Sftsv nucleic acid-lipid particle vaccine

Abstract

Provided is a vaccine for preventing and / or treating infection and symptom onset resulting from SFTSV.　These lipid particles encapsulate a nucleic acid capable of expressing Gn, Gc, or NP of SFTSV, wherein the lipid includes an amphiphilic lipid, a sterol, a PEG lipid, and a cationic lipid.

Description

SFTSV nucleic acid lipid particle vaccine

[0001] The present invention relates to a nucleic acid lipid particle vaccine encapsulating SFTSV mRNA.

[0002] Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease caused by Bandavirus dabiieense, commonly known as SFTS virus (SFTSV), of the Phenoviridae family. SFTS occurs in Asia, including Japan, Korea, and China, with approximately 100 cases reported annually in Japan. The mortality rate is 27-31% (Non-Patent Documents 1 and 2), and currently, no vaccine exists to prevent SFTSV infection or onset. While the effectiveness of favipiravir against SFTS has been reported in recent years (Non-Patent Document 3), no standard treatment that provides satisfactory therapeutic effects for SFTS has yet been established.

[0003] In recent years, the onset of SFTS has been reported in animals such as dogs and cats, and infection from affected animals to humans has become a public health problem (Non-Patent Document 4). As with humans, there are no effective preventive vaccines or therapeutic drugs for animals.

[0004] The glycoprotein precursor encoded by the SFTSV M segment is cleaved into glycoprotein N (Gn) and glycoprotein C (Gc) to form the spike complex, which is an envelope protein. Gn and Gc are important for SFTSV binding to receptors on the cell membrane and membrane fusion during endocytosis (Non-Patent Documents 5 and 6). Based on the results of SFTSV infection experiments using Type I Interferon Receptor knockout (IFNAR1 KO) mice, it has been reported that a live vaccinia virus strain vaccine expressing a glycoprotein precursor containing both Gn and Gc induces neutralizing activity against SFTSV and improves the survival rate of mice after SFTSV infection (Non-Patent Document 7). Furthermore, experimental results using wild-type or IFNAR1 KO mice have shown that LNP formulations (LNP-mRNA) in which mRNA encoding the full-length or head region of Gn is encapsulated in lipid nanoparticles (LNPs) induce anti-Gn antibody titers or improve survival rates after SFTSV infection (Patent Documents 1, 2, 3, Non-Patent Documents 8 and 9). However, there are no examples of LNP-mRNA or other vaccine formulations that use only Gc as a vaccine antigen for preventing or treating SFTSV infection and onset.

[0005] Nucleoprotein (NP) encoded by the SFTSV S segment is important for viral RNA encapsidation and ribonucleoprotein complex formation, and is essential for viral replication (Non-Patent Document 10). Based on the results of SFTSV infection experiments using IFNAR1 KO mice, it has been reported that a live vaccinia virus strain vaccine expressing NP improves the survival rate of mice after SFTSV infection (Non-Patent Document 7). However, there are no examples of LNP-mRNA using NP as a vaccine antigen for preventing or treating SFTSV infection or onset.

[0006] Known LNP-mRNAs include SARS-CoV-2 vaccines (Patent Documents 4, 5, and 6) and influenza vaccines (Patent Document 7).

[0007] WO2023 / 008881A1CN116024235AWO2019 / 038332A1WO2021 / 213945A1WO2021 / 154763A1WO2021 / 251453A1WO2015 / 164674A1

[0008] PLoS ONE (2016) 11 (10): e0165207. Emerg Infect Dis (2020) 26(4):692-699. PLoS Negl Trop Dis (2021) 15 (2): e0009103Jpn J Infect Dis (2023) 24; 76 (3): 211-214. J Virol (2013) 87(8):4384-94. J Virol (2016) 90:5292-5301. PLoS Pathog (2021) 17(2): n. npj Vaccines (2023) 8:16J Med Virol (2023) 95(11):e29203Protein Cell (2013) 4(6):445-455.

[0009] An object of the present invention is to provide a vaccine for preventing and / or treating infection and onset caused by SFTSV.

[0010] The present inventors discovered that nucleic acid lipid particles (LNP-mRNA) encapsulating mRNA encoding each of SFTSV Gn, Gc, and NP alone can be used to prevent SFTSV infection and onset, and further research led to the completion of the present invention. Specifically, they prepared LNP-mRNA encapsulating mRNA expressing SFTSV Gn, Gc, or NP (hereinafter referred to as LNP-mRNA Gn, LNP-mRNA Gc, or LNP-mRNA NP, respectively), and discovered that antibody production and cellular immunity against Gn, Gc, or NP were induced in mice administered LNP-mRNA Gn, LNP-mRNA Gc, or LNP-mRNA NP. We also found that administration of LNP-mRNA Gn or LNP-mRNA Gc induced anti-SFTSV neutralizing activity in the blood, and that administration of LNP-mRNA Gn, LNP-mRNA Gc, or LNP-mRNA NP improved survival and suppressed weight loss after SFTSV infection.

[0011] The gist of the present invention is as follows: [1] A lipid particle encapsulating a nucleic acid capable of expressing at least one antigen selected from the group consisting of Gn antigen, Gc antigen, and NP antigen of SFTSV, wherein the lipid particle comprises an amphipathic lipid, sterols, a PEG lipid, and a cationic lipid. [2] The particle of [1], wherein the nucleic acid is a nucleic acid capable of expressing two or more antigens selected from the group consisting of Gn antigen, Gc antigen, and NP antigen of SFTSV. [3] The particle of either [1] or [2], wherein the cationic lipid is one or more selected from the group consisting of the following cationic lipids: MC3: (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, ALC-0315: [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoic acid ester), and SM-102: heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoic acid ester [4] The particles of any of [1] to [3], wherein the amphiphilic lipid is at least one selected from the group consisting of distearoylphosphatidylcholine, dioleoylphosphatidylcholine, and dioleoylphosphatidylethanolamine. [5] The particles of any of [1] to [4], wherein the sterol is cholesterol. [6] A particle according to any one of [1] to [5], wherein the PEG lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol and / or N-[methoxy poly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine. [7] A particle according to any one of [1] to [6], wherein the lipid composition of the amphiphilic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 5 to 25% amphiphilic lipid, 10 to 55% sterols, 40 to 65% cationic lipid, and 1 to 5% PEG lipid. [7-2] A particle according to [7], wherein the amphiphilic lipid is 10 to 25%. [7-3] Particles according to any one of [1] to [6], wherein the lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is, in molar amounts, 5-15% amphipathic lipids, 35-50% sterols, 40-55% cationic lipids, and 1-3% PEG lipids. [7-4] Particles according to [7-3], wherein the amphipathic lipids are 10-15%, 35-45% sterols, 40-50% cationic lipids, and 1-2.5% PEG lipids. [7-5] Particles according to [7-4], wherein the PEG lipids are 1-2%. [7-6] Any of the particles of [1] to [6], wherein the lipid composition of amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 10 to 25% amphipathic lipid, 10 to 50% sterols, 40 to 65% cationic lipid, and 1 to 3% PEG lipid.[7-7] Particles according to [7-6], wherein the sterol content is 10 to 45%, the cationic lipid content is 42.5 to 65%, and the PEG lipid content is 1 to 2.5%. [7-8] Particles according to [7-7], wherein the PEG lipid content is 1 to 2%. [8] Particles according to any of [7] to [7-7], wherein the ratio of total lipid weight to nucleic acid weight is 15 to 30. [8-2] Particles according to [8], wherein the ratio of total lipid weight to nucleic acid weight is 15 to 25. [8-3] Particles according to [8-2], wherein the ratio of total lipid weight to nucleic acid weight is 17.5 to 22.5. [9] Particles according to any of [1] to [8-3], wherein the Gn antigen of SFTSV is a protein comprising an amino acid sequence having at least 95% identity with the amino acid sequence of a region consisting of amino acids 20 to 175 of SEQ ID NO: 1.

[10] The particle of [9], wherein the Gn antigen is a protein comprising an amino acid sequence having at least 95% identity with the amino acid sequence consisting of amino acid numbers 20 to 452 of SEQ ID NO: 1 or amino acid numbers 20 to 340 of SEQ ID NO: 28.

[11] The particle of any of [1] to [8], wherein the Gc antigen of SFTSV is a protein comprising an amino acid sequence having at least 95% identity with the amino acid sequence of a region including amino acid numbers 28 to 96 and / or amino acid numbers 190 to 241 of SEQ ID NO: 2.

[12] The particle of

[11] , wherein the Gc antigen is a protein comprising an amino acid sequence having at least 95% identity with the amino acid sequence of amino acid numbers 28 to 503 of SEQ ID NO: 2, amino acid numbers 28 to 241 of SEQ ID NO: 2, or amino acid numbers 28 to 369 of SEQ ID NO: 32.

[13] The particle of any of [1] to

[12] , wherein the NP antigen is a protein comprising an amino acid sequence having at least 95% identity with the amino acid sequence of amino acid numbers 19 to 262 of SEQ ID NO: 3.

[14] A particle according to any one of [1] to

[13] , characterized in that it has a signal sequence at the N-terminus of the antigen protein.

[15] A particle according to any one of [1] to

[14] , wherein the nucleic acid capable of expressing the Gn antigen, Gc antigen, or NP antigen of SFTSV is mRNA comprising a cap structure (Cap), a 5' untranslated region (5'-UTR), a translation region of the Gn antigen, Gc antigen, or NP antigen of SFTSV, and a 3' untranslated region (3'-UTR).

[16] The particle of

[15] , characterized in that the nucleic acid sequence capable of expressing the Gn antigen of SFTSV comprises a nucleotide sequence having at least 90% identity with the nucleotide sequence set forth in nucleotide numbers 71 to 1429 of SEQ ID NO: 9, nucleotide numbers 71 to 1429 of SEQ ID NO: 18, or nucleotide numbers 71 to 1093 of SEQ ID NO: 44 (preferably consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence set forth in SEQ ID NO: 9, 18, or 44).

[17] The particle of

[15] , characterized in that the nucleic acid sequence capable of expressing the Gc antigen of SFTSV comprises a nucleotide sequence having at least 90% identity with the sequence of nucleotide numbers 71 to 1582 of SEQ ID NO: 12, nucleotide numbers 71 to 1582 of SEQ ID NO: 21, or nucleotide numbers 71 to 1180 of SEQ ID NO: 56 (preferably consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence set forth in SEQ ID NO: 12, 21, or 56).

[18] The particle of

[15] , characterized in that the nucleic acid sequence capable of expressing the SFTSV NP antigen comprises a nucleotide sequence having at least 90% identity with the sequence of nucleotides 71 to 859 of SEQ ID NO: 15 or 71 to 859 of SEQ ID NO: 24 (preferably consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence set forth in SEQ ID NO: 15 or 24).

[19] The particle of any of [1] to

[18] , wherein the nucleic acid comprises at least one modified nucleotide. [19-2] The particle of

[19] , wherein the modified nucleotide comprises at least one pyrimidine nucleotide substituted at position 5 and / or pseudouridine optionally substituted at position 1. [19-3] The particle of

[19] , wherein the modified nucleotide comprises at least one nucleotide selected from the group consisting of 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, and 1-alkylpseudouridine.

[20] The particles of

[19] , wherein the modified nucleotide comprises at least one selected from the group consisting of 5-methylcytidine, 5-methyluridine, pseudouridine, and 1-methylpseudouridine.

[21] The particles of any of [1] to

[20] , having an average particle size of 30 to 300 nm.

[22] Use of the particles of any of [1] to

[21] for producing a composition for preventing and / or treating infection or onset caused by SFTSV.

[23] A pharmaceutical composition containing any of the particles of [1] to

[21] . [23-2] The composition of

[23] for expressing Gn, Gc, or NP of SFTSV in vivo or in vitro. [23-3] The composition of

[23] or [23-2] for use as a medicine. [23-4] The composition of [23-3] for inducing an immune response to SFTSV.

[24] The pharmaceutical composition of

[23] for preventing and / or treating SFTSV infection.

[25] The pharmaceutical composition of

[23] for preventing and / or treating severe fever with thrombocytopenia syndrome (SFTS) in a subject suspected of being infected with SFTSV. [25-2] A method for expressing SFTSV Gn, Gc, or NP in vitro, comprising introducing any of the compositions of

[23] to

[25] into a cell. [25-3] A method for expressing SFTSV Gn, Gc, or NP in vivo, comprising administering any of the compositions of

[23] to

[25] to a mammal. [25-4] A method for inducing an immune response to SFTSV, comprising administering any of the compositions of

[23] to

[25] to a mammal.

[26] A method for preventing and / or treating SFTSV infection, comprising administering any of the pharmaceutical compositions of

[23] to

[25] to a mammal. [26-2] A method for preventing and / or treating severe fever with thrombocytopenia syndrome (SFTS), comprising administering any of the compositions of

[23] to [25-4] to a subject suspected of SFTSV infection. This specification includes the disclosure of Japanese Patent Application No. 2024-073076, from which the present application claims priority.

[0012] According to the present invention, it is possible to prevent and / or treat infection by SFTSV, and also to prevent and / or treat symptoms and diseases caused by SFTSV infection.

[0013] 2A and 2B show blood anti-Gn antibody titers (A), anti-Gc antibody titers (B), and anti-NP antibody titers (C) in IFNAR1 KO mice. Bars indicate mean values, and error bars indicate SD. Circles indicate individual data. D50: 5 weeks after the second administration, D57: 1 week after the third administration. These figures show Gn-specific cellular immune responses in IFNAR1 KO mice. Figure 2-1A shows IFNγ production levels, and Figure 2-1B shows IL-13 production levels. Vertical bars indicate mean values, and error bars indicate SD. Circles on the bar graphs indicate individual data. These figures show Gc-specific cellular immune responses in IFNAR1 KO mice. Figure 2-2A shows IFNγ production levels, and Figure 2-2B shows IL-13 production levels. Vertical bars indicate mean values, and error bars indicate SD. Circles on the bar graphs indicate individual data. 2-3B show NP-specific cellular immune responses in IFNAR1 KO mice. Figures 2-3A and 2-3B show IFNγ production levels, and IL-13 production levels, respectively. Vertical bars indicate mean values, and error bars indicate SD. Circles on bar graphs represent individual data. Figures 2-3A and 2-3B show blood anti-Gn antibody titers (A), anti-Gc antibody titers (B), and anti-NP antibody titers (C) in IFNAR1 KO mice. Bars indicate mean values, and error bars indicate SD. Symbols in the graph represent individual data. Figure 2-3B shows blood anti-SFTSV neutralizing activity in IFNAR1 KO mice. Symbols in the graph represent mean values ​​for each individual at each dilution concentration, and error bars indicate SD. Figure 2-3B shows blood anti-SFTSV neutralizing activity in IFNAR1 KO mice. Symbols in the graph represent mean values ​​for each individual at each dilution concentration, and error bars indicate SD. 1 shows the anti-OVA antibody titer (A), anti-Gn antibody titer (B), anti-Gc antibody titer (C), and anti-NP antibody titer (D) in the blood of IFNAR1 KO mice. Bars indicate the mean, and error bars indicate SD. Symbols in the graphs represent individual data. The survival time (A) and body weight (B) of IFNAR1 KO mice after SFTSV infection are shown. The mean for each group is shown, and error bars indicate SD. The blood virus titer of IFNAR1 KO mice 4 days after SFTSV infection is shown. Bars indicate the mean, and error bars indicate SD. Symbols in the graphs represent individual data.

[0033] Figure 1 shows the amino acid sequence of Gn (SEQ ID NO: 1) and the amino acid sequence of Gc (SEQ ID NO: 2). Figure 2 shows the amino acid sequence of NP (SEQ ID NO: 3) and the amino acid sequence of Ova (SEQ ID NO: 4). Figure 3 shows the nucleotide sequence (SEQ ID NO: 5) of a DNA fragment containing a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a codon-optimized Gn sequence, and a human β-globin 3'-UTR sequence are linked in this order. Figure 4 shows the mRNA sequence of codon-optimized Gn (SEQ ID NO: 9). In the figure, C represents 5-methylcytidine and U represents 5-methyluridine. Figure 5 shows the nucleotide sequence (SEQ ID NO: 10) of a DNA fragment containing a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a codon-optimized Gc sequence, and a human β-globin 3'-UTR sequence are linked in this order.

[0033] Figure 1 shows the mRNA sequence of codon-optimized Gc (SEQ ID NO: 12). In the figure, C represents 5-methylcytidine, and U represents 5-methyluridine.

[0034] Figure 1 shows the nucleotide sequence (SEQ ID NO: 13) of a DNA fragment containing a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a codon-optimized NP sequence, and a human β-globin 3'-UTR sequence are linked in this order.

[0035] Figure 1 shows the mRNA sequence of codon-optimized NP (SEQ ID NO: 15). In the figure, C represents 5-methylcytidine, and U represents 5-methyluridine.

[0036] Figure 1 shows the nucleotide sequence (SEQ ID NO: 16) of a DNA fragment containing a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a Gn sequence, and a human β-globin 3'-UTR sequence are linked in this order.

[0037] Figure 1 shows the mRNA sequence of Gn (SEQ ID NO: 18). In the figure, C represents 5-methylcytidine, and U represents 5-methyluridine. This figure shows the nucleotide sequence (SEQ ID NO: 19) of a DNA fragment containing a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a Gc sequence, and a human β-globin 3'-UTR sequence are linked in this order. This figure shows the Gc mRNA sequence (SEQ ID NO: 21). In the figure, C represents 5-methylcytidine, and U represents 5-methyluridine.1 shows the nucleotide sequence (SEQ ID NO: 22) of a DNA fragment containing a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, an NP sequence, and a human β-globin 3'-UTR sequence are linked in this order. 1 shows the mRNA sequence of NP (SEQ ID NO: 24). In the figure, C represents 5-methylcytidine, and U represents 5-methyluridine. 1 shows the anti-Gn antibody titer (A) and the anti-Gc antibody titer (B) in IFNAR1 KO mice. 1 shows the mean value, and the error bars represent the SD. 1 shows individual data. 1 shows the pooled anti-SFTSV neutralizing activity in IFNAR1 KO mice. 1 shows the mean value for each individual at each dilution concentration, and the error bars represent the SD. 1 shows the individual anti-SFTSV neutralizing activity in IFNAR1 KO mice. Symbols in the graph represent individual data points, and error bars indicate SD. This is a schematic diagram showing the relationship of the four types of Gn partially deleted mutants used in Example 9 to the full-length Gn. The amino acid sequences of Gn1, Gn2, Gn3, and Gn4, with a 19-amino acid signal peptide added to the N-terminus, are set forth in SEQ ID NOS: 28, 29, 30, and 31, respectively. The numbers in the diagram indicate the position of the amino acid at the N-terminus of each domain. SP indicates the signal peptide, TM indicates the transmembrane region, I to III indicate domain numbers, and GG indicates a linker consisting of two glycines. This is a schematic diagram showing the relationship of the four types of Gc partially deleted mutants used in Example 10 to the full-length Gc. The amino acid sequences of Gc1, Gc2, Gc3, and Gc4, with a 19-amino acid signal peptide added to the N-terminus, are set forth in SEQ ID NOS: 32, 33, 34, and 35, respectively. The numbers in the diagram indicate the position of the amino acid at the N-terminus of each domain. SP represents a signal peptide, TM represents a transmembrane region, GG represents a linker consisting of two glycines, and GGGGS represents a linker consisting of four glycines and one serine. Figure 1 shows the amino acid sequences of Gn1 (SEQ ID NO: 28), Gn2 (SEQ ID NO: 29), Gn3 (SEQ ID NO: 30), and Gn4 (SEQ ID NO: 31).

[0033] Figure 1 shows the amino acid sequence of Gc1 (SEQ ID NO: 32), the amino acid sequence of Gc2 (SEQ ID NO: 33), the amino acid sequence of Gc3 (SEQ ID NO: 34), and the amino acid sequence of Gc4 (SEQ ID NO: 35).

[0034] Figure 1 shows the nucleotide sequence (SEQ ID NO: 36) of a DNA fragment containing a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a Gn1 sequence, and a human β-globin 3'-UTR sequence are linked in this order.

[0035] Figure 1 shows the nucleotide sequence (SEQ ID NO: 37) of a DNA fragment containing a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a Gn2 sequence, and a human β-globin 3'-UTR sequence are linked in this order.

[0033] Figure 1 shows the nucleotide sequence of a DNA fragment (SEQ ID NO: 38) comprising a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a Gn3 sequence, and a human β-globin 3'-UTR sequence are linked in this order.

[0034] Figure 1 shows the nucleotide sequence of a DNA fragment (SEQ ID NO: 39) comprising a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a Gn4 sequence, and a human β-globin 3'-UTR sequence are linked in this order.

[0035] Figure 1 shows the nucleotide sequence of a template DNA for IVT of Gn1 (SEQ ID NO: 40).

[0036] Figure 1 shows the nucleotide sequence of a template DNA for IVT of Gn2 (SEQ ID NO: 41).

[0037] Figure 1 shows the nucleotide sequence of a template DNA for IVT of Gn3 (SEQ ID NO: 42).

[0038] Figure 1 shows the nucleotide sequence of a template DNA for IVT of Gn4 (SEQ ID NO: 43).

[0033] Figure 1 shows the mRNA sequence of Gn1 (SEQ ID NO: 44). Figure 2 shows the mRNA sequence of Gn2 (SEQ ID NO: 45). Figure 3 shows the mRNA sequence of Gn3 (SEQ ID NO: 46). Figure 4 shows the mRNA sequence of Gn4 (SEQ ID NO: 47). Figure 5 shows the nucleotide sequence (SEQ ID NO: 48) of a DNA fragment comprising a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a Gc1 sequence, and a human β-globin 3'-UTR sequence are linked in this order.

[0033] Figure 1 shows the nucleotide sequence of a DNA fragment containing a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a Gc2 sequence, and a human β-globin 3'-UTR sequence are linked in this order (SEQ ID NO: 49). Figure 2 shows the nucleotide sequence of a DNA fragment containing a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a Gc3 sequence, and a human β-globin 3'-UTR sequence are linked in this order (SEQ ID NO: 50). Figure 3 shows the nucleotide sequence of a DNA fragment containing a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a Gc4 sequence, and a human β-globin 3'-UTR sequence are linked in this order (SEQ ID NO: 51). Figure 1 shows the nucleotide sequence of template DNA for IVT of Gc1 (SEQ ID NO:52). Figure 2 shows the nucleotide sequence of template DNA for IVT of Gc2 (SEQ ID NO:53). Figure 3 shows the nucleotide sequence of template DNA for IVT of Gc3 (SEQ ID NO:54). Figure 4 shows the nucleotide sequence of template DNA for IVT of Gc4 (SEQ ID NO:55). Figure 5 shows the mRNA sequence of Gc1 (SEQ ID NO:56). Figure 6 shows the mRNA sequence of Gc2 (SEQ ID NO:57). Figure 7 shows the mRNA sequence of Gc3 (SEQ ID NO:58). Figure 8 shows the mRNA sequence of Gc4 (SEQ ID NO:59).

[0014] Hereinafter, embodiments of the present invention will be described in more detail.

[0015] The present invention provides lipid particles encapsulating a nucleic acid capable of expressing glycoprotein N (Gn), glycoprotein C (Gc), or nucleoprotein (NP) encoded by the S segment of Bandavirus dabiieense (commonly known as SFTS virus (SFTSV)), which is a part of the glycoprotein precursor encoded by the SFTSV M segment of the Phenuiviridae virus. The lipids constituting the particles include at least four types: an amphipathic lipid, a sterol, a PEG lipid, and a cationic lipid.

[0016] The amphipathic lipid is a lipid that has affinity for both polar and nonpolar solvents, and specific examples thereof include distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, and combinations thereof. The amphipathic lipid used in the particles of the present invention is preferably distearoylphosphatidylcholine and / or dioleoylphosphatidylethanolamine, and more preferably distearoylphosphatidylcholine.

[0017] The sterols are sterols having a hydroxy group, and specific examples thereof include cholesterol.

[0018] The PEG lipid is a PEG-modified lipid, specifically, 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol and / or N-[methoxy poly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine, or a combination thereof, preferably 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol. The average molecular weight of the PEG lipid is not particularly limited, but is, for example, 1,000 to 5,000, preferably 1,500 to 3,000, and more preferably 1,800 to 2,200.

[0019] The cationic lipids herein are lipids in which some molecules have a net positive charge depending on the pKa of the lipid at a selected pH, such as physiological pH.The cationic lipid is an ionizable lipid, preferably a tertiary amine.Some cationic lipids are described in the public literature or are commercially available. For example, such cationic lipids include MC3 (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, disclosed in WO2010 / 144740, ALC-0315 (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoic acid ester), disclosed in WO2018 / 081480, and SM-102 (heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoic acid ester, disclosed in WO2017 / 049245.

[0020] The present invention also provides the above particles, wherein the cationic lipid is a cationic lipid represented by general formula (Ia) or a pharmaceutically acceptable salt thereof.

[0021] In the formula, R １ and R ２ are independently １ -C ３ represents an alkyl group; １ is C ２ -C ４ C which may have one or more alkanoyloxy groups １７ -C １９ represents an alkenyl group; ２ is C ２ -C ４ C which may have one or more alkanoyloxy groups １０ -C １９ Alkyl group, or C ２ -C ４ C which may have one or more alkanoyloxy groups １０ -C １９represents an alkenyl group; and p is 3 or 4.

[0022] R in general formula (Ia) １ and R ２ are independently １ -C ３ Preferably, both are methyl groups.

[0023] In the general formula (Ia), p is 3 or 4, preferably 3.

[0024] L in general formula (Ia) １ is C ２ -C ４ C which may have one or more alkanoyloxy groups １７ -C １９ It represents an alkenyl group, and preferably represents a C １７ -C １９ It is an alkenyl group. １ Specific examples of the alkyl group include an (R)-11-acetyloxy-cis-8-heptadecenyl group, a cis-8-heptadecenyl group, and a (8Z,11Z)-heptadecadienyl group.

[0025] L in general formula (Ia) ２ is C ２ -C ４ C which may have one or more alkanoyloxy groups １０ -C １９ Alkyl group, or C ２ -C ４ C which may have one or more alkanoyloxy groups １０ -C １９ It represents an alkenyl group, and preferably represents a C １０ -C １２ C which may have one or more alkyl groups or acetoxy groups １０ -C １９ Alternatively, L in general formula (Ia) is an alkenyl group. ２ C which may have one or more acetoxy groups １０ -C １２C which may have one or more alkyl groups or acetoxy groups １７ -C １９ It is also preferably an alkenyl group. ２ Specific examples of the alkyl group include a decyl group, a cis-7-decenyl group, a dodecyl group, and an (R)-11-acetyloxy-cis-8-heptadecenyl group.

[0026] Specific examples of cationic lipids that are components constituting the particles of the present invention include those having the following structural formula: Examples include (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate, 3-dimethylaminopropyl(9Z,12Z)-octacosa-19,22-dien-11-yl carbonate, and (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacos-9-en-7-yl acetate, each of which is represented by the formula: The cationic lipid is preferably (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate or (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacos-9-en-7-yl acetate.

[0027] The cationic lipid represented by general formula (Ia) may be one type of compound or a combination of two or more types of compounds.

[0028] A method for producing cationic lipids represented by general formula (Ia) is described in WO 2015 / 005253.

[0029] The lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is not particularly limited, but for example, in molar amounts, amphipathic lipids are 5 to 25%, sterols are 10 to 55%, cationic lipids are 40 to 65%, and PEG lipids are 1 to 5%. The lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is preferably in molar amounts of 10 to 25%, 10 to 55%, 40 to 65%, and 1 to 5%. The lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is more preferably in molar amounts of 10 to 22.5%, 15 to 55%, 40 to 65%, and 1 to 5%. In the lipid composition, the proportion of PEG lipid is preferably 1 to 3% in molar amount, more preferably 1 to 2%, even more preferably 1.2 to 2%, even more preferably 1.25 to 2%, even more preferably 1.3 to 2%, and particularly preferably 1.5 to 2%. In addition, the ratio of total lipid weight to nucleic acid weight in the lipid composition is not particularly limited, but may be 15 to 30, preferably 15 to 25, more preferably 15 to 22.5, and even more preferably 17.5 to 22.5.

[0030] When 3-dimethylaminopropyl (9Z,12Z)-octacosa-19,22-dien-11-yl carbonate or (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-dien-7,29-diyl diacetate is used as the cationic lipid, the lipid composition of the amphipathic lipid, sterols, cationic lipid, and PEG lipid is not particularly limited, but for example, in molar amounts, amphipathic lipid is 5 to 25%, sterols is 10 to 55%, cationic lipid is 40 to 65%, and PEG lipid is 1 to 5%, and it is preferable that amphipathic lipid is 15% or less, sterols is 20 to 55%, cationic lipid is 40 to 65%, and PEG lipid is 1 to 5%. It is more preferable that the lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is, in molar amounts, 5 to 15% amphipathic lipids, 35 to 50% sterols, 40 to 55% cationic lipids, and 1 to 3% PEG lipids; it is even more preferable that the lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is, in molar amounts, 10 to 15% amphipathic lipids, 35 to 45% sterols, 40 to 50% cationic lipids, and 1 to 2.5% PEG lipids; it is even more preferable that the lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is, in molar amounts, 10 to 15% amphipathic lipids, 35 to 45% sterols, 40 to 50% cationic lipids, and 1 to 2% PEG lipids. In the lipid composition, the PEG lipid is more preferably 1.2 to 2%, even more preferably 1.25 to 2%, even more preferably 1.3 to 2%, and even more preferably 1.5 to 2%. In the lipid composition, the ratio of total lipid weight to nucleic acid weight is not particularly limited, but may be 15 to 30, preferably 15 to 25, more preferably 15 to 22.5, and even more preferably 17.5 to 22.5.

[0031] When (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacos-9-en-7-yl acetate is used as the cationic lipid, the lipid composition of the amphipathic lipid, sterols, cationic lipid, and PEG lipid is not particularly limited, but for example, the molar amounts are 5 to 25% amphipathic lipid, 10 to 55% sterols, 40 to 65% cationic lipid, and 1 to 5% PEG lipid, and it is preferable that the molar amounts are 10 to 25% amphipathic lipid, 10 to 50% sterols, 40 to 65% cationic lipid, and 1 to 5% PEG lipid, and the lipid composition of the amphipathic lipid, sterols, cationic lipid, and PEG lipid is 10 to 25% amphipathic lipid, 10 to 50% sterols, 40 to 65% cationic lipid, and 1 to 3% PEG lipid. It is more preferable that the lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is, in molar amounts, 10 to 25% amphipathic lipids, 10 to 45% sterols, 42.5 to 65% cationic lipids, and 1 to 2.5% PEG lipids. It is even more preferable that the lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is, in molar amounts, 15 to 22.5% amphipathic lipids, 15 to 40% sterols, 45 to 65% cationic lipids, and 1 to 2% PEG lipids. It is even more preferable that the lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is, in molar amounts, 17.5 to 22.5% amphipathic lipids, 15 to 40% sterols, 45 to 65% cationic lipids, and 1 to 2% PEG lipids. In the lipid composition, the PEG lipid is more preferably 1.2 to 2%, even more preferably 1.25 to 2%, even more preferably 1.3 to 2%, and even more preferably 1.5 to 2%. In the lipid composition, the ratio of total lipid weight to nucleic acid weight is not particularly limited, but is preferably 15 to 30, more preferably 15 to 25, even more preferably 15 to 22.5, and even more preferably 17.5 to 22.5.

[0032] Specific lipid combinations in the present invention include distearoylphosphatidylcholine, dioleoylphosphatidylcholine, or dioleoylphosphatidylethanolamine as amphipathic lipids, cholesterol as sterols, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-dien-7,29-diyl diacetate, 3-dimethylaminopropyl(9Z,12Z)-octacosa-19,22-dien-11-yl carbonate, or (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacos-9-en-7-yl acetate as cationic lipids, and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol or N-[methoxy

[0039] Also preferred is a lipid combination using distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine as the amphipathic lipid, cholesterol as the sterol, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate or (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacos-9-en-7-yl acetate as the cationic lipid, and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol as the PEG lipid. A more preferred specific lipid combination in the present invention is distearoylphosphatidylcholine as the amphipathic lipid, cholesterol as the sterol, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate or (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacos-9-en-7-yl acetate as the cationic lipid, and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol as the PEG lipid.

[0033] In the present invention, the nucleic acid encapsulated in the lipid particle is capable of expressing the Gn antigen, Gc antigen, or NP antigen of SFTSV. "Gn antigen," "Gc antigen," and "NP antigen" refer to antigenic proteins that contain at least the region contained in each protein that is necessary for producing neutralizing antibodies.

[0034] The amino acid sequence of the extracellular domain of Gn of SFTSV is shown in SEQ ID NO: 1 (amino acid numbers 1 to 19 are the signal peptide). Furthermore, the amino acid sequence of Gn1, a partially deleted form of Gn, is shown in SEQ ID NO: 28 (amino acid numbers 1 to 19 are the signal peptide). The results of Test Example 4 showed that the group administered with mRNA encoding Gn1 exhibited neutralizing activity comparable to that of the group administered with mRNA of the Gn antigen extracellular domain, whereas no neutralizing antibody production was confirmed in other deleted forms, including Gn4, in which the N-terminus of Gn1 was further deleted. Therefore, the region of amino acid numbers 20 to 175 of SEQ ID NO: 1, which is the difference between Gn1 and Gn4, is identified as the region necessary for neutralizing antibody production. That is, the Gn antigen is a protein comprising at least the region of amino acids 20 to 175 of SEQ ID NO: 1, preferably a protein comprising a region comprising amino acids 20 to 340 of SEQ ID NO: 1, more preferably a protein comprising the amino acid sequence of amino acids 20 to 452 of SEQ ID NO: 1 or amino acids 20 to 340 of SEQ ID NO: 28, and optimally a protein consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 28. The nucleic acid to be encapsulated in the lipid particles may encode a protein consisting of an amino acid sequence that is at least 95%, preferably 96%, more preferably 97%, and more preferably 98% identical to the amino acid sequence of the Gn antigen. In one embodiment, the nucleic acid to be encapsulated in the lipid particles may encode an amino acid sequence in which the amino acid sequence of the Gn antigen may have several amino acid substitutions, deletions, insertions, and / or additions at several positions (preferably 5 positions or less, more preferably 3, 2, or 1 position), with several amino acids (preferably 10 positions or less, more preferably 7 positions or less, and even more preferably 5, 4, 3, 2, or 1 position) per position.

[0035] The amino acid sequence of the extracellular domain of Gc of SFTSV is shown in SEQ ID NO: 2 (amino acid numbers 1 to 27 are the signal peptide). Furthermore, the amino acid sequence of Gc1, a partially deleted form of Gc, is shown in SEQ ID NO: 32 (amino acid numbers 1 to 27 are the signal peptide). The results of Test Example 4 showed that the group administered with mRNA encoding Gc1 exhibited neutralizing activity comparable to that of the group administered with mRNA of the extracellular domain of Gc, whereas no neutralizing antibody production was observed in other deleted forms, including Gc2, in which 69 amino acids on the N-terminus and 52 amino acids in the middle of Gc1 were further deleted. Therefore, the region of amino acid numbers 28 to 96 and / or the region of amino acid numbers 190 to 241 of SEQ ID NO: 2, which are the difference between Gc1 and Gc2, are identified as the region necessary for neutralizing antibody production. That is, the Gc antigen is a protein comprising at least the region of amino acids 28 to 96 and / or the region of amino acids 190 to 241 of SEQ ID NO: 2, preferably a protein comprising a region including amino acids 28 to 241 of SEQ ID NO: 2, more preferably a protein comprising the amino acid sequence set forth in amino acids 28 to 503 of SEQ ID NO: 2, amino acids 28 to 241 of SEQ ID NO: 2, or amino acids 28 to 369 of SEQ ID NO: 32, and optimally a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 32. The nucleic acid to be encapsulated in the lipid particles may encode a protein consisting of an amino acid sequence that is at least 95%, preferably 96%, more preferably 97%, and more preferably 98% identical to the amino acid sequence of the Gc antigen. In one aspect, the nucleic acid encapsulated in the lipid particle may encode an amino acid sequence in which several amino acids (preferably 10 or less, more preferably 7 or less, and even more preferably 5, 4, 3, 2, or 1) may be substituted, deleted, inserted, and / or added at several positions (preferably 5 or less, more preferably 3, 2, or 1 position) in the amino acid sequence of the Gc antigen.

[0036] The full-length amino acid sequence of the fusion protein of SFTSV NP and IgE signal peptide is shown in SEQ ID NO: 3 (amino acid numbers 1 to 18 are the IgE signal peptide). The NP antigen is preferably a protein comprising amino acid numbers 19 to 262 of SEQ ID NO: 3, and optimally a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3. The nucleic acid to be encapsulated in the lipid particles may encode a protein consisting of an amino acid sequence that is at least 95%, preferably 96%, more preferably 97%, and more preferably 98% identical to the amino acid sequence of the NP antigen (amino acid numbers 19 to 262 of SEQ ID NO: 3). In one embodiment, the nucleic acid to be encapsulated in the lipid particles may encode an amino acid sequence in which several amino acids (preferably 5 or less, more preferably 3, 2, or 1) may be substituted, deleted, inserted, and / or added at several positions (preferably 5 or less, more preferably 3, 2, or 1) in the amino acid sequence of the NP antigen.

[0037] The Gn antigen, Gc antigen, and NP antigen may have a signal peptide sequence at their N-terminus for extracellular secretion. The signal peptide sequence is not particularly limited as long as it has the function of being secreted extracellularly after being translated intracellularly. Specific examples of such signal peptide sequences include amino acid sequences of amino acid numbers 1 to 19 of SEQ ID NO: 1, amino acid numbers 1 to 27 of SEQ ID NO: 2, and amino acid numbers 1 to 18 of SEQ ID NO: 3. The signal peptide sequence and the antigen may be linked directly or via several spacer amino acids (e.g., 15 or less, 10 or less, 8 or less, 5, 4, 3, 2, or 1).

[0038] The identity of an amino acid sequence is a numerical representation of the percentage of amino acid identity relative to the full-length sequence, where amino acids that completely match corresponding amino acids are considered to be the same amino acid. The sequence identity in the present invention is calculated using sequence analysis software GENETYX-SV / RC (manufactured by Genetyx Corporation), and this algorithm is commonly used in the technical field. The amino acids encoded by the nucleic acid encapsulated in the lipid particles of the present invention may contain amino acid mutations (substitutions), deletions, insertions, and / or additions, as long as they maintain a certain level of identity with any of SEQ ID NOS: 1 to 3.

[0039] The amino acids encoded by the nucleic acids to be encapsulated in the lipid particles of the present invention maintain the above-mentioned sequence identity, and may have several amino acids substituted, deleted, inserted, and / or added at several positions (preferably 5 positions or less, more preferably 3, 2, or 1 position) in the region of the amino acid sequence of any of SEQ ID NOs: 1 to 3, 28, and 32 corresponding to various antigens, with several amino acids per position (preferably 10 positions or less, more preferably 7 positions or less, and even more preferably 5, 4, 3, 2, or 1 position).

[0040] The nucleic acid capable of expressing the SFTSV Gn, Gc, or NP antigen may be an mRNA comprising a cap structure (Cap), a 5' untranslated region (5'-UTR), a translated region of the SFTSV Gn, Gc, or NP antigen, a 3' untranslated region (3'-UTR), and a polyA tail (polyA). It may also have a KOZAK sequence on the 5' side of the translated region of the SFTSV Gn, Gc, or NP. The cap structure (Cap) is present at the 5' end of many eukaryotic mRNAs and is a site containing a 7-methylguanosine structure. Examples of cap structures include cap0, cap1, cap2, and cap structures obtained using ARCA (Anti-Reverse Cap Analog), and the cap structure is as shown in the following structural formula:

[0041] (wherein Base represents any unmodified or modified nucleic acid base, and RNA represents any polynucleotide.)

[0042] (wherein Base represents any unmodified or modified nucleic acid base, and RNA represents any polynucleotide.)

[0043] (wherein Base represents any unmodified or modified nucleic acid base, and RNA represents any polynucleotide.)

[0044] (wherein Base represents any unmodified or modified nucleic acid base, and RNA represents any polynucleotide.)

[0045] The cap structure of the mRNA of the present invention is preferably cap0 or cap1, and more preferably cap1. The sequence of the 5'-untranslated region (5'-UTR) can be, for example, a sequence containing the 5'-untranslated region of the human β-globin gene. For example, the sequence of the 5'-untranslated region of the human β-globin gene is the sequence of bases 15 to 64 in SEQ ID NO: 9. The sequence of the Gn translation region is a sequence capable of expressing all or part of the amino acid sequence of the Gn antigen (which may have a desired signal peptide added to its N-terminus), and may include an initiation codon and / or a termination codon, such as the sequence of bases 71 to 1429 in SEQ ID NO: 9, 71 to 1429 in SEQ ID NO: 18, or 71 to 1093 in SEQ ID NO: 44. The sequence of the Gc translation region is a sequence capable of expressing all or part of the amino acid sequence of a Gc antigen (which may have a desired signal peptide added to its N-terminus), and may include a start codon and / or a stop codon, such as the sequence of nucleotides 71 to 1582 in SEQ ID NO: 12, 71 to 1582 in SEQ ID NO: 21, or 71 to 1180 in SEQ ID NO: 56. The sequence of the NP translation region is a sequence capable of expressing all or part of the amino acid sequence of a NP antigen (which may have a desired signal peptide added to its N-terminus), and may include a start codon and / or a stop codon. Examples of such sequences include those in which a sequence encoding an IgE secretory signal peptide for extracellular secretion of the NP antigen is linked, such as the sequence of nucleotides 71 to 859 in SEQ ID NO: 15 or the sequence of nucleotides 71 to 859 in SEQ ID NO: 24. The sequence of the 3' untranslated region (3'-UTR) can be, for example, the 3' untranslated region of the human β-globin gene, for example, the sequence from base numbers 1430 to 1561 in SEQ ID NO: 9. The sequence of the poly A tail (polyA) can be, for example, the sequence from base numbers 1562 to 1661 in SEQ ID NO: 9.The sequences of the cap structure (Cap), 5'-untranslated region (5'-UTR), Gn, Gc, or NP translated region, 3'-untranslated region (3'-UTR), and polyA tail (polyA) may be modified, and the nucleic acid sequence capable of expressing the Gn antigen may consist of a nucleotide sequence having at least 90%, preferably 95%, and more preferably 97% identity to the sequence of SEQ ID NO: 9, 18, or 44. The nucleic acid sequence capable of expressing the Gc antigen may consist of a nucleotide sequence having at least 90%, preferably 95%, and more preferably 97% identity to the sequence of SEQ ID NO: 12, 21, or 56. The nucleic acid sequence capable of expressing the NP antigen may consist of a nucleotide sequence having at least 90%, preferably 95%, and more preferably 97% identity to the sequence of SEQ ID NO: 15 or 24.

[0046] The length of the poly-A tail is not limited, but may be, for example, 10 to 250 bases in length, preferably 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or 95 or more bases in length, and may be 200 or less, 150 or less, 120 or less, 115 or less, or 110 or less bases in length. In one embodiment, the length of the poly-A tail is 50 to 150 bases in length, preferably 70 to 120 bases in length, and particularly preferably 90 to 110 bases in length. In yet another embodiment, the length of the poly-A tail is preferably 95, 100, 105, 110, 115, or 120 bases in length, and more preferably 100, 110, or 120 bases in length.

[0047] The mRNA of the present invention may be an mRNA having a sequence comprising a cap structure (Cap), a 5' untranslated region (5'-UTR), a translated region of Gn, Gc or NP, and a 3' untranslated region (3'-UTR), wherein the portion consisting of the cap structure (Cap), the 5' untranslated region (5'-UTR), the translated region of Gn antigen, Gc antigen or NP antigen, and the 3' untranslated region (3'-UTR) has at least 90%, preferably 95%, and more preferably 97% identity to the sequence from positions 1 to 1561 of SEQ ID NO: 9, the sequence from positions 1 to 1714 of SEQ ID NO: 12, or the sequence from positions 1 to 991 of SEQ ID NO: 15.

[0048] The nucleic acid to be encapsulated in the lipid particles may be in any form as long as it is capable of expressing the Gn antigen, Gc antigen, or NP antigen of SFTSV. Examples include single-stranded DNA, single-stranded RNA (e.g., mRNA), a single-stranded polynucleotide consisting of a mixture of DNA and RNA, double-stranded DNA, double-stranded RNA, a DNA-RNA hybrid polynucleotide, and a double-stranded polynucleotide consisting of two polynucleotides consisting of a mixture of DNA and RNA, with mRNA being preferred.

[0049] The nucleotides constituting the nucleic acid to be encapsulated in the lipid particles may be natural or modified nucleotides, but it is preferable that the nucleic acid contains at least one modified nucleotide.

[0050] The modified nucleotide may be one in which any of the base, sugar, and phosphodiester bond is modified, and the modification site may be one site or two or more sites.

[0051] Examples of base modifications include 5-methylation, 5-fluoroation, and N4-methylation of cytosine, 5-methylation (thymine) and 5-fluoroation of uracil, N6-methylation of adenine, and N2-methylation of guanine.

[0052] An example of a sugar modification is 2'-O-methylation of D-ribofuranose.

[0053] An example of a modification of a phosphodiester bond is a phosphorothioate bond.

[0054] The modified nucleotide is preferably one in which the base moiety is modified, and examples thereof include a pyrimidine nucleotide substituted at the 5th position and a pseudouridine optionally substituted at the 1st position. Specific examples include 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, and 1-alkylpseudouridine. The 1-alkylpseudouridine may be a 1-(C1-C6 alkyl)pseudouridine, preferably 1-methylpseudouridine or 1-ethylpseudouridine. More preferred examples of the modified nucleotide include 5-methylcytidine, 5-methyluridine, and 1-methylpseudouridine. Particularly preferred examples of the modified nucleotide include a combination of 5-methylcytidine and 5-methyluridine, or a combination of 5-methylcytidine and 1-methylpseudouridine.

[0055] The nucleic acids capable of expressing the Gn, Gc, or NP antigens of SFTSV of the present invention can be produced by in vitro transcription from DNA having the desired base sequence. Enzymes, buffer solutions, and nucleoside-5'-triphosphate mixtures (adenosine-5'-triphosphate (ATP), guanosine-5'-triphosphate (GTP), cytidine-5'-triphosphate (CTP), and uridine-5'-triphosphate (UTP), or nucleoside triphosphates incorporating corresponding modified bases) required for in vitro transcription are commercially available (e.g., AmpliScribe T7 High Yield Transcription Kit (Epicentre), mMESSAGE mMACHINE T7 Ultra Kit (Life Technologies)). The DNA used to produce single-stranded RNA is cloned DNA, for example, a plasmid DNA or a DNA fragment. Plasmid DNA or a DNA fragment may be commercially available or may be produced by a method generally known in the art (e.g., the methods described in Sambrook, J. et al., Molecular Cloning a Laboratory Manual second edition (1989); Rashtchian, A., Current Opinion in Biotechnology, 1995, 6(1), 30-36; Gibson, D. G. et al., Science, 2008, 319(5867), 1215-1220).

[0056] To obtain mRNA with improved stability and / or safety, some or all of the unmodified nucleotides in the mRNA can be replaced with modified nucleotides (e.g., 5-methylCTP, 5-methylUTP, N-methylpseudoUTP) by substituting some or all of the unmodified nucleoside-5'-triphosphates with modified nucleoside-5'-triphosphates in an in vitro transcription reaction (Kormann, M., Nature Biotechnology, 2011, 29, 154-157).

[0057] To obtain mRNA with improved stability and / or safety, a cap structure (the above-mentioned Cap0 structure) can be introduced at the 5' end of mRNA by using a capping enzyme after an in vitro transcription reaction. Furthermore, Cap0 can be converted to Cap1 by treating mRNA with Cap0 with 2'-O-methyltransferase. Commercially available capping enzymes and 2'-O-methyltransferases can be used (e.g., Vaccinia Capping System, M2080; mRNA Cap 2'-O-Methyltransferase, M0366, both manufactured by New England Biolab). When using commercially available products, mRNA with a cap structure can be produced according to the protocol provided with the product.

[0058] A cap structure at the 5' end of mRNA can also be introduced by methods other than those using enzymes. For example, by adding ARCA or CleanCap (registered trademark) to an in vitro transcription reaction, a cap analog structure possessed by ARCA or a Cap1 structure derived from CleanCap can be introduced into mRNA. Commercially available ARCA and CleanCap products can be used (ARCA, N-7003; CleanCap Reagent AG, N-7113, both manufactured by TriLink BioTechnologies). When using commercially available products, mRNA having a cap structure can be produced according to the protocol provided with the product.

[0059] In the present invention, the nucleic acid to be encapsulated in lipid particles may be purified by methods such as desalting, HPLC (reverse phase, gel filtration, ion exchange, affinity), PAGE, ultrafiltration, etc. By removing impurities through the purification process, the production of inflammatory cytokines in a living body to which the nucleic acid is administered can be reduced.

[0060] The nucleic acid-encapsulated lipid particles of the present invention can be produced by methods such as the thin film method, reverse-phase evaporation, ethanol injection, ether injection, dehydration-rehydration, surfactant dialysis, hydration, and freeze-thaw methods. For example, nucleic acid-encapsulated lipid particles can be produced by the method described in International Publication No. 2015 / 005253. Alternatively, the nucleic acid-encapsulated lipid particles of the present invention can be produced by pumping an aqueous solution of nucleic acid and a solution of lipid dissolved in an organic solvent and mixing the two solutions. The aqueous solution of nucleic acid may be a buffer solution, such as a buffer solution using an inorganic salt such as phosphate or carbonate, or a buffer solution using an organic salt such as acetate or citrate. The solution in which lipid is dissolved in an organic solvent may be any organic solvent as long as it dissolves cationic lipids, amphipathic lipids, sterols, and PEG lipids. However, an alcohol solution is preferred, and an ethanol solution is particularly preferred. The pump used to mix the aqueous solution of nucleic acid and the lipid solution is not particularly limited, and examples thereof include gear pumps, piston pumps, plunger pumps, diaphragm pumps, rotary pumps, vane pumps, peristaltic pumps, and centrifugal pumps. The aqueous solution of nucleic acid and the lipid solution may be mixed in a mixing chamber, or may be connected by a T- or Y-junction and mixed in a flow channel. For example, nucleic acid-encapsulated lipid particles can be produced by the methods described in International Publication No. 2009 / 127060 or International Publication No. 2004 / 002453. A preferred embodiment of the nucleic acid-encapsulated lipid particles of the present invention is a method in which a nucleic acid solution and a lipid solution are mixed in a microchannel. For example, the nucleic acid-encapsulated lipid particles of the present invention can be produced using Precision Nanosystems' NanoAssembler (registered trademark) and following the method described in the accompanying protocol.

[0061] The resulting nucleic acid-encapsulating lipid particles may be further subjected to steps such as purification and concentration. For example, the nucleic acid-encapsulating lipid particles may be purified and / or concentrated by tangential flow filtration using a membrane with an appropriate pore size. The purified and / or concentrated nucleic acid-encapsulating lipid particles may be substituted with a desired buffer solution, such as a phosphate buffer or a citrate buffer.

[0062] The particles of the present invention may have an average particle size of 30 to 300 nm, preferably 30 to 200 nm, more preferably 50 to 200 nm, more preferably 100 to 200 nm, more preferably 120 to 160 nm, and more preferably 125 to 150 nm. The average particle size can be obtained by measuring the volume average particle size based on the principle of dynamic light scattering using an instrument such as a Zeta Potential / Particle Sizer NICOMP (registered trademark) 380ZLS (PARTICLE SIZING SYSTEMS).

[0063] The particles of the present invention can be used to manufacture a composition for preventing and / or treating diseases caused by SFTSV infection, such as severe fever with thrombocytopenia syndrome (SFTS).

[0064] Furthermore, any one of the SFTSV Gc antigen, SFTSV Gn antigen, and SFTSV NP antigen may be expressed, or two or three of these may be expressed simultaneously. When any one of the SFTSV Gc antigen, SFTSV Gn antigen, and SFTSV NP antigen is expressed, it is preferable to express the SFTSV Gc antigen (more preferably SEQ ID NO: 2) or the SFTSV NP antigen (preferably SEQ ID NO: 3). When any two of the SFTSV Gc antigen, SFTSV Gn antigen, and SFTSV NP antigen are expressed, it is sufficient to select one of the combinations of Gn antigen and Gc antigen, Gn antigen and NP antigen, and Gc antigen and NP antigen, with the combination of Gc (SEQ ID NO: 2) and NP (SEQ ID NO: 3) being preferred. Lipid particles encapsulating a nucleic acid capable of expressing the Gn antigen of SFTSV, lipid particles encapsulating a nucleic acid capable of expressing the Gc antigen of SFTSV, or lipid particles encapsulating a nucleic acid capable of expressing the NP antigen of SFTSV may be formulated with the nucleic acid either in the same lipid particle or as a separate lipid particle, but are preferably prepared as separate lipid particles.

[0065] The particles of the present invention can be used to express the SFTSV Gn antigen, Gc antigen, or NP antigen in vivo or in vitro. That is, one, two, or three of lipid particles encapsulating a nucleic acid capable of expressing the SFTSV Gn antigen, lipid particles encapsulating a nucleic acid capable of expressing the SFTSV Gc antigen, or lipid particles encapsulating a nucleic acid capable of expressing the SFTSV NP antigen can be administered to a subject. Thus, the present invention provides a method for expressing one, two, or three of the SFTSV Gn antigen, SFTSV Gc antigen, and SFTSV NP antigen in vitro, which comprises introducing a composition containing the particles into cells. The present invention also provides a method for expressing one, two, or three of the SFTSV Gn antigen, SFTSV Gc antigen, and SFTSV NP antigen in vivo, comprising administering a composition containing the particles to a mammal. This antigen expression can induce an immune response against SFTSV, resulting in the prevention and / or treatment of SFTSV infection. Therefore, the present invention provides a method for inducing an immune response against SFTSV, comprising administering a composition containing the particles to a mammal. The present invention also provides a method for preventing and / or treating SFTSV infection, comprising administering a composition containing the particles to a mammal. The present invention also includes a kit comprising both lipid particles encapsulating nucleic acids capable of expressing one, two, or three of the SFTSV Gn antigen, SFTSV Gc antigen, and SFTSV NP antigen. Lipid particles encapsulating nucleic acids capable of expressing one, two or three of the SFTSV Gn antigen, the SFTSV Gc antigen and the SFTSV NP antigen can be used as vaccines against SFTSV infection.

[0066] The particles of the present invention can be used as pharmaceuticals or experimental reagents. The particles of the present invention are typically added to a carrier such as water, buffer solution, or physiological saline, and the resulting formulation (composition) can be introduced into cells (in vitro) or administered to mammals (in vivo). When administered to mammals, the carrier should be a pharmaceutically acceptable carrier (e.g., physiological saline). The particles of the present invention may also be formulated into creams, pastes, ointments, gels, lotions, and other formulations using fats, fatty oils, lanolin, petrolatum, paraffin, wax, resins, plastics, glycols, higher alcohols, glycerin, water, emulsifiers, suspending agents, and the like as base materials.

[0067] The particles of the present invention can be administered orally or parenterally to mammals such as humans, mice, rats, hamsters, guinea pigs, rabbits, pigs, monkeys, cats, dogs, horses, goats, sheep, and cows by methods such as intramuscular administration, intravenous administration, rectal administration, transdermal administration, transmucosal administration, subcutaneous administration, and intradermal administration.

[0068] When the particles of the present invention are administered to humans, for example, a single dose of about 0.001 to 1 mg, preferably 0.01 to 0.2 mg, in terms of the weight of mRNA per adult may be administered once or several times by intramuscular injection, subcutaneous injection, intradermal injection, intravenous drip injection, or intravenous injection, but the dose and number of administrations may be varied as appropriate depending on the type of disease, symptoms, age, administration method, etc. When administered to companion animals or livestock, the maximum dose is the same as that for humans, and the particles are administered by the same route as for humans.

[0069] When used as experimental reagents, the particles of the present invention can be introduced into cells in which SFTSV Gn, Gc, or NP antigens are to be expressed (e.g., HEK293 cells and their derivatives (HEK293T cells, FreeStyle 293 cells, and Expi293 cells), CHO cells, C2C12 mouse myoblasts, and immortalized mouse dendritic cells (MutuDC1940)), allowing the SFTSV Gn, Gc, or NP antigens to be expressed in vitro. Expression of SFTSV Gn, Gc, or NP antigens can be analyzed by detecting SFTSV Gn, Gc, or NP antigen proteins in a sample by Western blotting, or by detecting peptide fragments specific to SFTSV Gn, Gc, or NP antigens by mass spectrometry.

[0070] In the present invention, "treatment" means recovery, remission, alleviation, and / or delay of worsening of clinical symptoms of an infectious disease caused by a virus, bacteria, or the like, or a disease caused by such an infection (e.g., precancerous lesion, cancer, etc.) in a patient who has developed such a disease.

[0071] In the present invention, "prevention" means reducing the incidence of diseases caused by infectious diseases such as viruses or bacteria. Prevention includes reducing the risk of progression of diseases caused by infectious diseases such as viruses or bacteria, or reducing the severity of such diseases. The particles of the present invention are effective in preventing and / or treating the above-mentioned diseases because they induce a protective immune response. Typical forms of inoculation for the purpose of infection prevention include, but are not limited to, ingestion in advance by subjects (humans, livestock animals, companion animals, etc.) who plan to travel, move, or invade areas where there is a risk of SFTSV infection.

[0072] The particles of the present invention are expected to be used as a preventive and therapeutic agent for diseases caused by SFTSV in subjects suspected of SFTSV infection. Diseases caused by SFTSV include severe fever with thrombocytopenia syndrome (SFTS). A "subject suspected of SFTSV infection" refers to a subject (such as a human, livestock animal, or companion animal) who has been confirmed to have come into contact with a vector for SFTSV (typically, a tick, an animal that has come into contact with a tick, or the like), and it is not necessarily the case that the virus has been detected.

[0073] The present invention will be specifically described below with reference to examples. Note that these examples are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0074] The following abbreviations may be used in the examples. Gn: represents the protein in the N-terminal region of the envelope protein of SFTSV (amino acid sequence: SEQ ID NO: 1 (amino acid numbers 1 to 19 are the signal peptide)). Gc: represents the protein in the C-terminal region of the envelope protein of SFTSV (amino acid sequence: SEQ ID NO: 2 (amino acid numbers 1 to 27 are the signal peptide)). NP: represents the NP protein (amino acid sequence: amino acid numbers 19 to 262 of SEQ ID NO: 3). Ova: represents the Ovalbumin protein (SEQ ID NO: 4). Gn1: represents a partially truncated form of Gn (amino acid sequence: SEQ ID NO: 28 (amino acid numbers 1 to 19 are the signal peptide)). Gn2: represents a partially truncated form of Gn (amino acid sequence: SEQ ID NO: 29 (amino acid numbers 1 to 19 are the signal peptide)). Gn3: represents a partially truncated form of Gn (amino acid sequence: SEQ ID NO: 30 (amino acid numbers 1 to 19 are the signal peptide)). Gn4: represents a partially deleted Gn (amino acid sequence: SEQ ID NO: 31 (amino acid numbers 1 to 19 are the signal peptide)). Gc1: represents a partially deleted Gc (amino acid sequence: SEQ ID NO: 32 (amino acid numbers 1 to 27 are the signal peptide)). Gc2: represents a partially deleted Gc (amino acid sequence: SEQ ID NO: 33 (amino acid numbers 1 to 27 are the signal peptide)). Gc3: represents a partially deleted Gc (amino acid sequence: SEQ ID NO: 34 (amino acid numbers 1 to 27 are the signal peptide)). Gc4: represents a partially deleted Gc (amino acid sequence: SEQ ID NO: 35 (amino acid numbers 1 to 27 are the signal peptide)).

[0075] Example 1 Preparation of Gn mRNA-001 (1) Preparation of template DNA for Gn in vitro transcription (IVT) To prepare template DNA for IVT, Gn DNA was amplified by PCR and then purified. A DNA fragment (SEQ ID NO: 5) containing a sequence in which the T7 promoter sequence, human β-globin 5'-UTR sequence, KOZAK sequence, codon-optimized Gn sequence, and human β-globin 3'-UTR sequence were linked in this order was introduced into a plasmid. 10 ng of the plasmid was dissolved in nuclease-free water (283.2 μL) and diluted with 10x Buffer for KOD-Plus- Ver. 2 (40 μL, Toyobo Co., Ltd. catalog # KOD-211), 2 mM dNTP mix (40 μL, Toyobo Co., Ltd. catalog # KOD-211), 25 mM MgSO ４ To the mixture were added 24 μL of 50 μM sense primer (2.4 μL, SEQ ID NO: 6), 50 μM antisense primer (2.4 μL, SEQ ID NO: 7), and KOD Plus polymerase (8 μL, SEQ ID NO: 211). After incubation at 98°C for 15 seconds, 20 cycles of 98°C for 5 seconds, 55°C for 15 seconds, and 68°C for 90 seconds were performed, followed by a further incubation at 68°C for 1 minute to amplify the DNA. After the reaction, the template DNA (SEQ ID NO: 8) containing the PolyA sequence was purified using the Wizard SV Gel and PCR Clean-Up System (Promega catalog # A9281).

[0076] (2) Preparation of Gn mRNA-001 by IVT 2 μg of template DNA (4 μL) obtained in Example 1-(1), 100 mM CleanCap AG (10 μL, TriLink catalog # N-7113), 100 mM ATP (10 μL, Hongene catalog # R1331), 100 mM GTP (10 μL, Hongene catalog # R2331), 100 mM 5-methylCTP (10 μL, TriLink catalog # N-1014), 100 mM 5-methylUTP (10 μL, TriLink catalog # N-1024), Nuclease-free water (86 μL, Thermo Fisher Scientific catalog # AM9937), T7 Transcription 5x buffer (40 μL, Promega Scientific catalog # P140X), Enzyme mix, and T7 RNA Polymerase (20 μL, Promega Scientific catalog # P137X) were mixed and incubated at 37°C for 4 hours. RQ1 RNase-Free DNase (10 μL, Promega Scientific catalog # M6101) was added and incubated at 37°C for 15 minutes. This was mixed with 8 M LiCl solution (100 μL, Sigma-Aldrich catalog # L7026) and allowed to stand overnight at -20°C. After centrifugation (4°C, 5200 × g, 30 minutes), the supernatant was discarded. 70% ethanol was added, followed by centrifugation (4°C, 5200 × g, 10 minutes). The supernatant was discarded and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water (1000 μL) and purified using an RNeasy Midi kit (Qiagen catalog # 75144) according to the accompanying manual. The resulting solution (733 μL), rApid Alkaline Phosphatase (Roche catalog # 04 898 141 001) buffer solution (85 μL) and enzyme (32 μL) were mixed and incubated at 37°C for 30 minutes, followed by incubation at 75°C for 2 minutes.The resulting solution was purified using an RNeasy Midi kit (Qiagen catalog #75144) according to the accompanying manual to obtain the target mRNA. The resulting mRNA had the sequence of SEQ ID NO:9, in which all cytidines were substituted with 5-methylcytidines and all uridines were substituted with 5-methyluridines.

[0077] Example 2 Preparation of Gc mRNA-001 (1) Preparation of template DNA for Gc IVT To prepare template DNA for IVT, Gc DNA was amplified by PCR and then purified. A DNA fragment (SEQ ID NO: 10) containing a sequence in which the T7 promoter sequence, human β-globin 5'-UTR sequence, KOZAK sequence, codon-optimized Gc sequence, and human β-globin 3'-UTR sequence were linked in order was introduced into a plasmid. Using this plasmid as a template, DNA was amplified and purified in the same manner as in Example 1-(1), and template DNA (SEQ ID NO: 11) containing a PolyA sequence was prepared.

[0078] (2) Preparation of Gc mRNA-001 by IVT The target mRNA was obtained in the same manner as in Example 1-(2) using the template DNA obtained in Example 2-(1). The obtained mRNA had the sequence of SEQ ID NO: 12, in which all cytidines were substituted with 5-methylcytidines and all uridines were substituted with 5-methyluridines.

[0079] Example 3 Preparation of NP mRNA-001 (1) Preparation of template DNA for NP IVT To prepare template DNA for IVT, NP DNA was amplified by PCR and then purified. A DNA fragment (SEQ ID NO: 13) containing a sequence in which the T7 promoter sequence, human β-globin 5'-UTR sequence, KOZAK sequence, codon-optimized NP sequence, and human β-globin 3'-UTR sequence were linked in this order was introduced into a plasmid. DNA was amplified using this plasmid and purified in the same manner as in Example 1-(1), and template DNA (SEQ ID NO: 14) having a PolyA sequence was prepared.

[0080] (2) Preparation of NP mRNA-001 by IVT The target mRNA was obtained in the same manner as in Example 1-(2) using the template DNA obtained in Example 3-(1). The obtained mRNA had the sequence of SEQ ID NO: 15, in which all cytidines were substituted with 5-methylcytidines and all uridines were substituted with 5-methyluridines.

[0081] Example 4 Preparation of Gn mRNA-002 (1) Preparation of template DNA for Gn IVT To prepare template DNA for IVT, Gn DNA was amplified by PCR and then purified. A DNA fragment (SEQ ID NO: 16) containing a sequence in which the T7 promoter sequence, human β-globin 5'-UTR sequence, KOZAK sequence, Gn sequence, and human β-globin 3'-UTR sequence were linked in order was introduced into a plasmid. Using this plasmid as a template, DNA was amplified and purified in the same manner as in Example 1-(1), and template DNA (SEQ ID NO: 17) containing a PolyA sequence was prepared.

[0082] (2) Preparation of Gn mRNA-002 by IVT The template DNA obtained in Example 2-(1) was used to obtain the target mRNA in the same manner as in Example 1-(2). The obtained mRNA had the sequence of SEQ ID NO: 18, in which all cytidines were substituted with 5-methylcytidines and all uridines were substituted with 5-methyluridines.

[0083] Example 5 Preparation of Gc mRNA-002 (1) Preparation of template DNA for Gc IVT To prepare template DNA for IVT, Gc DNA was amplified by PCR and then purified. A DNA fragment (SEQ ID NO: 19) containing a sequence in which the T7 promoter sequence, human β-globin 5'-UTR sequence, KOZAK sequence, Gc sequence, and human β-globin 3'-UTR sequence were linked in order was introduced into a plasmid. Using this plasmid as a template, DNA was amplified and purified in the same manner as in Example 1-(1), and template DNA (SEQ ID NO: 20) containing a PolyA sequence was prepared.

[0084] (2) Preparation of Gc mRNA-002 by IVT The target mRNA was obtained in the same manner as in Example 1-(2) using the template DNA obtained in Example 2-(1). The obtained mRNA had the sequence of SEQ ID NO: 21, in which all cytidines were substituted with 5-methylcytidines and all uridines were substituted with 5-methyluridines.

[0085] Example 6 Preparation of NP mRNA-002 (1) Preparation of template DNA for NP IVT To prepare template DNA for IVT, NP DNA was amplified by PCR and then purified. A DNA fragment (SEQ ID NO: 22) containing a sequence in which the T7 promoter sequence, human β-globin 5'-UTR sequence, KOZAK sequence, NP sequence, and human β-globin 3'-UTR sequence were linked in order was introduced into a plasmid. DNA was amplified and purified using this plasmid in the same manner as in Example 1-(1), and template DNA (SEQ ID NO: 23) having a PolyA sequence was prepared.

[0086] (2) Preparation of NP mRNA-002 by IVT The target mRNA was obtained in the same manner as in Example 1-(2) using the template DNA obtained in Example 3-(1). The obtained mRNA had the sequence of SEQ ID NO: 24, in which all cytidines were substituted with 5-methylcytidines and all uridines were substituted with 5-methyluridines.

[0087] Example 7 Preparation of Ova mRNA-001 (1) Preparation of template DNA for IVT of Ova To prepare template DNA for IVT, Ova DNA was amplified by PCR and then purified. A DNA fragment (SEQ ID NO: 25) containing a sequence in which the T7 promoter sequence, human β-globin 5'-UTR sequence, KOZAK sequence, Ova sequence, and human β-globin 3'-UTR sequence were linked in order was introduced into a plasmid. DNA was amplified and purified using this plasmid in the same manner as in Example 1-(1), and template DNA (SEQ ID NO: 26) having a PolyA sequence was prepared.

[0088] (2) Preparation of Ova mRNA-001 by IVT The template DNA obtained in Example 3-(1) was used to obtain the target mRNA in the same manner as in Example 1-(2). The obtained mRNA had the sequence of SEQ ID NO: 27, in which all cytidines were substituted with 5-methylcytidines and all uridines were substituted with 5-methyluridines.

[0089] [Example 8] Preparation of mRNA-encapsulated nucleic acid-lipid particles using the mRNA described in Examples 1 to 7 (1) Preparation of mRNA-encapsulated nucleic acid-lipid particles Distearoylphosphatidylcholine (1,2-distearoyl-sn-glycero-3-phosphocholine: hereinafter referred to as DSPC, NOF Cholesterol (Sigma-Aldrich, Inc.), (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate (the compound described in Example 23 of WO 2015 / 005253) (hereinafter referred to as LP1), or (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacos-9-en-7-yl acetate (the compound described in Example 28 of WO 2015 / 005253) (hereinafter referred to as LP2), and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol having a molecular weight of about 2000. Glycol (1,2-Dimyristoyl-sn-Glycero-3-Methoxypolyethylene Glycol, hereinafter referred to as PEG-DMG, NOF CORPORATION, SUNBRIGHT GM-020) was dissolved in ethanol at the molar ratio shown in Table 1 to give a total lipid concentration of 5 mM.

[0090] On the other hand, the mRNA obtained in Examples 1 to 7 was diluted with citrate buffer (20 mM citrate buffer, pH 4.0).

[0091] The lipid solution and mRNA solution were mixed in a microchannel using a NanoAssembler Ignite (Precision Nanosystems Inc.) so that the total lipid to mRNA weight ratio was the value shown in Table 1 and the volume ratio was 1:3, yielding a crude dispersion of nucleic acid-lipid particles. The nucleic acid-lipid particle dispersion was dialyzed (Float-A-Lyzer G2, MWCO: 1,000 kD, Spectra / Por) against approximately 25 to 50 times the volume of buffer for 12 to 18 hours to remove ethanol, yielding a dispersion of purified mRNA-encapsulated nucleic acid-lipid particles.

[0092] LP1 was synthesized according to the method described in Example 23 of WO2015 / 005253, and LP2 was synthesized according to the method described in Example 28 of WO2015 / 005253.

[0093] (2) Evaluation of the Characteristics of the mRNA-Encapsulated Nucleic Acid-Lipid Particles The characteristics of the dispersion containing the nucleic acid-lipid particles prepared in (1) were evaluated. The methods for evaluating each characteristic are described below.

[0094] (2-1) mRNA Encapsulation Rate The mRNA encapsulation rate was measured using the Quant-iT RiboGreen RNA Assay kit (Invitrogen) according to the package insert. Specifically, mRNA in a dispersion of nucleic acid-lipid particles was quantified in the presence and absence of 0.015% Triton X-100 surfactant, and the encapsulation rate was calculated using the following formula: {([amount of mRNA in the presence of surfactant] - [amount of mRNA in the absence of surfactant]) / [amount of mRNA in the presence of surfactant]} x 100 (%)

[0095] (2-2) Average Particle Diameter The particle diameter of the nucleic acid-lipid particles was measured using a Zeta Potential / Particle Sizer NICOMP™ 380ZLS (PARTICLE SIZING SYSTEMS). The average particle diameter in the table represents the volume average particle diameter, and ± indicates deviation.

[0096] The results of the characteristic evaluation are shown in Table 2.

[0097]

[0098]

[0099] From the above results, it was revealed that these nucleic acid-lipid particles had 93% or more of the mRNA encapsulated within the lipid particles, and had an average particle size of about 125 nm to about 150 nm.

[0100] Example 9 Preparation of Gn1 mRNA-001 to Gn4 mRNA-001 (1) Preparation of template DNA for IVT of Gn1 to Gn4 To prepare template DNA for IVT, DNA fragments (SEQ ID NOS: 36 to 39) containing a sequence in which the T7 promoter sequence, the 5'-UTR sequence of human β-globin, the KOZAK sequence, the nucleotide sequence encoding a Gn partial deletion (Gn1, Gn2, Gn3, or Gn4), and the 3'-UTR sequence of human β-globin were linked in this order were introduced into a plasmid. Using this plasmid as a template, DNA was amplified and purified in the same manner as in Example 1-(1), and template DNAs (SEQ ID NOS: 40 to 43) having a PolyA sequence were prepared.

[0101] (2) Preparation of Gn1 mRNA-001 to Gn4 mRNA-001 by IVT Using the template DNA obtained in Example 9-(1), the target mRNAs were obtained in the same manner as in Example 1-(2). The obtained mRNAs had the sequences of SEQ ID NOS: 44 to 47, in which all cytidines were substituted with 5-methylcytidines and all uridines were substituted with 5-methyluridines.

[0102] Example 10 Preparation of Gc1 mRNA-001 to Gc4 mRNA-001 (1) Preparation of template DNA for IVT of Gc1 to Gc4 To prepare template DNA for IVT, DNA fragments (SEQ ID NOS: 48 to 51) containing a sequence in which the T7 promoter sequence, the 5'-UTR sequence of human β-globin, the KOZAK sequence, a nucleotide sequence encoding a Gc partial deletion (Gc1, Gc2, Gc3, or Gc4), and the 3'-UTR sequence of human β-globin were linked in this order were introduced into a plasmid. Using this plasmid as a template, DNA was amplified and purified in the same manner as in Example 1-(1), and template DNAs (SEQ ID NOS: 52 to 55) having a PolyA sequence were prepared.

[0103] (2) Preparation of Gc1 mRNA-001 to Gc4 mRNA-001 by IVT Using the template DNA obtained in Example 10-(1), the target mRNAs were obtained in the same manner as in Example 1-(2). The obtained mRNAs had the sequences of SEQ ID NOS: 56 to 59, in which all cytidines were substituted with 5-methylcytidines and all uridines were substituted with 5-methyluridines.

[0104] Example 11 Preparation of Gn mRNA-004 Template DNA for Gn IVT was prepared in the same manner as in Example 1-(1), and then the target mRNA was obtained in the same manner as in Example 1-(2). The obtained mRNA had the sequence of SEQ ID NO: 9, in which all cytidines were substituted with 5-methylcytidines and all uridines were substituted with 5-methyluridines.

[0105] Example 12 Preparation of Gc mRNA-004 Template DNA for IVT of Gc was prepared in the same manner as in Example 2-(1), and then the target mRNA was obtained in the same manner as in Example 2-(2). The obtained mRNA had the sequence of SEQ ID NO: 12, in which all cytidines were substituted with 5-methylcytidines and all uridines were substituted with 5-methyluridines.

[0106] Example 13 Preparation of Ova mRNA-002 Template DNA for IVT of Ova was prepared in the same manner as in Example 7-(1), and then the target mRNA was obtained in the same manner as in Example 7-(2). The obtained mRNA had the sequence of SEQ ID NO: 27, in which all cytidines were substituted with 5-methylcytidines and all uridines were substituted with 5-methyluridines. Example 14 mRNA encoding various antigens prepared by the methods of the above Examples (Gn1-4: Example 9, Gc1-4: Example 10, Gn: Example 1, Gc: Example 2, OVA: Example 7) was used to prepare mRNA nucleic acid-lipid particles in accordance with the method of Example 8. The lipid composition conformed to the conditions for using LP1 in Table 1.

[0107] The dispersion containing the prepared mRNA nucleic acid-lipid particles was characterized, and the results are shown in Table 3.

[0108]

[0109] From the above results, it was revealed that these nucleic acid-lipid particles had 98% or more of the mRNA encapsulated within the lipid particles, and had an average particle size of about 129 nm to about 145 nm.

[0110] (Test Example 1) Administration of various nucleic acid-lipid particles to IFNAR1 KO mice IFNAR1 KO mice (B6(Cg)-Ifnar1 ｔｍ１．２Ｅｅｓ / J) were obtained from Jackson Laboratory and acclimatized. LNP-mRNA Gn #1, LNP-mRNA Gc #1, or LNP-mRNA NP #1 prepared in Example 8 was administered to the disinfected triceps surae muscles of anesthetized mice at intervals of 2 to 5 weeks, a total of three times at 10 μg mRNA / 20 μL / mouse. The first and third administrations were into the right leg, and the second administration was into the left leg. Buffer was used to adjust the lipid particle concentration and for the negative control group.

[0111] Preparation of serum and spleen cells. Five weeks after the second administration of lipid particles and one week after the third administration, blood was collected from anesthetized mice. The blood was collected in a microtube containing a coagulant / serum separator (Becton Dickinson) and centrifuged at 10,000 RPM for 10 minutes to separate and collect serum. One week after the third administration, spleens were collected from mice euthanized by cervical dislocation under anesthesia. The spleens were mashed using a Cell Strainer (Falcon) mesh and a syringe (Terumo) gasket, adjusted to a single cell suspension in RPMI 1640 (Nacalai Tesque), and then centrifuged to collect cells. The supernatant was discarded, and the red blood cells were lysed using BD Pharm Lyse Lysing buffer (Becton Dickinson). After centrifugation, the cell suspension was resuspended in RPMI 1640 and passed through mini Cell Strainers (Hitec Corporation, Cat. HT-AMS-14002). After centrifugation, the supernatant was discarded, the cells were resuspended, and the cell concentration was measured. After centrifugation, the supernatant was discarded, and the cell concentration was adjusted with cell culture medium (RPMI 1640 supplemented with 10% (v / v) Fetal Bovine Serum (heat-inactivated and filtered, HyClone), 1% (v / v) Penicillin-Streptomycin Mixed Solution (Nacalai Tesque), 1 mM Sodium Pyruvate (Gibco), 1% (v / v) MEM Non-Essential Amino Acids (Gibco), 10 mM HEPES (Gibco), and 50 μM 2-Mercaptoethanol). Centrifugation was performed at 1500 RPM for 5 minutes at 4°C.

[0112] Anti-Gn, anti-Gc, and anti-NP antibody titers in mouse blood. Recombinant Gn protein (Immune Technology), recombinant Gc protein (Immune Technology), and recombinant NP protein (Native Antigen) were used as solid-phase antigens and added to a 96-well flat-bottom plate and incubated overnight at 4°C. A standard curve dilution series was prepared by diluting mouse serum containing anti-Gn antibodies, anti-Gc antibodies, or anti-NP antibodies in eight 3-fold dilutions starting from a maximum concentration of 1 arbitrary unit / mL. Each well was washed, and blocking solution was added and incubated at room temperature for 1 hour. Serum samples were prepared in seven 4-fold dilutions starting from a maximum concentration of 120-fold using blocking solution. The blocked plate was washed, and the diluted samples were added to the plate and incubated at room temperature for 1 hour. The detection antibody, Goat Anti-Mouse IgG, Human adz-HRP (SouthernBiotech), was diluted 4000-fold with blocking solution and added to the washed wells. After 1 hour, the wells were washed, and TMB Microwell Peroxidase Substrate System (Seracare) was added and left to stand for 10 minutes. ２ SO ４ The reaction was stopped using HCl. The corrected absorbance, calculated by subtracting the absorbance at 540 nm from the absorbance at 450 nm, was used for analysis on a plate reader to calculate the anti-Gn antibody titer, anti-Gc antibody titer, and anti-NP antibody titer. The blocking solution was 1X Dulbecco's Phosphate Buffered Saline supplemented with 1% (w / v) Bovine Serum Albumin and 0.05% (v / v) Tween 20 (BIO-RAD), and washing was performed three times with 1X Dulbecco's Phosphate Buffered Saline supplemented with 0.05% (v / v) Tween 20.

[0113] Gn-, Gc-, and NP-specific cellular immune responses in mice. Mouse splenocytes prepared 10 ６The cells were seeded at 1000 cells / well in a 96-well U-bottom plate (FALCON), and incubated at 37°C in 5% CO with Gn, Gc, and NP pooled peptide (Eurofins) adjusted to a final concentration of 10 μg / mL. ２ After 24 or 48 hours, the culture supernatant was collected, and IFN-γ (R&D Systems) and IL-13 (R&D Systems) production was measured according to the kit protocol.

[0114] (Results of Test Example 1) Mouse Blood Gn, Anti-Gc, and Anti-NP Antibody Titers The blood anti-Gn antibody response, blood anti-Gc antibody response, and blood anti-NP antibody response induced by lipid particle formulations of LNP-mRNA Gn #1, LNP-mRNA Gc #1, or LNP-mRNA NP #1 were evaluated ( FIG. 1 ). As a result, the LNP-mRNA Gn #1 group had higher blood anti-Gn antibody titers after two doses (D50) and three doses (D57) compared to the negative control group ( FIG. 1A ). The LNP-mRNA Gc #1 group had higher blood anti-Gc antibody titers after two doses (D50) and three doses (D57) compared to the negative control group ( FIG. 1B ). In the LNP-mRNA NP #1 group, the anti-NP antibody titers in the blood were higher after two doses (D50) and three doses (D57) compared to the negative control group (Fig. 1C).

[0115] Gn-, Gc-, and NP-specific cellular immune responses in mice. Gn-, Gc-, or NP-specific cellular immune responses induced by administration of LNP-mRNA Gn #1, LNP-mRNA Gc #1, or LNP-mRNA NP #1 lipid particles were evaluated using IFN-γ and IL-13 production levels (Figures 2-1 to 2-3). Compared to the negative control group, the LNP-mRNA Gn #1 group exhibited a higher Gn-specific cellular immune response (Figure 2-1). The LNP-mRNA Gc #1 group exhibited a higher Gc-specific cellular immune response (Figure 2-2). The LNP-mRNA NP #1 group exhibited a higher NP-specific cellular immune response (Figure 2-3).

[0116] Test Example 2: Administration of various nucleic acid lipid particles to IFNAR1 KO mice RBRC04021; IFNAR1 KO mice were used, which were generated by crossbreeding Ifnar1- / - Dnase2a- / - with Dnase2a+ / - Ifnar1- / -. At two-week intervals, LNP-mRNA Gn #1, LNP-mRNA Gc #1, or LNP-mRNA NP #1 prepared in Example 8 was administered twice at 10 μg mRNA / 20 μL / mouse to the disinfected triceps surae muscle of anesthetized mice. The first administration was to the right leg, and the second administration was to the left leg. Buffer was used to adjust the lipid particle concentration and for the negative control group.

[0117] Six days after the second administration of lipid particles, blood was collected from anesthetized mice in a 1.5 mL tube (Greiner Bio-One) and centrifuged at 4000 RPM for 10 minutes to separate and collect serum.

[0118] Anti-Gn, anti-Gc, and anti-NP antibody titers in mouse blood. Recombinant Gn protein (Immune Technology), recombinant Gc protein (Immune Technology), and recombinant NP protein (Native Antigen) were used as solid-phase antigens and added to a 96-well flat-bottom plate and incubated overnight at 4°C. A standard curve dilution series was prepared by diluting mouse serum containing anti-Gn antibodies, anti-Gc antibodies, or anti-NP antibodies in eight 3-fold dilutions starting from a maximum concentration of 1 arbitrary unit / mL. Each well was washed, and blocking solution was added and incubated at room temperature for 1 hour. Serum samples were prepared in seven 4-fold dilutions starting from a maximum concentration of 120-fold using blocking solution. The blocked plate was washed, and the diluted samples were added to the plate and incubated at room temperature for 1 hour. The detection antibody, Goat Anti-Mouse IgG, Human adz-HRP (SouthernBiotech), was diluted 4000-fold with blocking solution and added to the washed wells. After 1 hour, the wells were washed, and TMB Microwell Peroxidase Substrate System (Seracare) was added and left to stand for 10 minutes. ２ SO ４The reaction was stopped using HCl. The corrected absorbance, calculated by subtracting the absorbance at 540 nm from the absorbance at 450 nm, was used for analysis on a plate reader to calculate the anti-Gn antibody titer, anti-Gc antibody titer, and anti-NP antibody titer. The blocking solution was 1X Dulbecco's Phosphate Buffered Saline supplemented with 1% (v / v) Bovine Serum Albumin and 0.05% Tween 20 (BIO-RAD), and washing was performed three times with 1X Dulbecco's Phosphate Buffered Saline supplemented with 0.05% (v / v) Tween 20.

[0119] Measurement of neutralizing activity Vero76 cells were suspended in 10% FCS DMEM (Nacalai Tesque) and diluted to 1 × 10 4 The next day, 30 μL of serum diluted in 2% (w / v) FCS DMEM (Nacalai Tesque) in six serial dilutions (10-fold to 4-fold dilutions from the highest dilution) was mixed with 200 FFU / 30 μL of SFTSV YG1 and incubated at 37°C in CO ２ The cells were incubated for 1 hour in the presence of 50 μL of serum and SFTSV mixture. 50 μL of the serum and SFTSV mixture was added to Vero76 cells and incubated for 48 hours. Then, the cells were fixed with 4% (w / v) paraformaldehyde, and 100 μL of ice-cold 100% methanol was added to each well and incubated at -30°C for 10 minutes to permeabilize the membrane. The cells were washed with PBS (Nissui Pharmaceutical Co., Ltd.) and blocked for 60 minutes with Blocking Buffer (5% (w / v) Goat Serum, 0.3% (w / v) Triton X / PBS). 30 μL / well of 5000-fold diluted Rabbit Anti-NP Antibody was added and incubated overnight at 4°C. 30 μL / well of 1:1000 diluted Alexa488-labeled Goat Anti-Rabbit Antibody and 5:5000 diluted DAPI (Roche) were added and incubated for 1 hour in the dark. After washing with PBS, the cells were observed under a fluorescence microscope, and green fluorescent-positive cells were counted as infected cells, and the percentage of uninfected cells among the total cells was calculated.

[0120] (Results of Test Example 2) Mouse Blood Gn, Anti-Gc, and Anti-NP Antibody Titers The blood anti-Gn antibody response, blood anti-Gc antibody response, and blood anti-NP antibody response induced by lipid particle formulations of LNP-mRNA Gn #1, LNP-mRNA Gc #1, or LNP-mRNA NP #1 were evaluated (FIG. 3). As a result, the LNP-mRNA Gn #1 group had a higher blood anti-Gn antibody titer than the negative control group, LNP-mRNA Gc #1 group, and LNP-mRNA NP #1 group (FIG. 3A). The LNP-mRNA Gc #1 group had a higher blood anti-Gc antibody titer than the negative control group, LNP-mRNA Gn #1 group, and LNP-mRNA NP #1 group (FIG. 3B). The LNP-mRNA NP #1 group had a higher blood anti-NP antibody titer than the negative control group, the LNP-mRNA Gn #1 group, and the LNP-mRNA Gc #1 group (Fig. 3C).

[0121] Anti-SFTSV neutralizing activity in mouse blood. The anti-SFTSV neutralizing activity in blood induced by administration of lipid particles of LNP-mRNA Gn #1, LNP-mRNA Gc #1, or LNP-mRNA NP #1 was evaluated (Figure 4). Serum collected 6 days after the second administration showed neutralizing activity in the LNP-mRNA Gn #1 and LNP-mRNA Gc #1 groups compared to the negative control group (Figures 4B and 4C).

[0122] (Test Example 3) Administration of various nucleic acid-lipid particles to IFNAR1 KO mice IFNAR1 KO mice (B6(Cg)-Ifnar1 ｔｍ１．２Ｅｅｓ / J) were obtained from Jackson Laboratory and conditioned. Equal amounts of LNP-mRNA Gn #2, LNP-mRNA Gc #2, and LNP-mRNA NP #2 prepared in Example 8 were mixed and administered twice, at an interval of two weeks, to the disinfected triceps surae muscles of anesthetized mice at a total of 3 or 30 μg mRNA / 20 μL / dose. The first administration was into the right leg, and the second into the left leg. Buffer was used to adjust the lipid particle concentration and for the negative control group.

[0123] One week after the second administration of lipid particles, blood was collected from anesthetized mice in a microtube containing a coagulant / serum separator (Becton Dickinson) and centrifuged at 10,000 RPM for 10 minutes to separate and collect serum.

[0124] Measurement of neutralizing activity Vero76 cells were suspended in 10% FCS DMEM (Nacalai Tesque) and diluted to 1 × 10 4 The next day, 30 μL of serum diluted in 2% FCS DMEM (Nacalai Tesque) in six serial dilutions (10-fold to 4-fold dilutions from the highest dilution) was mixed with 200 FFU / 30 μL of SFTSV YG1-based recombinant SFTSV (SFTSV RG WT) and incubated at 37°C in CO ２ The cells were incubated for 1 hour in the presence of 50 μL of serum and SFTSV mixture. 50 μL of the serum and SFTSV mixture was added to Vero76 cells and incubated for 48 hours. Then, the cells were fixed with 4% (w / v) paraformaldehyde, and 100 μL of ice-cold 100% methanol was added to each well and incubated at -30°C for 10 minutes to permeabilize the membrane. The cells were washed with PBS (Nissui Pharmaceutical Co., Ltd.) and blocked for 60 minutes with Blocking Buffer (5% (w / v) Goat Serum, 0.3% (w / v) Triton X / PBS). 30 μL / well of 5000-fold diluted Rabbit Anti-NP Antibody was added and incubated overnight at 4°C. 30 μL / well of 1:1000 diluted Alexa488-labeled Goat Anti-Rabbit Antibody and 5:5000 diluted DAPI (Roche) were added and incubated for 1 hour in the dark. After washing with PBS, the cells were observed under a fluorescence microscope, and green fluorescent-positive cells were counted as infected cells, and the percentage of uninfected cells among the total cells was calculated.

[0125] (Results of Test Example 3) Anti-SFTSV Neutralizing Activity in Mouse Blood The anti-SFTSV neutralizing activity in blood induced by a mixed formulation of LNP-mRNA Gn #2, LNP-mRNA Gc #2, and LNP-mRNA NP #2 was evaluated ( FIG. 5 ). As a result of examining serum collected one week after the second administration, neutralizing activity was observed in the 3 μg and 30 μg administration groups compared to the negative control group ( FIG. 5 ).

[0126] (Test Example 4) Administration of various nucleic acid-lipid particles to IFNAR1 KO mice IFNAR1 KO mice (B6(Cg)-Ifnar1 ｔｍ１．２Ｅｅｓ LNP-mRNA Gn #3, LNP-mRNA Gc #3, LNP-mRNA NP #3, or LNP-mRNA OVA #1 prepared in Example 8 was administered to the disinfected triceps surae muscles of anesthetized mice at 13-day intervals, a total of three times at 10 μg mRNA / 20 μL / mouse. The first administration was into the right leg, and the second into the left leg. Buffer was used to adjust the concentration of the lipid particles. LNP-mRNA OVA #1 was used as a negative control.

[0127] Five days after the second administration of lipid particles, blood was collected from anesthetized mice in micro-collection tubes containing a coagulant / serum separator (Becton Dickinson), and centrifuged at 10,000 RPM for 10 minutes to separate and collect serum.

[0128] Anti-Gn, anti-Gc, and anti-NP antibody titers in mouse blood. Recombinant Gn protein (Immune Technology), recombinant Gc protein (Immune Technology), and recombinant NP protein (Native Antigen) were used as solid-phase antigens and added to a 96-well flat-bottom plate and incubated overnight at 4°C. A standard curve dilution series was prepared by diluting mouse serum containing anti-Gn antibodies, anti-Gc antibodies, or anti-NP antibodies in eight 3-fold dilutions starting from a maximum concentration of 1 arbitrary unit / mL. Each well was washed, and blocking solution was added and incubated at room temperature for 1 hour. Serum samples were prepared in seven 4-fold dilutions starting from a maximum concentration of 120-fold using blocking solution. The blocked plate was washed, and the diluted samples were added to the plate and incubated at room temperature for 1 hour. The detection antibody, Goat Anti-Mouse IgG, Human adz-HRP (SouthernBiotech), was diluted 4000-fold with blocking solution and added to the washed wells. After 1 hour, the wells were washed, and TMB Microwell Peroxidase Substrate System (Seracare) was added and left to stand for 10 minutes. ２ SO ４ The reaction was stopped using HCl. The corrected absorbance, calculated by subtracting the absorbance at 540 nm from the absorbance at 450 nm, was used for analysis on a plate reader to calculate the anti-Gn antibody titer, anti-Gc antibody titer, and anti-NP antibody titer. The blocking solution was 1X Dulbecco's Phosphate Buffered Saline supplemented with 1% (w / v) Bovine Serum Albumin and 0.05% (v / v) Tween 20 (BIO-RAD), and washing was performed three times with 1X Dulbecco's Phosphate Buffered Saline supplemented with 0.05% (v / v) Tween 20.

[0129] Nine days after the second administration of lipid particles, mice were intradermally inoculated with SFTSV (YG1, 100 FFU / 50 μL) into the footpad of the right foot under isoflurane anesthesia. The mice were weighed once daily.

[0130] Measurement of virus titer in mouse blood. Four days after SFTSV infection, blood was collected from anesthetized mice. Blood was collected in 1.5 mL tubes (Greiner Bio-One) and centrifuged at 4000 RPM for 10 minutes to separate and collect serum. Ten-fold serially diluted serum was added to Vero 76 cells and cultured at 37°C for 18 hours. Cells were then fixed with 4% (w / v) paraformaldehyde, and 100 μL of ice-cold 100% methanol was added to each well and incubated at -30°C for 10 minutes to permeabilize the membrane. After washing with PBS, the plates were blocked with Blocking Buffer (5% (w / v) Goat Serum, 0.3% (w / v) Triton X / PBS) for 60 minutes. Then, 30 μL / well of 5000-fold diluted Rabbit Anti-NP Antibody (Urata S et al., 2018) was added and incubated overnight at 4°C. 30 μL / well of 1000-fold diluted Alexa 488-labeled Goat Anti-Rabbit Antibody (Thermo Fisher Scientific) and 5000-fold diluted DAPI (Roche) were added and incubated for 1 hour in the dark. After washing with PBS, the cells were observed under a fluorescence microscope, green fluorescent positive cells were counted as infected cells, and the virus titer was calculated.

[0131] (Results of Test Example 4) Mouse Blood Gn, Anti-Gc, and Anti-NP Antibody Titers The blood anti-Gn antibody response, blood anti-Gc antibody response, and blood anti-NP antibody response induced by LNP-mRNA Gn #3, LNP-mRNA Gc #3, or LNP-mRNA NP #3 were evaluated (FIG. 6). As a result, the LNP-mRNA Gn #3 group had a higher blood anti-Gn antibody titer than the LNP-mRNA OVA #1 (negative control) group, the LNP-mRNA Gc #3 group, and the LNP-mRNA NP #3 group (FIG. 6B). The LNP-mRNA Gc #3 group had a higher blood anti-Gc antibody titer than the negative control group, the LNP-mRNA Gn #3 group, and the LNP-mRNA NP #3 group (FIG. 6C). The LNP-mRNA NP #3 group had a higher blood anti-NP antibody titer than the negative control group, the LNP-mRNA Gn #3 group, and the LNP-mRNA Gc #3 group (FIG. 6D).

[0132] Mouse Body Weight and Survival Period after SFTSV Infection Mice were inoculated intradermally with SFTSV, and survival periods and body weights were assessed 14 days after inoculation (Figure 7). All mice in the LNP-mRNA OVA #1 (negative control) group died within 6 days of infection, whereas all mice in the LNP-mRNA Gn #3, LNP-mRNA Gc #3, and LNP-mRNA NP #3 groups survived throughout the study period (Figure 7A). Furthermore, compared with the LNP-mRNA OVA #1 (negative control) group, weight loss after SFTSV inoculation was suppressed in the LNP-mRNA Gn #3, LNP-mRNA Gc #3, and LNP-mRNA NP #3 groups (Figure 7B).

[0133] Viral titers in mouse blood were measured 4 days after SFTSV infection (Figure 8). Compared with the LNP-mRNA OVA #1 (negative control) group, the LNP-mRNA Gn #3 group, the LNP-mRNA Gc #3 group, and the LNP-mRNA NP #3 group had lower viral titers in the blood (Figure 8).

[0134] (Test Example 5) Administration of various nucleic acid-lipid particles to IFNAR1 KO mice IFNAR1 KO mice (B6(Cg)-Ifnar1 ｔｍ１．２Ｅｅｓ The LNP-mRNA Gn #4, LNP-mRNA Gc #4, or LNP-mRNA OVA #2 prepared in Example 14, or LNP-mRNA Gn1 #1, LNP-mRNA Gn2 #1, LNP-mRNA Gn3 #1, LNP-mRNA Gn4 #1, LNP-mRNA Gc1 #1, LNP-mRNA Gc2 #1, LNP-mRNA Gc3 #1, or LNP-mRNA-Gc4 #1, prepared in Example 14, was administered twice at a two-week interval to the disinfected triceps surae muscles of anesthetized mice at 5 μg mRNA / 20 μL / mouse. The first administration was into the right leg, and the second administration was into the left leg. Buffer was used to adjust the concentration of lipid particles. LNP-mRNA OVA #2 was used as a negative control.

[0135] Preparation of serum 14 days after the second administration of lipid particles, blood was collected from the anesthetized mice. The serum was treated and collected in the same manner as in Test Example 4.

[0136] Anti-Gn and anti-Gc antibody titers in mouse blood Recombinant Gn protein (Immune Technology) and recombinant Gc protein (Immune Technology) were used as immobilized antigens, and measurements and calculations were carried out in the same manner as in Test Example 4.

[0137] Measurement of Neutralizing Activity in Blood The serum used was treated at 56° C. for 30 minutes. Measurement was carried out in the same manner as in Test Example 3.

[0138] (Results of Test Example 5) Anti-Gn and anti-Gc antibody titers in mouse blood The anti-Gn antibody response in the blood induced by LNP-mRNA Gn #4, or LNP-mRNA Gn1 #1, LNP-mRNA Gn2 #1, LNP-mRNA Gn3 #1, and LNP-mRNA Gn4 #1, and the anti-Gc antibody response in the blood induced by LNP-mRNA Gc #4, or LNP-mRNA Gc1 #1, LNP-mRNA Gc2 #1, LNP-mRNA Gc3 #1, and LNP-mRNA-Gc4 #1 were evaluated (Figure 23). As a result, the LNP-mRNA Gn1 #1, LNP-mRNA Gn2 #1, and LNP-mRNA Gn4 #1 administration groups had higher blood anti-Gn antibody titers than the LNP-mRNA Gn #4 administration group (Figure 23A). The LNP-mRNA Gc1 #1 administration group produced blood anti-Gc antibody titers equivalent to those of the LNP-mRNA Gc #4 administration group (Figure 23B).

[0139] Anti-SFTSV neutralizing activity in mouse blood The anti-SFTSV neutralizing activity in blood induced by LNP-mRNA Gn #4, LNP-mRNA Gn1 #1, LNP-mRNA Gn2 #1, LNP-mRNA Gn3 #1, LNP-mRNA Gn4 #1, LNP-mRNA Gc #4, LNP-mRNA Gc1 #1, LNP-mRNA Gc2 #1, LNP-mRNA Gc3 #1, and LNP-mRNA-Gc4 #1 was evaluated (Figure 24). The LNP-mRNA Gc1 #1 administration group showed high neutralizing activity equivalent to that of the LNP-mRNA Gn #4 administration group (FIG. 24A), and the LNP-mRNA Gc1 #1 administration group showed high neutralizing activity equivalent to that of the LNP-mRNA Gc #4 administration group (FIG. 24B). Furthermore, the neutralizing activity of the LNP-mRNA Gn #4 administration group, LNP-mRNA Gn1 #1 administration group, LNP-mRNA Gc #4 administration group, and LNP-mRNA Gc1 #1 administration group was evaluated for each individual. As a result, high neutralizing activity was observed in all individuals (FIG. 25).

[0140] SEQ ID NO: 1: Amino acid sequence of Gn SEQ ID NO: 2: Amino acid sequence of Gc SEQ ID NO: 3: Amino acid sequence of NP (amino acid numbers 1 to 18 are IgE signal peptide) SEQ ID NO: 4: Amino acid sequence of Ova SEQ ID NO: 5: Nucleotide sequence of a DNA fragment comprising a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a codon-optimized Gn sequence, and a human β-globin 3'-UTR sequence are linked in this order SEQ ID NO: 6: Nucleotide sequence of a sense primer SEQ ID NO: 7: Nucleotide sequence of an antisense primer SEQ ID NO: 8: Nucleotide sequence of a template DNA for in vitro transcription (IVT) of codon-optimized Gn SEQ ID NO: 9: mRNA sequence of codon-optimized Gn SEQ ID NO: 10: T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a codon-optimized Gc sequence, and a human β-globin 3'-UTR sequence are linked in this order SEQ ID NO: 11: Nucleotide sequence of template DNA for IVT of codon-optimized Gc SEQ ID NO: 12: mRNA sequence of codon-optimized Gc SEQ ID NO: 13: Nucleotide sequence of DNA fragment comprising a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a codon-optimized NP sequence, and a human β-globin 3'-UTR sequence are linked in order SEQ ID NO: 14: Nucleotide sequence of template DNA for IVT of codon-optimized NP SEQ ID NO: 15: mRNA sequence of codon-optimized NP SEQ ID NO: 16: Nucleotide sequence of DNA fragment comprising a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a Gn sequence, and a human β-globin 3'-UTR sequence are linked in order SEQ ID NO: 17: Nucleotide sequence of template DNA for IVT of Gn SEQ ID NO: 18: Gn mRNA sequence SEQ ID NO: 19: Nucleotide sequence of a DNA fragment comprising a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a Gc sequence, and a human β-globin 3'-UTR sequence are linked in this order SEQ ID NO: 20: Nucleotide sequence of template DNA for IVT of Gc SEQ ID NO: 21: Gc mRNA sequenceSEQ ID NO: 22: Nucleotide sequence of a DNA fragment comprising a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, an NP sequence, and a human β-globin 3'-UTR sequence are linked in this order SEQ ID NO: 23: Nucleotide sequence of template DNA for NP IVT SEQ ID NO: 24: NP mRNA sequence SEQ ID NO: 25: Nucleotide sequence of a DNA fragment comprising a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, an Ova sequence, and a human β-globin 3'-UTR sequence are linked in this order SEQ ID NO: 26: Nucleotide sequence of template DNA for Ova IVT SEQ ID NO: 27: Ova mRNA sequence SEQ ID NO: 28: Amino acid sequence of Gn1 SEQ ID NO: 29: Amino acid sequence of Gn2 SEQ ID NO: 30: Amino acid sequence of Gn3 SEQ ID NO: 31: Amino acid sequence of Gn4 SEQ ID NO: 32: Amino acid sequence of Gc1 SEQ ID NO: 33: Amino acid sequence of Gc2 SEQ ID NO: 34: Amino acid sequence of Gc3 SEQ ID NO: 35: Amino acid sequence of Gc4 SEQ ID NO: 36: Nucleotide sequence of a DNA fragment comprising a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a Gn1 sequence, and a human β-globin 3'-UTR sequence are linked in this order SEQ ID NO: 37: Nucleotide sequence of a DNA fragment comprising a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a Gn2 sequence, and a human β-globin 3'-UTR sequence are linked in this order SEQ ID NO: 38: T7 promoter sequence, human SEQ ID NO: 39: Nucleotide sequence of a DNA fragment comprising a sequence in which a β-globin 5'-UTR sequence, a KOZAK sequence, a Gn3 sequence, and a human β-globin 3'-UTR sequence are linked in this order SEQ ID NO: 40: Nucleotide sequence of template DNA for Gn1 IVT SEQ ID NO: 41: Nucleotide sequence of template DNA for Gn2 IVT SEQ ID NO: 42: Nucleotide sequence of template DNA for Gn3 IVTSEQ ID NO: 43: Nucleotide sequence of template DNA for IVT of Gn4 SEQ ID NO: 44: Gn1 mRNA sequence SEQ ID NO: 45: Gn2 mRNA sequence SEQ ID NO: 46: Gn3 mRNA sequence SEQ ID NO: 47: Gn4 mRNA sequence SEQ ID NO: 48: Nucleotide sequence of a DNA fragment comprising a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a Gc1 sequence, and a human β-globin 3'-UTR sequence are linked in this order SEQ ID NO: 49: Nucleotide sequence of a DNA fragment comprising a sequence in which a T7 promoter sequence, a human β-globin 5'-UTR sequence, a KOZAK sequence, a Gc2 sequence, and a human β-globin 3'-UTR sequence are linked in this order SEQ ID NO: 50: T7 promoter sequence, human SEQ ID NO:51: Nucleotide sequence of a DNA fragment comprising a sequence in which a β-globin 5'-UTR sequence, a KOZAK sequence, a Gc3 sequence, and a human β-globin 3'-UTR sequence are linked in this order SEQ ID NO:52: Nucleotide sequence of template DNA for IVT of Gc1 SEQ ID NO:53: Nucleotide sequence of template DNA for IVT of Gc2 SEQ ID NO:54: Nucleotide sequence of template DNA for IVT of Gc3 SEQ ID NO:55: Nucleotide sequence of template DNA for IVT of Gc4 SEQ ID NO:56: Gc1 mRNA sequence SEQ ID NO:57: Gc2 mRNA sequence SEQ ID NO:58: Gc3 mRNA sequence SEQ ID NO: 59: Gc4 mRNA sequence

[0141] Nucleic acid-lipid particles encapsulating SFTSV mRNA can be used for the prevention and treatment of SFTSV infection. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A lipid particle encapsulating a nucleic acid capable of expressing at least one antigen selected from the group consisting of Gn antigen, Gc antigen, and NP antigen of SFTSV, wherein the lipid particle comprises an amphipathic lipid, sterols, a PEG lipid, and a cationic lipid.

2. The particle according to claim 1, wherein the nucleic acid is capable of expressing two or more antigens selected from the group consisting of Gn antigen, Gc antigen and NP antigen of SFTSV.

3. The particle according to claim 1 or 2, wherein the cationic lipid is one or more selected from the following group of cationic lipids: MC3: (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, ALC-0315: [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoic acid ester), and SM-102: heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoic acid ester 4. Particles according to any one of claims 1 to 3, wherein the amphiphilic lipid is at least one selected from the group consisting of distearoylphosphatidylcholine, dioleoylphosphatidylcholine and dioleoylphosphatidylethanolamine.

5. Particles according to any one of claims 1 to 4, wherein the sterol is cholesterol.

6. Particles according to any one of claims 1 to 5, wherein the PEG lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol and / or N-[methoxy poly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine.

7. Particles according to any one of claims 1 to 6, wherein the lipid composition of amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 5 to 25% amphipathic lipid, 10 to 55% sterols, 40 to 65% cationic lipid, and 1 to 5% PEG lipid.

8. The particles according to claim 7, wherein the ratio of the total lipid weight to the nucleic acid weight is 15 to 30.

9. Particles described in any one of claims 1 to 8, wherein the Gn antigen of SFTSV is a protein comprising an amino acid sequence having at least 95% identity with the amino acid sequence of the region consisting of amino acid numbers 20 to 175 of SEQ ID NO:

1.

10. The particle described in claim 9, wherein the Gn antigen is a protein comprising an amino acid sequence having at least 95% identity with the amino acid sequence consisting of amino acid numbers 20 to 452 of SEQ ID NO: 1 or amino acid numbers 20 to 340 of SEQ ID NO:

28.

11. Particles described in any one of claims 1 to 8, wherein the Gc antigen of SFTSV is a protein comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of a region including amino acid numbers 28 to 96 and / or amino acid numbers 190 to 241 of SEQ ID NO:

2.

12. The particle described in claim 11, wherein the Gc antigen is composed of a protein comprising an amino acid sequence having at least 95% identity with the amino acid sequence of amino acids 28 to 503 of SEQ ID NO: 2, amino acids 28 to 241 of SEQ ID NO: 2, or amino acids 28 to 369 of SEQ ID NO:

32.

13. The particle according to any one of claims 1 to 12, wherein the SFTSV NP antigen consists of a protein comprising an amino acid sequence having at least 95% identity with the amino acid sequence of amino acids 19 to 262 of SEQ ID NO:

3.

14. The particle according to any one of claims 1 to 13, characterized in that the antigen protein has a signal sequence at the N-terminus.

15. A particle described in any one of claims 1 to 14, wherein the nucleic acid capable of expressing the Gn antigen, Gc antigen, or NP antigen of SFTSV is an mRNA comprising a cap structure (Cap), a 5' untranslated region (5'-UTR), a translation region of the Gn antigen, Gc antigen, or NP antigen of SFTSV, and a 3' untranslated region (3'-UTR).

16. The particle described in claim 15, characterized in that the sequence of the nucleic acid capable of expressing the Gn antigen of SFTSV comprises a base sequence having at least 90% identity with the base sequence set forth in base numbers 71 to 1429 of SEQ ID NO: 9, base numbers 71 to 1429 of SEQ ID NO: 18, or base numbers 71 to 1093 of SEQ ID NO:

44.

17. Particles according to claim 15, characterized in that the sequence of the nucleic acid capable of expressing the Gc antigen of SFTSV comprises a base sequence having at least 90% identity with the sequence of base numbers 71 to 1582 of SEQ ID NO: 12, base numbers 71 to 1582 of SEQ ID NO: 21, or base numbers 71 to 1180 of SEQ ID NO:

56.

18. Particles described in claim 15, characterized in that the sequence of the nucleic acid capable of expressing the NP antigen of SFTSV comprises a base sequence having at least 90% identity with the sequence of bases 71 to 859 of SEQ ID NO: 15 or bases 71 to 859 of SEQ ID NO:

24.

19. A particle according to any one of claims 1 to 18, wherein the nucleic acid comprises at least one modified nucleotide.

20. The particle of claim 19, wherein the modified nucleotide comprises at least one selected from the group consisting of 5-methylcytidine, 5-methyluridine, pseudouridine, and 1-methylpseudouridine.

21. Particles according to any one of claims 1 to 20, having an average particle size of 30 to 300 nm.

22. Use of particles according to any one of claims 1 to 21 for producing a composition for preventing and / or treating infection or disease caused by SFTSV.

23. A pharmaceutical composition comprising particles according to any one of claims 1 to 21.

24. A pharmaceutical composition according to claim 23 for preventing and / or treating SFTSV infection.

25. The pharmaceutical composition according to claim 23, for preventing and / or treating severe fever with thrombocytopenia syndrome (SFTS) in a subject suspected of being infected with SFTSV.

26. A method for preventing and / or treating SFTSV infection, comprising administering to a mammal a pharmaceutical composition according to any one of claims 23 to 25.

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