WO2024084785A1 - Composition appropriée pour être utilisée en tant que vaccin contre le virus rs - Google Patents

Composition appropriée pour être utilisée en tant que vaccin contre le virus rs Download PDF

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WO2024084785A1
WO2024084785A1 PCT/JP2023/029100 JP2023029100W WO2024084785A1 WO 2024084785 A1 WO2024084785 A1 WO 2024084785A1 JP 2023029100 W JP2023029100 W JP 2023029100W WO 2024084785 A1 WO2024084785 A1 WO 2024084785A1
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protein
vaccine
cpg
composition
virus
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靖雄 吉岡
岳彦 柴田
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一般財団法人阪大微生物病研究会
学校法人東京医科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs

Definitions

  • the present invention relates to a composition suitable for use as an RS virus vaccine.
  • Respiratory syncytial virus is an enveloped RNA virus classified in the Pneumovirus genus of the Paramyxovirus family, and is spherical or filamentous with a diameter of 80-350 nm.
  • RSV causes lifelong overt infection regardless of age, but is a particularly important pathogen in infancy, causing the most severe symptoms in the first few weeks to months after birth, despite the presence of maternal antibodies.
  • low birth weight infants, those with underlying cardiopulmonary disease, or immunodeficiency are at high risk of developing severe symptoms, and the clinical and public health impact is significant.
  • preventive vaccine Treatment is primarily 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 caused adverse events in which vaccinated individuals became more seriously ill than non-vaccinated individuals, and research is still ongoing.
  • Currently available preventive methods include human serum-derived anti-RSV immunoglobulins and palivizumab (genetically recombinant), a humanized monoclonal antibody preparation created using genetic engineering technology against the F (fusion) protein, one of the surface proteins of RSV.
  • F (fusion) protein F (fusion) protein
  • RSV G protein is localized on the surface of the RSV envelope and has an important function for the virus to enter host cells. G protein is important for infection, and vaccines have been developed using polypeptides derived from G protein as vaccine antigens (Patent Document 2, Non-Patent Document 1, Non-Patent Document 2).
  • Patent Document 2 Non-Patent Document 1
  • mG recombinant G protein
  • the 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.
  • the inventors have conducted intensive research in light of the above problems, and have found that the above problems can be solved by a composition that contains RS virus G protein and CpG oligodeoxynucleotide, and the G protein does not have modified glycans expressed in mammalian cells. Based on this finding, the inventors have conducted further research and have completed the present invention. In other words, the present invention encompasses the following aspects.
  • Item 1 A composition comprising a G protein of RS virus and a CpG oligodeoxynucleotide, the G protein having no modified glycan expressed in mammalian cells.
  • Item 2 The composition according to Item 1, wherein the G protein is a non-glycoprotein.
  • Item 3 The composition according to Item 1, wherein the G protein is an intracellular domain-deleted G protein.
  • 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.
  • Item 5 The composition according to any one of items 1 to 4, which is in a liquid or solid form.
  • Item 6 The composition according to any one of items 1 to 4, which is an RS virus vaccine.
  • a respiratory syncytial virus vaccine comprising a G protein of an respiratory syncytial virus and a CpG oligodeoxynucleotide, the G protein having no modified sugar chains expressed in mammalian cells, and the G protein and the CpG oligodeoxynucleotide being contained in separate containers.
  • 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.
  • the present invention provides a composition that is highly effective and safe and is suitable for use as an RS virus vaccine.
  • Lane 1 shows the results of SDS-PAGE in Test Example 1.
  • Lane 1 is the recombinant G protein (mG) purified using mammalian cells
  • lane 2 is the recombinant G protein (eG) purified using E. coli.
  • the results of measurement of total IgG, IgG1, IgG2a, and IgG2b in Test Example 2 are shown.
  • A shows the case where mG was administered alone or in combination with an adjuvant
  • B shows the case where eG was administered alone or in combination with an adjuvant.
  • PBS indicates the case where neither antigen nor adjuvant was administered.
  • the legend indicates the dilution ratio in the ELISA measurement.
  • p values between the groups at both ends of the bar are less than 0.05 with *, less than 0.01 with **, less than 0.001 with ***, and less than 0.0001 with ****.
  • the results of measuring the RSV N copy number in Test Example 3 are shown.
  • a and B show the cases when mG was administered alone or in combination with an adjuvant
  • C and D show the cases 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
  • B and D show the results of measuring the RSV N copy number in the nasal turbinates.
  • PBS indicates the case when neither antigen nor adjuvant was administered.
  • p values between the groups at both ends of the bars are indicated as * 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.
  • the cell marker measurement results of Test Example 4 are shown. 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 p value between the groups at both ends of the bar is less than 0.05 with *, less than 0.01 with **, less than 0.001 with ***, and less than 0.0001 with ****.
  • the results of evaluating pulmonary inflammation due to RSV infection in Test Example 5 are shown.
  • a and B show the cases where mG was administered alone or in combination with an adjuvant
  • C and D show the cases where eG was administered alone or in combination with an adjuvant.
  • a and C show the results of measuring lung weight
  • B and D show the results of measuring the number of specific cells.
  • PBS indicates the case where neither antigen nor adjuvant was administered.
  • the p value between the groups at both ends of the bar is less than 0.05 with *, less than 0.01 with **, less than 0.001 with ***, and less than 0.0001 with ****.
  • the results of measuring the RSV N copy number in Test Example 6 are shown.
  • PBS indicates the case where neither antigen nor adjuvant was administered.
  • p values between groups at both ends of the bars are indicated as * 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.
  • identity refers to the degree of agreement between the amino acid sequences of two or more comparable amino acid sequences. Thus, the greater the identity between two amino acid sequences, the greater the identity or similarity of those sequences.
  • the level of identity of amino acid sequences is determined, for example, using FASTA, a sequence analysis tool, 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.
  • conservative substitution means that an amino acid residue is replaced with an amino acid residue having a similar side chain.
  • substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine is a conservative substitution.
  • amino acid residues having acidic side chains such as aspartic acid and glutamic acid
  • amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine
  • amino acid residues having non-polar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan
  • amino acid residues having ⁇ -branched side chains such as threonine, valine, and isoleucine
  • amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.
  • the present invention relates to a composition (sometimes referred to as the "composition of the present invention” in this specification) that contains a G protein of RS virus and a CpG oligodeoxynucleotide, and the G protein does not have a modified sugar chain of a mammalian cell expression type. This is described below.
  • the Respiratory Syncytial Virus (RSV) (scientific name: Human orthopneumovirus) is an enveloped RNA virus.
  • the G protein and F protein are present on the surface of the RSV envelope and are involved in invading host cells.
  • 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 protein and deleted G protein (wherein a portion of the full-length G protein has been deleted).
  • G protein is a viral surface protein, and the extracellular domain is used as the antigen.
  • the G protein is preferably an intracellular domain-deleted G protein (wherein the intracellular domain of the full-length G protein has been deleted).
  • 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 an amino acid sequence shown in any one of SEQ ID NOs: 1 to 7. Examples include:
  • SEQ ID NOs:1 to 6 are the amino acid sequences of the G protein derived from RS virus A strain (A2 strain, Long strain, rsb1734 strain, rsb6190 strain, rsb5857 strain, rsb6256 strain, starting from SEQ ID NO:1)
  • SEQ ID NO:7 is the amino acid sequence of the G protein derived from RS virus B strain (B1 strain).
  • Table 1 shows the results of a comparison of the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:7
  • Table 2 shows the results of a comparison of the amino acid sequences of SEQ ID NO:1 to 6.
  • "*" indicates that the amino acid between the compared subjects is identical.
  • the amino acid identity of the highly conserved domain (CCD) in Table 1 (underlined part in Table 1) is 100%.
  • the amino acid identity of the highly conserved domain (CCD) in Table 2 (underlined part in Table 2) is approximately 95%, and the amino acid identity of the highly conserved domain is 100%.
  • 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 an amino acid sequence shown in any one of SEQ ID NOs: 8 to 14. Examples include:
  • SEQ ID NOs: 8 to 14 are the sequences of intracellular domain-deleted G proteins derived from SEQ ID NOs: 1 to 7, respectively.
  • 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.
  • the amino acid identity of the CCD is preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more, from the viewpoint of the efficacy, safety, etc. of the composition of the present invention as a vaccine.
  • the number of mutated amino acids in the CCD is preferably 1 to 4, more preferably 1 to 2, and even more preferably 1, from the viewpoint of the efficacy, safety, etc. of the composition of the present invention as a vaccine.
  • 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 viewpoint of the efficacy and safety of the composition of the present invention as a vaccine.
  • the number of mutated amino acids in the Highly CCD in the above (b) and (d) is preferably 1 to 4, more preferably 1 to 2, and even more preferably 1, from the viewpoint of the efficacy and safety of the composition of the present invention as a vaccine.
  • the G protein used in the composition of the present invention does not have modified glycans expressed in mammalian cells.
  • Modified glycans expressed in mammalian cells are glycans that are modified when the G protein is expressed in mammalian cells, and are not particularly limited in this respect.
  • a G protein having modified glycans expressed in mammalian cells has a high molecular weight due to the modified glycans.
  • the molecular weight of a G protein not having modified glycans 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, and preferably 30 kDa or more.
  • the molecular weight can be measured by comparing with a molecular weight marker on SDS-PAGE.
  • 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 (expressed and purified in bacteria such as E. coli, or synthesized in vitro).
  • a glycosylation mechanism e.g., insect cells or yeast
  • the G protein is not glycosylated, i.e., a non-glycoprotein.
  • the G protein is expressed in bacteria (preferably E. coli). By using these, it is possible to further improve the efficacy and safety of the composition of the present invention as a vaccine.
  • the G protein may contain other amino acid sequences (e.g., tag sequences such as a histidine tag, an HA tag, or a FLAG tag) as long as their performance as an RS virus vaccine antigen is not significantly impaired.
  • tag sequences such as a histidine tag, an HA tag, or a FLAG tag
  • the G protein may be chemically modified as long as its performance as an RS virus vaccine antigen is not significantly impaired.
  • the C-terminus of the G protein may be any of a carboxyl group (-COOH), a carboxylate ( -COO- ), an amide ( -CONH2 ) or an ester (-COOR).
  • examples of R in the ester include C1-6 alkyl groups such as methyl, ethyl, n-propyl, isopropyl, and n-butyl; C3-8 cycloalkyl groups such as cyclopentyl and cyclohexyl; C6-12 aryl groups such as phenyl and ⁇ -naphthyl; C1-2 phenyl- C1-2 alkyl groups such as benzyl and phenethyl; C7-14 aralkyl groups such as ⁇ -naphthyl- C1-2 alkyl groups such as ⁇ -naphthylmethyl; and pivaloyloxymethyl groups.
  • C1-6 alkyl groups such as methyl, ethyl, n-propyl, isopropyl, and n-butyl
  • C3-8 cycloalkyl groups such as cyclopentyl and cyclohexyl
  • C6-12 aryl groups such
  • Carboxyl groups (or carboxylates) of the G protein other than those at the C-terminus may be amidated or esterified.
  • the esters used may be, for example, the C-terminus esters described above.
  • G proteins include those in which the amino group of the N-terminal amino acid residue is protected with a protecting group (e.g., a C1-6 acyl group, such as a formyl group, a C1-6 alkanoyl group such as an acetyl group), those in which the N-terminal glutamine residue that may be generated by cleavage in the body is pyroglutamylated, and those in which substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino groups, imidazole groups, indole groups, guanidino groups, etc.) are protected with an appropriate protecting group (e.g., a C1-6 acyl group, such as a formyl group, a C1-6 alkanoyl group such as an acetyl group).
  • a protecting group e.g., a C1-6 acyl group, such as a formyl group, a C1-6 alkanoyl group such as an
  • the G protein may be in the form of a pharma- ceutically acceptable salt with an acid or base.
  • an acid salt or a basic salt can be used.
  • acid 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; amino acid salts such as aspartate and glutamate; and the like.
  • 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.
  • the G protein may be in the form of a solvate.
  • the solvent is not particularly limited as long as it is pharma- ceutically acceptable, and examples of the solvent include water, ethanol, glycerol, and acetic acid.
  • the G protein can be a single type or a combination of two or more types.
  • G proteins can be easily produced according to their amino acid sequence using known genetic engineering techniques. For example, they can be produced using PCR, restriction enzyme digestion, DNA ligation techniques, in vitro transcription/translation techniques, recombinant protein production techniques, etc.
  • G protein may be purified after synthesis.
  • G protein is extracted from cells harvested from the culture by centrifugation, filtration, etc.
  • methods such as enzyme digestion, osmotic destruction, rapid pressure/pressure reduction, ultrasound, various homogenizers, etc. can be used.
  • physical methods such as low-speed centrifugation, ultracentrifugation, filtration, molecular sieves, membrane concentration, chemical precipitants, solubilizers, adsorption/desorption agents, dispersants, etc.
  • physicochemical methods such as electrophoresis, column chromatography, supports, dialysis, salting out, etc. can be used in combination.
  • physicochemical conditions such as temperature, pressure, pH, ionic strength, etc. can be set appropriately.
  • CpG oligodeoxynucleotides 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 usable as vaccine adjuvants because they induce adaptive immune responses via TLRs (Toll-like receptors).
  • CpG oligodeoxynucleotides can contain at least one CpG motif, and can also contain multiple CpG motifs.
  • the number of nucleotides constituting a 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, even more preferably 15 to 25 bases, and particularly preferably 18 to 25 bases, etc.
  • CpG oligodeoxynucleotides are classified into class A (D type), class B (K type), class C, class P, and class S based on the sequence, secondary structure, effect on human peripheral blood mononuclear cells (PBMC), etc.
  • D type class A
  • K type class B
  • class C class P
  • class S class S based on the sequence, secondary structure, effect on human peripheral blood mononuclear cells (PBMC), etc.
  • preferred examples of CpG oligodeoxynucleotides include class B (K type) CpG oligodeoxynucleotides, etc.
  • the internucleotide bonds of CpG oligodeoxynucleotides can be phosphodiester or phosphorothioate bonds. 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 the poly-G motifs at both ends form a higher-order structure called a G-tetrad.
  • 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.
  • 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), etc.
  • CpG oligodeoxynucleotides that can be used are commercially available products, or those obtained according to known manufacturing methods can be used.
  • CpG oligodeoxynucleotides can be of one type alone or of two or more types in combination.
  • 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 per part by mass of G protein.
  • composition of the present invention may or may not contain other adjuvants besides CpG oligodeoxynucleotides.
  • adjuvants include Alum (aluminum compounds such as aluminum hydroxide gel), mineral oil, vegetable oil, alum, bentonite, silica, muramyl dipeptide derivatives, thymosin, interleukin, etc.
  • the composition of the present invention preferably contains a small amount of other adjuvants other than CpG oligodeoxynucleotide.
  • the content of other adjuvants is, 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 parts by mass, per part by mass of CpG oligodeoxynucleotide.
  • composition of the present invention it is preferable to use a combination of CpG oligodeoxynucleotide and another adjuvant.
  • the content of the other adjuvant is, for example, 1 to 200 parts by mass per part by mass of CpG oligodeoxynucleotide.
  • composition of the present invention may contain bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.
  • composition of the present invention is not particularly limited, and may be, for example, liquid or solid.
  • composition of the present invention is suitable for use as an RS virus vaccine.
  • the composition of the present invention can prevent the onset of RS virus infection and suppress symptoms (e.g., reducing the aggravation and risk of death) when RS virus infection does occur.
  • the target organisms of the composition of the present invention are not particularly limited, so 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.
  • the dosage form of the composition of the present invention is not particularly limited, and examples thereof include injectables such as aqueous injectables, non-aqueous injectables, suspension injectables, and solid injectables; oral preparations such as tablets, capsules, granules, powders, fine granules, syrups, enteric-coated formulations, sustained-release capsules, chewable tablets, drops, pills, oral liquids, lozenges, sustained-release preparations, and sustained-release granules; and external preparations such as nasal drops, inhalants, rectal suppositories, inserts, enemas, and jellies.
  • injectables such as aqueous injectables, non-aqueous injectables, suspension injectables, and solid injectables
  • oral preparations such as tablets, capsules, granules, powders, fine granules, syrups, enteric-coated formulations, sustained-release capsules, chewable tablets, drops, pills, oral liquids, lo
  • the content of G protein (antigen) in the composition of the present invention depends on the subject of administration, the route of administration, the dosage form, the condition of the patient, the doctor's judgment, etc., and is not limited, but can be, for example, 0.0001 to 95% by weight, preferably 0.001 to 50% by weight.
  • 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, 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 in one administration is, for example, 1 ⁇ g to 10 mg/kg (body weight), or 10 to 1000 ⁇ g/kg (body weight).
  • the administration interval and number of times are not particularly limited, but it is preferable to administer, for example, about 1 to 5 times at intervals of about 1 to 8 weeks.
  • the present invention also relates to an RS virus vaccine comprising a G protein of RS virus and a CpG oligodeoxynucleotide, the G protein having no modified glycan of a mammalian cell expression type, and the G protein and the CpG oligodeoxynucleotide being contained in separate containers.
  • the vaccine comprises at least two containers, namely, a container containing a G protein and a container containing a CpG oligodeoxynucleotide.
  • the vaccine can be used by administering to a subject a mixture obtained by mixing the G protein and the CpG oligodeoxynucleotide.
  • Test Example 1 Purification of mG and eG proteins The extracellular domain of the G protein, which is a vaccine antigen, was produced using mammalian or E. coli and purified.
  • the amino acid sequence of the G protein (SEQ ID NO: 1) is derived from the HRSV-A2 strain (UniProt: P03423). Specifically, the procedure was as follows.
  • ⁇ Recombinant G protein (mG) purified from mammalian cells The cDNA of the extracellular domain of G protein (amino acids 67-298: SEQ ID NO: 8) with an Ig ⁇ signal sequence and a 6x histidine tag at the N-terminus was optimized for human codons.
  • mG was expressed using the Expi293TM Expression System (Thermo Fisher Scientific) and purified on a Ni-SepharoseTM HisTrapTM FF column (GE Healthcare) followed by a SuperoseTM 6 Increase 10/300 GL column (GE Healthcare) using an AKTATM explorer chromatography system.
  • ⁇ Recombinant G protein (eG) purified using E. coli> The cDNA of the extracellular domain of G protein (amino acids 67-298) with a 6x histidine tag at the N-terminus was optimized for E. coli codons.
  • eG was prepared by transducing the vector into BL21(DE3) Competent E. coli (NEB). E. coli was collected by centrifugation at 8,000 x g and 4°C for 10 minutes and resuspended in buffer (20 mM NaH2PO4 , 20 mM Na2HPO4 , 0.5 M NaCl, 20 mM imidazole).
  • G proteins are known to undergo extensive glycosylation.
  • eG which does not have a glycosylation chain, was identified at a position of approximately 30 kDa predicted from the amino acid sequence ( Figure 1).
  • mG was identified at a position of approximately 90 kDa due to glycosylation.
  • Test Example 2 Antibody production by the addition of adjuvants to mG and eG vaccines
  • the protein obtained in Test Example 1 was mixed with an adjuvant and vaccinated into mice to compare the mG-specific IgG antibody induction ability.
  • the adjuvants used were Alum (aluminum hydroxide), CpG-ODN (B class CpG-ODN K3), and AddaVax (trademark) (squalene-based oil-in-water nanoemulsion).
  • Test Example 3 Protective effect against infection by the addition of adjuvant to mG and eG vaccines The protective effect against infection in the mG and eG vaccines was compared. Specifically, the procedure was as follows.
  • mice vaccinated with mG (A, B) or eG (C, D) (same as in Test Example 2) were intranasally administered 1 ⁇ 10 5 pfu RSV (/30 ⁇ L PBS) under anesthesia on the 31st day.
  • Five days after infection the right lung (A, C) and nasal turbinate (B, D) were collected and RNA was extracted using TRIzolTM Reagent (Thermo Fisher Scientific). ReverTra AceTM qPCR RT Master Mix (TOYOBO) was used for reverse transcription reaction.
  • Real-time quantitative PCR was performed using LightCyclerTM 480 SYBR Green I Master (Roche) to measure the expression level of the N protein of the HRSV-A2 strain.
  • the N protein gene was amplified by PCR using cDNA synthesized by reverse transcription from the mRNA of the RSV-A2 strain as a template, and cloned into the pcDNA3.1 vector (Thermo Fisher Scientific) to calculate the copy number of RSV N.
  • Test Example 4 T cell response by the addition of adjuvant to mG and eG vaccines
  • inflammatory T cells induced after vaccination were analyzed. Specifically, this was performed as follows.
  • mice vaccinated with mG (A) or eG (B) (as in Example 2) were spleens harvested on day 28 and spleen cells were purified and cultured at 37°C for 3 days in the presence of 10 ⁇ g/mL mG.
  • BD Cytofix/Cytoperm® Fixation/Permeablization Kit
  • Brilliant Violet 605TM anti-mouse IFN- ⁇ Antibody clone: XMG1.2; BioLegend
  • PE anti-mouse/human IL-5 Antibody clone: TRFK5; BioLegend
  • 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 FlowjoTM software (TreeStar).
  • mG vaccine induces Th2 cells, which are involved in the exacerbation of airway inflammation.
  • the mG vaccine induced IL-5 and IL-13 producing CD4 + T cells, which are Th2 cells, in the mG alone group compared to the non-vaccine group ( Figure 4A).
  • the mG vaccine with CpG-ODN suppressed the induction of Th2 cells and induced IFN- ⁇ producing CD4 + T cells, which are Th1 cells.
  • the eG vaccine induced Th2 cells in the eG alone group and the Alum-added group compared to the non-vaccine group, while the CpG-ODN-added group significantly induced Th1 cells (Figure 4B).
  • Test Example 5 Airway inflammation caused by the addition of adjuvant to mG and eG vaccines Pulmonary inflammation caused by RSV infection after mG and eG vaccination was evaluated. Specifically, the procedure was as follows.
  • mice 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 h 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 gentleMACSTM Tubes (Miltenyi Biotec) and a gentleMACS Dissociator (Miltenyi Biotec), and then subjected to hemolysis treatment with ACK buffer (8.3 g/L NH 4 Cl, 0.01 M Tris-HCl, pH 7.5).
  • CD8a Antibody (clone: 53-6.7; BioLegend), PE anti-mouse CD45 Antibody (clone: 30-F11; BioLegend), PE/DazzleTM 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).
  • 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).
  • the eG vaccine has superior antibody-inducing ability compared to the mG vaccine, and it has been shown that both efficacy and safety can be enhanced by using the Th1 cell-inducing adjuvant CpG-ODN.
  • Test Example 6 Comparison of the infection protective effects of eG+CpG vaccine and F protein vaccine The infection protective effect of the "eG + CpG-ODN" vaccine was compared with the F protein vaccine applied in human clinical trials to evaluate the usefulness of the eG vaccine. Specifically, the procedure was as follows.
  • mice 1 ⁇ g of mG, eG, and F (DS-Cav1: SEQ ID NO: 15) were mixed with 10 ⁇ g of CpG-ODN, and vaccinated mice (similar to Test Example 2) were intranasally administered 1 ⁇ 10 5 pfu RSV (/30 ⁇ L PBS) under anesthesia on the 31st day.

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Abstract

Le but de la présente invention est de fournir une composition ayant une efficacité et une sécurité plus élevées et appropriée pour une utilisation en tant que vaccin contre le virus RS.　L'invention concerne une composition contenant une protéine et un oligodésoxynucléotide CpG d'un virus RS, la protéine G n'ayant pas de chaîne de sucre modifiée d'un type exprimé par une cellule de mammifère.
PCT/JP2023/029100 2022-10-20 2023-08-09 Composition appropriée pour être utilisée en tant que vaccin contre le virus rs WO2024084785A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03502687A (ja) * 1987-09-29 1991-06-20 アメリカン　サイアナミド　カンパニー レスピラトリイ・シンシチアル・ウイルス：ワクチンおよび診断法
WO2002058725A2 (fr) * 2001-01-23 2002-08-01 Pierre Fabre Medicament Peptides non glycosyles derives de la proteine g du vrs et leur utilisation dans un vaccin
JP2005511010A (ja) * 2001-07-20 2005-04-28 ピエール・ファーブル・メディカマン 呼吸器合胞体ウイルス（ｒｓｖ）ｇタンパク質のペプチドとワクチンにおけるその利用
WO2015041318A1 (fr) * 2013-09-20 2015-03-26 独立行政法人医薬基盤研究所 Complexe contenant un oligonucléotide présentant une activité immunopotentialisatrice et son utilisation
US20210113683A1 (en) * 2019-08-12 2021-04-22 Advaccine (Suzhou) Biopharmaceuticals Co., Ltd. Immune composition comprising respiratory syncytial virus (rsv) g polypeptide

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03502687A (ja) * 1987-09-29 1991-06-20 アメリカン　サイアナミド　カンパニー レスピラトリイ・シンシチアル・ウイルス：ワクチンおよび診断法
WO2002058725A2 (fr) * 2001-01-23 2002-08-01 Pierre Fabre Medicament Peptides non glycosyles derives de la proteine g du vrs et leur utilisation dans un vaccin
JP2005511010A (ja) * 2001-07-20 2005-04-28 ピエール・ファーブル・メディカマン 呼吸器合胞体ウイルス（ｒｓｖ）ｇタンパク質のペプチドとワクチンにおけるその利用
WO2015041318A1 (fr) * 2013-09-20 2015-03-26 独立行政法人医薬基盤研究所 Complexe contenant un oligonucléotide présentant une activité immunopotentialisatrice et son utilisation
US20210113683A1 (en) * 2019-08-12 2021-04-22 Advaccine (Suzhou) Biopharmaceuticals Co., Ltd. Immune composition comprising respiratory syncytial virus (rsv) g polypeptide

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHU, R S et al. CpG Oligodeoxynucleotides Act as Adjuvants that Switch on T Helper 1 (Th1) Immunity. J Exp Med. 1997, vol. 186, no. 10, pp. 1623-1631 *

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