VACCINE COMPOSITION FOR PREVENTING OR TREATING INFECTION OF SARS-COV-2

Abstract

Provided is a recombinant protein for preventing or treating infection of SARS-Coronavirus-2 antigen comprising an extended receptor binding domain (RBD) of a spike protein of SARS-Coronavirus-2, and a vaccine composition comprising thereof. Also the present invention relates to a method for preventing infection of SARS-Coronavirus-2 by administering the recombinant antigen protein to a subject. The present invention can prevent COVID-19 infection. The present invention can be used as a vaccine.

Claims

1.-32. (canceled)

33. A recombinant protein for preventing or treating infection of SARS-Coronavirus-2 comprising a polypeptide that forms an extended receptor binding domain (RBD) of a spike protein of SARS-Coronavirus-2.

34. The recombinant protein according to claim 33, wherein a polypeptide forming a P2 domain of Tetanus toxin is optionally linked to the N-terminus or C-terminus of the extended receptor binding domain.

35. The recombinant protein according to claim 34, wherein a polypeptide forming the foldon domain of SEQ ID NO: 4 is linked between the polypeptide forming the extended receptor binding domain and the polypeptide forming the P2 domain of Tetanus toxin.

36. The recombinant protein according to claim 34, wherein the P2 domain of Tetanus toxin and the N-terminus or C-terminus of the extended receptor binding domain is linked by a linker consisting of at least three or more polypeptides.

37. The recombinant protein according to claim 33, wherein the polypeptide that forms an extended receptor binding domain of a spike protein of SARS-Coronavirus-2 contain the wild type RBD polypeptide sequence of SEQ ID NO: 33, and at least 5 to 25 optional polypeptide sequences are added to the polypeptide in the C-terminal and N-terminal directions.

38. The recombinant protein according to claim 33, wherein the recombinant protein is any one polypeptide selected from the group consisting of SEQ ID NOs: 1, 6 to 13, and 65.

39. A gene construct for producing a recombinant protein antigen for preventing or treating infection of SARS-Coronavirus-2, which comprises an open reading frame containing a polynucleotide sequence encoding the recombinant protein according to claim 33.

40. The gene construct according to claim 39, wherein a polynucleotide encoding a heterogonous signal peptide is sequentially operably linked to the open reading frame.

41. The gene construct according to claim 40, wherein a polynucleotide encoding a P2 domain of Tetanus toxin is linked so that the polynucleotides encoding each of the heterologous signal peptide, the open reading frame, and the P2 domain of Tetanus are linked.

42. The gene construct according to claim 41, wherein a polynucleotide encoding a foldon domain is further linked between the polynucleotides encoding the extended receptor binding domain and the P2 domain of Tetanus toxin.

43. The gene construct according to claim 41, wherein the linkage is linked by a polynucleotide encoding a linker consisting of at least three polypeptides.

44. The gene construct according to claim 39, wherein the gene construct consists of any one polynucleotide selected from the group consisting of SEQ ID NOs: 14 to 25, 49 to 64, 66, and 67.

45. A method for preventing or treating infection of SARS-Coronavirus-2, comprising: administering a subject in need thereof a therapeutically effective amount of the antigen protein according to claim 33.

46. The method according to claim 45, which further comprises administering a polypeptide forming any one SARS-Coronavirus-2 derived protein selected from the group consisting of the nucleocapsid (N) protein of SARS-Coronavirus-2 of SEQ ID NO: 26, a matrix (M) protein, and a small envelope (E) protein; an immunological adjuvant; or a mixture thereof.

47. The method according to claim 45, wherein i) a polypeptide forming the recombinant protein and ii) a polypeptide forming the N protein, or its fragments or analogues, and the mixing ratio of the polypeptides (recombinant protein:N protein) are administered in a weight ratio of 1:1 to 500.

48. The method according to claim 46, wherein the immunological adjuvant contains aluminum hydroxide, CpG oligodeoxynucleotide or a mixture thereof.

Description

DESCRIPTION OF DRAWINGS

[0046] The accompanying drawings illustrate a preferred embodiment of the present invention and together with the foregoing invention, serve to provide further understanding of the technical features of the present invention, and thus, the present invention is not construed as being limited to the drawing.

[0047] FIG. 1 is a schematic diagram showing a structure of SARS-CoV2 spike full-length protein domain.

[0048] FIG. 2 shows a schematic diagram of a construct for expression of a recombinant protein antigen (SK-RBD, SK-RBD-P2, SK-RBD-Ex1-P2, SK-RBD-Ex2-P2, SK-RBD-Ex3-P2) prepared based on the peptide sequence of an S protein. For example, in the case of the gene construct called SK-RBD, SP 1˜18 shows the form in which the open reading frame of the polynucleotide encoding the signal peptide having 18 polypeptide sequences is operably linked to the open reading frame of the polynucleotide encoding the peptide sequence of SK-RBD. In the case of the gene construct called SK-RBD-P2, SP 1˜18 shows the form in which the open reading frame of the polynucleotide encoding the signal peptide having 18 polypeptide sequences is operably linked to the open reading frame of the polynucleotide encoding the peptide sequence of SK-RBD, and the polynucleotide encoding the P2 domain is linked thereto.

[0049] FIG. 3 is an electrophoresis picture showing that the recombinant antigen prepared in one embodiment of the present invention forms a stable three-dimensional structure.

[0050] FIGS. 4A and 4B show the results of weight comparison and survival.

[0051] FIG. 5 shows (A) the results of cell-mediated immunity analysis of SK-RBD-P2 and (B) the results of activity analysis of T cells and B cells.

[0052] FIG. 6 is the result showing the degree of the increase in IFN-γ secreting T cells that specifically respond to RBD. It was confirmed that the immunized substance was memorized in T cells and the T cells secreted the cytokine IFN and were activated by a stimulating antigen.

[0053] FIG. 7 shows (A) the evaluation of binding force between ACE2 and RBD-Ex1-P2 antigen and (B) the evaluation of binding force between CR3022 and an antigen for vaccine through BLI.

[0054] FIG. 8 shows the results of anti-RBD ELISA in a RBD purified stock solution.

[0055] FIG. 9 shows the results of confirming the increase in IFN-gamma secreting T cells after immunization with the antigen obtained in one embodiment of the present invention.

MODE FOR INVENTION

[0056] Hereinafter, embodiments will be described in detail to aid understanding of the present invention. However, the embodiments according to the present invention may be modified in various forms, and the scope of the present invention should not be construed as being limited to the embodiments described below. The embodiments of the present invention are provided by way of example to aid in a specific understanding of the present invention. Embodiments of the present invention are provided to more completely explain the present invention to those with average knowledge in the field to which the present invention belongs.

[0057] 1. Preparation of Construct for Antigen Expressing Using Spike Protein of SARS-Coronavirus-2

[0058] In order to prepare an antigen protein used for a vaccine production, the S gene, N gene, M gene sequences were prepared by referring to the sequence of Genbank #MN908947 Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1.

[0059] FIG. 1 shows a schematic diagram showing a structure of SARS-CoV2 spike full-length protein domain, wherein the RBD is a domain consisting of the 331st to 524th polypeptides of the full-length peptide sequence.

[0060] Researchers designed a recombinant protein antigen using the newly designed extended RBD recombinant protein (SK-RBD (SEQ ID NO: 1), or (SK-RBD-ex1, SK-RBD-ex2, and SK-RBD-ex3 expressed by SEQ ID NOs: 6, 7, and 8, respectively)), and it was illustrated in FIG. 2 in detail. SP stands for a signal protein, P2 stands for a Tetanus P2 domain (CD4 T cell epitope), and foldon stands for a foldon protein domain. Herein, the P2 domain and the foldon protein domain were each linked through a GSGSG peptide linker. The recombinant protein antigen designed in this way is represented by SEQ ID NOs: 9 to 12. A recombinant protein antigen containing a foldon domain was prepared, and represented by SEQ ID NO: 13.

[0061] Expression constructs for expressing these recombinant protein antigens were designed by adding a polynucleotide encoding an appropriate signal peptide to each expression system so that the recombinant protein can be secreted into the periplasmic region or culture medium during expression or by replacing a polynucleotide so that a heterologous signal peptide could be expressed instead of the original signal peptide. In the Spike protein, the N-terminal 1 to 13 polypeptide (MFVFLVLLPLVSS) is its own signal peptide, and in the baculovirus system, CHO cell expression system, and mammalian cell expression system expressing the recombinant protein antigen, the polypeptide replaced with the human albumin signal peptide (SEQ ID NO: 2) was allowed to be expressed, or the original signal peptide was allowed to be expressed as it is.

[0062] Table 1 below shows the characteristics of the antigen proteins obtained from the gene constructs illustrated in FIG. 2.

TABLE-US-00001 TABLE 1 Charge Extinction Cys Cyc Section Residues Length MW PI at pH 7 Coefficient No. % Extended SK- 328-531 204 22.928 8.18 4.47 33850 8 3.9 RBD RBD(SEQ ID NO: 1) RBD- 321~554 225 25.276 8.26 5.40 33850 9 4.0 ex1(SEQ ID NO: 6) RBD- 321~591 271 30.311 7.69 2.34 33975 10 3.7 ex2(SEQ ID NO: 7) RBD- 321~537 217 24.380 8.36 5.47 33850 8 3.7 ex3(SEQ ID NO: 8)

[0063] PI in the above Table represents the isoelectric point. The length is the number of polypeptides, and the unit of molecular weight (MW) is kDa.

[0064] As can be seen in the above Table 1, it was found that the designed recombinant protein antigen has excellent adsorption to adjuvant and excellent refolding efficiency of the expressed protein.

[0065] In the case of SEQ ID NOs: 6, 7 and 8, the glycosylation pattern was observed as a stable single pattern upon BEV expression. On the other hand, the RBD-P2 protein obtained by the construct for RBD-P2 expression had a different glycosylation pattern, so it appeared in two bands, and the rest formed a single band. The protein formation of a single pattern with the same glycosylation means homogeneous antigenicity, and this represents important meaning for inducing immunogenicity. In addition, the N-/C-terminal portion of a protein is an important factor to be considered as the possibility of post-translational modification (PTM) is higher than that of polypeptides at other positions in the expression and purification process, and it may be related to the stability, activity and other immunorejection and the like of the protein.

[0066] The recombinant protein of the present invention was designed to stably maintain a three-dimensional structure in consideration of a single antigenicity of a protein, and its activity could be confirmed.

[0067] The structure of the extended RBD recombinant protein antigen was changed so that the N-terminus and C-terminus could be stabilized, and it was confirmed that the binding ability with ACE2 could be increased while the protein expression was maintained by this structural change.

[0068] BioLayer Interferometry (BLI) was used to evaluate the binding force of CR3022, ACE2 and the RBD protein.

[0069] In the case of SK-RBD (SEQ ID NO: 1), the protein yield was 17.1 mg/L, but in the case of RBD-P2, it was 58.5 mg/L, confirming the increased yield, and RBD-Ex1-P2 also showed a yield similar to that of RBD-P2.

[0070] On the other hand, while maintaining the protein expression yield of SK-RBD-Ex1-P2 antigen (SEQ ID NO: 10), the binding ability with ACE2 increased from 27.4 KD to 4.1 KD.

TABLE-US-00002 TABLE 2 Binding force with Antigen protein ACE2 protein(KD)-nM Reference (Sino-RBD) 4.4 SK-RBD (SEQ ID NO: 1) 13.3 SK-RBD-P2 (SEQ ID NO: 9) 27.4 SK-RBD-Ex1-P2(SEQ ID NO: 10) 4.1

[0071] 2. Antigen Preparation Using Other Proteins

[0072] The N protein antigen of SEQ ID NO: 26 was prepared based on the N protein gene of the SARS-corona-2 virus.

[0073] 3. Codon Optimization

[0074] The DNA sequence encoding the recombinant protein was synthesized in GenScript with codons optimized for insect cells and Chinese Hamster Ovary (CHO) cells, respectively.

[0075] The codon-optimized sequence for each expression system is as follows. The following sequence is a polynucleotide sequence.

TABLE-US-00003 TABLE 3 BEVS CHO Item SEQ ID NO: SEQ ID NO: SK-RBD 14 15 SK-RBD-P2 16 17 SK-RBD-Ex1-P2 18 19 SK-RBD-Ex2-P2 20 21 SK-RBD-Ex3-P2 22 23 SK-RBD-Foldon-P2 24 25 SK-S-trimer-P2 67 66

[0076] Further, a protein vaccine was designed with reference to the spike protein sequence (SEQ ID NOs:44 to 48) corresponding to the four popular Wuhan virus variants (B.1.1.7, B.1.351, B.1.1.248, B.1.429), and codons were optimized for the Insect and CHO expression system, and were represented by SEQ ID NOs: 49 to 64 and 66 to 67.

[0077] 4. Recombinant Protein Vaccine Preparation

[0078] A recombinant protein vaccine was produced by the following procedure using baculovirus and CHO cells.

[0079] 4-1. Production of Recombinant Protein Using Baculovirus Expression System

[0080] In order to express the recombinant protein (SK-RBD, SK-RBD-P2, SK-RBD-Ex1-P2, SK-RBD-Ex2-P2, SK-RBD-Ex3-P2, and SK-RBD-Foldon-P2) designed as shown in FIG. 2, and N protein with the baculovirus expression system, gene constructs represented by the codon-optimization SEQ ID NOs: 14, 16, 18, 20, 22 and 24, and SEQ ID NO: 28, respectively, were prepared. The construct gene prepared above was inserted into the transfer vector, pFastBac vector and cloned, and the gene sequence was analyzed.

[0081] The prepared plasmid was transformed into E. coli for bacmid production to prepare a recombinant bacmid, and the gene sequence was analyzed.

[0082] Recombinant baculovirus (P0) was prepared by inoculating the recombinant bacmid to Sf9 cells cultured as a monolayer for transfection and quantified by the plaque test method.

[0083] The recombinant baculovirus was infected to cultured Hi-5 cells to obtain P1 virus, and the antigen protein produced was confirmed in the supernatant.

[0084] The antigen protein produced by infecting the P1 virus into the Hi-5 cells was recovered.

[0085] The recovered recombinant protein was filtered using a filter, and the recombinant protein was purified using appropriate chromatography method (Ion Exchange, Size Exclusion and the like).

[0086] 4-2. Production of Recombinant Protein Using CHO Cell Expression

[0087] In order to express the recombinant protein (SK-RBD, SK-RBD-P2, SK-RBD-Ex1-P2, SK-RBD-Ex2-P2, SK-RBD-Ex3-P2, and SK-RBD-Foldon-P2) designed as shown in FIG. 2, and N protein with the baculovirus expression system, gene constructs represented by the codon-optimization SEQ ID NOs: 15, 17, 19, 21, 23 and 25, and SEQ ID NO: 29, respectively, were prepared.

[0088] The synthesized gene was inserted into an expression vector and cloned, and the gene sequence was analyzed.

[0089] The recombinant plasmid was transfected into CHO cells for protein production (CHO K-1 cell line).

[0090] The transfected cells expressing the recombinant protein were identified using antibiotics.

[0091] The identified transfected CHO cells were mass-cultured and the recombinant protein was recovered.

[0092] The recovered recombinant protein was filtered using a filter, and the recombinant protein was purified using appropriate chromatography method (Ion Exchange, Size Exclusion and the like).

[0093] 4-3. Recombinant Protein Identification and Quantification

[0094] The expression of the recombinant protein was confirmed using SDS-PAGE and Western blot method. The recombinant protein was quantified using a basic total protein quantification method (Lowry method, BCA method and the like).

[0095] 5. Recombinant Antigen Protein Evaluation

[0096] 5-1. Immunogenicity Test

[0097] The purified recombinant protein was combined with an adjuvant (e.g., Aluminum hydroxide) and injected into an animal model 2 to 3 times at intervals of 2 to 3 weeks. Safety was confirmed by measuring changes in body weight and body temperature. 2 to 3 weeks after the final injection, serum isolated from whole blood and splenocytes were obtained.

[0098] 5-2. Protection Test

[0099] The purified recombinant protein was combined with an adjuvant (e.g., Aluminum hydroxide) and injected into an animal model 2 to 3 times at intervals of 2 to 3 weeks. 2 to 3 weeks after the final injection, the animal was infected with a lethal amount of wild type SARS-Coronavirus-2 virus. For one week after the infection, virus shedding was evaluated in the nasal cavity, airways, organs and the like. For two weeks after the infection, changes in body weight and body temperature, survival rate and the like were evaluated.

[0100] 5-3. Immunogenicity Evaluation Analysis

[0101] For immunogenicity evaluation analysis, IgG ELISA assay was used. An antigen for coating (RBD, 51, S2, N and the like) was coated on a 96 well-plate, and the plate was blocked with a blocking buffer. The sample (serum) was reacted on the plate. An IgG detection antibody was reacted on the plate. A substrate buffer was added to develop color, and the absorbance was measured.

[0102] 5-4. Pseudovirus Preparation

[0103] An S protein gene of SARS-Coronavirus-2 was cloned into an expression vector. A reporter gene was cloned into a transfer vector. The two genes were transfected into cells for pseudovirus production to prepare a pseudovirus expressing the reporter protein.

[0104] 5-5. Neutralizing Antibody Titer Evaluation

[0105] The serially diluted sample (serum) was reacted with the pseudovirus. Cells for infection cultured in a 96 well-plate (Vero E6 and the like) were infected with the reacted pseudovirus and cultured. After 4 to 6 hours, it was washed with PBS and replaced with a new medium. After culturing for 24 to 72 hours, the expression level of the reporter protein was compared to evaluate the neutralizing antibody titer.

[0106] 5-6. Cell-Mediated Immunity Evaluation

[0107] An anti-IFN-γ antibody was coated on a 96 well-plate. The plate was blocked with a blocking buffer. Splenocytes and a stimulating antigen (Stimulate) were added thereto and cultured for 24 to 36 hours. An Interferon-gamma detection antibody was reacted, and a substrate was added and reacted. Immune cells were evaluated using an ELISPOT reader.

[0108] For the analysis of immune characteristics, an immune cell-specific antibody and a cytokine antibody were reacted with the isolated splenocytes for 2 hours. T cell distribution and cytokine expression rate were measured through flow cytometry.

[0109] 5-7. Antigenicity Evaluation of Antigen for Vaccine

[0110] BioLayer Interferometry (BLI) was used to evaluate the binding force with CR3022. CR3022 is a human monoclonal antibody against the recombinant SARS-CoV-2 Spike Glycoprotein 51. (Abcam, CAT #: ab273073)

[0111] BLI measures the affinity constant KD value (Kdis/Kon) through association and dissociation between an antibody and an antigen, and the smaller value, the higher affinity. Coronal 9 S-specific antibody was immobilized on ProA sensor chip (ForteBio) using Octet K2. The association was measured by dipping the sensor chip into an antigen sample diluted 2-fold from 100 nM, and the dissociation was measured by dipping into a well containing only a kinetic buffer. The data obtained by subtracting the reference from the result value was analyzed by fitting thereto to a 1:1 binding model using Octet Data Analysis software (11.0).

[0112] Enzymatic immunoassay was performed to demonstrate the biological activity and structural robustness of the antigen. Immune specific response was confirmed using the RBD protein as a main antigen of the recombinant Coronal 9 vaccine manufactured by our company and anti-SARS-CoV-2 neutralizing antibody, Human IgG1 (Acrobiosystems, Cat No. SAD-S53) neutralizing antibody or SARS-CoV-2 Spike neutralizing antibody, Mouse Mab (SinoBio, Cat No. MM57).

[0113] 6. Immunogenicity Test Result Through Total Antibody Titer/Neutralizing Antibody Titer Analysis 6-1. Result of Comparison Test of Immunogenicity Between SK-RBD and SK-RBD-P2 Using BALB/c

[0114] A 6-week-old female mouse was immunized with immunogenic substances, SK-RBD (SEQ ID NO: 1) and SK-RBD-P2 (SEQ ID NO: 9) by intramuscular injection (IM) 2 times at 3 weeks intervals. Then, blood was collected, serum was isolated and immunogenicity was analyzed. As a result of the analysis, it was confirmed that the antibody titer was formed by SK-RBD (SEQ ID NO: 1) and SK-RBD-P2 (SEQ ID NO: 9). Groups 1 and 2 were administered with PBS and aluminum hydroxide (=Alum. H), respectively, in the same amount as in groups 3 to 6 without administration of the antigen. As can be seen in Table 4, both groups showed high IgG antibody titer at weeks 6 and 8, but SK-RBD-P2 (SEQ ID NO: 9) showed saturation pattern at week 8. The total IgG value of the immune sample of week 8 was 2581 in SK-RBD (SEQ ID NO: 1) and 136462 in SK-RBD-P2 (SEQ ID NO: 9). The total antibody value induced by SK-RBD-P2 (SEQ ID NO: 9) was more than 5 times higher antibody titer, demonstrating better immunogenicity. N protein specific IgG antibody was also found in groups 4 and 6 immunized together with the N protein (SEQ ID NO: 26) (Table 4).

TABLE-US-00004 TABLE 4 Analysis of total antibody titer and neutralizing antibody titer of mouse immunized with RBD and RBD-P2 (BALB/c mouse) Antigen amount Subject RBD-specific total IgG N-specific total IgG No. Antigen (μg/dose) No. Adjuvant 4 w 6 W 8 W 4 W 6 W 8 W 1 Vehicle 1 0 10 PBS 67 25 25 189 128 385 2 Vehicle 2 0 10 Alum. H 25 25 25 70 83 276 3 SK-RBD- 10 10 Alum. H 26796 146153 136462 73 81 229 P2(SEQ ID NO: 9) 4 SK-RBD- 10 + 1 10 Alum. H 2923 8291 89642 560185 3318538 486428 P2(SEQ ID NO: 9) + N(SEQ ID NO: 26) 5 SK- 10 10 Alum. H 3916 7041 25182 108 110 210 RBD(SEQ ID NO: 1) 6 SK- 10 + 1 10 Alum. H 3489 9029 12076 154435 317386 301893 RBD(SEQ ID NO: 1) + N(SEQ ID NO: 26)

[0115] 6-2. Analysis of Total Antibody Titer and Neutralizing Antibody Titer of Mouse Immunized with SK-RBD-P2 (SEQ ID NO: 9) (BALB/c Mouse)

[0116] A 6-week-old female mouse was immunized with SK-RBD-P2 (SEQ ID NO: 9) and N (SEQ ID NO: 26) antigens by IM 2 times at 3 weeks intervals. Then, blood was collected, serum was isolated and immunogenicity was analyzed. The total antibody titer was measured by performing ELISA with the mouse immune serum at week 5 and week 6. As a result of analysis, as the amount of the administered antigen SK-RBD-P2 (SEQ ID NO: 9) increased (5, 10, 30 μg), the antibody titer increased dose-dependently. It was confirmed that N-specific antibody titer was formed in the serum immunized together with the N antigen. Looking at the groups 3 and 6 in Table 5 below, when the N protein antigen is administered together, there is no difference in the value of neutralizing antibody, but the ability to induce cell-mediated immunity is excellent. Therefore, this enables effective protection in the early stage of virus infection.

TABLE-US-00005 TABLE 5 Analysis of total antibody titer and neutralizing antibody titer of mouse immunized with SK-RBD-P2 (SEQ ID NO: 9) (BALB/c mouse) Neutralizing Antigen Total IgG titer antibody amount Subject 4 w 6 w 4 w 6 w titer No. Antigen (μg/dose) No. Adjuvant RBD-specific N-specific PBNA.sub.50 1 Vehicle 1 0 5 PBS 25 25 114 223 10 2 SK-RBD-P2(SEQ 5 5 Alum. H 1666 2536 159 467 ND ID NO: 9)-5 3 SK-RBD-P2(SEQ 10 5 Alum. H 7323 20336 252 781 10 ID NO: 9)-10 4 SK-RBD-P2(SEQ 30 5 Alum. H 30499 215966 252 781 320 ID NO: 9)-30 5 SK-RBD-P2(SEQ 5 + 0.5 5 Alum. H 100 313 84210 566945 20 ID NO: 9)-5 + N(SEQ ID NO: 26)-0.5 6 SK-RBD-P2(SEQ 10 + 1 5 Alum. H 1504 7044 86971 877576 10 ID NO: 9)-10 + N(SEQ ID NO: 26)-1 7 SK-RBD-P2(SEQ 30 + 3 5 Alum. H 7399 27697 176099 1394533 160 ID NO: 9)-30 + N(SEQ ID NO: 26)-3 *ND : Not Detected

[0117] 6-3. Analysis of Total Antibody Titer and Neutralizing Antibody Titer of Mouse Immunized with RBD-Ex1-P2 (SEQ ID NO: 10) and RBD-Ex2-P2 (SEQ ID NO: 11) (BALB/c Mouse)

[0118] 6-Week-old female BALB/c mouse was prepared, and immunized to the muscle with 0.1 ml of RBD-Ex1-P2 (SEQ ID NO: 10), RBD-Ex2-P2 (SEQ ID NO: 11) and N (SEQ ID NO: 26) proteins mixed with aluminum hydroxide 2 times at 3 weeks intervals. Then, blood was collected, serum was isolated and analyzed. As a result of the analysis, it was confirmed that RBD-Ex1-P2 (SEQ ID NO: 10) and RBD-Ex2-P2 (SEQ ID NO: 11) formed RBD-specific antibody titer and N-specific antibody titer, and as the amount of the administered antigen increased (5, 10, 30 μg), the antibody titer increased dose-dependently. Further, when N was administered together in an amount of 1/10, the RBD-specific IgG antibody titer tended to be slightly lower, but the neutralizing antibody titer was induced to the same level. RBD-specific IgG antibody titer and neutralizing antibody titer were shown in the group immunized with Alum+CpG adjuvant rather than Alum alone. As the CpG, Dynavax's brand name CpG 1018 adjuvant was used.

[0119] When 10 μg was administered, in the case of the alum adjuvant, the RBD-specific antibody titer was 4221, and the neutralizing antibody titer was similar to that of the vehicle, so it was hardly induced, but in the case of the alum+CpG, the RBD specific antibody titer was 5389108 and the neutralizing antibody titer was 320 or higher, which were very high (Table 6).

TABLE-US-00006 TABLE 6 Analysis of total antibody titer and neutralizing antibody titer of mouse immunized with RBD-Ex1-P2 (SEQ ID NO: 10) and RBD-Ex2-P2 (SEQ ID NO: 11) (BALB/c mouse) Antigen Total IgG titer amount Subject 3 w 5 w 3 w 5 w No. Antigen (ug/dose) No. Adjuvant RBD-specific N-specific PBNA.sub.50 1 Vehicle 1 0 5 Alum 25 25 23995 592 40 2 RBD-Ex1-P2(SEQ 5 5 Alum. H 25 2706 123 119 10 ID NO: 10)-5 3 RBD-Ex1-P2(SEQ 10 5 Alum. H 25 4221 151 127 40 ID NO: 10)-10 4 RBD-Ex1-P2(SEQ 30 5 Alum. H 1207 104476 177 353 80 ID NO: 10)-30 5 RBD-Ex1-P2(SEQ 5 + 0.5 5 Alum. H 74 3686 930 542933 0 ID NO: 10)-5 + N(SEQ ID NO: 26)-0.5 6 RBD-Ex1-P2(SEQ 10 + 1 5 Alum. H 25 2174 3460 282976 10 ID NO: 10)-10 + N(SEQ ID NO: 26)-1 7 RBD-Ex1-P2(SEQ 30 + 3 5 Alum. H 61 29738 15264 388091 160 ID NO: 10)-30 + N(SEQ ID NO: 26)-3 8 RBD-Ex2-P2(SEQ 5 5 Alum. H 25 1104 98 25 0 ID NO: 11)-5 9 RBD-Ex2-P2(SEQ 10 5 Alum. H 25 2839 137 60 0 ID NO: 11)-10 10 RBD-Ex2-P2(SEQ 30 5 Alum. H 2600 43961 161 25 80 ID NO: 11)-30 11 RBD-Ex2-P2(SEQ 5 + 0.5 5 Alum. H 167 25 772 61379 0 ID NO: 11)-5 + N(SEQ ID NO: 26)-0.5 12 RBD-Ex2-P2(SEQ 10 + 1 5 Alum. H 58 704 2640 536857 0 ID NO: 11)-10 + N(SEQ ID NO: 26)-1 13 RBD-Ex2-P2(SEQ 30 + 3 5 Alum. H 25 6329 5593 1314727 80 ID NO: 11)-30 + N(SEQ ID NO: 26)-3 14 RBD-Ex1-P2(SEQ 10 5 Alum. 112478 5389108 121 115 >320 ID NO: 10)-10 H + CpG 15 RBD-Ex1-P2(SEQ 10 + 1 5 Alum. 13988 900862 198650 235167 >320 ID NO: 10)-10 + H + CpG N(SEQ ID NO: 26)-1

[0120] Through the above result, it was found that the recombinant protein antigen of the group 14 and the like was excellent in generating a neutralizing antibody. In addition, when the N protein was administered together, it was found that it was effective in inducing cell-mediated immunity response required for initial virus protection as well as the generation of a neutralizing antibody.

[0121] 6-4. Total Antibody Titer and Neutralizing Antibody Titer Analysis According to Ratio of RBD-Ex1-P2 (SEQ ID NO: 10) and N (SEQ ID NO: 26) (BALB/c Mouse)

[0122] A 6-week-old female mouse was immunized with an antigen by IM 2 times at 3 weeks intervals. Then, blood was collected, serum was isolated and immunogenicity was analyzed. As a result of analysis, it was confirmed that the antibody titer was formed by the RBD-Ex1-P2 (SEQ ID NO: 10) and the N (SEQ ID NO: 26). In order to confirm the difference in immunogenicity according to the N protein injection, the N (SEQ ID NO: 26) antigen was immunized with two doses of 1/10 and 1/50 of the amount of the RBD-Ex1-P2 (SEQ ID NO: 10) antigen, and the RBD-specific antibody titer, the N-specific antibody titer and the neutralizing antibody titer were analyzed. As a result of analysis, when the N (SEQ ID NO: 26) was administered at a level of 1/10 of the amount of the RBD-Ex1-P2 (SEQ ID NO: 10) antigen, the RBD-specific antibody titer tended to decrease slightly, but the neutralizing antibody was similar or slightly increased, and when administered at a level of 1/50, both the RBD-specific antibody and the neutralizing antibody titer were significantly increased. When the RBD-Ex1-P2 (SEQ ID NO: 10) was administered alone, the RBD-specific antibody titer and the neutralizing antibody titer increased in a dose-dependent manner in the range of 5 to 50 ug, but when the N (SEQ ID NO: 26) was co-administered at a level of 1/50 of the amount of the RBD-Ex1-P2 (SEQ ID NO: 10) antigen, in the case of administering 30 ug of the RBD-Ex1-P2 (SEQ ID NO: 10), higher level of the RBD-specific antibody and neutralizing antibody titer were induced than the case of administering 50 ug of the RBD-Ex1-P2 (SEQ ID NO: 10).

TABLE-US-00007 TABLE 7 Analysis of total antibody titer and neutralizing antibody titer of mouse immunized with combination of RBD-Ex1-P2 (SEQ ID NO: 10) and N (SEQ ID NO: 26) (BALB/c mouse) Antigen Total IgG titer amount Subject 3 w 6 w 3 w 6 w No. Antigen (ug/dose) No. Adjuvant RBD-specific N-specific PBNA.sub.50 1 Vehicle 1 0 5 Alum. H 30 25 33 25 0 2 RBD-Ex1-P2(SEQ 30 5 Alum. H 121 57364 39 25 80 ID NO: 10)-30 3 RBD-Ex1-P2(SEQ 30 + 3 5 Alum. H 35 52590 4089 190572 320 ID NO: 10)-30 + N(SEQ ID NO: 26)- 3 4 RBD-Ex1-P2(SEQ 30 + 0.6 5 Alum. H 498 109455 1503 84553 1280 ID NO: 10)-30 + N(SEQ ID NO: 26)- 0.6

[0123] 6-5. Analysis of Total Antibody Titer of RBD-Ex1-P2 (SEQ ID NO: 10) and N (SEQ ID NO: 26) (SD-Rat)

[0124] A 7-week-old female rat was immunized with an antigen by IM 2 times at 3 weeks intervals. Then, blood was collected, serum was isolated and immunogenicity was analyzed. As a result of analysis, it was confirmed that the RBD-Ex1-P2 (SEQ ID NO: 10)-specific antibody titer and the N (SEQ ID NO: 26)-specific antibody titer were formed. In order to confirm the difference in immunogenicity according to the co-administered N protein injection, the total antibody titer and the neutralizing antibody were analyzed with the serum of the fully immunized mouse. As a result of analysis, it was confirmed that the RBD-specific antibody titer and N-specific IgG antibody titer were formed as shown in the graph below, and it was confirmed that the highest level of the total antibody titer was formed in the group 5 immunized with the RBD-Ex1-P2 (SEQ ID NO: 10) and the N (SEQ ID NO: 26) proteins at 50 ug and 5 ug, respectively.

TABLE-US-00008 TABLE 8 Analysis of total antibody titer of rat immunized with combination of RBD-Ex1-P2 (SEQ ID NO: 10) and N (SEQ ID NO: 26) Antigen amount Subject RBD-specific N-specific No. Antigen (ug/dose) No. Adjuvant IgG titer IgG titer 1 Vehicle 1 0 10 Alum. H 28 172 2 RBD-Ex1-P2(SEQ ID 30 10 Alum. H 6875 71 NO: 10)-30 3 RBD-Ex1-P2(SEQ ID 50 10 Alum. H 13145 71 NO: 10)-50 4 RBD-Ex1-P2(SEQ ID 30 + 3 10 Alum. H 6392 16220 NO: 10)-30 + N (SEQ ID NO: 26)-3 5 RBD-Ex1-P2(SEQ ID 50 + 5 10 Alum. H 31943 107793 NO: 10)-50 + N (SEQ ID NO: 26)-5

[0125] 6-6. Analysis of Cellular Immunogenicity of RBD-Ex1-P2 (SEQ ID NO: 10) and N (SEQ ID NO: 26) (SD-Rat)

[0126] In order to confirm the induction of cellular immunogenicity of a rat in the same group as in Table 8, the spleen was isolated from the fully immunized rat and ELISPot was performed. As a result of analysis, an increase in the number of IFN-gamma secreting T cell specifically responding to the RBD-Ex1-P2 (SEQ ID NO: 10) antigen stimulation was confirmed in the immune groups (G2 to G5). Further, an increase in the number of IFN-gamma secreting T cell specifically responding to the stimulating antigen N (SEQ ID NO: 26) was confirmed in the groups G4 and G5 immunized with the N (SEQ ID NO: 26) antigen.

[0127] 6-7. Analysis of Total Antibody Titer and Neutralizing Antibody Titer of Transgenic Mouse Immunized with RBD-Ex1-P2 (SEQ ID NO: 10) (hACE2 TG Mouse)

[0128] The total antibody titer was measured by performing ELISA with the immune serum of week 5 and week 6 from a TG mouse expressing a Human ACE2 gene. As a result of analysis, it was confirmed that the RBD-specific antibody titer was formed at week 6 at the level of 136077 as shown in the graph below. PBNA neutralizing antibody titer analysis was performed with the serum of week 6 from the mouse immunized with the RBD-Ex1-P2 (SEQ ID NO: 10) antigen. It was confirmed that in the hACE2 TG mouse susceptible to the wild-type SARS-CoV-2, the serum at week 6 showed PBNA.sub.50 value of 320 and the neutralizing antibody titer was formed.

TABLE-US-00009 TABLE 9 Analysis of total antibody titer and neutralizing antibody titer of transgenic mouse immunized with SK-RBD-P2 (SEQ ID NO: 9) (25 hACE2 TG mice) Antigen RBD specific amount Subject total IgG No. Antigen (ug/dose) No. Adjuvant 5 w 6 w PBNA.sub.50 1 Vehicle 1  0 5 Alum. H 25 25 20 2 SK-RBD- 20 5 Alum. H 28307 161692 640 P2(SEQ ID NO: 9)

TABLE-US-00010 TABLE 10 Analysis of total antibody titer and neutralizing antibody titer of transgenic mouse immunized with RBD-Ex1-P2 (SEQ ID NO: 10) antigen (hACE2 TG mouse) Antigen RBD specific amount Subject total IgG PBNA.sub.50 Group Antigen (ug/dose) No. Adjuvant 5 w 6 w (Pseudovirus) 1 Vehicle  0 5 Alum. H 25 25 ND* 2 RBD-Ex1-P2 20 5 Alum. H 22158 136077 320 (SEQ ID NO: 10) *ND: Not Detected

TABLE-US-00011 TABLE 11 Analysis of challenge test result of transgenic mouse immunized with RBD-Ex1-P2 (SEQ ID NO: 10) (25 hACE2 TG mice) Antigen amount Subject No. Antigen (ug/dose) No. Adjuvant 1 Vehicle 1 0 5 Alum. H 2 SK-RBD-P2 (SEQ ID NO: 9) 20 5 Alum. H 3 RBD-Ex1-P2(SEQ ID NO: 20 5 Alum. H 10)

[0129] After nasal infection with the wild type SARS-CoV-2 virus (NCCP 43326) in an amount of 5×10.sup.4 pfu/mouse, weight change and death rate were investigated for 12 days. As a result, in the case of vehicle 1 group, one died on day 6, two died on day 8, and one died on day 11, resulting in a total of 100% deaths excluding one without infection. However, 80% of the animals of the group administered with the RBD-P2 and all animals of the group administered with the RBD-Ex1-P2 vaccine survived. In other words, the survival rate was 80% or more. Therefore, it was confirmed that the recombinant protein antigen of the present invention could act as an excellent immunogen. Further, in the change of body weight after infection, in the vaccine group, it was showed an aspect that the body weight decreased within 20% and then gradually recovered, but in the vehicle group, death occurred with a rapid weight loss of about 30%. The vaccine was 100% protective in the TG mouse susceptibly modified to SARS-CoV-2 virus (FIG. 4a and FIG. 4b).

[0130] 7. Analysis of Mouse Cell-Mediated Immunity Result

[0131] 7-1. Result of Cellular Immunogenicity Analysis of BALB/c Mouse Immunized with RBD-P2

[0132] In an animal experiment using C57BL/6, IgG subtype analysis and cell-mediated immunity induction pattern analysis were performed. As a result of performing isotype antibody analysis of IgG1 and IgG2c in the serum, it was confirmed that both IgG1 and IgG2 subtype antibody titers were increased in the serum injected with the RBD-P2 antigen, and the tendency of increasing CD4+, CD8+ T cells was confirmed through FACS analysis (FIG. 5A).

[0133] Activated CD8+ cells and CD4+ cells were analyzed for analysis of T cell immunity and B cell immunity. As shown in FIG. 11, RBD-specific T cell activity tended to increase in the RBD-P2 immune group compared to the vehicle group. Further, the pattern of B cell increase in the germinal center was confirmed (FIG. 5B).

[0134] 7-2. Result of Cellular Immunogenicity Analysis of BALB/c Mouse Immunized with RBD-Ex1-P2

[0135] In order to confirm the induction of cellular immunogenicity in the immunization experiment using BALB/c, splenocytes of some subjects were isolated at week 3 after the second immunization and ELISPot measuring IFN-γ secreting T cell was performed. As a result, it was confirmed that the number of T cells responding specifically to the RBD-Ex1-P2 protein antigen was significantly increased in the vaccine administered group (Table 12, FIG. 6).

TABLE-US-00012 TABLE 12 Mouse immunogenicity group information (RBD-Ex1-P2 immunized mouse (BALB/c mouse) test) Antigen amount Subject Group Antigen (ug/dose) No. Adjuvant 1 Vehicle 0 5 Alum. H 2 RBD-Ex1-P2(SEQ ID NO: 10)- 10 5 Alum. H 10 3 RBD-Ex1-P2(SEQ ID NO: 10)- 10 5 Alum. H 10 + N (SEQ ID NO: 26)-1 4 RBD-Ex1-P2 10 5 Alum. H + (SEQ ID NO: 10)-10 CpG 5 RBD-Ex1-P2(SEQ ID NO: 10)- 10 5 Alum. H + 10 + N (SEQ ID NO: 26)-1 CpG

[0136] 8. Binding Force Evaluation Result

[0137] The Bio-layer Interferometry (BLI) principle was used to check whether the prepared antigen binds well to its receptor, ACE2. The binding force between the antigen for a vaccine and ACE2 (FIG. 7A) and CR3022 (FIG. 7B) was evaluated. It was confirmed that the binding force was the following Dissociation constant (KD) values, which was similar to the binding force (KD=4.4 nM) of the reference RBD (sino, Cat. 40592-V08B, Sino-RBD), and it was confirmed that the RBD-Ex1-P2 (SEQ ID NO: 10) had no problem with the ACE2 binding site and no problem with the binding function (FIG. 7).

[0138] Specifically, FIG. 7 shows (A) the evaluation of binding force between ACE2 and RBD-Ex1-P2 antigen and (B) the evaluation of binding force between CR3022 and an antigen for vaccine through BLI. The terminal structure of the extended RBD of the present invention was stabilized when compared to the RBD before the modification. Due to this, the protein expression level increased and the binding with the ACE2 increased, resulting in increased cell-mediated immunity. Low KD value indicates excellent binding (KD=Koff/Kon), and as shown in the result of FIG. 7A, it was confirmed that the KD value was higher than that of CR3022, so that the binding force with the ACE2 was excellent.

[0139] The RBD protein, which is the main antigenic site of the RBD-Ex1-P2 (SEQ ID NO: 10), was immunospecifically confirmed through enzyme immunoassay. By confirming the protein binding using a neutralizing antibody, it was confirmed that there was no abnormality in the biological activity and immunological activity of the RBD-Ex1-P2 (SEQ ID NO: 10) antigen (FIG. 8). RBDPC stands for Sino biological RBD reference (SinoBiologinal, 40592-VO8H).

[0140] Through this, synthetic sequence and information, protein expression confirmation, protein isolation and purification, and recombinant protein vaccine candidates were secured.

[0141] This can induce a sufficient antibody and protective immunity to prevent corona infection.

[0142] 9. Result of Comparison Test of Immunogenicity of SK-RBD-P2 (SEQ ID NO: 9), SK-RBD-P2 (SEQ ID NO: 9)+N(SEQ ID NO: 26), S-Trimer-P2 (SEQ ID NO: 65)+N(SEQ ID NO: 26) Using BALB/c

[0143] A 6-week-old female mouse was immunized with SK-RBD-P2 (SEQ ID NO: 9), SK-RBD-P2 (SEQ ID NO: 9)+N(SEQ ID NO: 26), S-Trimer-P2 (SEQ ID NO: 65)+N(SEQ ID NO: 26) antigen by IM 2 times at 2 weeks intervals. Then, blood was collected, serum was isolated and immunogenicity was analyzed. As a result of analysis, it was confirmed that RBD protein-specific antibody titer was formed in all immune group (G2-G4). The N protein-specific antibody showed high IgG titer at week 4 in both immune groups (G3, G4) and demonstrated excellent immunogenicity (Table 13).

TABLE-US-00013 TABLE 13 Analysis of total antibody titer and neutralizing antibody titer of mouse immunized with SK-RBD-P2 (SEQ ID NO: 9), SK-RBD-P2 (SEQ ID NO: 9) + N(SEQ ID NO: 26), S-Trimer-P2 (SEQ ID NO: 65) + N(SEQ ID NO: 26) (BALB/c mouse) Antigen Total IgG titer amount Subject 2 w 4 w 2 w 4 w No. Antigen (ug/dose) No. Adjuvant RBD-specific N-specific PBNA.sub.50 1 Vehicle 1 0 5 Alum. H 25 25 233 190 ND 2 SK-RBD- 10 5 Alum. H 52 12564 109 67 ND P2(SEQ ID NO: 9) 3 SK-RBD-P2 10 + 1 5 Alum. H 25 5423 1321 152911 ND (SEQ ID NO: 9) + N(SEQ ID NO: 26) 4 S-Trimer-P2 10 + 1 5 Alum. H 25 730 291 3949 40 (SEQ ID NO: 65) + N(SEQ ID NO: 26)

[0144] 10. Analysis of Cellular Immunogenicity of SK-RBD-P2 (SEQ ID NO: 9), SK-RBD-P2 (SEQ ID NO: 9)+N(SEQ ID NO: 26), S-Trimer-P2 (SEQ ID NO: 65)+N(SEQ ID NO: 26) (Balb/c Mouse)

[0145] In order to confirm the induction of cellular immunogenicity in the mouse immunized with the antigen of Table 13, the spleen was isolated from the fully immunized mouse and ELISPot was performed. As a result of analysis, the increase in IFN-gamma-secreting T cells specifically responding to the immunized antigen, N-peptide, and p2 peptide stimulation in the immune group (No. 2 to 4 above) excluding the vehicle was confirmed. The results are shown in FIG. 9. As can be seen from these results, the antigens of the present invention exhibited excellent effects of cell-mediated immunity response.

[0146] 11. Analysis of Total Antibody Titer and Neutralizing Antibody Titer of Transgenic Rat Immunized with SK-RBD (SEQ ID NO: 1), S-Trimer-P2 (SEQ ID NO: 65), N(SEQ ID NO: 26), Respectively

[0147] As a result of analysis of the serum of the RBD immunized groups of Table 14, the RBD-specific IgG antibody titer increased on Days 14, 28 and 43, and decreased on Day 57, compared to the vehicle group (G1). In the case of the S-Trimer-P2 (SEQ ID NO: 65), the S-Trimer-P2 (SEQ ID NO: 65)-specific antibody titer increased until Day 43, and then the antibody titer decreased, compared to the vehicle group (G1). As a result of analyzing the generation of the N-specific antibody in the serum of the groups immunized together with the N (G3, G5), the antibody titer increased by 227-2106 times until Day 43, compared to the vehicle group (G1), and then saturated.

TABLE-US-00014 TABLE 14 IgG Titer Group Day Day Day Day Day Antigen No. Group No. 0 14 28 43 57 SK RBD(SEQ G1 Vehicle 25 25 29 25 25 ID NO: 1) G2 SK-RBD (SEQ ID NO: 1) 25 52 6373 166915 77863 G3 SK-RBD (SEQ ID NO: 1) + 25 83 4663 169083 74617 N(SEQ ID NO: 26) S-Trimer- G1 Vehicle 25 25 33 38 37 P2(SEQ ID G4 S-Trimer-P2 (SEQ ID NO: 65) 25 1094 39042 116311 99781 NO: 65) G5 S-Trimer-P2 (SEQ ID NO: 65) + 25 1887 53134 146681 110747 N (SEQ ID NO: 26) N(SEQ ID G1 Vehicle 25 25 25 25 32 NO: 26) G2 SK-RBD (SEQ ID NO: 1) 25 25 25 25 25 G3 SK-RBD (SEQ ID NO: 1) + 25 245 12611 52646 41609 N(SEQ ID NO: 26) G4 S-Trimer-P2 (SEQ ID NO: 65) 25 25 36 179 242 G5 S-Trimer-P2 (SEQ ID NO: 65) + 25 85 1604 5683 5128 N (SEQ ID NO: 26)

TABLE-US-00015 TABLE 15 Sequence information SEQ ID NO: Item Note 1 Peptide SK RBD (328-531) (Recombinant antigen) 2 Peptide Human albumin signal peptide 3 Peptide P2 domain 4 Peptide Foldon domain 5 Peptide Linking signal peptide to SEQ ID NO: 1 6 Peptide RBD-ex1 (321-545) (Recombinant antigen) 7 Peptide RBD-ex2 (321-591) (Recombinant antigen) 8 Peptide RBD-ex3 (321-537) (Recombinant antigen) 9 Peptide SK RBD-P2 (Recombinant antigen) 10 Peptide RBD-ex1-P2 (Recombinant antigen) 11 Peptide RBD-ex2-P2 (Recombinant antigen) 12 Peptide RBD-ex3-P2 (Recombinant antigen) 13 Peptide RBD-Foldon-P2 (Recombinant antigen) 14 (BEVS) Polynucleotide Construct for expression of SEQ ID NO: 1 in BEVS 15 (CHO) Polynucleotide Construct for expression of SEQ ID NO: 1 in CHO 16 (BEVS) Polynucleotide Construct for expression of SEQ ID NO: 9 in BEVS 17 (CHO) Polynucleotide Construct for expression of SEQ ID NO: 9 in CHO 18 (BEVS) Polynucleotide Construct for expression of SEQ ID NO: 10 in BEVS 19 (CHO) Polynucleotide Construct for expression of SEQ ID NO: 10 in CHO 20 (BEVS) Polynucleotide Construct for expression of SEQ ID NO: 11 in BEVS 21 (CHO) Polynucleotide Construct for expression of SEQ ID NO: 11 in CHO 22 (BEVS) Polynucleotide Construct for expression of SEQ ID NO: 12 in BEVS 23 (CHO) Polynucleotide Construct for expression of SEQ ID NO: 12 in CHO 24 (BEVS) Polynucleotide Construct for expression of SEQ ID NO: 13 in BEVS 25 (CHO) Polynucleotide Construct for expression of SEQ ID NO: 13 in CHO 26 Peptide N protein 27 Peptide N protein (SP linkage) (Recombinant antigen) 28 (BEV) Polynucleotide Construct for expression of N protein in BEVS 29 (CHO) Polynucleotide Construct for expression of N protein in CHO 30 Polynucleotide Spike full length 31 Polynucleotide S1 domain 32 Polynucleotide S2 domain 33 Polynucleotide RBD domain 34 Peptide Spike full length 35 Peptide S1 domain 36 Peptide S2 domain 37 Peptide RBD domain 38 (BEVS) Polynucleotide Construct for expression of SEQ ID NO: 6 in BEVS 39 (CHO) Polynucleotide Construct for expression of SEQ ID NO: 6 in CHO 40 (BEVS) Polynucleotide Construct for expression of SEQ ID NO: 7 in BEVS 41 (CHO) Polynucleotide Construct for expression of SEQ ID NO: 7 in CHO 42 (BEVS) Polynucleotide Construct for expression of SEQ ID NO: 8 in BEVS 43 (CHO) Polynucleotide Construct for expression of SEQ ID NO: 8 in CHO 44 Peptide Spike protein sequence of original Wuhan strain 45 Peptide Spike protein sequence of B.1.1.7 46 Peptide Spike protein sequence of B.1.351 47 Peptide Spike protein sequence of B.1.1.248 48 Peptide Spike protein sequence of B.1.429 49 (CHO) Polynucleotide RBD ext1-P2_B.1.1.7 (CHO codon optimization) 50 (CHO) Polynucleotide RBD ext1-P2_B.1.351(CHO codon optimization) 51 (CHO) Polynucleotide RBD ext1-P2_B.1.1.248(CHO codon optimization) 52 (CHO) Polynucleotide RBD ext1-P2_B.1.429(CHO codon optimization) 53 (CHO) Polynucleotide RBD ext3-foldon-P2_B.1.1.7(CHO codon optimization) 54 (CHO) Polynucleotide RBD ext3-foldon-P2_B.1.351 (CHO codon optimization) 55 (CHO) Polynucleotide RBD ext3-foldon-P2_B.1.1.248(CHO codon optimization) 56 (CHO) Polynucleotide RBD ext3-foldon-P2_B.1.429(CHO codon optimization) 57 (CHO) Polynucleotide RBD ext1-P2_B.1.1.7 (CHO codon optimization) 58 (CHO) Polynucleotide RBD ext1-P2_B.1.351(CHO codon optimization) 59 (CHO) Polynucleotide RBD ext1-P2_B.1.1.248(CHO codon optimization) 60 (CHO) Polynucleotide RBD ext1-P2_B.1.429(CHO codon optimization) 61 (BEVS) Polynucleotide RBD-ext3-foldon-P2_B.1.1.7(Insect codon optimization) 62 (BEVS) Polynucleotide RBD-ext3-foldon-P2_B.1.351 (Insect codon optimization) 63 (BEVS) Polynucleotide RBD-ext3-foldon-P2_B.1.1.248(Insect codon optimization) 64 (BEVS) Polynucleotide RBD-ext3-foldon-P2_B.1.429(Insect codon optimization) 65 Polypeptide SK-S-trimer-P2 recombinant antigen protein 66 (CHO) Polynucleotide CHO codon optimization of SK-S-trimer-P2 67 (BEVS) Polynucleotide BEV codon optimization of SK-S-trimer-P2

[0148] (CHO) refers to a polynucleotide optimized for the CHO expression system, (BEVS) refers to a polynucleotide optimized for the BEVS expression system, and those are represented by _CHO and _BEVS, respectively, in the sequence list.

INDUSTRIAL APPLICABILITY

[0149] The present invention can prevent COVID-19 infection. The present invention can be used as a vaccine.

[0150] This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted concurrently herewith as the sequence listing text file entitled “000072usnp_SequenceListing.TXT”, file size 183 kilobytes (KB), created on 26 Oct. 2022. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e)(5).