WO2012124631A1 - シトルリンを含有するアジュバント組成物 - Google Patents
シトルリンを含有するアジュバント組成物 Download PDFInfo
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- WO2012124631A1 WO2012124631A1 PCT/JP2012/056132 JP2012056132W WO2012124631A1 WO 2012124631 A1 WO2012124631 A1 WO 2012124631A1 JP 2012056132 W JP2012056132 W JP 2012056132W WO 2012124631 A1 WO2012124631 A1 WO 2012124631A1
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- peptide
- citrulline
- antigen
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- vaccine
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- 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
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- 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
- A61K39/0005—Vertebrate antigens
- A61K39/0007—Nervous system antigens; Prions
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- 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
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- 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
- A61K39/145—Orthomyxoviridae, e.g. influenza virus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- 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
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- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
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- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55516—Proteins; Peptides
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- 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
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
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- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16171—Demonstrated in vivo effect
Definitions
- the present invention relates to an adjuvant composition containing citrulline, an adjuvant composition containing citrulline and an antioxidant (for example, ascorbic acid), and a vaccine composition containing the above adjuvant composition and an antigen.
- the vaccine may contain an adjuvant, a diluent, a preservative, a stabilizer, and a buffer as necessary together with the antigenic substance.
- an adjuvant is a substance that is administered together with an antigen and enhances the immune response to the administered antigen, and its action is (1) adsorbing the antigen to increase uptake into antigen-presenting cells, or (2) localizing the antigen locally.
- an adjuvant is very useful in terms of reducing the dose and frequency of administration of the vaccine and the amount of antigen in the vaccine. For this reason, various studies on adjuvants that enhance the action of vaccines have been conducted so far, but very few are actually used in the medical field.
- Alum adjuvant aluminum hydroxide
- squalene squalene
- MPL monophosphoryl lipid
- the adjuvant activity is strong, but the side reaction is strong and it is difficult to dissolve in water. Therefore, in the medical field, development of an adjuvant that elicits a high immune response to the human body, has few side effects, and has improved convenience is eagerly desired.
- citrulline is a kind of amino acid and one of the compounds constituting the urea cycle, and is widely present in animals, particularly mammals (chemical formula C 6 H 13 N 3 O 3 , also known as 2-amino-5- ( Carbamoylamino) pentanoic acid, molecular weight 175.2 g / mol). Citrulline was discovered in 1930 from watermelons in Japan and was named after the watermelon scientific name Citrullus vulgaris.
- Citrulline is not an amino acid that constitutes proteins in the body, but is one of the intermediates of the urea cycle. It is produced from arginine together with nitric oxide (NO), which is known as a substance that has a vasodilatory effect. It is condensed and regenerated to arginine.
- NO nitric oxide
- citrulline has been newly approved in Japan as a food material for supplements since 2007.
- it has been used overseas for some time, supplements aimed at improving blood flow, preventing arteriosclerosis, and enhancing vigor in the United States, and citrulline-malate in Europe as a medicine to relieve fatigue.
- citrulline has been reported to have various effects as described above, it is not known that citrulline has an adjuvant activity, and a vaccine containing citrulline and an antigen and an adjuvant containing citrulline have not yet been reported. Not done. Moreover, the knowledge which uses a citrulline and an antioxidant substance together as an adjuvant is not reported.
- JP 2002-226370 A Japanese Patent Laid-Open No. 53-075387 JP 63-068091 A International publication 2011024748 pamphlet International Publication No. 200833208 Pamphlet
- an object of the present invention is to provide an adjuvant that enhances the immunogenicity of an antigen, is rich in safety to the human body, and has improved convenience.
- citrulline exhibits an excellent adjuvant effect. I found very new knowledge that was not in the previous report.
- citrulline is a water-soluble substance, it can be easily formulated with an antigen, and since it is contained in the body of mammals and various foods, it is excellent in safety to the human body. Therefore, in the present invention, it has become possible to provide a vaccine or an adjuvant that is excellent in antibody production ability, safety, and convenience by using citrulline.
- the present invention is as follows.
- the citrulline is selected from the group consisting of L-citrulline, D-citrulline, L-thiocitrulline, L-homothiocitrulline, S-methyl-L-thiocitrulline and S-ethyl-L-thiocitrulline.
- An adjuvant composition containing the citrulline according to any one of [1] to [9] and a vaccine composition containing an antigen [11] The vaccine composition according to [10], wherein the weight ratio of the antigen to citrulline is 1: N, and N is 100 or more. [12] The vaccine composition according to [10], wherein the antigen is an influenza virus antigen. [13] The influenza virus antigen is at least one or two selected from the group consisting of hemagglutinin (HA), neuraminidase (NA), matrix 1 (M1), matrix 2 (M2), and nucleoprotein (NP). The vaccine composition according to [12], which is the above antigen.
- HA hemagglutinin
- NA neuraminidase
- M1 matrix 1
- M2 matrix 2
- NP nucleoprotein
- influenza virus antigen is hemagglutinin (HA).
- influenza virus antigen is Matrix 2 (M2).
- M2 Matrix 2
- the peptide is an amyloid ⁇ (A ⁇ ) peptide, a peptide consisting of a partial amino acid sequence of the amyloid ⁇ (A ⁇ ) peptide, or a peptide in which one or several cysteines are added or inserted into the peptide.
- the antigen is a peptide consisting of at least one or two or more amino acid sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
- the vaccine composition as described in. [26] A method for producing a vaccine having enhanced antibody-producing ability against an antigen contained in the vaccine, comprising adding citrulline as an adjuvant to the vaccine.
- the citrulline is selected from the group consisting of L-citrulline, D-citrulline, L-thiocitrulline, L-homothiocitrulline, S-methyl-L-thiocitrulline and S-ethyl-L-thiocitrulline.
- the method according to [26], wherein one or more of the above are used.
- the method according to [27], wherein the citrulline is L-citrulline or D-citrulline.
- influenza virus antigen is at least one selected from the group consisting of hemagglutinin (HA), neuraminidase (NA), matrix 1 (M1), matrix 2 (M2), and nucleoprotein (NP).
- HA hemagglutinin
- NA neuraminidase
- M1 matrix 1
- M2 matrix 2
- NP nucleoprotein
- influenza virus antigen is Matrix 2 (M2).
- M2 Matrix 2
- the weight ratio of the influenza virus antigen to citrulline is 1: N, and N is 300 or more.
- the antigen is a peptide.
- the weight ratio of the peptide to citrulline is 1: N, and N is 100 or more.
- peptide is an M2e peptide, a peptide in which one or several cysteines are added or inserted into the M2e peptide, or a peptide in which a peptide containing cysteine is added to the M2e peptide.
- the peptide is an amyloid ⁇ (A ⁇ ) peptide, a peptide consisting of a partial amino acid sequence of the amyloid ⁇ (A ⁇ ) peptide, or a peptide obtained by adding or inserting one or several cysteines to the peptide.
- the method according to [41], wherein the peptide is a peptide obtained by adding one cysteine residue to the C-terminus of a peptide consisting of 28 amino acids from the N-terminus of the amyloid ⁇ (A ⁇ ) peptide.
- the antigen is a peptide consisting of at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, 26].
- the citrulline is selected from the group consisting of L-citrulline, D-citrulline, L-thiocitrulline, L-homothiocitrulline, S-methyl-L-thiocitrulline and S-ethyl-L-thiocitrulline.
- citrulline has the same level of antibody production effect as compared to the existing adjuvant (Alum adjuvant), and when the peptide is used as an antigen, an excellent antibody production effect is found compared to Alum adjuvant. It was. Furthermore, it has been found that the combined use of citrulline and an antioxidant (for example, ascorbic acid) as an adjuvant improves the antibody production effect compared to the use of citrulline alone as an adjuvant. It was.
- an antioxidant for example, ascorbic acid
- citrulline is a water-soluble substance originally present in the living body, so that it can be easily formulated with an antigen and the risk of side effects is reduced as compared with conventional Alum adjuvant and oil-based adjuvant. Therefore, the adjuvant composition and vaccine composition containing the citrulline of the present invention are improved in convenience and superior in safety to the human body as compared with conventional adjuvants and vaccines containing the same.
- citrulline can be mass-produced by chemical synthesis, microorganisms, and the like, and therefore can be provided as an adjuvant for vaccines on a pharmaceutical production scale.
- FIG. 6 shows the measurement results of antibody titer against HA antigen when citrulline was administered to mice at 1 to 5 mg / body. It is a measurement result of the antibody titer with respect to M2e at the time of administering a citrulline to a mouse
- the first aspect of the present invention is an adjuvant composition containing citrulline.
- Citrullines contained in the adjuvant composition of the present invention include citrulline, citrulline derivatives, and citrulline salts.
- the citrulline includes L-citrulline and D-citrulline, preferably L-citrulline.
- Examples of the citrulline derivative include L-thiocitrulline, L-thiohomocitrulline, S-methyl-L-thiocitrulline, S-ethyl- And L-thiocitrulline.
- the citrulline of the present invention includes L-citrulline, D-citrulline, L-thiocitrulline, L-homothiocitrulline, S-methyl-L-thiocitrulline, S-ethyl-L-thiocitrulline.
- the citrulline contained in the adjuvant composition of the present invention may be a citrulline salt.
- citrulline salts include acid addition salts, metal salts, ammonium salts, organic amine addition salts, amino acid addition salts, and the like.
- Acid addition salts include inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate, acetate, maleate, fumarate, citrate, malate, lactate, ⁇ -ketoglutarate , Organic acid salts such as gluconate and caprylate.
- the metal salt include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, zinc salt and the like.
- ammonium salts include salts such as ammonium and tetramethylammonium.
- organic amine addition salt include salts of morpholine, piperidine and the like.
- amino acid addition salts include salts of glycine, phenylalanine, lysine, aspartic acid, glutamic acid and the like.
- Citrulline can be obtained by a chemical synthesis method, a fermentation production method, or the like.
- Methods for chemically synthesizing citrulline include, for example, Gastroenterology, 1997, Vol. 112, p. 1250-1259 (Non-Patent Document 4) and The Journal of Chemistry, 1938, Vol. 122, p. 477-p484 (Non-Patent Document 5).
- Examples of the method for fermentative production of L-citrulline include the methods described in JP-A No. 53-075387 (Patent Document 2) and JP-A No. 63-068091 (Patent Document 3).
- citrulline can be obtained by purchasing a commercially available product.
- L-citrulline (Sigma-Aldrich: Code ⁇ ⁇ No.27510 and C7629), L-thiocitrulline (Sigma-Aldrich: Code No) .88544, Wako Pure Chemical Industries, Ltd .: Code No. 205-13861), and S-methyl-L-thiocitrulline (Wako Pure Chemical Industries, Ltd .: Code No. 139-12611).
- the concentration of citrulline contained in the adjuvant composition of the present invention may be appropriately changed according to the type of antigen, dosage form, administration method, target patient, etc., but citrulline is contained at Amg / mL to Bmg / mL.
- a and B are 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100
- Different numerical values selected from the group consisting of: A is a numerical value smaller than B).
- the dose of citrulline using the adjuvant composition of the present invention may be appropriately changed according to the type of antigen, the drug dosage form, the administration method, and the like.
- the adjuvant composition of the present invention may contain an antioxidant in addition to citrulline.
- antioxidants include ascorbic acid (vitamin C), ⁇ -tocopherol (vitamin E), glutathione, N-acetylcysteine, butylhydroxyanisole, catechin, quercetin, uric acid, bilirubin, glucose, flavonoids, ceruloplasmin, Examples include albumin, ferritin, metallothionein, superoxide dismutase, glutathione peroxidase, glutathione transferase, catalase, and thioredoxin. Ascorbic acid and ⁇ -tocopherol are preferred.
- the weight ratio of citrulline and antioxidant may be 1: 2 to 2: 1, preferably 1: 1.
- the antioxidant contained in the adjuvant composition of the present invention may be 1 to 10 mg / mL, preferably 5 mg / mL.
- the second aspect of the present invention is a vaccine composition containing an adjuvant composition containing the above citrulline and an antigen.
- the antigen may be any antigen as long as it is usually contained in a vaccine.
- a vaccine for example, carbohydrate, glycolipid, glycoprotein, lipid, lipoprotein, phospholipid, peptide, polypeptide, protein, polynucleotide , Oligonucleotides, or derivatives thereof.
- peptide refers to those consisting of 2 to several tens of amino acids
- polypeptide refers to those consisting of several tens or more amino acids.
- proteins, polypeptides or peptides are preferred.
- the adjuvant composition containing the citrulline of the present invention is particularly preferred to use a peptide as an antigen because a particularly excellent antibody production effect is observed when the peptide is used as an antigen.
- the method for obtaining the antigen may be any of genetic recombination, chemical synthesis, or a method of obtaining from a natural product.
- the antigen may be an antigen derived from a pathogen (virus, bacteria, fungus, parasitic microorganism), virus-like particle, virosome, cancer cell, allergen, or self molecule.
- a pathogen virus, bacteria, fungus, parasitic microorganism
- virus-like particle virus-like particle
- virosome cancer cell
- allergen or self molecule
- the antigen derived from the pathogen may be a subunit antigen, a peptide antigen, an inactivated pathogen, an attenuated pathogen, or a recombinant antigen.
- virus examples include hepatitis virus, RS virus, adenovirus, abra virus, isa virus, canine distemper virus, influenza virus AC, equine arteritis virus, Ebola virus, enterovirus, calicivirus, coronavirus, monkey immunity Deficiency virus, sogotovirus, dengue virus, togavirus, avian infectious synovial sac disease virus, avian pneumonia virus (formerly turkey rhinotracheitis virus), nipper virus, newcastle disease virus, pneumovirus, feline infectious peritonitis virus, feline leukemia Virus, Norwalk virus, Papilloma virus, Papova virus, Parainfluenza virus type 1-3, Parvovirus, Picornavirus, Human cytomegalovirus , Human immunodeficiency virus, porcine respiratory injury / reproductive syndrome virus, flavivirus, henipavirus, hepadnavirus, herpes virus, hendra virus, poliovirus, Marek's disease virus
- the influenza virus is an RNA envelope virus having a particle size of about 100 nm in diameter belonging to the Orthomyxoviridae family, and is classified into A, B and C types based on the antigenicity of the internal protein. Among them, it is type A that infects both humans and animals and has a great variety.
- the type A has two types of envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA), and there are 16 types in HA and 9 types in NA due to the difference in antigenicity.
- HA hemagglutinin
- NA neuraminidase
- influenza virus used as an antigen in the present invention is not particularly limited in type, subtype, and strain type.
- antigens derived from influenza virus include HA, NA, M1, M2, and NP. Particularly preferred are HA and M2.
- HA a vaccine containing HA as an antigen is highly versatile.
- the M2 protein having a small variation in amino acid sequence among influenza viruses is useful as an antigen from the viewpoint of conferring immunity against a wide range of influenza viruses.
- the M2 protein is a peptide corresponding to a region consisting of a 23 amino acid sequence from which the hydrophobic transmembrane domain has been removed (M2e, SEQ ID NO: 1 N-terminal-SLLTEVETPIRNEWGCRCNDSSD-C-terminal), and for the purpose of enhancing the antigenicity of the M2e peptide, 1 or It may be an M2e peptide to which several cysteines are added or inserted, or an M2e peptide to which a peptide containing cysteine is added. Examples of the M2e peptide modified with cysteine include peptides described in International Publication No.
- Patent Document 4 specifically, between the 20th and 21st positions from the N-terminus, the 21st position, and the like.
- a synthetic peptide M2eC212223 (SEQ ID NO: 2: N-terminal-SLLTEVETPIRNEWGCRCNCDCSCSD-C-terminal) having a cysteine residue inserted between the 22nd and 22nd and 23rd positions, respectively, can be mentioned.
- the method for preparing the influenza virus antigen is not particularly limited, and any known method can be used.
- a virus strain isolated from an influenza-infected animal or influenza patient may be infected with chicken eggs or cells, cultured by a conventional method, and an antigen may be prepared from a purified virus stock solution. It is also possible to prepare an antigen using a genetically engineered recombinant virus or a specific antigen produced or expressed in various cells as a material.
- Examples of the self-molecule used for the antigen include amyloid ⁇ peptide or a peptide consisting of a partial amino acid sequence of amyloid ⁇ peptide.
- the amyloid ⁇ peptide or the peptide consisting of the partial amino acid sequence may be a peptide to which one or several cysteines are added or inserted, or a peptide to which a peptide containing cysteine is added.
- the peptide currently disclosed by the international publication 20080081208 (patent document 5) is mentioned.
- amyloid ⁇ peptide is amyloid ⁇ peptide consisting of 42 amino acids (SEQ ID NO: 3: N-terminal-DAEFRHDSGYEVHHQKLVFFFAEDVGSNKGAIIGLMVVGVVI-C-terminal), and a peptide consisting of a partial amino acid sequence of amyloid ⁇ peptide is 28 amino acids counted from the N-terminal.
- a peptide added with cysteine is a peptide 28AACys with one cysteine residue added to the C-terminal side of a 28-amino acid peptide counted from the N-terminus
- SEQ ID NO: 5 N-end-DAEFRHDSGYEVHHQKLVFFAFADGVGNK-C end.
- bacterium examples include Actinobacillus pleuropneumoniae, Aroiocox otiditis, Haemophilus influenzae (both typeable and nontypeable), Yersinia bacteria, Parrot disease Chlamydia, Campylobacter, Chlamydia pneumonia Pathogens, Clostridia species, Vibrio cholerae, Salmonella choleresius, Giardia, Diphtheria, Pseudomonas species, Streptococcus gordonii, Streptococcus thermophilus, Streptococcus bovis, Streptococcus agalactiae, Trachoma chlamydia, T.
- tuberculosis Hemolytica, Pasteurella multocida, Mycobacterium tuberculosis, Streptococcus pneumoniae, Proteus bulgaris, Proteus mirabilis, Haemophilus somnus, Helicoba Terpylori, Borrelia burgdorferi, Mycoplasma galericepticum, Moraxella catararis, Leptospira interrogans, Staphylococcus aureus, Streptococcus pyogenes, Shigella, Streptococcus, Escherichia coli, Bacillus anthracis, Salmonella typhi Examples include tetanus, Streptococcus pneumoniae, Bordetella pertussis, Staphylococcus epidermidis, Streptococcus faeces, Green streptococci, and Neisseria gonorrhoeae.
- Examples of the parasites include Shigella amoeba, Plasmodium genus, Forest type tropical Leishmania, Ascaris genus, Trichinella genus, Giardia genus, Schistosoma genus, Cryptosporidium genus, Trichomonas genus, Toxoplasma, Pneumocystis carini Can be mentioned.
- the concentration of the antigen contained in the vaccine composition of the present invention may be appropriately changed according to the type of antigen, the dosage form and dosage form of the vaccine composition, and the like.
- the weight ratio of the antigen and citrulline contained in the vaccine composition of the present invention is 1: N, and preferably N is 100 or more, 200 or more, 300 or more, 333 or more, 400 or more, 800 or more, 3000 or more, 3333. These are 6000 or more, 6666 or more, 10,000 or more, 13000 or more, 13333 or more, 160000 or more, or 16666 or more.
- the weight ratio of antigen to citrulline is also 1: A to 1: B (A and B are 100, 200, 300, 333, 400, 800, 3000, 3333, 6000, 6666, 10,000, 13000, 13333, A different number selected from 16000, 16666, where A is a smaller number than B).
- the weight ratio of the antigen to citrulline contained in the vaccine composition is 1: N, preferably N is 300 or more, 333 or more, 3000 or more, 3333 or more, 6000 or more, 6666 or more. It is 10,000 or more, 13000 or more, 13333 or more, 160000 or more, or 16666 or more.
- the weight ratio of antigen to citrulline is also 1: A to 1: B (A and B are selected from 300, 333, 3000, 3333, 6000, 6666, 10,000, 13000, 13333, 16000, 16666) And A is a smaller number than B).
- the weight ratio of the antigen to citrulline contained in the vaccine composition is 1: N, and preferably N is 100 or more, 200 or more, 400 or more, 800 or more.
- the weight ratio of antigen to citrulline is also 1: A to 1: B (A and B are different numbers selected from 100, 200, 400, 800, and A is a number smaller than B) ).
- the weight ratio of the antigen to citrulline contained in the vaccine composition is 1: N, and preferably N is 200 or more.
- the weight ratio of the antigen and citrulline contained in the vaccine composition is 1: N, and preferably N is 100 or more, 200 or more, 400 or more, 800 or more.
- the weight ratio of antigen to citrulline is also 1: A to 1: B (A and B are different numbers selected from 100, 200, 400, 800, and A is a number smaller than B) ).
- the dose of the antigen in the vaccine composition of the present invention may be appropriately changed according to the type of antigen, the dosage form or dosage form of the vaccine composition, and the like.
- the vaccine composition of the present invention may contain a single antigen or a combination of a plurality of types of antigens.
- a combination of a plurality of types of antigens is included, a plurality of different types of antigens may be included in the same virus or bacterium, or a plurality of types of antigens such as different viruses or bacteria may be included.
- an influenza virus vaccine composition it preferably contains antigens of different types, influenza virus types A and B.
- the dosage form of the vaccine composition may be one in which citrulline and antigen are formulated in a single container, or an adjuvant composition containing citrulline and an immunogenic composition containing antigen. Each may be formulated in separate containers.
- the dosage form at the time of formulating may be, for example, liquid, powder (freeze-dried powder, dry powder), capsule, tablet, frozen state, and the like.
- the dosage form of the adjuvant composition and vaccine composition may be, for example, liquid, powder (freeze-dried powder, dry powder), capsule, tablet, frozen state, and the like.
- the adjuvant composition and the vaccine composition may contain a pharmaceutically acceptable carrier in addition to citrullines and antigens.
- a pharmaceutically acceptable carrier any carrier that is usually used in the production of vaccines can be used. Specific examples include saline, buffered saline, dextrose, water, glycerol, isotonic aqueous buffers and combinations thereof, and further emulsifiers, preservatives (eg, thimerosal), isotonic agents, pH adjustment.
- An agent, an inactivating agent (eg, formalin) and the like may be appropriately blended.
- the administration target of the adjuvant composition and vaccine composition includes any organism that can be immunized, in particular, humans and other mammals (for example, domestic animals, pets, and wild animals).
- administration routes of the adjuvant composition and vaccine composition include transdermal administration, sublingual administration, eye drop administration, intradermal administration, intramuscular administration, oral administration, enteral administration, nasal administration, intravenous administration, Subcutaneous administration, intraperitoneal administration, inhalation administration from the mouth to the lung, and the like can be mentioned.
- the adjuvant composition and the vaccine composition can be administered by, for example, a stent, a catheter, a transcutaneous patch, a microneedle, an implantable sustained-release device, a syringe, a syringe with a microneedle, a needle-free apparatus, or a spray. It may be a thing.
- the antigen and the adjuvant composition are formulated in separate containers, the formulated antigen and the adjuvant composition may be administered at the same time, or the antigen and the adjuvant composition may be administered and fixed. The other may be administered after the period.
- the citrulline solution used as an adjuvant was prepared as follows. L-citrulline (Sigma, C7629-1G) was dissolved in distilled water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) to a final concentration of 80 mg / mL. This was sterile filtered at 0.22 ⁇ m and stored frozen at ⁇ 20 ° C. until use.
- HA antigen was prepared as an antigen (strain: A / Solomon strain, composition: 1200 ⁇ g / mL HA). HA antigens were mixed at various concentrations of citrulline to prepare compositions 1 to 4 in Table 1 (note that composition 1 does not contain citrulline).
- a mixed solution of HA antigen and Alum adjuvant Imject Alum (PIERCE) or ALHYDROGEL (BRENNTAG) was prepared (comparative control in Table 1).
- compositions 1 to 4 or comparative control were administered at 100 ⁇ L per mouse by the following method (the dose per mouse is described in Table 1).
- BALB / c mice 6 weeks old females were weighed and divided equally into 3 groups per group and 4 groups. Each individual was identified with an animal marker.
- blood was collected from the orbital venous plexus under isoflurane inhalation anesthesia using a capillary. After centrifugation, serum was dispensed into tubes. 0.1 mL of the test substance was placed in a disposable 1 mL syringe (manufactured by Terumo) and administered subcutaneously to the back of the neck of the mouse.
- Blocker TM Casein in PBS (Thermo) was added at 200 ⁇ L / well and allowed to react at room temperature for 1 hour.
- antiserum day 0, 14
- 50 ⁇ L / well of an anti-mouse IgG-POD labeled antibody (manufactured by Thermo) diluted 2000 times with Blocker TM Casein in PBS was added and allowed to react at room temperature for 1 hour.
- Example 1 From the results of Example 1, since citrulline had an adjuvant effect, the optimum amount was examined. In Example 1, as a result of changing the dosage of citrulline from 10 to 1000 ⁇ g / body, 1000 ⁇ g / body showed the highest adjuvant effect, so the effect of further increasing the dosage of citrulline was examined.
- (1) Preparation of citrulline-containing composition Compositions 5 to 10 having different concentrations of citrulline were prepared in the same manner as the preparation method of Example 1 (Table 2; composition 5 does not contain citrulline) .
- citrulline-containing composition As an antigen, in the region consisting of 23 amino acids presented on the virus surface of M2 protein of influenza A virus (M2e, SEQ ID NO: 1 N-terminal-SLLTEVETPIRNEWGCRCNDSSD-C-terminal), N Synthetic peptide M2eC212223 in which cysteine residues are inserted between the 20th and 21st positions, between the 21st and 22nd positions, and between the 22nd and 23rd positions from the end (SEQ ID NO: 2: N-terminal-SLLTVETPIRNEWGCRCNCDCSCSD-C The end) was used.
- M2eC212223 peptide was synthesize
- mice and serum collection Immunization to mice was performed as follows. BALB / c mice 7-week-old females were divided into 5 groups per group and 2 groups. Each individual was identified with an animal marker. The test substance was placed in a disposable 1 mL syringe (manufactured by Terumo Corporation), and 0.1 mL per mouse was subcutaneously administered to the back of the neck of the mouse (the dose per mouse is described in Table 3). The test substance was administered twice at an interval of 2 weeks, and 1 week after the second administration, the abdomen was opened under somnopentyl (Kyoritsu Pharmaceutical) anesthesia, and whole blood was collected from the intraperitoneal posterior vena cava. Serum separation was performed as in Example 1.
- each well was washed three times with 300 ⁇ L of 0.05% Tween20-containing phosphate buffer (PBST), and 300 ⁇ L of monoethanolamine (Wako Pure Chemical Industries) diluted to 10 mM with 0.1 M Carbonate buffer, pH 9.6. / Well was added and allowed to stand at room temperature for 1 hour. Thereafter, 10 mM monoethanolamine was sufficiently removed, and the sample was diluted with PBST and added at 100 ⁇ L / well (each sample was duplicated). After the reaction at room temperature for 1 hour, each diluted serum added was discarded and washed 3 times with 300 ⁇ L / well PBST.
- PBST 0.05% Tween20-containing phosphate buffer
- citrulline-containing composition As an antigen, an amyloid ⁇ (A ⁇ ) peptide consisting of 42 amino acid residues (SEQ ID NO: 3: N-terminal-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA-C-terminal) peptide consisting of 28 amino acids from the N-terminal A synthetic peptide 28AACys (SEQ ID NO: 5: N-end-DAEFRHDSGYEVHHQKLVFFAEDVGSNKC-C-end) in which one cysteine residue was added to the C-terminal of (SEQ ID NO: 4: N-end-DAEFRHDSGYEVHHQKLVFFFAEDVGSNK-C-end) was used.
- a ⁇ amyloid ⁇
- the citrulline solution used as an adjuvant was prepared as follows. L-citrulline (Sigma, C7629-1G) was dissolved in physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) to a final concentration of 50 mg / mL. The sample was stored frozen at ⁇ 30 ° C. until use. Similarly, L-ascorbic acid (Wako Pharmaceutical Co., Ltd.) was dissolved in physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) to a final concentration of 50 mg / mL, and refrigerated at 4 ° C. until use. Alum (ALHYDROGEL) was used as a control. The above citrulline solution, 28 AACys, and ascorbic acid solution were mixed to prepare Comparative Controls 1 and 2 and Compositions 13 to 18 in Table 4.
- mice and serum collection Immunization to mice was performed as follows. C57BL / 6 mice 7 weeks old males were divided into 4 groups per group and 8 groups. Each composition was administered subcutaneously in the abdomen using a 1 mL tuberculin syringe (Terumo, SS-01T2613S) in an amount of 200 ⁇ L per mouse (the dose per mouse is shown in Table 4). Mice were immunized twice at 2-week intervals. On the 14th day after the second immunization, blood was collected from the abdominal vena cava and killed under anesthesia with pentobarbital sodium (Kyoritsu Seiyaku, Somnopentyl).
- the collected blood was transferred to Microtina (BECTON DICKINSON), sufficiently coagulated at room temperature, and then centrifuged (5000 rpm, 10 minutes).
- the separated sera were each dispensed into two 0.5 mL tubes and stored at ⁇ 80 ° C. until measurement.
- the antibody against the A ⁇ peptide in serum was measured by the following ELISA method.
- each well was washed 3 times with 300 ⁇ L of 0.05% Tween20-containing PBS (PBST), 10 mM ethanolamine was added in 300 ⁇ L / well, and the mixture was allowed to stand at room temperature for 1 hour. Thereafter, 10 mM ethanolamine was sufficiently removed, and the sample was diluted 50 to 10,000 times with PBST and added at 100 ⁇ L / well (each sample was duplicated). After the reaction at room temperature for 1 hour, each diluted serum added was discarded and washed 3 times with 300 ⁇ L / well PBST.
- PBST 0.05% Tween20-containing PBS
- an HRP-labeled anti-mouse IgG goat antibody (American Qualax, A131PS) diluted 2000-fold with a sample diluent is added at 100 ⁇ L / well and reacted at room temperature for 1 hour. did.
- the labeled antibody dilution is discarded, washed twice with 300 ⁇ L / well of PBST and twice with the same amount of distilled water, added with 100 ⁇ L / well of chromogenic substrate solution TMB + (Dako), and protected from light at room temperature. Reacted for 30 minutes. Thereafter, 100 ⁇ L / well of 1N sulfuric acid was added to stop the color development, and the absorbance at 450 nm (OD450 value) was measured.
- a commercially available monoclonal antibody against A ⁇ (CHEMICON; MAB1560) was used as a standard serum.
- Standard serum was diluted with PBST to 0.156, 0.3125, 0.625, 1.25, 2.5, 5, 10 ng / mL to prepare a standard for antibody titer measurement.
- the OD450 value of each diluted specimen was measured in duplicate.
- the anti-A ⁇ IgG antibody titer of each mouse serum was calculated from the standard units obtained and the standard line of OD450 values.
- Table 4 shows the calculated anti-A ⁇ antibody titers in mouse serum in each immunization group.
- compositions 13 to 16 and 18 In the group to which citrulline was added (compositions 13 to 16 and 18), the antibody production effect was enhanced as compared with the group not administered with adjuvant (Comparative Control 1). That is, about 11.6 times (composition 13) in the 1 mg / body administration group, about 42.2 times (composition 14) in the 2 mg / body administration group (composition 14), and 4 mg / body administration group, compared to the adjuvant non-administration group. An extremely remarkable antibody production effect of about 125.8 times (Composition 15) and about 141.7 times (Composition 16) in the 8 mg / body administration group was observed.
- composition 13 is approximately 9.8 times
- composition 14 is approximately 35.5 times
- Composition 15 was about 105.6 times
- composition 16 was about 119 times
- composition 18 was about 39.4 times
- the antibody production effect was extremely superior compared to the existing Alum adjuvant.
- the present invention provides an adjuvant composition containing citrulline, and a vaccine composition containing the adjuvant composition and an antigen.
- Citrulline is also useful for living organisms and is a water-soluble substance. Therefore, it is safer than conventional Alum adjuvants and oil-based adjuvants, and is an adjuvant composition and vaccine composition that is superior in terms of preparation. Can be provided.
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Abstract
Description
[1]シトルリン類を含有するアジュバント組成物。
[2]前記シトルリン類が、L-シトルリン、D-シトルリン、L-チオシトルリン、L-ホモチオシトルリン、S-メチル-L-チオシトルリンおよびS-エチル-L-チオシトルリンからなる群より選択される1種または2種以上である、[1]に記載のアジュバント組成物。
[3]前記シトルリン類が、L-シトルリンまたはD-シトルリンである、[2]に記載のアジュバント組成物。
[4]前記シトルリン類が1mg/mL~50mg/mLで含まれる、[1]から[3]のいずれかに記載のアジュバント組成物。
[5]アジュバント組成物が、ペプチド抗原用のアジュバント組成物である、[1]から[4]のいずれかに記載のアジュバント組成物。
[6]抗酸化物質をさらに含む、[1]から[5]のいずれかに記載のアジュバント組成物。
[7]前記抗酸化物質が、アスコルビン酸である、[6]に記載のアジュバント組成物。
[8]前記シトルリン類と抗酸化物質の重量比が1:2~2:1である、[6]または[7]に記載のアジュバント組成物。
[9]前記抗酸化物質が1~10mg/mLで含まれる、[6]から[8]のいずれかに記載のアジュバント組成物。
[10][1]から[9]のいずれかに記載のシトルリン類を含有するアジュバント組成物と抗原を含有するワクチン組成物。
[11]前記抗原とシトルリン類の重量比が1:Nであって、Nが100以上である、[10]に記載のワクチン組成物。
[12]前記抗原が、インフルエンザウイルス抗原である、[10]に記載のワクチン組成物。
[13]前記インフルエンザウイルス抗原が、ヘマグルチニン(HA)、ノイラミニダーゼ(NA)、マトリックス1(M1)、マトリックス2(M2)、および核タンパク(NP)からなる群より選ばれた少なくとも1種または2種以上の抗原である、[12]に記載のワクチン組成物。
[14]前記インフルエンザウイルス抗原が、ヘマグルチニン(HA)である、[13]に記載のワクチン組成物。
[15]前記インフルエンザウイルス抗原が、マトリックス2(M2)である、[13]に記載のワクチン組成物。
[16]前記インフルエンザウイルス抗原とシトルリン類の重量比が1:Nであって、Nが300以上である、[12]から[15]のいずれかに記載のワクチン組成物。
[17]前記抗原がペプチドである、[10]に記載のワクチン組成物。
[18]前記ペプチドとシトルリン類の重量比が1:Nであって、Nが100以上である、[17]に記載のワクチン組成物。
[19]前記ペプチドが、M2eペプチド、M2eペプチドに1もしくは数個のシステインが付加もしくは挿入されたペプチド、またはM2eペプチドにシステインを含むペプチドが付加されたペプチドである、[17]に記載のワクチン組成物。
[20]前記ペプチドが、M2eペプチドにおいて、N末端から数えて20番目と21番目の間、21番目と22番目の間、および22番目と23番目の間にそれぞれシステイン残基を挿入した合成ペプチドである、[19]に記載のワクチン組成物。
[21]前記M2eペプチドとシトルリン類の重量比が1:Nであって、Nが200以上である、[19]または[20]に記載のワクチン組成物。
[22]前記ペプチドが、アミロイドβ(Aβ)ペプチド、アミロイドβ(Aβ)ペプチドの一部のアミノ酸配列からなるペプチド、または前記ペプチドに1もしくは数個のシステインが付加もしくは挿入されたペプチドである、[17]に記載のワクチン組成物。
[23]前記ペプチドがアミロイドβ(Aβ)ペプチドのN末端より28個のアミノ酸からなるペプチドのC末端にシステイン残基を1個付加したペプチドである、[22]に記載のワクチン組成物。
[24]前記アミロイドβペプチドとシトルリン類の重量比が1:Nであって、Nが100以上である、[22]または[23]に記載のワクチン組成物。
[25]前記抗原が配列番号1、配列番号2、配列番号3、配列番号4および配列番号5からなる群より選ばれた少なくとも1種または2種以上のアミノ酸配列からなるペプチドである、[10]に記載のワクチン組成物。
[26]ワクチンにアジュバントとしてのシトルリン類を添加することからなる、該ワクチンに含まれる抗原に対する抗体産生能の増強したワクチンの製造方法。
[27]前記シトルリン類が、L-シトルリン、D-シトルリン、L-チオシトルリン、L-ホモチオシトルリン、S-メチル-L-チオシトルリンおよびS-エチル-L-チオシトルリンからなる群より選択される1種または2種以上である、[26]に記載の方法。
[28]前記シトルリン類が、L-シトルリンまたはD-シトルリンである、[27]に記載の方法。
[29]前記シトルリン類を1mg/mL~50mg/mLで添加する、[26]から[28]のいずれかに記載の方法。
[30]前記抗原とシトルリン類の重量比が1:Nであって、Nが100以上である、[26]から[29]のいずれかに記載の方法。
[31]前記抗原が、インフルエンザウイルス抗原である、[26]から[29]のいずれかに記載の方法。
[32]前記インフルエンザウイルス抗原が、ヘマグルチニン(HA)、ノイラミニダーゼ(NA)、マトリックス1(M1)、マトリックス2(M2)および核タンパク(NP)からなる群より選ばれた少なくとも1種または2種以上の抗原である、[31]に記載の方法。
[33]前記インフルエンザウイルス抗原が、ヘマグルチニン(HA)である、[32]に記載の方法。
[34]前記インフルエンザウイルス抗原が、マトリックス2(M2)である、[32]に記載の方法。
[35]前記インフルエンザウイルス抗原とシトルリン類の重量比が1:Nであって、Nが300以上である、[31]から[34]のいずれかに記載の方法。
[36]前記抗原がペプチドである、[26]に記載の方法。
[37]前記ペプチドとシトルリン類の重量比が1:Nであって、Nが100以上である、[36]に記載の方法。
[38]前記ペプチドが、M2eペプチド、M2eペプチドに1もしくは数個のシステインが付加もしくは挿入されたペプチド、またはM2eペプチドにシステインを含むペプチドが付加されたペプチドである、[36]に記載の方法。
[39]前記ペプチドが、M2eペプチドにおいて、N末端から数えて20番目と21番目の間、21番目と22番目の間、および22番目と23番目の間にそれぞれシステイン残基を挿入した合成ペプチドである、[38]に記載の方法。
[40]前記M2eペプチドとシトルリン類の重量比が1:Nであって、Nが200以上である、[38]または[39]に記載の方法。
[41]前記ペプチドが、アミロイドβ(Aβ)ペプチド、アミロイドβ(Aβ)ペプチドの一部のアミノ酸配列からなるペプチド、または前記ペプチドに1もしくは数個のシステインが付加もしくは挿入されたペプチドである、[36]に記載の方法。
[42]前記ペプチドがアミロイドβ(Aβ)ペプチドのN末端より28個のアミノ酸からなるペプチドのC末端にシステイン残基を1個付加したペプチドである、[41]に記載の方法。
[43]前記アミロイドβペプチドとシトルリン類の重量比が1:Nであって、Nが100以上である、[41]または[42]に記載の方法。
[44]前記抗原が配列番号1、配列番号2、配列番号3、配列番号4、および配列番号5からなる群より選ばれた少なくとも1種または2種以上のアミノ酸配列からなるペプチドである、[26]に記載の方法。
[45]シトルリン類とともに抗酸化物質をも添加する、[26]から[44]のいずれかに記載の方法。
[46]前記抗酸化物質が、アスコルビン酸である、[45]に記載の方法。
[47]前記シトルリン類と抗酸化物質を重量比1:2~2:1で添加する、[45]または[46]に記載の方法。
[48]前記抗酸化物質を1~10mg/mLで添加する、[45]から[47]のいずれかに記載の方法。
[49]アジュバントとしてのシトルリン類の使用。
[50]前記シトルリン類が、L-シトルリン、D-シトルリン、L-チオシトルリン、L-ホモチオシトルリン、S-メチル-L-チオシトルリンおよびS-エチル-L-チオシトルリンからなる群より選択される1種または2種以上である、[49]に記載の使用。
[51]前記シトルリン類が、L-シトルリンまたはD-シトルリンである、[50]に記載の使用。
[52]1mg/mL~50mg/mLの前記シトルリン類をワクチンに添加する、[49]から[51]のいずれかに記載の使用。
[53]抗酸化物質を併用する、[49]から[52]のいずれかに記載の使用。
[54]前記抗酸化物質が、アスコルビン酸である、[53]に記載の使用。
[55]前記シトルリン類と抗酸化物質を重量比1:2~2:1でワクチンに添加する、[53]または[54]に記載の使用。
[56]1~10mg/mLの前記抗酸化物質をワクチンに添加する、[53]から[55]のいずれかに記載の使用。
抗原として、インフルエンザウイルスHA抗原を作成した(株:A/ソロモン株、組成:1200μg/mL HA)。HA抗原を様々なシトルリン濃度となるよう混合して、表1の組成物1~4を作成した(なお、組成物1はシトルリンを含まない)。比較対照として、HA抗原とAlumアジュバント(Imject Alum(PIERCE社)またはALHYDROGEL(BRENNTAG社))の混合液を作成した(表1の比較対照)。
上記の組成物1~4、または比較対照を下記の方法にてマウス1匹あたり100μLで投与した(マウス1匹あたりの投与量は表1に記載)。BALB/cマウス6週齢の雌の体重を測定し、均等になるように1群3匹、4群に分けた。各個体はアニマルマーカーで個体識別した。被験物投与の当日、イソフルラン吸入麻酔下で眼窩静脈叢より、キャピラリーを用いて採血した。遠心分離後、血清をチューブに分取した。被験物はディスポーザブルの1mL用シリンジ(テルモ社製)に0.1mLとり、マウスの頸背部皮下に投与した。被験物投与後14日目にイソフルラン吸入麻酔下で開腹し、腹腔内後大静脈より全採血した。血液は室温で30分以上静置したのち、遠心して(3000rpm、10分間)血清を分離した。このようにして得られた血清中のHA抗原に対する抗体を下記のELISA法にて測定した。
(3)ELISAによる抗HA抗体価の測定
免疫した抗原と同じものを1μg/mL濃度で、ELISAプレートへ50μL/well投入し、自然吸着により4℃で一晩静置して固相化した。PBS(当日調製)にて3回洗浄後、BlockerTM Casein in PBS(Thermo社製)を200μL/wellで投入し、室温で1時間反応させた。PBSにて3回洗浄後、抗血清(day0、14)をBlockerTM Casein in PBSにて200倍希釈し、50μL/wellで投入し室温で2時間反応させた。抗マウスIgG-POD標識抗体(Thermo社製)をBlockerTM Casein in PBSで2000倍に希釈したものを50μL/well投入し、室温で1時間反応させた。PBSにて4回洗浄後、基質TMB(BioFX社製)を50μL/well投入し、室温で15分反応後、1N H2SO4を50μL/well投入し反応を停止させた。450nmの吸光度を測定した。
(4)結果
ELISAの結果を表1に示した。シトルリンと抗原を共に免疫した群において、特にシトルリンを100μg/body、1000μg/bodyにて投与した群では、抗原だけを免疫した群と比較して明らかにシトルリンのアジュバント効果が認められ、100μg/body投与群では約1.3倍、1000μg/body投与群では約1.9倍の抗体産生効果が認められた。特に、シトルリンを1000μg/body投与した群では、Alumアジュバントと同等程度のアジュバント効果が認められた。また、シトルリン投与量の増加に応じて抗体の増加も認められた。
(1)シトルリン含有組成物の調製
実施例1の調製方法と同様の方法にて、シトルリンの濃度が異なる組成物5~10を作成した(表2;なお、組成物5はシトルリンを含まない)。
実施例1と同様の方法にて、マウス1匹あたりに組成物5~10を100μL投与し、血清を回収した(マウス1匹当たりの投与量は表2に記載)。
実施例1のELISAと同様の方法で抗体価を測定した。
(4)結果
ELISAの結果を図1に示した。全てのシトルリン投与群においてアジュバント効果が認められ、この範囲内ではある程度の用量依存性が認められた。
抗原として、A型インフルエンザウイルスのM2タンパク質のうちウイルス表面に提示される23アミノ酸からなる領域(M2e、配列番号1:N末-SLLTEVETPIRNEWGCRCNDSSD-C末)において、N末端から数えて20番目と21番目の間、21番目と22番目の間、および22番目と23番目の間にそれぞれシステイン残基を挿入した合成ペプチドM2eC212223(配列番号2:N末-SLLTEVETPIRNEWGCRCNCDCSCSD-C末)を使用した。
マウスへの免疫は次のように行った。BALB/cマウス7週齢の雌を1群5匹、2群に分けた。各個体はアニマルマーカーで個体識別した。被験物はディスポーザブルの1mL用シリンジ(テルモ社製)にとり、マウスの頸背部皮下に1匹あたり0.1mL投与した(マウス1匹当たりの投与量は表3に記載)。被験物を2週間間隔で2回投与し、2回目の投与から1週間後にソムノペンチル(共立製薬)麻酔下で開腹し、腹腔内後大静脈より全採血した。血清分離は実施例1と同様に行った。
(3)ELISAによる抗M2e抗体価の測定
血清中のM2eに対する抗体価を国際公開2011024748号(特許文献4)に記載した方法にて測定した。すなわち、M2eを0.1M Carbonate buffer,pH9.6で2μg/mLに希釈し、96-well plate(Nunc社、Immobilizer Amino)に100μL/well加え、4℃で一夜静置して固相化した。翌日、各wellを300μLの0.05%Tween20含有リン酸緩衝液(PBST)で3回洗浄し、0.1M Carbonate buffer,pH9.6で10mMに希釈したモノエタノールアミン(和光純薬)を300μL/wellずつ添加して、室温で1時間静置した。その後、10mMモノエタノールアミンを十分に除き、PBSTで検体を希釈して100μL/wellにて添加した(各検体ともduplicate)。室温、1時間の反応の後、添加した各希釈血清を捨て、300μL/wellのPBSTで3回洗浄した。洗浄後、well内の洗浄液を十分に除き、PBSTで2000倍希釈したHRP標識抗マウスIgGヤギ抗体(American Qualax社、A131PS)を100μL/well添加し、室温、1時間反応した。反応後、標識抗体希釈液を捨て、300μL/wellのPBSTで2回、同量の蒸留水で2回洗浄し、発色基質液TMB+(Dako社)を100μL/well添加して遮光下、室温で30分間反応した。その後、1N硫酸を100μL/well添加して発色を停止し、450nmの吸光度(OD450値)を測定した。
(4)結果
ELISAの結果を図2に示した。シトルリンをアジュバントとして使用した場合は、シトルリンを含まない場合と比べて、約14倍の抗体産生効果が認められた。これにより、シトルリンはインフルエンザウイルスHA抗原だけでなく、合成ペプチドであるM2eC212223に対してもアジュバント効果を示すことが確認できた。
抗原として、42アミノ酸残基からなるアミロイドβ(Aβ)ペプチド(配列番号3:N末-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA-C末)のうち、N末端より28個のアミノ酸からなるペプチド(配列番号4:N末-DAEFRHDSGYEVHHQKLVFFAEDVGSNK-C末)のC末端にシステイン残基を1個付加した合成ペプチド28AACys(配列番号5:N末-DAEFRHDSGYEVHHQKLVFFAEDVGSNKC-C末)を使用した。
マウスへの免疫は次のように行った。C57BL/6マウス7週齢の雄を1群4匹、8群に分けた。各組成物をマウス1匹あたり200μLずつ1mLツベルクリン用注射器(テルモ、SS-01T2613S)を用い、腹部皮下内に投与した(マウス1匹当たりの投与量は表4に示した)。マウスへの免疫は2週間隔で2回の免疫を行った。2回目免疫から14日目にペントバルビタールナトリウム(共立製薬、ソムノペンチル)麻酔下、腹部大静脈より採血して殺処分した。採取した血液はマイクロティナ(BECTON DICKINSON社)に移し、室温にて十分に凝固させた後、遠心分離した(5000回転、10分間)。分離した血清は各々0.5mLチューブ2本に分注し、測定まで-80℃にて保存した。血清中のAβペプチドに対する抗体を下記のELISA法にて測定した。
Aβペプチド(1-40のアミノ酸配列:N末-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV(配列番号6)、北海道システム・サイエンス社合成)を0.1M Carbonate buffer,pH9.6で10μg/mLに希釈し、8well strips(Nalgen-Nunc社、Immobilizer Amino)に100μL/well加え、4℃で一夜静置して固相化した。翌日、各wellを300μLの0.05%Tween20含有PBS(PBST)で3回洗浄し、10mM ethanolamineを300μL/wellずつ添加して、室温で1時間静置した。その後、10mM ethanolamineを十分に除き、PBSTで検体を50倍~10000倍に希釈して100μL/wellにて添加した(各検体ともduplicate)。室温で1時間の反応の後、添加した各希釈血清を捨て、300μL/wellのPBSTで3回洗浄した。洗浄後、well内の洗浄液を十分に除き、検体希釈液を用いて2000倍希釈したHRP標識抗マウスIgGヤギ抗体(American Qualax社、A131PS)を100μL/wellにて添加し、室温で1時間反応した。反応後、標識抗体希釈液を捨て、300μL/wellのPBSTで2回、同量の蒸留水で2回洗浄し、発色基質液TMB+(Dako社)を100μL/well添加して遮光下、室温で30分間反応した。その後、1N硫酸を100μL/well添加して発色を停止し、450nmの吸光度(OD450値)を測定した。
(4)結果
算出した各免疫群におけるマウス血清中の抗Aβ抗体価を表4に示した。シトルリンを添加した群(組成物13~16、18)では、アジュバント未投与群(比較対照1)と比べて、抗体産生効果が増強していた。すなわち、アジュバント未投与群と比べて、1mg/body投与群で約11.6倍(組成物13)、2mg/body投与群で約42.2倍(組成物14)、4mg/body投与群で約125.8倍(組成物15)、8mg/body投与群で約141.7倍(組成物16)といった極めて顕著な抗体産生効果が認められた。
Claims (56)
- シトルリン類を含有するアジュバント組成物。
- 前記シトルリン類が、L-シトルリン、D-シトルリン、L-チオシトルリン、L-ホモチオシトルリン、S-メチル-L-チオシトルリンおよびS-エチル-L-チオシトルリンからなる群より選択される1種または2種以上である、請求項1に記載のアジュバント組成物。
- 前記シトルリン類が、L-シトルリンまたはD-シトルリンである、請求項2に記載のアジュバント組成物。
- 前記シトルリン類が1mg/mL~50mg/mLで含まれる、請求項1から請求項3のいずれか一項に記載のアジュバント組成物。
- アジュバント組成物が、ペプチド抗原用のアジュバント組成物である、請求項1から請求項4のいずれか一項に記載のアジュバント組成物。
- 抗酸化物質をさらに含む、請求項1から請求項5のいずれか一項に記載のアジュバント組成物。
- 抗酸化物質が、アスコルビン酸である、請求項6に記載のアジュバント組成物。
- 前記シトルリン類と抗酸化物質の重量比が1:2~2:1である、請求項6または請求項7に記載のアジュバント組成物。
- 前記抗酸化物質が1~10mg/mLで含まれる、請求項6から請求項8のいずれか一項に記載のアジュバント組成物。
- 請求項1から請求項9のいずれか一項に記載のシトルリン類を含有するアジュバント組成物と抗原を含有するワクチン組成物。
- 前記抗原とシトルリン類の重量比が1:Nであって、Nが100以上である、ワクチン組成物。
- 前記抗原が、インフルエンザウイルス抗原である、請求項10に記載のワクチン組成物。
- 前記インフルエンザウイルス抗原が、ヘマグルチニン(HA)、ノイラミニダーゼ(NA)、マトリックス1(M1)、マトリックス2(M2)および核タンパク(NP)からなる群より選ばれた少なくとも1種または2種以上の抗原である、請求項12に記載のワクチン組成物。
- 前記インフルエンザウイルス抗原が、ヘマグルチニン(HA)である、請求項13に記載のワクチン組成物。
- 前記インフルエンザウイルス抗原が、マトリックス2(M2)である、請求項13に記載のワクチン組成物。
- 前記インフルエンザウイルス抗原とシトルリン類の重量比が1:Nであって、Nが300以上である、請求項12から請求項15のいずれか一項に記載のワクチン組成物。
- 前記抗原がペプチドである、請求項10に記載のワクチン組成物。
- 前記ペプチドとシトルリン類の重量比が1:Nであって、Nが100以上である、請求項17に記載のワクチン組成物。
- 前記ペプチドが、M2eペプチド、M2eペプチドに1もしくは数個のシステインが付加もしくは挿入されたペプチド、またはM2eペプチドにシステインを含むペプチドが付加されたペプチドである、請求項17に記載のワクチン組成物。
- 前記ペプチドが、M2eペプチドにおいて、N末端から数えて20番目と21番目の間、21番目と22番目の間、および22番目と23番目の間にそれぞれシステイン残基を挿入した合成ペプチドである、請求項19に記載のワクチン組成物。
- 前記M2eペプチドとシトルリン類の重量比が1:Nであって、Nが200以上である、請求項19または請求項20に記載のワクチン組成物。
- 前記ペプチドが、アミロイドβ(Aβ)ペプチド、アミロイドβ(Aβ)ペプチドの一部のアミノ酸配列からなるペプチド、または前記ペプチドに1もしくは数個のシステインが付加もしくは挿入されたペプチドである、請求項17に記載のワクチン組成物。
- 前記ペプチドがアミロイドβ(Aβ)ペプチドのN末端より28個のアミノ酸からなるペプチドのC末端にシステイン残基を1個付加したペプチドである、請求項22に記載のワクチン組成物。
- 前記アミロイドβペプチドとシトルリン類の重量比が1:Nであって、Nが100以上である、請求項22または請求項23に記載のワクチン組成物。
- 前記抗原が配列番号1、配列番号2、配列番号3、配列番号4および配列番号5からなる群より選ばれた少なくとも1種または2種以上のアミノ酸配列からなるペプチドである、請求項10に記載のワクチン組成物。
- ワクチンにアジュバントとしてのシトルリン類を添加することからなる、該ワクチンに含まれる抗原に対する抗体産生能の増強したワクチンの製造方法。
- 前記シトルリン類が、L-シトルリン、D-シトルリン、L-チオシトルリン、L-ホモチオシトルリン、S-メチル-L-チオシトルリンおよびS-エチル-L-チオシトルリンからなる群より選択される1種または2種以上である、請求項26に記載の方法。
- 前記シトルリン類が、L-シトルリンまたはD-シトルリンである、請求項27に記載の方法。
- 前記シトルリン類を1mg/mL~50mg/mLで添加する、請求項26から28のいずれかに記載の方法。
- 前記抗原とシトルリン類の重量比が1:Nであって、Nが100以上である、請求項26から29のいずれかに記載の方法。
- 前記抗原が、インフルエンザウイルス抗原である、請求項26から29のいずれかに記載の方法。
- 前記インフルエンザウイルス抗原が、ヘマグルチニン(HA)、ノイラミニダーゼ(NA)、マトリックス1(M1)、マトリックス2(M2)および核タンパク(NP)からなる群より選ばれた少なくとも1種または2種以上の抗原である、請求項31に記載の方法。
- 前記インフルエンザウイルス抗原が、ヘマグルチニン(HA)である、請求項32に記載の方法。
- 前記インフルエンザウイルス抗原が、マトリックス2(M2)である、請求項32に記載の方法。
- 前記インフルエンザウイルス抗原とシトルリン類の重量比が1:Nであって、Nが300以上である、請求項31から34のいずれかに記載の方法。
- 前記抗原がペプチドである、請求項26に記載の方法。
- 前記ペプチドとシトルリン類の重量比が1:Nであって、Nが100以上である、請求項36に記載の方法。
- 前記ペプチドが、M2eペプチド、M2eペプチドに1もしくは数個のシステインが付加もしくは挿入されたペプチド、またはM2eペプチドにシステインを含むペプチドが付加されたペプチドである、請求項36に記載の方法。
- 前記ペプチドが、M2eペプチドにおいて、N末端から数えて20番目と21番目の間、21番目と22番目の間、および22番目と23番目の間にそれぞれシステイン残基を挿入した合成ペプチドである、請求項38に記載の方法。
- 前記M2eペプチドとシトルリン類の重量比が1:Nであって、Nが200以上である、請求項38または39に記載の方法。
- 前記ペプチドが、アミロイドβ(Aβ)ペプチド、アミロイドβ(Aβ)ペプチドの一部のアミノ酸配列からなるペプチド、または前記ペプチドに1もしくは数個のシステインが付加もしくは挿入されたペプチドである、請求項36に記載の方法。
- 前記ペプチドがアミロイドβ(Aβ)ペプチドのN末端より28個のアミノ酸からなるペプチドのC末端にシステイン残基を1個付加したペプチドである、請求項41に記載の方法。
- 前記アミロイドβペプチドとシトルリン類の重量比が1:Nであって、Nが100以上である、請求項41または42に記載の方法。
- 前記抗原が配列番号1、配列番号2、配列番号3、配列番号4、および配列番号5からなる群より選ばれた少なくとも1種または2種以上のアミノ酸配列からなるペプチドである、請求項26に記載の方法。
- シトルリン類とともに抗酸化物質をも添加する、請求項26から44のいずれかに記載の方法。
- 前記抗酸化物質が、アスコルビン酸である、請求項45に記載の方法。
- 前記シトルリン類と抗酸化物質を重量比1:2~2:1で添加する、請求項45または46に記載の方法。
- 前記抗酸化物質を1~10mg/mLで添加する、請求項45から47のいずれかに記載の方法。
- アジュバントとしてのシトルリン類の使用。
- 前記シトルリン類が、L-シトルリン、D-シトルリン、L-チオシトルリン、L-ホモチオシトルリン、S-メチル-L-チオシトルリンおよびS-エチル-L-チオシトルリンからなる群より選択される1種または2種以上である、請求項49に記載の使用。
- 前記シトルリン類が、L-シトルリンまたはD-シトルリンである、請求項50に記載の使用。
- 1mg/mL~50mg/mLの前記シトルリン類をワクチンに添加する、請求項49から51のいずれかに記載の使用。
- 抗酸化物質を併用する、請求項49から52のいずれかに記載の使用。
- 前記抗酸化物質が、アスコルビン酸である、請求項53に記載の使用。
- 前記シトルリン類と抗酸化物質を重量比1:2~2:1でワクチンに添加する、請求項53または54に記載の使用。
- 1~10mg/mLの前記抗酸化物質をワクチンに添加する、請求項53から55のいずれかに記載の使用。
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US10100008B2 (en) | 2014-04-25 | 2018-10-16 | Ajinomoto Co., Inc. | Immunostimulating agent |
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WO2020138217A1 (ja) * | 2018-12-26 | 2020-07-02 | 大日本住友製薬株式会社 | ワクチンアジュバントを含む製剤 |
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US10100008B2 (en) | 2014-04-25 | 2018-10-16 | Ajinomoto Co., Inc. | Immunostimulating agent |
EP3395365A1 (en) | 2017-04-28 | 2018-10-31 | Ajinomoto Co., Inc. | Immunostimulating agent |
WO2020138217A1 (ja) * | 2018-12-26 | 2020-07-02 | 大日本住友製薬株式会社 | ワクチンアジュバントを含む製剤 |
JPWO2020138217A1 (ja) * | 2018-12-26 | 2021-11-11 | 大日本住友製薬株式会社 | ワクチンアジュバントを含む製剤 |
JP7410883B2 (ja) | 2018-12-26 | 2024-01-10 | 住友ファーマ株式会社 | ワクチンアジュバントを含む製剤 |
Also Published As
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JP5914458B2 (ja) | 2016-05-11 |
EP2684572A1 (en) | 2014-01-15 |
US20170239348A1 (en) | 2017-08-24 |
CN103561764B (zh) | 2015-09-16 |
US10556005B2 (en) | 2020-02-11 |
JPWO2012124631A1 (ja) | 2014-07-24 |
US20140065179A1 (en) | 2014-03-06 |
CN103561764A (zh) | 2014-02-05 |
US9381242B2 (en) | 2016-07-05 |
TR201910409T4 (tr) | 2019-08-21 |
EP2684572A4 (en) | 2014-09-10 |
EP2684572B1 (en) | 2019-05-22 |
KR20140012115A (ko) | 2014-01-29 |
ES2731636T3 (es) | 2019-11-18 |
KR101847848B1 (ko) | 2018-04-12 |
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