Coronavirus Disease 2019(COVID -19) Recombinant Spike Protein Forming Trimer, Method for Mass Producing Recombinant Spike Protein in Plants, and Method for Preparing Vaccine Composition on Basis Thereof

Abstract

The present invention relates to a recombinant spike protein of the COVID-19 virus forming a trimer and a method for mass-producing the recombinant spike protein, and more specifically to a method for designing a recombinant gene expressing a recombinant spike protein of the COVID-19 virus forming a trimer for the purposes of enhancing immunogenicity and effective antigen delivery, and a method for mass-producing the recombinant spike protein in plants.

Claims

1. A recombinant vector for producing a recombinant spike protein of a coronavirus forming a trimer, comprising: (i) a gene encoding a protein lacking an amino acid sequence from the transmembrane domain to the C-terminus of the spike protein of a coronavirus; a protein including an amino acid sequence from the N-terminus to sub-domain 2 (SD 2) of the spike protein of a coronavirus; or a protein lacking an amino acid sequence from the transmembrane domain in subunit 2 to the C-terminus of the spike protein of a coronavirus; and (ii) a gene encoding a protein of a trimeric motif region of Coronin 1 (mCor1).

2. The recombinant vector of claim 1, wherein the coronavirus is any one selected from the group consisting of SARS-CoV (SARS-Coronavirus), MERS-CoV (MERS-Coronavirus) and SARS-CoV-2 (SARS-Coronavirus-2).

3. The recombinant vector of claim 2, wherein the protein lacking an amino acid sequence from the transmembrane domain to the C-terminus of the spike protein of SARS-CoV-2 comprises the amino acid sequence of SEQ ID NO: 2.

4. The recombinant vector of claim 2, wherein the gene encoding a protein lacking an amino acid sequence from the transmembrane domain to the C-terminus of the spike protein of SARS-CoV-2 comprises the nucleotide sequence of SEQ ID NO: 1.

5. The recombinant vector of claim 2, wherein the protein including an amino acid sequence from the N-terminus to sub-domain 2 (SD 2) of the spike protein of SARS-CoV-2 comprises the amino acid sequence of SEQ ID NO: 4.

6. The recombinant vector of claim 2, wherein the gene encoding a protein including an amino acid sequence from the N-terminus to sub-domain 2 (SD 2) of the spike protein of SARS-CoV-2 comprises the nucleotide sequence of SEQ ID NO: 3.

7. The recombinant vector of claim 1, wherein the protein of a trimeric motif region of Coronin 1 (mCor1) comprises the amino acid sequence of SEQ ID NO: 6.

8. The recombinant vector of claim 1, wherein the gene encoding a protein of a trimeric motif region of Coronin 1 (mCor1) comprises the nucleotide sequence of SEQ ID NO: 5.

9. The recombinant vector of claim 1, wherein the vector is a binary vector.

10. The recombinant vector of claim 1, wherein the recombinant vector further comprises any one promoter selected from the group consisting of a 35S promoter derived from cauliflower mosaic virus, a 19S RNA promoter derived from cauliflower mosaic virus, a Mac promoter, an actin protein promoter and ubiquitin protein promoter of a plant.

11. A transgenic organism which is transformed by the recombinant vector of claim 1.

12. A transformant, in which the transgenic organism of claim 11 which is transformed is a prokaryote or a eukaryote.

13. A method for producing a recombinant spike protein of a coronavirus forming a trimer in a plant, comprising the steps of: (a) constructing the recombinant vector of claim 1; (b) preparing a transgenic organism by introducing the recombinant vector into an organism; (c) culturing the transgenic organism; (d) infiltrating the culture product into a plant; and (e) pulverizing the plant to obtain a recombinant spike protein of a coronavirus forming a trimer.

14. A recombinant protein which is prepared according to the method of claim 13.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] FIG. 1 is a diagram showing the construction of several recombinant vectors used in the present invention.

[0050] FIG. 2 shows the results of Western blot analysis using an anti-His antibody and staining with Coomassie brilliant blue after isolating by SDS/PAGE the total extract of N. benthamiana, which expressed SfΔ (TMD-CT), S1s and S2sΔ (TMD-CT) proteins having a His tag and HDEL, and these three types of proteins additionally expressed mCor1 protein.

[0051] FIG. 3 shows the results of pure isolation and purification of S.sub.full-t and S2s-t from N. benthamiana extract using Ni.sup.2+-NTA affinity column chromatography, development by SDS/PAGE, Western blot analysis using an anti-His antibody, and staining of the membrane with Coomassie brilliant blue.

[0052] FIG. 4 shows the results of fractionation of S.sub.full-t isolated from N. benthamiana extract using Ni.sup.2+-NTA affinity column chromatography by size-exclusion column chromatography, development by SDS/PAGE, and staining with Coomassie brilliant blue.

[0053] FIGS. 5A-5D show the results of Western blot analysis using an anti-His antibody after pure isolation and purification of S.sub.full-t and S.sub.full-m isolated from N. benthamiana extract using Ni.sup.2+-NTA affinity column chromatography, fractionation of S.sub.full-t and S.sub.full-m by size exclusion column chromatography, and negative staining of the isolated and purified S.sub.full-t, and confirmation of the morphology of the protein using an electron microscope, confirming that a trimer was formed.

[0054] FIG. 6 shows the results of fractionation of S2s-t isolated from the N. benthamiana extract using Ni.sup.2+-NTA affinity column chromatography by size-exclusion column chromatography, development by SDS/PAGE and staining with Coomassie brilliant blue.

[0055] FIGS. 7A-7D show the results of evaluation of the immunogenicity of COVID-19 plant vaccine candidate materials in experimental animals (mouse, Balb/c). For this, the results were shown after performing a humoral immune response analysis (FIG. 7B), a neutralizing antibody induction analysis (FIGS. 7C and 7D), and a cell-mediated immune response analysis (FIG. 7E).

[0056] FIGS. 8A-8E show the results of evaluation of the protective efficacy of the COVID-19 plant vaccine candidate materials in experimental animals (TG mouse), in which each immune antigen was immunized twice in total at an interval of 2 weeks by an intramuscular route, and in order to confirm the production of antibodies against the immune antigen, the results of evaluation of the immunogenicity such as the production of antibodies (IgGs) and the production of neutralizing antibodies in serum by performing blood collection before immunization, after primary immunization, and after 2 weeks of secondary immunization were shown (FIG. 8A), and the results of analysis of antibody induction according to plant vaccine immunization (FIG. 8B), and the results of analysis of the survival rate and tissue titer according to hamster challenge inoculation according to plant vaccine immunization (FIGS. 8C, 8D and 8E) were shown.

DETAILED DESCRIPTION

[0057] Hereinafter, the present invention will be described in detail.

[0058] As a membrane protein of the COVID-19 virus, the spike protein, which is known to be important for infiltrating cells, is considered as a vaccine candidate that can prevent infection of the COVID-19 virus. In the present invention, various recombinant proteins were made based on the spike protein, and they were used to develop vaccines. The full length of the spike protein forms a trimer when present on the surface of the virus, but when a recombinant protein is made using a portion encoding only the ectodomain of all or part of the spike gene, it was expected that the recombinant protein will not form a trimer well. Therefore, in order to utilize these recombinant proteins as vaccines, it was thought that it was important to form a trimer as when the protein was present on the surface of the virus, and it was attempted to develop a technique of maintaining a trimer when these exist in a soluble form. In the present invention, when the recombinant protein of the entire ectodomain of the spike protein [SfΔ(TMD-CT)], S1s or the S2 region without TMD and C-terminus [S2sΔ(TMD-CT)] was expressed and produced in a plant, it was attempted to develop a technique for inducing the formation of a trimer. Accordingly, the ectodomain of the spike protein lacking from the spike transmembrane domain to the C-terminus of the COVID-19 virus that can be mass-produced (Se; a total of 1,198 residues from the 16.sup.th amino acid to the 1,213.sup.rd amino acid), a site including from the N-terminus to the SD2 domain (S1s; a total of 666 residues from the 16.sup.th amino acid to the 681.sup.st amino acid), or a site without TMD and the C-terminus in the S2 subunit (S2sΔ(TMD-CT); a total of 532 residues from the 682.sup.nd amino acid to the 1,213.sup.rd amino acid) was fused to the trimeric motif of mouse Coronin 1 forming a trimeric structure, and additionally, a construct without mCor1 was constructed as a control group for the recombinant protein. A binary vector capable of the high expression of recombinant genes including parts of the above various spike proteins in plants was constructed.

[0059] The present invention may prove a recombinant vector for producing a recombinant spike protein of a coronavirus forming a trimer, including (i) a gene encoding a protein (SfΔ(TMD-CT)) lacking an amino acid sequence from the transmembrane domain to the C-terminus in the spike protein of a coronavirus; an S1 subunit protein (S1s) including the N-terminus to sub-domain 2 (SD2) in the spike protein of a coronavirus; or a protein (S2sΔ(TMD-CT)) lacking an amino acid sequence from the transmembrane domain to the C-terminus in an S2 subunit of the spike protein of a coronavirus spike; and (ii) a gene encoding a protein of the trimeric motif region of Coronin 1 (mCor 1).

[0060] In order to develop a vaccine by using the recombinant spike protein of the COVID-19 virus, the ectodomain of the spike protein with the transmembrane domain to the C-terminus removed (full length ectodomain of S; from the 16.sup.th amino acid to the 1213.sup.rd amino acid, a total of 1,198 residues), or a region from the N-terminus to sub domain 2 (SD2) (from the 16.sup.th amino acid to the 681.sup.st amino acid, a total of 666 residues), or the ectodomain of S2 (the 682.sup.nd amino acid to the 1,213.sup.rd amino acid, a total of 532 residues) was used to create a recombinant protein, and it was used to develop a vaccine. The full length of the spike protein forms a trimer when present on the surface of the virus, but it was expected that a trimer will not be formed well if the recombinant protein is expressed using the ectodomain part of all or part of the gene encoding the spike protein, and it was expected to have low immunogenicity due to the non-formation of the trimer. Therefore, in the present invention, it was originally intended to make a recombinant protein of the entire trimeric form of the spike protein as the full-length spike protein exists on the surface of the virus, or the S1c ectodomain from the N-terminus to the SD2 domain, or the S2 ectodomain. To this end, in the present invention, COVID-19 was selected and the 32 residues from the 122.sup.nd amino acid to the 153.sup.rd amino acid of mouse Coronin1 (mCor1) were fused by using a linker having 12 amino acid residues to construct a construct.

[0061] Subsequently, a His tag with five His residues was fused for isolation and purification of the recombinant protein, and finally a HDEL motif was fused to accumulate in the ER to complete the construct (SfΔ(TMD-CT):mCor1:Hisx5:HDEL, S1s:mCor1:Hisx5:HDEL, S2sΔ(TMD-CT):mCor1:Hisx5:HDEL, FIG. 1). In addition, constructs without mCor1 were constructed from the above three types of the spike recombinant proteins and used as control groups.

[0062] The coronavirus may be any one selected from the group consisting of SARS-CoV (SARS-Coronavirus), MERS-CoV (MERS-Coronavirus) and SARS-CoV-2 (SARS-Coronavirus-2).

[0063] In the spike protein of the COVID-19 virus, the protein lacking from the transmembrane domain to the C-terminus may include the amino acid sequence of SEQ ID NO: 2.

[0064] The gene encoding the protein lacking from the transmembrane domain to the C-terminus in the spike protein of the COVID-19 virus may include the nucleotide sequence of SEQ ID NO: 1, and specifically, the gene may include a nucleotide sequence having a sequence homology of 70% or more, more preferably, 80% or more, still more preferably, 90% or more, and most preferably, 95% or more to the nucleotide sequence of SEQ ID NO: 1. The “% of sequence homology” to a polynucleotide is determined by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region may include additions or deletions (i.e., gaps) compared to a reference sequence (not including additions or deletions) to the optimal alignment of the two sequences.

[0065] The S1s protein including from the N-terminus to sub-domain 2 (SD2) in the spike protein of the COVID-19 virus may include the amino acid sequence of SEQ ID NO: 4.

[0066] The gene encoding the S1s protein including the N-terminus to sub-domain 2 (SD2) in the spike protein of the COVID-19 virus may include the nucleotide sequence of SEQ ID NO: 3, and specifically, the gene may include a nucleotide sequence having a sequence homology of 70% or more, more preferably, 80% or more, still more preferably, 90% or more, and most preferably, 95% or more to the nucleotide sequence of SEQ ID NO: 3. The “% of sequence homology” to a polynucleotide is determined by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region may include additions or deletions (i.e., gaps) compared to a reference sequence (not including additions or deletions) to the optimal alignment of the two sequences.

[0067] In the spike protein of the COVID-19 virus, the S2 protein may include the amino acid sequence of SEQ ID NO: x.

[0068] The protein of the trimeric motif region of Coronin 1 (mCor 1) may include the amino acid sequence of SEQ ID NO: 6.

[0069] The gene encoding the protein of the trimeric motif region of Coronin 1 (mCor1) may include the nucleotide sequence of SEQ ID NO: 5, and specifically, the gene may include a nucleotide sequence having a sequence homology of 70% or more, more preferably, 80% or more, still more preferably, 90% or more, and most preferably, 95% or more to the nucleotide sequence of SEQ ID NO: 5. The “% of sequence homology” to a polynucleotide is determined by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region may include additions or deletions (i.e., gaps) compared to a reference sequence (not including additions or deletions) to the optimal alignment of the two sequences.

[0070] The recombinant genes were introduced into a plant expression vector, pTEX1, to make a plant expression vector. Six types of ectodomain recombinant protein genes were constructed from the spike gene of COVID-19 (FIG. 1), and the expression of the recombinant protein was induced by introducing them into plants (Nicotiana benthamiana). In order to confirm the expression of the protein in the leaf extract of N. benthamiana into which these genes were introduced, Western blot analysis was performed by using an anti-His antibody. As shown in FIG. 2, S.sub.full-t was confirmed at about 180 kD. Since it forms N-glycosylation of the spike protein, it was determined that it appeared to be larger than the calculated protein position. S1 and S2 appeared at the positions of 100 kD and 75 kD, respectively, and since these two proteins also appeared to be larger than the calculated size, it was also determined to be N-glycosylation (FIG. 2).

[0071] Further, in order to isolate these two types of proteins purely, a total extract of N. benthamiana was prepared, and pure isolation and purification were performed by using Ni.sup.2+-NTA affinity column chromatography therefrom, and afterwards, it was isolated and purified by SDS/PAGE and stained by Coomassie brilliant blue. Through this, it was confirmed that the recombinant proteins of the two types of the spike proteins could be purely isolated and purified (FIG. 3).

[0072] The recombinant vector may further include any one promoter selected from the group consisting of a 35S promoter derived from cauliflower mosaic virus, a 19S RNA promoter derived from cauliflower mosaic virus, a Mac promoter, an actin protein promoter and ubiquitin protein promoter of a plant, preferably, it may be a Mac promoter, and more preferably, a MacT promoter.

[0073] The MacT promoter may be a promoter in which A, which is the 3′ terminal nucleotide of the Mac promoter nucleotide sequence, is substituted with T, and the MacT promoter may include the nucleotide sequence of SEQ ID NO: 17, and specifically, the gene may include a nucleotide sequence having a sequence homology of 70% or more, more preferably, 80% or more, still more preferably, 90% or more, and most preferably, 95% or more to the nucleotide sequence of SEQ ID NO: 17. The “% of sequence homology” to a polynucleotide is determined by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region may include additions or deletions (i.e., gaps) compared to a reference sequence (not including additions or deletions) to the optimal alignment of the two sequences.

[0074] The recombinant vector may further include an RD29B-t termination site, and the RD29B-t termination site may include the nucleotide sequence of SEQ ID NO: 18, and specifically, the gene may include a nucleotide sequence having a sequence homology of 70% or more, more preferably, 80% or more, still more preferably, 90% or more, and most preferably, 95% or more to the nucleotide sequence of SEQ ID NO: 18. The “% of sequence homology” to a polynucleotide is determined by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region may include additions or deletions (i.e., gaps) compared to a reference sequence (not including additions or deletions) to the optimal alignment of the two sequences.

[0075] By including the BiP signal sequence and the ER retention signal HDEL at the N-terminus and C-terminus of the recombinant protein gene, respectively, it may have an effect of inducing accumulation in the endoplasmic reticulum (ER) at a high concentration. The recombinant vector may further include any one selected from the group consisting of a chaperone binding protein (BiP) and a His-Asp-Glu-Leu (HDEL) peptide, and the chaperone binding protein (BiP) may include the nucleotide sequence of SEQ ID NO: 12, and HDEL (His-Asp-Glu-Leu) may include the nucleotide sequence of SEQ ID NO: 10.

[0076] The recombination refers to a cell in which the cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, a heterologous peptide or a protein encoded by the heterologous nucleic acid. Recombinant cells can express genes or gene segments that are not found in the native form of the cell in either the sense or antisense form. In addition, recombinant cells can express genes found in the native form of cells, but the genes are modified and re-introduced into cells by artificial means.

[0077] The term “recombinant expression vector” means a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus or other vectors. In general, any plasmid and vector may be used as long as it is capable of replication and stabilization in the host. An important characteristic of the expression vector is that it has an origin of replication, a promoter, a marker gene and a translation control element. The recombinant expression vector and the expression vector including appropriate transcriptional/translational control signals may be constructed by a method well known to those skilled in the art. The method includes in vitro recombinant DNA technology, DNA synthesis technology and in vivo recombination technology.

[0078] A preferred example of the recombinant vector of the present invention is a Ti-plasmid vector capable of transferring a part of itself, the so-called T-region, into a plant cell when present in a suitable host. Another type of the Ti-plasmid vector is currently being used to transfer hybrid DNA sequences into plant cells, or protoplasts from which new plants can be produced that properly insert the hybrid DNA into the genome of the plant. A particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0120 516 B1 and U.S. Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host may be selected from viral vectors such as those that can be derived from double-stranded plant viruses (e.g., CaMV) and single-stranded viruses, geminiviruses and the like, and for example, incomplete plant viral vectors. The use of such vectors may be advantageous, especially when it is difficult to adequately transform a plant host.

[0079] In addition, the present invention may provide a transgenic organism which is transformed by the recombinant vector.

[0080] The transformed transgenic organism may be a prokaryote or a eukaryote, and for example, yeast (Saccharomyces cerevisiae), fungi such as E. coli, insect cells, human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cells may be used, and preferably it may be Agrobacterium. In the case of insect cells and human cells, the gene encoding a recombinant spike protein forming a trimer may be expressed by using an expression vector necessary for the expression of each of these types of animal cells.

[0081] Methods for delivering the vector of the present invention into a host cell may be carried out by the CaCl.sub.2 method, the Hanhan method (Hanahan, D., J. Mol. Biol., 166:557-580 (1983)), the electroporation method or the like, when the host cell is a prokaryotic cell. In addition, when the host cell is a eukaryotic cell, the vector may be injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, gene bombardment and the like.

[0082] The present invention may provide a method for producing a recombinant spike protein of a coronavirus forming a trimer in a plant, including the steps of:

[0083] (a) constructing the aforementioned recombinant vector;

[0084] (b) preparing a transgenic organism by introducing the recombinant vector into an organism;

[0085] (c) culturing the transgenic organism;

[0086] (d) infiltrating the culture product into a plant; and

[0087] (e) pulverizing the plant to obtain a recombinant spike protein of a coronavirus forming a trimer.

[0088] The method of infiltrating the plant may include a chemical cell method, a vacuum or syringe infiltration method, and most preferably, it may be a syringe infiltration method, but is not limited thereto.

[0089] The plant may be selected from food crops including rice, wheat, barley, corn, soybean, potato, wheat, red bean, oat and sorghum; vegetable crops including Arabidopsis thaliana, Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, Korean melon, pumpkin, green onion, onion and carrot; special crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut and rapeseed; fruit trees including apple tree, pear tree, date tree, peach, grape, tangerine, persimmon, plum, apricot and banana; and flowers including rose, carnation, chrysanthemum, lily and tulip.

[0090] The spike proteins of the COVID-19 virus produced in this way may be isolated and purified by Ni.sup.2+-NTA affinity column chromatography using the C-terminal His tag. In addition, the isolated protein may be further isolated by using size exclusion gel chromatography to increase the purity.

[0091] Another object of the present invention is to provide a vaccine composition using the spike protein of these plant-produced COVID-19. S.sub.full-t or S2s-t of the trimer of the spike protein may be used with or without alum to constitute a vaccine composition.

[0092] Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

<Example 1> Design of Gene Encoding Recombinant Spike Protein of COVID-19 Virus Forming Trimer

[0093] In order to induce the expression of the spike (S) derived from the COVID-19 virus surface protein in a soluble form in the ER lumen, the ectodomain of the spike protein with the transmembrane domain to the C-terminus removed (from the 16.sup.th amino acid to the 1,213.sup.rd amino acid, a total of 1,198 residues, SfΔ(TMD-CT)), or an S1 subunit region including the N-terminus to the SD2 domain (from the 16.sup.th amino acid to the 681.sup.st amino acid, a total of 666 residues), or the ectodomain of S2 (the 682.sup.nd amino acid to the 1,213.sup.rd amino acid, a total of 532 residues) was obtained. The ER targeting signal obtained from BiP, which is a protein from Arabidopsis thaliana, was fused to the 5′-end of the spark gene of the COVID-19 virus to enable ER targeting. Further, in order to induce trimer formation of the recombinant spike protein thus made, a Hisx5 tag and HDEL, which is an ER retention motif, were sequentially fused to the C-terminus of mCor1 for the purification of the spike protein and high accumulation in the ER to construct a construct. For expression, MacT was used, and the end of Rd29b was used as a transcription terminator. The transcription terminator was confirmed to show high transcription efficiency as a result of previous studies. The nucleotide sequences used in the experiment are shown in Table 1 below (FIG. 1).

[0094] Then, these constructs were introduced into pTEX to construct 9 final plant expression vectors.

TABLE-US-00001 TABLE 1 Sequence (5′-3′) Nucleotide gttaaccttaccaccagaactcagttacccccagcatacactaattctttcacacgtggtgtttactaccctgacaaagtt SEQ ID NO: 1 sequence of ttcagaagcagcgttttacacagcactcaggatttattcctacctttcttttccaacgtgacctggttccatgctatacatg SfΔ(TMD-CT) tatctgggaccaatggtaccaagaggtttgataacccggtcctaccatttaatgatggagtctattttgcctccactgag aagtctaatataataagaggctggatttttggaactactcttgattcgaagacccagagtctacttattgttaataacgct acaaatgttgttatcaaagtatgtgaatttcaattctgtaatgatccattcttgggtgtttactaccacaaaaacaacaaaa gttggatggaaagtgagtttcgggtttatagcagtgcgaataattgcacttttgagtacgtctcccaaccttttcttatgg accttgaaggaaagcagggaaatttcaagaatcttcgcgaatttgtgtttaagaatatcgatggttatttcaagatatatt ctaagcacacgcctattaatttagtgcgagatctccctcagggtttttcggcgctggaaccattggtagatttgccgata ggaatcaatatcactaggttccagactttacttgctctgcatagaagttacttgacccctggagatagctcatcaggttg gacagctggtgcggcagcttattacgtggggtatcttcagcctaggacgttcctattaaaatataatgaaaatggaac cattacagatgctgtagactgtgcacttgaccctctctcagaaacaaagtgtacgttgaaatccttcacggtagaaaaa gggatctaccaaacgtctaacttcagagtccagccaacagaatctattgtgagatttcccaatattacaaacttgtgcc ctttcggagaagtttttaacgccaccaggtttgcatcggtttatgcttggaacaggaaaagaatcagcaactgtgttgct gattatagtgtcctatataactccgcatccttttccactttcaagtgttacggagtttctcctactaaattaaatgatctctgc tttactaatgtctatgcagattcatttgtaatcagaggtgatgaggtcagacaaatcgctccagggcagactggaaag attgctgattataattataagcttcctgatgattttacaggctgcgttatagcatggaattctaataatcttgactctaaggt ggggggaaattataattacctgtatagactgtttaggaagagcaatctcaagcctttcgagagagacatttcaactga gatctaccaggcgggaagcactccgtgtaatggtgttgagggttttaattgttactttcctttacagtcatacggtttcca acccacgaatggggttggttaccaaccgtaccgagtagtagtactttctttcgagcttctacatgccccagcaactgttt gtggacctaagaagtctactaatttggttaaaaataagtgtgtcaattttaatttcaatggacttacgggcacaggagttc ttactgagtctaacaagaagtttctgcctttccagcagttcggcagagatattgctgacactactgatgctgtgcgtgat ccacagacacttgaaattcttgacattacaccatgttcttttggtggcgtgagtgttataactcccggaacaaatacctc caaccaggtggctgttctgtatcaggacgtgaactgtacagaagtccctgttgcaattcatgcagatcagcttactcct acctggcgtgtttattctacgggttccaatgtttttcaaacacgtgcaggctgcttgataggggctgaacatgtcaacaa ctcatatgaatgcgacatacccataggtgcaggtatatgcgctagttatcagactcagaccaattctccgcggcgggc acgaagtgtagctagtcaatccatcattgcctacactatgtcacttggtgcagaaaattcagttgcttactctaataactc tattgccatacccacaaattttactattagtgttaccacagaaattctaccagtgtctatgaccaagacatcagttgactgt acaatgtatatttgcggggattcaactgagtgctcgaatctgttgttgcaatacggcagtttttgtacccaattgaaccg ggctctgactggaatagctgtggaacaagataaaaacacccaagaagtttttgcacaagtcaaacaaatttataaaac accaccaattaaagatttcggtggtttcaacttctcacaaatactgccagatccgagcaaaccaagcaagaggtcatt cattgaagacctacttttcaacaaagtgacacttgcagatgctggcttcattaaacagtatggtgattgcttgggggata ttgctgctagagacctcatttgtgcacaaaagtttaacgggctgacagtgttgccacctttgttgacagatgagatgatt gctcagtacacttctgcactgctcgctggtacaatcacatctgggtggacctttggtgcaggtgctgccttacaaatac catttgctatgcagatggcttataggttcaatggtatcggagttacacagaacgttctctatgagaaccaaaaattgatt gccaaccaattcaatagtgccattggcaagattcaggactcactttcaagcacagcgagtgcacttggaaagttgca agatgtggtcaaccagaatgcacaagctttaaacacgcttgtgaaacaactcagctccaactttggggcaatttcaag tgttttgaatgatatcctttcacgtcttgataaagtggaagccgaggtgcaaattgacaggttgatcacaggccgacttc aaagtttgcagacttatgtgactcaacaattaattagggcagcagaaatccgcgcttcggctaatctggcggctacta aaatgtcagagtgtgtacttggacaatctaaacgagttgatttttgcggaaagggctatcatctcatgtccttccctcagt cagcgcctcacggtgtagtgttcttgcacgtgacttacgttcctgcacaagaaaagaatttcacaactgctccggccat ttgtcatgatggaaaagcccactttccgcgtgaaggtgtctttgtttcgaatggcacacactggtttgtaacccaaagg aatttttatgagccacaaatcattacgacggacaacacttttgtgtctggtaattgtgatgttgtaatcggaatcgtcaac aacaccgtttacgatcctttgcagcctgagttagattctttcaaagaggagctggataagtatttcaagaatcatacatc acccgatgttgatctcggtgatatctctggaattaatgcttcagttgtgaacattcaaaaggagattgaccgcctcaatg aggttgccaagaatttgaatgaatcgctcatcgatctccaagaacttggaaagtatgagcagtatatcaagtggcca Amino acid vnlttrtqlppaytnsftrgvyypdkvfrssvlhstqdlflpffsnvtwfhaihvsgtngtkrfdnpvlpfndgvyfa SEQ ID NO: 2 sequence of steksniirgwifgttldsktqsllivnnatnvvikvcefqfcndpflgvyyhknnkswmesefrvyssannctfe SfΔ(TMD-CT) yvsqpflmdlegkqgnfknlrefvfknidgyfkiyskhtpinlvrdlpqgfsaleplvdlpiginitrfqtllalhrs yltpgdsssgwtagaaayyvgylqprtfllkynengtitdavdcaldplsetkctlksftvekgiyqtsnfrvqpte sivrfpnitnlcpfgevfnatrfasvyawnrkrisncvadysvlynsasfstfkcygvsptklndlcftnvyadsfv irgdevrqiapgqtgkiadynyklpddftgcviawnsnnldskvggnynylyrlfrksnlkpferdisteiyqag stpcngvegfncyfplqsygfqptngvgyqpyrvvvlsfellhapatvcgpkkstnlvknkcvnfnfngltgtg vltesnkkflpfqqfgrdiadttdavrdpqtleilditpcsfggvsvitpgtntsnqvavlyqdvnctevpvaihad qltptwrvystgsnvfqtragcligaehvnnsyecdipigagicasyqtqtnsprrarsvasqsiiaytmslgaens vaysnnsiaiptnftisvtteilpvsmtktsvdctmyicgdstecsnlllqygsfctqlnraltgiaveqdkntqevfa qvkqiyktppikdfggfnfsqilpdpskpskrsfiedllfnkvtladagfikqygdclgdiaardlicaqkfngltvl pplltdemiaqytsallagtitsgwtfgagaalqipfamqmayrfngigvtqnvlyenqklianqfnsaigkiqds lsstasalgklqdvvnqnaqalntlvkqlssnfgaissvlndilsrldkveaevqidrlitgrlqslqtyvtqqliraae irasanlaatkmsecvlgqskrvdfcgkgyhlmsfpqsaphgvvflhvtyvpaqeknfttapaichdgkahfpr egvfvsngthwfvtqrfyepqiittdntfvsgncdvvigivnntvydplqpeldsfkeeldkyfknhtspdvdl gdisginasvvniqkeidrlnevaknlneslidlqelgkyeqyikwp Nucleotide gttaaccttaccaccagaactcagttacccccagcatacactaattctttcacacgtggtgtttactaccctgacaaagtt SEQ ID NO: 3 sequence of S1s ttcagaagcagcgttttacacagcactcaggatttattcctacctttcttttccaacgtgacctggttccatgctatacatg tatctgggaccaatggtaccaagaggtttgataacccggtcctaccatttaatgatggagtctattttgcctccactgag aagtctaatataataagaggctggatttttggaactactcttgattcgaagacccagagtctacttattgttaataacgct acaaatgttgttatcaaagtatgtgaatttcaattctgtaatgatccattcttgggtgtttactaccacaaaaacaacaaaa gttggatggaaagtgagtttcgggtttatagcagtgcgaataattgcacttttgagtacgtctcccaaccttttcttatgg accttgaaggaaagcagggaaatttcaagaatcttcgcgaatttgtgtttaagaatatcgatggttatttcaagatatatt ctaagcacacgcctattaatttagtgcgagatctccctcagggtttttcggcgctggaaccattggtagatttgccgata ggaatcaatatcactaggttccagactttacttgctctgcatagaagttacttgacccctggagatagctcatcaggttg gacagctggtgcggcagcttattacgtggggtatcttcagcctaggacgttcctattaaaatataatgaaaatggaac cattacagatgctgtagactgtgcacttgaccctctctcagaaacaaagtgtacgttgaaatccttcacggtagaaaaa gggatctaccaaacgtctaacttcagagtccagccaacagaatctattgtgagatttcccaatattacaaacttgtgcc ctttcggagaagtttttaacgccaccaggtttgcatcggtttatgcttggaacaggaaaagaatcagcaactgtgttgct gattatagtgtcctatataactccgcatccttttccactttcaagtgttacggagtttctcctactaaattaaatgatctctgc tttactaatgtctatgcagattcatttgtaatcagaggtgatgaggtcagacaaatcgctccagggcagactggaaag attgctgattataattataagcttcctgatgattttacaggctgcgttatagcatggaattctaataatcttgactctaaggt ggggggaaattataattacctgtatagactgtttaggaagagcaatctcaagcctttcgagagagacatttcaactga gatctaccaggcgggaagcactccgtgtaatggtgttgagggttttaattgttactttcctttacagtcatacggtttcca acccacgaatggggttggttaccaaccgtaccgagtagtagtactttctttcgagcttctacatgccccagcaactgttt gtggacctaagaagtctactaatttggttaaaaataagtgtgtcaattttaatttcaatggacttacgggcacaggagttc ttactgagtctaacaagaagtttctgcctttccagcagttcggcagagatattgctgacactactgatgctgtgcgtgat ccacagacacttgaaattcttgacattacaccatgttcttttggtggcgtgagtgttataactcccggaacaaatacctc caaccaggtggctgttctgtatcaggacgtgaactgtacagaagtccctgttgcaattcatgcagatcagcttactcct acctggcgtgtttattctacgggttccaatgtttttcaaacacgtgcaggctgcttgataggggctgaacatgtcaacaa ctcatatgaatgcgacatacccataggtgcaggtatatgcgctagttatcagactcagaccaattctccg Amino acid vnlttrtqlppaytnsftrgvyypdkvfrssvlhstqdlflpffsnvtwfhaihvsgtngtkrfdnpvlpfndgvyfa SEQ ID NO: 4 sequence of S1s steksniirgwifgttldsktqsllivnnatnvvikvcefqfcndpflgvyyhknnkswmesefrvyssannctfe protein yvsqpflmdlegkqgnfknlrefvfknidgyfkiyskhtpinlvrdlpqgfsaleplvdlpiginitrfqtllalhrs yltpgdsssgwtagaaayyvgylqprtfllkynengtitdavdcaldplsetkctlksftvekgiyqtsnfrvqpte sivrfpnitnlcpfgevfnatrfasvyawnrkrisncvadysvlynsasfstfkcygvsptklndlcftnvyadsfv irgdevrqiapgqtgkiadynyklpddftgcviawnsnnldskvggnynylyrlfrksnlkpferdisteiyqag stpcngvegfncyfplqsygfqptngvgyqpyrvvvlsfellhapatvcgpkkstnlvknkcvnfnfngltgtg vltesnkkflpfqqfgrdiadttdavrdpqtleilditpcsfggvsvitpgtntsnqvavlyqdvnctevpvaihad qltptwrvystgsnvfqtragcligaehvnnsyecdipigagicasyqtqtnsp Nucleotide cggcgggcacgaagtgtagctagtcaatccatcattgcctacactatgtcacttggtgcagaaaattcagttgcttact SEQ ID NO: 5 sequence of S2sΔ ctaataactctattgccatacccacaaattttactattagtgttaccacagaaattctaccagtgtctatgaccaagacatc (TMD-CT) agttgactgtacaatgtatatttgcggggattcaactgagtgctcgaatctgttgttgcaatacggcagtttttgtaccca attgaaccgggctctgactggaatagctgtggaacaagataaaaacacccaagaagtttttgcacaagtcaaacaaa tttataaaacaccaccaattaaagatttcggtggtttcaacttctcacaaatactgccagatccgagcaaaccaagcaa gaggtcattcattgaagacctacttttcaacaaagtgacacttgcagatgctggcttcattaaacagtatggtgattgctt gggggatattgctgctagagacctcatttgtgcacaaaagtttaacgggctgacagtgttgccacctttgttgacagat gagatgattgctcagtacacttctgcactgctcgctggtacaatcacatctgggtggacctttggtgcaggtgctgcct tacaaataccatttgctatgcagatggcttataggttcaatggtatcggagttacacagaacgttctctatgagaaccaa aaattgattgccaaccaattcaatagtgccattggcaagattcaggactcactttcaagcacagcgagtgcacttgga aagttgcaagatgtggtcaaccagaatgcacaagctttaaacacgcttgtgaaacaactcagctccaactttggggc aatttcaagtgttttgaatgatatcctttcacgtcttgataaagtggaagccgaggtgcaaattgacaggttgatcacag gccgacttcaaagtttgcagacttatgtgactcaacaattaattagggcagcagaaatccgcgcttcggctaatctgg cggctactaaaatgtcagagtgtgtacttggacaatctaaacgagttgatttttgcggaaagggctatcatctcatgtcc ttccctcagtcagcgcctcacggtgtagtgttcttgcacgtgacttacgttcctgcacaagaaaagaatttcacaactg ctccggccatttgtcatgatggaaaagcccactttccgcgtgaaggtgtctttgtttcgaatggcacacactggtttgta acccaaaggaatttttatgagccacaaatcattacgacggacaacacttttgtgtctggtaattgtgatgttgtaatcgg aatcgtcaacaacaccgtttacgatcctttgcagcctgagttagattctttcaaagaggagctggataagtatttcaaga atcatacatcacccgatgttgatctcggtgatatctctggaattaatgcttcagttgtgaacattcaaaaggagattgacc gcctcaatgaggttgccaagaatttgaatgaatcgctcatcgatctccaagaacttggaaagtatgagcagtatatcaa gtggcca Amino acid RRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKT SEQ ID NO: 6 sequence of S2sΔ SVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQ (TMD-CT) VKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQ protein YGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGW TFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQD SLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDK VEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQ SKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDG KAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNN TVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRL NEVAKNLNESLIDLQELGKYEQYIKWP Nucleotide gtgtctaggcttgaggaagatgttagaaatctcaacgcaattgtccagaaacttcaggaaaggttggataggctgga SEQ ID NO: 7 sequence of ggaaactgttcaagctaag mCOr1 Amino acid vsrleedvrnlnaivqklqerldrleetvqak SEQ ID NO: 8 sequence of mCor1 Nucleotide ggaggttcaggaggttcaggaggttcaggaggttca SEQ ID NO: 9 sequence of Linker Amino acid ggsggsggsggs SEQ ID NO: sequence of 10 Linker Nucleotide atggctcgct cgtttggagc taacagtacc gttgtgttgg cgatcatctt cttcggtgag tgattttccg SEQ ID NO: sequence of BiP atcttcttct ccgatttaga tctcctctac attgttgctt aatctcagaa ccttttttcg ttgttcctgg atctgaatgt 11 gtttgtttgc aatttcacga tcttaaaagg ttagatctcg attggtattg acgattggaa tctttacgat ttcaggatgt ttatttgcgt tgtcctctgc aatagaagag gctacgaagt ta Nucleotide cacgatgagctc SEQ ID NO: sequence of 12 HDEL Amino acid His-Asp-Glu-Leu SEQ ID NO: sequence of 13 HDEL Nucleotide aattattacatcaaaacaaaaa SEQ ID NO: sequence of 14 5'UTR Nucleotide agagatctcctttgccccagagatcacaatggacgacttcctctatctctacgatctagtcaggaagttcgacggaga SEQ ID NO: sequence of MacT aggtgacgataccatgttcaccactgataatgagaagattagccttttcaatttcagaaagaatgctaacccacagatg 15 gttagagaggcttacgcagcaggtctcatcaagacgatctacccgagcaataatctccaggagatcaaataccttcc caagaaggttaaagatgcagtcaaaagattcaggactaactgcatcaagaacacagagaaagatatatttctcaaga tcagaagtactattccagtatggacgattcaaggcttgcttcacaaaccaaggcaagtaatagagattggagtctctaa aaaggtagttcccactgaatcaaaggccatggagtcaaagattcaaatagaggacctaacagaactcgccgtaaag actggcgaacagttcatacagagtctcttacgactcaatgacaagaagaaaatcttcgtcaacatggtggagcacga cacgcttgtctactccaaaaatatcaaagatacagtctcagaagaccaaagggcaattgagacttttcaacaaagggt aatatccggaaacctcctcggattccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggc tcctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgacagtggtcccaaagatgg acccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtga cgcaagacgtgacgtaagtatctgagctagtttttatttttctactaatttggtcgtttatttcggcgtgtaggacatggca accgggcctgaatttcgcgggtattctgtttctattccaactttttcttgatccgcagccattaacgacttttgaatagata cgctgacacgccaagcctcgctagtcaaaagtgtaccaaacaacgctttacagcaagaacggaatgcgcgtgacg ctcgcggtgacgccatttcgccttttcagaaatggataaatagccttgcttcctattatatcttcccaaattaccaatacat tacactagcatctgaatttcataaccaatctcgatacaccaaatcgt Nucleotide aattttactcaaaatgttttggttgctatggtagggactatggggttttcggattccggtggaagtgagtggggaggca SEQ ID NO: sequence of gtggcggaggtaagggagttcaagattctggaactgaagatttggggttttgcttttgaatgtttgcgtttttgtatgatg 16 RD29B terminator cctctgtttgtgaactttgatgtattttatctttgtgtgaaaaagagattgggttaataaaatatttgcttttttggataagaaa ctcttttagcggcccattaataaaggttacaaatgcaaaatcatgttagcgtcagatatttaattattcgaagatgattgtg atagatttaaaattatcctagtcaaaaagaaagagtaggttgagcagaaacagtgacatctgttgtttgtaccatacaaa ttagtttagattattggttaacatgttaaatggctatgcatgtgacatttagaccttatcggaattaatttgtagaattattaat taagatgttgattagttcaaacaaaaat

<Example 2> Expression and Confirmation of Gene Encoding Recombinant Spike Protein of COVID-19 Virus Forming Trimer

[0095] 6 types of constructs shown in FIG. 1 [SfΔ(TMD-CT):mCor1:Hisx5:HDEL (S.sub.full-t), SfΔ(TMD-CT):Hisx5:HDEL (S.sub.full-m), S1s:mCor1:Hisx5:HDEL (S1s-t), S1s:Hisx5:HDEL (S1s-m), S2sΔ(TMD-CT):mCor1:Hisx5:HDEL (S2s-t), S2sΔ(TMD-CT):Hisx5:HEDL (S2s-m)] were induced for expression through transient expression in 4 to 5 week-old Nicotiana benthamiana plant leaves by using the vacuum infiltration method. Infiltrated leaves were harvested 5 days after infiltration (dpi), thoroughly ground in liquid nitrogen and dissolved in 3 volumes of buffer. The total soluble protein from the infiltrated leaf extract was developed by SDS-PAGE, followed by Western blot analysis. In the spike (S) recombinant proteins, S.sub.full-t and S.sub.full-m were identified at about the 180 kD position, and S1s-t and S1s-m were identified at the 100 kD position by an anti-His antibody. In addition, S2s-t and S2s-m were identified at the 75 kD position. Comparing the positions of the constructs without mCor and the proteins with mCor1, it can be seen that the difference between them was due to mCor1, as those with mCor1 were slightly smaller than those without mCor1. In addition, since all of these six recombinant proteins appeared to be larger than the calculated sizes, it was determined that this was due to N-glycosylation (FIG. 2). In addition, when the bands were confirmed by staining the same membrane with Coomassie Brilliant Blue, they were also observed here. Judging from the band intensity, the expression level of these recombinant proteins was estimated to be on the order of 20 to 50 μg/g fresh weight in infiltrated leaves.

<Example 3> Isolation and Purification of Recombinant Spark Protein of COVID-19 Virus Forming Trimer

[0096] S.sub.full-t and S2s-t, which are the recombinant proteins of two types of COVID-19 virus S protein, were expressed in N. benthamiana, isolated and purified by Ni.sup.2+-NTA affinity column, and developed through SDS/PAGE, and afterwards, Western blot was performed using an anti-His antibody to confirm isolation and purification. Subsequently, the membrane was stained with Coomassie brilliant blue. Through this, it was confirmed that the recombinant proteins derived from the two types of spark proteins could be purified by pure isolation (FIG. 3).

<Example 4> Isolation and Purification of S.SUB.full-t .Recombinant Protein Forming Trimer Through Size Exclusion Column Chromatography

[0097] The recombinant protein of S.sub.full-t isolated and purified by Ni.sup.2+-NTA affinity column was concentrated, and it was isolated and purified again by using size exclusion column chromatography. The fractions obtained from the column were developed through SDS/PAGE from No. 7 to No. 20, and Western blot was performed using an anti-His antibody to confirm the location and elution amount of the Se recombinant protein. Afterwards, the membrane was stained with Coomassie brilliant blue to confirm the contamination of other protein bands (FIG. 4).

<Example 5> Confirmation of Difference in Fractionation Through Size Exclusion Column of Recombinant Protein with and without mCor1 of SfΔ (TMD-CT) of COVID-19 Virus and Trimer Formation of S.SUB.full-t .Recombinant Protein by mCor1

[0098] After isolating and purifying proteins by Ni.sup.2+-NTA affinity column from N. benthamiana leaf extracts expressing S.sub.full-t it and S.sub.full-m, respectively (FIGS. 5A and B), these proteins were analyzed by using size exclusion column chromatography, and after fractionation (FIG. 6C), these fractions were purified using SDS/PAGE and analyzed by Western blotting using an anti-His antibody (FIG. 5D). After negative staining of the isolated S.sub.full-t protein, the shape of the protein was observed using an electron microscope to confirm the formation of a trimer (FIG. 5E).

<Example 6> Isolation and Purification of Recombinant Protein of S2e Forming Trimer Through Size Exclusion Column Chromatography

[0099] The recombinant protein of S2s-t isolated and purified by Ni.sup.2+-NTA affinity column was concentrated, and it was isolated and purified again by using size exclusion column chromatography. The fractions obtained from the column were developed through SDS/PAGE from No. 7 to No. 20, and Western blot was performed using an anti-His antibody to confirm the location and elution amount of the S2s-t recombinant protein (FIG. 6).

<Example 7> Immunogenicity Evaluation in Experimental Animals of COVID-19 Plant Vaccine Candidates

[0100] (1) Evaluation of Immunogenicity in Experimental Animals (Mouse, Balb/c) of COVID-19 Plant Vaccine Candidates

[0101] For animal experiments to evaluate the immunogenicity of the prepared plant vaccine, 6-week-old female mice (Balb/c) were used, 5 mice were assigned per experimental group, and four doses (1, 5, 15, 30 μg) were carried out for immunization. Each immune antigen (S.sub.full-t) was immunized 3 times at intervals of 2 weeks by an intramuscular route, and in order to confirm the production of antibodies to the immune antigen, blood was collected before immunization, 2 weeks after primary immunization, secondary immunization and tertiary immunization to carry out the evaluation of immunogenicity such as antibody (IgGs) production and neutralizing antibody production in serum. In this case, the negative control group was immunized with PBS.

[0102] 1) Humoral Immune Response Analysis

[0103] In the evaluation of the immunogenicity of the prepared plant vaccine, it was confirmed through ELISA whether antigen-specific antibodies were generated in the serum. It was confirmed that sufficient IgG titers appeared after a total of 2 or 3 mouse immunizations, and it was confirmed that both S1 and S2 specific antibodies, which are subdomains of the S antigen, were generated by this vaccine, and it was particularly confirmed that the titer against S2 was high. In addition, it was confirmed that the group immunized with an immune adjuvant (aluminum hydroxide) at the same time showed somewhat higher IgG titers and neutralizing antibody titers compared to the group not administered simultaneously.

[0104] 2) Neutralizing Antibody Induction Analysis

[0105] After separating the serum from the blood of the mice immunized with the prepared plant vaccine, the plaque reduction neutralization test (PRNT), which is generally known as the “gold standard” among the methods of measuring virus-neutralizing antibodies, was used to confirm the neutralizing ability of the antibody against SARS-CoV-2. In order to analyze the neutralizing antibody titer, the virus was treated in mouse serum (antibody) by using SARS-CoV-2 virus (BetaCoV/Korea/KCDC03/2020) received from the National Culture Collection for Pathogens, and afterwards, the change in the infection rate was confirmed. In addition, cross-reactions analysis for the recently reported UK mutant (B.1.1.7) and South African mutant (B.1.351) was also performed.

[0106] The neutralizing antibody induction showed higher neutralizing antibody titers in the group immunized with the immune adjuvant at the same time than the group injected with the immune antigen alone, similar to the result of IgG production, and it was confirmed that a higher neutralizing antibody titer was shown in the serum collected after immunization at a high concentration than in the serum immunized with a low concentration. In addition, it was confirmed that neutralizing antibodies were not induced in the PBS group, which was a negative control group (FIG. 7C). In addition, it was confirmed that cross-reactions also appeared in the UK and South African mutants, which have recently become a problem (FIG. 7D).

[0107] 3) Cell-Mediated Immune Response Analysis

[0108] Cell-mediated immune response according to plant vaccine immunization was analyzed using the ELISPOT test method. ELISPOST analysis was performed using a commercial IFN-γ ELISPOT kit. In the cell-mediated immune response, the number of splenocytes secreting IFN-γ was increased in the group immunized with the plant vaccine compared to the control group (PBS group), and it was confirmed that it was not significantly proportional to the amount of antigen. In addition, there was no increase effect according to the immune adjuvant. In conclusion, it could be confirmed that the cellular immune response was induced together with the humoral immunity by plant vaccine immunization (FIG. 7E).

Example 8> Evaluation of Protective Efficacy in Experimental Animals (TG Mice) of COVID-19 Plant Vaccine Candidates

[0109] An animal experiment to evaluate the protective efficacy of the plant vaccine prepared according to the present invention was performed by using 6-week-old transgenic mice (B6.Cg-Tg(K18-ACE2)2Primn/J) expressing the SARS-CoV-2 human receptor ACE2, and 14 mice were assigned per experimental group (12 mice in the control group), and immunization was performed at two doses (15 and 30 μg). Each immune antigen was immunized twice at intervals of 2 weeks by intramuscular route, and in order to confirm the production of antibodies to the immune antigen, blood was collected before immunization, 2 weeks after primary immunization and 2 weeks after secondary immunization to perform the evaluation of immunogenicity such as antibody (IgGs) production and neutralizing antibody production. In this case, the negative control group was immunized with PBS. Virus challenge inoculation was performed after nasal infection at a concentration of 2×10.sup.4 pfu/mouse 28 days after initial immunization, and the survival rate and tissue titer were measured (FIG. 8A).

[0110] 1) Antibody Induction Analysis According to Plant Vaccine Immunization

[0111] After separating the serum from the blood of the mice immunized with the prepared plant vaccine, it was confirmed by ELISA whether antigen-specific antibodies were generated in the serum. It was confirmed that sufficient IgG titers appeared after a total of two immunizations, and it was confirmed that both S1 and S2 specific antibodies, which are subdomains of the S antigen, were generated by this vaccine, and it was confirmed that the group immunized with an immune adjuvant (aluminum hydroxide) at the same time showed a higher IgG titer than the group that was not administered simultaneously (FIG. 8B).

[0112] 2) Analysis of Survival Rate and Tissue Titer According to Hamster Challenge Inoculation According to Plant Vaccine Immunization

[0113] 28 days after initial immunization, the virus was intranasally infected to confirm protection against SARS-CoV-2 infection in transgenic mice whose immunogenicity was confirmed, and the body weight, body temperature and clinical symptoms were observed for 14 days after infection (FIG. 8C). As a result of measuring the change in body weight for 2 weeks after infection, the hamsters immunized with the plant vaccine showed less weight loss compared to the PBS group which was a negative control group. As a result of the survival rate analysis of the plant vaccine immunization group, the 15 and 30 μg immunization groups survived 100%, and the 15 and 30 μg and immune adjuvant-concurrently administered group showed an 80% survival rate. In addition, the survival rate of the control (PBS) immune group was confirmed to be 1 out of 3 survivals (33%) (FIG. 8D). In order to confirm the virus proliferation pattern and histopathological findings in the lung tissue according to the virus challenge inoculation, autopsies were performed on days 3, 5 and 7 after infection, and the virus titer in the autopsied lung tissue was measured. In the plant vaccine immunization group, the inhibition of virus proliferation was confirmed in proportion to the administered dose compared to the control group, and on day 7, no virus was detected in the group administered with 30 μg and the immune adjuvant at the same time (FIG. 8E).