Composition suitable for use as RS virus vaccine

Abstract

To provide a composition suitable for use as an RS virus vaccine that achieves enhanced efficacy and safety.SOLUTION: A composition comprising a G protein of an RS virus and a CpG oligodeoxynucleotide, wherein the G protein lacks a modified glycan of a mammalian cell expression type.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition suitable for use as an RS virus vaccine. [Background technology]

[0002] Respiratory syncytial virus (RSV) (scientific name: human orthopneumovirus) (hereafter referred to as RSV) is an enveloped RNA virus classified in the genus Pneumovirus of the family Paramyxovirus. It is spherical or filamentous, with a diameter of 80–350 nm. RSV causes lifelong overt infection in people of all ages, but it is a particularly important pathogen in infancy, causing the most severe symptoms in the first few weeks to months of life despite the presence of maternal antibodies. Low birth weight infants, those with underlying cardiopulmonary disease, or immunodeficiency are at higher risk of developing severe symptoms, making it a significant clinical and public health impact.

[0003] Treatment primarily consists of supportive care, such as oxygen administration, fluid infusion, and respiratory management. Efforts to develop a preventive vaccine have been ongoing for the past 30 years, but past inactivated vaccines have resulted in adverse events in which vaccinated individuals became more seriously ill than unvaccinated individuals, and research is still ongoing. Currently available preventive methods include human serum-derived anti-RSV immunoglobulins and palivizumab (genetic recombinant), a humanized monoclonal antibody preparation created using genetic engineering technology against the F (fusion) protein, one of the RSV surface proteins. Currently, there is no licensed RSV vaccine, and the development of a safe and effective RSV vaccine is needed.

[0004] The RSV G protein is localized on the surface of the RSV envelope and plays an important role in the virus's entry into host cells. The G protein is important for infection, and vaccines using polypeptides derived from the G protein as vaccine antigens have been developed (Patent Document 2, Non-Patent Document 1, Non-Patent Document 2). However, in a study using mice, vaccination with recombinant G protein (mG) produced in mammalian cells induced eosinophilia (vaccine-associated disease exacerbation) in the lungs after RSV infection. This raised safety concerns, and development of vaccines targeting the G protein has not progressed. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6162751 [Patent Document 2] Patent No. 4310184 [Non-patent literature]

[0006] [Non-Patent Document 1] The Journal of Infectious Diseases 2001;184:1456-60 [Non-patent document 2] The Journal of Infectious Diseases 1997;176:560-9 Summary of the Invention [Problem to be solved by the invention]

[0007] An objective of the present invention is to provide a composition that is highly effective and safe and is suitable for use as an RS virus vaccine. [Means for solving the problem]

[0008] The present inventors have conducted extensive research in light of the above-mentioned problems and have found that the above-mentioned problems can be solved by a composition containing a G protein of RS virus and a CpG oligodeoxynucleotide, wherein the G protein does not have modified sugar chains expressed in mammalian cells. Based on this finding, the present inventors have conducted further research and have completed the present invention. Specifically, the present invention encompasses the following aspects.

[0009] Item 1. A composition comprising a G protein of RS virus and a CpG oligodeoxynucleotide, wherein the G protein does not have modified sugar chains expressed in mammalian cells.

[0010] Item 2. The composition according to Item 1, wherein the G protein is a non-glycoprotein.

[0011] Item 3. The composition according to Item 1, wherein the G protein is an intracellular domain-deleted G protein.

[0012] Item 4. The composition according to Item 1, wherein the content of the CpG oligodeoxynucleotide is 1 to 200 parts by mass per part by mass of the G protein.

[0013] Item 5. The composition according to any one of Items 1 to 4, which is in a liquid or solid form.

[0014] Item 6. The composition according to any one of Items 1 to 4, which is an RS virus vaccine.

[0015] Item 7. A respiratory syncytial virus vaccine comprising a G protein of an respiratory syncytial virus and a CpG oligodeoxynucleotide, wherein the G protein does not have a modified sugar chain expressed in mammalian cells, and the G protein and the CpG oligodeoxynucleotide are contained in separate containers.

[0016] Item 8. The RS virus vaccine according to Item 7, which is used by administering to a subject a mixture obtained by mixing the G protein and the CpG oligodeoxynucleotide. [Effects of the Invention]

[0017] According to the present invention, it is possible to provide a composition that is highly effective and safe and is suitable for use as an RS virus vaccine. [Brief explanation of the drawings]

[0018] [Figure 1] The SDS-PAGE results of Test Example 1 are shown. Lane 1 is the recombinant G protein (mG) purified using mammalian cells, and lane 2 is the recombinant G protein (eG) purified using E. coli. [Figure 2] The figures show the measurement results for total IgG, IgG1, IgG2a, and IgG2b in Test Example 2. A shows the case where mG was administered alone or in combination with an adjuvant, and B shows the case where eG was administered alone or in combination with an adjuvant. On the horizontal axis, PBS indicates the case where neither antigen nor adjuvant was administered. The legend indicates the dilution factor in the ELISA measurement. p values ​​between groups at both ends of the bars are indicated by * if they are less than 0.05, ** if they are less than 0.01, *** if they are less than 0.001, and **** if they are less than 0.0001. [Figure 3] The results of measuring the RSV N copy number in Test Example 3 are shown. A and B show the results when mG was administered alone or in combination with an adjuvant, and C and D show the results when eG was administered alone or in combination with an adjuvant. A and C show the results of measuring the RSV N copy number in the lungs, and B and D show the results of measuring the RSV N copy number in the nasal turbinates. On the horizontal axis, PBS indicates the case where neither antigen nor adjuvant was administered. P values ​​between the groups at both ends of the bars are less than 0.05, less than 0.01, less than 0.001, less than ***, and less than 0.0001, respectively. [Figure 4] The results of cell marker measurement in Test Example 4 are shown. A indicates the case where mG was administered alone or in combination with an adjuvant, and B indicates the case where eG was administered alone or in combination with an adjuvant. On the horizontal axis, PBS indicates the case where neither antigen nor adjuvant was administered. p values ​​between the groups at both ends of the bar are indicated by * if they are less than 0.05, ** if they are less than 0.01, *** if they are less than 0.001, and **** if they are less than 0.0001. [Figure 5] The figures show the results of evaluating pulmonary inflammation caused by RSV infection in Test Example 5. A and B show the results when mG was administered alone or in combination with an adjuvant, and C and D show the results when eG was administered alone or in combination with an adjuvant. A and C show the results of measuring lung weight, and B and D show the results of measuring the number of specific cells. On the horizontal axis, PBS indicates the case where neither antigen nor adjuvant was administered. p values ​​between groups at both ends of the bars are indicated by * if they are less than 0.05, ** if they are less than 0.01, *** if they are less than 0.001, and **** if they are less than 0.0001. [Figure 6] The results of the RSV N copy number measurement in Test Example 6 are shown. On the horizontal axis, PBS indicates the case where neither antigen nor adjuvant was administered. P values ​​between the groups at both ends of the bar are indicated by * if they are less than 0.05, ** if they are less than 0.01, *** if they are less than 0.001, and **** if they are less than 0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0019] 1.Definition In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0020] As used herein, the "identity" of an amino acid sequence refers to the degree of correspondence between two or more comparable amino acid sequences. Therefore, the greater the identity between two amino acid sequences, the greater the identity or similarity between those sequences. The level of identity between amino acid sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes," Proc. Natl. Acad. Sci. USA. 87:2264-2268 (1990); Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc. Natl. Acad. Sci. USA. 90:5873-7 (1993)). A program called BLASTX, based on the BLAST algorithm, has been developed. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of nucleotide sequences is also defined in the same manner as above.

[0021] As used herein, "conservative substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine constitutes a conservative substitution. Other examples of conservative substitution include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0022] 2. Composition In one aspect, the present invention relates to a composition (sometimes referred to herein as the "composition of the present invention") containing a G protein of respiratory syncytial virus and a CpG oligodeoxynucleotide, wherein the G protein does not have modified sugar chains expressed in mammalian cells. This is described below.

[0023] Respiratory syncytial virus (RSV) (scientific name: human orthopneumovirus) is an enveloped RNA virus. The surface of the RSV envelope contains G and F proteins, which are involved in entry into host cells.

[0024] The G protein is not particularly limited as long as it can be used as an antigen in an RS virus vaccine, and includes both full-length G proteins and deleted G proteins (wherein a portion of the full-length G protein has been deleted). G proteins are viral surface proteins, and the extracellular domain is used as the antigen. For this reason, intracellular domain-deleted G proteins (wherein the intracellular domain of the full-length G protein has been deleted) are preferred.

[0025] Examples of full-length G proteins include: (a) a protein comprising an amino acid sequence shown in any one of SEQ ID NOs: 1 to 7, or (b) a protein having 70% or more identity to the amino acid sequence shown in any one of SEQ ID NOs: 1 to 7; Examples include:

[0026] SEQ ID NOs: 1 to 6 are the amino acid sequences of the G protein derived from respiratory syncytial virus A strain (strains A2, Long, rsb1734, rsb6190, rsb5857, and rsb6256, in order from SEQ ID NO: 1), and SEQ ID NO: 7 is the amino acid sequence of the G protein derived from respiratory syncytial virus B strain (strain B1). Table 1 shows the comparison results of the amino acid sequences of SEQ ID NOs: 1 and 7, and Table 2 shows the comparison results of the amino acid sequences of SEQ ID NOs: 1 to 6. In the tables, an "*" indicates that the amino acid between the compared subjects is identical. The amino acid identity of the CCD (central conserved domain) in Table 1 (the underlined portion in Table 1) to the highly CCD is 100%. The amino acid identity of the CCD (central conserved domain) in Table 2 (the underlined portion in Table 2) is approximately 95%, and the amino acid identity of the highly CCD is 100%.

[0027] [Table 1]

[0028] [Table 2]

[0029] Examples of intracellular domain-deleted G proteins include: (c) a protein comprising an amino acid sequence shown in any one of SEQ ID NOs: 8 to 14, or (d) a protein having 70% or more identity to the amino acid sequence shown in any one of SEQ ID NOs: 8 to 14; Examples include:

[0030] SEQ ID NOs: 8 to 14 are the sequences of intracellular domain-deleted G proteins derived from SEQ ID NOs: 1 to 7, respectively.

[0031] In the above (b) and (d), the identity is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 99% or more. The number of mutated amino acids in the above (b) and (d) is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 2.

[0032] In (b) and (d) above, the amino acid identity of the CCD (central conserved domain) is preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more, from the viewpoints of efficacy, safety, etc. of the composition of the present invention as a vaccine. Furthermore, in (b) and (d) above, the number of mutated amino acids in the CCD (central conserved domain) is preferably 1 to 4, more preferably 1 to 2, and even more preferably 1, from the viewpoints of efficacy, safety, etc. of the composition of the present invention as a vaccine.

[0033] In the above (b) and (d), the amino acid identity of the Highly CCD is preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more, from the viewpoints of efficacy, safety, etc. of the composition of the present invention as a vaccine. Furthermore, in the above (b) and (d), the number of mutated amino acids in the Highly CCD is preferably 1 to 4, more preferably 1 to 2, and even more preferably 1, from the viewpoints of efficacy, safety, etc. of the composition of the present invention as a vaccine.

[0034] The G proteins used in the compositions of the present invention do not have modified sugar chains expressed in mammalian cells.

[0035] The modified sugar chains expressed in mammalian cells are sugar chains that are modified when a G protein is expressed in mammalian cells, and are not particularly limited thereto. A G protein with modified sugar chains expressed in mammalian cells has a high molecular weight due to the modified sugar chains. From this perspective, the molecular weight of a G protein with no modified sugar chains expressed in mammalian cells is, for example, 50 kDa or less, preferably 40 kDa or less, and more preferably 35 kDa or less. The molecular weight is, for example, 25 kDa or more, preferably 30 kDa or more. The molecular weight can be measured by comparing with molecular weight markers on SDS-PAGE.

[0036] The G protein can be one that has been expressed and purified in cells other than mammalian cells that have a glycosylation mechanism (e.g., insect cells or yeast), or one that has been obtained by a method that does not utilize the glycosylation mechanism (e.g., one that has been expressed and purified in bacteria such as Escherichia coli, or one that has been synthesized in vitro). From the viewpoints of the efficacy and safety of the composition of the present invention as a vaccine, it is particularly preferred that the G protein is not glycosylated, i.e., a non-glycoprotein. From the same viewpoint, it is particularly preferred that the G protein is expressed in bacteria (preferably Escherichia coli). By using these, the efficacy and safety of the composition of the present invention as a vaccine can be further improved.

[0037] The G protein may contain other amino acid sequences (for example, tag sequences such as a histidine tag, an HA tag, or a FLAG tag) as long as the activity as an RS virus vaccine antigen is not significantly impaired.

[0038] The G protein may be chemically modified as long as its performance as an RS virus vaccine antigen is not significantly impaired.

[0039] G proteins have a C-terminal carboxyl group (-COOH) and a carboxylate (-COO - ), amide (-CONH2) or ester (-COOR).

[0040] Here, R in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1-6 Alkyl groups; for example, C groups such as cyclopentyl and cyclohexyl 3-8 Cycloalkyl groups such as phenyl and α-naphthyl 6-12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1-2 Alkyl groups; α-naphthyl-C such as α-naphthylmethyl 1-2 C such as alkyl group 7-14 Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0041] The G protein may be amidated or esterified at a carboxyl group (or carboxylate) other than the C-terminus. In this case, the ester used may be, for example, the C-terminal ester described above.

[0042] Furthermore, in G proteins, the amino group of the N-terminal amino acid residue is protected by a protecting group (e.g., a formyl group, an acetyl group, or the like). 1-6 C such as alkanoyl 1-6those in which the N-terminal glutamine residue that can be generated by cleavage in vivo is pyroglutamated; those in which the substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected by an appropriate protecting group (e.g., C group such as formyl group, acetyl group, etc.); 1-6 C such as alkanoyl group 1-6 Also included are those protected with an alkyl group (e.g., acyl group).

[0043] The G protein may be in the form of a pharmaceutically acceptable salt with an acid or base. The salt is not particularly limited as long as it is pharmaceutically acceptable, and both acidic and basic salts can be used. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

[0044] The G protein may be in the form of a solvate. The solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include water, ethanol, glycerol, and acetic acid.

[0045] The G protein may be one type alone or a combination of two or more types.

[0046] G proteins can be easily produced using known genetic engineering techniques depending on their amino acid sequences, such as PCR, restriction enzyme digestion, DNA ligation, in vitro transcription / translation, and recombinant protein production techniques.

[0047] The G protein may be purified after synthesis. For example, the G protein is extracted from bacterial cells collected by centrifugation, filtration, or the like from the culture. The first step of extraction involves disrupting the bacterial cells, which can be achieved by enzymatic digestion, osmotic disruption, rapid pressure / pressure / vacuum application, ultrasound, various homogenizers, and the like. The disrupted bacterial cells can then be fractionated using a combination of physical methods such as low-speed centrifugation, ultracentrifugation, filtration, molecular sieving, and membrane concentration; chemical methods such as the use of precipitants, solubilizers, adsorbents, and dispersants; and physicochemical methods such as electrophoresis, column chromatography, support, dialysis, and salting out. When applying these methods, physicochemical conditions such as temperature, pressure, pH, and ionic strength can be appropriately adjusted.

[0048] CpG oligodeoxynucleotides (CpG-ODNs) are not particularly limited as long as they are single-stranded oligodeoxynucleotides containing an unmethylated cytosine-guanine dinucleotide (5'-CpG-3') motif (CpG motif). CpG oligodeoxynucleotides are known to be useful as vaccine adjuvants because they induce adaptive immune responses via TLR (Toll-like receptor). CpG oligodeoxynucleotides can contain at least one CpG motif, or can contain multiple CpG motifs.

[0049] The number of nucleotides constituting the CpG oligodeoxynucleotide is not particularly limited, but is, for example, 8 to 50 bases, preferably 8 to 40 bases, more preferably 8 to 30 bases, even more preferably 10 to 25 bases, still more preferably 15 to 25 bases, and particularly preferably 18 to 25 bases.

[0050] CpG oligodeoxynucleotides are classified into class A (D type), class B (K type), class C, class P, and class S based on sequence, secondary structure, effect on human peripheral blood mononuclear cells (PBMC), etc. Among these, from the viewpoint of efficacy, safety, etc. of the composition of the present invention as a vaccine, preferred CpG oligodeoxynucleotides include class B (K type) CpG oligodeoxynucleotides.

[0051] The internucleotide linkages of CpG oligodeoxynucleotides can be phosphodiester or phosphorothioate. Phosphorothioate bonds can improve nuclease resistance. Class B CpG oligodeoxynucleotides usually have a linear structure with a phosphorothioate backbone and typically do not form higher-order structures. Class A CpG oligodeoxynucleotides usually have a central phosphodiester bond and poly-G motifs at both ends form a higher-order structure called a G-tetrad.

[0052] Specific examples of CpG oligodeoxynucleotides are as follows: Class A: D35-CpG, ODN1585, ODN2216, ODN2336, etc.; Class B: K3-CpG, ODNBW006, ODN D-SL01, ODN1668, ODN1826, ODN2006 (CpG7909, PF-3512676), ODN2007, ODN684, etc.; Class C: ODN D-SL03, ODN 2395, ODN M362, etc.

[0053] Other examples include CpG-28, CpG-685 (GNKG-168), CpG-ODN C274, KSK-13 (KSK-CpG), CpG ODN 10104 (CpG-10104), CpG ODN-1585, ODN-5890, 1018-ISS, EMD-1201081 (HYB-2055, IMO-2055), and the like.

[0054] As the CpG oligodeoxynucleotide, commercially available products can be used, or those obtained according to known production methods can be used.

[0055] The CpG oligodeoxynucleotide may be one type alone or a combination of two or more types.

[0056] The content of CpG oligodeoxynucleotide is, for example, 1 to 200 parts by mass, preferably 2 to 150 parts by mass, and more preferably 5 to 100 parts by mass relative to 1 part by mass of G protein.

[0057] The compositions of the present invention may or may not contain adjuvants other than CpG oligodeoxynucleotides, such as alum (aluminum compounds such as aluminum hydroxide gel), mineral oil, vegetable oil, alum, bentonite, silica, muramyl dipeptide derivatives, thymosin, and interleukin.

[0058] In one embodiment, the composition of the present invention preferably contains a small amount of other adjuvants other than CpG oligodeoxynucleotides, for example, 0.1 parts by mass or less, preferably 0.01 parts by mass or less, more preferably 0.001 parts by mass or less, and even more preferably 0 part by mass, per part by mass of CpG oligodeoxynucleotides.

[0059] In another embodiment, the composition of the present invention is preferably used in combination with a CpG oligodeoxynucleotide and another adjuvant, in which case the content of the other adjuvant is, for example, 1 to 200 parts by mass per part by mass of the CpG oligodeoxynucleotide.

[0060] In addition to the above, the composition of the present invention may contain a base, carrier, solvent, dispersant, emulsifier, buffer, stabilizer, excipient, binder, disintegrant, lubricant, thickener, moisturizer, colorant, fragrance, chelating agent, etc.

[0061] The form of the composition of the present invention is not particularly limited, and may be, for example, liquid or solid.

[0062] The composition of the present invention is suitable for use as an RS virus vaccine, and can prevent the onset of RS virus infection and suppress symptoms (e.g., reduce the likelihood of aggravation and death) once RS virus infection has occurred.

[0063] The target organisms for the compositions of the present invention are not particularly limited as long as they are organisms that can be infected with RS virus. Examples of such organisms include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits. Of these, humans are preferred.

[0064] The dosage form of the composition of the present invention is not particularly limited, and examples thereof include injectable preparations such as aqueous injectable preparations, non-aqueous injectable preparations, suspension injectable preparations, and solid injectable preparations; oral preparations such as tablets, capsules, granules, powders, fine granules, syrups, enteric-coated preparations, sustained-release capsules, chewable tablets, drops, pills, oral liquid preparations, confectionery tablets, sustained-release preparations, and sustained-release granules; and external preparations such as nasal drops, inhalants, rectal suppositories, inserts, enemas, and jellies.

[0065] The content of G protein (antigen) in the composition of the present invention depends on the subject of administration, the administration route, the dosage form, the condition of the patient, the judgment of the physician, etc., and is not limited, but can be, for example, 0.0001 to 95% by weight, preferably 0.001 to 50% by weight.

[0066] The amount of the composition of the present invention to be used can be determined by a clinician based on various factors, such as the route of administration, the subject's health condition, the subject's age, sex, and body weight, pharmacological knowledge such as pharmacokinetics and toxicological characteristics, whether a drug delivery system is used, and whether the composition is administered as part of a combination of other drugs. The composition of the present invention is not particularly limited, but is preferably used so that the antigen dose per administration is, for example, 1 μg to 10 mg / kg (body weight), or 10 to 1000 μg / kg (body weight). The interval and number of administrations are not particularly limited, but are preferably administered 1 to 5 times, for example, at intervals of about 1 to 8 weeks.

[0067] The present invention also relates to an RS virus vaccine comprising a G protein of RS virus and a CpG oligodeoxynucleotide, wherein the G protein does not have a modified sugar chain expressed in mammalian cells, and the G protein and the CpG oligodeoxynucleotide are contained in separate containers.

[0068] The vaccine comprises at least two containers, namely, a container containing a G protein and a container containing a CpG oligodeoxynucleotide, and can be used by mixing the G protein and the CpG oligodeoxynucleotide to administer the mixture to a subject. [Example]

[0069] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0070] Test Example 1. Purification of mG and eG proteins The extracellular domain of the G protein, a vaccine antigen, was produced using mammalian or E. coli and purified. The amino acid sequence of the G protein (SEQ ID NO: 1) was derived from the HRSV-A2 strain (UniProt: P03423). Specifically, the procedure was as follows.

[0071] <Recombinant G protein (mG) purified using mammalian cells> The cDNA encoding the extracellular domain of G protein (amino acids 67-298: SEQ ID NO: 8) containing an N-terminal Igκ signal sequence and a 6x histidine tag was optimized for human codons. mG was expressed using the Expi293™ Expression System (Thermo Fisher Scientific) and purified using an AKTA® explorer chromatography system with a Ni-Sepharose® HisTrap® FF column (GE Healthcare) followed by a Superose® 6 Increase 10 / 300 GL column (GE Healthcare).

[0072] <Recombinant G protein (eG) purified using E. coli> The cDNA encoding the extracellular domain of G protein (amino acids 67-298) with an N-terminal hexa-histidine tag was optimized for E. coli codons. The resulting vector was transfected into BL21(DE3) Competent E. coli (NEB). E. coli cells were harvested by centrifugation at 8,000 × g for 10 minutes at 4°C and resuspended in buffer (20 mM NaH2PO4, 20 mM Na2HPO4, 0.5 M NaCl, 20 mM imidazole). After sonication, the soluble fraction was collected by centrifugation at 8,000 × g for 60 minutes at 4°C. eG was purified using a column in the same manner as mG. Endotoxin was then removed using EndoTrap™ HD 5 / 1 (BioVendor Laboratory Medicine).

[0073] G proteins are known to undergo extensive glycosylation. eG, which is not glycosylated, was identified at a position of approximately 30 kDa predicted from the amino acid sequence (Fig. 1). On the other hand, mG was identified at a position of approximately 90 kDa due to glycosylation.

[0074] Test Example 2. Antibody production by adding adjuvants to mG and eG vaccines The protein obtained in Test Example 1 was mixed with an adjuvant and used as a vaccine in mice to compare the ability to induce mG-specific IgG antibodies. Specifically, the procedure was as follows. The adjuvants used were Alum (aluminum hydroxide), CpG-ODN (B-class CpG-ODN K3), and AddaVax™ (a squalene-based oil-in-water nanoemulsion).

[0075] 1 μg of antigen ((A) mG, (B) eG) alone or 1 μg of antigen mixed with 50 μg Alum or 10 μg CpG-ODN (B-class CpG-ODN K3) and 50% v / v AddaVax in 50 μL of PBS was subcutaneously injected into the base of the tail of 6-week-old female BALB / c mice on days 0 and 21. Plasma was collected on day 28, and total IgG, IgG1, IgG2a, and IgG2b were measured by ELISA.

[0076] As a result, the mG vaccine showed increased mG-specific IgG antibody production in the Alum or AddaVax-added groups compared to the mG-only group (Figure 2A).On the other hand, the eG vaccine showed increased mG-specific IgG antibody production in the eG-only group compared to the mG-only group, and furthermore, the CpG-ODN and AddaVax-added groups showed significantly increased mG-specific IgG antibody production compared to the eG-only group (Figure 2B).

[0077] Test Example 3. Protective effect of mG and eG vaccines with adjuvants The protective effects of the mG and eG vaccines on infection were compared as follows.

[0078] Mice vaccinated with mG (A, B) or eG (C, D) (similar to Test Example 2) were given 1×10 5pfu RSV (in 30 μL PBS) was administered intranasally. Five days after infection, the right lung (A, C) and nasal turbinates (B, D) were collected, and RNA was extracted using TRIzol™ Reagent (Thermo Fisher Scientific). Reverse transcription was performed using ReverTra Ace™ qPCR RT Master Mix (TOYOBO). Real-time quantitative PCR was performed using a LightCycler™ 480 SYBR Green I Master (Roche) to measure the expression level of the N protein of the HRSV-A2 strain. For a standard curve, the N protein gene was amplified by PCR using cDNA synthesized by reverse transcription from RSV-A2 mRNA as a template, and cloned into the pcDNA3.1 vector (Thermo Fisher Scientific). The copy number of RSV N was calculated using this.

[0079] The mG vaccine significantly reduced viral load in the lower respiratory tract in the Alum- or AddaVax-added groups compared with the unvaccinated group (Figure 3A). Furthermore, all adjuvanted groups showed a slight protective effect against upper respiratory tract infection after the mG vaccine (Figure 3B). On the other hand, the eG vaccine significantly protected both the upper and lower respiratory tract in all adjuvanted groups (Figures 3C and 3D).

[0080] Test Example 4. T cell responses with adjuvant-added mG and eG vaccines To evaluate the safety of the mG and eG vaccines, we analyzed the inflammatory T cells induced after vaccination. Specifically, we performed the following procedure.

[0081] Spleens were harvested on day 28 from mice vaccinated with mG (A) or eG (B) (as in Experimental Example 2). Spleen cells were purified and cultured at 37°C for 3 days in the presence of 10 μg / mL mG. After culture, cells were surface stained with anti-mouse CD16 / CD32 antibody (clone: ​​93; BioLegend), APC anti-mouse CD3 antibody (clone: ​​145-2C11; BioLegend), Alexa Fluor® 700 anti-mouse CD4 antibody (clone: ​​GK1.5; BioLegend), Brilliant Violet 510™ anti-mouse / human CD44 antibody (clone: ​​IM7; BioLegend), and eBioscience™ Fixable Viability Dye eFluor™ 780 (Thermo Fisher Scientific). For intracellular staining, cells were fixed and permeabilized using the BD Cytofix / Cytoperm® Fixation / Permeablization Kit (BD) and then labeled with Brilliant Violet 605™ anti-mouse IFN-γ antibody (clone: ​​XMG1.2; BioLegend), PE anti-mouse / human IL-5 antibody (clone: ​​TRFK5; BioLegend), and PE-Cyanine7 IL-13 monoclonal antibody (clone: ​​eBio13A; Thermo Fisher Scientific). Flow cytometry analysis was performed using an Attune NxT Flow Cytometer (Thermo Fisher Scientific) and Flowjo™ software (TreeStar).

[0082] The mG vaccine is known to induce Th2 cells, which are involved in the exacerbation of airway inflammation. Restimulation of spleen cells after vaccination showed that the mG vaccine significantly increased the number of Th2 cells, IL-5 and IL-13-producing CD4 T cells, in the mG-only group compared to the non-vaccine group. +However, the CpG-ODN-added mG vaccine suppressed the induction of Th2 cells and suppressed the induction of Th1 cells, IFN-γ-producing CD4 T cells (Figure 4A). + Similarly, in the eG vaccine, Th2 cells were induced in the eG alone group and the Alum-added group compared to the non-vaccine group, while Th1 cells were significantly induced in the CpG-ODN-added group (Figure 4B).

[0083] Test Case 5. Airway inflammation caused by adjuvanting of mG and eG vaccines We evaluated lung inflammation caused by RSV infection after mG and eG vaccination as follows.

[0084] Lungs were harvested 5 days after infection from mice vaccinated with mG (A, B) or eG (C, D) (as in Example 3). (A, C) The right lung was weighed. (B, D) The left lung was shaken for 1 hour at 37°C in the presence of 100 U / mL Deoxyribonuclease 1 (Wako) and 200 U / mL Collagenase Type IV (Thermo Fisher Scientific). Single-cell suspensions were prepared using gentleMACS™ Tubes (Miltenyi Biotec) and a gentleMACS Dissociator (Miltenyi Biotec), followed by hemolysis in ACK buffer (8.3 g / L NH4Cl, 0.01 M Tris-HCl, pH 7.5).These cells were treated with anti-mouse CD16 / CD32 antibody (clone: ​​93; BioLegend), FITC anti-mouse Ly-6G Antibody (clone: ​​1A8; BioLegend), PerCP / Cyanine5.5 anti-mouse CD11c Antibody (clone: ​​N418; BioLegend), APC-R700 Rat Anti-Mouse Siglec-F (clone: E50-2440; BD), Alexa Fluor 700 anti-mouse CD4 Antibody(clone: ​​GK1.5; BioLegend), Brilliant Violet 421(trademark) anti-mouse IA / IE Antibody(clone: ​​M5 / 114.15.2; BioLegend), Brilliant Violet 510 anti-mouse / human CD11b Antibody(clone: M1 / 70; BioLegend), Brilliant Violet Surface staining was performed with 605™ anti-mouse CD8a antibody (clone: ​​53-6.7; BioLegend), PE anti-mouse CD45 antibody (clone: ​​30-F11; BioLegend), PE / Dazzle™ 594 anti-mouse CX3CR1 antibody (clone: ​​SA011F11; BioLegend), PE / Cyanine7 anti-mouse CD3 antibody (clone: ​​17A2; BioLegend), and eBioscience Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific). Flow cytometry analysis was performed using an Attune NxT Flow Cytometer (Thermo Fisher Scientific) and Flowjo software (TreeStar).

[0085] In both vaccines, the Alum or AddaVax-added groups had increased lung weight and enhanced infiltration of immune cells and eosinophils compared to the non-vaccine group, whereas in the CpG-ODN-added group, lung weight, immune cell and eosinophil infiltration were similar to those in the non-vaccine group (Figures 5A-D).

[0086] These results suggest that it was difficult to improve both the efficacy and safety of the mG vaccine using adjuvants. On the other hand, the eG vaccine has superior antibody induction ability compared to the mG vaccine, and furthermore, it was shown that both efficacy and safety can be enhanced by using CpG-ODN, a Th1 cell-inducing adjuvant.

[0087] Test Example 6: Comparison of the infection protective effects of eG+CpG vaccine and F protein vaccine The efficacy of the eG + CpG-ODN vaccine in preventing infection was compared with that of the F protein vaccine currently being used in human clinical trials, and the usefulness of the eG vaccine was evaluated.

[0088] 1 μg of mG, eG, or F (DS-Cav1: SEQ ID NO: 15) was mixed with 10 μg of CpG-ODN, and vaccinated mice (similar to Test Example 2) were injected with 1×10 5 pfu RSV (in 30 μL PBS) was administered intranasally. Five days after infection, the right lung (A) and nasal turbinates (B) were collected, and RNA was extracted using TRIzol Reagent (Thermo Fisher Scientific). Reverse transcription was performed using ReverTra Ace qPCR RT Master Mix (TOYOBO). Real-time quantitative PCR was performed using a LightCycler 480 with LightCycler 480 SYBR Green I Master (Roche) to measure the expression level of the HRSV-A2 N protein. For the standard curve, the N protein gene was amplified by PCR using cDNA synthesized by reverse transcription from RSV-A2 mRNA as a template, and cloned into the pcDNA3.1 vector (Thermo Fisher Scientific). The copy number of RSV N was calculated using this.

[0089] In the F protein vaccine, antibody production was most enhanced in the CpG-ODN-added group compared to other adjuvants, and the induction of inflammatory cells in the lungs was suppressed. Therefore, in this study, CpG-ODN was used as the adjuvant in all vaccine groups. The results showed that the eG vaccine had the same level of protective ability against upper and lower respiratory tract infections as the F protein vaccine (Figure 6).

Claims

1. A composition comprising a G protein of an RS virus and a CpG oligodeoxynucleotide, wherein the G protein does not have modified sugar chains expressed in mammalian cells.

2. The composition of claim 1 , wherein the G protein is a non-glycoprotein.

3. The composition of claim 1, wherein the G protein is an intracellular domain-deleted G protein.

4. The composition according to claim 1, wherein the content of the CpG oligodeoxynucleotide is 1 to 200 parts by mass per 1 part by mass of the G protein.

5. The composition according to any one of claims 1 to 4, which is in a liquid or solid form.

6. The composition according to any one of claims 1 to 4, which is an RS virus vaccine.

7. An RS virus vaccine comprising a G protein of RS virus and a CpG oligodeoxynucleotide, wherein the G protein does not have a modified sugar chain expressed in mammalian cells, and the G protein and the CpG oligodeoxynucleotide are contained in separate containers.

8. The RS virus vaccine according to claim 7, which is used to administer a mixture obtained by mixing the G protein and the CpG oligodeoxynucleotide to a subject.

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