the inflammatory process, while others are synthesized and/or released from cellular sources in response to acute inflammation or by other soluble inflammatory mediators. Examples of inflammatory mediators of the inflammatory response include, but are not limited to, plasma proteases, complement, kinins, clotting and fibrinolytic proteins, lipid mediators, prostaglandins, leukotrienes, platelet-activating factor (PAF), peptides and amines, including, but not limited to, histamine, serotonin, and neuropeptides, and proinflammatory cytokines, including, but not limited to, interleukin-1-beta (IL-1β), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor- alpha (TNF-α), interferon-gamma (IF-γ), and interleukin-12 (IL-12). Among the pro- inflammatory mediators, IL-1, IL-6, and TNF-α are known to activate hepatocytes in an acute phase response to synthesize acute-phase proteins that activate complement. [000412] The term “inhalation” as used herein refers to the act of drawing in a medicated vapor with the breath. [000413] The term “inhalation delivery device” as used herein refers to a machine/apparatus or component that produces small droplets or an aerosol from a liquid or dry powder aerosol formulation and is used for administration through the mouth in order to achieve pulmonary administration of a drug, e.g., in solution, powder, and the like. Examples of inhalation delivery device include, but are not limited to, a nebulizer, a metered-dose inhaler, and a dry powder inhaler (DPI). [000414] The terms “inhibiting”, “inhibit” or “inhibition” are used herein to refer to reducing the amount or rate of a process, to stopping the process entirely, or to decreasing, limiting, or blocking the action or function thereof. Inhibition may include a reduction or decrease of the amount, rate, action function, or process of a substance by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%. The term “injury” as used herein refers to damage or harm to a structure or function of the body caused by an outside agent or force, which may be physical or chemical. [000415] The term “insufflation” as used herein refers to the act of delivering air, a gas, or a powder under pressure to a cavity or chamber of the body. For example, nasal insufflation relates to the act of delivering air, a gas, or a powder under pressure through the nose. [000416] The term “interleukin (IL)” as used herein refers to a cytokine secreted by, and acting on, leukocytes. Interleukins regulate cell growth, differentiation, and motility, and stimulates immune responses, such as inflammation. Examples of interleukins include, interleukin-1 (IL-1), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-12 (IL-12). [000417] The term “isolated” is used herein to refer to material, such as, but not limited to, a nucleic acid, peptide, polypeptide, or protein, which is: (1) substantially or essentially free from components that normally accompany or interact with it as found in its naturally occurring environment. The terms “substantially free” or “essentially free” are used herein to refer to considerably or significantly free of, or more than about 95% free of, or more than about 99% free of such components. The isolated material optionally comprises material not found with the material in its natural environment; or (2) if the material is in its natural environment, the material has been synthetically (non-naturally) altered by deliberate human intervention to a composition and/or placed at a location in the cell (e.g., genome or subcellular organelle) not native to a material found in that environment. [000418] The term “lectin” as used herein refers to a class of proteins that bind specifically to certain sugars and so cause agglutination of particular cell types. [000419] The term “LINGO” as used herein refers to the “leucine-rich repeat and immunoglobulin-like domain-containing NoGo” family of proteins. [000420] The term “lymphocyte” refers to a small white blood cell formed in lymphatic tissue throughout the body and in normal adults making up about 22-28% of the total number of leukocytes in the circulating blood that plays a large role in defending the body against disease. Individual lymphocytes are specialized in that they are committed to respond to a limited set of structurally related antigens. This commitment, which exists before the first contact of the immune system with a given antigen, is expressed by the presence on the lymphocyte’s surface membrane of receptors specific for determinants (epitopes) on the antigen. Each lymphocyte possesses a population of receptors, all of which have identical combining sites. One set, or clone, of lymphocytes differs from another clone in the structure of the combining region of its receptors and thus differs in the epitopes that it can recognize. Lymphocytes differ from each other not only in the specificity of their receptors, but also in their functions. [000421] Two broad classes of lymphocytes are recognized: the B-lymphocytes (B-cells), which are precursors of antibody-secreting cells, and T-lymphocytes (T-cells), B-lymphocytes [000422] B-lymphocytes are derived from hematopoietic cells of the bone marrow. A mature B-cell can be activated with an antigen that expresses epitopes that are recognized by its cell surface. The activation process may be direct, dependent on cross-linkage of membrane Ig molecules by the antigen (cross-linkage-dependent B-cell activation), or indirect, via interaction with a helper T-cell, in a process referred to as cognate help. In many physiological situations, receptor cross-linkage stimuli and cognate help synergize to yield more vigorous B- cell responses. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)). [000423] Cross-linkage dependent B-cell activation requires that the antigen express multiple copies of the epitope complementary to the binding site of the cell surface receptors because each B-cell expresses Ig molecules with identical variable regions. Such a requirement is fulfilled by other antigens with repetitive epitopes, such as capsular polysaccharides of microorganisms or viral envelope proteins. Cross-linkage-dependent B-cell activation is a major protective immune response mounted against these microbes. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)). [000424] Cognate help allows B-cells to mount responses against antigens that cannot cross-link receptors and, at the same time, provides costimulatory signals that rescue B cells from inactivation when they are stimulated by weak cross-linkage events. Cognate help is dependent on the binding of antigen by the B-cell’s membrane immunoglobulin (Ig), the endocytosis of the antigen, and its fragmentation into peptides within the endosomal/lysosomal compartment of the cell. Some of the resultant peptides are loaded into a groove in a specialized set of cell surface proteins known as class II major histocompatibility complex (MHC) molecules. The resultant class II/peptide complexes are expressed on the cell surface and act as ligands for the antigen-specific receptors of a set of T-cells designated as CD4+ T- cells. The CD4+ T-cells bear receptors on their surface specific for the B-cell’s class II/peptide complex. B-cell activation depends not only on the binding of the T cell through its T cell receptor (TCR), but this interaction also allows an activation ligand on the T-cell (CD40 ligand) to bind to its receptor on the B-cell (CD40) signaling B-cell activation. In addition, T helper cells secrete several cytokines that regulate the growth and differentiation of the stimulated B- cell by binding to cytokine receptors on the B cell. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippincott- Raven Publishers, Philadelphia (1999)). [000425] During cognate help for antibody production, the CD40 ligand is transiently expressed on activated CD4+ T helper cells, and it binds to CD40 on the antigen-specific B cells, thereby transducing a second costimulatory signal. The latter signal is essential for B cell growth and differentiation and for the generation of memory B cells by preventing apoptosis of germinal center B cells that have encountered antigen. Hyperexpression of the CD40 ligand in both B and T cells is implicated in the pathogenic autoantibody production in human SLE patients. (Desai-Mehta, A. et al., “Hyperexpression of CD40 ligand by B and T cells in human lupus and its role in pathogenic autoantibody production,” J. Clin. Invest., 97(9): 2063-2073 (1996)). T-lymphocytes [000426] T-lymphocytes derive from precursors in hematopoietic tissue, undergo differentiation in the thymus, and are then seeded to peripheral lymphoid tissue and to the recirculating pool of lymphocytes. T-lymphocytes or T cells mediate a wide range of immunologic functions. These include the capacity to help B cells develop into antibody- producing cells, the capacity to increase the microbicidal action of monocytes/macrophages, the inhibition of certain types of immune responses, direct killing of target cells, and mobilization of the inflammatory response. These effects depend on their expression of specific cell surface molecules and the secretion of cytokines. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)). [000427] T cells differ from B cells in their mechanism of antigen recognition. Immunoglobulin, the B cell’s receptor, binds to individual epitopes on soluble molecules or on particulate surfaces. B-cell receptors see epitopes expressed on the surface of native molecules. Antibody and B-cell receptors evolved to bind to and to protect against microorganisms in extracellular fluids. In contrast, T cells recognize antigens on the surface of other cells and mediate their functions by interacting with, and altering, the behavior of these antigen- presenting cells (APCs). There are three main types of antigen-presenting cells in peripheral lymphoid organs that can activate T cells: dendritic cells, macrophages and B cells. The most potent of these are the dendritic cells, whose only function is to present foreign antigens to T cells. Immature dendritic cells are located in tissues throughout the body, including the skin, gut, and respiratory tract. When they encounter invading microbes at these sites, they endocytose the pathogens and their products, and carry them via the lymph to local lymph nodes or gut associated lymphoid organs. The encounter with a pathogen induces the dendritic cell to mature from an antigen-capturing cell to an antigen-presenting cell (APC) that can activate T cells. APCs display three types of protein molecules on their surface that have a role in activating a T cell to become an effector cell: (1) MHC proteins, which present foreign antigen to the T cell receptor; (2) costimulatory proteins which bind to complementary receptors on the T cell surface; and (3) cell-cell adhesion molecules, which enable a T cell to bind to the antigen-presenting cell (APC) for long enough to become activated. (“Chapter 24: The adaptive immune system,” Molecular Biology of the Cell, Alberts, B. et al., Garland Science, NY, 2002). [000428] T-cells are subdivided into two distinct classes based on the cell surface receptors they express. The majority of T cells express T cell receptors (TCR) consisting of α and β chains. A small group of T cells express receptors made of γ and δ chains. Among the α/β T cells are two important sublineages: those that express the coreceptor molecule CD4 (CD4+ T cells); and those that express CD8 (CD8+ T cells). These cells differ in how they recognize antigen and in their effector and regulatory functions. [000429] CD4+ T cells are the major regulatory cells of the immune system. Their regulatory function depends both on the expression of their cell-surface molecules, such as CD40 ligand whose expression is induced when the T cells are activated, and the wide array of cytokines they secrete when activated. [000430] T cells also mediate important effector functions, some of which are determined by the patterns of cytokines they secrete. The cytokines can be directly toxic to target cells and can mobilize potent inflammatory mechanisms. [000431] In addition, T cells particularly CD8+ T cells, can develop into cytotoxic T- lymphocytes (CTLs) capable of efficiently lysing target cells that express antigens recognized by the CTLs. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)). [000432] T cell receptors (TCRs) recognize a complex consisting of a peptide derived by proteolysis of the antigen bound to a specialized groove of a class II or class I MHC protein. The CD4+ T cells recognize only peptide/class II complexes while the CD8+ T cells recognize peptide/class I complexes. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)). [000433] The TCR’s ligand (i.e., the peptide/MHC protein complex) is created within antigen-presenting cells (APCs). In general, class II MHC molecules bind peptides derived from proteins that have been taken up by the APC through an endocytic process. These peptide-loaded class II molecules are then expressed on the surface of the cell, where they are available to be bound by CD4+ T cells with TCRs capable of recognizing the expressed cell surface complex. Thus, CD4+ T cells are specialized to react with antigens derived from extracellular sources. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)). [000434] In contrast, class I MHC molecules are mainly loaded with peptides derived from internally synthesized proteins, such as viral proteins. These peptides are produced from cytosolic proteins by proteolysis by the proteosome and are translocated into the rough endoplasmic reticulum. Such peptides, generally nine amino acids in length, are bound into the class I MHC molecules and are brought to the cell surface, where they can be recognized by CD8+ T cells expressing appropriate receptors. This gives the T cell system, particularly CD8+ T cells, the ability to detect cells expressing proteins that are different from, or produced in much larger amounts than, those of cells of the remainder of the organism (e.g., vial antigens) or mutant antigens (such as active oncogene products), even if these proteins in their intact form are neither expressed on the cell surface nor secreted. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)). [000435] T cells can also be classified based on their function as helper T cells; T cells involved in inducing cellular immunity; suppressor T cells; and cytotoxic T cells. Helper T cells [000436] Helper T cells are T cells that stimulate B cells to make antibody responses to proteins and other T cell-dependent antigens. T cell-dependent antigens are immunogens in which individual epitopes appear only once or a limited number of times such that they are unable to cross-link the membrane immunoglobulin (Ig) of B cells or do so inefficiently. B cells bind the antigen through their membrane Ig, and the complex undergoes endocytosis. Within the endosomal and lysosomal compartments, the antigen is fragmented into peptides by proteolytic enzymes and one or more of the generated peptides are loaded into class II MHC molecules, which traffic through this vesicular compartment. The resulting peptide/class II MHC complex is then exported to the B-cell surface membrane. T cells with receptors specific for the peptide/class II molecular complex recognize this complex on the B-cell surface. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)). [000437] B-cell activation depends both on the binding of the T cell through its TCR and on the interaction of the T-cell CD40 ligand (CD40L) with CD40 on the B cell. T cells do not constitutively express CD40L. Rather, CD40L expression is induced as a result of an interaction with an APC that expresses both a cognate antigen recognized by the TCR of the T cell and CD80 or CD86. CD80/CD86 is generally expressed by activated, but not resting, B cells so that the helper interaction involving an activated B cell and a T cell can lead to efficient antibody production. In many cases, however, the initial induction of CD40L on T cells is dependent on their recognition of antigen on the surface of APCs that constitutively express CD80/86, such as dendritic cells. Such activated helper T cells can then efficiently interact with and help B cells. Cross-linkage of membrane Ig on the B cell, even if inefficient, may synergize with the CD40L/CD40 interaction to yield vigorous B-cell activation. The subsequent events in the B-cell response, including proliferation, Ig secretion, and class switching (of the Ig class being expressed) either depend or are enhanced by the actions of T cell-derived cytokines. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)). [000438] CD4+ T cells tend to differentiate into cells that principally secrete the cytokines IL-4, IL-5, IL-6, and IL-10 (TH2 cells) or into cells that mainly produce IL-2, IFN-γ, and lymphotoxin (TH1 cells). The TH2 cells are very effective in helping B-cells develop into antibody-producing cells, whereas the TH1 cells are effective inducers of cellular immune responses, involving enhancement of microbicidal activity of monocytes and macrophages, and consequent increased efficiency in lysing microorganisms in intracellular vesicular compartments. Although the CD4+ T cells with the phenotype of TH2 cells (i.e., IL-4, IL-5, IL-6 and IL-10) are efficient helper cells, TH1 cells also have the capacity to be helpers. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)). T cells involved in Induction of Cellular Immunity [000439] T cells also may act to enhance the capacity of monocytes and macrophages to destroy intracellular microorganisms. In particular-gamma (IFN-γ) produced by helper T cells enhances several mechanisms through which mononuclear phagocytes destroy intracellular bacteria and parasitism including the generation of nitric oxide and induction of tumor necrosis factor (TNF) production. The TH1 cells are effective in enhancing the microbicidal action because they produce IFN-γ. By contrast, two of the major cytokines produced by TH2 cells, IL-4 and IL-10, block these activities. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)). Suppressor or Regulatory T (Treg) cells [000440] A controlled balance between initiation and downregulation of the immune response is important to maintain immune homeostasis. Both apoptosis and T cell anergy (a tolerance mechanism in which the T cells are intrinsically functionally inactivated following an antigen encounter (Scwartz, R. H., “T cell anergy,” Annu. Rev. Immunol., 21: 305-334 (2003)) are important mechanisms that contribute to the downregulation of the immune response. A third mechanism is provided by active suppression of activated T cells by suppressor or regulatory CD4+ T (Treg) cells. (Reviewed in Kronenberg, M. et al., “Regulation of immunity by self-reactive T cells,” Nature 435: 598-604 (2005)). CD4+ Tregs that constitutively express the IL-2 receptor alpha (IL-2Rα) chain (CD4+ CD25+) are a naturally occurring T cell subset that are anergic and suppressive. (Taams, L. S. et l., “Human anergic/suppressive CD4+CD25+ T cells: a highly differentiated and apoptosis-prone population,” Eur. J. Immunol., 31: 1122-1131 (2001)). Depletion of CD4+CD25+ Tregs results in systemic autoimmune disease in mice. Furthermore, transfer of these Tregs prevents development of autoimmune disease. Human CD4+CD25+ Tregs, similar to their murine counterpart, are generated in the thymus and are characterized by the ability to suppress proliferation of responder T cells through a cell-cell contact-dependent mechanism, the inability to produce IL-2, and the anergic phenotype in vitro. Human CD4+CD25+ T cells can be split into suppressive (CD25high) and nonsuppressive (CD25low) cells, according to the level of CD25 expression. A member of the forkhead family of transcription factors, FOXP3, has been shown to be expressed in murine and human CD4+CD25+ Tregs and appears to be a master gene controlling CD4+CD25+ Treg development. (Battaglia, M. et al., “Rapamycin promotes expansion of functional CD4+CD25+Foxp3+ regulator T cells of both healthy subjects and type 1 diabetic patients,” J. Immunol., 177: 8338-8347 (200)). Cytotoxic T Lymphocytes (CTL) [000441] The CD8+ T cells that recognize peptides from proteins produced within the target cell have cytotoxic properties in that they lead to lysis of the target cells. The mechanism of CTL-induced lysis involves the production by the CTL of perforin, a molecule that can insert into the membrane of target cells and promote the lysis of that cell. Perforin-mediated lysis is enhanced by a series of enzymes produced by activated CTLs, referred to as granzymes. Many active CTLs also express large amounts of fas ligand on their surface. The interaction of fas ligand on the surface of CTL with fas on the surface of the target cell initiates apoptosis in the target cell, leading to the death of these cells. CTL-mediated lysis appears to be a major mechanism for the destruction of virally infected cells. [000442] The term “macrophage” as used herein refers to a mononuclear, actively phagocytic cell arising from monocyte stem cells in the bone marrow. These cells are widely distributed in the body and vary in morphology and motility. Phagocytic activity is typically mediated by serum recognition factors, including certain immunoglobulins and components of the complement system, but also may be nonspecific. Macrophages also are involved in both the production of antibodies and in cell-mediated immune responses, particularly in presenting antigens to lymphocytes. They secrete a variety of immunoregulatory molecules. [000443] The terms “Major Histocompatibility Complex (MHC), MHC-like molecule” and “HLA” are used interchangeably herein to refer to cell-surface molecules that display a molecular fraction known as an epitope or an antigen and mediate interactions of leukocytes with other leukocyte or body cells. MHCs are encoded by a large gene group and can be organized into three subgroups- class I, class II, and class III. In humans, the MHC gene complex is called HLA (“Human leukocyte antigen”); in mice, it is called H-2 (for “histocompatibility”). Both species have three main MHC class I genes, which are called HLA- A, HLA-B, and HLA-C in humans, and H2-K, H2-D and H2-L in the mouse. These encode the α chain of the respective MHC class I proteins. The other subunit of an MHC class I molecule is β2-microglobulin. The class II region includes the genes for the α and β chains (designated A and B) of the MHC class II molecules HLA-DR, HLA-DP, and HLA-DQ in humans. Also in the MHC class II region are the genes for the TAP1:TAP2 peptide transporter, the PSMB (or LMP) genes that encode proteasome subunits, the genes encoding the DMα and BMβ chains (DMA and DMB), the genes encoding the α and β chains of the DO molecule (DOA and DOB, respectively), and the gene encoding tapasin (TAPBP). The class II genes encode various other proteins with functions in immunity. The DMA and DMB genes encoding the subunits of the HLA-DM molecule that catalyzes peptide binding to MHC class II molecules are related to the MHC class II genes, as are the DOA and DOB genes that encode the subunits of the regulatory HLA-DO molecule. [Janeway’s Immunobiology. 9th ed., GS, Garland Science, Taylor & Francis Group, 2017. pps.232-233]. MHC-like molecules, while not encoded by the same gene group as true MHCs, have the same folding and overall structure of MHCs, and specifically MHC class I molecules, and thus possesses similar biological functions such as antigen presentation. The CD1 family of molecules is an example of a MHC- like molecule. It consists of two groups based on amino acid homology: group 1, which includes CD1a, b, and c; and group 2, which consists of CD1d. Group 1 CD1s can present antigens to a wide variety of T cells, whereas CD1d presents antigens mostly to NKT cells. (Brutkiewicz. “CD1d Ligands: The Good, the Bad, and the Ugly.” The Journal of Immunology (2006) 177 (2) 769-775). While CD1d structurally resembles MHC Class I molecules, it traffics through the endosome of the exogenous antigen presentation pathway. The binding groove of the CD1d molecules tethers the lipid tail of a glycolipid antigen, while the carbohydrate head group of the antigen projects out of the groove for recognition by the TCR of the NKT cell. (Wah, MakTak, et al. “Chapter 11: NK, γδ T and NKT Cells.” Primer to the Immune Response. Elsevier, 2014). [000444] CD1d presents lipid antigens, and requires the presence of particular mechanisms to induce uptake of these molecules by APCs and subsequent loading onto CD1d molecules. Lipid transfer protein such as apolipoprotein E and fatty acid amide hydrolase (FAAH) have been shown to enhance the presentation of certain antigens by CD1d. Loading efficiency can be enhanced by specific proteins, such as saposins and microsomal triglyceride transfer protein , present in the endosomal and lysosomal compartments of cells by promoting lipid antigen exchange. Similar to MHC antigens, lipid antigens can also be processed by lysosomal enzymes to yield active compounds, as demonstrated in the case of CD1d for synthetic antigens, microbial antigens, and self-antigens. [Giradi and Zajonc (2012). “Molecular basis of lipid antigen presentation by CD1d and recognition by natural killer T cells.” Immunol Rev.250(1): 167-179.] [000445] MHC Class I-like molecules are nonclassical MHC type molecules; while including Cd1d CD1a, CD1b, CD1c, CD1e, and MR1 are also expressed on APCs and can activate various subsets of T cells. [Kumar and Delovitch (2014) “Different subsets of natural killer T cells may vary in their roles in health and disease.” Immunology 142: 321-336]. Other non-classical histocompatibility molecules include MR1, which activate MAIT cells. [000446] The term “mass median aerodynamic diameter” or “MMAD” as used herein refers to the median of the distribution of airborne particle mass with respect to the aerodynamic diameter. MMADs are usually accompanied by the geometric standard deviation (g or sigma g), which characterizes the variability of the particle size distribution. [000447] The term “matrix” as used herein refers to a three-dimensional network of fibers that contains voids (or “pores”) where the woven fibers intersect. The structural parameters of the pores, including the pore size, porosity, pore interconnectivity/ tortuosity and surface area, affect how substances (e.g., fluid, solutes) move in and out of the matrix. According to some embodiments, tuning of the drug delivery system comprises controlling the porosity and rate of pore formation. According to some embodiments, the polymer is porous. According to some embodiments, the polymer is nonporous. According to some embodiments, the polymer comprises a channel forming agent or porogen, e.g., CaCl2. [000448] The term “metered-dose inhaler”, “MDI”, or “puffer” as used herein refers to a pressurized, hand-held device that uses propellants to deliver a specific amount of medicine (“metered dose”) to the lungs of a patient. The term “propellant” as used herein refers to a material that is used to expel a substance usually by gas pressure through a convergent, divergent nozzle. The pressure may be from a compressed gas, or a gas produced by a chemical reaction. The exhaust material may be a gas, liquid, plasma, or, before the chemical reaction, a solid, liquid or gel. Propellants used in pressurized metered dose inhalers are liquefied gases, traditionally chlorofluorocarbons (CFCs) and increasingly hydrofluoroalkanes (HFAs). Suitable propellants include, for example, a chlorofluorocarbon (CFC), such as trichlorofluoromethane (also referred to as propellant 11), dichlorodifluoromethane (also referred to as propellant 12), and 1,2-dichloro-1,1,2,2-tetrafluoroethane (also referred to as propellant 114), a hydrochlorofluorocarbon, a hydrofluorocarbon (HFC), such as 1,1,1,2- tetrafluoroethane (also referred to as propellant 134a, HFC-134a, or HFA-134a) and 1,1,1,2,3,3,3-heptafluoropropane (also referred to as propellant 227, HFC-227, or HFA-227), carbon dioxide, dimethyl ether, butane, propane, or mixtures thereof. In other embodiments, the propellant includes a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or mixtures thereof. In other embodiments, a hydrofluorocarbon is used as the propellant. In other embodiments, HFC-227 and/or HFC-134a are used as the propellant.\ [000449] The term “microparticle” is used herein to refer generally to structures including microcapsules, microparticles, nanoparticles, nanocapsules, and nanospheres. The particles may contain therapeutic agent(s) in a core surrounded by a coating. Therapeutic agent(s) also may be dispersed throughout the particles. Therapeutic agent(s) also may be adsorbed into the particles. The particles may be of any order release kinetics, including zero order release, first order release, second order release, delayed release, sustained release, immediate release, etc., and any combination thereof. The particles may include, in addition to therapeutic agent(s), any of those materials routinely used in the art of pharmacy and medicine, including, but not limited to, erodible, nonerodible, biodegradable, or nonbiodegradable material or combinations thereof. The particles may be microcapsules that contain the therapeutic agent in a solution or in a semi-solid state. The particles may be of virtually any shape. [000450] The term “microparticulate composition”, as used herein, refers to a composition comprising a microparticulate formulation and a pharmaceutically acceptable carrier, where the microparticulate formulation comprises a therapeutic agent and a plurality of microparticles. [000451] The terms “minimum effective concentration”, “minimum effective dose”, or “MEC” are used interchangeably to refer to the minimum concentration of the recombinant fusion protein of the present disclosure required to produce a desired pharmacological effect in most patients. [000452] The term “mitogen” as used herein refers to a substance that stimulates mitosis. [000453] The term “modulate” as used herein means to regulate, alter, adapt, or adjust to a certain measure or proportion. [000454] The term “molecule” as used herein refers to a chemical unit composed of one or more atoms, whereby the atoms of the molecule are held together by chemical bonds. [000455] The term “monomer” refers to a molecule that may bind chemically to other molecules to form a polymer. [000456] The term “motogen” and its other grammatical forms as used herein refer to a substance that enhances cell migratory processes (motogenic effect), show anti-apoptotic effects and are inflammatory modulators. [000457] The term “morphogen” as used herein refers to a substance that governs the movement and development of cells during morphogenesis by forming a concentration gradient in the developing tissue. [000458] The term “mucosa-associated lymphoid tissue” or MALT, as used herein is a generic term for all organized lymphoid tissue found at mucosal surfaces in which an adaptive immune response can be initiated. It comprises GALT, NALT and BALT (when present). The term “mucosa-associated invariant T cells (MAIT)” as used herein refers primarily to γδT cells with limited diversity present in the mucosal immune system that respond to bacterially derived folate derivatives presented by the nonclassical MHC class 1b molecule MR1. [000459] The term “mucosal epithelia” as used herein refers to mucus-coated epithelia lining the body’s internal cavities that connect with the outside (e.g., the gut, airways, and vaginal tract). [000460] The term “mucosal mast cells” as used herein refers to specialized mast cells present in mucosa. They produce little histamine but large amounts of prostaglandins and leukotrienes. [000461] As used herein, the term "mutation" refers to a change of the DNA sequence within a gene or chromosome of an organism resulting in the creation of a new character or trait not found in the parental type, or the process by which such a change occurs in a chromosome, either through an alteration in the nucleotide sequence of the DNA coding for a gene or through a change in the physical arrangement of a chromosome. Three mechanisms of mutation include substitution (exchange of one base pair for another), addition (the insertion of one or more bases into a sequence), and deletion (loss of one or more base pairs). [000462] The term “myeloid” as used herein means of or pertaining to bone marrow. Granulocytes and monocytes, collectively called myeloid cells, are differentiated descendants from common progenitors derived from hematopoietic stem cells in the bone marrow. Commitment to either lineage of myeloid cells is controlled by distinct transcription factors followed by terminal differentiation in response to specific colony-stimulating factors and release into the circulation. Upon pathogen invasion, myeloid cells are rapidly recruited into local tissues via various chemokine receptors, where they are activated for phagocytosis as well as secretion of inflammatory cytokines, thereby playing major roles in innate immunity. [Kawamoto, H., Minato, N. Intl J. Biochem. Cell Biol. (2004) 36 (8): 1374-9]. [000463] The term “nanoparticle” as used herein refers to a particle whose extension in all three dimensions lies between 1 and 1000 nanometers. Because of their small size, nanoparticles have a very large surface area to volume ratio when compared to bulk material, which enables nanoparticles to possess unexpected optical, physical and chemical properties that are not found in bulk samples of the same material. They can be classified into different classes based on their properties, shapes or sizes. The different groups include fullerenes, metal NPs, ceramic NPs, and polymeric NPs. Fullerenes and carbon nanotubes (CNTs) represent two major classes of carbon-based NPs. Fullerenes contain nanomaterial that are made of globular hollow cage such as allotropic forms of carbon. [000464] The abbreviation "NFκB" as used herein refers to a proinflammatory transcription factor that switches on multiple inflammatory genes, including cytokines, chemokines, proteases, and inhibitors of apoptosis, resulting in amplification of the inflammatory response [Barnes, PJ, (2016) Pharmacol. Rev. 68: 788-815]. The molecular pathways involved in NF-κB activation include several kinases. The classic (canonical) pathway for inflammatory stimuli and infections to activate NF-κB signaling involve the IKK (inhibitor of κB kinase) complex, which is composed of two catalytic subunits, IKK-α and IKK-β, and a regulatory subunit IKK-γ (or NFκB essential modulator [Id., citing Hayden, MS and Ghosh, S (2012) Genes Dev. 26: 203-234]. The IKK complex phosphorylates Nf-κB- bound IκBs, targeting them for degradation by the proteasome and thereby releasing NF-κB dimers that are composed of p65 and p50 subunits, which translocate to the nucleus where they bind to κB recognition sites in the promoter regions of inflammatory and immune genes, resulting in their transcriptional activation. This response depends mainly on the catalytic subunit IKK-β (also known as IKK2), which carries out IκB phosphorylation. The noncanonical (alternative) pathway involves the upstream kinase NF-κB-inducing kinase (NIK) that phosphorylates IKK-α homodimers and releases RelB and processes p100 to p52 in response to certain members of the TNF family, such as lymphotoxin-β [Id., citing Sun, SC. (2012) Immunol. Rev.246: 125-140]. This pathway switches on different gene sets and may mediate different immune functions from the canonical pathway. Dominant-negative IKK-β inhibits most of the proinflammatory functions of NF-κB, whereas inhibiting IKK-α has a role only in response to limited stimuli and in certain cells, such as B-lymphocytes. The noncanonical pathway is involved in development of the immune system and in adaptive immune responses. The coactivator molecule CD40, which is expressed on antigen-presenting cells, such as dendritic cells and macrophages, activates the noncanonical pathway when it interacts with CD40L expressed on lymphocytes [Id., citing Lombardi, V et al. (2010) Int. Arch. Allergy Immunol.151: 179-89]. [000465] The term “natural killer (NK) cells” as used herein is meant to refer to lymphocytes in the same family as T and B cells, classified as group I innate lymphocytes. They have an ability to kill tumor cells without any priming or prior activation, in contrast to cytotoxic T cells, which need priming by antigen presenting cells. NK cells secrete cytokines such as IFNγ and TNFα, which act on other immune cells, like macrophages and dendritic cells, to enhance the immune response. Activating receptors on the NK cell surface recognize molecules expressed on the surface of cancer cells and infected cells and switch on the NK cell. Inhibitory receptors act as a check on NK cell killing. Most normal healthy cells express MHCI receptors, which mark them as “self.” Inhibitory receptors on the surface of the NK cell recognize cognate MHCI, which switches off the NK cell, preventing it from killing. Once the decision is made to kill, the NK cell releases cytotoxic granules containing perforin and granzymes, which leads to lysis of the target cell. Natural killer reactivity, including cytokine secretion and cytotoxicity, is controlled by a balance of several germ-line encoded inhibitory and activating receptors such as killer immunoglobulin-like receptors (KIRs) and natural cytotoxicity receptors (NCRs). The presence of the MHC Class I molecule on target cells serves as one such inhibitory ligand for MHC Class I-specific receptors, the Killer cell Immunoglobulin-like Receptor (KIR), on NK cells. Engagement of KIR receptors blocks NK activation and, paradoxically, preserves their ability to respond to successive encounters by triggering inactivating signals. Therefore, if a KIR is able to sufficiently bind to MHC Class I, this engagement may override the signal for killing and allows the target cell to live. In contrast, if the NK cell is unable to sufficiently bind to MHC Class I on the target cell, killing of the target cell may proceed. [000466] The term “nebulizer” as used herein refers to a device used to administer liquid medication in the form of a mist inhaled into the lungs. Nebulizers, which actively aerosolize a liquid formulation and operate continuously once loaded, require either compressed air or an electrical supply. Exemplary nebulizers include, a vibrating mesh nebulizer, a jet nebulizer (also known as an atomizer) and an ultrasonic wave nebulizer. Exemplary vibrating mesh nebulizers include, but are not limited to, Respironics i-Neb, Omron MicroAir, Beurer Nebulizer IHSO and Aerogen Aeroneb. Acorn-I, Acorn-II, AquaTower, AVA-NEB, Cirrhus, Dart, DeVilbiss 646, Downdraft, Fan Jet, MB-5, Misty Neb, Salter Labs 8900, Sidestream, Updraft-II, and Whisper Jet are examples of a jet nebulizer. Exemplary ultrasonic nebulizers include, but are not limited to, an Omron NE-U17 nebulizer and a Beurer Nebulizer IH30. [000467] The term “neutrophils” or “polymorphonuclear neutrophils (PMNs)” as used herein refers to the most abundant type of white blood cells in mammals, which form an essential part of the innate immune system. They form part of the polymorphonuclear cell family (PMNs) together with basophils and eosinophils. Neutrophils are normally found in the blood stream. During the beginning (acute) phase of inflammation, particularly as a result of bacterial infection and some cancers, neutrophils are one of the first-responders of inflammatory cells to migrate toward the site of inflammation. They migrate through the blood vessels, then through interstitial tissue, following chemical signals such as interleukin- 8 (IL-8) and C5a in a process called chemotaxis, the directed motion of a motile cell or part along a chemical concentration gradient toward environmental conditions it deems attractive and/or away from surroundings it finds repellent. [000468] The term “normal healthy subject” as used herein refers to a subject having no symptoms or other evidence of a fibrotic condition. [000469] The term “Nucleotide-binding Oligomerization Domain (NOD)-like receptors (NLRs)” as used herein refers to innate sensors that detect microbial products or cellular damage in the cytoplasm or activate signaling pathways, and are expressed in cells that are routinely exposed to bacteria, such as epithelial cells, macrophages and dendritic cells. Some NLRs activate NFκB to initiate the same inflammatory responses as the TLRs, while others trigger a distinct pathway that induces cell death and the production of pro-inflammatory cytokines. [Janeway’s Immunobiology.9th ed., GS, Garland Science, Taylor & Francis Group, 2017, at 96]. [000470] Subfamilies of NLRs can be distinguished based on the other protein domains they contain. For example the NOD subfamily has an amino-terminal caspase recruitment domain (CARD), which is structurally related to the T1R death domain in MyD88, and can dimerize with CARD domains on other proteins to induce signaling. NOD proteins recognize fragments of bacterial cell wall peptidoglycans, although it is not known if they do so through direct binding or through accessory proteins. Id. At 96. NOD1 senses γ-glutamyl diaminopimelic acid (iE-DAP), a breakdown product of peptidoglycans of Gram negative and some Gram positive bacteria, whereas NOD2 recognizes muramyl dipeptide (MDP), which is present in the peptidoglycans of most bacteria. Id. Other members of the NOD family, including NLRX1 and NLRC5, have been identified, but their function is less well understood. [Janeway’s Immunobiology.9th ed., GS, Garland Science, Taylor & Francis Group, 2017, at 96-98] [000471] When NOD1 or NOD2 recognizes its ligand, it recruits the CARD-containing serine-threonine kinase RIP2 (also known as RICK and RIPK2), which associates with the E3 ligases cIAP1, CIAP2, and XIAP, whose activity generates a polyubiquitin scaffold, which recruits TAK1 and IKK and results in activation of NFκB. NFκB then induces the expression of genes for inflammatory cytokines and for enzymes involved in the production of NO. [Janeway’s Immunobiology.9th ed., GS, Garland Science, Taylor & Francis Group, 2017, at 97]. [000472] Macrophages and dendritic cells express both TLRs and NOD1 and NOD2, and are activated by both pathways. In epithelial cells, NOD1 may also function as a systemic activator of innate immunity. NOD2 is strongly expressed in the Paneth cells of the gut where it regulates the expression of potent anti-microbial peptides such as the α- and β- defensins. [Janeway’s Immunobiology.9th ed., GS, Garland Science, Taylor & Francis Group, 2017, at 97]. [000473] Other members of the NOD family, including NLRX1 and NLRC5, have been identified, but their function is less well understood. [Janeway’s Immunobiology.9th ed., GS, Garland Science, Taylor & Francis Group, 2017, at 96-98] [000474] The NLRP family, another subfamily of NLR proteins, has a pyrin domain in place of the CARD domain at their amino termini. Humans have 14 NLR proteins containing pyrin domains, of which NLRP3 (also known as NAPL3 or cryopyrin) is the best characterized. NLRP3 resides in an inactive form in the cytoplasm, where its leucine rich repeat (LRR) domains are thought to bind the head-shock chaperone protein HSP90 and the co-chaperone SGT1. NRLP3 signaling is induced by reduced intracellular potassium, the generation of reactive oxygen species, or the disruption of lysosomes by particulate or crystalline matter. For example, death of nearby cells can release ATP into the extracellular space, which would activate the purinergic receptor P2X7, which is a potassium channel, and allow potassium ion efflux. A model proposed for ROS-induced NLRP3 activation involves intermediate oxidation of sensor proteins collectively called thioredoxin (TRX). Normally TRX proteins are bound to thioredoxin-interacting protein (TXNIP). Oxidation of TRX by ROS causes dissociation of TXNIP from TRX. The free TXNIP may then displace HSP90 and SGT1 from NLRP3, again causing its activation. In both cases, NLRP3 activation involves aggregation of multiple monomers via their leucine-rich repeat (LRR) and NOD domains to induce signaling. Phagocytosis of particulate matter (e.g. the adjuvant alum), may lead to the rupture of lysosomes and release of the active protease cathepsin B, which can activate NLRP3. [Janeway’s Immunobiology.9th ed., GS, Garland Science, Taylor & Francis Group, 2017, at 98-99]. [000475] NLR signaling, as exemplified by NLRP3, leads to the generation of pro- inflammatory cytokines and to cell death through formation of an inflammasome, a multiprotein complex. Activation of the inflammasome proceeds in several stages. Aggregation of NLRP molecules triggers autocleavage of procaspase I, which releases active caspase 1 - Aggregation of LRR domains of several NLRP3 molecules, or other NLRP molecules by a specific trigger or recognition event, which induces the pyrin domains of NLRP3 to interact with pyrin domains of ASC (also called PYCARD), an adaptor protein composed of an amino terminal pyrin domain and a carboxy terminal CARD domain, which further drives the formation of a polymeric ASC filament, with the pyrin domains in the center and the CARD domains facing outward; the CARD domains then interact with CARD domains of the inactive protease pro-caspase 1, initiating its CARD-dependent polymerization into discrete caspase 1 filaments. Active caspase 1 then carries out ATP-dependent proteolytic processing of proinflammatory cytokines, particularly 1L-1β and IL-18, into their active forms, and induces a form of cell death (pyroptosis) associated with inflammation because of the release of these pro-inflammatory cytokines upon cell rupture. [Janeway’s Immunobiology.9th ed., GS, Garland Science, Taylor & Francis Group, 2017, at 99-100]. [000476] A priming step, which can result from TLR signaling, must first occur in which cells inducer and translate the mRNAs that encode the pro-forms of IL-1, IL-18 or other cytokines for inflammasome activation to produce inflammatory cytokines. For example, the TLR-3 agonist poly I:C can be used experimentally to prime cells for triggering of the inflammasome. Janeway’s Immunobiology. 9th ed., GS, Garland Science, Taylor & Francis Group, 2017, at 100]. [000477] Inflammasome activation also can involve proteins of the PYHIN family, which have an H inversion (HIN) domain in place of an LRR domain. There are four PYIN proteins in humans. Id. At 100. A noncanonical inflammasome (caspase I-independent) pathway uses the protease caspase 11, which therefore is both a sensor and an effector molecule, to detect intracellular LPS. [Janeway’s Immunobiology.9th ed., GS, Garland Science, Taylor & Francis Group, 2017, at 101]. [000478] Besides activating effector functions and cytokine production, another outcome of the activation of innate sensing pathways is the induction of co-stimulatory molecules on tissue dendritic cells and macrophages. B7.1 (CD80) and B7.2 (CD86), for example, which are induced on macrophages and tissue dendritic cells by innate sensors such as TLRs in response to pathogenic recognition, are recognized by specific co-stimulatory receptors expressed by cells of the adaptive immune response, particularly CD4 T cells, and their activation by B7 is an important step in activating adaptive immune responses. [Janeway’s Immunobiology.9th ed., GS, Garland Science, Taylor & Francis Group, 2017, at 105]. [000479] The term “operatively linked” as used herein refers to a linkage in which two or more protein domains or peptides are ligated or combined via recombinant DNA technology or chemical reaction such that each protein domain or polypeptide of the resulting fusion peptide retains its original function. [000480] The term “oxygen saturation” (SpO2) as used herein refers to a measurement of how much oxygen the blood is carrying as a percentage of the maximum it could carry. For a healthy individual, the normal SpO2 should be between 96% to 99%. [000481] The term "parenteral" as used herein refers to introduction into the body by means other than through the digestive tract, for example, without limitation, by way of an injection (i.e., administration by injection), including, for example, subcutaneously (i.e., an injection beneath the skin), intramuscularly (i.e., an injection into a muscle), intravenously (i.e., an injection into a vein), or infusion techniques. [000482] The term "particles" as used herein refers to refers to an extremely small constituent (e.g., nanoparticles, microparticles, or in some instances larger) in or on which is contained the composition as described herein. [000483] The term “pathogenesis” as used herein refers to the development of a disease and the chain of events leading to that disease and its sequelae. [000484] The term “pathological” as used herein refers to indicative of or caused by disease. [000485] The term “pathophysiology” and its various grammatical forms as used herein refers to derangement of function in an individual or organ due to a disease. [000486] The term “pattern recognition receptors” or “PRRs” as used herein, is meant to refer to receptors that are present at the cell surface to recognize extracellular pathogens; in the endosomes where they sense intracellular invaders, and finally in the cytoplasm. They recognize conserved molecular structures of pathogens, called pathogen associated molecular patterns (PAMPs) specific to the microorganism and essential for its viability. PRRs are divided into four families: toll-like receptors (TLR); nucleotide oligomerization receptors (NLR); C-type leptin receptors (CLR), and RIG-1 like receptors (RLR). [000487] The term "peptide" is used herein to refer to two or more amino acids joined by a peptide bond. The terms "polypeptide" and "protein" are used herein in their broadest sense to refer to a sequence of subunit amino acids, amino acid analogs, or peptidomimetics. The subunits are linked by peptide bonds, except where noted. FDA considers any polymer composed of 40 or fewer amino acids to be a peptide. The peptides, polypeptides or proteins described herein may be chemically synthesized or recombinantly expressed. Synthetic polypeptides, prepared using the well-known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can include natural and unnatural amino acids. Amino acids used for peptide synthesis may be standard Boc (N-α-amino protected N- α-t-butyloxycarbonyl) amino acid resin with the standard deprotecting, neutralization, coupling and wash protocols of the original solid phase procedure of Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154), or the base-labile N-α-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpino and Han (1972, J. Org. Chem.37:3403-3409). Both Fmoc and Boc N-α-amino protected amino acids can be obtained from Sigma, Cambridge Research Biochemical, or other chemical companies familiar to those skilled in the art. In addition, the polypeptides can be synthesized with other N-α-protecting groups that are familiar to those skilled in this art. Solid phase peptide synthesis may be accomplished by techniques familiar to those in the art and provided, for example, in Stewart and Young, 1984, Solid Phase Synthesis, Second Edition, Pierce Chemical Co., Rockford, Ill.; Fields and Noble, 1990, Int. J. Pept. Protein Res. 35:161-214, or using automated synthesizers. The polypeptides useful in the described invention may comprise D-amino acids (which are resistant to L-amino acid- specific proteases in vivo), a combination of D- and L-amino acids, and various "designer" amino acids (e.g., β-methyl amino acids, C-α-methyl amino acids, and N-α-methyl amino acids, etc.) to convey special properties. Synthetic amino acids include ornithine for lysine, and norleucine for leucine or isoleucine. In addition, the polypeptides can have peptidomimetic bonds, such as ester bonds, to prepare peptides with novel properties. For example, a peptide may be generated that incorporates a reduced peptide bond, i.e., R1-CH2-NH-R2, where R1 and R2 are amino acid residues or sequences. A reduced peptide bond may be introduced as a dipeptide subunit. Such a polypeptide would be resistant to protease activity, and would possess an extended half-live in vivo. Accordingly, these terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids. The terms "polypeptide", "peptide" and "protein" also are inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. It will be appreciated, as is well known and as noted above, that polypeptides may not be entirely linear. For instance, polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslational events, including natural processing event and events brought about by human manipulation which do not occur naturally. Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well. In some embodiments, the peptide is of any length or size. [000488] The term "peptidomimetic" as used herein refers to a small protein-like chain designed to mimic a peptide. A peptidomimetic typically arises from modification of an existing peptide in order to alter the molecule's properties. [000489] The term “pharmaceutical composition” is used herein to refer to a composition that is employed to prevent, reduce in intensity, cure or otherwise treat a target condition or disease. [000490] The term “polymer” refers to a large molecule, or macromolecule, composed of many repeated subunits. [000491] The term “polymer matrix” as used herein refers to a matrix formed by polymers. [000492] The term “potentiate” and its other grammatical forms as used herein means to increase the power, effect, or potency, of; to enhance, to augment the activity of. [000493] The term “priming” as used herein refers to the process whereby T cells and B cell precursors encounter the antigen for which they are specific. The term “unprimed cells” (also referred to as virgin, naïve, or inexperienced cells) as used herein refers to T cells and B cells that have generated an antigen receptor (TCR for T cells, BCR for B cells) of a particular specificity, but have never encountered the antigen. [000494] For example, before helper T cells and B cells can interact to produce specific antibody, the antigen-specific T cell precursors must be primed. Priming involves several steps: antigen uptake, processing, and cell surface expression bound to class II MHC molecules by an antigen presenting cell, recirculation and antigen-specific trapping of helper T cell precursors in lymphoid tissue, and T cell proliferation and differentiation. [Janeway, CA, Jr., “The priming of helper T cells, Semin. Immunol. (1989) 1(1): 13-20]. Helper T cells express CD4, but not all CD4 T cells are helper cells. Id. The signals required for clonal expansion of helper T cells differ from those required by other CD4 T cells. The critical antigen-presenting cell for helper T cell priming appears to be a macrophage; and the critical second signal for helper T cell growth is the macrophage product interleukin 1 (IL-1). Id. If the primed T cells and/or B cells receive a second, co-stimulatory signal, they become activated T cells or B cells. [000495] The term “progression” as used herein refers to the course of a disease as it becomes worse or spreads in the body. [000496] The term “(human) peripheral blood mononuclear cells” or “hPBMCs” refers to lymphocytes (B cells, T cells and NK cells), monocytes and dendritic cells. [000497] The term “recombinant” as used herein refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques. For example, the term “recombinant” and grammatical variations thereof are meant to relate to or denote an organism, protein, or genetic material formed by or using recombined DNA comprising DNA pieces from different sources or from different parts of the same source. For example, the term “recombinant DNA” means a DNA molecule formed through recombination methods to splice fragments of DNA from a different source or from different parts of the same source. According to some embodiments, two or more different sources of DNA are cleaved using restriction enzymes and joined together using ligases. As another example, the term “recombinant protein” or “recombinant domains” and grammatical variations thereof means a protein molecule formed through recombination methods originating from spliced fragments of DNA from a different source or from different parts of the same source. As another example, the term “recombinant microbe” or “recombinant bacteria” and grammatical variations thereof mean a microbe/bacteria that comprises one or more recombinant DNA/protein molecules. [000498] The term “reduce” and its various grammatical forms as used herein refers to a diminution, a decrease, an attenuation or abatement of a degree, intensity, extent, size, amount, density or number. [000499] The term “release” and its various grammatical forms, refers to dissolution of an active drug component and diffusion of the dissolved or solubilized species by a combination of the following processes: (1) hydration of a matrix, (2) diffusion of a solution into the matrix; (3) dissolution of the drug; and (4) diffusion of the dissolved drug out of the matrix. [000500] The Renin-Angiotensin-aldosterone System (RAAS) or renin-angiotensin system (RAS) is a critical regulator of blood volume and systemic vascular resistance. It is composed of three major compounds: renin, angiotensin II, and aldosterone, which act to elevate arterial pressure in response to decreased renal blood pressure, decreased salt delivery to the distal convoluted tubule, and/or beta agonism. [000501] Angiotensin II (Ang II), the primary physiological product of the RAAS system, is a potent vasoconstrictor. Angiotensin converting enzyme (ACE) catalyzes the transformation of angiotensin I (Ang I) to Ang II. Ang II elicits its effects by activating two receptors: type 1 angiotensin II (AT1) receptor and type 2 angiotensin II (AT2) receptor [Ingraham, NE, et al. Eur. Respir. J. (2020); DOI: 10.1183/13993003.00912-2020, citing Balakumar, P. & Jagadeesh, G. Cell Signal (2014) 26: 2147-60]. Ang II action through AT1 receptor causes a cascade with resultant inflammation, vasoconstriction, and atherogenesis [Id., citing Strawn, WB & Ferrario, CM., Curr Opin. Lipido. (2002) 13: 505-12]. These effects also promote insulin resistance and thrombosis [Id., citing Dandona, P. et al. J. Hum. Hypertens. (2007) 21: 20-27]. In contrast, AT2 receptor stimulation causes vasodilation, decreased platelet aggregation, and the promotion of insulin action. However, the expression of AT2 receptor is low in healthy adults [Id., citing Dandona, P. et al. J. Hum. Hypertens. (2007) 21: 20-27]. As such, Ang II's effects in adults are modulated and balanced indirectly by angiotensin II converting enzyme (ACE2), which converts Ang II into lung-protective Angiotensin-(1–7) (Ang- [1–7]), similar to effects seen from AT2 receptor stimulation [Id., citing Ghazi, L. & Grawz, P. F1000Research 2017; 6: F1000, Faculty Rev-1297. doi:10.12688/f1000research.9692.1; Warner, FJ et al. Cell Mol. Life Sci. (2004) 61: 2704-13]. [000502] The term “restore” and its various grammatical forms as used herein refers to bringing back to a former or normal condition, to recover or renew. [000503] The term “secondary lymphoid tissues” as used herein refers to sites where lymphocytes interact with each other and nonlymphoid cells to generate immune responses to antigens. These include the spleen, lymph nodes, and mucosa-associated lymphoid tissues (MALT). [000504] The term “sequelae” and its various grammatical forms as used herein means a pathological condition resulting from a prior disease, injury or attack. [000505] The term “shock” as used herein refers to a critical condition brought on by a sudden drop in blood flow through the body, where the circulatory system fails to maintain adequate blood flow, sharply curtailing the delivery of oxygen and nutrients to vital organs. [000506] The term “sign” as used herein refers to a healthcare provider’s evidence of disease. [000507] The term “splice variant” as used herein refers to a recombinant DNA molecule derived from cutting and resealing of DNA from different sources that can result in an altered protein-coding sequence from the wild-type sequence. [000508] The term “stability” and its other grammatical forms as used herein with respect to a pharmaceutical product refers to the capability of a particular formulation to remain within its physical, chemical, microbiological, therapeutic and toxicological specifications. Stabilizers may be used to help an active pharmaceutical ingredient (API) maintain the desirable properties of the product until it is consumed by the patient. [000509] The term “susceptible subject” as used herein refers to an individual vulnerable to developing infection when their body is invaded by an infectious agent. Examples of individuals vulnerable to developing a serious lung infection include, without limitation, the very young, the elderly, those who are ill; those who are receiving immunosuppressants; those with long term health conditions; those that are obese; and those who are physically weak, e.g., due to malnutrition or dehydration. [000510] The term "sustained release" (also referred to as "extended release") is used herein in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant blood levels of a drug over an extended time period. Alternatively, delayed absorption of a parenterally administered drug form Is accomplished by dissolving or suspending the drug in an oil vehicle. Nonlimiting examples of sustained release biodegradable polymers include polyesters, polyester polyethylene glycol copolymers, polyamino-derived biopolymers, polyanhydrides, hydrogels, polyorthoesters, polyphosphazenes, SAIB, photopolymerizable biopolymers, protein polymers, collagen, polysaccharides, chitosans, and alginates. [000511] The term “symptom” as used herein refers to a patient’s subjective evidence of disease. [000512] The term “T cell exhaustion” as used herein refers to a state of T cell dysfunction that arises during many chronic infections and cancer. It is defined by poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Modulating pathways overexpressed in exhaustion — for example, by targeting programmed cell death protein 1 (PD1) and cytotoxic T lymphocyte antigen 4 (CTLA4) — can reverse this dysfunctional state and reinvigorate immune responses [Wherry EJ and Kurachi, M. Nature (2015) 15: 486-99, citing Wherry EJ. Nat. Immunol. (2011) 131:492–499; Schietinger A, Greenberg PD. Trends Immunol. (2014) 35:51–60; , Barber DL, et al. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature. (2006) 439:682–687; Nguyen LT, Ohashi PS. Nat. Rev. Immunol. (2014) 15:45–56]. The level and duration of chronic antigen stimulation and infection seem to be key factors that lead to T cell exhaustion and correlate with the severity of dysfunction during chronic infection. Examples of inhibitory receptors include the inhibitory pathways mediated by PD1 in response to binding of PD1 ligand 1 (PDL1) and/or PDL2. [Id., citing Okazaki T, et al., Nature Immunol. (2013) 14:1212–1218, Odorizzi PM, Wherry EJ. J. Immunol. (2012) 188:2957–2965, Araki K, et al. Cold Spring Harb. Symp. Quant. Biol. (2013) 78:239–247]. Exhausted T cells can co- express PD1 together with lymphocyte activation gene 3 protein (LAG3), 2B4 (also known as CD244), CD160, T cell immunoglobulin domain and mucin domain-containing protein 3 (TIM3; also known as HAVCR2), CTLA4 and many other inhibitory receptors [Id., citing Blackburn SD, et al. Nat. Immunol. (2009) 10:29–37]. Typically, the higher the number of inhibitory receptors co-expressed by exhausted T cells, the more severe the exhaustion. It has been suggested that inhibitory receptors such as PD1 might regulate T cell function in several ways [Id., citing Schietinger A, Greenberg PD. Trends Immunol. (2014) 35:51–60; Odorizzi PM, Wherry EJ. J. Immunol. (2012) 188:2957–2965], e.g., by ectodomain competition, which refers to inhibitory receptors sequestering target receptors or ligands and/or preventing the optimal formation of microclusters and lipid rafts (for example, CTLA4); second, through modulation of intracellular mediators, which can cause local and transient intracellular attenuation of positive signals from activating receptors such as the TCR and co-stimulatory receptors [Id., citing Parry RV, et al. Molec. Cell. Biol. (2005) 25:9543–9553; Yokosuka T, et al. J. Exp. Med. (2012) 209:1201–1217; Clayton KL, et al. J. Immunol. (2014) 192:782–791]; and third, through the induction of inhibitory genes [Id., citing Quigley M, et al. Nat. Med. (2010) 16:1147–1151]. Co-stimulatory receptors also are involved in T cell exhaustion [Id., citing Odorizzi PM, Wherry EJ. J. Immunol. (2012) 188:2957–2965]. For example, desensitization of co-stimulatory pathway signaling through the loss of adaptor molecules can serve as a mechanism of T cell dysfunction during chronic infection. The signaling adaptor tumor necrosis factor receptor (TNFR)-associated factor 1 (TRAF1) is downregulated in dysfunctional T cells in HIV progressors, as well as in chronic LCMV infection [Id., citing Wang C, et al. J. Exp. Med. (2012) 209:77–91]. Adoptive transfer of CD8+ T cells expressing TRAF1 enhanced control of chronic LCMV infection compared with transfer of TRAF1- deficient CD8+ T cells, which indicates a crucial role for TRAF1-dependent co-stimulatory pathways in this setting [Id., citing Wang C, et al. J. Exp. Med. (2012) 209:77–91]. It has also been possible to exploit the potential beneficial role of co-stimulation to reverse exhaustion by combining agonistic antibodies to positive co-stimulatory pathways with blockade of inhibitory pathways. 4-1BB (also known as CD137 and TNFRSF9) is a TNFR family member and positive co-stimulatory molecule that is expressed on activated T cells. Combining PD1 blockade and treatment with an agonistic antibody to 4-1BB dramatically improved exhausted T cell function and viral control [Id, citing Vezys V, et al. J. Immunol. (2011) 187:1634–1642]. Soluble molecules are a second class of signals that regulate T cell exhaustion; these include immunosuppressive cytokines such as IL-10 and transforming growth factor-β (TGFβ) and inflammatory cytokines, such as type I interferons (IFNs) and IL-6. [Id.] [000513] The term “targeted drug delivery” as used herein refers to a system of specifying a drug moiety directly into its targeted body area (organ, cellular, and subcellular level of specific tissue) to overcome a specific toxic effects of conventional drug delivery, thereby reducing the amount of drug required for therapeutic efficacy. [000514] The term “Tbet” as used herein refers to a Th1 cell transcription factor. Differential expression of the Th1 cell transcription factor T bet and a closely related T-box family transcription, factor particularly in CD8+ T cells, Eomesodermin (Eomes) facilitates the cooperative maintenance of the pool of antiviral CD8+ T cells during chronic viral infection. [Paley, MA et a., Science (2012) 338: 1220-125]. During chronic infections, T-bet is reduced in virus-specific CD8+ T cells; this reduction correlates with T cell dysfunction. In contrast, Eomes mRNA expression is up-regulated in exhausted CD8+ T cells during chronic infection. [Id.] [000515] The term “T follicular helper (Tfh) cells” as used herein refers to a distinct subset of CD4+ T lymphocytes, specialized in B cell help and in regulation of antibody responses. They develop within secondary lymphoid organs (SLO) and can be identified based on their unique surface phenotype, cytokine secretion profile, and signature transcription factor. They support B cells to produce high-affinity antibodies toward antigens, in order to develop a robust humoral immune response and are crucial for the generation of B cell memory. They are essential for infectious disease control and optimal antibody responses after vaccination. Stringent control of their production and function is critically important, both for the induction of an optimal humoral response against thymus-dependent antigens but also for the prevention of self-reactivity. [Gensous, N. et al. Front. Immunol. (2018) doi.org/10.3389/fimmu.2018.01637). [000516] The term “Th1 cells” as used herein refers to a lineage of CD4+ effector T cells that promotes cell-mediated immune responses and is required for host defense against intracellular viral and bacterial pathogens. Th1 cells secrete IFN-gamma, IL-2, IL-10, and TNF-alpha/beta. IL-12 and IFN-γ make naive CD4+ T cells highly express T-bet and STAT4 and differentiate to Th1 cells. (Zhang, Y. et al. Adv. Exp. Med. Bio. (2014) 841: 15-44)/ [000517] The term “Th2 cells” as used herein refers to a lineage of CD4+ effector T cells that secrete IL-4, IL-5, IL-9, IL-13, and IL-17E/IL-25. These cells are required for humoral or antibody-mediated immunity and play an important role in coordinating the immune response to large extracellular pathogens. IL-4 make naive CD4+ T cells highly express STAT6 and GATA3 and differentiate to Th2 cells. (Zhang, Y. et al. Adv. Exp. Med. Bio. (2014) 841: 15- 44)/ [000518] The term “Th17 cells” as used herein refers to a CD4+ T-cell subset characterized by production of interleukin-17 (IL-17). IL-17 is a highly inflammatory cytokine with robust effects on stromal cells in many tissues, resulting in production of inflammatory cytokines and recruitment of leukocytes, especially neutrophils, thus creating a link between innate and adaptive immunity. [Tesmer, LA, et al., Immunol. Rev. (2008) 223: 87-113]. The key transcription factor in Th17 cell development is RORγt. [000519] The term “thrombosis” as used herein refers to the formation of a blood clot (thrombus) within a blood vessel, which prevents blood from flowing normally through the circulatory system. For example, endothelial infection with influenza virus has been shown to increase the adhesion of human platelets to primary human lung microvascular endothelial cells via fibronectin, contributing to mortality from acute lung injury. [Sugiyama, MG et al. J. Virol. (2016) 90 (4): 1812-21] A blood clot that forms in the veins (a venous thromboembolism) can cause deep vein thrombosis and pulmonary embolisms. Deep vein thrombosis (DVT) occurs when a blood clot forms in a major vein, usually in the leg, which stops blood from flowing easily through the vein, which can lead to swelling, discoloration and pain. Patients with DVT are at risk for developing post-thrombotic syndrome (PTS), which can involve chronic leg swelling, calf pain calf heaviness/fatigue, skin discoloration and/or venous ulcers. A pulmonary embolism (PE) is a blood clot that has traveled to the lungs. It often starts as a DVT where a piece of the clot breaks off and is carried to the lungs. PE can block the flow of blood to the lungs, causing serious damage to the lungs and affecting a person’s ability to breath, which can lead to serious injury and death A blood clot that forms in the arteries (atherothrombosis) can lead to heart attack and stroke. [000520] The term “tissue-resident memory T cell” or “TRM” as used herein refers to memory lymphocytes that do not migrate after taking up residence in barrier tissues, where they are retained long term. They appear to be specialized for rapid effector function after restimulation with antigen or cytokines at sites of pathogen entry. [000521] The term “toll-like receptors (TLR)” innate receptors on macrophages, dendritic cells, and some other cells that recognize pathogens and their products, such as bacterial lipopolysaccharide. Recognition stimulates the receptor-bearing cells to produce cytokines that help initiate immune responses. [000522] The term “TRAIL” as used herein refers to tumor necrosis factor-related apoptosis-inducing ligand, a member of the TNF cytokine family expressed on the cell surface of some cells, e.g., NK cells, that induces cell death in target cells by ligation of the “death” receptors DR4 and DR5. [000523] The term “TRIM21” as used herein refers to tripartite motif-containing 21, a cytosolic Fc receptor and E3 ligase that is activated by IgG and can ubiquitinate viral proteins after an antibody coated virus enters the cytoplasm. [000524] The term “TRIM25” as used herein refers to an E3 ubiquitin ligase involved in signaling by RIG-1 and MDA-5 for the activation of MAVs. [000525] The terms “variants”, “mutants”, and “derivatives” are used herein to refer to nucleotide or polypeptide sequences with substantial identity to a reference nucleotide or polypeptide sequence. The differences in the sequences may be the result of changes, either naturally or by design, in sequence or structure. Natural changes may arise during the course of normal replication or duplication in nature of the particular nucleic acid sequence. Designed changes may be specifically designed and introduced into the sequence for specific purposes. Such specific changes may be made in vitro using a variety of mutagenesis techniques. Such sequence variants generated specifically may be referred to as “mutants” or “derivatives” of the original sequence. A skilled artisan likewise can produce polypeptide variants having single or multiple amino acid substitutions, deletions, additions or replacements, but biologically equivalent to the wild type sequence. These variants may include inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non- conservative amino acids; (b) variants in which one or more amino acids are added; (c) variants in which at least one amino acid includes a substituent group; (d) variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at conserved or non-conserved positions; and (d) variants in which a target protein is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the target protein, for example, an epitope for an antibody. The techniques for obtaining such variants, including, but not limited to, genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques, are known to the skilled artisan. [000526] The term “vascular permeability” as used herein means the net amount of a solute, typically a macromolecule, that has crossed a vascular bed and accumulated in the interstitium in response to a vascular permeabilizing agent or at a site of pathological angiogenesis. [Nagy, JA, et al. Angiogenesis (2008) 11(2): 1009-119]. [000527] The term “viral load” as used herein refers to a measurement of the amount of a virus in an organism, typically in the bloodstream, usually stated in virus particles per milliliter. [000528] The term “wild-type” as used herein refers to the most common phenotype of an organism, strain, gene, protein, nucleic acid, or characteristic as it occurs in nature. The terms “wild-type” and “naturally occurring” are used interchangeably. [000529] The term “WNT proteins” as used herein refers to a group of secreted lipid- modified signaling proteins that activate various pathways in different types of cells. These pathways can be classified as canonical and non-canonical Wnt pathways [Qu, F. et al. Frontiers in Biosci. (2013) 18: 493-503, citing Du, SJ et al. Mol. Cell Biol. (1995) 15: 2625- 34; Kuhl, M. et al., Trends Genet (2000) 16: 279-83; Komiya, Y., and Habas, R. Organogenesis (2008) 42: 68-75; Miller, JR, et al. Oncogene (1999) 1855: 7860-7]), and play a variety of important roles in physiological processes such as embryonic development and cell differentiation [Id., citing Logan, CY, and Nusse, R. Annu. Rev. Cell Dev. Biol. (2004) 20: 781-810; Moon, RT, et al. Nat. Rev. Genet. (2004) 5: 691-701; Murdoch, B. et al. Proc. Natl Acad. Sci. USA (2003) 100: 3422-27]. The interaction of extracellular Wnt ligands with their receptors initiates Wnt signaling. In the canonical Wnt pathway, some members of the Wnt family, such as Wnt1 and WNT3A, interact with Frizzled (FZD) receptors and their co- receptors, low-density lipoprotein receptor-related receptor 5/6 (LRP5/6), activating Dishevelled (DSH) family proteins, and leading to a change in the amount of nuclear β-catenin [Id., cigin Komiya, Y., Habas, R. Organogenesis (2008) 42: 68-75; Miller, JR, et al. Oncogene (1999) 1855: 7860-72; Kawano, Y., Kypta, R. J. Cell Sci. (2003) 116: 2627-34; Nelson, WJ, Nusse, R. Science (2004) 303: 1483-87]. DSH protects β-catenin from proteolytic degradation triggered by another complex comprising axin, glycogen synthase kinase-3 and the adenomatous polyposis coli protein. The blockade of β-catenin destruction raises the levels of cytoplasmic β-catenin, thus increasing the likelihood of some β-catenin translocating to the nucleus. This nuclear β-catenin then binds to T-cell factor (TCF)/lymphoid enhancer-binding factor transcription factors and promotes specific gene expression [ id., citing Macdonald, BT, et al. Cell (2007) 131: 1204.e1-1204.e2]. The canonical and non-canonical pathways differ in terms of the specific ligands that activate each pathway, and whether or not β-catenin is activated. The canonical pathway employs β-catenin to transmit signals, while the non- canonical pathway utilizes calcium/calmodulin-dependent kinase II (CaMKII) or Rho to exert its effects. In the non-canonical Wnt pathway, Wnt4, WNT5A, and Wnt11 activate the planar cell polarity (PCP) and the Wnt/Ca2+/CaMKII pathways. WNT3A has been regarded as an activator of the canonical Wnt signaling pathway. Recent evidence showed that treatment of primary adult human articular chondrocytes (AHACs) with recombinant WNT3A resulted in the activation of the non-canonical Wnt/Ca2+/CaMKII pathway [Id., citing Nalesso, G. et al., J. Cell Biol. (2011) 193: 551-54]. The PCP pathway is involved in the regulation of cytoskeletal structure, while the Wnt/Ca2+/CaMKII pathway regulates intracellular Ca2+ levels. Ligand binding causes an increase in intracellular Ca2+, which in turn activates CaMKII. CaMKII activates transforming growth factor (TGF)-β-activated kinase and Nemo-like kinase, which can interfere with TCF/β-catenin signaling in the canonical pathway [Id., citing Semenov, MV et al. Cell (2007) 131: 1378.e1-1378.e2]. Both the canonical and non-canonical Wnt pathways are involved in skeletal development, chondrodysplasia in embryonic life, and in the postnatal development of osteoarthritis [Id., citing Church, V. et al. J. Cell Sci. (2002z) 115: 4809-18; Akiyama, H. e t al. Genes Dev. (2004) 18: 1072-87; Lories, RJ et al., Arthritis Rheum. (2007) 56: 4095-4103; Chen, M. et al. J. Cell Sci. (2008) 121: 1455-65]. Wnt pathways also play a role in the multilineage commitment of adult MSCs (DeBoer, J. et al. Bone (2004) 34: 818-26; Etheridge, SL et al. Stem Cells (2004) 22: 849-60). Embodiments [000530] According to one aspect the described invention provides a method for reducing damaging effects of a severe virus infection in a subject comprising administering a pharmaceutical composition comprising a vehicle/carrier and a recombinant bifunctional fusion protein comprising a recombinant biologically active immunomodulatory component operatively linked to a recombinant biologically active antiviral component. [000531] According to some embodiments, the recombinant immunomodulatory component comprises a recombinant human trefoil factor 1, a sequence variant or a splice- variant thereof. According to some embodiments, the recombinant immunomodulatory component comprises a recombinant human trefoil factor 2, a sequence variant, or a splice- variant thereof. According to some embodiments, the recombinant immunomodulatory component comprises a recombinant human trefoil factor 2, a sequence variant or a splice- variant thereof. [000532] According to some embodiments, the recombinant fusion protein comprises a recombinant biologically active human TFF1 molecule, fragment or variant thereof, a recombinant human TFF2 molecule, fragment or variant thereof, or a recombinant human TFF3 molecule, fragment or variant thereof joined by its C-terminal end to a linker sequence, which is joined to an N-terminal end of a recombinant biologically active interferon molecule, fragment or variant. According to some embodiments, the linker can be from 6-20 amino acids in length, inclusive. According to some embodiments, the recombinant interferon component is pegylated. According to some embodiments the C-terminal end of the recombinant interferon molecule, fragment or variant sequence is further joined to a recombinant Fc derived antibody domain comprising a constant region of a human immunoglobulin heavy chain. According to some embodiments, the Fc component can: increase stability and aggregation resistance of the recombinant fusion protein; . extend serum half-life of the recombinant fusion protein; enhance Fc-mediated effector functions of the recombinant fusion protein; or all of the above. [000533] According to some embodiments, activity of the recombinant TFF1, TFF2 or TFF3 component of the recombinant fusion protein may be assayed in an animal model of inflammation. [See, e.g., Hoffmann, W, W. Intl. J. Mol. Sci. (2021) 22 (9): 4909. doi.og/10.3390/ijms22094909]. According to some embodiments, activity of the IFN component of the recombinant fusion protein may be assayed by showing inhibition of virus replication, activation of APCs, notably DCs [See, e.g., Kolumam, et al. J. Exp. Med. (2005) 202 (5): 637-50, citing Luft, T. et al. J. Immunol. (1998) 161: 1947-56; Biron, CA Immunity (2001) 14: 661-64; Le Bon, A. et al. Nat. Immunol. (2003) 4: 1009-15; Krug, A.et al, J. Exp. Med. (2003) 197: 899-906] and TH1 cytokine production, effector differentiation, proliferation etc. [Id., citing Parronchi, P. et al. J. Immunol. (1992) 149: 2977-83; Wenner, CA et al., J. Immunol. (1996) J. Immunol.156: 1442-7; Marrack, P. et al. J. Exp. Med. (1999) 189: 521- 30; Curtsinger, JM, et al. J. Immunol. (2005) 174: 4465-9]. [000534] According to some embodiments, the recombinant interferon molecule is a recombinant type I interferon or biologically active fragment thereof selected from IFN-α, IFN- β, IFN-ε, IFN-ω, IFN-κ, IFN-δ, IFN-τ and IFN-ζ. [Li, S. et al. Cell Physiol. Biochem. (2018) 51: 2377-96]. According to some embodiments, the recombinant IFN is a human interferon. According to some embodiments, the recombinant interferon molecule is a recombinant human IFN-α of SEQ ID NO: 57 (NCBI Ref NM_000605.4)._According to some embodiments, the recombinant interferon molecule is a recombinant human IFN-κ of SEQ ID NO: 54 (NCBI Ref NM_020124.3). According to some embodiments, the recombinant interferon is a recombinant human IFN-ω of SEQ ID NO: 55 (NCBI Ref. NM 0021773). According to some embodiments, the recombinant interferon is a recombinant human interferon-τ as disclosed by Chon, TW and Bixler, S, J. Interferon & Cytokine Res. (2010) 30 (7): 477-85: [000535] MAFVLSLLMALVLVSYGPGGSLGCDLSQNHVLVGRKNLRLLDEMRR LSPHFCLQDRKDFALPQEMVEGGQLQEAQAISVLHEMLQQSFNLFHTEHSSAAWDT TLLEPCRTGLHQQLDNLDACLGQVMGEEDSALGRTGPTLALKRYFQGIHVLKEKGY SDCAWETVRLEIMRSFSSLISLQERLRMMDGDLSSP (SEQ ID NO: 34) [000536] According to some embodiments, the recombinant Fc-derived antibody domain is a recombinant human protein of SEQ ID NO: 56 (NCBI Ref: 4CDH_A). [000537] According to some embodiments, the amino acid sequence of the recombinant hTFF1-IFNκ-IgG1 fusion protein is SEQ ID NO: 66:
Figure imgf000185_0001
RRKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK where Green = Homo sapiens TFF1 ORF w/o Leader, Blue = linker sequence, Gold = homo sapiens interferon kappa w/o Leader, Black = Homo sapiens human IgG1 Fc domain. [000539] According to some embodiments, the amino acid sequence of the recombinant hTFF2-IFNκ-IgG1 Fc fusion protein is SEQ ID NO: 35:
Figure imgf000185_0002
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK where Green = Homo sapiens TFF2 ORF w/o Leader, Blue = linker sequence, Gold = homo sapiens interferon kappa w/o Leader, Black = Homo sapiens human IgG1 Fc domain. [000541] According to some embodiments, the amino acid sequence of the recombinant hTFF3-IFNκ-IgG1 fusion protein is SEQ ID NO: 67:
Figure imgf000186_0001
RKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK where Green = Homo sapiens TFF3 ORF w/o Leader, Blue = linker sequence, Gold = homo sapiens interferon kappa w/o Leader, Black = Homo sapiens human IgG1 Fc domain. [000543] According to some embodiments, the sequences are codon-optimized to improve gene expression. According to some embodiments, the recombinant fusion protein is produced in CHO cells. According to some embodiments, the severe virus infection is a severe respiratory virus infection. According to some embodiments the respiratory virus is a cytomegalovirus. According to some embodiments the respiratory virus is Hanta virus. According to some embodiments the respiratory virus is an influenza virus. According to some embodiments, the respiratory virus is a MERS virus. According to some embodiments the respiratory virus is a respiratory syncytial virus. According to some embodiments, the respiratory virus is a SARS-CoV virus. According to some embodiments, the respiratory virus is Zika virus. According to some embodiments, the respiratory virus is a West Nile virus. According to some embodiments, the respiratory virus is a dengue virus. According to some embodiments, the respiratory virus is a Japanese encephalitis virus. According to some embodiments, the respiratory virus is a tick-borne encephalitis virus. According to some embodiments, the respiratory virus is a yellow fever virus. According to some embodiments, the respiratory virus is a rhinovirus. According to some embodiments, the respiratory virus is an adenovirus. According to some embodiments, the respiratory virus is a herpes virus. According to some embodiments, the respiratory virus is an Epstein Barr virus. According to some embodiments, the respiratory virus is a measles virus. According to some embodiments, the respiratory virus is a mumps virus. According to some embodiments, the respiratory virus is a rotavirus. According to some embodiments, the respiratory virus is a cocksackie virus. According to some embodiments, the respiratory virus is a norovirus. According to some embodiments, the respiratory virus is an encephalomyocarditis virus (EMCV). [000544] According to some embodiments, the composition of the described invention is cytoprotective, meaning it protects cells from damaging effects of a severe virus infection. According to some embodiments, the composition is cytoprotective to one or more of gastrointestinal tissue, lung tissue, heart tissue, kidney tissue, brain tissue or vascular tissue According to some embodiments, the composition of the described invention is cytoprotective to mucosal tissue. According to some embodiments, the mucosal tissue is lung tissue. According to some embodiments, the mucosal tissue is gastrointestinal tissue. [000545] According to some embodiments, the damaging effects of a severe virus infection comprise primary viral pneumonia. According to some embodiments, the damaging effects of a severe virus infection comprise superimposed bacterial pneumonia. According to some embodiments, the damaging effects of a severe virus infection comprise disruption or injury to alveolar epithelium, endothelium or both. According to some embodiments, the damaging effects of a severe virus infection comprise acute lung injury (ALI). According to some embodiments, the damaging effects of a severe virus infection comprise acute respiratory distress syndrome (ARDS). According to some embodiments, the damaging effects of a severe virus infection comprise symptoms of shock, including low blood pressure, lightheadedness, shortness of breath, and rash. According to some embodiments, the damaging effects of a severe virus infection comprise excessive complement activation. According to some embodiments, the effects comprise a pathological increase in vascular permeability. According to some embodiments, the damaging effects of a severe virus infection comprise thrombotic complications. According to some embodiments, the thrombotic complications include one or more of formation of pulmonary microthrombi, acute pulmonary embolism, deep-vein thrombosis, ischemic stroke, myocardial infarction, or systemic arterial embolism. According to some embodiments, the damaging effects of a severe virus infection comprise kidney damage. According to some embodiments, the damaging effects of a severe virus infection comprise elevated concentrations of one or more inflammatory mediator in plasma (hypercytokinemia), compared to a normal healthy subject. According to some embodiments, the inflammatory mediator is one or more of complement, prostaglandin D2, vasoactive intestinal peptide (VIP), interleukin-1-beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 (IL-12), IL-17, or tumor necrosis factor-alpha (TNF-α). [000546] According to some embodiments, the composition stimulates repair of a mucosal injury. According to some embodiments, the repair comprises epithelial proliferation. According to some embodiments, the repair restores an epithelial barrier, an endothelial barrier or both. According to some embodiments, the composition modulates an immune response. According to some embodiments, the immune response comprises recruitment of innate and adaptive immune cells. According to some embodiments, the innate immune cells comprise macrophages, dendritic cells (DCs), innate lymphoid cells (ILCs), and natural killer cells (NKs). According to some embodiments, the adaptive immune cells include αβ T cells, γδT cells, and B cells. [000547] According to some embodiments, the damaging effects comprise one or more of endothelial activation, a loss of barrier function and consequent microvascular leak. The term “endothelial activation” refers to changes to the endothelium under the stimulation of agents that allow it to participate in the inflammatory response. [Hunt, B.J., K.M. Jurd, BMJ (1998) 316 (7141): 1328-29]. The five core changes of endothelial cell activation are loss of vascular integrity; expression of leucocyte adhesion molecules; change in phenotype from antithrombotic to prothrombotic; cytokine production; and upregulation of HLA molecules. Loss of vascular integrity can expose subendothelium and cause the efflux of fluids from the intravascular space. Upregulation of leucocyte adhesion molecules such as E-selectin, ICAM- 1, and VCAM-1 allows leucocytes to adhere to endothelium and then move into the tissues. [Id., citing Adams, DH, Shaw, S. Lancet (1994) 343: 831-36] The prothrombotic effects of endothelial cell activation include loss of the surface anticoagulant molecules thrombomodulin and heparan sulphate; reduced fibrinolytic potential due to enhanced plasminogen activator inhibitor type 1 release; loss of the platelet antiaggregatory effects of ecto-ADPases and prostacyclin; and production of platelet activating factor, nitric oxide, and expression of tissue factor. [Id., citing Bach, FH et al. Nature Medicine (1995) 1: 869-73] Cytokines are synthesized, including interleukin [Id., citing Pober, JS, et al. Transplantation (1996) 61: 343- 49], which regulates the acute phase response, and chemoattractants such as interleukin [Id., citing Rajavashisth, TB et al. Arterioscler. Thromb. Vasc. Biol. (1995) 15: 1591-98] and monocyte chemoattractant protein 1 [Id., citing Mantovani, A. et al. Thromb. Haemost. (1997) 78: 406-14]. Expression of class II HLA molecules allows endothelial cells to act as antigen presenting cells, especially important in transplant rejection. [Id., citing Pober, JS, et al. Transplantation (1996) 61: 343-49]. Two stages of endothelial cell activation exist [Id., citing Bach, FH et al. Nature Medicine (1995) 1: 869-73]; the first, endothelial cell stimulation or endothelial cell activation type I, does not require de novo protein synthesis or gene upregulation and occurs rapidly. Effects include the retraction of endothelial cells, expression of P selectin, and release of von Willebrand factor. The second response, endothelial cell activation type II, requires time for the stimulating agent to cause an effect via gene transcription and protein synthesis. The genes involved are those for adhesion molecules, cytokines, and tissue factor. is induced by a wide range of agents such as certain bacteria and viruses, interleukin 1 and tumor necrosis factor, physical and oxidative stress, oxidized low density lipoproteins, [Id., citing Rajavashisth, TB et al. Arterioscler. Thromb. Vasc. Biol. (1995) 15: 1591-98] and antiendothelial cell antibodies (found in systemic autoimmune diseases such as the vasculitides, systemic lupus erythematosus, and antiphospholipid syndrome [Id., citing Meroni, P. et al. Lupus (1995) 4: 95-99]. Endothelial cell activation is a graded rather than an all or nothing response—for example, changes in endothelial cell integrity range from simple increases in local permeability to major endothelial cell contraction, exposing large areas of subendothelium. Activation may occur locally, as in transplant rejection, [Id., citing Bach, FH et al. Nature Medicine (1995) 1: 869-73] or systemically, as in septicemia and the systemic inflammatory response. [000548] The described invention relates to all routes of administration including intramuscular, subcutaneous, sublingual, intravenous, intraperitoneal, intranasal, intratracheal, topical, intradermal, intramucosal, intracavernous, intrarectal, into a sinus, gastrointestinal, intraductal, intrathecal, intraventricular, intrapulmonary, into an abscess, intraarticular, subpericardial, into an axilla, into the pleural space, intradermal, intrabuccal, transmucosal, transdermal, via inhalation, via insufflation, via nebulizer, and via subcutaneous injection. According to some embodiments, the administering is parenterally. According to some embodiments, the administering occurs by inhalation. According to some embodiments, the administering is by insufflation. [000549] Respiratory Delivery. The respiratory tract originates from the nose and terminates deep in the lung at the alveolar sac. The upper respiratory tract is composed of the nose, pharynx and nasal cavity, whereas the lower tract is composed of the larynx, bronchi and alveoli. On the basis of functional zones, the respiratory system can be divided into two regions, the conducting airway and the respiratory region. [Thakur, AK et al. Ch. 22 in Targeting Chronic Inflammatory Lung Diseases Using Advanced Drug Delivery Systems (2020) pp.475- 491]. The conducting airway includes the nasal cavity, sinuses, nasopharynx, oropharynx, larynx, trachea, bronchi and bronchioles. The main physiological functions of this airway are filtering and conditioning of the air. The respiratory airway includes the bronchioles, terminal bronchioles, alveolar ducts and alveolar sacs. Around 300 million alveoli are present in the lungs. The entire pulmonary region is lined by a continuous layer of pulmonary epithelial cells, which performs such functions as acting as a barrier and protection of the region by secreting mucus, surfactant proteins and antimicrobial peptides, repairing and regenerating epithelial cells; and modulating the response of smooth muscle and inflammatory cells. Goblet cells are the mucus-secreting cells that secrete mucus glycoproteins responsible for trapping and removing particles. [000550] Some approaches to enhance the residence time of a delivery system in the lungs include molecular engineering, and use of penetration enhancers, enzyme inhibitors, and bioadhesive polymers. [000551] Particle deposition in the lungs is governed by size, shape and density of the particles. The principle mechanisms guiding inhaled particle deposition in the respiratory airway include sedimentation due to gravity, Brownian diffusion, and impaction by inertial force (meaning the condition of being pressed closely together and firmly fixed). Larger particles (>10μm) are retained in the oropharyngeal region and larynx due to impaction. Particles between 2μm and 10 μm normally are deposited in the tracheobronchial region. Particles of 0.5 μm -2μm normally are deposited in the alveoli and small conducting airways due to gravitational sedimentation. Particles < 0.5μm generally are not deposited and are expelled in exhalation [Id., citing Moreno-Sastre, M. et al. J. Antimicrob. Chemother. (2015) 70: 2945-55; Yu, CP and Taulbee, DB. Inhaled Part. (1975) 4 (Pt.1): 35-47]. The magnitude of deposition of inhaled particles in lungs varies with particle parameters (diameter and density), breathing parameters (particle velocity and residence time), and morphometric parameters (branching angle airway radius and gravity angle. Impaction is most effective when air and particle velocities are higher than in the peripheral region of the lungs. In the alveolar region, particle deposition is mainly guided by diffusion and sedimentation due to smaller velocities and hence longer residence times [Id., citing Hoffmann, W. J. Aerosol Sci. (2011) 42: 693- 724]. [000552] Numerous natural polymers, synthetic polymers, and copolymers have been studied as carriers for pulmonary drug delivery [Vinjamuri, BP et al. Chapter 12 in Applications of Polymers in Drug Delivery (2021) pp. 333-354, doi.org/10.1016/B978-0-12-819659- 5.00012-4, citing Sheth, P. and Myrdal, PB. In Smyth, HDC, Hickey, AJ Eds, Controlled Pulmonary Drug Delivery, Springer, New York ( 2011)]. Examples of widely used natural polymers include albumin, chitosan, gelatin, and hyaluronic acid. Albumin facilitates high tissue retention of respiratory therapeutics, thereby providing high localized or systemic drug concentration in lung tissue [Id., citing Li, Q. et al J. Microencapsul. (2001) 18: 825-9; Choi, SH et al. J. Controlled Release (2015) 197: 199-207; Woods, A. et al J. Controlled Release (2015) 210: 1-9; Seo, J. et al. Pulmon. Pharacol. Ther. (2016) 36: 53-61], Spray-dried alginate microparticle formulations comprising sodium alginate and sodium carboxymethyl cellulose in 1:1 weight ratio produced inhalable microspheres with an aerodynamic diameter of 4.68 ± 0.23 μm [Id., citing Shahin, HI et al. J. Controlled Release (2019) 302: 126-139]. Chitosan, which is obtained by partial deactylation of chitin, is a copolymer linked by β-1,4 of D- glucosamine and N-acetylglucosamine carrying a cationic charge, thereby adhering to negatively charged surfaces and chelating metal ions; it is biocompatible, biodegradable, mucoadhesive, antibacterial and nontoxic, although poorly water soluble [Id., citing Yamamoto, H. et al. J. Controlled Release (2005) 102: 373-81]. Commonly used synthetic polymers include polylactic acid (PLA), polyethylene glycol (PEG), polyvinyl alcohol, and acrylyic acid derivatives. Polylactic-co-glycolic acid (PLGA) is chemically composed of PLA and polyglycolic acid (PGA). PLGA particles degrade by bulk erosion, undergoing hydrolysis to PLA and PGA. [Id., citing Ungaro, F. et al. J. Pharm. Pharmacol. (2012) 64: 1217-35]. PLGA and gelatin-based nanoparticles have been used for pulmonary protein and DNA delivery [Id., citing Menon, JU et al, Acta Biomater. (2014) 10: 2643-52]. [000553] According to some embodiments, the carrier is a controlled release carrier. The term “controlled release” is intended to refer to any drug-containing formulation in which the manner and profile of drug release from the formulation are controlled. This includes immediate as well as non-immediate release formulations, with non-immediate release formulations including, but not limited to, sustained release and delayed release formulations. According to some embodiments, the controlled release of the pharmaceutical formulation is mediated by changes in temperature. According to some other embodiments, the controlled release of the pharmaceutical formulation is mediated by changes in pH. [000554] According to some embodiments, the carrier is a delayed release carrier. According to another embodiment, the delayed release carrier comprises a biodegradable polymer. According to another embodiment, the biodegradable polymer is a synthetic polymer. According to another embodiment, the biodegradable polymer is a naturally occurring polymer. [000555] According to some embodiments, the carrier is a sustained release carrier. According to another embodiment, the sustained-release carrier comprises a biodegradable polymer. According to another embodiment, the biodegradable polymer is a synthetic polymer. According to another embodiment, the biodegradable polymer is a naturally occurring polymer. [000556] According to some embodiments, the carrier is a short-term release carrier. The term “short-term” release, as used herein, means that the carrier is constructed and arranged to deliver therapeutic levels of the biologically active fusion protein for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. According to some other embodiments, the short term release carrier delivers therapeutic levels of the biologically active fusion protein for about 1, 2, 3, or 4 days. [000557] According to some embodiments, the carrier is a long-term release carrier. The term “long-term” release, as used herein, means that the carrier is constructed and arranged to deliver therapeutic levels of the recombinant fusion protein for at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. According to another embodiment, the long-term-release carrier comprises a biodegradable polymer. According to another embodiment, the biodegradable polymer is a synthetic polymer. [000558] According to some embodiments, the recombinant fusion protein of the present disclosure can be covalently attached to polyethylene glycol (PEG) polymer chains. According to some other embodiments, the recombinant fusion protein of the present disclosure is stapled with hydrocarbons to generate hydrocarbon-stapled fusion proteins that are capable of forming stable alpha-helical structure (Schafmeister, C. et al., J. Am. Chem. Soc., 2000, 122, 5891- 5892, incorporated herein by reference in its entirety). [000559] According to some embodiments, the carrier comprises particles. According to some other embodiments, the recombinant fusion protein of the present invention is encapsulated or entrapped into microspheres, nanocapsules, liposomes, or microemulsions, or comprises d-amino acids in order to increase stability, to lengthen delivery, or to alter activity of the recombinant fusion protein. These techniques can lengthen the stability and release simultaneously by hours to days, or delay the uptake of the drug by nearby cells. [000560] According to some embodiments, the composition is formulated as a solution. According to some embodiments, a carbohydrate, e.g., a polyol (e.g., mannitol, sorbitol, or xylitol) trehalose, or lactose may be used as an excipient to protect the protein from denaturation/degradation upon lyophilization, spray drying and reconstitution and to improve aerosol performance. [See, e.g., Andya, JD et al. Pharm. Res. (1999) 16 (30): 350-8; Constantino, HR et al. J. Pharm Sci. (1998) 87 (11): 2406-11]. [000561] For administration by inhalation, the compositions for use according to the described invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. [000562] According to some embodiments, the pharmaceutical composition is packaged in an inhalation device, including, for example, but not limited to a nebulizer, a metered-dose inhaler (MDI), and a dry powder inhaler (DPI). [000563] According to some embodiments, the pharmaceutical composition is a liquid for aerosolized delivery using a nebulizer. According to some such embodiments, the flow-rate of the pharmaceutical composition is at least 0.3 ml/min, and the pharmaceutical composition is delivered as 2 mm particles, with distribution into deepest alveoli. [000564] According to some other embodiments, the therapeutic amount of the pharmaceutical composition is administered via an inhalation device. Examples of the inhalation device that can be used for administering the pharmaceutical composition include, but are not limited to, a nebulizer, a metered-dose inhaler (MDI), a dry powder inhaler (DPI), and a dry powder nebulizer. [000565] According to some embodiments, the dry powder is produced by a spray drying process. According to some other embodiments, the dry powder is produced by micronization. According to another embodiment, the dry powder comprises microparticles with Mass Median Aerodynamic Diameter (MMAD) of 1 to 5 microns. [000566] Particle characteristics play a role in performance of polymeric particulate systems, which is dependent on the amount of drug loaded into the particle. For example, if the load percent is low, drug diffusion from a particulate delivery system comprising large particle size may be slow compared to that from small particles or a colloidal dispersion, because in the latter, the drug has a shorter path to cover for diffusion to take place. If the load percent is high, however, then the larger particles have a faster diffusion rate than small particles due to the amount of drug loaded into the microparticle available space. Higher load or amount of drug leads to a faster rate of diffusion since flux (diffusion) is directly proportional to the concentration gradient. Small particles tend to have a low drug load, and the very small may actually be empty. [000567] Nanoparticles are solid particles ranging in size from 1 nm to 1000 nm Some potential advantages of nanoparticles for pulmonary delivery include sustained release, drug targeting, dose reduction, and improved patient compliance. Prolonged residence time of nanoparticles in pulmonary regions may be achieved through the use of mucoadhesive polymeric materials. [000568] Microparticles are solid particles ranging in size from 1 μm to 1000 μm. Porous microparticles possess improved aerolization due to small aerodynamic diameters and low bulk densities, thereby providing better deposition in pulmonary regions. Mucoadhesion is commonly defined as the adhesion between two materials, at least one of which is a mucosal surface. Mucoadhesive microparticles have the capacity to prolong the residence time and reduce mucociliary clearance. [000569] According to some embodiments, the pharmaceutical composition further comprises at least one additional therapeutic agent. According to some embodiments, the additional therapeutic agent is selected from a supportive therapy, an immunomodulatory agent, an analgesic agent, an anti-inflammatory agent, an anti-infective agent, an anti-malarial agent, an anti-viral agent or an anti-fibrotic agent. [000570] According to some embodiments, the supportive therapy is therapeutic apheresis comprising a virion removing step. According to some embodiments, the therapeutic apheresis reduces viral load. [000571] According to some embodiments, each of these agents can be used alone as a monotherapy or in combination with a second agent, e.g., an immunomodulatory agent with a second immunomodulatory agent, e.g., methotrexate; an immunomodulatory agent with an analgesic agent; an immunomodulatory agent with an anti-inflammatory agent; an immunomodulatory agent with an anti-infective agent; an immunomodulatory agent with an anti-malarial agent; an immunomodulatory agent with an anti-viral agent; an immunomodulatory agent with an antifibrotic agent; or an immunomodulatory agent with a supportive therapy. [000572] Exemplary approaches to the inhibition of specific cytokines include drugs that inhibit cytokine synthesis (e.g., glucocorticoids, cyclosporine A, tacrolimus, myophnolate- helper lymphocyte (Th2)-selective inhibitors), humanized blocking antibodies to cytokines or their receptors; soluble receptors that mop up secreted cytokines, low molecular weight receptor antagonists, and drugs that block the signal transduction pathways activated by cytokines. [000573] Cyclosporine, tacrolimus and sirolimus are immunosuppressive medications that inhibit T cell activation through a series of calcium-dependent signal events involved in cytokine gene transcription. Cyclosporine inhibits the activation of helper T cells. Tacrolimus also interferes with T cell receptor-dependent cell activation. CSA and tacrolimus inhibit IL- 2, IL-3, Il-4, IFN-γ, GM-CSF, and TNFα production. Transcriptional factors nuclear factor of activated T cells (NF-AT), NF-κB, and PU-box are inhibited by tacrolimus. T cell receptor mediated apoptosis of lymphocytes and thymocytes is augmented by tacrolimus. Sirolimus (rapamycin) is a macrocyclic lactone produced by Streptomyces hygroscopicis that inhibits T lymphocyte activation and proliferation that occurs in response to antigenic and cytokine stimulation; it binds intracellularly to the immunophilin, FK binding protein-12, which becomes an immunosuppressive complex, which in turn binds to and inhibits activation of mammalian regulatory kinase (target of rapamycin, mTOR). This inhibition suppresses cytokine-driven T cell proliferation, inhibiting the progression from the G1 to S phases of the cell cycle. [Nelson, RP, and Ballow, M. J. Allergy Clin. Immunol. (2003) 111 (2): S720- 732]. [000574] According to some embodiments, the immunomodulatory agent is a recombinant interferon, e.g., one or more of IFN-α; IFN-α-2b, IFN-β, IFN-γ, IFN-κ, or IFN-ω. According to some embodiments, the recombinant interferon is in a PEGylated form. [000575] According to some embodiments, the immunomodulatory agent is a recombinant IL-2 receptor inhibitor (e.g., denileukin diftitox). [000576] According to some embodiments, the immunomodulatory agent is a phosphodiesterase 4 (PDE4) inhibitor, e.g., cilomilast, a second generation PDE4 inhibitor with anti-inflammatory effects that target bronchoconstriction, mucus hypersecretion and airway remodeling. [Giembycz, MA. Br. J. Clin. Pharmacol. (2006) 62(2): 138-52]. [000577] According to some embodiments, the immunomodulatory agent is a hyperimmune globulin prepared from a donor with high titers of a desired antibody. Examples include cytomegalovirus immunoglobulin iv; respiratory syncytial virus immune globulin iv (RSV-IGIV); and palivizumab, a humanized mouse monoclonal antibody given IM. [000578] According to some embodiments, the immunomodulatory agent targets pro- inflammatory cytokines. According to some embodiments, the immunomodulatory agent is a TNFα inhibitor/antagonist [e.g., etanercept; adalimumab; infliximab, certolizumab pegol, golimumab]. [000579] According to some embodiments, the immunomodulatory agent is an IL-1β inhibitor [e.g., rilonacept; canakinumab; Anakinra]. [000580] According to some embodiments, the immunomodulatory agent is chimeric IL- 1Ra. This molecule is a fusion of the N-terminal peptide of IL-1β and IL-1Ra, resulting in inactive IL-1Ra. Because the IL-1β N-terminal peptide contains several protease sites clustered around the caspase-1 site, local proteases at sites of inflammation can cleave chimeric IL-1Ra and activate IL-1Ra. [Rider, P. et al. J. Immunol. (2015) 195.doi: 10.4049/jimmunol.1501168]. [000581] According to some embodiments, the immunomodulatory agent is an IL-6 inhibitor [e.g., tocilizumab, siltuximab, sarilumab, olokizumab, or sirukumab]. [000582] According to some embodiments, the immunomodulatory agent is an IL-12/ IL- 23 inhibitor (e.g., ustekinumab, briakinumab), or an IL23 inhibitor (e.g., guselkumab, tildrakizumab). [000583] According to some embodiments, the immunomodulatory agent targets cytokine signaling pathways, e.g., compounds targeting TLR-4 signaling, e.g., enamionone E121 (ethyl 4-(4’-chlorophenyl) amino-6 methyl-2-oxocytlohex-3-en-1-aote), an aniline enaminone); JODI 18b and 19, [Szollosi, DE et al. J. Pharmacy & Pharmacol. (2018) 70: 18- 26]; TAK-242 (resatorvid, Takeda), TLR-C34, a 2-acetamidopyranoside that inhibits TLR4 signaling [Olusayo, A., et al. J. Applied Toxicol (2019) 39(4). Doi: 10/1002/jat.3771]; C35 [Neal, MD. Et al. PLoS One (2013) 8 (6): e65779]. [000584] According to some embodiments, the immunomodulatory agent is a p38 MAPK inhibitor, e.g., SB203580, 4-(4’-Fluorophenyl)-2-(4’-methylsulfinylphenyl)-5- (4’-pyridyl)- imidazole, a pyridinyl imidazole inhibitor used to elucidate the roles of p38 mitogen-activated protein (MAP) kinase [Cuenda, A. et al. FEBS Lett. (1995) 364: 229-33]; SB203580 inhibits also the phosphorylation and activation of protein kinase B (PKB, also known as Akt) [Lali, F.V. et al. J. Biol. Chem. (2000) 275 (10): 7395-402]. SB239063 [trans-4-[4-(4-Fluorophenyl)- 5-(2-methoxy-4-pyrimidinyl)-1H-imidazol-1-yl]cyclohexanol; Barone. Et al J. Pharmacol. Exp. Ther (2001) 296: 312 [PMID 11160612], and RWJ 67657 (4-[4-(4-Fluorophenyl)-1-(3- phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol). Wadsworth, SA et al. J. Pharmacol. Exp. Ther. (1999) 291 (2): 680-7). [000585] According to some embodiments, the immunomodulatory agent targets Janus kinase signaling (e.g., tofacitinub, baricitinib, or upadacitinib). [000586] According to some embodiments, the immunomodulatory agent targets cell adhesion molecules to reduce leukocyte recruitment, e.g., molecules that are α4 integrin inhibitors [e.g., vedolizumab, natalizumab]. [Szollosi, DE et al. J. Pharmacy & Pharmacol. (2018) 70: 18-26]. [000587] According to some embodiments, the immunomodulatory agent is a recombinant anti-inflammatory cytokine, e.g., IL-4, IL-10, IL-11. [000588] Immunomodulation also includes therapies that boost an individual’s defenses by providing physiologic or supraphysiologic doses of exogenous cytokines, e.g., to treat viral infections. [000589] According to some embodiments, a parameter for measuring activation state of lymphocytes is cytokine release profile. For example, ELISPOT, or enzyme linked immunospot, is a technique that was developed for the detection of secreted proteins, such as cytokines and growth factors. It is performed using a PVDF or nitrocellulose membrane 96- well plate pre-coated with an antibody specific to the secreted protein. Cells are added to the plate and attach to the coated membrane. Cells are then stimulated and the secreted protein binds to the antibody. Next, a detection antibody is added that binds specifically to the bound protein. The resulting antibody complex can be detected either through enzymatic action to produce a colored substrate or with fluorescent tags. The membrane can be analyzed by manually counting the spots or with an automated reader designed for this purpose. Each secreting cell appears as a spot of color or fluorescence. [000590] According to some embodiments, another parameter for measuring activation of lymphocytes is by quantifying cellular subset differentiation. For example, the differentiation of CD45+/CD3+ T-lymphocytes to CD45+/CD3+/CD4+ helper T-lymphocytes, CD45+/CD3+/ CD8+ cytotoxic T-lymphocytes, and CD45+/CD3+/CD25+ activated T- lymphocytes can be quantified by flow cytometry analysis. [000591] According to some embodiments, the immunomodulatory agent is a corticosteroid. According to some embodiments, the corticosteroid is selected from prednisone, dexamethasone, azathioprine, mycophenolate, and mycophenolate mofetil. [000592] The term “analgesic agent” as used herein refers to an agent producing diminished sensation to pain without loss of consciousness. According to some embodiments, the analgesic agent is selected from codeine, hydrocodone, oxycodone, methadone, hydromorphone, morphine, fentanyl, and combinations thereof. [000593] An “anti-inflammatory agent” is a substance that reduces inflammation (redness, swelling, and pain) in the body by inhibiting inflammatory mediators in the body that cause inflammation. According to some embodiments, the anti-inflammatory agent is selected from aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac nabumetone, naproxen, nintedanib, oxaprozin, pirfenidone, piroxicam, salsalate, sarilumab (Kevzara®) sulindac, tolmetin, and combinations thereof. [000594] The term “anti-viral agent” as used herein means any of a group of chemical substances having the capacity to inhibit the replication of or to destroy viruses used chiefly in the treatment of viral diseases. According to some embodiments, the antiviral agent inhibits viral entry, thereby decreasing viral load. According to some embodiments, the anti-viral agent is selected from acyclovir, gancidovir, foscarnet; ribavirin; amantadine, azidodeoxythymidine /zidovudine), nevirapine, a tetrahydroimidazobenzodiazepinone (TIBO) compound; efavirenz; remdecivir, lopinavir/ritonavir, umifenovir, favipiravir, ivermectin, and delavirdine. [000595] According to some embodiments, the agent that blocks viral entry is a peptide inhibitor. According to some embodiments, the peptide inhibitor is a fusion peptide. [000596] As described in Xia, et al., Sci. Advisor (2019) 5: eaav4580, the S glycoprotein is a type 1 transmembrane glycoprotein common to all HCoVs. The S proteins consist of two subunits S1 and S2. The S1 subunit binds the cellular receptor through its receptor binding domain (RBD), followed by conformational changes in the S2 subunit, which allows the fusion peptide to insert into the host target cell membrane. The heptad repeat 1 (HR1) region in the SR2 unit forms a homotrimeric assembly, which exposes three highly conserved grooves on the surface that bind heptad repeat 2 (HR2). This six-helix bundle (6-HB) core structure formed during the fusion process helps bring the viral and cellular membranes into close proximity for viral fusion and entry. The HR region in the S2 subunit is conserved among various HCoVs and plays a pivotal role in HCoV infections by forming the 6-HB that mediates viral fusion. Furthermore, the mode of interaction between HR1 and HR2 is conserved among CoVs such that certain residues in the HR1 helices interact with certain residues in the HR2 helices. [000597] According to some embodiments, the fusion peptide is a synthetic peptide derived from HR1 and HR2 regions of SARS-CoV spike protein as described in Liu, S. et al. Lancet (2004) 363: 938-47, and as shown in Table 1 below. [000598] Table 1
Figure imgf000200_0001
[000599] As described in Liu, S. et al. Lancet (2004) 363: 938-47, the HR1 and H2 sequences tend to form a coiled-coil structure, and the amino acid sequences of peptides derived from the HR1 and HR2 regions of SARS-CoV spike protein are similar to those from the HIV-1 gp41 HR1 and HR2 regions. Peptides derived from the HR2 regions of many other enveloped viruses, including Ebola virus [Id., citing Watanabe, S. et al. J. Virol. (2000) 74: 10194-201], Newcastle disease virus [Id., citing Yu, M. et al. J. Gen. Virol. (2002) 83: 623- 29], parainfluenza virus [Id., citing Yao, Q & Compans, RW. Virology (1996) 223: 103-12], and respiratory syncytial virus [Id., citing Yu, M. et al. J. Gen. Virol. (2002) 83: 623-29] inhibit the corresponding virus infection in the micromolar range; these viruses have similar fusogenic mechanisms to HIV-1. [Id., citing Watanabe, S. et al. J. Virol. (2000) 74: 10194-201]. [000600] As described in Liu, S. et al. Lancet (2004) 363: 938-47, two major factors can affect the potency of a fusion-inhibitory peptide: the sensitivity of a virus to the corresponding antiviral peptides; and the sequence and conformation of the inhibitory peptide. [Id.]. The anti-viral activity of the peptide fusion inhibitors depends on their optimum peptide sequences and conformations. Changes in their sequences or conformations could substantially affect their antiviral activity and the stability of the complexes they form. [Id., citing 17, 47, 48]. According to some embodiments, optimization of the peptide sequence and conformation of a fusion peptide inhibitor may improve antiviral activity. According to some embodiments, a fusion peptide of Table 1 may be used as a lead in designing more potent anti-SARS-CoV peptides. [000601] As described in Lu, L. et al. Nat. Communic. (2014) 5: 3067, two peptides HR1P and HR2P, spanning residues 998-1039 in the HR1 domain [ANKFNQALGAMQTGFTTTNEAFQKVQDAVNNNAQALSKLASE, SEQ ID NO: 24] and 1251-1286 in the HR2P domain [SLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKEL, SEQ ID NO: 25], respectively, from MERS-CoV can form a stable six-helix bundle fusion core structure. HR2P can effectively inhibit MERS-CoV replication and its spike protein-mediated cell–cell fusion. Introduction of hydrophilic residues into HR2P results in significant improvement of its stability, solubility and antiviral activity. Therefore, the HR2P analogues can be further developed into effective viral fusion inhibitors. [000602] According to some embodiments, the viral entry inhibitor is peptide OC43- HR2P [LAEADANVVAQIKVLASNTADFGEQIADLANNFANAIL, SEQ ID NO: 26] as described in Xia, et al., Sci. Advisor (2019) 5: eaav4580. According to some embodiments, the viral entry inhibitor is EK1 [SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL, SEQ ID NO: 27] as described in Xia, et al., Sci. Advisor (2019) 5: eaav4580. EK1 is a modified derivative of OC43-HR2P. [000603] In order to improve the inhibitory activity of EK1, cholesterol (Chol) and palmitic acid (Palm) were covalently attached to the C-terminus of EK1 sequence under the help of a flexible polyethylene glycol (PEG) spacer, and the corresponding lipopeptides EK1C and EK1P were constructed. According to some embodiments, the viral entry inhibitor is EK1P [SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL, SEQ ID NO: 28] or a PEG-based spacer-containing derivative [PEG4-C (Palm) thereof as disclosed by Xia, S. et al. Cell Res. (2020) 30(4): 343-55. According to some embodiments, the viral entry inhibitor is EK1C [SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL, SEQ ID NO: 29] or a PEG-based spacer-containing derivative [PEG4-C (Chol)] thereof as disclosed by Xia, S. et al. Cell Res. (2020) 30(4): 343-55. [000604] As described by Xia et al. Cell Res. (2020) 30(4): 343-55, which is incorporated herein by reference, on the basis of the structure of EK1C, a series of cholesteryl EK1 with multiple linkers were constructed, where the glycine/serine-based linker, i.e., GSG, or PEG- based spacer was employed between EK1 and the cholesterol moiety. Compared with EK1C1, EK1C2 and EK1C showed similar inhibitory activities. EK1C3 peptide with both the 3-amino acid linker “GSG” and the PEG4-based spacer, exhibited 4-fold more potency than EK1C1. It is noteworthy that changing “GSG” in EK1C3 to a longer 5-amino acid linker “GSGSG” (SEQ ID NO: 30) significantly increased the inhibitory potency of the hybrid molecule, and EK1C4 had IC50 value of 1.3 nM, which was 43-fold more potent than EK1C1. These findings indicate that the linker length has a significant effect on the overall activity of lipopeptides. Comparison of increasing PEG-based arm lengths in EK1C4 shows that inhibitors potency slightly decreased in the cell–cell fusion assay. The data suggest that “GSGSG-PEG4” linker (SEQ ID NO: 30) was optimal to bridge both parts of the conjugates. Similarly, EK1C4 showed the most potent inhibitory activity against SARS-CoV-2 PsV infection, with IC50 value of 15.8 nM, providing 149-fold stronger anti-SARS-CoV-2 activity than that of EK1 (IC50 = 2,375 nM) [000605] According to some embodiments, the agent that blocks viral entry is a dipeptidyl peptidase 4 (DPP4) inhibitor. Drugs in the DPP-4 inhibitor class that are approved for use to treat type 2 diabetes include sitagliptin, saxagliptin, linagliptin, and alogliptin. [000606] According to some embodiments, the agent that blocks viral entry is an ACE2 inhibitor. Shortly after the identification of the angiotensin-converting enzyme 2 (ACE2), a metallocarboxypeptidase that mediates various cardiovascular and renal functions, peptide inhibitors of the enzyme were developed by selection of constrained peptide libraries displayed on phage [McKee, DL et al., Pharmacol. Res. (2020) 157: 104859, citing Huang, L. et al. J. Biol. Chem. (2003) 278 (18): 15532-40], the most potent inhibitor of which, termed DX600, with the amino acid sequence of Ac-GDYSHCSPLRYYPWWKCTYPDPEGGG-NH2 [SEQ ID NO: 31] had a Ki of 2.8 nm and an IC50 of 10.1 μM. Subsequent experimental studies in mice and in human cell lines revealed that DX600 is a potent ACE2 inhibitor specific for only human ACE2 [Id., citing Pedersen, KB et al. Am J. Physiol. Regul. Integr. Comp. Physiol. (2011) 301(5): R1293-99; Ye, M. et aql. Hypertension (2012) 60(3): 730-40]. Other small- peptide and tripeptide inhibitors have been developed for potent and selective inhibition of human ACE2 and inhibition of SARS-CoV cell entry in vitro [Id., citing Guy, JL et al. FEBS J. (2005) 272 (14): 3512-20; Han, DP, et al., Virology (2006) 350 (1): 15-25; Mores, A. et al. J. Med. Chem. (2008) 51(7): 2216-26]]. Synthetic small-molecule inhibitors of human ACE2, including MLN-4760 (CAS number: 305335−31-3) [Id., citing Ye, M. et al. Hypertension (2012) 60(3): 730-40; Trask, AJ et al., Am. J. Hypertens. (2010) 23(6): 687-93;], N-(2- aminoethyl)-1 aziridine-ethanamine [Id., citing Huentelmann, MJ et al. Hypertension (2004) 44(6): 903-6] and the TNF-α converting enzyme (TACE) small-molecule inhibitor TAPI-2 that blocks SARS-CoV S protein-induced shedding of ACE2 [Id., citing Haga, S. et al. Antiviral Res. (2010) 85(3): 551-55; Mohler, JM, et al., Nature (1994) 370 (6486): 218-20] have been developed for experimental inhibition of SARS-CoV cell entry. Moreover, the phytochemical nicotianamine (CAS number: 34441−14-0), a metal chelator ubiquitously present in higher plants [Id., citing Takahashi, M. et al., Plant Cell (2003) 15(6): 1263-80], was identified in high concentrations in soybean, and was shown as a potent inhibitor of human ACE2 with an IC50 of 84 nM [Id., citing Takahashi, S. et al. Biomed Res. (2015) 36(3): 219- 224]. Because dietary phytochemicals as naturally occurring compounds display a wide safety profile and less pharmacological side effects [Id., citing Naujokat, C. & McKee, DL. Curr. Med. Chem. (2020) doi: 10.2174/0929867327666200228110738], nicotianamine constitutes a candidate drug for ACE2 inhibition and thus blockade of SARS-CoV-2 cell entry. Finally, a recent study demonstrates that a clinical-grade soluble recombinant human ACE2 protein (hrsACE2) inhibits attachment of SARS-CoV-2 to simian Vero-E 6 cells, and inhibits infection of engineered human capillary organoids and kidney organoids by SARS-CoV-2 isolated from a nasopharyngeal sample of a patient with confirmed COVID-19 disease [Id., citing Monteil, V. et al. Cell (2020) doi: 10.1016/j.cell.2020.04.004], suggesting that hrsACE2 can block host cell entry of SARS-CoV-2 and early stages of SARS-CoV-2 infections. [000607] According to some embodiments, the anti-viral agent is a protease inhibitor that inhibits a host cell protease to block viral entry. According to some embodiments, the anti- viral agent is a serine protease TMPRSS2 inhibitor, e.g., camostat (FOY-305), [N,N- dimethylcarbamoylmethyl 4-(4-guanidinobenzoyloxy)-phenylacetate] methanesulfate and camostat mesilate (Foipan™), alternatively termed camostat mesylate, (NI-03), (CAS number: 59721−28-7); or Nafamostat mesilate (Buipel™), (6-amidino-2-naphthyl-4-guanidino benzoate-dimethanesulfonate) (FUT-175), (CAS number: 81525−10-2). [McKee, DL, et al. Pharmacol. Res. (2020) 157: 104859] Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases, which entails S protein cleavage at the S1/S2 and the S2’ site and allows fusion of viral and cellular membranes, a process driven by the S2 subunit. [Hoffman, M. et al. Cell (2020) 181 (2): 271-80]. SARS-CoV can use the endosomal cysteine proteases cathepsin B and L (CatB/L) [Id., citing Simmons et al., 2005) and the transmembrane serine protease TMPRSS2 [Id., citing Glowacka, I. et al. J. Virol. (2011) 85: 4122-34, Matsuyama, S. et al. J. Virol. (2010) 84: 12658-664, Shulla, K. et al. J. Virol. (2011) 85: 873-82] for S protein priming in cell lines, and inhibition of both proteases is required for robust blockade of viral entry [Id., citing Kawase, M et al. J. Virol. (2012) 86: 6537-45]. However, only TMPRSS2 activity is essential for viral spread and pathogenesis in the infected host whereas CatB/L activity is dispensable [Id., citing Iwata-Yoshikawa, N. et al. J. Virol. (2019) 93: 10.1128/JVL01815-18, Shirato, K. et al. Virology (2016) 91: 10.1128/JVL01387-16, Shirato, K. et al. Virology (2018) 517: 9-15, Zhou, P et al. Antiviral Res. (2015) 116: 76-84]. SARSCoV-2 uses the SARS- CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. [Hoffman, M. et al. Cell (2020) 181 (2): 271-80] According to some embodiments, the protease inhibitor is cysteine protease inhibitor K11777, ((2S)-N-[(1E,3S)-1- (benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{€-4-methylpiperazine-1-carbonyl]amino}-3- phenylpropanamide, or a P3 derivative thereof which inhibits SARS-CoV and Ebola virus entry. [Zhou, P et al. Antiviral Res. (2015) 116: 76-84]. [000608] According to some embodiments the anti-viral agent comprises sera from a convalescent patient, e.g., a coronavirus patient, such as a SARSCoV, a MERS, or a COVID- 19 patient. [000609] The term “anti-malarial agent as used herein refers to a substance used for treatment of clinical Plasmodium falciparum malaria According to some embodiments, the anti-malarial agent is selected from an aryl aminoalcohol compound selected from quinine, quinidine, chloroquine, amodiaquine, mefloquine, halofantrine, lumefantrine, piperaquine, and tafenoquine; an antifolate compound, selected from pyrimethamine, proguanil, chlorproguanil, trimethoprim; an artemisinin compound selected from artemisinin, dihydroartemisinin, artemether, artesunate; and atovaquone. Chloroquine phosphate inhibits terminal phosphorylation of ACE2, and hydroxychloroquine elevates the pH in endosomes which are involved in virus cell entry [McKee, DL, et al. Pharmacol. Res. (2020) 157: 104859, citing 44, 45]. The triple combination of cepharanthine (an anti-inflammatory alkaloid from Stephania cepharantha Hayata), (CAS number: 48,104,902), selamectin (an avermectin isolated from Streptomyces avermitilis and used as an anti-helminthic and parasiticide drug in veterinary medicine), (CAS number.220119−17-5), and mefloquine hydrochloride (Lariam™, used for the prophylaxis and treatment of malaria) [[57], [58], [59]] has recently been shown to inhibit infection of simian Vero E6 cells with pangolin coronavirus GX_P2V/2017/Guangxi (GX_P2V), whose S protein shares 92.2 % amino acid identity with that of SARS-CoV-2. [000610] The term “anti-malarial agent as used herein refers to a substance used for treatment of clinical Plasmodium falciparum malaria. According to some embodiments, the anti-malarial agent is an aryl aminoalcohol compound selected from quinine, quinidine, chloroquine, amodiaquine, mefloquine, halofantrine, lumefantrine, piperaquine, and tafenoquine. According to some embodiments, the anti-malarial agent is an antifolate compound selected from pyrimethamine, proguanil, chlorproguanil, and trimethoprim. According to some embodiments, the anti-malarial agent is an artemisinin compound selected from artemisinin, dihydroartemisinin, artemether, artesunate; and atovaquone. Chloroquine phosphate inhibits terminal phosphorylation of ACE2, and hydroxychloroquine elevates the pH in endosomes which are involved in virus cell entry [McKee, DL, et al. Pharmacol. Res. (2020) 157: 104859, citing Vincent, MJ et al. (2005) Virol. J.2: 69; Bari, MAA. Pharmacol. Res. Perspect. (2017) 5(1): e0029344, 45]. The triple combination of cepharanthine (an anti- inflammatory alkaloid from Stephania cepharantha Hayata), (CAS number: 48,104,902), selamectin (an avermectin isolated from Streptomyces avermitilis and used as an anti- helminthic and parasiticide drug in veterinary medicine), (CAS number. 220119−17-5), and mefloquine hydrochloride (Lariam™, used for the prophylaxis and treatment of malaria) [Id., citing Bailly, C. Phytomedicine (2019) 62: 152956; Kjoha, S. et al. Psychopharmacology (Berl.) (2018) 235 (6): 1697-1709; Tickell-Painter, M. et al. Cochrane Database Syst. Rev. (2017) 10. Doi:10.1002/14651858.CD006491.pub4. CD006491].[57], [58], [59]] has recently been shown to inhibit infection of simian Vero E6 cells with pangolin coronavirus GX_P2V/2017/Guangxi (GX_P2V), whose S protein shares 92.2 % amino acid identity with that of SARS-CoV-2. [Id., citing Fan, HH et al. China Med. J. (2020). Doi: 10.1097/CM9.0000000000000797]. [000611] The term “anti-infective agent” as used herein means any of a group of chemical substances having the capacity to inhibit the growth of, or to destroy microorganisms, used chiefly in the treatment of infectious diseases. According to some embodiments, the anti- infective agent is selected from amoxicillin, doxycycline, demeclocycline; eravacycline, minocycline, ormadacycline, tetracycline, cephalexin, defotaxime, cetazidime, cefuroxime, ceftaroline; ciprofloxacin, levofloxacin, moxifloxacin clindamycin, lincomycin, metronidazole, azithromycin; clarithromycin, erythromycin, sulfamethoxazle and trimethoprim; sulfasalazine, amoxicillin and clavulanate; vancomycin, dalbavancin, oritavancin, telavancin, gentamycin, tobramycin, amikacin, imipenem and cilastatin, meropenem, doripenem, or ertapenem. [000612] The term “anti-fibrotic agent” as used herein refers to a substance that inhibits or reduces tissue scarring According to some embodiments, the anti-fibrotic agent is selected from nintedanib, pirfenidone, and combinations thereof. [000613] According to another aspect, the described invention provides a method for reducing progression of symptoms of a severe virus infection in a subject comprising administering a pharmaceutical composition comprising a vehicle/carrier, a bifunctional fusion protein comprising a biologically active immunomodulatory component operatively linked to. A biologically active antiviral component, wherein the immunomodulatory component comprising a recombinant trefoil factor 2, a sequence variant, or a splice variant thereof. [000614] According to some embodiments, the severe virus infection is a severe respiratory virus infection. According to some embodiments the respiratory virus is a cytomegalovirus. According to some embodiments the respiratory virus is Hanta virus. According to some embodiments the respiratory virus is an influenza virus. According to some embodiments, the respiratory virus is a MERS virus. According to some embodiments the respiratory virus is a respiratory syncytial virus. According to some embodiments, the respiratory virus is a SARS-CoV virus. According to some embodiments, the respiratory virus is a Zika virus. According to some embodiments, the respiratory virus is a West Nile virus. According to some embodiments, the respiratory virus is a dengue virus. According to some embodiments, the respiratory virus is a Japanese encephalitis virus. According to some embodiments, the respiratory virus is a tick-borne encephalitis virus. According to some embodiments, the respiratory virus is a yellow fever virus. According to some embodiments, the respiratory virus is a rhinovirus. According to some embodiments, the respiratory virus is an adenovirus. According to some embodiments, the respiratory virus is a herpes virus. According to some embodiments, the respiratory virus is an Epstein Barr virus. According to some embodiments, the respiratory virus is a measles virus. According to some embodiments, the respiratory virus is a mumps virus. According to some embodiments, the respiratory virus is a rotavirus. According to some embodiments, the respiratory virus is a Cocksackie virus. According to some embodiments, the respiratory virus is a norovirus. According to some embodiments, the respiratory virus is an encephalomyocarditis virus (EMCV). [000615] According to some embodiments, the symptoms of a severe virus infection comprise primary viral pneumonia. According to some embodiments, the symptoms of a severe virus infection comprise superimposed bacterial pneumonia. According to some embodiments, symptoms of a severe virus infection comprise disruption or injury to alveolar epithelium, endothelium or both. According to some embodiments, the symptoms of a severe virus infection comprise acute lung injury (ALI). According to some embodiments, the symptoms of a severe virus infection comprise acute respiratory distress syndrome (ARDS). According to some embodiments, the symptoms of a severe virus infection comprise symptoms of shock, including low blood pressure, lightheadedness, shortness of breath, and rash. According to some embodiments, the symptoms of a severe virus infection comprise excessive complement activation. According to some embodiments, the symptoms comprise a pathological increase in vascular permeability. According to some embodiments, the symptoms of a severe virus infection comprise thrombotic complications. According to some embodiments, the thrombotic complications include one or more of formation of pulmonary microthrombi, acute pulmonary embolism, deep-vein thrombosis, ischemic stroke, myocardial infarction, or systemic arterial embolism. According to some embodiments, the symptoms of a severe virus infection comprise kidney damage. According to some embodiments, the symptoms of a severe virus infection comprise elevated concentrations of one or more inflammatory mediator in plasma (hypercytokinemia), compared to a normal healthy subject. According to some embodiments, the inflammatory mediator is one or more of interferon α, interferon β, interferon-κ, interferon-γ, interferon-γ-1b, complement, prostaglandin D2, vasoactive intestinal peptide (VIP), interleukin-1-beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 (IL-12), IL-17, tumor necrosis factor-alpha (TNF-α). [000616] According to some embodiments, the composition stimulates repair of a mucosal injury. According to some embodiments, the repair comprises epithelial proliferation. According to some embodiments, the repair restores an epithelial barrier, an endothelial barrier or both. According to some embodiments, the composition modulates an immune response. According to some embodiments, the immune response comprises recruitment of innate and adaptive immune cells. According to some embodiments, the innate immune cells comprise macrophages, dendritic cells (DCs), innate lymphoid cells (ILCs), and natural killer cells (NKs). According to some embodiments, the adaptive immune cells include αβ T cells, γδ T cells, and B cells. [000617] According to some embodiments, the step of administering occurs nasally, intratracheally, orally, parenterally, topically, or by inhalation. According to some embodiments, the administering is parenterally (by injection). According to some embodiments, the administering occurs by inhalation. According to some embodiments, the administering is by insufflation. [000618] According to some embodiments, the pharmaceutical composition further comprises at least one additional therapeutic agent or a supportive therapy. According to some embodiments, the additional therapeutic agent is selected from one or more of an immunomodulatory agent, an analgesic agent, an anti-inflammatory agent, an anti-infective agent, an anti-malarial agent, an anti-viral agent or an anti-fibrotic agent. [000619] According to some embodiments, each of these agents can be used alone as a monotherapy or in combination with a second agent, e.g., an immunomodulatory agent with a second immunomodulatory agent, e.g., methotrexate; an immunomodulatory agent with an analgesic agent, an immunomodulatory agent with an anti-inflammatory agent, an immunomodulatory agent with an anti-infective agent, an immunomodulatory gent with an anti-malarial agent, an immunomodulatory agent with an antiviral, an immunomodulatory agent with an antifibrotic agent; an immunomodulatory agent with supportive therapy. [000620] Exemplary approaches to the inhibition of specific cytokines include drugs that inhibit cytokine synthesis (e.g., glucocorticoids, cyclosporine A, tacrolimus, myophnolate- helper lymphocyte (Th2)-selective inhibitors), humanized blocking antibodies to cytokines or their receptors; soluble receptors that mop up secreted cytokines, low molecular weight receptor antagonists, and drugs that block the signal transduction pathways activated by cytokines. [000621] According to some embodiments, the immunomodulatory agent is a corticosteroid selected from prednisone, azathioprine, dexamethasone, mycophenolate, mycophenolate mofetil, and combinations thereof. [000622] According to some embodiments, cyclosporine, tacrolimus and sirolimus are immunosuppressive medications that inhibit T cell activation through a series of calcium- dependent signal events involved in cytokine gene transcription. Cyclosporine inhibits the activation of helper T cells. Tacrolimus also interferes with T cell receptor-dependent cell activation. CSA and tacrolimus inhibit IL-2, IL-3, Il-4, IFN-γ, GM-CSF, and TNF-α production. Transcriptional factors nuclear factor of activated T cells (NF-AT), NF-κB, and PU-box are inhibited by tacrolimus. T cell receptor mediated apoptosis of lymphocytes and thymocytes is augmented by tacrolimus. Sirolimus (rapamycin) is a macrocyclic lactone produced by Streptomyces hygroscopicis that inhibits T lymphocyte activation and proliferation that occurs in response to antigenic and cytokine stimulation; it binds intracellularly to the immunophilin, FK binding protein-12, which becomes an immunosuppressive complex, which in turn binds to and inhibits activation of mammalian regulatory kinase (target of rapamycin, mTOR). This inhibition suppresses cytokine-driven T cell proliferation, inhibiting the progression from the G1 to S phases of the cell cycle. Nelson, RP, and Ballow, M. J. Allergy Clin. Immunol. (2003) 111 (2): S720-732]. [000623] According to some embodiments, the immunomodulatory agent is a recombinant interferon, e.g., TFN-α; IFN-α-2b, IFN-β, IFN-γ, IFN-κ, IFN-ω, IFN-δ, IFN-τ and IFN-ζ. According to some embodiments, the recombinant interferon is in a PEGylated form. [000624] According to some embodiments, the immunomodulatory agent is a recombinant IL-2 receptor inhibitor (e.g., denileukin diftitox). [000625] According to some embodiments, the immunomodulatory agent is a PDE4 inhibitor, e.g., cilomilast, a second generation PDE4 inhibitor with anti-inflammatory effects that target bronchoconstriction, mucus hypersecretion and airway remodeling. [Giembycz, MA. Br. J. Clin. Pharmacol. (2006) 62(2): 138-52]. [000626] According to some embodiments, the immunomodulatory agent is a hyperimmune globulin prepared from a donor with high titers of a desired antibody. Examples include cytomegalovirus immunoglobulin iv; respiratory syncytial virus immune globulin iv (RSV-IGIV); and palivizumab, a humanized mouse monoclonal antibody given IM. [000627] According to some embodiments, the immunomodulatory agent targets pro- inflammatory cytokines. According to some embodiments, the immunomodulatory agent is a TNFα inhibitor/antagonist [e.g., etanercept; adalimumab; infliximab, certolizumab pegol, or golimumab]. [000628] According to some embodiments, the immunomodulatory agent is an IL-1β inhibitor [e.g., rilonacept; canakinumab; or Anakinra]. [000629] According to some embodiments, the immunomodulatory agent is chimeric IL- 1Ra. This molecule is a fusion of the N-terminal peptide of IL-1β and IL-1Ra, resulting in inactive IL-1Ra. Because the IL-1β N-terminal peptide contains several protease sites clustered around the caspase-1 site, local proteases at sites of inflammation can cleave chimeric IL-1Ra and turn IL-1Ra active. [Rider, P. et al. J. Immunol. (2015) 195:doi: 10.4049/jimmunol.1501168. [000630] According to some embodiments, the immunomodulatory agent is an IL-6 inhibitor (e.g., tocilizumab, siltuximab, sarilumab, olokizumab, or sirukumab). [000631] According to some embodiments, the immunomodulatory agent is an IL-12/ IL- 23 inhibitor (e.g., ustekinumab, briakinumab) an IL23 inhibitor (e.g., guselkumab, or tildrakizumab). [000632] According to some embodiments, the immunomodulatory agent targets cytokine signaling pathways, e.g., compounds targeting TLR4 signaling, e.g., enamionone E121 (ethyl 4-(4’-chlorophenyl) amino-6 methyl-2-oxocytlohex-3-en-1-aote), an aniline enaminone); JODI 18b and 19, [Szollosi, DE et al. J. Pharmacy & Pharmacol. (2018) 70: 18- 26]; TAK-242 (resatorvid, Takeda), TLR-C34, a 2-acetamidopyranoside that inhibits TLR4 signaling [Olusayo, A., et al. J. Applied Toxicol (2019) 39(4). Doi: 10/1002/jat.3771]; or C35 [Neal, MD. Et al. PLoS One (2013) 8 (6): e65779]. [000633] According to some embodiments, the immunomodulatory agent is a p38 MAPK inhibitor, e.g., SB203580, 4-(4’-fluorophenyl)-2-(4’-methylsulfinylphenyl)-5- (4’-pyridyl)- imidazole, a pyridinyl imidazole inhibitor used to elucidate the roles of p38 mitogen-activated protein (MAP) kinase [Cuenda, A. et al. FEBS Lett. (1995) 364: 229-33]; SB203580 inhibits also the phosphorylation and activation of protein kinase B (PKB, also known as Akt [Lali, F.V. et al. J. Biol. Chem. (2000) 275 (10): 7395-402]. SB239063 [trans-4-[4-(4-fluorophenyl)- 5-(2-methoxy-4-pyrimidinyl)-1H-imidazol-1-yl] cyclohexanol; Barone. Et al J. Pharmacol. Exp. Ther (2001) 296: 312 [PMID 11160612], and RWJ 67657 (4-[4-(4-fluorophenyl)-1-(3- phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol). Wadsworth, SA et al. J. Pharmacol. Exp. Ther. (1999) 291 (2): 680-7). [000634] According to some embodiments, the immunomodulatory agent targets Janus kinase signaling (e.g., tofacitinub, baricitinib, or upadacitinib). [000635] According to some embodiments, the immunomodulatory agent targets cell adhesion molecules to reduce leukocyte recruitment, e.g., molecules that are α4 integrin inhibitors, e.g., vedolizumab, or natalizumab. [Szollosi, DE et al. J. Pharmacy & Pharmacol. (2018) 70: 18-26]. [000636] According to some embodiments, the immunomodulatory agent is a recombinant anti-inflammatory cytokine, e.g., IL-4, IL-10,or IL-11. [000637] Immunomodulation also includes therapies that boost an individual’s defenses by providing physiologic or supraphysiologic doses of exogenous cytokines, e.g., to treat viral infections. [000638] According to some embodiments, a parameter for measuring activation state of lymphocytes is cytokine release profile. For example, ELISPOT, or enzyme linked immunospot, is a technique that was developed for the detection of secreted proteins, such as cytokines and growth factors. It is performed using a PVDF or nitrocellulose membrane 96- well plate pre-coated with an antibody specific to the secreted protein. Cells are added to the plate and attach to the coated membrane. Cells are then stimulated and the secreted protein binds to the antibody. Next, a detection antibody is added that binds specifically to the bound protein. The resulting antibody complex can be detected either through enzymatic action to produce a colored substrate or with fluorescent tags. The membrane can be analyzed by manually counting the spots or with an automated reader designed for this purpose. Each secreting cell appears as a spot of color or fluorescence. [000639] According to some embodiments, another parameter for measuring activation of lymphocytes is by quantifying cellular subset differentiation. For example, the differentiation of CD45+/CD3+ T-lymphocytes to CD45+/CD3+/CD4+ helper T-lymphocytes, CD45+/CD3+/ CD8+ cytotoxic T-lymphocytes, and CD45+/CD3+/CD25+ activated T- lymphocytes can be quantified by flow cytometry analysis. [000640] According to some embodiments, the immunomodulatory agent is a corticosteroid. According to some embodiments, the corticosteroid is selected from prednisone, azathioprine, dexamethasone, mycophenolate, mycophenolate mofetil, and combinations thereof. [000641] The term “analgesic agent” as used herein refers to an agent producing diminished sensation to pain without loss of consciousness. According to some embodiments, the analgesic agent is selected from codeine, hydrocodone, oxycodone, methadone, hydromorphone, morphine, fentanyl, and combinations thereof. [000642] An “anti-inflammatory agent” is a substance that reduces inflammation (redness, swelling, and pain) in the body by inhibiting inflammatory mediators in the body that cause inflammation. According to some embodiments, the anti-inflammatory agent is selected from aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac nabumetone, naproxen, nintedanib, oxaprozin, pirfenidone, piroxicam, salsalate, sarilumab (Kevzara®) sulindac, tolmetin, and combinations thereof. [000643] The term “anti-viral agent” as used herein means any of a group of chemical substances having the capacity to inhibit the replication of or to destroy viruses used chiefly in the treatment of viral diseases. According to some embodiments, the anti-viral agent is selected from acyclovir, gancidovir, foscarnet; ribavirin; amantadine, azidodeoxythymidine /zidovudine), nevirapine, a tetrahydroimidazobenzodiazepinone (TIBO) compound; efavirenz; remdecivir, lopinavir/ritonavir (Kaletra™), umifenovir (Arbidol™), favipiravir (Avigan™) and delavirdine. [000644] According to some embodiments, the anti-viral agent is an agent that inhibits virus entry and thereby reduces viral load. [000645] According to some embodiments, the agent that blocks viral entry is a peptide inhibitor. According to some embodiments, the peptide inhibitor is a fusion protein. [000646] As described in Xia, et al., Sci. Advisor (2019) 5: eaav4580, the S glycoprotein is a type 1 transmembrane glycoprotein common to all HCoVs. The S proteins consist of two subunits S1 and S2. The S1 subunit binds the cellular receptor through its receptor binding domain (RBD), followed by conformational changes in the S2 subunit, which allows the fusion peptide to insert into the host target cell membrane. The heptad repeat 1 (HR1) region in the SR2 unit forms a homotrimeric assembly, which exposes three highly conserved grooves on the surface that bind heptad repeat 2 (HR2). This six-helix bundle (6-HB) core structure formed during the fusion process helps bring the viral and cellular membranes into close proximity for viral fusion and entry. The HR region in the S2 subunit is conserved among various HCoVs and plays a pivotal role in HCoV infections by forming the 6-HB that mediates viral fusion. Furthermore, the mode of interaction between HR1 and HR2 is conserved among CoVs such that certain residues in the HR1 helices interact with certain residues in the HR2 helices. [000647] According to some embodiments, the fusion peptide is a synthetic peptide derived from HR1 and HR2 regions of SARS-CoV spike protein as described in Liu, S. et al. Lancet (2004) 363: 938-47, and as shown in Table 2 below. [000648] Table 2
Figure imgf000213_0001
[000649] As described in Liu, S. et al. Lancet (2004) 363: 938-47, the HR1 and H2 sequences tend to form a coiled-coil structure, and the amino acid sequences of peptides derived from the HR1 and HR2 regions of SARS-CoV spike protein are similar to those from the HIV-1 gp41 HR1 and HR2 regions. Peptides derived from the HR2 regions of many other enveloped viruses, including Ebola virus [Id., citing Watanabe, S. et al. J. Virol. (2000) 74: 10194-201], Newcastle disease virus [Id., citing Yu, M. et al. J. Gen. Virol. (2002) 83: 623- 29], parainfluenza virus [Id., citing Yao, Q & Compans, RW. Virology (1996) 223: 103-12], and respiratory syncytial virus [Id., citing Yu, M. et al. J. Gen. Virol. (2002) 83: 623-29] inhibit the corresponding virus infection in the micromolar range; these viruses have similar fusogenic mechanisms to HIV-1. [Id., citing Watanabe, S. et al. J. Virol. (2000) 74: 10194-201]. [000650] As described in Liu, S. et al. Lancet (2004) 363: 938-47, two major factors can affect the potency of a fusion-inhibitory peptide: the sensitivity of a virus to the corresponding antiviral peptides; and the sequence and conformation of the inhibitory peptide. [Id.]. The anti-viral activity of the peptide fusion inhibitors depends on their optimum peptide sequences and conformations. Changes in their sequences or conformations could substantially affect their antiviral activity and the stability of the complexes they form. [Id., citing 17, 47, 48]. According to some embodiments, optimization of the peptide sequence and conformation of a fusion peptide inhibitor may improve antiviral activity. According to some embodiments, a fusion peptide of Table 2 may be used as a lead in designing more potent anti-SARS-CoV peptides. [000651] As described in Lu, L. et al. Nat. Communic. (2014) 5: 3067, two peptides HR1P and HR2P, spanning residues 998-1039 in the HR1 domain [ANKFNQALGAMQTGFTTTNEAFQKVQDAVNNNAQALSKLASE, SEQ ID NO: 24] and 1251-1286 in the HR2P domain [SLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKEL, SEQ ID NO: 25], respectively, from MERS-CoV can form a stable six-helix bundle fusion core structure. HR2P can effectively inhibit MERS-CoV replication and its spike protein-mediated cell–cell fusion. Introduction of hydrophilic residues into HR2P results in significant improvement of its stability, solubility and antiviral activity. Therefore, the HR2P analogues can be further developed into effective viral fusion inhibitors. [000652] According to some embodiments, the viral entry inhibitor is peptide OC43- HR2P [LAEADANVVAQIKVLASNTADFGEQIADLANNFANAIL, SEQ ID NO: 26] as described in Xia, et al., Sci. Advisor (2019) 5: eaav4580. According to some embodiments, the viral entry inhibitor is EK1 [SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL, SEQ ID NO: 27] as described in Xia, et al., Sci. Advisor (2019) 5: eaav4580. EK1 is a modified derivative of OC43-HR2P. [000653] In order to improve the inhibitory activity of EK1, cholesterol (Chol) and palmitic acid (Palm) were covalently attached to the C-terminus of EK1 sequence under the help of a flexible polyethylene glycol (PEG) spacer, and the corresponding lipopeptides EK1C and EK1P were constructed. According to some embodiments, the viral entry inhibitor is EK1P [SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL, SEQ ID NO: 28] or a PEG-based spacer-containing derivative [PEG4-C (Palm) thereof as disclosed by Xia, S. et al. Cell Res. (2020) 30(4): 343-55. According to some embodiments, the viral entry inhibitor is EK1C [SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL, SEQ ID NO: 29] or a PEG-based spacer-containing derivative [PEG4-C (Chol)] thereof as disclosed by Xia, S. et al. Cell Res. (2020) 30(4): 343-55. [000654] As described by Xia et al. Cell Res. (2020) 30(4): 343-55, which is incorporated herein by reference, on the basis of the structure of EK1C, a series of cholesteryl EK1 with multiple linkers were constructed, where the glycine/serine-based linker, i.e., GSG, or PEG- based spacer was employed between EK1 and the cholesterol moiety. Compared with EK1C1, EK1C2 and EK1C showed similar inhibitory activities. EK1C3 peptide with both the 3-amino acid linker “GSG” and the PEG4-based spacer, exhibited 4-fold more potency than EK1C1. It is noteworthy that changing “GSG” in EK1C3 to a longer 5-amino acid linker “GSGSG” (SEQ ID NO: 30) significantly increased the inhibitory potency of the hybrid molecule, and EK1C4 had IC50 value of 1.3 nM, which was 43-fold more potent than EK1C1. These findings indicate that the linker length has a significant effect on the overall activity of lipopeptides. Comparison of increasing PEG-based arm lengths in EK1C4 shows that inhibitors potency slightly decreased in the cell–cell fusion assay. The data suggest that “GSGSG-PEG4” linker (SEQ ID NO: 30) was optimal to bridge both parts of the conjugates. Similarly, EK1C4 showed the most potent inhibitory activity against SARS-CoV-2 PsV infection, with IC50 value of 15.8 nM, providing 149-fold stronger anti-SARS-CoV-2 activity than that of EK1 (IC50 = 2,375 nM) [000655] According to some embodiments, the agent that blocks viral entry is a dipeptidyl peptidase 4 (DPP4) inhibitor. Drugs in the DPP-4 inhibitor class that are approved for use to treat type 2 diabetes include sitagliptin, saxagliptin, linagliptin, and alogliptin. [000656] According to some embodiments, the agent that blocks viral entry is an ACE2 inhibitor. Shortly after the identification of the angiotensin-converting enzyme 2 (ACE2), a metallocarboxypeptidase that mediates various cardiovascular and renal functions, peptide inhibitors of the enzyme were developed by selection of constrained peptide libraries displayed on phage [McKee, DL et al., Pharmacol. Res. (2020) 157: 104859, citing Huang, L. et al. J. Biol. Chem. (2003) 278 (18): 15532-40], the most potent inhibitor of which, termed DX600, with the amino acid sequence of Ac-GDYSHCSPLRYYPWWKCTYPDPEGGG-NH2 [SEQ ID NO: 31] had a Ki of 2.8 nm and an IC50 of 10.1 μM. Subsequent experimental studies in mice and in human cell lines revealed that DX600 is a potent ACE2 inhibitor specific for only human ACE2 [Id., citing Pedersen, KB et al. Am J. Physiol. Regul. Integr. Comp. Physiol. (2011) 301(5): R1293-99; Ye, M. et aql. Hypertension (2012) 60(3): 730-40]. Other small- peptide and tripeptide inhibitors have been developed for potent and selective inhibition of human ACE2 and inhibition of SARS-CoV cell entry in vitro [Id., citing Guy, JL et al. FEBS J. (2005) 272 (14): 3512-20; Han, DP, et al., Virology (2006) 350 (1): 15-25; Mores, A. et al. J. Med. Chem. (2008) 51(7): 2216-26]]. Synthetic small-molecule inhibitors of human ACE2, including MLN-4760 (CAS number: 305335−31-3) [Id., citing Ye, M. et al. Hypertension (2012) 60(3): 730-40; Trask, AJ et al., Am. J. Hypertens. (2010) 23(6): 687-93;], N-(2- aminoethyl)-1 aziridine-ethanamine [Id., citing Huentelmann, MJ et al. Hypertension (2004) 44(6): 903-6] and the TNF-α converting enzyme (TACE) small-molecule inhibitor TAPI-2 that blocks SARS-CoV S protein-induced shedding of ACE2 [Id., citing Haga, S. et al. Antiviral Res. (2010) 85(3): 551-55; Mohler, JM, et al., Nature (1994) 370 (6486): 218-20] have been developed for experimental inhibition of SARS-CoV cell entry. Moreover, the phytochemical nicotianamine (CAS number: 34441−14-0), a metal chelator ubiquitously present in higher plants [Id., citing Takahashi, M. et al. Plant Cell (2003) 15(6): 1263-80], was identified in high concentrations in soybean, and was shown as a potent inhibitor of human ACE2 with an IC50 of 84 nM [Id., citing Takahashi, S. et al. Biomed Res. (2015) 36(3): 219- 224]. Because dietary phytochemicals as naturally occurring compounds display a wide safety profile and less pharmacological side effects [Id., citing Naujokat, C. & McKee, DL. Curr. Med. Chem. (2020) doi: 10.2174/0929867327666200228110738], nicotianamine constitutes a candidate drug for ACE2 inhibition and thus blockade of SARS-CoV-2 cell entry. Finally, a recent study demonstrates that a clinical-grade soluble recombinant human ACE2 protein (hrsACE2) inhibits attachment of SARS-CoV-2 to simian Vero-E 6 cells, and inhibits infection of engineered human capillary organoids and kidney organoids by SARS-CoV-2 isolated from a nasopharyngeal sample of a patient with confirmed COVID-19 disease [Id., citing Monteil, V. et al. Cell (2020) doi: 10.1016/j.cell.2020.04.004], suggesting that hrsACE2 can block host cell entry of SARS-CoV-2 and early stages of SARS-CoV-2 infections. [000657] According to some embodiments, the anti-viral agent is a protease inhibitor that inhibits a host cell protease to block viral entry. According to some embodiments, the anti- viral agent is a serine protease TMPRSS2 inhibitor, e.g., camostat (FOY-305), [N,N- dimethylcarbamoylmethyl 4-(4-guanidinobenzoyloxy)-phenylacetate] methanesulfate and camostat mesilate (Foipan™), alternatively termed camostat mesylate, (NI-03), (CAS number: 59721−28-7); or Nafamostat mesilate (Buipel™), (6-amidino-2-naphthyl-4-guanidino benzoate-dimethanesulfonate) (FUT-175), (CAS number: 81525−10-2). [McKee, DL, et al. Pharmacol. Res. (2020) 157: 104859] Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases, which entails S protein cleavage at the S1/S2 and the S2’ site and allows fusion of viral and cellular membranes, a process driven by the S2 subunit. [Hoffman, M. et al. Cell (2020) 181 (2): 271-80]. SARS-CoV can use the endosomal cysteine proteases cathepsin B and L (CatB/L) [Id., citing Simmons et al., 2005) and the transmembrane serine protease TMPRSS2 [Id., citing Glowacka, I. et al. J. Virol. (2011) 85: 4122-34, Matsuyama, S. et al. J. Virol. (2010) 84: 12658-664, Shulla, K. et al. J. Virol. (2011) 85: 873-82] for S protein priming in cell lines, and inhibition of both proteases is required for robust blockade of viral entry [Id., citing Kawase, M et al. J. Virol. (2012) 86: 6537-45]. However, only TMPRSS2 activity is essential for viral spread and pathogenesis in the infected host whereas CatB/L activity is dispensable [Id., citing Iwata-Yoshikawa, N. et al. J. Virol. (2019) 93: 10.1128/JVL01815-18, Shirato, K. et al. Virology (2016) 91: 10.1128/JVL01387-16, Shirato, K. et al. Virology (2018) 517: 9-15, Zhou, P et al. Antiviral Res. (2015) 116: 76-84]. SARSCoV-2 uses the SARS- CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. [Hoffman, M. et al. Cell (2020) 181 (2): 271-80] According to some embodiments, the protease inhibitor is cysteine protease inhibitor K11777, ((2S)-N-[(1E,3S)-1- (benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{€-4-methylpiperazine-1-carbonyl]amino}-3- phenylpropanamide, or a P3 derivative thereof which inhibits SARS-CoV and Ebola virus entry. [Zhou, P et al. Antiviral Res. (2015) 116: 76-84]. [000658] According to some embodiments the anti-viral agent comprises sera from a convalescent patient, e.g., a coronavirus patient, e.g., a SARSCoV, a MERs, or a COVID-19 patient. [000659] The term “anti-malarial agent as used herein refers to a substance used for treatment of clinical Plasmodium falciparum malaria According to some embodiments, the anti-malarial agent is selected from an aryl aminoalcohol compound selected from quinine, quinidine, chloroquine, hydroxychloroquine, amodiaquine, mefloquine, halofantrine, lumefantrine, piperaquine, and tafenoquine; an antifolate compound, selected from pyrimethamine, proguanil, chlorproguanil, trimethoprim; an artemisinin compound selected from artemisinin, dihydroartemisinin, artemether, artesunate; and atovaquone. Chloroquine phosphate inhibits terminal phosphorylation of ACE2, and hydroxychloroquine elevates the pH in endosomes which are involved in virus cell entry [McKee, DL, et al. Pharmacol. Res. (2020) 157: 104859, citing Vincent, MJ et al. (2005) Virol. J.2: 69; Bari, MAA. Pharmacol. Res. Perspect. (2017) 5(1): e00293]. The triple combination of cepharanthine (an anti- inflammatory alkaloid from Stephania cepharantha Hayata), (CAS number: 48,104,902), selamectin (an avermectin isolated from Streptomyces avermitilis and used as an anti- helminthic and parasiticide drug in veterinary medicine), (CAS number. 220119−17-5), and mefloquine hydrochloride (Lariam™, used for the prophylaxis and treatment of malaria) [Id., citing Bailly, C. Phytomedicine (2019) 62: 152956; Kjoha, S. et al. Psychopharmacology (Berl.) (2018) 235 (6): 1697-1709; Tickell-Painter, M. et al. Cochrane Database Syst. Rev. (2017) 10. Doi:10.1002/14651858.CD006491.pub4. CD006491].[57], [58], [59]] has recently been shown to inhibit infection of simian Vero E6 cells with pangolin coronavirus GX_P2V/2017/Guangxi (GX_P2V), whose S protein shares 92.2 % amino acid identity with that of SARS-CoV-2. [Id., citing Fan, HH et al. China Med. J. (2020). Doi: 10.1097/CM9.0000000000000797]. [000660] The term “anti-infective agent” as used herein means any of a group of chemical substances having the capacity to inhibit the growth of, or to destroy microorganisms, used chiefly in the treatment of infectious diseases. According to some embodiments, the anti- infective agent is selected from amoxicillin, doxycycline, demeclocycline; eravacycline, minocycline, ormadacycline, tetracycline, cephalexin, defotaxime, cetazidime, cefuroxime, ceftaroline; ciprofloxacin, levofloxacin, moxifloxacin clindamycin, lincomycin, metronidazole, azithromycin; clarithromycin, erythromycin, sulfamethoxazle and trimethoprim; sulfasalazine, amoxicillin and clavulanate; vancomycin, dalbavancin, oritavancin, telavancin, gentamycin, tobramycin, amikacin, imipenem and cilastatin, meropenem, doripenem, or ertapenem. [000661] The term “anti-fibrotic agent” as used herein refers to a substance that inhibits or reduces tissue scarring According to some embodiments, the anti-fibrotic agent is selected from nintedanib, pirfenidone, and combinations thereof. [000662] According to some embodiments, the supportive therapy is therapeutic apheresis comprising a virion removing step. According to some embodiments, the therapeutic apheresis reduces viral burden. [000663] Therapeutic apheresis is an extracorporeal treatment that can separate blood components (plasma and/or cellular components) from the patient's blood for the treatment of conditions in which a pathogenic substance in the blood is causing morbidity. According to some embodiments, the therapeutic apheresis is consistent with then current guidelines of the American Society for Apheresis (ASFA). See, e.g., Szczepiorkowski ZM, Bandarenko N, Kim HC, et al. Apheresis Applications Committee of the American Society for Apheresis Guidelines on the use of therapeutic apheresis in clinical practice-evidence-based approach from the Apheresis Applications Committee of the American Society for Apheresis. J Clin Apher.2007;22:106–175; Szczepiorkowski M, Winters J, Bandarenko N, et al. Guidelines on the use of therapeutic apheresis in clinical practice-evidence-based approach from the Apheresis Applications Committee of the American Society Apheresis. J Clin Apher. 2010;25:83–177, each of which is incorporated herein by reference. [000664] According to some embodiments, viral particles in the blood can be labeled with a reagent that sensitizes the particles to UV light, much like photopheresis, so that the virion- containing blood can be treated with UV light inside the apheresis machine, and the treated blood then returned to the patient. [000665] According to some embodiments, virions can be physically removed from the blood by passing an infected patient’s blood through a filter or membrane comprising appropriately sized pores and then returning the filtered blood to the patient; the SARSCoV-2 virion for example has a reported diameter of 100 nm [Bar-On, YM et al. eLife(2020) 9: e57309]. Most human cells fall within a size range of 1-120 microns: platelets are about 2 microns, red cells about 3 microns by about 8 microns, neutrophils about 8-10 microns, lymphocytes about 6-12 microns, exocrine cells about 10 microns, fibroblasts 10-15 microns; osteocytes about 10-20 microns including processes; chondrocytes and liver cells about 20 microns, goblet and ciliated cells about 50 microns long and 5-10 microns wide; macrophages about 20-80 microns; hematopoietic stem cells about 30-40 microns and adipocytes filled with stored lipids typically 70-120 microns in diameter, but may be up to five time larger in very obese individuals. [Feitas, Jr., RA. Nanomedicine, Vol. 1: Basic Capabilities, Landes Biosciences, Georgetown TX (1999)]. [000666] According to some embodiments, the principles of affinity chromatography are used to remove viral particles from the infected patient’s blood. For example, the infected blood is passed through a field comprising anti-S protein binding antibody fragments immobilized directly or indirectly on magnetic particles where it remains for a time sufficient to allow the antibody fragments to bind to S protein spikes on virions in the blood. After a suitable time, the virions are removed by a magnetic surface before the treated blood is returned to the patient. According to some embodiments, the magnetic particles can comprise recombinant protein A (Uniprot P38507), recombinant protein G (Uniprot P19909), or recombinant protein A/G [Rockland-Inc.], which contain five homologous Ig-binding domains, each of which is able to bind the Fc fragment of immunoglobulins. Other exemplary semiselective or selective therapeutic apheresis methods available are discussed in detail by Bambauer R, Schiel R, Lehmann B, Bambauer C. Therapeutic apheresis, technical overview. ARPN J Sci Technol.20122:399–421, which is incorporated herein by reference. Compositions [000667] According to some embodiments, the described invention provides pharmaceutical compositions comprising a recombinant trefoil factor protein-Fc conjugate. According to some embodiments, the recombinant protein-Fc conjugate comprises a recombinant TFF1, a recombinant TFF2, a recombinant TFF3, a recombinant interferon, or a recombinant fusion protein. According to some embodiments, the recombinant fusion protein comprises a recombinant TFF1 and a recombinant interferon. According to some embodiments, the recombinant fusion protein comprises a recombinant TFF2 and a recombinant interferon. According to some embodiments, the recombinant fusion protein comprises a recombinant TFF3 and a recombinant interferon. [000668] According to some embodiments, the recombinant interferon is selected from, e.g., a recombinant IFN-α; IFN-α-2b, IFN-β, IFN-γ, IFN-κ, IFN-ω, IFN-δ, IFN-τ and IFN-ζ. According to some embodiments, the recombinant interferon is in a PEGylated form. According to some embodiments, the recombinant TFF1, TFF2, or TFF3 is glycosylated. According to some embodiments, the recombinant TFF1-IFN, TFF2-IFN or TFF3-IFN bi- functional protein can be generated by preparing separate recombinant TFF1-Fc, TFF2-Fc, or TFF3-Fc and IFN-Fc fusion proteins that can form an Fc-mediated dimer comprising arms of both TFF1, TFF2, or TFF3, and the recombinant IFN. According to some embodiments, the recombinant bifunctional proteins are biologically active, meaning they exhibit the biological activity of a naturally occurring TFF1, TFF2, or TFF3, a naturally occurring IFN, or both a naturally occurring TFF1, TFF2 or TFF3 and a naturally occurring IFN. According to some embodiments, the recombinant TFF1-Fc, TFF2-Fc or TFF3-Fc monomer may decrease the viscosity of mucin, elasticity of mucin or both. According to some embodiments, the recombinant TFF1-Fc, TFF2-Fc, or TFF3-Fc fusion protein may form a TFF1-Fc, TFF2-Fc, or TFF3-Fc homodimer. According to some embodiments, the recombinant TFF1-Fc, TFF2-Fc, or TFF3-Fc homodimer may increase the viscosity of mucin, the elasticity of mucin or both. [000669] According to some embodiments, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type such as a cell of the respiratory tract. Tissue-specific regulatory elements are known in the art. [000670] According to some embodiments, the composition of the described invention may be prepared in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions). According to some embodiments, the recombinant fusion protein is encapsulated within a polymer matrix of a population of microparticles. [000671] According to some embodiments, the composition of the described invention can be administered as a solution by inhalation. According to some embodiments, administering by inhalation is with an inhalation delivery device. [000672] The described composition can be aerosolized in a variety of forms, including, without limitation, dry powder inhalants, metered dose inhalants, or liquid/liquid suspensions. The respirable particles may be liquid or solid. The particles may optionally contain other therapeutic ingredients such as amiloride, benzamil or phenamil, with the selected compound included in an amount effective to inhibit the reabsorption of water from airway mucous secretions, as described in U.S. Pat. No. 4,501,729, incorporated by reference in its entirety herein. [000673] The particulate pharmaceutical composition may optionally be combined with a carrier to aid in dispersion or transport. A suitable carrier such as a sugar (i.e., lactose, sucrose, trehalose, mannitol) may be blended with the active compound or compounds in any suitable ratio (e.g., a 1 to 1 ratio by weight). [000674] Particles comprising a recombinant TFF-IFN fusion protein according to the present disclosure should include particles of respirable size, that is, particles of a size sufficiently small to pass through the mouth or nose and larynx upon inhalation and into the bronchi and alveoli of the lungs. In general, particles ranging from about 1 to 10 microns, inclusive, in size (e.g., less than about 5 microns in size) are respirable. Particles of non- respirable size which are included in the aerosol tend to deposit in the throat and be swallowed, and the quantity of non-respirable particles in the aerosol may be minimized. For nasal administration (insufflation), a particle size in the range of 10-500 µM, inclusive, ensures retention in the nasal cavity. [000675] Liquid pharmaceutical compositions for producing an aerosol may be prepared by combining the active compound with a suitable vehicle, such as sterile pyrogen free water. The hypertonic saline solutions used to carry out the present invention are sterile, pyrogen-free solutions, comprising from one to fifteen percent (by weight) of the physiologically acceptable salt, e.g., from three to seven percent by weight of the physiologically acceptable salt. [000676] Aerosols of liquid particles comprising a recombinant TFF-IFN fusion protein may be produced by any suitable means, such as with a pressure-driven jet nebulizer or an ultrasonic nebulizer. See, e.g., U.S. Pat. No.4,501,729, incorporated by reference in its entirety herein. Nebulizers are commercially available devices which transform solutions or suspensions of the active ingredient into a therapeutic aerosol mist either by means of acceleration of compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation. [000677] Exemplary formulations for use in nebulizers consist of the recombinant fusion protein in a liquid carrier, the recombinant fusion protein comprising up to 40% w/w of the formulation, for example, less than 20% w/w. The carrier is typically water (and most preferably sterile, pyrogen-free water) or a dilute aqueous alcoholic solution, preferably made isotonic, but may be hypertonic with body fluids by the addition of, for example, sodium chloride. Optional additives include preservatives if the formulation is not made sterile, for example, methyl hydroxybenzoate, antioxidants, flavoring agents, volatile oils, buffering agents and surfactants. [000678] Aerosols of solid particles comprising the active compound may likewise be produced with any solid particulate therapeutic aerosol generator. Aerosol generators for administering solid particulate therapeutics to a subject, produce particles which are respirable and generate a volume of aerosol containing a predetermined metered dose of a therapeutic at a rate suitable for human administration. One illustrative type of solid particulate aerosol generator is an insufflator. Suitable formulations for administration by insufflation include finely comminuted powders which may be delivered by means of an insufflator or taken into the nasal cavity in the manner of a snuff. In the insufflator, the powder (e.g., a metered dose thereof effective to carry out the treatments described herein) is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn through the device upon inhalation or by means of a manually-operated pump. The powder employed in the insufflator consists either solely of the active ingredient or of a powder blend comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant. [000679] According to some embodiments, the compositions of the described invention may be formulated as a dispersible dry powder for delivery by inhalation or insufflation (either through the mouth or through the nose, respectively). Dry powder compositions may be prepared by processes known in the art, such as lyophilization and jet milling, as disclosed in International Patent Publication No. WO 91/16038 and as disclosed in U.S. Pat. No.6,921,527, the disclosures of which are incorporated by reference. For example, the composition of the described invention may be placed within a suitable dosage receptacle in an amount sufficient to provide a subject with a unit dosage treatment. The dosage receptacle can be one that fits within a suitable inhalation device to allow for the aerosolization of the dry powder composition by dispersion into a gas stream to form an aerosol and then capturing the aerosol so produced in a chamber having a mouthpiece attached for subsequent inhalation by a subject in need of treatment. Such a dosage receptacle includes any container enclosing the composition known in the art such as gelatin or plastic capsules with a removable portion that allows a stream of gas (e.g., air) to be directed into the container to disperse the dry powder composition. Such containers are exemplified by those shown in U.S. Pat. Nos. 4,227,522; 4,192,309; and 4,105,027. Further exemplary containers also include those used in conjunction with Glaxo's Ventolin® Rotohaler brand powder inhaler or Fison's Spinhaler® brand powder inhaler. Another suitable unit-dose container which provides a superior moisture barrier is formed from an aluminum foil plastic laminate. The pharmaceutical-based powders is filled by weight or by volume into the depression in the formable foil and hermetically sealed with a covering foil-plastic laminate. Such a container for use with a powder inhalation device is described in U.S. Pat. No. 4,778,054 and is used with Glaxo's Diskhaler® (U.S. Pat. Nos. 4,627,432; 4,811,731; and 5,035,237). Each of the above cited references is incorporated by reference herein in its entirety. [000680] According to some embodiments, the dry powder may be produced by a spray drying process. [000681] According to some embodiments, the composition of the invention may be formulated as a solution. Aqueous droplet inhalers (ADI) deliver a pre-metered dose of liquid formulation without using a propellant. ADIs actively aerosolize liquid producing a soft mist of fine particles. Berodual Respimat® (Boehringer Ingelheim Pharma Gmbh & Co.) is an exemplary aqueous droplet inhaler. An exemplary formulation is sterile, contains a sufficient amount of the recombinant fusion protein, preserves stability of the recombinant fusion protein, and is not harmful for the proposed application. For example, the compositions of the described invention may be formulated as aqueous suspensions wherein the active(s) are in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include, without limitation, stabilizers (e.g., mannitol, sorbitol, xylitol, trehalose, lactose) suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia), dispersing or wetting agents including, a naturally-occurring phosphatide (e.g., lecithin), or condensation products of an alkylene oxide with fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyethylene sorbitan monooleate). [000682] Compositions of the described invention also may be formulated in the form of dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water. The active ingredient in such powders and granules is provided in admixture with a dispersing or wetting agent, suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients also may be present. [000683] According to another embodiment, the pharmaceutical composition is packaged in an inhalation device, including, for example, but not limited to a nebulizer, a metered-dose inhaler (MDI), and a dry powder inhaler (DPI). [000684] According to some other embodiments, the pharmaceutical composition is a liquid for aerosolized delivery using a nebulizer. Methods of preparing a biologically active fusion protein comprising a recombinant TFF1 (rTFF1), recombinant TFF2 (rTFF2), or recombinant TFF3 (rTFF3) molecule, fragment or variant operationally linked to a recombinant IFN [000685] According to another aspect, the described invention provides a method of preparing a biologically active recombinant trefoil factor 1 (rTFF1), recombinant trefoil factor 2 (TFF2), or recombinant trefoil factor 3 (rTFF3) operationally linked to a biologically active recombinant IFN. [000686] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, it is understood that other forms of expression vectors may be used, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. [000687] Methods for preparing recombinant constructs/vectors herein can follow standard recombinant DNA and molecular cloning techniques as described by J. Sambrook and D. Russell (Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001); T. J. Silhavy et al. (Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y, 1984); and F. M. Ausubel et al. (Short Protocols in Molecular Biology, 5th Ed. Current Protocols, John Wiley and Sons, Inc., NY, 2002), for example. [000688] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (2001), Ausubel et al. (2002), and other laboratory manuals. [000689] The term “transformation” as used herein refers to the transfer of a nucleic acid molecule into a host organism or host cell by any method. A nucleic acid molecule that has been transformed into an organism/cell may be one that replicates autonomously in the organism/cell, or that integrates into the genome of the organism/cell, or that exists transiently in the cell without replicating or integrating. Non-limiting examples of nucleic acid molecules suitable for transformation are disclosed herein, such as plasmids and linear DNA molecules. [000690] The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel (1990) GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. Additional vectors include minichromosomes such as bacterial artificial chromosomes, yeast artificial chromosomes, or mammalian artificial chromosomes. For other suitable expression systems for both prokaryotic and eukaryotic cells, see, e.g., Chapters 16 and 17 of Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. [000691] According to some embodiments, recombinant mouse TFF1, TFF2, or TFF3 can be expressed in CHO-K1 and human Jurkat clone E6-1 cancer cells purchased from American Type Culture Collection (Manassas, VA) using a retroviral vector pMIG as described by Dubeykovskaya, Z. et al. J. Biol. Chem. (2009) 284 (6): 3650-62]. The pMIG vector contains a multiple cloning site followed by an internal ribosome binding site and the green fluorescent protein (GFP) gene. A strong viral long terminal repeat promoter controls transcription of both the cloned and GFP genes. The coding region of immature mouse TFF1 along with the noncoding 5’ flanking region and EcoR1/SalI flanking restriction sites can be amplified from MGC clone (BC050086; Open Biosystems, Baltimore, MD) by PCR and introduced into corresponding restriction sites of the pMIG polylinker. The integrity of the resultant construct, pMIG-mTFF2 (GFP) is verified by sequencing. The stable CHO-K1 cell line secreting recombinant mouse TFF1, recombinant mouse TFF2 or recombinant TFF3 is generated by infection with the pMIG-mTFF2 retrovirus in the presence of Polybrene (5 μg/mL). The GFP-positive pool of cells is collected by flow cytometric sorting. Stable pools or Jurkat cells bearing empty pMIG or pMIG-mTFF2 can be selected in identical fashion. [000692] Recombinant human TFF1, TFF2 or TFF3 purified from E coli can be obtained commercially, e.g., from Peprotech (Rocky Hills, NJ). [000693] The described invention provides nucleic acid constructs that encode one or more polypeptides that can be expressed in prokaryotic and eukaryotic cells. For example, the described invention provides expression vectors (e.g., DNA- or RNA-based vectors) containing nucleotide sequences that encode TFF2. In addition, the described invention provides methods for making the vectors described herein, as well as methods for introducing the vectors into appropriate host cells for expression of the encoded polypeptides. In general, the methods provided herein include constructing nucleic acid sequences encoding a polypeptide and cloning the sequences into an expression vector. The expression vector can be introduced into host cells or incorporated into virus particles. [000694] cDNA or DNA sequences encoding the polypeptide can be obtained (and, if desired, modified) using conventional DNA cloning and mutagenesis methods, DNA amplification methods, and/or synthetic methods. In general, a sequence encoding a polypeptide can be inserted into a cloning vector for genetic modification and replication purposes prior to expression. Each coding sequence can be operably linked to a regulatory element, such as a promoter, for purposes of expressing the encoded protein in suitable host cells in vitro and in vivo. [000695] Expression vectors can be introduced into host cells for producing secreted polypeptides. There are a variety of techniques available for introducing nucleic acids into viable cells. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, polymer-based systems, DEAE-dextran, viral transduction, the calcium phosphate precipitation method, etc. For in vivo gene transfer, a number of techniques and reagents may also be used, including liposomes; and natural polymer-based delivery vehicles, such as chitosan and gelatin; viral vectors are also suitable for in vivo transduction. In some situations, it may be desirable to provide a targeting agent, such as an antibody or ligand specific for a cell surface membrane protein. Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem.262, 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990). [000696] Where appropriate, gene delivery agents such as, e.g., integration sequences can also be employed. Numerous integration sequences are known in the art (see, e.g., Nunes-Duby et al., Nucleic Acids Res. 26:391-406, 1998; Sadwoski, J. Bacteriol., 165:341-357, 1986; Bestor, Cell, 122(3):322-325, 2005; Plasterk et al., TIG 15:326-332, 1999; Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These include recombinases and transposases. Examples include Cre (Sternberg and Hamilton, J. Mol. Biol., 150:467-486, 1981), lambda (Nash, Nature, 247, 543-545, 1974), FIp (Broach, et al., Cell, 29:227-234, 1982), R (Matsuzaki, et al., J. Bacteriology, 172:610-618, 1990), cpC31 (see, e.g., Groth et al., J. Mol. Biol.335:667- 678, 2004), sleeping beauty, transposases of the mariner family (Plasterk et al., supra), and components for integrating viruses such as AAV, retroviruses, and antiviruses having components that provide for virus integration such as the LTR sequences of retroviruses or lentivirus and the ITR sequences of AAV (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413- 439, 2003). [000697] Cells may be cultured in vitro or genetically engineered, for example. Host cells can be obtained from normal or affected subjects, including healthy humans, cancer patients, private laboratory deposits, public culture collections such as the American Type Culture Collection, or from commercial suppliers. [000698] Cells that can be used for production and secretion of the polypeptide in vivo include, without limitation, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, or granulocytes, various stem or progenitor cells, such as hematopoietic stem or progenitor cells (e.g., as obtained from bone marrow), umbilical cord blood, peripheral blood, fetal liver, etc., and tumor cells (e.g., human tumor cells). The choice of cell type depends on the type of infectious disease being treated or prevented, and can be determined by one of skill in the art. [000699] Different host cells have characteristic and specific mechanisms for post- translational processing and modification of proteins. A host cell may be chosen which modifies and processes the expressed gene products in a specific fashion similar to the way the recipient processes its heat shock proteins (hsps). [000700] According to some embodiments, an expression construct as provided herein can be introduced into an antigenic cell. As used herein, antigenic cells can include preneoplastic cells that are infected with a cancer-causing infectious agent, such as a virus, but that are not yet neoplastic, or antigenic cells that have been exposed to a mutagen or cancer- causing agent, such as a DNA-damaging agent or radiation, for example. Other cells that can be used are preneoplastic cells that are in transition from a normal to a neoplastic form as characterized by morphology or physiological or biochemical function. According to some embodiments, an expression construct as provided herein can be introduced into a non- antigenic cell, for example a serial killer cell, such as NK cells, NKTs, CIKs, GDTs, DCs, MAIT cells, and CD8+ and/or CD4+ CTL cells. [000701] Typically, the cancer cells and preneoplastic cells used in the methods provided herein are of mammalian origin. According to some embodiments, cancer cells (e.g., human tumor cells) can be used in the methods described herein. Cell lines derived from a preneoplastic lesion, cancer tissue, or cancer cells also can be used. Cancer tissues, cancer cells, cells infected with a cancer-causing agent, other preneoplastic cells, and cell lines of human origin can be used. According to some embodiments, a cancer cell can be from an established tumor cell line or tumor cell line variant such as, without limitation, an established non-small cell lung carcinoma (NSCLC), bladder cancer, melanoma, ovarian cancer, renal cell carcinoma, prostate carcinoma, sarcoma, breast carcinoma, squamous cell carcinoma, head and neck carcinoma, hepatocellular carcinoma, pancreatic carcinoma, or colon carcinoma cell line. [000702] According to some embodiments, the mammalian cell line is A293 (human embryonic kidney). According to some embodiments, the mammalian cell line is A549 (human lung epithelial cell line derived from lung carcinomatous tissue. According to some embodiments, the mammalian cell line is human Jurkat clone E6-1. According to some embodiments, the mammalian cell line is a Chinese Hamster Ovary (CHO) cell line. [000703] Both prokaryotic and eukaryotic vectors can be used for expression of polypeptide in the methods provided herein. Prokaryotic vectors include constructs based on E. coli sequences (see, e.g., Makrides, Microbiol Rev 1996, 60:512-538). Non-limiting examples of regulatory regions that can be used for expression in E. coli include lac, trp, 1pp, phoA, recA, tac, T3, T7 and lambda PL. Non-limiting examples of prokaryotic expression vectors may include the Agt vector series such as .lambda.gt11 (Huynh et al., in "DNA Cloning Techniques, Vol. I: A Practical Approach," 1984, (D. Glover, ed.), pp. 49-78, IRL Press, Oxford), and the pET vector series (Studier et al., Methods Enzymol 1990, 185:60-89). [000704] A variety of regulatory regions can be used for expression of the exogenous immunomodulators in mammalian host cells. For example, the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter can be used. Inducible promoters that may be useful in mammalian cells include, without limitation, promoters associated with the metallothionein II gene, mouse mammary tumor virus glucocorticoid responsive long terminal repeats (MMTV- LTR), the n-interferon gene, and the hsp70 gene (see, Williams et al., Cancer Res 1989, 49:2735-42; and Taylor et al., Mol Cell Biol 1990, 10:165-75). Heat shock promoters or stress promoters also may be advantageous for driving expression of the recombinant fusion proteins in recombinant host cells. [000705] Animal regulatory regions that exhibit tissue specificity and have been utilized in transgenic animals also can be used in tumor cells of a particular tissue type: the elastase I gene control region that is active in pancreatic acinar cells (Swift et al., Cell 1984, 38:639-646; Ornitz et al., Cold Spring Harbor Symp Quant Biol 1986, 50:399-409; and MacDonald, Hepatology 1987, 7:425-515); the insulin gene control region that is active in pancreatic beta cells (Hanahan, Nature 1985, 315:115-122), the immunoglobulin gene control region that is active in lymphoid cells (Grosschedl et al., Cell 1984, 38:647-658; Adames et al., Nature 1985, 318:533-538; and Alexander et al., Mol Cell Biol 1987, 7:1436-1444), the mouse mammary tumor virus control region that is active in testicular, breast, lymphoid and mast cells (Leder et al., Cell 1986, 45:485-495), the albumin gene control region that is active in liver (Pinkert et al., Genes Devel, 1987, 1:268-276), the alpha-fetoprotein gene control region that is active in liver (Krumlauf et al., Mol Cell Biol 1985, 5:1639-1648; and Hammer et al., Science 1987, 235:53-58); the alpha 1-antitrypsin gene control region that is active in liver (Kelsey et al., Genes Devel 1987, 1:161-171), the beta-globin gene control region that is active in myeloid cells (Mogram et al., Nature 1985, 315:338-340; and Kollias et al., Cell 1986, 46:89-94); the myelin basic protein gene control region that is active in oligodendrocyte cells in the brain (Readhead et al., Cell 1987, 48:703-712); the myosin light chain-2 gene control region that is active in skeletal muscle (Sani, Nature 1985, 314:283-286), and the gonadotropic releasing hormone gene control region that is active in the hypothalamus (Mason et al., Science 1986, 234:1372-1378). [000706] An expression vector also can include transcription enhancer elements, such as those found in SV40 virus, Hepatitis B virus, cytomegalovirus, immunoglobulin genes, metallothionein, and beta-actin (see, Bittner et al., Meth Enzymol 1987, 153:516-544; and Gorman, Curr Op Biotechnol 1990, 1:36-47). In addition, an expression vector can contain sequences that permit maintenance and replication of the vector in more than one type of host cell, or integration of the vector into the host chromosome. Such sequences include, without limitation, to replication origins, autonomously replicating sequences (ARS), centromere DNA, and telomere DNA. [000707] In addition, an expression vector can contain one or more selectable or screenable marker genes for initially isolating, identifying, or tracking host cells that contain DNA encoding the immunogenic proteins as described herein. For long term, high yield production of gp96-Ig and T cell costimulatory fusion proteins, stable expression in mammalian cells can be useful. A number of selection systems can be used for mammalian cells. For example, the Herpes simplex virus thymidine kinase (Wigler et al., Cell 1977, 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalski and Szybalski, Proc Natl Acad Sci USA 1962, 48:2026), and adenine phosphoribosyltransferase (Lowy et al., Cell 1980, 22:817) genes can be employed in tk-, hgprf-, or aprf- cells, respectively. In addition, antimetabolite resistance can be used as the basis of selection for dihydrofolate reductase (dhfr), which confers resistance to methotrexate (Wigler et al., Proc Natl Acad Sci USA 1980, 77:3567; O'Hare et al., Proc Natl Acad Sci USA 1981, 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, Proc Natl Acad Sci USA 1981, 78:2072); neomycin phosphotransferase (neo), which confers resistance to the aminoglycoside G-418 (Colberre- Garapin et al., J Mol Biol 1981, 150:1); and hygromycin phosphotransferase (hyg), which confers resistance to hygromycin (Santerre et al., Gene 1984, 30:147). Other selectable markers such as histidinol and Zeocin™ also can be used. [000708] A number of viral-based expression systems also can be used with mammalian cells. Vectors using DNA virus backbones have been derived from simian virus 40 (SV40) (Hamer et al., Cell 1979, 17:725), adenovirus (Van Doren et al., Mol Cell Biol 1984, 4:1653), adeno-associated virus (McLaughlin et al., J Virol 1988, 62:1963), and bovine papillomas virus (Zinn et al., Proc Natl Acad Sci USA 1982, 79:4897). When an adenovirus is used as an expression vector, the donor DNA sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This fusion gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) can result in a recombinant virus that is viable and capable of expressing heterologous products in infected hosts. (See, e.g., Logan and Shenk, Proc Natl Acad Sci USA 1984, 81:3655-3659). [000709] Bovine papillomavirus (BPV) can infect many higher vertebrates, including man, and its DNA replicates as an episome. A number of shuttle vectors have been developed for recombinant gene expression, which exist as stable, multicopy (20-300 copies/cell) extrachromosomal elements in mammalian cells. Typically, these vectors contain a segment of BPV DNA (the entire genome or a 69% transforming fragment), a promoter with a broad host range, a polyadenylation signal, splice signals, a selectable marker, and "poisonless" plasmid sequences that allow the vector to be propagated in E. coli. Following construction and amplification in bacteria, the expression gene constructs are transfected into cultured mammalian cells by, for example, calcium phosphate coprecipitation. For those host cells that do not manifest a transformed phenotype, selection of transformants is achieved by use of a dominant selectable marker, such as histidinol and G418 resistance. [000710] Alternatively, the vaccinia 7.5K promoter can be used. (See, e.g., Mackett et al., Proc Natl Acad Sci USA 1982, 79:7415-7419; Mackett et al., J Virol 1984, 49:857-864; and Panicali et al., Proc Natl Acad Sci USA 1982, 79:4927-4931.) In cases where a human host cell is used, vectors based on the Epstein-Barr virus (EBV) origin (OriP) and EBV nuclear antigen 1 (EBNA-1; a trans-acting replication factor) can be used. Such vectors can be used with a broad range of human host cells, e.g., EBO-pCD (Spickofsky et al., DNA Prot Eng Tech 1990, 2:14-18); pDR2 and lambdaDR2 (available from Clontech Laboratories). [000711] Retroviruses, such as Moloney murine leukemia virus, can be used since most of the viral gene sequence can be removed and replaced with exogenous coding sequence while the missing viral functions can be supplied in trans. In contrast to transfection, retroviruses can efficiently infect and transfer genes to a wide range of cell types including, for example, primary hematopoietic cells. Moreover, the host range for infection by a retroviral vector can be manipulated by the choice of envelope used for vector packaging. [000712] For example, a retroviral vector can comprise a 5' long terminal repeat (LTR), a 3' LTR, a packaging signal, a bacterial origin of replication, and a selectable marker. The gp96-Ig fusion protein coding sequence, for example, can be inserted into a position between the 5' LTR and 3' LTR, such that transcription from the 5' LTR promoter transcribes the cloned DNA. The 5' LTR contains a promoter (e.g., an LTR promoter), an R region, a U5 region, and a primer binding site, in that order. Nucleotide sequences of these LTR elements are well known in the art. A heterologous promoter as well as multiple drug selection markers also can be included in the expression vector to facilitate selection of infected cells. See, McLauchlin et al., Prog Nucleic Acid Res Mol Biol 1990, 38:91-135; Morgenstern et al., Nucleic Acid Res 1990, 18:3587-3596; Choulika et al., J Virol 1996, 70:1792-1798; Boesen et al., Biotherapy 1994, 6:291-302; Salmons and Gunzberg, Human Gene Ther 1993, 4:129-141; and Grossman and Wilson, Curr Opin Genet Devel 1993, 3:110-114. [000713] Any of the cloning and expression vectors described herein may be synthesized and assembled from known DNA sequences using techniques that are known in the art. The regulatory regions and enhancer elements can be of a variety of origins, both natural and synthetic. Some vectors and host cells may be obtained commercially. Non-limiting examples of useful vectors are described in Appendix 5 of Current Protocols in Molecular Biology, 1988, ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, which is incorporated herein by reference; and the catalogs of commercial suppliers such as Clontech Laboratories, Stratagene Inc., and Invitrogen, Inc. Expression systems in S. cerevisiae [000714] A recombinant human TFF1 glycosylated at the N-terminal Ser residue produced in Brevebacillus choshiinensis has been described. [Cheng, Y-M et al. BMC Biotechnology (2015) 15: 32]. In vivo, human TFF2 is glycosylated via an N-linkage, presumably on Asn(15), which forms part of the single consensus site for N-glycosylation in [000715] human TFF2. [May, FEB, et al., Gut (2000) 46 (4): 454-59]. Recombinant human glycosylated TFF2 can be purified from yeast. Yeast are single-celled microorganisms that are classified, along with molds and mushrooms, as members of the Kingdom Fungi. [000716] Expression systems in yeast are well established. Both plasmid vectors and chromosomal integration are widely used to introduce genes and control copy number in S. cerevisiae; the choice depends on the overall goal (e.g. overexpression, precise control of gene number). While the plasmids available for use in yeast are much more limited than those for Escherichia coli, they have been successfully employed for many metabolic engineering applications. They are useful for gene expression; however, plasmids offer limited control of copy number, and segregational stability can be a significant issue even in selective medium. As homologous recombination is very efficient in S. cerevisiae, integration of genes into the genome offers an alternate, straightforward mechanism for gene introduction. Chromosomal integration also allows the insertion of precise numbers of the same or different genes. [Da Silva, NA, Sriskrishnan, S. FEMS Yeast Res. (2012) 12 (2): 197-214] [000717] The three classes of autonomously replicating plasmids in yeast are YRp, YEp, and YCp. All are S. cerevisiae/E. coli shuttle vectors that typically carry a multiple cloning site (MCS) for the insertion of expression cassettes. The widely used YCp and YEp vectors have proven successful for many applications. YCp (CEN/ARS) vectors carry both an origin of replication and a centromere sequence, have high segregational stability in selective medium, and are maintained at 1–2 copies per cell [Id., citing Clarke, L. & Carbon J. Nature (1980) 287: 504-0]. YEp vectors are based on the S. cerevisiae native 2μ episomal plasmid and contain either the full 2μ sequence or, more commonly, a 2μ sequence including both the origin and the REP3 (STB) stability locus [Id., citing Futcher, AB & Cox, BS J. Bacteriol. (1986) 154: 612-2; Kikuchi, Y. Cell (1983) 35: 487-93]. For the full sequence, use of a cir0 strain lacking the native plasmid is recommended to prevent the recombination between the vectors and to keep copy number of the recombinant vector high. For the partial 2μ plasmids, a cir+ host carrying the native 2μ is required to provide the transacting factors (REP1 and REP2) required for stability. These vectors are generally more structurally stable than the full 2μ plasmids, but may be maintained at lower copy number. Although the maintenance of YEp vectors at 10–40 copies [Id., citing Romanos, MA, et al., Yeast (1992) 8: 23—88] is generally assumed, copy number is not controlled and can vary widely with the gene product and level of expression. [000718] Several vector series have been developed carrying a series of selection markers on YEp and YCp plasmids and YIp integrating vectors. A series of YCp and YEp plasmids have been constructed with LEU2, HIS3, LYS2, URA3, and TRP1 selection markers. [Id., citing Ma, H. et al. Gene (1987) 58: 201-16]. The YEplac and YCplac plasmids [Id., citing Gietz, RD, Sugino, A. Gene (1988) 74: 527-34] carry a MCS and URA3, TRP1, and LEU2 selection markers on both 2μ- and CEN/ARS-based plasmids, respectively. The pRS series [Id., citing Sikorski RS & Hieter, P. Genetics (1989) 122: 19-27; Christianson, TW et al. Gene (1992) 110: 119-22] are similar useful cloning vectors with URA3, TRP1, HIS3, and LEU2 markers on both 2μ and CEN/ARS plasmids. The pRS series was extended to include the MET15, ADE2, kanMX, hphNT1, and natNT2 selectable markers. [Id., citing Brachmann, CB et al. Yeast (1998) 115-32; Taxis, C. & Knop, M. BioTechniques (2006) 40: 73-78] The various vector series have been widely used for gene expression in yeast. [000719] Groups of vectors carrying constitutive and inducible promoters have also been developed. These include the variants of the pRS series carrying ADH1, TEF1, GPD1, MET25, CYC1, GAL1, and GALL or GALS (GAL1 variant) promoters [Id., citing Mumberg, D. et al., Gene (1995) 156: 119-22; Mumberg, D. et al. Nucleic Acids Res. (1994) 22: 5767-68] and the CUP1 promoter [Id., citing Labbe, S. & Thiele, SJ Expression of Recombinant Genes in Eukaryotic Systems, In Methods in Enzymology, Vol 306; Abelson J Simon M Glorioso J Schmidt M pp. 145–153. Academic Press, New York]. YEp and YCp expression vectors carrying a URA3 selection marker and the PGK, GAL1, GAL10, PHO5, and CUP1 promoters have been developed. [Id., citing Cartwright, CP et al. Yeast (1994) 10: 497-508]. The YEplac and YCplac plasmids were modified to carry the tetracycline-responsive tet-on/off promoters [Id., citing Gari, E. et al., Yeast (1997) 13: 837-48]. The commercially available pYES and pYC series (Invitrogen) offer expression from the GAL1 promoter on 2μ or CEN/ARS vectors, respectively. These vectors are available with URA3, TRP1, and blasticidin resistance selection markers. The pGREG vectors [Id., citing Jansen, G. et al. Gene (2005) 344: 43-51] are derivatives of the pRS series with five different selectable markers on CEN/ARS-based plasmids with a GAL1 promoter and can be useful tools for plasmid construction via in vivo recombination and for the expression of N- and C-terminal-tagged fusion proteins (nine tags available). Vectors series [e.g., Id. Citing Funk, M. et al. Guide to Yeast Genetics and Molecular and Cell Biology, Pt B Methods Enzymol (2002) 350: 248–257, Van Mullem, V. et al. Yeast (2003) 20: 739-46, Geiser, JR, BioTechniques (2005) 38: 378-82; and Alberti, S et al. Yeast (2007) 24: 913-19] have been constructed utilizing the Gateway™ cloning technology [Invitrogen, Id., citing Walhout, AJM et al. Methods Enzymol. (2007) 328: 575- 92], which allows for the insertion of ORFs into vectors by in vitro recombination using the bacteriophage lambda att sites. The vector series contain various promoters and selection markers, 2μ or CEN/ARS sequences, and additional features such as epitope tags. A series of 32 pXP shuttle vectors, utilizing three constitutive promoters, PGK1, TEF1, and HXT7-391, and six reusable selection markers on both 2μ and CEN/ARS vectors that can be used as templates for gene integration also have been constructed. [Id., citing Fang, F. et al. Yeast (2011) 28: 123-36]. This series was later extended this series to include the GAL1, ADH2, and CUP1 promoters. [Id., citing M.W.Y. Shen, F. Fang, S. Sandmeyer & N.A. Da Silva (unpublished data)]. [000720] In S. cerevisiae, homologous recombination requires only limited flanking homology [Id., citing Manivasakam, P. et al. Nucleic Acid Res. (1991) 23: 2799-2800]. Efficient targeting increases with the length of the homology, and an overlap of approximately 50 bp (25 bp on each side) is sufficient to easily screen and recover integrants in specific genomic locations. Standard length primers can thus be used to amplify a desired gene cassette with flanking target regions, and the PCR product can be transformed into the yeast for insertion by double-crossover. The efficiency of recombination in yeast also allows the use of nested primers or the assembly of two or more fragments [ Id., citing Erdeniz, N et al. Genome Res. (1997) 7: 1174-83; Hawkins, KM & Smolke, CD. J. Biol. Chem. (2006) 281: 13485-92; Flagfeldt, DB et al. Yeast (2009) 26: 545-51; Shao, ZY et al. Nucleic Acids Res. (2009) 37: e16]. [000721] A wide variety of promoters are available for the control of transcription level in S. cerevisiae, including constitutive and inducible promoters of various strengths. The regulated promoters include those for which a higher level of control is possible or a clear inducer/repressor exists. Choice of promoter in combination with gene number allows a very wide range of gene expression levels. [000722] The most widely used constitutive promoters have often been from the yeast glycolytic pathway. These glucose-dependent promoters include those for phosphoglycerate kinase PPGK1 [Id., citing Holland, JP & Holland, MJ (1980) J. Biol. Chem.255: 2596-2605; Ogden, JE et al. (1989) Mol. Cell Biol. (1986) 6: 4335-43] pyruvate decarboxylase PPDC1 [Id., citing Kellermann, E. et al., (1986) Nucleic Acids Res. 14: 8963-77], triosephosphate isomerase PTPI1 [Id., citing Alber, T. & Kawasaki, G (1982) J. Mol. Appl. Genet.1: 419-34], alcohol dehydrogenase I PADH1 [Id., citing Hitzeman, RA et al. (1981) Nature 293: 717-722 (expressing a human gene for interferon in yeast); Denis, CL et al. (1983) J. Biol. Chem.258: 1165-71], glyceraldehyde-3-phosphate dehydrogenase PTDH3 (GAP491) or PGPD [Id., citing Holland, JP & Holland, MJ (1980) J. Biol. Chem.255: 2596-2605]. The constitutive promoter for translation elongation factor PTEF1 (Id., citing Gatignol, A. et al. (1990) Gene 91: 35-41) has also been widely used and has been modified by error-prone PCR to provide a group of promoters offering a range of expression levels (Id., citing Alper, H. et al. (2006) Proc. Natl Acad. Sci. USA 102: 12678-83; Nevoigt, E. et al. (2007) Biotechnol. Bioengin.96: 550-58). Other commonly used native promoters include PCYC1 [Id., citing Guarente, L. et al. (1984) Cell 36: 503-11], PACT1 (Id., citing Gallwitz, D. & Seidel, R. (1980) Nucleic Acids Res.8: 1043-59, PMFα1 [Id., citing Brake, AJ et al. (1984) Proc. Natl. Acad. Sci USA 81: 4642-46], and those for hexose transport, for example PHXT7 [Id., citing Reifenberger, E. et al. (1995) Mol. Microbiol.16: 157-67; Diderich, JA et al. (1999) Microbiology 145: 3447-54]. [000723] Regulated promoters enable control over the timing and level of gene expression. They are thus more suitable when expression of genes is desired at a specific stage of cell growth, or to prevent the build-up of toxic pathway intermediates. The use of inducible promoters is limited by the sensitivity of the promoter to the inducer (including the strength and time for response to the inducer or repressor), background levels of expression because of ‘leaky’ promoters, and the cost of metabolites for induction. In addition, strain response to the inducer may add to the complexity of expression control. [000724] A large variety of regulated native or engineered promoters have been successfully used to control the gene expression in S. cerevisiae. The most tightly regulated native promoters are from the galactose-inducible S. cerevisiae genes GAL1, GAL7, and GAL10 [Id., citing Douglas, HC & Hawthorne DC. Genetics (1964) 49: 837-44, 1964; Bassel, J. & Mortimer, R. J. Bacteriol. (1971) 108: 179-83]. These promoters are induced approximately 1000-fold in the presence of galactose and strongly repressed in the presence of glucose [Adams, BG J. Bacteriol (1972) 111: 308-15]. While several genes have been identified to be involved in the regulation of these promoters, the GAL4 gene encoding a transactivator and GAL80 gene encoding the repressor for Gal4 control the central regulation mechanism along with the GAL upstream activation site (UAS) [Id., citing Johnston, M et al. Mol Cell Biol. (1994) Mol Cell Biol. 14: 3834-41]. Modifications in the GAL1 and GAL10 promoters [Id., citing Li, AM et al. (2008) EMS Yeast Res. 8: 6-9] and engineering key enzymes involved in galactose catabolism and transport [Id., citing Hawkins, KM & Smolke, CD (2006) J. Biol. Chem.281: 13485-492; Hawkins, KM & Smolke, CD (20008) Nat. Chem. Biol. 4: 564-73] have provided tunable control to galactose-driven expression under these promoters. The GAL-regulated promoters have been widely used in yeast metabolic pathway engineering, including for artemisinic acid synthesis [Id., citing Ro, D-K et al. (2006) Nature 440: 940-43; Ro, D-K et al (2008) BMC Biotechnol.8: 83], increased acetyl-CoA synthesis for isoprenoid production [Id., citing Shiba, Y. et al. (2007) Metab. Eng. 9: 160-68 et al.], expression of the bacterial isoprenoid pathway in S. cerevisiae [Id., citing Maury, J. et al. (2008) FEBS Lett.582: 4032-38], and n-butanol synthesis [Id., citing Steen, EJ et al., (2008) Microb. Cell Fact.7: 36]. [000725] Several inducible S. cerevisiae promoters have been employed in yeast including PPHO5, PMET25, and PMET3. [Id.] The PHO5 promoter of the acid phosphatase gene is regulated by inorganic phosphate (Pi) in the medium with approximately 200-fold repression in the presence of phosphate. The MET25 gene [Id., citing Sangsoda, S. et al (1985) Mol. Gen. Genet. 200: 407-11; Kerjan, P. et al. (1986) Nucleic Acids Res. 14: 7861-71] encodes O-acetyl homoserine sulfhydrolase, and the MET3 gene encodes ATP sulfurylase [Id., citing Cherest, H. et al. (1987) Mol. Gen. Genet.210: 307-13]. Both promoters are repressed in the presence of methionine or S-adenosylmethionine. A useful heterologous promoter system in yeast utilizes the bacterial tetracycline operator (tetO) and hybrid transactivator, based on the expression system developed for mammalian cells [Id., citing Gossen, M. & Bujard, H. (1992) Proc. Nat. Acad. Sci. USA 89: 5547-51; Gossen, M. et al. (1995) Science 268: 1766-69] and shown to be active in yeast [Id., citing by Dingermann, T et al. (1992) EMBO J.11: 1487-92]. [000726] Although the preferred medium – or large-scale – method of breaking yeast cells is mechanical shearing, lysis on ice with the aid of acid-washed glass beads, e.g., in a BeadBeater, is described in Green, MR & Sambrook, J. Molecular Cloning, 4th Ed. Vol. 3, pages 1564-65 Cold Spring Harbor, New York (2012). Other methods, including enzymatic cell wall digestion with zymolase, a French press (Lerner-Marmarosh, BN. et al. J. Biol. Chem. (1999) 274: 34711-718), and microfluidization (Aller, SG et al. Science (2009) 323: 1718-22), can be used. Exemplary protease inhibitors should be included during cell lysis to protect the target protein against degradation by serum proteases (PMSF, leupeptiin, benzamidine), cysteine proteases (PMSF, E-64), aspartic proteases (pepstatin A), metalloproteases (EDTA, EGTA) or chymotrypsin (chymostatin). [000727] Alternatively a signal sequence may be used to secrete the recombinant human TFF2 product into the culture medium with selective removal of domain 1 of Vps10 to prevent the protein from being diverted to the vacuole. (See Fitzgerald, I. & Glick, BS. Microb. Cell Fact. (2014) 13: 125 A number of signal sequences have been used to secrete foreign proteins from yeasts, but the most popular is the secretion signal from S. cerevisiae pre-pro-α-factor, which is the precursor to a peptide mating pheromone [Id., citing Gasser, B. et al., Future Microbiol. (2013) 8: 191-208; Braake, AJ et al. Proc. Nat. Acad. Sci. USA (1984) 81: 4642- 46; Oka, C. et al. Biosci. Biotechnol. Biochem. (1999) 63: 1977-83]. Pre-pro-α-factor contains a 19-residue signal sequence that terminates in a signal peptidase cleavage site. Following the signal sequence is a 66-residue pro region, which is removed in the late Golgi by the Kex2 endoprotease [Id., citing Fuller, RS et al. Annu. Rev. Physiol. (1988) 50: 345-62]. Finally, downstream of the dibasic Kex2 cleavage signal is an EAEA tetrapeptide (SEQ ID NO: 32), which is trimmed by the dipeptidyl aminopeptidase Ste13 [Id., Julius, D. et al. Cell (1983) 32: 839-52]. Efficient secretion of foreign proteins requires the entire pre-pro-α-factor secretion signal, with or without the downstream EAEA tetrapeptide (SEQ ID NO: 32) but the secreted products are often heterogeneous due to incomplete processing by Kex2 or Ste13 [Id., citing Gasser, B. et al. Future Microbiol. (2013) 8: 191-208; Lin Cereghino, J. & Cregg, JM. FEMS Microbiol. Rev. (2000) 24: 45-66]. [000728] If a protein is poorly secreted or incompletely processed with existing approaches, the Ost1 signal sequence drives efficient translocation into the ER, and avoids the incomplete processing caused by the α-factor pro region. If incomplete processing is not a concern, a hybrid secretion signal consisting of the Ost1 signal sequence followed by the α- factor pro region might be beneficial for ensuring both efficient translocation and efficient ER export. The S. cerevisiae OstI signal sequence or similar hydrophobic signal sequences, which has been shown to direct cotranslational translocation [Id., citing Willer, M. et al. J. Biol. Chem. (2008) 283: 33883-888; Forte, GM et al., PLoS Biol. (2011) 9: e1001073], therefore can be used to bypass the pro region and its associated complications. Compared to the α-factor secretion signal, the Ost1 signal sequence has two advantages. First, because translocation with the Ost1 signal sequence is cotranslational, even rapidly folding proteins should enter the ER efficiently. Second, because the Ost1 signal sequence requires cleavage only by signal peptidase, the N-termini of the mature proteins are likely to be homogeneous. [000729] A foreign protein that crosses the ER membrane must then be exported to the Golgi. As described above, ER export can be accelerated by receptors such as Erv29, which recognizes the α-factor pro region. In yeast, the vacuolar sorting receptor Vps10 targets misfolded proteins from the Golgi to the vacuole [Id., citing Jorgensen, MU et al. Eur. J. Biochem. (1999) 260: 461-9; Westphal, V. et al. J. Biol. Chem. (1996) 271: 11865-70; Hong, E. et al. J. Cell Biol. (1996) 135: 623-33]. For some foreign proteins, deleting Vps10 may enhance secretion by preventing vacuolar targeting. However, a problem with deleting Vps10 is that some vacuolar hydrolases will be missorted to the culture medium and contaminate a secreted foreign protein and may contribute to its degradation. Selective removal of domain 1 of Vps10 can prevent a foreign protein from being diverted to the vacuole with no significant effect on vacuolar function. [Id, citing Jorgensen, MU et al. Eur. J. Biochem. (1999) 260: 461- 9]. [000730] For S. cerevisiae, secretion can be measured in rich glucose medium (YPD). A 5-mL culture in YPD is inoculated from a preculture, and grown overnight at 30°C with shaking in a baffled flask to an OD600 of 0.7-0.8. Then 1.75 OD600 units are transferred to a 15-mL tube, washed twice with deionized water by centrifugation and resuspension, and resuspended in 5 mL of fresh YPD to an OD600 of 0.35. This culture is incubated with shaking at 30°C for 3 h, an incubation period that enables secretion of a detectable amount of foreign protein while the cells remained in mid-log phase. Then 1.6 mL of culture is transferred to a microcentrifuge tube. Each culture is centrifuged at 3000×g (5600 rpm) for 5 min in a microcentrifuge to separate the cells from the secreted proteins. [000731] Secreted proteins can be precipitated with cold TCA, centrifuged, resuspended in SDS-PAGE sample buffer, and loaded onto a gel for SDS-PAGE and for immunoblotting using human TFF2 as a control. Recombinant polypeptide [000732] According to some embodiments, the recombinant polypeptide may be cloned into two or more plasmid constructs for transfection (via, e.g., lipids, calcium phosphate, cationic polymers, DEAE-dextran, activated dendrimers, magnetic beads, electroporation, biolistic technology, microinjection, laserfection/optoinjection) or transduction (via, e.g., retrovirus, lentivirus, adenovirus, adeno-associated virus) into cells of tumor cell line or tumor cell line variants. According to some embodiments, recombinant DNA encoding the polypeptide may be cloned into a lentiviral vector plasmid for integration into the genome of cells of tumor cell line or tumor cell line variants. According to some embodiments, recombinant DNA encoding the polypeptide may be cloned into a plasmid DNA construct encoding a selectable trait, such as an antibiotic resistance gene. Lentiviral Constructs [000733] According to some embodiments, the DNA sequences coding for a polypeptide may be cloned into a lentiviral vector for transduction into mammalian cells. According to some embodiments, the lentiviral system may comprise a lentiviral transfer plasmid encoding the polypeptide, packaging plasmids encoding the GAG, POL, TAT, and REV sequences, and an envelope plasmid encoding the ENV sequences. According to some embodiments, the lentiviral transfer plasmid uses a viral LTR promoter for gene expression. According to some embodiments, the lentiviral transfer plasmid uses a hybrid promoter, or other specialized promoter. According to some embodiments, the promoter of the lentiviral transfer plasmid is selected to express the two or more immune modulator sequences at a desired level relative to other immunomodulatory sequences. According to some embodiments, the relative level is measured on the level of transcription as mRNA transcripts. According to some embodiments, the relative level is measured on the level of translation as protein expression. Multicistronic plasmid constructs [000734] According to some embodiments, the polypeptide sequence may be cloned in a multicistronic vector for co-expression of another recombinant sequence. According to some embodiments, a polypeptide sequence may be cloned into a plasmid comprising an IRES element to promote translation of two or more proteins from a single transcript. According to some embodiments, one or more polypeptides is cloned into a multicistronic vector comprising sequences for a self-cleaving 2A peptide to produce two molecules from a single transcript. [000735] According to some embodiments, the vector constructs further comprise one or more tags, as described herein. [000736] According to some embodiments, the recombinant proteins of the present disclosure comprise an affinity tag. Affinity tags are unique proteins/peptides that can be attached at the N- or C-terminus of a recombinant protein. According to some embodiments, these tags help in protein purification. According to some embodiments, the affinity tag can act as a solubility enhancer, aid in affinity purification, or both. Exemplary protein affinity tags are shown in Table 3. [000737] Table 3.
Figure imgf000243_0001
[000738] Note: Tags can be removed from the tagged protein by proteolysis if a protease cleavage site is included between the native sequence and the tag. Common proteases include (but are not limited to) TEV protease, thrombin and Factor Xa. [000739] Note: Many functionally inert synthetic tags with corresponding high-affinity monoclonal antibodies for detection and/or affinity purification have been developed (too numerous to list). SELECT REFERENCES 1. Bornhorst JA and Falke JJ (2000) Purification of proteins using polyhistidine affinity tags. Methods in Enzymology 326: 245-254. 2. Einhauer A and Jungbauer A (2001) The FLAG™ peptide, a versatile fusion tag for the purification of recombinant proteins. Journal of Biochemical and Biophysical Methods 49 (1– 3): 455–65. 3. Götzke, H, Kilisch M, Martínez-Carranza M, Sograte-Idrissi S, Rajavel A, Schlichthaerle T, Engels N, Jungmann R, Stenmark P, Opazo F, Frey S (2019) The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications. Nature Communications 10 (1): 4403 4. Keefe AD, Wilson DS, Seelig B, Szostak JW (2001) One-Step Purification of Recombinant Proteins Using a Nanomolar-Affinity Streptavidin-Binding Peptide, the SBP- Tag. Protein Expression and Purification 23 (3): 440–6. 5. Maertens B, Spriestersbach A, Kubicek J, Schäfer F (2015) Strep-Tagged Protein Purification. Methods in Enzymology 559: 53-69. 6. Pina AS, et alb. (2014) Affinity tags in protein purification and peptide enrichment: An overview. Methods in Molecular Biology 1129: 147-168. 7. Schmidt TGM, Koepke J, Frank R, Skerra A (1996) Molecular Interaction Between the Strep-tag Affinity Peptide and its Cognate Target, Streptavidin. Journal of Molecular Biology 255 (5): 753–66. 8. Wood DW (2014) New trends and affinity tag designs for recombinant protein purification. Curr Opin Struct Biol 26: 54-61. Lentiviral System [000740] According to some embodiments, the lentiviral system may be employed where the transfer vector with polypeptide sequences, an envelope vector, and a packaging vector are each transfected into host cells for virus production. According to some embodiments, the lentiviral vectors may be transfected into 293T cells by any of calcium phosphate precipitation transfection, lipid based transfection, or electroporation, and incubated overnight. For embodiments where the i polypeptide sequence may be accompanied by a fluorescence reporter, inspection of the 293T cells for florescence may be checked after overnight incubation. The culture medium of the 293T cells comprising virus particles may be harvested 2 or 3 times every 8-12 hours and centrifuged to sediment detached cells and debris. The culture medium may then be used directly, frozen or concentrated as needed. Lipid Based Transfection [000741] According to some embodiments, host cells may be transfected with polypeptide sequences using a lipid based transfection method. According to some embodiments, established lipid based transfection reagents, such as LIPOFECTAMINE, may be used. A cell line may be grown to about 70-90% confluence in a tissue culture vessel. Appropriate amounts of Lipofectamine® and plasmid construct comprising the polypeptide sequence may be separately diluted in tissue culture media and briefly incubated at room temperature. The diluted Lipofectamine® and plasmid constructs in media may be mixed together and incubated briefly at room temperature. The plasmid LIPOFECTAMINE mixture may then be added to the cells of the cell line in the tissue culture vessel and incubated for 1-3 days under standard tissue culture conditions. Selection of Expressing Clones [000742] According to some embodiments, cell populations of the cell line that have been transfected with polypeptide sequences may be selected for various levels of expression. [000743] According to some embodiments, the polypeptide sequences may be accompanied by antibiotic resistance genes, which may be used to select for clones with stable integration of the recombinant DNA encoding the polypeptide sequences. According to some embodiments, the polypeptide sequences may be cloned into a plasmid construct comprising antibiotic resistance, such as the Neomycin/Kanamycin resistance gene. Transfected cells are treated with antibiotics according to the manufacturer’s protocol for 1-2 weeks or more with daily media changes. At some point during antibiotic treatment, there is massive cell death of all cells that have not stably integrated the antibiotic resistance gene, leaving behind small colonies of stably expressing clones. Each of the stably expressing clones may be picked, cultured in a separate tissue culture container, and tested for levels of polypeptide expression by any established method, such as western blot, flow cytometry, and fluorescence microscopy. [000744] According to some embodiments, transfected cells may be selected for high expression of the polypeptide by fluorescence activated cell sorting (FACS). According to some embodiments, polypeptide sequences may be accompanied by one or more fluorescent proteins (e.g. GFP), which can be used to quantify expression of immune modulator. For example, a bicistronic plasmid comprising a polypeptide sequence connected to a GFP sequence via IRES sequence would result in both polypeptide and GFP protein translated from the same transcript. Thus, the GFP expression level would act as a proxy for the expression level of polypeptide. Single cell suspensions of polypeptide /GFP transfected cells could be selected for the desired level of expression by FACS based on the fluorescence intensity. Any fluorescent protein may be used in this regard. For example, any of the following recombinant fluorescent proteins (rXFP) may be used: EBFP, ECFP, EGFP, YFP, mHoneydew, mBanana, mOrange, tdTomato, mTangerine, mStrawberry, mCherry, mGrape, mRasberry, mGrape2, mPlum. [000745] Alternatively, the expression of the recombinant polypeptide may be directly observed by fluorescent antibodies specific to each polypeptide or specific to a tag engineered onto each polypeptide. For example, according to some embodiments the extracellular region of a polypeptide sequence may be fused with a FLAG tag or HA tag. Anti-FLAG or anti-HA antibodies may be used, along with a fluorophore attached to the primary antibody or a secondary antibody) to detect the expression of the polypeptide on the surface of the transfected tumor cells. Tumor cells expressing the desired level of polypeptide may be selected by FACS sorting and cultured separately. [000746] The relative amount of recombinant polypeptide expressed within each clonally derived cell line, and between cell lines, can be measured on the level of transcription or translation. For example, the relative amount of recombinant polypeptide can be quantified by western blot, RT-PCR, flow cytometry, immunofluorescence, and northern blot, among others. [000747] According to some embodiments, the differences in the amount of expressed polypeptide relative to one another may be a result of random integration into more or less transcriptionally active regions of the genome of the cell line. According to some embodiments, the relative differences in the amount of expressed polypeptide may be achieved by elements engineered into the transfected or transduced DNA used to create the cell line. [000748] For example, according to some embodiments, the level of expression of the polypeptide molecules may be achieved on the transcriptional level by engineering stronger or weaker gene promoter sequences to control expression of the immune modulator gene. According to some embodiments, one or more of the following promoters may be used to control expression of immunomodulators: simian virus 40 early promoter (SV40), cytomegalovirus immediate-early promoter (CMV), human Ubiquitin C promoter (UBC), human elongation factor 1α promoter (EF1A), mouse phosphoglycerate kinase 1 promoter (PGK), and chicken β-Actin promoter coupled with CMV early enhancer (CAGG). [000749] According to some embodiments, the level of expression of the polypeptide molecules may be achieved on the translational level by engineering stronger or weaker Kozak consensus sequences around the start codon of the polypeptide transcript. According to some embodiments, the following nucleotide sequences may be provided to control polypeptide translation: GCCGCC(A/G)CCAUGG (SEQ ID NO: 15). According to some embodiments, a sequence that is at least 60% identical to SEQ ID NO: 15 may be provided to control polypeptide translation. According to some embodiments, a sequence that is at least 70% identical to SEQ ID NO: 15 may be provided to control polypeptide translation. According to some embodiments, a sequence that is at least 80% identical to SEQ ID NO: 15 may be provided to control polypeptide translation. According to some embodiments, a sequence that is at least 90% identical to SEQ ID NO: 15 may be provided to control polypeptide translation. According to some embodiments, a sequence that is at least 95% identical to SEQ ID NO: 15 may be provided to control polypeptide translation. According to some embodiments, a sequence that is at least 96% identical to SEQ ID NO: 15 may be provided to control polypeptide translation. According to some embodiments, a sequence that is at least 97% identical to SEQ ID NO: 15 may be provided to control polypeptide translation. According to some embodiments, a sequence that is at least 98% identical to SEQ ID NO: 15 may be provided to control polypeptide translation. According to some embodiments, a sequence that is at least 99% identical to SEQ ID NO: 15 may be provided to control polypeptide translation. [000750] Non-viral approaches can also be employed for the introduction of a vector encoding one or more polypeptide molecules to a cell derived from a patient. For example, a nucleic acid molecule encoding a polypeptide molecule can be introduced into a cell by administering the nucleic acid molecule in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A.84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by micro- injection under surgical conditions (Wolff et al., Science 247:1465, 1990). According to some embodiments, the nucleic acids are administered in combination with a liposome and protamine. [000751] Methods for accomplishing transfection in vitro include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. [000752] According to some embodiments, polypeptides whose functionality has been modified by genetic engineering are intended to be included within the scope of the claimed invention. For example, a polypeptide may be modified by genetic engineering to change a signal sequence, to make the polypeptide product a secreted product, to increase stability of the polypeptide; to alter key amino acids, or to codon optimize sequences for humans. All such modifications are included within the scope of the claimed invention. [000753] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention. [000754] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, exemplary methods and materials have been described. All publications mentioned herein are incorporated herein by reference to disclose and described the methods and/or materials in connection with which the publications are cited. [000755] It must be noted that as used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural references unless the context clearly dictates otherwise. [000756] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application and each is incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. EXAMPLES [000757] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. Example 1. Establishment of a mouse model infected with severe/highly pathogenic H7N9 influenza virus and low pathogenicity/mild H9N2 influenza virus [000758] C57 mice were anesthetized with ketamine (40 µl/mouse) before infection, and then infected with an influenza virus H7N9 virus strain A/Shanghai/4664T/2013 (3.5 x105 of 50% tissue culture infective dose TCID50/50 ul of volume) and an H9N2 virus strain A/Chicken/Shanghai/F/98 (1.7 x 107 of 50% egg infective dose EID50/50µl of volume) at a high dose by nasal drip. The mice were placed in an IVC cage, and for 14 consecutive days, the mice were weighed, and the survival and survival status of the mice were observed. Lung tissues were taken at 6 hours, 1 day, 2 days, 3 days, 7 days, and 14 days after infection, and quick-frozen in liquid nitrogen for use (FIG. 4A). As shown in FIG. 4B, the weight of the H7N9-infected mice continued to decrease, and as of Day 7, most mice lost 30% or more of their weight; whereas, the weight of the H9N2-infected mice decreased by 12.6% in the first 3 days after infection, the weight gradually recovered from Day 4, the weight returned to the level before infection on Day 8, and the weight gradually increased from Day 9. Analysis of survival rate (FIG.4C) showed that the H7N9-infected mice died beginning from Day 4 after infection, mortality reached 37% on Day 7, and the mice all died by Day 8; whereas, after H9N2 infection, 12% of the mice died from being bitten (wounded) caused by rage and irritability due to viral infection rather than dying from direct viral infection, and the remaining 88% of the mice could survive for 2 weeks or more. The H7N9 test was completed in the P3 laboratory, and the H9N2 test was completed in the P2 laboratory. Example 2: Differential gene expression in lung tissue of mice infected with severe and mild influenza with a transcriptome chip [000759] The following experiment describes screening of TFF2 as a protective molecule by analyzing and screening significantly different gene expression in lung tissues of mice infected with severe influenza and mild influenza with a transcriptome chip. [000760] Whole-genome expression profiles in lung tissues of H7N9- and H9N2-infected mice were obtained at time points of 0 day, 6 hours, 1 day, 2 days, 3 days, 7 days, and 14 days using an Agilent 2100 system. As shown in FIG. 5A, the P value was set at 0.01, and 188 significantly different genes were screened, among which 85 genes were highly expressed in the H7N9 group and 103 genes were highly expressed in the H9N2 group. Typical genes that were highly expressed in the H7N9 group include 01fr11, IFN13, IFNb1 and Krt17, which are related to inflammation; genes that were highly differentially expressed in the H9N2 group include TFF2, Gp2, Muc5b, Muc5a and Clca3, which are closely related to host defense and epithelial cell repair. Among them, TFF2 had the largest difference between the two groups, reaching a level of expression 106.7 times the control, and the P value was 0.002. The TFF2 mRNA decreased slightly in the first 2 days of H9N2 infection, and gradually increased from Day 3; whereas, it continued to decrease in H7N9 infection (FIG.5B). In order to further verify the expression of TFF2 in H7N9 and H9N2, a qPCR test was performed with primers [TFF2- F (SEQ ID NO: 1): GAGAAACCTTCCCCCTGTCG; and TFF2-R (SEQ ID NO: 2): TTTCGACTGGCACAGTC CTC] using the SYBR Real-time PCR method according to the instructions of GoTaq qPCR Master Mix (Promega, A6001). Changes in level of TFF2 mRNA were verified in 3 mice at each time point in both groups, and the change trend was consistent with the chip data (FIG.5C). Next, the level of expression of the TFF2 protein was detected. The TFF2 protein was very weakly expressed in the control group, and gradually disappeared after H7N9 infection. However, after H9N2 infection, the level of expression of the TFF2 protein in lung tissues became more and more over time (FIG.5D). Example 3: Cloning of the TFF2 gene and eukaryotic expression of the TFF2 protein. [000761] In this Example, the TFF2 gene was first cloned based on the TFF2 sequence (the nucleotide sequence of the human-derived TFF2 protein (hTFF2) is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4; and the nucleotide sequence of the murine-derived TFF2 protein (mTFF2) is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6). A recombinant plasmid (either pSV1.0-TFF2 or pSV1.0- TFF2-6xHis ("6xHis" disclosed as SEQ ID NO: 33)) was transfected into 293T cells. Then, eukaryotic expression of the TFF2 protein was detected via Western Blot. The product was eluted with imidazole of different concentration gradients through a Ni-column. The target protein was collected, filtered, and washed to obtain a high-purity TFF2 protein. The specific steps were as follows. [000762] PCR amplification was performed using cDNA generated from reverse transcription of RNA extracted from lung tissues of H9N2-infected mice as a template. The upstream primer was SEQ ID NO: 7, i.e., 5'-CGCTCTAGAATGCGACCTCGAGGTGCCCC- 3'; and the downstream primer was SEQ ID NO: 8, i.e., 5'- CCTGGATCCTCAGTAGTGACAATCTTCCA-3'. The primer sequences for TFF2-6xHis ("6xHis" disclosed as SEQ ID NO: 33) were as follows. The upstream primer was SEQ ID NO: 9, i.e., 5'-CGCTCTAGAATGCGACCTCGAGGTGCCCC-3'; and the downstream primer was SEQ ID NO: 10, i.e., 5'- CGGGATCCTCAGTGATGATGATGATGATGGTAGTGACAATCTTCCA-3'. The amplification procedure was as follows: pre-denaturation at 95°C for 2 minutes; denaturation at 95°C for 15 seconds; annealing at 55°C for 30 seconds; extension at 72°C for 30 seconds; and final extension at 72°C for 10 minutes; the number of cycles was 30. After amplification was completed, the target gene was isolated on a 1% agarose gel. The amplified product TFF2 was then recovered using the Sanprep column DNA gel recovery kit. Both the recovered product TFF2 and the vector pSV1.0 were recovered by double digestion with endonucleases BamHI and XbaI. Digestion was performed in a 37°C water bath for 7 hours. The fragments were recovered using the Sanprep column DNA gel recovery kit after 1% agarose gel electrophoresis. The target fragment TFF2 was ligated with the vector pSV1.0 at 4°C overnight to form recombinant plasmids pSV1.0-TFF2 and pSV1.0-TFF2-6xHis ("6xHis" disclosed as SEQ ID NO: 33). The recombinant plasmids were transformed into E. coli TOP10; positive clones were identified by colony PCR and double digestion (BamHI and XbaI). The target sequence was identified by sequencing to be completely correct, and no mutation occurred. The constructed cloning map is shown in FIG.6A. [000763] Recombinant plasmid pSV1.0-TFF2 (identified by sequencing as correct) was transfected into 293F T cells. The transfection reagent was TurboFect, and the medium was DMEM complete medium (10% FBS and 1% P.S.). The cells were incubated in an incubator at 37°C for 72 hours, then collected into a pre-chilled EP tube, and lysed with RIPA lysis buffer. 5 X SDS Loading Buffer was added to the supernatant. The supernatant was heated in a boiling water bath for 10 minutes to denature the protein, and the supernatant was used as the loading sample after brief centrifugation. Then, proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) through a 15% separation gel at a voltage of 70 V, for 30-40 minutes (i.e., until the marker began to separate). After bromophenol blue had migrated out of the concentrated gel position, the voltage was adjusted to 110 V; after 1 h 30 min, the power was turned off, and the protein was transferred onto a membrane at a constant current of 200 mA for 1.5 hours. After the protein had been transferred onto the membrane, the PVDF front film (the surface in contact with the gel) was well marked and blocked in 5% skimmed milk powder for 1 hour at room temperature. Then, a primary antibody was added at a suitable dilution ratio (TFF2:1:400 or β-actin:1:1000), and the mixture was diluted with 5% skimmed milk powder and incubated in a shaker at 4°C overnight. After washing the membrane with 0.05% PBST, a secondary antibody was added (for TFF2, a goat anti-rabbit antibody (1:3000); and for β-Actin, a goat anti-mouse antibody (1:3000)), and the mixture was diluted with 5% skimmed milk powder, and incubated in a shaker for 1 hour at room temperature. Then, the membrane was washed and developed. The film was exposed for 2 minutes with a quantitative analyzer, and the development results were recorded and analyzed. The results are shown in FIG.6B. The TFF2 protein was abundantly expressed in the cells and secreted into the culture supernatant. After concentration, the TFF2 protein was not contained in the filtered waste solution. In order to quantify the TFF2 concentration, a standard curve of TFF2 was generated using the SEA748MU ELISA Kit for Trefoil Factor 2 (TFF2) kit (USCN). The TFF2 concentration was quantified according to a standard curve using optical density (OD). [000764] The preparation method of TFF2-6xHis ("6xHis" disclosed as SEQ ID NO: 33) was similar to that of TFF2. pSV1.0-TFF2-6xHis ("6xHis" disclosed as SEQ ID NO: 33) was transfected into 293F T cells. After 48 hours, the supernatant was collected, and purified by Ni-column affinity chromatography. More specifically, a 1 ml Ni-column was manufactured with Ni-NTA agarose gel. After the liquid was allowed to dry, the column was washed by adding 10 column volumes of water. After the column was equilibrated with 10 column volumes of 5 mM imidazole solution, the TFF2-containing cell supernatant was added, and passed through the column. Then, the column was successively washed with 5 column volumes of imidazole with concentrations of 5 mM, 10 mM, 20 mM, 100 mM, 200 mM and 500 mM imidazole, respectively, and the washing solutions were collected. After performing SDS- PAGE according to the above method, the gels were stained with Coomassie G250 for 30 min, and washed with a decolorizing solution overnight. The following day, it was observed that the eluates of 10 mM, 20 mM and 100 mM imidazole contained the target band. The three tubes of imidazole eluates were collected, added to a 10,000 KD protein concentration tube Amicon Ultra-15 (Minipore), and concentrated at 3000 g for 10 minutes. Then, the concentrate was washed twice by adding PBS, and imidazole was removed from the solution. The purified protein was re-identified by SDS-PAGE. The purity was > 99%, and no impurity protein band was observed (FIG. 6C). The concentration was detected with the Nanodrop software; the sample was frozen at -80°C after dispensing. Example 4: Protective effect of the TFF2 protein against severe influenza. [000765] In this Example, the TFF2 purified protein (20 μg/50 µl) or a control supernatant was dosed once by nasal dripping 2 hours before infection as well as 3 days and 8 days after infection, respectively. Then, influenza virus challenge models exactly the same as in Example 1 were used. The mice were weighed continuously, and the survival and survival status of the mice were observed (FIG.7A). FIG.7B shows that after the addition of the TFF2 protein for treatment, the survival rate of the H7N9-infected mice can be significantly improved (from 33.3% to 87.5%), the weight loss of the mice can be reduced (the weight loss was reduced by 9.24% on average), and the survival status of the mice can be improved. Moreover, the protective effect of the human-derived TFF2 protein (hTFF2) on the H7N9-challenged mice (100%, n = 5) was similar to that of the murine-derived TFF2 (mTFF2) (87.5%, n = 8). In the H9N2 infection model, treatment with the TFF2 protein also reduced weight loss (-0.9% Vs - 6.1%), and the weight returned to the level before infection on Day 2; whereas, the untreated group required 4 days to recover. [000766] TFF2 gene-knockout mice were prepared by homologous recombination, and used to perform the above H9N2 infection test. The results showed that the weight loss of the H9N2-infected TFF2 knockout mice was more severe than that of the wild-type mice (-9.3% Vs- 6.1%), and these mice required at least 7 days to return to the level before infection (FIG. 7C). In addition, the protective effect was verified again with the influenza virus PR8, a murine adapted H1N1 strain. The dose of PR8 virus used was 10000 of TCID50, and the remaining conditions were consistent with the foregoing. It was found that the TFF2 protein also plays a role in influenza infection with the murine adapted strain PR8. The degree of weight loss was not only greatly reduced (the weight loss was reduced by 10.44% on average), but also 51.4% of the mice can be protected from PR8-induced death (FIG.7D). Example 5: The TFF2 protein cannot inhibit virus replication, but promotes the damage repair of lung tissues by inhibiting inflammation. [000767] In this Example, lung tissue samples were collected from Example 41 day, 3 days, and 7 days after H7N9 infection, and lysed using the Trizol lysis solution. The lung tissue RNA was extracted, and then reverse-transcribed into cDNA using a RNA reverse transcription kit (Promega). H7N9 viral load was detected by the Taqman fluorescence quantitative PCR method. The materials required for detecting H7 were as follows. Primer F (SEQ ID NO: 11): GAAGAGGCAATGCAAAATAGAAT ACA, Primer R (SEQ ID NO: 12): CCCGAAGCTAAACCARAGTATCA, and Taqman probe (SEQ ID NO: 13): 5'-FAM- CCAGTCAAACTAAGCAGYGGCTACAAA-BHQ-3'. [000768] By comparing the viral loads in lung tissues of both groups of mice, it was found that the viral load increased significantly after infection, reaching an order of magnitude of 105. However, there was no significant difference in viral load between the TFF2 treated group and the untreated group at 1 day, 3 days, and 7 days after infection (FIG.8A). [000769] The effect of TFF2 on virus replication was further verified in vitro by using A549 cells as a model. The expression of the nuclear protein (NP protein) of the virus was detected using Western Blot. It was found that pSV1.0-TFF2 was over-expressed in the cells after transfection. However, the expression of the NP protein was not significantly different between the two groups, whether H7N9 infection or H9N2 infection (FIG. 8B). This fully illustrates that the protective mechanism of TFF2 against influenza virus infection is not through inhibition of influenza virus replication. [000770] Next, differences in lung tissue microstructure were compared between the TFF2 treated group and the control group. It was found that after TFF2 treatment, the inflammatory cell infiltration was significantly reduced on Day 1 and Day 3 in the early stage of H7N9 infection, and the lung tissue morphology was relatively complete (FIG. 8C). Moreover, in the early stage of infection, the secretion of the inflammatory factor TNF-α in the TFF2 treated group was significantly lower than that of the control group (the secretion was reduced by 61.9% 1 day after infection) (FIG.8D). Therefore, after the addition of TFF2, in the host, the occurrence of excessive inflammatory reactions were reduced, the degree of damage to the lung mucosal tissues was reduced, and the damage repair of lung tissues was promoted, thereby protecting the host from dying of a severe influenza virus infection. [000771] In order to further confirm the mechanism of action of TFF2, a mouse model of lung tissue inflammatory disease induced by nasal dripping LPS was used. 20 μg of purified TFF2 protein (50 μ) was first dripped into lung tissue by nasal dripping, and after 2 h, the mice were treated with LPS at 5 mg/kg by nasal dripping. After 24 hours, lung tissues of the mice were taken, prepared into pathological sections and subjected to RNA extraction, for analyzing the histopathological microstructure and inflammatory cytokine expression respectively. It was found that after the addition of the TFF2 protein for treatment, the alveolar structure of lung tissues was clear, the degree of inflammatory infiltration was lower, and there was no erythrocyte diapedesis (FIG. 8E). Moreover, the levels of expression of TNF-α and IL-6 in lung tissues were significantly reduced (TNF-α was reduced by 56.6%, and IL-6 was reduced by 74.1%) (FIG. 8F). This further illustrates that the protective effect of TFF2 against acute lung damage caused by influenza viruses is not influenza virus-specific, and it also plays a role in acute inflammatory diseases caused by other factors (e.g., LPS). Therefore, TFF2 plays an important role in preventing and treating acute pulmonary/bronchial inflammatory diseases. Example 6. Results reported from Synairgen clinical trial using nebulized interferon β [000772] SNG001 (Synairgen, plc) is a formulation of interferon beta (IFN-β) delivered to the lung using a nebulizer. A Phase II double-blind placebo-controlled trial called SG016 recruited 101 patients from 9 specialists sites in the UK during the period 30 March to 27 May 2020. 50% of the patients received SNG001, and 50% received placebo. The Company reported that SNG001 significantly reduced breathlessness, and that the trial found that patients who received SNG001 were at least twice as likely to recover to the point where their everyday activities were not compromised through having been infected by SARS-CoV-2, that patients receiving SNG001 had a 79% lower risk of developing severe disease compared to placebo, and that patients who received SNG001 were more than twice as likely to recover from COVID-19 as those on placebo (www.synairgen.com). Example 7. Clinical study of combined application of IFN-κ + TFF2 in the treatment of new coronavirus infection to investigate the safety and efficacy of IFN-κ + TFF2 in the treatment of new coronavirus infection. Rationale for this study [000773] IFN-κ is a type I interferon that can activate interferon-stimulating genes and inhibit the replication of encephalomyocarditis virus (ECMV) and human papilloma virus (HPV). Many members of the type I interferon family have completed clinical drug trials as antiviral drug candidates. For example, recombinant IFN-α2 has been used to treat hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. The drug, IFN-β is used to treat multiple sclerosis (MS). The combination of hormone and IFN-α treatment in patients with severe acute respiratory syndrome during the acute phase can shorten the length of hospital stay, promote the absorption of lung lesions, reduce the demand for hormones, but not shorten the duration of fever. However, IFN-α treatment may be related to the body's persistent inflammatory response, which is not conducive to patient recovery. Significantly different from IFN-α, IFN- κ is a relatively mild type I interferon that can effectively inhibit the replication of enveloped viruses. Our results suggest that IFN-κ is one of the constitutively expressed interferon members, which forms the natural immune barrier against the virus. It has no obvious toxicity in the respiratory tract and can effectively inhibit the influenza viruses PR8, H9N2 and H7N9 and Zika virus duplication, and plays an important protective role in reducing the pathogenicity of the virus (patent application number: CN 201710668628.2; PCT/CN2017/116350). Similar to IFN-α, its mechanism of action is mainly to inhibit viral replication by up-regulating interferon-stimulated genes. [000774] TFF2 protein is composed of 106 amino acids and has a molecular weight of about 7-12kD. It contains 4 exons and 2 symmetrical three-leaf domains, so its structure is extremely stable, resistant to acid, heat and protease. Results suggest that the TFF2 protein molecule can reduce the symptoms of respiratory infections and play an important protective role in reducing the morbidity and mortality of influenza H7N9, PR8 and H9N2 infections, and has applied for an invention patent (patent application number: 201610104936.8). Further research shows that TFF2 protein does not inhibit influenza virus replication, but rather inhibits the inflammatory response, reduces respiratory tissue damage, and promotes repair of respiratory mucosal tissues. Because other respiratory pathogens cause respiratory tissue damage similar to influenza virus infection, the protective effect of TFF2 is not limited to respiratory tissue damage caused by influenza viruses, but also includes respiratory damage caused by other respiratory pathogens. TFF2 may play an important role in responding to the outbreak of toxic infections in the respiratory tract (such as a new type of coronavirus infection), especially for the prevention and treatment of viral infections and severe respiratory infections for which no effective treatment is available. [000775] This study comprises the combined use of IFN-κ and TFF2; on the one hand, IFN-κ can inhibit the replication of new coronaviruses in vivo; on the other hand, TFF2 may suppress the host's inflammatory response, reduce the damage caused by viral infection, and promoted the repair of respiratory mucosa. The goal of this study is to improve patient prognosis. Patient inclusion criteria [000776] 1) Age 18-70 years, regardless of gender. Those who meet the criteria for diagnosis of pneumonia in the Chinese Adult Community Acquired Pneumonia Guidelines for Respiratory Diseases of the Chinese Medical Association in 2015; [000777] 2) Meet the clinical diagnosis of viral pneumonia: i. Fever (oral temperature ≥38 °C, or axillary temperature ≥37.5 °C; or fever history within 24 hours before baseline, whether or not taking antipyretics; or fever symptoms within 48 hours before baseline), with respiratory symptoms, accompanied by or without dyspnea (breathing frequency> 30 beats / min); [000778] ii. White blood cell count are normal or low, with or without thrombocytopenia; [000779] iii. Chest imaging (CT of the chest): Unilateral or bilateral chest imaging shows multiple (at least 2 lesions) or diffuse patchy or ground glass infiltration (with or without consolidation) [000780] 3) Those with positive pathogenicity of new coronavirus. After screening for respiratory viruses, the oropharyngeal test is positive for one or more viral nucleic acids, and can be selected. All cases were further subjected to digital PCR for quantitative virus detection; [000781] 4) Patients can receive nebulized inhalation Exclusion criteria [000782] Patients who meet any of the following criteria are not eligible for this study: [000783] 1) Evidence of pneumonia caused by other non-new coronaviruses, such as Streptococcus pneumoniae, Legionella pneumophila urine antigen, Mycoplasma pneumoniae, Chlamydia, adenovirus, respiratory syncytial virus, rhinovirus, and influenza virus; [000784] 2) There is clear evidence of bacterial infection, PCT> 1μg / L; [000785] 3) Screening of subjects who have used antiviral drugs during the week prior to the study and who may need another antiviral treatment during the study; [000786] 4) Patients with severe non-infectious underlying pulmonary diseases, including: tuberculosis, lung tumors, pulmonary edema, atelectasis, pulmonary embolism, pulmonary eosinophil infiltration, and patients with pulmonary vasculitis; [000787] 5) Severe liver and kidney dysfunction: a) ALT and AST exceed the upper limit of normal value by more than 10 times; b) Serum creatinine value exceeds the upper limit of normal value by more than 1.5 times; [000788] 6) Patients who are participating in or participating in other clinical studies within 30 days before administration; [000789] 7) Patients with a history of allergies to interferon; or other interferon contraindications: history of angina pectoris, myocardial infarction and other serious cardiovascular diseases; history of epilepsy or other central nervous system dysfunction; patients with obvious hematopoietic system abnormalities (platelet < 30 × 109 cells / L, neutrophils <0.5 × 109 cells / L); [000790] 8) pregnant women (positive urine or serum pregnancy test) or lactating women; [000791] 9) Other investigators consider it inappropriate to enroll in this trial, or the investigator believes that there may be any situation that increases the risk of the subject or interferes with the clinical trial. [000792] Patients can be excluded from the study if one or more of the following conditions occur: 1) Researchers clearly violate experimental protocols 2) Patient refuses to continue treatment or observation 3) Unacceptable toxicity 4) Researchers believe that termination of study patients will get the best medical benefits 5) Unrelated medical illness or complications 6) Lost follow-up [000793] Candidates who have completed the informed consent process and undergo pre- treatment examinations, including clinical observation, body temperature, blood routine, blood biochemistry, urine routine, inflammatory factors, blood oxygen examination, chest imaging examination, and whether there are underlying diseases (such as obesity) , Diabetes, hypertension, etc.), whether it is severe (respiratory support required). Combine the above results to further determine whether the patient is suitable for enrollment; [000794] The number of patients ultimately participating in the IFN-κ + TFF2 combination treatment is based on the actual number of 2019-nCoV infected persons who are willing to accept this study; [000795] Initiate combined treatment of IFN-κ + TFF2. The patients will be divided into 2 groups. The first group is aerosolized once a day and the second group is aerosolized 2 times a day in the morning and evening respectively; [000796] Make daily clinical observations after treatment: observe the patient's mobility, breathing rate, fever, cough, sore throat, nasal congestion, discomfort, headache, muscle pain or discomfort, need supportive breathing treatment, and whether other organs are damaged. [000797] Imaging examinations: Imaging examinations are performed at the time of physical examination before the start of treatment; imaging examinations are performed on the 4th and 8th days after treatment to evaluate the patient's response to treatment. If the clinician considers it necessary according to the patient's condition, the number of examinations can be increased; [000798] Pharyngeal test virus load measurement: Take pharyngeal test samples before and on the 4th and 8th day after treatment for new coronavirus load measurement; [000799] Blood routine, blood biochemistry, urine routine, and inflammatory factor detection: peripheral blood was collected for blood routine, blood biochemistry, inflammatory factor examination, and urine for routine urine examination before and on the 4th and 8th day after treatment. Further evaluation of the occurrence of acute respiratory distress syndrome and sepsis; [000800] [Protein preparation]: GMP production will be commissioned by Inshore Biotechnology Co., Ltd., protein form: lyophilized powder, dosage is 1 mg / bottle, protein purity> 95%, endotoxin <100EU / mg [000801] [Quantity]: IFN-κ (107U / mg, 1 mg / penicillin bottle); TFF2 (1 mg / penicillin bottle) [000802] [Usage]: Atomization inhalation [000803] [Production Date]: XX, XX, XX, [Expiration date]: XX, XX, XX, [000804] [Storage]: dry, low temperature storage [000805] Administration method and scheme a) The dose of IFN-κ is 100,000 U / kg / time, 50kg adult is 5x106U / time, the reference is based on the comparison of the clinical efficacy of different doses of recombinant human interferon α2 b nebulized inhalation for the treatment of pediatric viral pneumonia (Lihua Li, Practical Journal of Cardio-Cerebro-Pulmonary Vascular Disease, 2017); The dose of TFF2 is 0.092 mg / kg / time, and 50 kg for adults is 4.6 mg / time. , Alleviate allergic respiratory diseases in mice (Trefoil Factor–2 Reverses Airway Remodeling Changes in Allergic Airways Disease. Am J Respir Cell Mol Biol.2013 Jan; 48 (1): 135-44.). According to the Meeh-Rubner formula Calculate the dose relative to animals the human body with the dose conversion factor per kilogram of human body; b) Take 5 vials containing 1 mg of TFF2 protein and add 1 mL of saline, take 1 vial containing 1 mg of IFN-κ protein, add 5 mL of saline and mix; c) 5 mL of TFF2 protein and 5 mL of IFN-κ protein (mass ratio of 5: 1) are added to the atomizer; d) Connect the nebulizer generator to the nasal cavity of the subject. After all liquid nebulization is completed, the treatment is terminated. e) For patients with severe respiratory diseases, such as acute respiratory distress syndrome, sepsis, and septic shock, in addition to targeted treatment, supportive supplemental oxygen therapy can be supplemented with IFN-κ + TFF2 combined with atomization Inhalation treatment. [000806] Efficacy evaluation 9.1 Quality of life: evaluation before medication and at the end of the trial 9.1.1 Evaluate the effects of daily life, mood, walking ability, normal work, relationship with others, sleep and fun before and after treatment; 9.1.2 ECOG scoring criteria for physical fitness Zubrod-ECOG-WHO (ZPS, 5-point scale, Annex 1). 9.2 Efficacy judgment indicators 9.2.1 Body temperature, blood pressure, length of hospital stay; 9.2.2 Blood routine and blood oxygen; 9.2.3 Chest imaging (CT, MRI); 9.2.4 Throat swab virus load; 9.2.5 Comprehensive Evaluation □ Remission □ Stable disease □ Progression Safety Evaluation [000807] The safety evaluation standard adopts the Chinese Drug Clinical Trial Management Standards and the Biological Toxicity Clinical Use Management Methods Common Toxicity Standard (CTC). Adverse events were assessed as mild (level 1), moderate (level 2), severe (level 3), life-threatening (level 4), and death (level 5). The relationship between the adverse event and the research agent should be analyzed to determine whether they are positively related, may be related, may not be related, not related, or cannot be determined. Vital signs observation and laboratory inspection [000808] Blood routine, blood biochemistry, body temperature, blood oxygen, electrocardiogram and imaging examination, and coronavirus load test must be performed within one week before enrollment; [000809] Record the subject's breathing, heart rate, temperature, and blood pressure at 1 and 3 hours after medication; Concomitant reactions within 24 hours after nebulization: □ None □ Nausea and vomiting □ Diarrhea □ Allergic reaction □ Fever □ Other reactions; Relevance to treatment: □ Yes □ No [000810] Table 4. ECOG performance status score:
Figure imgf000262_0001
[000811] Table 5:
Figure imgf000262_0002
Figure imgf000263_0001
[000812] An open-label, randomized clinical trial involving hospitalized adult patients with confirmed SARS-CoV-2 infection, which causes the mild or moderate respiratory illness of COVID-19, and eligible COVID-19 patients with written consent were enrolled according to a single arm protocol. Patients were assigned in a 1:2 ratio to receive either inhalation treatment with 5 mg TFF2 plus 1-2 mg [1.16 million U/mg] IFN-κ protein, once every 48 hours for three doses, in addition to standard care, or standard care alone. The end point was the time to discharge from the hospital. Clinical characteristics including effectiveness and safety indicators were collected and analyzed to evaluate the safety and effectiveness of the treatment regimen. [000813] A total of 28 patients with laboratory-confirmed SARS-CoV-2 infection were enrolled. 10 were assigned to the IFN-κ plus TFF2 group, and 18 to the control group. The clinical indicators of the enrolled patients were evaluated for 10 days. CT imaging improvement reached 100% in the IFNκ plus TFF2 group, and 76.47% for the control group after 9 days of treatment, with an average improvement time of 3.5 days versus 7.4 days, respectively (p<0.05), accompanied with a significant shortened average cough remission time 3.3 versus 9.9 days (p=XXX), respectively. The proportion of patients with undetectable viral RNA in throat swab samples significantly differed in the IFN-κ plus TFF2 group and in the control group by 57.13% vs 18.75% at day 6, and 71.43% vs 37.5% at day 9, accordingly, average hospitalized stays were 11 vs 14 days (p=XXX), respectively. Importantly, no severe adverse events (SAE) were observed in the IFN-κ plus TFF2 group and no significant differences in the safety indicators, including AST, ALT, platelet, TBil, between the IFN-κ plus TFF2 group and the control group. [000814] In conclusion, aerosol inhalation of IFN-κ plus TFF2 was found to be a safe treatment that was able to significantly accelerate clinical improvement, including cough relief, imaging improvement, viral negative reversion in patients with moderate COVID-19, thereby achieves an early release from hospitalization. These data support further exploring the application of IFN-κ plus TFF2 in an expanded clinical trial. Chinese Clinical Trial Register number, ChiCTR2000030262.” [000815] Examples 8, 9, 11, and 12, which follow, provide the amino acid and nucleic acid sequences for a recombinant TFF2-IFN-alpha-IgG1Fc (Example 8), a recombinant TFF2- INF-κ-IgG1-Fc (Example 9), a recombinant TFF1-IFN-κ-IgG1Fc (Example 11) and a recombinant TFF3-IFN-κ-IgG1Fc fusion protein according to the present disclosure. Example 10 describes recombinant TFF2/IFNk bi-functional proteins prepared as separate TFF2-Fc and IFNκ-Fc proteins for forming an Fc-mediated dimer comprising arms of both TFF2 and IFN- κs. Likewise, separate TFF1-Fc and IFNκ-Fc proteins and TFF3-Fc and IFNκ-Fc proteins can be prepared for forming an Fc-mediated dimer comprising arms of both TFF1 and IFN-κs, and of both TFF3 and IFN-κs. [000816] For each recombinant fusion protein, the signal sequence (Mus musculus Ig light chain, ACCESSION AAT76275, VERSION AAT76275.1, METDTLLLWVLLLWVPGSTG), which is part of the bi-functional protein is essential for getting the recombinant proteins into the ER and onto the secretory pathway of the mammalian host cell, and thus will precede the sequences of each recombinant protein as shown in para. [000533], [000535] and [000537]. That is, the synthetic gene used for expression of each of these proteins in a mammalian cell will always encode this signal peptide sequence at the beginning of the recombinant product. Accordingly, the sequences of ALL of the recombinant trefoil factor proteins described below (rTFF1, rTFF2 or rTFF3-based) will begin with the amino acid sequence METDTLLLWVLLLWVPGSTG, which is then followed by the recombinant TFF sequences sans their native signal sequences. Example 8. TFF2-IFN-alpha-IgG1Fc fusion protein [000817] Color Key: Green=Homosapiens TFF2 ORF w/o Leader, Blue=linker sequence, Gold=Homosapiens interferon alpha 2 w/o Leader, Black=Homo sapiens human IGG1 Fc domain
Figure imgf000265_0002
[000819] Linker: GGGGSGGGGSGGGGS (SEQ ID NO: 38) [000820] TFF2 ACCESSION NM_005423 VERSION NM_005423.5 MGRRDAQLLAALLVLGLCALAGSEKPSPCQCSRLSPHNRTNCGFPGITSDQCFDNGCCFDSSVTGVPWCFHPLPK QESDQCVMEVSDRRNCGYPGISPEECASRKCCFSNFIFEVPWCFFPKSVEDCHY (SEQ ID NO: 44) [000821] Nucleotide sequence
Figure imgf000265_0001
[000822] MGRRDAQLLAALLVLGLCALAGS (SEQ ID NO: 46) (leader) EKPSPCQCSRLSPHNRTNCGFPGITSDQCFDNGCCFDSSVTGVPWCFHPLPKQESDQCVMEVSDRRNCGYPGISP EECASRKCCFSNFIFEVPWCFFPKSVEDCHY (SEQ ID NO: 36) (coding sequence) [000823] Interferon alpha- 2 ACCESSION NM_000605 VERSION NM_000605.4 MALTFALLVALLVLSCKSSCSVGCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIP VLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKE KKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE (SEQ ID NO: 47) [000824] Homo sapiens interferon kappa (IFNK) ACCESSION NM_020124 VERSION NM_020124.3 [000825] MSTKPDMIQKCLWLEILMGIFIAGTLSLDCNLLNVHLRRVTWQNLRHLSSMSNSFPVECLRENI AFELPQEFLQYTQPMKRDIKKAFYEMSLQAFNIFSQHTFKYWKERHLKQIQIGLDQQAEYLNQCLEEDKNENEDM KEMKENEMKPSEARVPQLSSLELRRYFHRIDNFLKEKKYSDCAWEIVRVEIRRCLYYFYKFTALFRRK (SEQ ID NO: 48) [000826] Nucleotide sequence Green = Leader Sequence (SignalP 4.1), Blue = body sequence, Red = STOP codon
Figure imgf000266_0002
[000827] Note that for creation of the bifunctional fusion protein, the leader sequence is removed from the IFN sequence and the sequence from the 3’ end to the leader is fused to the 3’ end of TFF2. [000828] Mouse leader-TTF2-Linker-IFN-Alpha-Fc Fusion Protein
Figure imgf000266_0001
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K [SEQ ID 39] Example 9 TFF2-INF-κ-IgG1-Fc fusion protein [000830] Sequence Provided: = Homo sapiens TFF2 ORF w/o Leader, Blue = linker sequence, Gold = homo sapiens interferon kappa w/o Leader, Black = Homo sapiens human IgG1 Fc domain
Figure imgf000267_0001
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 50) [000832] For the TFF2 sequence, any well-characterized mammalian leader sequence precedes the coding sequence of the TFF2 gene at the 5’ end of the recombinant fusion protein. According to some embodiments, mouse light chain kappa leader sequence is used at the 5’ end of the recombinant fusion protein. [000833] Mus musculus Ig light chain ACCESSION AAT76275 VERSION AAT76275.1
Figure imgf000267_0002
[000834] Mouse Leader + TFF2 + IFN kappa + Linker + FC fusion
Figure imgf000267_0003
YFYKFTALFRRKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK [SEQ ID NO: ____] References 1. Guler-Gane G, Kidd S, Sridharan S, Vaughan TJ, Wilkinson TC, Tigue NJ. Overcoming the Refractory Expression of Secreted Recombinant Proteins in Mammalian Cells through Modification of the Signal Peptide and Adjacent Amino Acids. PloS one 2016;11:e0155340. [PubMed: 27195765] 2. Liu H, Zou X, Li T, Wang X, Yuan W, Chen Y, et al. Enhanced production of secretory glycoprotein VSTM1-v2 with mouse IgG kappa signal peptide in optimized HEK293F transient transfection. J Biosci Bioeng 2016;121:133–9. [PubMed: 26140918] 3. Wang X, Liu H, Yuan W, Cheng Y, Han W. Efficient production of CYTL1 protein using mouse IgGkappa signal peptide in the CHO cell expression system. Acta Biochim Biophys Sin (Shanghai) 2016;48:391–4. [PubMed: 26922322] [000836] Example 10. Generation of recombinant TFF2/IFNk bi-functional proteins by preparing separate TFF2-Fc and IFNκ-Fc proteins for forming an Fc-mediated dimer comprising arms of both TFF2 and IFN-κ. [000837] Mus musculus Ig light chain leader sequence (ACCESSION AAT76275, VERSION AAT76275.1) –METDTLLLWVLLLWVPGSTG (SEQ ID NO: 51) [000838] TFF2 without native leader sequence (Homo sapiens, Seq ID AAX4293.1, ACCESSION NM_005423, VERSION NM_005423.5):
Figure imgf000268_0001
[000842] IgG1 Fc fragment (Homo sapiens, Domain Seq ID 4CDH_A)
Figure imgf000269_0001
[000844] Assembled Mouse Leader-TFF2-linker-Fc
Figure imgf000269_0002
[000845] Mouse leader + IFN kappa+ linker + Fc fusion protein
Figure imgf000269_0003
VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK [SEQ ID NO: 40] [000847] For the TFF2-Fc sequence, any well-characterized mammalian leader sequence precedes the coding sequence of the TFF2 gene at the 5’ end of the recombinant fusion protein. According to some embodiments, mouse light chain kappa leader sequence is used at the 5’ end of the recombinant fusion protein. [000848] Assembled IFN-κ-Fc [
Figure imgf000269_0004
[000850] For the IFN-κ-Fc sequence, any well-characterized mammalian leader sequence precedes the coding sequence of the IFN gene at the 5’ end of the recombinant fusion protein. According to some embodiments, mouse light chain kappa leader sequence is used at the 5’ end of the recombinant fusion protein. Example 11. Human TFF1-IFN-κ-Fc fusion protein [000851] Homo sapiens trefoil factor 1 (TFF1), mRNA NCBI Reference Sequence: NM_003225.3 [000852] Select Reference: Zhu R, Liu Y, Yan J, Tian Y, Yan W, Aryal S, Tan F, Chen Y, Tang Y and Bai Y.2021. Overexpression of trefoil factor 1 and trefoil factor 3 in primary extramammary Paget's disease and implication of a novel therapeutic target. J Dermatol 48 (11): e549-e550 1 atccctgact cggggtcgcc tttggagcag agaggaggca atggccacca tggagaacaa 61 ggtgatctgc gccctggtcc tggtgtccat gctggccctc ggcaccctgg ccgaggccca 121 gacagagacg tgtacagtgg ccccccgtga aagacagaat tgtggttttc ctggtgtcac 181 gccctcccag tgtgcaaata agggctgctg tttcgacgac accgttcgtg gggtcccctg 241 gtgcttctat cctaatacca tcgacgtccc tccagaagag gagtgtgaat tttagacact 301 tctgcaggga tctgcctgca tcctgacgcg gtgccgtccc cagcacggtg attagtccca 361 gagctcggct gccacctcca ccggacacct cagacacgct tctgcagctg tgcctcggct 421 cacaacacag attgactgct ctgactttga ctactcaaaa ttggcctaaa aattaaaaga 481 gatcgatatt aa (SEQ ID NO: 84) [000853] Start to Stop Translation: atggccaccatggagaacaaggtgatctgcgccctggtcctggtgtccatgctggccctcggcaccctggccgaggccca gacagagacgtgtacagtggccccccgtgaaagacagaattgtggttttcctggtgtcacgccctcccagtgtgcaaata agggctgctgtttcgacgacaccgttcgtggggtcccctggtgcttctatcctaataccatcgacgtccctccagaagag gagtgtgaattttag (SEQ ID NO: 85) [000854] MATMENKVICALVLVSMLALGTLAEAQTETCTVAPRERQNCGFPGVT PSQCANKGCCFDDTVRGVPWCFYPNTIDVPPEEECEF*(SEQ ID NO: 86) [000855] Leader (signal) sequence: Atggccaccatggagaacaaggtgatctgcgccctggtcctggtgtccatgctggccctcggcaccctggcc (SEQ ID NO: 87) MATMENKVICALVLVSMLALGTLA (SEQ ID NO: 88) [000856] TFF1 Sequence sans Leader for fusion: EAQTETCTVAPRERQNCGFPGVTPSQCANKGCCFDDTVRGVPWCFYPNTIDVPPEEE CEF (SEQ ID NO: 89) [000857] Leader Sequence for TFF1/IFN-k/IgG1 Fc) According to some embodiments, consider using this well-characterized leader sequence at the 5’ end of the recombinant fusion protein. [000858] Mus musculus Ig light chain ACCESSION AAT76275 VERSION AAT76275.1 METDTLLLWVLLLWVPGSTG (SEQ ID NO: 51) [000859] Mouse leader + TFF1 + leader+ IFNκ + Fc fusion protein: [000860] Sequence Provided: Purple: Mus musculus Ig light chain, Green = Homo sapiens TFF1 ORF w/o Leader, Blue = linker sequence, Gold = homo sapiens interferon kappa w/o Leader, Black = Homo sapiens human IgG1 Fc domain
Figure imgf000271_0001
EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 90) [000861] Note that for creation of the bifunctional TFF1-IFN fusion protein, the leader sequence is removed from the IFN sequence and the sequence from the 3’ end to the leader is fused to the 3’ end of TFF1. Example 12. Human TFF3-IFN-κ-Fc fusion protein [000862] Homo sapiens trefoil factor 3 (TFF3), mRNA NCBI Reference Sequence: NM_003226.4 [000863] Select Reference: Yang Y, Lin Z, Lin Q, Bei W and Guo J.2022. Pathological and therapeutic roles of bioactive peptide trefoil factor 3 in diverse diseases: recent progress and perspective. Cell Death Dis 13 (1): 62 (2022) 1 gagtcctgag ctgcgtcccg gagcccacgg tggtcatggc tgccagagcg ctctgcatgc 61 tggggctggt cctggccttg ctgtcctcca gctctgctga ggagtacgtg ggcctgtctg 121 caaaccagtg tgccgtgcca gccaaggaca gggtggactg cggctacccc catgtcaccc 181 ccaaggagtg caacaaccgg ggctgctgct ttgactccag gatccctgga gtgccttggt 241 gtttcaagcc cctgcaggaa gcagaatgca ccttctgagg cacctccagc tgcccccggc 301 cgggggatgc gaggctcgga gcacccttgc ccggctgtga ttgctgccag gcactgttca 361 tctcagcttt tctgtccctt tgctcccggc aagcgcttct gctgaaagtt catatctgga 421 gcctgatgtc ttaacgaata aaggtcccat gctccacccg aggacagttc ttcgtgcctg 481 agactttctg aggttgtgct ttatttctgc tgcgtcgtgg gagagggcgg gagggtgtca 541 ggggagagtc tgcccaggcc tcaagggcag gaaaagactc cctaaggagc tgcagtgcat 601 gcaaggatat tttgaatcca gactggcacc cacgtcacag gaaagcctag gaacactgta 661 agtgccgctt cctcgggaaa gcagaaaaaa tacatttcag gtagaagttt tcaaaaatca 721 caagtctttc ttggtgaaga cagcaagcca ataaaactgt cttccaaagt ggtcctttat 781 ttcacaacca ctctcgctac tgttcaatac ttgtactatt cctgggtttt gtttctttgt 841 acagtaaaca ttatgaacaa acaggca (SEQ ID NO: 91) [000864] START to STOP Translation: atggctgccagagcgctctgcatgctggggctggtcctggccttgctgtcctccagctctgctgaggagtacgtgggcct gtctgcaaaccagtgtgccgtgccagccaaggacagggtggactgcggctacccccatgtcacccccaaggagtgcaaca accggggctgctgctttgactccaggatccctggagtgccttggtgtttcaagcccctgcaggaagcagaatgcaccttc tga (SEQ ID NO: 92) [000865] Translation: MAARALCMLGLVLALLSSSSAEEYVGLSANQCAVPAKDRVDCGYPHVTPKECNNR GCCFDSRIPGVPWCFKPLQEAECTF* (SEQ ID NO: 93) [000866] Leader (signal) sequence: Atggctgccagagcgctctgcatgctggggctggtcctggccttgctgtcctccagctctgct (SEQ ID NO: 94) MAARALCMLGLVLALLSSSSA (SEQ ID NO: 95) [000867] TFF3 Sequence sans Leader for fusion: EEYVGLSANQCAVPAKDRVDCGYPHVTPKECNNRGCCFDSRIPGVPWCFKPLQEAE CTF (SEQ ID NO: 96) [000868] Leader Sequence for TFF3/INF-k/IgG1 Fc) Consider using this well-characterized leader sequence at the 5’ end of the recombinant fusion protein. [000869] Mus musculus Ig light chain ACCESSION AAT76275 VERSION AAT76275.1 METDTLLLWVLLLWVPGSTG (SEQ ID NO: 51) [000870] Mouse leader + TFF3 + linker+ IFN-kappa + Fc fusion protein: [000871] Sequence Provided: Purple: Mus musculus Ig light chain, Green = Homo sapiens TFF3 ORF w/o Leader, Blue = linker sequence, Gold = homo sapiens interferon kappa w/o Leader, Black = Homo sapiens human IgG1 Fc domain
Figure imgf000272_0001
VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 97) [000872] Note that for creation of the bifunctional fusion protein, the leader sequence is removed from the IFN sequence and the sequence from the 3’ end to the leader is fused to the 3’ end of TFF3. [000873] Example 13. Pilot run transfection of EpiCHO cells in vitro with TFF2- IFNκ-Fc fusion protein [000874] Gel electrophoresis and Western blot of cell lysates and supernatants [000875] 0.5 mL of supernatants was harvested from 25.0 mL Erlenmeyer flask cultures of transfected and non-transfected (control) ExpiCHO cells every 24h. [000876] Cells were gently pelleted at 200 x g, and the supernatants clarified at 15000 x g for 10 min; both cells and supernatants were stored at -80°C. [000877] 8.0 μL supernatants were combined with 10 μL Tris-Glycine SDS Sample Buffer (2X) (Novex, LC676) and 2.0 μL NuPAGE Sample Reducing Agent (10X) (Invitrogen, NP0009) and resolved by SDS PAGE (10% gel). [000878] Cells from 0.5mL harvests were lysed in 150.0 µl RIPA Lysis buffer (ThermoFisher Cat# 89900, Lot# XI352172) and 20.0 µg lysates (quantified via BCA assay) were combined with 10.0 mL of 2X Laemmli SDS Sample Buffer ((with 2.0 mL 10x DTT) and resolved by SDS PAGE (10% gel). [000879] Resolved proteins were either visualized with Coomassie Blue or transferred to PVDF membranes for western blot analysis. [000880] Proteins transferred to PVDF membranes for western blot analysis: Membranes were blocked with 5.0 % nonfat dry milk in PBST. Membranes were then probed with mouse- anti-human Fc monoclonal antibodies (Mouse anti-Fc; GenScript, Cat #A01797-40) at 1:500 and then with Goat-anti-mouse HRP polyclonal antibodies (Anti-mouse HRP; Promega Cat #W402B), 1:5000 in PBS containing 0.1% tween 20 (pH 7.4) (PBST) with 5.0 % nonfat dry milk for visualization of Fc-containing protein species. Reactive species were visualized with Pierce ECL Western Blotting Substrate (ThermoFisher Scientific, Cat #32209) and imaged via iBright 1500 imager (ThermoFisher). [000881] Membranes were probed with Rabbit-anti-human GAPDH monoclonal antibodies (Rabbit anti-GAPDH; Cell Signaling Cat #5174T) at 1:1000 and then with HRP- conjugated goat-anti-rabbit polyclonal antibodies (Anti-rabbit HRP; Promega Cat#W401B), 1:13000 in PBST with 5.0 % nonfat dry milk for visualization of GAPDH. Reactive species were visualized with Pierce ECL Western Blotting Substrate (ThermoFisher Scientific, Cat #32209) and imaged via iBright 1500 imager (ThermoFisher). [000882] Results [000883] Results are shown in FIG.9 and FIG.10. [000884] FIG.9A shows transfected epiCHO cell supernatants resolved by 10% SDS PAGE and stained with coomassie blue on days 0, 3, 4, 7 and 10 after transfection of epiCHO cells with a TFF2-IFNκ-Fc construct. FIG.9B shows a Western blot in which TFF2-IFNκ- Fc was detected by anti-Fc antibodies (primary antibody: mouse anti-human Fc monoclonal; secondary antibody: HRP-conjugated goat anti-mouse polyclonal). [000885] Side-by-side Coomassie gel analysis of the resolved control and TFF2-IFNκ-Fc transfected supernatants shows similar amounts of total protein in both samples. [000886] Western blot of resolved supernatants with anti-Fc antibodies shows a prominent species at ~ 70 kD that is present only in transfected cell supernatants (some minor reactive species also visible) at days 0, 3, 5, 7 and 10. Intensity of the band reached an apparent maximum by day 5, indicating that while cells remained viable until day 9, accumulation of Fc-reactive species in supernatants reached a maximum several days prior. [000887] FIG. 10A shows a Western blot of resolved epiCHO cell lysates after transfection with a TFF2- IFNκ-Fc construct. TFF2-IFNκ-Fc was detected by anti-Fc antibodies (primary antibody: mouse anti-human Fc monoclonal; secondary antibody: HRP- conjugated goat anti-mouse polyclonal). The anti-Fc intensity was maximum at day 5, consistent with the maximum viable cell number in culture. The Fc positive species in the supernatants confirms secretion. [000888] FIG. 10B shows a Western blot of resolved epiCHO cell lysates after transfection with a GAPDH construct probed with rabbit anti-human anti-GAPDH antibodies (secondary antibody HRP-conjugated goat-anti-rabbit polyclonal antibodies). The Western blot of resolved lysates with anti-GAPDH antibodies shows a prominent species at ~ 40 kD present in both transfected and control cell supernatants at days 0, 3, 5, 7 and 10, indicating that similar amounts of control and transfected cellular proteins are present in resolved samples at each time point. Note: less material is apparent at day 10 likely due to cellular degradation. [000889] Pilot protein purification by Protein A affinity chromatography. [000890] A 240.0 mL day 7 cell supernatant was brought to pH 7.4 with 1.0 M phosphate buffer (pH 8.0). [000891] 4.0 mL protein A-conjugated Sepharose beads (ThermoFisher, Cat# 20334, Lot# XG353744) were added to the buffered supernatant at room temperature and gently swirled for 2.0 hr [000892] The supernatant with beads was added to a disposable gravity column at a 1.0 mL per minute flow rate. [000893] Beads were washed 2X with 10.0 mL of 0.1M PBS pH 7.6. [000894] Proteins were eluted with 10.0 mL PBS pH 2.5 and collected in 1.0 mL fractions. [000895] Fractions were neutralized with 111.1 μL 1.0M PBS pH 8.0 [000896] OD280 and BCA protein quantification methods were employed for analysis of fractions, and a rough estimate suggests ~ 1.0 mg total protein was obtained. [000897] 8.0 μL eluates were then resolved by SDS PAGE and visualized by Coomassie Blue and western blot as described above. [000898] FIG.11A shows a 10% SDS gel stained with coomassie blue containing elution fractions 1 through 9 from Protein A affinity chromatography of the pilot run expressed fusion product. The expected species has a molecular weight of about 70 kDa. Smaller Fc reactive species indicate proteolysis that will be addressed in subsequent runs. Larger species likely represent multimers. [000899] FIG. 11B shows a Western blot of the resolved elution fractions 1 through 9 from Protein A affinity chromatography of the pilot run expressed fusion product detected by anti-Fc antibodies (primary antibody: mouse anti-human Fc monoclonal; secondary antibody: HRP-conjugated goat anti-mouse polyclonal). Analysis of flow-through by western with anti- Fc antibodies indicates a purification efficiency of less than 30.0% for this pilot run. [000900] Coomassie Blue and western analysis of Day 7 cell lysates (corresponding to about max. TFF2-IFNκ-Fc observed in supernatants) with anti-Fc and Anti-GADPH antibodies indicates (i) that similar amounts of total protein were resolved from lysates of both control and transfected cells; and (ii) that substantially less Fc-reactive species are seen in lysates versus supernatants, suggesting that proteolysis observed in supernatants occurs post-secretion. [000901] While the present invention has been described with reference to the specific embodiments thereof it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adopt a particular situation, material, composition of matter, process, process step or steps, to the objective spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMS What is claimed is: 1. A method for reducing damaging effects of a severe respiratory virus infection in a susceptible subject comprising administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant bifunctional fusion protein comprising a biologically active recombinant immunomodulatory component operatively linked to a biologically active recombinant anti-viral component, the immunomodulatory component comprising a recombinant human trefoil factor protein, wherein the pharmaceutical composition is cytoprotective.
2. The method according to claim 1, wherein the recombinant human trefoil protein is a recombinant biologically active human trefoil factor 1 protein (rhTFF1), a recombinant biologically active human trefoil factor 2 protein (rhTFF2), or a recombinant biologically active trefoil factor 3 protein (rhTFF3) fragment or variant joined by its C-terminal end to a linker sequence, which is joined to an N-terminal end of a recombinant biologically active human interferon molecule, fragment or variant.
3. The method according to claim 2, wherein the C-terminal end of the recombinant interferon molecule, fragment or variant sequence is further joined to a recombinant Fc derived antibody domain comprising a recombinant constant region of a human immunoglobulin heavy chain.
4. The method according to claim 2, wherein the recombinant interferon molecule is a recombinant type I interferon or biologically active fragment thereof selected from recombinant IFN-α, IFN-β, IFN-ε, IFN-ω, IFN-κ, and IFN-τ.
5. The method according to claim 2, wherein a. the recombinant hTFF1 molecule is a protein of SEQ ID NO:89; or b. the recombinant hTFF2 molecule is a protein of SEQ ID NO: 52; or c. .the recombinant hTFF3 molecule is a protein of SEQ ID NO:96.
6. The method according to claim 2, wherein a. the recombinant interferon molecule is a human IFN-α of SEQ ID NO: 52 (NCBI Ref: NP_005414); or b. the recombinant interferon molecule is a human IFN-κ of SEQ ID NO: 54 (NCBI Ref NM_020124.3); or c. the recombinant interferon is a human IFN-ω of SEQ ID NO: 55 (NCBI Ref. NM 002177.3); or d. the recombinant interferon is a human interferon-τ of SEQ ID NO: 34 MAFVLSLLMALVLVSYGPGGSLGCDLSQNHVLVGRKNLRLLDEMRRLSPHFCLQDR KDFALPQEMVEGGQLQEAQAISVLHEMLQQSFNLFHTEHSSAAWDTTLLEPCRTGL HQQLDNLDACLGQVMGEEDSALGRTGPTLALKRYFQGIHVLKEKGYSDCAWETVR LEIMRSFSSLISLQERLRMMDGDLSSP.
7. The method according to claim 3, wherein the recombinant Fc-derived antibody domain is of SEQ ID NO: 56 (NCBI Ref 4CDH_A).
8. The method according to claim 3, wherein the amino acid sequence of the recombinant fusion protein is SEQ ID NO: 35.
9. The method according to claim 1, further comprising encapsulating the recombinant fusion protein into particles.
10. The method according to claim 1, a. wherein the respiratory virus is a respiratory syncytial virus (RSV), an Ebola virus, a cytomegalovirus, a Hanta virus, an influenza virus, a coronavirus, a Zika virus, A West Nile virus, a dengue virus, a Japanese encephalitis virus, a tick-borne encephalitis virus, a yellow fever virus, a rhinovirus, an adenovirus, a herpes virus, an Epstein Barr virus, a measles virus, a mumps virus, a rotavirus, a cocksackie virus, a norovirus, or an encephalomyocarditis virus (EMCV); or b. wherein the method stimulates repair of a mucosal injury, modulates an immune response, or both; or c. wherein the administering occurs parenterally, by inhalation, or by insufflation; or d. . wherein the susceptible patient includes a very young subject, an elderly subject, a subject who is ill; an immunocompromised subject, a subject with long term health conditions, a subject who is obese, or a subject that is physically weak due to malnutrition or dehydration.
11. The method according to claim 1, wherein the damaging effects of the severe respiratory virus infection include one or more of: primary viral pneumonia; or superimposed bacterial pneumonia; or disruption or injury to alveolar epithelium, endothelium or both; or acute lung injury (ALI); or acute respiratory distress syndrome (ARDS); or symptoms of shock; or excessive complement activation; or a pathological increase in vascular permeability; or endothelial activation, loss of barrier function and consequent microvascular leak; or thrombotic complications; or kidney damage; or elevated concentrations of one or more inflammatory mediators in plasma (hypercytokinemia), compared to a normal healthy subject.
12. The method according to claim 11, wherein (a) symptoms of shock include low blood pressure, lightheadedness, shortness of breath, and rash; or (b) the thrombotic complications include one or more of formation of pulmonary microthrombi, acute pulmonary embolism, deep-vein thrombosis, ischemic stroke, myocardial infarction, or systemic arterial embolism; or (c) the inflammatory mediator is one or more of interferon α, interferon β, interferon-κ, interferon-γ, complement, prostaglandin D2, vasoactive intestinal peptide (VIP), nterleukin-1-beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 (IL-12), IL-17, tumor necrosis factor-alpha (TNF-α).
13. The method according to claim 10, wherein a. repair of a mucosal injury comprises epithelial proliferation; or b. repair of a mucosal injury restores an epithelial barrier, an endothelial barrier or both; or c. the immune response comprises recruitment of innate and adaptive immune cells.
14. The method according to claim 13, wherein (a) the innate immune cells comprise macrophages, dendritic cells (DCs), innate lymphoid cells (ILCs), and natural killer cells (NKs); or (b) the adaptive immune cells include αβ T cells, γδT cells, and B cells.
15. The method according to claim 1, wherein the method comprises aerosolizing the composition in a form selected from a dry powder, a suspension or a solution and administering the aerosolized composition to the respiratory system.
16. The method according to claim 15, wherein the composition is a solution.
17. The method according to claim 15, wherein the administering to the respiratory system is by an inhalation delivery device or a solid particulate therapeutic aerosol generator.
18. The method according to claim 17, a. wherein the solid particulate aerosol generator is an insufflator; or b. wherein the inhalation delivery device is a nebulizer, a metered-dose inhaler, or a dry powder inhaler (DPI); or c. wherein the respirable particles range in size from about 1 to 10 microns, inclusive; or the particles for nasal administration (insufflation), range in size from 10-500 µM, inclusive.
19. The method according to claim 1, wherein the pharmaceutical composition further comprises a supportive therapy or an additional therapeutic agent selected from one or more of an immunomodulatory agent, an analgesic agent, an anti-inflammatory agent, an anti-infective agent, an anti-malarial agent, an anti-viral agent or an anti-fibrotic agent.
20. The method according to claim 19, a. wherein the immunomodulatory agent can be used as a monotherapy or in combination with the supportive therapy, the analgesic agent, the anti- inflammatory agent, the anti-infective agent, the anti-malarial agent, the anti- viral agent or the anti-fibrotic agent; or b. wherein the immunomodulatory agent is selected from the group consisting of i. methotrexate; ii. a glucocorticoid, iii. cyclosporine, tacrolimus and sirolimus; iv. a recombinant interferon selected from IFN-α in a PEGylated form; IFN-α-2b, IFN-β, IFN-γ, IFN-κ, IFN-ω; and IFN-τ; v. a recombinant IL-2 receptor inhibitor; vi. a PDE4 inhibitor; vii. a hyperimmune globulin prepared from a donor with high titers of a desired antibody; viii. a TNFα inhibitor/antagonist; ix. an IL-1β inhibitor; x. a chimeric IL-1Ra; xi. an IL-6 inhibitor; xii. an IL-12/ IL-23 inhibitor selected from ustekinumab, briakinumab, or an IL-23 inhibitor selected from guselkumab, tildrakizumab; xiii. a compound that targets TLR4 signaling; xiv. a p38 MAPK inhibitor, xv. a compound that targets Janus kinase signaling; xvi. a compound that targets cell adhesion molecules to reduce leukocyte recruitment; and xvii. a recombinant anti-inflammatory cytokine. c. wherein the analgesic agent is selected from the group consisting of codeine, hydrocodone, oxycodone, methadone, hydromorphone, morphine, and fentanyl, or d. wherein the anti-inflammatory agent is selected from aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac nabumetone, naproxen, nintedanib, oxaprozin, pirfenidone, piroxicam, salsalate, sarilumab (Kevzara®) sulindac, tolmetin, and combinations thereof; or e. wherein the anti-infective agent is amoxicillin, doxycycline, demeclocycline; eravacycline, minocycline, ormadacycline, tetracycline, cephalexin, defotaxime, cetazidime, cefuroxime, ceftaroline; ciprofloxacin, levofloxacin, moxifloxacin, clindamycin, lincomycin, metronidazole, azithromycin; clarithromycin, erythromycin, sulfamethoxazle and trimethoprim; sulfasalazine, amoxicillin and clavulanate; vancomycin, dalbavancin, oritavancin, telavancin, gentamycin, tobramycin, amikacin, imipenem and cilastatin, meropenem, doripenem, or ertapenem; or f. wherein the anti-malarial agent is selected from quinine, quinidine, chloroquine, hydroxychloroquine, amodiaquine, mefloquine, halofantrine, lumefantrine, piperaquine, and tafenoquine; an antifolate compound selected from pyrimethamine, proguanil, chlorproguanil, trimethoprim; an artemisinin compound selected from artemisinin, dihydroartemisinin, artemether, artesunate; and atovaquone; or g. wherein the anti-viral agent is selected from acyclovir, gancidovir, foscarnet; ribavirin; amantadine, azidodeoxythymidine /zidovudine), nevirapine, a tetrahydroimidazobenzodiazepinone (TIBO) compound; efavirenz; remdecivir, lopinavir/ritonavir, umifenovir, favipiravir, ivermectin, and delavirdine; or h. wherein the anti-fibrotic agent is selected from nintedanib, pirfenidone, and combinations thereof; or i. wherein the supportive therapy is therapeutic apheresis comprising a virion removing step.
21. The method according to claim 20, wherein a. the glucocorticoid is a corticosteroid selected from prednisone, dexamethasone, azathioprine, mycophenolate, mycophenolate mofetil, and combinations thereof; or b. the recombinant IL-2 inhibitor is denileukin diftitox; or c. the PDE4 inhibitor is cilomilast; or d. the TNFα inhibitor/antagonist is selected from the group consisting of etanercept; adalimumab; infliximab, certolizumab pegol, or golimumab; or e. the IL-1β inhibitor is selected from rilonacept; canakinumab; and Anakinra; or f. the IL-6 inhibitor is selected from tocilizumab, siltuximab, sarilumab, olokizumab, and sirukumab; or g. the compound that targets TLR4 signaling is selected from (ethyl 4-(4’- chlorophenyl) amino-6 methyl-2-oxocyclohex-3-en-1-aote (enamionone E121),t; JODI 18b; JODI 19, resatorvid, TLR-C34; and C35; h. the p38 MAPK inhibitor is selected from the group consisting of 4-(4’- fluorophenyl)-2-(4’-methylsulfinylphenyl)-5- (4’-pyridyl)-imidazole (SB203580), trans-4-[4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)-1H- imidazol-1-yl]cyclohexanol (SB239063), and 4-[4-(4-fluorophenyl)-1-(3- phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol (RWJ 67657); or i. the compound that targets Janus kinase signaling is tofacitinub, baricitinib, or upadacitinib; or j. the compound that targets a cell adhesion molecule to reduce leukocyte recruitment is an α4 integrin inhibitor selected from vedolizumab and natalizumab; or k. the recombinant anti-inflammatory cytokine is IL-4, IL-10, or IL-11; or l. the interferon is in a PEGylated form.
22. The method according to claim 20, wherein a physiologic or supraphysiological dose of the recombinant interferon selected from recombinant IFN-α; IFN-α-2b, IFN-β, IFN-γ, IFN- κ, IFN-ω, and IFN-τ or a PEGylated form thereof boosts immune defenses of the subject.
23. The method according to claim 19, (a) wherein the anti-viral agent is an agent that inhibits viral entry and decreases viral load.; or (b) wherein the anti-viral agent that inhibits or blocks viral entry is a synthetic peptide selected from the group consisting of: a. NP-1, SEQ ID NO: 18; b. NP-2; SEQ ID NO: 19; c. NP-3; SEQ ID NO: 20; d. NP-4; SEQ ID NO: 21; e. CP-1; SEQ ID NO: 22; f. CP-2; SEQ ID NO: 23; g. HR2P; SEQ ID NO: 25; h. OC43-HR2-, SEQ ID NO: 26; i. EK1, SEQ ID NO: 27; j. EK1P, SEQ ID NO: 28; k. EK1C, SEQ ID NO: 29; or (c) wherein the anti-viral agent that inhibits or blocks viral entry is a dipeptidyl peptidase 4 (DPP4) inhibitor; an ACE2 inhibitor; a transmembrane serine protease TMRSS2 inhibitor; a cathepsin B inhibitor, a cathepsin L inhibitor or a cathepsin B/ L inhibitor.
24. The method according to claim 20, wherein the therapeutic apheresis reduces viral load.
25. A method for reducing progression of symptoms of a severe respiratory virus infection in a susceptible human subject, comprising administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant bifunctional fusion protein comprising a recombinant biologically active immunomodulatory component operatively linked to a recombinant biologically active anti-viral component, wherein the recombinant immunomodulatory component comprises a recombinant human trefoil protein, and a vehicle, wherein the method rescues symptoms of the severe virus infection.
26. The method according to claim 25, wherein the recombinant human trefoil protein is a recombinant biologically active human trefoil factor 1 protein (rhTFF1), a recombinant biologically active human trefoil factor 2 protein (rhTFF2), or a recombinant biologically active trefoil factor 3 protein (rhTFF3)fragment or variant joined by its C-terminal end to a linker sequence, which is joined to an N-terminal end of a recombinant biologically active recombinant human interferon molecule, fragment or variant.
27. The method according to claim 26, wherein the C-terminal end of the recombinant interferon molecule, fragment or variant sequence is further joined to a recombinant Fc derived antibody domain comprising a recombinant constant region of a human immunoglobulin heavy chain.
28. The method according to claim 26, wherein the recombinant interferon (IFN) molecule is a recombinant type I interferon or biologically active fragment thereof selected from recombinant IFN-α, IFN-β, IFN-ε, IFN-ω, IFN-κ, and IFN-τ.
29. The method according to claim 26, wherein a. the recombinant hTFF1 molecule is a protein of SEQ ID NO:89; b. the recombinant hTFF2 molecule is a protein of SEQ ID NO: 52; c. the recombinant hTFF3 molecule is a protein of SEQ ID NO:96.
30. The method according to claim 26, wherein a. the recombinant interferon molecule is a human IFN-α of SEQ ID NO: 53 (NCBI Ref NP_000596.2); or b. the recombinant interferon molecule is a human IFN-κ of SEQ ID NO: 54 (NCBI Ref NM_020124.3); or c. the recombinant interferon is a human IFN-ω of SEQ ID NO: 55 (NCBI Ref. NM 002177.3); or d. the recombinant interferon is a human interferon-τ of SEQ ID NO: 34: MAFVLSLLMALVLVSYGPGGSLGCDLSQNHVLVGRKNLRLLDEMRRLSPHFCLQDR KDFALPQEMVEGGQLQEAQAISVLHEMLQQSFNLFHTEHSSAAWDTTLLEPCRTGL HQQLDNLDACLGQVMGEEDSALGRTGPTLALKRYFQGIHVLKEKGYSDCAWETVR LEIMRSFSSLISLQERLRMMDGDLSSP.
31. The method according to claim 27, wherein the recombinant Fc-derived antibody domain is of SEQ ID NO: 56 (NCBI Ref Seq 4CDH_A):
32. The method according to claim 27, wherein the amino acid sequence of the recombinant bifunctional fusion protein is SEQ ID NO: 35.
33. The method according to claim 25, further comprising encapsulating the recombinant bifunctional fusion protein into particles.
34. The method according to claim 25, (a) wherein the respiratory virus is a respiratory syncytial virus (RSV), an Ebola virus, a cytomegalovirus, a Hanta virus, an influenza virus, a coronavirus, a Zika virus, a West Nile virus, a dengue virus, a Japanese encephalitis virus, a tick-borne encephalitis virus, a yellow fever virus, a rhinovirus, an adenovirus, a herpes virus, an Epstein Barr virus, a measles virus, a mumps virus, a rotavirus, a cocksackie virus, a norovirus, or an encephalomyocarditis virus (EMCV); or (b) wherein the method stimulates repair of a mucosal injury, modulates an immune response, or both; or (c) wherein the administering occurs parenterally, by inhalation, or by insufflation; or (d) wherein the susceptible patient includes a very young subject, an elderly subject, a subject who is ill; an immunocompromised subject, a subject with long term health conditions, a subject who is obese, or a subject that is physically weak due to malnutrition or dehydration.
35. The method according to claim 25, wherein symptoms of the severe respiratory virus infection include one or more of: primary viral pneumonia; superimposed bacterial pneumonia; disruption or injury to alveolar epithelium, endothelium or both; acute lung injury (ALI); acute respiratory distress syndrome (ARDS); symptoms of shock; excessive complement activation; a pathological increase in vascular permeability; endothelial activation, loss of barrier function and consequent microvascular leak; thrombotic complications; kidney damage; or elevated concentrations of one or more inflammatory mediators in plasma (hypercytokinemia), compared to a normal healthy subject.
36. The method according to claim 35, wherein a. symptoms of shock include low blood pressure, lightheadedness, shortness of breath, and rash; or. b. the thrombotic complications include one or more of formation of pulmonary microthrombi, acute pulmonary embolism, deep-vein thrombosis, ischemic stroke, myocardial infarction, or systemic arterial embolism; or c. the inflammatory mediator is one or more of interferon α, interferon β, interferon-κ, interferon-γ, complement, prostaglandin D2, vasoactive intestinal peptide (VIP), nterleukin-1-beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin- 12 (IL-12), IL-17, tumor necrosis factor-alpha (TNF-α).
37. The method according to claim 34, wherein a. repair of a mucosal injury comprises epithelial proliferation; or b. repair of a mucosal injury restores an epithelial barrier, an endothelial barrier or both; or c. the immune response comprises recruitment of innate and adaptive immune cells.
38. The method according to claim 37, wherein a. the innate immune cells comprise macrophages, dendritic cells (DCs), innate lymphoid cells (ILCs), and natural killer cells (NKs); or b. the adaptive immune cells include αβ T cells, γδT cells, and B cells.
39. The method according to claim 25, wherein the method comprises aerosolizing the composition in a form selected from a dry powder, a suspension or a solution and administering the aerosolized composition to the respiratory system.
40. The method according to claim 39, wherein the composition is a solution.
41. The method according to claim 39, wherein the administering is by an inhalation delivery device or a solid particulate therapeutic aerosol generator.
42. The method according to claim 39, (a) wherein the solid particulate aerosol generator is an insufflator; or (b) wherein the inhalation delivery device is a nebulizer, a metered-dose inhaler, or a dry powder inhaler (DPI); or (c) wherein respirable particles range in size from about 1 to 10 microns, inclusive; or particles for nasal administration (insufflation), range in size from 10-500 µM, inclusive.
43. The method according to claim 25, wherein the pharmaceutical composition further comprises a supportive therapy or an additional therapeutic agent selected from one or more of an immunomodulatory agent, an analgesic agent, an anti-inflammatory agent, an anti-infective agent, an anti-malarial agent, an anti-viral agent or an anti-fibrotic agent.
44. The method according to claim 43, a. wherein the immunomodulatory agent can be used as a monotherapy or in combination with the supportive therapy, the analgesic agent, the anti-inflammatory agent, the anti-infective agent, the anti-malarial agent, the anti-viral agent or the anti- fibrotic agent; or b. wherein the immunomodulatory agent is selected from the group consisting of i. methotrexate; ii. a glucocorticoid, iii. cyclosporine, tacrolimus and sirolimus; iv. a recombinant interferon selected from IFN-α; IFN-α-2b, IFN-β, IFN-γ, IFN-κ, IFN-ω, and IFN-τ; v. a recombinant IL-2 receptor inhibitor; vi. a PDE4 inhibitor; vii. a hyperimmune globulin prepared from a donor with high titers of a desired antibody; viii. a TNFα inhibitor/antagonist; ix. an IL-1β inhibitor; x. a chimeric IL-1Ra; xi. an IL-6 inhibitor; xii. an IL-12/ IL-23 inhibitor selected from ustekinumab or briakinumab, or an IL-23 inhibitor selected from guselkumab or tildrakizumab; xiii. a compound that targets TLR4 signaling; xiv. a p38 MAPK inhibitor, xv. a compound that targets Janus kinase signaling selected from the group consisting of tofacitinub, baricitinib, and upadacitinib; xvi. a compound that targets cell adhesion molecules to reduce leukocyte recruitment; and xvii. a recombinant anti-inflammatory cytokine selected from the group consisting of IL-4, IL-10, and IL-11; or c. wherein the analgesic agent is selected from the group consisting of codeine, hydrocodone, oxycodone, methadone, hydromorphone, morphine, and fentanyl; or d. wherein the anti-inflammatory agent is selected from aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac nabumetone, naproxen, nintedanib, oxaprozin, pirfenidone, piroxicam, salsalate, sarilumab (Kevzara®) sulindac, tolmetin, and combinations thereof; or e. wherein the anti-infective agent is amoxicillin, doxycycline, demeclocycline; eravacycline, minocycline, ormadacycline, tetracycline, cephalexin, defotaxime, cetazidime, cefuroxime, ceftaroline; ciprofloxacin, levofloxacin, moxifloxacin, clindamycin, lincomycin, metronidazole, azithromycin; clarithromycin, erythromycin, sulfamethoxazle and trimethoprim; sulfasalazine, amoxicillin and clavulanate; vancomycin, dalbavancin, oritavancin, telavancin, gentamycin, tobramycin, amikacin, imipenem and cilastatin, meropenem, doripenem, or ertapenem; or f. wherein the anti-malarial agent is selected from quinine, quinidine, chloroquine, hydroxycloroquine, amodiaquine, mefloquine, halofantrine, lumefantrine, piperaquine, and tafenoquine; an antifolate compound, selected from pyrimethamine, proguanil, chlorproguanil, trimethoprim; cepharanthine/selamectin/mefloquine hydrochloride; an artemisinin compound selected from artemisinin, dihydroartemisinin, artemether, artesunate; and atovaquone; or g. wherein the anti-viral agent is selected from acyclovir, gancidovir, foscarnet; ribavirin; amantadine, azidodeoxythymidine /zidovudine), nevirapine, a tetrahydroimidazobenzodiazepinone (TIBO) compound; efavirenz; remdecivir, lopinavir/ritonavir, umifenovir, favipiravir, ivermectin, and delavirdine; or h. wherein the anti-fibrotic agent is selected from nintedanib, pirfenidone, and combinations thereof; or i. wherein the supportive therapy is therapeutic apheresis comprising a virion removing step.
45. The method according to claim 36, wherein a. The glucocorticoid is a corticosteroid selected from prednisone, dexamethasone, azathioprine, mycophenolate, mycophenolate mofetil,, and combinations thereof; or b. The recombinant IL-2 inhibitor is denileukin diftitox; or c. The PDE4 inhibitor is cilomilast; or d. The TNFα inhibitor/antagonist is selected from the group consisting of etanercept; adalimumab; infliximab, certolizumab pegol, or golimumab; or e. The IL-1β inhibitor is selected from rilonacept; canakinumab; and Anakinra; or f. the IL-6 inhibitor is selected from tocilizumab, siltuximab, sarilumab, olokizumab, and sirukumab; or g. the compound that targets TLR4 signaling is selected from (ethyl 4-(4’- chlorophenyl) amino-6 methyl-2-oxocytlohex-3-en-1-aote (enamionone E121), JODI 18b; JODI 19, resatorvid, TLR-C34 and C35; or h. the p38 MAP inhibitor is selected from the group consisting of 4-(4’- fluorophenyl)-2-(4’-methylsulfinylphenyl)-5- (4’-pyridyl)-imidazole (SB203580), trans-4-[4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)-1H-imidazol-1- yl]cyclohexanol (SB239063); and 4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4- pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol (RWJ 67657); or i. the compound that targets Janus kinase signaling is tofacitinub, baricitinib, or upadacitinib; or j. the compound that targets a cell adhesion molecule to reduce leukocyte recruitment is an α4 integrin inhibitor selected from vedolizumab and natalizumab; or k. the recombinant anti-inflammatory cytokine is IL-4, IL-10, or IL-11; l. the recombinant interferon is in a PEGylated form.
46. The method according to claim 36, wherein a physiologic or supraphysiological dose of the recombinant interferon selected from IFN-α in a PEGylated form; IFN-α-2b, IFN-β, IFN- γ, IFN-κ, IFN-ω, and IFN-τ boosts the subject’s defenses.
47. The method according to claim 35, (a) wherein the antiviral agent inhibits viral entry and decreases viral load; or (b) wherein the anti-viral agent that blocks viral entry is a synthetic peptide selected from the group consisting of: (i) NP-1, SEQ ID NO: 18; (ii) NP-2; SEQ ID NO: 19; (iii) NP-3; SEQ ID NO: 20; (iv) NP-4; SEQ ID NO: 21; (v) CP-1; SEQ ID NO: 22; (vi) CP-2; SEQ ID NO: 23; (vii) HR2P; SEQ ID NO: 25; (viii) OC43-HR2-, SEQ ID NO: 26; (ix) EK1, SEQ ID NO: 27; (x) EK1P, SEQ ID NO: 28; and (xi) EK1C, SEQ ID NO: 29; or (c) wherein the anti-viral agent that inhibits viral entry is a dipeptidyl peptidase 4 (DPP4) inhibitor; an ACE2 inhibitor; a transmembrane serine protease TMRSS2 inhibitor; a cathepsin B inhibitor, a cathepsin L inhibitor or a cathepsin B/ L inhibitor.
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