Nucleic acid-lipid particle vaccine

Abstract

Provided is a vaccine for preventing and / or treating infection caused by the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2).　The lipid particles encapsulate a nucleic acid capable of expressing the S protein of the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2) and / or a fragment thereof. The lipid contains a cationic lipid represented by general formula (Ia) or a pharmaceutically acceptable salt thereof. In the formula, R1 and R2 independently indicate a C1–C3 alkyl group; L1 indicates a C17–C19 alkenyl group that may contain one or a plurality of C2–C4 alkanoyloxy groups; L2 indicates a C10–C19 alkyl group that may contain one or a plurality of C2–C4 alkanoyloxy groups or a C10–C19 alkenyl group that may contain one or a plurality of C2–C4 alkanoyloxy groups; and p is 3 or 4.

Description

[Technical field]

[0001] The present invention relates to a nucleic acid-lipid particle vaccine encapsulating SARS-CoV-2 mRNA. [Background technology]

[0002] COVID-19 (coronavirus disease 2019: COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes symptoms mainly due to acute inflammation in the respiratory tract. In particular, high-risk individuals are burdened with symptoms mainly due to inflammation in the lower respiratory tract, such as invasive pneumonia and acute respiratory distress syndrome (Non-Patent Document 1). There are more than six known types of coronavirus (CoV) that infect humans and mainly cause respiratory symptoms. SARS-CoV-2 is classified as a betacoronavirus and is virologically similar to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), which have caused outbreaks in the past.

[0003] The spike protein (S) expressed on the surface of SARS-CoV-2 virus particles plays an important role in the initial infection mechanism. S is a type I membrane protein consisting of two subunits, S1 and S2, which form a trimer (approximately 500 kDa, approximately 20 nm). The receptor-binding domain (RBD) present in S1 interacts with angiotensin-converting enzyme 2 (ACE2) expressed on the host cell surface. Compared to SARS-CoV S, SARS-CoV-2 S has been suggested to have 10-20 times higher affinity for ACE2 and higher thermodynamic stability, suggesting that it is involved in the high transmissibility of SARS-CoV-2 (Non-Patent Documents 2, 3).

[0004] In the convalescent serum of SARS patients, IgG against RBD persists for at least 3 years, and after the serum is treated with RBD protein to adsorb anti-RBD antibodies, the neutralizing activity is reduced to 50% or less, suggesting that anti-RBD antibodies are responsible for the main neutralizing activity (Non-Patent Documents 4, 5). In fact, it has been reported that isolated anti-SARS-CoV-2 RBD monoclonal antibodies have neutralizing activity against SARS-CoV-2 (Non-Patent Documents 6, 7).

[0005] Analysis of peripheral blood from COVID-19 patients in the convalescent phase about three weeks after becoming asymptomatic suggests that induction of specific CD4+ and CD8+ T cells is important for protection against SARS-CoV-2 infection (Non-Patent Document 8). Specifically, analysis of blood samples from 10-20 COVID-19 patients revealed that anti-SARS-CoV-2 RBD antibody responses and SARS-CoV-2-specific CD4+ T cell responses were confirmed in all cases, and SARS-CoV-2-specific CD8+ T cell responses were confirmed in approximately 70% of cases. In addition, the correlation between anti-SARS-CoV-2 RBD IgG titers in the blood and the frequency of S-specific CD4+ T cells (R = 0.8109) suggests that T cell epitopes exist in S, and that S-specific CD4+ T cells may play an important role in inducing antibody responses (Non-Patent Document 8). Furthermore, it has been shown that blood anti-SARS-CoV-2 neutralizing activity correlates with blood anti-SARS-CoV-2 S IgG titer (R = 0.9279) (Non-Patent Document 9).

[0006] It is assumed that cellular immunopathology and antibody-dependent enhancement (ADE) may be involved in the mechanism of "immune enhancement" that aggravates COVID-19 symptoms (Non-Patent Document 10). In the case of SARS, it has been suggested that fatal patients have a T helper (Th) type-2 dominant blood cytokine profile compared to patients who recovered with mild symptoms (Non-Patent Document 11). In a mouse SARS-CoV infection model, it has been suggested that a Th2-dominant immune response to S induces pulmonary immunopathology accompanied by an eosinophil-based inflammatory response (Non-Patent Document 12). On the other hand, ADE has been reported for other viruses such as dengue virus and respiratory syncytial virus, but there have been no reports of specific antibodies against SARS-CoV inducing ADE in SARS patients. Regarding vaccine antigen candidates for SARS-CoV, data has been reported suggesting that even antigens consisting of only the RBD rather than the full-length S may be able to avoid the risk of lung damage (Non-Patent Document 13). Regarding SARS-CoV-2, there is no direct clinical evidence that antibodies against S are involved in ADE, but it has been pointed out that in order to avoid the risk, it is necessary to induce an appropriate cellular immune response (Non-Patent Document 14). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Viruses 12:372 2020 [Non-Patent Document 2] Science 367:1260 2020 [Non-Patent Document 3] Viruses 12:428 2020 [Non-Patent Document 4] Virol J 7:299 2010 [Non-Patent Document 5] Virology 334:74 2005 [Non-Patent Document 6] Nat Commun 11:2251 2020 [Non-Patent Document 7] Nature 583:290 2020 [Non-Patent Document 8] Cell 181:1 2020 [Non-Patent Document 9] Nat Med 26:1033 2020 [Non-Patent Document 10] Nat Rev Immunol 20:347 2020 [Non-Patent Document 11] J Immunol 181:5490 2008 [Non-Patent Document 12] PLoS One 7:e35421 2012 [Non-Patent Document 13] Vaccine 25:2832 2007 [Non-Patent Document 14] PNAS 117:8218 2020 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention aims to provide a vaccine for preventing and / or treating infection with the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2). [Means for solving the problem]

[0009] The inventors administered lipid particles encapsulating mRNA encoding the RBD of SARS-CoV-2 to mice, observed the induction of SARS-CoV-2 S protein IgG in the blood, and discovered that the immune response was Th1-dominant, thus completing the present invention.

[0010] The gist of the present invention is as follows. (1) A lipid particle encapsulating a nucleic acid capable of expressing the S protein of the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2) and / or a fragment thereof, wherein the lipid comprises a cationic lipid represented by general formula (Ia) or a pharma- ceutical acceptable salt thereof. [ka] During the ceremony, R 1 and R 2 each independently represents a C1-C3 alkyl group; L 1 may have one or more C2-C4 alkanoyloxy groups; 17 -C 19 represents an alkenyl group; L 2 may have one or more C2-C4 alkanoyloxy groups; 10 -C 19 C which may have one or more alkyl groups or C2-C4 alkanoyloxy groups 10 -C 19 represents an alkenyl group; p is 3 or 4. (2) R in general formula (Ia) 1 and R 2 and are both methyl groups. (3) The particle according to (1) or (2), wherein p in general formula (Ia) is 3. (4) L in general formula (Ia) 1 C may have one or more acetoxy groups 17 -C 19 The particle according to any one of (1) to (3), wherein the group is an alkenyl group. (5) L in general formula (Ia) 2 C may have one or more acetoxy groups 10 -C 12 C which may have one or more alkyl groups or acetoxy groups 10 -C 19The particle according to any one of (1) to (4), wherein the group is an alkenyl group. (6) L in general formula (Ia) 2 C may have one or more acetoxy groups 10 -C 12 C which may have one or more alkyl groups or acetoxy groups 17 -C 19 The particle according to any one of (1) to (4), wherein the group is an alkenyl group. (7) L in general formula (Ia) 1 The particle according to any one of (1) to (6), wherein is a (R)-11-acetyloxy-cis-8-heptadecenyl group, a cis-8-heptadecenyl group, or a (8Z,11Z)-heptadecadienyl group. (8) L in general formula (Ia) 2 is a decyl group, a cis-7-decenyl group, a dodecyl group, or a (R)-11-acetyloxy-cis-8-heptadecenyl group. (9) The cationic lipid has the following structural formula: [ka] The particle according to (1), (10) The cationic lipid has the following structural formula: [ka] The particle according to (1), (11) The cationic lipid has the following structural formula: [ka] The particle according to (1), (12) The particle according to any one of (1) to (11), wherein the lipid further comprises an amphipathic lipid, a sterol, and a PEG lipid. (13) The particles according to (12), wherein the amphiphilic lipid is at least one selected from the group consisting of distearoylphosphatidylcholine, dioleoylphosphatidylcholine, and dioleoylphosphatidylethanolamine. (14) The particles according to (12) or (13), wherein the sterol is cholesterol. (15) The particles according to any one of (12) to (14), 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. (16) The particles according to any one of (12) to (15), wherein the lipid composition of amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 15% or less of amphipathic lipid, 20 to 55% of sterols, 40 to 65% of cationic lipid, and 1 to 5% of PEG lipid, and the ratio of the total lipid weight to the nucleic acid weight is 15 to 30. (17) The particles according to (16), wherein 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, and the ratio of the total lipid weight to the nucleic acid weight is 15 to 25. (18) The particles according to (17), wherein the lipid composition of amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 10 to 15% amphipathic lipid, 35 to 45% sterols, 40 to 50% cationic lipid, and 1 to 2% PEG lipid, and the ratio of the total lipid weight to the nucleic acid weight is 17.5 to 22.5. (19) The particles according to (18), wherein the lipid composition of amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 10 to 15% amphipathic lipid, 35 to 45% sterols, 45 to 50% cationic lipid, and 1.5 to 2% PEG lipid, and the ratio of the total lipid weight to the nucleic acid weight is 17.5 to 22.5. (20) A particle described in any one of (1) to (19), wherein the fragment of the S protein of the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2) includes a receptor binding domain. (21) A particle described in (20), wherein the receptor binding domain in a fragment of the S protein of the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2) consists of an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 11. (22) The particle described in (20), wherein the receptor binding domain in a fragment of the S protein of the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2) consists of an amino acid sequence having at least 95% identity to any of the amino acid sequences of SEQ ID NOs: 25, 29, 33, 37, and 94 to 107. (23) The particle described in (20), wherein the fragment of the S protein of the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2) consists of an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 10. (24) The particle described in (20), wherein the fragment of the S protein of the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2) consists of an amino acid sequence having at least 95% identity to any one of the amino acid sequences of SEQ ID NOs: 24, 28, 32, 36, and 80 to 93. (25) The particles described in (1) to (19), wherein the S protein of the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2) has an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 6. (26) A particle described in (25), wherein the receptor binding domain in the S protein of the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2) consists of an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 11. (27) A particle described in (25) or (26), wherein the nucleic acid capable of expressing the S protein of the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2) is an mRNA including a cap structure (Cap), a 5' untranslated region (5'-UTR), a translation region of the S protein, a 3' untranslated region (3'-UTR), and a polyA tail (polyA). (28) A particle described in any of (20) to (24), wherein the nucleic acid capable of expressing a fragment of the S protein of the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2) is an mRNA including a cap structure (Cap), a 5' untranslated region (5'-UTR), a leader sequence, a translation region of the receptor binding domain in the S protein, a 3' untranslated region (3'-UTR), and a polyA tail (polyA). (29) The particle described in (27), wherein the sequence of the translation region of the S protein consists of a nucleotide sequence having at least 90% identity with the sequence of the translation region of the S protein in the sequence of SEQ ID NO:5. (30) The particle described in (27), wherein the sequence of the translation region of the S protein consists of a nucleotide sequence having at least 90% identity with the sequence of the translation region of the S protein in the sequence of SEQ ID NO:16. (31) The particle according to (27), wherein the nucleic acid capable of expressing the S protein of the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2) has the nucleotide sequence of SEQ ID NO: 5. (32) The particle according to (27), wherein the nucleic acid capable of expressing the S protein of the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2) has the nucleotide sequence of SEQ ID NO: 16. (33) A particle described in (27), wherein the sequence of the translation region of the receptor binding domain in the S protein consists of a nucleotide sequence having at least 90% identity to the sequence of the translation region of the receptor binding domain in the S protein in the sequence of SEQ ID NO: 9. (34) A particle described in (27), wherein the sequence of the translation region of the receptor binding domain in the S protein consists of a nucleotide sequence having at least 90% identity to the sequence of the translation region of the receptor binding domain in the S protein in the sequence of SEQ ID NO: 19. (35) A particle described in (27), wherein the sequence of the translation region of the receptor binding domain in the S protein consists of a nucleotide sequence having at least 90% identity to the sequence of the translation region of the receptor binding domain in the S protein in any one of the sequences of SEQ ID NOs: 21, 23, 27, 31, 35, 66 to 79. (36) The particle described in (28), wherein the nucleic acid capable of expressing a fragment of the S protein of the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2) has the nucleotide sequence of SEQ ID NO: 9. (37) The particle described in (28), wherein the nucleic acid capable of expressing a fragment of the S protein of the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2) has the nucleotide sequence of SEQ ID NO: 19. (38) The particle according to any one of (1) to (37), wherein the nucleic acid contains at least one modified nucleotide. (39) The particle according to (38), wherein the modified nucleotide comprises at least one pyrimidine nucleotide substituted at the 5-position and / or a pseudouridine nucleotide optionally substituted at the 1-position. (40) The particle according to (38), wherein the modified nucleotide comprises at least one selected from the group consisting of 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, and 1-alkylpseudouridine. (41) The particles according to any one of (1) to (40), having an average particle size of 30 nm to 300 nm. (42) Use of the particles according to any one of (1) to (41) for producing a composition for preventing and / or treating infection with a novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2). (43) A composition comprising the particles according to any one of (1) to (41). (44) The composition according to (43) for expressing the S protein of the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2) and / or a fragment thereof in vivo or in vitro. (45) The composition according to (43) or (44) for use as a medicine. (46) The composition according to (45) for inducing an immune response against the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2). (47) The composition according to (45) or (46) for preventing and / or treating infection with a novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2). (48) A method for expressing the S protein of novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2) and / or a fragment thereof in vitro, comprising introducing the composition according to (43) or (44) into a cell. (49) A method for expressing the S protein of novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2) and / or a fragment thereof in vivo, comprising administering to a mammal the composition according to any one of (43) to (47). (50) A method for inducing an immune response against a novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2), comprising administering to a mammal the composition according to (45) or (46). (51) A method for preventing and / or treating infection with a novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2), comprising administering to a mammal the composition according to any one of (45) to (47). (52) The method according to any one of (49) to (51), wherein the mammal is a human. Effect of the Invention

[0011] The present invention makes it possible to prevent and / or treat infection with SARS-CoV-2. This specification includes the contents described in the specifications and / or drawings of Japanese patent applications, Patent Application No. 2020-101420 and Patent Application No. 2021-33278, which are the basis of the priority of this application. [Brief description of the drawings]

[0012] [Figure 1] RBD protein expression levels by the particles of Example 3 and Example 4. Buffer: 10 mM histidine buffer (pH 6.5) containing 300 mM sucrose. [Diagram 2] Anti-RBD antibody response induced by the particles of Example 3 and Example 4. Buffer: 10 mM histidine buffer (pH 6.5) containing 300 mM sucrose. The vertical bars indicate the geometric mean values, and the symbols 1 to 5 indicate the individual antibody levels. [Diagram 3] RBD-hACE2 binding inhibitory activity of the sera from Example 3 and Example 4. Buffer: 10 mM histidine buffer (pH 6.5) containing 300 mM sucrose. The horizontal bars indicate the geometric mean values, and the circle symbols indicate the individual inhibitory activity levels. [Figure 4]RBD-specific cellular immunity induced by the groups of Example 3 and Example 4. Buffer: 10 mM histidine buffer (pH 6.5) containing 300 mM sucrose. Vertical bars indicate the average value, and error bars indicate the standard error. In all treatment groups, the DMSO concentration was adjusted to 0.1% (v / v). No peptides: group without peptide. [Diagram 5] Anti-RBD antibody response in blood induced by administration of the particles of Example 4, Example 7, or Example 8. Buffer; 10 mM histidine buffer (pH 7.0) containing 300 mM sucrose. The vertical bars indicate the geometric mean values, and the symbols 1 to 4 indicate the individual antibody levels. [Figure 6] Anti-RBD antibody response in blood induced by administration of the particles of Example 8 or Example 10. Buffer; 10 mM histidine buffer (pH 7.0) containing 300 mM sucrose. The vertical bars indicate the geometric mean values, and the symbols 1 to 5 indicate the individual antibody levels. [Figure 7] Anti-SARS-CoV-2 neutralizing activity in blood induced by administration of the particles of Example 10. Buffer; 10 mM histidine buffer (pH 7.0) containing 300 mM sucrose. The vertical bars indicate the geometric mean values, and the symbols 1 to 5 indicate the individual neutralizing activities. [Figure 8] Anti-SARS-CoV-2 neutralizing activity in blood induced by administration of the particles of Example 8 and Example 10. Buffer; 10 mM histidine buffer (pH 7.0) containing 300 mM sucrose. The vertical bars indicate the geometric mean values, and the symbols 1 to 5 indicate the individual neutralizing activities. [Figure 9] Anti-RBD antibody response in blood induced by administration of the particles of Example 8 or Example 10. Buffer; 10 mM histidine buffer (pH 7.0) containing 300 mM sucrose. The vertical bars indicate the geometric mean value, and the error bars indicate the standard deviation. [Figure 10]RBD-specific cellular immunity induced by Example 10. Buffer: 10 mM histidine buffer (pH 7.0) containing 300 mM sucrose. Vertical bars indicate average values, and symbols 1 to 7 indicate individual cytokine induction levels. In all treatment groups, the DMSO concentration was adjusted to 0.1% (v / v). No peptides: group without peptide. [Figure 11] Strain-specific immunogenicity of mRNA vaccine against SARS-CoV-2 RBD. (ae, g, and h) Six-week-old C57BL / 6 and BALB / c mice were intramuscularly administered mock or LNP-mRNA-RBD (3 μg mRNA) twice at 2-week intervals. (a) Two weeks after the second administration, blood anti-RBD antibody titers were measured by ELISA. (be) Lymphocytes were prepared from mouse popliteal lymph nodes and analyzed by flow cytometry. (bd) Germinal center (GC) B cells were gated as GL7+CD38- CD19+ cells. (e) THF cells were gated as CD185+ PD-1+CD3ε+ CD4+ T cells. (f) Overlapping peptides of SARS-CoV-2 spike protein. 16 overlapping peptides were divided into 8 pool peptides, with 16 overlapping peptides as 1 pool peptide. (g and h) Spleen cells were prepared from mouse spleens and re-treated with pooled peptides for 24 h. IFN-γ levels in culture supernatants were measured by ELISA. (gh) Pie charts show the percentage of cytokine-producing CD8+ and CD4+ T cells after 6 h treatment of pools 2, 3, and 4 with protein transport inhibitors. 3+: IFN-γ+IL-2+TNF-α+, 2+: IFN-γ+IL-2+, IFN-γ+TNF-α+, IL-2+TNF-α+, 1+: IFN-γ+, IL-2+, TNF-α+. N = 4–5. Vertical bars indicate mean values, error bars indicate standard error. *P < 0.05, using Mann-Whitney test. [Figure 12]Immunogenicity of LNP-mRNA-RBD (mRNA-RBD(HPLC)) containing HPLC-purified mRNA. (a) Human peripheral blood mononuclear cells (PBMCs) from SARS-CoV-2 non-infected individuals were treated with LNP-mRNA-Full (0.4, 2, and 10 μg / mL in terms of mRNA), LNP-mRNA-RBD (0.4, 2, and 10 μg / mL in terms of mRNA), or mRNA-RBD(HPLC) (0.4, 2, and 10 μg / mL in terms of mRNA) for 24 hours, and IFN-α levels in the culture supernatant were measured by ELISA. (b) Bone marrow-derived dendritic cells (BM-DCs) from C57BL / 6 and BALB / c mice were treated with LNP-mRNA-Full (0.4, 2, and 10 μg / mL in terms of mRNA), LNP-mRNA-RBD (0.4, 2, and 10 μg / mL in terms of mRNA), or mRNA-RBD (HPLC) (0.4, 2, and 10 μg / mL in terms of mRNA) for 24 hours, and IFN-α levels in the culture supernatant were measured by ELISA. (ci) C57BL / 6 mice were intramuscularly administered mock, LNP-mRNA-RBD (3 μg mRNA), or mRNA-RBD (HPLC) (3 μg mRNA) on days 0 and 14. (c) Two weeks after the second administration, blood anti-RBD antibody titers were measured by ELISA. (d and e) Popliteal lymph nodes were harvested from the mice. (d) GC B cells were gated as GL7+CD38- CD19+ cells. (e) TFH cells were gated as CD185+ PD-1+CD3ε+CD4+ T cells. (f and g) Spleen cells were prepared from mouse spleens and treated with pooled peptides for 24 h. IFN-γ levels in culture supernatants were measured by ELISA. The percentages of cytokine-producing CD8+ and CD4+ T cells after 6 h treatment with peptide pools 3 and 4 together with protein transport inhibitors are shown in pie charts. 3+: IFN-γ+IL-2+TNF-α+, 2+: IFN-γ+IL-2+, IFN-γ+TNF-α+, and IL-2+TNF-α+, 1+: IFN-γ+, IL-2+, and TNF-α+. (h, i) Representative data from Fig. 12f, g, Fig. 21, 22 are shown.IFN-γ+IL-2+TNF-α+ and IFN-γ+TNF-α+CD8+ T cells are shown in scatter plots. N=4–5. Vertical bars indicate the mean and error bars indicate the standard error. *P<0.05, using ANOVA followed by Dunn's multiple comparison test. [Figure 13] Anti-RBD blood antibody responses in cynomolgus macaques administered LNP-mRNA-RBD encapsulated with HPLC-purified mRNA. (a) Schedule of LNP-mRNA-RBD administration, infection, and sample collection. (bc) Cynomolgus macaques were immunized intramuscularly with mock or LNP-mRNA-RBD (HPLC) (100 μg) on ​​days 0 and 21. (b) Anti-RBD blood antibody titers were measured by ELISA on days 0, 7, 14, 21, 28, and 7 days after infection. (c) Neutralizing antibodies were measured by neutralization assay. (d) Anti-RBD blood IgG titers were measured by ELISA in swab samples (conjunctiva, oral cavity, nasal cavity, trachea, and rectum). Black arrows indicate the days of vaccine administration, and red arrows indicate the days of SARS-CoV-2 infection. [Figure 14] Protective responses against SARS-CoV-2 infection in cynomolgus monkeys administered mRNA-RBD (HPLC). One week after the second dose, SARS-CoV-2 (2×107 PFU) was administered to the conjunctiva, nasal cavity, oral cavity, and trachea of ​​cynomolgus monkeys. (a) Viral RNA and (b) viral titers in swab samples were measured by RT-PCR and cell culture. (cd) Viral RNA in lung tissue was measured by RT-PCR. RU: right upper lobe, RM: right middle lobe, RL: right lower lobe, LU: left upper lobe, LM: left middle lobe, LL: left lower lobe. [Figure 15] Anti-spike protein ectodomain (ECD) antibody response in blood in mice administered LNP-mRNA-RBD. Mock or LNP-mRNA-RBD (3 μg mRNA) was administered intramuscularly to C57BL / 6 and BALB / c mice on days 0 and 14. Two weeks after the second administration, anti-ECD antibody titers in blood were measured by ELISA. N = 4-5. Horizontal bars indicate the mean value, and symbols indicate the data for each individual. *P < 0.05, using the Mann-Whitney test. [Figure 16]Gating for GC B and THF cells. Lymphocytes were prepared from mouse popliteal lymph nodes and immunostained for GC B and THF cells, followed by flow cytometry analysis. Cells were gated for lymphocyte size, singlets, live, T or B cells, and THF or GC B cells. [Figure 17] RBD-specific T cell responses. Spleen cells were prepared from mouse spleens and treated with spike protein peptide pool, ECD protein, or RBD protein for 24 hours. IFN-γ and IL-13 levels in the culture supernatant were measured by ELISA. N = 4–5 mice. Vertical bars indicate the mean and error bars indicate the standard error. *P < 0.05, using ANOVA and Sidak's multiple comparison test. [Figure 18] RBD-specific CD8+ T cell responses. Spleen cells were prepared from mouse spleens and treated with protein transport inhibitors and pooled peptides for 6 h. The percentage of cytokine-producing CD8+ T cells was analyzed by flow cytometry. N = 4–5. Vertical bars indicate the mean and error bars indicate the standard error. *P < 0.05, using the Mann-Whitney test. [Figure 19] RBD-specific CD4+ T cell responses. Spleen cells were prepared from mouse spleens and treated with protein transport inhibitors and pooled peptides for 6 h. The percentage of cytokine-producing CD4+ T cells was analyzed by flow cytometry. N = 4–5. Vertical bars indicate the mean and error bars indicate the standard error. *P < 0.05, using the Mann-Whitney test. [Figure 20]Spike protein-specific immune responses in mice treated with mRNA-RBD (HPLC). (a) Mock, mRNA-RBD, or mRNA-RBD (HPLC) (3 μg mRNA) was administered intramuscularly to C57BL / 6 and BALB / c mice on days 0 and 14. Two weeks after the second administration, anti-ECD antibody titers in the blood were measured by ELISA. (b) Spleen cells were prepared from mouse spleens and treated with spike protein, ECD, or RBD pool peptide for 24 hours. N = 4. Vertical bars indicate mean values, error bars indicate standard error. *P < 0.05, using ANOVA and Dunn or Sidak's multiple comparison test. [Figure 21] RBD-specific T cell responses in C57BL / 6 mice treated with mRNA-RBD (HPLC). Spleen cells were prepared from mouse spleens and treated with protein transport inhibitors and pooled peptides for 6 hours. The percentages of cytokine-producing CD8+ and CD4+ T cells were analyzed by flow cytometry. N = 4. Vertical bars indicate mean values, error bars indicate standard error. *P < 0.05, using ANOVA and Dunn's multiple comparison test. [Figure 22] RBD-specific T cell responses in BALB / c mice treated with mRNA-RBD (HPLC). Spleen cells were prepared from mouse spleens and treated with protein transport inhibitors and pooled peptides for 6 hours. The percentages of cytokine-producing CD8+ and CD4+ T cells were analyzed by flow cytometry. N = 4. Vertical bars indicate mean values, error bars indicate standard error, *P < 0.05, using ANOVA and Dunn's multiple comparison test. [Figure 23] Changes in body temperature before and after SARS-CoV-2 infection. One week after the second administration of mRNA-RBD (HPLC), cynomolgus monkeys were administered SARS-CoV-2 (2.2 × 106 PFU) into the oral cavity, nasal cavity, and trachea. Body temperature was recorded using a telemetry transmitter and computer from 2 days before SARS-CoV-2 administration. [Figure 24] Chest radiograph after SARS-CoV-2 infection in a cynomolgus macaque treated with mRNA-RBD (HPLC). [Diagram 25]Blood anti-RBD antibody response induced by Examples 10, 12, 14, 16, 18, and 20. Buffer: 10 mM histidine buffer (pH 7.0) containing 300 mM sucrose. N=4. Vertical bars indicate geometric mean values, and symbols 1 to 4 indicate individual anti-RBD antibody levels. [Figure 26] Blood anti-RBD antibody response induced by Examples 10 and 21 to 30. Buffer: 10 mM histidine buffer (pH 7.0) containing 300 mM sucrose. N=4. Vertical bars indicate geometric mean values, and symbols 1 to 4 indicate individual anti-RBD antibody levels. [Figure 27] Blood anti-RBD antibody response induced by Examples 8, 32a, 32b, 32c, 32d, 32f, and 33. Buffer: 10 mM histidine buffer (pH 7.0) containing 300 mM sucrose. N=4-5. Vertical bars indicate geometric mean values, and symbols 1-5 indicate individual anti-RBD antibody levels. The RBD antigens used for immobilization in ELISA were derived from the Wuhan strain (Original) and the B.1.351 strain (351). [Figure 28] RBD-hACE2 binding inhibitory activity of BALB / c mouse serum administered with Example 10. Buffer: 10 mM Histidine buffer (pH 7.0) containing 300 mM Sucrose. SARS-CoV-1 derived (Control), Wuhan strain derived (Original), and point mutants of Original (K417N, E484K, N501Y, K417N / E484K / N501Y) were used as RBD antigens. N=4. Horizontal bars indicate geometric mean values, and symbols 1 to 4 indicate individual inhibitory activity levels. [Figure 29] Anti-SARS-CoV-2 neutralizing activity of cynomolgus monkey plasma administered with Example 10. N=4. Pre indicates before administration of Example 10, and Post indicates after administration of Example 10. The vertical bars indicate the geometric mean values, and the circle symbols indicate individual neutralizing activities. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Hereinafter, the embodiment of the present invention will be described in more detail.

[0014] The present invention provides lipid particles encapsulating a nucleic acid capable of expressing the S protein of a novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2) and / or a fragment thereof, wherein the lipid comprises a cationic lipid represented by general formula (Ia) or a pharma- ceutically acceptable salt thereof. [ka] During the ceremony, R 1 and R 2 each independently represents a C1-C3 alkyl group; L 1 may have one or more C2-C4 alkanoyloxy groups; 17 -C 19 represents an alkenyl group; L 2 may have one or more C2-C4 alkanoyloxy groups; 10 -C 19 C which may have one or more alkyl groups or C2-C4 alkanoyloxy groups 10 -C 19 represents an alkenyl group; p is 3 or 4.

[0015] R in general formula (Ia) 1 and R 2 each independently represents a C1-C3 alkyl group, and preferably, both are a methyl group.

[0016] In the general formula (Ia), p is 3 or 4, and is preferably 3.

[0017] L in general formula (Ia) 1 may have one or more C2-C4 alkanoyloxy groups; 17 -C 19 It represents an alkenyl group, and preferably represents an alkenyl group which may have one or more acetoxy groups. 17 -C19 It is an alkenyl group. 1 Specific examples of such an alkyl group include an (R)-11-acetyloxy-cis-8-heptadecenyl group, a cis-8-heptadecenyl group, and a (8Z,11Z)-heptadecadienyl group.

[0018] L in general formula (Ia) 2 may have one or more C2-C4 alkanoyloxy groups; 10 -C 19 C which may have one or more alkyl groups or C2-C4 alkanoyloxy groups 10 -C 19 It represents an alkenyl group, and preferably represents an alkenyl group which may have one or more acetoxy groups. 10 -C 12 C which may have one or more alkyl groups or acetoxy groups 10 -C 19 Alternatively, L in formula (Ia) is an alkenyl group. 2 C may have one or more acetoxy groups 10 -C 12 C which may have one or more alkyl groups or acetoxy groups 17 -C 19 It is also preferable that L is 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.

[0019] Specific examples of cationic lipids that are components constituting the particles of the present invention include those having the following structural formula: [ka] [ka] [ka] Examples of the compound represented by the formula:

[0020] Pharmaceutically acceptable salt refers to the salt that can be used as medicine.The cationic lipid, which is the component that constitutes the particles of the present invention, can be a pharmaceutically acceptable salt, and such salts are preferably alkali metal salts such as sodium salt, potassium salt, lithium salt, alkaline earth metal salts such as calcium salt, magnesium salt, aluminum salt, iron salt, zinc salt, copper salt, nickel salt, cobalt salt and other metal salts; inorganic salts such as ammonium salt, t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl- Examples of the salt include amine salts, such as organic salts such as phenethylamine salts, piperazine salts, tetramethylammonium salts, and tris(hydroxymethyl)aminomethane salts; inorganic acid salts, such as hydrohalogen salts, such as hydrofluoride, hydrochloride, hydrobromide, and hydroiodide, nitrates, perchlorates, sulfates, and phosphates; organic acid salts, such as lower alkanesulfonates, such as methanesulfonates, trifluoromethanesulfonates, and ethanesulfonates, arylsulfonates, such as benzenesulfonates and p-toluenesulfonates, acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, and maleates; and amino acid salts, such as glycine salts, lysine salts, arginine salts, ornithine salts, glutamate salts, and aspartate salts.

[0021] The cationic lipid represented by general formula (Ia) may be one type of compound or a combination of two or more types of compounds.

[0022] A method for producing the cationic lipid represented by general formula (Ia) is described in WO 2015 / 005253.

[0023] The lipids of the present invention may further include amphipathic lipids, sterols and PEG lipids.

[0024] Amphipathic lipids are lipids that have affinity for both polar and nonpolar solvents, and specific examples thereof include distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, and combinations thereof.

[0025] The sterols are sterols having a hydroxyl group, and a specific example thereof is cholesterol.

[0026] The PEG lipid is a lipid modified with PEG, and specific examples thereof include 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol and / or N-[methoxy poly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine, and combinations thereof.

[0027] The lipid composition of the amphipathic lipid, sterols, cationic lipid, and PEG lipid is not particularly limited, but is preferably, in molar amounts, 15% or less of amphipathic lipid, 20 to 55% of sterols, 40 to 65% of cationic lipid, and 1 to 5% of PEG lipid, and the ratio of the total lipid weight to the nucleic acid weight is 15 to 30; more preferably, the lipid composition of the amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 5 to 15% of amphipathic lipid, 35 to 50% of sterols, 40 to 55% of cationic lipid, and 1 to 3% of PEG lipid, and the ratio of the total lipid weight to the nucleic acid weight is 15 to 25; It is 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 lipid, and the ratio of the total lipid weight to the nucleic acid weight is 17.5 to 22.5, and 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, 45 to 50% cationic lipids, and 1.5 to 2% PEG lipid, and the ratio of the total lipid weight to the nucleic acid weight is 17.5 to 22.5.

[0028] In the present invention, the nucleic acid encapsulated in the lipid particles is capable of expressing the S protein and / or a fragment thereof of the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2). The sequence of the Wuhan strain of SARS-CoV-2 has been published (NCBI ID NC_045512) (https: / / www.ncbi.nlm.nih.gov / nuccore / NC_045512).

[0029] The fragment of the SARS-CoV-2 S protein may contain the receptor-binding domain (RBD) present in the S protein.

[0030] The receptor-binding domain may be added with a secretory peptide (a peptide encoded by a leader sequence), such as the S protein signal sequence.

[0031] The amino acid sequence of the S protein of SARS-CoV-2 is shown in SEQ ID NO: 6. The nucleic acid to be encapsulated in the lipid particles may be capable of expressing the S protein of SARS-CoV-2, which consists of an amino acid sequence having at least 95%, preferably 96%, more preferably 97% identity to the amino acid sequence of SEQ ID NO: 6.

[0032] The amino acid sequence of the receptor binding domain present in the S protein of SARS-CoV-2 is shown in SEQ ID NO: 11. A secretory peptide (e.g., an S protein signal sequence) may be added to the receptor binding domain present in the S protein of SARS-CoV-2. The amino acid sequence of the receptor binding domain present in the S protein of SARS-CoV-2 to which the S protein signal sequence has been added is shown in SEQ ID NO: 10. The nucleic acid to be encapsulated in the lipid particles is preferably capable of expressing the receptor binding domain in the S protein of SARS-CoV-2, which consists of an amino acid sequence having at least 95%, preferably 96%, more preferably 97% identity to the amino acid sequence of SEQ ID NO: 11 or 10.

[0033] As used herein, identity refers to the relationship between sequences of two or more nucleotide or amino acid sequences, as determined by comparison of sequences, as known in the art. In the art, "identity" also means the degree of sequence relatedness between nucleic acid molecules or polypeptides, as the case may be, as determined by the match between two or more nucleotide sequences or two or more amino acid sequences in a row. Identity can be evaluated by calculating the percentage of identical matches between the smaller of two or more sequences and gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithm"). Specifically, it can be evaluated by using software such as ClustalW2 provided by the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI), but is not limited to software that can be used by those skilled in the art.

[0034] The sequence identity in the present invention is calculated using sequence analysis software GENETYX-SV / RC (Genetyx Corporation), and this algorithm is commonly used in the art. The amino acids encoded by the nucleic acids 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 the amino acid sequence of the target SARS-CoV-2 S protein and / or a fragment thereof.

[0035] The amino acids encoded by the nucleic acid to be encapsulated in the lipid particles of the present invention retain 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 amino acid sequence of the targeted SARS-CoV-2 S protein and / or the amino acid sequence of a fragment thereof, with several amino acids (preferably 10 positions or less, more preferably 7 positions or less, even more preferably 5, 4, 3, 2 or 1 position) per position.

[0036] The amino acid sequence of the receptor binding domain present in the S protein of SARS-Cov-2 may be deleted, substituted, or added, and examples thereof include a sequence in which the 538th cysteine ​​(the number is counted from the N-terminus of the S protein) is replaced with serine (SEQ ID NO: 25) (hereinafter, also referred to as "C538S type"); a sequence in which amino acids are deleted from the N-terminus and C-terminus of the full-length RBD sequence (R319-F541) (SEQ ID NO: 29); a sequence in which amino acids are added to the N-terminus and C-terminus of the full-length RBD sequence (R319-F541) (SEQ ID NO: 33); and a sequence in which a mutation is introduced in which multiple amino acid residues are substituted (SEQ ID NO: 37). A secretory peptide (e.g., S protein signal sequence) may be added to these sequences, and the amino acid sequences of SEQ ID NOs: 25, 29, 33, and 37 to which the S protein signal sequence is added are shown in SEQ ID NOs: 24, 28, 32, and 36, respectively.

[0037] The receptor binding domain present in the S protein of SARS-CoV-2 may be derived from a mutant strain, and the amino acid sequences of the receptor binding domains of the South African type, British type, Brazilian type, California type, Indian type, South African C538S type, British C538S type, Brazilian C538S type, California C538S type, Indian C538S type, combination mutant type (1) (see Example 33 described below), combination mutant type (2) (see Example 33 described below), combination mutant type (3) (see Example 33 described below), and combination mutant type (4) (see Example 33 described below) are shown in SEQ ID NOs: 94 to 107. The sequences in which the S protein signal sequence is added to the amino acid sequences of SEQ ID NOs: 94 to 107 are shown in SEQ ID NOs: 80 to 93.

[0038] The nucleic acid to be encapsulated in the lipid particles may be capable of expressing a receptor-binding domain in the S protein of SARS-CoV-2, which consists of an amino acid sequence having at least 95%, preferably 96%, more preferably 97% identity with the amino acid sequences of SEQ ID NOs: 25, 29, 33, 37, and 94 to 107 (excluding the S protein signal sequence). The nucleic acid to be encapsulated in the lipid particles may be capable of expressing a receptor-binding domain in the S protein of SARS-CoV-2, which consists of an amino acid sequence having at least 95%, preferably 96%, more preferably 97% identity with the amino acid sequences of SEQ ID NOs: 24, 28, 32, 36, and 80 to 93 (including the S protein signal sequence).

[0039] The nucleic acid capable of expressing the S protein of SARS-CoV-2 may be an mRNA including a cap structure (Cap), a 5' untranslated region (5'-UTR), a translation region of the S protein, a 3' untranslated region (3'-UTR) and a polyA tail (polyA). The cap structure (Cap) is present at the 5' end of many eukaryotic mRNAs and is a site having a 7-methylguanosine structure. Examples of the cap structure include cap0, cap1, cap2, ARCA, or CleanCap (registered trademark), and are preferably cap1 or CleanCap, and more preferably CleanCap. The sequence of the 5' untranslated region (5'-UTR) is, for example, the sequence of base numbers 19 to 88 in the sequence of SEQ ID NO: 4. The sequence of the translation region of the S protein is a sequence capable of expressing all or a part of the amino acid sequence of the S protein, and may include an initiation codon and / or a termination codon, and is, for example, the sequence of base numbers 89 to 3910 in the sequence of SEQ ID NO: 4. The sequence of the S protein translation region may be a nucleotide sequence having at least 90% identity with the sequence of the S protein translation region in the sequence of SEQ ID NO: 5. The sequence of the 3' untranslated region (3'-UTR) is, for example, the sequence of base numbers 3911 to 4042 in the sequence of SEQ ID NO: 4. The sequence of the polyA tail (polyA) is, for example, the sequence of base numbers 4043 to 4142 in the sequence of SEQ ID NO: 4. The cap structure (Cap), 5' untranslated region (5'-UTR), the S protein translation region, 3' untranslated region (3'-UTR) and polyA tail (polyA) sequences may be modified, and the sequence of the nucleic acid capable of expressing the S protein of SARS-CoV-2 may be a nucleotide sequence having at least 90%, preferably 95%, more preferably 97% identity with the sequence of SEQ ID NO: 5, most preferably the nucleotide sequence of SEQ ID NO: 5. The codons of the nucleic acid may be optimized. By optimizing the codons, the effect as a vaccine may be improved and side effects may be reduced. The codons can be optimized according to the codon usage of the target organism. For example, the codons can be optimized for the coding sequence. In the sequence of SEQ ID NO: 16, the codons in the sequence of the translated region of the S protein are optimized.The sequence of the nucleic acid capable of expressing the S protein of SARS-CoV-2 may consist of a nucleotide sequence having at least 90%, preferably 95%, more preferably 97% identity with the sequence of SEQ ID NO: 16.

[0040] The nucleic acid capable of expressing a fragment of the S protein of SARS-CoV-2 may be an mRNA including a cap structure (Cap), a 5' untranslated region (5'-UTR), a leader sequence, a translation region of the receptor binding domain in the S protein, a 3' untranslated region (3'-UTR) and a polyA tail (polyA). The cap structure (Cap) is present at the 5' end of many eukaryotic mRNAs and is a site having a 7-methylguanosine structure. Examples of the cap structure include cap0, cap1, cap2, ARCA, and CleanCap (registered trademark), and are preferably cap1 or CleanCap, and more preferably CleanCap. The sequence of the 5' untranslated region (5'-UTR) is, for example, the sequence of base numbers 19 to 88 in the sequence of SEQ ID NO: 8. The sequence of the leader sequence is, for example, the sequence of base numbers 89 to 127 in the sequence of SEQ ID NO: 8. The sequence of the translation region of the receptor binding domain in the S protein is a sequence capable of expressing all or a part of the amino acid sequence of the receptor binding domain in the S protein, and may include an initiation codon and / or a stop codon, for example, the sequence of base numbers 128 to 799 in the sequence of SEQ ID NO: 8. The sequence of the translation region of the receptor binding domain in the S protein may be a nucleotide sequence having at least 90% identity with the sequence of the translation region of the receptor binding domain in the S protein in the sequence of SEQ ID NO: 9. The sequence of the 3' untranslated region (3'-UTR) is, for example, the sequence of base numbers 800 to 931 in the sequence of SEQ ID NO: 8. The sequence of the polyA tail (polyA) is, for example, the sequence of base numbers 932 to 1031 in the sequence of SEQ ID NO: 8. The sequences of the cap structure (Cap), 5' untranslated region (5'-UTR), leader sequence, translated region of the receptor binding domain in the S protein, 3' untranslated region (3'-UTR) and polyA tail (polyA) may be modified, and the sequence of the nucleic acid capable of expressing the receptor binding domain in the S protein of SARS-CoV-2 may consist of a nucleotide sequence having at least 90%, preferably 95%, more preferably 97% identity to the sequence of SEQ ID NO: 9, and most preferably the nucleotide sequence of SEQ ID NO: 9.The codons of the nucleic acid may be optimized. By optimizing the codons, the effect as a vaccine may be improved and side effects may be reduced. The optimization may be performed according to the frequency of codon usage of the target organism. For example, the optimization of the codons may be performed on the coding sequence, and in the sequence of SEQ ID NO: 19, the codons of the sequence of the translation region of the receptor binding domain in the S protein are optimized. The sequence of the nucleic acid capable of expressing the receptor binding domain in the S protein of SARS-CoV-2 may be a nucleotide sequence having at least 90%, preferably 95%, more preferably 97% identity with the sequence of SEQ ID NO: 19. In addition, the sequence of the translation region of the receptor binding domain in the S protein may be a nucleotide sequence having at least 90%, preferably 95%, more preferably 97% identity with the sequence of the translation region of the receptor binding domain in the S protein in any of the sequences of SEQ ID NOs: 21, 23, 27, 31, 35, and 66 to 79.

[0041] SEQ ID NO: 21 is the nucleotide sequence of the mRNA of Example 11, which is an mRNA having the same sequence as that of Example 6 except for poly A. In the sequence of Example 6, poly A has 110 adenine nucleotides, whereas in the mRNA of Example 11, it has 50 adenine nucleotides. The nucleic acid contained in the lipid particles of the present invention may be an mRNA having a relatively short poly A portion, preferably 30 or more, 40 or more, more preferably 50 or more. The upper limit of poly A is not particularly limited, but is preferably 500 or less, 400 or less, 300 or less, 200 or less, or 110 or less.

[0042] Sequence number 23 is the nucleotide sequence of the mRNA of Example 13, and the mRNA of Example 13 is an mRNA capable of expressing a sequence in which the cysteine ​​at position 538 (the number is counted from the N-terminus of the S protein) is replaced with serine.

[0043] Sequence number 27 is the nucleotide sequence of the mRNA of Example 15, and the mRNA of Example 15 is an mRNA capable of expressing a sequence in which amino acids are deleted at the N-terminus and C-terminus of the full-length RBD sequence (R319-F541).

[0044] Sequence number 31 is the nucleotide sequence of the mRNA of Example 17, and the mRNA of Example 17 is an mRNA capable of expressing a sequence in which amino acids have been added to the N-terminus and C-terminus of the full-length RBD sequence (R319-F541).

[0045] SEQ ID NO: 35 is the nucleotide sequence of the mRNA of Example 19, and the mRNA of Example 19 is an mRNA capable of expressing the sequence of Example 6 in which amino acid residue substitutions have occurred at multiple positions.

[0046] SEQ ID NOs: 66 to 79 are nucleotide sequences of mRNA capable of expressing the amino acid sequences of the receptor binding domains of the South African type, British type, Brazilian type, California type, Indian type, South African C538S type, British C538S type, Brazilian C538S type, California C538S type, Indian C538S type, combined mutant type (1) (see Example 33 described below), combined mutant type (2) (see Example 33 described below), combined mutant type (3) (see Example 33 described below), and combined mutant type (4) (see Example 33 described below).

[0047] The nucleic acid to be encapsulated in the lipid particles may be in any form as long as it is capable of expressing the S protein of SARS-CoV-2 and / or a fragment thereof. Examples of such nucleic acids 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 hybrid polynucleotide of DNA-RNA, and a double-stranded polynucleotide consisting of two types of polynucleotide consisting of a mixture of DNA and RNA, and preferably, mRNA.

[0048] 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.

[0049] The modified nucleotide may be one in which any of the base, sugar, and phosphodiester bond has been modified. The modification site may be one or more.

[0050] 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.

[0051] An example of a sugar modification is 2'-O-methylation of D-ribofuranose.

[0052] An example of a modification of a phosphodiester bond is a phosphorothioate bond.

[0053] The modified nucleotide is preferably one in which the base portion is modified, and may be, for example, a pyrimidine nucleotide substituted at the 5th position or a pseudouridine which may be substituted at the 1st position, and specifically, 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, and 1-alkylpseudouridine can be exemplified. The 1-alkylpseudouridine may be 1-(C1-C6 alkyl)pseudouridine, and is preferably 1-methylpseudouridine or 1-ethylpseudouridine. The modified nucleotide with the modified base portion may be used alone or in combination in place of a natural nucleotide. The combination of modified nucleotides with modified base portions may be, for example, a combination of 5-methylcytidine and 5-methyluridine, a combination of 5-methylcytidine and pseudouridine, or a combination of 5-methylcytidine and 1-methylpseudouridine, and is preferably a combination of 5-methylcytidine and 5-methyluridine.

[0054] The nucleic acid capable of expressing the S protein of SARS-CoV-2 and / or a fragment thereof of the present invention can be produced by in vitro transcription reaction from DNA having a desired base sequence. Enzymes, buffers, and nucleoside-5'-triphosphate mixtures (adenosine-5'-triphosphate (ATP), guanosine-5'-triphosphate (GTP), cytidine-5'-triphosphate (CTP) and uridine-5'-triphosphate (UTP)) necessary for in vitro transcription are commercially available (e.g., AmpliScribe T7 High Yield Transcription Kit (Epicentre), mMESSAGE mMACHINE T7 Ultra Kit (Life Technologies), etc.). The DNA used to produce single-stranded RNA is cloned DNA, for example, a plasmid DNA or a DNA fragment. Plasmid DNA or DNA fragments may be commercially available or may be produced by methods 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 DG et al., Science, 2008, 319(5867), 1215-1220, etc.).

[0055] In order to obtain mRNA with improved stability and / or safety, some or all of the natural nucleotides in the mRNA can also be replaced with modified nucleotides by replacing some or all of the natural nucleoside-5'-triphosphates with modified nucleoside-5'-triphosphates in an in vitro transcription reaction (Kormann, M., Nature Biotechnology, 2011, 29, 154-157.).

[0056] In order to obtain mRNA with improved stability and / or safety, a cap structure (the above-mentioned Cap0 structure) can be introduced into the 5'-end of mRNA by a method using a capping enzyme after an in vitro transcription reaction. Cap0 can also be converted to Cap1 by a method of allowing 2'-O-methyltransferase to act on mRNA having Cap0. 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 having a cap structure can be produced according to the protocol attached to the product.

[0057] The cap structure at the 5' end of mRNA can also be introduced by a method other than using an enzyme. 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 (registered trademark) can be introduced into mRNA. Commercially available products can be used for ARCA and CleanCap (registered trademark) (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 attached to the product.

[0058] In the present invention, the nucleic acid to be encapsulated in the lipid particles may be purified by methods such as desalting, reverse phase column, gel filtration, HPLC, PAGE, etc. By removing impurities through the purification treatment, the production of inflammatory cytokines in a living body to which the nucleic acid is administered can be reduced.

[0059] An example of the above-mentioned impurity is double-stranded RNA (dsRNA). The amount of dsRNA contained in the nucleic acid encapsulated in the lipid particles is preferably 10% or less, more preferably 7.5% or less, even more preferably 5% or less, and particularly preferably 3% or less, in terms of mass percentage.

[0060] The nucleic acid-encapsulating lipid particles of the present invention can be produced by a method such as a thin film method, a reverse phase evaporation method, an ethanol injection method, an ether injection method, a dehydration-rehydration method, a surfactant dialysis method, a hydration method, a freeze-thaw method, etc. For example, the nucleic acid-encapsulating lipid particles can be produced by the method described in WO 2015 / 005253.

[0061] The particles of the present invention may have an average particle diameter of 30 nm to 300 nm, preferably 30 to 200 nm, more preferably 30 to 150 nm, and even more preferably 30 to 100 nm. The average particle diameter can be obtained by measuring the volume average particle diameter based on the principle of dynamic light scattering using an instrument such as Zeta Potential / Particle Sizer NICOMP (registered trademark) 380ZLS (PARTICLE SIZING SYSTEMS).

[0062] The particles of the present invention can be used to manufacture a composition for preventing and / or treating infection with a new coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2). The strain of SARS-CoV-2 is not particularly limited, but the Wuhan strain is preferred.

[0063] The particles of the present invention can be used to express the S protein of SARS-CoV-2 and / or a fragment thereof in vivo or in vitro. Thus, the present invention provides a method for expressing the S protein of SARS-CoV-2 and / or a fragment thereof in vitro, comprising introducing a composition containing the particles into a cell. The present invention also provides a method for expressing the S protein of SARS-CoV-2 and / or a fragment thereof in vivo, comprising administering a composition containing the particles to a mammal. By expressing the S protein of SARS-CoV-2 and / or a fragment thereof in vivo, an immune response against SARS-CoV-2 can be induced. As a result, SARS-CoV-2 infection can be prevented and / or treated. Thus, the present invention provides a method for inducing an immune response against SARS-CoV-2, comprising administering a composition containing the particles to a mammal. The present invention also provides a method for preventing and / or treating SARS-CoV-2 infection, comprising administering a composition containing the particles to a mammal.

[0064] The particles of the present invention can be used as medicines and as experimental reagents. The particles of the present invention are usually added to a carrier such as water, a buffer solution, or physiological saline, and the resulting mixture (composition) can be introduced into cells (in vitro) or administered to a mammal (in vivo). When administered to a mammal, the carrier should be a pharma- ceutically acceptable carrier (e.g., physiological saline). The particles of the present invention may also be formulated into creams, pastes, ointments, gels, lotions, and other dosage forms using fats, fatty oils, lanolin, petrolatum, paraffin, wax, resins, plastics, glycols, higher alcohols, glycerin, water, emulsifiers, suspending agents, and other base materials.

[0065] The particles of the present invention can be administered orally or parenterally, for example, via intramuscular, intravenous, rectal, transdermal, transmucosal, subcutaneous, or intradermal administration, to mammals such as humans, mice, rats, hamsters, guinea pigs, rabbits, pigs, monkeys, cats, dogs, horses, goats, sheep, and cows.

[0066] 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, of mRNA per dose for an adult may be administered once or several times by intramuscular injection, subcutaneous injection, intradermal injection, intravenous drip injection, or intravenous injection, although the dose and number of administrations may be appropriately changed depending on the type of disease, symptoms, age, administration method, etc.

[0067] When used as an experimental reagent, the particles of the present invention can be introduced into cells in which SARS-CoV-2 S protein and / or fragments thereof 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)) to express SARS-CoV-2 S protein and / or fragments thereof in vitro. The expression of SARS-CoV-2 S protein and / or fragments thereof can be analyzed by detecting SARS-CoV-2 S protein and / or fragments thereof in a sample by Western blotting or by detecting peptide fragments specific to SARS-CoV-2 S protein and / or fragments thereof by mass spectrometry.

[0068] As used herein, treatment refers to recovery, remission, alleviation, and / or delay in worsening of clinical symptoms of a disease caused by a virus, bacteria, or the like, or a disease caused by such an infection (e.g., pneumonia, etc.) in a patient who has developed such a disease.

[0069] As used herein, prevention means reducing the incidence of disease 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 by inducing a protective immune response. EXAMPLES

[0070] The present invention will be specifically described below with reference to examples. Note that these examples are for the purpose of explaining the present invention and are not intended to limit the scope of the present invention. [Example 1] Preparation of SARS-CoV-2 S full mRNA-001 (1) Preparation of template DNA for in vitro transcription (IVT) of SARS-CoV-2 S full To prepare template DNA for in vitro transcription (IVT), SARS-CoV-2 S full DNA was amplified by PCR and purified. A DNA fragment (SEQ ID NO: 1) containing the sequence of the T7 promoter sequence, human β-globin 5'-UTR sequence, KOZAK sequence, SARS-CoV-2 S full, and human β-globin 3'-UTR sequence linked in order was introduced into a plasmid (pUC57mini-S full). 6 ng of the plasmid was dissolved in nuclease-free water (849.6 μL), to which 10× Buffer for KOD-Plus- Ver.2 (120 μL, Toyobo Co., Ltd. catalog # KOD-211), 2 mM dNTP mix (120 μL, Toyobo Co., Ltd. catalog # KOD-211), 25 mM MgSO4 (72 μL, Toyobo Co., Ltd. catalog # KOD-211), 50 μM sense primer (7.2 μL, SEQ ID NO: 2), 50 μM antisense primer (7.2 μL, SEQ ID NO: 3), and KOD Plus polymerase (24 μL, Toyobo Co., Ltd. catalog # KOD-211) were added, and the mixture was incubated at 98°C for 1 minute, followed by 20 cycles of 98°C for 5 seconds, 55°C for 15 seconds, and 68°C for 4 minutes, and then further incubation at 68°C for 1 minute to amplify the S full DNA. After the reaction, the template DNA (sequence number 4) was purified using Wizard SV Gel and PCR Clean-Up System (Promega catalog # A9281).

[0071] (2) Preparation of SARS-CoV-2 S-full mRNA-001 by in vitro transcription The mixture contained 360.5 μg / mL template DNA obtained in Example 1-(1) (70 μL), 100 mM CleanCap AG (50 μL, TriLink catalog # T-7113), 100 mM ATP (50 μL, Hongene catalog # R1331), 100 mM GTP (50 μL, Hongene catalog # R2331), 100 mM 5-Me-CTP (50 μL, Hongene catalog # R3-029), 100 mM 5-methyluridine triphosphate (50 μL), Nuclease-free water (380 μL, Thermo Fisher catalog # AM9937), T7 Transcription 5× buffer (200 μL, Promega catalog # P140X), Enzyme mix, T7 RNA Polymerase (100 μL, Promega catalog # P137X) and incubated at 37°C for 4 hours. RQ1 RNase-Free DNase (25μL, Promega catalog # M6101) was added and incubated at 37°C for 15 minutes. 8M LiCl solution (500μL, Sigma-Aldrich catalog # L7026) was added and left to stand overnight at -20°C. After centrifugation (4°C, 4000×g, 30 minutes), the supernatant was discarded, 70% ethanol was added, and the mixture was centrifuged (4°C, 4000×g, 10 minutes), after which the supernatant was discarded and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water and purified using the RNeasy Maxi kit (Qiagen catalog # 75162) according to the attached manual. The resulting eluate (5.8 mL, 4906 μg in terms of UV) was mixed with nuclease-free water (419 μL), rApid Alkaline Phosphatase (Roche catalog # 04 898 141 001) buffer (800 μL), and enzyme (981 μL), and incubated at 37°C for 30 minutes, followed by incubation at 75°C for 3 minutes. 8M LiCl solution (8000 μL) was added, and the mixture was left to stand overnight at -20°C.After centrifugation (4°C, 4000×g, 30 min), the supernatant was discarded, 70% ethanol was added, and the mixture was centrifuged (4°C, 4000×g, 10 min), after which the supernatant was discarded and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water, and the target mRNA was obtained by purifying the mixture using the RNeasy Maxi kit according to the attached manual. The obtained mRNA has the sequence of SEQ ID NO: 5. It was analyzed using LabChip GX Touch Standard RNA Reagent Kit (PerkinElmer catalog #CLS960010) and confirmed to be of the desired length.

[0072] Example 2: Preparation of SARS-CoV-2 RBD mRNA-002 (1) Preparation of template DNA for in vitro transcription (IVT) of SARS-CoV-2 RBD To prepare template DNA for in vitro transcription (IVT), SARS-CoV-2 RBD DNA was amplified by PCR and purified. A DNA fragment (SEQ ID NO: 7) containing the sequence of the T7 promoter sequence, the 5'-UTR sequence of human β-globin, the KOZAK sequence, the signal sequence of the SARS-CoV-2 S protein, the SARS-CoV-2 RBD, and the 3'-UTR sequence of human β-globin was introduced into a plasmid (pUC57mini-RBD). 6 ng of the plasmid was dissolved in nuclease-free water (849.6 μL), to which 10× Buffer for KOD-Plus- Ver.2 (120 μL, Toyobo Co., Ltd. catalog # KOD-211), 2 mM dNTP mix (120 μL, Toyobo Co., Ltd. catalog # KOD-211), 25 mM MgSO4 (72 μL, Toyobo Co., Ltd. catalog # KOD-211), 50 μM sense primer (7.2 μL, SEQ ID NO: 2), 50 μM antisense primer (7.2 μL, SEQ ID NO: 3), and KOD Plus polymerase (24 μL, Toyobo Co., Ltd. catalog # KOD-211) were added, and the mixture was incubated at 98°C for 1 minute, followed by 20 cycles of 98°C for 5 seconds, 55°C for 15 seconds, and 68°C for 1 minute, and then further incubation at 68°C for 1 minute to amplify the RBD DNA. After the reaction, the template DNA (sequence number 8) was purified using Wizard SV Gel and PCR Clean-Up System (Promega catalog # A9281).

[0073] (2) Preparation of SARS-CoV-2 RBD mRNA-002 by in vitro transcription Instead of the template DNA obtained in Example 1-(1), the template DNA obtained in Example 2-(1) was used to obtain mRNA in the same manner as in Example 1-(2). The obtained mRNA has the sequence of SEQ ID NO: 9. It was analyzed using the LabChip GX Touch Standard RNA Reagent Kit and confirmed to be of the desired length.

[0074] [Example 3] Preparation of mRNA-encapsulated nucleic acid-lipid particles using SARS-CoV-2 S full mRNA described in Example 1 (1) Preparation of mRNA-encapsulated nucleic acid-lipid particles Distearoylphosphatidylcholine (1,2-Distearoyl-sn-glycero-3-phosphocholine, hereinafter referred to as DSPC, NOF CORPORATION), cholesterol (Cholesterol, hereinafter referred to as Chol, Sigma-Aldrich, Inc.), (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate (compound described in Example 23 of WO2015 / 005253) (hereinafter referred to as LP), and 1,2-Dimyristoyl-sn-Glycerol Methoxypolyethylene Glycol (1,2-Dimyristoyl-sn-Glycero-3-Methoxypolyethylene Glycol, hereinafter referred to as PEG-DMG, NOF CORPORATION) having a molecular weight of about 2000 were used. CORPORATION) was dissolved in ethanol at a molar ratio of DSPC:Chol:LP:PEG-DMG=12.5:41:45:1.5 to a total lipid concentration of 5 mM. Meanwhile, the SARS-CoV-2 S-full mRNA-001 obtained in Example 1 was adjusted to 52.7 μg / mL in citrate buffer (20 mM citrate buffer, pH 4.0). The lipid solution and the mRNA solution were mixed in a microchannel using a NanoAssemblr BenchTop (Precision Nanosystems Inc.) so that the volume ratio was 1:3, and a crude dispersion of nucleic acid-lipid particles was obtained. The nucleic acid-lipid particle dispersion was dialyzed (Float-A-Lyzer G2, MWCO: 1,000 kD, Spectra / Por) for 12-18 hours against approximately 25-50 times the amount of 300 mM sucrose, 10 mM histidine buffer (pH 6.5) to remove ethanol, and a dispersion of purified mRNA-encapsulated nucleic acid-lipid particles was obtained. LP was synthesized according to the method described in Example 23 of WO2015 / 005253.

[0075] (2) Characterization of 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 explained below. (2-1) Encapsulation rate of mRNA The mRNA encapsulation rate was measured using the Quant-iT RiboGreen RNA Assay kit (Invitrogen) in accordance with the attached instructions. That is, the amount of mRNA in the dispersion of the nucleic acid-lipid particles was quantified in the presence and absence of 0.015% Triton X-100 surfactant, and the encapsulation rate was calculated by 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(%)

[0076] (2-2) Ratio of mRNA to lipid The amount of mRNA in the nucleic acid-lipid particle dispersion was measured by reverse phase chromatography (System: Agilent 1100 series, Column: Bioshell A400 Protein C4 (10 cm × 4.6 mm, 3.4 μm) (SUPELCO), Buffer A: 0.1 M triethylamine acetate (pH 7.0), Buffer B: acetonitrile, (B%): 5-50% (0-15 min), Flow Rate: 1 mL / min, Temperature: 70 °C, Detection: 260 nm). The amount of each lipid in the nucleic acid-lipid particle dispersion was measured by reverse phase chromatography (System: DIONEX UltiMate 3000, Column: XSelect CSH C18 (150 mm × 3 mm, 3.5 μm, 130 Å) (Waters catalog # 186005263), Buffer A: 0.2% formic acid, Buffer B: 0.2% formic acid, methanol, (B%): 75-100% (0-6 min), 100% (6-15 min), Flow Rate: 0.45 mL / min, Temperature: 50 °C, Detection: Corona CAD (Charged Aerosol Detector)). The ratio of the total lipid amount to the mRNA was calculated by the following formula. [Total lipid concentration] / [mRNA concentration] (wt / wt)

[0077] (2-3) Average particle diameter The particle size of the nucleic acid-lipid particles was measured using a Zeta Potential / Particle Sizer NICOMP™ 380ZLS (PARTICLE SIZING SYSTEMS). The average particle size in the table represents the volume average particle size, and ± indicates deviation. The results are shown in Table 1.

[0078] [Example 4] Preparation of mRNA-encapsulated nucleic acid-lipid particles using SARS-CoV-2 RBD mRNA described in Example 2 In the same manner as in Example 3, Using mRNA The mRNA-encapsulated nucleic acid-lipid particles were prepared and their characteristics were evaluated. The results are shown in Table 1. (Table 1) JPEG0007672657000009.jpg24164The above results demonstrated that these nucleic acid-lipid particles had 90% or more of the mRNA encapsulated within the lipid particles, and had an average particle size of approximately 100 nm to approximately 130 nm.

[0079] [Example 5] Preparation of SARS-CoV-2 S full optimized mRNA-003 (1) Preparation of template DNA for in vitro transcription (IVT) of SARS-CoV-2 S fully optimized A DNA fragment (sequence number 12) containing a sequence in which the T7 promoter sequence, the 5'-UTR sequence of human β-globin, the KOZAK sequence, the SARS-CoV-2 S fully optimized sequence, and the 3'-UTR sequence of human β-globin were linked in order was artificially synthesized and introduced into a plasmid (S_opt2 EcoRI). 1 ng of the plasmid was dissolved in nuclease-free water (69 μL), and 5× SuperFi Green Buffer (20 μL, ThermoFisher Scientific catalog # 12357-010), 2.5 mM dNTP mix (8 μL, Takara Bio Inc. catalog # 4030), 50 μM sense primer 2 (1 μL, SEQ ID NO: 13), 50 μM antisense primer 2 (1 μL, SEQ ID NO: 14), and Platinum SuperFi DNA Polymerase (1 μL, ThermoFisher Scientific catalog # 12357-010) were added. After incubation at 98°C for 30 seconds, 20 cycles of 98°C, 5 seconds, 60°C, 10 seconds, and 72°C, 2 minutes were performed, and further incubation was performed at 72°C for 1 minute to amplify the SARS-CoV-2 S full optimized template DNA (SEQ ID NO: 15). The template DNA was cleaved with the restriction enzymes NheI and HindIII, and then introduced into a plasmid cleaved with the same restriction enzymes to prepare a template plasmid (pUCKIVT1 S fully optimized). The plasmid was cleaved with the restriction enzyme BspQI, and the DNA was purified by isopropanol precipitation to prepare a linear plasmid DNA.

[0080] (2) Preparation of SARS-CoV-2 S full optimized mRNA-003 by in vitro transcription Instead of the template DNA obtained in Example 1-(1), the linear plasmid DNA obtained in Example 5-(1) was used to obtain mRNA in the same manner as in Example 1-(2). The obtained mRNA has the sequence of SEQ ID NO: 16. It was analyzed using the LabChip GX Touch Standard RNA Reagent Kit and confirmed to be of the desired length.

[0081] [Example 6] Preparation of SARS-CoV-2 RBD optimized mRNA-004 (1) Preparation of template DNA for in vitro transcription (IVT) of SARS-CoV-2 RBD optimized A DNA fragment (sequence number 17) containing a sequence in which the T7 promoter sequence, human β-globin 5'-UTR sequence, KOZAK sequence, SARS-CoV-2 RBD optimized, and human β-globin 3'-UTR sequence were linked in order was artificially synthesized and introduced into a plasmid (S_RBD_opt2 EcoRI). 1 ng of the plasmid was dissolved in nuclease-free water (69 μL), and 5× SuperFi Green Buffer (20 μL, ThermoFisher Scientific catalog # 12357-010), 2 mM dNTP mix (8 μL, Takara Bio Inc. catalog # 4030), 50 μM sense primer 2 (1 μL, SEQ ID NO: 13), 50 μM antisense primer 2 (1 μL, SEQ ID NO: 14), and Platinum SuperFi DNA Polymerase (1 μL, ThermoFisher Scientific catalog # 12357-010) were added. After incubation at 98°C for 30 seconds, 20 cycles of 98°C, 5 seconds, 60°C, 10 seconds, and 72°C, 1 minute were performed, and the mixture was further incubated at 72°C for 1 minute to amplify the SARS-CoV-2 RBD optimized DNA (SEQ ID NO: 18). The template DNA was cleaved with the restriction enzymes NheI and HindIII, and then introduced into a plasmid cleaved with the same restriction enzymes to produce a template plasmid (pUCKIVT1-RBD optimized). The plasmid was cleaved with the restriction enzyme BspQI, and the DNA was purified by isopropanol precipitation to prepare a linear plasmid DNA.

[0082] (2) Preparation of SARS-CoV-2 RBD optimized mRNA-004 by in vitro transcription Instead of the template DNA obtained in Example 1-(1), the linear plasmid DNA obtained in Example 6-(1) was used to obtain mRNA in the same manner as in Example 1-(2). The obtained mRNA has the sequence of SEQ ID NO: 19. It was analyzed using the LabChip GX Touch Standard RNA Reagent Kit and confirmed to be of the desired length.

[0083] [Example 7] Preparation of mRNA-encapsulated nucleic acid lipid particles using SARS-CoV-2 S full optimized mRNA described in Example 5 The preparation and characteristic evaluation of mRNA-encapsulated nucleic acid lipid particles using the mRNA described in Example 5 were carried out in the same manner as in Example 3. However, dialysis was carried out using 300 mM sucrose, 10 mM histidine buffer (pH 7.0) instead of 300 mM sucrose, 10 mM histidine buffer (pH 6.5), to obtain a dispersion of mRNA-encapsulated nucleic acid lipid particles. The results are shown in Table 2.

[0084] [Example 8] Preparation of mRNA-encapsulated nucleic acid-lipid particles using the SARS-CoV-2 RBD optimized mRNA described in Example 6 Preparation and characteristic evaluation of mRNA-encapsulated nucleic acid lipid particles using the mRNA described in Example 6 were carried out in the same manner as in Example 3. However, dialysis was carried out using 300 mM sucrose, 10 mM histidine buffer (pH 7.0) instead of 300 mM sucrose, 10 mM histidine buffer (pH 6.5), to obtain a dispersion of mRNA-encapsulated nucleic acid lipid particles. The results are shown in Table 2.

[0085] [Example 9] HPLC purification of SARS-CoV-2 RBD optimized mRNA-004 The mRNA obtained by the method described in Example 6-(2) was separated and purified by reverse phase chromatography (YMC-Triart Bio C4 (YMC catalog # TB30S05-1510WT), 5% acetonitrile, 400 mM triethylamine acetate (pH 7.0) / 25% acetonitrile, 400 mM triethylamine acetate (pH 7.0), 75°C).

[0086] [Example 10] Preparation of mRNA-encapsulated nucleic acid-lipid particles using SARS-CoV-2 RBD optimized mRNA described in Example 6 In the same manner as in Example 8, the mRNA-encapsulated nucleic acid lipid particles were prepared using the mRNA described in Example 9, and the characteristics were evaluated. The results are shown in Table 2. (Table 2) JPEG0007672657000010.jpg36170 From the above results, it was revealed that these nucleic acid-lipid particles had 90% or more of the mRNA encapsulated within the lipid particles, and had an average particle size of approximately 90 nm to approximately 130 nm.

[0087] [Example 11] Preparation of SARS-CoV-2 RBD S2000 mRNA (1) Preparation of template DNA for in vitro transcription (IVT) of SARS-CoV-2 RBD S2000 A plasmid was constructed to prepare template DNA for in vitro transcription (IVT). A DNA fragment (SEQ ID NO: 20) was introduced containing the sequence of GCTAGC (NheI site), T7 promoter sequence, human β-globin 5'-UTR sequence, KOZAK sequence, SARS-CoV-2 S protein signal sequence, SARS-CoV-2 RBD translation region, human β-globin 3'-UTR sequence, poly A tail, and GAAGAGC (BspQI site) linked in order to prepare a plasmid (pUC57-S2000). Plasmid (100μg) was dissolved in nuclease-free water (860μL, Thermo Fisher, catalog # AM9937), 10X NEB Buffer 3.1 (100μL, New England Biolabs, catalog # R7203S), BspQI (40μL, New England Biolabs, catalog # R0712) were added, and the mixture was incubated at 50℃ for 1 hour. After that, isopropanol (1400μL) was added and the mixture was left to stand at -80℃ overnight. After centrifugation (-8℃, 15,000rpm, 10min), the supernatant was discarded, 70% ethanol was added, and the mixture was centrifuged (-8℃, 15,000rpm, 10min), the supernatant was discarded, and the mixture was air-dried. The resulting residue was dissolved in TE-Buffer (pH8.0) to prepare a 500μg / mL solution. (2) Preparation of SARS-CoV-2 RBD S2000 mRNA by in vitro transcription Instead of the template DNA obtained in Example 1-(1), the template DNA obtained in Example 11-(1) was used to obtain mRNA in the same manner as in Example 1-(2). The obtained mRNA has the sequence of SEQ ID NO: 21. It was analyzed using the LabChip GX Touch Standard RNA Reagent Kit and confirmed to be of the desired length.

[0088] [Example 12] Preparation of mRNA-encapsulated nucleic acid-lipid particles using SARS-CoV-2 RBD S2000 mRNA described in Example 11 In the same manner as in Example 8, the preparation and characteristic evaluation of mRNA-encapsulated nucleic acid lipid particles using the mRNA described in Example 11 were carried out. However, the amount of mRNA was measured by the following method. The nucleic acid-lipid particle dispersion was diluted and dissolved in 90% methanol, and the amount of mRNA in the nucleic acid-lipid particles was measured using an ultraviolet-visible spectrophotometer (PerkinElmer, LAMBDA TM 465) The mRNA concentration was calculated using the following formula. {[Absorbance at 260 nm]-[Absorbance at 350 nm]} x 40 x dilution factor (μg / mL) The results are shown in Table 3. The results of the characteristic evaluation revealed that the present nucleic acid-lipid particles had 95% or more of the mRNA encapsulated within the lipid particles and had an average particle size of approximately 150 nm.

[0089] [Example 13] Preparation of SARS-CoV-2 RBD S2001 mRNA (1) Preparation of template DNA for in vitro transcription (IVT) of SARS-CoV-2 RBD S2001 A plasmid was constructed to prepare template DNA for in vitro transcription (IVT). A DNA fragment (SEQ ID NO: 22) containing the sequence of GCTAGC (NheI site), T7 promoter sequence, human β-globin 5'-UTR sequence, KOZAK sequence, SARS-CoV-2 S protein signal sequence, SARS-CoV-2 RBD translation region, human β-globin 3'-UTR sequence, poly A tail, and GAAGAGC (BspQI site) was introduced into the plasmid (pUC57-S2001). Plasmid (100μg) was dissolved in nuclease-free water (860μL, Thermo Fisher, catalog # AM9937), 10X NEB Buffer 3.1 (100μL, New England Biolabs, catalog # R7203S), BspQI (40μL, New England Biolabs, catalog # R0712) were added, and the mixture was incubated at 50℃ for 1 hour. After that, isopropanol (1400μL) was added and the mixture was left to stand at -80℃ overnight. After centrifugation (-8℃, 15,000rpm, 10min), the supernatant was discarded, 70% ethanol was added, and the mixture was centrifuged (-8℃, 15,000rpm, 10min), the supernatant was discarded, and the mixture was air-dried. The resulting residue was dissolved in TE-Buffer (pH8.0) to prepare a 500μg / mL solution. (2) Preparation of SARS-CoV-2 RBD S2001 mRNA by in vitro transcription Instead of the template DNA obtained in Example 1-(1), the template DNA obtained in Example 13-(1) was used to obtain mRNA in the same manner as in Example 1-(2). The obtained mRNA has the sequence of SEQ ID NO: 23. It was analyzed using the LabChip GX Touch Standard RNA Reagent Kit and confirmed to be of the desired length.

[0090] [Example 14] Preparation of mRNA-encapsulated nucleic acid-lipid particles using SARS-CoV-2 RBD S2001 mRNA described in Example 13 In the same manner as in Example 12, mRNA-encapsulated nucleic acid lipid particles were prepared using the mRNA described in Example 13, and their characteristics were evaluated. The results are shown in Table 3. The results of the characteristic evaluation revealed that the nucleic acid lipid particles had 95% or more of the mRNA encapsulated within the lipid particles, and had an average particle size of approximately 140 nm.

[0091] [Example 15] Preparation of SARS-CoV-2 RBD S2002 mRNA (1) Preparation of template DNA for in vitro transcription (IVT) of SARS-CoV-2 RBD S2002 A plasmid was constructed to prepare template DNA for in vitro transcription (IVT). A DNA fragment (SEQ ID NO: 26) containing the sequence of GCTAGC (NheI site), T7 promoter sequence, human β-globin 5'-UTR sequence, KOZAK sequence, SARS-CoV-2 S protein signal sequence, SARS-CoV-2 RBD translation region, human β-globin 3'-UTR sequence, poly A tail, and GAAGAGC (BspQI site) was introduced into the plasmid (pUC57-S2002). Plasmid (100μg) was dissolved in nuclease-free water (860μL, Thermo Fisher, catalog # AM9937), 10X NEB Buffer 3.1 (100μL, New England Biolabs, catalog # R7203S), BspQI (40μL, New England Biolabs, catalog # R0712) were added, and the mixture was incubated at 50℃ for 1 hour. After that, isopropanol (1400μL) was added and the mixture was left to stand at -80℃ overnight. After centrifugation (-8℃, 15,000rpm, 10min), the supernatant was discarded, 70% ethanol was added, and the mixture was centrifuged (-8℃, 15,000rpm, 10min), the supernatant was discarded, and the mixture was air-dried. The resulting residue was dissolved in TE-Buffer (pH8.0) to prepare a 500μg / mL solution. (2) Preparation of SARS-CoV-2 RBD S2002 mRNA by in vitro transcription Instead of the template DNA obtained in Example 1-(1), the template DNA obtained in Example 15-(1) was used to obtain mRNA in the same manner as in Example 1-(2). The obtained mRNA has the sequence of SEQ ID NO: 27. It was analyzed using the LabChip GX Touch Standard RNA Reagent Kit and confirmed to be of the desired length.

[0092] [Example 16] Preparation of mRNA-encapsulated nucleic acid-lipid particles using SARS-CoV-2 RBD S2002 mRNA described in Example 15 In the same manner as in Example 12, mRNA-encapsulated nucleic acid lipid particles were prepared using the mRNA described in Example 15, and their characteristics were evaluated. The results are shown in Table 3. The results of the characteristic evaluation revealed that the nucleic acid lipid particles had 95% or more of the mRNA encapsulated within the lipid particles, and had an average particle size of approximately 140 nm.

[0093] [Example 17] Preparation of SARS-CoV-2 RBD S2003 mRNA (1) Preparation of template DNA for in vitro transcription (IVT) of SARS-CoV-2 RBD S2003 A plasmid was constructed to prepare template DNA for in vitro transcription (IVT). A DNA fragment (SEQ ID NO: 30) containing the sequence of GCTAGC (NheI site), T7 promoter sequence, human β-globin 5'-UTR sequence, KOZAK sequence, SARS-CoV-2 S protein signal sequence, SARS-CoV-2 RBD translation region, human β-globin 3'-UTR sequence, poly A tail, and GAAGAGC (BspQI site) was introduced into the plasmid (pCC1-S2003). Plasmid (100μg) was dissolved in nuclease-free water (860μL, Thermo Fisher, catalog # AM9937), 10X NEB Buffer 3.1 (100μL, New England Biolabs, catalog # R7203S), BspQI (40μL, New England Biolabs, catalog # R0712) were added, and the mixture was incubated at 50℃ for 1 hour. After that, isopropanol (1400μL) was added and the mixture was left to stand at -80℃ overnight. After centrifugation (-8℃, 15,000rpm, 10min), the supernatant was discarded, 70% ethanol was added, and the mixture was centrifuged (-8℃, 15,000rpm, 10min), the supernatant was discarded, and the mixture was air-dried. The resulting residue was dissolved in TE-Buffer (pH8.0) to prepare a 500μg / mL solution. (2) Preparation of SARS-CoV-2 RBD S2003 mRNA by in vitro transcription Instead of the template DNA obtained in Example 1-(1), the template DNA obtained in Example 17-(1) was used to obtain mRNA in the same manner as in Example 1-(2). The obtained mRNA has the sequence of SEQ ID NO: 31. It was analyzed using the LabChip GX Touch Standard RNA Reagent Kit and confirmed to be of the desired length.

[0094] [Example 18] Preparation of mRNA-encapsulated nucleic acid-lipid particles using SARS-CoV-2 RBD S2003 mRNA described in Example 17 In the same manner as in Example 12, mRNA-encapsulated nucleic acid lipid particles were prepared using the mRNA described in Example 17, and their characteristics were evaluated. The results are shown in Table 3. The results of the characteristic evaluation revealed that the nucleic acid lipid particles had 95% or more of the mRNA encapsulated within the lipid particles, and had an average particle size of approximately 140 nm.

[0095] [Example 19] Preparation of SARS-CoV-2 RBD S2004 mRNA (1) Preparation of template DNA for in vitro transcription (IVT) of SARS-CoV-2 RBD S2004 A plasmid was constructed to prepare template DNA for in vitro transcription (IVT). A DNA fragment (SEQ ID NO: 34) containing the sequence of GCTAGC (NheI site), T7 promoter sequence, human β-globin 5'-UTR sequence, KOZAK sequence, SARS-CoV-2 S protein signal sequence, SARS-CoV-2 RBD translation region, human β-globin 3'-UTR sequence, poly A tail, and GAAGAGC (BspQI site) was introduced into the plasmid (pCC1-S2004). Plasmid (100μg) was dissolved in nuclease-free water (860μL, Thermo Fisher, catalog # AM9937), 10X NEB Buffer 3.1 (100μL, New England Biolabs, catalog # R7203S), BspQI (40μL, New England Biolabs, catalog # R0712) were added, and the mixture was incubated at 50℃ for 1 hour. After that, isopropanol (1400μL) was added and the mixture was left to stand at -80℃ overnight. After centrifugation (-8℃, 15,000rpm, 10min), the supernatant was discarded, 70% ethanol was added, and the mixture was centrifuged (-8℃, 15,000rpm, 10min), the supernatant was discarded, and the mixture was air-dried. The resulting residue was dissolved in TE-Buffer (pH8.0) to prepare a 500μg / mL solution. (2) Preparation of SARS-CoV-2 RBD S2004 mRNA by in vitro transcription Instead of the template DNA obtained in Example 1-(1), the template DNA obtained in Example 19-(1) was used to obtain mRNA in the same manner as in Example 1-(2). The obtained mRNA has the sequence of SEQ ID NO: 35. It was analyzed using the LabChip GX Touch Standard RNA Reagent Kit and confirmed to be of the desired length.

[0096] [Example 20] Preparation of mRNA-encapsulated nucleic acid-lipid particles using SARS-CoV-2 RBD S2004 mRNA described in Example 19 In the same manner as in Example 12, mRNA-encapsulated nucleic acid lipid particles were prepared using the mRNA described in Example 19, and their characteristics were evaluated. The results are shown in Table 3. The results of the characteristic evaluation revealed that the nucleic acid lipid particles had 95% or more of the mRNA encapsulated within the lipid particles, and had an average particle size of approximately 180 nm.

[0097] [Examples 21 to 30] Preparation of mRNA-encapsulated nucleic acid lipid particles using the mRNA described in Example 6 (1) Preparation of mRNA-encapsulated nucleic acid-lipid particles Distearoylphosphatidylcholine (DSPC), cholesterol, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate (LP), and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG) having a polyethylene glycol molecular weight of approximately 2000 were dissolved in ethanol in the molar ratios shown in Table 4 to a total lipid concentration of 5 mM. On the other hand, the mRNA obtained in Example 6 was diluted with citrate buffer (20 mM citrate buffer, pH 4.0). The lipid solution and mRNA solution were mixed in a microchannel using a NanoAssemblr BenchTop (Precision Nanosystems Inc.) so that the total lipid weight ratio to mRNA was the value shown in Table 4 and the volume ratio was 1:3, to obtain 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) for 12 to 18 hours against approximately 25 to 50 times the amount of buffer solution to remove ethanol, to obtain a dispersion of purified mRNA-encapsulated nucleic acid-lipid particles. (2) Characterization of 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 explained below. (2-1) Encapsulation rate of mRNA The mRNA encapsulation rate was measured using the Quant-iT RiboGreen RNA Assay kit (Invitrogen) in accordance with the attached instructions. That is, the amount of mRNA in the dispersion of the nucleic acid-lipid particles was quantified in the presence and absence of 0.015% Triton X-100 surfactant, and the encapsulation rate was calculated by 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(%) (2-2) Ratio of mRNA to lipid The amount of mRNA in the nucleic acid-lipid particle dispersion was measured using an ultraviolet-visible spectrophotometer. The nucleic acid-lipid particle dispersion was diluted and dissolved in 90% methanol, and the amount of mRNA in the nucleic acid-lipid particle was measured using an ultraviolet-visible spectrophotometer (PerkinElmer, LAMBDA (trademark) 465). The mRNA concentration was calculated using the following formula. {[Absorbance at 260 nm]-[Absorbance at 350 nm]} x 40 x dilution factor (μg / mL) The amount of each lipid in the nucleic acid-lipid particle dispersion was measured by reverse phase chromatography (System: DIONEX UltiMate 3000, Column: XSelect CSH C18 (130 Å, 3.5 μm, 3.0 mm × 150 mm,) (Waters catalog # 186005263), Buffer A: 0.2% formic acid, Buffer B: 0.2% formic acid, methanol, (B%): 75-100% (0-6 min), 100% (6-15 min), Flow Rate: 0.45 mL / min, Temperature: 50 °C, Detection: Corona CAD (Charged Aerosol Detector)). The ratio of the total lipid amount to the mRNA was calculated by the following formula. [Total lipid concentration] / [mRNA concentration] (wt / wt) (2-3) Average particle diameter The particle size of the nucleic acid-lipid particles was measured using a Zeta Potential / Particle Sizer NICOMP™ 380ZLS (PARTICLE SIZING SYSTEMS). The average particle size in the table represents the volume average particle size, and ± indicates deviation. The results of the characteristic evaluation are shown in Table 5. It was revealed that these nucleic acid-lipid particles had 95% or more of the mRNA encapsulated within the lipid particles and had an average particle size of about 90 nm to about 140 nm.

[0098] [Example 31] Preparation of mutant SARS-CoV-2 RBD mRNA SARS-CoV-2 RBD mRNA was produced for RBDs having the mutations listed in Table 6. The symbols following the Example numbers in Table 7 correspond to the respective mutation types as listed in Table 6. For example, Example 32-a represents the nucleic acid-lipid particles obtained in Example 32 encapsulating mRNA having a South African mutation. (1) Preparation of template DNA for in vitro transcription (IVT) of mutant SARS-CoV-2 RBD To prepare template DNA for in vitro translation (IVT), mutant SARS-CoV-2 RBD DNA was amplified by PCR and purified. A DNA fragment (sequence number 38) containing the sequence of the T7 promoter sequence, human β-globin 5'-UTR sequence, KOZAK sequence, SARS-CoV-2 S protein signal sequence, mutant SARS-CoV-2 RBD, and human β-globin 3'-UTR sequence linked in order was introduced into a plasmid (pUC57mini-mutant RBD). 10 ng of the plasmid was dissolved in nuclease-free water (566.4 μL), to which 10× Buffer for KOD-Plus- Ver.2 (80 μL, Toyobo Co., Ltd. catalog # KOD-211), 2 mM dNTP mix (80 μL, Toyobo Co., Ltd. catalog # KOD-211), 25 mM MgSO4 (48 μL, Toyobo Co., Ltd. catalog # KOD-211), 50 μM sense primer (4.8 μL, SEQ ID NO: 2), 50 μM antisense primer (4.8 μL, SEQ ID NO: 3), and KOD Plus polymerase (16 μL, Toyobo Co., Ltd. catalog # KOD-211) were added. The mixture was incubated at 98°C for 15 seconds, followed by 20 cycles of 98°C for 5 seconds, 55°C for 15 seconds, and 68°C for 1 minute, and then further incubated at 68°C for 1 minute to amplify the RBD DNA. After the reaction, the template DNA (sequence number 52) was purified using Wizard SV Gel and PCR Clean-Up System (Promega catalog # A9281). Template DNAs of SEQ ID NOs: 53 to 55, 57, and 62 to 65 were obtained by a similar method using DNA fragments of SEQ ID NOs: 39 to 41, 43, and 48 to 51, respectively, instead of the DNA fragment (SEQ ID NO: 38). (2) Preparation of mutant SARS-CoV-2 RBD mRNA by in vitro transcription Instead of the template DNA obtained in Example 1-(1), the template DNA (SEQ ID NO: 52) obtained in Example 31-(1) was used to obtain mRNA in the same manner as in Example 1-(2). The obtained mRNA has the sequence of SEQ ID NO: 66. It was analyzed using the LabChip GX Touch Standard RNA Reagent Kit and confirmed to be of the desired length. Instead of the template DNA (SEQ ID NO: 52), the template DNAs of SEQ ID NOs: 53 to 55, 57, and 62 to 65 were used, respectively, to obtain mRNAs of SEQ ID NOs: 67 to 69, 71, and 76 to 79, respectively, by a similar method.

[0099] [Example 32] Preparation of mRNA-encapsulated nucleic acid-lipid particles using SARS-CoV-2 RBD mRNA described in Example 31 In the same manner as in Example 8, preparation and characteristic evaluation of mRNA-encapsulated nucleic acid lipid particles were carried out using the mRNA described in Example 31. The results are shown in Table 7. Characterization revealed that these nucleic acid-lipid particles had an average particle size of approximately 110 nm to approximately 130 nm, with more than 95% of the mRNA encapsulated within the lipid particles.

[0100] [Example 33] Preparation of mRNA-encapsulated nucleic acid-lipid particles using SARS-CoV-2 RBD mRNA described in Example 6 The preparation and characteristic evaluation of mRNA-encapsulated nucleic acid lipid particles using the mRNA described in Example 6 were carried out in the same manner as in Example 8. The results are shown in Table 7. The results of the characterization revealed that the nucleic acid-lipid particles had 95% or more of the mRNA encapsulated within the lipid particles and had an average particle size of approximately 110 nm.

[0101] [Table 3]

[0102] [Table 4]

[0103] [Table 5]

[0104] [Table 6]

[0105] [Table 7]

[0106] [Test Example 1] Administration (Figures 2-4) The test substance was administered to the calf of the hind limb of the mouse under 1-4% (v / v) vaporized isoflurane anesthesia. In the three-times administration test, additional administration was performed 7 and 21 days after the initial administration (first administration: right hind limb, second administration: left hind limb, third administration: right hind limb), and in the two-times administration test, additional administration was performed 13 days after the initial administration (first administration: right hind limb, second administration: left hind limb). The test substance was administered at 3 μg mRNA / 20 μL / body or 1 μg mRNA / 20 μL / body per administration (referred to as Example No._3 and Example No._1 in Figures 2-4, respectively. For example, the description of Example 3_3 in Figures 2-4 means that the group was administered 3 μg mRNA / 20 μL / body of the particles of Example 3). The administration solution was prepared using a 10 mM histidine buffer containing 300 mM sucrose, pH 6.5. S1 protein (Sino Biological, Cat#40591-V08H) supplemented with a commercially available saponin adjuvant (Quil-A Adjuvant, Invivogen, Cat#vac-quil) was used as a positive control for anti-RBD antibody response (S1 / Quil-A group). S1 protein and Quil-A were administered at 1 μg S1 and 10 μg Quil-A / 20 μL / body per administration.

[0107] Preparation of serum and spleen cells Blood obtained from the tail vein at the time of administration of the test substance was collected in a tube containing a serum separator (BD, Cat#365967), and serum was collected after centrifugation (15,000 rpm, 4°C, 5 minutes, centrifuge: TOMY, MX-205). Blood obtained from the heart 14 days after the final administration of the three-dose test was collected in a tube and left at room temperature for 3 hours, then left at room temperature for 22 hours in a refrigerator set at 4°C, and centrifuged (1700×g, 4°C, 5 minutes) to collect serum. In addition, spleens were collected from mice killed by exsanguination under isoflurane anesthesia, and a cell suspension was prepared using a cell strainer (CORNING, Cat#352350), and hemolysis was performed using ACK solution (Lysing Buffer, BD, Cat#555899) to prepare spleen cells.

[0108] Protein Expression Analysis The particles of Example 3 or 4 were added to Expi293F cells (Thermo Fisher Scientific, Cat# A14527) so that the mRNA concentration in the medium was 10 μg / mL. In addition, as a negative control, an equal amount of buffer was added to the amount of the particles of Example 4. The culture supernatant and cell pellet were collected 3 days after the addition. The cell pellet was dissolved in M-PER (Thermo Fisher Scientific, Cat# 78501) supplemented with 1× Protease / Phosphatase inhibitor (Thermo Fisher Scientific, Cat# 78443), and the cell lysate was collected after centrifugation (9100×g, 4° C., 10 minutes). The culture supernatant diluted 810-fold and 2430-fold with D-PBS and the cell lysate diluted 10-fold and 30-fold with D-PBS were immobilized on a 96 half-well plate (Coaster, Cat# 3690), and the protein expressed by the particles of Example 3 or 4 was detected by Enzyme-Linked Immunosorbent Assay (ELISA) using an anti-RBD antibody (Sino Biological, Cat# 40592-T62).

[0109] Anti-RBD antibody titers in blood (Figures 2-4) Recombinant RBD protein (Sino Biological, Cat#40592-V08H) was added to Ni plates (QIAGEN, Cat#35061) at 0.25 μg / mL in blocking solution (1% BSA, 0.05% Tween 20 in PBS) (50 μL / well), left at room temperature for 2 hours, and then washed three times with 300 μL / well of washing solution (0.05% Tween 20 in PBS). The sample dilution series was made in eight stages of 4-fold dilution from the highest concentration of 100-fold diluted serum using blocking solution. The standard serum dilution series was made in eight stages of 3-fold dilution from the highest concentration of 2 DS UNIT / mL using blocking solution. The sample dilution solution and standard serum dilution solution were added (50 μL / well), left at room temperature for 1 hour, and then washed three times with washing solution. The detection antibody was HRP-labeled anti-mouse IgG antibody (Southern Biotech, Cat#1030-05), which was diluted 4000-fold with blocking solution and added to the plate (50 μL / well), and then left to stand at room temperature for 1 hour. After washing three times with washing solution, TMB Microwell Peroxidase Substrate System (SERACARE Life Sciences, Cat#5120-0047) was added (50 μL / well) and left to stand for 10 minutes. TMB Stop Solution (SERACARE Life Sciences, Cat#5150-0021, 50 μL / well) was used as the reaction stop solution. The absorbance at a wavelength of 450 nm (control wavelength 540 nm) was measured using a plate reader, and the corrected absorbance (Delta) obtained by subtracting the absorbance measured at 540 nm from the absorbance measured at 450 nm was used for analysis. A standard curve was created from the anti-RBD antibody concentration and Delta of the standard serum using Nonlinear Regression: 4 Parameters. The anti-RBD antibody concentration of the sample was calculated from the standard curve, the dilution ratio of the measurement sample, and Delta. The average antibody concentration of the wells where Delta was 0.5 to 1.5 was calculated as the anti-RBD antibody concentration of the measurement sample. If Delta of the highest sample concentration well was less than 0.5, 20 DS UNIT / mL was substituted for the data.

[0110] RBD-hACE2 binding inhibitory activity 10 μg / mL Streptavidin (Thermo Fisher Scientific, Cat#21125, dissolved in PBS) was added to a 96 half-well plate (Coaster, Cat# 3690), and the plate was left to stand overnight at 4°C, after which it was washed three times with washing solution (PBS containing 0.05% Tween 20). Blocking solution (1% BSA, PBS containing 0.05% Tween 20) was added, the plate was left to stand for 1 hour at room temperature, and then it was washed three times with washing solution. Then, 0.2 μg / mL recombinant RBD protein (Acro Biosystems, Cat#SPD-C82E9) solution prepared in blocking solution was added to the plate, the plate was left to stand for 1 hour at room temperature, and then it was washed three times with washing solution. Mouse serum diluted 20-fold with blocking solution was added to the plate, the plate was left to stand for 1 hour at room temperature, and then it was washed three times with washing solution. A 1 μg / mL solution of recombinant hACE2 protein (Acro Biosystems, Cat#AC2-H5257) prepared in blocking solution was added to the plate, left to stand at room temperature for 1 hour, and then washed three times with washing solution. The detection antibody was an HRP-labeled anti-human IgG1 antibody (CYGNUS TECHNOLOGIES, Cat#IM50) diluted 500-fold in blocking solution, added to the plate, and left to stand at room temperature for 1 hour. After washing three times with washing solution, TMB Microwell Peroxidase Substrate System (SERACARE Life Sciences, Cat#5120-0047) was added and left to stand for 10 minutes. TMB Stop Solution (SERACARE Life Sciences, Cat#5150-0021) was used as the reaction stop solution. The absorbance at a wavelength of 450 nm was measured and analyzed using a plate reader.

[0111] SARS-CoV-2 epitope peptide pool We commissioned the synthesis of 253 overlapping peptides (#1 to #253) to cover the entire S region of SARS-CoV-2 (Eurofins). Each peptide was dissolved in 200 μL of dimethyl sulfoxide (DMSO, Nacalai Tesque, Cat#13408-64). Equal amounts of #1 to #62, #63 to #107, and #108 to #253 were mixed to prepare three epitope peptide pools (Euro1, Euro2, and Euro3, respectively) to cover the RBD and the regions before and after it. In addition, a commercially available epitope peptide pool (JPT, Cat#PM-WCPV-S-1, 2 vials, a peptide pool covering the N-terminal region is JPT-N, and a peptide pool covering the C-terminal region is JPT-C) covering the entire S region of SARS-CoV-2 was dissolved in 40 μL of DMSO per vial.

[0112] RBD-specific cellular immune response Spleen cells were prepared in RPMI Complete medium (containing 10% FBS [Sigma-Aldrich, Cat#172012-500ML], 1% PS [Penicilin-Streptomycin Mixed Solution, Nacalai Tesque, Cat#26253-84], 1 mM Sodium Pyruvate [Thermo Fisher Scientific, Cat#11360-070], 10 mM HEPES [Thermo Fisher Scientific, Cat#15630080], 1x StemSure [Fujifilm Wako Pure Chemical Industries, Ltd., Cat#195-15791], and 1x MEM Non-Essential Amino Acids Solution [Thermo Fisher Scientific, Cat#11140-050]) at a concentration of 1x107 cells / mL and seeded into a U-bottom 96-well plate. Epitope peptide pool Euro 1-3 solutions prepared in RPMI Complete medium to a final concentration of 0.1% (v / v) and commercial epitope peptide pools JPT-N and JPT-C prepared to a final concentration of 0.025% (v / v) were added to spleen cells and cultured for 48 hours under conditions of 37°C and 5% CO2. The amounts of IFN-γ and IL-13 cytokines in the cell culture supernatant were measured using Mouse IFN-γ DuoSet ELISA (R&D Systems, Cat#DY485) and Mouse IL-13 Duoset ELISA (R&D systems, Cat#DY413). The absorbance at a wavelength of 450 nm (control wavelength 540 nm) was measured using a plate reader, and the corrected absorbance (Delta) obtained by subtracting the absorbance measured at 540 nm from the absorbance at 450 nm was used for analysis. A standard curve was created using nonlinear regression: 4 parameters from the cytokine concentrations and delta values ​​of the standard solutions, and the cytokine concentrations of the measurement samples were calculated from the standard curve. When the IL-13 concentration was less than 0.000 (<0.000), a cutoff value of 0.005 was substituted for the data.

[0113] statistical analysis To compare the anti-RBD antibody response in blood and the RBD-hACE2 binding inhibitory activity, a t-test was performed for the three-dose test, and a Dunnett test was performed for the two-dose test using the Buffer group as a control. To compare the RBD-specific cellular immune response, a Dunnett test was performed for each peptide treatment using the S1 / Quil-A group as a control. All analyses were performed using SAS ver. 9.2.

[0114] Administration to mice (Figures 5-9, 25-28) Under 1-4% (v / v) vaporized isoflurane anesthesia, the test substance was administered to the hind calf of BALB / c mice (FIGS. 5-8, 25, 26, and 28) or C57BL / 6 mice (FIG. 9) twice at 2-week intervals (FIG. 5) or twice at 3-week intervals (FIGS. 6-9, 25, 26, and 28). In FIG. 27, the test substance was administered only once to the hind calf of a BALB / c mouse. The test substance was administered at 0.03, 0.3, or 3 μg mRNA / 20 μL / body per administration (for example, the description of Example 8_0.03 in FIG. 5 means that the particles of Example 8 were administered at 0.03 μg mRNA / 20 μL / body). 2 μg mRNA / 20 μL / body was administered per time in Figures 25 and 27, and 3 μg mRNA / 20 μL / body was administered per time in Figures 26 and 28. The administration solution was prepared using a 10 mM histidine buffer solution containing 300 mM sucrose, pH 7.0.

[0115] Administration to monkeys (Figure 29) Example 10 was administered to the deltoid muscle of the upper arm of a cynomolgus monkey three times at two-week intervals. Example 10 was administered at 50 μg mRNA / 200 μL / body per administration. The administration solution was prepared using a 10 mM histidine buffer solution containing 300 mM sucrose, pH 7.0.

[0116] Anti-RBD antibody titers in blood (Figures 5, 6, 9, and 25-27) Streptavidin (Thermo Fisher Scientific Inc.) solid phase solution was added to the ELISA plate at 25 μL / well and left to stand overnight in a refrigerator set at 4°C. The plate was washed three times with Wash Buffer (180 μL / well) using a plate washer (AMW-96SX, Biotech Co., Ltd.), 1% BSA / PBST was added (150 μL / well), and the plate was left to stand at room temperature for more than 1 hour for blocking. After washing three times with Wash Buffer (180 μL / well) using a plate washer, RBD solution (Original strain RBD: Acro Biosystems, Cat# SPD-C82E9, 351 strain RBD: Sino Biological, Cat# 40592-V08H85-B) was added (25 μL / well) and the plate was left to stand at room temperature for more than 1 hour. After washing three times with Wash Buffer (180 μL / well) using a plate washer, serial dilutions of the measurement sample and serial dilutions of the standard serum (Figures 5, 6, 9, 25, and 26) were added (25 μL / well) and left to stand at room temperature for more than 1 hour. As the standard sample in Figure 27, serial dilutions of an anti-RBD antibody (clone #3) that binds equally to the Original-derived RBD and the B.1.351 strain-derived RBD were used. After washing three times with Wash Buffer (180 μL / well) using a plate washer, a detection antibody dilution of HRP-labeled anti-mouse IgG antibody (Southern Biotech, Cat#1030-05) was added (25 μL / well) and left to stand at room temperature for 1 hour. After washing three times with washing solution, TMB Microwell Peroxidase Substrate System (SERACARE Life Sciences, Cat#5120-0047) was added (30 μL / well) and left to stand for 10 minutes. The reaction was stopped with TMB Stop Solution (SERACARE Life Sciences, Cat# 5150-0021, 30 μL / well). The absorbance at a wavelength of 450 nm (control wavelength 540 nm) was measured using a plate reader, and the corrected absorbance (Delta) obtained by subtracting the absorbance measured at 540 nm from the absorbance measured at 450 nm was used for analysis.A standard curve was created from the anti-RBD antibody concentration and Delta of the standard serum using Nonlinear Regression: 4 Parameters. The anti-RBD antibody concentration of the sample was calculated from the standard curve, the dilution ratio of the measurement sample, and Delta.

[0117] Anti-SARS-CoV-2 neutralizing activity in blood (Figures 7 and 8) VeroE6 cells were seeded on plates and cultured overnight in an incubator set at 37 ± 2°C and 5 ± 1% CO2. A dilution series of mouse serum was mixed with the SARS-CoV-2 WA1 / 2020 strain and left to stand for 2 to 2.5 hours in an incubator set at 37 ± 2°C and 5 ± 1% CO2. The mixture of mouse serum and SARS-CoV-2 WA1 / 2020 strain was then added to VeroE6 cells and cultured for 72 ± 8 hours in an incubator set at 37 ± 2°C and 5 ± 1% CO2. The amount of viable cells was then measured using CellTiter-Glo (Promega), and the anti-SARS-CoV-2 neutralizing activity titer of the mouse serum was calculated.

[0118] RBD-specific cellular immune responses (Figure 10) Spleen cells were cultured in RPMI Complete medium at 1 × 10 7 The MHC class II subunits of RBD were adjusted to a final concentration of 0.1% (v / v) in RPMI Complete medium and seeded in a U-bottom 96-well plate. The epitope peptide pool was added to spleen cells and cultured for 48 hours under conditions of 37℃ and 5% CO2. The amounts of IFN-γ and IL-13 cytokines in the cell culture supernatant were measured using Mouse IFN-γ DuoSet ELISA and Mouse IL-13 Duoset ELISA. The absorbance at a wavelength of 450 nm (control wavelength 540 nm) was measured using a plate reader, and the absorbance measured at 540 nm was subtracted from the absorbance at 450 nm for analysis. A calibration curve was created from the cytokine concentrations of the standard solutions and the measured values ​​using Nonlinear Regression: 4 Parameters, and the cytokine concentrations of the measurement samples were calculated from the calibration curve.

[0119] statistical analysis For the blood anti-RBD antibody response shown in Figure 5, a Wilcoxon test was performed to compare two groups at doses of 0.03 μg mRNA / body and 0.3 μg mRNA / body. A Steel test was performed to compare three groups at a dose of 3 μg mRNA / body, using Example 8 as a control. For the anti-RBD antibody responses in blood shown in FIG. 6, the Wilcoxon test was performed to compare the two groups at each dose of 0.03 μg mRNA / body, 0.3 μg mRNA / body, and 3 μg mRNA / body. For the anti-SARS-CoV-2 neutralizing activity in blood shown in Figure 7, a Steel test was performed using the Buffer group as a comparison control. For the comparison between the two groups in Figure 8, a Wilcoxon test was performed. For the anti-RBD antibody response in blood shown in FIG. 9, a Steel assay was performed using the Buffer group as a comparative control. Steel-Dwass assay was performed for RBD-specific cellular immunity shown in FIG. All analyses were performed using SAS version 9.2.

[0120] RBD-hACE2 binding inhibitory activity (Figure 28) Anti-His tag antibody (Wako Pure Chemical Industries, Cat# 017-23211) was added to a 96 half-well plate, and the plate was left to stand overnight at 4°C, and then washed three times with washing solution (PBS containing 0.05% Tween 20). Blocking solution (PBS containing 1% BSA and 0.05% Tween 20) was added, and the plate was left to stand at room temperature for 1 hour, and then washed three times with washing solution. Then, 0.2 μg / mL of recombinant RBD protein (Control: Acro Biosystems, Cat# SPD-S52H6, Original: Sino Biological, Cat# 40592-V08H, K417N: Sino Biological, Cat# 40592-V08H59, E484K: ACRO Biosystems, Cat# SRD-C52H3, N501Y: Sino Biological, Cat# 40592-V08H82, K417N / E484K / N501Y: ACRO Biosystems, Cat# SPD-C52Hp) solution prepared in blocking solution was added to the plate, left at room temperature for 1 hour, and then washed three times with washing solution. A dilution series of mouse serum diluted in blocking solution was added to the plate, left at room temperature for 1 hour, and then washed three times with washing solution. A 1 μg / mL solution of recombinant hACE2 protein (Acro Biosystems, Cat#AC2-H5257) prepared in blocking solution was added to the plate, left to stand at room temperature for 1 hour, and then washed three times with washing solution. The detection antibody was an HRP-labeled anti-human IgG1 antibody (CYGNUS TECHNOLOGIES, Cat#IM50) diluted 500-fold in blocking solution, added to the plate, and left to stand at room temperature for 1 hour. After washing three times with washing solution, TMB Microwell Peroxidase Substrate System (SERACARE Life Sciences, Cat#5120-0047) was added and left to stand for 10 minutes. TMB Stop Solution (SERACARE Life Sciences, Cat#5150-0021) was used as the reaction stop solution.The absorbance at a wavelength of 450 nm (control wavelength 540 nm) was measured using a plate reader, and the corrected absorbance (Delta) obtained by subtracting the absorbance measured at 540 nm from the absorbance measured at 450 nm was used for analysis. The data shows the mouse dilution ratio showing 50% inhibition (IC). 50 ).

[0121] Blood anti-SARS-CoV-2 neutralizing activity (Figure 29) Vero-TMPRSS2 cells were plated. Dilution series of monkey plasma and 100TCID 50 SARS-CoV-2 strains (D614G: HP095, B.1.1.7 lineage: QHN001, P.1 lineage: TY7-501, B.1.351 lineage: TY8-612) were mixed and placed in a CO2 incubator. The mixture of monkey plasma and SARS-CoV-2 was then added to Vero-TMPRSS2 cells and cultured for 3 days in a CO2 incubator. The highest dilution ratio at which cytopathic effect (CPE) was no longer observed was calculated as the neutralizing antibody titer.

[0122] result Example 4: RBD protein expression induction ability It has been suggested that the mechanism of action of the nucleic acid lipid particle vaccine of the present invention is that after administration into the body, an antigen protein is produced from mRNA encoding an antigen gene, and a specific immune response to the antigen is induced. It is assumed that the delivery of the active ingredient mRNA into tissues and cells and the translation from the mRNA are important factors in the efficacy of the nucleic acid lipid particle vaccine of the present invention. In order to comprehensively evaluate this series of factors, the titer was evaluated using cultured cells as an index of the ability to induce expression of the antigen protein. The particles of Example 3, the particles of Example 4, or Buffer were added to Expi293F cells, and the RBD protein expressed in the culture supernatant and in the cells after 3 days was quantified by ELISA. The results are shown in Figure 1. The RBD protein expressed by the particles of Example 4 was found in the culture supernatant and in the cells. The full-length S protein expressed by the particles of Example 3 was found only in the cells.

[0123] Circulating anti-RBD antibody response The blood anti-RBD antibody response induced by administration of the particles of Example 3 or Example 4 was evaluated. The results are shown in Figure 2. The 3-times administration group of Example 4 had a higher blood anti-RBD antibody titer than the 2-times administration group and 3-times administration group of Example 3 (P = 0.0346). Furthermore, the 2-times administration group of Example 4 had a higher blood anti-RBD antibody titer than the Buffer group (Example 4_3; P = 0.0019, Example 4_1; P = 0.0313).

[0124] RBD-hACE2 binding inhibitory activity The RBD-hACE2 binding inhibitory activity induced by administration of the particles of Example 3 or Example 4 was evaluated. The results are shown in Figure 3. Compared with the two-time administration group and the three-time administration group of Example 3, the serum of the three-time administration group of Example 4 had higher RBD-hACE2 binding inhibitory activity (P = 0.0005). In addition, compared with the Buffer group, the serum of the two-time administration group of Example 4 had higher RBD-hACE2 binding inhibitory activity (Example 4_3; P < 0.0001, Example 4_1; P = 0.0006).

[0125] RBD-specific cellular immune response Spleen cells were prepared, and RBD-specific cellular immune responses from the cultured spleen cells were evaluated. The results are shown in FIG. 4. Compared to the S1 / Quil-A group, the Example 4 group had a higher IFN-γ production level in response to treatment with Euro2 and JPT-N epitope peptide pools covering the RBD (P < 0.001). On the other hand, compared to the S1 / Quil-A group, the Example 4 group had a lower IL-13 production level in response to treatment with Euro2 and JPT-N epitope peptide pools (P < 0.005). These results demonstrated that the nucleic acid lipid particle vaccine of the present invention induces an immune response in which Th1 type is predominant.

[0126] Circulating anti-RBD antibody responses in BALB / c mice The blood anti-RBD antibody response induced by administration of the particles of Example 8 or Example 4 was evaluated. The results are shown in Figure 5. At doses of 0.03 μg mRNA / body and 0.3 μg mRNA / body, Example 8 had higher blood anti-RBD antibody titers than Example 4 (both P = 0.0286). Furthermore, at a dose of 3 μg mRNA / body, no significant difference was observed in blood anti-RBD antibody titers among Examples 4, 7, and 8 (both P = 0.061). The blood anti-RBD antibody response induced by administration of the particles of Example 10 or Example 8 was evaluated. The results are shown in Figure 6. No significant difference was observed in the blood anti-RBD antibody titer between Example 10 and Example 8 at any dose (0.03 μg mRNA / body: P = 0.8413, 0.3 μg mRNA / body: P = 0.0952, 3 μg mRNA / body: P = 0.6905).

[0127] Blood anti-SARS-CoV-2 neutralizing activity The blood anti-SARS-CoV-2 neutralizing activity induced by administration of the particles of Example 10 was evaluated. The results are shown in FIG. 7. Compared to the Buffer group, the group administered 3 μg mRNA / body of Example 10 had higher blood anti-SARS-CoV-2 neutralizing activity (P = 0.0374). In addition, a comparison of the blood anti-SARS-CoV-2 neutralizing activity induced by administration of the particles of Example 8 and Example 10 showed no significant difference (FIG. 8, P = 1).

[0128] Circulating anti-RBD antibody responses in C57BL / 6 mice The blood anti-RBD antibody response induced by administration of the particles of Example 8 or Example 10 was evaluated. The results are shown in Figure 9. At doses of 3 μg mRNA / body and 10 μg mRNA / body, the blood anti-RBD antibody titers were higher in Example 8 and Example 10 compared to the Buffer group (P < 0.05 for both doses of Example 10).

[0129] RBD-specific cellular immune response Spleen cells were prepared and RBD-specific cellular immune responses from the cultured spleen cells were evaluated. The results are shown in Figure 10A. Compared to the group receiving 0.1 μg / body of RBD protein plus 100 μg / body of Alum adjuvant, the group receiving 3 μg / body of Example 10 showed higher IFN-γ induction (P < 0.05). Also, compared to the group receiving 1.0 μg / body of RBD protein plus 100 μg / body of Alum adjuvant, the groups receiving 0.03 μg / body and 3 μg / body of Example 10 showed higher IFN-γ induction (both P < 0.05). To evaluate the Th cell profile of Example 10, the IFN-γ level / IL-5 level ratio and the IFN-γ level / IL-13 level ratio were analyzed. The results are shown in FIG. 10B. Compared with the Alum-adjuvanted RBD protein group, Example 10 showed a higher IFN-γ level / IL-13 level ratio (P < 0.05 for all three groups of Example 10 compared to the two groups of Alum-adjuvanted RBD protein). This result demonstrated that the nucleic acid lipid particle vaccine of the present invention induces an immune response in which Th1 type is predominant.

[0130] Circulating anti-RBD antibody responses in BALB / c mice (Figures 25-27) The anti-RBD antibody response in the blood induced by administration of the particles of Example 10, 12, 14, 16, 18, or Example 20 was evaluated. The results are shown in Figure 25. All groups showed higher levels of anti-RBD antibody in the blood compared to the Buffer group. The anti-RBD antibody response in the blood induced by administration of the particles of Example 10 or Examples 21-30 was evaluated. The results are shown in Figure 26. Higher anti-RBD antibody titers in the blood were observed in all particles compared to the Buffer group. The blood anti-RBD antibody response induced by administration of the particles of Example 10, 32a, 32b, 32c, 32d, 32f, or Example 33 was evaluated. The results are shown in Figure 27. Compared to Example 32a, Examples 10, 32b, 32c, 32d, 32f, and Example 33 showed higher blood anti-RBD antibody levels.

[0131] RBD-hACE2 binding inhibitory activity (Figure 28) The RBD-hACE2 binding inhibitory activity induced by the particles of Example 10 was evaluated. The results are shown in Figure 28. Compared to the control RBD, the binding of the original RBD, K417N, E484K, N501Y, or K417N / E484K / N501Y RBD mutants to hACE2 was inhibited to the same extent by the serum of Example 10 group.

[0132] Blood anti-SARS-CoV-2 neutralizing activity (Figure 29) The anti-SARS-CoV-2 neutralizing activity in blood induced by the particles of Example 10 was evaluated. The results are shown in Figure 29. Infection of Vero-TMPRSS2 cells with the D614G strain, B.1.1.7 strain, P.1 strain, and B.1.351 strain was neutralized to the same extent by the serum of Example 10 group.

[0133] [Test Example 2] Optimization of LNP-mRNA vaccine candidates encoding the SARS-CoV-2 RBD Following the outbreak of the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), two types of mRNA vaccines encoding the full-length SARS-CoV-2 spike protein have been launched (1, 2). However, there are still areas that need improvement regarding side effects such as fever.

[0134] We optimized an mRNA vaccine candidate (LNP-mRNA-RBD) in which mRNA encoding the receptor-binding domain (RBD) contained in the SARS-CoV-2 spike protein was encapsulated in lipid nanoparticles (LNPs) using immunogenicity as an indicator.

[0135] First, 6-8 week-old C57BL / 6 mice or BALB / c mice were intramuscularly administered 3 μg of LNP-mRNA-RBD (equivalent to mRNA) twice at two-week intervals, and the anti-RBD antibody response in the blood was evaluated. As a result, BALB / c mice showed higher anti-RBD antibody responses in the blood compared to C57BL / 6 mice (Figure 11a, Figure 15). To compare the RBD-specific B cell response induced by LNP-mRNA-RBD between mouse strains, T cells in the popliteal lymph nodes (pLN) of LNP-mRNA-RBD-administered mice were analyzed. FH and GC B cells were analyzed by flow cytometry (Figure 16). The results showed that T and GC B cells in pLNs correlated with the anti-RBD antibody response in the blood. FH (CD4 + CD185 + PD-1 + cells) and GC B cells (CD38 - GL7 + CD19 + The number of cells was higher in LNP-mRNA-RBD-treated BALB / c mice compared with C57BL / 6 mice (Fig. 11b-e).

[0136] Antigen-specific CD8 induced by LNP-mRNA-RBD + and CD4 +To analyze T cells, a peptide library of the spike protein was designed. This peptide library consisted of 128 peptides of 20 amino acids each designed to overlap by 10 amino acids. This peptide library was divided into a total of eight pooled peptides, with 16 peptides per pooled peptide (Fig. 11f). When spleen cells prepared from mice administered LNP-mRNA-RBD were treated with pooled peptides 3 and 4, IFN-γ production was induced in C57BL / 6 mouse spleen cells, and IFN-γ production was confirmed in BALB / c mouse spleen cells treated with peptide pool 3 (Fig. 11g and h, Fig. 17a and b). IL-13 was not induced in either C57BL / 6 or BALB / c mouse spleen cells (Fig. 17c and d). To analyze the antigen-specific T cells induced by LNP-mRNA-RBD administration, spleen cells were treated with pooled peptides 2, 3, or 4, and T cells producing three cytokines (IL-2, IFN-γ, and TNF-α) were analyzed by flow cytometry. As a result, spike antigen-specific polyfunctional CD8 + and CD4 + T cells were observed in BALB / c mouse spleen cells treated with pool peptides 3 and 4 (Fig. 11h, Figs. 18b and 19b). In C57BL / 6 mouse spleen cells, polyfunctional CD8 + T cells and weak CD4 + 11g, 18a and 19a). These data suggest that LNP-mRNA-RBD-administered BALB / c mice may induce higher B and T cell responses than C57BL / 6 mice.

[0137] It has been reported that nucleic acid-based vaccines, DNA or RNA, act as intrinsic adjuvants (14-16). In LNP-mRNA vaccines, mRNA is recognized by toll-like receptor (TLR) 3, TLR7, TLR8, RIG-I, or MDA5 and acts as an intrinsic adjuvant (17). Kariko et al. have used methylated bases and other modified bases (e.g., pseudouridine) to control innate immune activation and improve the expression efficiency of antigen proteins (18, 19). Other studies have shown that type I IFN induced by LNP-mRNA enhances CD8 + It has been shown that it affects T cell responses and the expression efficiency of antigen proteins (20, 21, 22). Regarding SARS-CoV-2 vaccines, the frequency of side effects was higher in the LNP-mRNA-RBD group compared to the LNP-mRNA-Full group, which encodes the full-length spike, so LNP-mRNA-Full was evaluated in a phase III clinical trial and launched (13). The reason for the difference in the frequency of side effects is unclear, but we believe that the natural immune stimulatory effect of LNP-mRNA may be involved (13).

[0138] To analyze the innate immune stimulatory effect of LNP-mRNA, the level of type I IFN production from human PBMCs treated with LNP-mRNA-RBD was measured by ELISA. As a result, LNP-mRNA-RBD showed higher IFN-α induction ability in PBMCs from three healthy individuals compared to LNP-mRNA-Full (Figure 12a). Next, a similar experiment was performed using bone marrow-derived dendritic cells (BM-DCs) from C57BL / 6 mice or BALB / c mice. As a result, BM-DCs from C57BL / 6 mice treated with LNP-mRNA-full or LNP-mRNA-RBD showed higher IFN-α production compared to BM-DCs from BALB / c mice (Figure 12b). The manufacturing process of mRNA encapsulated in LNP-mRNA contains RNA as a contaminant, such as double-stranded RNA as a TLR3 ligand, which may activate innate immunity (22). Therefore, to remove impurities resulting from RNA production, we purified mRNA by HPLC and prepared LNP-mRNA (mRNA-RBD (HPLC)) containing the HPLC-purified mRNA. As a result, type I IFN production from human PBMCs and mouse BM-DCs treated with mRNA-RBD (HPLC) was significantly reduced compared to LNP-mRNA-RBD (Figures 12a and b).

[0139] We administered mRNA-RBD (HPLC) to C57BL / 6 or BALB / c mice to evaluate its immunogenicity. As a result, the mRNA-RBD (HPLC) group enhanced the anti-RBD IgG1, IgG2, and total IgG titers in the blood in both BALB / c and C57BL / 6 mice (Fig. 12c and Fig. 20a). Furthermore, compared with the LNP-mRNA-RBD group, the pLN of C57BL / 6 mice administered mRNA-RBD (HPLC) induced a high number of GC B cells (Fig. 12d and e). Furthermore, compared with the LNP-mRNA-RBD group, the mRNA-RND (HPLC) group showed a high number of RBD-specific polyfunctional CD8 cells that produce IFN-γ and other type I cytokines. + and CD4 + A large number of T cells were induced (Figs. 12f-i, and Figs. 20b-e, 21, 22). The protective effect of the mRNA-RBD (HPLC) vaccine against SARS-CoV-2 infection was evaluated in a non-human primate (NHP) cynomolgus monkey model. In this study, mRNA-RBD (HPLC) was administered intramuscularly to four monkeys, with two monkeys serving as negative controls. As a result, compared with the negative control group, the mRNA-RBD (HPLC) group showed a higher anti-RBD antibody response (Figure 13b). The mRNA-RBD (HPLC) group also showed anti-SARS-CoV-2 neutralizing activity in the blood (Figure 13c). Furthermore, compared with the negative control group, the mRNA-RBD (HPLC) group showed a higher anti-RBD IgG response in the mucosal tissues of the conjunctiva, nasal cavity, oral cavity, trachea, and rectum (Figure 13d).

[0140] The mRNA-RBD (HPLC) group dramatically reduced SARS-CoV-2 (Figure 14a) and viral RNA (Figure 14b) in swabs on day 1 after SARS-CoV-2 infection. The mRNA-RBD (HPLC) group also reduced viral RNA in the trachea, bronchi, and lungs on day 7 after infection (Figure 14c, Figure 25). Furthermore, the negative control group showed fever and pneumonia after SARS-CoV-2 infection (Figures 23, 24). Lung tissue analysis after SARS-CoV-2 infection revealed infiltration of lymphocytes and neutrophils, thickening of the alveolar walls, and viral antigens in the negative control group, but these phenomena were not observed in the mRNA-RBD (HPLC) group (Fig. 14d, 14e). In addition, bronchus-associated lymphoid tissue (BALT) was confirmed to have been formed in the mRNA-RBD (HPLC) group (Fig. 14d). These results suggest that antibodies induced through the formation of BLAT in mucous membranes such as the nasal mucosa and tracheal mucosa may neutralize SARS-CoV-2 by binding to it, resulting in a decrease in viral RNA and infectious virus in swabs on the first day after infection.

[0141] Materials and Methods mouse Six- to eight-week-old C57BL / 6 and BALB / c mice were purchased from CLEA, Japan. Mice were housed under specific pathogen-free conditions. All mouse studies were approved by the Animal Experiment Committee of the Institute of Medical Science, The University of Tokyo.

[0142] Crab-eating macaque Female cynomolgus monkeys aged 7–10 years, born at Shiga University of Medical Science and native to the Philippines, Vietnam, and China, were used. All procedures were performed under ketamine and xylazine anesthesia, and efforts were made to minimize suffering. CMK-2 (CLEA Japan, Tokyo, Japan) food pellets were given once a day after recovery from anesthesia, and drinking water was available ad libitum. Animals were housed singly in cages under controlled light conditions (12-h light / 12-h dark cycle, lights on at 8:00 a.m.). SARS-CoV-2 (2 × 10) was infused into the conjunctiva (0.05 mL × 2), naris (0.5 mL × 2), oral cavity (0.9 mL), and trachea (5 mL) with a pipette and catheter under ketamine / xylazine anesthesia. 7 Monkeys were inoculated with 1000 PFU / 7mL HBSS. Under ketamine / xylazine anesthesia, fluid samples from the conjunctiva, nasal cavity, oral cavity, and trachea were collected using two cotton swabs (Eiken Chemical Co., Ltd., Tokyo, Japan), which were then dipped into 1 mL of Dulbecco's modified Eagle's medium (DMEM, Nacalai Tesque, Kyoto, Japan) containing 0.1% bovine serum albumin (BSA) and antibiotics. Bronchial samples were collected using a bronchoscope (MEV-2560; Machida Endoscope Co., Ltd., Tokyo) and a cytology brush (BC-203D-2006; Olympus Corporation, Tokyo, Japan).

[0143] LNP-mRNA vaccine The mRNA-encapsulated nucleic acid-lipid particles of Example 10 were used.

[0144] reagent Overlapping 20-aa peptides of the spike protein were synthesized and purchased from Eurofins Genomics (Ebersberg, Germany). SARS-CoV-2 spike protein (ECD) and RBD were purchased from GenScript (Piscataway, NJ, USA).

[0145] virus SARS-CoV-2 isolates were propagated in VeroE6 cells at 37°C in Opti-MEM I (Invitrogen, Carlsbad, CA, USA) containing 0.3% bovine serum albumin (BSA) and 1 μg L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin / mL.

[0146] Methods of immunization Six to eight week old C57BL / 6 and BALB / c mice were immunized intramuscularly with mock, LNP-mRNA-RBD (3 μg) or LNP-mRNA-RBD (HPLC) (3 μg) on ​​days 0 and 14. Popliteal lymph nodes, spleens and blood were collected 2 weeks after the second immunization. Cynomolgus monkeys were immunized intramuscularly with mock or LNP-mRNA-RBD (HPLC) (100 μg) on ​​days 0 and 21. Blood was collected on days 0, 7, 14, 21 and 28.

[0147] ELISA method ECD and RBD specific antibody titers were measured by ELISA. Briefly, half-area 96-well plates were coated with ECD (1 μg / mL) or RBD (1 μg / mL) in bicarbonate buffer at 4°C. Plates were blocked with PBS containing 1% BSA for 60 min at room temperature. Plates were washed three times with PBST and incubated with diluted plasma or swab samples for 120 min at room temperature. Plates were washed three times with PBST and incubated with HRP-conjugated goat anti-mouse IgG, IgG1, IgG2a, IgG2c, or mouse anti-monkey IgG for 120 min at room temperature. After washing three times with PBST, TMB substrate buffer was added and incubated for 10 min at room temperature. The reaction was then stopped by adding 1 N H2SO4. OD values ​​at 450 nm and 540 or 560 nm were measured using a spectrophotometer. OD 450 -OD 540 or O.D. 450 -OD 560 The reciprocal of the plasma dilution at which the antibody titer was 0.2 was taken as the antibody titer. Single cell suspensions of spleen cells from immunized mice were stimulated with peptide pools 1-8, ECD, and RBD proteins for 24 h. IFN-γ and IL-13 levels in the supernatants were measured by ELISA (R&D).

[0148] GC B cells and T FH staining Single cell suspensions from popliteal lymph nodes were stained with LIVE / DEAD Aqua, anti-CD279 (29F.1A12), anti-CD8a (53-6.7), anti-CD3e (145-2C11), anti-GL7 (GL7), anti-CD4 (RM4-5), anti-CD185 (L138D7), anti-CD38 (90), and anti-CD19 (6D5) antibodies. All antibodies were purchased from BioLegend, San Diego, CA, USA. GC B and T cells FH The percentage of cells was analyzed by flow cytometry.

[0149] Cytokine intracellular staining assay Single cell suspensions of splenocytes were stimulated with peptide pools 2, 3, and 4 with protein transport inhibitor (eBioscience, San Diego, CA, USA) for 6 h. After stimulation, dead cells were stained with LIVE / DEAD Aqua. After washing, cells were stained with anti-CD8a (53-6.7), anti-CD4 (RM4-5: Invitrogen), anti-TCRβ (H57-597), anti-F4 / 80 (RM8), anti-TER-119 (TER-119), anti-CD11b (M1 / 70), anti-CD19 (6D5), anti-CD11c (N418), anti-NK-1.1 (PK136), and anti-CD45R / B220 (RA3-6B2) antibodies. All antibodies were purchased from BioLegend unless otherwise noted. After fixation, permeabilization with IC Fixation Buffer (eBioscience), intracellular cytokines, and CD3 were stained with anti-IFN-γ (XMG1.2), anti-IL-2 (JES6-5H4), anti-TNF-α (MP6-XT22), and anti-CD3 (17A2) antibodies. All antibodies were purchased from BioLegend. Cytokine-producing CD8 + and CD4 +The percentage of T cells was determined by flow cytometry.

[0150] Preparation and stimulation of human peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMCs) were obtained with informed consent from three healthy adult volunteers uninfected with SARS-CoV-2. All experiments using human PBMCs were approved by the ethical review board of the Institute of Medical Science, University of Tokyo. PBMCs were prepared using Ficoll Histopaque and then stimulated with LNP-mRNA-Full (0.4, 2, 10 μg / mL), LNP-mRNA-RBD (0.4, 2, 10 μg / mL), or LNP-mRNA-RBD(HPLC) (0.4, 2, 10 μg / mL) for 24 h, and IFN-α levels in the culture supernatants were measured using ELISA (Mabtech, Stockholm, Sweden).

[0151] Bone marrow-derived dendritic cells and stimulation Bone marrow-derived dendritic cells (BM-DCs) were differentiated by culturing in mouse GM-CSF for 7 days. The cells were stimulated with LNP-mRNA-Full (0.4, 2, 10 μg / mL), LNP-mRNA-RBD (0.4, 2, 10 μg / mL), or LNP-mRNA-RBD (HPLC) (0.4, 2, 10 μg / mL) for 24 hours, and IFN-α in the culture supernatant was measured using ELISA (Invitrogen).

[0152] Neutralizing antibody titers Thirty-five microliters of virus (140 tissue culture infectious doses 50) was incubated with 35 μL of two-fold serially diluted serum for 1 h at room temperature, and 50 μL of the mixture was added to confluent VeroE6 / TMPRS2 cells in 96-well plates and incubated for 1 h at 37°C. After adding 50 μL of DMEM containing 5% FCS, the cells were further incubated at 37°C for 3 days. Viral cytopathic effect (CPE) was observed under an inverted microscope, and the virus neutralization titer was determined as the reciprocal of the highest serum dilution that completely prevented CPE (24).

[0153] Virus titration using VeroE6 / TMPRSS2 against SARS-CoV-2 Confluent Vero E6 cell lines expressing TMPRSS2 (JCRB Cell Bank, Japan) were incubated with diluted swab samples and 10% w / v tissue homogenate samples for 1 h. Cells were washed with HBSS and incubated in DMEM containing 0.1% BSA for 3 days (25). Viral titers were monitored microscopically and calculated using the Reed-Muench method.

[0154] Real-time RT-PCR of viral RNA Viral RNA from swab samples and tissues (20 mg) was collected using the QIAmp viral RNA Mini kit and RNeasy Mini kit, respectively. Viral RNA was measured by real-time RT-PCR (2019-nCoV_N1-F, 2019-nCoV_N1-R, 2019-nCoV_N1-P, TaqMan Fast Virus 1-step Master Mix) using a CFX-96 (Bio-Rad, Hercules, CA, USA).

[0155] Body temperature Two weeks before virus inoculation, two body temperature data loggers (iButton, Maxim Integrated, San Jose, CA) were implanted into the peritoneal cavity or subcutaneous tissue of each monkey under ketamine / xylazine anesthesia followed by inhaled isoflurane to monitor body temperature.

[0156] X-ray Lung radiographs were taken using an I-PACS system (Komica Minolta) and a PX-20BT (Kenko Tokina).

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[0158] The present invention can be used to prevent and / or treat infection by SARS-CoV-2. [Sequence List Free Text]

[0159] <Sequence number 1> (DNA fragment containing SARS-CoV-2 S full) <SEQ ID NO:2> (sense primer) GTAATACGACTCACTATAA <SEQ ID NO:3> (antisense primer) TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCAATGAAAATAAATGTTTTTTATTAGGC <Sequence number 4> (SARS-CoV-2 S-full template DNA) GTAATACGATCACTACACTATA A T7 promoter: base numbers 1 to 18 A :Transcription start site: base number 19 5'-UTR (including transcription initiation site and KOZAK sequence from nucleotides 89 to 94): nucleotides 19 to 88 Spike protein full length sequence: nucleotides 89 to 3910 3'-UTR: base numbers 3911 to 4042 PolyA sequence (A100): base numbers 4043 to 4142 <SEQ ID NO: 5>(SARS-CoV-2 S-full mRNA-001) <SEQ ID NO:6> (Amino acid sequence of SARS-CoV-2 S-full) RBD sequence: amino acid numbers 319 to 541 <SEQ ID NO: 7> (DNA fragment containing SARS-CoV-2 RBD) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTTGTTTTTCTTGTTTTATTGCCACTAGTCTCTAGTAGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <SEQ ID NO: 8> (Template DNA for SARS-CoV-2 RBD) GTAATACGACTCACTATA A T7 promoter: base numbers 1 to 18 A :Transcription start site: base number 19 5'-UTR (including transcription initiation site and KOZAK sequence from nucleotides 89 to 94): nucleotides 19 to 88 Spike protein signal sequence: nucleotides 89-127 RBD sequence: base numbers 128 to 799 3'-UTR: base numbers 800 to 931 PolyA sequence (A100): nucleotide numbers 932 to 1031 <SEQ ID NO:9> (SARS-CoV-2 RBD mRNA-002) <SEQ ID NO: 10> (SARS-CoV-2 RBD amino acid sequence (including S protein signal sequence)) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNY KLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF S protein signal sequence: amino acids 1-13 RBD sequence: amino acid numbers 14 to 236 <SEQ ID NO: 11> (SARS-CoV-2 RBD amino acid sequence (not including S protein signal sequence)) RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF <Sequence number 12>(S_opt2 EcoRI) NheI sequence: nucleotide numbers 1 to 6 T7 promoter: base numbers 7 to 24 A: Transcription start site: base number 25 5'-UTR (including the transcription start site and the KOZAK sequence at base numbers 89 to 94): base numbers 25 to 94 Spike protein full length sequence: nucleotides 95 to 3916 3'-UTR: base numbers 3917 to 4048 EcoRI sequence: base numbers 4049 to 4054 <SEQ ID NO: 13> (sense primer 2) TGATGCTAGCGTAATACGACTCACTATAAG NheI sequence: nucleotide numbers 5 to 10 <SEQ ID NO: 14> (antisense primer 2) GCCAAAGCTTGCTCTTCGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT HindIII sequence: base numbers 5 to 10 BspQI sequence: nucleotides 11 to 17 <Sequence number 15> (SARS-CoV-2 S fully optimized template DNA) NheI sequence: nucleotide numbers 5 to 10 T7 promoter: nucleotide numbers 11 to 28 A: Transcription start site: base number 29 5'-UTR (including the transcription start site and the KOZAK sequence at base numbers 89 to 94): base numbers 29 to 98 Spike protein full length sequence: base numbers 99 to 3920 3'-UTR: base numbers 3921 to 4052 PolyA sequence (A110): base numbers 4053 to 4162 BspQI sequence: base numbers 4164 to 4170 HindIII sequence: base numbers 4171 to 4176 <Sequence number 16>(SARS-CoV-2 S full optimized mRNA-003) <SEQ ID NO: 17> (S_RBD_opt2 EcoRI) GCTAGCGTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCGAATTC NheI sequence: base numbers 1 - 6 T7 promoter: base numbers 7 to 24 A: Transcription start site: base number 25 5'-UTR (including the transcription start site and the KOZAK sequence at base numbers 89 to 94): base numbers 25 to 94 RBD sequence: base numbers 95 to 805 3'-UTR: base numbers 806 to 937 EcoRI sequence: nucleotide numbers 938 to 943 <Sequence number 18> (SARS-CoV-2 RBD optimized template DNA) NheI sequence: nucleotide numbers 5 to 10 T7 promoter: nucleotide numbers 11 to 28 A: Transcription start site: base number 29 5'-UTR (including the transcription start site and the KOZAK sequence at base numbers 89 to 94): base numbers 29 to 98 Spike protein signal sequence: nucleotides 99-137 RBD sequence: base numbers 138 to 809 3'-UTR: base numbers 810 to 941 PolyA sequence (A110): nucleotide numbers 942 to 1051 BspQI sequence: nucleotide numbers 1053 to 1059 HindIII sequence: base numbers 1060 to 1065 <Sequence number 19>(SARS-CoV-2 RBD optimized mRNA-004) <SEQ ID NO: 20> SARS-CoV-2 RBD S2000 template DNA GCTAGCGTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAAAGCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACGAAGAGC NheI sequence: nucleotide numbers 1 to 6 T7 promoter: base numbers 7 to 24 A: Transcription start site: base number 25 5'-UTR sequence (including the transcription start site and the KOZAK sequence at base numbers 89 to 94): base numbers 25 to 94 Spike protein signal sequence: nucleotides 95-133 RBD sequence: base numbers 134 to 805 3'-UTR: base numbers 806 to 937 PolyA sequence (A50): nucleotide numbers 938 to 987 BspQI sequence: nucleotide numbers 989 to 995 <SEQ ID NO: 21> SARS-CoV-2 RBD S2000 mRNA sequence AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAAAGCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUGGAAGGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACAAACGGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAAUGCGUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO:22> SARS-CoV-2 RBD S2001 template DNA GCTAGCGTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAAAGCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAGAGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACGAAGAGC NheI sequence: nucleotide numbers 1 to 6 T7 promoter: base numbers 7 to 24 A: Transcription start site: base number 25 5'-UTR sequence (including the transcription start site and the KOZAK sequence at base numbers 89 to 94): base numbers 25 to 94 Spike protein signal sequence: nucleotides 95-133 RBD sequence: base numbers 134 to 805 3'-UTR: base numbers 806 to 937 PolyA sequence (A50): nucleotide numbers 938 to 987 BspQI sequence: nucleotide numbers 989 to 995 <SEQ ID NO: 23> SARS-CoV-2 RBD S2001 mRNA sequence AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAAAGCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUGGAAGGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACAAACGGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAGAGCGUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 24> Amino acid sequence of SARS-CoV-2 RBD S2001 (including S protein signal sequence) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNY KLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNF S protein signal sequence: amino acids 1-13 RBD sequence: amino acid numbers 14 to 236 <SEQ ID NO: 25> Amino acid sequence of SARS-CoV-2 RBD S2001 (without the S protein signal sequence) RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNF <SEQ ID NO:26> SARS-CoV-2 RBD S2002 template DNA GCTAGCGTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACAAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAAAGCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGACCCAAATGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACGAAGAGC NheI sequence: Base numbers 1 - 6 T7 promoter: Base numbers 7 - 24 A: Transcription start site: base number 25 5'-UTR sequence (including the transcription start site and the KOZAK sequence at base numbers 89 to 94): base numbers 25 to 94 Spike protein signal sequence: nucleotides 95-133 RBD sequence: base numbers 134 to 736 3'-UTR: base numbers 737 to 868 PolyA sequence (A50): nucleotide numbers 869 to 918 BspQI sequence: nucleotides 920 to 926 <SEQ ID NO: 27> SARS-CoV-2 RBD S2002 mRNA sequence AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACAAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAAAGCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUGGAAGGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACAAACGGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGACCCAAAUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 28> Amino acid sequence of SARS-CoV-2 RBD S2002 (including the S protein signal sequence). MFVFLVLLPLVSSFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADY NYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPK S protein signal sequence: amino acids 1-13 RBD sequence: amino acid numbers 14 to 213 <SEQ ID NO: 29> Amino acid sequence of SARS-CoV-2 RBD S2002 (without the S protein signal sequence) FPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPK <SEQ ID NO: 30> SARS-CoV-2 RBD S2003 template DNA NheI sequence: nucleotide numbers 1 to 6 T7 promoter: base numbers 7 to 24 A: Transcription start site: base number 25 5'-UTR sequence (including the transcription start site and the KOZAK sequence at base numbers 89 to 94): base numbers 25 to 94 Spike protein signal sequence: nucleotides 95-133 RBD sequence: base numbers 134 to 874 3'-UTR: base numbers 875 to 1006 PolyA sequence (A50): nucleotide numbers 1007 to 1056 BspQI sequence: nucleotide numbers 1058 to 1064 <SEQ ID NO: 31> SARS-CoV-2 RBD S2003 mRNA sequence <SEQ ID NO: 32> Amino acid sequence of SARS-CoV-2 RBD S2003 (including S protein signal sequence) MFVFLVLLPLVSSEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYK LPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLTGTGVLTE S protein signal sequence: amino acids 1-13 RBD sequence: amino acid numbers 14 to 259 <SEQ ID NO: 33> Amino acid sequence of SARS-CoV-2 RBD S2003 (not including the S protein signal sequence) EKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG CVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLTGTGVLTE <SEQ ID NO:34> SARS-CoV-2 RBD S2004 template DNA GCTAGCGTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAACAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCACCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACACCCTGGACAGCAAAGTCGGCGGCAACTACACCTACCTGTACCGGCTGTTCAGAAAGAGCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAGAGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACGAAGAGC NheI sequence: nucleotide numbers 1 to 6 T7 promoter: base numbers 7 to 24 A: Transcription start site: base number 25 5'-UTR sequence (including the transcription start site and the KOZAK sequence at base numbers 89 to 94): base numbers 25 to 94 Spike protein signal sequence: nucleotides 95-133 RBD sequence: base numbers 134 to 805 3'-UTR: base numbers 806 to 937 PolyA sequence (A50): nucleotide numbers 938 to 987 BspQI sequence: nucleotide numbers 989 to 995 <SEQ ID NO: 35> SARS-CoV-2 RBD S2004 mRNA sequence AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAACAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACACCCUGGACAGCAAAGUCGGCGGCAACUACACCUACCUGUACCGGCUGUUCAGAAAGAGCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUGGAAGGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACAAACGGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAGAGCGUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 36> Amino acid sequence of SARS-CoV-2 RBD S2004 (including S protein signal sequence) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRINNCVADYSVLYNSTSFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNY KLPDDFTGCVIAWNSNTLDSKVGGNYTYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNF S protein signal sequence: amino acids 1-13 RBD sequence: amino acid numbers 14 to 236 <SEQ ID NO: 37> Amino acid sequence of SARS-CoV-2 RBD S2004 (without the S protein signal sequence) RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRINNCVADYSVLYNSTSFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDF TGCVIAWNSNTLDSKVGGNYTYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNF <SEQ ID NO: 38> DNA fragment containing mutant SARS-CoV-2 RBD (South African type) (mutated codons are underlined, and mutation sites are in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGC AAC ATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTG AAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TAC GGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <Accession No. 39> DNA fragment containing mutant SARS-CoV-2 RBD (UK type) (Mutated codons are underlined and mutation sites are shown in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <SEQ ID NO: 40> DNA fragment containing mutant SARS-CoV-2 RBD (Brazilian type) (mutated codons underlined, mutated sites in bold). GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGC ACG ATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTG AAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TAC GGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <SEQ ID NO: 41> DNA fragment containing mutant SARS-CoV-2 RBD (California type) (mutated codons underlined, mutated sites in bold). GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTAC CGGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCAC GCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <SEQ ID NO: 42> DNA fragment containing mutant SARS-CoV-2 RBD (Indian type) (mutated codons are underlined, and mutation sites are in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTAC CGG TACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTG CAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <Array No. 43> DNA fragment containing mutant SARS-CoV-2 RBD (South African C538S type) (Mutated codons are underlined and the mutation sites are in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGC AAC ATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTG AAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TAC GGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAA AGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <SEQ ID NO: 44> DNA fragment containing mutant SARS-CoV-2 RBD (UK C538S type) (mutated codons underlined, mutation sites in bold). GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAA AGC GTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <SEQ ID NO: 45> DNA fragment containing mutant SARS-CoV-2 RBD (Brazil C538S type) (mutated codons underlined, mutation sites in bold). GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGC ACGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCCCCCCTGCAACGGCGTG AAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TAC GGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAA AGC GTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <SEQ ID NO: 46> DNA fragment containing mutant SARS-CoV-2 RBD (California C538S type) (mutated codons are underlined, and mutation sites are in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTAC CGG TACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAA AGC GTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <Array No. 47> DNA fragment containing mutant SARS-CoV-2 RBD (Indian C538S type) (Mutated codons are underlined and the mutation sites are in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTAC CGG TACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTG CAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAA AGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <SEQ ID NO: 48> DNA fragment containing mutant SARS-CoV-2 RBD ( Combination Mutations (1) )(Mutated codons are underlined and the mutation sites are shown in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGC AAG AACCTGGACAGCAAAGTCGGCGGCAACTACAACTAC CGG TACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAG GTC GGCAGCACCCCCTGCAACGGC GCGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCA AGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <SEQ ID NO: 49> A DNA fragment containing mutant SARS-CoV-2 RBD ( Combination Mutations (2) ) (The mutated codon is underlined, and the mutation site is in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGC AGA ACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTAC CTC CCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <Array number 50> DNA fragment containing mutant SARS-CoV-2 RBD( Combination Mutations (3) )(Mutated codons are underlined and mutation sites are shown in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGC AAC ATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGC AGA ACCCCCTGCAACGGCGTG AAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <SEQ ID NO: 51> DNA fragment containing mutant SARS-CoV-2 RBD( Combination Mutations (4) )(Mutated codons are underlined and the mutation sites are shown in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGC AGC AACTGCTAC CTC CCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <SEQ ID NO: 52> Template DNA of mutant SARS-CoV-2 RBD (South African type) (Mutated codons are underlined and the mutation sites are in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGC AACATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCCACCCCTGCAACGGCGTG AAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TAC GGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCC AACTACTAAACTGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: base numbers 1 to 18 A: Transcription start site: base number 19 5'-UTR sequence (including the transcription start site and the KOZAK sequence at base numbers 83 to 88): base numbers 19 to 88 Spike protein signal sequence: nucleotides 89-127 RBD sequence: base numbers 128 to 799 3'-UTR: base numbers 800 to 931 PolyA sequence (A100): nucleotide numbers 932 to 1031 <SEQ ID NO: 53> Mutant SARS-CoV-2 RBD template DNA (UK type) (mutated codons are underlined, and mutation sites are in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCC AACTACTAAACTGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: base numbers 1 to 18 A: Transcription start site: base number 19 5'-UTR sequence (including the transcription start site and the KOZAK sequence at base numbers 83 to 88): base numbers 19 to 88 Spike protein signal sequence: nucleotides 89-127 RBD sequence: base numbers 128 to 799 3'-UTR: base numbers 800 to 931 PolyA sequence (A100): nucleotide numbers 932 to 1031 <SEQ ID NO: 54> Template DNA of mutant SARS-CoV-2 RBD (Brazilian type) (mutated codons are underlined, and mutation sites are in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGC ACG ATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTG AAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCC AACTACTAAACTGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: base numbers 1 to 18 A: Transcription start site: base number 19 5'-UTR sequence (including the transcription start site and the KOZAK sequence at base numbers 83 to 88): base numbers 19 to 88 Spike protein signal sequence: nucleotides 89-127 RBD sequence: base numbers 128 to 799 3'-UTR: base numbers 800 to 931 PolyA sequence (A100): nucleotide numbers 932 to 1031 <SEQ ID NO: 55> Template DNA of mutant SARS-CoV-2 RBD (California type) (mutated codons are underlined, and mutation sites are in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTAC CGGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCGGCAGCACCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAG CTACGGCTTCCAGCCCACAAACGGCGTGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCGCCACAGTGTCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAA CAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: base numbers 1 to 18 A: Transcription start site: base number 19 5'-UTR sequence (including the transcription start site and the KOZAK sequence at base numbers 83 to 88): base numbers 19 to 88 Spike protein signal sequence: nucleotides 89-127 RBD sequence: base numbers 128 to 799 3'-UTR: base numbers 800 to 931 PolyA sequence (A100): nucleotide numbers 932 to 1031 <SEQ ID NO: 56> Mutant SARS-CoV-2 RBD template DNA (Indian type) (mutated codons are underlined, and mutation sites are in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTAC CGG TACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTG CAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCGCCACAGTGTCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATT AAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: base numbers 1 to 18 A: Transcription start site: base number 19 5'-UTR sequence (including the transcription start site and the KOZAK sequence at base numbers 83 to 88): base numbers 19 to 88 Spike protein signal sequence: nucleotides 89-127 RBD sequence: base numbers 128 to 799 3'-UTR: base numbers 800 to 931 PolyA sequence (A100): nucleotide numbers 932 to 1031 <SEQ ID NO: 57> Template DNA of mutant SARS-CoV-2 RBD (South African C538S type) (mutated codons are underlined, and mutation sites are in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGC AAC ATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTG AAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TAC GGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAA AGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: base numbers 1 to 18 A: Transcription start site: base number 19 5'-UTR sequence (including the transcription start site and the KOZAK sequence at base numbers 83 to 88): base numbers 19 to 88 Spike protein signal sequence: nucleotides 89-127 RBD sequence: base numbers 128 to 799 3'-UTR: base numbers 800 to 931 PolyA sequence (A100): nucleotide numbers 932 to 1031 <SEQ ID NO: 58> Template DNA of mutant SARS-CoV-2 RBD (UK C538S type) (mutated codons are underlined, and mutation sites are in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TAC GGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAA AGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: base numbers 1 to 18 A: Transcription start site: base number 19 5'-UTR sequence (including the transcription start site and the KOZAK sequence at base numbers 83 to 88): base numbers 19 to 88 Spike protein signal sequence: nucleotides 89-127 RBD sequence: base numbers 128 to 799 3'-UTR: base numbers 800 to 931 PolyA sequence (A100): nucleotide numbers 932 to 1031 <SEQ ID NO: 59> Template DNA of mutant SARS-CoV-2 RBD (Brazilian C538S type) (mutated codons are underlined, and mutation sites are in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGC ACG ATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTG AAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TAC GGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAA AGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: base numbers 1 to 18 A: Transcription start site: base number 19 5'-UTR sequence (including the transcription start site and the KOZAK sequence at base numbers 83 to 88): base numbers 19 to 88 Spike protein signal sequence: nucleotides 89-127 RBD sequence: base numbers 128 to 799 3'-UTR: base numbers 800 to 931 PolyA sequence (A100): nucleotide numbers 932 to 1031 <SEQ ID NO: 60> Template DNA of mutant SARS-CoV-2 RBD (California C538S type) (mutated codons are underlined, and mutation sites are in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTAC CGG TACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAA AGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: base numbers 1 to 18 A: Transcription start site: base number 19 5'-UTR sequence (including the transcription start site and the KOZAK sequence at base numbers 83 to 88): base numbers 19 to 88 Spike protein signal sequence: nucleotides 89-127 RBD sequence: base numbers 128 to 799 3'-UTR: base numbers 800 to 931 PolyA sequence (A100): nucleotide numbers 932 to 1031 <SEQ ID NO: 61> Mutant SARS-CoV-2 RBD template DNA (Indian C538S type) (mutated codons are underlined, and mutation sites are in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTAC CGG TACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTG CAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAA AGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: base numbers 1 to 18 A: Transcription start site: base number 19 5'-UTR sequence (including the transcription start site and the KOZAK sequence at base numbers 83 to 88): base numbers 19 to 88 Spike protein signal sequence: nucleotides 89-127 RBD sequence: base numbers 128 to 799 3'-UTR: base numbers 800 to 931 PolyA sequence (A100): nucleotide numbers 932 to 1031 <SEQ ID NO: 62> Mutant SARS-CoV-2 RBD template DNA ( Combination Mutations (1) ) (The mutated codon is underlined, and the mutation site is in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGC AAG AACCTGGACAGCAAAGTCGGCGGCAACTACAACTAC CGG TACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAG GTC GGCAGCACCCCCTGCAACGGC GCGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCGCCACAGTTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTA TTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: base numbers 1 to 18 A: Transcription start site: base number 19 5'-UTR sequence (including the transcription start site and the KOZAK sequence at base numbers 83 to 88): base numbers 19 to 88 Spike protein signal sequence: nucleotides 89-127 RBD sequence: base numbers 128 to 799 3'-UTR: base numbers 800 to 931 PolyA sequence (A100): nucleotide numbers 932 to 1031 <SEQ ID NO: 63> Mutant SARS-CoV-2 RBD template DNA ( Combination Mutations (2) ) (The mutated codon is underlined, and the mutation site is in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGC AGA ACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTAC CTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCC TTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: base numbers 1 to 18 A: Transcription start site: base number 19 5'-UTR sequence (including the transcription start site and the KOZAK sequence at base numbers 83 to 88): base numbers 19 to 88 Spike protein signal sequence: nucleotides 89-127 RBD sequence: base numbers 128 to 799 3'-UTR: base numbers 800 to 931 PolyA sequence (A100): nucleotide numbers 932 to 1031 <SEQ ID NO: 64> Mutant SARS-CoV-2 RBD template DNA ( Combination Mutations (3) ) (The mutated codon is underlined, and the mutation site is in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGC AAC ATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGC AGA ACCCCCTGCAACGGCGTG AAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCC AACTACTAAACTGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: base numbers 1 to 18 A: Transcription start site: base number 19 5'-UTR sequence (including the transcription start site and the KOZAK sequence at base numbers 83 to 88): base numbers 19 to 88 Spike protein signal sequence: nucleotides 89-127 RBD sequence: base numbers 128 to 799 3'-UTR: base numbers 800 to 931 PolyA sequence (A100): nucleotide numbers 932 to 1031 <SEQ ID NO: 65 Mutant SARS-CoV-2 RBD template DNA ( Combination Mutations (4) ) (The mutated codon is underlined, and the mutation site is in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGC AGC AACTGCTAC CTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCC TTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: base numbers 1 to 18 A: Transcription start site: base number 19 5'-UTR sequence (including the transcription start site and the KOZAK sequence at base numbers 83 to 88): base numbers 19 to 88 Spike protein signal sequence: nucleotides 89-127 RBD sequence: base numbers 128 to 799 3'-UTR: base numbers 800 to 931 PolyA sequence (A100): nucleotide numbers 932 to 1031 <SEQ ID NO: 66> Mutant SARS-CoV-2 RBD mRNA (South African type) (The mutated codon is underlined, and the mutated site is in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGC AAC AUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUG AAA GGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACA UACGGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAAUGCGUGAACUUCUGAGCUCGCUUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCC AACUACUAAACUGGGGGAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 67> Mutant SARS-CoV-2 RBD mRNA (UK type) (The mutated codon is underlined and the mutated site is in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUGGAAGGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACA UACGGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAAUGCGUGAACUUCUGAGCUCGCUUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCC AACUACUAAACUGGGGGAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 68> Mutant SARS-CoV-2 RBD mRNA (Brazilian type) (The mutated codon is underlined, and the mutated site is in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAG CGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGC ACGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCCCCCUGCAACGGCGUG AAA GGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACA UAC GGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAAUGCGUGAACUUCUGAGCUCGCUUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCC AACUACUAAACUGGGGGAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 69> Mutant SARS-CoV-2 RBD mRNA (California type) (The mutated codon is underlined, and the mutated site is in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUAC CGGUACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGACAUCAGCACCGAGAUCUACCAGGCGGCAGCACCCCUGCAACGGCGUGGAAGGCUUCAACUGCUACUUCCCACUGCAGAG CUACGGCUUCCAGCCCACAAACGGCUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAA CAAAUGCGUGAACUUCUGAGCUCGCUUUUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 70> Mutant SARS-CoV-2 RBD mRNA (Indian type) (The mutated codon is underlined, and the mutated site is in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUAC CGG UACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUG CAAGGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACAAACGGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAAUGCGUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 71> Mutant SARS-CoV-2 RBD mRNA (South African C538S type) (Mutated codons are underlined and the mutation sites are in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGC AACAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCCCCCUGCAACGGCGUG AAA GGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACA UAC GGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAA AGC GUGAACUUCUGAGCUCGCUUUUUGCUGUCCAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 72> Mutant SARS-CoV-2 RBD mRNA (UK C538S type) (The mutated codon is underlined and the mutated site is in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUGGAAGGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACA UAC GGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAA AGCGUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 73> Mutant SARS-CoV-2 RBD mRNA (Brazil C538S type) (Mutated codons are underlined and the mutation sites are shown in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGC ACG AUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUG AAAGGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACA UAC GGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAA AGC GUGAACUUCUGAGCUCGCUUUUUGCUGUCCAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 74> Mutant SARS-CoV-2 RBD mRNA (California C538S type) (The mutated codon is underlined, and the mutated site is in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACGCCCACAUGUUCGUGUUCCUGGUGCUGCUGCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAAUUCGCCAGCGUGUACCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCG UGCUGUACAACAGCCCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGCAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUAC CGG UACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCGGCAGCACCCCCUGCAACGGCGUGGAAGGCUUCAACUGCUACUUCCCACUGCAGAGCUACGCUUCCAGCCCCACAAACGGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCACGCCCCCGCCACAGUGUGCGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAAA AGCGUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 75> Mutant SARS-CoV-2 RBD mRNA (Indian C538S type) (Mutated codons are underlined and the mutation sites are in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUAC CGG UACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUG CAAGGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACAAACGGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAA AGC GUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 76> Mutant SARS-CoV-2 RBD mRNA (Combined Mutant (1)) (Mutated codons are underlined and mutation sites are in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGC AAG AACCUGGACAGCAAAGUCGGCGGCAACUACAACUAC CGG UACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAG GUC GGCAGCACCCCCUGCAACGGC GCG GAAGGCUUCAACUGCUCUUCCCACUGCAGAGCUACGGCUUCCAGCCCACAAACGGCUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAAUGCGUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUA UUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 77> Mutant SARS-CoV-2 RBD mRNA (combination mutation type (2)) (The mutated codon is underlined, and the mutation site is in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGC AGA ACCCCCUGCAACGGCGUGGAAGGCUUCAACUGCUAC CUCCCACUGCAGAGCUACGGCUUCCAGCCCACAAACGGCUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAAUGCGUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCC UUUGUUCCCUAAGUCCAACUACUAAACUGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 78> Mutant SARS-CoV-2 RBD mRNA (combination mutation type (3)) (The mutated codon is underlined, and the mutation site is in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAG CGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGC AACAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGC AGA ACCCCCUGCAACGGCGUG AAA GGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACA UAC GGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAAUGCGUGAACUUCUGAGCUCGCUUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCC AACUACUAAACUGGGGGAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 79> Mutant SARS-CoV-2 RBD mRNA (combination mutation type (4)) (The mutated codon is underlined and the mutation site is in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUGGAAGGC AGC AACUGCUAC CUCCCACUGCAGAGCUACGGCUUCCAGCCCACAAACGGCUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAAUGCGUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCC UUUGUUCCCUAAGUCCAACUACUAAACUGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 80> Amino acid sequence of mutant SARS-CoV-2 RBD (South African variant) (mutated amino acids, including the S protein signal sequence, are underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG N IADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGV K GFNCYFPLQSYGFQPT Y GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF S protein signal sequence: amino acids 1-13 RBD sequence: amino acid numbers 14 to 236 <SEQ ID NO: 81> Amino acid sequence of mutant SARS-CoV-2 RBD (UK type) (mutated amino acids, including the S protein signal sequence, are underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPT Y GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF S protein signal sequence: amino acids 1-13 RBD sequence: amino acid numbers 14 to 236 <SEQ ID NO: 82> Amino acid sequence of mutant SARS-CoV-2 RBD (Brazilian type) (mutated amino acids, including the S protein signal sequence, are underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG T IADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGV K GFNCYFPLQSYGFQPT Y GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF S protein signal sequence: amino acids 1-13 RBD sequence: amino acid numbers 14 to 236 <SEQ ID NO: 83> Amino acid sequence of mutant SARS-CoV-2 RBD (California type) (mutated amino acids, including the S protein signal sequence, are underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNY R YRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF S protein signal sequence: amino acids 1-13 RBD sequence: amino acid numbers 14 to 236 <SEQ ID NO: 84> Amino acid sequence of mutant SARS-CoV-2 RBD (Indian type) (mutated amino acids, including the S protein signal sequence, are underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNY R YRLFRKSNLKPFERDISTEIYQAGSTPCNGV Q GFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF S protein signal sequence: amino acids 1-13 RBD sequence: amino acid numbers 14 to 236 <SEQ ID NO: 85> Amino acid sequence of mutant SARS-CoV-2 RBD (South African C538S type) (mutated amino acids, including the S protein signal sequence, are underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG N IADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGV K GFNCYFPLQSYGFQPT Y GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK S VNF S protein signal sequence: amino acids 1-13 RBD sequence: amino acid numbers 14 to 236 <SEQ ID NO: 86> Amino acid sequence of mutant SARS-CoV-2 RBD (UK C538S type) (mutated amino acids, including the S protein signal sequence, are underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPT Y GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK S VNF S protein signal sequence: amino acids 1-13 RBD sequence: amino acid numbers 14 to 236 <SEQ ID NO: 87> Amino acid sequence of mutant SARS-CoV-2 RBD (Brazilian C538S type) (mutated amino acids, including the S protein signal sequence, are underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG T IADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGV K GFNCYFPLQSYGFQPT Y GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK S VNF <SEQ ID NO: 88> Amino acid sequence of mutant SARS-CoV-2 RBD (California C538S type) (mutated amino acids, including the S protein signal sequence, are underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNY R YRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK S VNF S protein signal sequence: amino acids 1-13 RBD sequence: amino acid numbers 14 to 236 <SEQ ID NO: 89> Amino acid sequence of mutant SARS-CoV-2 RBD (Indian C538S type) (mutated amino acids, including the S protein signal sequence, are underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNY R YRLFRKSNLKPFERDISTEIYQAGSTPCNGV Q GFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK S VNF S protein signal sequence: amino acids 1-13 RBD sequence: amino acid numbers 14 to 236 <SEQ ID NO: 90> Amino acid sequence of mutant SARS-CoV-2 RBD (combination mutant type (1)) (mutated amino acids, including the S protein signal sequence, are underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNS K NLDSKVGGNYNY R YRLFRKSNLKPFERDISTEIYQ V GSTPCNG A EGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF S protein signal sequence: amino acids 1-13 RBD sequence: amino acid numbers 14 to 236 <SEQ ID NO: 91> Amino acid sequence of mutant SARS-CoV-2 RBD (combination mutant type (2)) (mutated amino acids, including the S protein signal sequence, are underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAG R TPCNGVEGFNCY L PLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF S protein signal sequence: amino acids 1-13 RBD sequence: amino acid numbers 14 to 236 <SEQ ID NO: 92> Amino acid sequence of mutant SARS-CoV-2 RBD (combination mutant type (3)) (mutated amino acids, including the S protein signal sequence, are underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG N IADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAG R TPCNGV K GFNCYFPLQSYGFQPT Y GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF S protein signal sequence: amino acids 1-13 RBD sequence: amino acid numbers 14 to 236 <SEQ ID NO: 93> Amino acid sequence of mutant SARS-CoV-2 RBD (combination mutant type (4)) (mutated amino acids, including the S protein signal sequence, are underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEG S NCY L PLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF S protein signal sequence: amino acids 1-13 RBD sequence: amino acid numbers 14 to 236 <SEQ ID NO: 94> Amino acid sequence of mutant SARS-CoV-2 RBD (South African type) (not including S protein signal sequence. Mutated amino acids are underlined.) RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG N IADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGV K GFNCYFPLQSYGFQPT Y GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF <SEQ ID NO: 95> Amino acid sequence of mutant SARS-CoV-2 RBD (UK type) (not including S protein signal sequence. Mutated amino acids are underlined.) RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPT Y GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF <SEQ ID NO: 96> Amino acid sequence of mutant SARS-CoV-2 RBD (Brazilian type) (not including S protein signal sequence. Mutated amino acids are underlined.) RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG T IADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGV K GFNCYFPLQSYGFQPT Y GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF <SEQ ID NO: 97> Amino acid sequence of mutant SARS-CoV-2 RBD (California type) (not including S protein signal sequence. Mutated amino acids are underlined.) RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNY R YRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF <SEQ ID NO: 98> Amino acid sequence of mutant SARS-CoV-2 RBD (Indian type) (not including S protein signal sequence. Mutated amino acids are underlined.) RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNY R YRLFRKSNLKPFERDISTEIYQAGSTPCNGV Q GFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF <SEQ ID NO: 99> Amino acid sequence of mutant SARS-CoV-2 RBD (South African C538S type) (not including S protein signal sequence. Mutated amino acids are underlined.) RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG N IADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGV K GFNCYFPLQSYGFQPT Y GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK S VNF <SEQ ID NO: 100> Amino acid sequence of mutant SARS-CoV-2 RBD (UK C538S type) (not including the S protein signal sequence. Mutated amino acids are underlined.) RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPT Y GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK S VNF <SEQ ID NO: 101> Amino acid sequence of mutant SARS-CoV-2 RBD (Brazilian C538S type) (not including S protein signal sequence. Mutated amino acids are underlined.) RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG T IADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGV K GFNCYFPLQSYGFQPT Y GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK S VNF <SEQ ID NO: 102> Amino acid sequence of mutant SARS-CoV-2 RBD (California C538S type) (not including the S protein signal sequence. Mutated amino acids are underlined.) RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNY RYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK S VNF <SEQ ID NO: 103> Amino acid sequence of mutant SARS-CoV-2 RBD (Indian C538S type) (not including S protein signal sequence. Mutated amino acids are underlined.) RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNY R YRLFRKSNLKPFERDISTEIYQAGSTPCNGV Q GFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK S VNF <SEQ ID NO: 104> Amino acid sequence of mutant SARS-CoV-2 RBD (combination mutant type (1)) (not including the S protein signal sequence. Mutated amino acids are underlined.) RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNS K NLDSKVGGNYNY R YRLFRKSNLKPFERDISTEIYQ V GSTPCNG A EGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF <SEQ ID NO: 105> Amino acid sequence of mutant SARS-CoV-2 RBD (combination mutant type (2)) (not including the S protein signal sequence. Mutated amino acids are underlined.) RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAG R TPCNGVEGFNCY L PLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF <SEQ ID NO: 106> Amino acid sequence of mutant SARS-CoV-2 RBD (combination mutant type (3)) (not including the S protein signal sequence. Mutated amino acids are underlined.) RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG N IADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAG R TPCNGV K GFNCYFPLQSYGFQPT Y GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF <SEQ ID NO: 107> Amino acid sequence of mutant SARS-CoV-2 RBD (combination mutant type (4)) (not including the S protein signal sequence. Mutated amino acids are underlined.) RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEG S NCY LPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

Claims

1. A lipid particle encapsulating a nucleic acid capable of expressing a fragment of the S protein of the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2), the fragment of the S protein comprises a signal sequence and a receptor binding domain of the S protein; the lipid particles comprise cationic lipids, amphipathic lipids, sterols, and PEG lipids; The cationic lipid has the following structural formula: or a pharma- ceutically acceptable salt thereof, The amphipathic lipid is distearoylphosphatidylcholine (DSPC), The sterol is cholesterol, The particle, wherein the PEG lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol.

2. The particle according to claim 1, wherein the lipid composition of amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 15% or less of amphipathic lipid, 20 to 55% of sterols, 40 to 65% of cationic lipid, and 1 to 5% of PEG lipid, and the ratio of the total lipid weight to the nucleic acid weight is 15 to 30.

3. The particle according to claim 2, wherein the lipid composition of amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 10 to 15% amphipathic lipid, 35 to 45% sterols, 40 to 50% cationic lipid, and 1 to 2% PEG lipid, and the ratio of the total lipid weight to the nucleic acid weight is 17.5 to 22.

5.

4. The particle described in any one of claims 1 to 3, wherein the nucleic acid capable of expressing a fragment of the S protein of the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2) is an mRNA comprising a cap structure (Cap), a 5' untranslated region (5'-UTR), a leader sequence, a translation region of the receptor binding domain of the S protein, a 3' untranslated region (3'-UTR) and a polyA tail (polyA).

5. 5. The particle according to claim 4, wherein the 5' untranslated region (5'-UTR) and the 3' untranslated region (3'-UTR) are the 5'-UTR sequence of human β-globin and the 3'-UTR sequence of human β-globin, respectively.

6. The particle according to any one of claims 1 to 5, wherein the nucleic acid comprises at least one modified nucleotide.

7. 7. The particle according to claim 6, wherein the modified nucleotide is at least one selected from the group consisting of 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, and 1-alkylpseudouridine.

8. The particles according to any one of claims 1 to 7, wherein the average particle size of the particles is from 30 nm to 300 nm.

9. Use of the particles according to any one of claims 1 to 8 for producing a composition for preventing and / or treating infection with a new coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2).

10. A composition comprising the particles according to any one of claims 1 to 9.

11. The composition according to claim 10 for use as a medicine.

12. The composition of claim 11 for inducing an immune response against the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2).

13. The composition according to claim 11 or 12, for preventing and / or treating infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

14. An ethanol solution containing cationic lipids, amphipathic lipids, sterols, and PEG lipids. A buffer solution containing a nucleic acid capable of expressing a fragment of the S protein of the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2) The method for producing lipid particles according to claim 1 , comprising a step of mixing in a flow channel.

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