vaccine

Abstract

To provide an animal vaccine.SOLUTION: A specific cationic lipid is mixed with an antigen to produce a vaccine.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to vaccines for animals. [Background technology]

[0002] Cationic lipids have been reported as carriers for delivering nucleic acids and the like into cells (Patent Documents 1 to 3). Specifically, as the cationic lipid, COATSOME (registered trademark) SS-E is used in the Examples of Patent Document 1, COATSOME (registered trademark) SS-EC is used in the Examples of Patent Document 2, and COATSOME (registered trademark) SS-OP is used in the Examples of Patent Document 3.

[0003] Non-Patent Document 1 discloses COATSOME (registered trademark) SS-OP and COATSOME (registered trademark) SS-EC as cationic lipids. Non-Patent Document 1 reports that COATSOME (registered trademark) SS-EC may be applicable as an adjuvant or DNA vaccine. However, the adjuvant function of COATSOME (registered trademark) SS-EC when combined with an antigen has not actually been confirmed. It has also been reported that lipid nanoparticles (LNPs) have an adjuvant effect (Non-Patent Document 2).

[0004] The adjuvant activity of cationic lipids has attracted attention since a report was published showing that liposomes containing cationic lipids have adjuvant activity. The cationic lipids used in the report are cationic lipids that are positively charged over a wide range from neutral to acidic pH, and are highly toxic compared to their adjuvant activity, preventing their practical application. The mechanism of their adjuvant effect is also unclear. On the other hand, the cationic lipids contained in LNPs used in mRNA vaccines are characterized by being positively charged only in acidic pH ranges, and because they clearly have different sites of action on cells, it is speculated that their adjuvant activity is also different (Non-Patent Document 3). The adjuvant effect of mRNA-LNP was reported in 2018, but it was unclear whether this effect was due to the mRNA or the cationic lipid. Non-Patent Document 4 revealed that the use of mRNA-free LNP had an adjuvant effect, and further showed that LNP without cationic lipids had no adjuvant activity. Non-Patent Document 5 also showed that administration of mRNA-free LNP induced an inflammatory reaction (immune response). These findings demonstrated that the adjuvant activity of mRNA-LNP was due to the cationic lipid. Therefore, cationic lipids are expected to be used as adjuvants.

[0005] Adjuvants, when used in combination with an antigen, can enhance the immunogenicity of the antigen, accelerate the immune response to the antigen, prolong the immune response to the antigen, and / or switch the immune response to a different immune response from that induced by the antigen alone (e.g., a switch from a Th1 immune response to a Th2 immune response or a switch from humoral to cellular immunity). Therefore, adjuvants are useful for reducing the vaccine dose or administration frequency, or the required amount of antigen in a vaccine. Furthermore, in the veterinary industry, adjuvants are essential components for simultaneous immunization with multiple antigens. Currently, the most commonly used adjuvants in Japan are mineral acid salts (e.g., adjuvants based on aluminum salts such as aluminum hydroxide and aluminum phosphate (also known as "alum adjuvants"). Alum adjuvants are the oldest and most widely used adjuvants, but they are difficult to mix uniformly with antigens due to their insolubility. They induce humoral immunity but only a low level of cellular immunity, and they have been problematic in terms of side effects such as fever and allergic reaction (IgE). In addition, oil adjuvants are used exclusively for veterinary purposes. Oil adjuvants have a strong adjuvant effect. However, the problem was that it caused strong side effects in administered animals (for example, swelling at the injection site, systemic symptoms such as fever, and weight loss). For this reason, the veterinary medical industry desperately needed to develop an adjuvant that was more effective and caused fewer side effects than alum adjuvants and oil adjuvants. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 6093710 [Patent Document 2] Patent No. 6875685 [Patent Document 3] WO2019 / 188867 [Non-patent literature]

[0007] [Non-Patent Document 1] Hidemasa Akita, Pharmaceutics, 82(2), 79-83(2022) [Non-patent document 2] Joanna L. Turley and Ed C. Lavelle, Immunity 54 2695-2697, December 14, 2021 [Non-patent document 3] R, Verbeke et al., Immunity 55, 1993-2005, November 8, 2022 [Non-patent document 4] Mohamad-Gabriel Alameh et. al., Immunity 54 2877-2892, December 14, 2021 [Non-patent document 5] S. Ndeupen et al., iScience 24, 103479, December 17, 2021 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a vaccine containing an adjuvant that has excellent immunostimulatory activity and few side effects. [Means for solving the problem]

[0009] The present inventors have found that cationic lipids represented by the general formula (I) described below function successfully as adjuvants, and have completed the present invention.

[0010] That is, the present invention can be exemplified as follows. [1] A vaccine comprising a cationic lipid and an antigen, A vaccine wherein the cationic lipid is a compound represented by general formula (I) described below. [In the formula, na and nb each independently represent an integer of 1 to 4; X a and X b are each independently a group having the structure X 1 or structure X as described below 2 represents R 7 represents an alkyl group having 1 to 6 carbon atoms, p represents an integer of 1 to 4; R 1a and R 1b are each independently, R 3 or R 4 represents R 3 is -(CH2) n1 - represents R 4 is -C6H4-(CH2) n3 -COO-(CH2) n2 - represents n1 represents an integer of 1 to 5, n2 represents an integer of 1 to 5, n3 represents an integer of 1 to 3, R 2a and R 2b are each independently R 5 or R6 represents R 5 represents a functional group obtained by removing a carboxy group from a monoester of succinic acid or glutaric acid with a fat-soluble vitamin having a hydroxyl group, R 6 represents an aliphatic hydrocarbon group having 12 to 22 carbon atoms.] [2] The vaccine described in [1] satisfies any one of the following conditions (1) to (4) and any one of the following conditions (5) to (8): (1) na is 2 and X a is X 1 and R 1a is R where n1 is 3 3 and R 2a is R 5 is; (2) na is 2, and X a is X 2 and R 1a R where n1 is 2 3 and R 2a is R 5 is; (3) na is 2 and X a is X 2 and R 1a R where n1 is 2 3 and R 2a is R 6 is; (4) na is 2 and X a is X 2 and R 1a is R where n2 is 2 and n3 is 1 4 and R 2a is R 6 is; (5) nb is 2 and X b is X 1 and R 1b is R where n1 is 3 3 and R 2b is R 5 is; (6) nb is 2 and X b is X 2 and R 1b R where n1 is 2 3 and R2b is R 5 is; (7) nb is 2 and X b is X 2 and R 1b R where n1 is 2 3 and R 2b is R 6 is; (8) nb is 2 and X b is X 2 and R 1b is R where n2 is 2 and n3 is 1 4 and R 2b is R 6 is. [3] The vaccine according to [1] or [2], wherein p is 3 or 4. [4] na is identical to nb, and X a is X b is identical to R 1a R 1b is identical to and R 2a R 2b The vaccine according to any one of [1] to [3], which is identical to [5] The vaccine according to any one of [1] to [4], wherein the cationic lipid is a component of ssPalmEC and / or ssPalmE. [6] The vaccine according to any one of [1] to [5], wherein the cationic lipid is a constituent lipid of a lipid membrane structure. [7] The vaccine according to [6], wherein the lipid membrane structure further contains a non-cationic lipid.[8] The vaccine described in [7], wherein the non-cationic lipid is one or more components selected from the group consisting of phospholipids, PEG-lipids, and steroids. [9] The vaccine described in [8], wherein the steroid is one or more components selected from the group consisting of cholesterol and its derivatives.

[10] The vaccine according to any one of [1] to [9], wherein the antigen is one or more components selected from the group consisting of pathogens and components derived therefrom.

[11] The vaccine according to any one of [1] to

[10] , wherein the antigen is an inactivated antigen.

[12] The vaccine according to

[11] , wherein the inactivated antigen is an inactivated virus.

[13] The vaccine according to

[11] , wherein the inactivated antigen is an inactivated bacterium.

[14] The vaccine according to

[11] , wherein the inactivated antigen is a toxoid.

[15] The virus is bovine viral diarrhea virus.

[12] The vaccine described in.

[16]

[13] The vaccine according to

[13] , wherein the bacterium is Avibacterium paragallinarum.

[17] The vaccine according to

[14] , wherein the toxoid is a toxoid of Clostridium septicum.

[18] A vaccine according to any one of [1] to

[17] for preventing, mitigating, or treating an infectious disease in an animal.

[19] The vaccine according to

[18] , wherein the animal is a livestock animal or a pet animal.

[20] The vaccine according to any one of [1] to

[19] , which is administered by a route selected from the group consisting of intramuscular administration, intradermal administration, subcutaneous administration, and intraocular administration. [twenty one] The vaccine according to any one of [1] to

[19] , which is administered intramuscularly. [twenty two] A method for producing a vaccine, comprising: The vaccine is a vaccine according to any one of [1] to

[21] , mixing said cationic lipid and said antigen. [twenty three] The method according to

[22] , wherein the vaccine is a vaccine with improved functionality. [Effects of the Invention]

[0011] The present invention provides a vaccine containing an adjuvant that has excellent immunostimulatory activity and few side effects. In one aspect, the vaccine can be used to prevent, alleviate, or treat infectious diseases in animals. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the results of measuring neutralizing antibody titers after administration of a vaccine containing ssPalmEC-LNP. [Figure 2] FIG. 1 shows the reaction inhibition rate in chickens administered with a vaccine containing ssPalmEC-LNP. DETAILED DESCRIPTION OF THE INVENTION

[0013] <1> vaccine The vaccines described herein are vaccines that contain a cationic lipid and an antigen.

[0014] The cationic lipid contained in the vaccine described herein (hereinafter also simply referred to as "cationic lipid") is a compound represented by the following general formula (I): "Cationic lipid" may refer to a lipid that has no net charge at neutral pH but has a net positive charge at acidic pH. The cationic nature of the cationic lipid may be due, for example, to the tertiary amine contained in the cationic lipid. As the cationic lipid, one type of cationic lipid may be used, or two or more types of cationic lipids may be used in combination.

[0015] [ka]

[0016] In formula (I), na and nb each independently represent an integer of 1 to 4 (ie, 1, 2, 3, or 4).

[0017] In formula (I), X a and X b are each independently represented by the following structure X 1 or the following structure X 2 represents R 7 represents an alkyl group having 1 to 6 carbon atoms, p represents an integer of 1 to 4 (ie, 1, 2, 3, or 4).

[0018] [ka]

[0019] [ka]

[0020] In formula (I), R 1a and R 1b are each independently R 3 or R 4 represents R 3 is -(CH2) n1 - represents R 4 is -C6H4-(CH2) n3 -COO-(CH2) n2 - represents n1 represents an integer from 1 to 5 (i.e., 1, 2, 3, 4, or 5); n2 represents an integer from 1 to 5 (i.e., 1, 2, 3, 4, or 5); n3 represents an integer of 1 to 3 (ie, 1, 2, or 3).

[0021] In formula (I), R 2a and R 2b are each independently R5 or R 6 represents R 5 represents a functional group obtained by removing a carboxy group from a monoester of succinic acid or glutaric acid with a fat-soluble vitamin having a hydroxyl group, R 6 represents an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

[0022] na may or may not be the same as nb. na may particularly be the same as nb. Both na and nb may particularly be 2.

[0023] X a is X b It may or may not be the same as X. a In particular, X b may be the same as

[0024] R 1a is R 1b It may or may not be the same as R 1a In particular, R 1b may be the same as

[0025] R 2a is R 2b It may or may not be the same as R 2a In particular, R 2b may be the same as

[0026] For example, R 1a R 3 If R 2a is R 5 For example, R 1b R 3 If R 2b is R 5 For example, R 1a R 3 If R 2a is R 6 For example, R 1b R 3 If R 2b is R6 For example, R 1a R 4 If R 2a is R 6 For example, R 1b R 4 If R 2b is R 6 It may be.

[0027] n1 may in particular be 2 or 3.

[0028] For example, X a is X 1 If R 1a is R where n1 is 3 3 For example, X b is X 1 If R 1b is R where n1 is 3 3 For example, X a is X 2 If R 1a is R where n1 is 2 3 For example, X b is X 2 If R 1b is R where n1 is 2 3 It may be.

[0029] n2 may in particular be 2.

[0030] n3 may in particular be 1.

[0031] For example, X a is X 2 If R 1a is R where n2 is 2 and n3 is 1 4 For example, X b is X 2 If R 1b is R where n2 is 2 and n3 is 1 4 It may be.

[0032] R 4 About R4 is "*-C6H4-(CH2) n3 -COO-(CH2) n2 -**" can also be expressed, where * is R 2a -COO or R 2b -COO bond position, ** is X a or X b The bond position with R 4 For R, -C6H4- can be 1,2-phenylene, 1,3-phenylene, or 1,4-phenylene. 4 For -C6H4-, in particular, may be 1,4-phenylene.

[0033] The alkyl group having 1 to 6 carbon atoms may be linear (acyclic) or cyclic. The alkyl group having 1 to 6 carbon atoms may be linear or branched. The number of carbon atoms in the alkyl group having 1 to 6 carbon atoms may be preferably 1 to 3. Examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, an n-pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a 1,2-dimethylpropyl group, a 2-methylbutyl group, a 2-methylpentyl group, a 3-methylpentyl group, a 2,2-dimethylbutyl group, a 2,3-dimethylbutyl group, and a cyclohexyl group. Examples of the alkyl group having 1 to 6 carbon atoms are preferably a methyl group, an ethyl group, an n-propyl group, and an isopropyl group. Examples of the alkyl group having 1 to 6 carbon atoms are more preferably a methyl group.

[0034] p may in particular be 3 or 4. p may more in particular be 4.

[0035] X 2 Regarding the nitrogen atom (N) (CH2) na or (CH2) nb and the carbon atom in the ring structure is R 1a or R 1a Any carbon atom in the ring structure is bonded to R 1a or R1a For example, when p is 2 or more (for example, 3 or 4, particularly 4), the carbon atom at the 4th position when the nitrogen atom (N) is at the 1st position may be bonded to R 1a or R 1a may be combined with

[0036] "Monoesters of succinic acid or glutaric acid with fat-soluble vitamins having a hydroxyl group" (hereinafter simply referred to as "monoesters") means compounds obtained by esterifying one of the carboxyl groups of succinic acid or glutaric acid with fat-soluble vitamins having a hydroxyl group. 2a or R 2b R 5 If R 2a -COO- or R 2b -COO -(i.e., R 5 -COO-) is an acyloxy group obtained by removing a hydrogen atom from the carboxy group of a monoesterified product. The above description of the functional group obtained by removing a carboxy group from a monoesterified product indicates the structure of the functional group, unless otherwise specified, and does not necessarily indicate the method for producing the functional group. That is, the monoesterified product may or may not be one obtained by reacting a fat-soluble vitamin having a hydroxyl group with succinic acid or glutaric acid. For example, the monoesterified product may be one obtained by reacting a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride. Furthermore, the functional group obtained by removing a carboxy group from a monoesterified product or the acyloxy group obtained by removing a hydrogen atom from the carboxy group of a monoesterified product may or may not be derived from the monoesterified product.

[0037] "Vitamins" are not limited to vitamins themselves, but may also include provitamins and vitamin derivatives. Examples of fat-soluble vitamins having a hydroxyl group include retinol, ergosterol, 7-dehydrocholesterol, calciferol, corcalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol, and tocotrienol. A preferred example of a fat-soluble vitamin having a hydroxyl group is tocopherol.

[0038] The monoesters are preferably monoesters of succinic acid with tocopherol (i.e., succinic acid monotocopherol ester) or monoesters of glutaric acid with tocopherol (i.e., glutaric acid monotocopherol ester), and more preferably succinic acid monotocopherol ester.

[0039] The aliphatic hydrocarbon group having 12 to 22 carbon atoms may be linear (acyclic) or cyclic. The aliphatic hydrocarbon group having 12 to 22 carbon atoms may particularly be linear (acyclic). The aliphatic hydrocarbon group having 12 to 22 carbon atoms may be linear or branched. The aliphatic hydrocarbon group having 12 to 22 carbon atoms may be saturated or unsaturated. When the aliphatic hydrocarbon group having 12 to 22 carbon atoms is unsaturated, the number of unsaturated bonds contained in the aliphatic hydrocarbon group may be, for example, usually 1 to 6, preferably 1 to 3, and more preferably 1 to 2. The position of the unsaturated bond is not particularly limited. The configuration of the unsaturated bond (i.e., EZ) is not particularly limited. Examples of the unsaturated bond include a carbon-carbon double bond and a carbon-carbon triple bond. Examples of a preferred unsaturated bond include a carbon-carbon double bond. The number of carbon atoms in the aliphatic hydrocarbon group having 12 to 22 carbon atoms may be preferably 13 to 19, and more preferably 13 to 17. Examples of the aliphatic hydrocarbon group include an alkyl group, an alkenyl group, and an alkynyl group. Examples of the aliphatic hydrocarbon group include an alkyl group and an alkenyl group. Examples of the aliphatic hydrocarbon group having 12 to 22 carbon atoms include a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a nonadecyl group, an icosyl group, a henicosyl group, a docosyl group, a dodecenyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl group, a hexadecenyl group, a heptadecenyl group, an octadecenyl group, a nonadecenyl group, an icosenyl group, a henicosyl group, a docosenyl group, a dodecadienyl group, a tridecadienyl group, and a tetradecenyl group. Examples of the alkyl group include a dienyl group, a pentadecadienyl group, a hexadecadienyl group, a heptadecadienyl group, an octadecadienyl group, a nonadecadienyl group, an icosadienyl group, a henicosadienyl group, a docosadienyl group, an octadecatrienyl group, an icosatrienyl group, an icosatetraenyl group, an icosapentaenyl group, a docosahexaenyl group, an isostearyl group, a 1-hexylheptyl group, a 1-hexylnonyl group, a 1-octylnonyl group, a 1-octylundecyl group, and a 1-decylundecyl group.Preferred examples of the aliphatic hydrocarbon group having 12 to 22 carbon atoms include a tridecyl group, a pentadecyl group, a heptadecyl group, a nonadecyl group, a heptadecenyl group, a heptadecadienyl group, and a 1-hexylnonyl group. More preferred examples of the aliphatic hydrocarbon group having 12 to 22 carbon atoms include a tridecyl group, a heptadecyl group, a heptadecenyl group, and a heptadecadienyl group. A particularly preferred example of the aliphatic hydrocarbon group having 12 to 22 carbon atoms is a heptadecenyl group. The heptadecenyl group may be, for example, an 8-heptadecenyl group. The heptadecenyl group may be, for example, particularly, a (Z)-8-heptadecenyl group.

[0040] Specifically, the aliphatic hydrocarbon group having 12 to 22 carbon atoms may be a functional group obtained by removing a carboxy group from a fatty acid having 13 to 23 carbon atoms. 2a or R 2b R 6 If R 2a -COO- or R 2b -COO- (i.e., R 6 -COO-) may be an acyloxy group obtained by removing a hydrogen atom from the carboxy group of a fatty acid having 13 to 23 carbon atoms. For example, when the fatty acid is linoleic acid, the aliphatic hydrocarbon group having 12 to 22 carbon atoms is an 8,11-heptadecadienyl group (specifically, an (8Z,11Z)-8,11-heptadecadienyl group). Furthermore, when the fatty acid is oleic acid, the aliphatic hydrocarbon group having 12 to 22 carbon atoms is an 8-heptadecenyl group (specifically, a (Z)-8-heptadecenyl group). Unless otherwise specified, the above description of the aliphatic hydrocarbon group having 12 to 22 carbon atoms indicates the structure of the functional group, and does not necessarily indicate the method for producing the functional group. That is, for example, a functional group obtained by removing a carboxy group from a fatty acid having 13 to 23 carbon atoms or an acyloxy group obtained by removing a hydrogen atom from the carboxy group of a fatty acid having 13 to 23 carbon atoms may or may not be derived from a fatty acid.

[0041] In one embodiment, na may be identical to nb, and X a is X bmay be identical to R 1a is R 1b and R 2a is R 2b may be the same as

[0042] In one embodiment, X a is X 1 R may be 1a is R 3 R may be 2a is R 5 In one embodiment, na may be 2 and X a is X 1 R may be 1a is R where n1 is 3 3 R may be 2a is R 5 In one embodiment, na may be 2 and X a is R 7 X is methyl 1 R may be 1a is R where n1 is 3 3 R may be 2a may be a residue obtained by removing the carboxy group from a monotocopherol succinate ester.

[0043] In one embodiment, X b is X 1 R may be 1b is R 3 R may be 2b is R 5 In one embodiment, nb may be 2 and X b is X 1 R may be 1b is R where n1 is 3 3 R may be 2b is R 5 In one embodiment, nb may be 2 and X b is R 7 X is methyl 1 R may be 1b is R where n1 is 3 3 R may be 2bmay be a residue obtained by removing the carboxy group from a monotocopherol succinate ester.

[0044] In one embodiment, X a is X 2 R may be 1a is R 3 R may be 2a is R 5 In one embodiment, na may be 2 and X a is X 2 R may be 1a R where n1 is 2 3 R may be 2a is R 5 In one embodiment, na may be 2 and X a is x where p is 4 2 R may be 1a R where n1 is 2 3 R may be 2a may be a residue obtained by removing the carboxy group from a monotocopherol succinate ester.

[0045] In one embodiment, X b is X 2 R may be 1b is R 3 R may be 2b is R 5 In one embodiment, nb may be 2 and X b is X 2 R may be 1b R where n1 is 2 3 R may be 2b is R 5 In one embodiment, nb may be 2 and X b is x where p is 4 2 R may be 1b R where n1 is 2 3 R may be 2b may be a residue obtained by removing the carboxy group from a monotocopherol succinate ester.

[0046] In one embodiment, X a is X2 R may be 1a is R 3 R may be 2a is R 6 In one embodiment, na may be 2 and X a is X 2 R may be 1a R where n1 is 2 3 R may be 2a is R 6 In one embodiment, na may be 2 and X a is x where p is 4 2 R may be 1a R where n1 is 2 3 R may be 2a may be an 8-heptadecenyl group (e.g., a (Z)-8-heptadecenyl group).

[0047] In one embodiment, X b is X 2 R may be 1b is R 3 R may be 2b is R 6 In one embodiment, nb may be 2 and X b is X 2 R may be 1b R where n1 is 2 3 R may be 2b is R 6 In one embodiment, nb may be 2 and X b is x where p is 4 2 R may be 1b R where n1 is 2 3 R may be 2b may be an 8-heptadecenyl group (e.g., a (Z)-8-heptadecenyl group).

[0048] In one embodiment, X a is X 2 R may be 1a is R 4 R may be 2a is R 6 In one embodiment, na may be 2 and Xa is X 2 R may be 1a is R where n2 is 2 and n3 is 1 4 R may be 2a is R 6 In one embodiment, na may be 2 and X a is x where p is 4 2 R may be 1a is R where n2 is 2 and n3 is 1 4 R may be 2a may be an 8-heptadecenyl group (e.g., a (Z)-8-heptadecenyl group).

[0049] In one embodiment, X b is X 2 R may be 1b is R 4 R may be 2b is R 6 In one embodiment, nb may be 2 and X b is X 2 R may be 1b is R where n2 is 2 and n3 is 1 4 R may be 2b is R 6 In one embodiment, nb may be 2 and X b is x where p is 4 2 R may be 1b is R where n2 is 2 and n3 is 1 4 R may be 2b may be an 8-heptadecenyl group (e.g., a (Z)-8-heptadecenyl group).

[0050] Specific examples of cationic lipids include ssPalmE, ssPalmEC, ssPalmOC, and ssPalmOP. Preferred examples of cationic lipids include ssPalmE and ssPalmEC, and more preferably ssPalmEC. ssPalmEC is also referred to as "ssPalmE-P4C2." The structures of ssPalmE, ssPalmEC, ssPalmOC, and ssPalmOP are shown below.

[0051] [ka]

[0052] [ka]

[0053] The cationic lipid may be a commercially available product or may be obtained by appropriate production. For example, a commercially available ssPalmE-P4C2 may be COATSOME (registered trademark) SS-EC (manufactured by Yuka Sangyo Co., Ltd.). The method for producing the cationic lipid is not particularly limited. The cationic lipid can be produced, for example, by chemical synthesis. Specifically, the cationic lipid can be produced, for example, by the method described in Japanese Patent No. 6093710, Japanese Patent No. 6875685, or WO2019 / 188867.

[0054] Cationic lipids, for example, may function as adjuvants. The term "adjuvant" may refer to a substance that, when used in combination with an antigen, enhances the immunogenicity of the antigen, accelerates the immune response to the antigen, prolongs the immune response to the antigen, and / or switches the immune response to a different immune response from that induced by the antigen alone (e.g., switches from a Th1 immune response to a Th2 immune response, or switches from humoral immunity to cellular immunity). An adjuvant may be a single substance or a combination of multiple substances. That is, cationic lipids may function as adjuvants alone or in combination with other components.

[0055] The cationic lipid may, for example, constitute a lipid membrane structure. In other words, the cationic lipid may, for example, be a constituent lipid of a lipid membrane structure. Specifically, the cationic lipid may, for example, be a constituent lipid of a lipid membrane structure. The lipid membrane structure may or may not consist of a cationic lipid. That is, the lipid membrane structure may contain other components in addition to the cationic lipid. In other words, the cationic lipid may constitute the lipid membrane structure alone or in combination with other components. In other words, the vaccine described herein may contain other components in addition to the cationic lipid and the antigen. Examples of other components include non-cationic lipids, surfactants, polyethylene glycol (PEG), and proteins. Examples of other components include non-cationic lipids in particular. "Non-cationic lipid" refers to lipids other than cationic lipids. "Non-cationic lipid" specifically refers to lipids that do not have a net positive charge at a selected pH, such as physiological pH. Examples of non-cationic lipids include phospholipids, PEG lipids, glycolipids, peptide lipids, and steroids. Non-cationic lipids include, in particular, phospholipids, PEG lipids, and steroids. Other components include cationic lipids other than the cationic lipid of formula (I).

[0056] Phospholipids include natural or synthetic phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), phosphatidic acid (PA), dicetyl phosphate, sphingomyelin (SPM), and cardiolipin; partially or fully hydrogenated versions of these phospholipids; natural lecithins (natural lecithins contain phospholipids), such as soybean lecithin, corn lecithin, cottonseed oil lecithin, and egg yolk lecithin; and hydrogenated versions of natural lecithins, such as hydrogenated soybean lecithin and hydrogenated egg yolk lecithin. Phospholipids may have acyl groups at the C1 and C2 positions of the glycerol. The acyl groups at the C1 and C2 positions can be independently selected. That is, the acyl groups at the C1 and C2 positions may or may not be the same. Examples of acyl groups constituting phospholipids include acyl groups having 8 to 24 carbon atoms. Examples of acyl groups having 8 to 24 carbon atoms include residues obtained by removing a hydroxyl group from fatty acids such as caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, nonadecanoic acid, arachidic acid, heneicosanoic acid, behenic acid, tricosanoic acid, lignoceric acid, myristoleic acid, palmitoleic acid, oleic acid, eicosenoic acid, erucic acid, hexadecadienoic acid, linoleic acid, eicosadienoic acid, docosadienoic acid, hexadecatrienoic acid, α-linolenic acid, γ-linolenic acid, eicosatrienoic acid, arachidonic acid, eicosapentaenoic acid, docosatetraenoic acid, and docosahexaenoic acid. Preferred phospholipids include synthetic phospholipids. More preferred examples of the phospholipid include synthetic phospholipids containing an unsaturated bond in the acyl group. More preferred examples of the phospholipid include dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE). Particularly preferred examples of the phospholipid include dioleoylphosphatidylethanolamine (DOPE).

[0057] "PEG lipid" may refer to a lipid modified with PEG. Examples of PEG lipids include PEG phospholipids and diacylglycerol PEGs. "PEG phospholipid" may refer to a phospholipid to which PEG is bound. The phospholipid is as described above. "Diacylglycerol PEG" may refer to a diacylglycerol to which PEG is bound. Diacylglycerol PEG may have acyl groups at the C1 and C2 positions or the C1 and C3 positions of the glycerol. The acyl groups at the C1 and C2 positions or the C1 and C3 positions can be independently selected. That is, the acyl groups at the C1 and C2 positions or the C1 and C3 positions may be the same or different. Examples of acyl groups constituting diacylglycerol PEG include those exemplified as acyl groups constituting phospholipids. The molecular weight of the PEG constituting the PEG lipid is not particularly limited. The molecular weight of the PEG constituting the PEG lipid may be preferably 200 to 10,000, more preferably 2,000 to 5,000. The PEG lipid preferably includes diacylglycerol PEG in which each acyl group is saturated. The PEG lipid more preferably includes diacylglycerol PEG in which each acyl group is a myristoyl group or a stearoyl group. The PEG lipid further preferably includes diacylglycerol PEG (DMG-PEG2000) in which a myristoyl group is bonded to a PEG having a molecular weight of about 2000, or diacylglycerol PEG (DSG-PEG) in which a stearoyl group is bonded to a PEG having a molecular weight of about 2000. EG2000).

[0058] Examples of steroids include sterols and derivatives thereof. Examples of sterols include cholesterol, phytosterols, and dihydrocholesterol. A preferred sterol is cholesterol. Examples of sterol derivatives include sterol fatty acid esters. Specific examples of sterol derivatives (specifically fatty acid esters) include cholesterol derivatives (specifically fatty acid esters) such as cholesteryl stearate, cholesteryl nonanoate, cholesteryl hydroxystearate, and dihydrocholesteryl oleate. A preferred example of a sterol derivative is cholesteryl stearate. A preferred example of a steroid is cholesterol or a derivative thereof. A more preferred example of a steroid is cholesterol.

[0059] Examples of surfactants include 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, cholic acid sodium salt, octyl glycoside, and ND-gluco-N-methylalkanamides.

[0060] As the other components, commercially available products may be used, or those obtained by appropriate manufacturing may be used.

[0061] The term "lipid membrane structure" may refer to particles having a membrane structure in which the hydrophilic groups of amphipathic lipids are aligned toward the aqueous phase side of the interface. The term "amphipathic lipid" may refer to a lipid having both hydrophilic and hydrophobic groups. Examples of amphipathic lipids include cationic lipids and phospholipids.

[0062] The quantitative ratio of each component constituting the lipid membrane structure is not particularly limited, as long as a lipid membrane structure is formed. The content of the cationic lipid in the lipid membrane structure may be, for example, usually 5 to 100 mol%, preferably 5 to 95 mol%, more preferably 10 to 90 mol%, and particularly preferably 20 to 70 mol%. The content of the cationic lipid in the lipid membrane structure may be, for example, usually 5 to 100 mol%, preferably 10 to 90 mol%, and more preferably 20 to 70 mol%, relative to the total content of lipids in the lipid membrane structure.

[0063] Lipid membrane structures can be prepared, for example, by dissolving or dispersing the components of the lipid membrane structure (cationic lipids and optionally other components) in an appropriate solvent or dispersion medium, and then, if necessary, carrying out a procedure to induce assembly. Examples of solvents or dispersion mediums include aqueous and alcoholic solvents. Procedures to induce assembly include the ethanol dilution method, simple hydration method, ultrasonic treatment, heating, vortexing, ether injection, French press method, cholic acid method, and Ca 2+ Examples of the ethanol dilution method include the fusion method, the freeze-thaw method, and the reverse phase evaporation method. The ethanol dilution method can be performed using, for example, a microchannel or a vortex.

[0064] The cationic lipid may exist in any form, such as a lipid nanoparticle (LNP), liposome, emulsion, or micelle. In other words, the lipid membrane structure may exist in any form, such as a LNP, liposome, emulsion, or micelle. The term "lipid nanoparticle (LNP)" may refer to a particle containing a lipid as a constituent and having a particle diameter of 100 nm or less. The term "lipid nanoparticle (LNP)" may specifically refer to a lipid membrane structure having a particle diameter of 100 nm or less. Unless otherwise specified, the term "particle diameter" refers to the particle diameter measured by dynamic light scattering (DLS). The term "lipid nanoparticle (LNP)" may also refer to a particle containing a lipid as a constituent and having an average particle diameter of 100 nm or less. The term "lipid nanoparticle (LNP)" may specifically refer to a lipid membrane structure having an average particle diameter of 100 nm or less. That is, the lipid membrane structures may have an average particle diameter of 100 nm or less. Unless otherwise specified, "average particle diameter" refers to the number-average particle diameter measured by dynamic light scattering (DLS). Measurement of particle diameter or average particle diameter by dynamic light scattering can be performed using a commercially available DLS device such as Zetasizer nano ZS (Malvern).

[0065] The antigens contained in the vaccines described herein (hereinafter simply referred to as "antigens") are not particularly limited. An "antigen" may refer to a substance that induces antibody production in an animal body and specifically reacts with the antibody. However, as used herein, nucleic acids are not included in the term "antigen." Antigens can be selected appropriately depending on various conditions, such as the intended use of the vaccines described herein. Antigens include pathogens and components derived therefrom. Examples of pathogens include viruses, bacteria, fungi, parasitic microorganisms, and mycoplasmas. The pathogens exemplified above may be, for example, pathogens that cause infectious diseases to which the vaccines described herein are applied. That is, examples of viruses, bacteria, fungi, parasitic microorganisms, and mycoplasmas include viruses, bacteria, fungi, parasitic microorganisms, and mycoplasmas that cause infectious diseases to which the vaccines described herein are applied, respectively. The infectious diseases to which the vaccines described herein are applied and the pathogens that cause them will be described below. Examples of components derived from pathogens include constituents of pathogens and products of pathogens. Specific examples of antigens include inactivated antigens, attenuated antigens, subunit antigens, and recombinant antigens. Inactivated antigens include inactivated viruses, inactivated bacteria, and inactivated toxins (also called "toxoids"). Specific examples of viruses (which may be inactivated) include enveloped viruses. An example of an enveloped virus is bovine viral diarrhea virus. Specific examples of bacteria (which may be inactivated) include gram-negative bacteria. Examples of gram-negative bacteria include Escherichia coli. Another example of gram-negative bacteria is Avibacterium paragallinarum (Haemophilus paragallinarum), which is known as the causative agent of infectious coryza, as described below. Examples of toxoids include inactivated toxins produced by bacteria of the genus Clostridium.Examples of Clostridium bacteria include Clostridium septicum. Antigens, in terms of chemical structure, include sugars, lipids, peptides, proteins, and their chemical or recombinant conjugates (glycolipids, glycoproteins, lipoproteins, etc.). A single antigen may be used, or two or more antigens may be combined.

[0066] Inactivated antigens can be prepared, for example, by subjecting the target substance (e.g., a virus, bacterium, or toxin) that serves as the raw material to an inactivation treatment. The inactivation treatment is not particularly limited, as long as it does not impair the immunogenicity of the target substance (in other words, does not impair the immunogenicity of the vaccine described herein) and inactivates the target substance. Examples of inactivation of the target substance include the elimination of proliferation and toxicity. The inactivation treatment can be appropriately selected depending on various conditions, such as the type of target substance. Examples of inactivation treatments include treatment with a detoxifying agent, heat treatment, and ultraviolet irradiation. In particular, examples of inactivation treatments include treatment with a detoxifying agent. A single type of inactivation treatment may be used, or two or more types of treatments may be used in combination. Examples of detoxifying agents include formalin, β-propiolactone (BPL), and binary ethyleneimine (BEI). A single component of the detoxifying agent may be used, or two or more components may be used in combination.

[0067] The vaccines described herein may or may not consist of cationic lipids and antigens. That is, the vaccines described herein may contain other components in addition to cationic lipids and antigens. Examples of other components include the other components exemplified as components of lipid membrane structures. Examples of other components also include detoxifying agents. The vaccines described herein may contain other components exemplified as components of lipid membrane structures, for example, by containing lipid membrane structures containing other components exemplified as components of lipid membrane structures. The vaccines described herein may contain a detoxifying agent, for example, by containing an antigen containing a detoxifying agent (e.g., a detoxified antigen). The other components may be one type of component, or two or more types of components.

[0068] The vaccines described herein can be produced, for example, by appropriately mixing the raw materials (i.e., cationic lipids, antigens, and optionally other components). That is, the present specification discloses a method for producing the vaccines described herein, which includes mixing cationic lipids and antigens. Both the cationic lipids and the antigens may or may not be pre-mixed with additional components before mixing. That is, in the expression "mixing cationic lipids and antigens," both the cationic lipids and the antigens may be pre-mixed with other components. For example, the cationic lipids may be pre-mixed with other components to form a lipid membrane structure. Furthermore, after mixing the cationic lipids and the antigens, other components may be further mixed. When the antigen is an inactivated antigen, for example, a pre-prepared inactivated antigen may be mixed with a cationic lipid or the like, or the target substance (e.g., a virus, bacterium, or toxin) that will be the raw material for the inactivated antigen may be mixed with a cationic lipid or the like and then inactivated. When the antigen is an inactivated antigen, preferably a pre-prepared inactivated antigen may be mixed with a cationic lipid or the like.

[0069] When the cationic lipid constitutes the lipid membrane structure, the antigen may or may not be encapsulated in the lipid membrane structure. A lipid membrane structure encapsulating an antigen can be produced, for example, by producing the lipid membrane structure in the coexistence of the antigen.

[0070] Furthermore, mixing a cationic lipid with an antigen can improve the function of a vaccine, i.e., produce a vaccine with improved function. Specifically, the present specification discloses a method for improving the function of a vaccine, comprising mixing a cationic lipid with an antigen. The present specification also discloses a method for producing a vaccine with improved function, comprising mixing a cationic lipid with an antigen. In other words, the vaccine described herein may be a vaccine with improved function. Specifically, mixing a cationic lipid with an antigen can improve the function of the vaccine compared to a vaccine that does not contain a cationic lipid. Improvements in vaccine function include enhancing the immunogenicity of the vaccine (specifically, the immunogenicity of the antigen contained in the vaccine), accelerating the immune response to the vaccine (specifically, the immune response to the antigen contained in the vaccine), prolonging the immune response to the vaccine (specifically, the immune response to the antigen contained in the vaccine), and switching to an immune response different from that induced by the antigen alone contained in the vaccine (e.g., switching from a Th1 immune response to a Th2 immune response or switching from humoral immunity to cellular immunity). Improvements in vaccine function, such as enhanced immunogenicity of the antigen contained in the vaccine, can be confirmed, for example, using as an indicator the enhanced immune response to the vaccine (specifically, the immune response to the antigen contained in the vaccine) when the vaccine is administered to an animal. Enhancement of the immune response can be measured, for example, using as an indicator the increased production of antibodies against the antigen contained in the vaccine. Examples of increased antibody production include an increased amount of antibody production, an increased rate of antibody production, and an extended period of antibody production. Switching of immune responses can be confirmed, for example, by confirming the type of immune response induced.

[0071] The content and content ratio of each component (i.e., cationic lipid, antigen, and optionally other components) in the vaccines described herein are not particularly limited, as long as the effects of the vaccines described herein are obtained. The content and content ratio of each component (i.e., cationic lipid, antigen, and optionally other components) in the vaccines described herein may vary depending on the type of each component and the characteristics of the vaccines described herein. This can be set appropriately depending on various conditions such as the intended use of the vaccine as described in the specification.

[0072] The content of cationic lipid in the vaccines described herein may be, for example, an amount capable of improving the function of the vaccine. The content of cationic lipid in the vaccines described herein may be, for example, 0.1 μmol / mL or more, 0.2 μmol / mL or more, 0.5 μmol / mL or more, 1 μmol / mL or more, 2 μmol / mL or more, 5 μmol / mL or more, 10 μmol / mL or more, 20 μmol / mL or more, 50 μmol / mL or more, or 100 μmol / mL or more, or 200 μmol / mL or less, 100 μmol / mL or less, 50 μmol / mL or less, 20 μmol / mL or less, 10 μmol / mL or less, 5 μmol / mL or less, 2 μmol / mL or less, 1 μmol / mL or less, 0.5 μmol / mL or less, or 0.2 μmol / mL or less, or a compatible combination thereof. The content of the cationic lipid in the vaccines described herein may be, for example, 0.1 to 0.2 μmol / mL, 0.2 to 0.5 μmol / mL, 0.5 to 1 μmol / mL, 1 to 2 μmol / mL, 2 to 5 μmol / mL, 5 to 10 μmol / mL, 10 to 20 μmol / mL, 20 to 50 μmol / mL, 50 to 100 μmol / mL, or 100 to 200 μmol / mL. The content of the cationic lipid in the vaccines described herein may be, for example, 0.1 to 200 μmol / mL, 0.5 to 50 μmol / mL, or 2 to 10 μmol / mL. When the vaccines described herein are in a form other than a liquid, " / mL" may be interpreted as " / g."

[0073] The content of the antigen in the vaccine described herein may be, for example, an amount that can elicit an immune response against the antigen in an animal when the vaccine described herein is administered to an animal.

[0074] The form of the vaccines described herein is not particularly limited. The vaccines described herein may be in any form, such as a liquid or a powder. The vaccines described herein may particularly be in a liquid form.

[0075] The vaccines described herein can be used, for example, by administering to animals. The vaccines described herein can be used, for example, by administering to animals to prevent, alleviate, or treat infectious diseases in animals. That is, the vaccines described herein may be, for example, for preventing, alleviating, or treating infectious diseases in animals. "Preventing, alleviating, or treating an infectious disease" also includes preventing, alleviating, or treating complications caused by the infectious disease, respectively. The infectious disease to which the vaccines described herein are applied may be one type of infectious disease, or two or more types of infectious diseases. The animal to which the vaccines described herein are applied may be one type of animal, or two or more types of animals.

[0076] The type of animal is not particularly limited. Examples of animals include livestock animals and pet animals. Examples of livestock animals include cows, pigs, sheep, goats, deer, buffalo, rabbits, horses, chickens, ducks, turkeys, guinea chickens, quails, pheasants, and ostriches. Examples of livestock animals include cows, pigs, and chickens. Examples of pet animals include dogs and cats.

[0077] The type of infection is not particularly limited.

[0078] Infectious diseases include the following. The "pathogen" written in parentheses indicates an example of a pathogen that can cause each infectious disease. Infectious bovine rhinotracheitis (pathogen: infectious bovine rhinotracheitis virus) Bovine viral diarrhea (bovine viral diarrhea-mucosal disease) (pathogen: bovine viral diarrhea virus) Bovine parainfluenza (parainfluenza in cattle) (pathogen: bovine parainfluenza virus 3) Bovine respiratory syncytial virus infection (pathogen: bovine respiratory syncytial virus) Bovine adenovirus infection (pathogens: bovine adenoviruses A to C, human adenovirus C, and ovine adenovirus A) Histophilus somni infection (pathogen: Histophilus somni) Bovine pasteurellosis (Mannheimia) (pathogens: Pasteurella multocida, Mannheimia haemolytica, and Bibersteinia trehalosi (Pasteurella trehalosi)) bovine ephemeral fever (pathogen: bovine epidemic virus) Ibaraki disease (pathogen: Ibaraki virus) Bovine rotavirus disease (pathogen: rotavirus A to C) Bovine coronavirus disease (pathogen: bovine coronavirus) Bovine colibacillosis (pathogen: Escherichia coli) Akabane disease (pathogen: Akabane virus) Chuzan disease (pathogen: Chuzan virus) Aino virus infection (pathogen: Aino virus) Peeton virus infection (pathogen: Peeton virus) Emphysema (blackleg) (pathogen: Clostridium chauvoei) Malignant edema (pathogen: Clostridium septicum) Bovine Clostridium perfringens infection (formerly known as bovine necrotic enteritis) (pathogen: Clostridium perfringens) Enzootic encephalitis (pathogens: Japanese encephalitis virus, West Nile virus, Eastern Equine Encephalitis (EEE) virus, Western Equine Encephalitis (WEE) virus, and Venezuelan Equine Encephalitis (VEE) virus) Porcine parvovirus infection (pathogen: parvovirus) Porcine Getah virus infection (pathogen: Getah virus) swine erysipelas (pathogen: Erysipelothrix rhusiopathiae) Actinobacillus pleuropneumoniae infection in pigs (pathogen: Actinobacillus pleuropneumoniae types 1, 2, and 5) Swine flu (pathogen: influenza A virus) Infectious bursal disease (pathogen: infectious bursal disease virus) Newcastle disease (pathogen: Newcastle disease virus) Infectious bronchitis (pathogen: gamma coronavirus) Infectious laryngotracheitis (pathogen: Irtovirus) Infectious coryza (pathogen: Avibacterium paragallinarum (Haemophilus paragallinarum)) Mycoplasma gallisepticum infection (pathogen: Mycoplasma gallisepticum) Egg Drop Syndrome-1976 (EDS-76) (Pathogen: Egg Drop Syndrome Virus) Chicken colibacillosis (pathogen: Escherichia coli)

[0079] Infectious diseases also include livestock infectious diseases (legal infectious diseases) and notifiable infectious diseases as defined in the Livestock Infectious Diseases Prevention Act. Livestock infectious diseases (legal infectious diseases) and notifiable infectious diseases as defined in the Livestock Infectious Diseases Prevention Act, as well as the pathogens that can cause them, can be found, for example, on the NARO website (https: / / www.naro.affrc.go.jp / org / niah / disease_fact / kansi.html).

[0080] The method of administering the vaccines described herein is not particularly limited as long as the effects of the vaccines described herein can be obtained. The method of administering the vaccines described herein can be appropriately selected depending on various conditions such as the type of animal, the type of infectious disease, and the type of antigen. Administration routes include intramuscular administration, intradermal administration, subcutaneous administration, intraocular administration, intratracheal administration, and intranasal administration. Administration routes for the vaccines described herein particularly include intramuscular administration, intradermal administration, subcutaneous administration, and intraocular administration. In one aspect, the vaccines described herein are expected to be able to suppress adverse reactions at the injection site, for example, and therefore may be suitable for administration routes such as intramuscular administration, intradermal administration, subcutaneous administration, and intraocular administration. The vaccines described herein can be administered to animals as is, or after being appropriately prepared into a form suitable for administration. For example, liquid vaccines described herein may be administered to animals as is, or after being appropriately diluted. Furthermore, for example, non-liquid vaccines described herein may be administered to animals as is, or after being appropriately prepared into a liquid form.

[0081] The vaccines described herein may be used alone or as a mixed vaccine preparation with other vaccines. That is, the present invention also discloses mixed vaccine preparations of the vaccines described herein with other vaccines. The other vaccines include vaccines for preventing, alleviating, or treating infectious diseases in animals. The infectious disease to which the other vaccines are applied may or may not be the same as the infectious disease to which the vaccines described herein are applied. The infectious disease to which the other vaccines are applied may be one type of infectious disease, or two or more types of infectious diseases. The animals to which the other vaccines are applied may or may not be the same as the animals to which the vaccines described herein are applied. Typically, the other vaccines may be applied to at least some or all of the animals to which the vaccines described herein are applied. The animals to which the other vaccines are applied may be one type of animal, or two or more types of animals. As the other vaccines, one type of vaccine may be used, or two or more types of vaccines may be used.

[0082] The dose of the vaccine described herein is, in terms of the dose of cationic lipid per dose, for example, 1 nmol or more, 2 nmol or more, 5 nmol or more, 10 nmol or more, 20 nmol or more, 50 nmol or more, The amount of the cationic lipid per dose of the vaccine described herein may be, for example, 1 to 2 nmol, 2 to 5 nmol, 5 to 10 nmol, 10 to 20 nmol, 20 to 50 nmol, 50 to 100 nmol, 100 to 200 nmol, 200 to 500 nmol, or 500 to 1000 nmol. The dose of the vaccine described herein may be, specifically, for example, 1 to 1000 nmol, 2 to 500 nmol, or 5 to 200 nmol, converted into the dose of cationic lipid per dose.

[0083] The vaccines described herein may be administered, for example, once, twice or more times.

[0084] <2> Vaccine availability The vaccines described herein can be used, for example, to prevent, reduce, or treat infectious diseases in animals. That is, the present specification provides a method for preventing, reduce, or treat infectious diseases in an animal, the method comprising the step of administering to the animal a vaccine described herein.

[0085] The animals and infectious diseases to which the vaccines described herein are applicable are as described above.

[0086] The administration route, dosage, and other administration methods for the vaccines described herein are as described above. [Example]

[0087] The present invention will now be described in more detail with reference to the following non-limiting examples.

[0088] Example 1 Preparation of adjuvants and evaluation of their physical properties 1. Adjuvant Preparation ssPalmEC-LNP was used as the adjuvant in Examples 1 to 3. ssPalmEC-LNP was prepared by the following procedure.

[0089] (1) Preparation of ethanol solutions of each lipid ssPalmE-P4C2 (COATSOME® SS-EC, Yuka Sangyo Co., Ltd.), cholesterol (Sigma), and DOPE (18:1 deruta9-cis 1,2-dioleolyl-sn-glycero-3-phosphoethanolamine, Avanti) were each dissolved in 99.5% ethanol to prepare a 5 mM ethanol solution. DMG-PEG 2000 (SUN BRIGHT®, Yuka Sangyo Co., Ltd.) was dissolved in 99.5% ethanol to prepare a 1 mM ethanol solution. Each lipid ethanol solution was stored at -20°C.

[0090] (2) Preparation of buffer solution Malic acid (Nacalai Tesque) and sodium chloride (Nacalai Tesque) were dissolved in distilled water and adjusted to pH 3.0 with NaOH to prepare a 20 mM malic acid / 30 mM sodium chloride aqueous solution (pH 3.0) (hereinafter also referred to as "malic acid buffer"). The malic acid buffer was filtered through a 0.2 μm pore size filter and stored at 4°C.

[0091] MES (2-(N-morpholino)ethanesulfonic acid, manufactured by Nacalai Tesque, Inc.) was dissolved in distilled water and adjusted to pH 5.5 with NaOH to prepare a 20 mM MES aqueous solution (pH 5.5) (hereinafter also referred to as "MES acid buffer"). The MES buffer was filtered through a 0.2 μm pore size filter and stored at 4°C.

[0092] (3) Preparation of ssPalmEC-LNPs by Ethanol Dilution Method 0.473 mL of 5 mM ssPalmE-P4C2 ethanol solution, 0.237 mL of 5 mM DOPE ethanol solution, 0.079 mL of 5 mM cholesterol ethanol solution, and 0.118 mL of 1 mM DMG-PEG 2000 ethanol solution (molar ratio ssPalmE-P4C2:DOPE:cholesterol:DMG-PEG 2000 = 60:30:10:3) were mixed and diluted with 3.093 mL of 99.5% ethanol to prepare 4 mL of lipid-ethanol mixed solution. 0.4 mL of the lipid-ethanol mixed solution and 0.6 mL of malic acid buffer were mixed using a microchannel (iLiNP® 1.0, Lilac Pharma) at 0.3 mL / min for the lipid-ethanol mixed solution and 0.45 mL / min for the malic acid buffer, for a total of 0.75 mL / min for 80 seconds. This was repeated 10 times to obtain 10 mL of ssPalmEC-LNP solution. After adding an equal volume of MES acid buffer and mixing, the buffer and ethanol were replaced with D-PBS(-) (phosphate-buffered saline, manufactured by Nacalai Tesque, Inc.) using an ultrafiltration membrane. Specifically, the ssPalmEC-LNP solution diluted with MES buffer was placed in the cup of an Amicon Ultra-15 tube (molecular weight cutoff 50 kDa, manufactured by Sigma) and centrifuged at 1000 × g to concentrate. D-PBS(-) was added to the concentrate up to the marked line and centrifuged again under the same conditions. This procedure was repeated once more to recover the concentrate. The remaining ssPalmEC-LNP was recovered by washing the ultrafiltration membrane with D-PBS(-) to a total volume of 1 mL. The recovered ssPalmEC-LNP was stored at 4 °C. The lipid concentration of the recovered ssPalmEC-LNP was 4 μmol / mL.

[0093] 2. Evaluation of adjuvant properties The particle size, polydispersity index (PdI), and zeta potential of ssPalmEC-LNP were measured by dynamic light scattering (Zetasizer nano ZS, Malvern). The measurement results are shown in Table 1.

[0094] [Table 1]

[0095] Example 2 Vaccine Preparation and Evaluation (1) 1. Vaccine Preparation (1) Preparation of inactivated bovine viral diarrhea virus type 1 (Nose-KB strain) MDBK cells (obtained from the RIKEN Cell Bank and adapted to suspension culture) were cultured in suspension at 37°C in a cell growth medium (Eagle's MEM (Nissui Pharmaceutical Co., Ltd.) containing 3% tryptose phosphate broth and 0.03% L-glutamine) supplemented with 3 vol% fetal bovine serum (Hyclone). The cell density was 8.6 × 10 5 Adjust to 10 cells / mL. 4.0 TCID 50 The Nose-KB strain of bovine viral diarrhea virus type 1 was inoculated to the above volume. The cell and virus suspension was seeded into a virus growth medium (Eagle's MEM (Nihon Pharmaceuticals) containing 2.5 vol% lactalbumin hydrolysate solution, 0.1 w / v% Bacto-peptone, and 0.15 w / v% glucose) supplemented with 1 vol% fetal bovine serum (Low IgG) (Invitrogen) and cultured at 37°C for 3 days. The medium was then centrifuged at 12,800 × g and the supernatant was collected. The culture supernatant was concentrated to approximately 1 / 10 its original volume using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa (KrosFlo, K25S-300-01N). Formalin (Kenei Pharmaceutical) was added to the resulting virus suspension to a concentration of 0.1 vol%. The virus was inactivated by sensitization at 2–5°C for 4 weeks. This was used as the immunization antigen BVD1-NoK.

[0096] (2) Vaccine preparation Pre-inactivation viral load of 10 8.72 TCID 50ssPalmEC-LNP was added to the BVD1-NoK antigen at 50 nmol, 150 nmol, and 450 nmol (as total lipid) per dose, respectively, and the resulting solutions were adjusted to 12 mL with phosphate buffer and Eagle's MEM supplemented with 0.1 vol% formalin, respectively, to create investigational vaccines A, B, and C. Furthermore, investigational vaccine D was created by adjusting the same amount of BVD1-NoK antigen as investigational vaccines A to C to 12 mL with phosphate buffer and Eagle's MEM supplemented with 0.1 vol% formalin.

[0097] 2. Safety Assessment The vaccines A to D were each inoculated into rats as follows, and ssPalmEC-LNP was evaluated as an adjuvant.

[0098] Twenty-two rats weighing approximately 100 g were used. Five rats each received 1.0 mL of each of the test vaccines A to D intramuscularly in the left and right thighs, forming groups A to D. The remaining two rats served as an unvaccinated control group (group E). Each rat was housed for 21 days. No abnormalities were observed in either clinical symptoms or local reactions at the injection site 21 days after vaccination in any of groups A to D. These results demonstrated the high safety of ssPalmEC-LNP.

[0099] 3.Efficacy evaluation In the safety evaluation, blood was collected from rats 21 days after vaccination to obtain serum. The serum (4 groups x 5 rats + 2 rats in the control group, a total of 22 samples) was used to measure the neutralizing antibody titer. Neutralizing antibody titers were measured in accordance with 3.5.7.2.2 of the "Infectious Bovine Rhinotracheitis / Bovine Viral Diarrhea Bivalent / Bovine Parainfluenza / Bovine Respiratory Syncytial Virus Infection Combined (Adjuvanted) Inactivated Vaccine" (Ministry of Agriculture, Forestry and Fisheries Notification No. 1567, October 3, 2002) biological preparation standard. MDBK-NST cells (purchased from the RIKEN Cell Bank) were used. The highest dilution of serum that inhibited CPE in two wells of cultured cells was defined as the neutralizing antibody titer. The geometric mean (GM) of the neutralizing antibody titer for each group was calculated, and a t-test was performed.

[0100] The results are shown in Figure 1. Antibody titers did not increase in Group D (vaccinated without ssPalmEC-LNP) and Group E (unvaccinated), but did increase in the vaccinated groups (Groups A to C) with ssPalmEC-LNP. Furthermore, a t-test showed that the antibody titer in Group B was significantly higher than that in Group D. These results demonstrated the effectiveness of ssPalmEC-LNP as an adjuvant. Furthermore, the results in Group B exceeded the standard values ​​set forth in the Standards for Veterinary Biological Products, "Infectious Bovine Rhinotracheitis, Bovine Viral Diarrhea Bivalent, Bovine Parainfluenza, and Bovine Respiratory Syncytial Virus Infection Combined (Adjuvanted) Inactivated Vaccine" (Ministry of Agriculture, Forestry and Fisheries Notification No. 1567, October 3, 2002), passing the national certification test.

[0101] Example 3 Vaccine Preparation and Evaluation (2) 1. Vaccine Preparation The antigen used was Clostridium septicum (C. septicum) toxoid. C. septicum No. 44T strain was cultured in cooked meat medium (Becton Dickinson and Company) for 20 hours at 37°C, then subcultured in porcine BHI medium (Becton Dickinson and Company) containing 0.3 w / v% glucose (Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.05 w / v% L-cysteine ​​hydrochloride monohydrate (Nacalai Tesque, Inc.) for 24 hours. The culture medium was centrifuged (12,800 × g) to collect the culture supernatant. The toxin in the culture supernatant was measured using the 3.1.2 toxicity test method for the Bovine Clostridium Infection Five-Type Mixed Toxoid (Adjuvanted) in accordance with the Standards for Biological Products for Animals (Ministry of Agriculture, Forestry and Fisheries Notification No. 646, April 22, 2010), and was found to be 12,800 CU / mL. The culture supernatant was detoxified by adding 0.4 vol% formalin (Kenei Pharmaceutical Co., Ltd.) and treating at 37°C for 3 days. The treated product was concentrated 26-fold, added 0.1 vol% formalin, and treated overnight at 37°C to obtain the toxoid used as an antigen. ssPalmEC-LNP was added in the amounts shown in Table 1 to the toxoid equivalent to 57,574 CU per dose, and phosphate-buffered saline (PBS) was added to make the total volume 0.4 mL to obtain experimental vaccines A to D.

[0102] [Table 2]

[0103] 2. Potency Test 0.4 mL of each of the test vaccines A to D was administered intramuscularly to the thigh of guinea pigs (Hartley, 5 weeks old, female) (first vaccination). Two weeks after the first vaccination, the same amount of the test vaccine was administered (second vaccination). Ten days after the second vaccination, blood was collected from the heart under appropriate anesthesia. The blood was centrifuged at 3500 rpm for 15 minutes to separate the serum. Serum neutralizing antibody titers were measured using the following procedure based on the Standards for Veterinary Biological Products: Bovine Clostridium Infection Five-Type Mixed (Adjuvanted) Toxoid (Ministry of Agriculture, Forestry and Fisheries Notification No. 646, April 22, 2010).

[0104] Serum was serially diluted 2-fold starting from a 5-fold dilution in cell culture medium (Eagle's MEM (Nihon Pharmaceutical Co., Ltd.) supplemented with 0.29% w / v% tryptose phosphate broth, 0.0292% w / v% L-glutamine, and 5% v / v% fetal bovine serum (heat-inactivated at 56°C for 30 minutes)). The neutralizing toxin was prepared by filtering C. septicum culture supernatant through a membrane filter (Millipore) with a pore size of 450 nm or less and diluting it with cell culture medium to a concentration of 4 CU. Equal volumes of the diluted serum and the neutralizing toxin were mixed and neutralized at 37°C for 1 hour to prepare the sample. 100 μL of the sample was added to each well of a Vero cell sheet in a 96-well plate and incubated at 37°C under 5% carbon dioxide for 1 day. Cytopathic effect (CPE) was observed. The highest dilution of serum that inhibited CPE in at least 50% of the cultured cells was defined as the neutralizing antibody titer.

[0105] The results are shown in Table 2. The neutralizing antibody titer induced increased with the amount of ssPalmEC-LNP added. Compared with the antigen-only group (vaccine D group), the 50 nmol lipid / day group (vaccine B group) and the 150 nmol lipid / day group (group C) showed approximately 8-fold and 18-fold higher neutralizing antibody titers, respectively, demonstrating the effectiveness of ssPalmEC-LNP as an adjuvant. Furthermore, the titers of groups B and C exceeded the standard values ​​set forth in the Standards for Veterinary Biological Products, Bovine Clostridium Infection Five-Type Mixed (Adjuvanted) Toxoid (Ministry of Agriculture, Forestry and Fisheries Notification No. 646, April 22, 2010), and were therefore eligible for national testing.

[0106] 3.Safety confirmation test The safety confirmation test was conducted simultaneously with the potency test. Each guinea pig was identified by an animal marker. The body weight was measured on the day, 1 day, and 2 days after the first vaccine administration in the potency test. Measurements and clinical observations were made.

[0107] As a result, no significant changes were observed in local or systemic clinical observations in any of the groups. Body weight measurements on the same day showed no significant differences between the weights of the group administered vaccine D and the groups administered vaccines A to C.

[0108] Example 4 Preparation of Adjuvant (2) The lipid ethanol solutions used to prepare ssPalmEC-LNP, SM-102-LNP, and ALC-0315-LNP were prepared as follows: SM-102 and ALC-0315 are the cationic lipids contained in the novel coronavirus vaccines Spikebax and Comirnaty, respectively. ssPalmEC-LNP: ssPalmE-P4C2 (COATSOME® SS-EC, NOF Corporation), cholesterol (Tokyo Chemical Industry Co., Ltd.), DOPE (COATSOME ME-8181, NOF Corporation), and DMG-PEG-2000 (SUNBRIGHT GM-020, NOF Corporation) were dissolved in 99.5% ethanol to prepare a stock ethanol solution containing 10 mg / mL ssPalmE-P4C2, 1.935 mg / mL cholesterol, 3.72 mg / mL DOPE, and 2.503 mg / mL DMG-PEG-2000 (middle column of Table 3). The stock ethanol solution was mixed with ethanol in the volume shown in the "LNP preparation" column of Table 3 to prepare a lipid ethanol solution for ssPalmEC-LNP.

[0109] [Table 3]

[0110] SM-102-LNP: SM-102 (Cayman Chemical), cholesterol (Tokyo Chemical Industry Co., Ltd.), DSPC (COATSOME MC-8080, NOF Corporation), and DMG-PEG-2000 (SUNBRIGHT GM-020, NOF Corporation) were dissolved in 99.5% ethanol to prepare a stock ethanol solution containing 100 mg / mL SM-102, 7.731 mg / mL cholesterol, 15.8 mg / mL DSPC, and 25.03 mg / mL DMG-PEG-2000 (Table 4, middle column). The stock ethanol solution was mixed with ethanol in the volume shown in the "LNP preparation" column of Table 4 to prepare lipid ethanol solutions for SM-102-LNP.

[0111] [Table 4]

[0112] ALC-0315-LNP: ALC-0315 (Cayman Chemical), cholesterol (Tokyo Chemical Industry Co., Ltd.), DSPC (COATSOME MC-8080, NOF Corporation), and ALC-0159 (Cayman Chemical) were dissolved in 99.5% ethanol to prepare a stock ethanol solution containing 50 mg / mL ALC-0315, 7.731 mg / mL cholesterol, 15.8 mg / mL DSPC, and 100 mg / mL ALC-0159 (middle column of Table 5). The stock ethanol solution was mixed with ethanol in the volume shown in the "LNP preparation" column of Table 5 to prepare the lipid ethanol solution for ALC-0315-LNP.

[0113] [Table 5]

[0114] Each LNP was prepared according to the ethanol dilution method. Specifically, LNPs were prepared using the lipid ethanol solution for each LNP prepared as described above using a microchannel (iLiNP® 1.0, Lilac Pharma). Two YSP-101 syringe pumps (standard type, YAC Corporation) were used. The lipid ethanol solution and PBS (Nacalai Tesque, Inc.) were loaded into glass syringes, respectively, and the lipid ethanol solution for LNP and PBS were injected into the microchannel at a flow rate of 0.45 mL / min and 0.30 mL / min, respectively, followed by mixing to obtain the LNP solution. The prepared LNP solution was subjected to buffer replacement with PBS using ultrafiltration (Amicon Ultra-15 tubing, 100 kDa, Merck) until the calculated ethanol concentration was less than 1%. The prepared LNP was stored at 4°C. The physical property data of each LNP are shown in Table 6.

[0115] [Table 6]

[0116] Example 5 Vaccine Preparation and Evaluation (3) 1. Vaccine Preparation The antigen used was a Clostridium septicum toxoid prepared by the same method as in Example 3. Toxoid equivalent to 57,574 CU per dose was mixed with ssPalmEC-LNP, SM-102-LNP, and ALC-0315-LNP prepared in Example 4 according to the adjuvant column in Table 7. Because each LNP has a different lipid composition, cationic lipid was added to the mixture at 400 nmol per dose. Phosphate-buffered saline (PBS) was added to a total volume of 0.4 mL to prepare experimental vaccines E to G. Experimental vaccine H, a control without adjuvant, was prepared using the same amount of antigen as experimental vaccines E to G and PBS alone (Table 7).

[0117] 2. Potency Test Five guinea pigs were inoculated with experimental vaccines E to H using the same procedure as in Example 3, and serum neutralizing antibody titers were measured. The results are shown in the neutralizing antibody titer column in Table 7. A Student's t-test (significance level <0.05) was performed using the individual neutralizing antibody titers. Experimental vaccine E was significantly higher than experimental vaccine H, but no significant difference was observed between experimental vaccines F and G. Experimental vaccine E also had a significantly higher neutralizing antibody titer than experimental vaccines F and G. These results demonstrate that ssPalmEC-LNP, when the molar number of cationic lipids is the same, induces significantly higher neutralizing antibody titers than ALC-0315-LNP and SM-102-LNP. The neutralizing antibody titers of equivalent pooled sera from each group were higher for experimental vaccines E and G than for experimental vaccine H. The neutralizing antibody titer of experimental vaccine F was the same.

[0118] [Table 7]

[0119] 3.Safety confirmation test As in Example 3, a safety confirmation test was carried out simultaneously with the potency test. As a result, no significant changes were observed in local or systemic clinical observations in any of the groups. Body weight measurements on the same day showed no significant differences between the weights of the group administered investigational vaccine H and the groups administered investigational vaccines E to G.

[0120] Example 6 Preparation of Adjuvant (3) The lipid ethanol solutions for each LNP used in preparing ssPalmEC-LNP, ssPalmOP-LNP, and LNP w / o cationic lipid were prepared as follows (note that w / o means without). ssPalmEC-LNP: ssPalmE-P4C2 (COATSOME® SS-EC, NOF Corporation), cholesterol (Tokyo Chemical Industry Co., Ltd.), DOPE (COATSOME ME-8181, NOF Corporation), and DMG-PEG-2000 (SUNBRIGHT GM-020, NOF Corporation) were dissolved in 99.5% ethanol to prepare a stock ethanol solution containing 7.01 mg / mL ssPalmE-P4C2, 1.935 mg / mL cholesterol, 3.72 mg / mL DOPE, and 2.503 mg / mL DMG-PEG-2000 (middle column of Table 8). The stock ethanol solution was mixed with ethanol in the volume shown in the "LNP preparation" column of Table 8 to prepare lipid ethanol solutions for ssPalmEC-LNPs.

[0121] [Table 8]

[0122] ssPalmOP-LNP: ssPalmOP (COATSOMESS-OP, NOF Corporation), cholesterol (Tokyo Chemical Industry Co., Ltd.), DOPE (COATSOME ME-8181, NOF Corporation), and DMG-PEG-2000 (SUNBRIGHT GM-020, NOF Corporation) were dissolved in 99.5% ethanol to prepare a stock ethanol solution containing 10 mg / mL ssPalmOP, 1.935 mg / mL cholesterol, 3.72 mg / mL DOPE, and 2.503 mg / mL DMG-PEG-2000 (middle column of Table 9). The stock ethanol solution was mixed with ethanol in the volume shown in the "LNP preparation" column of Table 9 to prepare a lipid ethanol solution for ssPalmOP-LNP.

[0123] [Table 9]

[0124] LNP w / o cationic lipid: Cholesterol (Tokyo Chemical Industry Co., Ltd.), DOPE (COATSOME ME-8181, NOF Corporation), and DMG-PEG-2000 (SUNBRIGHT GM-020, NOF Corporation) were dissolved in 99.5% ethanol to prepare stock ethanol solutions containing 1.935 mg / mL cholesterol, 4 mg / mL DOPE, and 2.503 mg / mL DMG-PEG-2000 (middle column of Table 10). The stock ethanol solution was mixed with ethanol in the volume shown in the "LNP preparation" column of Table 10 to prepare lipid ethanol solutions for LNP w / o cationic lipid.

[0125] [Table 10]

[0126] Each LNP was prepared using the ethanol dilution method as in Example 4. Specifically, LNPs were prepared using the lipid ethanol solutions for each LNP prepared as described above, using a microchannel (iLiNP® 1.0, Lilac Pharma). The LNP solution was obtained at a flow rate of 0.36 mL / min for the lipid ethanol solution for LNP and 0.12 mL / min for PBS, for a total of 0.48 mL / min. The prepared LNP solution was replaced with 0.01 mol / L Tris-HCl 10% sucrose buffer (pH 7.5) using an ultrafiltration membrane. The prepared LNPs were stored at 4°C. The physical property data at this time are shown in Table 11.

[0127] [Table 11]

[0128] Example 7 Vaccine Preparation and Evaluation (4) 1. Vaccine Preparation The antigen used was a Clostridium septicum (C. septicum) toxoid obtained by the same method as in Example 3. Toxoid equivalent to 57,574 CU per dose was mixed with ssPalmEC-LNP and ssPalmOP-LNP prepared in Example 4 according to the adjuvant column in Table 12. Because the lipid compositions of the two LNPs are different, a total lipid amount of 800 μg per dose was added. Phosphate-buffered saline (PBS) was added to a total volume of 0.4 mL to prepare experimental vaccines I to K. Experimental vaccine L, which did not contain any adjuvant, was used as a control (Table 12).

[0129] 2. Potency Test Guinea pigs were inoculated with experimental vaccines I to L using the same procedure as in Example 3, and serum neutralizing antibody titers were measured. A Student's t-test was performed at a significance level of 5%, demonstrating that ssPalmEC-LNP (investigative vaccine I) induced significantly higher neutralizing antibody titers than ssPalmOP-LNP (investigative vaccine J) and LNP w / o cationic lipid (investigative vaccine K) (Table 12). The neutralizing antibody titers of equal-volume pooled sera from each group were slightly higher for experimental vaccines J and K than for experimental vaccine L. Furthermore, experimental vaccine J was slightly higher than experimental vaccine K, and although the difference was not significant, this suggested a weak adjuvant effect.

[0130] [Table 12]

[0131] 3.Safety confirmation test A safety confirmation test was conducted using the same procedures as in Example 3. As a result, no significant changes were observed in local or systemic clinical observations in any of the groups. Furthermore, when a Student's t-test was performed for each group at a significance level of 5%, no significant difference was observed between the weight gain ratios of the non-adjuvanted control (investigative vaccine L) group and the experimental vaccine J and K groups. In the experimental vaccine I group, the weight gain ratio on day 1 after the first vaccination was significantly lower than that of the experimental vaccine L group, but was equivalent to that of the experimental vaccine L group from day 2 after the first vaccination onwards.

[0132] Example 8 Preparation of Adjuvant (4) The lipid ethanol solutions used in the preparation of ssPalmEC-LNP were prepared as in Example 6. Ta. ssPalmEC-LNP was prepared according to the ethanol dilution method as in Example 6. Specifically, the lipid solutions were mixed in the amounts shown in Table 13 to prepare a lipid-ethanol solution. A microchannel was used to prepare the LNP. The lipid-ethanol solution was mixed at a flow rate of 0.36 mL / min and PBS at 0.12 mL / min, for a total of 0.48 mL / min, until the entire lipid-ethanol solution was used. The prepared LNP solution was subjected to buffer exchange with 0.01 mol / L Tris-HCl in 10% sucrose buffer (pH 7.5) using an ultrafiltration membrane. The prepared LNP was stored at 4°C. The physical property data of this LNP are shown in Table 14.

[0133] [Table 13]

[0134] [Table 14]

[0135] Example 9 Vaccine Preparation and Evaluation (5) 1. Preparation of inactivated bacterial cells In accordance with the Standards for Veterinary Biological Products, a combined inactivated vaccine (seed) for Newcastle disease, infectious bronchitis, infectious coryza (types A and C), and Mycoplasma gallisepticum infections (with oil-based adjuvant) (Ministry of Agriculture, Forestry and Fisheries Notification No. 134, January 27, 2023) was prepared as follows: Avibacterium paragallinarum type C KA strain was inoculated into 3.7 w / v% porcine-derived BHI medium containing 5 v / v% inactivated chicken serum negative for infectious coryza types A and C and 0.01 w / v% β-NAD, and cultured for 24 hours at 37°C. The culture solution was treated with a surfactant, centrifuged, concentrated, and inactivated by adding 0.1 vol% β-propiolactone and allowing to stand at 2–5°C for 44 hours. This was used as the antigen in the tests described below.

[0136] 2. Vaccine Preparation Pre-inactivation viable bacterial count: 3.6 x 10 per dose 7 The ssPalm EC-LNP prepared in Example 6 was added as an adjuvant at 50 nmol, 200 nmol, and 800 nmol per dose, respectively, and adjusted with phosphate-buffered saline (PBS) to form experimental vaccines A, B, and C. Experimental vaccine D was also prepared by adjusting the same amount of antigen as experimental vaccines A to C in PBS without using an adjuvant (Table 15).

[0137] [Table 15]

[0138] 3. Vaccination and evaluation Ten SPF chickens were used in each test group. Four-week-old SPF chickens were immunized with the four vaccines prepared by injecting 0.5 mL of each immunogen into the leg muscle of each chicken, and then reared for five weeks. Clinical observations were also conducted throughout the test period, and the inoculation site was also observed. Blood samples were collected 5 weeks after immunization, and serum samples were obtained. Antibody titers (reaction inhibition rates) against the antigen were measured using an enzyme-linked immunosorbent assay (competitive ELISA) according to the following procedure. All samples were measured in duplicate, and the average value was calculated for each test group. The significance of the difference from the control group was tested using the Student t-test.

[0139] The antigen (inactivated Avibacterium paragallinarum type C KA strain) was sonicated and diluted to 5 μg / mL in 50 mM carbonate / bicarbonate buffer. 100 μL of the solution was added to each well of a 96-well ELISA plate (Fisher Scientific) and coated overnight. The plate was washed with PBS containing 0.05% Tween (PBS-T) and blocked with PBS-T containing 1% bovine serum albumin (BSA) (Wako) for 1 hour at 30°C. After washing with PBS-T, 100 μL of each serum sample, reference negative serum, and reference positive serum were added to two wells (duplicate assays) and incubated for 1 hour at 30°C. The plate was then washed with PBS-T and 100 μL / well of 6 ng / mL anti-IC type C monoclonal antibody was added for 1 hour at 30°C. The plate was washed with PBS-T, and 100 μL / well of HRP-labeled anti-mouse IgG antibody diluted 1:16,000 was added for 1 hour at 30°C. After washing with PBS-T, 100 μL / well of TMB substrate kit (Thermo Fisher) was added and the reaction was allowed to proceed for 30 minutes at 30°C. The reaction was stopped by adding 100 μL / well of 1M sulfuric acid. The absorbance at 450 nm was measured, and the reaction inhibition rate was calculated using the following formula, which was used as the titer. Reaction inhibition rate (%) = 100 - [(OD of test serum × 100) ÷ (OD of negative serum)] Statistical differences between titers were tested using the Student's t-test at a significance level of 1%.

[0140] 4. Safety Assessment Throughout the test period, no abnormalities were observed in the chickens due to vaccination, and no side effects such as swelling or induration were observed at the injection site after immunization.

[0141] 5. Evaluation of antibody induction ability Groups A to C, which received a vaccine using ssPalm EC-LNP as an adjuvant, showed a statistically significant increase in antibody titers compared to group D, which did not use an adjuvant. There was a positive correlation with the concentration of EC-LNP (Table 16, Figure 2). These results demonstrated that ssPalm EC-LNP has a dose-dependent adjuvant effect in chickens.

[0142] [Table 16]

Claims

1. A vaccine comprising a cationic lipid and an antigen, the antigen is one or more components selected from the group consisting of pathogens and components derived therefrom, A vaccine wherein the cationic lipid is ssPalmE-P4C2.

2. The vaccine according to claim 1, wherein the cationic lipid is a constituent lipid of a lipid membrane structure.

3. The vaccine according to claim 2 , wherein the lipid membrane structure further contains a non-cationic lipid.

4. 4. The vaccine of claim 3, wherein the non-cationic lipid is one or more components selected from the group consisting of phospholipids, PEG lipids, and steroids.

5. 5. The vaccine of claim 4, wherein the steroid is one or more components selected from the group consisting of cholesterol and its fatty acid esters.

6. The vaccine of claim 1 , wherein the antigen is an inactivated antigen.

7. The vaccine of claim 6, wherein the inactivated antigen is an inactivated virus.

8. 7. The vaccine of claim 6, wherein the inactivated antigen is an inactivated bacterium.

9. The vaccine of claim 6, wherein the inactivated antigen is a toxoid.

10. The virus is bovine viral diarrhea virus. The vaccine of claim 7.

11. The bacterium is Avibacterium paragallinarum. The vaccine of claim 8.

12. 10. The vaccine of claim 9, wherein the toxoid is a toxoid of Clostridium septicum.

13. 10. The vaccine of claim 1 for preventing, reducing, or treating an infectious disease in an animal.

14. 14. The vaccine of claim 13, wherein the animal is a livestock animal or a companion animal.

15. 10. The vaccine of claim 1, administered by a route of administration selected from the group consisting of intramuscular administration, intradermal administration, subcutaneous administration, and intraocular administration.

16. 10. The vaccine of claim 1, which is administered intramuscularly.

17. A method for producing a vaccine, comprising: The vaccine is the vaccine of claim 1, mixing said cationic lipid and said antigen.

18. 18. The method of claim 17, wherein the vaccine is an improved vaccine.

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