Nucleic acid lipid particle vaccine

Abstract

To provide a vaccine for preventing and / or treating infection by new coronavirus.SOLUTION: A lipid particle encapsulating nucleic acid in which S protein of new coronavirus and / or a fragment thereof can be expressed, where a peptide consists of a mutated receptor-binding domain made up of a sequence in which mutation is introduced into a signal sequence of S protein and an amino acid sequence of a receptor-binding domain of S protein, the lipid particle contains cationic lipid, amphipathic lipid, sterols, and PEG lipid, the cationic lipid is cationic lipid represented by a structural formula, the amphipathic lipid is distearoylphosphatidylcholine (DSPC), the sterols is cholesterol, and the PEG lipid is 1,2-dimyristolyl-sn-glycerol methoxypolyethylene glycol.SELECTED DRAWING: None

Description

Technical Field

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

Background Art

[0002] Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2), presenting a pathological condition mainly characterized by acute inflammation in the respiratory tract. In particular, pathological conditions mainly characterized by inflammation in the lower respiratory tract such as invasive pneumonia and acute respiratory distress syndrome in high-risk individuals have become the disease burden (Non-Patent Document 1). More than six types of coronaviruses (CoV) that infect humans and mainly present respiratory symptoms are known. SARS-CoV-2 is classified into the genus Betacoronavirus and is virologically similar to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) that have caused outbreaks in the past.

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

[0004] In the convalescent sera of SARS patients, IgG against RBD persists for at least three years or more. After treating the sera with RBD protein to adsorb anti-RBD antibodies, the neutralizing activity decreases to 50% or less, suggesting that anti-RBD antibodies play a major role in neutralizing activity (Non-Patent Documents 4 and 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 and 7).

[0005] Analysis using peripheral blood from the convalescent phase of COVID-19 patients about three weeks after becoming asymptomatic has suggested that the induction of specific CD4+ and CD8+ T cells is important for the defense against SARS-CoV-2 infection (Non-Patent Document 8). Specifically, as a result of analyzing blood samples from 10-20 COVID-19 patients, in all cases, a blood anti-SARS-CoV-2 RBD antibody response and a SARS-CoV-2-specific CD4+ T cell response were confirmed, and in about 70% of the cases, a SARS-CoV-2-specific CD8+ T cell response was confirmed. Also, since the blood anti-SARS-CoV-2 RBD IgG titer correlates with the frequency of S-specific CD4+ T cells (R = 0.8109), it was suggested that there are T cell epitopes in S and that S-specific CD4+ T cells may play an important role in the induction of the antibody response (Non-Patent Document 8). Furthermore, it has been shown that the blood anti-SARS-CoV-2 neutralizing activity correlates with the blood anti-SARS-CoV-2 S IgG titer (R = 0.9279) (Non-Patent Document 9).

[0006] As mechanisms of "immune enhancement" in which COVID-19 symptoms worsen, cellular immunopathology and antibody-dependent enhancement (ADE) are assumed to be involved (Non-Patent Document 10). In the case of SARS, it has been suggested that in fatal patients, the cytokine profile in the blood is Th2-dominant compared to patients who recovered from mild cases (Non-Patent Document 11). In a mouse SARS-CoV infection model, it has been suggested that a Th2-dominant immune response against S induces pulmonary immunopathology accompanied by an eosinophil-dominated inflammatory response (Non-Patent Document 12). On the other hand, regarding ADE, although it has been reported for other viruses such as dengue virus and respiratory syncytial virus, there is no report that specific antibodies against SARS-CoV induce ADE in SARS patients. Regarding vaccine antigen candidates against SARS-CoV, data suggesting that antigens consisting only of RBD rather than full-length S may be able to avoid the risk of lung injury have been reported (Non-Patent Document 13). Regarding SARS-CoV-2, although there is no direct clinical evidence that antibodies against S are involved in ADE, it has been pointed out that it is necessary to induce an appropriate cellular immune response to avoid the risk (Non-Patent Document 14).

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide a vaccine for preventing and / or treating infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Means for Solving the Problems

[0009] When the present inventors administered lipid particles encapsulating mRNA encoding the RBD of SARS-CoV-2 to mice, induction of SARS-CoV-2 S protein IgG in the blood was observed, and it was found that the immune response became Th1 type dominant, leading to the completion of the present invention.

[0010] The gist of the present invention is as follows. A lipid particle encapsulating a nucleic acid capable of expressing the S protein and / or a fragment thereof of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), wherein the lipid contains a cationic lipid represented by the general formula (Ia) or a pharmaceutically acceptable salt thereof. [Chemical formula] In the formula, R 1 and R 2 each independently represents a C1-C3 alkyl group; L 1 represents a C 17 -C 19 alkenyl group which may have one or more C2-C4 alkanoyloxy groups; L 2 represents a C 10 -C 19 alkyl group which may have one or more C2-C4 alkanoyloxy groups, or a C 10 -C 19 alkenyl group which may have one or more C2-C4 alkanoyloxy groups; p is 3 or 4. (2) The particle according to (1), wherein R 1 and R 2 in the general formula (Ia) are both methyl groups. (3) The particle according to (1) or (2), wherein p in the general formula (Ia) is 3. (4) The particle according to any one of (1) to (3), wherein L 1 in the general formula (Ia) is a C 17 -C 19 alkenyl group which may have one or more acetoxy groups. (5) The particle according to any one of (1) to (3), wherein L 2 in the general formula (Ia) is a C 10 -C 12 alkyl group which may have one or more acetoxy groups, or a C 10 -C 19The particle according to any one of (1) to (4), which is an alkenyl group. (6) L in the general formula (Ia) 2 which may have one or more acetoxy groups and is a C 10 -C 12 alkyl group, or which may have one or more acetoxy groups and is a C 17 -C 19 The particle according to any one of (1) to (4), which is an alkenyl group. (7) L in the general formula (Ia) 1 which is an (R)-11-acyloxy-cis-8-heptadecenyl group, a cis-8-heptadecenyl group, or an (8Z,11Z)-heptadecadienyl group, and is the particle according to any one of (1) to (6). (8) L in the general formula (Ia) 2 which is a decyl group, a cis-7-decenyl group, a dodecyl group, or an (R)-11-acyloxy-cis-8-heptadecenyl group, and is the particle according to any one of (1) to (7). (9) The cationic lipid has the following structural formula:

Chemical formula

Chemical formula

Chemical formula

Advantages of the Invention

[0011] According to the present invention, it becomes possible to prevent and / or treat infection by SARS-CoV-2. This specification includes the contents described in the specifications and / or drawings of Japanese Patent Application Nos. 2020-101420 and 2021-33278, which are the basis of the priority of the present application.

Brief Description of the Drawings

[0012]

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Mode for Carrying Out the Invention

[0013] Hereinafter, embodiments 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 and / or a fragment thereof of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), wherein the lipid particles contain a cationic lipid represented by the general formula (Ia) or a pharmaceutically acceptable salt thereof.

Chemical formula

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

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

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

[0018] L in the general formula (Ia) 2 is a C which may have one or more C2-C4 alkanoyloxy groups 10 -C 19 alkyl group, or a C which may have one or more C2-C4 alkanoyloxy groups 10 -C 19 alkenyl group, preferably a C which may have one or more acetoxy groups 10 -C 12 alkyl group, or a C which may have one or more acetoxy groups 10 -C 19 alkenyl group. Alternatively, L in the general formula (Ia) 2 is also preferably a C which may have one or more acetoxy groups 10 -C 12 alkyl group, or a C which may have one or more acetoxy groups 17 -C 19 alkenyl group. As L 2 specific examples thereof include a decyl group, a cis-7-decenyl group, a dodecyl group, or a (R)-11-acyloxy-cis-8-heptadecenyl group.

[0019] Specific examples of the cationic lipid which is a component constituting the particles of the present invention include the following structural formula:

Chemical formula

Chemical formula

Chemical formula

[0020] A pharmaceutically acceptable salt refers to a salt that can be used as a medicine. The cationic lipid, which is a component constituting the particles of the present invention, may be a pharmaceutically acceptable salt. Such salts preferably include alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; metal salts such as aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; inorganic salts such as ammonium salt; amine salts such as 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-benzylphenethylamine salt, piperazine salt, tetramethylammonium salt, and tris(hydroxymethyl)aminomethane salt; halogen atom hydrohalic acid salts such as hydrofluoride salt, hydrochloride salt, hydrobromide salt, and hydroiodide salt; inorganic acid salts such as nitrate salt, perchlorate salt, sulfate salt, and phosphate salt; lower alkane sulfonate salts such as methanesulfonate salt, trifluoromethanesulfonate salt, and ethanesulfonate salt; aryl sulfonate salts such as benzenesulfonate salt and p-toluenesulfonate salt; organic acid salts such as acetate salt, malate salt, fumarate salt, succinate salt, citrate salt, tartrate salt, oxalate salt, and maleate salt; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate salt, and aspartate salt.

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

[0022] The method for producing the cationic lipid represented by the general formula (Ia) is described in the pamphlet of International Publication No. 2015 / 005253.

[0023] The lipid of the present invention may further contain an amphiphilic lipid, sterols, and PEG lipids.

[0024] Amphiphilic lipids are lipids that have an affinity for both polar and non-polar solvents. Specifically, examples thereof include distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, and combinations thereof.

[0025] Sterols are sterols having a hydroxy group. Specifically, examples thereof include cholesterol.

[0026] PEG lipids are lipids modified with PEG. Specifically, 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 amphiphilic lipid, sterols, cationic lipid, and PEG lipid is not particularly limited, but in terms of molar amount, the amphiphilic lipid is 15% or less, sterols are 20 - 55%, cationic lipid is 40 - 65%, PEG lipid is 1 - 5%, and the ratio of the total lipid weight to the nucleic acid weight is preferably 15 - 30. It is more preferable that the lipid composition of the amphiphilic lipid, sterols, cationic lipid, and PEG lipid is, in terms of molar amount, the amphiphilic lipid is 5 - 15%, sterols are 35 - 50%, cationic lipid is 40 - 55%, PEG lipid is 1 - 3%, and the ratio of the total lipid weight to the nucleic acid weight is 15 - 25. It is even more preferable that the lipid composition of the amphiphilic lipid, sterols, cationic lipid, and PEG lipid is, in terms of molar amount, the amphiphilic lipid is 10 - 15%, sterols are 35 - 45%, cationic lipid is 40 - 50%, PEG lipid is 1 - 2%, and the ratio of the total lipid weight to the nucleic acid weight is 17.5 - 22.5. It is even more preferable that the lipid composition of the amphiphilic lipid, sterols, cationic lipid, and PEG lipid is, in terms of molar amount, the amphiphilic lipid is 10 - 15%, sterols are 35 - 45%, cationic lipid is 45 - 50%, PEG lipid is 1.5 - 2%, and the ratio of the total lipid weight to the nucleic acid weight is 17.5 - 22.5.

[0028] In the present invention, the nucleic acid encapsulated in the lipid particle 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 S protein of SARS-CoV-2 preferably contains the receptor-binding domain (RBD) present in the S protein.

[0030] A secretion peptide (a peptide encoded by a leader sequence) may be added to the receptor-binding domain. Examples of the leader sequence include 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 encapsulated in the lipid particle is preferably one that can express 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 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 secretion peptide (e.g., the 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 with the S protein signal sequence added is shown in SEQ ID NO: 10. The nucleic acid encapsulated in the lipid particle is preferably one that can express 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 with the amino acid sequence of SEQ ID NO: 11 or 10.

[0033] As used herein, identity refers to the relationship between two or more nucleotide sequences or amino acid sequences determined by sequence comparison, as is known in the art. In the art, "identity" also refers, in some cases, to the degree of sequence relatedness between nucleic acid molecules or polypeptides 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 the gap alignment (if any) addressed by a specific 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 it is not limited to this as long as it is used by those skilled in the art.

[0034] The identity of the sequences in the present invention is calculated using the sequence analysis software GENETYX-SV / RC (manufactured by Genetics Co., Ltd.), and this algorithm is commonly used in the art. The amino acids encoded by the nucleic acid encapsulated in the lipid particles of the present invention may have 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 S protein of the target SARS-CoV-2 and / or its fragment.

[0035] The amino acids encoded by the nucleic acid encapsulated in the lipid particles of the present invention maintain the above-described sequence identity, and in the amino acid sequence of the S protein of the target SARS-CoV-2 and / or its fragment, at several positions (preferably 5 or fewer, more preferably 3, 2, or 1 position), several (preferably 10 or fewer, more preferably 7 or fewer, still more preferably 5, 4, 3, 2, or 1) amino acids per position may be substituted, deleted, inserted, and / or added.

[0036] The amino acid sequence of the receptor binding domain present in the S protein of SARS-CoV-2 may have deletions, substitutions, or additions. For example, a sequence in which cysteine at position 538 (numbered from the N-terminus of the S protein) is substituted with serine (SEQ ID NO: 25) (hereinafter sometimes referred to as "C538S type"), a sequence in which amino acids are deleted at the N-terminus and C-terminus of the full-length sequence of RBD (R319 - F541) (SEQ ID NO: 29), a sequence in which amino acids are added at the N-terminus and C-terminus of the full-length sequence of RBD (R319 - F541) (SEQ ID NO: 33), and a sequence into which mutations with substitution of a plurality of amino acid residues are introduced (SEQ ID NO: 37) can be exemplified. Secretory peptides (for example, the S protein signal sequence) may be added to these sequences, and the amino acid sequences of those with the S protein signal sequence added to SEQ ID NOs: 25, 29, 33, and 37 are shown as 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. The amino acid sequences of the receptor binding domains of the South African type, UK type, Brazilian type, California type, Indian type, South African C538S type, UK C538S type, Brazilian C538S type, California C538S type, Indian C538S type, combined mutant type (1) (see Example 33 below), combined mutant type (2) (see Example 33 below), combined mutant type (3) (see Example 33 below), and combined mutant type (4) (see Example 33 below) are shown as SEQ ID NOs: 94 - 107. The sequences with the S protein signal sequence added to the amino acid sequences of SEQ ID NOs: 94 - 107 are shown as SEQ ID NOs: 80 - 93.

[0038] The nucleic acid encapsulated in the lipid particle is capable of expressing the receptor-binding domain in the S protein of SARS-CoV-2, and 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, 94 to 107 (excluding the S protein signal sequence). The nucleic acid encapsulated in the lipid particle may also be capable of expressing the receptor-binding domain in the S protein of SARS-CoV-2, and 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, 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 containing 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), etc., preferably cap1 or CleanCap, and more preferably CleanCap. The sequence of the 5' untranslated region (5'-UTR) is, for example, the sequence of nucleotide 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 part of the amino acid sequence of the S protein, and may contain a start codon and / or a stop codon. For example, it is the sequence of nucleotide numbers 89 to 3910 in the sequence of SEQ ID NO: 4. Also, the sequence of the translation region of the S protein may be 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. The sequence of the 3' untranslated region (3'-UTR) is, for example, the sequence of nucleotide 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 nucleotide numbers 4043 to 4142 in the sequence of SEQ ID NO: 4. The sequences of the cap structure (Cap), 5' untranslated region (5'-UTR), translation region of the S protein, 3' untranslated region (3'-UTR), and polyA tail (polyA) may be modified. The sequence of the nucleic acid capable of expressing the S protein of SARS-CoV-2 is preferably a nucleotide sequence having at least 90%, preferably 95%, more preferably 97% identity with the sequence of SEQ ID NO: 5, and most preferably consists of the nucleotide sequence of SEQ ID NO: 5. The codons of the nucleic acid are preferably optimized. By optimizing the codons, the effect as a vaccine can be improved and side effects can be reduced. It can be optimized according to the codon usage frequency of the target organism. The codon optimization is preferably performed, for example, on the coding sequence. In the sequence of SEQ ID NO: 16, the codons of the sequence of the translation region of the S protein are optimized.The nucleotide 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 containing 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 a site present at the 5' end of many eukaryotic mRNAs and has a 7-methylguanosine structure. Examples of the cap structure include cap0, cap1, cap2, ARCA, or CleanCap (registered trademark), etc., but preferably cap1 or CleanCap, and more preferably CleanCap. The sequence of the 5' untranslated region (5'-UTR) is, for example, the sequence of nucleotide numbers 19 to 88 in the sequence of SEQ ID NO: 8. The sequence of the leader sequence is, for example, the sequence of nucleotide 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 part of the amino acid sequence of the receptor-binding domain in the S protein, and may contain a start codon and / or a stop codon. For example, it is the sequence of nucleotide numbers 128 to 799 in the sequence of SEQ ID NO: 8. Also, 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 nucleotide 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 nucleotide 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, translation region of the receptor-binding domain in the S protein, 3' untranslated region (3'-UTR), and polyA tail (polyA) may be modified. The sequence of the nucleic acid capable of expressing the receptor-binding domain in the S protein of SARS-CoV-2 is a nucleotide sequence having at least 90%, preferably 95%, more preferably 97% identity with the sequence of SEQ ID NO: 9, and most preferably consists of the nucleotide sequence of SEQ ID NO: 9.It is advisable to optimize the codons of nucleic acids. By optimizing the codons, the effectiveness as a vaccine can be improved and side effects can be reduced. Optimization can be carried out according to the codon usage frequency of the target organism. Codon optimization is preferably performed, for example, on the coding sequence. 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 nucleic acid sequence 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 with the sequence of SEQ ID NO: 19. Also, 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, 66 to 79.

[0041] SEQ ID NO: 21 is the nucleotide sequence of the mRNA of Example 11, and the mRNA of Example 11 is the same mRNA except for the sequence other than polyA of the sequence of Example 6. In the sequence of Example 6, polyA has 110 adenine nucleotides, while in the mRNA of Example 11, the number of adenine nucleotides is 50. The nucleic acid contained in the lipid particles of the present invention may be mRNA with a relatively short polyA portion, preferably 30 or more, 40 or more, more preferably 50 or more. The upper limit of polyA is not particularly limited, but 500 or less, 400 or less, 300 or less, 200 or less, 110 or less is preferred.

[0042] SEQ ID NO: 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 the 538th position (the number is counted from the N-terminus of the S protein) is replaced with serine.

[0043] SEQ ID NO: 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 sequence (R319 - F541) of RBD.

[0044] SEQ ID NO: 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 with amino acids added to the N-terminus and C-terminus of the full-length sequence (R319 - F541) of RBD.

[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 a sequence in which amino acid residues are substituted at multiple positions in the sequence of Example 6.

[0046] SEQ ID NOs: 66 to 79 are the nucleotide sequences of mRNAs 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 later), combined mutant type (2) (see Example 33 described later), combined mutant type (3) (see Example 33 described later), and combined mutant type (4) (see Example 33 described later).

[0047] The nucleic acid encapsulated in the lipid particle may be in any form as long as it can express the S protein of SARS-CoV-2 and / or its fragment. For example, single-stranded DNA, single-stranded RNA (e.g., mRNA), single-stranded polynucleotide mixed with DNA and RNA, double-stranded DNA, double-stranded RNA, DNA-RNA hybrid polynucleotide, double-stranded polynucleotide composed of two types of polynucleotides mixed with DNA and RNA, etc. may be mentioned, and preferably it is mRNA.

[0048] The nucleotides constituting the nucleic acid encapsulated in the lipid particle may be natural or modified nucleotides, but it is preferable to contain at least one modified nucleotide.

[0049] Modified nucleotides may have any of the base, sugar, and phosphodiester bond moieties modified. The modification site may be one or two or more.

[0050] Examples of base modifications include 5-methylation of cytosine, 5-fluorination, N4-methylation, 5-methylation (thymine) of uracil, 5-fluorination, N6-methylation of adenine, N2-methylation of guanine, and the like.

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

[0052] An example of the modification of the phosphodiester bond can be a phosphorothioate bond.

[0053] Modified nucleotides are preferably those with a modified base moiety. For example, they may be pyrimidine nucleotides substituted at the 5-position and pseudouridine which may be substituted at the 1-position. Specifically, 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, 1-alkylpseudouridine can be exemplified. Also, 1-alkylpseudouridine may be 1-(C1-C6 alkyl)pseudouridine, preferably 1-methylpseudouridine or 1-ethylpseudouridine. Modified nucleotides with a modified base moiety may be used alone or in combination instead of natural nucleotides. Combinations of modified nucleotides with a modified base moiety 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, preferably a combination of 5-methylcytidine and 5-methyluridine.

[0054] The nucleic acid capable of expressing the S protein and / or its fragment of SARS-CoV-2 of the present invention can be produced by an 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)) required for in vitro transcription are commercially available (for example, AmpliScribe T7 High Yield Transcription Kit (Epicentre), mMESSAGE mMACHINE T7 Ultra Kit (Life thechnologies), etc.). The DNA used for producing single-stranded RNA is cloned DNA, for example, plasmid DNA or DNA fragments. Plasmid DNA or DNA fragments may be commercially available ones, or can be produced by methods generally known in the art (for example, the methods described in Sambrook, J. et al., Molecular Cloning a Laboratory Manual second edition (1989), Rashtchian, A., Current Opinion in Biotechnology, 1995, 6(1), 30-36, Gibson D. G. et al., Science, 2008, 319(5867), 1215-1220, etc.).

[0055] In order to obtain mRNA with improved stability and / or safety, in the in vitro transcription reaction, some or all of the natural nucleoside-5'-triphosphates can be replaced with modified nucleoside-5'-triphosphates, thereby replacing some or all of the natural nucleotides in the mRNA with modified nucleotides (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 Cap0 structure) can be introduced at the 5'-end of mRNA by a method using a capping enzyme after an in vitro transcription reaction. Further, Cap0 in mRNA can be converted to Cap1 by a method of allowing 2'-O-methyltransferase to act on the mRNA having Cap0. As the capping enzyme and 2'-O-methyltransferase, commercially available products can be used (for example, 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 different from 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. As ARCA and CleanCap (registered trademark), commercially available products can be used (ARCA, N-7003; CleanCap Reagent AG, N-7113, both manufactured by TriLink BioTechnologies). When using commercially available products, mRNA having a cap structure can be produced according to the protocol attached to the product.

[0058] In the present invention, the nucleic acid encapsulated in lipid particles may be purified by methods such as desalting, reverse-phase column, gel filtration, HPLC, PAGE, etc. By the purification treatment, impurities are removed, and thus the production of inflammatory cytokines in a living body administered with the nucleic acid may be reduced.

[0059] Examples of the impurities include 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-encapsulated lipid particles of the present invention can be produced by methods such as the thin film method, reverse phase evaporation method, ethanol injection method, ether injection method, dehydration-rehydration method, surfactant dialysis method, hydration method, freeze-thaw method, etc. For example, the nucleic acid-encapsulated lipid particles can be produced by the method described in WO2015 / 005253 pamphlet.

[0061] The particles of the present invention preferably have an average particle diameter of 30 nm to 300 nm, more preferably 30 to 200 nm, even more preferably 30 to 150 nm, and still 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 such as the dynamic light scattering method 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 for producing a composition for preventing and / or treating infection by 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] Using the particles of the present invention, the S protein of SARS-CoV-2 and / or a fragment thereof can be expressed in vivo or in vitro. Therefore, the present invention provides a method for expressing the S protein of SARS-CoV-2 and / or a fragment thereof in vitro, which includes introducing a composition containing the above particles into cells. Further, the present invention also provides a method for expressing the S protein of SARS-CoV-2 and / or a fragment thereof in vivo, which includes administering a composition containing the above 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. Therefore, the present invention provides a method for inducing an immune response against SARS-CoV-2, which includes administering a composition containing the above particles to a mammal. Further, the present invention provides a method for preventing and / or treating SARS-CoV-2 infection, which includes administering a composition containing the above particles to a mammal.

[0064] The particles of the present invention can be used as a medicine and also as a reagent for experiments. The particles of the present invention are usually added to carriers such as water, buffer solution, and physiological saline, and this formulation (composition) can be introduced into cells (in vitro) or administered to mammals (in vivo). When administered to a mammal, the carrier is preferably a pharmaceutically acceptable carrier (for example, physiological saline). Further, the particles of the present invention may be formulated into dosage forms such as creams, pastes, ointments, gels, and lotions using fats, fatty oils, lanolin, petrolatum, paraffin, waxes, resins, plastics, glycols, higher alcohols, glycerin, water, emulsifiers, suspending agents, etc. as base materials.

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

[0066] When administering the particles of the present invention to humans, for example, the dose of mRNA per adult administration may be about 0.001 to 1 mg, preferably 0.01 to 0.2 mg, and it may be administered intramuscularly, subcutaneously, intradermally, by intravenous drip injection, or by intravenous injection once or several times. However, the dose and the number of administrations can be appropriately changed depending on the type of disease, symptoms, age, administration method, and the like.

[0067] When used as an experimental reagent, the particles of the present invention are introduced into cells that are desired to express the S protein of SARS-CoV-2 and / or a fragment thereof (for example, HEK293 cells and its derivative cells (HEK293T cells, FreeStyle 293 cells, and Expi293 cells), CHO cells, C2C12 mouse myoblasts, immortalized mouse dendritic cells (MutuDC1940)), and the S protein of SARS-CoV-2 and / or a fragment thereof can be expressed in vitro. The expression of the S protein of SARS-CoV-2 and / or a fragment thereof can be analyzed by detecting the S protein of SARS-CoV-2 and / or a fragment thereof in the sample by Western blotting, or by detecting a peptide fragment specific to the S protein of SARS-CoV-2 and / or a fragment thereof by mass spectrometry.

[0068] In this specification, treatment means the recovery, remission, alleviation and / or delay of the progression of clinical symptoms of these diseases in patients who have developed an infectious disease caused by a virus or bacteria, or a disease caused by the infection (for example, pneumonia).

[0069] In this specification, prevention means reducing the incidence of a disease caused by an infectious disease caused by a virus or bacteria. Prevention includes reducing the risk of progression of a disease caused by an infectious disease caused by a virus or bacteria, or reducing the severity of those diseases. Since the particles of the present invention induce a protective immune response, they are effective for the prevention and / or treatment of the above diseases.

Examples

[0070] Hereinafter, the present invention will be specifically described by way of examples. These examples are for explaining the present invention and do not 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 the template DNA for in vitro transcription (IVT), SARS-CoV-2 S full DNA was amplified by PCR and then purified. A DNA fragment (SEQ ID NO: 1) containing a sequence in which the T7 promoter sequence, the 5'-UTR sequence of human β-globin, the Kozak sequence, SARS-CoV-2 S full, and the 3'-UTR sequence of human β-globin were ligated in sequence was introduced into a plasmid (pUC57mini-S full). To 6 ng of the plasmid dissolved in nuclease-free water (849.6 μL), 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. After incubation at 98°C for 1 minute, 20 cycles of 98°C for 5 seconds, 55°C for 15 seconds, and 68°C for 4 minutes were performed, followed by incubation at 68°C for 1 minute to amplify the S full DNA. After the reaction, the template DNA (SEQ ID NO: 4) was purified using the 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 360.5 μg / mL template DNA (70 μL) obtained in Example 1-(1), 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) were mixed and incubated at 37 °C for 4 hours. RQ1 RNase-Free DNase (25 μL, Promega catalog # M6101) was mixed and incubated at 37 °C for 15 minutes. 8M LiCl solution (500 μL, Sigma-Aldrich catalog # L7026) was mixed and left standing at -20 °C overnight. After centrifugation (4 °C, 4000×g, 30 minutes), the supernatant was discarded, 70% ethanol was added, and after centrifugation (4 °C, 4000×g, 10 minutes), the supernatant was discarded and air-dried. The obtained residue was dissolved in Nuclease-free water and purified using the RNeasy Maxi kit (Qiagen catalog # 75162) according to the attached manual. The obtained eluate (5.8 mL, 4906 μg in terms of UV) was mixed with Nuclease-free water (419 μL), buffer (800 μL) and enzyme (981 μL) of rApid Alkaline Phosphatase (Roche catalog # 04 898 141 001), incubated at 37 °C for 30 minutes, and then incubated at 75 °C for 3 minutes. 8M LiCl solution (8000 μL) was mixed and left standing at -20 °C overnight.After centrifugation (4°C, 4000×g, 30 minutes), the supernatant was discarded, 70% ethanol was added, and after centrifugation (4°C, 4000×g, 10 minutes), the supernatant was discarded and air-dried. The obtained residue was dissolved in Nuclease-free water and purified using the RNeasy Maxi kit according to the attached manual to obtain the target mRNA. The obtained mRNA has the sequence of SEQ ID NO: 5. It was analyzed using the LabChip GX Touch Standard RNA Reagent Kit (PerkinElmer catalog #CLS960010) to confirm that it was 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 the template DNA for in vitro transcription (IVT), SARS-CoV-2 RBD DNA was amplified by PCR and then purified. A DNA fragment (SEQ ID NO: 7) containing a sequence in which 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, SARS-CoV-2 RBD, and the 3'-UTR sequence of human β-globin were ligated in sequence was introduced into a plasmid (pUC57mini-RBD). 6 ng of the plasmid was added to nuclease-free water (849.6 μL) in which it was dissolved, 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. After incubation at 98°C for 1 minute, 20 cycles of 98°C for 5 seconds, 55°C for 15 seconds, and 68°C for 1 minute were performed, followed by incubation at 68°C for 1 minute to amplify the RBD DNA. After the reaction, the template DNA (SEQ ID NO: 8) was purified using the 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, and mRNA was obtained in the same manner as in Example 1-(2). The obtained mRNA had 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 target length.

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

[0075] (2) Characterization of mRNA-encapsulated nucleic acid lipid particles (1) The characteristics of the dispersion containing the nucleic acid-lipid particles prepared were evaluated. The methods for each characteristic evaluation will be described. (2-1) Encapsulation efficiency of mRNA The encapsulation efficiency of mRNA was measured using the Quant-iT RiboGreen RNA Assay kit (Invitrogen) according to the attached instructions. That is, in the presence and absence of 0.015% Triton X-100 surfactant, the mRNA in the dispersion of the nucleic acid-lipid particles was quantified, and the encapsulation efficiency 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 dispersion of the nucleic acid-lipid particles 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 dispersion of the nucleic acid-lipid particles 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 mRNA was calculated by the following formula. [Total lipid concentration] / [mRNA concentration] (wt / wt)

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

[0078] [Example 4] Preparation of mRNA-encapsulated nucleic acid lipid particles using the SARS-CoV-2 RBD mRNA described in Example 2 Using the same method as in Example 3, the Using mRNA preparation and property evaluation of the mRNA-encapsulated nucleic acid lipid particles described in Example 2 were carried out. The results are shown in Table 1. (Table 1) From the results of JPEG2025094115000009.jpg24164 and above, it was revealed that in these nucleic acid lipid particles, more than 90% of the mRNA was encapsulated within the lipid particles and they 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 full optimized A DNA fragment (SEQ ID NO: 12) containing a T7 promoter sequence, a 5'-UTR sequence of human β-globin, a Kozak sequence, a fully optimized SARS-CoV-2 S, and a 3'-UTR sequence of human β-globin linked in sequence was artificially synthesized and introduced into a plasmid (S_opt2 EcoRI). To 69 μL of nuclease-free water in which 1 ng of the plasmid was dissolved, 20 μL of 5× SuperFi Green Buffer (ThermoFisher Scientific catalog # 12357-010), 8 μL of 2.5 mM dNTP mix (Takara Bio Inc. catalog # 4030), 1 μL of 50 μM sense primer 2 (SEQ ID NO: 13), 1 μL of 50 μM antisense primer 2 (SEQ ID NO: 14), and 1 μL of Platinum SuperFi DNA Polymerase (ThermoFisher Scientific catalog # 12357-010) were added. After incubation at 98°C for 30 seconds, 20 cycles of 98°C for 5 seconds, 60°C for 10 seconds, and 72°C for 2 minutes were performed, followed by incubation at 72°C for 1 minute to amplify the template DNA of fully optimized SARS-CoV-2 S (SEQ ID NO: 15). After cutting the template DNA with restriction enzymes NheI and HindIII, it was introduced into a plasmid cut with the same restriction enzymes to prepare a template plasmid (pUCKIVT1 S full optimized). After cutting the plasmid with the restriction enzyme BspQI, the DNA was purified by isopropanol precipitation to prepare linear plasmid DNA.

[0080] (2) Preparation of SARS-CoV-2 S full optimized mRNA-003 by in vitro transcription Using the linear plasmid DNA obtained in Example 5-(1) instead of the template DNA obtained in Example 1-(1), mRNA was obtained in the same manner as in Example 1-(2). The obtained mRNA had the sequence of SEQ ID NO: 16. It was analyzed with 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 (SEQ ID NO: 17) containing a sequence in which the T7 promoter sequence, the 5'-UTR sequence of human β-globin, the Kozak sequence, SARS-CoV-2 RBD optimized, and the 3'-UTR sequence of human β-globin are linked in sequence was artificially synthesized and introduced into a plasmid (S_RBD_opt2 EcoRI). To 69 μL of nuclease-free water in which 1 ng of the plasmid was dissolved, 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 for 5 seconds, 60°C for 10 seconds, and 72°C for 1 minute were performed, followed by incubation at 72°C for 1 minute to amplify SARS-CoV-2 RBD optimized DNA (SEQ ID NO: 18). After cutting the template DNA with restriction enzymes NheI and HindIII, it was introduced into a plasmid cut with the same restriction enzymes to prepare a template plasmid (pUCKIVT1-RBD optimized). After cutting the plasmid with the restriction enzyme BspQI, the DNA was purified by isopropanol precipitation to prepare linear plasmid DNA.

[0082] (2) Preparation of SARS-CoV-2 RBD optimized mRNA-004 by in vitro transcription Using the linear plasmid DNA obtained in Example 6-(1) instead of the template DNA obtained in Example 1-(1), mRNA was obtained 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 to confirm that it was of the desired length.

[0083] [Example 7] Preparation of mRNA-encapsulated nucleic acid lipid particles using the SARS-CoV-2 S full optimized mRNA described in Example 5 Using the same method as in Example 3, the preparation and property evaluation of mRNA-encapsulated nucleic acid lipid particles were carried out using the mRNA described in Example 5. However, instead of 300 mM sucrose, 10 mM histidine buffer (pH 6.5), dialysis was performed using 300 mM sucrose, 10 mM histidine buffer (pH 7.0) 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 Using the same method as in Example 3, the preparation and property evaluation of mRNA-encapsulated nucleic acid lipid particles were carried out using the mRNA described in Example 6. However, instead of 300 mM sucrose, 10 mM histidine buffer (pH 6.5), dialysis was performed using 300 mM sucrose, 10 mM histidine buffer (pH 7.0) 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 fractionated 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 the SARS-CoV-2 RBD optimized mRNA described in Example 6 Using the same method as in Example 8, the preparation and property evaluation of mRNA-encapsulated nucleic acid lipid particles were carried out using the mRNA described in Example 9. The results are shown in Table 2. (Table 2) JPEG2025094115000010.jpg36170 From the above results, it was revealed that in these nucleic acid-lipid particles, more than 90% of the mRNA was encapsulated within the lipid particles and they had an average particle diameter of about 90 nm to about 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 the template DNA for in vitro transcription (IVT). A DNA fragment (SEQ ID NO: 20) containing a sequence in which GCTAGC (NheI site), T7 promoter sequence, 5'-UTR sequence of human β-globin, Kozak sequence, signal sequence of SARS-CoV-2 S protein, translation region of SARS-CoV-2 RBD, 3'-UTR sequence of human β-globin, polyA tail, and GAAGAGC (BspQI site) were ligated in sequence was introduced into a plasmid (pUC57-S2000). Nuclease-free water (860 μL, Thermo Fisher, catalog # AM9937) in which the plasmid (100 μg) was dissolved was added with 10X NEB Buffer 3.1 (100 μL, New England Biolabs, catalog # R7203S) and BspQI (40 μL, New England Biolabs, catalog #R0712), incubated at 50 °C for 1 hour, then isopropanol (1400 μL) was added, and it was left standing at -80 °C overnight. After centrifugation (-8 °C, 15,000 rpm, 10 minutes), the supernatant was discarded, 70% ethanol was added, after centrifugation (-8 °C, 15,000 rpm, 10 minutes), the supernatant was discarded and air-dried. The obtained residue was dissolved in TE-Buffer (pH 8.0) and prepared into a 500 μg / mL solution. (2) Preparation of SARS-CoV-2 RBD S2000 mRNA by in vitro transcription Using the template DNA obtained in Example 11-(1) instead of the template DNA obtained in Example 1-(1), mRNA was obtained 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 to confirm that it was of the desired length.

[0088] [Example 12] Preparation of mRNA-encapsulated nucleic acid lipid particles using the SARS-CoV-2 RBD S2000 mRNA described in Example 11 Using the same method as in Example 8, the preparation and property evaluation of mRNA-encapsulating 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 with an ultraviolet-visible spectrophotometer (manufactured by PerkinElmer, LAMBDA TM 465). The mRNA concentration was calculated by 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. From the results of the property evaluation, it became clear that more than 95% of the mRNA was encapsulated in the lipid particles, and the nucleic acid lipid particles had an average particle diameter of about 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 a sequence in which GCTAGC (NheI site), T7 promoter sequence, 5'-UTR sequence of human β-globin, Kozak sequence, signal sequence of SARS-CoV-2 S protein, translation region of SARS-CoV-2 RBD, 3'-UTR sequence of human β-globin, polyA tail, and GAAGAGC (BspQI site) were ligated in sequence was introduced into a plasmid (pUC57-S2001). 10X NEB Buffer 3.1 (100 μL, New England Biolabs, catalog # R7203S) and BspQI (40 μL, New England Biolabs, catalog # R0712) were added to Nuclease-free water (860 μL, Thermo Fisher, catalog # AM9937) in which the plasmid (100 μg) was dissolved. After incubation at 50 °C for 1 hour, isopropanol (1400 μL) was added and left standing at -80 °C overnight. After centrifugation (-8 °C, 15,000 rpm, 10 minutes), the supernatant was discarded, 70% ethanol was added, and after centrifugation (-8 °C, 15,000 rpm, 10 minutes), the supernatant was discarded and air-dried. The obtained residue was dissolved in TE-Buffer (pH 8.0) and prepared into a 500 μg / mL solution. (2) Preparation of SARS-CoV-2 RBD S2001 mRNA by in vitro transcription Using the template DNA obtained in Example 13-(1) instead of the template DNA obtained in Example 1-(1), mRNA was obtained 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 target length.

[0090] [Example 14] Preparation of mRNA-encapsulated nucleic acid lipid particles using the SARS-CoV-2 RBD S2001 mRNA described in Example 13 Using the same method as in Example 12, the preparation and property evaluation of mRNA-encapsulated nucleic acid lipid particles using the mRNA described in Example 13 were carried out. The results are shown in Table 3. From the results of the property evaluation, it was revealed that more than 95% of the mRNA was encapsulated in the lipid particles, and the nucleic acid lipid particles had an average particle diameter of about 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 the template DNA used for in vitro transcription (IVT). A plasmid (pUC57-S2002) was prepared by introducing a DNA fragment (SEQ ID NO: 26) containing a sequence in which GCTAGC (NheI site), T7 promoter sequence, 5'-UTR sequence of human β-globin, Kozak sequence, signal sequence of SARS-CoV-2 S protein, translation region of SARS-CoV-2 RBD, 3'-UTR sequence of human β-globin, polyA tail, and GAAGAGC (BspQI site) were linked in sequence. To Nuclease-free water (860 μL, Thermo Fisher, catalog # AM9937) in which the plasmid (100 μg) was dissolved, 10X NEB Buffer 3.1 (100 μL, New England Biolabs, catalog # R7203S) and BspQI (40 μL, New England Biolabs, catalog # R0712) were added, and after incubation at 50 °C for 1 hour, isopropanol (1400 μL) was added and left standing at -80 °C overnight. After centrifugation (-8 °C, 15,000 rpm, 10 minutes), the supernatant was discarded, 70% ethanol was added, and after centrifugation (-8 °C, 15,000 rpm, 10 minutes), the supernatant was discarded and air-dried. The obtained residue was dissolved in TE-Buffer (pH 8.0) and prepared into a 500 μg / mL solution. (2) Preparation of SARS-CoV-2 RBD S2002 mRNA by in vitro transcription Using the template DNA obtained in Example 15-(1) instead of the template DNA obtained in Example 1-(1), mRNA was obtained 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 the SARS-CoV-2 RBD S2002 mRNA described in Example 15 Using the same method as in Example 12, preparation and property evaluation of mRNA-encapsulated nucleic acid lipid particles using the mRNA described in Example 15 were carried out. The results are shown in Table 3. From the results of the property evaluation, it was revealed that more than 95% of the mRNA was encapsulated in the lipid particles and the nucleic acid lipid particles had an average particle diameter of about 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 a sequence in which GCTAGC (NheI site), T7 promoter sequence, 5'-UTR sequence of human β-globin, KOZAK sequence, signal sequence of SARS-CoV-2 S protein, translation region of SARS-CoV-2 RBD, 3'-UTR sequence of human β-globin, polyA tail, and GAAGAGC (BspQI site) were ligated in sequence was introduced into a plasmid (pCC1-S2003). 10X NEB Buffer 3.1 (100 μL, New England Biolabs, catalog # R7203S) and BspQI (40 μL, New England Biolabs, catalog # R0712) were added to Nuclease-free water (860 μL, Thermo Fisher, catalog # AM9937) in which the plasmid (100 μg) was dissolved. After incubation at 50 °C for 1 hour, isopropanol (1400 μL) was added and left standing at -80 °C overnight. After centrifugation (-8 °C, 15,000 rpm, 10 minutes), the supernatant was discarded, 70% ethanol was added, and after centrifugation (-8 °C, 15,000 rpm, 10 minutes), the supernatant was discarded and air-dried. The obtained residue was dissolved in TE-Buffer (pH 8.0) and prepared into a 500 μg / mL solution. (2) Preparation of SARS-CoV-2 RBD S2003 mRNA by in vitro transcription Using the template DNA obtained in Example 17-(1) instead of the template DNA obtained in Example 1-(1), mRNA was obtained in the same manner as in Example 1-(2). The obtained mRNA has the sequence of SEQ ID NO: 31. It was analyzed with the LabChip GX Touch Standard RNA Reagent Kit and confirmed to be of the target length.

[0094] [Example 18] Preparation of mRNA-encapsulated nucleic acid lipid particles using the SARS-CoV-2 RBD S2003 mRNA described in Example 17 Using the same method as in Example 12, the preparation and property evaluation of mRNA-encapsulated nucleic acid lipid particles using the mRNA described in Example 17 were carried out. The results are shown in Table 3. From the results of the property evaluation, it was revealed that more than 95% of the mRNA was encapsulated in the lipid particles, and the nucleic acid lipid particles had an average particle diameter of about 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 the template DNA for in vitro transcription (IVT). A plasmid (pCC1-S2004) was prepared by introducing a DNA fragment (SEQ ID NO: 34) containing a sequence in which GCTAGC (NheI site), T7 promoter sequence, 5'-UTR sequence of human β-globin, Kozak sequence, signal sequence of SARS-CoV-2 S protein, translation region of SARS-CoV-2 RBD, 3'-UTR sequence of human β-globin, polyA tail, and GAAGAGC (BspQI site) were linked in sequence. To 10X NEB Buffer 3.1 (100 μL, New England Biolabs, catalog # R7203S) and BspQI (40 μL, New England Biolabs, catalog # R0712) were added to Nuclease-free water (860 μL, Thermo Fisher, catalog # AM9937) in which the plasmid (100 μg) was dissolved, and after incubation at 50 °C for 1 hour, isopropanol (1400 μL) was added and left standing at -80 °C overnight. After centrifugation (-8 °C, 15,000 rpm, 10 minutes), the supernatant was discarded, 70% ethanol was added, and after centrifugation (-8 °C, 15,000 rpm, 10 minutes), the supernatant was discarded and air-dried. The obtained residue was dissolved in TE-Buffer (pH 8.0) and prepared into a 500 μg / mL solution. (2) Preparation of SARS-CoV-2 RBD S2004 mRNA by in vitro transcription Using the template DNA obtained in Example 19-(1) instead of the template DNA obtained in Example 1-(1), mRNA was obtained 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 to confirm that it has the desired length.

[0096] [Example 20] Preparation of mRNA-encapsulated nucleic acid lipid particles using the SARS-CoV-2 RBD S2004 mRNA described in Example 19 Using the same method as in Example 12, the preparation and property evaluation of mRNA-encapsulated nucleic acid lipid particles using the mRNA described in Example 19 were carried out. The results are shown in Table 3. From the results of the property evaluation, it was revealed that more than 95% of the mRNA was encapsulated in the lipid particles, and the nucleic acid lipid particles had an average particle diameter of about 180 nm.

[0097] [Examples 21 - 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 Distearoyl phosphatidylcholine (DSPC), cholesterol, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)penta-9,26-diene-7,29-diyl diacetate (LP), and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG) with a polyethylene glycol molecular weight of about 2000 were dissolved in ethanol at a total lipid concentration of 5 mM at the molar ratios described in Table 4. On the other hand, the mRNA obtained in Example 6 was diluted and prepared with citrate buffer (20 mM Citrate Buffer, pH 4.0). The above lipid solution and mRNA solution were mixed in a microchannel using NanoAssemblr BenchTop (Precision Nanosystems Inc.) such that the total lipid weight ratio to mRNA was the value described in Table 4 and the volume ratio was 1:3, to obtain a crude dispersion of nucleic acid-lipid particles. The dispersion of nucleic acid-lipid particles was dialyzed (Float-A-Lyzer G2, MWCO: 1,000 kD, Spectra / Por) against approximately 25 to 50 volumes of buffer for 12 to 18 hours to remove ethanol, and a dispersion of purified mRNA-encapsulated nucleic acid-lipid particles was obtained. (2) Characterization of mRNA-encapsulated nucleic acid-lipid particles Characterization of the dispersion containing the nucleic acid-lipid particles prepared in (1) was performed. The methods for each characterization will be described. (2-1) Encapsulation efficiency of mRNA The encapsulation efficiency of mRNA was measured using the Quant-iT RiboGreen RNA Assay kit (Invitrogen) according to the attached instructions. That is, in the presence and absence of 0.015% Triton X-100 surfactant, the mRNA in the dispersion of nucleic acid-lipid particles was quantified, and the encapsulation efficiency 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 dispersion of nucleic acid-lipid particles 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 particles was measured using an ultraviolet-visible spectrophotometer (manufactured by PerkinElmer, LAMBDA (trademark) 465). The mRNA concentration was calculated by 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 dispersion of nucleic acid-lipid particles 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 mass to mRNA was calculated by the following formula. [Total lipid concentration] / [mRNA concentration] (wt / wt) (2 - 3) Average particle size The particle size of the nucleic acid-lipid particles was measured using a Zeta Potential / Particle Sizer NICOMPTM 380ZLS (PARTICLE SIZING SYSTEMS). The average particle size in the table represents the volume-average particle size, and ± below represents the deviation. The results of the characterization are shown in Table 5. It was revealed that these nucleic acid-lipid particles encapsulate more than 95% of the mRNA and have an average particle size of approximately 90 nm to approximately 140 nm.

[0098] [Example 31] Preparation of mutant SARS-CoV-2 RBD mRNA For the RBD with the mutations described in Table 6, SARS-CoV-2 RBD mRNA was prepared. The symbols after the example numbers in Table 7 correspond to the respective mutant types as described in Table 6. For example, Example 32-a represents the nucleic acid-lipid particles encapsulating the mRNA having the South African type mutation obtained in Example 32. (1) Preparation of template DNA for in vitro transcription (IVT) of mutant SARS-CoV-2 RBD To prepare the template DNA for in vitro translation (IVT), the mutant SARS-CoV-2 RBD DNA was amplified by PCR and then purified. A DNA fragment (SEQ ID NO: 38) containing a sequence in which 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 mutant SARS-CoV-2 RBD, and the 3'-UTR sequence of human β-globin were ligated in sequence was introduced into a plasmid (pUC57mini-mutant RBD). To 566.4 μL of nuclease-free water in which 10 ng of the plasmid was dissolved, 80 μL of 10× Buffer for KOD-Plus- Ver.2 (Toyobo Co., Ltd. catalog # KOD-211), 80 μL of 2 mM dNTP mix (Toyobo Co., Ltd. catalog # KOD-211), 48 μL of 25 mM MgSO4 (Toyobo Co., Ltd. catalog # KOD-211), 4.8 μL of 50 μM sense primer (SEQ ID NO: 2), 4.8 μL of 50 μM antisense primer (SEQ ID NO: 3), and KOD Plus polymerase (16 μL, Toyobo Co., Ltd. catalog # KOD-211) were added. After incubation at 98°C for 15 seconds, 20 cycles of 98°C for 5 seconds, 55°C for 15 seconds, and 68°C for 1 minute were performed, followed by incubation at 68°C for 1 minute to amplify the RBD DNA. After the reaction, the template DNA (SEQ ID NO: 52) was purified using the Wizard SV Gel and PCR Clean-Up System (Promega catalog # A9281). Using the DNA fragments of SEQ ID NOs: 39 to 41, 43, and 48 to 51 instead of the DNA fragment (SEQ ID NO: 38) and by the same method, the template DNAs of SEQ ID NOs: 53 to 55, 57, and 62 to 65 were obtained respectively. (2) Preparation of Mutant SARS-CoV-2 RBD mRNA by in vitro Transcription Using the template DNA (SEQ ID NO: 52) obtained in Example 31-(1) instead of the template DNA obtained in Example 1-(1), mRNA was obtained 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 to confirm that it was of the target length. Using the template DNAs of SEQ ID NOs: 53 to 55, 57, and 62 to 65 respectively instead of the template DNA (SEQ ID NO: 52), mRNAs of SEQ ID NOs: 67 to 69, 71, and 76 to 79 were obtained respectively by the same method.

[0099] [Example 32] Preparation of mRNA-Encapsulated Nucleic Acid Lipid Particles Using the SARS-CoV-2 RBD mRNA Described in Example 31 Using the same method as in Example 8, the preparation and property evaluation of mRNA-encapsulated nucleic acid-lipid particles using the mRNA described in Example 31 were carried out. The results are shown in Table 7. From the results of the property evaluation, it was revealed that in these nucleic acid-lipid particles, 95% or more of the mRNA was encapsulated within the lipid particles and they had an average particle diameter of about 110 nm to about 130 nm.

[0100] [Example 33] Preparation of mRNA-Encapsulated Nucleic Acid Lipid Particles Using the SARS-CoV-2 RBD mRNA Described in Example 6 Using the same method as in Example 8, the preparation and property evaluation of mRNA-encapsulated nucleic acid-lipid particles using the mRNA described in Example 6 were carried out. The results are shown in Table 7. From the results of the property evaluation, it was revealed that in these nucleic acid-lipid particles, 95% or more of the mRNA was encapsulated within the lipid particles and they had an average particle diameter of about 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) Under 1-4% (v / v) vaporized isoflurane anesthesia, the test substance was administered to the gastrocnemius region of the hind limb of the mouse. In the three-dose test, additional doses were given 7 days and 21 days after the first dose (right hind limb at the first dose, left hind limb at the second dose, right hind limb at the third dose), and in the two-dose test, an additional dose was given 13 days after the first dose (right hind limb at the first dose, left hind limb at the second dose). The test substance was administered at 3 μg mRNA / 20 μL / body or 1 μg mRNA / 20 μL / body per administration (described as Example No._3 and Example No._1 in FIGS. 2-4, respectively. For example, the description of Example 3_3 in FIGS. 2-4 means the group in which the particles of Example 3 were administered at 3 μg mRNA / 20 μL / body). As the buffer for preparing the administration solution, 10 mM Histidine buffer containing 300 mM sucrose, pH 6.5 was used. S1 protein (Sino Biological, Cat#40591-V08H) added with a commercially available saponin adjuvant (Quil-A Adjuvant, Invivogen, Cat#vac-quil) was set as the positive control group for the 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 The blood obtained from the tail vein at the time of administration of the test substance was collected into a tube containing a serum separator (BD, Cat#365967), and the serum was collected after centrifugation (15,000 rpm, 4°C, 5 minutes, centrifuge: TOMY, MX-205). The blood obtained from the heart 14 days after the final administration of the 3-dose test was collected into a tube, left standing at room temperature for 3 hours, then left standing in a refrigerator set at 4°C for 22 hours, and then centrifuged (1700×g, 4°C, 5 minutes) to collect the serum. In addition, the spleen was collected from a mouse sacrificed by exsanguination under isoflurane anesthesia, a cell suspension was prepared using a cell strainer (CORNING, Cat#352350), and hemolysis treatment 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 would be 10 μg / mL. Also, as a negative control, an equal volume of Buffer as the amount of the particles of Example 4 added was added. The culture supernatant and cell pellet were collected 3 days after the addition. The cell pellet was lysed with 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 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 the Enzyme-Linked Immunosorbent Assay (ELISA) method using an anti-RBD antibody (Sino Biological, Cat#40592-T62).

[0109] Serum Anti-RBD Antibody Titer (Figures 2 - 4) Recombinant RBD protein (Sino Biological, Cat#40592-V08H) was added at 0.25 μg / mL (50 μL / well) to Ni plates (QIAGEN, Cat#35061) with blocking solution (PBS containing 1% BSA and 0.05% Tween 20), and left standing at room temperature for 2 hours. Then, it was washed three times with 300 μL / well of washing solution (PBS containing 0.05% Tween 20). The sample dilution series was prepared in 8 steps with 4-fold dilution from the 100-fold diluted serum of the highest concentration using the blocking solution. The standard serum dilution series was prepared in 8 steps with 3-fold dilution from 2 DS UNIT / mL of the highest concentration using the blocking solution. The sample dilution solution and the standard serum dilution solution were added (50 μL / well), left standing at room temperature for 1 hour, and then washed three times with the washing solution. The detection antibody was prepared by diluting HRP-labeled anti-mouse IgG antibody (Southern Biotech, Cat#1030-05) 4000-fold with the blocking solution, adding it to the plate (50 μL / well), and leaving it standing at room temperature for 1 hour. After washing three times with the washing solution, TMB Microwell Peroxidase Substrate System (SERACARE Life Sciences, Cat#5120-0047) was added (50 μL / well) and left standing 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 (reference wavelength 540 nm) was measured using a plate reader, and the corrected absorbance (Delta), which was the absorbance measured at 450 nm minus the absorbance measured at 540 nm, was used for analysis. A calibration curve was created using Nonlinear Regression: 4 Parameter from the anti-RBD antibody concentration and Delta of the standard serum. The anti-RBD antibody concentration of the sample was calculated from the calibration curve, the dilution factor of the measured sample, and Delta. The average value of the antibody concentration in the wells where Delta was 0.5 - 1.5 was calculated as the anti-RBD antibody concentration of the measured sample. When Delta in the well of the highest sample concentration was less than 0.5, 20 DS UNIT / mL was substituted as the data.

[0110] RBD-hACE2 Binding Inhibitory Activity 10 μg / mL of Streptavidin (Thermo Fisher Scientific, Cat# 21125, dissolved in PBS) was added to a 96-well plate (Coaster, Cat# 3690) and left standing overnight at 4 °C, then washed three times with a washing solution (PBS containing 0.05% Tween 20). A blocking solution (PBS containing 1% BSA and 0.05% Tween 20) was added and left standing at room temperature for 1 hour, then washed three times with the washing solution. Then, a 0.2 μg / mL solution of recombinant RBD protein (Acro Biosystems, Cat# SPD-C82E9) prepared with the blocking solution was added to the plate and left standing at room temperature for 1 hour, then washed three times with the washing solution. Mouse serum diluted 20-fold with the blocking solution was added to the plate, left standing at room temperature for 1 hour, and then washed three times with the washing solution. A 1 μg / mL solution of recombinant hACE2 protein (Acro Biosystems, Cat# AC2-H5257) prepared with the blocking solution was added to the plate and left standing at room temperature for 1 hour, then washed three times with the washing solution. The detection antibody was a HRP-labeled anti-human IgG1 antibody (CYGNUS TECHNOLOGIES, Cat# IM50) diluted 500-fold with the blocking solution, added to the plate, and left standing at room temperature for 1 hour. After washing three times with the washing solution, TMB Microwell Peroxidase Substrate System (SERACARE Life Sciences, Cat# 5120-0047) was added and left standing 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 A total of 253 overlapping peptides (#1 to #253) were custom-synthesized (Eurofins) to cover the full length of the SARS-CoV-2 S protein. Each peptide was dissolved in 200 μL of dimethyl sulfoxide (DMSO, Nacalai Tesque, Cat#13408-64). To cover the RBD and the regions before and after it, #1 to #62, #63 to #107, and #108 to #253 were mixed in equal amounts to prepare three epitope peptide pools (Euro1, Euro2, and Euro3 in that order). Additionally, commercially available epitope peptide pools that cover the full length of the SARS-CoV-2 S protein (JPT, Cat#PM-WCPV-S-1, 2 vials, the peptide pool covering the N-terminal region is JPT-N, and the peptide pool covering the C-terminal region is JPT-C) were dissolved in 40 μL of DMSO per vial.

[0112] RBD-Specific Cellular Immune Response Spleen cells were prepared to 1×107 cells / mL with 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], 1×StemSure [FUJIFILM Wako Pure Chemical Corporation, Cat#195-15791], 1×MEM Non-Essential Amino Acids Solution [Thermo Fisher Scientific, Cat#11140-050]) and seeded in a U-bottom 96-well plate. Epitope peptide pool Euro1-3 solution prepared to a final concentration of 0.1% (v / v) and commercially available epitope peptide pools JPT-N and JPT-C prepared to a final concentration of 0.025% (v / v) with RPMI Complete medium were added to the spleen cells, and the cells were cultured at 37°C under 5% CO2 for 48 hours. 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 (reference 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 calibration curve was created using Nonlinear Regression: 4 Parameter from the cytokine concentrations of the standard solution and the Delta values, and the cytokine concentrations of the measurement samples were calculated from the calibration curve. When the IL-13 concentration was less than 0.000 (<0.000), the cut-off value of 0.005 was substituted as the data.

[0113] Statistical Analysis For the comparison of anti-RBD antibody response in blood and RBD-hACE2 binding inhibitory activity, a t-test was performed for the three-dose study, and a Dunnett test was performed for the two-dose study using the Buffer group as a control. For the comparison of RBD-specific cellular immune responses, a Dunnett test was performed for each peptide treatment using the S1 / Quil-A group as a control. SAS ver. 9.2 was used for all analyses.

[0114] Administration to Mice (Figures 5 - 9, Figures 25 - 28) Under 1-4% (v / v) isoflurane vapor anesthesia, the test substance was administered to the hindlimb gastrocnemius of BALB / c mice (Figs. 5-8, 25, 26, and 28) or C57BL / 6 mice (Fig. 9) twice at two-week intervals (Fig. 5) or twice at three-week intervals (Figs. 6-9, 25, 26, and 28). Fig. 27 shows that the test substance was administered only once to the hindlimb gastrocnemius of BALB / c mice. 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 the group in which the particles of Example 8 were administered at 0.03 μg mRNA / 20 μL / body). Figs. 25 and 27 were administered at 2 μg mRNA / 20 μL / body per administration, and Figs. 26 and 28 were administered at 3 μg mRNA / 20 μL / body per administration. The buffer for preparing the administration solution used was 10 mM Histidine buffer containing 300 mM sucrose, pH 7.0.

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

[0116] Serum Anti-RBD Antibody Titer (Figures 5, 6, 9, and Figures 25 - 27) The streptavidin (Thermo Fisher Scientific Inc.) solid-phase solution was added to the ELISA plate at 25 μL / well and left standing overnight in a refrigerator set at 4°C. It was washed three times with Wash Buffer (180 μL / well) using a plate washer (AMW-96SX, Biotech Co., Ltd.), blocking was performed by adding 1% BSA / PBST (150 μL / well) and leaving it standing at room temperature for 1 hour or more. After washing three times with Wash Buffer (180 μL / well) using a plate washer, an 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 left standing at room temperature for 1 hour or more. After washing three times with Wash Buffer (180 μL / well) using a plate washer, the measurement sample stage diluent and the standard serum stage diluent (Figures 5, 6, 9, 25, 26) were added (25 μL / well) and left standing at room temperature for 1 hour or more. As the standard sample in Figure 27, a serial diluent of an anti-RBD antibody (clone #3) that binds equivalently to the RBD derived from Original and the RBD derived from the B.1.351 strain was used. After washing three times with Wash Buffer (180 μL / well) using a plate washer, an HRP-labeled anti-mouse IgG antibody (Southern Biotech, Cat#1030-05) detection antibody diluent was added (25 μL / well) and left standing at room temperature for 1 hour. After washing three times with the washing solution, TMB Microwell Peroxidase Substrate System (SERACARE Life Sciences, Cat#5120-0047) was added (30 μL / well) and left standing for 10 minutes. As the reaction stop solution, TMB Stop Solution (SERACARE Life Sciences, Cat# 5150-0021, 30 μL / well) was used. The absorbance at a wavelength of 450 nm (reference 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 the analysis.Using the anti-RBD antibody concentration of the standard serum and Delta, a calibration curve was created using Nonlinear Regression: 4 Parameter. The anti-RBD antibody concentration of the sample was calculated from the calibration curve, the dilution factor of the measurement sample, and Delta.

[0117] Serum Anti-SARS-CoV-2 Neutralizing Activity (Figures 7 and 8) VeroE6 cells were seeded onto plates and cultured overnight in an incubator set at 37 ± 2°C and a CO2 concentration of 5 ± 1%. A dilution series of mouse serum was mixed with the SARS-CoV-2 WA1 / 2020 strain and left standing in an incubator set at 37 ± 2°C and a CO2 concentration of 5 ± 1% for 2 - 2.5 hours. Then, the mixture of mouse serum and the SARS-CoV-2 WA1 / 2020 strain was added to the VeroE6 cells and cultured in an incubator set at 37 ± 2°C and a CO2 concentration of 5 ± 1% for 72 ± 8 hours. Subsequently, the amount of viable cells was 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 Response (Figure 10) Spleen cells were prepared at 1×10 7 cells / mL in RPMI Complete medium and seeded into a U-bottom 96-well plate. The MHC class II epitope peptide pool of RBD prepared to a final concentration of 0.1% (v / v) in RPMI Complete medium was added to the spleen cells and cultured at 37°C and 5% CO2 for 48 hours. 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 (reference wavelength 540 nm) was measured using a plate reader, and the value obtained by subtracting the absorbance measured at 540 nm from the absorbance measured at 450 nm was used for analysis. A calibration curve was created using Nonlinear Regression: 4 Parameter from the cytokine concentration and measured values of the standard solution, and the cytokine concentration of the measurement sample was calculated from the calibration curve.

[0119] Statistical Analysis Regarding the blood anti-RBD antibody response shown in Fig. 5, the comparison between the two groups at each dose of 0.03 μg mRNA / body and 0.3 μg mRNA / body was performed by Wilcoxon test. For the comparison among the three groups at the 3 μg mRNA / body dose, Steel test was performed using Example 8 as the comparative control. Regarding the blood anti-RBD antibody response shown in Fig. 6, the comparison between the two groups at each dose of 0.03 μg mRNA / body, 0.3 μg mRNA / body, and 3 μg mRNA / body was performed by Wilcoxon test. Regarding the blood anti-SARS-CoV-2 neutralizing activity shown in Fig. 7, Steel test was performed using the Buffer group as the comparative control. For the comparison between the two groups in Fig. 8, Wilcoxon test was performed. Regarding the blood anti-RBD antibody response shown in Fig. 9, Steel test was performed using the Buffer group as the comparative control. Regarding the RBD-specific cellular immunity shown in Fig. 10, Steel-Dwass test was performed. SAS ver. 9.2 was used for all the analyses.

[0120] RBD-hACE2 Binding Inhibitory Activity (Figure 28) Anti-His tag antibody (Wako Pure Chemical Industries, Cat# 017-23211) was added to a 96-well plate and left standing at 4 °C overnight, then washed three times with a washing solution (PBS containing 0.05% Tween 20). A blocking solution (PBS containing 1% BSA and 0.05% Tween 20) was added and left standing at room temperature for 1 hour, then washed three times with the washing solution. Subsequently, a 0.2 μg / mL recombinant RBD protein solution (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) prepared with the blocking solution was added to the plate, left standing at room temperature for 1 hour, and then washed three times with the washing solution. A mouse serum dilution series diluted with the blocking solution was added to the plate, left standing at room temperature for 1 hour, and then washed three times with the washing solution. A 1 μg / mL recombinant hACE2 protein (Acro Biosystems, Cat# AC2-H5257) solution prepared with the blocking solution was added to the plate, left standing at room temperature for 1 hour, and then washed three times with the washing solution. The detection antibody was a 500-fold dilution of an HRP-labeled anti-human IgG1 antibody (CYGNUS TECHNOLOGIES, Cat# IM50) with the blocking solution, added to the plate, and left standing at room temperature for 1 hour. After washing three times with the washing solution, TMB Microwell Peroxidase Substrate System (SERACARE Life Sciences, Cat# 5120-0047) was added and left standing for 10 minutes. The reaction stop solution used was TMB Stop Solution (SERACARE Life Sciences, Cat# 5150-0021).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 the analysis. The data indicate the mouse dilution ratio showing 50% inhibition (IC. 50 )。

[0121] Serum Anti-SARS-CoV-2 Neutralizing Activity (Figure 29) Vero-TMPRSS2 cells were seeded in plates. A dilution series of monkey plasma was mixed with 100 TCID 50 of SARS-CoV-2 strains (D614G: HP095, B.1.1.7 lineage strain: QHN001, P.1 lineage strain: TY7-501, B.1.351 lineage strain: TY8-612) and left standing in a CO2 incubator. Subsequently, the mixture of monkey plasma and SARS-CoV-2 was added to Vero-TMPRSS2 cells and cultured in a CO2 incubator for 3 days. Then, the highest dilution ratio at which no cytopathic effect (CPE) was observed was calculated as the neutralizing antibody titer.

[0122] Results RBD Protein Expression Inducing Ability of Example 4 The mechanism of action of the nucleic acid-lipid particle vaccine of the present invention is suggested to produce an antigen protein from the mRNA encoding the antigen gene after administration in vivo and induce a specific immune response against the antigen. The efficacy of the nucleic acid-lipid particle vaccine of the present invention is assumed that the delivery of the mRNA, which is the active ingredient, into tissues and cells and the translation from the mRNA are important factors. For the purpose of comprehensively evaluating this series of factors, the titer was evaluated using cultured cells with the ability to induce the expression of the antigen protein as an index. 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 cells after 3 days of culture was quantified by ELISA. The results are shown in Figure 1. The RBD protein expressed by the particles of Example 4 was observed in the culture supernatant and cells. The full-length S protein expressed by the particles of Example 3 was only observed intracellularly.

[0123] Serum 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. Compared with the two-dose group and three-dose group of Example 3, the three-dose group of Example 4 had a higher blood anti-RBD antibody titer (P = 0.0346). Also, compared with the Buffer group, the two-dose group of Example 4 had a higher blood anti-RBD antibody titer (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-dose group and three-dose group of Example 3, the serum of the three-dose group of Example 4 had a higher RBD-hACE2 binding inhibitory activity (P = 0.0005). Also, compared with the Buffer group, the serum of the two-dose group of Example 4 had a 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 the RBD-specific cellular immune response from cultured spleen cells was evaluated. The results are shown in Figure 4. Compared with the S1 / Quil-A group, the Example 4 group had a higher IFN-γ production level against the Euro2 and JPT-N epitope peptide pool treatment covering RBD (P < 0.001). On the other hand, compared with the S1 / Quil-A group, the Example 4 group had a lower IL-13 production level against the Euro2 and JPT-N epitope peptide pool treatment (P <0.005). From this result, it was found that the nucleic acid-lipid particle vaccine of the present invention induces an immune response with a dominant Th1 type.

[0126] Serum Anti-RBD Antibody Response 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 Fig. 5. At each dose of 0.03 μg mRNA / body and 0.3 μg mRNA / body, compared with Example 4, Example 8 had a higher blood anti-RBD antibody titer (both P = 0.0286). Also, at a dose of 3 μg mRNA / body, no significant difference was observed in the blood anti-RBD antibody titers among Example 4, Example 7, and Example 8 (all 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 Fig. 6. At any dose, no significant difference was observed in the blood anti-RBD antibody titers between Example 10 and Example 8 (0.03 μg mRNA / body: P = 0.8413, 0.3 μg mRNA / body: P = 0.0952, 3 μg mRNA / body: P = 0.6905).

[0127] Serum 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 with the Buffer group, the group administered 3 μg mRNA / body of Example 10 had a higher blood anti-SARS-CoV-2 neutralizing activity (P = 0.0374). Also, as a result of comparing the blood anti-SARS-CoV-2 neutralizing activities induced by administration of the particles of Example 8 and Example 10, no significant difference was observed (Fig. 8, P = 1).

[0128] Serum Anti-RBD Antibody Response 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 Fig. 9. At each dose of 3 μg mRNA / body and 10 μg mRNA / body, compared with the Buffer group, Example 8 and Example 10 had higher blood anti-RBD antibody titers (P < 0.05 for both doses of Example 10).

[0129] RBD-Specific Cellular Immune Response Spleen cells were prepared and the RBD-specific cellular immune response from the cultured spleen cells was evaluated. The results are shown in Fig. 10A. Compared with the group in which 100 μg / body of Alum adjuvant was added to 0.1 μg / body of RBD protein, the group administered with 3 μg / body of Example 10 showed high IFN-γ induction (P < 0.05). Also, compared with the group in which 100 μg / body of alum adjuvant was added to 1.0 μg / body of RBD protein, the groups administered with 0.03 μg / body and 3 μg / body of Example 10 showed high 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 RBD protein group with Alum adjuvant added, Example 10 showed a high IFN-γ level / IL-13 level ratio (P < 0.05 for all three groups of Example 10 compared to the two groups of RBD protein with Alum adjuvant added). From this result, it was found that the nucleic acid-lipid particle vaccine of the present invention induces an immune response with Th1 type predominance.

[0130] Serum Anti-RBD Antibody Response in BALB / c Mice (Figures 25 - 27) The blood anti-RBD antibody response induced by administration of the particles of Example 10, 12, 14, 16, 18, or Example 20 was evaluated. The results are shown in Fig. 25. All showed high blood anti-RBD antibody levels compared to the Buffer group. The blood anti-RBD antibody response induced by administration of the particles of Example 10 or Example 21 - 30 was evaluated. The results are shown in Fig. 26. In all particles, a high blood anti-RBD antibody titer was observed 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 Fig. 27. Compared with Example 32a, Example 10, 32b, 32c, 32d, 32f, and Example 33 showed high blood anti-RBD antibody levels.

[0131] RBD-hACE2 Binding Inhibitory Activity (Figure 28) The inhibitory activity of the particles of Example 10 on RBD-hACE2 binding was evaluated. The results are shown in Fig. 28. Compared with 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 sera of the Example 10 group.

[0132] Serum Anti-SARS-CoV-2 Neutralizing Activity (Figure 29) The neutralizing activity against SARS-CoV-2 in blood induced by the particles of Example 10 was evaluated. The results are shown in Fig. 29. The infection of Vero-TMPRSS2 cells with the D614G strain, B.1.1.7 lineage strain, P.1 lineage strain, and B.1.351 lineage strain was neutralized to the same extent by the sera of the Example 10 group.

[0133] [Test Example 2] Optimization of an LNP-mRNA Vaccine Candidate Encoding SARS-CoV-2 RBD The pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occurred, and two types of mRNA vaccines encoding the full-length SARS-CoV-2 spike protein were launched (1, 2). However, there are still points to be improved 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 (LNP) using immunogenicity as an index.

[0135] First, 6- to 8-week-old C57BL / 6 mice or BALB / c mice were administered 3 μg of LNP-mRNA-RBD intramuscularly twice at 2-week intervals, and the anti-RBD antibody response in the blood was evaluated. As a result, compared with C57BL / 6 mice, BALB / c mice showed a higher anti-RBD antibody response in the blood (Figure 11a, Figure 15). To compare the RBD-specific B cell responses induced by LNP-mRNA-RBD among mouse strains, T FH and GC B cells in the popliteal lymph nodes (pLNs) of LNP-mRNA-RBD-administered mice were analyzed by flow cytometry (Figure 16). As a result, correlating with the anti-RBD antibody response in the blood, T FH (CD4 + CD185 + PD-1 + cells) and GC B cells (CD38 - GL7 + CD19 + cells) were higher in LNP-mRNA-RBD-administered BALB / c mice compared with C57BL / 6 mice (Figure 11b-e).

[0136] The antigen-specific CD8 + and CD4 +To analyze T cells, a peptide library of spike proteins was designed. This peptide library consists of 128 20 - amino - acid peptides designed such that 10 amino acids overlap. This peptide library was divided into 8 pool peptides, with 16 peptides in each pool peptide (Figure 11f). When spleen cells prepared from LNP - mRNA - RBD - administered mice were treated with pool peptides 3 and 4, C57BL / 6 mouse spleen cells induced IFN - γ production, and BALB / c mouse spleen cells showed IFN - γ production upon treatment with peptide pool 3 (Figures 11g and h, Figures 17a and b). IL - 13 was not induced in spleen cells of either C57BL / 6 or BALB / c mice (Figures 17c and d). To analyze antigen - specific T cells induced by LNP - mRNA - RBD administration, spleen cells were treated with pool 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 multifunctional CD8 + and CD4 + T cells were observed in BALB / c mouse spleen cells treated with pool peptides 3 and 4 (Figures 11h, Figures 18b and 19b). In C57BL / 6 mouse spleen cells, multifunctional CD8 + T cells and weak CD4 + T cell responses were shown (Figures 11g, Figures 18a and 19a). These data suggest that, compared with C57BL / 6 mice, BALB / c mice administered with LNP - mRNA - RBD are likely to induce higher B - cell and T - cell responses.

[0137] Vaccines using nucleic acids have been reported to have DNA or RNA acting as endogenous adjuvants (14-16). In LNP-mRNA vaccines, mRNA has been reported to be recognized by toll-like receptor (TLR) 3, TLR7, TLR8, RIG-I, or MDA5 and act as an endogenous adjuvant (17). Kariko et al. have used methylated bases and other modified bases (e.g., pseudouridine, etc.) for the control of innate immune activation and the improvement of antigen protein expression efficiency (18,19). In other studies, it has been clarified that type I IFN induced by LNP-mRNA affects the CD8 + T cell response and the antigen protein expression efficiency (20,21,22). Regarding the SARS-CoV-2 vaccine, compared with the LNP-mRNA-Full group encoding the full length of the spike, the LNP-mRNA-RBD group had a higher frequency of side effects. Therefore, LNP-mRNA-Full was evaluated in a phase III clinical trial and marketed (13). Although the reason for the difference in the frequency of side effects is unclear, we consider that the innate immune activation effect by LNP-mRNA may be involved (13).

[0138] To analyze the innate immune activation effect by LNP-mRNA, the levels of type I IFN production from human PBMCs treated with LNP-mRNA-RBD were measured by ELISA. As a result, in PBMCs from three healthy individuals, LNP-mRNA-RBD showed higher IFN-α inducing ability compared to LNP-mRNA-Full (Figure 12a). Next, similar experiments were conducted 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). In the manufacturing process of the mRNA encapsulated in LNP-mRNA, it has been shown that it contains contaminants such as RNA, for example, double-stranded RNA as a TLR3 ligand, which may activate innate immunity (22). Therefore, to remove contaminants resulting from RNA production, the mRNA was purified by HPLC, and LNP-mRNA (mRNA-RBD (HPLC)) encapsulating the HPLC-purified mRNA was prepared. As a result, type I IFN production from human PBMCs and mouse BM-DCs treated with mRNA-RBD (HPLC) was significantly decreased compared to LNP-mRNA-RBD (Figures 12a and b).

[0139] C57BL / 6 or BALB / c mice were administered mRNA-RBD (HPLC) to evaluate immunogenicity. As a result, the mRNA-RBD (HPLC) group enhanced the blood anti-RBD IgG1 titer, IgG2 titer, and total IgG titer in both BALB / c mice and C57BL / 6 mice (Figures 12c and 20a). Also, compared to the LNP-mRNA-RBD group, more GC B cells were induced in the pLNs of C57BL / 6 mice administered mRNA-RBD (HPLC) (Figures 12d and e). Furthermore, compared to the LNP-mRNA-RBD group, in the mRNA-RND (HPLC) group, multifunctional CD8 + and CD4 + T cells that specifically produce IFN-γ and other type I cytokines were induced more (Figures 12f-i, and Figures 20b-e, 21, 22). In the cynomolgus monkey model, which is a non-human primate (NHP), the protective effect of the mRNA-RBD (HPLC) vaccine against SARS-CoV-2 was evaluated. In this study, two monkeys were used as the negative control group, and four monkeys were intramuscularly administered with mRNA-RBD (HPLC). As a result, compared with the negative control group, the mRNA-RBD (HPLC) group showed a high anti-RBD antibody response (Figure 13b). In addition, 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 high 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 in the swab (Figure 14a) and viral RNA (Figure 14b) on the first day after SARS-CoV-2 infection. Also, on the seventh day after infection, the mRNA-RBD (HPLC) group also reduced the viral RNA in the trachea, bronchi, and lungs (Figure 14c, Figure 25). Furthermore, the negative control group showed fever and pneumonia after SARS-CoV-2 infection (Figure 23, 24). When histological analysis of the lungs was performed after SARS-CoV-2 infection, infiltration of lymphocytes and neutrophils was observed in the negative control group, thickening of the alveolar wall and viral antigen were also confirmed, and these phenomena were not confirmed in the mRNA-RBD (HPLC) group (Figure 14d, 14e). In addition, it was confirmed that bronchus-associated lymphoid tissue (BALT) was formed in the mRNA-RBD (HPLC) group (Figure 14d). These results suggest that antibodies induced through the formation of BLAT in mucosae such as nasal mucosa and tracheal mucosa bind to SARS-CoV-2 and neutralize it, resulting in a decrease in viral RNA and infectious virus in the swab on the first day after infection.

[0141] Materials and Methods Mice C57BL / 6 and BALB / c mice at 6 - 8 weeks of age were purchased from CLEA Japan, Inc. in Japan. The mice were bred under specific pathogen - free conditions. All mouse experiments were approved by the Animal Experiment Committee of the Institute of Medical Science, The University of Tokyo.

[0142] Cynomolgus monkeys Female cynomolgus monkeys, 7 - 10 years old, born at Shiga University of Medical Science and native to the Philippines, Vietnam, and China were used. All procedures were performed under ketamine anesthesia and xylazine anesthesia, and efforts were made to minimize pain. After recovery from anesthesia, CMK - 2 (CLEA Japan, Inc., Tokyo, Japan) hood pellets were given once a day, and drinking water was available ad libitum. The animals were housed singly in cages under light - controlled conditions (12 - hour light / 12 - hour dark cycle, lights on at 8:00 am). Under ketamine / xylazine anesthesia, SARS - CoV - 2 (2×10 7 PFU / 7 mL HBSS) was inoculated into the monkeys via pipette and catheter into the conjunctiva (0.05 mL×2), nostrils (0.5 mL×2), oral cavity (0.9 mL), and trachea (5 mL). After collection of body fluid samples from the conjunctiva, nasal cavity, oral cavity, and trachea using two cotton swabs (Eiken Chemical Co., Ltd., Tokyo, Japan) under ketamine / xylazine anesthesia, the cotton swabs were immersed in 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).

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

[0144] Reagents 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 The SARS-CoV-2 isolate was 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 of L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin / mL.

[0146] Immunization method 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. Two weeks after the second immunization, popliteal lymph nodes, spleens, and blood were collected. Cynomolgus monkeys were immunized intramuscularly with mock or LNP-mRNA-RBD (HPLC) (100 μg) on days 0 and 21. Blood samples were collected on days 0, 7, 14, 21, and 28.

[0147] ELISA method ECD- and RBD-specific antibody titers were measured by ELISA. Briefly, ECD (1 μg / mL) or RBD (1 μg / mL) was coated in bicarbonate buffer on a half-area 96-well plate at 4 °C. The plate was blocked with PBS containing 1% BSA at room temperature for 60 minutes. The plate was washed three times with PBST, and diluted plasma or swab samples were incubated at room temperature for 120 minutes. The plate was washed three times with PBST, and incubated with HRP-labeled goat anti-mouse IgG, IgG1, IgG2a, IgG2c, or mouse anti-monkey IgG at room temperature for 120 minutes. After washing three times with PBST, TMB substrate buffer was added and incubated at room temperature for 10 minutes. Then, 1 N H2SO4 was added to stop the reaction. OD values at 450 nm and 540 or 560 nm were measured using a spectrophotometer. The reciprocal value of the plasma dilution at which OD 450 -OD 540 or OD 450 -OD 560 reached 0.2 was taken as the antibody titer. The single-cell suspension of splenocytes from immunized mice was stimulated with peptide pools 1-8, ECD, and RBD protein for 24 hours. The IFN-γ and IL-13 levels in the supernatant were measured by ELISA (R&D).

[0148] GC B cells and T FH Staining The single-cell suspension of popliteal lymph nodes was 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. The percentages of GC B cells and T FH cells were analyzed by flow cytometry.

[0149] Intracellular cytokine staining assay The single-cell suspension of splenocytes was stimulated with peptide pools 2, 3, 4 for 6 hours together with a protein transport inhibitor (eBioscience, San Diego, CA, USA). After stimulation, dead cells were stained with LIVE / DEAD Aqua. After washing, the 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), anti-CD45R / B220 (RA3-6B2) antibodies. All antibodies were purchased from BioLegend unless otherwise specified. After fixation, permeabilization with IC Fixation Buffer (eBioscience), intracellular cytokines, CD3 were stained with anti-IFN-γ (XMG1.2), anti-IL-2 (JES6-5H4), anti-TNF-α (MP6-XT22), anti-CD3 (17A2) antibodies. All antibodies were purchased from BioLegend. Cytokine-producing CD8 + and CD4 +The proportion 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 from three healthy adult volunteers uninfected with SARS-CoV-2 after obtaining informed consent. All experiments using human PBMCs were approved by the Ethics Review Committee of the Institute of Medical Science, The University of Tokyo. After preparing PBMCs using Ficoll Histopaque, they 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 the IFN-α levels in the culture supernatant were measured using ELISA (Mabtech, Stockholm, Sweden).

[0151] Stimulation with bone marrow-derived dendritic cells Bone marrow-derived dendritic cells (BM-DCs) were differentiated by culturing with 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 titer 35 microliters of virus (140 tissue culture infectious doses 50) was incubated with 35 μL of 2-fold serial dilutions of serum at room temperature for 1 hour, and 50 μL of the mixture was added to confluent VeroE6 / TMPRS2 cells in a 96-well plate and incubated at 37°C for 1 hour. After adding 50 μL of DMEM containing 5% FCS, the cells were further incubated at 37°C for 3 days. The viral cytopathic effect (CPE) was observed under an inverted microscope, and the virus neutralizing 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 line expressing TMPRSS2 (JCRB Cell Bank, Japan) was incubated with diluted swab samples and 10% w / v tissue homogenate samples for 1 hour. The cells were washed with HBSS and incubated with DMEM containing 0.1% BSA for 3 days (25). Virus 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 CFX-96 (Bio-Rad, Hercules, CA, USA).

[0155] Body temperature Two body temperature data loggers (iButton, Maxim Integrated, San Jose, California) were implanted into the peritoneal cavity or subcutaneous tissue of each monkey under ketamine / xylazine anesthesia 2 weeks before virus inoculation, and then isoflurane was inhaled to monitor body temperature.

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

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[0158] The present invention can be used for the prevention and / or treatment of infections caused by SARS-CoV-2. [Sequence Listing Free-Text]

[0159] <SEQ ID NO:1> (DNA fragment containing SARS-CoV-2 S full) <SEQ ID NO: 2> (Sense Primer) GTAATACGACTCACTATAA <SEQ ID NO: 3> (Antisense Primer) TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCAATGAAAATAAATGTTTTTTATTAGGC <SEQ ID NO: 4> (Template DNA of SARS-CoV-2 S-full) GTAATACGACTCACTATA A T7 promoter: Base numbers 1 to 18 A : Transcription start point: Base number 19 5’-UTR (including the transcription start point and the KOZAK sequence of base numbers 89 to 94): Base numbers 19 to 88 Full-length Spike protein sequence: Base numbers 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) <Accession No. 6> (Amino acid sequence of SARS-CoV-2 S-full) RBD sequence: Amino acid numbers 319 - 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 - 18 A : Transcription start point: Base number 19 5’-UTR (including the transcription start point and the KOZAK sequence of base numbers 89 - 94): Base numbers 19 - 88 Spike protein signal sequence: Base numbers 89 - 127 RBD sequence: Base numbers 128 - 799 3’-UTR: Base numbers 800 - 931 polyA sequence (A100): Base numbers 932 - 1031 <SEQ ID NO:9> (SARS-CoV-2 RBD mRNA-002) <SEQ ID NO: 10> (Amino acid sequence of SARS-CoV-2 RBD (including S protein signal sequence)) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF S protein signal sequence: Amino acid numbers 1 - 13 RBD sequence: Amino acid numbers 14 - 236 <SEQ ID NO: 11> (Amino acid sequence of SARS-CoV-2 RBD (excluding S protein signal sequence)) RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF <SEQ ID NO: 12> (S_opt2 EcoRI) NheI sequence: base numbers 1 - 6 T7 promoter: base numbers 7 - 24 A: transcription start point: base number 25 5’-UTR (including the transcription start point and the KOZAK sequence at base numbers 89 - 94): base numbers 25 - 94 Full-length Spike protein sequence: base numbers 95 - 3916 3’-UTR: base numbers 3917 - 4048 EcoRI sequence: base numbers 4049 - 4054 <SEQ ID NO: 13> (Sense primer 2) TGATGCTAGCGTAATACGACTCACTATAAG NheI sequence: base numbers 5 - 10 <SEQ ID NO: 14> (Antisense primer 2) GCCAAAGCTTGCTCTTCGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCAATGAAAATAAATGTTTTTT HindIII sequence: base numbers 5 - 10 BspQI sequence: base numbers 11 - 17 <SEQ ID NO: 15> (Template DNA for SARS-CoV-2 S full optimized) NheI sequence: base numbers 5 - 10 T7 promoter: base numbers 11 - 28 A: transcription start point: base number 29 5’-UTR (including the transcription start point and the KOZAK sequence at base numbers 89 - 94): base numbers 29 - 98Full-length Spike protein sequence: base numbers 99 - 3920 3’-UTR: base numbers 3921 - 4052 polyA sequence (A110): base numbers 4053 - 4162 BspQI sequence: base numbers 4164 - 4170 HindIII sequence: base numbers 4171 - 4176 <SEQ ID NO: 16> (SARS-CoV-2 S full optimized mRNA-003) <SEQ ID NO: 17> (S_RBD_opt2 EcoRI) GCTAGCGTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCGAATTC NheI sequence: base numbers 1 to 6 T7 promoter: Base numbers 7 - 24 A: Transcription start point: Base number 25 5’-UTR (including the transcription start point and the KOZAK sequence at base numbers 89 - 94): Base numbers 25 - 94 RBD sequence: Base numbers 95 - 805 3’-UTR: Base numbers 806 - 937 EcoRI sequence: Base numbers 938 - 943 <SEQ ID NO: 18> (Template DNA for SARS-CoV-2 RBD optimized) NheI sequence: base numbers 5 - 10 T7 promoter: base numbers 11 - 28 A: transcription start point: base number 29 5’-UTR (including the transcription start point and the Kozak sequence at base numbers 89 - 94): base numbers 29 - 98 Spike protein signal sequence: base numbers 99 - 137 RBD sequence: base numbers 138 - 809 3’-UTR: base numbers 810 - 941 polyA sequence (A110): base numbers 942 - 1051 BspQI sequence: base numbers 1053 - 1059 HindIII sequence: base numbers 1060 - 1065 <SEQ ID NO:19> (SARS-CoV-2 RBD optimized mRNA-004) Template DNA of SARS-CoV-2 RBD S2000 with Array Number 20 GCTAGCGTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAAAGCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACGAAGAGC NheI sequence: Base numbers 1 - 6 T7 promoter: Base numbers 7 - 24 A: Transcription start point: Base number 25 5’-UTR sequence (including the transcription start point and the KOZAK sequence at base numbers 89 - 94): Base numbers 25 - 94 Spike protein signal sequence: Base numbers 95 - 133 RBD sequence: Base numbers 134 - 805 3’-UTR: Base numbers 806 - 937 polyA sequence (A50): Base numbers 938 - 987 BspQI sequence: Base numbers 989 - 995 <SEQ ID NO: 21> mRNA sequence of SARS-CoV-2 RBD S2000 AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAAAGCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUGGAAGGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACAAACGGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAAUGCGUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <Array No. 22> Template DNA of SARS-CoV-2 RBD S2001 GCTAGCGTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAAAGCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAGAGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACGAAGAGC NheI sequence: nucleotide numbers 1 - 6 T7 promoter: nucleotide numbers 7 - 24 A: transcription start point: nucleotide number 25 5’-UTR sequence (including the transcription start point and the KOZAK sequence at nucleotide numbers 89 - 94): nucleotide numbers 25 - 94 Spike protein signal sequence: nucleotide numbers 95 - 133 RBD sequence: nucleotide numbers 134 - 805 3’-UTR: nucleotide numbers 806 - 937 polyA sequence (A50): nucleotide numbers 938 - 987 BspQI sequence: nucleotide numbers 989 - 995 <SEQ ID NO: 23> mRNA sequence of SARS-CoV-2 RBD S2001 AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAAAGCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUGGAAGGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACAAACGGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAGAGCGUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <Sequence number 24> Amino acid sequence of SARS-CoV-2 RBD S2001 (including the S protein signal sequence). MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNF S protein signal sequence: Amino acid numbers 1-13 RBD sequence: Amino acid numbers 14-236 <Sequence number 25> Amino acid sequence of SARS-CoV-2 RBD S2001 (not including the S protein signal sequence). RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNF <Sequence number 26> Template DNA of SARS-CoV-2 RBD S2002 GCTAGCGTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACAAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAAAGCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGACCCAAATGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACGAAGAGC NheI sequence: base numbers 1 - 6 T7 promoter: base numbers 7 - 24 A: Transcription start point: Base number 25 5’-UTR sequence (including the transcription start point and the KOZAK sequence at base numbers 89 - 94): Base numbers 25 - 94 Spike protein signal sequence: Base numbers 95 - 133 RBD sequence: Base numbers 134 - 736 3’-UTR: Base numbers 737 - 868 polyA sequence (A50): Base numbers 869 - 918 BspQI sequence: Base numbers 920 - 926 <SEQ ID NO:27> mRNA sequence of SARS-CoV-2 RBD S2002 AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACAAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAAAGCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUGGAAGGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACAAACGGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGACCCAAAUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 28> Amino acid sequence of SARS-CoV-2 RBD S2002 (including the S protein signal sequence). MFVFLVLLPLVSSFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPK S protein signal sequence: Amino acid numbers 1 to 13 RBD sequence: Amino acid numbers 14 to 213 <SEQ ID NO:29> Amino acid sequence of SARS-CoV-2 RBD S2002 (excluding the S protein signal sequence.) FPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPK <SEQ ID NO:30> Template DNA of SARS-CoV-2 RBD S2003 NheI sequence: base numbers 1 - 6 T7 promoter: base numbers 7 - 24 A: transcription start point: base number 25 5’-UTR sequence (including the transcription start point and the KOZAK sequence at base numbers 89 - 94): base numbers 25 - 94 Spike protein signal sequence: base numbers 95 - 133 RBD sequence: base numbers 134 - 874 3’-UTR: base numbers 875 - 1006 polyA sequence (A50): base numbers 1007 - 1056 BspQI sequence: base numbers 1058 - 1064 <SEQ ID NO: 31> mRNA sequence of SARS-CoV-2 RBD S2003 <SEQ ID NO: 32> Amino acid sequence of SARS-CoV-2 RBD S2003 (including the S protein signal sequence). MFVFLVLLPLVSSEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLTGTGVLTE S protein signal sequence: Amino acid numbers 1-13 RBD sequence: Amino acid numbers 14-259 <SEQ ID NO: 33> Amino acid sequence of SARS-CoV-2 RBD S2003 (not including the S protein signal sequence). EKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLTGTGVLTE <SEQ ID NO: 34> Template DNA of SARS-CoV-2 RBD S2004 GCTAGCGTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAACAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCACCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACACCCTGGACAGCAAAGTCGGCGGCAACTACACCTACCTGTACCGGCTGTTCAGAAAGAGCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAGAGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACGAAGAGC NheI sequence: base numbers 1 - 6 T7 promoter: base numbers 7 - 24 A: transcription start point: base number 25 5’-UTR sequence (including the transcription start point and the KOZAK sequence at base numbers 89 - 94): base numbers 25 - 94 Spike protein signal sequence: base numbers 95 - 133 RBD sequence: base numbers 134 - 805 3’-UTR: base numbers 806 - 937 polyA sequence (A50): base numbers 938 - 987 BspQI sequence: base numbers 989 - 995 <SEQ ID NO:35> mRNA sequence of SARS-CoV-2 RBD S2004 AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAACAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACACCCUGGACAGCAAAGUCGGCGGCAACUACACCUACCUGUACCGGCUGUUCAGAAAGAGCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUGGAAGGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACAAACGGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAGAGCGUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <Sequence number 36> Amino acid sequence of SARS-CoV-2 RBD S2004 (including the S protein signal sequence). MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRINNCVADYSVLYNSTSFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNTLDSKVGGNYTYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNF S protein signal sequence: Amino acid numbers 1 to 13 RBD sequence: Amino acid numbers 14 to 236 <Sequence number 37> Amino acid sequence of SARS-CoV-2 RBD S2004 (not including the S protein signal sequence). RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRINNCVADYSVLYNSTSFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNTLDSKVGGNYTYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNF <Sequence number 38> DNA fragment containing mutant SARS-CoV-2 RBD (South African type) (mutated codons underlined, mutation sites in bold). GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGC AAC ATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTG AAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TAC GGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <Accession No. 39> DNA fragment containing mutant SARS-CoV-2 RBD (UK strain) (Mutated codons are underlined and mutation sites are in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TAC GGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <SEQ ID NO: 40> DNA fragment containing mutant SARS-CoV-2 RBD (Brazilian type) (mutated codons underlined, mutation sites in bold). GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGC ACG ATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTG AAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TAC GGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <SEQ ID NO: 41> DNA fragment containing mutant SARS-CoV-2 RBD (California type) (mutated codons underlined, mutated sites in bold). GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTAC CGG TACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <SEQ ID NO: 42> DNA fragment containing mutant SARS-CoV-2 RBD (Indian type) (mutated codons underlined, mutated sites 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 AGC GTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <SEQ ID NO: 44> DNA fragment containing mutant SARS-CoV-2 RBD (UK C538S type) (mutated codons underlined, mutated sites in bold). GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TAC GGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAA 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 ACG ATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTG AAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TAC GGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAA AGC GTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <SEQ ID NO: 46> DNA fragment containing mutant SARS-CoV-2 RBD (California C538S type) (mutated codons underlined, mutated sites in bold). GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTAC CGG TACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAA AGC GTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <Array number 47> DNA fragment containing mutant SARS-CoV-2 RBD (India C538S type) (Mutated codons are underlined, and the mutation sites are in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTAC CGG TACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTG CAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAA AGC GTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <SEQ ID NO: 48> DNA fragment containing mutant SARS-CoV-2 RBD( Combined Mutant (1) )(Mutated codons are underlined and mutation sites are shown in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGC AAG AACCTGGACAGCAAAGTCGGCGGCAACTACAACTAC CGG TACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAG GTC GGCAGCACCCCCTGCAACGGC GCG GAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <SEQ ID NO: 49> DNA fragment containing mutant SARS-CoV-2 RBD( Combined Mutant (2) )(Mutated codons are underlined and the mutation sites are shown in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGC AGA ACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTAC CTC CCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <Array No. 50> DNA fragment containing mutant SARS-CoV-2 RBD( Combined Mutant (3) )(Mutated codons are underlined and mutation sites are in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGC AAC ATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGC AGA ACCCCCTGCAACGGCGTG AAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TAC GGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC <SEQ ID NO: 51> DNA fragment containing mutant SARS-CoV-2 RBD( Combined Mutant (4) )(Mutated codons are underlined and mutated sites are shown in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCAGC 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 AAC ATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTG AAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TAC GGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: Base numbers 1 - 18 A: Transcription start point: Base number 19 5’ - UTR sequence (including the transcription start point and the KOZAK sequence at base numbers 83 - 88): Base numbers 19 - 88 Spike protein signal sequence: Base numbers 89 - 127 RBD sequence: Base numbers 128 - 799 3’ - UTR: Base numbers 800 - 931 polyA sequence (A100): Base numbers 932 - 1031 <SEQ ID NO: 53> Template DNA of mutant SARS - CoV - 2 RBD (UK type) (Mutated codons are underlined and mutation sites are in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TAC GGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: Base numbers 1-18 A: Transcription start point: Base number 19 5’-UTR sequence (including the transcription start point and the KOZAK sequence at base numbers 83-88): Base numbers 19-88 Spike protein signal sequence: Base numbers 89-127 RBD sequence: Base numbers 128-799 3’-UTR: Base numbers 800-931 polyA sequence (A100): Base numbers 932-1031 <SEQ ID NO: 54> Template DNA of mutant SARS-CoV-2 RBD (Brazilian type) (Mutated codons are underlined and mutation sites are shown in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGC ACG ATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTG AAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TAC GGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: Base numbers 1 - 18 A: Transcription start point: Base number 19 5’ - UTR sequence (including the transcription start point and the KOZAK sequence at base numbers 83 - 88): Base numbers 19 - 88 Spike protein signal sequence: Base numbers 89 - 127 RBD sequence: Base numbers 128 - 799 3’ - UTR: Base numbers 800 - 931 polyA sequence (A100): Base numbers 932 - 1031 <SEQ ID NO: 55> Template DNA of mutant SARS - CoV - 2 RBD (California type) (Mutated codons are underlined and mutation sites are shown in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTAC CGG TACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: Base numbers 1 - 18 A: Transcription start point: Base number 19 5’-UTR sequence (including the transcription start point and the KOZAK sequence at base numbers 83 - 88): Base numbers 19 - 88 Spike protein signal sequence: Base numbers 89 - 127 RBD sequence: Base numbers 128 - 799 3’-UTR: Base numbers 800 - 931 polyA sequence (A100): Base numbers 932 - 1031 <SEQ ID NO: 56> Template DNA of mutant SARS-CoV-2 RBD (Indian type) (Mutated codons are underlined and the mutation sites are shown in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTAC CGG TACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTG CAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: Base numbers 1-18 A: Transcription start point: Base number 19 5’-UTR sequence (including the transcription start point and the KOZAK sequence at base numbers 83-88): Base numbers 19-88 Spike protein signal sequence: Base numbers 89-127 RBD sequence: Base numbers 128-799 3’-UTR: Base numbers 800-931 polyA sequence (A100): Base numbers 932-1031 <SEQ ID NO: 57> Template DNA of mutant SARS-CoV-2 RBD (South African C538S type) (Mutated codons are underlined and the mutation sites are shown in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGC AAC ATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTG AAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TAC GGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAA AGC GTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: Base numbers 1 - 18 A: Transcription start point: Base number 19 5’-UTR sequence (including the transcription start point and the KOZAK sequence at base numbers 83 - 88): Base numbers 19 - 88 Spike protein signal sequence: Base numbers 89 - 127 RBD sequence: Base numbers 128 - 799 3’-UTR: Base numbers 800 - 931 polyA sequence (A100): Base numbers 932 - 1031 <SEQ ID NO:58> Template DNA of mutant SARS-CoV-2 RBD (UK C538S type) (Mutated codons are underlined and the mutation sites are shown in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TAC GGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAA AGC GTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: Base numbers 1 - 18 A: Transcription start point: Base number 19 5’ - UTR sequence (including the transcription start point and the KOZAK sequence at base numbers 83 - 88): Base numbers 19 - 88 Spike protein signal sequence: Base numbers 89 - 127 RBD sequence: Base numbers 128 - 799 3’ - UTR: Base numbers 800 - 931 polyA sequence (A100): Base numbers 932 - 1031 <SEQ ID NO:59> Template DNA of mutant SARS - CoV - 2 RBD (Brazil C538S type) (Mutated codons are underlined and the mutation sites are shown in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGC ACG ATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTG AAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TAC GGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAA AGC GTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: Base numbers 1-18 A: Transcription start point: Base number 19 5’-UTR sequence (including the transcription start point and the KOZAK sequence at base numbers 83-88): Base numbers 19-88 Spike protein signal sequence: Base numbers 89-127 RBD sequence: Base numbers 128-799 3’-UTR: Base numbers 800-931 polyA sequence (A100): Base numbers 932-1031 <SEQ ID NO: 60> Template DNA of mutant SARS-CoV-2 RBD (California C538S type) (Mutated codons are underlined and the mutation sites are shown in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTAC CGG TACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAA AGC GTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: Base numbers 1-18 A: Transcription start point: Base number 19 5’-UTR sequence (including the transcription start point and the KOZAK sequence at base numbers 83-88): Base numbers 19-88 Spike protein signal sequence: Base numbers 89-127 RBD sequence: Base numbers 128-799 3’-UTR: Base numbers 800-931 polyA sequence (A100): Base numbers 932-1031 <SEQ ID NO: 61> Template DNA of mutant SARS-CoV-2 RBD (India C538S type) (Mutated codons are underlined and the mutation sites are shown in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTAC CGG TACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTG CAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAA AGC GTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: Base numbers 1 - 18 A: Transcription start point: Base number 19 5’-UTR sequence (including the transcription start point and the KOZAK sequence at base numbers 83 - 88): Base numbers 19 - 88 Spike protein signal sequence: Base numbers 89 - 127 RBD sequence: Base numbers 128 - 799 3’-UTR: Base numbers 800 - 931 polyA sequence (A100): Base numbers 932 - 1031 <SEQ ID NO: 62> Template DNA of mutant SARS-CoV-2 RBD( Combined Mutant (1) )(Mutated codons are underlined and the mutation sites are shown in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGC AAG AACCTGGACAGCAAAGTCGGCGGCAACTACAACTAC CGG TACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAG GTC GGCAGCACCCCCTGCAACGGC GCG GAAGGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: Base numbers 1 - 18 A: Transcription start point: Base number 19 5’-UTR sequence (including the transcription start point and the KOZAK sequence at base numbers 83 - 88): Base numbers 19 - 88 Spike protein signal sequence: Base numbers 89 - 127 RBD sequence: Base numbers 128 - 799 3’-UTR: Base numbers 800 - 931 polyA sequence (A100): Base numbers 932 - 1031 <SEQ ID NO: 63> Template DNA of mutant SARS-CoV-2 RBD( Combined Mutant (2) )(Mutated codons are underlined and mutation sites are shown in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGC AGA ACCCCCTGCAACGGCGTGGAAGGCTTCAACTGCTAC CTC CCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: Base numbers 1 - 18 A: Transcription start point: Base number 19 5’-UTR sequence (including the transcription start point and the KOZAK sequence at base numbers 83 - 88): Base numbers 19 - 88 Spike protein signal sequence: Base numbers 89 - 127 RBD sequence: Base numbers 128 - 799 3’-UTR: Base numbers 800 - 931 polyA sequence (A100): Base numbers 932 - 1031 <SEQ ID NO: 64> Template DNA of mutant SARS-CoV-2 RBD( Combined Mutant (3) )(Mutated codons are underlined and mutation sites are shown in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGC AAC ATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGC AGA ACCCCCTGCAACGGCGTG AAA GGCTTCAACTGCTACTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TAC GGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: Base numbers 1 - 18 A: Transcription start point: Base number 19 5’-UTR sequence (including the transcription start point and the KOZAK sequence at base numbers 83 - 88): Base numbers 19 - 88 Spike protein signal sequence: Base numbers 89 - 127 RBD sequence: Base numbers 128 - 799 3’-UTR: Base numbers 800 - 931 polyA sequence (A100): Base numbers 932 - 1031 <Template DNA of mutant SARS-CoV-2 RBD ( Combined Mutant (4) )(Mutated codons are underlined and mutation sites are shown in bold.) GTAATACGACTCACTATAAGGAGACCCAAGCTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCCCCCGGACAGACAGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAAGGC AGC AACTGCTAC CTC CCACTGCAGAGCTACGGCTTCCAGCCCACAAACGGCGTGGGCTACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACAGTGTGCGGCCCCAAGAAAAGCACCAACCTGGTCAAGAACAAATGCGTGAACTTCTGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA T7 promoter: Base numbers 1 - 18 A: Transcription start point: Base number 19 5’ - UTR sequence (including the transcription start point and the KOZAK sequence at base numbers 83 - 88): Base numbers 19 - 88 Spike protein signal sequence: Base numbers 89 - 127 RBD sequence: Base numbers 128 - 799 3’ - UTR: Base numbers 800 - 931 polyA sequence (A100): Base numbers 932 - 1031 <SEQ ID NO: 66> Mutant SARS - CoV - 2 RBD mRNA (South African type) (Mutated codons are underlined and mutation sites are in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGC AAC AUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUG AAA GGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACA UAC GGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAAUGCGUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 67> Mutant SARS-CoV-2 RBD mRNA (UK strain) (Mutated codons are underlined and the mutation sites are in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUGGAAGGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACA UAC GGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAAUGCGUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 68> Mutant SARS-CoV-2 RBD mRNA (Brazilian type) (Mutated codons are underlined and mutation sites are in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGC ACG AUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUG AAA GGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACA UAC GGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAAUGCGUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 69> Mutant SARS-CoV-2 RBD mRNA (California type) (Mutated codons are underlined, mutated sites are in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUAC CGG UACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUGGAAGGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACAAACGGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAAUGCGUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 70> Mutant SARS-CoV-2 RBD mRNA (Indian type) (Mutated codons are underlined and the mutation sites are shown in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUAC CGG UACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUG CAA GGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACAAACGGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAAUGCGUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 71> Mutant SARS-CoV-2 RBD mRNA (South African C538S type) (Mutated codons underlined, mutated sites in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGC AAC AUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUG AAA GGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACA UAC GGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAA AGC GUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 72> Mutant SARS-CoV-2 RBD mRNA (UK C538S type) (Mutated codons are underlined, and the mutation sites are in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUGGAAGGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACA UAC GGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAA AGC GUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 73> Mutant SARS-CoV-2 RBD mRNA (Brazil C538S type) (Mutated codons are underlined, and the mutation sites are in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGC ACG AUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUG AAA GGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACA UAC GGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAA AGC GUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 74> Mutant SARS-CoV-2 RBD mRNA (California C538S type) (Mutated codons are underlined and the mutation sites are in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUAC CGG UACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUGGAAGGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACAAACGGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAA AGC GUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 75> Mutant SARS-CoV-2 RBD mRNA (India C538S type) (Mutated codons are underlined and the mutation sites are in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUAC CGG UACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUG CAA GGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACAAACGGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAA AGC GUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 76> Mutant SARS-CoV-2 RBD mRNA (Combined Mutant (1)) (Mutated codons are underlined and the mutation sites are in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAAG AACCUGGACAGCAAAGUCGGCGGCAACUACAACUAC CGG UACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAG GUC GGCAGCACCCCCUGCAACGGC GCG GAAGGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACAAACGGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAAUGCGUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 77> Mutant SARS-CoV-2 RBD mRNA (Combined Mutant (2)) (Mutated codons are underlined and mutation sites are shown in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGC AGA ACCCCCUGCAACGGCGUGGAAGGCUUCAACUGCUAC CUCCCACUGCAGAGCUACGGCUUCCAGCCCACAAACGGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAAUGCGUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 78> Mutant SARS-CoV-2 RBD mRNA (Combined mutant (3)) (Mutated codons are underlined and mutation sites are shown in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGC AACAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGC AGA ACCCCCUGCAACGGCGUG AAA GGCUUCAACUGCUACUUCCCACUGCAGAGCUACGGCUUCCAGCCCACA UAC GGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAAUGCGUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 79> Mutant SARS-CoV-2 RBD mRNA (Combined mutant (4)) (Mutated codons are underlined and mutation sites are shown in bold.) AGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACCGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGGCGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCGCCAGCUUCAGCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUCGCCCCCGGACAGACAGGCAAGAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAACCUGGACAGCAAAGUCGGCGGCAACUACAACUACCUGUACCGGCUGUUCCGGAAGUCCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAGCACCCCCUGCAACGGCGUGGAAGGC AGC AACUGCUAC CUCCCACUGCAGAGCUACGGCUUCCAGCCCACAAACGGCGUGGGCUACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCCGCCACAGUGUGCGGCCCCAAGAAAAGCACCAACCUGGUCAAGAACAAAUGCGUGAACUUCUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA <SEQ ID NO: 80> Amino acid sequence of mutant SARS-CoV-2 RBD (South African type) (including the S protein signal sequence, with mutated amino acids underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG N IADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGV K GFNCYFPLQSYGFQPT Y GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF S protein signal sequence: amino acid numbers 1 - 13 RBD sequence: amino acid numbers 14 - 236 <SEQ ID NO: 81> Amino acid sequence of mutant SARS-CoV-2 RBD (UK type) (including the S protein signal sequence, with mutated amino acids underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPT Y GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF S protein signal sequence: amino acid numbers 1 - 13 RBD sequence: amino acid numbers 14 - 236 <SEQ ID NO:82> Amino acid sequence of mutant SARS-CoV-2 RBD (Brazil type) (including the S protein signal sequence, with mutated amino acids underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG T IADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGV K GFNCYFPLQSYGFQPT Y GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF S protein signal sequence: amino acid numbers 1 - 13 RBD sequence: amino acid numbers 14 - 236 <SEQ ID NO:83> Amino acid sequence of mutant SARS-CoV-2 RBD (California type) (including the S protein signal sequence, with mutated amino acids underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNY R YRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF S protein signal sequence: amino acid numbers 1 - 13 RBD sequence: amino acid numbers 14 - 236 <SEQ ID NO:84> Amino acid sequence of mutant SARS-CoV-2 RBD (Indian type) (including the S protein signal sequence, with mutated amino acids underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNY R YRLFRKSNLKPFERDISTEIYQAGSTPCNGV Q GFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF S protein signal sequence: amino acid numbers 1 - 13 RBD sequence: amino acid numbers 14 - 236 <SEQ ID NO:85> Amino acid sequence of mutant SARS-CoV-2 RBD (South African C538S type) (including the S protein signal sequence, with mutated amino acids underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG N IADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGV K GFNCYFPLQSYGFQPT Y GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK S VNF S protein signal sequence: Amino acid numbers 1 - 13 RBD sequence: Amino acid numbers 14 - 236 <SEQ ID NO:86> Amino acid sequence of mutant SARS-CoV-2 RBD (UK C538S type) (including the S protein signal sequence, with mutated amino acids underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPT Y GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK S VNF S protein signal sequence: Amino acid numbers 1 - 13 RBD sequence: Amino acid numbers 14 - 236 <SEQ ID NO:87> Amino acid sequence of mutant SARS-CoV-2 RBD (Brazil C538S type) (including the S protein signal sequence, with mutated amino acids 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) (including the S protein signal sequence, with mutated amino acids underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNY R YRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK S VNF S protein signal sequence: Amino acid numbers 1 - 13 RBD sequence: Amino acid numbers 14 - 236 <SEQ ID NO: 89> Amino acid sequence of mutant SARS-CoV-2 RBD (India C538S type) (including the S protein signal sequence, with mutated amino acids underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNY R YRLFRKSNLKPFERDISTEIYQAGSTPCNGV Q GFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK S VNF S protein signal sequence: Amino acid numbers 1 - 13 RBD sequence: Amino acid numbers 14 - 236 <SEQ ID NO:90> Amino acid sequence of mutant SARS-CoV-2 RBD (combination mutant (1)) (including the S protein signal sequence, with mutated amino acids underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNS K NLDSKVGGNYNY R YRLFRKSNLKPFERDISTEIYQ V GSTPCNG A EGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF S protein signal sequence: Amino acid numbers 1 - 13 RBD sequence: Amino acid numbers 14 - 236 <SEQ ID NO:91> Amino acid sequence of mutant SARS-CoV-2 RBD (combination mutant (2)) (including the S protein signal sequence, with mutated amino acids underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAG R TPCNGVEGFNCY L PLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF S protein signal sequence: Amino acid numbers 1 - 13 RBD sequence: Amino acid numbers 14 - 236 <SEQ ID NO:92> Amino acid sequence of mutant SARS-CoV-2 RBD (combination mutant (3)) (including the S protein signal sequence, with mutated amino acids underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG N IADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAG R TPCNGV K GFNCYFPLQSYGFQPT Y GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF S protein signal sequence: Amino acid numbers 1 - 13 RBD sequence: Amino acid numbers 14 - 236 <SEQ ID NO:93> Amino acid sequence of mutant SARS-CoV-2 RBD (combination mutant (4)) (including the S protein signal sequence, with mutated amino acids underlined.) MFVFLVLLPLVSSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEG S NCY L PLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF S protein signal sequence: amino acid numbers 1 - 13 RBD sequence: amino acid numbers 14 - 236 <SEQ ID NO:94> Amino acid sequence of mutant SARS-CoV-2 RBD (South African type) (excluding the 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) (excluding the 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) (excluding the 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) (excluding the 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) (excluding the 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) (excluding the 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) (excluding 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 (Brazil C538S type) (excluding the 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) (excluding 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 (India C538S type) (excluding the 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 (1)) (excluding 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 (2)) (excluding 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 (3)) (excluding 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 (4)) (excluding 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 peptide having a sequence in which a mutation has been introduced into the amino acid sequence of a fragment of the S protein of the novel coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2), The peptide comprises a signal sequence of the S protein and a mutant receptor-binding domain having a sequence in which a mutation has been introduced into the amino acid sequence of the 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. 2. The particle according to claim 1, wherein one or more and up to 10 or less amino acids in the amino acid sequence of the receptor binding domain of the S protein have been substituted, deleted, inserted and / or added.

3. 3. The particle according to claim 1, wherein at least one cysteine ​​in the amino acid sequence of the receptor binding domain of the S protein is substituted with serine.

4. 4. The particle according to any one of claims 1 to 3, 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.

5. The particle according to claim 4, 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.

6. The particle described in any one of claims 1 to 5, wherein the nucleic acid capable of expressing a peptide consisting of a sequence in which a mutation has been introduced into the amino acid sequence of 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 a sequence in which a mutation has been introduced into the amino acid sequence of the receptor binding domain of the S protein, a 3' untranslated region (3'-UTR) and a poly A tail (poly A).

7. 7. The particle according to claim 6, 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.

8. The particle according to any one of claims 1 to 7, wherein the nucleic acid comprises at least one modified nucleotide.

9. 9. The particle according to claim 8, wherein the modified nucleotide is at least one selected from the group consisting of 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, and 1-alkylpseudouridine.

10. The particles according to any one of claims 1 to 9, wherein the average particle size of the particles is from 30 nm to 300 nm.

11. Use of the particles according to any one of claims 1 to 10 for producing a composition for preventing and / or treating infection with a new coronavirus (severe acute respiratory syndrome coronavirus 2: SARS-CoV-2).

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

13. 13. The composition of claim 12, further comprising a buffer comprising sucrose and histidine.

14. The composition according to claim 12 or 13, for use as a medicine.

15. The composition of claim 14 for inducing an immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

16. The composition according to claim 14 or 15, for preventing and / or treating infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

17. An ethanol solution containing cationic lipids, amphipathic lipids, sterols, and PEG lipids. A buffer solution containing a nucleic acid capable of expressing a peptide having a sequence in which a mutation has been introduced into the amino acid sequence of 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 obtaining a dispersion of nucleic acid-lipid particles by mixing in a flow channel.

18. A production method further comprising a step of dialyzing the dispersion of nucleic acid-lipid particles obtained by the step described in claim 17 against a buffer solution containing sucrose and histidine.

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