TW201617363A - Virosomes containing respiratory syncytial virus strain line 19 fusion protein and uses thereof - Google Patents

Abstract

The invention provides Respiratory Syncytial Virus (RSV) vaccines and, in particular, RSV virosomes and related compositions and methods.

Description

Virus-like particle containing respiratory fusion virus 19 fusion protein and use thereof

The present invention relates to respiratory syncytial virus (RSV) vaccines, and more particularly to RSV-like viral particles and related compositions and methods.

Respiratory tract fusion virus (RSV) is a respiratory pathogen that infects human lungs of all ages. Although most other healthy humans typically recover from RSV infection after 1 to 2 weeks after only mild symptoms, infections are particularly severe in babies and the elderly, such as those with chronic lung disease or cardiovascular disease. Indeed, in these humans, RSV is an important cause of hospitalization and even death.

Currently, no drug approval is available for the treatment of RSV. In general, therapies are supportive and may include, for example, moisturization methods, utilizing intravenous fluids, humidified oxygen, mechanical ventilation, and aerosolized tracheal dilators. The current medical regimen for preventing RSV infection is limited to the use of Palivizumab (Symagis®), an IgG monoclonal antibody against the RSV F glycoprotein. The use of palivizumab is limited to babies at high risk because of premature birth or other medical problems, such as congenital heart disease.

There are no vaccines approved for combating RSV. The development of RSV vaccine based on killed virus and subunit protein has been related to immunity The problem of the sufficiency of the originality and the induction of the enhanced respiratory disease (ERD) are hindered. Regarding the latter, early development of ESV vaccines, including formalin-inactivated RSV and alum adjuvants, usually have a high percentage of vaccinated babies that cause ERD to be severe enough to require hospitalization during subsequent natural RSV exposure. The use of this vaccine is likely to cause two deaths. The development of live attenuated and recombinant viral vaccines against RSV has been challenged, for example, over-attenuation and the difficulty of passage, as well as potential confounding factors that may be replied.

Due to the potential severity of RSV infection in these humans, and the limited use of palivizumab, there is a need for vaccines against RSV infection.

The present invention provides a pseudovirus particle comprising a fusion (F) protein of the respiratory syncytial virus (RSV) line 19 (for example, a protein having the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having the sequence number: Proteins greater than 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity). For example, the F protein may have one or more substitutions relative to the sequence of SEQ ID NO: 1. Exemplary substitutions include, for example, the substitution of I557 of SEQ ID NO: 1 (eg, I557V substitution).

In addition, or in the alternative, the F protein may have one or more of the following positions (eg, two, three or four) compared to, for example, SEQ ID NO: 1, 79, 191, 357, and 371 (eg, M79I) , R191K, K358T and Y371N). In certain embodiments, the F protein comprises one or more of the following amino acids (eg, two or more, three or more): methionine at residue 79, Arginine of base 191, lysine at residue 357, and tyrosine at residue 371. In addition, or alternatively, the F The protein may comprise one or more of the following amino acids (eg, two, three, four, five, six, seven, eight, nine or ten): proline at residue 4, at residue Alanine of 16 , serine at residue 25, valine at residue 76, alanine at residue 103, threonine at residue 122 Threonine, isoleucine at residue 152, arginine at residue 213, asparagine at residue 515, and glycine at residue 519 Acid (glycine). In all of the foregoing specific examples, the F protein does not have the complete sequence of the FV protein of the RSV A2 strain.

The pseudoviral particle may further comprise a G protein of RSV, which is a small hydrophobic (SH) protein, and/or a matrix (M) protein. The pseudoviral particles of the present invention may also comprise RSV extracted from an RSV strain (e.g., including an RSV strain (e.g., RSV strain A2) in which the F protein is replaced by the F protein of RSV virus line 19 as described herein. Lipids and proteins of the membrane of the chimeric RSV strain of the virus strain.

The F protein, like the viable viral particles present in the present invention, can be substantially in a pre-fusion conformation. For example, the ratio of 5C4/palivizumab of the pseudoviral particle calculated by dividing the slope of the curve can be at least 20% greater than (eg, at least 25%, 30%, 35%, 40%) , 45%, 50% or more) The ratio of 5C4/palivizumab calculated by dividing the slope of the curve by the virus-like particles produced by the RSV A2 strain.

The pseudoviral particles of the present invention may also include adjuvants such as saponin, PHAD (phosphorylated hexavalent disaccharide), 3-D-PHAD (3-O-defluorenyl derivative of phosphorylated hexamethylene disaccharide) 3-OD MPLA (3-O-dehydrazinyl derivative of mono-o-decyl lipid A) or MPLA (mono-o-decyl lipid A).

Additionally, or alternatively, the pseudoviral particles may further comprise one or more additional lipids. For example, the pseudoviral particles can include phospholipids such as, for example, phospholipid choline (PC) (eg, one or more synthetic or intrinsically pure PCs) and/or phospholipids, ethanolamine (PE) (eg, , one or more synthetic or intrinsically pure PEs). In some embodiments, at least one of the one or more synthetic or intrinsically pure PCs and the one or more synthetic or intrinsically pure PEs are the only non-viral phospholipids in the pseudoviral particles. Furthermore, the pseudoviral particles may optionally include sterol or sterol derivatives (eg, steroids in a ratio of from 0 to 30 mole percent of total added phospholipids (eg, 5 to 30 mole percent, 10 to 25 mole percent) Or about 20 moles).

The PC type and the PE type may be, for example, a molar ratio of 3:1 to 1:3 (for example, between 2:1 to 1:2, 3:1 to 1:1, 2:1 to 1:1, 1) : 1 to 1:3 or 1:1 to 1:2) and/or containing a thiol chain having an unsaturated bond (for example, each of the thiol chains of PC and PE contains an unsaturated bond or a thiol chain) The total number of unsaturated bonds in the middle can be 4). Additionally, or alternatively, the thiol chain can have from 14 to 18 carbon atoms (eg, 16 or 18 carbon atoms).

In some embodiments, the pseudoviral particles of the present invention may comprise one or more (eg, two or more) of: 1,2-diolenyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycerol-phosphoethanolamine (PPPE), and 1-palmitoyl-2-olerene Base-sn-glycerol-phosphoethanolamine (PLPE). In such specific examples, the pseudoviral particles of the present invention may include, for example, about 400 to 450 nmol of DOPC per mg of viral membrane protein, about 800 to 900 nmol of DOPE, about 250 to 350 nmol of 3-D-PHAD, and about 200 to 300 nmol of cholesterol.

The pseudoviral particles of the present invention may include an adjuvant (for example, a synthetic adjuvant), and the adjuvant may include 0.01 to 2 mg of adjuvant per mg of viral protein (for example, a ratio of 0.1 to 2 mg per mg of viral protein). The agent is in a ratio of 0.5 to 2 mg of adjuvant per mg of viral protein or a ratio of about 1 mg of adjuvant per mg of viral protein). The molar ratio of total synthetic phospholipid to adjuvant may be, for example, between 3 and 6 or between 3.5 and 5. In a specific example, the molar ratio of total synthetic phospholipid to adjuvant is between 1.5 and 10.

In any of the foregoing specific examples of the present invention, the pseudoviral particles may have, for example, a mode particle diameter of less than 15% having a particle size of more than 150 nm and less than 15% and less than 50 nm of between 55 and 90 nm. A narrow size distribution of the modal diameter.

In another aspect, the invention provides a pharmaceutical composition (e.g., a vaccine) comprising any of the pseudoviral particles of the invention (e.g., comprising a pharmaceutically acceptable carrier, diluent, excipient, and/or adjuvant).

The invention also provides methods comprising administering a pharmaceutical composition of the invention to an individual (e.g., an individual who is not at risk of developing RSV, but at risk of development) to induce an immune response to RSV in the subject.

In another aspect, the invention provides a method of making the virus-like particles of the invention. The methods include (i) dissolving a viral outer membrane comprising an RS protein of the RSV 19F F protein (eg, a chimeric RSV comprising an A2 F protein via an RSV strain 19F protein replacement), and (ii) The virus outer membrane is recombined in the absence of viral nucleic acid.

In another aspect, the invention features the use of the pseudoviral particles of the invention as a medicament for inducing an immune response to RSV, for use in prevention or Methods of treating RSV infection, and/or for individual vaccination against RSV.

The fusion (F) protein (RSV line 19F protein, RSV L 19F protein or abbreviated as L19F) of the "Respiratory fusion virus (RSV) line 19" means that the sequence having the sequence number: 1 or the substantial amino acid sequence is identical to (for example) , greater than 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity) Sequence number: 1 sequence (but without the sequence of the RSV A2 F protein) protein. The RSV L19F protein may optionally include, for example, one or more substitutions selected from the group consisting of M79I, R191K, K371N, and I557V (with the restriction that it does not have the complete sequence of the RSV A2 F protein).

It will be appreciated that, for example, residues at the N-terminus may be undesirably lost during purification, and thus the RSV L19F protein also includes such truncated forms.

The phrase "percent (%) amino acid sequence identity" is defined relative to a reference polypeptide sequence as an amine in the candidate sequence after alignment of the sequence and introduction of the gap, and, if necessary, without considering any retention substitutions as partial sequence identity. The base acid residue is the same percentage as the amino acid residue in the reference polypeptide sequence. Alignment for the purpose of determining percent amino acid sequence identity can be achieved by various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. . Those of ordinary skill in the art can determine suitable parameters for the alignment sequence, including any algorithms needed to achieve maximal alignment in the full length of the sequence of fish-sweet comparisons. For example, for a reference polypeptide of Sequence A, when compared to the various polypeptides of Sequence B, the percent amino acid sequence identity is calculated as: 100 times the fraction X/Y, where X is the same ratio between A and B. The number of amino acid residues given, and wherein Y is the total number of amino acid residues in the B polypeptide sequence.

With respect to "reserved amino acid substitution" as used herein, it is meant that in the amino acid sequence, one amino acid is replaced by another amino acid in the same family of amino acids based on the chemical nature of its side chain. . In general, the encoded amino acids can be divided into four families: acidic (aspartic acid, glutamic acid); basic (iso-acid, arginine, histidine); non-polar (alanine) , proline, leucine, isoleucine, valine, phenylalanine, methionine, tryptophan); and no charge polarity (glycine, aspartame, glutamine, Cysteine, serine, threonine, tyrosine). Amphetamine, tryptophan and tyrosine are sometimes grouped as aromatic amino acids. In a similar manner, the amino acids may also be the following groups: acidic (aspartic acid, glutamic acid), basic (aspartic acid, arginine, histidine); aliphatic (glycine) , alanine, valine, leucine, isoleucine, serine, threonine, if necessary, the samaric and sulphonic acid groups are aliphatic-hydroxy; aromatic (phenylalanine) , tyrosine, tryptophan); guanamine (asparagine, glutamine); and sulfur (cysteine, methionine).

The present invention provides several advantages. For example, the virus-like particles of the invention, including the RSV L19F protein, induce a higher degree of neutralizing antibodies than the virus-like particles comprising the RSV A2F protein. This increase in neutralizing potency is important from a clinical point of view because the increased response to vaccination is associated with a reduced likelihood of RSV-related and respiratory disease (see, for example, Falsey et al., J. Infect. Dis. 198: 1317-1326, 2008). Furthermore, the pseudoviral particles of the present invention exhibit increased stability, as described herein, which is useful, for example, to consider the shelf life of a pharmaceutical composition comprising the pseudoviral particles. In general, the use of pseudoviral particles provides other advantages. For example, the manufacture of pseudoviral particles does not involve the use of chemicals (eg, formalin) that may modify protective antigens Determining the base, resulting in reduced immunogenicity and possible ERD (see above).

Other features and advantages of the present invention will be apparent from the description, appended claims, appended claims.

Figure 1 is a panel of three graphs showing the size distribution of pseudoviral particles produced by three well-known virus strains determined by particle size ranging from 0 to 400 nm particle size (particles/ml). The size distribution refers to the pseudoviral particles of RSV A2 (top panel), RSV A2 L19F (bottom left panel) and RSV A2 L19F I557V mutant strain (bottom right panel).

Figure 2 is a panel of three graphs showing the density (g/ml; triangle), protein concentration (μg/ml;) of the pseudoviral particles derived from each of the specified centrifugation (Fr) for each of the three designated strains; Square) and the results of equilibrium density gradient centrifugation as determined by phosphate concentration (nmol/ml; round). The virus-like particle lines were derived from RSV A2 (top panel), RSV A2 L19F (bottom left panel) and RSV A2 L19F I557V mutant strain (bottom right panel).

Figure 3 shows the log _10- transformed TCID ₅₀ per milliliter of homogeneous lung tissue in mice infected with control (HNE), A2 virus-like particles, A2 L19F virus-like particles and A2 L19F I557V-like virus particles. Calculated RSV titer chart.

Figure 4 shows the log ₂ transformation calculated from the serum of mice infected with control (HNE), A2 virus-like particles, A2 L19F virus-like particles and A2 L19F I557V-like virus particles 2 weeks after the initial vaccination. In vitro neutralizing titer map.

Figure 5 shows the log ₂ conversion calculated from the serum of mice infected with control (HNE), A2 virus-like particles, A2 L19F virus-like particles and A2 L19F I557V-like virus particles 2 weeks after the second vaccination. Shape of the in vitro neutralization titer.

Figure 6 shows that RSV in serum was set by ELISA for mice infected with control (HNE), A2 virus-like particles, A2 L19F virus-like particles and A2 L19F I557V-like virus particles 2 weeks after the second vaccination. - Geometric mean titer (GMT) map of the log ₂ transformation of specific IgG antibodies.

Figure 7 is a graph showing the serum collected from mice infected with control (HNE), A2 virus-like particles, A2 L19F virus-like particles and A2 L19F I557V-like virus particles 2 weeks after the second vaccination. /ml IgG1 isotype concentration map.

Figure 8 is a graph showing the serum collected from mice infected with control (HNE), A2 virus-like particles, A2 L19F-like virus particles, and A2 L19F I557V-like virus particles 2 weeks after the second vaccination. Concentration plot of /ml IgG2A isotype.

Figure 9 shows mice infected with control (HNE), A2 virus-like particles, A2 L19F virus-like particles and A2 L19F I557V-like virus particles on study day 0 (primary vaccination), day 14 (second time) Weight chart for vaccination +1 day), 30/31 day and 35th day.

Figure 10 shows the absorbance as determined at 492 nm, wherein RSV strain A2 F protein (A2), RSV A2 L19F protein (19F) and RSV A2 mutant strain I557V strain 19F protein (557) to increase virus concentration (μg/ml) ELISA results map as determined by Synagis® antibody.

Figure 11 shows the absorption as measured at 492 nm, where the RSV strain A2 F Protein (A2), RSV A2 L19F protein (19F) and RSV A2 mutant strain I557V are 19F protein (557) to increase the virus concentration (μg/ml) by ELISA results as determined by 5C4 antibody.

Figure 12 shows the absorbance as determined at 492 nm, wherein RSV strain A2 F protein (A2), RSV A2 L19F protein (19F) and RSV A2 mutant strain I557V strain 19F protein (557) to increase the concentration of the pseudoviral particles (μg/ Ml) ELISA results map as determined by 5C4 antibody.

Figure 13 shows the absorbance as determined at 492 nm, in which RSV strain A2 F protein (A2), RSV A2 L19F protein (19F) and RSV A2 mutant strain I557V strain 19F protein (557) to increase the concentration of pseudoviral particles (μg/ Ml) ELISA results graph as determined by Synagis® antibody.

The present invention provides a pseudovirus particle comprising a fusion protein of a respiratory syncytial virus (RSV) line 19 (L19 protein), a pharmaceutical composition comprising the same, and a method of using and manufacturing the same. Optionally, the RSV L19F protein of the viruvirus particle of the invention comprises one or more substitutions (e.g., I557V), as further explained below. The virus-like particles, methods and compositions of the present invention are described herein further in detail with reference to RSV structural proteins as described below.

Respiratory fusion virus (RSV) structural protein

RSV is included in the membrane, fusion protein (F), small hydrophobic protein (SH), and matrix protein (M) or four related proteins. Specific examples of the invention include viable viral particles comprising RSV L19F protein, optionally with RSV G protein, SH protein and M protein (and lipid components, as described below) One or more combinations. The RSV G protein, the SH protein, and the M protein may be 19 of the RSV system or may be one or more of the different RSV strains derived from the Ruth RSV A2 strain. In the latter case, the pseudoviral particles can be made from a chimeric RSV strain, such as the RSV A2 virus in which the A2F sequence is replaced with the RSV 19F sequence. An example of such a chimera is disclosed in Moore et al., J. Virol. 83(9): 4185-4194, 2009.

The RSV L19F protein sequence of EMBL FJ614814 is as follows: (sequence number: 1)

As described above, the RSV L19F protein, such as the virion-containing granules included in the present invention, may optionally include one or more substitution mutations. The sequence of SEQ ID NO: 1 is different from the F protein sequence of RSV strain A2 and has a longitude (Long) at 5 amino acid positions (79, 191, 357, 371 and 557; annotated in the sequence by subscript and broad body; Moore Et al., JVI 2009). The invention includes the use of RSV L19F (as in the pseudoviral particles described herein), comprising one or more of the amino acids corresponding to the amino acids present in the amino acid of the intact RSV A2 strain. Substitution, or retention substitution (with the proviso that the resulting F protein sequence is not the F protein sequence of RSV strain A2). Therefore, the present invention includes the RSV L19F For use, the RSV L19F has isoleucine at position 557 substituted with lysine (I557V) or, for example, alanine, leucine, valine, phenylalanine, methionine, tryptophan; Methionine is substituted with isoleucine (M79I) or, for example, alanine, valine, leucine, valine, phenylalanine or tryptophan; arginine at position 191 is lysine (R191K) Or substituted with, for example, histidine, serine, aspartame or glutamine; the lysine at position 357 is substituted with threonine (K357T) or, for example, arginine, histidine; and/or The tyrosine at position 371 is substituted with aspartame (Y371N) or, for example, glycine, glutamine, cysteine, serine, threonine, phenylalanine or tyrosine. The RSV L19F protein may optionally be included in one, two, three, four, or all five of these positions, or any combination thereof, with the proviso that the resulting F protein sequence is not the F protein sequence of RSV strain A2. Mutations can be made using methods well known in the art, including, for example, the bacterial artificial chromosome (BAC) method disclosed by Hotard et al., Virology 434(1): 129-136, 2012.

In addition to the substitution mutations described above, the invention may include the use of an RSV L19F protein having a substantially amino acid sequence identical to the sequence described above (SEQ ID NO: 1), with or without a designation Substitution, the restriction is that the sequence is not a complete RSV A2 F protein sequence. Thus, for example, an RSV L19F protein for use in a virus-like particle of the invention may have greater than 80% amino acid sequence identity (eg, greater than 80) for the above sequence (SEQ ID NO: 1; with or without a particular substitution). %, 85%, 90%, 95%, 96%, 97%, 98% or 99%, or greater sequence identity), with the proviso that the resulting F protein sequence is not a complete RSV A2F protein sequence. The selection of locus resistant strains for sequence variability can be considered by, for example, different strains The known structure and sequence configuration are carried out (for example, considering between strains; see, for example, Johnson et al., J. Gen. Virol. 69: 2623-2628, 1988), as is done in the art. By. Furthermore, the maintenance of the fusion ability can be determined by methods known in the art (for example, see below).

As noted above, the improved efficacy of the virulent virions of the present invention may result from, at least in part, increased stability of the pre-fusion conformation of the RSV L19F protein. Thus, in a particular embodiment of the invention, as described above, for example, the variant RSV L19F protein maintains an increased or substantial pre-fusion configuration compared to the pre-fusion configuration of the RSV A2 F protein. Maintaining an increased pre-fusion conformation means, for example, that when compared to a virus-like particle containing RSV A2F in the assay of the invention, for example, maintaining a pre-fusion conformation of the F protein of at least 20% (eg, at least 25%) , 30%, 35%, 40%, 45%, 50% or more).

Individual antibodies against RSV-F unique in pre-fusion formats, such as C54, AM22 and D25, have been found and provide a means of assay in pre-fusion conformation assays (McLellan, J Set al., Science 2013, May 31; 340 (6136): 1113). Other antibodies are only capable of determining the pre-fusion conformation or the epitope that does not respond to changes in conformation. By exposure to low permeation or high temperatures, RSV-F can be induced to change to a post-fusion configuration. Synagis® (Pacliizumab; Medimmune) is a humanized mouse monoclonal antibody that recognizes the conformation-independent epitope of the F protein presented in most RSV strains. 5C4 is a mouse monoclonal antibody that specifically recognizes the pre-fusion conformation of the RSV F protein. The ratio of pre-fusion to post-fusion, or the ratio of pre-fusion to constant epitope, can be used to quantitatively quantify the pre-fusion conformation. Such ratios can be known by comparison of the results of ELISA assays as described herein. For example, it can be visualized when measured according to the method described in Example 2. Proteins with a 5C4/palivizumab ratio greater than 1 can be calculated by dividing the slope of the curve as described below. Fusion can also be measured directly in the fusion assay; the virus is labeled with the self-quenching concentration of the fluorescent dye octadecylrhodamine, and the fusion of the virus with the cells is quenched by measuring fluorescence ( Quantitative dequenching) (Srinivasakuma, N. et al., J. Virol. 65 (1991) 4063-69).

Other RSV structural proteins (along with RSV L19 F, as described herein) that may be included in the pseudoviral particles of the invention include RSV G proteins, SH proteins, and/or M proteins. These proteins may be from the RSV virus line 19 or from another RSV strain (eg, RSV strain A2, strain A, Long strain, or B strain). In the latter case, a non-F-protein derived from a non-lineage 19 RSV strain of the present invention may be produced by a chimeric RSV strain (e.g., RSV A2 L19 F) as described above. In a specific example, the virulent virion particles of the invention are produced by a chimeric RSV A2 L19 F process, comprising an I557V substitution mutation (i.e., RSV A2 L19 F I557V). Further, in addition to the naturally occurring sequence, the non-F structural protein included in the pseudoviral particle of the present invention, as described above, may have a sequence substantially identical to a naturally occurring sequence (see, for example, the following).

An exemplary sequence of the RSV A2 protein that can be included in the viruvirus particle of the invention, together with the RSV line 19F protein as described herein, is provided below.

RSV A2 G protein (UniprotKB P03423): (sequence number: 2)

RSV A2 SH Protein (UniprotKB P04852) (sequence number: 3)

RSV A2 M protein (UniprotKB P03419) (sequence number: 4)

Imitation virus particle

In some embodiments, the pseudoviral particles are lipid bilayers containing viral surface proteins (eg, from glycoproteins and surface proteins with enveloped viruses or truncated or fragmented fragments thereof). Thus, in general, viable viral particles can be considered as recombination membranes with outer membrane viruses. The virus-like particles can be obtained by using a detergent (for example, C12E8, Triton X-100 or n-octyl-β-D-glucopyranoside) or a short-chain phospholipid (for example, phospholipid choline or a derivative thereof, such as 1,2-hept-acyl - sn - phosphatidylcholine (DHPC) and 1,2-dihexyl acyl - sn - phosphatidylcholine (DCPCs)) was extracted from the viral membrane with the outer membrane protein is produced. Typically, viral nucleocapsids are removed from the extracted proteins and lipids by centrifugation. Optionally, other ingredients (eg, adjuvants, additional lipids and/or cholesterol; dissolved in a solution comprising detergent or short chain phospholipids as needed) are mixed with the extracted protein and lipid. The detergent or short chain phospholipid is then removed by, for example, dialysis, thereby allowing for the composition and reformation of the straight bilayer outer membrane. This results in the formation of pseudoviral particles including the extracted membrane proteins and lipids as well as any additional ingredients as described above.

Detailed methods for making the virus-like particles of the present invention are disclosed in WO 2004/071492; Stegmann et al., EMBO J. 6: 2651-2659, 1987; and Kamphuis et al., Plos One 7 (5): E36812, 1-11, 2012, the entire contents of which are incorporated herein by reference. A particular example, Briefly, purified virus pellet formed by centrifugation-based and was dissolved in 1,2-dihexyl include acyl - sn - phosphatidylcholine buffer (DCPCs) of. The nucleocapsid is then removed by centrifugation. Subsequently, lecithin choline (PC) and lecithin 醯 ethanolamine (PE) are, for example, a 2:1 molar mixture which is evaporated into a dry film in a glass tube. The supernatant, including the extracted lipid and protein, is then added to the lipid mixture. Optionally, adjuvants or other ingredients (eg, additional lipids or cholesterol) are dissolved in DCPC and added to the protein/lipid mixture, filtered and dialyzed after culture to remove DCPC, resulting in the formation of pseudoviral particles (Kamphuis et Al., supra).

In addition to the specific methods described above, in general, the pseudoviral particles can be assembled from any integral membrane protein or peripheral membrane protein, or a protein conjugated to a lipid anchor. The main features of the pseudoviral particles include those taken from phagocytes of sufficient size for the immune system, as well as compositions, surface structures and functional activities (especially membrane fusion activities) that closely mimic the outer membrane of the natural virus. Thus, viral-derived lipids and integrated membrane proteins used to make viru-like viral particles will typically be presented to a pseudoviral particle membrane.

A virus-like particle of the invention comprising an RSV line 19F protein (as described herein) will typically comprise a lipid from a virus from which it is obtained (e.g., RSV L19, RSV A2 L19 F or RSV A2 L19 F I557V). apart from Such lipids, pseudoviral particles may optionally be incorporated into other ingredients such as, for example, amphiphilic adjuvants (e.g., see WO 2004/110486), other lipids (e.g., phospholipid choline (PC) and/or Phospholipids, ethanolamine (PE), and/or sterols or derivatives thereof. In order to incorporate such other ingredients into the viable viral particles of the invention, and consistent with the foregoing, the virus is solubilized with detergent or short chain phospholipids, the viral nucleocapsid protein is removed, and then dissolved as needed in the same cleansing The other component of the agent or short-chain phospholipid is added to the melted viral membrane. The detergent or short chain phospholipids are then removed, resulting in the formation of virus-like particles including viral membrane proteins and lipids as well as other components.

In this manner may be incorporated into viral particles imitation of adjuvants include MPLA (monophosphoryl lipid acyl A) and MPLA derivatives (e.g., 3'-O- to acyl MPLA, 3D MPL ^TM (3'- A composition of O-deindenyl monophosphonyl lipid A), MPLA and MPLA derivatives (eg, phosphonium hexamethylene disaccharide (PHAD, also known as glucopyranoside lipid A or GLA) And 3'-O-dedecyl derivatives of PHAD (3-D-PHAD)), saponins (for example, OS21), lipidated imidazoquinolines (for example, see WO 2010/048520 and US 2011/0282061), lipids Stimulating peptides (for example, Pam ₃ CSK ₄ and L-18 cell wall dipeptide (L 18-MDP)) and vitamin D and vitamin D derivatives. The use of MPLA herein is disclosed, for example, in WO 2004/110486 Stegmann et al., as described above; and Kamphois et al., as described above. The use of Pam ₃ CSK ₄ and L18-MDP in the pseudoviral particles herein is disclosed in Shafique et al., PlosOne 8 ( 4): e61287, 1-12, 2013.

In addition to viral lipids, the functional recombination viral membranes of the invention may comprise purified lipids from other sources, for example, purified lipids or synthetic lipids. Other lipids can be added to the membrane of the pseudoviral particles during the preparation process. Generally In other words, when a lipid mixture similar to a virus-derived lipid or a lipid composition close to the assembled outer membrane of a virus is added, the fusion activity of the pseudoviral particles is optimally maintained. Thus, in accordance with the present invention, a wide range of lipids can be included in the viral membrane. The lipid group comprises neutral and charged phospholipids, sterol-derived lipids, and neutral and charged synthetic lipids. Thus, the lipid composition provided with the fusion-active pseudoviral particles can be a composition obtained or obtainable from a natural viral membrane. Thus, the lipid composition for use in the present invention includes a composition consisting only of a viral natural lipid, a composition consisting of a virus natural lipid supplemented with a lipid derived from another source, and a composition composed of lipids of various origins, It mimics the lipid composition of a natural viral membrane. The pseudoviral particles of the present invention may comprise one or more lipids selected from the group consisting of cationic lipids, synthetic lipids, glycolipids, phospholipids, sterols, and the like. Preferred lipids include sterols such as cholesterol, and phospholipids such as phospholipid choline (PC), sphingomyelin (SPM), phospholipid oxime ethanolamine (PE), and phospholipid lysine (PS). However, other phospholipids may also be added. These include, but are not limited to, phospholipid glycerol (PG), phospholipid citrate (PA), heart fat (CL), and phospholipid creatinine (PI), with varying fatty sulfhydryl compositions and natural and/or synthetic Source, and cetyl phosphate. Ceramide and various glycolipids such as cerebrosides and gangliosides may also be added. Examples of sterol derivatives which may be incorporated into the pseudoviral particles of the present invention include cholesterol hemisuccinates, phytosterols such as lanosterol, ergosterol, and vitamin D and vitamin D related compounds.

In various embodiments, the virulent virions of the present invention have a mode particle size significantly less than 220 nm to aid in filter sterilization. Therefore, the pseudovirus particles of the present invention may have a diameter (particle size) ranging from 40 to 200 nm, for example, from 50 nm to 100 nm, and from 70 nm to 130 nm. In various examples, RSV imitation The viral particles have a homogeneous size distribution of less than 10 to 15% of viable virus particles having a particle size greater than 150 nm, and less than 10 to 15% having a particle size of less than 50 nm. Thus, in various examples, the mode particle size can be less than 90 nm or less than 85 nm. For example, the mode particle size can range from 55 to 90 nm, 58 to 82 nm, 65 to 80 nm, 65 to 77 nm, 68 to 78 nm, 68 to 78 nm, or 69 to 74 nm.

The remainder of this paragraph discloses a composition of a pseudoviral particle comprising a specific component, including adjuvants, lipids and cholesterol in specific amounts. This pseudoviral particle composition can be used in the RSV pseudoviral particles herein, including the F protein of the RSV line 19 (including substitutions as disclosed herein (eg, I557V; see above), and including F as needed) The sequence was made by chimeric RSV of RSV A2 of the RSV line 19F sequence (including substitution mutations as described herein; eg, RSV A2 L19F I557V). The use of this particular type of pseudoviral particle composition other than the content of the RSV L19 F protein-containing pseudoviral particles as described herein is not encompassed by the present invention.

The pseudoviral particles according to this example comprise: (i) lipids and proteins extracted from the RSV membrane (having an F protein of the RSV line 19 F, as described above); (ii) selected from the group consisting of PHAD and 3-D-PHAD a group of synthetic adjuvants, preferably in a ratio of 0.01 to 2 mg adjuvant per mg of viral protein; (iii) at least one synthetic or essentially pure phospholipid choline (PC) class and at least one synthetic or essential Pure phospholipid oxime ethanolamine (PE) in a molar ratio of from 3:1 to 1:3, characterized in that the thiol chain has from 14 to 18 carbon atoms and the total number of unsaturated bonds in the thiol chain is four, and The molar ratio of the total synthetic phospholipid to the adjuvant is between 1.5 and 10; and (iv) the sterol or sterol derivative in a proportion of from 0 to 30 mol% of the total added phospholipid.

As used herein, the expression "synthetic or intrinsically pure" means the addition of a non-viral phospholipid of defined quality, purity and chemical structure to the externalities. It does not necessarily mean a purified or semi-purified phospholipid extracted from a mixed fatty acid composition of natural origin, such as tissue-derived, plant-derived or egg-derived PC and PE. In one embodiment of this example, the synthetic or intrinsically pure PC and PE systems are manufactured in accordance with Good Manufacturing Practices (cGMP). Synthetic or essentially pure PC and PE grades are commercially available, for example, from Avanti Polar Lipids, Alabaster, AL. In one embodiment, the at least one synthetic or intrinsically pure PC and at least one synthetic or intrinsically pure PE are the only non-viral phospholipids in the pseudoviral particles.

When the synthesized PE and PC have between 14 and 18 carbon atoms and the total number of unsaturated bonds in the thiol chain is four, RSV-like virus particles having the desired properties are prepared according to this example. Preferably, both PC and PE contain a thiol chain having an unsaturated bond. The thiol chain can be monounsaturated or diunsaturated. From the standpoint of reducing possible oxidation, a monounsaturated thiol chain is preferred. For example, an RSV-like virus particle (having an RSV line 19F protein, as described herein) is provided wherein each of the thiol chains of PC and PE contains an unsaturated bond. In a specific example, the PC type and/or the PE type are symmetric phospholipids, that is, the sulfhydryl chains having the same positions of sn -1 and sn -2 in the glycerol trunk. Although two or more different synthetic PC-based and/or PE-based blends are contemplated, the RSV-like viral particles (including the RSV-based 19F protein, as described herein) according to this example of the invention typically comprise a synthetic Or intrinsically pure PCs and a synthetic or essentially pure PE class.

In one embodiment, the thiol chain in the synthesized PC and/or PE has 16 or 18 carbon atoms, preferably 18 carbon atoms. Preferably, PC and PE The total number of carbon atoms in the base chain is at least 70. Very good results are obtained if the total number of carbon atoms is 72.

Excellent results were obtained with RSV-like virus particles comprising one or more selected from the group consisting of 1,2-dioleryl- sn -glycero-3-phosphocholine (DOPC), 1,2-dione yl - Sn - glycero-3-phosphoethanolamine (DOPE), 1,2- dipalmitoyl acyl - Sn - glycerol - phosphoethanolamine (PPPE) and 1-palmitoyl-2-oleyl acyl acyl - Sn - glycerol - phosphate Ethanolamine (PLPE). Preferably, RSV-like virus particles (including RSV line 19F proteins, as described herein) are provided, comprising two or more selected from the group consisting of synthetic DOPC, DOPE, PLPE, and PLPC.

In a particular aspect, the RSV-like viral particles (including the RSV-based 19F protein, as described herein) comprise a synthetic PC consisting of synthetic DOPC and a synthetic PE consisting of DOPE.

The improved RSV-like viral particles of this example of the invention (including the RSV-based 19F protein, as described herein) are re-characterized between synthetic or essentially pure PCs and synthetic or essentially pure PEs. The molar ratio is between 3:1 and 1:3. Preferably, the ratio is between 2:1 and 1:2.

In one embodiment, the amount of PC synthesized is equal to or exceeds the amount of synthetic PE used. Thus, RSV-like virus particles (including RSV-based 19F proteins, as described herein) are provided comprising synthetic PCs and synthetic PEs in a molar ratio of from 3:1 to 1:1, preferably from 2:1 to 1:1. In a particular aspect, the PC is present in excess of PE, for example in a molar ratio of from 3:1 up to 1:1, preferably from 2:1 up to 1:1.

In another preferred embodiment, the synthesized PC is used in an amount equal to or less than the amount of synthetic PE used. Therefore, provide RSV-like virus particles (including The RSV line 19F protein, as described herein, comprises a synthetic PC class and a synthetic PE class with a molar ratio of from 1:1 to 1:3, preferably from 1:1 to 2:1. In a particular preferred embodiment, the PE is present in excess of PC, for example in a molar ratio of from 3:1 up to 1:1, preferably from 2:1 up to 1:1.

The addition of further viral lipids enhances one or more desirable properties of the pseudoviral particles. For example, cholesterol can be added to increase the storage stability of the virus-like particles. In a specific embodiment, the RSV-like viral particles (including the RSV-based 19F protein, as described herein) comprise cholesterol in the range of from 5 to 30 mol%, preferably from 10 to 25 mol%, more preferably, based on total phospholipid added. It is about 20% by mole. In a preferred embodiment, the pseudoviral particles comprise DOPC, DOPE and cholesterol, preferably wherein the DOPE is present in excess of, for example at least 1.5 times, DOPC.

It has been found that the molar ratio of total synthesized phospholipids to synthetic adjuvants ranging from 1.5 to 10 is related to the nature of the RSV pseudoviral particles according to the examples of the present invention. Good results are obtained when the ratio is between 3 and 6, preferably between 3.5 and 5. In a particular aspect, the ratio is between 3.4 and 4.5, such as 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4 or 4.5.

The RSV pseudoviral particles of this example of the invention (including the RSV line 19F protein, as described herein) are characterized by the presence of a synthetic adjuvant selected from the group consisting of PHAD (phosphorylated hexamethylene disaccharide) and its 3-O- Demethylated derivative, 3-D-PHAD. Both are synthetic TLR-4 agonists well known in the art. PHAD is also referred to in the art as glucopyranoside lipid A or GLA. Reference is made to Lousada-Dietrich et al., Vaccine. 2011 Apr 12;29(17):3284-92.

In a specific example, the RSV pseudoviral particles contain PHAD having the following structure (name 14 indicates the total number of carbon atoms in each thiol chain):

In a preferred embodiment, the pseudoviral particles contain 3-D-PHAD having the following structure:

Synthetic adjuvants The RSV-like viral particles (including the RSV-based 19F protein, as described herein) used in this example of the invention are preferably at a ratio of 0.01 to 2 mg per mg of viral protein, more preferably per mg of viral protein. The ratio of 0.5 to 2 mg, for example about 1 mg per mg of viral protein, is optionally between 3 and 6, preferably between 3.5 and 5, based on the molar ratio of the total synthetic phospholipid to the adjuvant.

In a particular embodiment, the RSV-like viral particles of this example of the invention (including the RSV-based 19F protein, as described herein) comprise from about 400 to 450 nmol DOPC, from about 800 to 900 nmol DOPE, from about 250 to 350 nmol 3-mg per mg of viral membrane protein. DPHAD and about 200 to 300 nmol of cholesterol.

Pharmaceutical composition and method

The invention also provides a pharmaceutical composition (eg, a vaccine) comprising one or more of the pseudoviral particles of the invention (including RSV line 19F protein, as described herein) and a pharmaceutically acceptable carrier, diluent, excipient, and / or adjuvant. A pharmaceutically acceptable tranquilizer, penetrant, buffer, dispersant or the like may also be included in the pharmaceutical composition of the present invention. The form of use depends on the purpose of the administration and therapeutic application. The pharmaceutical composition can include any compatible, non-toxic substance suitable for delivery of the virus-like particles to the patient.

As used herein, the term "medical acceptable" relates to compounds, components, materials, compositions, dosage forms, etc., which are suitable for use in questionable objects in the context of well-thought-out medical decisions. For example, humans have tissue exposure without excessive toxicity, irritation, allergic response, or other problematic complications, with a reasonable proportion of benefits/risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other components of the formulation. Suitable carriers, diluents, excipients, etc., as well as methods of use thereof, can be found in standard pharmaceutical documents, e.g., Remington's Pharmaceutical Science s, 18 th edition, Mack Pyblishing Company, Easton, PA, 1990; and Handbook of Pharmaceutical Excipients , 5 ^th edition, 2005, their respective entirety for all purposes by reference herein.

Formulations are selected based on or based in part on the route of delivery of the pharmaceutical composition. The pharmaceutical compositions of the present invention can be delivered by an acceptable route, for example, a mucosal route (eg, intranasal, intrapulmonary or intraoral) and a parenteral route (eg, by intramuscular, intravascular (eg, intravenous) Or intra-arterial) or intraperitoneal injection).

Formulations may be suitable for liquids, solutions (eg, aqueous, water-based), suspensions (eg, aqueous, non-aqueous), emulsions (eg, water-based, oil-in-water), elixirs, syrups, elixirs. , capsules (including, for example, hard and soft gelatin capsules), cachets, pills, ampoules, large pills, suppositories, uterine agents, tinctures, gels, patches, ointments, emulsions, lotions, oils, hair A foaming agent, spray or aerosol can be selected by those of ordinary skill in the art.

A formulation suitable for intranasal administration, the carrier of which is a liquid, can be administered in the form of intranasal spray, intranasal drip or nebulizer aerosol. The pharmaceutically acceptable carrier for intranasal delivery may, for example, be a powder containing Wu Jun painted prion particles, water, buffered physiological saline solution, glycerin, polysorbate 20, cremophor EL, octyl/anthracene glyceride, and may be buffered. To provide a neutral pH environment.

Formulations suitable for pulmonary administration (for example, by inhalation or inhalation therapy) include those in the form of aerosol sprays that are self-pressurized, using suitable propellants such as dichlorodifluoromethane, trichlorofluoromethane , dichloro-tetrafluoroethane, carbon dioxide or other suitable gas.

Formulations suitable for parenteral administration (for example, by intramuscular, intravascular or intraperitoneal injection), including aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (eg, solutions, suspensions) Liquid), wherein the pseudoviral particles are provided by suspension or other methods. The liquid may additionally contain other pharmaceutically acceptable components such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, Suspending agents, thickening agents, and blood (or other related bodily fluids) that impart a formulation to the intended recipient are isotonic solutes. Examples of the excipient include, for example, water, alcohols, polyols, glycerin, vegetable oil, and the like. Examples of suitable isotonic carriers for use in the formulation include sodium chloride injection, Ringer's solution or lactated Ringer's injection.

Formulations may be presented in single or multiple doses in sealed containers, such as ampoules and vials, and may be stored in lyophilized (lyophilized) conditions, and only need to be added with sterile liquid carriers immediately after use, for example, water for injection .

For oral administration, the pseudoviral particles can be administered in liquid dosage forms such as elixirs, syrups and suspensions. Liquid dosage forms for oral administration may contain excipients such as coloring agents and flavoring agents to increase patient acceptability.

As noted above, adjuvants may optionally be included in the membrane of the virus-like particles of the present invention. Further, the adjuvant may optionally be included in the pharmaceutical composition including the pseudo-viral particles, but on the outer side of the pseudo-viral particle film. Adjuvants for this purpose may be selected based on, for example, the route of administration to be used. Exemplary adjuvants include aluminum salts (eg, potassium aluminum sulfate, alum, aluminum phosphate, aluminum hydroxyphosphate, and aluminum hydroxide), immunostimulating matrices (eg, saponins containing liquid and cholesterol complexes), CpG oligos, 3D -MPL, MF-59, QS21, cell wall thiol dipeptide, MPLA and related forms and derivatives (see above) and polyphosphazine.

Suitable amounts of pseudoviral particles to be administered to an individual such as a human can be determined by one of ordinary skill in the art. Typically, about 1-100 μg of antigenic protein, for example 15-50 μg of antigenic protein, is administered in a single dose. Individuals such as human patients can be treated in a single dose or as needed Multi-dose treatment. In the case of multiple doses, a suitable course of treatment can be determined by one of ordinary skill in the art. In various examples, the course of treatment may include from 2 to 10 doses (eg, 3 to 4 doses), each being separated from each other for 1 to 4 days or 1 to 2 weeks, depending on the condition of the patient (eg, regardless of The patient has an existing RSV infection (and its severity) or the patient is treated prophylactically).

Individuals treatable in accordance with the methods of the present invention include human patients in need of such treatment as determined by those of ordinary skill in the art. Examples of such patients include (i) older patients, over about 65 years old, with or without potential heart or respiratory illnesses; (ii) patients, over about 50 years old, with potential heart or respiratory illnesses; (iii) women or pregnancy Prevention of infant infections 4 to 6 months after birth; (iv) infants less than 6 months; (v) infants of approximately 6 to 12 months; (vi) children of approximately 12 months to 5 years; (vii) Immunocompromised patients.

[Examples]

In the following examples, RSV L19 F with SEQ ID NO: 1 was used, with and without substitution mutations (I557V) as indicated.

Example 1: Animal test

This example describes the investigation of a virus-like particle vaccine derived from three different RSV strains: RSV-A2, RSV-A2 line 19F, and RSV-A2 line 19F I557V. The virus-like particle vaccine strains were all adjuvanted with 3-dedecyl-phosphonium hexamethylene disaccharide (3D PHAD). The virus-like particles are connected at intervals of 2 weeks. Comparison of vaccines in mice. Protection of vaccinated mice after challenge with RSV virus and immune response after vaccination (neutralizing antibody titer) were evaluated as compared to the negative control group (vehicle).

Method and material Preparation of virus-like particles

Tangential flow filtration (TFF) column (GE UFP-30-C-MMOA1, 26 cm2, 30 kDa cut-off), stored in 20% ethanol, prior to the use of sterile phosphate buffer solution for concentrated virus (PBS does not have Ca ²⁺ and Mg ²⁺ ) rinse.

28 vials (about 39 ml) of RSV-A2 were rapidly thawed, concentrated by TFF and diafiltered to a volume of 3.4 ml relative to 30 ml of phosphate buffered saline (PBS). 15 vials (about 22.5 ml) of RSV-A2 line 19F were similarly concentrated to 3.7 ml. 10 vials (about 15 ml) of RSV-A2 line 19F I557V were similarly concentrated to a volume of 3.2 ml. The protein concentration was 1.8 mg/ml (RSV-A2), 0.81 mg/ml (RSV-A2 line 19F) and 1.25 mg/ml (RSV-A2 line 19F I557V) according to Bradford. To make virus-like particles, 170 μl of DCPC solution was added to 1.7 ml of RSV-A2; 411 μl of DCPC solution was added to 3.7 ml of RSV-A2 line 19F; and 356 μl of DCPC solution was added to 3.2 ml of RSV-A2 line 19F I557V.

After 30 minutes on ice, the samples were spun at 40,000 rpm for 30 minutes on a tabletop S100 AT 4 rotor (Sorvall discovery M120-SE tabletop centrifuge). The supernatant was collected, filtered through a 0.1 μm Pall Acrodics filter, and observed to contain 2.3 mg/ml of RSV-A2; 0.57 mg/ml of RSV-A2 line 19F; and 0.83 mg/ml of RSV-A2 line 19F I557V.

By DOPC, DOPE, 3D PHAD and cholesterol (all from Avanti

The dry lipid membrane prepared from the mixture of Polar Lipids contained 425 nmol of DOPC, 850 nmol of DOPE, 300 nmol of 3D PHAD and 255 nmol of cholesterol per mg of viral supernatant protein. The membrane specifically contains a lipid as follows:

‧ Solution 1: For RSV-A2 supernatant: 995 nmol DOPC, 1989 nmol DOPE, 702 nmol 3D PHAD, 597 nmol cholesterol

‧ Solution 2: For the RSV-A2 line 19F supernatant: 850 nmol DOPC, 1700 nmol DOPE, 600 nmol 3D PHAD, 510 nmol cholesterol

‧ Solution 3: For the RSV-A2 line 19F I557V supernatant: 1063 nmol DOPC, 2125 nmol DOPE, 750 nmol 3D PHAD, 638 nmol cholesterol

The lipid film was evaporated on a glass tube wall by argon gas (chloroform/methanol) followed by a vacuum desiccator for 30 minutes.

Prepared by diluting a 500 mM stock of DCPC with HNE buffer (5 mM HEPES, 145 mM sodium chloride, 1 mM ethylenediamine-tetraacetic acid (EDTA), pH 7.4, containing 50 IU of penicillin and 50 μg/ml streptomycin). 100 mM DCPC solution. 200 μl of DCPC solution was added to each mash and the tube was immersed in hot tap water for 30 to 60 seconds. Some turbidity was observed in all solutions. After the virus supernatant was added, the solution was shaken, incubated on ice for 15 minutes, and filtered through a 0.22 μm filter of Whatman FP30. All solutions showed little turbidity before and after filtration. The solution was then injected into a gamma-ray sterile dialyzed slide-a-lyzer (Thermo, 10 kDa cut-off, size 0.5 to 3 ml) and dialyzed against 6 x 2 L PBS for 48 hours at 4 °C. All solutions were then collected after overnight dialysis against HNE buffer at 4 °C. Bradford Protein Assay (Biorad) 1.2 mg (in 1.63 ml) was produced for solution 1; 1.2 mg (in 3.66 ml) for solution 2; and 2.1 mg (in 4.6 ml) for solution 3. 2 x 75 μg from each solution was kept cold until the start of the animal test (4 to 10 ° C).

Particle size fractions were evaluated by a Nanosign® meter. All analyses were performed on samples stored at 4 to 10 ° C. For Nanosign® LM-10 instruments, 405 nm lasers, software version 3 (beta), shutter set to 1200 and gain to 500, threshold at 20 ° C "Automatic" is accomplished by Peliter cooling/heating regulation by single particle tracking. The imitation viral particle dilution was made in HNE buffer such that between 10 and 100 particles were visible in the observation chamber. Therefore, the absolute number of particles cannot be compared between solutions. Each sample accumulated 10 observations of 90 seconds, for a total of approximately 50,000 measurement trace lengths. The result is adjusted for the length of the cable trace. The sample stick holiday was set at 1.00 cP.

Samples of virus-like particles were analyzed by equilibrium density gradient centrifugation of a 10-60% sucrose gradient in HNE buffer. Samples were spun in a Sorvall AH650 rotor at 50,000 rpm for 64 hours and 30 minutes, and samples from the gradient were analyzed for protein (Bradford analysis; Bio-Rad), phosphate and density (by refractometer).

Test mobility, group assignment, dose level, and dosing

Six groups of female Balb/c mice (6 to 8 weeks old) received mock-like virus particles (5 μg protein per dose) composition and control composition on days 2 and 13. More specifically, the animals were anesthetized with 3 to 4.5% isoflurane/O ₂ for administration. The protein concentration was adjusted to 5 μg of IM injection per 50 μl with HNE. The pseudoviral particle composition and the control composition were administered to the two calf muscles in an IM injection at 25 μg per limb (total 5 μg of protein). Table 1 illustrates the treatment schedule.

Blood and serum samples

Sera from study animals were used to measure total immunoglobulin G (IgG), IgGl, IgG2a and viral microneutralization titers at different time points. The blood sample was collected from the animal to be terminated on the 0th, 13th, and 30th day before the vaccination by the submandibular bleeding or retro-orbital puncture for the neutralization antibody assay. And collect blood before the RSV challenge on the 31st. On the day of the end of the death (day 35), blood was collected from the posterior vena cava. The blood was allowed to solidify at room temperature during the test procedure and placed in the refrigerator. The coagulated blood samples were centrifuged and serum was taken from all samples and stored frozen at <-10 °C in polypropylene tubes.

Lung samples and virus titration

On day 35, lungs were harvested, processed, and counted from a total of 5 animals from each study group. The right lobe of the lung is used for the determination of the RSV virus titer. More specifically, the lungs harvested from animals sacrificed on day 35 were aseptically removed from the right lobe, and then the lung tissue was automated using a Potter homogenizer Polytron-Aggregate H (Thomas Scientific, Swedesboro, NJ, USA) homogenized in 1 ml of DMEM medium containing 2% fetal bovine serum (FBS). Lung homogenate was centrifuged at 1,400 rpm for 10 minutes at 4 °C, and the supernatant was diluted to a 1:5 starting dilution, which was used to determine viral titer using the TCID50 method.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 5.00 (GraphPad Software, San Diego, California, USA). Statistical significance was assessed using the Mann-Whitney U test. A P value of 0.05 or lower is considered to represent a statistically significant difference.

result Imitation virus particle size and size distribution

Figure 1 shows that the size fraction of the pseudoviral particles is between 0 and 400 nm. Table 2 summarizes the modal enthalpy, standard deviation and the percentage of particles >150 nm. The results of equilibrium density gradient centrifugation analysis of the pseudoviral particles are shown in Fig. 2.

Virus titer

Viral titers were determined by fractions from lung homogenate supernatants from 5 mice per group. Figure 3 shows the pneumovirus titer four days after the challenge (day 35). The titer is shown as the 50% endpoint titer as determined by TCID ₅₀ . Control of live virus challenge was observed for all virus-like particle vaccine formulations, while control mice were unprotected. The viral titers observed in the control group were significantly higher relative to the titers of all groups receiving the vaccine. There was no difference in the protection conferred by the virus-like particle vaccine in this study. Statistical analysis was performed using a two-sided Mann Whitney U test.

In vitro neutralization

Serum from each group of individual mice was analyzed by virus neutralization for anti-RSV antibodies. The neutralizing antibody titer (log ₂ ) 2 weeks after the first vaccination and 2 weeks after the subsequent vaccination are shown in Fig. 4 and Fig. 5, respectively. Statistical analysis was performed using a two-sided Mann Whitney U test.

Mice that received pseudoviral particles derived from RSV-A2 L 19F (p < 0.01) or RSV-A2 L 19F I557V (p < 0.001) were observed significantly compared to mice receiving RSV-A2 virus-like particles. The virus neutralized antibody titer (log ₂ ) 2 weeks after the initial vaccination. For RSV-A2 L 19F-like virus particles and RSV-A2 L 19F I557V-like virus particles, the average neutralizing titers were 2.5 ± 1.4 and 3.4 ± 1.6, respectively. For the imitation virus particles derived from RSV-A2 virus-like particles, the average titer was 0.32 ± 0.70. Serum from control mice did not show detectable neutralizing activity.

Two weeks after the second vaccination, all mice receiving the virus-like particle vaccine showed neutralizing antibody titers (log2). The titers were significantly higher (p < 0.001) in mice receiving virions derived from RSV-A2 L 19F or RSV-A2 L 19F I557V compared to all other groups. The mean neutralizing antibody titers for the pseudoviral particles derived from RSV-A2 L 19F and RSV-A2 L 19F I557V were similar (9.0 ± 0.6 and 9.19 ± 0.70, respectively). The average titer for RSV-A2 virus-like particles was 6.61 ± 0.86.

An increase in neutralizing antibody titers was observed in all vaccinated animals throughout the course of this four-week study. Although accepting RSV-A2 L 19F imitation The majority of animals with viral particles (8/10) or RSV-A2 L 19F I557V-like virus particles (9/10) showed an early increase of 2 weeks after the first vaccination, and animals receiving RSV-A2 virus-like particles ( 1/10) only a few have an early increase in neutralizing antibodies. Neutralizing antibodies were further increased in all animals after the second vaccination. For RSV-A2 L 19F-like virus particles, the increase after the second vaccination (effective price is log 2) is 2.5 ± 4.5 to 9.0 ± 0.6; and for RSV-A2 L 19F I557V virus-like particles is 3.4 ± 6.4 To 9.19 ± 0.71. For animals receiving RSV-A2 virus-like particles, the observed increase in neutralizing antibody titer was 0.54 ± 0.93 to 6.1 ± 0.9. At the second time point, 14 days after vaccination 1 and vaccination 2, the highest neutralizing antibody titer was observed for animals vaccinated with RSV-A2 L 19F and RSV-A2 L 19F I557V.

ELISA RSV-specific serum IgG

Sera from individual mice were analyzed for anti-RSV antibodies by ELISA 2 weeks after the second vaccination (30/31). The geometric mean titer (GMT) ± standard deviation (SD) for RSV-specific total IgG was obtained after the second vaccination. The statistics were finely divided using the two-sided Mann Whitney U test.

RSV-specific IgG titers were observed in all animals receiving the RSV vaccine, while control animals showed no RSV-specific IgG titers 2 weeks after the second vaccination (Figure 6). The titer (log 10) for the group receiving the virus-like particle vaccine ranged from 4.3 ± 0.79 to 5.48 ± 0.24. The highest and similar GMT were observed in animals receiving RSV-A2 L 19F-like virus particles and RSV-A2 L 19F I557V-like virus particles (5.32 ± 0.19 and 5.4 ± 0.24, respectively). The titers from the two groups were significantly higher than the titers of the group receiving the RSV-A2 virus-like particles.

RSV-specific IgG isotype response

The RSV-specific IgG isotype concentration was determined in each group of 10 mice 2 weeks after the second vaccination (day 30/31). The concentrations of RSV-specific IgG1 and IgG2a antibodies are shown in Figures 7 and 8, respectively, and are expressed as μg/ml serum. Statistical analysis was performed using a two-sided Mann Whitney U test.

Figure 7 shows that IgGl antibodies were increased 2 weeks after the second vaccination in all groups of the vaccinated vaccine compared to the control group. The average titer (μg/ml) for animals receiving RSV-A2 virus-like particles was 5.27 ± 3.86. The highest GMT was observed in animals vaccinated with RSV-A2 L 19F-like virus particles or RSV-A2 L 19F I557V-like virus particles, but there were large changes in the groups (11.08 ± 12.87 and 15.04 ± 12.00, respectively). Significant differences in IgGl titers were observed in animals receiving RSV-A2 L 19F I557V-like virus particles compared to animals receiving RSV-A2 virus-like particles (p < 0.05).

Figure 8 shows the IgG2a concentration 2 weeks after the second vaccination. All groups receiving the imitation virus particles showed an increased potency with a large change in the group, but the control mice showed no increase. The highest GMT (μg/ml) was observed in animals vaccinated with RSV-A2 L 19F-like virus particles (30.19 ± 13.85) or RSV-A2 L 19F I557V-like virus particles (28.64 ± 13.28). However, the range of titers in individual mice in the two groups was high (12.36 to 54.32 and 13.91 to 55.37, respectively, and the IgG2 titer for animals receiving RSV-A2 virus-like particles was 18.13 ± 9.96 for RSV-A2 L The 19F virus-like particle was 30.19±13.85, and for RSV-A2 L 19F I557V was 28.64±13.28).

Clinical Observation

Animals were weighed on days 0, 14, 30/31 and 35 (Figure 9). One in the day During the general health check and weighing period, no changes were observed even after the virus challenge. All mice from different groups gained weight during each period after the second vaccination. The minimum weight loss after challenge was observed in animals receiving the HNE control. No animal death or dying was studied during the study. All animals survive until the end of the step (30th or 35th).

in conclusion

The degree of virion derived from the L 19F A2 virus or the L 19F I557V A2 virus induces a higher degree of neutralizing antibody and ELISA serum IgG compared to the extent induced by the aviral particles derived from the A2 virus. No adverse effects were observed in general health and weight changes in any of the vaccinated groups in this study.

Example 2: RSV-F protein stability

As described above, Synagis® (Pacliizumab; Medimmune) is a humanized mouse monoclonal antibody that recognizes the conformation-independent epitope of the F protein presented in most RSV strains. 5C4 is a mouse monoclonal antibody that specifically recognizes the pre-fusion configuration of the RSV F protein. The palivizumab is used to quantify the F protein in intact virus, and the ratio of 5C4 to palivizumab is used to determine the relative concentration of F protein in urban pre-fusion configuration.

96-well ELISA plate (Greiner high binding) was coated overnight at 4 °C with a 1:100 dilution of rabbit-anti-RSV serum in coating buffer (0.2 M sodium bicarbonate/sodium carbonate, pH 9.6). . The plates were washed with a phytate buffered physiological saline solution (PBS, without Ca ²⁺ and Mg ²⁺ ) and at room temperature (RT) for 2% bovine serum albumin (BSA) in the coating buffer. After blocking for 1 hour, it was washed with PBS. Sequence 2 dilutions of purified viral stocks from RSV strains A2, 19F and I557V, or pseudoviral particles produced by such strains (preparations and compositions as disclosed in Example 1) In PBS), applied to the disks. After incubation for 1.5 hours at room temperature, the plates were washed with PBS and incubated with 100 μl of 4 μg/ml palivizumab or 4 μg/ml of 5C4 in PBS with 1% BSA for 1.5 hours at room temperature. The plates were washed with PBS and incubated with 1:2000 dilutions in antibody PBS coupled to horseradish peroxidase (HRP): goat-anti-human-HRP and goat for palivizumab and 5C4, respectively - Anti-mouse HRP (both from Bethyl Labs). After incubation at room temperature for 1 hour, the plates were washed again and developed with o-phenyl-diamine (OPD). After 30 minutes the reaction was stopped with H2SO4 and the absorbance at 492 nm was read on an ELISA reader. The plots of absorbance relative to viral protein concentration are shown as palivizumab (Figure 10) and 5C4 (Figure 11). The line indicates the semi-logarithm of the data point; for all matches, r2 is >0.95, except for I557V (0.92). For the C54 (Fig. 12) and palivizumab (Fig. 13), the relative virion particle protein concentration was plotted.

Table 3 shows the ratio of 5C4/palivizumab calculated by the slope of the curve, or the absorbance calculated by fitting a protein concentration of 50 μg/ml. These data show an increased stability of the pre-fusion form of the RSV F protein in the pseudoviral particles made from RSV I557V and RSV 19F. Repeated assays during the two-month course showed the stability of the 5C4 epitope of the pseudoviral particles.

Other specific examples

Although the present invention has been described in connection with the specific embodiments thereof, it is understood that the invention can be further modified and the present invention is intended to cover any variations, uses, or applications of the present invention. In general, the principles of the present invention and including such It will be appreciated by those of ordinary skill in the art that the present invention can be applied in accordance with the present invention.

All documents and patent applications mentioned in the specification are hereby incorporated herein by reference in their entirety as if individually individually individually individually individually individually individually individually individually .

As described herein, the present invention does not include such pseudoviral particles, compositions and related methods that do not include the RSV 19F protein (whether having the sequence of SEQ ID NO: 1, and having one or more substitution mutations relative to SEQ ID NO: 1. The sequence or sequence having the substantial sequence identical to SEQ ID NO: 1 (as one of the substitutions as needed), as described herein).

The symbols used in the text, such as "a" and "the", are not intended to exclude the plural. Similarly, the use of most words does not exclude the representation of a single form.

Other specific examples are included in the scope of the following patent application.

<110> RSV Corporation Emory University

<120> Viable virus particles containing respiratory fusion virus 19 fusion protein and use thereof

<130> 51011-003TW2

<150> US 62/049,705

<151> 2014-09-12

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 574

<212> PRT

<213> respiratory syncytial virus

<400> 1

<210> 2

<211> 298

<212> PRT

<213> respiratory syncytial virus

<400> 2

<210> 3

<211> 64

<212> PRT

<213> respiratory syncytial virus

<400> 3

<210> 4

<211> 256

<212> PRT

<213> respiratory syncytial virus

<400> 4

Claims (34)

A pseudoviral particle comprising a fusion (F) protein of the respiratory syncytial virus (RSV) line 19. For example, the pseudo-viral particles of claim 1 further comprise a G protein of RSV, a small hydrophobic (SH) protein or a matrix (M) protein. A virus-like particle according to claim 1 or 2, wherein the F protein comprises at least one substitution mutation. The pseudoviral particle of claim 3, wherein the substitution mutation comprises an amino acid substitution at I557. The virion particle of claim 4, wherein the substitution is an I557V amino acid substitution. The virion particle according to any one of claims 1 to 5, wherein the amino acid sequence of the F protein comprises one or more of the following amino acids: methionine at residue 79, The arginine at residue 191, the lysine at residue 357, and the tyrosine at residue 371. The imitation virus particle of claim 6, wherein the amino acid sequence of the F protein comprises two or more of the following amino acids: methionine at residue 79, and residue at residue 191 Aminic acid, an amide acid at residue 357, and tyrosine at residue 371. The imitation virus particle of claim 7, wherein the amino acid sequence of the F protein comprises three or more of the following amino acids: methionine at residue 79, and residue at residue 191 Aminic acid, an amide acid at residue 357, and tyrosine at residue 371. The virion particle according to any one of claims 1 to 8, wherein The amino acid sequence of the F protein comprises one or more of the following amino acids: proline at residue 4, alanine at residue 16, serine at residue 25, at residue 76. Proline, alanine at residue 103, threonine at residue 122, isoleucine at residue 152, arginine at residue 213, aspartate at residue 515 And glycine at residue 519. The pseudoviral particle of claim 9, wherein the amino acid sequence of the F protein comprises the following amino acid: proline at residue 4, alanine at residue 16, at residue 25 Serine, proline at residue 76, alanine at residue 103, threonine at residue 122, isoleucine at residue 152, arginine at residue 213, Aspartate at residue 515 and glycine at residue 519. The virus-like particle according to any one of claims 1 to 10, wherein the pseudo-viral particle comprises a lipid and a protein extracted from a membrane of the RSV strain. The virus-like particle of claim 11, wherein the RSV strain from which the lipid and the protein are extracted is a chimeric RSV strain comprising an RSV strain in which the F protein is replaced with an RSV-based 19F protein. The virus-like particle according to claim 11, wherein the RSV strain from which the lipid and the protein are extracted is a chimeric RSV strain comprising the RSV A2 strain in which the A2 F protein is replaced with the RSV-based 19F protein. The virion particle according to any one of claims 1 to 13, wherein the F protein is substantially a pre-fusion conformation. The virus-like particle according to any one of claims 1 to 14, wherein the pseudoviral particle has a large proportion of 5C4/palivizumab The ratio of 5C4/palilizumab to the pseudoviral particles produced from the RSV A2 strain was at least 20%, and the ratio of 5C4/palivizumab was calculated by dividing the slope of the curve. The virion particle according to any one of claims 1 to 15, wherein the F protein comprises at least one substitution mutation selected from the group consisting of M79I, R191K, K357T and Y371N. The virus-like particle according to any one of claims 1 to 16, further comprising an adjuvant. The virus-like particle of claim 17, wherein the adjuvant is selected from the group consisting of saponin, PHAD (phosphorylated hexavalent disaccharide), 3-D-PHAD (phosphorylated hexamethylene) 3-O-dethiol derivative of disaccharide), 3-OD MPLA (3-O-descapto derivative of mono-o-decyl lipid A) and MPLA (mono-o-decyl lipid A). The pseudoviral particles according to any one of claims 1 to 18, further comprising phospholipid choline (PC), phospholipid oxime ethanol (PE), and/or sterol or sterol derivatives. The virion particle according to any one of claims 1, 2, 6 to 15, or 17 to 19, wherein the F protein comprises the sequence of SEQ ID NO: 1. The virion particle according to any one of claims 1 to 15 or 17 to 19, wherein the F protein comprises a sequence having the substitution of SEQ ID NO: 1 of 1557. The imitation virus particle of claim 21, wherein the substitution at I557 is I557V. A pharmaceutical composition comprising as claimed in claims 1 to 22 A pseudoviral particle, and a pharmaceutically acceptable carrier, diluent, excipient, and/or adjuvant. A method of inducing an immune response to an RSV in an individual, the method comprising administering to the individual a pharmaceutical composition as claimed in claim 23 of the patent application. The method of claim 24, wherein the individual is not infected with RSV but is at risk of developing RSV infection. A method of reducing infection and/or replication of RSV in an individual, the method comprising administering to the individual a pharmaceutical composition as claimed in claim 23 of the patent. The method of any one of claims 24 to 26, wherein the individual is a human individual. A method of producing a virus-like particle according to any one of claims 1 to 22, which comprises (i) dissolving a viral outer membrane of an RSV strain comprising an F protein of RSV line 19, and (ii) In the absence of viral nucleic acid, the outer membrane of the virus is further composed. The method of claim 28, wherein the lysed outer membrane of the virus is a viral outer membrane of a chimeric RSV comprising an RSV strain A2 in which the A2F protein is replaced with an RSV-based 19F protein. The method of claim 28, wherein the RSV 19F protein comprises an I557V amino acid substitution. A use of the pseudoviral particles of any one of claims 1 to 22, which is used as a medicine. Use of a virus-like particle according to any one of claims 1 to 22 for inducing an immune response against RSV. Use of a virus-like particle according to any one of claims 1 to 22 It is a method for preventing or treating RSV infection. Use of a virus-like particle according to any one of claims 1 to 22 for use in an individual vaccination against RSV.