AD35-VECTORED VACCINE FOR PREVENTING SARS-COV-2 INFECTION

Abstract

Disclosed is an Ad35-vectored vaccine for preventing SARS-CoV-2 infection, comprising an Ad35 vector, wherein the Ad35 vector is loaded with a nucleic acid sequence shown in SEQ ID NO: 1. Some embodiments of the present disclosure have better safety and use convenience. Experiments have shown that the vaccine can produce more S proteins in human cells, which is expected to be developed as a vaccine for preventing SARS-CoV-2 infection. Some embodiments of the present disclosure may be used in combination with another vaccine or may also be used as a therapeutic vaccine for Corona Virus Disease 2019. When a patient is vaccinated with the Ad35-vectored vaccine of the present disclosure at the initial stage of infection, the vaccine quickly induces an immune response in the human body, thereby achieving a therapeutic effect.

Claims

1. An Ad35-vectored vaccine for preventing SARS-CoV-2 infection, comprising an Ad35 vector, wherein the Ad35 vector is loaded with a nucleic acid sequence shown in SEQ ID NO: 1.

2. The Ad35-vectored vaccine of claim 1, wherein the Ad35 vector is a replication-defective Ad35 vector.

3. The Ad35-vectored vaccine of claim 2, wherein the replication-defective Ad35 vector is a replication-defective Ad35 vector with genes in E1 and E3 regions deleted.

4. The Ad35-vectored vaccine of claim 1, wherein the Ad35 vector has an element for regulating the expression of the nucleic acid sequence shown in SEQ ID NO: 1.

5. The Ad35-vectored vaccine of claim 1, wherein the transcription direction of the nucleic acid sequence shown in SEQ ID NO: 1 is opposite to the transcription direction of the other genes of the Ad35 vector.

6. The Ad35-vectored vaccine of claim 1, wherein the nucleic acid sequence can be expressed as a protein in a human-derived cell or the human body.

7. The Ad35-vectored vaccine of claim 6, wherein the protein is capable of, in the human body: inducing an immune response; or generating a biological reporter molecule; or generating a trace molecule for detection; or regulating a gene function; or acting as a therapeutic molecule.

8. The Ad35-vectored vaccine of claim 1, wherein the vaccine further comprises at least one selected from the group consisting of pharmaceutically acceptable adjuvant, carrier, diluent or excipient.

9. The Ad35-vectored vaccine of claim 1, wherein the vaccine further comprises at least one drug that has a therapeutic effect on COVID-19.

10. The Ad35-vectored vaccine of claim 2, wherein the Ad35 vector has an element for regulating the expression of the nucleic acid sequence shown in SEQ ID NO: 1.

11. The Ad35-vectored vaccine of claim 3, wherein the Ad35 vector has an element for regulating the expression of the nucleic acid sequence shown in SEQ ID NO: 1.

12. The Ad35-vectored vaccine of claim 2, wherein the transcription direction of the nucleic acid sequence shown in SEQ ID NO: 1 is opposite to the transcription direction of the other genes of the Ad35 vector.

13. The Ad35-vectored vaccine of claim 3, wherein the transcription direction of the nucleic acid sequence shown in SEQ ID NO: 1 is opposite to the transcription direction of the other genes of the Ad35 vector.

14. The Ad35-vectored vaccine of claim 2, wherein the nucleic acid sequence can be expressed as a protein in a human-derived cell or the human body.

15. The Ad35-vectored vaccine of claim 3, wherein the nucleic acid sequence can be expressed as a protein in a human-derived cell or the human body.

16. The Ad35-vectored vaccine of claim 2, wherein the vaccine further comprises at least one selected from the group consisting of pharmaceutically acceptable adjuvant, carrier, diluent or excipient.

17. The Ad35-vectored vaccine of claim 3, wherein the vaccine further comprises at least one selected from the group consisting of pharmaceutically acceptable adjuvant, carrier, diluent or excipient.

18. The Ad35-vectored vaccine of claim 2, wherein the vaccine further comprises at least one drug that has a therapeutic effect on COVID-19.

19. The Ad35-vectored vaccine of claim 3, wherein the vaccine further comprises at least one drug that has a therapeutic effect on COVID-19.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 shows the detection results of the expression of S protein.

[0033] FIG. 2 shows the detection results of binding antibodies in the sera of macaque monkeys at different times after immunization.

[0034] FIG. 3 shows the results of an ELISpot assay on the peripheral blood cells (PBMCs) of macaque monkey 18 days after immunization.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] The amino acid sequence of Spike (S) protein of SARS-CoV-2 is shown in YP_009724390.1, with a full length of 1273 aa, denoted as NB1.

[0036] pre-mRNA transcribed by eukaryotic cells can produce various mRNA splicing isoforms by various splicing modes (by selecting different splicing site combinations), which ultimately leads to various proteins resulting from the same gene sequence. This is very unfavorable for the expression of the protein. By performing codon optimization on the wild-type natural nucleic acid sequence while removing potential variable splicing sites based on self-owned technology, the inventors ensured the uniqueness of the expression of the protein and reduced the difficulty in the subsequent purification of the protein. The optimized nucleic acid sequence is denoted as NB2, and the specific sequence thereof is shown in SEQ ID NO: 1:

TABLE-US-00001 (SEQ ID NO.: 1) ATGTTCGTGTTTCTGGTGCTGCTGCCTCTGGTGAGCTCCCAGTGCGTGAA CCTGACCACAAGGACCCAGCTGCCACCTGCCTATACCAATAGCTTCACAC GGGGCGTGTACTATCCCGACAAGGTGTTTAGATCTAGCGTGCTGCACTCC ACCCAGGATCTGTTTCTGCCTTTCTTTTCTAACGTGACATGGTTCCACGC CATCCACGTGTCCGGCACCAATGGCACAAAGCGGTTCGACAATCCAGTGC TGCCCTTTAACGATGGCGTGTACTTCGCCTCCACCGAGAAGTCTAACATC ATCAGAGGCTGGATCTTTGGCACCACACTGGACAGCAAGACCCAGTCCCT GCTGATCGTGAACAATGCCACAAACGTGGTCATCAAGGTGTGCGAGTTCC AGTTTTGTAATGATCCCTTCCTGGGCGTGTACTATCACAAGAACAATAAG TCTTGGATGGAGAGCGAGTTTAGGGTGTATTCCTCTGCCAACAATTGCAC CTTTGAGTACGTGAGCCAGCCTTTCCTGATGGACCTGGAGGGCAAGCAGG GCAATTTCAAGAACCTGAGGGAGTTCGTGTTTAAGAATATCGATGGCTAC TTCAAGATCTACTCCAAGCACACACCAATCAACCTGGTGCGCGACCTGCC ACAGGGCTTCTCTGCCCTGGAGCCACTGGTGGATCTGCCCATCGGCATCA ACATCACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGACC CCAGGCGACAGCTCCTCTGGATGGACAGCAGGAGCTGCCGCCTACTATGT GGGCTATCTGCAGCCCCGCACCTTCCTGCTGAAGTACAACGAGAATGGCA CCATCACAGACGCAGTGGATTGCGCCCTGGACCCCCTGTCTGAGACCAAG TGTACACTGAAGAGCTTTACAGTGGAGAAGGGCATCTACCAGACCAGCAA CTTCAGGGTGCAGCCAACAGAGTCCATCGTGCGCTTTCCCAATATCACCA ACCTGTGCCCTTTTGGCGAGGTGTTCAATGCCACACGCTTCGCCAGCGTG TACGCCTGGAATAGGAAGCGCATCTCCAACTGCGTGGCCGACTATTCTGT GCTGTACAACAGCGCCTCCTTCTCTACCTTTAAGTGTTATGGCGTGAGCC CCACCAAGCTGAATGATCTGTGCTTTACAAACGTGTACGCCGATTCCTTC GTGATCAGGGGCGACGAGGTGCGCCAGATCGCACCAGGACAGACCGGCAA GATCGCAGACTACAATTATAAGCTGCCTGACGATTTCACAGGCTGCGTGA TCGCCTGGAACTCTAACAATCTGGATAGCAAAGTGGGCGGCAACTACAAT TATCTGTACCGGCTGTTTAGAAAGTCTAATCTGAAGCCATTCGAGCGGGA CATCTCCACCGAGATCTACCAGGCCGGCTCTACACCCTGCAATGGCGTGG AGGGCTTTAACTGTTATTTCCCTCTGCAGTCCTACGGCTTCCAGCCAACC AACGGCGTGGGCTATCAGCCCTACAGAGTGGTGGTGCTGTCTTTTGAGCT GCTGCACGCACCTGCAACCGTGTGCGGCCCAAAGAAGAGCACAAATCTGG TGAAGAACAAGTGCGTGAACTTCAACTTCAACGGACTGACCGGCACAGGC GTGCTGACCGAGAGCAACAAGAAGTTCCTGCCATTTCAGCAGTTCGGCAG GGACATCGCAGATACCACAGACGCCGTGCGCGACCCTCAGACCCTGGAGA TCCTGGACATCACACCATGTTCCTTCGGCGGCGTGTCTGTGATCACCCCA GGCACCAATACATCCAACCAGGTGGCCGTGCTGTATCAGGACGTGAATTG CACAGAGGTGCCCGTGGCAATCCACGCAGATCAGCTGACCCCTACATGGC GGGTGTACTCTACCGGCAGCAACGTGTTCCAGACAAGAGCCGGATGCCTG ATCGGAGCAGAGCACGTGAACAATAGCTATGAGTGCGACATCCCTATCGG CGCCGGCATCTGTGCCTCCTACCAGACCCAGACAAACTCCCCAAGGAGAG CCCGGTCTGTGGCCAGCCAGTCCATCATCGCCTATACCATGAGCCTGGGC GCCGAGAACAGCGTGGCCTACTCCAACAATTCTATCGCCATCCCTACCAA CTTCACAATCAGCGTGACCACAGAGATCCTGCCAGTGAGCATGACCAAGA CATCCGTGGACTGCACCATGTATATCTGTGGCGATTCCACAGAGTGTTCT AACCTGCTGCTGCAGTACGGCTCCTTTTGCACCCAGCTGAATAGAGCCCT GACAGGCATCGCCGTGGAGCAGGACAAGAACACCCAGGAGGTGTTCGCCC AGGTGAAGCAGATCTACAAGACACCACCCATCAAGGACTTTGGCGGCTTC AACTTCAGCCAGATCCTGCCCGATCCTAGCAAGCCATCCAAGCGGTCTTT TATCGAGGACCTGCTGTTCAACAAGGTGACCCTGGCCGATGCCGGCTTCA TCAAGCAGTATGGCGATTGTCTGGGCGACATCGCCGCCAGAGACCTGATC TGCGCCCAGAAGTTTAATGGCCTGACCGTGCTGCCTCCACTGCTGACAGA TGAGATGATCGCACAGTACACCTCTGCCCTGCTGGCCGGCACCATCACAA GCGGATGGACATTCGGCGCAGGAGCCGCCCTGCAGATCCCCTTTGCCATG CAGATGGCCTATCGGTTCAACGGCATCGGCGTGACCCAGAATGTGCTGTA CGAGAACCAGAAGCTGATCGCCAATCAGTTTAACAGCGCCATCGGCAAGA TCCAGGACTCTCTGAGCTCCACCGCCAGCGCCCTGGGCAAGCTGCAGGAT GTGGTGAATCAGAACGCCCAGGCCCTGAATACACTGGTGAAGCAGCTGTC TAGCAACTTCGGCGCCATCTCCTCTGTGCTGAATGACATCCTGAGCCGGC TGGACAAGGTGGAGGCAGAGGTGCAGATCGACCGGCTGATCACCGGCAGA CTGCAGTCCCTGCAGACCTACGTGACACAGCAGCTGATCAGGGCAGCAGA GATCAGGGCCTCTGCCAATCTGGCCGCCACAAAGATGAGCGAGTGCGTGC TGGGACAGTCCAAGAGGGTGGACTTTTGCGGCAAGGGCTATCACCTGATG AGCTTCCCACAGTCCGCCCCTCACGGAGTGGTGTTTCTGCACGTGACCTA CGTGCCAGCCCAGGAGAAGAACTTCACCACAGCCCCCGCCATCTGTCACG ATGGCAAGGCCCACTTTCCTAGGGAGGGCGTGTTCGTGAGCAACGGCACC CACTGGTTTGTGACACAGCGCAATTTCTACGAGCCACAGATCATCACCAC AGACAATACCTTCGTGTCCGGCAACTGCGACGTGGTCATCGGCATCGTGA ACAATACAGTGTATGATCCTCTGCAGCCAGAGCTGGACTCTTTTAAGGAG GAGCTGGATAAGTACTTCAAGAATCACACCAGCCCCGACGTGGATCTGGG CGACATCTCTGGCATCAATGCCAGCGTGGTGAACATCCAGAAGGAGATCG ACAGACTGAACGAGGTGGCCAAGAATCTGAACGAGAGCCTGATCGATCTG CAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCCTGGTATATCTG GCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGC TGTGCTGTATGACAAGCTGCTGTTCCTGCCTGAAGGGCTGCTGTTCTTGT GGCAGCTGCTGTAAGTTTGATGAGGACGATTCCGAGCCTGTGCTGAAGGG CGTGAAGCTGCACTACACCTAA.

[0037] Construction of Ad35 vector for S protein expression pAd35-NB2:

[0038] With NB1 and NB2 as templates, respectively, PCR amplification was carried out by using NB1-F and NB1-R as primers to obtain an NB1 fragment and by using NB2-F and NB2-R as primers to obtain an NB2 fragment; furthermore, with CMV-R and BGH-F as primers, respectively, and pGA351-EGFP plasmid as a template, the vector plasmid backbone pGA351 was subjected to PCR amplification and then to in vitro two-fragment recombination with the NB1 and NB2 fragments, respectively, using a homologous recombinase (Exnase) to obtain pGA351-NB1 and pGA351-NB2. The pGA351-NB2 was linearized using Bstzl7I and SgarAI, and was subjected to homologous recombination with pAd35ΔE1ΔE3 (i.e., Ad35 empty vector), which was digested to delete the unique restriction site in the E1 region and linearized, by using competent BJ5183 to construct a pAd35-NB2 vaccine vector.

[0039] Primer sequence for amplifying NB1:

TABLE-US-00002 NB1-F: (SEQ ID NO.: 2) GCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCGCCACCATGTTTGT TTTTCTTGT NB1-R: (SEQ ID NO.: 3) AGAATAGGGCCCTCTAGACTAGTTTATGTGTAATGTAATTTG

[0040] Primer sequence for amplifying NB2:

TABLE-US-00003 NB2-F: (SEQ ID NO.: 4) GCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCGCCACCATGTTCGT GTTTCTGGT NB2-R: (SEQ ID NO.: 5) AGAATAGGGCCCTCTAGACTAGTTTATCAGGTGTAGTGCAGCTTC

TABLE-US-00004 BGH-F: (SEQ ID NO.: 6) TCTAGAGGGCCCTATTCTATAGTGTC CMV-R: (SEQ ID NO.: 7) GGATCCGAGCTCGGTACCAAGCTTAAGTTTAAACGCTAGAGTCCGG

[0041] PCR conditions: 95° C., 3 min; 95° C., 30 s; 60° C., 30 s; 72° C., 2 min; cycles 30; 72° C., 5 min.

[0042] Rescue and production of Ad35-NB2 vector [0043] 1) According to a conventional method, pAd35-NB2 was linearized with AsiSI, recovered by means of precipitation in ethanol, and transfected into 293 cells with cationic liposome transfection method; [0044] 2) 8 hours after transfection, 2 ml of a DMEM medium containing 5% fetal bovine serum was added, incubated for 7-10 days, and cytopathic effect observation was carried out; [0045] 3) after the cytopathic effect was observed, the cells and culture supernatant were collected, freeze-thawed for three times in a water bath at 37° C. and liquid nitrogen, and centrifugated to remove cell debris, and then a 10 cm dish was infected with the supernatant; [0046] 4) after 2 or 3 days, the cells and culture supernatant were collected, freeze-thawed for 3 times, centrifugated to remove cell debris, and then 3-5 15 cm dishes were infected with the supernatant; [0047] 5) after 2 or 3 days, the cells were collected, freeze-thawed for 3 times, and centrifugated t to remove cell debris; [0048] 6) 30 15 cm dishes were infected with the supernatant; after 2 or 3 days, the cells were collected, freeze-thawed for 3 times, and centrifugated to remove cell debris; [0049] 7) the supernatant was added to a cesium chloride density gradient centrifuge tube, and centrifuged at 4° C., 40000 rpm, for 4 hours, and a virus band was pipetted out, desalted, and subpackaged; and [0050] 8) the virion titer was determined by means of OD260 absorbance, with the calculation formula being: virus concentration =OD260×dilution multiple×36/genome length (Kb), and the virus stock solution was cryopreserved at −80° C.

[0051] Detection of Spike gene expression:

[0052] According to a conventional method, 2.5 μg of pGA351-NB1 and pGA351-NB2 were respectively transfected into A549 cells using a cationic liposome, and after 48 hours, the cells were collected. The A549 cells were infected with Ad35-NB2 virus, and after 36 h, the cells were collected. The four samples mentioned above were treated with the conventional Western Blot method, and were detected for the protein (FIG. 1).

[0053] It could be seen from FIG. 1 that no S protein expression was detected in the pGA351-NB1 sample, while the S protein expression could be observed in the codon-optimized pGA351-NB2 and vaccine candidate strain Ad35-NB2 samples, indicating that the NB2 sequence has unexpected effects.

[0054] Immunogenicity evaluation:

[0055] Macaque monkeys were obtained from Guangdong Landau Biotechnology Co. Ltd. The vaccinated macaque monkeys were 2 or 3 years old. The macaque monkeys were randomly divided into 3 groups, with 2 monkeys in each experimental group and 4 monkeys in a control group, specifically as shown in the following table:

TABLE-US-00005 Group Macaque Immunization No. monkey Sex dose/mode 1 170060 Female Ad35-NB2 (1 × 10.sup.11 vp) 170040 Female Intramuscular (I.M.) injection 2 170025 Male Ad35-NB2 (1 × 10.sup.11 vp) 170051 Male intranasal (I.N.) immunization 3 180026 Female — 180024 Female 180023 Male 180039 Male

[0056] Macaque monkeys were immunized with the vaccine prepared from the Ad35-NB2 virus strain, blood was taken at days 14 and 18, respectively, after immunization, so as to determine the antibody binding titer by using an ELISA method. The peripheral blood was separated and detected for a cellular immune response by using the ELISpot method.

Experimental Results:

(1) Binding Antibodies

[0057] At days 14 and 18 after vaccination through intramuscular injection, significant presence of S-specific IgG and RBD-specific IgG could be detected in the sera of all the macaque monkeys intramuscularly injected with 1×10.sup.11 VP, and among the two macaque monkeys in the immunization group, significant presence of anti-S2 IgG titers was detected in one macaque monkey (FIG. 2).

[0058] At day 14 after intranasal immunization n vaccination, significant presence of S-specific IgG and RBD-specific IgG could be detected in the sera of all the macaque monkeys vaccinated with 1×10.sup.11 VP, and anti-S2 IgG titers could also be detected, but was relatively weak (FIG. 2).

[0059] The difference between the antibody titers induced by intramuscular injection immunization and intranasal immunization was small.

(2) Cellular Immunity

[0060] In order to determine if the Ad35-NB2 could also induce a cellular immune response in non-human primates (NHPs), the inventors detected the response of S-specific IFN-γ secreting cells from peripheral blood mononuclear cells (PBMCs) to S1 and S2 peptide libraries.

[0061] The results showed that:

[0062] 1) At day 18 after intramuscular injection vaccination, all the macaque monkeys intramuscularly injected with 1×10.sup.11 VP had cellular immune responses to the S1 and S2 peptide libraries (FIG. 3).

[0063] 2) At day 18 after intranasal immunization vaccination, among the two vaccinated macaque monkeys, one macaque monkey had a weak cellular immune response to the S1 peptide library, and neither of them had an obvious response to the S2 peptide library at day 18 (FIG. 3).

[0064] Therefore, in the macaque monkeys, the cellular immune response was mainly directed at the S1 region. These results indicated that immunization through intramuscular injection could cause a systemic cellular immune response to the S protein, especially to the S1 region, while the systemic cellular immune response caused by the mucosal vaccination of the vaccine was relatively weak.

[0065] The above experimental results showed that the vaccine could stimulate macaque monkeys to produce cellular immunity, which could further improve the protection of the body.