WO2017170712A1 - Low-molecular-weight-compound adjuvant and vaccine using same - Google Patents

Abstract

The present invention addresses the problem of providing a chemosynthetic adjuvant for selectively inducing TSLP. The present invention provides a vaccine adjuvant including a RAR γ agonist, and a vaccine obtained by combining the vaccine adjuvant and an antigen.

Description

Low molecular weight compound adjuvant and vaccine using the same

The present invention relates to a low molecular compound adjuvant and a vaccine using the same.

Recently, threats such as re-emerging infectious diseases and new infectious diseases are increasing, but the most effective countermeasure is the administration of vaccines.

Conventionally, inactivated or attenuated pathogens are used for many vaccines, but quality control is difficult and there are problems such as non-specific inflammation and side effects. Therefore, an adjuvant made of a low molecular weight compound is eagerly desired.

On the other hand, Thymic Stromal Lymphopoietin (TSLP) is a cytokine mainly produced by epithelial cells and activates dendritic cells to enhance humoral immunity. It has been reported that TSLP protein itself enhances antibody production against HIV antigen, and that TSLP protein itself can be used as an adjuvant (Non-patent Documents 1 and 2). However, for the same reason as described above, it is desirable not to administer the TSLP protein itself but to use a low molecular weight compound that induces the TSLP protein.

So far, the present inventor has found that a plurality of low molecular compounds such as xylene induce TSLP (Non-patent Document 3).
However, in order to use it as an adjuvant, it not only induces TSLP from the viewpoint of reducing side effects, but also other proteins (particularly TNF-α, IL-1β, IL-4 etc. involved in allergic reactions) It is important to selectively induce TSLP, not protein). Conventionally, certain cytokines cannot be selectively induced not only by TSLP but also by low molecular weight compounds, and such low molecular weight compounds have been desired.

Eur.J.Immnol. 2012. 42: 293-295 (2012) Eur.J.Immnol. 2012. 42: 353-363 (2012) International Archives of Allergy and Immunology 157 (2): 194-201 ・ February 2012 Immunobiology 221 (2016) 161-165

An object of the present invention is to provide a chemically synthesized adjuvant that selectively induces TSLP.

Under such circumstances, the present inventor has found that, among a wide variety of low molecular weight compounds, an agonist of RARγ selectively induces TSLP and can produce neutralizing antibodies when formulated in a vaccine. Based on this finding, the present inventor has further studied the timing of administration of an RARγ agonist and the antigen contained in the vaccine, and as a result, completed the present invention.

Accordingly, the present invention provides the inventions described in the following sections:
Item 1. A vaccine adjuvant comprising an agonist of RARγ.

Item 2. Item 2. The vaccine adjuvant according to Item 1, wherein the RARγ agonist is a retinoid.

Item 3. Item 3. The vaccine adjuvant according to Item 1 or 2, for use in an administration schedule in which the antibody is administered at least 4 hours after administration of the RARγ agonist.

Item 4. Item 4. The vaccine adjuvant according to Item 3, wherein the RARγ agonist is administered as an external preparation.

Item 5. Target diseases are influenza, HIV, chlamydia, malaria, tuberculosis, tumor, measles, measles, mumps, chickenpox, yellow fever, rotavirus gastroenteritis, smallpox, pneumococci, rabies, cholera, diphtheria, pertussis, Item 5. Any one of Items 1 to 4, which is at least one selected from the group consisting of tetanus, Japanese encephalitis, papillomavirus, hepatitis (types A, B, C) and Ebola hemorrhagic fever, dengue fever, Zika fever, etc. Vaccine adjuvant.

Item 6. Item 3. A vaccine comprising a combination of the vaccine adjuvant according to Item 1 or 2 and an antigen.

Item 7. Item 7. The vaccine according to Item 6, for use in an administration schedule in which the antibody is administered at least 4 hours after administration of the RARγ agonist.

Item 8. Item 8. The vaccine according to Item 7, wherein the RARγ agonist is administered as an external preparation.

Item 9. Target diseases are influenza, HIV, chlamydia, malaria, tuberculosis, tumor, measles, measles, mumps, chickenpox, yellow fever, rotavirus gastroenteritis, smallpox, pneumococci, rabies, cholera, diphtheria, pertussis, Item 9. The item according to any one of Items 6 to 8, which is at least one selected from the group consisting of tetanus, Japanese encephalitis, papillomavirus, hepatitis (A type, B type, C type) and Ebola hemorrhagic fever, dengue fever, Zika fever, etc. Vaccine.

Item 10. Item 9. The vaccine according to any one of Items 6 to 8, wherein the RARγ agonist is a retinoid and the target disease is influenza.

Item 11. 3. A combination of an antigen and a vaccine adjuvant according to item 1 or 2 for use as a vaccine.

Item 12. Item 12. The combination according to Item 11, wherein the RARγ agonist is a retinoid and the target disease is influenza.

Item 13. Item 12. The combination according to Item 11, wherein the RARγ agonist is a retinoid.

Item 14. Item 14. The combination according to any one of Items 11 to 13, for use in an administration schedule in which the antibody is administered at least 4 hours after administration of the RARγ agonist.

Item 15. Item 15. The combination according to Item 14, wherein the RARγ agonist is administered as an external preparation.

Item 16. Target diseases are influenza, HIV, chlamydia, malaria, tuberculosis, tumor, measles, measles, mumps, chickenpox, yellow fever, rotavirus gastroenteritis, smallpox, pneumococci, rabies, cholera, diphtheria, pertussis, Any one of Items 11, 13 to 15, which is at least one selected from the group consisting of tetanus, Japanese encephalitis, papillomavirus, hepatitis (types A, B, C) and Ebola hemorrhagic fever, dengue fever, Zika fever, etc. Combination described in.

Item 17. 3. Use of an antigen and the vaccine adjuvant according to item 1 or 2 for producing a vaccine.

Item 18. The use according to Item 17, wherein the agonist of RARγ is a retinoid and the target disease is influenza.

Item 19. Item 18. The use according to Item 17, wherein the agonist of RARγ is a retinoid.

Item 20. Item 20. The use according to any one of Items 17 to 19, for use in an administration schedule in which the antibody is administered at least 4 hours after administration of the RARγ agonist.

Item 21. Item 21. The use according to Item 20, wherein the RARγ agonist is administered as an external preparation.

Item 22. Target diseases are influenza, HIV, chlamydia, malaria, tuberculosis, tumor, measles, measles, mumps, chickenpox, yellow fever, rotavirus gastroenteritis, smallpox, pneumococci, rabies, cholera, diphtheria, pertussis, Any one of Items 17, 19 to 21, which is at least one selected from the group consisting of tetanus, Japanese encephalitis, papillomavirus, hepatitis (A type, B type, C type) and Ebola hemorrhagic fever, dengue fever, Zika fever, etc. Use as described in.

Item 23. A method for preventing or treating a disease, comprising a step of administering a vaccine adjuvant according to Item 1 or 2 to a mammal, and a step of administering an antigen to the mammal.

Item 24. The method according to Item 23, wherein the agonist of RARγ is a retinoid and the target disease is influenza.

Item 25. Item 24. The method according to Item 23, wherein the RARγ agonist is a retinoid.

Item 26. Item 26. The method according to any one of Items 23 to 25, for use in an administration schedule in which the antibody is administered at least 4 hours after administration of the RARγ agonist.

Item 27. Item 27. The method according to Item 26, wherein the RARγ agonist is administered as an external preparation.

Item 28. Target diseases are influenza, HIV, chlamydia, malaria, tuberculosis, tumor, measles, measles, mumps, chickenpox, yellow fever, rotavirus gastroenteritis, smallpox, pneumococci, rabies, cholera, diphtheria, pertussis, Item 23, 25 to 27, which is at least one selected from the group consisting of tetanus, Japanese encephalitis, papillomavirus, hepatitis (types A, B, C) and Ebola hemorrhagic fever, dengue fever, zika fever, etc. The method described in 1.

According to the present invention, a chemically synthesized adjuvant capable of producing neutralizing antibodies when TSLP is selectively induced and blended with a vaccine can be provided.

The result of Example 1 is shown. * P <0.05, ** P <0.01, *** P <0.001. The result of Example 2 is shown. The result of Example 3 is shown. The result of Example 4 is shown. The result of Example 5 is shown. The result of Example 6 is shown. ** P <0.01, *** P <0.001. The result of Example 7 is shown. The result of Example 9 is shown. The result of Example 10 is shown.

Vaccine Adjuvant The present invention provides a vaccine adjuvant comprising an agonist of retinoic acid receptor γ (RARγ). Retinoic acid receptors (RAR) and retinoid X receptors (RXR) are known as retinoic acid receptors. In the present invention, an agonist of RARγ means a compound having agonist activity for RARγ belonging to the retinoic acid receptor (RAR) among these receptors.

In the present invention, RARγ agonists include retinoids and RARγ agonists other than retinoids, and retinoids are preferred. In the present invention, examples of the retinoid include retinoic acid or a salt thereof, retinal, retinol and the like, and retinoic acid or a salt thereof is preferable. The retinoin chain or a salt thereof typically has the following formula:

All-trans-retinoic acid (atRA) represented by: 9-cis-retinoic acid, 13-cis-retinoic acid, 9,13-cis-retinoic acid, and salts thereof.

RARγ agonists other than retinoids include 3-fluoro-4-[[2-hydroxy-2- (5,5,8,8-tetramethyl-5,6,7,8, -tetrahydro-2-naphthalenyl) acetyl ] Amino] -benzoic acid (3-fluoro-4-[[2-hydroxy-2- (5,5,8,8-tetramethyl-5,6,7,8, -tetrahydro-2-naphthalenyl) acetyl] amino ] -benzoic acid) (BMS189961, CAS number: 185629-22-5) or a salt thereof; 3-fluoro-4-[[(2R) -hydroxy-2- (5,5,8,8-tetramethyl-5 , 6,7,8, -Tetrahydro-2-naphthalenyl) acetyl] amino] -benzoic acid (3-fluoro-4-[[(2R) -hydroxy (5,5,8,8-tetramethyl-5,6, 7,8-tetrahydro-2-naphthalenyl) acetyl] amino] -benzoic acid (BMS-270394, CAS number: 262433-54-5) or a salt thereof.

When the agonist of RARγ, which is the active ingredient of the present invention, is in the form of a salt, the salt includes an acid addition salt and a salt with a base. Specific examples of acid addition salts include hydrochloride, hydrobromide, hydroiodide, sulfate, perchlorate, phosphate and other inorganic acid salts, oxalate, malonate, succinic acid Salt, maleate, fumarate, lactate, malate, citrate, tartrate, benzoate, trifluoroacetate, acetate, methanesulfonate, p-toluenesulfonate, trifluoromethane Examples include organic acid salts such as sulfonates, and acidic amino acid salts such as glutamates and aspartates. Specific examples of salts with bases include alkali metal or alkaline earth metal salts such as sodium salt, potassium salt or calcium salt, salts with organic bases such as pyridine salt and triethylamine salt, bases such as lysine and arginine. And salts with sexual amino acids.

These RARγ agonists are known per se or can be appropriately produced from known compounds. In addition, these RARγ agonists can be used alone or in combination of two or more. In the present invention, the case where the agonist of RARγ is present in the form of hydrate or solvate is also included. When the RARγ agonist can give stereoisomers, geometric isomers and the like, unless otherwise specified, the stereoisomers and geometric isomers are included in the RARγ agonists which are the active ingredients of the present invention.

In the present invention, the active ingredient RARγ agonist itself may be used as an adjuvant, but various pharmaceutically acceptable carriers (for example, isotonic agents, chelating agents, stabilizers, pH regulators, preservatives). , Antioxidants, solubilizers, thickeners, etc.) may be used as an adjuvant composition. Further, in the present invention, it is preferable to use a RARα agonist in combination with a RARγ agonist because a synergistic action can be brought about.

The RARα agonist is not particularly limited, and examples thereof include 4-[[(2,3-dihydro-1,1,3,3-tetramethyl-2-oxo-1H-indene-5-nyl) carbonyl] amino]. -Benzoic acid (4-[[(2,3-Dihydro-1,1,3,3-tetramethyl-2-oxo-1H-inden-5-yl) carbonyl] amino] -benzoic acid) (BMS753, CAS number : 215307-86-1); 4-[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) amino] carbonyl] benzoic acid salt (4-[[( 5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) amino] carbonyl] benzoic acid) (Tamibarotene, CAS 94497-51-5), 4-[(5,6, 7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) carboxamide] benzoic acid (4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl- 2-naphthalenyl) carboxamido] benzoic acid (CAS-102121-60-8). The blending ratio of the RARγ agonist and the RARα agonist is not particularly limited. For example, the latter is in the range of 0.1 to 10 mol, preferably 0.5 to 6 mol, preferably 1 to 5 mol with respect to 1 mol of the former. Can be set as appropriate.

Examples of isotonic agents include sugars such as glucose, trehalose, lactose, fructose, mannitol, xylitol, and sorbitol, polyhydric alcohols such as glycerin, polyethylene glycol, and propylene glycol, sodium chloride, potassium chloride, and calcium chloride. Examples include inorganic salts. These tonicity agents can be used alone or in combination of two or more.

Examples of chelating agents include edetate salts such as disodium edetate, disodium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, ethylenediaminetetraacetate, nitrilotriacetic acid or salts thereof, hexametalin Examples include acid soda and citric acid. These chelating agents can be used alone or in combination of two or more.

Examples of the stabilizer include sodium bisulfite.

Examples of the pH regulator include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, and further alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates such as sodium carbonate, or hydrogen carbonate. Salts, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, bases such as trometamol, and the like. These pH regulators can be used singly or in combination of two or more.

Examples of preservatives include paraoxybenzoic acid esters such as sorbic acid, potassium sorbate, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and butyl paraoxybenzoate, chlorhexidine gluconate, benzalkonium chloride, chloride Quaternary ammonium salts such as benzethonium and cetylpyridinium chloride, alkyl polyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, chlorhexidine and the like can be mentioned. These preservatives can be used singly or in combination of two or more.

Examples of the antioxidant include sodium bisulfite, dry sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherol and the like. These antioxidants can be used alone or in combination of two or more.

Examples of the solubilizer include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, D-mannitol and the like.
Examples of the thickening agent include polyethylene glycol, methyl cellulose, ethyl cellulose, carmellose sodium, xanthan gum, sodium chondroitin sulfate, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol and the like. These solubilizing agents can be used alone or in combination of two or more. These thickening agents can be used alone or in combination of two or more.

In addition to the RARγ agonist, the adjuvant composition may further contain a compound known to be used as an adjuvant.

In the embodiment of the adjuvant composition, the content of the RARγ agonist in the composition is not particularly limited, and is, for example, 90% by mass or more, 70% by mass or more, 50% by mass or more in terms of the content of the RARγ agonist. It can be appropriately set from conditions such as 30% by mass or more, 10% by mass or more, 5% by mass or more, 1% by mass or more.

In the present invention, the dosage form for administering the agonist of RARγ is not particularly limited, and for example, oral administration agents such as tablets, pills, capsules, powders, granules, syrups; injections (intravenous injection, intramuscular injection) , Topical injections, etc.), mouthwashes, drops, external preparations (ointments, creams, patches, inhalants), parenteral administration agents such as suppositories, and the like. Among the above-mentioned preparation forms, preferable examples include oral administration agents, injections, and external preparations. The agonist of RARγ, which is an active ingredient of the present invention, can exert TSLP production-inducing action even by application. From the viewpoints of low invasiveness, suppression of non-specific adjuvant effect expression (adjuvant effect near the administration site can be expected), convenience, etc., it is particularly preferable to use it as an external preparation such as an ointment, cream or patch. . When administering as an external preparation, the administration site is not particularly limited, and can be used at a place where the external preparation is usually applied. Examples thereof include skin, oral cavity, and the like, and skin is preferable.

The vaccine adjuvant of the present invention can be used by combining with an antigen to make a vaccine and administering it to a subject such as a mammal. Accordingly, the present invention also provides a method for preventing or treating a disease, comprising the steps of administering the vaccine adjuvant to a mammal and administering an antigen to the mammal. As will be described later, the method of the present invention includes not only a method of administering the vaccine adjuvant and the antigen at intervals, but also a method of administering both at the same time. Therefore, in the method of the present invention, the step of administering the vaccine adjuvant to a mammal and the step of administering an antigen to the mammal are performed separately (for example, before, simultaneously with and / or after the adjuvant administration step). And a method of performing both steps simultaneously.
Examples of mammals include humans, monkeys, mice, rats, rabbits, cats, dogs, pigs, cows, horses, sheep and the like. The vaccine adjuvant of the present invention can be used for any of the conventional diseases for vaccine treatment. Examples of typical target diseases include influenza (types A, B and C), HIV, chlamydia, malaria, tuberculosis, tumor, measles, mumps, chickenpox, yellow fever, rotavirus gastroenteritis, smallpox, pneumococci, rabies, cholera, diphtheria, pertussis, tetanus, Japanese encephalitis, papillomavirus , Hepatitis (A type, B type, C type) and Ebola hemorrhagic fever, dengue fever, Zika fever, and combinations of these diseases. The present invention can be used for the prevention and treatment of the above diseases, particularly for prevention.

The content of the RARγ agonist in the preparation varies depending on the administration route, patient age, body weight, symptoms, etc., and cannot be specified unconditionally, but the RARγ agonist single dose is usually about 0.001 to 10 mg.

In the present invention, the RARγ agonist, which is an active ingredient of the adjuvant, may be administered at the same time as the vaccine, but is preferably administered separately at regular intervals. The present inventor has found that when a RARγ agonist is administered to a patient, the TSLP production-inducing action is maximized after a certain period of time (typically, about 24 hours after administration in the case of an external preparation). It was. Therefore, in a preferred embodiment of the present invention, the vaccine adjuvant of the present invention comprises at least 4 hours, preferably at least 12 hours, more preferably 23 hours, more preferably 24 hours after administration of the RARγ agonist. It can be used in an administration schedule in which an antigen is administered after the course.

Further, in a preferred embodiment of the present invention, the vaccine adjuvant of the present invention is administered with an antigen before the lapse of 50 hours, preferably before the lapse of 36 hours, more preferably before the lapse of 25 hours after administration of the RARγ agonist. Can be used in an administration schedule. In an embodiment of an external preparation such as an ointment, cream or patch, the active ingredient is continuously absorbed from the skin for a predetermined time after application of the ointment or cream or after application of the patch. In this case, in the present invention, “after elapse of t time after administration of RARγ agonist” means after elapse of t time from the time of applying ointment or cream or the time of applying the patch.

In the present invention, when the antigen is divided into a plurality of days (typically at intervals of several days) and administered, a RARγ agonist is administered to at least one of the plurality of antigen administrations. However, it is preferable to administer an agonist of RARγ for all of the multiple administrations of antigen.

The antigen is not particularly limited, and examples thereof include carbohydrates, lipids, glycolipids, phospholipids, polypeptides, proteins, glycoproteins, lipoproteins, oligonucleotides, polynucleotides, and chemical or recombinant conjugates thereof. Can be mentioned. Antigens also include viruses, fungi, bacteria, parasitic microorganisms, allergens, self-molecules and the like. Examples of antigens used for influenza vaccines include hemagglutinin (HA), neuraminidase (NA), M1 protein, M2 protein, and nucleoprotein (NP). These antigens can be used alone or in combination of two or more.

By using the vaccine adjuvant of the present invention, TSLP can be selectively induced and humoral immunity can be mainly enhanced. Further, in the present invention, it is considered that RARγ agonists selectively induce TSLP using epithelial cells as an action point, not dendritic cells, lymphocytes and the like.

Vaccine The present invention provides a vaccine comprising a combination of the aforementioned vaccine adjuvant and the aforementioned antigen. The vaccine of the present invention may be in the form of a preparation containing both the vaccine adjuvant and the antigen, or may be in the form of a kit provided with the vaccine adjuvant and the preparation containing the antigen separately. In the present invention, “a vaccine comprising a combination of a vaccine adjuvant and an antigen” includes both of the above forms.

In the present invention, a vaccine preparation containing both a vaccine adjuvant and the antigen, or an antigen-containing preparation provided in a vaccine kit is prepared by using various pharmaceutically acceptable carriers (for example, isotonic agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizers, thickeners, etc.) may be further blended.

Examples of isotonic agents include sugars such as glucose, trehalose, lactose, fructose, mannitol, xylitol, and sorbitol, polyhydric alcohols such as glycerin, polyethylene glycol, and propylene glycol, sodium chloride, potassium chloride, and calcium chloride. Examples include inorganic salts. These tonicity agents can be used alone or in combination of two or more.

Examples of chelating agents include edetate salts such as disodium edetate, disodium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, ethylenediaminetetraacetate, nitrilotriacetic acid or salts thereof, hexametalin Examples include acid soda and citric acid. These chelating agents can be used alone or in combination of two or more.

Examples of the stabilizer include sodium bisulfite.

Examples of the pH regulator include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, and further alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates such as sodium carbonate, or hydrogen carbonate. Salts, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, bases such as trometamol, and the like. These pH regulators can be used singly or in combination of two or more.

Examples of preservatives include paraoxybenzoic acid esters such as sorbic acid, potassium sorbate, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and butyl paraoxybenzoate, chlorhexidine gluconate, benzalkonium chloride, chloride Quaternary ammonium salts such as benzethonium and cetylpyridinium chloride, alkyl polyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, chlorhexidine and the like can be mentioned. These preservatives can be used singly or in combination of two or more.

Examples of the antioxidant include sodium bisulfite, dry sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherol and the like. These antioxidants can be used alone or in combination of two or more.

Examples of the solubilizer include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, D-mannitol and the like.
Examples of the thickening agent include polyethylene glycol, methyl cellulose, ethyl cellulose, carmellose sodium, xanthan gum, sodium chondroitin sulfate, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol and the like. These solubilizing agents can be used alone or in combination of two or more. These thickening agents can be used alone or in combination of two or more.

Among the present invention, in the embodiment of the vaccine preparation containing both the vaccine adjuvant and the antigen, the upper limit of the content of the antigen in the preparation is not particularly limited, but the antigen amount can be reduced by using the method of the present invention. For example, 1 mass% or less, preferably 0.1 mass% or less, more preferably 0.01 mass% or less, more preferably 0.001 mass% or less. The lower limit of the antigen content in the preparation is not particularly limited, but it can be preferably set based on conditions such as 0.1% by mass or more, 0.001% by mass or more, 0.000001% by mass or more.

In the present invention, the dosage form of the vaccine preparation is not particularly limited. For example, oral administration agents such as tablets, pills, capsules, powders, granules, syrups; injections (intravenous injection, intramuscular injection, local injection, etc.) And various preparation forms such as a mouthwash, an instillation, an external preparation (ointment, cream, patch, inhalant), a parenteral preparation such as a suppository, and the like. Among the above-mentioned preparation forms, preferable examples include oral administration agents, injections, and external preparations. The antigen content in the preparation varies depending on the route of administration, patient age, body weight, symptoms, etc., and cannot be generally specified, but the daily dose of the antigen is usually about 10 to 5000 mg, more preferably about 100 to 1000 mg. It can be an amount. When it is administered once a day, it is sufficient that this amount is contained in one preparation, and when it is administered three times a day, it is sufficient that this one-third amount is contained in one preparation.

In the embodiment of the vaccine kit, the content of the antigen in the preparation is not particularly limited, and can be appropriately set, for example, from an amount equivalent to a normal use amount to an amount of about 1/1000. In another preferred embodiment of the present invention, the upper limit of the content of the antigen in the preparation is, for example, 1% by mass or less, preferably 0.1% by mass or less, more preferably 0.01% by mass or less, more preferably It can be set from conditions such as 0.001% by mass or less. In one preferred embodiment, the lower limit of the content of the antigen in the preparation can be set, for example, based on conditions such as preferably 0.1% by mass or more, 0.001% by mass or more, 0.000001% by mass or more.

The formulation form of the antigen preparation provided in the vaccine kit in the present invention is not particularly limited, and is an oral administration agent such as a tablet, pill, capsule, powder, granule, syrup, etc .; injection (intravenous injection, intramuscular injection) , Topical injections, etc.), mouthwashes, drops, external preparations (ointments, creams, patches, inhalants), parenteral administration agents such as suppositories, and the like. Among the above-mentioned preparation forms, preferable examples include injections.

The antigen content in the preparation varies depending on the route of administration, patient age, body weight, symptoms, etc., and cannot be generally specified, but the daily dose of the antigen is usually about 10 to 5000 mg, more preferably about 100 to 1000 mg. It can be an amount. When it is administered once a day, it is sufficient that this amount is contained in one preparation, and when it is administered three times a day, it is sufficient that this one-third amount is contained in one preparation.

Also, in the embodiment of the vaccine kit, the present invention can include other components as necessary. Examples of other components include, but are not limited to, a tool for collecting an adjuvant (for example, a syringe) and the like. Moreover, the document etc. which wrote down the above-mentioned administration schedule can also be included.

The target disease, administration schedule, etc. of the vaccine of the present invention are the same as those described above for the vaccine adjuvant of the present invention. Of the vaccines according to the present invention, those in which the RARγ agonist is a retinoid and the target disease is influenza are preferred.

The present invention is not limited to compounds that have TSLP-producing ability, and RARγ agonists are not only capable of producing TSLP but also other proteins (particularly TNF-α, IL-1β, IL-4 involved in allergic reactions). It has been found that TSLP can be selectively induced over a protein such as a protein, and that neutralizing antibodies are produced when it is actually mixed with a vaccine and administered to a subject.

On the other hand, Non-Patent Document 4 (Immunobiology 221 (2016) 161-165) merely describes that retinoic acid induced TSLP, and selectively used TSLP rather than other proteins. There is also no disclosure about guiding to this. In the first place, the non-patent document 4 is not limited to the measurement of blood TSLP in mice sensitized with ovalbumin, and the administration of TSLP together with an antigen associated with some disease is also neutralizing the disease. There is no specific disclosure that the antibody was produced. Therefore, it cannot be said that Non-Patent Document 4 describes that a vaccine containing retinoic acid can be used.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

Example 1 TSLP expression inducing action of retinoic acid (FIG. 1)
20 μl of acetone solution containing 0.2 to 20 nmol of All-trans retinoic acid (atRA, Sigma Aldrich) was applied to the auricle of ICR mice (Japan SLC, male, SPF, 6 weeks old). After 24 hours, the mice were exsanguinated and the auricles were collected. Weigh it, put it in a sample tube (TOMY), add 10 times the volume (w / v) of homogenizing buffer, and use a bead-type cell crusher (Precyllys 24, Bertin tech.). Homogenization was performed twice for 30 seconds. The obtained homogenate was centrifuged at 21,600 × g, 4 ° C. for 20 minutes, and the amount of TSLP in the supernatant was measured with an ELISA kit (R & D Systems) (left). In addition, the collected skin sample was homogenized in 500 μl of RNAiso Plus (Takara Bio) in the same manner using a bead-type cell crusher, and then RNA was extracted according to the protocol attached to RNAiso Plus. The concentration of the obtained RNA solution was measured using NanoDrop ^(R) spectrophotometer ND-1000 (Thermo Fisher Scientific). Thereafter, in accordance with ^{PrimeScript (R) RT Master Mix (} perfect Real Time) accessory protocol, to prepare a cDNA solution using a ^{PCR thermal Cycler Dice (R) Gradient} ( Takara Bio). In Thermal Cycle Dice ^TM Real Time System (Takara Bio), using ^{SYBR (R) Premix Ex Taq TM} II (Tli RNaseH Plus), was carried out PCR reactions. The following primers were used.
TSLP
Forward: 5'-AGCTTGTCTCCTGAAAATCGAG -3 '(SEQ ID NO: 1) Reverse: 5'-AGGTTTGATTCAGGCAGATGTT -3' (SEQ ID NO: 2) GAPDH
Forward: 5′-TGT GTC CGT CGT GGA TCT GA-3 ′ (SEQ ID NO: 3) Reverse: 5′-TTG CTG TTG AAG TCG CAG GAG-3 ′ (SEQ ID NO: 4)

The threshold cycle (Ct) value of each sample was calculated by the 2nd derivative maximum method, and after creating a relative calibration curve and confirming that the PCR reaction conditions for each gene were close to 100% amplification efficiency, RNA was obtained by the Ct method. The amount was converted into a relative amount (right side of FIG. 1).

As is clear from FIG. 1, atRA increased TSLP expression in a concentration-dependent manner at the protein level (FIG. 1, left) and the mRNA level (FIG. 1, right).

Example 2 Temporal change of TSLP production by retinoic acid (FIG. 2)
All-trans retinoic acid (atRA, Sigma Aldrich) was applied to the auricle of ICR mice (Japan SLC, male, SPF, 6 weeks old) with acetone solution containing 2 nmol or 20 μl of acetone. After a certain period of time, the mice were exsanguinated and the auricles were collected. Weigh it, put it in a sample tube (TOMY), add 10 times the volume (w / v) of homogenizing buffer, and use a bead-type cell crusher (Precyllys 24, Bertin tech.). Homogenization was performed twice for 30 seconds. The obtained homogenate was centrifuged at 21,600 × g, 4 ° C. for 20 minutes, and the amount of TSLP in the supernatant was measured by ELISA kit (R & D Systems). atRA group, black circle, acetone group, white circle.

As is clear from FIG. 2, TSLP production by atRA reached a maximum at 24 hours after application and then decreased.

Example 3 TSLP selective expression inducing action of retinoic acid (FIG. 3)
All-trans retinoic acid (atRA, Sigma Aldrich) was applied to the auricle of ICR mice (Japan SLC, male, SPF, 6 weeks old) with acetone solution containing 2 nmol or 20 μl of acetone. Mice were bled to death after 8 and 24 hours, and the auricles were collected. Weigh it, put it in a sample tube (TOMY), add 10 times the volume (w / v) of homogenizing buffer, and use a bead-type cell crusher (Precyllys 24, Bertin tech.). Homogenization was performed twice for 30 seconds. The obtained homogenate was centrifuged at 21,600 × g, 4 ° C. for 20 minutes, and the amount of cytokine in the supernatant was measured by ELISA. The ELISA kit used is as follows. TSLP (R & D Systems), IL-1β (R & D Systems), TNF-α (eBioscience), and IL-4 (eBioscience).

As is clear from FIG. 3, atRA induced the expression of TSLP, but did not induce the expression of IL-1β, TNF-α and IL-4. Black column, acetone application group, white column, atRA (2 nmol) application group, * P <0.05,

Example 4 TSLP producing tissue (FIG. 4)
All-trans retinoic acid (atRA, Sigma Aldrich) was applied to the auricle of ICR mice (Japan SLC, male, SPF, 6 weeks old) with acetone solution containing 2 nmol or 20 μl of acetone. After 24 hours, the mice were exsanguinated and the auricles were collected. 1 Frozen sections were prepared and fluorescent immunostaining was performed using an anti-TSLP antibody (SantaCruz).

As is clear from FIG. 4, it was revealed that TSLP is mainly expressed in epithelial tissues.

Example 5 Auricular tissue 24 hours after application of retinoic acid (FIG. 5)
All-trans retinoic acid (atRA, Sigma Aldrich) was applied to the auricle of ICR mice (Japan SLC, male, SPF, 6 weeks old) with acetone solution containing 2 nmol or 20 μl of acetone. After 24 hours, the mice were exsanguinated and the auricles were collected. Frozen sections were prepared and Hematoxylin-Eosin staining was performed.

Even when atRA was applied, there was no finding that induced an inflammatory reaction such as leukocyte infiltration.

Example 6 Action of RARγ agonist (FIG. 6)
20 μl of an acetone solution containing RARα agonist BMS753 (100 nnoml) and / or RARγ agonist BMS189961 (20, 100 nmol) was applied to the ears of ICR mice (Japan SLC, male, SPF, 6 weeks old). After 24 hours, the mice were exsanguinated and the auricles were collected. Weigh it, put it in a sample tube (TOMY), add 10 times the volume (w / v) of homogenizing buffer, and use a bead-type cell crusher (Precyllys 24, Bertin tech.). Homogenization was performed twice for 30 seconds. The obtained homogenate was centrifuged at 21,600 × g, 4 ° C. for 20 minutes, and the amount of TSLP in the supernatant was measured with an ELISA kit (R & D Systems).

RARγ agonist BMS189961 alone enhanced TSLP production. The RARα agonist BMS753 alone did not increase TSLP production, but the effect was enhanced by using it together with the RARγ agonist BMS189961.

Example 7 Retinoic acid enhances production of anti-OVA antibody (IgG1, IgE) (FIG. 7)
All-trans retinoic acid (atRA, Sigma Aldrich) was applied to the back of ICR mice (Japan SLC, male, SPF, 6 weeks old) with acetone solution containing 4, 12, 40 nmol or 40 μl of acetone. 24 hours later, 50 μl of physiological saline containing 3 μg of ovalbumin (OVA) was intradermally injected into the same site. Two weeks later, blood was collected, and the serum anti-OVA IgG1 and anti-OVA IgE levels were measured by the respective ELISA kit (Cayman).

When OVA was injected alone, IgG1 production was slightly observed and IgE production was hardly observed. The production of IgG1 was significantly induced from the pretreatment with atRA-4 nmol. On the other hand, IgE production increased slightly with application of 12 nmol or more.

Example 8 Retinoic acid enhances neutralizing antibody production against recombinant influenza hemagglutinin A (H1N1) strain (A / California / 7/2009 (H1N1) or B Victoria strain (B / Brisbane 60/2008) Vaccine activity was evaluated using the influenza hemagglutinin gene recombinant as an antigen All-trans retinoic acid (atRA, Sigma Aldrich) 40 nmol in acetone solution or 40 μl acetone in BALB / c mice (Japan SLC, female, SPF, 6 weeks old) 24 hours later, 0.1 ml of physiological saline containing 3 μg of recombinant influenza hemagglutinin was injected subcutaneously, 3 weeks later, all-trans retinoic acid (atRA, Sigma Aldrich) ) Was applied to the back of an acetone solution containing 40 nmol or 40 μl of acetone, and 24 hours later, 0.1 ml of physiological saline containing 3 μg of recombinant influenza hemagglutinin was subcutaneously injected. Two weeks later, blood was collected, and the inhibitory effect of serum on hemagglutinin by the recombinant influenza vaccine was evaluated as neutralizing antibody titer using 5 mice per group, and antibody titer (GMT) was calculated using Geomean function. The results are shown in Table 1 below.

As is clear from Table 1, neutralizing antibody production by intradermal injection of any recombinant influenza vaccine hemagglutinin was increased 8.6-fold and 12.2-fold, respectively, by pretreatment with atRA.

Example 9 Retinoic acid reduces the amount of recombinant influenza hemagglutinin necessary for neutralizing antibody production (FIG. 8)
All-trans retinoic acid (atRA, Sigma Aldrich) was applied to the back of BALB / c mice (Japan SLC, female, SPF, 6 weeks old) with acetone solution containing 40 nmol or 40 μl of acetone. 24 hours later, recombinant influenza hemagglutinin A (H1N1) strain (A / California / 7/2009 (H1N1) [0.0003, 0.03 and 3 μg] or B Victoria strain (B / Brisbane 60/2008) [0.03, 0.3 And 3 μg] of saline containing 3 μg] was subcutaneously injected, and after 3 weeks, all-trans retinoic acid (atRA, Sigma Aldrich) was again applied to the back with 40 nmol in acetone or 40 μl, and 24 hours later. Then, 0.1 ml of physiological saline containing the corresponding antigen and amount was injected subcutaneously, and blood was further collected after 2 weeks, and the inhibitory effect of serum on hemagglutination by the recombinant influenza vaccine hemagglutinin was evaluated as a neutralizing antibody titer. The antibody titer (GMT) was calculated using the Geomean function using 5 mice per group.

When A (H1N1) strain (A / California / 7/2009 (H1N1) (A) and B Victoria strain (B / Brisbane 60/2008) (B) are administered alone by pretreatment with atRA Thus, equivalent antibody production was induced in an amount 10 to 100 times less.

Example 10 Effect of retinoic acid on neutralizing antibody and IgE production in the case of low concentration antigen stimulation (FIG. 9)
All-trans retinoic acid (atRA, Sigma Aldrich) was applied to the back of BALB / c mice (Japan SLC, female, SPF, 6 weeks old) with acetone solution containing 4, 12, 40 nmol or 40 μl of acetone. 24 hours later, 0.1 ml of physiological saline containing recombinant influenza hemagglutinin A (H1N1) strain (A / California / 7/2009 (H1N1) [0.003 μg] was injected subcutaneously at the same site. An acetone solution containing the corresponding concentration of all-trans retinoic acid (atRA, Sigma Aldrich) or 40 μl of acetone was applied to the back, and 24 hours later, 0.1 ml of physiological saline containing the corresponding antigen was injected subcutaneously. Blood was collected after a week, and the inhibitory effect of serum on hemagglutinin by the recombinant influenza vaccine was evaluated as neutralizing antibody titer, using 5 mice per group, and antibody titer (GMT) was calculated using Geomean function. In addition, total IgE was measured by ELISA (Levis IgE-ELISA kit (mouse), Shiba Goat).

Even when the dose of recombinant influenza hemagglutinin A (H1N1) strain (A / California / 7/2009 (H1N1)) was reduced to 0.003 μg, the neutralizing antibody titer increased in a dose-dependent manner with retinoic acid ( A) did not show an increase in total IgE (B).

The present invention is the first invention that a low-molecular-weight organic compound having high purity and extremely low flammability can be used as a vaccine. In addition, the present invention can be expected to reduce the side effects caused by the unpurified antigen itself that has been conventionally used as a vaccine, and to reduce the amount of antigen to be used. Expected to be. Furthermore, since the vaccine adjuvant of the present invention can be applied as an external preparation, it is useful in terms of low invasiveness, suppression of nonspecific adjuvant effect expression, convenience, and the like. Therefore, the present invention is extremely useful in industry.

Claims (13)

A vaccine adjuvant comprising an agonist of RARγ. The vaccine adjuvant according to claim 1, wherein the agonist of RARγ is a retinoid. The vaccine adjuvant according to claim 1 or 2, for use in an administration schedule in which an antibody is administered at least 4 hours after administration of an agonist of RARγ. The vaccine adjuvant according to claim 3, wherein an agonist of RARγ is administered as an external preparation. Target diseases are influenza, HIV, chlamydia, malaria, tuberculosis, tumor, measles, measles, mumps, chickenpox, yellow fever, rotavirus gastroenteritis, smallpox, pneumococci, rabies, cholera, diphtheria, pertussis, The vaccine adjuvant according to any one of claims 1 to 4, which is at least one selected from the group consisting of tetanus, Japanese encephalitis, papillomavirus, hepatitis and Ebola hemorrhagic fever, dengue fever, and Zika fever. A vaccine comprising a combination of the vaccine adjuvant according to claim 1 or 2 and an antigen. The vaccine according to claim 6, for use in an administration schedule in which an antibody is administered at least 4 hours after administration of an agonist of RARγ. The vaccine according to claim 7, wherein the RARγ agonist is administered as an external preparation. Target diseases are influenza, HIV, chlamydia, malaria, tuberculosis, tumor, measles, measles, mumps, chickenpox, yellow fever, rotavirus gastroenteritis, smallpox, pneumococci, rabies, cholera, diphtheria, pertussis, The vaccine according to any one of claims 6 to 8, which is at least one selected from the group consisting of tetanus, Japanese encephalitis, papillomavirus, hepatitis and Ebola hemorrhagic fever, dengue fever and Zika fever. The vaccine according to any one of claims 6 to 8, wherein the agonist of RARγ is a retinoid, and the target disease is influenza. A method for preventing or treating a disease, comprising a step of administering the vaccine adjuvant according to claim 1 or 2 to a mammal, and a step of administering an antigen to the mammal. A combination of an antigen and a vaccine adjuvant according to claim 1 or 2 for use as a vaccine. Use of an antigen and a vaccine adjuvant according to claim 1 or 2 for the manufacture of a vaccine.

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