RU2757013C2 - Recombinant anti-influenza vaccine with wide range of protection and method for its preparation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to the field of medicine, namely to a recombinant protein molecule intended for the preparation of a vaccine against influenza, an amino acid sequence of which includes an amino acid sequence of flagellin of Salmonella typhimurium bacterium, to the C-end of which an amino acid sequence of a conservative fragment of the second subunit of hemagglutinin of influenza viruses of the first phylogenetic group RLENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRMQLRDNA is attached, or an amino acid sequence of a conservative fragment of the second subunit of hemagglutinin of influenza viruses of the second phylogenetic group RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENA is attached, and includes 2 copies of an amino acid sequence of M2e peptides of a human influenza A virus SLLTEVETPIRNEWGCRCNDSSD and 2 copies of an amino acid sequence of M2e peptides of H1N1pdm09 virus SLLTEVETPTRSEWECRCSDSSD, as well as glycine-rich linkers connecting the above-mentioned elements, as well as relates to a candidate vaccine against the infection caused by an influenza virus including a recombinant protein molecule.

EFFECT: group of inventions provides for an increase in immunogenicity of the anti-influenza vaccine and an expansion of the protection range.

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Description

Application area

The invention relates to the field of medical biotechnology and is intended to create anti-influenza vaccines of a wide range of protection ("universal"), which will find application in the control of pandemics and epidemics of influenza.

Relevance

Vaccination of the population is the most effective and affordable way to reduce the damage caused by influenza epidemics. Modern inactivated influenza vaccines provide a preventive effect, mainly due to the induction of the body's immune response to the surface proteins of the influenza virus: hemagglutinin (HA) and neuraminidase (NA). The constant mutational drift of influenza viruses requires an annual update of the strain composition of the vaccines. Despite careful global monitoring of the antigenic variability of influenza viruses, in many epidemic seasons one of the viral components of the vaccines did not coincide with the circulating strain, which reduced the epidemiological effectiveness of traditional vaccines. In addition, traditional influenza vaccines produced in chicken embryos have a number of contraindications, primarily the presence of allergic reactions. The most common in practice subunit vaccines, being strain-specific, do not induce an immune response to all viral proteins and form vaccine dependence. These disadvantages can be overcome by creating recombinant vaccines with a broad spectrum of protection.

The creation of a "universal" vaccine against the most variable in antigenic and pathogenic properties of the infectious agent - the influenza virus - is one of the most urgent, but not yet resolved, tasks for medical science and healthcare practice.

Candidate proteins for the creation of such vaccines are conserved viral proteins, primarily the matrix protein (M1 and M2), the NP nucleoprotein, and the second subunit of the hemagglutinin molecule (HA2). The object of close attention for vaccine developers is the small size (23 amino acids) highly conserved ectodomain of the M2 protein, M2e, the sequence of which is almost identical for all influenza type A viruses circulating in the human population, including pandemic viruses A / Singapore / 1/57 and A / Hong Kong / 1/68, which caused pandemics in 1957 and 1968, respectively. Only the 2009 pandemic virus A (H1N1) pdm09 has differences in 4 amino acid residues.

The inclusion of the human influenza A virus consensus M2e peptide in the vaccine preparation should provide protection against infection by all human influenza A virus subtypes, and the inclusion of the M2e peptide of the A (H1N1) pdm09 virus will provide protection not only against the virus that caused the 2009 pandemic, but also against high the pathogenic and similar in M2e peptide of the avian influenza A (H5N1) virus. One of the essential characteristics of the M2 protein is its abundant expression on infected cells and availability for effectors of the immune system of the macroorganism [Lamb, 1985, Holsinger, 1991; DeFillete, 2005]. As shown in 1990-2000, passive transfer of monoclonal anti-M2e antibodies leads to restriction of viral replication and protection of experimental animals from infection [Zebedee, 1988; Treanor 1990; Fan, 2004; Zharikova, 2005; Liu, 2005]. At the same time, after influenza infection and vaccination, antibodies to low immunogenic M2e are formed in small amounts, but there are many ways to increase the immunogenicity of this peptide. As vaccine preparations, various constructs are proposed, including the M2e peptide [Tompkins, 2007; Park, 2011; Bessa, 2008; Denis, 2008; Neirynck 1999; DeFilette, 2005, 2006; Hulleatt, 2008, Mozdanovska, 2003]. Several candidate "generic" vaccines are currently undergoing clinical trials. Their immunogenicity and safety for humans has been shown [Fiers, 2009; Rudolph and Ben-Yedidia, 2011].

The second variant of actively developed cross-reactive vaccines are vaccines that include the second hemagglutinin subunit (or its fragments) of the influenza A virus. The HA2 region (aa 76-130) includes a large α-helix of the second HA subunit, partially accessible from the surface of the molecule.

Immunization with traditional vaccines and natural influenza infection does not lead to the formation of a significant amount of anti-HA2 antibodies, which is associated with the low immunogenicity of the HA2 stem region in the presence of immunodominant HA1 receptor-binding regions [Kwong, 2009]. Recently, however, a number of monoclonal antibodies (from mice, humans) have been isolated that react with epitopes localized in the stem part of HA. These antibodies are cross-reactive and neutralize influenza virus subtypes within one or two phylogenetic groups, thus providing a wide range of protection [Trosby, 2008; Gocnik, 2007; Prabhu, 2008; Wang, 2010; Wei, 2010; Corti, 2011; Wrammert, 2011; Kalleward, 2016].

It is assumed that the construction of a protein that forms an immune response to conserved epitopes of HA2 can serve as the basis for a broad-spectrum vaccine. In recent years, a number of candidate vaccines based on HA2 viruses of influenza A I or II phylogenetic groups (ak 38-59, 23-185, 1-172, 76-130) have been developed [Wang, 2010; Bommakanti, 2010; Stanekova, 2013; Schneemann, 2012; Chen, 2015]. Their immunogenicity and effectiveness in protecting against infection with lethal doses of homologous and heterologous viruses of the same phylogenetic group was shown.

The effect of the use of a recombinant cross-protective vaccine will be evident in influenza caused both by drift variants of the influenza A virus, caused by point mutations in the genes of surface proteins, and by viral reassortants with pandemic spread. In addition to the use of such a vaccine as a "barricade" in the event of a new pandemic, there are two more categories of the population for which the relevance of the proposed vaccine is indisputable. Firstly, people in need of sparing low-allergenic vaccine preparations: pregnant women, young children, people with somatic and chronic infectious diseases. For these categories, recombinant influenza vaccines will be the vaccines of choice. Secondly, children whose reward with a recombinant "universal" vaccine will prevent the development of vaccine dependence that occurs during annual immunization with subunit vaccines.

State of the art

1. Modern influenza vaccines

In Russia, to date, only strain-specific influenza vaccines (live and inactivated) are produced and used in practice.

Live vaccine Ultravac - influenza allantoic live dry intranasal vaccine. Ultravac contains attenuated epidemically relevant strains of influenza virus types A (H1N1, H3N2) and B, obtained from virus-containing allantoic fluid of chicken embryos.

Inactivated domestic vaccines: Grippol, Grippol plus, Sovigrip, Ultrix. Polymer-subunit vaccines Grippol and Sovigrip are a solution of protective surface antigens of hemagglutinin and neuraminidase in combination with a water-soluble high-molecular immunostimulant, Polyoxidonium and Sovidon, respectively. Grippol Plus is an improved analogue of the Grippol vaccine without the addition of thiomersal preservative, which reduces the frequency of post-vaccination reactions. The Ultrix vaccine is a preparation with an inactivated, split virus, partially purified from internal proteins and polysaccharides. All of these vaccines are produced in chicken embryos. The full production cycle of these vaccines, starting with the production of reassortant strains, takes a significant amount of time - 6-8 months.

Genetic engineering methods allow creating vaccines in a shorter time compared to traditional ones, as well as with strictly defined properties, including pronounced cross-reactivity.

An analogue of the presented invention is a recombinant universal vaccine against avian influenza A / H5N1 (RF patent 2358981). The recombinant virus-like particle based on the nuclear antigen of the hepatitis B virus (HBc) presents on its surface peptides of the extracellular domain M2 of the avian influenza virus protein. The resulting virus-like particles are highly immunogenic. The proposed vaccine is active against various strains of the avian influenza A / H5N1 virus and can be considered as a candidate for a universal vaccine against these strains. At the same time, a vaccine against influenza viruses circulating in the human population, including possible pandemic viruses, is more urgent for public health.

The closest analogue, selected as a prototype, is a recombinant vaccine against pandemic influenza A (H1N1) pdm09 (RF patent No. 2451027). Was designed a recombinant protein molecule based on HBc for obtaining a vaccine against infection caused by the influenza A (H1N1) pdm09 virus. The molecule consists of a methionine residue, a sequence of the extracellular domain M2 of the influenza A (H1N1) pdm09 protein from 2 to 24 amino acids and a sequence of the hepatitis B virus nuclear antigen from 4 to 149 amino acids. The molecule is capable of forming virus-like particles. Also disclosed is a recombinant nucleic acid encoding such a molecule, a vector for its expression, virus-like particles formed by such molecules, and a vaccine based on the resulting virus-like particles. A prototype vaccine can be considered as a candidate for a recombinant swine influenza virus vaccine.

The disadvantages of both the analogue and the prototype of the present invention include their lack of immunogenicity and protection against influenza caused by viruses of the subtypes AH1, AH2, AH3. It is to eliminate the limited use of vaccines presented in RF patents 2358981 and 2451027 that the present invention is directed. This disadvantage of vaccines - analogues in the present invention is eliminated by genetic fusion of four copies of the ectodomain of the viral protein M2, including two copies of the M2e amino acid sequence, consensus for human influenza A viruses of the AH1, AH2, AH3 subtypes and two copies of M2e of the influenza A (H1N1) virus pdm09 with the flagellin protein, as well as by using two viral peptides M2e and a fragment ak 76-130 HA2 in the construction.

2.Flagellin as a carrier protein and an adjuvant for targeted proteins in vaccine formulations

One of the most promising carriers for the presentation of foreign peptides is the TLR5 ligand flagellin, the main protein component of the bacterial flagellum, which imparts motility to cells and ensures their adhesion to the tissues of the host's mucous membranes. The flagellin monomer consists of 4 domains. Domains D0 and D1 are composed of tandem long alpha chains and are highly conserved among various bacteria. D2 and D3 domains are highly variable and are located on the surface of the bacterial flagellum; antibodies actively interact with them [Kim, 2015]. Purified or recombinant flagellin induces an immune response in vitro and in vivo after systemic administration. It has been shown that genetic fusion of flagellin with an antigen enhances the ability of the antigen to induce a specific immune response [Hajam, 2017].

In our previous studies, we have shown that immunization of mice with recombinant flagellin containing 4 copies of the M2e peptide at the C-terminus provides an effective immune response during intranasal immunization and protects mice from lethal influenza infection [Stepanova, 2015].

Numerous studies have shown that flagellin makes a significant contribution to the effectiveness of the latest vaccines against various bacterial and viral infections, including influenza. Flagellin promotes the emergence of adaptive immunity in various ways. First, it can directly activate human dendritic cells. Secondly, it transmits a signal that stimulates T-lymphocytes, promoting their proliferation and cytokine production. Finally, as a result of recent work, it was found that TLR5 is expressed in activated B-lymphocytes and plasma cells and can directly induce an immune response in mice. [Kim, 2015].

Flagellin has several advantages over other adjuvants. Thus, a recombinant protein based on flagellin and a target antigen is easily produced in large quantities in Escherichia coli cells. Preexisting immunity to flagellin does not reduce its effectiveness as an adjuvant [Simon, 2011]. An important advantage of flagellin is the possibility of intranasal administration. Thus, flagellin can be used simultaneously as a carrier protein and an adjuvant in the creation of antiviral vaccines.

Disclosure of invention

The objective of the present invention was to develop recombinant vaccine preparations directed against all currently known human influenza A viruses, as well as against avian viruses AH5N1, AH7N9 posing a threat to humans.

The problem was solved by:

a) the design of the construct of several variants of recombinant proteins, including the flagellin sequence of the bacterium Salmonella typhimurium, conserved fragments (aa76-130) of the second hemagglutinin subunit of influenza viruses of the first or second phylogenetic groups and M2e peptide, consensus for different subtypes of influenza A.

b) synthesis of chimeric genes encoding hybrid proteins, including the flagellin sequence of the bacterium Salmonella typhimurium, to the end of which conserved fragments (ak 76-130) of the second hemagglutinin subunit of influenza viruses of the first or second phylogenetic groups and 4 copies of M2e peptide, consensus for influenza viruses, are attached A / H1N1, A / H3N2, A / H2N2 - M2eh or М2е of the virus A / H1N1pdm09 - M2es in order of M2eh-M2es-M2eh-M2es.

c) synthesis of a chimeric gene encoding the FlgSh-HA2-2-4M2e protein containing the flagellin sequence, the hypervariable domain of which is replaced by a fragment of the second subunit (ak 76-130) of the HA subunit of influenza II phylogenetic group II viruses, and 4 copies of M2e (M2eh -M2es-M2eh-M2es).

d) creating strains of Escherichia coli - producers of hybrid proteins,

e) isolation and purification of recombinant proteins,

f) studies of the specific activity (immunogenicity and protectiveness) of candidate vaccines in laboratory animals.

The originality of the solution to the problem, which is the subject of the invention, lies in the following aspects:

The first is the simultaneous incorporation of two viral proteins into the flagellin molecule: the M2e peptide and a fragment (ak 76-130) of the second HA subunit of influenza viruses, which stimulates various antiviral defense mechanisms.

The following were chosen as conservative peptides of the influenza virus intended for inclusion in recombinant vaccines:

M2h - consensus sequence of human influenza A virus strains SLLTEVETPIRNEWGCRCNDSSD

M2s - sequence of the pandemic strain "swine flu" H1N1pdm09 SLLTEVETPTRSEWECRCSDSSD

HA2-1 - a fragment of the second subunit of HA (ak 76-130), consensus for influenza viruses AH1N1, H2N2, H1N1pdm09 (I phylogenetic group)

RLENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRMQLRDNA HA2-2 - fragment of the second HA subunit (ak 76-130), consensus for influenza A viruses H3N2 and H7N9 (II phylogenetic group)

RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENA

The second is the simultaneous use of both the hypervariable domain and the C terminus of the flagellin molecule as the site of insertion of target peptides.

The third is the study of the specific properties (immunogenicity, protectiveness, cross-protectiveness) of candidate vaccine preparations with two methods of administration into the host's body - intranasal and subcutaneous.

Fourth, the study on experimental animals of the duration of the preservation of humoral and cellular immunity to recombinant proteins.

The technical result consists in increasing the immunogenicity of the influenza vaccine and expanding the range of protection. It has been demonstrated that, in addition to stimulating the formation of specific IgGs, immunization induces the formation of virus-specific lymphocytes, both CD4 ⁺ and CD8 ⁺ types. It was shown that polypeptide ak 76-130 in the composition of recombinant proteins causes an additional immune response due to CD8 ⁺ T - cells

The essence of the invention is illustrated by drawings.

Brief Description of Figures

FIG. 1 - Scheme and theoretical modeling of the 3-D structure of the recombinant proteins Flg-4M2e, Flg-HA2-1-4M2e, Flg-HA2-2-4M2e, FlgSh-HA2-2-4M2e.

FIG. 2 - Electrophoresis in PAGE and Western blot of recombinant proteins with anti-Flagellin antibodies 93713 (lanes 4, 5, 6) and anti-4M2e monoclonal antibodies 14C2 (lanes 7, 8, 9) of recombinant proteins Flg-HA2-1-4M2e ( lanes 1, 4, 7), Flg-HA2-2-4M2e (lanes 2, 5, 8), FlgSh-HA2-2-4M2e (lanes 3, 6, 9) after chromatographic purification on Ni sorbent.

FIG. 3 - Geometric mean titers (SGT) of anti-M2e specific IgG in the blood serum of mice after subcutaneous immunization with recombinant proteins: (A) SGT IgG to M2eh after the second and third immunizations; (B) CGT IgG to M2es after the second and third immunizations; (B) CGT IgG to Flg after the third immunization; BALB / c mice (n = 10 / group) were immunized on days 0, 14, 28 with a dose of 10 μg of one of the recombinant proteins: Mice of the control groups were injected with phosphate buffered saline (PBS) or Flg-his protein.

The Mann-Whitney test was used to calculate the p-value. * - significant difference from the control groups (Flg-his, PBS) with p <0.01

FIG. 4 - Geometric mean titers (SGT) of anti-M2e specific IgG in the blood serum of mice after intranasal immunization with recombinant proteins: (A) SGT IgG to M2eh after the second and third immunizations; (B) CGT IgG to M2es after the second and third immunizations; (B) CGT IgA to M2e after the third immunization. BALB / c mice (n = 10 / group) were immunized on days 0, 14, 28 with a dose of 10 μg of one of the recombinant proteins: Mice of the control groups were injected with phosphate buffered saline (PBS) or Flg-his protein.

The Mann-Whitney test was used to calculate the p-value. * - significant difference from the control groups (Flg-his, PBS) with p <0.01

FIG. 5 - Geometric mean titers (SGT) of specific IgG to influenza viruses in the blood serum of mice after (A, B) subcutaneous and (C) intranasal immunization with proteins Flg-H2-1-4M2ehs, Flg-H2-2-4M2ehs, FlgSh-H2- 2-4M2ehs. (A) - SGT IgG to the A / H2N2 virus. (B) CGT IgG to the A / H7N9 virus. (C) SGT IgG to influenza viruses A / H2N2, A / H7N9, A / H3N2, A / H1N1pdm09, A / H5N1.

BALB / c mice (n = 10 / group) were immunized on days 0, 14, 28 with a dose of 10 μg of one of the recombinant proteins: Mice of the control groups were injected with phosphate buffered saline (PBS) or Flg-his protein.

The Mann-Whitney test was used to calculate the p-value. * - significant difference from the control groups (Flg-his, PBS) with p <0.01

Fig. 6 - Mucosal immune response. Geometric mean titers of (A) anti-M2e and (B) anti-viral IgA in BAL fluid and nasal washes of mice after intranasal immunization with recombinant proteins Flg-H2-1-4M2ehs, Flg-H2-2-4M2ehs, Flg-4M2ehs. BALB / c mice (n = 10 / group) were immunized on days 0, 14, 28 with a dose of 10 μg of one of the recombinant proteins: Mice of the control groups were injected with phosphate buffered saline (PBS) or Flg-his protein.

The Mann-Whitney test was used to calculate the p-value. Significant differences from the control group (PBS) are shown.

FIG. 7 - T-cell immune response after subcutaneous immunization. (A, B) M2e-specific T-cell response in the spleen of mice. (A) CD4 + T lymphocytes and (B) CD8 + T lymphocytes producing cytokines after activation with the M2e peptide. (C, D) Virus-specific CD4 + T-cell response in the spleen of mice. (B) activation of splenocytes by influenza A / H1N1 virus. (D) activation of splenocytes by influenza A / H3N2 virus. (E, F) Virus-specific CD8 + T cell response in the spleen of mice. (E) activation of splenocytes by influenza A / H1N1 virus. (E) activation of splenocytes by influenza A / H3N2 virus.

достоверное отличие от опытных групп с р≤0.05;

достоверное отличие от опытных групп с р<0.01To calculate the p-value, the Mann-Whitney test (U-test) was used. Significant differences between the experimental groups are shown. * significant difference from the control groups (Flg-his, PBS) with p≤0.05; ** significant difference from the control groups (Flg-his, PBS) with p≤0.01;

significant difference from the experimental groups with p≤0.05;

significant difference from the experimental groups with p <0.01

FIG. 8 - T-cell immune response after intranasal immunization (A, B) M2e-specific and (C, D) virus-specific T-cell response in the lungs of mice. (A) CD4 + and (B) CD8 + T-lymphocytes producing IFN-γ or TNF-α after cell activation with the M2e peptide. (C) CD4 + and (D) CD8 + T-lymphocytes producing IFN-γ or TNF-α after cell activation by influenza viruses A / H1N1 (Flg-HA-2-1-4M2ehs group) and A / H3N2 (Flg-HA group -2-2-4M2ehs).

достоверное отличие от опытных групп с р<0.01To calculate the p-value, the Mann-Whitney test (U-test) was used. Significant differences between the experimental groups are shown. * significant difference from the control group with p≤0.05;

significant difference from the experimental groups with p <0.01

FIG. 9 - Effectiveness of immunization. Survival and dynamics of the weight of immunized and control mice after infection with lethal doses of viruses.

(A, B) subcutaneous immunization (C, D, E) intranasal immunization. Recombinant proteins used for immunization are indicated in the corresponding groups. 2 weeks after the third immunization, mice were infected with influenza virus A / Shanghai / 2/2013-PR8-IDCDC (H7N9) or A / Aichi / 2/68 (H3N2) or A / California / 1/66 (H2N2) at a dose of 10LD50 or A / Kurgan / 5 / 05RG (H5N1). Changes in body weight (left) and death of animals (right) were recorded within 14 days after infection. Significant differences were shown (Montel-Cox test) between the experimental and control groups.

Example 1. Alignment and analysis of sequences of target peptides.

The amino acid sequences of HA2 for analysis were taken from the GenBank and GISAID databases. To build consensus, the sequences were aligned using the MAFFT server and the FFT-NS-i, FFT-NS-2 algorithms (depending on the number of sequences) [Katoh, 2002] and analyzed in the Unipro UGENE v. 1.14.0 [Okonechnikov, 2012]. Alignment and analysis of a small number of sequences was performed using the Vector NTI software package (Invitrogen, USA).

The search for probable T-cell epitopes was performed using the NetCTLpan1.1 Server [Stranzl, 2010] and default search parameters. The search for experimental B- and CD4 + T-cell epitopes homologous to the HA2 regions was performed in the Immune Epitope Database [Vita, 2010]. Visualization of the three-dimensional structure of proteins was performed using the Chimera 1.5.3 software [Pettersen, 2004]. For homologous modeling of the three-dimensional structure of the protein by the primary sequence, the open web resource Phyre2 was used [Kelley, 2009].

In the HA2 region (76-130), the consensus sequences of hemagglutinins of influenza viruses of phylogenetic group I (subtypes H1, H1pdm, H2, and H5) are 80% identical. In addition, substitutions in this region at all positions except 124 and 128 occur for amino acids with similar properties. Taking into account the substitutions for amino acids with similar properties, the homology is 96.4%.

The consensus sequences of hemagglutinins of influenza viruses of the II phylogenetic group (subtypes H3 and H7) in the HA2 region (76-130) are 63.6% identical. Taking into account the substitutions for amino acids with similar properties, the homology will be 80%.

For influenza viruses of the I phylogenetic group, most of the experimentally detected B- and T-cell epitopes are concentrated in the middle of the HA2 region (76-130) - amino acids 84-116. For influenza viruses of the II phylogenetic group, epitopes are concentrated in the first half of the site - amino acids 76-103. The HA2 region (76-130) of influenza viruses of both phylogenetic groups contains potential CD8 + T cell epitopes for various HLA alleles.

Example 2. Recombinant protein molecules Flg-4M2e, Flg-HA2-1-4M2, Flg-HA2-2-4M2, FlgSh-HA2-2-4M2 and their encoding nucleic acids.

We have created chimeric genes encoding fusion proteins, including the flagellin sequence of the bacterium Salmonella typhimurium, to the C end of which or in the hypervariable part are attached the M2e peptide sequences of different influenza virus subtypes, as well as conserved fragments of the second hemagglutinin subunit of influenza viruses of the first or second phylogenetic groups, in different combinations (Fig. 1). To assess the immunological properties of the target peptides, the genes of the following chimeric proteins were obtained separately and together:

1) Flg-4M2e - contains the flagellin sequence and four copies of the M2e peptide in the order M2h-M2s-M2h-M2s at the C end of the molecule.

2) Flg-HA2-1-4M2e - contains the flagellin sequence, to which a fragment (ak 76-130) of the second subunit of HA of influenza viruses of phylogenetic group I is attached at the C end, followed by 4 copies of M2e (M2h-M2s-M2h-M2s ).

3) Flg-HA2-2-4M2e - contains the flagellin sequence, to which a fragment (ak 76-130) of the second HA subunit of influenza viruses of phylogenetic group II is attached at the C end, followed by 4 copies of M2e (M2h-M2s-M2h-M2s ).

4) FlgSh-HA2-2-4M2e - contains the flagellin sequence, the hypervariable domain of which is replaced by a fragment of the second subunit (ak 76-130) of the HA of influenza viruses of the II phylogenetic group, and 4 copies of M2e are attached to the C end (M2h-M2s-M2h-M2s ).

In all M2e-containing constructs, the M2e sequences were delimited from each other by glycine-rich linkers. The "assembly" of chimeric genes from individual components was carried out directly in the expression vector pQE30. The flagellin gene without its own start codon was cloned into the BamHI site of the vector, which ensured its expression with an N-terminal 6-histidine tag attached to it, an encoded vector, intended for the purification of recombinant proteins by metal affinity chromatography.

Standard genetic engineering techniques were used to create chimeric genes. The flagellin gene was previously obtained by us using PCR on genomic DNA of Salmonella typhimurium and cloned. Nucleotide sequences encoding HA2-1, HA2-2 and tandem copies of M2e were synthesized in vitro; in the cases of HA2-1 and HA2-2, the codon composition was optimized for expression in E. coli. Thus, the expression vectors pQE30_Flg_4M2e, pQE30_Flg_HA2-1, pQE30_Flg_HA2-2, pQE30_Flg_HA2-1_4M2e and pQE30_Flg_HA2-2-4M2e were created to express the corresponding recombinant proteins. As a control, an expression vector pQE-Flg was obtained for the expression of full-length flagellin without target peptides.

Additionally, we created a chimeric gene encoding a flagellin-based fusion protein containing the insertion of a fragment of the second HA subunit (ak 76-130) of phylogenetic group II influenza viruses into the internal variable region of flagellin and 4 copies of the M2e peptide at its C-terminus. Flagellin consists of three domains: two conserved and one variable domain located in the inner part of the molecule (amino acids 154 to 431). Based on the structure of the protein, it can be assumed that the removal of the variable domain will not change the properties of flagellin as an adjuvant. However, this will more than halve its length, and a different sequence can be included instead of the variable region. We constructed a chimeric gene encoding a fusion protein FlgSh-HA2-2-4M2e in which the central variable domain of flagellin was replaced by a hemagglutinin fragment of influenza viruses of the second phylogenetic group, and 4 copies of the M2e peptide were attached to the C-terminus (in order of M2h-M2s-M2h -M2s). The corresponding gene was cloned into the pQE30 vector, resulting in the expression vector pQE30-FlgSh-HA2-2-4M2e.

Example 3. Creation of E. coli strains - producers of recombinant proteins.

To create strains producing recombinant proteins, the corresponding expression vectors pQE30 were introduced into E. coli strain DLT1270, a derivative of the DH10B strain, into the chromosome of which the lactose operon repressor gene lacI is integrated. This strain was used instead of the SG strain designed to work with vectors of the pQE series and containing lacI on the plasmid. Cultures of producer strains were grown in LB at 37C, when the optical density of the culture OD600 ~ 0.4-0.7, IPTG (up to 0.1 mM) was added to the medium, then the culture was grown at 30C for 3-12 hours. Protein preparations were analyzed by SDS-PAGE (Fig. 2). The obtained results show that all proteins (Flg-4M2e, Flg-HA2-1-4M2e, Flg-HA2-2-4M2e, FlgSh-HA2-2-4M2e and control Flg-his) are expressed at the level of 10-30% of the total protein ...

Since all fusion proteins contained the N-terminal 6-histidine tag, the purification of the recombinant proteins produced in the cells of the producing strains was carried out by metal affinity chromatography according to standard protocols.

Example 4. Humoral response with subcutaneous and intranasal immunization

The humoral response to subcutaneous immunization. All recombinant proteins (Flg-HA2-1-4M2ehs, Flg-HA2-2-4M2ehs, FlgSh-HA2-2-4M2ehs, Flg-4M2ehs) stimulated the formation of a strong humoral M2e-specific and moderate virus-specific immune response upon subcutaneous immunization. Anti-M2e IgG efficiently bound to synthetic peptides of both M2eh and M2es (Fig. 3A, B). There were no significant differences in anti-M2e IgG titers between the recombinant proteins. The level of IgG antibodies to Flg in mice immunized with the recombinant protein with truncated flagellin (FlgSh-HA2-2-4M2ehs) was more than 2 times lower (Fig. 3B) than among mice after immunization with recombinant proteins based on full-length flagellin. Investigation of the profile of anti-M2e IgG subclasses showed that all recombinant proteins stimulated predominantly IgG1 production.

The selected HA2-2 and HA2-1 consensus sequences (ak76-130) are highly conserved among influenza viruses of phylogenetic groups II and I; therefore, it was important to evaluate the formation of cross-reactive antibodies after immunization of mice with recombinant proteins. FIG. 5A, B show SGT IgG to influenza virus A / H2N2 (first phylogenetic group) and A / H7N9 (second phylogenetic group) after immunization of mice with recombinant proteins Flg-HA2-1-4M2ehs, Flg-HA2-2-4M2ehs, FlgSh-HA2 -2-4M2ehs. The recombinant protein based on truncated flagellin (FlgSh-HA2-2-4M2ehs) had the greatest potential in inducing the formation of cross-reactive antiviral antibodies. In mice, after immunization with FlgSh-HA2-2-4M2ehs, antibodies were detected that were able to bind both with the homologous influenza A / H7N9 virus and with the heterologous A / H2N2 virus. Moreover, the level of such antibodies was significantly higher for influenza viruses A / H2N2 and A / H7N9 than for a similar protein based on full-length flagellin. In addition, after immunization of mice with the FlgSh-HA2-2-4M2ehs protein, the level of antibodies to the influenza virus of the first phylogenetic group (A / H2N2) was comparable to that in mice immunized with the Flg-HA2-1-4M2ehs protein. Thus, the recombinant protein based on truncated flagellin (FlgSh-HA2-2-4M2ehs) had the greatest potential in the induction of cross-reactive antiviral antibodies.

The humoral response in intranasal immunization. With respect to the M2e peptide, the immunogenicity of the recombinant proteins Flg-HA2-2-4M2ehs and Flg-HA2-1-4M2ehs was comparable to the Flg-4M2ehs protein. All three proteins stimulated the production of high levels of serum anti-M2e IgG and IgA (Fig. 4). At the same time, high titers of antibodies were formed against both M2eh and M2es peptides. All three proteins induced the formation of anti-M2e IgG predominantly of the IgG1 subclass.

The production of anti-HA antibodies to various subtypes of influenza A viruses in the serum was significantly lower than the level of anti-M2e antibodies. Nevertheless, intranasal immunization with both recombinant proteins Flg-HA2-2-4M2ehs and Flg-HA2-1-4M2ehs led to the formation of antibodies to the region of the second hemagglutinin subunit, which were able to bind to influenza viruses of both phylogenetic groups (Fig.5B), with However, no significant differences were found between proteins in their ability to form anti-HA2 antibodies to different viruses.

The level of IgA to M2e peptide and influenza viruses A / H2N2 and A / H7N9 was also assessed in bronchoalveolar lavages (BAL) and nasal washes of immunized mice. Both drugs induced the formation of anti-M2e (Fig. 6A) and anti-viral IgA (Fig. 6B) in BAL fluid and nasal washes in the same titers. In general, no differences in the levels of specific antibodies were found between the Flg-HA2-2-4M2ehs and Flg-HA2-1-4M2ehs proteins.

Example 5. T-cell response with subcutaneous and intranasal immunization

T cell response to subcutaneous immunization

Antigen-specific T-cell (CD4 + and CD8 + T cells producing IL-4, TNFα +, IFNγ +) response after subcutaneous immunization with recombinant proteins Flg-HA2-1-4M2ehs, Flg-HA2-2-4M2ehs, FlgSh-HA2 -2-4M2ehs, Flg-4M2ehs were assessed in the spleen 2 weeks after the third immunization (in 5 mice of each group) after activation of splenocytes by target antigens: peptide М2е, influenza viruses A / H1N1 (phylogenetic group 1) or A / H3N2 (phylogenetic group 2). Control mice were injected with the flagellin carrier protein (Flg-his) or phosphate buffered saline (PBS).

Activation of splenocytes with M2e peptide showed that after immunization of mice with recombinant proteins based on full-length flagellin Flg-HA2-1-4M2ehs, Flg-HA2-2-4M2ehs, Flg-4M2ehs, M2e-specific CD4 + T lymphocytes were formed, producing predominantly TNFα + (0.23%; 0.32%; 0.19%, respectively). After immunization of mice with a recombinant protein based on the truncated flagellin FlgSh-HA2-2-4M2ehs, the formation of M2e-specific CD4 + T cells producing IFNγ + (0.43%) was revealed, which was significantly higher than in the control and other experimental groups (p ≤0.05).

Immunization with Flg-HA2-1-4M2ehs, Flg-HA2-2-4M2ehs, Flg-4M2ehs proteins did not lead to the formation of a significant number of M2e-specific CD8 + T lymphocytes (Fig. 7B), whereas after immunization with FlgSh-HA2-2- 4M2ehs revealed the formation of М2е-specific CD8 + cells producing IFNγ + (0.87%), which was significantly higher than in the control and experimental groups (p <0.05).

Activation of splenocytes by influenza viruses A / H1N1 (phylogenetic group 1) or A / H3N2 (phylogenetic group 2). Splenocytes from mice immunized with recombinant proteins were activated by homologous and heterologous influenza virus. Activation of splenocytes from mice immunized with the recombinant Flg-HA2-1-4M2ehs protein by both homologous (A / H1N1) and heterologous (A / H3N2) viruses did not result in the detection of virus-specific CD4 + T cells (Fig. 7B). In the spleen of mice immunized with Flg-HA2-2-4M2ehs, virus-specific CD4 + cells producing mainly TNFα + were detected (0.16%, significant difference from the control groups and from Flg-HA2-1-4M2ehs, p <0.05). The most pronounced virus-specific CD4 + response was found in mice immunized with FlgSh-HA2-2-4M2ehs. When splenocytes were activated by a homologous virus (A / H3N2), virus-specific CD4 + producing IFNγ + (0.97%; significant difference from the control and experimental groups with p <0.05), as well as CD4 +, simultaneously producing two cytokines (TNFα + IFNγ + - 0.07%) (Fig.7D). Upon activation with a heterologous virus (A / H1N1), virus-specific CD4 + cells, predominantly producing IFNγ +, were also detected in this group of mice, although their number was significantly lower (0.19%) than upon activation with a homologous virus.

Virus-specific CD8 + response was expressed only in mice immunized with the recombinant protein FlgSh-HA2-2-4M2ehs, both after activation of splenocytes by homologous and heterologous viruses. When splenocytes were activated by a heterologous virus (A / H1N1), CD8 + T cells were detected, predominantly producing IFNγ + (0.34%) (Fig. 7D). When splenocytes were activated by a homologous virus (A / H3N2), CD8 + T cells were detected, producing both one cytokine (IFNγ - 1.73%) and two cytokines (TNFα + IFNγ - 0.18%) (Fig. 7E).

Thus, significant differences were found in the formation of an antigen-specific T-cell response in the spleen of mice after subcutaneous immunization with recombinant proteins. The recombinant protein based on truncated flagellin (FlgSh-HA2-2-4M2ehs) had the highest activity and induced the formation of M2e-specific CD4 + and CD8 + T cells, producing mainly IFNγ +, as well as virus-specific CD4 + and CD8 + T cells after activation by viruses of both phylogenetic groups. At the same time, restimulation of splenocytes with a homologous virus revealed not only T cells producing 1 cytokine (TNFα + or IFNγ +), but also cells producing both cytokines. Activation by a heterologous virus resulted in the formation of only IFNγ + -producing CD4 + and CD8 + T cells. The antigen-specific T-cell response in the spleen after immunization with other proteins (Flg-HA2-1-4M2ehs, Flg-HA2-2-4M2ehs, Flg-4M2ehs) was characterized by a significantly lower level and narrower range of cytokines produced.

T-cell response in intranasal immunization.

Activation of lung cells with M2e peptide. Intranasal immunization with the recombinant proteins Flg-4M2ehs and Flg-HA2-2-4M2ehs stimulated the formation of cytokine producing CD4 + cells in the lungs of mice (0.34% and 0.44%, respectively) (Fig. 8A). Moreover, the majority of CD4 + cells in both groups of mice produced TNF-α. Both recombinant proteins were characterized by the absence of M2e-specific CD4 + IFN-γ +, as well as a significant number of M2e-specific CD8 + T cells (Fig. 8B). The recombinant protein Flg-HA2-1-4M2ehs practically did not induce the formation of M2e-specific CD4 + and CD8 + T cells in the lungs of mice. Activation of lung cells by influenza viruses A / H1N1 (phylogenetic group 1) and A / H3N2 (phylogenetic group 2). The effect of activation of lung cells by influenza virus A / Aichi / 2/68 (H3N2) or A / California / 07/09 (H1N1pdm09) showed that (Fig.8B) intranasal immunization of mice with the recombinant protein Flg-HA2-2-4M2ehs led to the formation a significant number of H3N2-specific CD4 + and CD8 + T cells in the lungs, producing mainly TNF-α (0.66% and 0.44%, respectively). Virus specific cells producing IFN-γ + were detected in insignificant quantities. The T cell response to the recombinant protein Flg-HA2-1-4M2ehs was much weaker than that to Flg-HA2-2-4M2ehs. The production of H1N1-specific CD4 + and CD8 + T cells was 0.032% and 0.066%, respectively (Fig. 8D).

Thus, the recombinant Flg-HA2-2-4M2ehs protein with the HA2 consensus sequence of influenza viruses of the second phylogenetic group during intranasal immunization was more efficient in the formation of virus-specific CD4 + and CD8 + T cells than the recombinant Flg-HA2-1-4M2ehs protein.

Example 6. Evaluation of the duration of the immune response after immunization

Duration of specific immune response after subcutaneous immunization

and the cytokine profile of M2- and virus-specific CD4 + and CD8 + effector (Tem CD44 + / CD62L-) and central (Tcm CD44 + CD62L +) memory T cells in spleens was assessed 6 months after immunization of mice. The study of blood sera from mice taken 6 months after immunization with recombinant proteins (Flg-HA2-1-4M2ehs, Flg-HA2-2-4M2ehs, FlgSh-HA2-2-4M2ehs, Flg-4M2ehs) revealed rather high titers of M2e-specific antibodies of the IgG and IgA classes. In the long term after immunization, there were no significant changes in the levels of IgG subclasses (IgG2a / b), realizing antibody-dependent cytotoxicity. In the spleen of mice, M2e-specific CD4 + effector memory cells, predominantly producing TNF-α, as well as CD4 + central memory cells, producing TNF-α and IFN-γ, and CD8 + central T-memory cells, mainly producing IFN-γ. Virus-specific T-cell response was also characterized by the formation of CD4 + effector memory cells, predominantly producing TNF-α, CD4 + central memory cells, producing IFN-γ, and CD8 + central T-memory cells, producing IFN-γ and TNF-α ... The most significant pool of antigen-specific effector and central T-memory cells was found in mice immunized with the recombinant FlgSh-HA2-2-4M2ehs protein.

Duration of specific immune response after intranasal immunization

After 6 months. after intranasal immunization with the recombinant proteins Flg-HA2-1-4M2ehs, Flg-HA2-2-4M2ehs, Flg-4M2ehs, in the blood of M2e-specific IgG and IgA mice circulated in rather high titers. The virus-specific T-cell response was characterized by the formation of CD4 + and CD8 + effector memory cells, predominantly producing TNF-α.

Example 7. Protectiveness of recombinant proteins obtained in a bacterial expression system in a model of lethal influenza infection

Subcutaneous immunization

To compare the protective effect of recombinant proteins with different designs (Flg-HA2-1-4M2ehs, Flg-HA2-2-4M2ehs, FlgSh-HA2-2-4M2ehs, Flg-4M2ehs), two weeks after the last immunization, mice were infected with lethal doses of influenza viruses of various subtypes (A / Shanghai / 2/2013-PR8-IDCDC (H7N9), A / Aichi / 2/68 (H3N2)) at a dose of 10LD ₅₀ . As shown in FIG. 9A, mice immunized with all four recombinant proteins were almost completely protected (90-100% survival rate) from infection with a high dose of influenza A / Shanghai / 2/2013-PR8-IDCDC (H7N9) virus. The smallest weight loss was found in mice immunized with the recombinant FlgSh-HA2-2-4M2ehs protein. Complete protection (100% survival) of experimental animals was also observed after infection with the influenza A / Aichi / 2/68 (H3N2) virus (Fig. 9B). In both cases, minimal weight loss was found in mice immunized with the recombinant FlgSh-HA2-2-4M2ehs protein.

We also observed a significant decrease in viral titers in the lungs of immunized mice compared to naive mice after infection with influenza viruses A / Aichi / 2/68 (H3N2), A / Shanghai / 2/2013 (H7N9) -PR8-IDCDC), A / California / 07/09 H1N1pdm09 and A / Kurgan / 05/05 RG (H5N1).

Intranasal immunization

Intranasal immunization with recombinant proteins Flg-HA2-1-4M2ehs, Flg-HA2-2-4M2ehs, Flg-4M2ehs provided 60-100% protection of mice (Fig. 9 C, D, E) from lethal infection with influenza A / California / 1 viruses / 66 (H2N2), A / Kurgan / 05/05 RG (H5N1) or A / Aichi / 2/68 (H3N2). The strongest protective effect was observed after immunization of mice with the recombinant protein Flg-HA2-2-4M2ehs - 90-100% survival. The protective effect of the recombinant Flg-HA2-1-4M2ehs protein was 70-80%, and the recombinant Flg-4M2ehs protein containing only one target antigen (M2e) - 60-70%. Minimal weight loss was also found in mice immunized with the recombinant Flg-HA2-2-4M2ehs protein.

The results obtained confirm a wide range of protection of candidate vaccines based on recombinant proteins, including conserved epitopes of two viral proteins M2 and the second hemagglutinin subunit. Vaccines provide protection for 90-100% of immunized animals after infection with influenza A viruses of different subtypes (H1N1, H2N2, H3N2, H5N1, H7N9) with high lethal doses (10 LD ₅₀ ) and lead to a significant decrease in the reproduction of influenza viruses in the lungs.

Obviously, in order to obtain a strong “universal” response (to human and avian viruses), two viral peptides 4M2e and HA2 should be included in the recombinant protein, which makes it possible to increase the infecting dose to 10 LD.

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Claims (2)

1. Recombinant protein molecule designed to create a vaccine against influenza, the amino acid sequence of which includes the amino acid sequence of Salmonella typhimurium flagellin, to the C-end of which is attached the amino acid sequence of the conserved fragment of the second hemagglutinin subunit of influenza viruses of the first phylogenetic group RLENLNKKMEDGELLMLFDSHENERTY the second subunit of the influenza virus hemagglutinin second phylogenetic group RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENA, and comprises two copies of the amino acid sequence of M2e peptide of influenza a virus and human SLLTEVETPIRNEWGCRCNDSSD 2 copies of the amino acid sequence of M2e virus H1N1pdm09 SLLTEVETPTRSEWECRCSDSSD peptides as well as connecting the above elements are glycine-rich linkers. 2. Candidate vaccine against influenza virus infection, comprising a recombinant protein molecule according to claim 1.

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