RECOMBINANT CHIMERIC ADENOVIRAL VECTOR SUBSTITUTED BY KNOB GENE OF CHIMPANZEE ADENOVIRUS SEROTYPE 6, AND APPLICATION THEREOF

Abstract

The present invention is a chimeric adenovirus vector in which the knob domain of the end of the fiber protein of human adenovirus type 5 is replaced with the knob gene of chimpanzee adenovirus serotype 6 and/or in addition the hexon protein of human adenovirus type 5 is replaced with hypervariable regions 1-7 of human adenovirus serotype 28. The present invention not only provides the optimal adenovirus vector in the development of treatments or vaccines for various diseases, but also when the chimeric adenovirus vector produced in the present invention is infected with a host cell for production, it can contribute to improved productivity by exhibiting superior cell infection ability compared to the recombinant HAdV-5 vector-based vaccine.

Claims

1. A chimeric adenovirus vector in which the knob domain of the end of the fiber protein of human adenovirus type 5 has been replaced with the knob gene of chimpanzee adenovirus serotype 6.

2. The chimeric adenovirus vector according to claim 1, wherein the hexon protein of human adenovirus type 5 is further replaced with hypervariable regions 1-7 of human adenovirus serotype 28.

3. The chimeric adenovirus vector according to claim 1, wherein the protein expressed by the knob gene of chimpanzee adenovirus serotype 6 comprises the amino acid sequence of SEQ ID NO: 1.

4. The chimeric adenovirus vector according to claim 1, wherein the knob gene sequence of chimpanzee adenovirus serotype 6 comprises the nucleic acid sequence of SEQ ID NO: 2.

5. The chimeric adenovirus vector according to claim 1, wherein the vector further comprises a fiber protein combined with the tail and shaft domains of human adenovirus type 5 and the knob domain of chimpanzee adenovirus serotype 6.

6. The chimeric adenovirus vector according to claim 2, wherein the chimeric protein in which the hexon protein of human adenovirus type 5 is replaced with the hypervariable region 1-7 of human adenovirus serotype 28 comprises the amino acid sequence of SEQ ID NO: 3.

7. The chimeric adenovirus vector according to claim 2, wherein the gene encoding a chimeric protein in which the hexon protein of human adenovirus type 5 is replaced with hypervariable regions 1-7 of human adenovirus serotype 28 comprises the nucleic acid sequence of SEQ ID NO: 4.

8. The chimeric adenovirus vector according to claim 1, wherein the vector is shown in FIG. 6 or FIG. 8.

9. The chimeric adenovirus vector of claim 1, wherein the vector comprises as an insert a) a gene encoding the spike protein of the coronavirus, or b) a gene encoding the E6 and E7 proteins of human papillomavirus type 16 or 18.

10. A host cell transformed with the chimeric adenovirus vector of claim 1.

11. The host cell according to claim 10, wherein the cell is HEK293, PERC6 or 911 cell.

12. An isolated polypeptide encoded by the chimeric adenovirus vector of claim 1.

13-15. (canceled)

Description

DESCRIPTION OF DRAWINGS

[0088] FIG. 1 shows the results of sequence analysis of the amino acid sequences of the fiber proteins of HAdV-5 and ChAdV-6 in the present invention through Clustal Omega and ESPript programs (A) and a schematic diagram of the fiber proteins of the chimeric adenovirus vector (B),

[0089] FIG. 2 shows the results of sequence analysis of the amino acid sequences of the hexon proteins of HAdV-5 and HAdV-28 in the present invention through Clustal Omega and ESPript programs (A) and a schematic diagram of the hexon proteins of the chimeric adenovirus vector (B),

[0090] FIG. 3 schematically shows the sequence of the vector regulator synthetic gene produced in the present invention,

[0091] FIG. 4 is a schematic diagram showing the HAdV-5 vector construction process in the present invention,

[0092] FIGS. 5 and 6 are schematic diagrams of the ChimAd vector production process (FIG. 5) and the final ChimAd vector structure (FIG. 6) produced in the present invention.

[0093] FIGS. 7 and 8 are schematic diagrams showing the production process of the ChimAd-H28 vector (FIG. 7) and the final structure of the ChimAd-H28 vector (FIG. 8) produced in the present invention,

[0094] FIG. 9 shows the result of confirming the chimeric capsid gene in the two chimeric adenovirus vectors (ChimAd, ChimAd-H28) prepared in the present invention by amplifying the knob gene (0.5 Kb) of ChAdV-6 and the hexon HVR gene of HAdV-28 (1 Kb) by PCR, separating them on a 0.8% agarose gel, and passing ultraviolet rays through them,

[0095] FIG. 10 is a photograph at 200 magnification using an optical microscope to show the formation of cytopathic effect (CPE) after induction of transformation in HEK293 cells with the HAdV-5 vector and two types of chimeric adenovirus vectors prepared in the present invention,

[0096] FIG. 11 shows the expression of hexon (about 108 kDa), fiber (about 61 kDa), and penton (about 68 kDa) proteins through Western blot after HAdV-5 and two kinds of chimeric adenovirus were produced in HEK293 cells,

[0097] FIG. 12 is a graph showing the number of positive cells infected with adenovirus taken at 200-fold magnification of an optical microscope, counting, and converting to virus titer through immunocytochemistry using an anti-hexon polyclonal antibody 48 hours after infecting HEK293 cells with 0, 5, 10, 25, or 50 MOI virus doses of HAdV-5 and chimeric adenoviruses prepared in the present invention. The X-axis represents the dose of virus infected cells, and the Y-axis represents the HAdV-5, ChimAd, and ChimAd-H28 virus titers according to each virus dose. * indicates the significance level between the HAdV-5 virus and the chimeric adenovirus verified using a paired t-test (* p<0.05, ** p<0.01),

[0098] FIG. 13 shows the in vitro neutralizing antibody test in HEK293 cells by reacting HAdV-5 and chimeric adenoviruses prepared in the present invention with anti-HAdV-5 polyclonal antibodies, and then measuring the number of adenovirus-infected cells and the measured values are plotted graphically. The X axis represents the dilution factor of the anti-HAdV-5 polyclonal antibody serially diluted from 10.sup.1 to 10.sup.4, and the Y axis represents the number of cells infected by HAdV-5, ChimAd and ChimAd-H28 viruses. * indicates the level of significance between the HAdV-5 virus and the chimeric adenovirus verified using a paired t-test (* p<0.05, ** p<0.01, *** p<0.005, **** p<0.0005),

[0099] FIG. 14 shows the result of in vitro neutralizing antibody test in HEK293 cells by reacting HAdV-5 and chimeric adenoviruses prepared in the present invention with healthy human serum (n=7) and the values expressed as reciprocal numbers by measuring the number of cells infected with adenovirus shown as a dot plot. The X-axis represents the adenovirus reacted with the serum, and the Y-axis represents the adenovirus neutralizing antibody (Ad Nab) titer for 7 human serum samples. The significance level between the HAdV-5 virus, the ChimAd virus, and the ChimAd-H28 virus, verified using a paired t-test, is shown as a P value,

[0100] FIG. 15 is a schematic diagram of cloning ChimAd-CV and ChimAd-CVN using the Gibson assembly gene synthesis method in a ChimAd vector obtained through gene synthesis,

[0101] FIG. 16 shows the SARS-Coronavirus-2 S protein results (A) and beta variant S protein results (B) for virus-binding antibody titers induced by ChimAd-CV and ChimAd-CVN using BALB/c female mice,

[0102] FIG. 17 shows the neutralizing antibody titers induced by ChimAd-CV and ChimAd-CVN in BALB/c female mice as neutralizing antibody measurement results (A, B) using a beta variant (B.1.351 variant type) pseudovirus.

[0103] FIG. 18 shows the results of cellular immunization (A, B) using S peptides of ChimAd-CV and ChimAd-CVN as stimulating antigens and the results of cellular immunization (C, D) using NP peptides as stimulating antigens, using BALB/c female mice,

[0104] FIG. 19 shows the results of S protein of SARS-coronavirus-2 (A) and the S protein of delta variant strain (B) induced by ChimAd-CV and ChimAd-CVN using hACE2 transgenic female mice,

[0105] FIG. 20 shows the results of neutralizing antibody measurement using FRNT of SARS-coronavirus-2 (A, B) and of the delta variant strain (C,D) as a result of confirming the viral neutralizing antibody titer induced by ChimAd-CV and ChimAd-CVN using hACE2 transgenic female mice,

[0106] FIG. 21 shows survival rate (A, B) and the average body weight (C,D) after SARS-coronavirus-2 (Wuhan-Hu-1), delta variant (B.1.617.2) challenge test in mouse animal models immunized by ChimAd-CV and ChimAd-CVN using hACE2 transgenic female mice,

[0107] FIG. 22 shows coronavirus titers detected in the lungs after the challenge test,

[0108] FIG. 23 is a schematic diagram of cloning ChimAd-HPV by using the Gibson assembly gene synthesis method in a ChimAd vector from a human papillomavirus gene obtained through gene synthesis,

[0109] FIG. 24 shows the results of cellular immunization (A, B) using the HPV16-type E7 peptide as a stimulating antigen, as a result of ELISPOT, showing the specific T cell immune response to the ChimAd-HPV vaccine candidate.

[0110] FIG. 25 shows the results of measuring the size of tumors, after subcutaneously injecting TC-1 cells, a tumor cell line, into C57BL/6 mice, and vaccine candidates, including a control group were injected before and after tumor cell injection to measure the size of cancer over time Results showing the lowest tumor volume in the ChimAd-HPV group (A, B) are shown.

MODE FOR INVENTION

[0111] Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are described with the intention of illustrating the present invention, and the scope of the present invention is not to be construed as being limited by the following examples.

EXAMPLE

Example 1: Gene Synthesis for Construction of Chimeric Adenoviral Vectors

1-1) Synthetizing ChAdV-6 Knob Gene

[0112] In order to construct a chimeric adenoviral vector by replacing the knob domain at the end of the fiber protein of HAdV-5 with the corresponding gene of ChAdV-6, a non-human adenovirus, genetic homology of the amino acid sequence to the fiber protein of HAdV-5 (Genebank: AY339865.1) and ChAdV-6 (Genebank: AY530877) was analyzed using the Clustal Omega program. Finally, the Clustal Omega results visualized the homology region through ESPript figure (FIG. 1A), and among the knob domains of ChAdV-6 (SEQ ID NO: 1), a base sequence corresponding to 405 to 574 amino acids (SEQ ID NO: 1), which is a region with low homology to the gene sequence of HAdV-5, was obtained by gene synthesis. (FIG. 1B, SEQ ID NO: 2).

1-2) Synthetizing HAdV-28 Hypervariable Region (HVR) Gene

[0113] To construct a chimeric adenovirus vector by replacing the hypervariable region in the hexon protein of HAdV-5 with the corresponding gene of HAdV-28, an adenovirus serotype with low seroprevalence in humans, genetic homology of the amino acid sequence to the hexon protein of HAdV-5 (Genebank: AY339865.1) and HAdV-28 (Genebank: DQ149626.1) were analyzed using the Clustal Omega program. Finally, the Clustal Omega results were visualized through the ESPript figure, and the HVR1 region to the HVR7 region with low genetic homology of amino acid sequences between HAdV-5 and HAdV-28 were set (Table 1, FIG. 2A). Subsequently, to replace the HVR region in the hexon protein of HAdV-5 with the corresponding gene of HAdV-28, the nucleotide sequence from the beginning of the HVR1 region of HAdV-28 (132 amino acids) to the end of the HVR7 region (449 amino acids) was gene synthesized. (FIG. 2B, SEQ ID NO: 4). the genetic homology of amino acid sequences between HAdV-5 and HAdV-28 was set from the HVR1 region to the HVR7 region (Table 1, FIG. 2A). Subsequently, to replace the HVR region in the hexon protein of HAdV-5 with the corresponding gene of HAdV-28, the nucleotide sequence from the beginning of the HVR1 region of HAdV-28 to the end of the HVR7 region was gene synthesized. (FIG. 2B, SEQ ID NO: 4).

TABLE-US-00001 TABLE1 HVR Target region Position Sequence HAdV-5 HVR1 132-168 PCEWDEAATALEINLEEEDDDNEDEV DEQAEQQKTHV (SEQIDNO:15) HVR2 188-195 VEGQTPKY (SEQIDNO:16) HVR3 209-220 SQWYETEINHAA (SEQIDNO:17) HVR4 248-265 GILVKQQNGKLESQVEMQ (SEQIDNO:18) HVR5 268-284 STTEATAGNGDNLTPKV (SEQIDNO:19) HVR6 305-316 TIKEGNSRELMG (SEQIDNO:20) HVR7 418-458 GGVINTETLTKVKPKTGQENGWEKDA TEFSDKNEIRVGNNF (SEQIDNO:21) HAdV- HVR1 132-153 SSQWDAQEKSGQGSDMVTKTHT 28 (SEQIDNO:22) HVR2 173-185 TEITADNQKKEIF (SEQIDNO:23) HVR3 199-209 ENWQENEVFYG (SEQIDNO:24) HVR4 237-256 AKFKTPAEGQEPKELDIDLA (SEQIDNO:25) HVR5 259-273 DTDGGTADTEYKADI (SEQIDNO:26) HVR6 294-305 GKEDDSSEINLV (SEQIDNO:27) HVR7 407-449 DGLGTNATYQGVKVSTGDGATQSGWA KDDTMARQNQICRGNIY (SEQIDNO:28)

[0114] Table 1 shows HVR regions selected through nucleotide sequence analysis between HAdV-5 and HAdV-28.

1-3) Synthesis of Vector Regulators

[0115] Vector regulators to be inserted into the chimeric adenovirus vector were synthesized by combining vector regulators known to date (e.g., promoter, origin of replication, etc.). In more detail, corresponding vector regulators were obtained by synthesizing genes in the order of ITR (inverted terminal repeat, Genebank: AY339865.1), BR322 origin of replication (Genebank: MT612434.1), kanamycin resistance gene (Genebank: MT084773.1), ITR sequence, encapsidation signal, Genebank: AY339865.1), CMV enhancer (Genebank: MT267310.1), CMV promoter (Genebank: MT267310.1), and SV40 poly(A) (Genebank: MT612432.1) (FIG. 3).

Example 2: Preparation of Chimeric Adenoviral Vectors

2-1) Preparing HAdV-5 Vector

[0116] Using DNA of HAdV-5 (ATCC, VR-1516) as a template, PCR amplification was performed using primers except for some of the E1 and E3 genes to obtain the amplified products (fragments A-G) (Table 2). The synthetic gene (4.1 Kb) comprising the 7 vector PCR amplification products and the synthesized CMV promoter sequence has the same sequence (about 20 bp) at each end, and were combined through a gibson assembly (NEB, E2611S) reaction to construct HAdV-5 vectors (FIG. 4). A ChimAd vector was constructed using this as a basic vector.

TABLE-US-00002 TABLE2 Primer PCR name Sequence(5to3direction) product FragmentA F:TAAGGGTGGGAAAGAATATATAAGGTGGGGGTC(SEQID 4Kb NO:29) R:GCCTTCCATCAAGGGCATCCCG(SEQIDNO:30) FragmentB F:GTAAAGTTCCAAGAAGCGCGGGATGC(SEQIDNO:31) 4Kb R:AGTTAATCTCCTGGTTCACCGTCTG(SEQIDNO:32) FragmentC F:GTAACCGCATACGAGCAGACGGTG(SEQIDNO:33) 4Kb R:CGTGGAGCGGAAGGTCACG(SEQIDNO:34) FragmentD F:GCCAGACATGATGCAAGACCCCGTG(SEQIDNO:35) 4Kb R:CGTACCACTGAGATTCTCCTATTTGAGGTTC(SEQIDNO: 36) FragmentE F:CAACCTGAACCTCAAATAGGAGAATCTCAG(SEQIDNO: 4Kb 37) R:GGATAACGATCTAAAGGCGAACTTCAAAC(SEQIDNO: 38) FragmentF F:GTCAGGCAGTAGTTTGAAGTTCGC(SEQIDNO:39) 4.7Kb R: CTCTAGTTATAACTAGAGGATCTTGATGTAATCCAGGGTTAG (SEQIDNO:40) FragmentG F: 5Kb CCTCTAGTTATAACTAGAGTACCCGGGATCTTATTCCCTTTA AC(SEQIDNO:41) R:CGGAGTAACTTGTATGTGTTGGGAATTG(SEQIDNO:42) Gene F:CAATTCCCAACACATACAAGTTACTCCG(SEQIDNO:43) 5Kb synthesis R:TATATTCTTTCCCACCCTTACGCGTTAAGATACATTGATG (promoter, (SEQIDNO:44) etc.)

[0117] Table 2 shows PCR primers used to construct HAdV-5 vector

2-2) Preparation of ChimAd Vector

[0118] A vector PCR amplification product was obtained by PCR amplification using the HAdV-5 vector prepared above as a template and using the primers shown in Table 3, excluding the knob gene sequence. The synthesized ChAdV-6 knob gene was PCR amplified to have the same sequence (about 20 bp) as the vector PCR amplification product at the 3 and 5 ends to obtain an insert gene, and was combined with the vector PCR amplification product through gibson assembly reaction to construct a ChimAd vector (FIGS. 5 and 6).

TABLE-US-00003 TABLE3 Primer PCR name Sequence(5to3direction) product 6Knob F:CCTTTGCTACCAACTCATTCTCTTTTTCA 33.4Kb vector TACATTGCCC(SEQIDNO:45) R: ACAATTAGGGCTTGGGTCAGGTGTGGTCCACA AAGTTAGC(SEQIDNO:46) 6Knob F: 0.5Kb insert GCTAACTTTGTGGACCACACCTGACCCAAGCC CTAATTGT(SEQIDNO:47) R: GGGCAATGTATGAAAAAGAGAATGAGTTGGTA GCAAAGG(SEQIDNO:48)

[0119] Table 3 shows the PCR primers used to amplify the vector and the knob gene for the construction of the ChimAd vector

2-3) Preparation of the ChimAd-H28 Vector

[0120] Using the ChimAd vector as a template, the vector sequence excluding the HVR region (19,879-20,858 bp) of HAdV-5 was PCR amplified using the primers in Table 4 by PCR, and the amplified product (32.9 Kb) and the PCR amplified product of the HVR sequences of HAdV-28 (1 Kb) synthesized above were combined through Gibson assembly reaction to construct a ChimAd-H28 vector (FIGS. 7 and 8).

TABLE-US-00004 TABLE4 Primer PCR name Sequence(5to3direction) product 28HVR F: 32.9Kb vector TGCAGGGGTAACATCTACGCCATGGAAAT CAATCTAAATG(SEQIDNO:49) R: TGCGCATCCCACTGACTGGAATTTGGGGC ACCCTTGGGAG(SEQIDNO:50) 28HVR F: 1Kb insert CTCCCAAGGGTGCCCCAAATTCCAGTCAG TGGGATGCGCA(SEQIDNO:51) R: CATTTAGATTGATTTCCATGGCGTAGATG TTACCCCTGCA(SEQIDNO:52)

[0121] Table 4 shows the PCR primers used to amplify the vector and the 28-type hexon HVR gene for the construction of the ChimAd-H28 vector.

[0122] As shown in FIG. 9, only the ChAdV-6 knob gene inserted in 0.5 Kb is amplified in the ChimAd vector by the PCR method, In the ChimAd-H28 vector using this as a template, the amplification of the ChAdV-6 knob gene and the 1 Kb HAdV-28 hexon HVR gene were simultaneously confirmed. Through this, two final chimeric adenoviral vectors (ChimAd, ChimAd-H28) were obtained by confirming the presence or absence of the chimeric capsid gene in the HAdV-5 vector.

Example 3: Production of Adenovirus

[0123] To evaluate the virus formation of HAdV-5 and the two chimeric adenoviruses designed and produced in the present invention, each vector was cut and removed from the pBR322 replication start point and kanamycin resistance gene sequence unnecessary for adenovirus formation using Pac I restriction enzyme, and HEK293 cells (human embryonic kidney; ATCC, CRL-1573) expressing E1 and E3 genes subcultured the previous day at 1.610.sup.6 cells in a 100 mm cell culture dish were transformed using Lipofectamine 2000 (Invitrogen, #11668027).

[0124] Transformed cells were cultured for 10-20 days in an incubator maintained at 37 C. and 5.0% CO.sub.2 partial pressure, and the formation of a cytopathic effect by adenovirus infection was monitored (FIG. 10). To harvest HAdV-5 and two chimeric adenoviruses from whole CPE, the cells attached to the bottom of the flask were detached using a scraper, and the supernatant was removed by centrifugation at 4 C. and 500g together with the culture medium, and after washing with 1PBS, adenovirus was released by repeating the freezing-thawing process three times in the resulting pellet. Cell debris was centrifuged at 14,000g at room temperature, and the adenovirus-containing supernatant was dispensed into 1.5 mL microcentrifuge tubes and stored at 80 C.

[0125] As shown in FIG. 10, the formation of CPE by transformation in HEK293 cells using the HAdV-5, ChimAd, and ChimAd-H28 vectors prepared in the present invention was confirmed at 200 magnification through an optical microscope. From these results, it is considered that adenovirus replication was achieved in the host cell by the three vectors constructed above, and cytotoxicity was induced by the virus propagation or the expressed adenovirus protein.

Example 4: Western Blot Analysis for Confirming Adenovirus Envelope Protein

[0126] To confirm the expression of adenoviral structural proteins for HAdV-5 and chimeric adenoviruses produced in the HEK293 cell line, Western blot analysis was performed as follows. After adding SDS-PAGE loading dye to HAdV-5, ChimAd, and ChimAd-H28 viruses and bathing at 100 C. for 15 minutes, and were separated by electrophoresis on 10% SDS-PAGE at 100 V, and then transferred to a PVDF membrane. After removing the activity of the PVDF membrane with 5% skim milk solution, the primary antibody, anti-HAdV-5 polyclonal antibody (Abcam, #ab6982) was diluted 1:1000 with TBS solution and reacted at room temperature for 1 hour. After washing three times with TBS-T buffer solution, the secondary antibody, HRP-attached goat-derived anti-mouse immunoglobulin G was diluted 1:5000 with TBS solution, reacted at room temperature for 1 hour, and then washed with TBS-T buffer solution repeatedly. Confirmation of Western results was confirmed after color development using diaminobenzidine (Sigma, #34002) (FIG. 11).

[0127] As shown in FIG. 11, in HAdV-5, ChimAd and ChimAd-H28 viruses, In the virus, hexon (about 108 kDa), fiber (about 61 kDa), and penton (about 68 kDa) proteins were detected in response to all anti-HAdV-5 polyclonal antibodies specifically. When comparing the detected fiber protein of the HAdV-5 virus and the detected fiber protein of the ChimAd virus, since fiber proteins of the same size were confirmed in ChimAd virus, it can be seen that the chimAd virus knob protein replaced with the ChAdV-6 knob gene was normally expressed. In addition, hexon protein of the same size was confirmed in HAdV-5 and ChimAd-H28, indicating that the hexon protein of the ChimAd-H28 virus replaced with the HVR gene of HAdV-28 was normally expressed. In addition, it can be confirmed that the main coat proteins (hexon, fiber, and penton) of adenovirus are stably expressed by the two chimeric adenovirus vectors.

Example 5: Analysis of Adenovirus Infectivity in HEK293 Cells

[0128] To analyze the cell infectivity of two chimeric adenoviruses in which the knob protein sequence that binds to the CAR receptor of the host cell and acts on cell entry is replaced with the gene sequence of ChAdV-6, cells infected with HAdV-5, ChimAd and ChimAd-H28 viruses were observed under the same MOI virus dose condition.

[0129] Adenovirus infection titer was measured using the QuickTiter titer immunoassay kit (Cell biolabs, #VPK-109) of immunocytochemistry using an anti-hexon polyclonal antibody that binds to adenovirus hexon protein. The adenovirus to be used for titer measurement was diluted from 10.sup.1 to 10.sup.6 by 10-fold serial dilution using 1PBS, and infection was induced by dispensing 100 l of adenovirus dilution into HEK293 cells in a 24-well plate (SPL, #30024) prepared with a cell count of 2.510.sup.5 by subculture 1 hour before. Immunostaining for detection of adenovirus was performed 48 hours after infection in an incubator maintained at a temperature of 37 C. and a partial pressure of CO.sub.2 of 5.0%. Cells infected for 48 hours were fixed by removing the supernatant, dispensing cold methanol, and leaving the cells at 20 C. for 20 minutes. After sufficiently removing methanol from the fixed cells with 1PBS solution, the activity was inhibited with 1% skim milk (Difco, #232100) solution, and the primary antibody, anti-hexon antibody, was diluted with 1PBS and reacted at room temperature for 1 hour. After washing three times with 1PBS, the HRP-conjugated secondary antibody was diluted to 1 and reacted. Then, using 3.3-diaminobenzidine stain kit (DAB) (Thermo Fisher, #34002), color was developed for 10 minutes, and then, using an optical microscope, after select the virus dilution interval in which brown spots, which are positive cells infected with adenovirus, are observed between 50 and 100, the infection titer (pfu/mL) of HAdV-5, ChimAd and ChimAd-H28 viruses was measured by applying the adenovirus titer conversion formula provided by Cell Biolabs.

[0130] For the infectivity analysis, HEK293 cells subcultured in a 24-well plate at 2.510.sup.5 cells were incubated for 1 hour in an incubator at a temperature of 37 C. and a CO.sub.2 partial pressure of 5.0%, and then based on the pfu/mL value obtained from the virus titer measurement, HAdV-5 and chimeric adenoviruses were diluted with 1PBS at 0, 5, 10, 25, and 50 MOI virus doses, respectively, to induce infection in the cells. 48 hours after infection, the number of adenovirus-infected cells at each MOI virus dose was counted in at least 4 fields at 200 magnification under an optical microscope through immunocytochemical staining using an anti-hexonpolyclonal antibody (Table 5), and the number of positive cells (brown spots) was converted into pfu/mL values, and the infectivity of HAdV-5 and chimeric adenoviruses for each MOI virus dose was compared (FIG. 12).

TABLE-US-00005 TABLE 5 Virus dose (MOI) Ad infected cell spots 5 10 25 50 HAdV-5 4.0 8.1 16.6 22.2 ChimAd 10.1 17.9 21.6 34.6 ChimAd-H28 9.5 17.7 19.8 32.3

[0131] Table 5 shows the number of cells infected by HAdV-5 and chimeric adenovirus at the same MOI. As shown in Table 5, when HEK293 cells were infected with the same time and MOI virus dose, the number of cells infected with ChimAd and ChimAd-H28 viruses was higher than that of HAdV-5 virus.

[0132] In addition, when comparing the virus titers for each MOI virus dose in FIG. 12, the ChimAd virus and ChimAd-H28 virus had 1.3-3 times and 1-2 times more virus titers than HAdV-5 virus, respectively. showed a scientifically significant difference. Through these results, it was confirmed that not only cell infectivity but also virus productivity was increased due to replacement of the ChAdV-6 knob gene and the HAdV-28 hexon HVR gene.

Example 6: In Vitro Neutralizing Antibody Test Using Anti-HAdV-5 Polyclonal Antibody

[0133] To evaluate the existing immune evasion ability of the HAdV-5 vector due to replacement of the knob gene and HVR region of hexon, which is the main neutralizing antibody recognition site, in the two chimeric adenoviruses produced, an in vitro neutralizing antibody test was performed using an anti-HAdV-5 polyclonal antibody (Abcam, #ab6982) as a neutralizing antibody against HAdV-5.

[0134] In the in vitro neutralizing antibody test, the anti-HAdV-5 antibody was diluted using 1PBS solution to a 10-4 dilution factor through serial dilution, and after neutralizing with 1.2510.sup.7 VP of HAdV-5, ChimAd, and ChimAd-H28 viruses corresponding to a 50 MOI virus dose for 1 hour at room temperature, 2.510.sup.5 cells were dispensed into HEK293 cells in a prepared 24-well cell culture plate, respectively and cultured in an incubator maintained at a temperature of 37 C. and a CO.sub.2 partial pressure of 5.0%, and viral infection was induced for 48 hours. 48 hours after infection, immunocytochemical staining using an anti-hexon polyclonal antibody was performed, and by counting the number of positive cells infected with HAdV-5, ChimAd and ChimAd-H28 viruses in at least 4 fields at 200 magnification using an optical microscope for each anti-HAdV-5 polyclonal antibody dilution factor (10.sup.1-10.sup.4), the ability to avoid neutralization against anti-HAdV-5 antibodies was analyzed (Table 6).

TABLE-US-00006 TABLE 6 Dilution of anti-HAdV-5 antibody Ad infected cell spots 10.sup.1 10.sup.2 10.sup.3 10.sup.4 HAdV-5 1.0 12.5 44.1 57.8 ChimAd 4.4 40.1 97.0 100.8 ChimAd-H28 2.6 13.5 69.5 100.1

[0135] Table 6 shows the number of cells infected with HAdV-5 and chimeric adenovirus after reaction with anti-HAdV-5 polyclonal antibody. As shown in FIG. 13, compared to the HAdV-5 virus, by ChimAd virus about 3.1 to about 4.5 times at low antibody dilution factor (10.sup.1-10.sup.2) and about 1.8 to about 2.2 times more adenovirus-infected cells at high antibody dilution factor (10.sup.3 to 10.sup.4). ChimAd-H28 virus also confirmed about 2.8 times more adenovirus-infected cells than HAdV-5 virus at a low antibody dilution factor and about 1.5 to about 1.7 times at a high antibody dilution factor, indicating a statistically significant difference. Through this, it was obtained that the neutralizing antibody reaction can be avoided up to 4.5 times only by replacing the knob gene in the adenovirus capsid protein. In addition, the above results are 2 times higher avoidance activity than the result of in vitro neutralizing antibody test of a chimeric adenoviral vector (2 fold avoidance compared to HAdV-5 vector) in which the recently developed HAdV-5 structure was replaced with the HAdV-3 knob gene.

[0136] As a result, the ChimAd vector constructed in the present invention is considered to be an adenoviral vector with superior immune evasion ability against HAdV-5 than conventional chimeric adenoviral vectors.

Example 7: In Vitro Neutralizing Antibody Test Using Human Serum

[0137] To evaluate the neutralizing antibody titer in human serum capable of recognizing ChimAd and ChimAd-H28 viruses, which are the chimeric adenoviral vectors constructed in the present invention, an in vitro neutralizing antibody test was conducted using the serum of 7 healthy adults. The complement cascade was inactivated in human serum by heat-inactivation at 56 C., for 30 minutes, and then serially diluted two-fold with 1PBS. Thereafter, the inactivated sera were neutralized with adenovirus under the same 50 MOI virus dose condition at room temperature for 1 hour, and then through the in vitro neutralizing antibody test method described in Example 6, the number of positive cells (brown spots) was measured in at least four fields at 200 magnification using an optical microscope and expressed as an average to determine whether HAdV-5 and chimeric adenoviruses were infected in HEK293 cells on day 2 of infection (Table 7). The number of stained cells was converted into a reciprocal number and then multiplied by 100 to derive neutralizing antibody titers for HAdV-5, ChimAd, and ChimAd-H28 viruses in human serum, and expressed as a dot plot (FIG. 14).

TABLE-US-00007 TABLE 7 Ad infected Human serum (N = 7) cell spots #1 #2 #3 #4 #5 #6 #7 HAdV-5 18.5 14.4 1.4 8.6 19.25 11.4 8.6 ChimAd 45.2 50.8 23.2 22 39 31.2 22 ChimAd-H28 44 44 6 27.2 43.6 27.6 23.8

[0138] Table 7 shows the number of cells infected by HAdV-5 and the chimeric adenovirus following reaction with human sera. As can be seen in Table 7, in the seven human serogroups, we identified cells infected on average 4.6 times more by ChimAd virus than by HAdV-5 virus and by average 2.9 times more cells infected by ChimAd-H28 virus than by HAdV-5 virus, which was a statistically significant difference. In addition, as can be seen in FIG. 13, which shows the number of stained cells as the reciprocal number, it can be seen that the average 2.6-2.7 times lower Ad neutralizing antibody (NAb) titer in the ChimAd virus than in the HAdV-5 virus. These results indicate that vector immunity is lower when administering ChimAd vector-based or ChimAd-H28 vector-based vaccines to humans than HAdV-5 vector-based vaccines, so it will be an optimal adenoviral vector that can deliver foreign genes more efficiently.

Example 8: Application of ChimAd Vector-Based Corona Vaccine

8-1) Genetic Modification Encoding SARS-COV-2 S Protein and N Protein

[0139] To develop a vaccine using the coronavirus structural protein, two S polypeptide synthetic genes comprising NTD and CTD of Spike protein and N protein including modifications such as amino acid substitution were obtained.

[0140] Specifically, in the case of the CV gene, SARS-Coronavirus-2 of the beta-variant S protein was introduced with glycine at 614, acidic amino acid substitution of residues 682 to 685, and consecutive proline substitution mutations at 986 and 987. [Pallesen, Jesper, et al. Proceedings of the National Academy of Sciences 114.35 (2017): E7348-E7357. Bangaru, Sandhya, et al. Science 370.6520 (2020): 1089-1094. Plante, Jessica A., et al. Nature 592.7852 (2021): 116-121.].

[0141] 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 through a (GGGGS) 3 linker, respectively, and at this time, additional structure prediction was performed through prediction tools such as trRosetta and Alphafold. [Du, Zongyang, et al. Nature Protocols (2021): 1-18. Jumper, John, et al. Nature 596.7873 (2021): 583-589.]

8-2) Gene Synthesis to be Used as SARS-COV-2 Vaccine Material

[0142] To develop a vaccine using coronavirus structural proteins, synthetic genes encoding Spike and Nucleocapsid proteins among structural proteins were obtained. As shown in Table 8, 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 comprising the S protein was named CV, and the chimera form in which a portion of the N protein and the S protein were fused was named CVN.

TABLE-US-00008 TABLE 8 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)

8-3) Cloning Chimeric Adenovirus Comprising SARS-COV-2 Gene

[0143] In order to clone the two SARS-COV-2 vaccine antigens synthesized above into the ChimAd vector, which is a chimeric adenovirus vector, using the plasmid DNA of the ChimAd vector as a template, PCR amplification was performed except for a part of the GOI binding region gene, and the amplified product (about 33.4 Kb) was purified through gel extraction. The insertion gene for cloning was PCR amplified to comprise 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 amplification product CV (about 3.8 Kb) and CVN (about 4.7 Kb) were obtained, and then combined with the previously purified DNA PCR amplification products through a gibson assembly reaction, respectively, to finally obtain two types of ChimAd-CV and ChimAd-CVN plasmids (FIG. 15).

TABLE-US-00009 TABLE9 Primer PCR name Sequence(5to3direction) product ChimAd F:CTAAGCTTCTAGATAAGATATCCGATCCAC 33.4Kb vector (SEQIDNO:53) R:CGCGTCGACGGTACCAGATCTCTAGCGGATC (SEQIDNO:54) CV F:GATCTGGTACCGTCGACGCGATGTTTGTGTT 3.8Kb region TCTGGTGCTG(SEQIDNO:55) R: TATCTTATCTAGAAGCTTAGTCAGGTATAGTGCA GTTTCAC(SEQIDNO:56) CVN F:GATCTGGTACCGTCGACGCGATGGGCCTGCC 4.7Kb region CAACAACAC(SEQIDNO:57) R: TATCTTATCTAGAAGCTTAGTCAGGGGAAGGTCT TGTAGGC(SEQIDNO:58)

8-4) Production and Purification of Chimeric Adenovirus-Based Corona Vaccine Candidate

[0144] To produce a chimeric adenovirus-based coronavirus vaccine candidate using the ChimAd-CV and ChimAd-CVN plasmids produced in the present invention, each plasmid was treated with Pac I restriction enzyme (NEB, R0547L) to cut and remove the pBR322 replication start point and kanamycin resistance gene sequence that are unnecessary for adenovirus formation, and HEK293 cells were transformed with Lipofectamine 2000 (Invitrogen, 11668027) and cultured for 10-20 days in an incubator at 37 C. and CO.sub.2 partial pressure of 5.0% to obtain a primary virus stock.

[0145] ChimAd-CV and ChimAd-CVN virus production was performed by infecting HEK293 cells at a cell density of about 110.sup.6 cells/ml with a titer of 2 MOI using the primary virus stock obtained above, and after 48 hours, 0.45 m 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 m and 0.6 m ULTA GF filters (Cytiva, KGF-A-0210TT, KGF-A-9606TT), and virus concentration and buffer exchange were performed through tangential flow filtration (TFF). ChimAd-CV and ChimAd-CVN virus were isolated from virus samples in PBS buffer 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), and formulated into a vaccine buffer, and finally filtered with a 0.2 m ULTA GF filter (Cytiva, KGF-A-9610TT) to obtain two vaccine candidates.

8-5) Immunogenicity Test Results Through Binding Antibody Titer/Neutralizing Antibody Titer/Cellular Immunity Analysis

(1) Immune Induction Using BALB/c Female

[0146] 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{circumflex over ()}9 ifu/dose. At 4-week intervals, the first and second vaccinations were administered by intramuscular injection twice (days 0 and 28), and blood was collected 4 and 6 weeks after the first vaccination (days 27 and 42), serum was separated and the spleen was removed.

(2) ELISA; Binding Antibody Titer of Mouse Anti-Serum by ChimAd-CV and ChimAd-CVN

[0147] A binding antibody titer test was performed to confirm the presence of antibodies in the serum through a mouse immunity induction test. To perform the test, coronavirus SARS-coronavirus-2 and beta variant S protein were dispensed into microplates at a concentration of 1 g/ml, 100 ng/well each, and coated at 4 C. for more than 16 hours. After reaction, the plate was washed at least 4 times with 1PBS comprising Tween 20 (PBS-T) and 100 l 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-fold to approximately 110.sup.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, to proceed with color development, 50 l 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 450 nm.

[0148] As a result of the binding antibody titer, it was confirmed that all experimental groups inoculated with ChimAd-CV and ChimAd-CVN showed high binding antibody titers of 10.sup.4 or more to SARS-coronavirus-2 and beta variant strains compared to the control group. In the experimental group inoculated with the control group, binding antibody titers to both SARS-coronavirus-2 and beta variant strains were measured, but the values were confirmed to be invalid (FIG. 16).

(3) pVNT (Pseudovirus Neutralization Test);

Measurement of Neutralizing Antibody Titers Against Pseudoviruses in Mouse Anti-Serum by ChimAd-CV and ChimAd-CVN

[0149] Neutralizing antibodies in serum were tested through a mouse immune induction test. The pseudovirus system is a virus that uses the backbone of Lentivirus to express the luciferase gene and the Spike protein of SARS-coronavirus-2 or beta variants. In this experiment, Takara's Lenti-X Packaging Mix product was used to produce pseudoviruses comprising the Spike protein of SARS-coronavirus-2 and beta variant strains. 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 comprising 10% FBS was used. The serum was diluted by 1/8 and then serially diluted 2-fold to about 10.sup.3. Pseudoviruses with a certain RLU value were mixed with the same amount of diluted serum and neutralization reaction was performed at 37 C. and 5% CO.sub.2 incubator for 1 time. hACE2-293T cells cultured a day earlier were treated with 100 l of mixture of the virus and serum and cultured in an incubator at 37 C. and 5% CO.sub.2 for about 72 hours. After completion of incubation, about 100 l of the culture medium was removed, 30 l of One-Glo luciferase reagent (Promega) was added, and reacted for 3-5 minutes. Then, 60 l 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 IC.sub.50 value according to the serum dilution factor using a statistical program.

[0150] As shown in Table 10, the results of neutralizing antibodies against the beta variant virus showed IC.sub.50=1253 for the ChimAd-CV inoculated group and IC.sub.50=1131 for the ChimAd-CVN inoculated group, which were all higher than 10.sup.3, confirming a higher neutralizing antibody titer than the negative control group (FIG. 17).

TABLE-US-00010 TABLE 10 pVNT(IC50) No Group 4 wk 6 wk 1 PBS 7 42 2 ChimAd 9 0 3 ChimAd-CV 244 1253 4 ChimAd-CVN 409 1131

(4) ELISpot; Specific T Cell Immune Response Caused by ChimAd-CV, ChimAd-CVN Adenovirus

[0151] 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, 110.sup.5 cells, 210.sup.4 cells of spleen cells and Genscript's SARS-coronavirus-2 Spike protein peptide pool or Nucleprotein peptide pool were used as stimulating antigens and incubated together for 16 hours in a 37 C., 5% CO.sub.2 incubator. After incubation, the cells were washed with a washing solution, and 100 l 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 l of streptavidin-AKP (alkaline phosphate) was added and incubated at room temperature for 2 hours. After the final washing, 100 l of BCIP/NBT substrate was added and reacted at room temperature for about 45 minutes. When color development occurred due to the reaction, the spot was washed with sterilized water, the number of colored spots was counted, and the number of cells in the Spot Forming Unit (SFU) was calculated.

[0152] 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). Particularly 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. In addition, it was confirmed that ChimAd-CVN induces a specific immune response to Nucleocapsid peptide (FIG. 18).

TABLE-US-00011 TABLE 11 SPF/10{circumflex over ()}6 splenocytes (Spike peptide pool) No Group 4 wk 6 wk 1 PBS 34 17 2 ChimAd 61 42 3 ChimAd-CV 2367 3824 4 ChimAd-CVN 3020 6949
8-6) Antibody Titer, Neutralizing Antibody Titer and Challenge Test after ChimAd-CV, ChimAd-CVN Animal Immunization
A. Immune Induction and Challenge Test Using hACE2 Transgenic Female Mouse

[0153] The immune induction and challenge tests conducted at IVI used 6-week-old female human ACE2 transgenic mice (Tg(K18-ACE2)2Prlmn) purchased from Jackson Laboratory (ME, USA). All 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. Mice were inoculated intramuscularly twice at 4-week intervals, and blood was collected 4 and 6 weeks after the first priming inoculation.

B. ELISA; Measurement of Antibody Titers of Mouse Anti-Serum by ChimAd-CV, ChimAd-CVN

[0154] After the mouse immune induction test, it was performed to measure the antibody titer in serum. In a 96-well plate, 100 ng/well of SARS-coronavirus-2 S protein and delta variant S protein were dispensed at a concentration of 2 g/ml and coated at 4 C. for more than 16 hours. After the reaction, 100 l of PBS containing 1% BSA was dispensed and allowed to react. Each serum was diluted 1:100, then serially diluted 5-fold and reacted at 4 C. 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.

[0155] 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.sup.5 or more against SARS-coronavirus-2 and delta variant strains compared to the control group (FIG. 19).

C. FRNT (Focus Reduction Neutralization Test); Measurement of Neutralizing Antibody Titers Against Coronavirus in ChimAd-CV and ChimAd-CVN Mouse Anti-Serum

[0156] 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 was mixed with 4.510.sup.2 PFU/25 l of SARS-coronavirus-2 coronavirus (NCCP #43326 (BetaCoV/Korea/KCDC03/2020) and 4.010.sup.2 PFU/25 l of delta variant coronavirus (NCCP #43390 (hCoV-19/Korea119861)/KDCA/2021) and reacted for 30 minutes at 37 C. After reaction, 50 l of virus and serum mixture was treated with Vero cells (1.510.sup.4 cells/well) cultured one day before and incubated at 37 C. in a CO.sub.2 incubator for 4 hours. After 4 hours of reaction, the supernatant was removed, washed with a PBS buffer solution, and 300 l of 4% formaldehyde solution (Sigma) was added and fixed in a place without light for more than 16 hours. After the reaction, it was washed with PBS. After washing, 100% cold methanol was added and reacted for 10 minutes, followed by reaction at room temperature for 1 hour using blocking solution. Afterwards, it was treated with anti-SARS-COV-2 NP rabbit monoclonal antibody (Sino Biological) diluted 1:3000 and incubated at 37 C. for 1 hour. After incubation, it was washed with a PBS solution containing 0.1% Tween20 (PBS-T), and it was treated with HRP-conjugated goat anti-rabbit IgG (Bio-Rad) diluted 1:2000 and incubated at 37 C. for 1 hour. After washing with PBS-T, the TMB solution was reacted at room temperature for 30 minutes in the absence of light, the reaction was stopped with water, and then 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 is a method of measuring neutralizing antibodies by identifying virus-infected cells by attaching a virus-derived monoclonal antibody to a virus-infected cell and then attaching a secondary antibody labeled with HRP to develop a color as a substrate.

[0157] As a result of the neutralizing antibody against the SARS-coronavirus-2 virus, the neutralizing antibody titer can be confirmed as shown in Table, which is more than 100 times higher than the negative control group in both the ChimAd-CV and ChimAd-CVN groups after the first vaccination. 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.

[0158] In addition, as a result of neutralizing antibodies against the delta variant virus, it was confirmed that both the ChimAd-CV vaccinated group and the ChimAd-CVN vaccinated group showed neutralizing antibody titers more than 40 times higher than the negative control group after the second vaccination (FIG. 20).

TABLE-US-00012 TABLE 12 SARS-coronavirus- Delta variant 2(FRNT50) virus (FRNT50) No Group 4 wk 6 wk 4 wk 6 wk 1 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
D. Virus Challenge Test after ChimAd-CV, ChimAd-CVN Immunization

[0159] The challenge test was intranasal infection with 510.sup.5 PFU SARS-COV-2 virus (SARS-coronavirus-2, delta variant strain) 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.

[0160] As a result of confirming the vaccine protective ability of ChimAd-CV and ChimAd-CVN against SARS-Coronavirus-2 (Wuhan-Hu-1) attack, both survival rates were found to be 100%, and it was confirmed that the ChimAd-CVN vaccinated group did not lose weight and the body weight of the ChimAd-CV vaccinated group was maintained without loss in all but one animal, and that there were individuals whose body weight decreased by more than 10% and then recovered.

[0161] In addition, as a result of confirming the vaccine protective ability of ChimAd-CV and ChimAd-CVN against Delta variant attack, ChimAd-CVN showed a survival rate of 40% and average body weight reduction of about 20% after 7 days of infection. Confirmed what was visible. The ChimAd-CV results showed that the survival rate was 100% and the average body weight was maintained at 100% after 7 days of infection (FIG. 21).

E. Lung Virus Titer for ChimAd-CV, ChimAd-CVN Immune Induction and Challenge Test (Plaque Assay)

[0162] 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.sub.2 for more than 16 hours. The virus isolated from the lungs collected from all groups was stepwise diluted 4-fold up to 10.sup.6, and 200 l 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 a 37 C., 5% CO.sub.2 incubator 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.

[0163] As a result of measuring the residual virus titer in the lungs after the challenge test, in the ChimAd-CV vaccinated group against SARS-coronavirus-2 and delta variant attack, the lung virus titer decreased by more than 20,000 times 2 days after the attack compared to the control group. It was confirmed that the virus was cleared and not measured 4 and 7 days after the attack. 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 (FIG. 22).

Example 9: Application of Chimeric Adenovirus-Based HPV Treatment and Prevention Vaccine

9-1) Genetic Modification Encoding the Proteins of HPV16 Type E6 and E7 and HPV Type 18 E6 and E7 Proteins.

[0164] For genetic modification of the anti-carcinogenic protein of HPV type 16, in the case of the E6 protein, a deletion mutation of residues 113 to 122 was induced to inhibit the interaction with carcinogenic proteins caused by the binding domain of the anti-cancer protein p53. In the case of the E7 protein, a deletion mutation of residues 21 to 26 was induced to strongly inhibit the interaction with pRb, the main target, and to suppress the transforming activity ability of E7 itself. In addition, for genetic modification of the anti-cancer protein of HPV type 18, in the case of the E6 protein, a deletion mutation of residues 113 to 122 was induced to inhibit the interaction with carcinogenic proteins caused by the binding domain of the anti-cancer protein p53. In the case of the E7 protein, a deletion mutation of residues 24 to 29 was induced to strongly inhibit the interaction with pRb, the main target, and to suppress the transforming activity ability of E7 itself.

[0165] Linear polypeptide (E6E7) in which the amino acids of E6 and E7, tumor proteins derived from human papillomavirus type 16, have been deleted, and linear polypeptide (Linear; E6E7) in which the amino acids of E6 and E7, tumor proteins derived from human papillomavirus type 18, have been deleted. were combined with Linker.

9-2) Chimeric Adenovirus-Based HPV Type 16, Type 18 E6, E7 Gene Synthesis and Chimeric Adenovirus Cloning

[0166] The E6 and E7 genes, which are anticancer proteins of human papillomavirus types 16 and 18, were modified and synthetic genes encoding these proteins were obtained. For each protein, the strain was selected as shown in Table 13 and the codon was optimized to facilitate expression in HEK cells.

TABLE-US-00013 TABLE 13 Accession no. HPV 16E6E7 E6; AAV91660.1, E7; BAO18680.1 HPV 18 E6E7 E6; 1ADC35716.1, E7; 1ADC35717.1

[0167] Cloning of the HPV vaccine antigen cloned into the chimeric adenovirus vector was performed in the same manner as the SARS-coronavirus vaccine production (Example 8-3), and finally the ChimAd-HPV plasmid was obtained (FIG. 23).

TABLE-US-00014 TABLE14 Primer PCR name Sequence(5to3direction) product ChimAd F:CTAAGCTTCTAGATAAGATATCCGATCCAC 33.4Kb vector (SEQIDNO:59) R:CGCGTCGACGGTACCAGATCTCTAGCGGATC (SEQIDNO:60) HPV16/ F:GATCTGGTACCGTCGACGCGATGCACCAGAA 1.4Kb 18 GAGAACA(SEQIDNO:61) E6E7 R: TATCTTATCTAGAAGCTTAGTTATTGTTGACTAG CGCACCATGG(SEQIDNO:62)

9-3) Immunization Induction Using C57BL/6 Female

[0168] Mouse immunization was performed using the ChimAd-HPV vaccine candidate obtained by the above preparing method. The test group includes three groups: buffer, ChimAd used as a mock-up, and ChimAd-HPV. For the immune induction test, 5-week-old C57BL/6 female mice were used, and each adenovirus test group was injected at a concentration of 2*10{circumflex over ()}7 PFU/ml. At 4-week intervals, the first and second vaccinations were administered by intramuscular injection twice (days 0 and 14), and blood was collected and serum was separated and the spleen was removed at 2 and 4 weeks after the first vaccination (days 14 and 28). In addition, to confirm the effect of treating cervical cancer, 110.sup.5 TC-1 tumor cells were subcutaneously injected into the flank of 6-week-old C57BL/6 female mice 7 days before the first vaccination, and 6 mice were used for each group. In addition, to confirm the effect of preventing cervical cancer, 110.sup.5 TC-1 tumor cells were subcutaneously injected into the flanks of 6-week-old C57BL/6 female mice 7 days after the second vaccination, and 6 mice were used in each group.

9-4) ELISPOT; Specific T Cell Immune Response by ChimAd-HPV Adenovirus

[0169] On the 35th day after the second vaccination, spleens were removed from each group. The spleen was crushed into small pieces using a Cell strainer (Falcon) and the spleen cells were recovered and washed with a PBS buffer solution. Afterwards, red blood cells were removed using red blood cell lysis buffer (Sigma), and the number of spleen cells was counted and diluted to the number of cells to be 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.

[0170] Then, 210.sup.5 cells and 410.sup.4 cells of splenocytes recovered from each mouse and the 49-57 amino acid peptide of Genscript's HPV E7 protein were used as stimulating antigens on a plate coated with mouse IFN-gamma monoclonal antibody and incubated for 16 hours at 37 C. in a 5% CO.sub.2 incubator. After incubation, the cells were washed four times using 1 Wash buffer, 100 l of streptavidin-AKP (alkaline phosphate) was added and incubated at room temperature for 2 hours. After the final washing, 100 l of BCIP/NBT substrate was added and incubated at room temperature for 45 minutes. When color development due to the reaction appeared, it was washed with sterilized water and the number of colored dots was counted.

[0171] As a result of measuring the specific T cell immune response to the HPV E7 protein antigen using ELISPOT, the ChimAd-HPV group showed higher results than the control group, confirming that it induced a specific immune response to the antigen. (FIG. 24)

9-5) Tumor Inhibition Assay; Confirmation of Tumor Treatment and Prevention Effects by ChimAd-HPV Adenovirus

[0172] To confirm the treatment effect of ChimAd-HPV adenovirus on cervical cancer, 110.sup.5 TC-1 tumor cells were subcutaneously injected into the flank of 6-week-old C57BL/6 female mice, and 6 mice were used per group. To confirm treatment efficacy, 7 days after injection of TC-1 cells, intramuscular injection of control group and ChimAd-HPV adenovirus was performed, and a second injection was performed on the 21st day after injection of tumor cells. In addition, to confirm the preventive efficacy, control and ChimAd-HPV adenoviruses were injected intramuscularly twice at two-week intervals, and 14 days after the second injection, TC-1 tumor cells (110.sup.5 cells) were injected subcutaneously into the flank. Tumor cells were measured every 4-5 days by measuring their longest (length) and shortest dimensions (width) using digital calipers to estimate tumor size in mice. Tumor volume was calculated by the following equation: tumor volume=(lengthwidth.sup.2)/2.

[0173] As shown in FIG. 24, as a result of measuring the tumor volume using ChimAd-HPV adenovirus, the tumor volume was found to be lower in the ChimAd-HPV adenovirus group compared to the negative control group and positive control group in both therapeutic and preventive efficacy. It was confirmed that no tumor cells occurred in the ChimAd-HPV adenovirus group. (FIG. 25)

TABLE-US-00015 [Sequencelistpretext] SEQIDNO:1 Chimpanzeeadenovirusserotype6Fiberknobdomaina.a sequence(synthesizedChAdV-6knoba.asequencesareunderlined) PDPSPNCQLLSDRDAKFTLCLTKCGSQILGTVAVAAVTVGSALNPINDTVKSAIVFLR FDSDGVLMSNSSMVGDYWNFREGQTTQSVAYTNAVGFMPNIGAYPKTQSKTPKNSI VSQVYLTGETTMPMTLTITFNGTDEKDTTPVSTYSMTFTWQWTGDYKDKNITFATN SFS SEQIDNO:2 Chimpanzeeadenovirusserotype6/Pan6/Sad23Fiberknobdomain nucleotidesequence CCTGACCCAAGCCCTAATTGTCAATTACTTTCAGACAGAGATGCCAAATTTACTC TCTGTCTTACAAAATGCGGTAGTCAAATACTAGGCACTGTGGCAGTGGCGGCTGT TACTGTAGGATCAGCACTAAATCCAATTAATGACACAGTCAAAAGCGCCATAGT TTTCCTTAGATTTGATTCCGATGGTGTACTCATGTCAAACTCATCAATGGTAGGTG ATTACTGGAACTTTAGGGAGGGACAGACCACTCAAAGTGTAGCCTATACAAATG CTGTGGGATTCATGCCAAATATAGGTGCATATCCAAAAACCCAAAGTAAAACAC CTAAAAATAGCATAGTCAGTCAGGTATATTTAACTGGAGAAACTACTATGCCAAT GACACTAACCATAACTTTCAATGGCACTGATGAAAAAGACACAACCCCAGTTAG CACCTACTCTATGACTTTTACATGGCAGTGGACTGGAGACTATAAGGACAAAAAT ATTACCTTTGCTACCAACTCATTCTCT SEQIDNO:3 ChimerichexonHVR1to7regiona.asequence(HAdV-5hexon+HAdV- 28HVR1-7region) SSQWDAQEKSGQGSDMVTKTHTFGVAAMGGENITKNGLQIGTEITADNQKKEIFAN KTYQPEPQVGEENWQENEVFYGGRALKKETKMKPCYGSFARPTNENGGQAKFKTP AEGQEPKELDIDLAFFDTDGGTADTEYKADIVMYAENVNLETPDTHVVYKPGKEDD SSEINLVQQSMPNRPNYIGFRDNFVGLMYYNSTGNMGVLAGQASQLNAVVDLQDR NTELSYQLLLDSLGDRTRYFSMWNSAVDSYDPDVRIIENHGVEDELPNYCFPLDGLG TNATYQGVKVSTGDGATQSGWAKDDTMARQNQICRGNIY SEQIDNO:4 ChimerichexonHVR1to7regionnucleotidesequence(HAdV-5hexon+ HAdV-28HVR1-7region) TCCAGTCAGTGGGATGCGCAAGAAAAAAGTGGACAAGGAAGTGACATGG TTACTAAAACTCACACATTTGGGCAGGCGCCTTATTCTGGTATAAATATTACAAA GGAGGGTATTCAAATAGGTACTGAAATTACCGCCGACAATCAAAAGAAAGAAAT TTTTGCCGATAAAACATTTCAACCTGAACCTCAAATAGGAGAAGAGAACTGGCA AGAAAATGAAGTCTTCTATGGCGGGAGAGTCCTAAAAAAGACTACCCCAATGAA ACCATGTTACGGTTCATATGCAAAACCCACAAATGAAAATGGAGGGCAAGCAAA ATTCAAAACACCTGCTGAAGGACAGGAACCTAAAGAACTTGACATTGACCTTGC TTTTTTCGACACTGACGGCGGAACAGCTGACACTGAATATAAAGCAGATATTGTA TTGTACAGTGAAGATGTAGATATAGAAACCCCAGACACTCATATTTCTTACATGC CCGGCAAGGAAGATGACAGTTCAGAAATCAATCTAGTTCAACAATCTATGCCCA ACAGGCCTAATTACATTGCTTTTAGGGACAATTTTATTGGTCTAATGTATTACAAC AGCACGGGTAATATGGGTGTTCTGGCGGGCCAAGCATCGCAGTTGAATGCTGTT GTAGATTTGCAAGACAGAAACACAGAGCTTTCATACCAGCTTTTGCTTGATTCCA TTGGTGATAGAACCAGGTACTTTTCTATGTGGAATCAGGCTGTTGACAGCTATGA TCCAGATGTTAGAATTATTGAAAATCATGGAACTGAAGATGAACTTCCAAATTAC TGCTTTCCACTGGACGGATTGGGAACTAATGCTACCTATCAAGGTGTAAAAGTAT CAACTGGAGATGGTGCTACGCAAAGCGGATGGGCAAAAGACGACACAATGGCC AGGCAAAACCAAATATGCAGGGGTAACATCTAC SEQIDNO:5 Chimericfibergenesequence(HAdV-5tail,shiftdomain+ChAdV- 6knobdomain) ATGAAGCGCGCAAGACCGTCTGAAGATACCTTCAACCCCGTGTATCCATATGAC ACGGAAACCGGTCCTCCAACTGTGCCTTTTCTTACTCCTCCCTTTGTATCCCCCAA TGGGTTTCAAGAGAGTCCCCCTGGGGTACTCTCTTTGCGCCTATCCGAACCTCTA GTTACCTCCAATGGCATGCTTGCGCTCAAAATGGGCAACGGCCTCTCTCTGGACG AGGCCGGCAACCTTACCTCCCAAAATGTAACCACTGTGAGCCCACCTCTCAAAA AAACCAAGTCAAACATAAACCTGGAAATATCTGCACCCCTCACAGTTACCTCAG AAGCCCTAACTGTGGCTGCCGCCGCACCTCTAATGGTCGCGGGCAACACACTCAC CATGCAATCACAGGCCCCGCTAACCGTGCACGACTCCAAACTTAGCATTGCCACC CAAGGACCCCTCACAGTGTCAGAAGGAAAGCTAGCCCTGCAAACATCAGGCCCC CTCACCACCACCGATAGCAGTACCCTTACTATCACTGCCTCACCCCCTCTAACTA CTGCCACTGGTAGCTTGGGCATTGACTTGAAAGAGCCCATTTATACACAAAATGG AAAACTAGGACTAAAGTACGGGGCTCCTTTGCATGTAACAGACGACCTAAACAC TTTGACCGTAGCAACTGGTCCAGGTGTGACTATTAATAATACTTCCTTGCAAACT AAAGTTACTGGAGCCTTGGGTTTTGATTCACAAGGCAATATGCAACTTAATGTAG CAGGAGGACTAAGGATTGATTCTCAAAACAGACGCCTTATACTTGATGTTAGTTA TCCGTTTGATGCTCAAAACCAACTAAATCTAAGACTAGGACAGGGCCCTCTTTTT ATAAACTCAGCCCACAACTTGGATATTAACTACAACAAAGGCCTTTACTTGTTTA CAGCTTCAAACAATTCCAAAAAGCTTGAGGTTAACCTAAGCACTGCCAAGGGGT TGATGTTTGACGCTACAGCCATAGCCATTAATGCAGGAGATGGGCTTGAATTTGG TTCACCTAATGCACCAAACACAAATCCCCTCAAAACAAAAATTGGCCATGGCCT AGAATTTGATTCAAACAAGGCTATGGTTCCTAAACTAGGAACTGGCCTTAGTTTT GACAGCACAGGTGCCATTACAGTAGGAAACAAAAATAATGATAAGCTAACTTTG TGGACCACACCTGACCCAAGCCCTAATTGTCAATTACTTTCAGACAGAGATGCCA AATTTACTCTCTGTCTTACAAAATGCGGTAGTCAAATACTAGGCACTGTGGCAGT GGCGGCTGTTACTGTAGGATCAGCACTAAATCCAATTAATGACACAGTCAAAAG CGCCATAGTTTTCCTTAGATTTGATTCCGATGGTGTACTCATGTCAAACTCATCAA TGGTAGGTGATTACTGGAACTTTAGGGAGGGACAGACCACTCAAAGTGTAGCCT ATACAAATGCTGTGGGATTCATGCCAAATATAGGTGCATATCCAAAAACCCAAA GTAAAACACCTAAAAATAGCATAGTCAGTCAGGTATATTTAACTGGAGAAACTA CTATGCCAATGACACTAACCATAACTTTCAATGGCACTGATGAAAAAGACACAA CCCCAGTTAGCACCTACTCTATGACTTTTACATGGCAGTGGACTGGAGACTATAA GGACAAAAATATTACCTTTGCTACCAACTCATTCTCT SEQIDNO:6 Chimerichexongenesequence(HAdV-5hexon+HAdV-28HVR1- 7region) ATGGCTACCCCTTCGATGATGCCGCAGTGGTCTTACATGCACATCTCGGGCCAGG ACGCCTCGGAGTACCTGAGCCCCGGGCTGGTGCAGTTTGCCCGCGCCACCGAGA CGTACTTCAGCCTGAATAACAAGTTTAGAAACCCCACGGTGGCGCCTACGCACG ACGTGACCACAGACCGGTCCCAGCGTTTGACGCTGCGGTTCATCCCTGTGGACCG TGAGGATACTGCGTACTCGTACAAGGCGCGGTTCACCCTAGCTGTGGGTGATAAC CGTGTGCTGGACATGGCTTCCACGTACTTTGACATCCGCGGCGTGCTGGACAGGG GCCCTACTTTTAAGCCCTACTCTGGCACTGCCTACAACGCCCTGGCTCCCAAGGG TGCCCCAAATTCCAGTCAGTGGGATGCGCAAGAAAAAAGTGGACAAGGAAGTGA CATGGTTACTAAAACTCACACATTTGGGCAGGCGCCTTATTCTGGTATAAATATT ACAAAGGAGGGTATTCAAATAGGTACTGAAATTACCGCCGACAATCAAAAGAAA GAAATTTTTGCCGATAAAACATTTCAACCTGAACCTCAAATAGGAGAAGAGAAC TGGCAAGAAAATGAAGTCTTCTATGGCGGGAGAGTCCTAAAAAAGACTACCCCA ATGAAACCATGTTACGGTTCATATGCAAAACCCACAAATGAAAATGGAGGGCAA GCAAAATTCAAAACACCTGCTGAAGGACAGGAACCTAAAGAACTTGACATTGAC CTTGCTTTTTTCGACACTGACGGCGGAACAGCTGACACTGAATATAAAGCAGATA TTGTATTGTACAGTGAAGATGTAGATATAGAAACCCCAGACACTCATATTTCTTA CATGCCCGGCAAGGAAGATGACAGTTCAGAAATCAATCTAGTTCAACAATCTAT GCCCAACAGGCCTAATTACATTGCTTTTAGGGACAATTTTATTGGTCTAATGTATT ACAACAGCACGGGTAATATGGGTGTTCTGGCGGGCCAAGCATCGCAGTTGAATG CTGTTGTAGATTTGCAAGACAGAAACACAGAGCTTTCATACCAGCTTTTGCTTGA TTCCATTGGTGATAGAACCAGGTACTTTTCTATGTGGAATCAGGCTGTTGACAGC TATGATCCAGATGTTAGAATTATTGAAAATCATGGAACTGAAGATGAACTTCCAA ATTACTGCTTTCCACTGGACGGATTGGGAACTAATGCTACCTATCAAGGTGTAAA AGTATCAACTGGAGATGGTGCTACGCAAAGCGGATGGGCAAAAGACGACACAAT GGCCAGGCAAAACCAAATATGCAGGGGTAACATCTACGCCATGGAAATCAATCT AAATGCCAACCTGTGGAGAAATTTCCTGTACTCCAACATAGCGCTGTATTTGCCC GACAAGCTAAAGTACAGTCCTTCCAACGTAAAAATTTCTGATAACCCAAACACCT ACGACTACATGAACAAGCGAGTGGTGGCTCCCGGGCTAGTGGACTGCTACATTA ACCTTGGAGCACGCTGGTCCCTTGACTATATGGACAACGTCAACCCATTTAACCA CCACCGCAATGCTGGCCTGCGCTACCGCTCAATGTTGCTGGGCAATGGTCGCTAT GTGCCCTTCCACATCCAGGTGCCTCAGAAGTTCTTTGCCATTAAAAACCTCCTTCT CCTGCCGGGCTCATACACCTACGAGTGGAACTTCAGGAAGGATGTTAACATGGTT CTGCAGAGCTCCCTAGGAAATGACCTAAGGGTTGACGGAGCCAGCATTAAGTTT GATAGCATTTGCCTTTACGCCACCTTCTTCCCCATGGCCCACAACACCGCCTCCA CGCTTGAGGCCATGCTTAGAAACGACACCAACGACCAGTCCTTTAACGACTATCT CTCCGCCGCCAACATGCTCTACCCTATACCCGCCAACGCTACCAACGTGCCCATA TCCATCCCCTCCCGCAACTGGGCGGCTTTCCGCGGCTGGGCCTTCACGCGCCTTA AGACTAAGGAAACCCCATCACTGGGCTCGGGCTACGACCCTTATTACACCTACTC TGGCTCTATACCCTACCTAGATGGAACCTTTTACCTCAACCACACCTTTAAGAAG GTGGCCATTACCTTTGACTCTTCTGTCAGCTGGCCTGGCAATGACCGCCTGCTTAC CCCCAACGAGTTTGAAATTAAGCGCTCAGTTGACGGGGAGGGTTACAACGTTGC CCAGTGTAACATGACCAAAGACTGGTTCCTGGTACAAATGCTAGCTAACTATAAC ATTGGCTACCAGGGCTTCTATATCCCAGAGAGCTACAAGGACCGCATGTACTCCT TCTTTAGAAACTTCCAGCCCATGAGCCGTCAGGTGGTGGATGATACTAAATACAA GGACTACCAACAGGTGGGCATCCTACACCAACACAACAACTCTGGATTTGTTGG CTACCTTGCCCCCACCATGCGCGAAGGACAGGCCTACCCTGCTAACTTCCCCTAT CCGCTTATAGGCAAGACCGCAGTTGACAGCATTACCCAGAAAAAGTTTCTTTGCG ATCGCACCCTTTGGCGCATCCCATTCTCCAGTAACTTTATGTCCATGGGCGCACTC ACAGACCTGGGCCAAAACCTTCTCTACGCCAACTCCGCCCACGCGCTAGACATG ACTTTTGAGGTGGATCCCATGGACGAGCCCACCCTTCTTTATGTTTTGTTTGAAGT CTTTGACGTGGTCCGTGTGCACCAGCCGCACCGCGGCGTCATCGAAACCGTGTAC CTGCGCACGCCCTTCTCGGCCGGCAACGCCACAACATAA [ChimAd-basedcoronavirusvaccinesequence] SEQIDNO:7 SARS-COV-2_B.1.351(beta)spikemodifiedaminoacidsequence MFVFLVLLPLVSSQCVNFTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPF FSNVTWFHAIHVSGTNGTKRFANPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQS LLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVS QPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRGLPQGFSALEPLVDLP IGINITRFQTLHISYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDC ALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASV YAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEV RQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKP FERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAP ATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVR DPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRV YSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPQQAQSVASQSIIA YTMSLGVENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLL LQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSK PSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMI AQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRENGIGVTQNVLYENQKLIANQ FNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRL DPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDF CGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSN GTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYF KNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPW YIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLH YTSEQ SEQIDNO:8 SARS-COV-2_B.1.351(beta)spikemodifiednucleicacidsequence ATGTTTGTGTTTCTGGTGCTGCTGCCTCTGGTGAGCAGCCAGTGCGTG AACTTCACCACCAGAACCCAGCTGCCTCCTGCCTACACCAACAGCTTT ACCAGAGGCGTGTATTACCCCGACAAAGTGTTTAGATCCTCCGTGCTGCAC AGCACCCAGGACCTGTTTCTGCCCTTTTTCAGCAACGTGACCTGGTTC CATGCCATCCACGTGAGCGGCACCAACGGCACCAAGAGATTCGCCAAT CCCGTGCTGCCTTTTAATGACGGCGTGTACTTTGCCAGCACCGAGAAG AGCAATATCATCAGAGGCTGGATCTTCGGCACCACCCTGGATAGCAAGACC CAGAGCCTGCTGATCGTGAATAATGCCACCAATGTGGTGATCAAAGTG TGCGAGTTCCAGTTTTGTAACGACCCCTTTCTGGGCGTGTATTACCAC AAGAACAACAAAAGCTGGATGGAGAGCGAATTCAGAGTGTACAGCAGC GCCAATAACTGCACCTTTGAGTACGTGAGCCAGCCCTTTCTGATGGACCTG GAGGGCAAGCAGGGCAACTTCAAGAACCTGAGAGAGTTCGTGTTTAAA AATATCGACGGCTACTTCAAGATCTACAGCAAGCATACACCCATCAAC CTGGTGAGAGGCCTGCCCCAGGGCTTCAGCGCCCTGGAGCCCCTGGTG GACCTGCCCATCGGCATCAACATTACCCGGTTTCAGACCCTGCACAGA AGCTATCTGACCCCCGGCGACAGCAGCAGCGGCTGGACCGCCGGAGCC GCCGCCTATTATGTGGGCTACCTGCAGCCCAGAACCTTCCTGCTGAAATAC AACGAGAACGGCACCATCACCGACGCCGTGG?CTGCGCCCTGGACCCC CTGAGCGAGACCAAGTGTACCCTGAAGAGCTTTACCGTGGAGAAGGGC ATCTACCAGACCAGCAACTTTAGAGTGCAGCCCACCGAGAGCATCGTG AGATTTCCCAACATCACCAACCTGTGCCCCTTCGGGGAGGTGTTTAAT GCCACCAGATTCGCCAGCGTGTACGCCTGGAATAGAAAAAGAATCAGC AACTGCGTGGCCGACTACAGCGTGCTGTACAACTCTGCCAGCTTTTCC ACCTTTAAGTGCTACGGCGTGAGCCCAACCAAACTGAACGACCTGTGCTTC ACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAGGTGAGA CAGATCGCCCCCGGCCAGACCGGCAATATCGCCGATTACAACTACAAG CTGCCCGACGACTTTACCGGCTGCGTGATCGCCTGGAACAGCAACAAC CTGGACAGCAAGGTGGGCGGCAACTACAACTACCTGTACAGACTGTTT AGAAAGAGCAACCTGAAGCCCTTTGAGAGAGACATCAGCACCGAGATC TACCAGGCCGGCAGCACCCCCTGCAACGGCGTGAAAGGCTTTAATTGT TACTTCCCACTGCAGTCCTACGGCTTCCAGCCTACCTACGGCGTGGGATAT CAGCCCTATAGAGTGGTGGTGCTGTCCTTCGAGCTGCTGCACGCCCCC GCCACCGTGTGCGGCCCAAAGAAGAGCACCAACCTGGTGAAGAACAAG TGCGTGAACTTCAACTTCAACGGCCTGACCGGCACCGGCGTGCTGACC GAGTCCAACAAGAAATTTCTGCCCTTCCAGCAGTTCGGAAGAGACATTGCC GATACCACCGACGCCGTGAGAGACCCCCAGACCCTGGAGATCCTGGAC ATCACCCCCTGTAGCTTCGGCGGCGTGTCCGTGATCACCCCAGGCACC AACACCTCCAACCAGGTGGCCGTGCTGTACCAGGGCGTGAACTGTACC GAGGTGCCTGTGGCCATCCACGCCGATCAGCTGACCCCAACCTGGAGA GTGTACTCCACCGGCTCCAACGTGTTCCAGACCAGAGCCGGCTGTCTG ATCGGCGCCGAGCACGTGAACAACTCCTACGAGTGCGACATCCCTATCGGC GCCGGCATCTGCGCCAGCTACCAGACCCAGACCAATAGCCCCCAG CAGGCCCAG TCCGTGGCCAGCCAGTCCATCATTGCCTACACCATGTCCCTGGGCGTG GAGAACAGCGTGGCCTACTCCAACAACAGCATCGCCATCCCCACCAATTTC ACCATCAGCGTGACCACCGAAATCCTGCCAGTGAGCATGACCAAAACC TCCGTGGATTGCACCATGTACATCTGCGGAGACTCCACCGAGTGCTCC AACCTGCTGCTGCAGTACGGCTCCTTTTGCACCCAGCTGAACAGAGCC CTGACCGGAATCGCCGTGGAGCAGGATAAAAACACCCAGGAAGTGTTC GCCCAGGTGAAACAGATCTACAAGACCCCCCCCATCAAAGATTTTGGA GGCTTTAATTTTAGCCAGATCCTGCCTGATCCCTCCAAACCCAGCAAGAGA TCCTTTATCGAGGATCTGCTGTTCAACAAAGTGACACTGGCCGATGCC GGATTTATCAAACAGTACGGAGACTGCCTGGGCGACATCGCCGCCAGA GACCTGATCTGCGCCCAGAAGTTTAACGGCCTGACCGTGCTGCCACCC CTGCTGACCGACGAGATGATCGCCCAGTATACCTCCGCCCTGCTGGCCGGC ACCATCACCTCCGGCTGGACCTTTGGCGCCGGAGCCGCCCTGCAGATC CCATTTGCCATGCAGATGGCCTACAGATTTAATGGAATCGGAGTGACC CAGAACGTGCTGTATGAGAACCAGAAACTGATCGCCAATCAGTTCAAC AGCGCCATCGGCAAGATCCAGGATTCTCTGAGCTCCACCGCCTCTGCCCTG GGCAAGCTGCAGGACGTGGTGAATCAGAACGCCCAGGCCCTGAACACC CTGGTGAAACAGCTGAGCAGCAACTTTGGCGCCATCAGCAGCGTGCTG AATGATATCCTGAGCAGACTGGATCCTCCAGAGGCCGAGGTGCAGATC GACAGACTGATCACAGGGAGACTGCAGAGCCTGCAGACCTATGTGACA CAGCAGCTGATCAGAGCCGCCGAAATTAGAGCCAGCGCCAACCTGGCC GCCACCAAAATGAGCGAGTGTGTGCTGGGACAGAGCAAAAGAGTGGAC TTCTGCGGCAAAGGCTACCATCTGATGAGCTTCCCCCAGAGCGCCCCC CACGGCGTGGTGTTCCTGCATGTGACCTACGTGCCCGCCCAGGAAAAA AATTTTACCACCGCCCCTGCCATCTGTCACGACGGCAAAGCCCATTTTCCC AGAGAGGGCGTGTTTGTGAGCAACGGCACCCACTGGTTTGTGACCCAG AGAAACTTTTACGAACCCCAGATCATCACCACAGATAATACCTTTGTG AGCGGAAATTGTGATGTGGTGATCGGCATCGTGAATAATACCGTGTAT GATCCTCTGCAGCCAGAGCTGGACAGCTTTAAGGAGGAGCTGGACAAG GATTTTAAAAATCATACCAGCCCCGACGTGGATCTGGGAGATATCAGCGGA ATCAACGCCTCCGTGGTGAATATCCAGAAGGAGATCGACAGACTGAAC GAGGTGGCCAAAAATCTGAATGAGTCCCTGATCGACCTGCAGGAGCTG GGCAAATATGAACAGTACATCAAGTGGCCATGGTACATCTGGCTGGGG TTTATCGCCGGACTGATCGCCATCGTGATGGTGACCATCATGCTGTGT TGCATGACCAGCTGTTGTAGCTGTCTGAAAGGCTGTTGTAGCTGCGGC AGCTGCTGCAAATTTGACGAGGATGATTCCGAGCCAGTGCTGAAGGGA GTGAAACTGCACTATACCTGA

[0174] The following describes the sequence of the fusion CVN polypeptide insert of the invention.

TABLE-US-00016 (SEQIDNO:9) MGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATR RIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTP KDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGS (SEQIDNO:10) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSTKKSAAEASKKPRQKRTAT KAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFF GMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFP*

[0175] In the above sequence, SEQ ID NO: 9 is the sequence bound to the N-terminus of the SARS-CoV-2-B.1.1.351 Spike protein, SEQ ID NO: 10 is the sequence bound to the C-terminus, the underline indicates the linker, and the arrow indicates the SARS-Indicates the location where the CoV-2-B.1.1.351 Spike protein is inserted.

TABLE-US-00017 (SEQIDNO:11) ATGGGCCTGCCCAACAACACCGCCAGCTGGTTTACCGCCCTGACCCAGCA CGGCAAGGAGGACCTGAAGTTCCCCAGAGGCCAGGGCGTGCCCATTAACA CCAACAGCAGCCCCGACGACCAGATCGGCTACTACAGAAGAGCCACCAGA AGAATCAGAGGCGGCGACGGCAAGATGAAGGACCTGAGCCCCAGATGGTA CTTCTACTATCTGGGAACCGGCCCCGAAGCCGGCCTGCCCTATGGCGCCA ACAAAGATGGCATCATCTGGGTGGCCACCGAAGGCGCCCTGAACACCCCC AAGGATCACATCGGAACCAGAAATCCCGCCAACAATGCCGCCATCGTGCT GCAGCTGCCCCAGGGAACCACCCTGCCTAAAGGCTTTTATGCCGAAGGAA GCAGAGGAGGAAGC (SEQIDNO:12) GGCGGCGGAGGAAGCGGCGGAGGAGGCAGCGGCGGAGGCGGAAGCGGCG GAGGAGGAAGCGGCGGAGGCGGAAGCGGCGGCGGAGGAAGCACCAAAAAG AGCGCCGCCGAGGCCAGCAAAAAGCCCAGACAGAAAAGAACCGCCACCAA GGCCTACAATGTGACCCAGGCCTTTGGCAGAAGAGGACCCGAGCAGACCC AGGGCAATTTTGGCGACCAGGAGCTGATCAGACAGGGCACCGACTACAAA CACTGGCCCCAGATCGCCCAGTTCGCCCCCAGCGCCAGCGCCTTCTTCGG CATGAGCAGAATCGGCATGGAGGTGACCCCCAGCGGCACCTGGCTGACCT ACACCGGCGCCATCAAGCTGGACGACAAGGACCCCAACTTCAAGGACCAG GTGATCCTGCTGAACAAGCACATCGACGCCTACAAGACCTTCCCCTGA

[0176] In the above sequence, SBQ ID NO: 11 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: 12 is a gene sequence encoding a polypeptide bound to the C-termins, 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.

TABLE-US-00018 [ChimAdvector-basedHPVvaccinesequence] SEQIDNO:13 Humanpapillomavirustype16,E6,E7&Human pappillomavirustype18,E6,E7modifiedamino acidsequence(Linkderunderlined) MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVY DFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYN KPLCDLLIRCIKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLMH GDTPTLHEYMLDLQPETTQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFC CKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPRGRKRRSMAR FEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLF VVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLINTGLYNLLI RCLKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQVMHGPK ATLQDIVLHLEPQNEIPVQLSDSEEENDEIDGVNHQHLPARRAEPQRHTM LCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVCPWCASQQ SEQIDNO:14 Humanpapillomavirustype16,E6,E7&Human papillomavirustype18,E6,E7modifiednucleic acidsequence(linkerunderlined) ATGCACCAGAAGAGAACAGCCATGTTCCAGGACCCTCAGGAACGGCCACG GAAGCTCCCACAACTTTGTACAGAGCTGCAAACAACCATTCATGACATCA TCCTTGAATGCGTTTACTGCAAGCAGCAACTTCTGCGCCGGGAAGTCTAC GATTTCGCTTTCAGAGATCTGTGCATCGTGTATAGAGACGGGAACCCCTA CGCCGTGTGTGACAAATGCCTTAAATTCTACAGTAAGATTTCAGAGTACC GGCATTATTGCTACAGCCTTTATGGAACTACATTGGAGCAGCAGTACAAC AAACCTTTGTGCGATCTTCTTATCCGGTGCATTAAGCAACGGCACTTGGA CAAAAAACAACGGTTCCATAACATCAGGGGCAGGTGGACTGGACGCTGCA TGAGTTGCTGTCGCTCAAGCCGCACTCGGAGAGAGACTCAACTGATGCAT GGTGATACTCCAACTTTGCACGAATATATGTTGGACCTCCAACCCGAAAC TACCCAACTGAATGACAGCTCTGAGGAAGAGGACGAAATCGACGGCCCTG CAGGGCAAGCCGAACCTGACAGGGCCCATTATAACATTGTGACATTTTGC TGCAAATGCGATAGTACTTTGAGACTCTGTGTGCAGTCAACTCATGTTGA CATTAGAACACTTGAAGACCTCCTCATGGGGACACTGGGGATTGTTTGCC CAATCTGTTCACAGAAACCCAAGAGGCCGCAAAAGGCGGTCTATGGCTCG CTTTGAGGACCCTACAAGGAGGCCCTACAAGCTCCCCGATTTGTGTACAG AACTGAATACCAGTCTTCAGGATATTGAAATCACTTGCGTCTACTGCAAA ACTGTGCTCGAACTTACCGAAGTCTTTGAATTTGCCTTCAAGGACCTTTT TGTTGTGTATCGCGATTCAATCCCACACGCAGCCTGTCACAAATGTATTG ACTTCTACTCTAGGATCAGAGAATTGAGGCACTATTCAGACTCTGTGTAT GGCGACACCTTGGAAAAATTGACCAACACTGGATTGTATAATCTGCTTAT TCGGTGTCTTAAGCTCCGGCACTTGAACGAAAAACGCAGATTCCACAACA TTGCCGGGCATTATCGCGGGCAATGCCATTCTTGTTGCAATCGCGCCCGC CAGGAGCGGCTGCAAAGGCGCAGAGAGACTCAGGTGATGCATGGACCCAA GGCCACTCTCCAGGACATCGTCCTTCATCTTGAACCCCAGAACGAAATTC CCGTTCAACTTAGCGACTCAGAAGAAGAAAATGATGAAATCGACGGAGTG AACCATCAGCATCTCCCAGCAAGAAGAGCCGAGCCACAAAGACACACAAT GCTCTGTATGTGCTGCAAGTGTGAAGCACGGATCGAACTCGTCGTCGAAT CTAGTGCAGATGATCTCAGAGCCTTTCAACAGCTCTTCTTGAATACACTG AGCTTTGTCTGTCCATGGTGCGCTAGTCAACAA