Composition for use in vaccine

Abstract

The present invention relates to a composition for use in vaccines, the composition containing a cationic lipid represented by formula (1).

Description

Compositions for use in vaccines

[0001] The present invention relates to compositions for use in vaccines.

[0002] Vaccines (mRNA-LNP vaccines) composed of lipid nanoparticles (LNPs) encapsulating messenger RNA (mRNA) are expected to be a highly effective modality because they can be used for 100-day missions. However, while current mRNA-LNP vaccines potently induce vaccine effects, side effects such as fever, localized swelling, and fatigue due to the production of inflammatory cytokines have been frequently observed, potentially triggering vaccine aversion. Therefore, to elevate the vaccine as a modality applicable not only in emergencies but also in peacetime, it is essential to maintain vaccine efficacy while reducing side effects caused by inflammatory properties. Furthermore, Patent Document 1 discloses cationic lipids with improved intracellular dynamics, but does not disclose the use of these cationic lipids in vaccines.

[0003] International Publication No. 2019 / 188867

[0004] Therefore, the present invention has been made in consideration of the above problems, and an object of the present invention is to provide a composition for use in a vaccine that can reduce side reactions.

[0005] As a result of extensive research, the present inventors have discovered a composition for use in a vaccine that can reduce side effects, and have completed the present invention.

[0006] That is, the present invention includes the following aspects: [1] A compound represented by the following formula (1): (In formula (1), R １ａ and R １ｂ each independently represents an alkylene group having 1 to 6 carbon atoms; ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups; R２ａ and R ２ｂ each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms; Y ａ and Y ｂ each independently represents an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond; Z ａ and Z ｂ each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally a heteroatom; R ３ａ and R ３ｂ each independently represent a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, a residue derived from a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms. [2] The composition according to [1], further comprising a nucleic acid. [3] The composition according to [2], wherein the ratio of the content of the cationic lipid to the content of the nucleic acid is 0.05 to 150, expressed as cationic lipid content (nmol) / nucleic acid content (μg). [4] The composition according to any of [1] to [3], further comprising one or more selected from the group consisting of an ionic lipid, a neutral lipid, a PEG-modified lipid, and a sterol. [5] The composition according to [1] to [4], which is in the form of a lipid particle. [6] The composition according to any of [1] to [5], for preventing or treating an infectious disease. [7] The composition according to any one of [1] to [6], which is for intradermal administration, subcutaneous administration, nasal administration, or intramuscular administration. [8] The composition according to [1] to [7], wherein preventing an infectious disease comprises inducing protective immunity against the infectious disease in a subject.

[0007] The present invention also includes the following aspects. [A1] A vaccine comprising a cationic lipid represented by the above formula (1). [A2] The vaccine according to [A1], further comprising a nucleic acid. [A3] The vaccine according to [A1] to [A2], wherein the ratio of the cationic lipid content to the nucleic acid content is 0.05 to 150, expressed as cationic lipid content (nmol) / nucleic acid content (μg). [A4] The vaccine according to any of [A1] to [A3], further comprising one or more selected from the group consisting of ionic lipids, neutral lipids, PEG-modified lipids, and sterols. [A5] The composition according to [A1] to [A4], which is a lipid particle. [A6] The vaccine according to any of [A1] to [A5], for preventing or treating an infectious disease. [A7] The vaccine according to any of [A1] to [A6], which is for intradermal, subcutaneous, nasal, or intramuscular administration. [A8] The vaccine according to any one of [A1] to [A7], wherein preventing an infectious disease comprises inducing protective immunity against the infectious disease in a subject.

[0008] [B1] A method for preventing a disease, comprising administering a composition comprising a cationic lipid represented by formula (1) and a nucleic acid. [B2] The method according to [B1], wherein the ratio of the cationic lipid content to the nucleic acid content is 0.05 to 150, expressed as cationic lipid content (nmol) / nucleic acid content (μg). [B3] The method according to [B1] or [B2], wherein the composition further comprises one or more selected from the group consisting of ionic lipids, neutral lipids, PEG-modified lipids, and sterols. [B4] The composition according to [B1] to [B3], wherein the composition is a lipid particle. [B5] The method according to any one of [B1] to [B4], wherein the disease is an infectious disease. [B6] The method according to any one of [B1] to [B5], wherein the composition is administered intradermally, subcutaneously, intranasally, or intramuscularly. [B7] The method according to any one of [B1] to [B6], wherein the composition induces protective immunity against the disease in a subject.

[0009] [C1] A method for inducing protective immunity against a disease in a subject, the method comprising administering a composition comprising a cationic lipid represented by formula (1) and a nucleic acid. [C2] The method according to [C1], wherein the ratio of the cationic lipid content to the nucleic acid content is 0.05 to 150, expressed as cationic lipid content (nmol) / nucleic acid content (μg). [C3] The method according to [C1] or [C2], wherein the composition further comprises one or more selected from the group consisting of ionic lipids, neutral lipids, PEG-modified lipids, and sterols. [C4] The composition according to [C1] to [C3], wherein the composition is a lipid particle. [C5] The method according to any of [C1] to [C4], wherein the disease is an infectious disease. [C6] The method according to any of [C1] to [C5], wherein the composition is administered intradermally, subcutaneously, intranasally, or intramuscularly.

[0010] [D1] Use of a composition comprising a cationic lipid represented by formula (1) and a nucleic acid for preventing a disease. [D2] The use according to [D1], wherein the ratio of the cationic lipid content to the nucleic acid content is 0.05 to 150, expressed as cationic lipid content (nmol) / nucleic acid content (μg). [D3] The use according to [D1] or [D2], wherein the composition further comprises one or more selected from the group consisting of ionic lipids, neutral lipids, PEG-modified lipids, and sterols. [D4] The composition according to [D1] to [D3], wherein the composition is a lipid particle. [D5] The use according to any of [D1] to [D4], wherein the disease is an infectious disease. [D6] The use according to any of [D1] to [D5], wherein the composition is administered intradermally, subcutaneously, intranasally, or intramuscularly. [D7] The use according to any of [D1] to [D6], wherein the prevention of an infectious disease comprises inducing protective immunity against the infectious disease in a subject.

[0011] [E1] Use of a cationic lipid represented by formula (1) in the manufacture of a composition for use in a vaccine. [E2] The use according to [E1], wherein the composition further comprises a nucleic acid. [E3] The use according to [E2], wherein the ratio of the cationic lipid content to the nucleic acid content, expressed as cationic lipid content (nmol) / nucleic acid content (μg), is 0.05 to 150. [E4] The use according to any of [E1] to [E3], wherein the composition further comprises one or more selected from the group consisting of ionic lipids, neutral lipids, PEG-modified lipids, and sterols. [E5] The composition according to [E1] to [E4], wherein the composition is a lipid particle. [E6] The composition according to any of [E1] to [E5], wherein the composition is for preventing or treating an infectious disease. [E7] The use according to any of [E1] to [E6], wherein the composition is for intradermal, subcutaneous, nasal, or intramuscular administration. [E8] The use according to [E1] to [E7], wherein the prevention of an infectious disease comprises inducing protective immunity against the infectious disease in a subject. The use in [E1] to [E8] may be use of the cationic lipid represented by formula (1) in the production of a vaccine.

[0012] According to the present invention, a composition for use in a vaccine that can reduce side reactions can be provided.

[0013] 1 is a diagram showing the results of measuring the encapsulation rate of mRNA-LNP. It is a cryo-electron microscope image of mRNA-LNP. It is a diagram showing the antibody titer after two subcutaneous immunizations. It is a diagram showing the antibody titer after prime subcutaneous immunization. It is a diagram comparing the antibody titer after two subcutaneous immunizations with that of the control. It is a diagram showing the antibody titer after two intramuscular immunizations. It is a diagram showing the T cell response after two subcutaneous immunizations or two intramuscular immunizations. It is a diagram showing the infection protection capability using body weight and survival rate as indicators. It is a diagram showing the mRNA transfer efficiency after one intramuscular immunization. It is a diagram showing the mRNA transfer efficiency after two intramuscular immunizations. It is a diagram showing HA expression after two intramuscular immunizations. It is a diagram showing the activation of antigen-presenting cells in regional lymph nodes after subcutaneous immunization. It is a diagram showing the amount of cytokines in the blood after subcutaneous immunization. It is a diagram showing the amount of cytokines in the blood after subcutaneous immunization. It is a diagram showing the amount of cytokines in the blood after intramuscular immunization. It is a diagram showing the amount of cytokines in the blood after intramuscular immunization. It is a diagram showing side reactions after intramuscular immunization. FIG. 10 is a diagram comparing blood cytokine levels and antibody titers between LNPs using different neutral lipids.

[0014] Hereinafter, embodiments of the present invention will be described in detail. Note that the present invention is not limited to the following embodiments, and various modifications can be made within the scope of the present invention.

[0015] The composition of the present embodiment is a compound represented by the following formula (1): (In formula (1), R １ａ and R １ｂ each independently represents an alkylene group having 1 to 6 carbon atoms; ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups; R ２ａ and R ２ｂ each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms; Y ａ and Y ｂ each independently represents an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond; Z ａ and Z ｂeach independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally a heteroatom; R ３ａ and R ３ｂ each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or a residue derived from a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

[0016] In this embodiment, R １ａ and R １ｂ are each independently an alkylene group having 1 to 6 carbon atoms, which may be linear or branched and is not particularly limited, but is preferably linear. The number of carbon atoms in the alkylene group is not particularly limited, but for example, it is preferably 1 to 4, and more preferably 1 to 2. Examples of alkylene groups having 1 to 6 carbon atoms include, but are not particularly limited to, methylene, ethylene, trimethylene, isopropylene, tetramethylene, isobutylene, pentamethylene, and neopentylene. Among these, methylene, ethylene, trimethylene, isopropylene, or tetramethylene is preferred, and ethylene is more preferred. R １ａ is R １ｂ may be the same as or different from, and are not particularly limited, but １ａ is R １ｂ It is preferable that the group is the same as

[0017] In this embodiment, X ａ and X ｂ are each independently a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups, and are not particularly limited, but are preferably each independently a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups. ａ is X ｂmay be the same as or different from, and are not particularly limited, but X ａ is X ｂ It is preferable that the group is the same as

[0018] The alkyl group having 1 to 6 carbon atoms in the acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group may be linear, branched, or cyclic. The number of carbon atoms in the alkyl group is not particularly limited, but is preferably 1 to 3, for example. Examples of the alkyl group having 1 to 6 carbon atoms include, but are not particularly limited to, a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a t-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. Among these, a methyl group, an ethyl group, a propyl group, or an isopropyl group is preferred, and a methyl group is more preferred.

[0019] A preferred specific structure of the non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group is: X １ It is shown as follows. X １ R ５ represents an alkyl group having 1 to 6 carbon atoms, which may be linear, branched, or cyclic. The number of carbon atoms in the alkyl group is not particularly limited, but is preferably 1 to 3, for example. Examples of the alkyl group having 1 to 6 carbon atoms include, but are not particularly limited to, a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a t-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. Among these, a methyl group, an ethyl group, a propyl group, or an isopropyl group is preferred, and a methyl group is more preferred.

[0020] The number of carbon atoms in the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups is not particularly limited, but is preferably, for example, 4 to 5. The cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups is not particularly limited, but examples thereof include an aziridylene group, an azetidylene group, a pyrrolidine group, a piperidylene group, an imidazolidylene group, and a piperaziylene group. Among these, a pyrrolidine group, a piperidylene group, or a piperaziylene group is preferred, and a piperidylene group is more preferred.

[0021] A preferred specific structure of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one tertiary amino group is X ２ It is shown as follows. X ２ In the formula, p is 1 or 2. When p is 1, X ２ is a pyrrolidine group, and when p is 2, X ２ is a piperidylene group.

[0022] A preferred specific structure of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and two tertiary amino groups is X ３ It is shown as follows. X ３ The w in is 1 or 2. When w is 1, X ３ is an imidazolidylene group, and when w is 2, X ３ is a piperazylene group.

[0023] In this embodiment, R ２ａ and R ２ｂ R each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms, and is not particularly limited, but is preferably an alkylene group having 8 or less carbon atoms. ２ａ is R ２ｂ may be the same as or different from, and are not particularly limited, but ２ａ is R ２ｂ It is preferable that the group is the same as

[0024] The alkylene group having 8 or less carbon atoms may be linear or branched and is not particularly limited, but is preferably linear. The number of carbon atoms in the alkylene group is not particularly limited, but is preferably 6 or less, and more preferably 4 or less. The alkylene group having 8 or less carbon atoms is not particularly limited, but examples thereof include a methylene group, an ethylene group, a propylene group, an isopropylene group, a tetramethylene group, an isobutylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, and an octamethylene group. Among these, a methylene group, an ethylene group, a propylene group, or a tetramethylene group is preferred, and an ethylene group is more preferred.

[0025] An oxydialkylene group having 8 or less carbon atoms refers to an alkylene group (alkylene-O-alkylene) linked via an ether bond, in which the total number of carbon atoms in the two alkylene groups is 8 or less. Here, the two alkylene groups may be the same or different, but are preferably the same. The oxydialkylene group having 8 or less carbon atoms is not particularly limited, but examples include an oxydimethylene group, an oxydiethylene group, an oxydipropylene group, and an oxydibutylene group. Among these, an oxydimethylene group, an oxydiethylene group, or an oxydipropylene group is preferred, and an oxydiethylene group is more preferred.

[0026] In this embodiment, Y ａ and Y ｂ each independently represents an ester bond, an amide bond, a carbamate bond, an ether bond, or a urea bond, and is not particularly limited, but is preferably each independently an ester bond, an amide bond, or a carbamate bond, more preferably each independently an ester bond or an amide bond, and even more preferably each independently an ester bond. ａ and Y ｂ The bond direction of Y is not particularly limited. ａ and Y ｂ When is an ester bond, -Z ａ -CO-O-R ２ａ - and -Z ｂ -CO-O-R２ｂ Preferably, Y is a group having the structure -. ａ Is Y ｂ may be the same as or different from, and are not particularly limited, ａ Is Y ｂ It is preferable that the group is the same as

[0027] In this embodiment, Z ａ and Z ｂeach independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally a heteroatom. The aromatic compound preferably has 6 to 12 carbon atoms, and more preferably 6 to 7 carbon atoms. The aromatic compound preferably contains one aromatic ring. The aromatic ring contained in the aromatic compound having 3 to 16 carbon atoms is not particularly limited, but examples thereof include an aromatic hydrocarbon ring and an aromatic heterocycle. The aromatic hydrocarbon ring is not particularly limited, but examples thereof include a benzene ring, a naphthalene ring, and an anthracene ring. The aromatic heterocycle is not particularly limited, but examples thereof include an imidazole ring, a pyrazole ring, an oxazole ring, an isoxazole ring, a thiazole ring, an isothiazole ring, a triazine ring, a pyrrole ring, a furanthiophene ring, a pyrimidine ring, a pyridazine ring, a pyrazine ring, a pyridine ring, a purine ring, a pteridine ring, a benzimidazole ring, an indole ring, a benzofuran ring, a quinazoline ring, a phthalazine ring, a quinoline ring, an isoquinoline ring, a coumarin ring, a chromone ring, a benzodiazepine ring, a phenoxazine ring, a phenothiazine ring, and an acridine ring. Among these, the aromatic ring is preferably a benzene ring, a naphthalene ring, or an anthracene ring, and more preferably a benzene ring. The aromatic ring may have a substituent. The substituent is not particularly limited, and examples thereof include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 4 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkylsulfanyl group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a ureido group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, and an aryloxy group having 6 to 10 carbon atoms.Among these, an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, an acetamido group, a methoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a methylsulfanyl group, a phenylsulfonyl group, a nitro group, a trifluoromethyl group, a cyano group, a methyl group, an ethyl group, a propyl group, an isopropyl group, a t-butyl group, a ureido group, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a t-butoxy group, a phenyl group, and a phenoxy group are preferred. ａ is Z ｂ may be the same as or different from, and are not particularly limited, ａ is Z ｂ It is preferable that the group is the same as

[0028] Z ａ and Z ｂ A preferred specific structure of Z １ It is shown as follows. Z １ s represents an integer of 0 to 3, t represents an integer of 0 to 3, u represents an integer of 0 to 4, and u R ４ each independently represents a substituent. １ In Z, s is preferably an integer of 0 to 1, and more preferably 0. １ The t in Z is preferably an integer of 0 to 2, and more preferably 1. １ The u in Z is preferably an integer of 0 to 2, more preferably an integer of 0 to 1. １ R ４is a substituent of an aromatic ring (benzene ring) contained in an aromatic compound having 3 to 16 carbon atoms that does not inhibit the reaction in the synthesis process of the cationic lipid represented by the above formula (1). Examples of the substituent include, but are not limited to, an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 4 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkylsulfanyl group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a ureido group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, and an aryloxy group having 6 to 10 carbon atoms. Among these, an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, an acetamide group, a methoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a methylsulfanyl group, a phenylsulfonyl group, a nitro group, a trifluoromethyl group, a cyano group, a methyl group, an ethyl group, a propyl group, an isopropyl group, a t-butyl group, a ureido group, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a t-butoxy group, a phenyl group, and a phenoxy group are preferred. ４ When there are multiple R ４ may be the same or different.

[0029] In this embodiment, R ３ａ and R ３ｂ each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or a residue derived from a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms, and is not particularly limited, but is preferably each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms, and more preferably each independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms. ３ａand / or R ３ｂ is a residue derived from a reaction product of succinic anhydride or glutaric anhydride, R ３ａ and R ３ｂ The carbonyl group which is a substituent bonded to may be derived from succinic anhydride or glutaric anhydride.

[0030] The fat-soluble vitamin having a hydroxyl group is not particularly limited, but examples thereof include retinol, ergosterol, 7-dehydrocholesterol, calciferol, corcalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol, and tocotrienol. Of these, tocopherol is preferred. The sterol derivative having a hydroxyl group is not particularly limited, but examples thereof include cholesterol, cholestanol, stigmasterol, β-sitosterol, lanosterol, and ergosterol. Of these, cholesterol or cholestanol is preferred.

[0031] The aliphatic hydrocarbon group having 12 to 22 carbon atoms may be linear or branched. The aliphatic hydrocarbon group may be saturated or unsaturated. In the case of an unsaturated aliphatic hydrocarbon group, the number of unsaturated bonds contained in the aliphatic hydrocarbon group is preferably 1 to 6, more preferably 1 to 3, and even more preferably 1 to 2. Examples of unsaturated bonds include carbon-carbon double bonds and carbon-carbon triple bonds, with carbon-carbon double bonds being preferred. The number of carbon atoms in the aliphatic hydrocarbon group is preferably 13 to 19, and more preferably 13 to 17. The aliphatic hydrocarbon group is not particularly limited, but examples include alkyl groups, alkenyl groups, and alkynyl groups. Among these, alkyl groups and alkenyl groups are preferred. The aliphatic hydrocarbon group having 12 to 22 carbon atoms is not particularly limited, and examples thereof include dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, icosyl group, henicosyl group, docosyl group, dodecenyl group, tridecenyl group, tetradecenyl group, pentadecenyl group, hexadecenyl group, heptadecenyl group, octadecenyl group, nonadecenyl group, icosenyl group, henicosyl group, docosenyl group, dodecadienyl group, and tridecadienyl group. , tetradecadienyl group, pentadecadienyl group, hexadecadienyl group, heptadecadienyl group, octadecadienyl group, nonadecadienyl group, icosadienyl group, henicosadienyl group, docosadienyl group, octadecatrienyl group, icosatrienyl group, icosatetraenyl group, icosapentaenyl group, docosahexaenyl group, isostearyl group, 1-hexylheptyl group, 1-hexylnonyl group, 1-octylnonyl group, 1-octylundecyl group, and 1-decylundecyl group. Among these, a tridecyl group, a pentadecyl group, a heptadecyl group, a nonadecyl group, a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group is preferred, and a tridecyl group, a heptadecyl group, a heptadecenyl group, or a heptadecadienyl group is more preferred.

[0032] In this embodiment, R ３ａ and R ３ｂThe aliphatic hydrocarbon group having 12 to 22 carbon atoms, represented by the formula (1), is preferably derived from a fatty acid. In this case, the carbonyl carbon derived from the fatty acid is included in -CO-O- in formula (1). The aliphatic hydrocarbon group is not particularly limited, but for example, when linoleic acid is used as the fatty acid, it becomes a heptadecadienyl group, and when oleic acid is used as the fatty acid, it becomes a heptadecenyl group.

[0033] R ３ａ is R ３ｂ may be the same as or different from, and are not particularly limited, but ３ａ is R ３ｂ It is preferable that the group is the same as

[0034] In this embodiment, R １ａ is R １ｂ is the same as X ａ is X ｂ is the same as R ２ａ is R ２ｂ is identical to Y ａ Is Y ｂ is identical to Z ａ is Z ｂ is the same as R ３ａ is R ３ｂ It is preferably the same as

[0035] The cationic lipid represented by the above formula (1) is preferably any one of the following cationic lipids (1-1) to (1-3): [Cationic lipid (1-1)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 6 carbon atoms (e.g., a methylene group, an ethylene group); ａ and X ｂ each independently represents an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group (e.g., —N(CH ３ )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 or 2 tertiary amino groups (e.g., a piperidylene group); R ２ａ and R ２ｂ are each independently an alkylene group having 8 or less carbon atoms (e.g., a methylene group, an ethylene group, a propylene group); ａ and Yｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ are each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a heteroatom (e.g., -C ６ H ４ -CH ２ -, -CH ２ -C ６ H ４ -CH ２ -); R ３ａ and R ３ｂ are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, a 1-hexylnonyl group); a cationic lipid.

[0036] [Cationic lipid (1-2)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 4 carbon atoms (e.g., a methylene group, an ethylene group); ａ and X ｂ each independently represents an acyclic alkyl tertiary amino group having 1 to 3 carbon atoms and one tertiary amino group (e.g., —N(CH ３ )-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one tertiary amino group (e.g., a piperidylene group); R ２ａ and R ２ｂ are each independently an alkylene group having 6 or less carbon atoms (e.g., a methylene group, an ethylene group, a propylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ are each independently a divalent group derived from an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and optionally having a heteroatom (e.g., -C ６ H ４ -CH ２ -, -CH ２ -C６ H ４ -CH ２ -); R ３ａ and R ３ｂ are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride, or an aliphatic hydrocarbon group having 13 to 19 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, a 1-hexylnonyl group); a cationic lipid.

[0037] [Cationic lipid (1-3)] R １ａ and R １ｂ are each independently an alkylene group having 1 to 2 carbon atoms (e.g., a methylene group, an ethylene group); ａ and X ｂ are each independently, X １ : (In the formula, R ５ is an alkyl group having 1 to 3 carbon atoms (e.g., a methyl group), or X ２ : (wherein p is 1 or 2); R ２ａ and R ２ｂ are each independently an alkylene group having 4 or less carbon atoms (e.g., a methylene group, an ethylene group, a propylene group); ａ and Y ｂ are each independently an ester bond or an amide bond; Z ａ and Z ｂ are each independently, Z １ : (wherein s is an integer of 0 to 1, t is an integer of 0 to 2, u is an integer of 0 to 2 (preferably 0), and u R ４ each independently represents a substituent; R ３ａ and R ３ｂ are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride, or an aliphatic hydrocarbon group having 13 to 17 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, a 1-hexylnonyl group); a cationic lipid.

[0038] In this embodiment, the cationic lipid represented by the above formula (1) is not particularly limited, but examples thereof include O-Ph-P3C1, O-Ph-P4C1, O-Ph-P4C2, O-Bn-P4C2, E-Ph-P4C2, L-Ph-P4C2, HD-Ph-P4C2, O-Ph-amide-P4C2, and O-Ph-C3M, with O-Ph-P4C2 (hereinafter also referred to as "ssPalmO") being preferred.

[0039] The ratio (mol%) of the cationic lipid represented by the above formula (1) to the total lipids present in the composition of this embodiment is not particularly limited, but is preferably 5% to 100%, more preferably 10% to 90%, even more preferably 20% to 70%, and even more preferably 35% to 65%. Of these, 40% to 60% is preferred, and 45% to 55% is more preferred. The cationic lipid represented by the above formula (1) may be used alone or in combination of two or more.

[0040] In this embodiment, the cationic lipid represented by the above formula (1) can be produced by a known method. Although not particularly limited, it can be produced by, for example, the method described in WO 2019 / 188867.

[0041] The composition of this embodiment may further contain a nucleic acid. Examples of nucleic acids include, but are not limited to, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), with RNA being preferred. Examples of RNA include, but are not limited to, mRNA, circular RNA, self-amplifying RNA, miRNA, piRNA, tsRNA, siRNA, tRNA, rRNA, snRNA, snoRNA, and lncRNA. Among these, mRNA, circular RNA, self-amplifying RNA, miRNA, piRNA, tsRNA, or siRNA is preferred, and mRNA, circular RNA, self-amplifying RNA, or siRNA is more preferred.

[0042] The nucleic acid is not particularly limited, but from the perspective of use in a vaccine, it is preferably a nucleic acid having preventive or therapeutic activity against a predetermined disease (prophylactic or therapeutic nucleic acid), and examples thereof include nucleic acids derived from pathogenic viruses, pathogenic bacteria, pathogenic fungi, and pathogenic organisms. Examples of pathogenic viruses include, but are not limited to, influenza virus, avian influenza virus, parainfluenza virus, adenovirus, SARS coronavirus, MERS coronavirus, SARS coronavirus 2, AIDS virus, respiratory syncytial virus, cytomegalovirus, hepatitis virus, Japanese encephalitis virus, measles virus, rubella virus, varicella-zoster virus, viruses belonging to the Picornaviridae family, viruses belonging to the Papillomaviridae family, herpes virus, mumps virus, rotavirus, cholera virus, rabies virus, and viruses that cause viral hemorrhagic fevers (e.g., Ebola hemorrhagic fever, Marburg disease, Lassa fever, and Crimean-Congo hemorrhagic fever). Pathogenic bacteria include, but are not limited to, diphtheria, tetanus, Mycobacterium tuberculosis, Streptococcus pneumoniae, Neisseria meningitidis, Staphylococcus aureus, Pseudomonas aeruginosa, Bordetella pertussis, Bacillus anthracis, Rickettsia, and Salmonella. Pathogenic fungi include, but are not limited to, Cryptococcus and Aspergillus. Pathogenic organisms include, but are not limited to, Plasmodium falciparum. This embodiment also encompasses nucleic acids derived from viruses, bacteria, fungi, and pathogenic organisms that will be newly detected in the future. Nucleic acids may be used singly or in combination of two or more. It is preferred that the nucleic acids be purified by a method commonly used by those skilled in the art.

[0043] In this embodiment, the nucleic acid base is not particularly limited, but may be, for example, the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0044] In this embodiment, the nucleobase may be partially or completely modified as known in the art. The nucleobase may be, for example, a modified nucleobase (e.g., 8-bromoadenyl group, 8-bromoguanyl group, 5-bromocytosyl group, 5-iodocytosyl group, 5-bromouracil group, 5-iodouracil group, 5-fluorouracil group, 5-methylcytosyl group, 8-oxoguanyl group, hypoxanthinyl group, etc.) substituted with one to three optional substituents (e.g., halogen atoms, alkyl groups, aromatic group-substituted alkyl groups, alkoxy groups, acyl groups, alkoxyalkyl groups, hydroxy groups, amino groups, monoalkylamino groups, dialkylamino groups, carboxy groups, aryl groups, heteroaryl groups, cyano groups, nitro groups, etc.) at any positions.

[0045] In this embodiment, the nucleobases may be partially or fully modified as known in the art. Modified nucleobases in a nucleic acid (e.g., an RNA nucleic acid such as an mRNA nucleic acid) include 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and / or pseudouridine (ψ). In some embodiments, modified nucleobases in a nucleic acid (e.g., an RNA nucleic acid such as an mRNA nucleic acid) include 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudouridine, 5-methylcytidine, and / or 5-methoxycytidine.

[0046] In the composition of this embodiment, the ratio of the content of the cationic lipid represented by formula (1) to the content of the nucleic acid, expressed as cationic lipid content (nmol) / nucleic acid content (μg), may be 0.05 to 150, 0.05 to 145, 0.1 to 140, 0.2 to 135, 0.25 to 130, 0.5 to 125, 1 to 120, 5 to 115, 10 to 110, or 15 to 105. By having the total lipid content within the above range, high vaccine efficacy can be maintained while side reactions can be suppressed. Furthermore, the suppression of side reactions can be evaluated using inflammatory potential, which is the main cause of side reactions, as an indicator, and a reduction in the production of inflammatory cytokines and chemokines may also be used as an indicator.

[0047] The composition of the present embodiment may further contain other components in addition to the cationic lipid represented by formula (1) and nucleic acid. Examples of other components include lipids other than the cationic lipid represented by formula (1), surfactants, polyethylene glycol, and proteins.

[0048] The lipids other than the cationic lipid represented by the above formula (1) are not particularly limited, but examples thereof include ionic lipids, neutral lipids, PEG-modified lipids, and sterols. Examples of ionic lipids include, but are not limited to, lipids that have a positive charge at a selected pH such as physiological pH, and examples thereof include 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA) (see, for example, U.S. Pat. No. 8,158,601), 2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), Merck-32 (see, for example, WO 2012 / 018754), Acuitas-5 (see, for example, WO 2015 / 199952), KL-10 (see, for example, U.S. Pat. App. Pub. No. 2012 / 0295832), and C12-200 (see, for example, Love, KT et al., al., PNAS, 107:1864 (2009)) and amino alcohol lipids. Neutral lipids include, but are not limited to, lipids that do not have a net positive charge at a selected pH, such as physiological pH. Neutral lipids are also called helper lipids or neutral phospholipids. Neutral lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal). Among these, DOPC, DPPC, DSPC, POPC, or DOPE is preferred, and DOPC or DSPC is more preferred. In the composition of this embodiment, the neutral lipid content may be 1 mol% to 20 mol%, and preferably 5 mol% to 15 mol%.PEG-modified lipids include, but are not limited to, lipids containing a polymer moiety such as polyethylene glycol, such as dimyristoylphosphatidylethanolamine-poly(ethylene glycol) 2000 (DMPE-PEG2000), DPPE-PEG2000, DMG-PEG2000, DPG-PEG2000, PEG2000-c-DOMG, and PEG2000-c-DOPG. Among these, DMG-PEG2000 is preferred. The molecular weight of the polyethylene glycol is, but is not limited to, for example, in the range of about 500 to about 10,000 Da, preferably in the range of about 1,000 to about 5,000 Da, and specific examples include DMG-PEG1000, DMG-PEG2000, and DMG-PEG5000. In the composition of this embodiment, the content of the PEG-modified lipid may be 0.01 mol% to 10 mol%, preferably 0.1 mol% to 5 mol%. The sterol is not particularly limited, but examples thereof include cholesterol, cholestanol, stigmasterol, β-sitosterol, lanosterol, and ergosterol. Among these, cholesterol, cholestanol, stigmasterol, or β-sitosterol is preferred. In the composition of this embodiment, the content of the sterol may be 10 mol% to 60 mol%, preferably 20 mol% to 50 mol%. Furthermore, the lipid other than the cationic lipid represented by formula (1) above may be used alone or in combination of two or more.

[0049] In the composition of this embodiment, the ratio of the total content of cationic lipids represented by formula (1) and lipids other than the cationic lipid represented by formula (1) (hereinafter also referred to as "total lipid content") to the content of nucleic acid, expressed as total lipid content (nmol) / nucleic acid content (μg), may be 0.05 to 250, 0.05 to 245, 0.1 to 240, 0.5 to 235, 1 to 230, 5 to 225, 10 to 220, 15 to 215, 25 to 210, or 30 to 205. By maintaining the total lipid content within the above range, high vaccine efficacy can be maintained while side reactions can be suppressed. Furthermore, the suppression of side reactions can be evaluated using inflammatory potential, which is the main cause of side reactions, as an indicator, and a decrease in the production of inflammatory cytokines and chemokines may also be used as an indicator.

[0050] From the viewpoint of use in a vaccine, the composition of this embodiment may be a lipid membrane structure containing the cationic lipid represented by the above formula (1) as a membrane constituent. Here, "lipid membrane structure" refers to a particle having a membrane structure in which the hydrophilic groups of the amphipathic lipid are aligned toward the aqueous phase side of the interface. Furthermore, the lipid membrane structure preferably encapsulates nucleic acid. The form of the lipid membrane structure is not particularly limited, but examples include liposomes (e.g., unilamellar liposomes and multilamellar liposomes), O / W emulsions, W / O emulsions, spherical micelles, wormlike micelles, LNPs, and unspecified layered structures. Among these, LNPs are preferred.

[0051] The composition of the present embodiment is not particularly limited, but can be prepared, for example, by dispersing the cationic lipid represented by formula (1) above and other components (e.g., lipids other than the cationic lipid represented by formula (1) above) in an appropriate solvent or dispersion medium (e.g., aqueous solvent, alcoholic solvent, etc.), and performing a procedure to induce organization as necessary. As the procedure to induce organization, known techniques can be used, and are not particularly limited, for example, ethanol dilution using a microchannel or vortex, simple hydration, ultrasonic treatment, heating, vortexing, ether injection, French press method, cholic acid method, Ca ２＋ These include methods such as fusion, freeze-thaw and reverse phase evaporation.

[0052] When the composition of this embodiment is a lipid membrane structure and further contains nucleic acid, lipid membrane structures encapsulating nucleic acid can be prepared by adding the desired nucleic acid to the composition during formation. For example, when liposomes are formed by the ethanol dilution method, an aqueous solution of nucleic acid and an ethanol solution of the components of the composition of this embodiment (e.g., the cationic lipid represented by the above formula (1) and other components) may be vigorously mixed using a vortex or a microchannel, and then the mixture may be diluted with an appropriate buffer solution. When liposomes are formed by the simple hydration method, the components of the composition of this embodiment are dissolved in an appropriate organic solvent, the solution is placed in a glass container, and the solvent is removed by drying under reduced pressure to obtain a lipid thin film. An aqueous solution of nucleic acid may be added to the obtained lipid thin film, hydration may be performed, and then ultrasonicated using a sonicator. Therefore, the composition of this embodiment may also be a lipid membrane structure encapsulating nucleic acid.

[0053] As used herein, the term "vaccine" is art-recognized and refers to, but is not limited to, a formulation for enhancing immunity against a disease and preventing (prophylactic vaccine) or treating (therapeutic vaccine) the disease. Thus, when the composition of the present embodiment further comprises a nucleic acid, the composition may be used to prevent or treat an infectious disease. As used herein, the term "prevention or treatment" refers to prophylactic and / or therapeutic treatment. The term "prophylactic and / or therapeutic treatment" is art-recognized and may include administration of the composition of the present embodiment to a subject. Therapeutic treatment may refer to administration after the onset of an undesirable condition (e.g., a disease or other undesirable condition in a subject) and may be intended to reduce, suppress, reverse, alleviate, or stabilize an existing undesirable condition or its side effects. It also includes attenuation of any direct or indirect pathological effects of a disease and prevention of metastasis. It may also refer to treatment to delay the onset of a disease or slow the progression of a disease. Prophylactic treatment may refer to administration prior to clinical manifestation of an undesirable condition and may be intended to protect against, inhibit recurrence of, and / or prevent an undesirable condition (e.g., an infectious disease). Preventing disease may also refer to inducing immune defenses in a subject. Inducing immune defenses refers to eliciting immunity or an immune response against a disease agent (e.g., an infectious agent) that can be displayed by a vertebrate (e.g., a human) and prevent or ameliorate the infection or reduce at least one symptom thereof.

[0054] Infectious diseases include, but are not limited to, infectious diseases caused by the above-mentioned pathogenic viruses, pathogenic bacteria, pathogenic fungi and pathogenic organisms.

[0055] The composition of the present embodiment is not particularly limited, but is preferably for oral administration or parenteral administration (e.g., intravenous administration, intramuscular administration, topical administration, nasal administration, transdermal administration, subcutaneous administration, intradermal administration, intraperitoneal administration, etc.), more preferably for parenteral administration, even more preferably for intradermal administration, subcutaneous administration, nasal administration, or intramuscular administration, and even more preferably for subcutaneous administration or intramuscular administration.

[0056] The subject to which the composition of the present embodiment is administered is a mammal. Examples of mammals include, but are not limited to, humans, non-human primates, domestic animals, laboratory animals, and livestock, with humans being preferred. Administration to a healthy subject is also preferred. Administration of the composition of the present embodiment may allow the composition to reach and contact target cells, resulting in the introduction of nucleic acids contained in the present embodiment into cells in vivo.

[0057] The dosage and administration interval of the composition of this embodiment can be appropriately selected depending on the subject to be administered, the administration route, and the subject's age, weight, and symptoms. For example, in the case of parenteral administration, the dosage for an adult is, but is not particularly limited to, 0.01 μg to 100 mg, or may be 0.1 μg to 10 mg, or may be 0.1 μg to 1 mg per dose. The administration interval of the composition of this embodiment may be such that a single dose is administered once a day, or may be administered in divided doses several times a day.

[0058] In order to explain the present invention in more detail, examples of the present invention are shown below, but the present invention is not limited to these examples.

[0059] Experimental animals, reagents, etc.: Mice: 6-week-old male C57BL6 mice. mRNA: DNA encoding hemagglutinin (HA) and neuraminidase (NA) derived from A / Viet Nam / 1203 / 2004 (H5N1) was amplified by PCR to prepare plasmid DNA serving as an mRNA template. This plasmid was linearized with restriction enzymes, extracted with phenol and chloroform, and purified by ethanol precipitation. The linearized plasmid was transcribed using the MEGAscript T7 transcription kit (Thermo Fisher Scientific) to obtain mRNA. Uridine in the mRNA was replaced with N1-methylpseudouridine. After removing the double-stranded RNA remaining in the prepared mRNA, the 5' Cap modification was added according to the protocol of the ScriptCap Cap 1 Capping System (C-SCCS1710, Madison, WI, USA), and the 3'-end poly(A) tail modification was added according to the protocol of the poly(A) Tailing Kit (AM1350, Thermo Fisher Scientific). LNP: LNP ｓｓＰａｌｍＯ－１ , LNP ｓｓＰａｌｍＯ－２ , LNP ＳＭ１０２ The quantitative ratio of nucleic acid to lipid is defined as the lipid / RNA ratio (nmol / μg), which is the amount of lipid (nmol) relative to the weight of nucleic acid (μg). LNPs were prepared with the following lipid composition ratio and L / R ratio. ｓｓＰａｌｍＯ－１ :ssPalmO / DOPC / cholesterol / DMG-PEG2000=52.5 / 7.5 / 40 / 1.5, L / R ratio=200 LNP ｓｓＰａｌｍＯ－２ :ssPalmO / DOPC / cholesterol / DMG-PEG2000=52.5 / 7.5 / 40 / 1.5, L / R ratio=33 LNP ＳＭ１０２: SM-102 / DSPC / cholesterol / DMG-PEG2000 = 50 / 10 / 38.5 / 1.5, L / R ratio = 33. ssPalmO is "COATSOME (trademark) SS-OP" purchased from NOF Corporation. SM-102 is purchased from Cayman Chemical (Formal Name: 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester). LNP was prepared as follows. LNP ｓｓＰａｌｍＯ－１ Lipid ethanol solutions were mixed so that the total lipid concentration of ssPalmO, DOPC, and cholesterol was 4.0 mM. The mRNA solution was diluted with malic acid / NaOH buffer (pH 3.0, 30 mM NaCl) so that the mRNA concentration was 0.0067 mg / mL. Both solutions were mixed using NanoAssembler Ignite (Precision Nanosystems, Vancouver, Canada) so that the ratio of nucleic acid solution to lipid solution was 3:1. The total flow rate of the solution was 4.0 mL / min. An equal amount of MES buffer (pH 6.5) was mixed with the solution and ultrafiltration was performed at room temperature at 1000 g. PBS was added to the particle solution and ultrafiltration was performed. PBS was again added to the obtained particle solution and ultrafiltration was performed to obtain a particle solution. - LNP ｓｓＰａｌｍＯ－２ Lipid ethanol solutions were mixed so that the total lipid concentration of ssPalmO, DOPC, and cholesterol was 1.2 mM. The mRNA solution was diluted with malic acid / NaOH buffer (pH 3.0, 30 mM NaCl) so that the mRNA concentration was 0.012 mg / mL. Both solutions were mixed using NanoAssembler Ignite (Precision Nanosystems, Vancouver, Canada) so that the ratio of nucleic acid solution to lipid solution was 3:1. The total flow rate of the solution was 4.0 mL / min. An equal amount of MES buffer (pH 6.5) was mixed with the solution and ultrafiltration was performed at room temperature at 1000 g. PBS was added to the particle solution and ultrafiltration was performed. PBS was again added to the obtained particle solution and ultrafiltration was performed to obtain a particle solution. - LNP ＳＭ－１０２Lipid ethanol solutions were mixed so that the total lipid concentration of SM-102, DSPC, cholesterol, and DMG-PEG2000 was 1.2 mM. The mRNA solution was diluted with acetic acid / NaOH buffer (pH 5.0) so that the mRNA concentration was 0.012 mg / mL. Both solutions were mixed using NanoAssembler Ignite (Precision Nanosystems, Vancouver, Canada) at a nucleic acid solution to lipid solution ratio of 3:1. The total flow rate of the solution at this time was 2.0 mL / min. An equal amount of MES buffer (pH 5.5) was mixed with the solution and subjected to ultrafiltration at room temperature at 1000 g. PBS was added to the particle solution and ultrafiltration was performed. PBS was again added to the obtained particle solution and ultrafiltration was performed to obtain a particle solution. The particle size and zeta potential of the mRNA-LNP were measured using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK), and the encapsulation rate of the mRNA-LNP was measured using a Quant-it (registered trademark) RiboGreen RNA Assay Kit (Thermo Fisher Scientific). The measurement results are shown in Figure 1. The prepared mRNA-LNP was observed with a cryo-electron microscope (Talos Arctica, FEI) and the results are shown in Figure 2. - Antigens for ELISA: Recombinant proteins of HA and NA derived from A / VietNam / 1203 / 2004 (H5N1) (referred to as rHA and rNA).

[0060] (Test Example 1: Evaluation of antibody titer after two subcutaneous immunizations) HA- or NA-derived mRNA-LNP (1 μg / mouse as mRNA amount) was administered subcutaneously on days 0 and 21. Blood and regional lymph nodes were collected on day 35. Plasma rHA- and rNA-specific IgG1, IgG2b, and IgG2c were evaluated by ELISA. In addition, antibody titers (titers) were calculated for plasma rHA-specific IgG1, IgG2b, and IgG2c using a two-fold serial dilution series. Germinal center B cells were detected using a flow cytometer (Attune NxT, Thermo Fisher Scientific). A hemagglutination assay (HI test) was performed using the following steps. After pretreating the serum with RDE (II) receptor-destroying enzyme (DENKA Co., Ltd., Tokyo, Japan), the serum was diluted 10-fold and then serially diluted 2-fold. After reacting with 8 HA units of viral antigen for 30 minutes, the serum was mixed with chicken erythrocytes (Japan Bioserum Co., Ltd., Hiroshima, Japan) and incubated for 30 minutes. The HI antibody titer was determined as the maximum serum dilution that completely inhibited hemagglutination. The HI test evaluated the HI antibody titers against the Vietnam strain (A / Viet Nam / 1203 / 2004), which is the same as the vaccine strain, and the Hokkaido strain (A / Ezo red fox / Hokkaido / 1 / 2022), which is a different H5N1 virus from the vaccine strain. At the amino acid level, the Vietnam strain and the Hokkaido strain share 91% homology in HA and 94% homology in NA.

[0061] In the following figures, LNP ｓｓＰａｌｍＯ－１ , LNP ｓｓＰａｌｍＯ－２ , LNP ＳＭ１０２are designated Palm1, Palm2, and SM102, respectively. The results of evaluating rHA- and rNA-specific IgG in plasma are shown in Figure 3. (a) shows the results of rHA-specific IgG induction in the group subcutaneously immunized twice with HA-derived mRNA, and (b) shows the results of rNA-specific IgG induction in the group subcutaneously immunized twice with NA-derived mRNA. In both HA and NA, higher antibody titers were observed in the mRNA-LNP compared to the non-vaccine group. Although significant differences were observed between LNPs, they were not remarkable. From the above results, it can be concluded that LNP ｓｓＰａｌｍＯ－１ , LNP ｓｓＰａｌｍＯ－２ is LNP ＳＭ１０２ It was found that the rHA-specific IgG antibody titer was equivalent to that of the non-vaccinated group. The antibody titer of rHA-specific IgG in plasma is shown in (c). In the group subcutaneously immunized twice with HA-derived mRNA, a higher antibody titer was observed compared to the non-vaccinated group. No significant difference was observed between LNPs. The number of germinal center B cells in the regional lymph nodes is shown in (d). The LNP group, which was subcutaneously immunized twice with HA-derived mRNA, ｓｓＰａｌｍＯ－１ and LNP ｓｓＰａｌｍＯ－２ is LNP ＳＭ１０２ A tendency was observed for the LNP to induce germinal center B cells more strongly than the non-vaccine group. HI antibody titers against the Vietnam strain and Hokkaido strain are shown in (e) and (f). The group subcutaneously immunized twice with HA-derived mRNA showed a higher HI antibody titer against the Vietnam strain compared to the non-vaccine group, and no significant difference was observed between LNPs. Because the Hokkaido strain is a different strain from the vaccine strain, the group subcutaneously immunized twice with HA-derived mRNA did not show an HI antibody titer against the Hokkaido strain. From the results of this test example, it can be seen that LNP ｓｓＰａｌｍＯ－２ is LNP ＳＭ１０２ It was shown that it induces antigen-specific antibody titers equivalent to those of the IgG4-dependent immunization.

[0062] (Test Example 2: Evaluation of antibody titer after prime subcutaneous immunization) Mice were subcutaneously immunized with HA- or NA-derived mRNA-LNP (1 μg / mouse as mRNA amount) on day 0. Blood was collected on day 14, and rHA- and rNA-specific IgG1, IgG2b, and IgG2c in the plasma were evaluated by ELISA.

[0063] The evaluation results of rHA- and rNA-specific IgG in plasma are shown in Figure 4. (a) shows the results of rHA-specific IgG induction in the group immunized subcutaneously once with HA-derived mRNA, and (b) shows the results of rNA-specific IgG induction in the group immunized subcutaneously once with NA-derived mRNA. For rHA-specific IgG, a higher antibody titer was observed with mRNA-LNP compared to the non-vaccine group, and no significant difference was observed between LNPs. For rNA-specific IgG, a higher antibody titer was observed with mRNA-LNP compared to the non-vaccine group. Although a significant difference was observed between LNPs, it was not a remarkable difference. From the results of this test example, it can be seen that LNP ｓｓＰａｌｍＯ－２ is LNP ＳＭ１０２ It was shown that it induces antigen-specific antibody titers equivalent to those of the IgG4-dependent immunization.

[0064] (Test Example 3: Comparison with Recombinant Protein + Alum) Mice were subcutaneously immunized with HA-derived mRNA-LNP (1 μg / mouse as mRNA amount) on days 0 and 21. As a control, rHA (recombinant HA protein, 1 μg / mouse) + alum (aluminum hydroxide salt, 50 μg / mouse) was also subcutaneously immunized. Blood and spleens were collected on day 35. rHA-specific IgG1, IgG2b, and IgG2c in the plasma were evaluated by ELISA. The spleen was restimulated in vitro with rHA, and then IFN-γ- or IL-13-producing CD4- and CD8-positive T cells were detected by intracellular staining.

[0065] The evaluation results of rHA-specific IgG in plasma are shown in Figure 5. (a) shows the results of rHA-specific IgG induction in groups immunized subcutaneously twice with HA-derived mRNA or rHA + alum. Higher antibody titers were observed with mRNA-LNP compared to the non-vaccine group. Compared to the rHA + alum vaccine group, mRNA-LNP showed significantly higher IgG2b and IgG2c induction. (b) to (d) show the results of T cell responses in groups immunized subcutaneously twice with HA-derived mRNA or rHA + alum. IFN-γ-producing CD4-positive T cells were significantly induced in the mRNA-LNP vaccine group compared to the non-vaccine group, but not in the rHA + alum vaccine group. On the other hand, IL-13-producing CD4-positive T cells and IFN-γ-producing CD8-positive T cells were not induced in either vaccine group compared to the non-vaccine group. From the results of this test example, it was clear that LNP ｓｓＰａｌｍＯ－２ It was found that rHA+alum showed superior antibody induction and IFN-γ-producing CD4-positive T cell induction.

[0066] Test Example 4: Evaluation of antibody titer after two intramuscular immunizations Mice were intramuscularly immunized with HA-derived mRNA-LNP (1 μg / mouse as mRNA amount) on days 0 and 21. Blood was collected on day 35, and rHA-specific IgG1, IgG2b, and IgG2c in the plasma were evaluated by ELISA. A hemagglutination test (HI test) was used to evaluate the HI antibody titer against the Vietnam strain (A / Viet Nam / 1203 / 2004), which is the same as the vaccine strain, and the Hokkaido strain (A / Ezo red fox / Hokkaido / 1 / 2022), an H5N1 virus different from the vaccine strain.

[0067] The evaluation results of rHA-specific IgG in plasma are shown in Figure 6(a). A higher antibody titer was observed in the group that received two intramuscular immunizations with HA-derived mRNA compared to the non-vaccine group. Although a significant difference was observed between LNPs, it was not a remarkable difference. HI antibody titers against the Vietnam strain and Hokkaido strain are shown in Figures 6(b) and (c). The group that received two intramuscular immunizations with HA-derived mRNA showed a higher HI antibody titer against the Vietnam strain compared to the non-vaccine group, and no significant difference was observed between LNPs. Because the Hokkaido strain is a different strain from the vaccine strain, the group that received two intramuscular immunizations with HA-derived mRNA did not show an HI antibody titer against the Hokkaido strain. The results of this test example show that LNPs, even when administered intramuscularly, ｓｓＰａｌｍＯ－２ is LNP ＳＭ１０２ It was found to induce antigen-specific antibody titers equivalent to those of the IgG4-dependent immunization.

[0068] Test Example 5: T cell response Mice were subcutaneously or intramuscularly immunized with HA- or NA-derived mRNA-LNP (1 μg / mouse as mRNA amount) on days 0 and 21. Spleens were collected on day 35. After restimulating the spleens in vitro with rHA or rNA, IFN-γ- or IL-13-producing CD4- and CD8-positive T cells were detected by intracellular staining.

[0069] The results of the T cell response are shown in Figure 7. (a) to (d) show the results of the T cell response in the group that received two subcutaneous immunizations with HA-derived mRNA. IFN-γ-producing CD4-positive T cells were significantly induced in the mRNA-LNP vaccine group compared to the non-vaccinated group, and LNP ｓｓＰａｌｍＯ－２ is LNP ｓｓＰａｌｍＯ－１ and LNP ＳＭ１０２ On the other hand, IL-13-producing CD4-positive T cells, IFN-γ-producing CD8-positive T cells, and IL-13-producing CD8-positive T cells were not induced in any of the vaccine groups compared to the non-vaccine group. (e) to (h) show the results of T cell responses in the group immunized twice subcutaneously with NA-derived mRNA. IFN-γ-producing CD4-positive T cells and IFN-γ-producing CD8-positive T cells were significantly induced in the LNP group compared to the non-vaccine group. ｓｓＰａｌｍＯ－２ It was significantly induced in the LNP group. ＳＭ１０２IFN-γ-producing CD4-positive T cells were not induced in any of the vaccine groups compared to the non-vaccine group. On the other hand, IL-13-producing CD4-positive T cells and IL-13-producing CD8-positive T cells were not induced in any of the vaccine groups compared to the non-vaccine group. (i) to (k) show the results of T cell responses in groups immunized intramuscularly twice with HA-derived mRNA. IFN-γ-producing CD4-positive T cells were significantly induced in the mRNA-LNP vaccine group compared to the non-vaccine group, and no significant difference was observed between LNPs. On the other hand, IL-13-producing CD4-positive T cells were not induced in any of the vaccine groups compared to the non-vaccine group. IFN-γ-producing CD8-positive T cells were significantly induced in the LNP vaccine group compared to the non-vaccine group. ｓｓＰａｌｍＯ－２ The LNP group showed significant induction. ＳＭ１０２ From the results of this test example, it was found that LNP ｓｓＰａｌｍＯ－２ is LNP ＳＭ１０２ It was found that the induction of IFN-γ-producing CD4-positive T cells was equal to or greater than that of the control group.

[0070] Test Example 6: Evaluation of infection protection ability using body weight and survival rate as indicators Mice were subcutaneously immunized with HA- or NA-derived mRNA-LNP (1 μg / mouse as mRNA amount) on days 0 and 21. On day 35, they were intranasally infected with the same Vietnam strain (A / Viet Nam / 1203 / 2004) as the vaccine strain, and then the infection protection ability was evaluated using body weight and survival rate as indicators. Furthermore, to evaluate cross-protection ability, mice were intranasally infected with the Hokkaido strain (A / Ezo red fox / Hokkaido / 1 / 2022), an H5N1 virus different from the vaccine strain, or the California strain (A / California / 07 / 2009), and then the infection protection ability was evaluated using body weight and survival rate as indicators.

[0071] The results of weight and survival rate are shown in Figure 8. (a), (c), and (e) show the group that was subcutaneously immunized twice with HA-derived mRNA-LNP, and (b), (d), and (f) show the group that was subcutaneously immunized twice with NA-derived mRNA-LNP. (a) and (b) show the results of observing the weight and survival rate of mice infected with the Vietnam strain. In the non-vaccinated group, significant weight loss was observed, and all mice died within 14 days. On the other hand, no weight loss was observed with any of the LNPs, whether HA or NA, and all mice survived. From the above results, it can be seen that LNP ｓｓＰａｌｍＯ－１ and LNP ｓｓＰａｌｍＯ－２ is LNP ＳＭ１０２ (c) and (d) show the results of observing the body weight and survival rate of mice infected with the Hokkaido strain. In mice infected with HA as an antigen, LNP ＳＭ１０２ Although the survival rate tended to be longer in the LNP group compared to the non-vaccinated group, all patients died within 14 days of infection. ｓｓＰａｌｍＯ－１ and LNP ｓｓＰａｌｍＯ－２ The survival rates were 80% and 60%, respectively. ｓｓＰａｌｍＯ－１ and LNP ｓｓＰａｌｍＯ－２ is LNP ＳＭ１０２ Furthermore, in mice treated with NA as an antigen, all mice died within 14 days of infection with either LNP, but a slight increase in survival rate was observed. ｓｓＰａｌｍＯ－１ , LNP ｓｓＰａｌｍＯ－２ , LNP ＳＭ１０２ From the above results, it was found that HA was superior to NA in terms of cross-protection against the Hokkaido strain, and LNP ｓｓＰａｌｍＯ－１ and LNP ｓｓＰａｌｍＯ－２ is LNP ＳＭ１０２ (e) and (f) show the results of observing the weight and survival rate of mice infected with the California strain. In mice infected with HA as an antigen, LNP ＳＭ１０２ Although the survival rate tended to be longer compared to the non-vaccinated group, the survival rate was still 40%. ｓｓＰａｌｍＯ－２In the mice treated with NA as an antigen, all mice survived regardless of the LNP used, but the degree of weight loss was significantly higher than that of the LNP. ｓｓＰａｌｍＯ－２ LNP is better ＳＭ１０２ These results indicate that NA is superior to HA in terms of cross-protection against the California strain, and LNP ｓｓＰａｌｍＯ－２ is LNP ＳＭ１０２ It was shown that it may have better cross-protective ability than

[0072] Test Example 7: Evaluation of mRNA introduction efficiency after a single intramuscular immunization. Luciferase-derived mRNA-LNP (1 μg / mouse as mRNA amount) was administered intramuscularly. 6, 24, 48, and 72 hours after immunization, D-luciferin (Fujifilm Wako Pure Chemical Industries, Ltd.) was administered intraperitoneally, and luminescence was measured using an in vivo imaging system (Lumina Series III, PerkinElmer). Six hours after immunization, the muscle, regional lymph nodes (dLN), liver, and spleen were collected and homogenized, and luminescence was measured using the ONE-Glo EX Luciferase Assay System (Promega).

[0073] The results of luminescence measurements are shown in Figure 9. (a) shows the results of observing the change in luminescence intensity over time using an in vivo imaging system for a group that received a single intramuscular immunization with luciferase-derived mRNA. In the LNP group, strong fluorescence was observed in the muscle and liver, and the fluorescence was observed to gradually disappear over time. (b) shows the quantified luminescence intensity at the administration site. Six hours after administration, luminescence was observed at the administration site in the LNP group compared to the PBS group, and no significant difference was observed between the LNPs. Luminescence intensity decreased after 24 hours, but no significant difference was observed between the LNPs at any time point. (c) to (f) show the results of measuring luciferase activity in the muscle, regional lymph nodes (dLN), liver, and spleen for a group that received a single intramuscular immunization with luciferase-derived mRNA. In the muscle and liver, the luminescence intensity was significantly increased in the LNP group compared to the PBS group, but no significant difference was observed between the LNPs. ｓｓＰａｌｍＯ－２is LNP ＳＭ１０２ It was found that the mRNA transfection efficiency was equivalent to that of the control group.

[0074] Test Example 8: Evaluation of mRNA introduction efficiency after two intramuscular immunizations Luciferase-derived mRNA-LNP (1 μg / mouse as mRNA amount) was administered intramuscularly on days 0 and 21. D-luciferin was administered intraperitoneally 6 hours, 24 hours, 48 ​​hours, and 72 hours after the second immunization, and luminescence was measured using an in vivo imaging system. In addition, 6 hours after the second immunization, the muscles were collected and homogenized, and luminescence was measured using the ONE-Glo EX Luciferase Assay System.

[0075] The results of luminescence measurements are shown in Figure 10. (a) shows the results of observing the change in luminescence intensity over time using an in vivo imaging system for a group that was immunized intramuscularly twice with luciferase-derived mRNA. In the LNP group, strong fluorescence was observed in the muscle and liver, and it was observed that the fluorescence gradually disappeared over time. (b) shows the quantified values ​​of luminescence intensity at the administration site. Luminescence was observed at the administration site in the LNP group 6 hours after administration, and no significant difference was observed between LNPs. Luminescence intensity decreased after 24 hours, but no significant difference was observed between LNPs at any time point. (c) shows the results of measuring luciferase activity in muscle for a group that was immunized intramuscularly twice with luciferase-derived mRNA. Luminescence intensity in the LNP group was significantly increased in muscle compared to the PBS group. Although slight, LNP ｓｓＰａｌｍＯ－２ is LNP ＳＭ１０２ The results of this test showed that LNP ｓｓＰａｌｍＯ－２ is LNP ＳＭ１０２ It was found that the mRNA transfection efficiency was equivalent to that of the control.

[0076] Test Example 9: Evaluation of HA expression after two intramuscular immunizations Mice were intramuscularly immunized with HA-derived mRNA-LNP (1 μg / mouse as mRNA amount) on days 0 and 21. Six hours after the second immunization, the muscles were collected, and HA expression by dendritic cells (DCs) in the muscles was evaluated using a flow cytometer.

[0077] The results of evaluating HA expression in dendritic cells at the administration site of a group that received two intramuscular immunizations with HA-derived mRNA are shown in Figure 11. Compared to the PBS group, the proportion of HA-expressing dendritic cells was increased in the LNP group, and no significant difference was observed between the LNPs. ｓｓＰａｌｍＯ－２ is LNP ＳＭ１０２ It was found that the mRNA transfection efficiency was equivalent to that of the control.

[0078] Test Example 10: Evaluation of activation of antigen-presenting cells in regional lymph nodes after subcutaneous immunization Mice were subcutaneously immunized with HA-derived mRNA-LNP (1 μg / mouse as mRNA amount), and regional lymph nodes were collected one day later. The expression of costimulatory molecules (CD86) in dendritic cells (plasmacytoid DCs, conventional DCs, migrative DCs), B cells, and macrophages was evaluated using a flow cytometer.

[0079] The activation of antigen-presenting cells was evaluated using CD86 expression as an index. ＳＭ１０２ In the LNP group, enhanced expression of CD86 was confirmed in dendritic cells, B cells, and macrophages compared to the non-vaccinated group. ｓｓＰａｌｍＯ－２ In this case, LNP ｓｓＰａｌｍＯ－１ and LNP ＳＭ１０２ The results of this test showed that LNP ｓｓＰａｌｍＯ－２ is LNP ｓｓＰａｌｍＯ－１ and LNP ＳＭ１０２ These results suggest that while LNPs induce immune responses (antibody production, T cell responses) equivalent to those of human immunocytes, they have a low ability to activate innate immunity.

[0080] Test Example 11: Evaluation of Blood Cytokine Levels After Subcutaneous Immunization Mice were subcutaneously immunized with HA-derived mRNA-LNP (1 μg / mouse as mRNA amount) on days 0 and 21. On days 0 and 21, blood was collected 6 hours after subcutaneous immunization, and the cytokine levels in the blood were measured using LEGENDplex (BioLegend).

[0081] The levels of cytokines in the blood 6 hours after (a) the first subcutaneous immunization (Prime) and (b) the second subcutaneous immunization (Boost) are shown in Figure 13. ｓｓＰａｌｍＯ－２ is LNP ＳＭ１０２The amounts of the inflammatory cytokines or chemokines IFN-α, IFN-β, IFN-γ, MCP-1, IP-10, and IL-6 were significantly lower in LNP compared to LNP. ｓｓＰａｌｍＯ－１ LNP ｓｓＰａｌｍＯ－２ Although not as much as LNP ＳＭ１０２ The results of this test showed that the LNP ｓｓＰａｌｍＯ－２ is LNP ＳＭ１０２ Compared to the previous study, it was shown that the vaccine may induce the same effect while reducing side effects.

[0082] Test Example 12 Evaluation of Blood Cytokine Levels After Subcutaneous Immunization Measurements were carried out in the same manner as in Test Example 11, except that the levels of cytokines and chemokines different from those in Test Example 11 were measured.

[0083] The levels of cytokines in the blood 6 hours after the first subcutaneous immunization (Prime) and the second subcutaneous immunization (Boost) are shown in Figure 14. The inflammatory cytokines or chemokines KC, TNF-α, IL-12 p70, RANTES, IL-1β, GM-CSF, and IL-10 were hardly induced by any of the LNPs.

[0084] Test Example 13: Evaluation of blood cytokine levels after intramuscular immunization Mice were intramuscularly immunized with HA-derived mRNA-LNP (1 μg / mouse as mRNA amount) on days 0 and 21. On days 0 and 21, blood was collected 6 hours after intramuscular immunization, and the cytokine levels in the blood were measured using LEGENDplex.

[0085] The levels of cytokines in the blood 6 hours after the first intramuscular immunization (Prime) and the second intramuscular immunization (Boost) are shown in Figure 15. ｓｓＰａｌｍＯ－２ is LNP ＳＭ１０２ The results of this test demonstrate that the amounts of inflammatory cytokines or chemokines IFN-α, IFN-β, IFN-γ, MCP-1, and IP-10 are significantly lower in LNP than in LNP administered intramuscularly. ｓｓＰａｌｍＯ－２ is LNP ＳＭ１０２ Compared to the previous study, it was shown that the vaccine may induce the same effect while reducing side effects.

[0086] Test Example 14: Evaluation of Adverse Reactions After Intramuscular Immunization Six hours after intramuscular immunization with HA-derived mRNA-LNP (1 μg / mouse as mRNA amount), Evans blue was administered intravenously. Six hours later, the muscle was collected and homogenized to measure the amount of Evans blue in the muscle, and vascular permeability at the administration site, i.e., inflammation at the administration site, was evaluated. When measuring body weight as an indicator of adverse reactions, body weight was measured 24 hours after intramuscular immunization with HA-derived mRNA-LNP (20 μg / mouse as mRNA amount). When measuring body temperature as an indicator of adverse reactions, HA-derived mRNA-LNP (5 μg / mouse as mRNA amount) was administered intramuscularly on days 0 and 21. Body temperature was measured 6 hours after intramuscular immunization on days 0 and 21.

[0087] The evaluation of side effects after intramuscular immunization is shown in Figure 16. (a) shows the amount of Evans blue in the muscle 6 hours after intramuscular immunization. ｓｓＰａｌｍＯ－２ Group: LNP ＳＭ１０２ The amount of Evans blue at the injection site was significantly lower than that of the control group, indicating a reduction in inflammation, a side effect. (b) shows the change in body weight 24 hours after intramuscular immunization. Both LNPs induced weight loss, but LNP ｓｓＰａｌｍＯ－２ Group: LNP ＳＭ１０２ (c) and (d) show body temperature 6 hours after the first intramuscular immunization (Prime) and the second intramuscular immunization (Boost). ｓｓＰａｌｍＯ－２ Group: LNP ＳＭ１０２ The results of this test showed that the increase in body temperature was significantly alleviated compared to the LNP group. ｓｓＰａｌｍＯ－２ is LNP ＳＭ１０２ It was shown that side effects were reduced compared to

[0088] (Test Example 15 Comparison of Neutral Lipids) Except for using DSPC instead of DOPC, LNP ｓｓＰａｌｍＯ－２ DSPC-containing LNPs were prepared in the same manner as in the preparation of ｓｓＰａｌｍＯ－２ In the following figures, DOPC-containing LNP was prepared. ｓｓＰａｌｍＯ－２ , DSPC-containing LNP ｓｓＰａｌｍＯ－２ , and DSPC-containing LNPs ＳＭ１０２are abbreviated as Palm2 (DOPC), Palm2 (DSPC), and SM102 (DSPC), respectively. HA-derived mRNA-LNP (1 μg / mouse as mRNA amount) was subcutaneously immunized on days 0 and 21. Blood was collected 6 hours after the second immunization, and cytokine levels were measured using LEGENDplex. Blood was collected on day 35, and rHA-specific IgG1, IgG2b, and IgG2c in plasma were evaluated by ELISA.

[0089] The amount of cytokines in the blood is shown in Figures 17(a) to 17(f). ｓｓＰａｌｍＯ－２ is a DOPC-containing LNP ｓｓＰａｌｍＯ－２ It was also shown that the amounts of IFN-γ and IP-10, which are inflammatory cytokines or chemokines, were significantly increased compared to those of DSPC-containing LNPs. ｓｓＰａｌｍＯ－２ indicates DSPC-containing LNPs ＳＭ１０２ The results showed that the amounts of IFN-α, IFN-β, IFN-γ, IL-6, MCP-1, and IP-10 were significantly reduced compared to the control group. The plasma rHA-specific IgG is shown in Figure 17(g). Higher antibody titers were observed in the mRNA-LNP compared to the non-vaccine group. No significant differences were observed between the LNPs. The results of this test example show that DOPC-containing LNPs ｓｓＰａｌｍＯ－２ indicates DSPC-containing LNPs ｓｓＰａｌｍＯ－２ It was shown that the vaccine effect was similar to that of DSPC-containing LNPs, while side effects were reduced. ｓｓＰａｌｍＯ－２ indicates DSPC-containing LNPs ＳＭ１０２ Since the inflammation was significantly reduced compared to LNP, ｓｓＰａｌｍＯ－２ The main reason for the low inflammatory activity of the above was thought to be due to the use of ssPalmO.

[0090] (Test Example 16: Evaluation of blood cytokine levels after intramuscular immunization) mRNA-LNPs were obtained according to the above-mentioned "Experimental animals, reagents, etc.", except for changing the mRNA as follows: mRNA: spike protein derived from the novel coronavirus (SARS-CoV-2: original strain and mutant strain). Uridine in the mRNA was replaced with N1-methylpseudouridine.

[0091] Mice are intramuscularly immunized with SARS-CoV-2-derived mRNA-LNP. Six hours after intramuscular immunization, blood samples are collected and the amount of cytokines in the blood is measured using LEGENDplex.

[0092] LNP ｓｓＰａｌｍＯ－２ is LNP ＳＭ１０２ Compared with the above, the amount of inflammatory cytokines or chemokines can be significantly reduced, indicating the possibility of reduced side effects.

[0093] Summary LNPs (LNPs) prepared using ssPalmO as an ionic lipid ｓｓＰａｌｍＯ Specifically, we used mRNA of hemagglutinin (HA) and neuraminidase (NA) derived from the highly pathogenic avian influenza virus of the H5N1 subtype (H5N1 virus) as model antigens, and prepared LNPs (LNPs) using SM102, an ionic lipid commonly used worldwide. ＳＭ１０２ ) and LNP ｓｓＰａｌｍＯ The vaccine effect and inflammation-causing activity of LNP were evaluated in mice. ｓｓＰａｌｍＯ is LNP ＳＭ１０２ Furthermore, when the mice were infected with the same strain of H5N1 virus as the vaccine strain after vaccination, a significant weight loss was observed in the non-vaccinated group, and all mice died within 14 days. ｓｓＰａｌｍＯ and LNP ＳＭ１０２ In the vaccine group, no weight loss was observed and all animals survived. After vaccination, the mice were infected with a strain of H5N1 virus different from the vaccine strain. ｓｓＰａｌｍＯ is LNP ＳＭ１０２ Furthermore, weight loss was improved compared to LNP. ｓｓＰａｌｍＯ is LNP ＳＭ１０２ Compared with LNP, it was found that the production of inflammatory cytokines and chemokines such as IL-6, IFN-γ, and MCP-1 after vaccination, which are indicators of inflammation that are the main cause of side effects, was significantly and markedly reduced. ｓｓＰａｌｍＯ is LNP ＳＭ１０２Compared to the above, it was found to be a novel LNP that can induce equivalent vaccine effects and infection protection capabilities while reducing side effects.

Claims

1. The following formula (1): (In formula (1), R １ａ and R １ｂ each independently represents an alkylene group having 1 to 6 carbon atoms; ａ and X ｂ each independently represents a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups; R ２ａ and R ２ｂ each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms; ａ and Y ｂ each independently represents an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond; Z ａ and Z ｂ each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a heteroatom; R ３ａ and R ３ｂ each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or a residue derived from a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

2. The composition of claim 1, further comprising a nucleic acid.

3. The composition according to claim 2, wherein the ratio of the cationic lipid content to the nucleic acid content is 0.05 to 150, expressed as cationic lipid content (nmol) / nucleic acid content (μg).

4. The composition according to any one of claims 1 to 3, further comprising one or more selected from the group consisting of ionic lipids, neutral lipids, PEG-modified lipids and sterols.

5. The composition of claim 4 which is a lipid particle.

6. A composition according to claim 2 or 3 for preventing or treating an infectious disease.

7. The composition according to claim 2, for intradermal, subcutaneous, intranasal or intramuscular administration.

Images