AD7-VECTORED VACCINE FOR PREVENTING SARS- COV-2 INFECTION

Abstract

Disclosed is an Ad7-vectored vaccine for preventing SARS-CoV-2 infection, which is an adenovirus vectored vaccine, comprising an Ad7 vector loaded with a nucleic acid sequence shown in SEQ ID NO: 1. Some embodiments of the disclosure have good safety and are convenient to use. Experiments have shown that the vaccine is capable of producing more S protein in human cells, which is expected to be developed as a vaccine for preventing SARS-CoV-2 infection. Some embodiments of the disclosure may be used in combination with other vaccines, and may also be used as therapeutic vaccines for COVID-19. When a patient is vaccinated with the Ad7-vectored vaccine of the present disclosure at the initial stage of infection, the vaccine can quickly induces immune response in the human body, thereby achieving a therapeutic effect.

Claims

1. An adenovirus vectored vaccine for preventing SARS-CoV-2 infection, comprising an Ad7 vector loaded with a nucleic acid sequence shown in SEQ ID NO: 1.

2. The adenovirus vectored vaccine of claim 1, wherein the Ad7 vector is a replication-defective Ad7 vector.

3. The adenovirus vectored vaccine of claim 2, wherein the replication-defective Ad7 vector is a replication-defective Ad7 vector with genes in E1 and E3 regions deleted.

4. The adenovirus vectored vaccine of claim 1, wherein the nucleotide sequence can be expressed as protein in a human-derived cell or the human body.

5. The adenovirus vectored vaccine of claim 4, wherein, in the human body, the protein is capable of: inducing an immune response; or generating a biology reporter molecule; or generating a trace molecule for detection; or regulating gene function; or functioning as a therapeutic molecule.

6. The adenovirus vectored vaccine of claim 1, further comprising a pharmaceutically acceptable adjuvant, vector, diluent or excipient.

7. The adenovirus vectored vaccine of claim 1, further comprising at least one medicament useful for treating COVID-19.

8. The adenovirus vectored vaccine of claim 1, wherein the Ad7 vector is capable of regulating the expression of the nucleic acid sequence shown in SEQ ID NO: 1.

9. The adenovirus vectored vaccine of claim 1, wherein the transcription direction of the nucleic acid sequence shown in SEQ ID NO: 1 is opposite to those of the other genes in the Ad7 vector.

10. The adenovirus vectored vaccine of claim 2, wherein the nucleotide sequence can be expressed as protein in a human-derived cell or the human body.

11. The adenovirus vectored vaccine of claim 3, wherein the nucleotide sequence can be expressed as protein in a human-derived cell or the human body.

12. The adenovirus vectored vaccine of claim 2, further comprising a pharmaceutically acceptable adjuvant, vector, diluent or excipient.

13. The adenovirus vectored vaccine of claim 3, further comprising a pharmaceutically acceptable adjuvant, vector, diluent or excipient.

14. The adenovirus vectored vaccine of claim 2, further comprising at least one medicament useful for treating COVID-19.

15. The adenovirus vectored vaccine of claim 3, further comprising at least one medicament useful for treating COVID-19.

16. The adenovirus vectored vaccine of claim 2, wherein the Ad7 vector is capable of regulating the expression of the nucleic acid sequence shown in SEQ ID NO: 1.

17. The adenovirus vectored vaccine of claim 3, wherein the Ad7 vector is capable of regulating the expression of the nucleic acid sequence shown in SEQ ID NO: 1.

18. The adenovirus vectored vaccine of claim 2, wherein the transcription direction of the nucleic acid sequence shown in SEQ ID NO: 1 is opposite to those of the other genes in the Ad7 vector.

19. The adenovirus vectored vaccine of claim 3, wherein the transcription direction of the nucleic acid sequence shown in SEQ ID NO: 1 is opposite to those of the other genes in the Ad7 vector.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

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

[0033] FIG. 2 shows detection results of binding antibodies in serum of mice 28 days after immunization.

[0034] FIG. 3 shows detection results of binding antibodies in serum of macaque monkeys at different times after immunization.

[0035] FIG. 4 shows ELISpot detection results of peripheral blood PBMCs of the macaque monkeys 18 days after immunization.

DETAILED DESCRIPTION

[0036] The amino acid sequence of Spike (S) protein of SARS-CoV-2 is shown in YP_009724390.1, which is denoted as NB1.

[0037] The 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 and 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 is shown in SEQ ID NO: 1:

TABLE-US-00001 (SEQ ID NO.: 1) ATGTTCGTGTTTCTGGTGCTGCTGCCTCTGGTGAGCTCCCAGTGCGTGA ACCTGACCACAAGGACCCAGCTGCCACCTGCCTATACCAATAGCTTCAC ACGGGGCGTGTACTATCCCGACAAGGTGTTTAGATCTAGCGTGCTGCAC TCCACCCAGGATCTGTTTCTGCCTTTCTTTTCTAACGTGACATGGTTCC ACGCCATCCACGTGTCCGGCACCAATGGCACAAAGCGGTTCGACAATCC AGTGCTGCCCTTTAACGATGGCGTGTACTTCGCCTCCACCGAGAAGTCT AACATCATCAGAGGCTGGATCTTTGGCACCACACTGGACAGCAAGACCC AGTCCCTGCTGATCGTGAACAATGCCACAAACGTGGTCATCAAGGTGTG CGAGTTCCAGTTTTGTAATGATCCCTTCCTGGGCGTGTACTATCACAAG AACAATAAGTCTTGGATGGAGAGCGAGTTTAGGGTGTATTCCTCTGCCA ACAATTGCACCTTTGAGTACGTGAGCCAGCCTTTCCTGATGGACCTGGA GGGCAAGCAGGGCAATTTCAAGAACCTGAGGGAGTTCGTGTTTAAGAAT ATCGATGGCTACTTCAAGATCTACTCCAAGCACACACCAATCAACCTGG TGCGCGACCTGCCACAGGGCTTCTCTGCCCTGGAGCCACTGGTGGATCT GCCCATCGGCATCAACATCACCCGGTTTCAGACACTGCTGGCCCTGCAC AGAAGCTACCTGACCCCAGGCGACAGCTCCTCTGGATGGACAGCAGGAG CTGCCGCCTACTATGTGGGCTATCTGCAGCCCCGCACCTTCCTGCTGAA GTACAACGAGAATGGCACCATCACAGACGCAGTGGATTGCGCCCTGGAC CCCCTGTCTGAGACCAAGTGTACACTGAAGAGCTTTACAGTGGAGAAGG GCATCTACCAGACCAGCAACTTCAGGGTGCAGCCAACAGAGTCCATCGT GCGCTTTCCCAATATCACCAACCTGTGCCCTTTTGGCGAGGTGTTCAAT GCCACACGCTTCGCCAGCGTGTACGCCTGGAATAGGAAGCGCATCTCCA ACTGCGTGGCCGACTATTCTGTGCTGTACAACAGCGCCTCCTTCTCTAC CTTTAAGTGTTATGGCGTGAGCCCCACCAAGCTGAATGATCTGTGCTTT ACAAACGTGTACGCCGATTCCTTCGTGATCAGGGGCGACGAGGTGCGCC AGATCGCACCAGGACAGACCGGCAAGATCGCAGACTACAATTATAAGCT GCCTGACGATTTCACAGGCTGCGTGATCGCCTGGAACTCTAACAATCTG GATAGCAAAGTGGGCGGCAACTACAATTATCTGTACCGGCTGTTTAGAA AGTCTAATCTGAAGCCATTCGAGCGGGACATCTCCACCGAGATCTACCA GGCCGGCTCTACACCCTGCAATGGCGTGGAGGGCTTTAACTGTTATTTC CCTCTGCAGTCCTACGGCTTCCAGCCAACCAACGGCGTGGGCTATCAGC CCTACAGAGTGGTGGTGCTGTCTTTTGAGCTGCTGCACGCACCTGCAAC CGTGTGCGGCCCAAAGAAGAGCACAAATCTGGTGAAGAACAAGTGCGTG AACTTCAACTTCAACGGACTGACCGGCACAGGCGTGCTGACCGAGAGCA ACAAGAAGTTCCTGCCATTTCAGCAGTTCGGCAGGGACATCGCAGATAC CACAGACGCCGTGCGCGACCCTCAGACCCTGGAGATCCTGGACATCACA CCATGTTCCTTCGGCGGCGTGTCTGTGATCACCCCAGGCACCAATACAT CCAACCAGGTGGCCGTGCTGTATCAGGACGTGAATTGCACAGAGGTGCC CGTGGCAATCCACGCAGATCAGCTGACCCCTACATGGCGGGTGTACTCT ACCGGCAGCAACGTGTTCCAGACAAGAGCCGGATGCCTGATCGGAGCAG AGCACGTGAACAATAGCTATGAGTGCGACATCCCTATCGGCGCCGGCAT CTGTGCCTCCTACCAGACCCAGACAAACTCCCCAAGGAGAGCCCGGTCT GTGGCCAGCCAGTCCATCATCGCCTATACCATGAGCCTGGGCGCCGAGA ACAGCGTGGCCTACTCCAACAATTCTATCGCCATCCCTACCAACTTCAC AATCAGCGTGACCACAGAGATCCTGCCAGTGAGCATGACCAAGACATCC GTGGACTGCACCATGTATATCTGTGGCGATTCCACAGAGTGTTCTAACC TGCTGCTGCAGTACGGCTCCTTTTGCACCCAGCTGAATAGAGCCCTGAC AGGCATCGCCGTGGAGCAGGACAAGAACACCCAGGAGGTGTTCGCCCAG GTGAAGCAGATCTACAAGACACCACCCATCAAGGACTTTGGCGGCTTCA ACTTCAGCCAGATCCTGCCCGATCCTAGCAAGCCATCCAAGCGGTCTTT TATCGAGGACCTGCTGTTCAACAAGGTGACCCTGGCCGATGCCGGCTTC ATCAAGCAGTATGGCGATTGTCTGGGCGACATCGCCGCCAGAGACCTGA TCTGCGCCCAGAAGTTTAATGGCCTGACCGTGCTGCCTCCACTGCTGAC AGATGAGATGATCGCACAGTACACCTCTGCCCTGCTGGCCGGCACCATC ACAAGCGGATGGACATTCGGCGCAGGAGCCGCCCTGCAGATCCCCTTTG CCATGCAGATGGCCTATCGGTTCAACGGCATCGGCGTGACCCAGAATGT GCTGTACGAGAACCAGAAGCTGATCGCCAATCAGTTTAACAGCGCCATC GGCAAGATCCAGGACTCTCTGAGCTCCACCGCCAGCGCCCTGGGCAAGC TGCAGGATGTGGTGAATCAGAACGCCCAGGCCCTGAATACACTGGTGAA GCAGCTGTCTAGCAACTTCGGCGCCATCTCCTCTGTGCTGAATGACATC CTGAGCCGGCTGGACAAGGTGGAGGCAGAGGTGCAGATCGACCGGCTGA TCACCGGCAGACTGCAGTCCCTGCAGACCTACGTGACACAGCAGCTGAT CAGGGCAGCAGAGATCAGGGCCTCTGCCAATCTGGCCGCCACAAAGATG AGCGAGTGCGTGCTGGGACAGTCCAAGAGGGTGGACTTTTGCGGCAAGG GCTATCACCTGATGAGCTTCCCACAGTCCGCCCCTCACGGAGTGGTGTT TCTGCACGTGACCTACGTGCCAGCCCAGGAGAAGAACTTCACCACAGCC CCCGCCATCTGTCACGATGGCAAGGCCCACTTTCCTAGGGAGGGCGTGT TCGTGAGCAACGGCACCCACTGGTTTGTGACACAGCGCAATTTCTACGA GCCACAGATCATCACCACAGACAATACCTTCGTGTCCGGCAACTGCGAC GTGGTCATCGGCATCGTGAACAATACAGTGTATGATCCTCTGCAGCCAG AGCTGGACTCTTTTAAGGAGGAGCTGGATAAGTACTTCAAGAATCACAC CAGCCCCGACGTGGATCTGGGCGACATCTCTGGCATCAATGCCAGCGTG GTGAACATCCAGAAGGAGATCGACAGACTGAACGAGGTGGCCAAGAATC TGAACGAGAGCCTGATCGATCTGCAGGAGCTGGGCAAGTATGAGCAGTA CATCAAGTGGCCCTGGTATATCTGGCTGGGCTTCATCGCCGGCCTGATC GCCATCGTGATGGTGACCATCATGCTGTGCTGTATGACAAGCTGCTGTT CCTGCCTGAAGGGCTGCTGTTCTTGTGGCAGCTGCTGTAAGTTTGATGA GGACGATTCCGAGCCTGTGCTGAAGGGCGTGAAGCTGCACTACACCTA A.

[0038] Construction of Ad7 vector for S protein expression (pAd7-NB2):

[0039] Using NB1 and NB2 as templates respectively, an NB1 fragment was obtained through PCR amplification with primers NB1-F and NB1-R, and an NB2 fragment was obtained through PCR amplification with primers NB2-F and NB2-R. A vector plasmid backbone pGK was obtained through PCR amplification by using CMV-R and BGH-F as primers and using pGA1-EGFP plasmid as a template. Then, in vitro two-fragment recombination with the NB1 and NB2 fragments was carried out, respectively, by using a homologous recombination enzyme (Exnase), to obtain pGK-NB1 and pGK-NB2. The pGK-NB2 was linearized by using Bstzl7I and SgarAI, and was subjected to homologous recombination with pAd7AE1AE3 (i.e., Ad7 empty vector), which was digested to delete the unique restriction site in the E1 region and linearized, by using competent BJ5183 to construct a pAd7-NB2 vaccine vector.

TABLE-US-00002 Primer sequences for amplifying NB1: NB1-F: (SEQ ID NO.: 2) GCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCGCCACCATGTTTG TTTTTCTTGT NB1-R: (SEQ ID NO: 3) AGAATAGGGCCCTCTAGACTAGTTTATGTGTAATGTAATTTG Primer sequences for amplifying NB2: NB2-F: (SEQ ID NO.: 4) GCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCGCCACCATGTTCGT GTTTCTGGT NB2-R: (SEQ ID NO.: 5) AGAATAGGGCCCTCTAGACTAGTTTATCAGGTGTAGTGCAGCTTC Primer sequences for amplifying pGK: BGH-F: (SEQ ID NO.: 6) TCTAGAGGGCCCTATTCTATAGTGTC CMV-R: (SEQ ID NO.: 7) GGATCCGAGCTCGGTACCAAGCTTAAGTTTAAACGCTAGAGTCCGG 
PCR conditions: 95° C. for 3 min; 95° C. for 30 s; 60° C. for 30 s; 72° C. for 2 min; 30 cycles; and 72° C. for 5 min.

[0040] Rescue and production of Ad7-NB2 vector:

[0041] 1) according to a conventional method, pAd7-NB2 was linearized with AsiSI, recovered by precipitation in ethanol, and transfected into 293 cells by means of cationic lipofection;

[0042] 2) 8 hours after the transfection, 2 ml of DMEM medium containing 5% fetal bovine serum was added, incubated for 7-10 days, and cytopathic effect observation was carried out; 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;

[0043] 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;

[0044] 5) after 2 or 3 days, the cells were collected, freeze-thawed for 3 times, and centrifugated to remove cell debris;

[0045] 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;

[0046] 7) the supernatant was added to a cesium chloride density gradient centrifuge tube;, and centrifuged at 4° C., 40000 rpm, for 4 hours, then a virus band was pipetted out, desalted, and subpackaged; and

[0047] 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.

[0048] Detection of Spike Gene Expression:

[0049] According to a conventional method, 2.5 μg of pGK-NB1 and pGK-NB2 were respectively transfected using cationic liposomes, and after 48 hours, the cells were collected. The HEK293 cells were infected with Ad7-NB2 and Ad7 empty vector viruses, and after 24 hours, the cells were collected. The four samples mentioned above were processed according to the conventional Western Blot method, and were detected for the protein (FIG. 1).

[0050] It could be seen from FIG. 1 that no S protein expression was detected in the pGK-NB1 sample, while the expression of S protein could be observed in the codon-optimized pGK-NB2 and vaccine candidate strain Ad7-NB2 samples, indicating that the NB2 sequence has unexpected effects.

[0051] Immunogenicity Evaluation of Ad7-NB2 Vaccine:

[0052] 1. Immunized Mice

[0053] An immunogenicity evaluation scheme of the Ad7-NB2 vaccine in mice was designed, as shown in Table 1, and immunization was carried out according to the designed immunization scheme.

TABLE-US-00003 TABLE 1 Primary immunization (0 day) Detection Grouping Vaccine type Vaccine dosage (28.sup.th day) G1 GT101 — 100 ul Detection of G2 Ad7-NB2 5 × 10.sup.9 VP/animal  1 ml binding antibody against S protein

[0054] Balb/c mice, 6 to 8 weeks old, were divided into 2 groups with 5 mice in each group. The G1 group was an experimental control group and mice in the G1 group were intramuscularly injected with GT101 (the virus stock solution) at a dosage of 5×10.sup.9VP/animal, while the G2 group was an experimental group and mice in the G2 group were intramuscularly injected with the Ad7-NB2 vaccine at a dosage of 5×10.sup.9VP/animal. At the 28.sup.th day after immunization, blood was taken from orbit, and serum was separated. Binding antibody levels of the S protein of SARS-ncov2 were detected. From FIG. 2, the experimental group could produce a high titer of binding antibody against the S protein of SARS-ncov2 after immunization.

[0055] 2. Immunized Macaque Monkeys

[0056] The macaque monkeys were obtained from Guangdong Landau Biotechnology Co. Ltd. The vaccinated macaque monkeys were 2 to 3 years old. The macaque monkeys were randomly divided into 3 groups, with 2 monkeys in each experimental group, and 4 monkeys in each control group, specifically as shown in Table 2:

TABLE-US-00004 TABLE 2 Groups Macaque No. monkeys Gender Immunization dose/mode 1 170082 Female Ad7-NB2( vp) 170018 Female Intramuscular (I.M.) injection 2 181045 Male Ad7-NB2(1 × 10.sup.11 vp) 180021 Male Intranasal (I.N.) immunization 3 180026 Female — 180024 Female 180023 Male 180039 Male

[0057] Macaque monkeys were immunized with the vaccine prepared from the Ad7-NB2 virus strain. Blood was taken at the 14.sup.th day and the 18.sup.th day after immunization to determine antibody binding titers by ELISA. The peripheral blood was separated and detected for a cellular immune response by ELISpot assay.

[0058] Experimental Results:

[0059] (1) Binding Antibodies

[0060] At the 14.sup.th day and the 18.sup.th day after intramuscular vaccination, 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.11VP, and among the two macaque monkeys in the immunization group, significant presence of anti-Ω IgG could be detected in one macaque monkey (FIG. 3).

[0061] At the 14.sup.th day after the intranasal vaccination, among the two macaque monkeys vaccinated with 1×10.sup.11 VP in the immunization group, significant presence of S-specific IgG and RBD-specific IgG could be detected in one macaque monkey. S2-specific IgG could be detected in all macaque monkeys in the immunization group. At the 18.sup.th day after immunization, S-specific IgG and S2-specific IgG could both be detected. Among the two macaque monkeys in the immunization group, significant presence of RBD-specific IgG could be detected in one monkey (FIG. 3).

[0062] The difference between the antibody titers induced by intramuscular injection immunization and intranasal immunization was small.

[0063] (2) Cellular Immunity

[0064] To determine whether Ad7-NB2 could also induce a cellular immune response in non-human primates (NHPs), the responses of S-specific IFN-γ secreting cells from the peripheral blood mononuclear cells (PBMCs) to S1 and S2 peptide libraries were detected.

[0065] The Results Showed that:

[0066] 1) At the 18.sup.th day after the intramuscular injection vaccination, all macaque monkeys intramuscularly injected with 1×10.sup.11VP had cellular immune responses to the S1 and S2 peptide libraries (FIG. 4).

[0067] 2) At the 18.sup.th day after the intranasal vaccination, among the two vaccinated macaque monkeys, one macaque monkey showed weak cellular immune response to the S1 peptide library, and neither of them had obvious response to the S2 peptide library at the 18.sup.th day (FIG. 4).

[0068] Therefore, in the macaque monkeys, the cellular immune response was mainly directed at the S1 region. These results indicate that the intramuscular injection immunization of the vaccine could cause systemic cellular immune response to S protein, especially to the S1 region, while the systemic cellular immune response caused by the mucosal immunization of the vaccine was relatively weak.

[0069] The above experimental results showed that the vaccine could induce cellular immunity in macaque monkeys, which may further enhance the protective effect on the body.