WO2023167487A1 - Recombinant expression vector for prevention of sars-cov-2 infection and application thereof - Google Patents

Abstract

The present invention relates to a chimeric adenoviral vector containing a spike protein or nucleocapsid (NP) of a beta variant (B.1.351) that prevents infection with SARS-CoV-2 virus. It has been confirmed that the vector of the present invention has a high neutralizing antibody titer and excellent T-cell immune response against not just the initial strain but also the delta variant and beta and omicron variants, and, against the initial strain of the virus, a 100% survival rate is exhibited and protective potency could be confirmed. The vector of the present invention shows a preventive effect against the SARS-CoV-2 virus and can thus be applied as a vaccine composition for prevention and treatment of the SARS-CoV-2 virus.

Description

Recombinant expression vector for prevention of SARS-CoV-2 infection and its application

The present invention relates to a recombinant expression vector for the prevention of SARS-coronavirus-2 infection and its application, and specifically to the application of the vector to the prevention of SARS-coronavirus-2, etc., and more particularly, to the beta mutant spike protein (S A COVID-19 preventive vaccine containing a recombinant chimeric adenoviral vector encoded by linking a nucleocapsid (N protein) to a nucleocapsid (N protein) and/or a spike protein, as well as SARS-coronavirus-2 strains (beta, delta, Omicron, etc.) suggests the possibility of preventive and therapeutic vaccines against infections.

Coronaviruses are positive-sense single-stranded RNA viruses that have an envelope on the surface of particles with a diameter of 100-200 mm. Seven types of coronaviruses are known to cause respiratory and digestive infections in humans, and two of the seven types are alpha- genus of coronaviruses, and the remaining five species were identified as genus beta-coronaviruses. Severe acute respiratory syndrome coronavirus 2 (hereinafter referred to as 'SARS-CoV-2'), which causes COVID-19 (novel coronavirus, 2019-nCov), is a beta-coronavirus It is a genus, and after it was first reported in Wuhan, China in December 2019 (Wuhan-Hu-1), confirmed cases are appearing one after another worldwide. (pandemic) is intensifying.

The viral envelope of SARS-CoV-2 is composed of spike glycoprotein (S protein) and hemagglutinin-esterase dimer (HE). The S ₁ region of the S protein mainly binds to the receptor (Angiotensin-converting enzyme 2, ACE2) through the RBD (Receptor binding domain), and the S ₂ region is a direct cell-virus fusion accompanied by a dramatic change in tertiary structure (infection initiation). Therefore, vaccine candidates targeting the S protein as a major antigen have been developed and commercialized.

Unlike DNA viruses, which have DNA repair pathways (base excision repair, nucleotide excision repair, mismatch repair) that correct errors during DNA replication, RNA viruses use RNA polymerase to correct errors that occur during genome replication. Since there is no related protein, mutations are more frequent compared to DNA viruses. In the case of the SARS-CoV-2 mutant virus, mutations frequently occurred in the N-terminal domain (NTD) and RBD domain of the S protein, and as a result, a licensed vaccine developed targeting the S protein of the initial state. The antibody-binding ability to the S protein of the mutant virus in which these structural changes occurred was reduced, and the protective efficacy against the mutant virus was lowered.

Adenovirus vector vaccine is a vaccine that induces an immune response by expressing an antigen protein in the body by inserting and delivering an antigen gene for a pathogen into an adenovirus template. Human adenoirus serotype 5 (HAdV-5) vector is the most widely used. Although used, there is a problem that vaccine efficacy is reduced due to high serum retention rates in the human population.

On the other hand, the chimeric adenovirus (ChimAd) vector is a recombinant key substituted with the projection gene of chimpanzee adenovirus serotype 6 (ChAdV-6), which has a low antiserum retention rate in the human population, based on HAdV-5. As a Meric adenovirus vector, the present inventors confirmed that the cell infectivity and virus productivity increased up to 2.5 times compared to the conventional HAdV-5 vector. In addition, the present inventors conducted a neutralizing antibody test using a polyclonal antibody to HAd-5 and human serum for the corresponding serotype, and found that the chimeric adenoviral vector had an average of 2.6 times lower anti-adenovirus-5 neutralization than the HAdV-5 viral vector. Antibody titer was shown, demonstrating that the ChimAd vector was optimized for transfer of foreign genes into cells by avoiding anti-vector immunity against adenovirus serotype-5.

In December 2020, a fatal mutation of SARS-CoV-2 occurred in South Africa and was classified as a beta mutation and designated as a variant of concern (VOC). The mutant was found to be the first mutant to have all three key mutations (E484K, K417N, N501Y) of VOCs in RBD as well as deletion in NTD, and in the case of AstraZeneca's AZD-1222 vaccine, beta mutant (B.1.351) In the group, a rapid decline in efficacy was confirmed for licensed vaccines, such as a decrease in vaccine efficacy by up to 10% compared to the previous initial week.

In addition, as cross-neutralization was confirmed for the initial strain and other mutant viruses in the serum of convalescent patients who were infected with the beta mutation, the S protein for the mutant virus, not the S protein for the initial strain, was targeted. The direction of vaccine development is changing, and the need for a universal vaccine capable of fighting against the continuous emergence of mutant viruses is emerging.

N protein (nucleocapsid protein, NP) is a protein that forms a nucleocapsid of about 50 kDa, and is known to be involved in signal transduction necessary for virus budding, RNA replication, and mRNA transcription. The N protein was predicted to be highly antigenic, as more than 80% of coronavirus-infected patients had neutralizing antibodies against the protein.

It is known that there is little variation in coronaviruses, but it has been reported that mutations in that area increase the infectivity of the mutant virus. In addition, a strong N-specific antibody response and a cellular immune response were induced in C57BL/6 mice vaccinated with a vaccine encoding the N protein of SARS-CoV-2. A high initial T-cell immune response is known to play a role in mitigating the severity of the coronavirus by itself, and in some cases, anti-N protein antibodies formed by N protein are not related to direct virus neutralization, but N protein specific T - It has been reported that the increase in cellular immunity is correlated with the neutralizing antibody titer, so it is expected to be more valuable as a vaccine antigen.

[Prior Patent Literature]

Korean Patent Publication No. 1020210108331

The present invention was developed in response to the above needs, and an object of the present invention is to provide amino acids containing the S sequence of the beta mutant strain and the N partial sequence of the initial strain in order to prevent infection of SARS-coronavirus-2.

Another object of the present invention is to provide a chimeric adenovirus vector containing the S gene sequence of the beta mutant to prevent infection with SARS-coronavirus-2.

Another object of the present invention is to provide a chimeric adenovirus vector containing partial sequences of the S gene of the beta mutant strain and the N gene of the initial strain.

Another object of the present invention is to provide a vaccine for further mitigating the infection of SARS-coronavirus-2 through the enhancement of T-cell immunity.

Another object of the present invention is to provide a vaccine for preventing infection of SARS-coronavirus-2.

In order to achieve the above object, the present invention includes amino acids 1 to 1270 of the spike protein of the beta mutant strain of SARS-coronavirus-2, wherein, based on the initial week, 614, 682 to 685, 986, It has a mutation at amino acid residue No. 987, where the aspartic acid amino acid of protein No. 614 of the spike protein is substituted with glycine, arginine amino acid No. 682 is glutamine, alanine amino acid No. 683 is glutamine, 684, 685 arginine The amino acids are alanine and glutamine, respectively, at 986 and 987 consecutive Provided is an insert containing a gene encoding a spike protein of a beta mutant strain of SARS-coronavirus-2 substituted with a proline substitution mutant.

In one embodiment of the present invention, amino acid residues from 44 to 180 of the nucleocapsid protein of SARS-coronavirus-2 are linked to the N-terminus of the spike protein of the beta mutant strain of SARS-coronavirus-2, , Contains a gene encoding a fusion polypeptide prepared by linking amino acid residues 247 to 364 of the nucleocapsid protein of SARS-Coronavirus-2 to the C-terminus of the spike protein of the beta mutant SARS-Coronavirus-2 It provides an insert that does.

In one embodiment of the present invention, the amino acid residues from positions 44 to 180 of the nucleocapsid protein of the SARS-coronavirus-2 consist of the amino acid sequence of SEQ ID NO: 3, and the nucleocapsid of the SARS-coronavirus-2 The amino acid residues at positions 247 to 364 of the capsid protein are preferably composed of the amino acid sequence of SEQ ID NO: 4, but all mutant sequences that achieve the object of the present invention through mutation such as one or more substitutions, deletions, inversions, etc. to the corresponding sequence are also present. included in the scope of the invention.

In another embodiment of the present invention, the gene encoding amino acid residues 44 to 180 of the nucleocapsid protein of SARS-coronavirus-2 linked to the N-terminus of the spike protein is the nucleotide sequence shown in SEQ ID NO: 5 The gene encoding amino acid residues 247 to 364 of the nucleocapsid protein of SARS-coronavirus-2 linked to the C-terminus of the spike protein is preferably composed of the nucleotide sequence shown in SEQ ID NO: 6, All mutant sequences that achieve the object of the present invention through mutation of one or more substitutions, deletions, inversions, etc. in the sequence are also included in the scope of the present invention.

In another embodiment of the present invention, the modified spike protein of the beta mutant of SARS-coronavirus-2 consists of the amino acid sequence shown in SEQ ID NO: 1, and the modified spike protein of the beta mutant of SARS-coronavirus-2 The gene encoding the spike protein is preferably composed of the nucleotide sequence shown in SEQ ID NO: 2, but all mutant sequences that achieve the object of the present invention through mutation such as one or more substitutions, deletions, inversions, etc. in the corresponding sequence are also within the scope of the present invention. included in

In addition, the present invention provides a recombinant expression vector comprising the insert of the present invention.

In one embodiment of the present invention, the expression vector is preferably a viral vector or a non-viral vector, and the viral vector is a group consisting of an adenovirus vector, an adeno-associated virus vector, a lentiviral vector, a retroviral vector, and a herpesvirus vector. It is preferably selected from, and in one embodiment, it is preferably a chimeric adenoviral vector, but is not limited thereto.

In another embodiment of the present invention, the non-viral vector is preferably a plasmid, cosmid, phagemid, or bacterial artificial chromosome (BAC), but is not limited thereto.

In another embodiment of the present invention, the adenovirus vector replaces the terminal domain of the fiber protein of human adenovirus type 5 with the spine gene of chimpanzee adenovirus serotype 6, and the tail of human adenovirus type 5 and a fibrous protein combined with a Shaft domain and a protrusion domain of chimpanzee adenovirus serotype 6, but is not limited thereto.

In another embodiment of the present invention, the protein expressed by the chimpanzee adenovirus serotype 6 protrusion gene preferably includes the amino acid sequence of SEQ ID NO: 7, but is not limited thereto.

In addition, the present invention provides a vaccine composition for preventing infection of SARS-CoV-2, comprising the recombinant expression vector of the present invention as an active ingredient.

In one embodiment of the present invention, the vaccine preferably has an effect on one or more viruses selected from the group consisting of SARS-CoV-2 virus initial strain, delta virus, beta and Omicron virus mutant strains, but is not limited thereto.

In addition, the vector of the present invention is preferably capable of infecting mammalian cells, but is not limited thereto.

In addition, the present invention provides a host cell transformed with a chimeric adenovirus vector comprising the S gene derived from the beta mutation of the present invention or a part of the N gene linked to the S gene.

In addition, the present invention provides a method for producing a recombinant virus using the transformed cell of the present invention.

In addition, the present invention provides a method of using the two recombinant chimeric adenoviruses obtained by the present invention as a vaccine in an animal model.

The present invention provides mouse immunity and virus neutralization efficacy using the recombinant chimeric adenovirus produced by the above method.

The present invention provides efficacy for immune response of specific T cells according to the recombinant chimeric adenovirus produced by the above method.

The present invention provides the protective efficacy of the initial strain after immunization with the recombinant chimeric adenovirus produced by the above method.

In addition, the present invention provides a vaccine composition for preventing or treating an infection of SARS-CoV-2, comprising the chimeric adenovirus vector of the present invention as an active ingredient.

The present invention is a vaccine composition for preventing SARS-coronavirus-2 infection, preferably a vaccine capable of mitigating or preventing the infection of beta, delta, and omicron mutant strains as well as the initial strain, but is not limited thereto.

The present invention will be described below.

The present inventors developed a vaccine composition effective against not only the initial strain but also the dominant strain, the delta mutant strain, based on the S protein of the beta mutant strain using the ChimAd vector as a delivery vehicle, and in addition, to maximize the T cell immune response, the form linked to the N protein The present invention was completed through the evaluation of cellular immunogenicity and protective ability against viral infection of the neutralizing antibody against the S protein of .

The present invention provides a chimeric adenovirus vector encoding the beta-mutant S protein as a vaccine composition for preventing SARS-coronavirus-2 infection.

In order to inhibit the post-fusion structural change of the S protein, substitution of glycine at 614, residues 682 to 685 with acidic amino acids, and consecutive proline substitution mutations at 986 and 987 were introduced based on the initial week. . [Pallesen, Jesper, et al. "Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen." Proceedings of the National Academy of Sciences 114.35 (2017): E7348-E7357. Bangaru, Sandhya, et al. "Structural analysis of full-length SARS-CoV-2 spike protein from an advanced vaccine candidate." Science　370.6520 (2020): 1089-1094. Plante, Jessica A., et al. "Spike mutation D614G alters SARS-CoV-2 fitness." Nature 592.7852 (2021): 116-121.]

In addition, the present invention provides a chimeric adenoviral vector encoding a protein in which the S protein and the N protein are fused, including a portion of the N protein capable of enhancing T-cell immunity to the beta-mutant S protein.

In fusion with the mutant S protein, structurally unstable regions and hyperphosphorylation domains were excluded, and the NTD region containing the T cell epitope and the CTD having structural stability through dimer formation while having the T cell epitope Linked to S protein via a linker [Peng, Y., Mentzer, A. J., Liu, G., Yao, X., Yin, Z., Dong, D., ... & Dong, T. ( 2020). Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19. Nature immunology, 21(11), 1336-1345, Zinzula, L., Basquin, J., Bohn, S., Beck, F., Klumpe, S., Pfeifer, G., ... & Baumeister, W. (2021). High-resolution structure and biophysical characterization of the nucleocapsid phosphoprotein dimerization domain from the Covid-19 severe acute respiratory syndrome coronavirus 2. Biochemical and biophysical research communications, 538, 54-62.] Finally, the NTD of NP (44-180) and CTD(247-364) was linked to both ends of the S protein via the (GGGGS)3 linker.

“Chimeric protein” or “chimeric polypeptide” generally refers to a protein composed of multiple protein components, each of which is a series of polypeptides linked through amino-terminal (N-terminal, NTD) and carboxy-terminal (C-terminal, CTD) bonds. means

In the present invention, a chimeric protein or polypeptide (NP (NTD )-Spike-NP (CTD)).

The peptide of the present invention is preferably expressed in various delivery vehicles such as recombinant adenovirus vectors, RNA, recombinant proteins, and non-viral vectors, and more preferably inserted into chimeric adenovirus vectors, but is not limited thereto.

The chimeric adenoviral vector of the present invention is in the state of patent application (Korean Patent Application No.: 10-2022-0034885), and reference is made to the patent for specific descriptions, and is omitted herein.

The chimeric adenovirus containing the polynucleotide S is called ChimAd-CV, and the chimeric adenovirus containing the NP(NTD)-Spike-NP(CTD) is called ChimAd-CVN.

Cells transfected with the chimeric adenovirus vector containing the genes of the present invention are preferably mammalian cells including HEK293, PERC6 or 911 cells, more preferably HEK293 cells, but are not limited thereto.

For example, an adenovirus-producing cell suitable for the present invention should be a cell capable of producing a virus by trans-complementation of the E1 and E3 gene deletion sites of a replication defective adenovirus, and the viral early transcription gene A number of cells transformed with . Cells into which the E1 and E3 genes are preferred include A549 (carcinomic human alveolar basal epithelial cell), HeLa (Human cervical cancer cell), CHO (Chinese hamster ovary cell), MRC5 (human lung fibroblast), and PC12 (rat pheochromocytoma cell) etc., but not limited thereto.

In the present invention, when producing ChimAd-CV and ChimAd-CVN viruses, it is preferable to infect host cells at a cell density of 1x10 ⁶ cells/ml with a titer of 2 MOI and recover the cells after 48 hours, but is not limited thereto.

In order to elute the virus from the recovered cells, 0.5% Tween20 and 20 U/ml benzonase to induce host cell DNA degradation are preferably treated for 4 hours, but are not limited thereto.

In addition to Tween20, surfactants capable of eluting viruses include Tween80, Triton X-100, Zwittergent™3-14, and CHAPS, but are not limited thereto.

The present invention provides ChimAd-CV and ChimAd-CVN produced by the above method.

The ChimAd-CV and ChimAd-CVN vaccine compositions produced in the present invention provide excellent neutralizing antibody titers against the initial strain, beta mutant strain and delta mutant strain.

The ChimAd-CV and ChimAd-CVN vaccine compositions produced according to an embodiment of the present invention provide excellent neutralizing antibody titers against pseudoviruses based on beta mutants.

The ChimAd-CV and ChimAd-CVN vaccine compositions produced in the present invention provide efficacy against the immune response of specific T cells.

In order to confirm the immune response of ChimAd-CV and ChimAd-CVN-specific T cells produced according to an embodiment of the present invention, ELISPOT (Enzyme-Linked ImmunoSpot, ELISPOT) experimental technique was used. Mouse splenocytes immunized with ChimAd-CV or ChimAd-CVN were isolated to confirm an immune response to S protein or NP peptide. As a result, as ChimAd-CVN confirmed cellular immunity more than twice as good as ChimAd-CV, it is thought that higher T-cell immunity is induced by linking N protein. [Referring to the literature, adenoviral vector As a result of the T-cell immune response of the COVID-19 vaccine using the delivery vehicle or DNA, it was confirmed that the prime 4-week results of ChimAd-CV and ChimAd-CVN were similar (Graham, Simon P., et al. "Evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOx1 nCoV-19." npj Vaccines 5.1 (2020): 1-6. Seo, Yong Bok, et al. "Soluble spike DNA vaccine provides long- term protective immunity against SARS-CoV-2 in mice and nonhuman primates." Vaccines 9.4 (2021): 307.).

A high initial T-cell immune response is known to play a role in mitigating the severity of the coronavirus by itself, and in some cases, anti-N protein antibodies formed by N protein are not related to direct virus neutralization, but N protein specific T -Enhancement of cellular immunity correlates with neutralizing antibody titer. [Tan, A. T., Linster, M., Tan, C. W., Le Bert, N., Chia, W. N., Kunasegaran, K., ... & Bertoletti, A. (2021). Early induction of functional SARS-CoV-2-specific T cells associates with rapid viral clearance and mild disease in COVID-19 patients. Cell reports, 　34(6), 108728. Wyllie, D., Jones, H. E., Mulchandani, R., Trickey, A., Taylor-Phillips, S., Brooks, T., ... & Oliver, I. ( 2021). SARS-CoV-2 responsive T cell numbers and anti-Spike IgG levels are both associated with protection from COVID-19: A prospective cohort study in keyworkers. MedRxiv, 2020-11. Ni, L., Ye, F., Cheng, M. L., Feng, Y., Deng, Y. Q., Zhao, H., ... & Dong, C. (2020). Detection of SARS-CoV-2-specific humoral and cellular immunity in COVID-19 convalescent individuals. Immunity, 52(6), 971-977]

In the present invention, the ChimAd-CV and ChimAd-CVN vaccine compositions produced by the above method provide protective efficacy against early strains.

Body weight change and survival rate after immunization and viral infection with ChimAd-CV and ChimAd-CVN produced according to an embodiment of the present invention were confirmed. Both the experimental groups inoculated with ChimAd-CV and ChimAd-CVN showed a 100% survival rate against the initial strain infection, confirming the excellent defense ability.

As can be seen through the present invention, the present invention can provide excellent efficacy as a preventive vaccine against the SARS-CoV-2 virus. In the present invention, a vaccine composition comprising a beta mutant S sequence or an S sequence fused with N based on a chimeric adenovirus vector was developed. It has excellent cross-immunogenicity with high neutralizing antibodies against the initial strain and mutant strain (beta, delta, omicron), and the vaccine containing the N sequence provided the efficacy of maximizing T-cell immunity. In addition, it is considered that it can be applied as a preventive vaccine for SARS-CoV-2 as it has excellent defense against early strains.

1 is a schematic diagram of cloning ChimAd-CV and ChimAd-CVN using the Gibson assembly gene synthesis method in a chimeric adenovirus (ChimAd) vector obtained through gene synthesis.

Figure 2 shows the virus-binding antibody titers induced by ChimAd-CV and ChimAd-CVN using BALB/c female mice as A for the initial strain S protein and as B for the beta mutant S protein.

Figure 3 shows the neutralizing antibody titers induced by ChimAd-CV and ChimAd-CVN using BALB/c female mice as neutralizing antibody measurement results using pseudovirus of beta mutant (B.1.351 variant type). A-B. SARS-coronavirus-2 (Wuhan-Hu-1) pseudovirus neutralizing antibody test, C-D. Beta mutant strain (B.1.351) Pseudovirus neutralizing antibody test.

Figure 4 shows the results of cellular immunity (A, B) using S peptides of ChimAd-CV and ChimAd-CVN as stimulating antigens using BALB/c female mice and the results of cellular immunization (C, B) using NP peptides as stimulating antigens. D).

Figure 5 shows the virus-binding antibody titers induced by ChimAd-CV and ChimAd-CVN using hACE2 transgenic female mice, with A for the initial strain S protein and B for the delta mutant S protein.

6 shows the results (A, B) of neutralizing antibody measurement using FRNT of the initial strain as a result of confirming the viral neutralizing antibody titer induced by ChimAd-CV and ChimAd-CVN using hACE2 transgenic female mice. In addition, the neutralizing antibody measurement results (C, D) using FRNT of the delta mutant strain are shown. A-B. SARS-coronavirus-2 (Wuhan-Hu-1) FRNT neutralizing antibody test, C-D. Delta mutant strain (B.1.617.2) FRNT neutralizing antibody test.

Figure 7 shows the results of comparison of body weights and mortality rates between ChimAd-CV and ChimAd-CVN for initial infection in mouse animal models immunized by ChimAd-CV and ChimAd-CVN using hACE2 transgenic female mice.

Figure 8 shows the coronavirus titers detected in the lungs after challenge with SARS-coronavirus-2 (Wuhan-Hu-1).

Figure 9 shows the antibody titer induced according to the inoculation dose of ChimAd-CV using BALB/c female mice as a result for the S protein of SARS-coronavirus-2.

Figure 10 shows the virus neutralizing antibody titer induced by the inoculation dose of ChimAd-CV using BALB/c female mice with various mutant viruses (Wuhan, beta mutant, delta, Omicron (BA.1)) VLP (Virus Like Particle ) is represented by the measurement results (A to D).

In FIG. 10, A is a SARS-coronavirus-2 (Wuhan-Hu-1) VLP neutralizing antibody test, B is a beta mutant (B.1.351) VLP neutralizing antibody test, and C is a delta mutant (B.1.617.2) VLP neutralization. Antibody test, D is Omicron (BA.1) VLP neutralizing antibody test.

11 shows the results of cellular immunity induced by the inoculation dose of ChimAd-CV using BALB/c female mice, and S peptide was used as a stimulating antigen.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for explaining the invention in more detail, and the scope of the present invention is not limited by the following examples.

Example 1: Genetic modification encoding SARS-CoV-2 S protein, N protein

In order to develop a vaccine using the coronavirus structural protein, two S polypeptide synthesis genes including NTD and CTD of Spike protein and N protein including modifications such as amino acid substitution were secured. (See Table 1)

Specifically, in the case of the CV gene, substitution of glycine at 614, residues 682 to 685 with acidic amino acids, and consecutive proline substitution mutations at 986 and 987 of the initial strain of the beta-mutant S protein were introduced. [Pallesen, Jesper, et al. "Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen." Proceedings of the National Academy of Sciences 114.35 (2017): E7348-E7357. Bangaru, Sandhya, et al. "Structural analysis of full-length SARS-CoV-2 spike protein from an advanced vaccine candidate." Science　370.6520 (2020): 1089-1094. Plante, Jessica A., et al. "Spike mutation D614G alters SARS-CoV-2 fitness." Nature 592.7852 (2021): 116-121.].

In the case of the CVN gene, positions 44 to 180 and 247 to 364 of the N protein were linked to the N-terminus and C-terminus (GGGGS) of the Spike protein through ₃ linkers, respectively, and at this time, through prediction tools such as trRosetta and Alphafold. Further structural predictions were made. [Du, Zongyang, et al. "The trRosetta server for fast and accurate protein structure prediction." Nature Protocols (2021): 1-18. Jumper, John, et al. "Highly accurate protein structure prediction with AlphaFold." Nature 596.7873 (2021): 583-589.]

Example 2: Gene synthesis to be used as SARS-CoV-2 vaccine material

In order to develop a vaccine using coronavirus structural proteins, synthetic genes encoding spike and nucleocapsid proteins among structural proteins were secured. As shown in Table 1, for each vaccine candidate, S protein derived from coronavirus B.1.351 and N protein derived from Wuhan-Hu-1 were selected and codon optimized for easy expression in adenovirus. The region containing the S protein is called CV, and the chimera form in which a part of the N protein is fused with the S protein is called CVN.

	Region	Accession no.
CV	Spike surface glycoprotein	QUT64557 (B.1.351)
CVN	NP(NTD)-Spike-NP(CTD)	QUT64557 (B.1.351), MN908947 (Wuhan-Hu-1)

Example 3: Cloning of Chimeric Adenovirus Containing the SARS-CoV-2 Gene

In order to clone the two SARS-CoV-2 vaccine antigens synthesized above into ChimAd vector, which is a chimeric adenovirus vector, PCR amplification was performed excluding part of the gene of the GOI binding region using the plasmid DNA of the ChimAd vector as a template, and the amplified The product (about 33.4 Kb) was purified through a gel extraction process. The inserted gene for cloning was PCR amplified to include the same sequence (homologous region, about 20 bp) as the 5' and 3' ends of the GOI binding region in the ChimAd vector, and the CV of the amplification product obtained using the primers in Table 2 below (about 3.8 Kb) and CVN (about 4.7 Kb), and then combined with the previously purified DNA PCR amplification products through a gibson assembly reaction, respectively, two types of ChimAd-CV and ChimAd-CVN plasmids were finally obtained (Fig. 1).

Primer name	Sequence (5' to 3' direction)	PCR products
ChimAd vector	F: CTAAGCTTCTAGATAAGATATCCGATCCAC (SEQ ID NO: 8)	33.4 Kb
	R: CGCGTCGACGGTACCAGATCTCTAGCGGATC (SEQ ID NO: 9)
CV region	F: GATCTGGTACCGTCGACGCGATGTTTGTGTTTCTGGTGCTG (SEQ ID NO: 10)	3.8 Kb
	R: TATCTTATCTAGAAGCTTAGTCAGGTATAGTGCAGTTTCAC (SEQ ID NO: 11)
CVN region	F: GATCTGGTACCGTCGACGCGATGGGCCTGCCCAACAACAC (SEQ ID NO: 12)	4.7 Kb
	R: TATCTTATCTAGAAGCTTAGTCAGGGGAAGGTCTTGTAGGC (SEQ ID NO: 13)

Example 4: Production and purification of chimeric adenovirus-based corona vaccine candidates

In order to produce a chimeric adenovirus-based coronavirus vaccine candidate using the ChimAd-CV and ChimAd-CVN plasmids produced in the present invention, each plasmid is treated with PacI restriction enzyme (NEB, R0547L) to eliminate unnecessary pBR322 for adenovirus formation. The origin of replication and kanamycin resistance gene sequences were cut and removed, and HEK293 cells were transformed using Lipofectamine 2000 (Invitrogen, 11668027), and maintained for 10-20 days in an incubator at 37°C and a _CO2 partial pressure of 5.0%. After culturing, a primary virus stock was obtained.

ChimAd-CV and ChimAd-CVN virus production was performed by infecting HEK293 cells at a cell density of about 1x 10 ⁶ cells/ml with a titer of 2 MOI using the primary virus stock obtained above, and after 48 hours, 0.45 ㎛ CFF microfiltration filter (Cytiva, CFP-4-E-6A) was used to enrich the cells. The recovered cells were treated with 0.5% Tween20 (Sigma-aldrich, P2287-500ML) and 20 U/ml benzonase (Milipore, E1014-25KU) for 4 hours to induce cell lysis and host cell DNA degradation. Thereafter, cell debris was removed using 2 ㎛ and 0.6 ㎛ ULTA GF filters (Cytiva, KGF-A-0210TT, KGF-A-9606TT), and virus concentration and buffer exchange were performed through tangential flow filtration (TFF). Virus samples in PBS buffer were subjected to anion exchange chromatography using a HiTrap Capto Q ImpRes column (Cytiva, 17547051) and fast protein liquid chromatography (FPLC) using a Capto core 700 column (Cytiva, 17548115) to detect ChimAd-CV and ChimAd-CVN. The virus was isolated, finally formulated into a vaccine buffer, and finally filtered with a 0.2 μm ULTA GF filter (Cytiva, KGF-A-9610TT) to secure two vaccine candidates.

Example 5: Immunogenicity test results through binding antibody/neutralizing antibody/cellular immunity assay

5-1) Induction of immunity using BALB/c female

Mouse immunization was performed using the ChimAd-CV and ChimAd-CVN vaccine candidates obtained by the above preparation method. A total of 4 groups were included in the test group: ChimAd used as buffer, mock-up, ChimAd-CV and ChimAd-CVN. For the immunity induction test, 5-week-old BALB/c female mice were used, and each adenovirus test group was injected at a concentration of 2.5*10^9 vp/dose. Inoculations were administered intramuscularly twice a total of 1st and 2nd vaccinations at 4-week intervals (day 0 and 28), and 4 and 6 weeks after the first vaccination (day 27 and 42), blood was collected and serum was collected. isolated and the spleen was removed.

5-2) ELISA; Mouse anti-serum binding antibodies by ChimAd-CV and ChimAd-CVN

In order to confirm the presence of antibodies in the serum through the mouse immunity induction test, a binding antibody test was performed. To perform the test, 100 ng / well of the coronavirus initial strain and beta mutant S protein were dispensed at a concentration of 1 ug / ml in a microplate and coated for more than 16 hours at 4 degrees. After the reaction, the plate was washed more than 4 times with 1XPBS containing 0.05% Tween20 (PBS-T), and 100ul of 0.5% casein was dispensed and reacted at 37 ° C for 1 hour. After washing with PBS-T, each serum was diluted 1:100 and then serially diluted 3 times to about 1x10 ⁶ . After dispensing, the mixture was reacted at 37° C. for 1 hour, washed with PBS-T, diluted with HRP-coupled goat anti-mouse IgG HRP (Thermo) at a ratio of 1:5000, and reacted for 1 hour. After the reaction was finished, 50 μl of TMB solution (Thermo) was dispensed to proceed with the color development, and the color development reaction proceeded for 15-20 minutes. After that, the reaction was stopped by adding an equal amount of Stop solution (Thermo), and the absorbance was measured at a wavelength of 450 nm. did

As a result of the binding antibody titer, it was confirmed that both the initial strain and the beta mutant showed a high binding antibody titer of 10 ⁴ or more compared to the control group inoculated with ChimAd-CV and ChimAd-CVN. In the experimental group inoculated with the control group, Binding antibody titers for both the initial strain and the beta mutant were measured, but were found to be invalid values (FIG. 2).

5-3) pVNT (pseudovirus neutralization test)

; Measurement of neutralizing antibody against pseudovirus in mouse anti-serum by ChimAd-CV and ChimAd-CVN

The neutralizing antibody of the antibody in serum through mouse immunity induction test was tested. The pseudovirus system is a virus that uses the backbone of Lentivirus to express the luminescent gene and Spike protein of the initial strain or beta mutant strain. In this experiment, pseudoviruses containing Spike proteins of the initial strain and the beta mutant strain were produced using Takara's Lenti-X™SARS-CoV-2 Packaging Mix product. The produced pseudovirus was transformed into 293T cells in which ACE2 protein was overexpressed, and luciferase expression was quantified and measured, and neutralizing antibody tests were performed using a constant RLU (Relative Light Units) value. For the neutralizing antibody test, each mouse serum was first inactivated at 56° C. for 30 minutes, and DMEM medium containing 10% FBS was used. Serum was diluted 1/8 and then serially diluted 2-fold to about 10 ³ . Pseudoviruses having a constant RLU value were mixed in equal amounts with the diluted serum at 37°C and 5% CO _{2 .} Neutralization was performed in an incubator for 1 hour. The hACE2-293T cells cultured one day before were treated with 100 μl of the virus and serum mixture, and cultured in a 37°C, 5% CO ₂ incubator for about 72 hours. After the incubation was completed, about 100 ul culture medium was removed, 30 ul of One-Glo luciferase reagent (Promega) was added, reacted for 3-5 minutes, and then 60 ul of the mixture was transferred to a white plate and the luminescence value was measured using a luminometer. The measured RLU value was calculated using a statistical program to calculate the IC50 value according to the serum dilution factor.

As a result of the neutralizing antibody against the beta-mutant virus, as shown in Table 3, the IC ₅₀ =1253 for the ChimAd-CV vaccinated group and the IC ₅₀ =1131 for the ChimAd-CVN vaccinated group were all higher than 10 ³ , confirming a higher neutralizing antibody titer than the negative control group. .

No	Group	pVNTs (IC50)
		4wk	6wk
One	PBS	7	42
2	ChimAd	9	0
3	ChimAd-CV	244	1253
4	ChimAd-CVN	409	1131

5-4) ELISpot; ChimAd-CV, ChimAd-CVN specific T cell immune response by adenovirus

In the mouse immunity induction test, spleens were removed from each group 4 weeks after the first inoculation and 2 weeks after the second inoculation (6 weeks after the first inoculation). Splenocytes were collected using a cell strainer (Falcon), and washed with PBS buffer. After washing, red blood cells were removed using red blood cell lysis buffer (Sigma), and the number of splenocytes was counted and diluted to the cell concentration used for the test. In this experiment, R&D system's mouse IFN-gamma ELISpot kit was used, and the test was performed according to the manufacturer's instructions. In each test, splenocytes of 1X10 ⁵ cells and 2X10 ⁴ cells were incubated for 16 hours in a 37°C, 5% CO2 incubator together using a peptide pool or Nucleprotein peptide pool of Genscript's initial stock Spike protein as stimulating antigens. After culturing, it was washed with a washing solution, and 100ul of IFN-gamma detection antibody bound to biotin was added thereto, followed by stirring at room temperature for 2 hours. After the reaction, it was washed 4 times using a washing solution, and 100 μl of streptavidin-AKP (alkaline phosphate) was added and incubated for 2 hours at room temperature. After the last wash, 100ul of BCIP/NBT substrate was added and reacted at room temperature for about 45 minutes. The number of cells in Unit (SFU) was converted.

As a result of measuring the induction of specific T-cell immunity to each stimulatory antigen of S protein and N protein through the ELISpot test, T-cell immunity was higher after the second vaccination (week 6) than after the first vaccination (week 4). In particular, it was confirmed that ChimAd-CVN induces a specific immune response about twice as high as ChimAd-CV against Spike protein peptides. In addition, it was confirmed that ChimAd-CVN induces a specific immune response to the Nucleocapsid peptide.

No	Group	SPF/10^6 splenocytes (Spike peptide pool)
		4wk	6wk
One	PBS	34	17
2	ChimAd	61	42
3	ChimAd-CV	2367	3824
4	ChimAd-CVN	3020	6949

Example 6: ChimAd-CV, ChimAd-CVN antibody titer after animal immunization, neutralizing antibody titer and challenge test

6-1) Immunity induction and challenge test using hACE2 transgenic female mice

For the immune induction test and challenge test conducted at IVI, 6-week-old female human ACE2 transgenic mice (tg(K18-ACE2)2Prlmn) purchased from Jackson Laboratory (ME, USA) were used. All of the above tests were conducted in a BSL-3 grade facility in the International Vaccine Institute. A total of 4 groups were included in the immunity induction test group: ChimAd used as buffer, mock-up, ChimAd-CV and ChimAd-CVN. The inoculum was injected at a concentration of 2.5 x 10^9 vp/dose. Mice were intramuscularly inoculated twice at 4-week intervals, and blood was collected 4 weeks and 6 weeks after the first priming inoculation.

6-2) ELISA; Antibody titer measurement of mouse anti-serum by ChimAd-CV, ChimAd-CVN

After mouse immunity induction test, it was performed to measure the antibody titer in serum. In a 96-well plate, the initial strain S protein and the delta mutant strain S protein were dispensed at a concentration of 2ug/ml and 100ng/well each, and coated at 4 degrees for 16 hours or more. After the reaction, 100ul of PBS containing 1% BSA was dispensed and reacted. After diluting each serum 1:100, it was serially diluted 5-fold and reacted at 4 degrees for more than 16 hours. HRP-coupled Goat anti-mouse IgG HRP (Southern Biotech) was diluted 1:3000 and reacted at 37°C for 1 hour. After the reaction was completed, TMB solution (Millipore) was dispensed to proceed with color development, and then, an equal amount of 0.5 N HCl solution (Merck) was added to stop the reaction, and absorbance was measured at a wavelength of 450 nm.

Compared to the control group, it was confirmed that both the experimental group inoculated with ChimAd-CV and the experimental group inoculated with ChimAd-CVN showed a high binding antibody titer of 10 ⁵ or more to the initial strain and the delta mutant strain (FIG. 5).

6-3) FRNT (Focus Reduction Neutralization Test);

Measurement of neutralizing antibodies against coronavirus in ChimAd-CV and ChimAd-CVN mouse anti-serum

After the mouse immune induction test, a test was performed to measure neutralizing antibodies in serum. For the neutralizing antibody test, first, each mouse serum was inactivated at 56° C. for 30 minutes, and serially diluted 2-fold using DMEM medium containing 2% FBS. Diluted serum and 4.5x10 ² PFU/25ul of primary strain coronavirus (NCCP #43326 (BetaCoV/Korea/KCDC03/2020) and 4.0x10 ² PFU/25ul of delta mutant coronavirus (NCCP #43390 (hCoV-19/Korea119861) /KDCA/2021) and reacted for 30 minutes at 37 ° C. After the reaction, Vero cells (1.5x10 ⁴ cells/well) cultured one day before were treated with 50 μl of the virus and serum mixture and kept at 37 ° C, 5% CO ₂ Incubated for 4 hours in an incubator After 4 hours of reaction, the supernatant was removed, washed with PBS buffer solution, treated with 300ul of 4% formaldehyde solution (Sigma), and fixed for more than 16 hours in a place without light. After washing with PBS, 100% cold methanol was added, reacted for 10 minutes, and then reacted for 1 hour at room temperature using a blocking solution, then anti-SARS-CoV diluted 1:3000. -2 NP rabbit monoclonal antibody (Sino Biological) was treated and incubated at 37 degrees for 1 hour, washed with PBS solution (PBS-T) containing 0.1% Tween20 after incubation, and then diluted 1:2000 HRP-conjugated It was treated with goat anti-rabbit IgG (Bio-Rad) and incubated for 1 hour at 37 ° C. After washing with PBS-T, the TMB solution was reacted for 30 minutes at room temperature in the absence of light, and the reaction was stopped with water. For FRNT50 analysis, a Spot reader (Cytation 7 Cell Imaging Multi-Mode Reader) was used, and the value was calculated according to the dilution factor of serum. After that, a secondary antibody labeled with HRP (horedish peroxidase) is attached to the substrate to develop color, and virus-infected cells are identified to measure neutralizing antibodies.

As a result of the neutralizing antibody against the initial strain virus, the neutralizing antibody titer was confirmed as shown in Table 5, which confirmed that both the ChimAd-CV and ChimAd-CVN groups showed more than 100 times higher neutralizing antibody titer than the negative control group after the first inoculation. did After the second inoculation, it was confirmed that the ChimAd-CV inoculation group showed 30-fold higher neutralizing antibody titers and the ChimAd-CVN-inoculated group showed 50-fold higher neutralizing antibody titers.

In addition, as a result of the neutralizing antibody to the delta mutant virus, it was confirmed that both the ChimAd-CV and ChimAd-CVN inoculated groups showed neutralizing antibody titers 40 times higher than the negative control group after the second inoculation.

No	Group	SARS-coronavirus-2 (FRNT50)		Delta mutant strain (FRNT50)
		4wk	6wk	4wk	6wk
One	PBS	10	10	10	10
2	ChimAd	10	50	10	26
3	ChimAd-CV	1288	1660	646	1202
4	ChimAd-CVN	1096	3020	513	1096

6-4) Virus challenge test after immunization with ChimAd-CV and ChimAd-CVN

In the challenge test, 5x10 ⁵ PFU SARS-CoV-2 virus (initial strain, delta mutant strain) was intranasally infected 4 weeks after boosting inoculation. After inoculation, survival and weight were monitored daily. On the 2nd, 4th and 7th days after infection, blood, lungs, liver, kidneys, and spleens were collected, organ weights were measured, and viral titers in the lungs were measured.

As a result of confirming the vaccine defense ability of ChimAd-CV and ChimAd-CVN against the initial strain (Wuhan-Hu-1) attack, both showed 100% survival rate, and the ChimAd-CVN inoculation group showed that body weight was maintained without loss. It was confirmed that all but one of the ChimAd-CV inoculated groups did not lose weight and maintained, and it was confirmed that there was an individual whose body weight decreased by more than 10% and then recovered.

6-5) ChimAd-CV, ChimAd-CVN immunity induction and lung virus titer for challenge test (plaque assay)

After induction of mouse immunity and challenge test, a Plague assay test was performed to measure the residual virus titer in the lung. For titer measurement, Vero cells (ATCC, Cat#CCL-81) were used and cultured for more than 16 hours in a 37°C, 5% CO ₂ incubator. The virus isolated from the lungs collected from all groups was diluted to 10 ⁶ in a 4-fold stepwise manner, and 200 ul of the diluted virus was added to the cultured Vero cells and allowed to react at room temperature for 30 minutes. After the reaction, the virus mixture was removed, covered with DMEM (Overlay media) containing 0.8% Agarose and 2% FBS, reacted at room temperature for 15 minutes, and then cultured for 72 hours in a 37°C, 5% CO ₂ incubator. After fixation with 10% formalin solution for 1 hour, overlay media was removed, and plaques were stained for 5 minutes using crystal violet (Sigma). To confirm the virus titer, the number of plaques was counted, and each dilution factor and total amount (ml) were calculated to calculate the final titer.

As a result of measuring the residual virus titer in the lungs after the challenge test, it was confirmed that the lung virus titer decreased more than 20,000 times 2 days after the challenge compared to the control group after the virus challenge in the ChimAd-CV vaccinated group, and 4 days after the challenge. and 7 days later, it was confirmed that virus clearance was not measured. In the ChimAd-CVN inoculation group, it was confirmed that no lung virus titer was measured until 2, 4, and 7 days after virus clearance.

Example 7: Immunogenicity test result through binding antibody/neutralizing antibody/cellular immunity assay according to DRF (Dose range finding) test of ChimAd-CV

7-1) Dose response test of ChimAd-CV using BALB/c female

A dose-response mouse immunization was performed using the ChimAd-CV vaccine candidate. For the immunity induction test, 5-week-old BALB/c female mice were used, and the doses of the ChimAd-CV vaccine candidate were divided into 4 groups as shown in Table 6, and were composed of stabilizer (Vehicle), mock up, and non-immunization groups. Considering the previously licensed vaccination concentration, the high and low concentrations were set at 2-fold from 5.0.E+09 VP/Dose (1/10 of the mouse standard human body). Inoculations were administered intramuscularly twice a total of 1st and 2nd vaccinations (Day 0, Day 28) at intervals of 4 weeks, and blood was collected 4 weeks and 6 weeks after the first vaccination (Day 27, Day 42) and serum was collected. isolated and the spleen was removed.

The isolated serum was measured for binding antibody titers and neutralizing antibody titers to various corona viruses (Wuhan, beta-mutant, delta, and Omicron (BA.1)), and T-cell immune responses were analyzed with isolated spleens.

No	Vaccine	Dose (VP/dose)	Mouse
One	Non-immunization	-	5
2	stabilizer		100ul	5
3	ChimAd (Mock up)	100ul	5
4	ChimAd-CV1	1.00.E+10	10
5	ChimAd-CV2	5.00.E+09	10
6	ChimAd-CV3	2.50.E+09	10
7	ChimAd-CV4	1.25.E+09	10

7-2) ELISA; Mouse anti-serum binding antibody by dose response of ChimAd-CV vaccine candidate

In order to confirm the presence of antibodies in the serum through a mouse immunity induction test for each dose response of the ChimAd-CV vaccine candidate, a binding antibody test was performed. To perform the test, the coronavirus SARS-Coronavirus-2 and the beta mutant S protein were dispensed at a concentration of 1ug/ml by 100ng/well in a microplate and coated for more than 16 hours at 4 degrees. After the reaction, the plate was washed more than 4 times with 1XPBS containing 0.05% Tween20 (PBS-T), and 100ul of 0.5% casein was dispensed and reacted at 37 ° C for 1 hour. After washing with PBS-T, each serum was diluted 1:100 and then serially diluted 3 times to about 1x10 ⁶ . After dispensing, the mixture was reacted at 37° C. for 1 hour, washed with PBS-T, diluted with HRP-coupled goat anti-mouse IgG HRP (Thermo) at a ratio of 1:5000, and reacted for 1 hour. After the reaction was finished, 50 μl of TMB solution (Thermo) was dispensed to proceed with the color development, and the color development reaction proceeded for 15-20 minutes. After that, the reaction was stopped by adding an equal amount of Stop solution (Thermo), and the absorbance was measured at a wavelength of 450 nm. did

As a result of the binding antibody titer, regardless of the inoculated dose, it was confirmed that all SARS-Coronavirus-2 and beta mutants exhibited a high binding antibody titer of 10 ⁴ or more compared to the experimental group control group (FIG. 9).

7-3) pVNT (pseudovirus neutralization test); Measurement of neutralizing antibody against pseudovirus in mouse anti-serum by ChimAd-CV

The neutralizing antibody of the antibody in serum through mouse immunity induction test was tested. This experiment was measured using Virongy's Rapid Cell-Based SARS-CoV-2 Neutralizing Antibody Assay Kit using Wuhan virus, beta mutant virus, delta virus, and Omicron (BA.1) virus. The virus used in this test is a hybrid SARS-CoV-2 virus-like particle (VLP) developed by Virongy, which is combined with the 4 structural proteins (S, M, N, E) of SARS-CoV-2 and has luciferase can be expressed.

In the neutralizing antibody test, each mouse serum was first inactivated at 56° C. for 30 minutes, and the serum was diluted 1/8 using Virongy infection medium and then serially diluted 2-fold to about 10 ³ . A sample diluted in a white plate (cell culture) and 45ul of HA-CoV2(Luc)VLP were mixed and neutralized for 30 minutes in a 37°C 5% CO ₂ incubator.

HEK293T (ACE2/TMPRSS2) cells were prepared at 2.5 x 10 ⁴ cells/15 μL, treated with 15 μL of the virus and serum mixture, and cultured in a 37°C 5% CO ₂ incubator for about 18 hours. After the incubation, add 7.5 ul of cell lysis buffer and leave on a shaker for 2 minutes. After adding 25ul of Luciferase solution and reacting for 1 minute, the luminescence value was measured using a luminometer. The measured RLU values were calculated using a statistical program to calculate IC50 values according to serum dilution factors.

As shown in Table 7, after boosting (week 6), neutralizing antibodies to coronavirus by inoculation dose were confirmed. As a result of confirming neutralizing antibodies against the Wuhan virus, CV1 had IC50=1177, CV2 had IC50=577, CV3 had IC50=311, and CV4 had IC50=607 neutralizing antibodies. The highest level of neutralizing antibodies was confirmed in CV1. As a result of confirming neutralizing antibody against beta virus, CV1 has IC50=965, CV2 has IC50=416, CV3 has IC50=627, and CV4 has IC50=304. A dose dependent neutralizing antibody titer was confirmed.

As a result of confirming neutralizing antibodies against delta virus, CV1 had IC50=517, CV2 had IC50=139, CV3 had IC50=798, and CV4 had IC50=115 neutralizing antibodies. confirmed. As a result of confirming neutralizing antibodies against Omicron virus, CV1 had IC50=76, CV2 had IC50=117, CV3 had IC50=110, and CV4 had IC50=93. A neutralizing antibody titer was confirmed (FIG. 10).

Target virus	pVNTs (IC50)
	CV1	CV2	CV3	CV4
Wuhan virus	1177	577	311	607
Beta variant	965	416	627	344
Delta variant	517	325	798	115
Omicron (BA.1)	76	117	110	93

7-4) ELISpot; Specific T cell immune response by ChimAd-CV adenovirus

In the mouse immunity induction test, spleens were removed from each group 4 weeks after the first inoculation and 2 weeks after the second inoculation (6 weeks after the first inoculation). Splenocytes were collected using a cell strainer (Falcon), and washed with PBS buffer. After washing, red blood cells were removed using red blood cell lysis buffer (Sigma), and the number of splenocytes was counted and diluted to the cell concentration used for the test. In this experiment, R&D system's mouse IFN-gamma ELISpot kit was used, and the test was performed according to the manufacturer's instructions. In each test, splenocytes of 1X10 ⁵ cells and 2X10 ⁴ cells and Genscript's SARS-coronavirus-2 Spike protein peptide pool were used as stimulating antigens and cultured for 16 hours in a 37°C 5% CO2 incubator. After culturing, it was washed with a washing solution, and 100ul of IFN-gamma detection antibody bound to biotin was added thereto, followed by stirring at room temperature for 2 hours. After the reaction, it was washed 4 times using a washing solution, and 100 μl of streptavidin-AKP (alkaline phosphate) was added and incubated for 2 hours at room temperature. After the last wash, 100ul of BCIP/NBT substrate was added and reacted at room temperature for about 45 minutes. The number of cells in Unit (SFU) was converted.

As shown in Table 8, as a result of measuring the induction of specific T-cell immunity to the S protein stimulating antigen through the ELISpot test, ChimAd-CV induces a high T-cell immune response at the 6th week compared to the 4th week by inoculation dose. It was confirmed. . Regardless of the inoculation dose, excellent cellular immunity induction ability was confirmed at 6 weeks in all inoculation ranges (FIG. 11).

test group parking	SPF/10^6 splenocytes (Spike peptide pool)
	CV1	CV2	CV3	CV4
Week 4	794	934	923	888
Week 6	1575	1691	1878	2491

[Sequence information]

SEQ ID NO: 1 - SARS-CoV-2_B.1.351(beta) spike modified amino acid sequence

MFVFLVLLPLVSSQCVNFTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS

NVTWFHAIHVSGTNGTKRFANPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIV

NNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDL

EGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRGLPQGFSALEPLVDLPIGINITRFQT

LHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK

CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISN

CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIAD

YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPC

NGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCV

NFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP

GTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNS

YECDIPIGAGICASYQTQTNSPqqaqSVASQSIIAYTMSLGVENSVAYSNNSIAIPTNFTI

SVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQE

VFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDC

LGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM

QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQAL

NTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA

SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAP

AICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDP

LQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL

QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDD

SEPVLKGVKLHYT*

ID NO: 2 - SARS-CoV-2_B.1.351(beta) spike modified nucleic acid sequence

The following describes the sequences of fusion polypeptide inserts of the invention

MGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKM

KDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQL

PQGTTLPKGFYAEGSRGGS (SEQ ID NO: 3)

GGGGSGGGGSGGGGS ↓ GGGGSGGGGSGGGGS

TKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIA

QFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFP* (SEQ ID NO: 4)

In the above sequences, SEQ ID NO: 3 is a sequence bound to the N-terminus of the SARS-CoV-2-B.1.1.351 Spike protein, SEQ ID NO: 4 is a sequence bound to the C-terminus, the underline indicates a linker, and an arrow indicates a SARS-CoV-2-B.1.1.351 Spike protein. -2-B.1.1.351 Indicates the site where Spike protein is inserted.

ATG GGC CTG CCC AAC AAC ACC GCC AGC TGG TTT ACC GCC CTG ACC

CAG CAC GGC AAG GAG GAC CTG AAG TTC CCC AGA GGC CAG GGC GTG

CCC ATT AAC ACC AAC AGC AGC CCC GAC GAC CAG ATC GGC TAC

TAC AGA AGA GCC ACC AGA AGA ATC AGA GGC GGC GAC GGC AAG ATG

AAG GAC CTG AGC CCC AGA TGG TAC TTC TAC TAT CTG GGA ACC GGC

CCC GAA GCC GGC CTG CCC TAT GGC GCC AAC AAA GAT GGC ATC

ATC TGG GTG GCC ACC GAA GGC GCC CTG AAC ACC CCC AAG GAT CAC ATC

GGA ACC AGA AAT CCC GCC AAC AAT GCC GCC ATC GTG CTG

CAG CTG CCC CAG GGA ACC ACC CTG CCT AAA GGC TTT TAT GCC GAA GGA AGC

AGA GGA GGA AGC (SEQ ID NO: 5) GGC GGC GGA GGA AGC GGC GGA GGA

GGC AGC GGC GGA GGC GGA AGC ↓ GGC GGA GGA GGA AGC GGC GGA

GGC GGA AGC GGC GGC GGA GGA AGC ACC AAA AAG AGC GCC GCC GAG GCC

AGC AAA AAG CCC AGA CAG AAA AGA ACC GCC ACC AAG

GCC TAC AAT GTG ACC CAG GCC TTT GGC AGA AGA GGA CCC GAG CAG

ACC CAG GGC AAT TTT GGC GAC CAG GAG CTG ATC AGA CAG GGC ACC

GAC TAC AAA CAC TGG CCC CAG ATC GCC CAG TTC GCC CCC AGC GCC

AGC GCC TTC TTC GGC ATG AGC AGA ATC GGC ATG GAG GTG ACC CCC

AGC GGC ACC TGG CTG ACC TAC ACC GGC GCC ATC AAG CTG GAC GAC

AAG GAC CCC AAC TTC AAG GAC CAG GTG ATC CTG CTG AAC AAG CAC

ATC GAC GCC TAC AAG ACC TTC CCC TGA (SEQ ID NO: 6)

In the above sequences, SEQ ID NO: 5 is a gene sequence encoding a polypeptide bound to the N-terminus of the SARS-CoV-2-B.1.1.351 Spike protein, SEQ ID NO: 6 is a gene sequence encoding a polypeptide bound to the C-terminus, The underline indicates the gene sequence encoding the linker, and the arrow indicates the insertion site of the SARS-CoV-2-B.1.1.351 Spike protein.

SEQ ID NO: 7: Chimpanzee adenovirus serotype 6 Fiber knob domain a.a sequence

PDPSPNCQLLSDRDAKFTLCLTKCGSQILGTVAVAAVTVGSALNPINDTVKSAIVFLRFDS

DGVLMSNSSMVGDYWNFREGQTTQSVAYTNAVGFMPNIGAYPKTQSKTPKNSIVSQVY

LTGETTMPMTLTITFNGTDEKDTTPVSTYSMTFTWQWTGDYKDKNITFATNSFSFSYIAQE

Claims (12)

1. It includes amino acids 1 to 1270 of the spike protein of the beta mutant strain of SARS-coronavirus-2, where it has mutations at amino acid residues 614, 682 to 685, 986, and 987 based on the initial week, Here, in the protein, the aspartic acid amino acid of protein No. 614 of the spike protein is substituted with glycine, arginine amino acid No. 682 is glutamine, alanine amino acid No. 683 is glutamine, and arginine amino acids No. 684 and 685 are alanine and glutamine, respectively, and No. 986 and 987 consecutive An insert containing a gene encoding a spike protein of a beta mutant strain of SARS-coronavirus-2 substituted with a proline substitution mutation.
2. The method of claim 1, wherein the insert connects amino acid residues 44 to 180 of the nucleocapsid protein of SARS-coronavirus-2 to the N-terminus of the spike protein of the beta mutant strain of SARS-coronavirus-2. and a gene encoding a fusion polypeptide prepared by linking amino acid residues 247 to 364 of the nucleocapsid protein of SARS-Coronavirus-2 to the C-terminus of the spike protein of the beta mutant SARS-Coronavirus-2. containing inserts.
3. The method of claim 2, wherein amino acid residues from positions 44 to 180 of the nucleocapsid protein of SARS-coronavirus-2 consist of the amino acid sequence of SEQ ID NO: 3, and the nucleocapsid of SARS-coronavirus-2 Amino acid residues at positions 247 to 364 of the protein are an insert consisting of the amino acid sequence of SEQ ID NO: 4.
4. The method of claim 2, wherein the gene encoding amino acid residues 44 to 180 of the nucleocapsid protein of SARS-coronavirus-2 linked to the N-terminus of the spike protein consists of the nucleotide sequence shown in SEQ ID NO: 5, , The insert, characterized in that the gene encoding amino acid residues 247 to 364 of the nucleocapsid protein of SARS-coronavirus-2 linked to the C-terminus of the spike protein consists of the nucleotide sequence shown in SEQ ID NO: 6.
5. The method of claim 1, wherein the modified spike protein of the beta mutant strain of SARS-coronavirus-2 consists of the amino acid sequence shown in SEQ ID NO: 1, and encodes the modified spike protein of the beta-mutant strain of SARS-coronavirus-2. The insert characterized in that the gene consists of the nucleotide sequence shown in SEQ ID NO: 2.
6. A recombinant expression vector comprising the insert of any one of claims 1 to 5.
7. 7. The recombinant expression vector according to claim 6, wherein the expression vector is a viral vector or a non-viral vector.
8. 8. The recombinant expression vector according to claim 7, wherein the viral vector is selected from the group consisting of adenovirus vectors, adeno-associated virus vectors, lentiviral vectors, retroviral vectors and herpesvirus vectors.
9. The method according to claim 8, wherein the adenovirus vector replaces the terminal domain of the fiber protein of human adenovirus type 5 with the spine gene of chimpanzee adenovirus serotype 6, and the tail and shaft of human adenovirus type 5 ( Shaft) domain and a protrusion domain of chimpanzee adenovirus serotype 6. A recombinant expression vector containing a fiber protein combined.
10. The recombinant expression vector according to claim 9, wherein the protein expressed by the chimpanzee adenovirus serotype 6 protrusion gene comprises the amino acid sequence of SEQ ID NO: 7.
11. A vaccine composition for preventing or treating infections of SARS-CoV-2, comprising the recombinant expression vector according to any one of claims 6 to 10 as an active ingredient.
12. The method of claim 11, wherein the vaccine is SARS-CoV-2, characterized in that it has an effect on one or more viruses selected from the group consisting of virus initial strain, delta virus, beta and Omicron virus mutant strains (SARS-coronavirus-2 ( A vaccine composition for preventing or treating an infection of SARS-CoV-2).

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