KR20230131439A - Recombinant expression vector for preventing SARS-coronavirus-2 infection and its application - Google Patents

Abstract

The present invention relates to a chimeric adenovirus vector containing the spike protein or nucleocapsid (NP) of a beta mutant strain (B.1.351) that prevents infection by the SARS-CoV-2 virus. The vector of the present invention It was confirmed that it had high neutralizing antibody titers and excellent T-cell immune responses against not only the initial strain but also delta virus, beta, and omicron viruses, and its protective ability was confirmed with a 100% survival rate against the initial strain virus. The vector of the present invention shows a preventive effect against the SARS-CoV-2 virus and can be applied as a vaccine composition for the prevention and treatment of the SARS-CoV-2 virus.

Description

{Recombinant expression vector for preventing SARS-coronavirus-2 infection and its application}

The present invention relates to a recombinant expression vector for preventing 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 specifically, to the beta mutant spike protein (S It is a COVID-19 preventive vaccine containing a recombinant chimeric adenovirus vector encoded by linking the nucleocapsid (N protein) to the spike protein and/or the spike protein, and is a vaccine against SARS-Coronavirus-2 as well as mutant strains (beta, delta, Omicron, etc.) suggests the possibility of a vaccine for prevention and treatment of infectious diseases.

Coronaviruses are positive-sense single stranded RNA viruses that have an outer envelope on the surface of particles with a diameter of 100-200 mm. Seven types of coronaviruses are known to cause respiratory and digestive system infections in humans, and two of the seven types are alpha- The remaining five species were found to be belonging to the genus Coronavirus and beta-coronavirus. Severe acute respiratory syndrome coronavirus 2 (hereinafter referred to as 'SARS-CoV-2'), which causes novel coronavirus infection (COVID-19, novel coronavirus, 2019-nCov), is a beta-coronavirus. Since it was first reported in Wuhan, China in December 2019 (Wuhan-Hu-1), confirmed cases have been increasing worldwide, and since its occurrence, it has become a pandemic through various mutations (alpha mutant, beta mutant, delta mutant, etc.). The pandemic is worsening.

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 RBD (Receptor binding domain), and the S ₂ region binds to direct cell-virus fusion (infection) accompanied by dramatic changes in tertiary structure. In charge of initiation). Accordingly, vaccine candidates targeting S protein as a major antigen have been developed and commercialized.

Unlike DNA viruses that undergo a DNA repair pathway (base excision repair, nucleotide excision repair, mismatch repair) that corrects errors during the DNA replication process, RNA viruses use RNA polymerase to correct errors occurring during the genome replication process. Because there is no related protein, mutations occur more frequently than 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 was developed targeting the initial S protein. The antibody binding ability to the S protein of the mutant virus in which structural changes occurred was reduced, resulting in a decrease in the protective efficacy against the mutant virus.

Adenovirus vector vaccines are vaccines that induce an immune response by expressing antigen proteins in the body by inserting and delivering antigen genes for pathogens into an adenovirus template. Human adenovirus serotype 5 (HAdV-5) vector is the most widely used. Although it is used, there is a problem that vaccine efficacy is reduced due to the high serum prevalence in the human population.

On the other hand, the chimeric adenovirus (ChimAd) vector is a recombinant key based on HAdV-5 replaced with the protruding gene of chimpanzee adenovirus serotype 6 (ChAdV-6), which has a low antiserum retention rate in the human population. As a Merrick adenovirus vector, the present inventors confirmed that the cell infection ability and virus productivity were increased by up to 2.5 times compared to the existing HAdV-5 vector. In addition, through a neutralizing antibody test using a polyclonal antibody against HAd-5 and human serum for the corresponding serotype, the present inventors found that the chimeric adenovirus vector had an average of 2.6 times lower anti-adenovirus-5 neutralization than the HAdV-5 viral vector. The antibody titer was shown, proving that the ChimAd vector is an optimized vector for transferring foreign genes into cells by evading anti-vector immunity against adenovirus serotype-5.

In December 2020, a fatal mutation of SARS-coronavirus-2 occurred in South Africa, and was classified as a beta mutation and designated as a variant of concern (VOC). The mutant strain was found to be the first mutant to have not only a deletion in the NTD but also all three core mutations of VOCs in the RBD (E484K, K417N, N501Y), and in the case of AstraZeneca's AZD-1222 vaccine, it is a beta mutant strain (B.1.351). In the military, a rapid decline in the effectiveness of already licensed vaccines was confirmed, with the vaccine effectiveness decreasing 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 a recovering patient infected with the beta mutant, the S protein targeting the mutant virus rather than the S protein for the initial strain was used. There is a trend toward vaccine development, and the need for a universal vaccine that can combat the continued emergence of mutated 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, RNA replication, and mRNA transcription required for virus budding. The N protein was predicted to have high antigenicity as more than 80% of coronavirus-infected patients have neutralizing antibodies to the protein.

It is known to be a part of the coronavirus with little mutation, but mutations in this area have been reported to increase the infectiousness of the mutated virus. In addition, strong N-specific antibody responses and cellular immune responses were induced in C57BL/6 mice vaccinated with a vaccine encoding the N protein of SARS-CoV-2. It is known that a high initial T-cell immune response in itself plays a role in alleviating the severity of coronavirus, and some say that 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 titer of neutralizing antibodies, so it is expected to have greater value as a vaccine antigen.

[Prior patent literature]

Republic of Korea Patent Publication No. 1020210108331

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

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

Another object of the present invention is to provide a vaccine to further alleviate SARS-coronavirus-2 infection by increasing T-cell immunity.

Another object of the present invention is to provide a vaccine for preventing infection with 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 amino acids 614, 682 to 685, and 986 based on the initial strain. It has a mutation at amino acid residue 987, where the aspartic acid amino acid of protein 614 of the spike protein is replaced with glycine, the arginine amino acid at position 682 is replaced with glutamine, the alanine amino acid at position 683 is glutamine, and the amino acid 684 and 685 are replaced with arginine. The amino acids are alanine and glutamine, respectively, at positions 986 and 987 in a row. An insert containing a gene encoding the spike protein of the beta mutant strain of SARS-coronavirus-2, which is replaced by a proline substitution mutation, is provided.

In one embodiment of the present invention, amino acid residues from positions 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, Containing 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 strain of SARS-coronavirus-2. An insert is provided.

In one embodiment of the present invention, 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 protein of SARS-coronavirus-2 Amino acid residues 247 to 364 of the capsid protein are preferably composed of the amino acid sequence of SEQ ID NO: 4, but any mutant sequence that achieves the purpose of the present invention through one or more mutations such as substitution, deletion, or inversion in the sequence can also be used. included in the scope of the invention.

In another embodiment of the present invention, the gene encoding amino acid residues from positions 44 to 180 of the nucleocapsid protein of SARS-coronavirus-2 linked to the N terminus of the spike protein is the base sequence shown in SEQ ID NO: 5. It is preferable 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 base sequence shown in SEQ ID NO: 6. All mutant sequences that achieve the purpose of the present invention through mutations such as one or more substitutions, deletions, and inversions in the sequence are also included within the scope of the present invention.

In another embodiment of the present invention, 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 the modified spike protein of the beta mutant strain of SARS-coronavirus-2 The gene encoding the spike protein is preferably composed of the base sequence shown in SEQ ID NO: 2, but all mutant sequences that achieve the purpose of the present invention through mutations such as one or more substitutions, deletions, and inversions in the sequence are also within the scope of the present invention. included in

Additionally, the present invention provides a recombinant expression vector containing 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 lentivirus vector, a retrovirus vector, and a herpesvirus vector. It is preferable that the vector is selected from, and in one embodiment, it is preferably a chimeric adenovirus vector, but is not limited thereto.

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

In another embodiment of the present invention, the adenovirus vector replaces the protruding domain at the end of the fiber protein of human adenovirus type 5 with the protruding 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 protruding domain of chimpanzee adenovirus serotype 6, but is not limited thereto.

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

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

In one embodiment of the present invention, the vaccine is preferably effective against 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 containing the S gene derived from the beta mutation of the present invention or a part of the N gene linked to the S gene.

Additionally, the present invention provides a method for producing a recombinant virus using the transformed cells of the present invention.

Additionally, the present invention provides a method of using the two types of recombinant chimeric adenoviruses obtained by the present invention as vaccines in animal models.

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 against the immune response of specific T cells according to the recombinant chimeric adenovirus produced by the above method.

The present invention provides initial protective efficacy 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 SARS-CoV-2 infection, 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, and is preferably a vaccine that can alleviate or prevent severe infection not only from the initial strain but also from beta, delta, and omicron mutant strains, but is not limited to this.

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, a vaccine composition linked to the N protein to maximize the T cell immune response. The present invention was completed by evaluating the cellular immunogenicity and protective ability against viral infection of a neutralizing antibody against the S protein.

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

In order to inhibit the structural change of the S protein to its post-fusion form, substitution of glycine at position 614, acidic amino acid at residues 682 to 685, and consecutive proline substitution mutations at positions 986 and 987 were introduced as of 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 adenovirus vector encoding a protein that is a fusion of the S protein and the N protein, 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, the structurally unstable region and hyperphosphorylation domain were excluded, and the NTD region containing the T cell epitope and the CTD, which has a T cell epitope and has structural stability through dimer formation, were used. It was connected to the S protein through 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 (44-180) and CTD(247-364) was linked to both ends of S protein through (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 bonds at the amino terminus (N terminus, NTD) and carboxy terminus (C terminus, CTD). means.

In the present invention, a chimeric protein or polypeptide (NP(NTD) containing the S polypeptide of a beta mutant strain between the amino-terminal N polypeptide (Nos. 44 to 180) and the carboxy-terminal N polypeptide (Nos. 247 to 364) is used. )-Spike-NP(CTD)).

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

The chimeric adenovirus vector of the present invention is a patent application (Korean patent application number: 10-2022-0034885), and detailed descriptions refer to the relevant patent and are omitted in this specification.

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.

The cells transformed 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 production cell suitable for the present invention must be a cell capable of producing a virus by trans-complementation of the E1 and E3 gene defects of a replication defective adenovirus, and the virus early transcription gene. Many transformed cells are being developed. Cells into which E1 and E3 genes are preferably introduced 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 is not limited thereto.

In the present invention, when producing ChimAd-CV and ChimAd-CVN viruses, it is preferable, but not limited to, to infect host cells with a cell density of 1x 10 ⁶ cells/ml at a titer of 2 MOI and recover the cells 48 hours later.

To elute the virus from the recovered cells, treatment with 0.5% Tween20 and 20 U/ml benzonase for 4 hours to induce host cell DNA degradation is preferred, but is not limited to this.

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

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 initial strains, beta mutant strains, and delta mutant strains.

ChimAd-CV and ChimAd-CVN vaccine compositions produced according to embodiments of the present invention provide excellent neutralizing antibody titers against beta mutant-based pseudoviruses.

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

The ELISPOT (Enzyme-Linked ImmunoSpot, ELISPOT) experimental technique was used to confirm the immune response of specific T cells of ChimAd-CV and ChimAd-CVN produced according to an embodiment of the present invention. Mouse spleen cells immunized with ChimAd-CV or ChimAd-CVN were isolated and the immune response to S protein or NP peptide was confirmed. As a result, as ChimAd-CVN was confirmed to have a cellular immunity capacity that is more than twice that of ChimAd-CV, it is believed that higher T-cell immunity is induced by linking the N protein. [When referring to the literature, adenovirus vector. The results of the T-cell immune response of the COVID-19 vaccine using a delivery vehicle or DNA were confirmed to be similar to the results of the prime 4th week of ChimAd-CV and ChimAd-CVN (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.).

It is known that a high initial T-cell immune response in itself plays a role in alleviating the severity of coronavirus, and some say that anti-N protein antibodies formed by N protein are not related to direct virus neutralization, but N protein-specific T -The increase in cellular immunity is correlated with the titer of neutralizing antibodies. [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 initial strains.

Body weight change and survival rate after immunization and viral infection were confirmed with ChimAd-CV and ChimAd-CVN produced according to an embodiment of the present invention. Both experimental groups inoculated with ChimAd-CV and ChimAd-CVN showed a 100% survival rate against initial infection, confirming excellent protective 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 to N based on a chimeric adenovirus vector was developed. It has excellent cross-immunogenicity due to high neutralizing antibody titers against initial strains and mutant strains (beta, delta, and omicron), and the vaccine containing the N sequence provided efficacy that maximized T-cell immunity. In addition, it is believed to be applicable as a preventive vaccine for SARS-CoV-2 as it has excellent protective ability against early strains.

Figure 1 is a schematic diagram of cloning ChimAd-CV and ChimAd-CVN using the Gibson assembly gene synthesis method from a coronavirus gene obtained through gene synthesis into a chimeric adenovirus (ChimAd) vector.
Figure 2 shows the virus-binding antibody titers induced by ChimAd-CV and ChimAd-CVN using BALB/c female mice, with the results for the initial strain S protein shown in A and the results for the beta mutant S protein shown in B.
Figure 3 shows the results of neutralizing antibody titers induced by ChimAd-CV and ChimAd-CVN using BALB/c female mice using a beta mutant (B.1.351 variant type) pseudovirus. AB. SARS-Coronavirus-2 (Wuhan-Hu-1) pseudovirus neutralizing antibody test, CD. Beta mutant strain (B.1.351) pseudovirus neutralizing antibody test.
Figure 4 shows the results of cellular immunization using the S peptides of ChimAd-CV and ChimAd-CVN as stimulating antigens using BALB/c female mice (A, B) and the results of cellular immunization using NP peptides as stimulating antigens (C, D).
Figure 5 shows the virus-binding antibody titers induced by ChimAd-CV and ChimAd-CVN using hACE2 transgenic female mice, with the results for the S protein of the initial strain as A and the results for the S protein of the delta mutant strain as B.
Figure 6 shows the results of virus neutralizing antibody titers induced by ChimAd-CV and ChimAd-CVN using hACE2 transgenic female mice, and shows the neutralizing antibody measurement results (A, B) using the initial FRNT. In addition, the neutralizing antibody measurement results using FRNT of the delta mutant strain are shown (C, D). AB. SARS-Coronavirus-2 (Wuhan-Hu-1) FRNT neutralizing antibody test, CD. Delta mutant strain (B.1.617.2) FRNT neutralizing antibody test.
Figure 7 shows the body weight comparison results and mortality rate of ChimAd-CV and ChimAd-CVN for initial infection in a mouse animal model immunized with ChimAd-CV and ChimAd-CVN using hACE2 transgenic female mice.
Figure 8 shows the coronavirus titer detected in the lungs after challenge with SARS-coronavirus-2 (Wuhan-Hu-1).
Figure 9 shows the binding antibody titer induced according to the inoculation dose of ChimAd-CV using BALB/c female mice against 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 for various mutant viruses (Wuhan, beta mutant, delta, omicron (BA.1)) VLP (Virus Like particle) ) are shown in the measurement results (A~D).
In Figure 10, A is the SARS-coronavirus-2 (Wuhan-Hu-1) VLP neutralizing antibody test, B is the beta mutant (B.1.351) VLP neutralizing antibody test, and C is the delta mutant (B.1.617.2) VLP neutralizing antibody test. Antibody test, D is Omicron (BA.1) VLP neutralizing antibody test.
Figure 11 shows the results of cellular immunity induced by an inoculation dose of ChimAd-CV using BALB/c female mice, using S peptide as a stimulating antigen.

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

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

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

Specifically, in the case of the CV gene, substitution of glycine at position 614, acidic amino acid at residues 682 to 685, and consecutive proline substitution mutations at positions 986 and 987 were introduced based on the initial week of the beta mutant S protein. [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 of the Spike protein, respectively, through (GGGGS) ₃ linker, and at this time, they were linked through prediction tools such as trRosetta and Alphafold. Additional structural predictions were performed. [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 the spike protein and Nucleocapsid protein among the structural proteins were secured. For each vaccine candidate, as shown in Table 1, the S protein derived from coronavirus B.1.351 and the N protein derived from Wuhan-Hu-1 were selected and codon optimized to facilitate expression in adenovirus. The region containing the S protein is named CV, and the chimeric form that fuses part of the N protein and the S protein is named CVN.

Example 3: Cloning of chimeric adenovirus containing SARS-CoV-2 genes

In order to clone the two SARS-CoV-2 vaccine antigens synthesized above into the ChimAd vector, a chimeric adenovirus vector, the plasmid DNA of the ChimAd vector was used as a template and PCR amplified excluding part of the gene in the GOI binding region. The product (approximately 33.4 Kb) was purified through gel extraction. The insertion gene for cloning is an amplification product CV (approximately 3.8 bp) obtained using the primers in Table 2 below to PCR amplify the same sequence (homologous region, approximately 20bp) as the 5' and 3' ends of the GOI binding region in the ChimAd vector. After securing Kb) and CVN (approximately 4.7 Kb), they were combined with the previously DNA-purified vector PCR amplification product through Gibson assembly reaction, respectively, to finally obtain two types of plasmids, ChimAd-CV and ChimAd-CVN (Figure 1).

Example 4: Production and purification of chimeric adenovirus-based coronavirus vaccine candidate

In order to produce chimeric adenovirus-based coronavirus vaccine candidates using the ChimAd-CV and ChimAd-CVN plasmids produced in the present invention, each plasmid was treated with PacⅠ restriction enzyme (NEB, R0547L) to produce pBR322, which is unnecessary for adenovirus formation. The replication origin and kanamycin resistance gene sequence were cut and removed, and HEK293 cells were transformed using Lipofectamine 2000 (Invitrogen, 11668027) and incubated for 10-20 days in an incubator maintained at a temperature of 37°C and a CO ₂ partial pressure of 5.0%. After culturing, primary virus stock was obtained.

For ChimAd-CV and ChimAd-CVN virus production, HEK293 cells with a cell density of approximately ^1x106 cells/ml were infected at a titer of 2 MOI using the primary virus stock obtained above, and after 48 hours, the virus was filtered through a 0.45 ㎛ CFF microfiltration filter (Cytiva, Cells were concentrated using CFP-4-E-6A). 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. Afterwards, 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 analyzed for ChimAd-CV and ChimAd-CVN through 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). The virus was separated and finally formulated into a vaccine buffer, and finally filtered through a 0.2 ㎛ ULTA GF filter (Cytiva, KGF-A-9610TT) to obtain two vaccine candidates.

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

5-1) Immune induction using BALB/c female

Mice were immunized using the ChimAd-CV and ChimAd-CVN vaccine candidates obtained by the above manufacturing method. The test group includes a total of 4 groups: buffer, ChimAd used as a mock-up, ChimAd-CV, and ChimAd-CVN. For the immune 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. At 4-week intervals, the first and second vaccinations were administered by intramuscular injection twice (days 0 and 28). Blood was collected 4 and 6 weeks after the first vaccination (days 27 and 42), and serum was obtained. It was separated and the spleen was removed.

5-2) ELISA; Binding antibodies of mouse anti-serum by ChimAd-CV and ChimAd-CVN

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

As a result of the binding antibody titer, it was confirmed that both the initial strain and the beta mutant strain 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, The binding antibody titers for both the initial strain and the beta mutant strain were measured, but were found to be invalid values (Figure 2).

5-3) pVNT (pseudovirus neutralization test)

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

Neutralizing antibodies in serum were tested through a mouse immune induction test. The pseudovirus system is a virus that uses the backbone of a lentivirus to express a light-emitting gene and the Spike protein of an early or beta mutant strain. In this experiment, Takara's Lenti-X™SARS-CoV-2 Packaging Mix product was used to produce pseudoviruses containing the Spike protein of the initial strain and beta mutant strain. The produced pseudovirus was transformed into 293T cells overexpressing the ACE2 protein, luciferase expression was quantified and measured, and a neutralizing antibody test was performed using a certain 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. The serum was diluted by 1/8 and then serially diluted 2-fold to about 10 ³ , and pseudoviruses with a certain RLU value were mixed in equal amounts with the diluted serum and incubated at 37°C and 5% CO ₂ A neutralization reaction was performed in an incubator for 1 hour. hACE2-293T cells cultured a day earlier were treated with 100ul of the virus and serum mixture and cultured in an incubator at 37°C and 5% CO ₂ for about 72 hours. After completion of incubation, about 100ul of the culture medium was removed, 30ul of One-Glo luciferase reagent (Promega) was added, and reacted for 3-5 minutes. Then, 60ul of the mixture was transferred to a white plate and the luminescence value was measured using a luminometer. The measured RLU value was used to calculate the IC50 value according to the serum dilution factor using a statistical program.

As shown in Table 3, the results of neutralizing antibodies against the beta mutant virus showed that the ChimAd-CV vaccinated group had IC ₅₀ = 1253, and the ChimAd-CVN vaccinated group had IC ₅₀ = 1131, which were all higher than 10 ³ , confirming higher neutralizing antibody titers compared to the negative control group. .

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

In the mouse immunity induction test, spleens were removed from each group 4 weeks after the first vaccination and 2 weeks after the second vaccination (6 weeks after the first vaccination). Spleen cells were recovered using Cell strainer (Falcon) and washed with PBS buffer solution. After washing, red blood cells were removed using red blood cell lysis buffer (Sigma), and the number of spleen cells was counted and diluted to the cell concentration used in the test. This experiment used R&D system's mouse IFN-gamma ELISpot kit, and the test was performed according to the manufacturer's instructions. For each test, 1X10 ⁵ cells, 2X10 ⁴ cells of splenocytes and Genscript's initial Spike protein peptide pool or Nucleprotein peptide pool were used as stimulating antigens and incubated at 37°C in a 5% CO2 incubator for 16 hours. After incubation, the cells were washed with a washing solution, and 100 μl of biotin-conjugated IFN-gamma detection antibody was added and reacted by stirring at room temperature for 2 hours. After the reaction, the cells were washed four times using a washing solution, and 100 ul of streptavidin-AKP (alkaline phosphate) was added and incubated at room temperature for 2 hours. After the final washing, 100 ul of BCIP/NBT substrate was added and reacted at room temperature for about 45 minutes. When color development appears due to the reaction, it is washed with sterilized water, the number of colored spots is counted, and spot forming is performed. The number of cells per unit (SFU) was converted.

As a result of measuring the induction of specific T-cell immunity to each stimulating 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 a specific immune response to the Spike protein peptide was induced in ChimAd-CVN, which was about two times higher than that in ChimAd-CV. Additionally, it was confirmed that ChimAd-CVN induces a specific immune response to 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, neutralizing antibody titer and challenge test after animal immunization

6-1) Immune induction and challenge test using hACE2 transgenic female mouse

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

6-2) ELISA; Measurement of antibody titers of mouse anti-serum by ChimAd-CV, ChimAd-CVN

After the mouse immune induction test, it was performed to measure the antibody titer in serum. Initial strain S protein and delta mutant strain S protein were dispensed into a 96-well plate at a concentration of 2ug/ml, 100ng/well each, and coated at 4 degrees for more than 16 hours. After the reaction, 100 ul of PBS containing 1% BSA was added and reacted. Each serum was diluted 1:100, then serially diluted 5-fold and reacted at 4 degrees for more than 16 hours. HRP-conjugated goat anti-mouse IgG HRP (Southern Biotech) was diluted 1:3000 and reacted at 37°C for 1 hour. After the reaction was completed, the TMB solution (Millipore) was dispensed to proceed with the color development. Afterwards, the reaction was stopped by adding an equal amount of 0.5 N HCl solution (Merck), and the 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 high binding antibody titers of 10 ⁵ or more against the initial strain and the delta mutant strain (FIG. 5).

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

Measurement of neutralizing antibody titers against coronavirus of ChimAd-CV, ChimAd-CVN mouse anti-serum

After the mouse immune induction test, a test was performed to measure neutralizing antibodies in the serum. For the neutralizing antibody test, each mouse serum was first 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 initial strain coronavirus (NCCP #43326 (BetaCoV/Korea/KCDC03/2020) and 4.0x10 ² PFU/25ul of delta strain coronavirus (NCCP #43390 (hCoV-19/Korea119861) /KDCA/2021) and reacted for 30 minutes at 37°C. After reaction, 50ul of virus and serum mixture was treated with Vero cells (1.5x10 ⁴ cells/well) cultured one day before and incubated at 37°C, 5% CO ₂ Cultured in an incubator for 4 hours. After 4 hours of reaction, the supernatant was removed, washed with PBS buffer solution, and treated with 300ul of 4% formaldehyde solution (Sigma) and fixed in a place without light for more than 16 hours. After reaction, After washing with PBS, 100% cold methanol was added and reacted for 10 minutes, followed by reaction at room temperature for 1 hour using blocking solution, followed by anti-SARS-CoV diluted 1:3000. -2 NP rabbit monoclonal antibody (Sino Biological) was treated and incubated at 37 degrees for 1 hour. After incubation, the cells were washed with a PBS solution (PBS-T) containing 0.1% Tween20, and then HRP-conjugated diluted at 1:2000. 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 at room temperature for 30 minutes in the absence of light, and the reaction was stopped with water. Then, it was dried. 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 the serum. The FRNT method involves attaching a virus-derived monoclonal antibody to a virus-infected cell. This is a method of measuring neutralizing antibodies by attaching a secondary antibody labeled with HRP (horedish peroxidase) and developing it as a substrate to identify cells infected with the virus.

As a result of the neutralizing antibody against the initial strain virus, the neutralizing antibody titer can be confirmed as shown in Table 5, which confirms that both the ChimAd-CV and ChimAd-CVN groups showed a neutralizing antibody titer more than 100 times higher after the first vaccination compared to the negative control group. did. After the second vaccination, neutralizing antibody titers were confirmed to be more than 30 times higher in the ChimAd-CV vaccinated group and more than 50 times higher in the ChimAd-CVN vaccinated group.

In addition, as a result of neutralizing antibodies against the delta mutant virus, it was confirmed that after the second vaccination, both the ChimAd-CV vaccinated group and the ChimAd-CVN vaccinated group showed neutralizing antibody titers that were more than 40 times higher than the negative control group.

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 ChimAd-CV, ChimAd-CVN immunization

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

As a result of confirming the vaccine protective ability of ChimAd-CV and ChimAd-CVN against attack by the initial strain (Wuhan-Hu-1), both survival rates were found to be 100%, and the body weight of the ChimAd-CVN vaccinated group was maintained without loss. It was confirmed that in the ChimAd-CV vaccinated group, all animals except one maintained their weight without loss, and that there were individuals whose body weight decreased by more than 10% and then recovered.

6-5) Lung virus titer for ChimAd-CV, ChimAd-CVN immune induction and challenge test (Plaque assay)

After the mouse immune induction and challenge test, a plaque assay test was performed to measure the remaining virus titer in the lungs. To measure titer, Vero cells (ATCC, Cat#CCL-81) were used and cultured in an incubator at 37°C and 5% CO ₂ for more than 16 hours. The virus isolated from the lungs collected from all groups was stepwise diluted 4-fold up to 10 ⁶ , and 200 ul of the diluted virus was added to the cultured Vero cells and reacted at room temperature for 30 minutes. After 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 in an incubator at 37°C and 5% CO ₂ for 72 hours. 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, each dilution factor and the 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 in the ChimAd-CV vaccinated group decreased by more than 20,000 times 2 days after the attack compared to the control group for the initial attack, and 4 days after the attack It was confirmed that the virus was cleared and not measured after 7 days. It was confirmed that the ChimAd-CVN vaccinated group achieved virus clearance and that the lung virus titer was not measured after 2 days, 4 days, and 7 days.

Example 7: Result of immunogenicity test through binding antibody/neutralizing antibody/cellular immunity analysis according to DRF (Dose range finding) test of ChimAd-CV

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

Mouse immunization was performed in a dose-response manner using the ChimAd-CV vaccine candidate. For the immune induction test, 5-week-old BALB/c female mice were used, and the doses of ChimAd-CV vaccine candidate were divided into 4 groups as shown in Table 6, and consisted of stabilizer (Vehicle), Mock up, and Non-immunization groups. Considering the previously approved vaccination concentration, the high and low concentrations were set at 2-fold from 5.0.E+09 VP/Dose (1/10 compared to human body based on mouse). At 4-week intervals, the first and second vaccinations were administered by intramuscular injection twice (days 0 and 28). Blood was collected 4 and 6 weeks after the first vaccination (days 27 and 42), and serum was obtained. It was separated and the spleen was removed.

The separated serum was measured for binding antibody titers and neutralizing antibody titers against various coronaviruses (Wuhan, beta variant, delta, and omicron (BA.1)), and T-cell immune responses were analyzed using the extracted spleen.

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; Binding antibodies of mouse anti-serum according to dose response of ChimAd-CV vaccine candidate

A binding antibody test was performed to confirm the presence of antibodies in the serum through a dose-response mouse immune induction test of the ChimAd-CV vaccine candidate. To perform the test, coronavirus SARS-coronavirus-2 and beta mutant S protein were dispensed into microplates at a concentration of 1ug/ml, 100ng/well each, and coated at 4 degrees for more than 16 hours. After reaction, the plate was washed at least 4 times with 1 After washing with PBS-T, each serum was diluted 1:100 and then serially diluted 3 times to about 1x10 ⁶ . After dispensing and reacting at 37°C for 1 hour, the mixture was washed with PBS-T, and HRP-conjugated Goat anti-mouse IgG HRP (Thermo) was diluted 1:5000 and reacted for 1 hour. After the reaction was completed and to proceed with color development, 50ul of TMB solution (Thermo) was dispensed and color development was performed for 15-20 minutes. Afterwards, the reaction was stopped by adding an equal amount of Stop solution (Thermo), and the absorbance was measured at a wavelength of 450nm. did.

As a result of the binding antibody titer, it was confirmed that regardless of the inoculation dose, all experimental groups showed high binding antibody titers of 10 ⁴ or more to SARS-coronavirus-2 and beta mutant strains compared to the control group (FIG. 9).

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

Neutralizing antibodies in serum were tested through a mouse immune induction test. This experiment was conducted using Virongy's Rapid Cell-Based SARS-CoV-2 Neutralizing Antibody Assay Kit to measure Wuhan virus, beta mutated 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 four structural proteins (S, M, N, E) of SARS-CoV-2 and luciferase. can be expressed.

For 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 . The diluted sample and 45ul of HA-CoV2(Luc)VLP were mixed on a white plate (cell culture) and neutralized for 30 minutes in an incubator at 37℃5% CO ₂ .

HEK293T (ACE2/TMPRSS2) cells were _prepared at ^2.5 After incubation, add 7.5 ul of Cell lysis buffer and leave on 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 value was used to calculate the IC50 value according to the serum dilution factor using a statistical program.

As shown in Table 7, neutralizing antibodies against coronavirus were confirmed for each dose after boosting (week 6). 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. The highest neutralizing antibodies were confirmed in CV1. As a result of confirming neutralizing antibodies against betavirus, CV1 had IC50=965, CV2 had IC50=416, CV3 had IC50=627, and CV4 had IC50=304, showing that CV1 had the highest neutralizing antibody titer. The dose dependent neutralizing antibody titer was confirmed.

As a result of confirmation of neutralizing antibodies against delta virus, IC50 = 517 for CV1, IC50 = 139 for CV2, IC50 = 798 for CV3, and IC50 = 115 for CV4, dose dependent neutralizing antibodies against delta virus excluding CV3. was confirmed. As a result of confirming neutralizing antibodies against omicron viruses, IC50 = 76 for CV1, IC50 = 117 for CV2, IC50 = 110 for CV3, and IC50 = 93 for CV4, and more than 100 omicron viruses were detected in CV2 and CV3. The neutralizing antibody titer was confirmed (Figure 10).

Target virus pVNT(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 caused by ChimAd-CV adenovirus

In the mouse immunity induction test, spleens were removed from each group 4 weeks after the first vaccination and 2 weeks after the second vaccination (6 weeks after the first vaccination). Spleen cells were recovered using Cell strainer (Falcon) and washed with PBS buffer solution. After washing, red blood cells were removed using red blood cell lysis buffer (Sigma), and the number of spleen cells was counted and diluted to the cell concentration used in the test. This experiment used R&D system's mouse IFN-gamma ELISpot kit, and the test was performed according to the manufacturer's instructions. For each test, 1X10 ⁵ cells, 2X10 ⁴ cells of splenocytes and the peptide pool of Genscript's SARS-coronavirus-2 Spike protein were used as stimulating antigens and incubated for 16 hours in a 37°C 5% CO2 incubator. After incubation, the cells were washed with a washing solution, and 100 μl of biotin-conjugated IFN-gamma detection antibody was added and reacted by stirring at room temperature for 2 hours. After the reaction, the cells were washed four times using a washing solution, and 100 ul of streptavidin-AKP (alkaline phosphate) was added and incubated at room temperature for 2 hours. After the final washing, 100 ul of BCIP/NBT substrate was added and reacted at room temperature for about 45 minutes. When color development appears due to the reaction, it is washed with sterilized water, the number of colored spots is counted, and spot forming is performed. The number of cells per unit (SFU) was converted.

As shown in Table 8, as a result of measuring the induction of specific T-cell immunity to S protein-stimulated antigens through the ELISpot test, it was confirmed that ChimAd-CV induced a higher T-cell immune response at the 6th week of vaccination compared to the 4th week for each vaccination dose. . 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]

AG GGC GTG ACC GTG GTG GTG GTG GTG GTG GCC GCC GCC GCC GCC GTG CTG CTG ACC ACC ACC AGA GTG AGA GTG ACC TCC ACC GTG ACC ACC ACC ACC AGA GGT GGT CTG CTG ATC GCC GA G CAC GTG AAC AAC TCC TAC GAG TGC GAC　ATC　CCT　ATC　GGC　GCC　GGC　ATC　TGC　GCC　AGC　TAC　CAG　ACC　CAG　ACC　AAT　AGC　CCC　cag cag gcc cag

The sequence of the fusion polypeptide insert of the invention is described below.

MGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGS (SEQ ID NO: 3)

GGGGSGGGGSGGGGS ↓ GGGGSGGGGSGGGGS

TKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFP* (SEQ ID NO: 4)

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

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

PDPSPNCQLLSDRDAKFTLCLTKCGSQILGTVAVAAVTVGSALNPINDTVKSAIVFLRFDSDGVLMSNSSMVGDYWNFREGQTTQSVAYTNAVGFMPNIGAYPKTQSKTPKNSIVSQVYLTGETTMPMTLTITFNGTDEKDTTPVSTYSMTFTWQWTGDYKDKNITFATNSFSFSYIAQE

Claims (12)

It contains amino acids 1 to 1270 of the spike protein of the beta mutant strain of SARS-coronavirus-2, where it has mutations in amino acid residues 614, 682 to 685, 986, and 987 based on the initial strain; Here, in the protein, the aspartic acid amino acid of protein 614 of the spike protein is replaced with glycine, the arginine amino acid at 682 is replaced with glutamine, the alanine amino acid at 683 is glutamine, the arginine amino acids at 684 and 685 are replaced with alanine and glutamine, respectively, and 986 and 987 consecutive An insert containing the gene encoding the spike protein of the beta mutant strain of SARS-coronavirus-2, which is replaced by a proline substitution mutation. The method of claim 1, wherein the insert connects amino acid residues from positions 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 from positions 247 to 364 of the nucleocapsid protein of SARS-coronavirus-2 to the C terminus of the spike protein of the beta mutant strain of SARS-coronavirus-2. Insert containing. 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 247 to 364 of the protein are inserts consisting of the amino acid sequence of SEQ ID NO: 4. The method of claim 2, wherein the gene encoding amino acid residues from positions 44 to 180 of the nucleocapsid protein of SARS-coronavirus-2 linked to the N terminus of the spike protein consists of the base sequence shown in SEQ ID NO: 5, , an 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 base sequence shown in SEQ ID NO: 6. 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 is characterized in that the gene consists of the base sequence shown in SEQ ID NO: 2. A recombinant expression vector comprising the insert of any one of claims 1 to 5. The recombinant expression vector according to claim 6, wherein the expression vector is a viral vector or a non-viral vector. 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, lentivirus vectors, retrovirus vectors, and herpesvirus vectors. The method of claim 8, wherein the adenovirus vector replaces the protruding domain at the end of the fiber protein of human adenovirus type 5 with the protruding gene of chimpanzee adenovirus serotype 6, and the tail and saft (Tail) of human adenovirus type 5 ( A recombinant expression vector containing a fibrous protein combined with a Shaft domain and a protruding domain of chimpanzee adenovirus serotype 6. The recombinant expression vector according to claim 9, wherein the protein expressed by the protrusion gene of chimpanzee adenovirus serotype 6 includes the amino acid sequence of SEQ ID NO: 7. A vaccine composition for preventing or treating SARS-CoV-2 infection, comprising the recombinant expression vector of any one of claims 6 to 10 as an active ingredient. The method of claim 11, wherein the vaccine is effective against one or more viruses selected from the group consisting of SARS-CoV-2 virus initial strain, delta virus, beta and omicron virus mutant strains (SARS-Coronavirus-2 ( Vaccine composition for preventing or treating SARS-CoV-2) infection.