WO2022077593A1 - 一种SARS-CoV-2冠状病毒疫苗及其制备方法 - Google Patents

一种SARS-CoV-2冠状病毒疫苗及其制备方法 Download PDF

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WO2022077593A1
WO2022077593A1 PCT/CN2020/125329 CN2020125329W WO2022077593A1 WO 2022077593 A1 WO2022077593 A1 WO 2022077593A1 CN 2020125329 W CN2020125329 W CN 2020125329W WO 2022077593 A1 WO2022077593 A1 WO 2022077593A1
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sars
cov
sequence
coronavirus
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黄文林
周晓鸿
田烁
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广州达博生物制品有限公司
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Definitions

  • the invention belongs to the field of biological genetic engineering, in particular to a SARS-CoV-2 coronavirus vaccine.
  • the new coronavirus has a strong ability to spread between people, and the transmission index R0 is about 2.5.
  • the virus mainly infects people through respiratory tract or contact, can cause acute pulmonary lesions, and can also cause serious damage to other systems such as the urinary system, digestive system and nervous system. There is currently no effective drug treatment. Therefore, it is very important to design and develop an effective vaccine against the virus.
  • the SARS-CoV-2 coronavirus is a single-stranded RNA virus, which is very unstable, subject to immune supervision and rejection in somatic cells, and is prone to mutation. Therefore, vaccines designed based on antigenic determinants with relatively conserved sequences can induce relatively stable antibodies, effectively reduce the phenomenon of vaccine failure caused by virus mutation, and achieve long-term protection.
  • SARS-CoV-2 virus is a non-segmented single-stranded positive-stranded RNA virus with 5 genomes and a molecular weight of 32 kb.
  • the SARS-CoV-2 virus particle is about 100nm in size and has multiple sparse rod-shaped proteins on its surface. Electron microscope photos show that the virus is shaped like a crown and belongs to the coronavirus.
  • the genome of SARS-CoV-2 virus mainly encodes four structural proteins, spike protein (spike, S protein), nucleoprotein (nucleoprotein, N protein), membrane protein (membrane, M protein), envelope protein (envelop, E protein).
  • spike protein spike protein
  • nucleoprotein nucleoprotein
  • membrane protein membrane protein
  • envelope protein envelope protein
  • the spike protein/S protein is a transmembrane glycoprotein containing important virus neutralizing epitopes.
  • SARS-CoV-2 virus S protein binds to host cell receptors (such as angiotensin converting enzyme 2 [angiotensin I covering enzyme, ACE2]), which can mediate viral entry through endocytosis.
  • host cell receptors such as angiotensin converting enzyme 2 [angiotensin I covering enzyme, ACE2]
  • ACE2 angiotensin I covering enzyme
  • the isolation of the SARS-CoV-2 virus provides raw material for the development of vaccines.
  • new coronavirus vaccines mainly include inactivated vaccines, subunit vaccines and DNA vaccines that are under development or entering the clinical stage.
  • Inactivated vaccines are technically easy to prepare and can efficiently induce humoral immunity. They are the most classic form of vaccines.
  • the mass production process of inactivated vaccines may pose a risk of leakage.
  • inactivated vaccines of the SARS-CoV-2 virus may induce virus-induced reactivation in immunized populations, causing a localized pandemic.
  • vaccines constructed with adenovirus vectors also have the potential to induce a combined humoral and cellular immune response, thereby enhancing the protective ability of the vaccine. Therefore, the development of new vaccines contributes to a comprehensive evaluation of different vaccine routes and helps us select the optimal vaccine from the perspective of safety and efficacy.
  • Adenoviruses are a class of non-integrating viruses with high affinity to the upper respiratory tract. It can enter epithelial cells through the CAR receptor (coxsackie/adenovirus receptor, coxsackie/adenovirus receptor), which is more likely to cause an immune response in the respiratory mucosa and produce IgM antibodies.
  • CAR receptor coxsackie/adenovirus receptor, coxsackie/adenovirus receptor
  • adenoviruses lacking E1 and E3 genes cannot be amplified in ordinary cells and can be used as carriers for carrying macromolecules.
  • Chinese patent 201610696322.3 discloses an Ebola virus vaccine based on a chimpanzee adenovirus vector.
  • the disclosed replication-deficient chimpanzee adenovirus vector includes an engineered chimpanzee adenovirus AdC68 genomic sequence in which El is deleted.
  • the chimpanzee adenovirus used in this patent has high immunogenicity and can not only induce specific humoral immune responses, but also specific cellular immune responses. In addition, it will not be affected by pre-existing anti-human serotype adenovirus neutralizing antibodies in the human body. , so it is an ideal vaccine carrier.
  • Chinese Patent 201811262788.8 discloses an adenovirus vector system and a recombinant adenovirus construction method, including: PCR amplification to obtain target gene fragments containing homologous overlapping regions on both sides, and DNA assembly with the PmeI linearized adenovirus plasmid to obtain Adenovirus plasmids containing exogenous target genes; or clone multiple gene fragments into shuttle plasmids, then use restriction endonucleases to cut out fragments containing all target genes, and perform DNA assembly with pKAd5f11pES-PmeI linearized with PmeI, Obtain an adenovirus plasmid containing the exogenous gene of interest.
  • the applicant analyzed each genome of the SARS-CoV-2 virus based on the results of the SARS-CoV-2 virus gene sequencing, and cloned the codon-optimized spike gene sequence (Spike) into a shuttle plasmid. Plasmids carrying the spike protein gene were co-transfected with adenovirus backbone plasmids into 293 cells and then plaque-selected. After the visible plaques were selected, the cells were lysed and transfected into 293 cells in a pre-cultured 24-well plate, and amplified to a concentration of 1 ⁇ 10 6 pfu/mL, identified by PCR and sequencing. An adenovirus type 5 vector carrying the SARS-CoV-2 spike gene was constructed, and a recombinant adenovirus vector vaccine against SARS-CoV-2 was obtained through amplification, purification and cross-protection tests.
  • Spike codon-optimized spike gene sequence
  • the S gene of SARS-CoV-2 coronavirus is optimized by codons, and the truncated or mutant sequence of the S gene is introduced into a secretion-deficient adenovirus vector, and the corresponding recombinant adenovirus is obtained by packaging.
  • the recombinant adenovirus can express different truncations or mutants of the SARS-CoV-2 virus S protein in vivo, and complete processing, folding, glycosylation and other modifications, basically maintaining the natural conformation of the S protein, and has high biological activity. , long half-life, long-lasting immunogenicity and so on.
  • this product uses a defective adenovirus vector carrying secreted peptides. Therefore, after the recombinant adenovirus vaccine is expressed in vivo, it can be secreted to the outside of cells, thereby activating humoral immunity.
  • the present invention provides a SARS-CoV-2 coronavirus vaccine.
  • the SARS-CoV-2 coronavirus vaccine comprises a fragment from the S gene of the SARS-CoV-2 coronavirus and a sequence from a replication-defective adenovirus.
  • the fragment of the S gene of the SARS-CoV-2 coronavirus is the full-length sequence or the truncated sequence of the S gene after the wild type or codon optimization or the mutant sequence thereof; the SARS-CoV-2 coronavirus
  • the full-length sequence of the viral S gene is SEQ ID NO:9.
  • the truncated sequence of the S gene of the SARS-CoV-2 coronavirus includes the full-length sequence or partial sequence of the S1 and/or S2 domains of the S gene.
  • the truncated sequence of the S gene of the SARS-CoV-2 coronavirus includes the N-terminal sequence of the S1 gene, the C-terminal sequence of the S1 gene, the intermediate fragment sequence of the S1 gene, the N-terminal sequence of the S2 gene, the C-terminal sequence of the S2 gene, One or more of the sequences of the intermediate segment of the S2 gene.
  • the described fragment from the S gene of the SARS-CoV-2 coronavirus includes the sequence of amino acid number 319-685 of the S gene.
  • the replication-deficient adenovirus is a type 5 adenovirus of subclass C with complete deletion and/or partial deletion of the E1 and/or E3 regions.
  • the replication deficient adenovirus is type 5 adenovirus of subtype C with complete deletion of E3 region, type 5 adenovirus of subtype C with partial deletion of E3 region, and type 5 adenovirus of subtype C with complete deletion of E1 region.
  • replication-deficient adenovirus contains a secretory peptide sequence.
  • replication deficient adenovirus contains CMV promoter and BGH gene polyA sequence.
  • the vaccine further includes thymopentin; the addition amount of thymopentin in the vaccine is 1-2 mg/mL, preferably 1.6 mg/mL.
  • the SARS-CoV-2 coronavirus vaccine comprises a fragment from the SARS-CoV-2 coronavirus S gene; the SARS-CoV-2 coronavirus S gene fragment is SARS-CoV -2 The 1-2055th gene fragment, the 1-1827th gene fragment, the 955th-2055th gene fragment or the 955th-1827th gene fragment of the full-length sequence of the coronavirus S gene; or the S gene for the above gene fragments The gene sequence in which the 454th amino acid of the S protein is mutated from R to A and/or the 466th amino acid is mutated from R to A.
  • the present invention also provides a preparation method of a SARS-CoV-2 coronavirus vaccine.
  • the described preparation method comprises the following steps:
  • the sequence of the S gene from the SARS-CoV-2 coronavirus is the full-length sequence or partial sequence of the S gene.
  • the sequence of the S gene from the SARS-CoV-2 coronavirus is obtained by PCR; the PCR primers are V1-V4, wherein the sequence of V1 is SEQ ID NO: 1, and the sequence of V2 is SEQ ID NO: 2 , the sequence of V3 is SEQ ID NO: 3, and the sequence of V4 is SEQ ID NO: 4.
  • V1 (SEQ ID NO: 1): TCC CCCGGG ATGTTCGTCTTCCTGGTCCT
  • V2 (SEQ ID NO: 2): TCC CCCGGG ATGAGGGTGCAGCCAACCGAG
  • V3 (SEQ ID NO: 3): CCC AAGCTT TTAGGCCACCTGGTTTGCTTGTAT
  • V4 (SEQ ID NO: 4): CCC AAGCTT TTACCGGGCTCTTCTGGGAGAGT
  • the underlined part of each primer is the restriction site.
  • the restriction sites of V1 and V2 are SmaI, and the restriction sites of V3 and V4 are HindIII.
  • V1 and V4 are a pair of primers to amplify the S1 gene, named Ad/S1, which is the 1-2055th position of the S gene.
  • V1 and V3 are a pair, and the N-terminal fragment of the S1 gene is amplified, named Ad/S1N, which is the 1-1827th position of the S gene.
  • V2 and V4 are a pair of primers to amplify the C-terminal fragment of the S1 gene, named Ad/S1C, which is the 955th-2055th position of the S gene.
  • V2 and V3 are a pair of primers to amplify the middle fragment of the S1 gene, named Ad/S1M, which is the 955th-1827th position of the S gene.
  • FIG. 1 A schematic diagram of the fragment amplification of the S gene is shown in Figure 1.
  • a mutation primer was designed to reduce the possible risk of SARS-CoV-2, and the mutation primer was V5-V8, where the sequence of V5 was SEQ ID NO: 5, and the sequence of V6 is SEQ ID NO: 6, the sequence of V7 is SEQ ID NO: 7, and the sequence of V8 is SEQ ID NO: 8.
  • V5 and V6 are a pair, which can mutate the 454th amino acid of the S gene from R to A;
  • V7 and V8 are a pair, which can mutate the 466th amino acid of the S gene from R to A. ;
  • the step (2) is: cloning the sequence of the S gene from the SARS-CoV-2 coronavirus into the pShuttle plasmid, and then recombining it with the adenovirus backbone plasmid.
  • the adenovirus backbone plasmid is pBHGlox(delta)E1,3Cre.
  • the step (2) is as follows: the purified target S gene fragment is digested with SmaI/HindIII and then connected to the pShuttle plasmid, transformed and screened for ampicillin resistance, and then combined with HEK293 cells.
  • the adenovirus backbone plasmid pBHGlox(delta)E1,3Cre was recombined.
  • the packaging cell in the step (3) is a cell line or cell line that integrates the E1 region gene of the C subtype 5 adenovirus (Ad5), preferably a cell line that integrates the C subtype 5 adenovirus (Ad5).
  • Ad5 C subtype 5 adenovirus
  • Ad5 C subtype 5 adenovirus
  • the purification method in the step (4) is plaque purification.
  • Figure 1 is a schematic diagram of fragment amplification of the S gene.
  • Figure 2 is a technical roadmap for the preparation of the vaccine of the present invention.
  • Figure 3 is the sequencing result of the recombinant adenovirus in step (3) of Example 1.
  • Figure 4 is the sequencing result of the recombinant adenovirus in step (3) of Example 2.
  • Figure 5 is the sequencing result of the recombinant adenovirus in step (3) of Example 3.
  • Figure 6 is the sequencing result of the recombinant adenovirus in step (3) of Example 4.
  • Figure 7 shows the expression results of RT-PCR detection of the SARS-CoV-2 vaccine prepared in Examples 1-12.
  • Figure 8 shows the results of cross-reaction between the antigens expressed by each SARS-CoV-2 vaccine and the sera of patients with new coronary pneumonia in convalescence.
  • Figure 9 shows the results of cross-reaction between the SARS-CoV-2 vaccine mutants expressing antigens and the sera of convalescent patients with new coronary pneumonia.
  • Figure 10 shows the results of the titer test of the SARS-CoV-2 vaccine animal immune neutralizing antibody.
  • Figure 11 shows the results of the titer detection of SARS-CoV-2 vaccine animal immune-specific antibody titers.
  • the pShuttle vector and the viral backbone plasmid pBHGlox(delta)E1,3Cre were purchased from Microbix Biosystems, cat. No. PD-01-64.
  • 293 cells were purchased from ATCC under the catalog number CRL-1573.
  • mice were purchased from Guangdong Provincial Laboratory Animal Center.
  • the S gene of SARS-CoV-2 coronavirus was optimized, and the sequence of the optimized S gene was shown in SEQ ID NO: 9, and the whole gene was synthesized for this gene as an amplification template for subsequent vaccine construction.
  • V1 (SEQ ID NO: 1): TCC CCCGGG ATGTTCGTCTTCCTGGTCCT
  • V4 (SEQ ID NO: 4): CCC AAGCTT TTACCGGGCTCTTCTGGGAGAGT
  • the S1 gene was amplified, that is, positions 1-2055 of the S gene of the basic example, and the amplified product was named S1.
  • the S1 fragment of the PCR amplification product obtained above was identified by running gel and recovered by gel cutting, and then digested with SmaI and HindIII at 37 degrees. pShuttle was digested with these two enzymes at the same time. Next, the digested PCR product was ligated with pShuttle at 16°C overnight with T4 ligase. The ligation product was transformed into E. coli DH5 ⁇ , positive clones were screened by ampicillin resistance, and clones were selected for colony PCR identification. Plasmids were extracted from positive colonies after culture.
  • the recombinant plasmid confirmed by sequencing was co-transfected with the viral backbone plasmid pBHGlox(delta)E1,3Cre into 293 cells to package the recombinant adenovirus.
  • Viruses are collected by picking plaques: low melting point agarose is added to the culture medium, and small plaques can be seen under the microscope on the 10th to 21st day after transfection. After plaque formation, the plaques were picked up with agarose and placed in 1 mL of fresh medium overnight. Usually 3-6 plaques are picked, and then the titers are compared, and the one with the highest titer is used for subsequent experiments.
  • the virus in the medium was added to the fresh 293 cell culture medium for a small amount of virus amplification. When the cells reappeared with plaques, the cells and supernatant were collected, and the virus was collected by repeated freezing and thawing three times.
  • the virus was used as the P1 generation virus, and the 293 cells were infected with the P1 generation virus, and the infection was carried out for three consecutive generations. , and the virus was collected after plaque formation and purified and concentrated in vitro.
  • the virus obtained after purification is the SARS-CoV-2 vaccine.
  • the SARS-CoV-2 vaccine contains the SARS-CoV-2 virus S gene and a defective adenovirus.
  • the defective adenovirus is a type 5 adenovirus of subclass C with complete deletion of the E1 region, and cannot replicate in ordinary human cells; the recombinant adenovirus is named Ad/S1 vaccine, and the inserted gene is sequenced, and the sequencing results are shown in the figure 3.
  • PCR amplification primers used in step (1) are:
  • V1 (SEQ ID NO: 1): TCC CCCGGG ATGTTCGTCTTCCTGGTCCT
  • V3 (SEQ ID NO: 3): CCC AAGCTT TTAGGCCACCTGGTTTGCTTGTAT
  • the amplified product was named S1N fragment, and the final SARS-CoV-2 vaccine was named Ad/S1N vaccine, and the inserted gene was sequenced.
  • the sequencing results are shown in Figure 4.
  • PCR amplification primers used in step (1) are:
  • V2 (SEQ ID NO: 2): TCC CCCGGG ATGAGGGTGCAGCCAACCGAG
  • V4 (SEQ ID NO: 4): CCC AAGCTT TTACCGGGCTCTTCTGGGAGAGT
  • the amplified product was named S1C fragment, and the final SARS-CoV-2 vaccine was named Ad/S1C vaccine, and the inserted gene was sequenced.
  • the sequencing results are shown in Figure 5.
  • PCR amplification primers used in step (1) are:
  • V2 (SEQ ID NO: 2): TCC CCCGGG ATGAGGGTGCAGCCAACCGAG
  • V3 (SEQ ID NO: 3): CCC AAGCTT TTAGGCCACCTGGTTTGCTTGTAT
  • the amplified product was named S1M fragment, and the final SARS-CoV-2 vaccine was named Ad/S1M vaccine, and the inserted gene was sequenced.
  • the sequencing results are shown in Figure 6.
  • Ad/S1R454A vaccine The difference between the Ad/S1R454A vaccine and the Ad/S1 vaccine is that the 454th amino acid of the S protein corresponding to the S gene is mutated from R to A; the mutation is achieved by PCR, and the PCR primers used are:
  • V5 (SEQ ID NO: 5): CTACAATTATCTGTACCGGCTGTTTAGAAAGAGCA
  • V6 (SEQ ID NO: 6): TGCTCTTTCTAAACAGCCGGTACAGATAATTGTAG
  • the final SARS-CoV-2 vaccine was named Ad/S1R454A vaccine.
  • Ad/S1R466A vaccine The difference between the Ad/S1R466A vaccine and the Ad/S1 vaccine is that the 466th amino acid of the S protein corresponding to the S gene is mutated from R to A; the mutation is achieved by PCR, and the PCR primers used are:
  • V7 (SEQ ID NO: 7): CTGAAGCCCTTCGAGAGGGACATCTCTACAGAA
  • V8 (SEQ ID NO: 8): TTCTGTAGAGATGTCCCTCTCGAAGGGCTTCAG recombinant plasmid are the same as in Example 1.
  • the final SARS-CoV-2 vaccine was named Ad/S1R466A vaccine.
  • Ad/S1NR454A vaccine The difference between the Ad/S1NR454A vaccine and the Ad/S1N vaccine is that the 454th amino acid of the S protein corresponding to the S gene is mutated from R to A; the mutation is achieved by PCR, and the PCR primers used are:
  • V5 (SEQ ID NO: 5): CTACAATTATCTGTACCGGCTGTTTAGAAAGAGCA
  • V6 (SEQ ID NO: 6): TGCTCTTTCTAAACAGCCGGTACAGATAATTGTAG
  • the final SARS-CoV-2 vaccine was named Ad/S1NR454A vaccine.
  • Ad/S1NR466A vaccine The difference between the Ad/S1NR466A vaccine and the Ad/S1N vaccine is that the 466th amino acid of the S protein corresponding to the S gene is mutated from R to A; the mutation is achieved by PCR, and the PCR primers used are:
  • V7 (SEQ ID NO: 7): CTGAAGCCCTTCGAGAGGGACATCTCTACAGAA
  • V8 (SEQ ID NO: 8): TTCTGTAGAGATGTCCCTCTCGAAGGGCTTCAG recombinant plasmid are the same as in Example 1.
  • the final SARS-CoV-2 vaccine was named Ad/S1NR466A vaccine.
  • Ad/S1CR454A vaccine The difference between the Ad/S1CR454A vaccine and the Ad/S1C vaccine is that the 454th amino acid of the S protein corresponding to the S gene is mutated from R to A; the mutation is achieved by PCR, and the PCR primers used are:
  • V5 (SEQ ID NO: 5): CTACAATTATCTGTACCGGCTGTTTAGAAAGAGCA
  • V6 (SEQ ID NO: 6): TGCTCTTTCTAAACAGCCGGTACAGATAATTGTAG
  • the final SARS-CoV-2 vaccine was named Ad/S1CR454A vaccine.
  • Ad/S1CR466A vaccine The difference between the Ad/S1CR466A vaccine and the Ad/S1C vaccine is that the 466th amino acid of the S protein corresponding to the S gene is mutated from R to A; the mutation is achieved by PCR, and the PCR primers used are:
  • V7 (SEQ ID NO: 7): CTGAAGCCCTTCGAGAGGGACATCTCTACAGAA
  • V8 (SEQ ID NO: 8): TTCTGTAGAGATGTCCCTCTCGAAGGGCTTCAG recombinant plasmid are the same as in Example 1.
  • the final SARS-CoV-2 vaccine was named Ad/S1CR466A vaccine.
  • Ad/S1MR454A vaccine The difference between the Ad/S1MR454A vaccine and the Ad/S1M vaccine is that the 454th amino acid of the S protein corresponding to the S gene is mutated from R to A; the mutation is achieved by PCR, and the PCR primers used are:
  • V5 (SEQ ID NO: 5): CTACAATTATCTGTACCGGCTGTTTAGAAAGAGCA
  • V6 (SEQ ID NO: 6): TGCTCTTTCTAAACAGCCGGTACAGATAATTGTAG
  • the final SARS-CoV-2 vaccine was named Ad/S1MR454A vaccine.
  • Ad/S1MR466A vaccine The difference between the Ad/S1MR466A vaccine and the Ad/S1M vaccine is that the 466th amino acid of the S protein corresponding to the S gene is mutated from R to A; the mutation is achieved by PCR, and the PCR primers used are:
  • V7 (SEQ ID NO: 7): CTGAAGCCCTTCGAGAGGGACATCTCTACAGAA
  • V8 (SEQ ID NO: 8): TTCTGTAGAGATGTCCCTCTCGAAGGGCTTCAG recombinant plasmid are the same as in Example 1.
  • the final SARS-CoV-2 vaccine was named Ad/S1MR466A vaccine.
  • Example 1-12 After each SARS-CoV-2 vaccine purified in Example 1-12 was infected with A549 cells at 1 ⁇ 10 7 pfu/mL for 2 hours, the virus suspension was discarded, and the cell culture medium was added, under the conditions of 37°C and 5% CO 2 . cultivate under. 48h after infection, cells were collected, RNA was extracted, and the expression level of each inserted gene mRNA was detected by RT-PCR. Blank control (without template) and negative control (template was RNA extracted from cells without vaccination) were set. The results are shown in Figure 7.
  • Example 5-12 After each mutant SARS-CoV-2 vaccine purified in Example 5-12 was infected with A549 cells at 1 ⁇ 10 7 pfu/mL for 2 hours, the virus suspension was discarded, and the cell culture medium was added, at 37° C., 5% CO 2 conditions. Cell lysates were collected 48 h after infection, and indirect ELISA was used to detect the cross-reaction of antigens expressed by each SARS-CoV-2 vaccine with the convalescent sera of COVID-19 patients. The specific results are shown in Figure 9.
  • mice The test subjects Balb/C mice were grouped as follows:
  • Intramuscular injection in the inner thigh Intramuscular injection in the inner thigh.
  • Vaccine stock solution concentration 1 ⁇ 10 11 vp/mL.
  • mice dose 1 ⁇ 10 9 vp/mice.
  • Immunization procedure immunization was performed once every 6 days, for a total of 3 times; the mouse serum was collected on the 12th day, the 18th day and the 24th day respectively.
  • mice serum was collected on the 12th day, the 18th day and the 24th day, respectively.
  • Neutralizing antibody titer detection method SARS-CoV-2 pseudovirus neutralization method.
  • This result is the experimental result of the SARS-CoV-2 vaccine obtained in Example 3.
  • the result shows that: after diluting the immune serum of mice by 25 times, the serum of mice immunized for 12 days can neutralize 59% of SARS-CoV-2 Pseudovirus, 18-day-immunized serum was able to neutralize 65% of SARS-CoV-2 pseudovirus, and 24-day-immunized serum was able to neutralize 91% of SARS-CoV-2 pseudovirus.
  • the Ad/S1C vaccine could induce high titers of SARS-CoV-2 neutralizing antibodies in mice, and the production of neutralizing antibodies could be detected on day 12.
  • Example 3 the SARS-CoV-2 vaccine obtained in Example 3 was selected.
  • mice The test subjects Balb/C mice were grouped as follows:
  • High-dose vaccine + thymopentin group vaccine stock solution 1 ⁇ 10 9 vp/only + thymopentin;
  • Low-dose vaccine group vaccine stock solution 2 ⁇ 10 8 vp/vaccine
  • High-dose vaccine group vaccine stock solution 1 ⁇ 10 9 vp/vaccine
  • Physiological saline control group inject the same volume of normal saline with other groups of reagents.
  • Vaccine stock solution concentration 1 ⁇ 10 11 vp/mL.
  • the concentration of thymopentin in the vaccine injection is 1.6 mg/mL.
  • Intramuscular injection in the inner thigh Intramuscular injection in the inner thigh.
  • Immunization procedure immunization was performed once every 6 days, for a total of 3 times; the mouse serum was collected on the 18th day and the 24th day respectively.
  • mice serum was collected on the 18th day and the 24th day, respectively.
  • Immunogenicity detection method ELISA method was used to determine the specific binding antibody titer against the S protein RBD region in mice, and the results are shown in Figure 11.
  • Serum titer against the new crown Spike RBD 18 days after immunization, the serum titer is about 2500-3400. The serum titer was about 8000-32000 24 days after immunization.

Abstract

提供了一种SARS-CoV－2冠状病毒的疫苗及其制备方法，通过对SARS-CoV-2冠状病毒的S基因进行密码子优化，并将S基因的截短体及突变体序列导入分泌型缺陷型腺病毒载体，包装得到相应重组腺病毒。该重组腺病毒能够在体内表达SARS-CoV-2病毒相关蛋白，并完成加工、折叠、糖基化等修饰，基本上保持5蛋白的天然构象，具有生物活性高、半衰期长、免疫原性持久等特点。所述携带分泌肽的缺陷型腺病毒载体使重组腺病毒疫苗在体内表达后，能被分泌至细胞外，从而激活体液免疫。

Description

一种SARS-CoV-2冠状病毒疫苗及其制备方法 技术领域
本发明属于生物基因工程领域，具体涉及一种SARS-CoV-2冠状病毒疫苗。
背景技术
新型冠状病毒在人与人中传播能力极强，传播指数R0约为2.5。该病毒主要通过呼吸道或接触传播感染人群，可引起急性肺炎性病变，对其他系统如泌尿系统，消化系统及神经系统也能造成严重损伤，目前尚无有效的药物治疗手段。因此，设计研发出一种有效针对该病毒的疫苗，非常重要。
此外，SARS-CoV-2冠状病毒是一种单链RNA病毒，十分不稳定，在体细胞内受到免疫监督与排斥，极易产生突变。因此，根据具有相对保守序列的抗原决定簇设计的疫苗能够诱导产生较为稳定的抗体，有效减少因病毒突变引起的疫苗失效现象，从而达到长期保护的作用。
世界范围内不同的科学家已分离出若干冠状病毒株，病毒基因序列分析结果提示，新冠病毒的DNA序列与新冠病毒、MERS的基因有80％的同源性，其中刺突蛋白基因的同源性与新冠病毒达74.2％，刺突中蛋白的RBD区域与血管紧张素转移酶受体2有4个结合域，世界卫生组织(WHO)已于1月10日公布，引起新冠病毒肺炎的病原是一种冠状病毒亚型变异株。SARS-CoV-2病毒是一种非节段性单股正链RNA病毒，有5个基因组组成，分子量达32kb。SARS-CoV-2病毒颗粒约100nm大小，其表面有多个稀疏的棒状蛋白。电镜照片显示病毒形如冠状，属于冠状病毒。SARS-CoV-2病毒的基因组主要编码了四种结构蛋白，刺突蛋白(spike，S蛋白)、核蛋白(nucleoprotein，N蛋白)、膜蛋白(membrane，M蛋白)、囊膜蛋白(envelop，E蛋白)。针对SARS-CoV-2病毒感染人群血清抗体检测结果表明，恢复期新冠病人血清中存在大量针对S蛋白及N蛋白的抗体。其中，刺突蛋白/S蛋白是一种穿膜糖蛋白，含有重要的病毒中和表位。SARS-CoV-2病毒S蛋白与宿主细胞的受体(如血管紧张素转化酶2[angiotensin I coverting enzyme,ACE2])结合后，可通过内吞作用介导病毒侵入。相关学者经过实验证明，S蛋白主要通过其RBD区域与ACE2受体结合。结构生物学研究显示，该区域有4个ACE2结合位点。该结合域结构保守，也是产生免疫性中和抗体的优势抗原区域。
SARS-CoV-2病毒的分离，为疫苗的研制提供了原始材料。目前，新冠病毒疫苗主要有灭活疫苗、亚单位疫苗和DNA疫苗几个类型正在研发或进入临床阶段。灭活疫苗在技术角度易于制备，能高效引起体液免疫，是最为经典的疫苗形式。但灭活疫苗的批量生产过程可能存在泄露风险。而且，SARS-CoV-2病毒的灭活疫苗可能在受免疫人群中产生病毒诱导复活，引起局部性大流行。另外，前期针对SARS与MERS的灭活疫苗引起的细胞免疫应答较低，且有研究表明MERS灭活疫苗导致小鼠肺部发生过敏性病理反应。此外，携带S基因全长的牛痘病毒在恒河猴攻毒实验中反而加剧了肺部损伤。同时，诸多研究也表明，全长的S基因可能引起严重的抗体增强效应(ADE，antibody dependent enhancement)。但在新型疫苗路线中，研究者能够利用生物信息学分析，并结合分子生物学、分子克隆、反向遗传重编码、免疫学等技术方法，筛选出免疫原性强及生物安全性好的疫苗。而且，以腺病毒载体构建的疫苗还具有诱导体液与细胞免疫组合反应的潜能，从而提高疫苗的保护能力。因此，新型疫苗的研发有助于对不同疫苗路线的全面评估，并帮助我们从安全性及有效性角度选取最优的疫苗。
腺病毒是一类上呼吸道亲和性高的非整合性病毒。它能够通过CAR受体(coxsackie/adenovirus receptor，柯萨奇/腺病毒受体)进入上皮细胞，较容易引起呼吸道粘膜免疫反应，产生IgM抗体。另外，E1与E3基因缺失的腺病毒，无法在普通细胞中扩增，能够作为携带大分子的载体。
中国专利201610696322.3中公开了一种基于黑猩猩腺病毒载体的埃博拉病毒疫苗。其公开的复制缺陷型黑猩猩腺病毒载体包括改造的黑猩猩腺病毒AdC68基因组序列，其中E1缺失。该专利采用的黑猩猩腺病毒具有高免疫原性，不仅能诱导特异性体液免疫反应，而且能诱导特异性细胞免疫反应，此外，不会受人体内预存抗人血清型腺病毒中和抗体的影响，因此是一种理想的疫苗载体。
中国专利201811262788.8中公开了一种腺病毒载体系统及重组腺病毒构建方法，包括：PCR扩增获得两侧含有同源重叠区的目的基因片段，与PmeI线性化的腺病毒质粒进行DNA组装，得到含有外源目的基因的腺病毒质粒；或者先将多个基因片段克隆到穿梭质粒，再使用限制性内切酶切下含全部目的基因的片段，与PmeI线性化的pKAd5f11pES-PmeI进行DNA组装，获得含有外源目的基因的腺病毒质粒。
目前尚未有关于通过腺病毒载体制备针对SARS-CoV-2病毒S蛋白截短体疫苗相关的研究报道。
发明内容
本申请人以SARS-CoV-2病毒基因测序结果，对SARS-CoV-2病毒各个基因组进行分析，将密码子优化后的刺突基因序列(Spike)克隆到穿梭质粒中。带有刺突蛋白基因的质粒，与腺病毒骨架质粒共转染293细胞后，进行空斑挑选。挑选可视型空斑后，进行细胞裂解，并将其转染到预先培养的24孔板的293细胞中，并使之扩增到1×10 6pfu/mL浓度，经PCR及测序鉴定，构建得到一种携带SARS-CoV-2刺突基因的腺病毒5型载体，并经放大、纯化及交叉保护性试验，获得针对SARS-CoV-2的重组腺病毒载体疫苗。
本发明通过对SARS-CoV-2冠状病毒的S基因进行密码子优化，并将S基因的截短体或突变体序列导入分泌型缺陷型腺病毒载体，包装得到相应重组腺病毒。该重组腺病毒能够在体内表达SARS-CoV-2病毒S蛋白的不同截短体或突变体，并完成加工、折叠、糖基化等修饰，基本上保持S蛋白的天然构象，具有生物活性高、半衰期长、免疫原性持久等特点。此外，本品采用了携带分泌肽的缺陷型腺病毒载体。因此，该重组腺病毒疫苗在体内表达后，能被分泌至细胞外，从而激活体液免疫。
一方面，本发明提供了一种SARS-CoV-2冠状病毒疫苗。
所述的SARS-CoV-2冠状病毒疫苗包含来自SARS-CoV-2冠状病毒S基因的片段和复制缺陷型腺病毒的序列。
所述的SARS-CoV-2冠状病毒S基因的片段为野生型或密码子优化后的S基因的全长序列或截短体序列或它们的突变体序列；所述的SARS-CoV-2冠状病毒S基因的全长序列为SEQ ID NO：9。
所述的SARS-CoV-2冠状病毒S基因的截短体序列中包括S基因的S1和/或S2结构域的全长序列或部分序列。
所述的SARS-CoV-2冠状病毒S基因的截短体序列包括S1基因的N端序列、S1基因C端序列、S1基因中间片段序列、S2基因的N端序列、S2基因C端序列、S2基因中间片段序列中的一种或多种。
所述的来自SARS-CoV-2冠状病毒S基因的片段包括S基因氨基酸号为319-685的序列。
所述的复制缺陷型腺病毒为E1和/或E3区完全缺失和/或部分缺失的C亚类的5型腺病毒。
优选地，所述的复制缺陷型腺病毒为E3区完全缺失的C亚类的5型腺病毒、E3区部分缺失的C亚类的5型腺病毒、E1区完全缺失的C亚类的5型腺病毒或E1区部分缺失的C亚类的5型腺病毒。
进一步地，所述的复制缺陷型腺病毒内装分泌肽序列。
进一步地，所述的复制缺陷型腺病毒内装CMV启动子和BGH基因polyA序列。
优选地，所述的疫苗中还包括胸腺五肽；胸腺五肽在疫苗中的添加量为1-2mg/mL，优选为1.6mg/mL。
作为一些优选的实施例，所述的SARS-CoV-2冠状病毒疫苗包含来自SARS-CoV-2冠状病毒S基因的片段；所述的SARS-CoV-2冠状病毒S基因的片段为SARS-CoV-2冠状病毒S基因全长序列的第1-2055位基因片段、第1-1827位基因片段、第955-2055位基因片段或第955-1827位基因片段；或针对以上基因片段的S基因的S蛋白的第454个氨基酸由R突变为A和/或第466个氨基酸由R突变为A的基因序列。
另一方面，本发明还提供了一种SARS-CoV-2冠状病毒疫苗的制备方法。
所述的制备方法包括以下步骤：
(1)取得来自SARS-CoV-2冠状病毒的S基因的序列；
(2)将来自SARS-CoV-2冠状病毒的S基因的序列与缺陷型腺病毒重组结合；
(3)转染包装细胞；
(4)经扩增、分离、纯化，制成制剂。
所述的步骤(1)中来自SARS-CoV-2冠状病毒的S基因的序列为S基因的全长序列或部分序列。
所述的来自SARS-CoV-2冠状病毒的S基因的序列通过PCR获得；所述的PCR引物为V1-V4，其中V1的序列为SEQ ID NO：1，V2的序列为SEQ ID NO：2，V3的序列为SEQ ID NO：3，V4的序列为SEQ ID NO：4。
V1(SEQ ID NO：1)：TCC CCCGGGATGTTCGTCTTCCTGGTCCT
V2(SEQ ID NO：2)：TCC CCCGGGATGAGGGTGCAGCCAACCGAG
V3(SEQ ID NO：3)：CCC AAGCTTTTAGGCCACCTGGTTGCTTGTAT
V4(SEQ ID NO：4)：CCC AAGCTTTTACCGGGCTCTTCTGGGAGAGT
每条引物中的下划线部分为酶切位点。V1及V2的酶切位点为SmaI，V3及V4的酶切位点为HindIII。
V1和V4为一对引物，扩增S1基因，命名为Ad/S1，为S基因的第1-2055位。
V1和V3为一对，扩增S1基因的N端片段，命名为Ad/S1N，为S基因的第1-1827位。
V2和V4为一对引物，扩增S1基因C端片段，命名为Ad/S1C，为S基因的第955-2055位。
V2与V3为一对引物，扩增S1基因中间片段，命名为Ad/S1M，为S基因的第955-1827位。
S基因的片段扩增示意图见图1。
进一步地，根据前期对SARS疫苗的研究结果，设计突变引物，以减轻SARS-CoV-2可能带来的风险，突变引物为V5-V8，其中V5的序列为SEQ ID NO：5，V6的序列为SEQ ID NO：6，V7的序列为SEQ ID NO：7，V8的序列为SEQ ID NO：8。
所述的突变引物中，V5和V6为一对，能将S基因的第454个氨基酸由R突变为A；V7和V8为一对，能将S基因的第466个氨基酸由R突变为A；利用突变引物分别对Ad/S1、Ad/S1N、Ad/S1C和Ad/S1M进行突变。
优选地，所述步骤(2)为：将来自SARS-CoV-2冠状病毒的S基因的序列克隆到pShuttle质粒，再将其与腺病毒骨架质粒重组结合。
优选地，所述的腺病毒骨架质粒为pBHGlox(delta)E1,3Cre。
优选地，所述的步骤(2)为：将纯化好的目的S基因片段经SmaI/HindIII酶切后连接至pShuttle质粒中，转化并经氨苄抗性筛选后，再将其在HEK293细胞中与腺病毒骨架质粒pBHGlox(delta)E1,3Cre重组结合。
进一步地，所述的步骤(3)中的包装细胞为整合了C亚类5型腺病毒(Ad5)E1区基因的细胞系或细胞株，优选为整合了C亚类5型腺病毒(Ad5)E1区基因的HEK293细胞。
具体地，所述的步骤(4)中的纯化方法为挑空斑纯化。
本发明优选的疫苗制备技术线路参考图2。
附图说明
图1为S基因的片段扩增示意图。
图2为本发明疫苗制备的技术路线图。
图3为实施例1步骤(3)重组腺病毒的测序结果。
图4为实施例2步骤(3)重组腺病毒的测序结果。
图5为实施例3步骤(3)重组腺病毒的测序结果。
图6为实施例4步骤(3)重组腺病毒的测序结果。
图7为RT-PCR检测实施例1-12中制备的SARS-CoV-2疫苗的表达结果。
图8为各SARS-CoV-2疫苗表达抗原与新冠肺炎恢复期病人血清交叉反应的结果。
图9为各SARS-CoV-2疫苗突变型表达抗原与新冠肺炎恢复期病人血清交叉反应的结果。
图10为SARS-CoV-2疫苗动物免疫中和抗体效价检测结果。
图11为SARS-CoV-2疫苗动物免疫特异抗体效价检测结果。
具体实施方式
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
以下实施例中：
pShuttle载体与病毒骨架质粒pBHGlox(delta)E1,3Cre购自Microbix Biosystems，货号为PD-01-64。
293细胞购自ATCC，货号为CRL-1573。
Balb/C小鼠购自广东省实验动物中心。
基础实施例SARS-CoV-2冠状病毒的S基因进行优化
对SARS-CoV-2冠状病毒的S基因进行优化，优化后的S基因的序列为SEQ ID NO：9所示，针对该基因进行全基因合成，作为后续疫苗构建的扩增模板。
实施例1 SARS-CoV-2冠状病毒疫苗Ad/S1的构建
(1)S基因片段的扩增：
以基础实施例获得的S基因为模板，以下述引物扩增：
V1(SEQ ID NO：1)：TCC CCCGGGATGTTCGTCTTCCTGGTCCT
V4(SEQ ID NO：4)：CCC AAGCTTTTACCGGGCTCTTCTGGGAGAGT
扩增S1基因，即基础实施例S基因的的第1-2055位，扩增产物命名为S1。
(2)构建重组质粒：
以上获得的PCR扩增产物S1片段经跑胶鉴定与切胶回收后，用SmaI与HindIII在37度条件下进行酶切。同时用这两种酶对pShuttle进行酶切。接下来用T4连接酶将酶切后的PCR产物与pShuttle在16度条件进行过夜连接。将连接产物转化大肠杆菌DH5α，利用氨苄抗性筛选阳性克隆，并挑选克隆进行菌落PCR鉴定。阳性菌落经培养后提取质粒。
(3)共转染：
将测序确认过的重组质粒，与病毒骨架质粒pBHGlox(delta)E1,3Cre共同转染至293细胞中以包装重组腺病毒。病毒的收集采用挑空斑的方式：在培养液中加入低溶点琼脂糖，转染后一般在第10-21天可以在显微镜下看到小的空斑。空斑形成后将空斑与琼脂糖一起挑起， 放入1mL新鲜培养基中过夜。通常挑取3-6个空斑不等，然后比较滴度，使用滴度最高的一个空斑进行后续实验。将培养基中病毒加入新鲜293细胞培养液中进行病毒少量扩增。至细胞再次出现空斑，收集细胞及上清，反复冻融三次收集病毒，以此病毒为P1代病毒，以P1代病毒感染293细胞，连续进行三代感染，至P4代进行病毒的大量扩增，待空斑形成后收集病毒并对病毒进行体外纯化和浓缩。纯化后获得的病毒即为SARS-CoV-2疫苗。SARS-CoV-2疫苗包含SARS-CoV-2病毒S基因和缺陷型腺病毒。缺陷型腺病毒为E1区完全缺失的C亚类的5型腺病毒，不能在普通的人体细胞内复制；该重组腺病毒命名为Ad/S1疫苗，并对插入基因进行测序，测序结果见图3。
实施例2 SARS-CoV-2冠状病毒疫苗Ad/S1N的构建
与实施例1的区别在于：步骤(1)使用的PCR扩增引物为：
V1(SEQ ID NO：1)：TCC CCCGGGATGTTCGTCTTCCTGGTCCT
V3(SEQ ID NO：3)：CCC AAGCTTTTAGGCCACCTGGTTGCTTGTAT
其余相同。
扩增产物命名为S1N片段，最终得到的SARS-CoV-2疫苗命名为Ad/S1N疫苗，并对插入基因进行测序，测序结果见图4。
实施例3 SARS-CoV-2冠状病毒疫苗Ad/S1C的构建
与实施例1的区别在于：步骤(1)使用的PCR扩增引物为：
V2(SEQ ID NO：2)：TCC CCCGGGATGAGGGTGCAGCCAACCGAG
V4(SEQ ID NO：4)：CCC AAGCTTTTACCGGGCTCTTCTGGGAGAGT
其余相同。
扩增产物命名为S1C片段，最终得到的SARS-CoV-2疫苗命名为Ad/S1C疫苗，并对插入基因进行测序，测序结果见图5。
实施例4 SARS-CoV-2冠状病毒疫苗Ad/S1M的构建
与实施例1的区别在于：步骤(1)使用的PCR扩增引物为：
V2(SEQ ID NO：2)：TCC CCCGGGATGAGGGTGCAGCCAACCGAG
V3(SEQ ID NO：3)：CCC AAGCTTTTAGGCCACCTGGTTGCTTGTAT
其余相同。
扩增产物命名为S1M片段，最终得到的SARS-CoV-2疫苗命名为Ad/S1M疫苗，并对插入基因进行测序，测序结果见图6。
实施例5 SARS-CoV-2冠状病毒疫苗Ad/S1R454A的构建
Ad/S1R454A疫苗与Ad/S1疫苗的区别在于S基因对应的S蛋白的第454个氨基酸由R突变为A；通过PCR来实现突变，使用的PCR引物为：
V5(SEQ ID NO：5)：CTACAATTATCTGTACCGGCTGTTTAGAAAGAGCA
V6(SEQ ID NO：6)：TGCTCTTTCTAAACAGCCGGTACAGATAATTGTAG
重组质粒的构建以及共转染同实施例1。
最终得到的SARS-CoV-2疫苗命名为Ad/S1R454A疫苗。
实施例6 SARS-CoV-2冠状病毒疫苗Ad/S1R466A的构建
Ad/S1R466A疫苗与Ad/S1疫苗的区别在于S基因对应的S蛋白的第466个氨基酸由R突变为A；通过PCR来实现突变，使用的PCR引物为：
V7(SEQ ID NO：7)：CTGAAGCCCTTCGAGAGGGACATCTCTACAGAA
V8(SEQ ID NO：8)：TTCTGTAGAGATGTCCCTCTCGAAGGGCTTCAG重组质粒的构建以及共转染同实施例1。
最终得到的SARS-CoV-2疫苗命名为Ad/S1R466A疫苗。
实施例7 SARS-CoV-2冠状病毒疫苗Ad/S1NR454A的构建
Ad/S1NR454A疫苗与Ad/S1N疫苗的区别在于S基因对应的S蛋白的第454个氨基酸由R突变为A；通过PCR来实现突变，使用的PCR引物为：
V5(SEQ ID NO：5)：CTACAATTATCTGTACCGGCTGTTTAGAAAGAGCA
V6(SEQ ID NO：6)：TGCTCTTTCTAAACAGCCGGTACAGATAATTGTAG
重组质粒的构建以及共转染同实施例1。
最终得到的SARS-CoV-2疫苗命名为Ad/S1NR454A疫苗。
实施例8 SARS-CoV-2冠状病毒疫苗Ad/S1NR466A的构建
Ad/S1NR466A疫苗与Ad/S1N疫苗的区别在于S基因对应的S蛋白的第466个氨基酸由R突变为A；通过PCR来实现突变，使用的PCR引物为：
V7(SEQ ID NO：7)：CTGAAGCCCTTCGAGAGGGACATCTCTACAGAA
V8(SEQ ID NO：8)：TTCTGTAGAGATGTCCCTCTCGAAGGGCTTCAG重组质粒的构建以及共转染同实施例1。
最终得到的SARS-CoV-2疫苗命名为Ad/S1NR466A疫苗。
实施例9 SARS-CoV-2冠状病毒疫苗Ad/S1CR454A的构建
Ad/S1CR454A疫苗与Ad/S1C疫苗的区别在于S基因对应的S蛋白的第454个氨基酸由R突变为A；通过PCR来实现突变，使用的PCR引物为：
V5(SEQ ID NO：5)：CTACAATTATCTGTACCGGCTGTTTAGAAAGAGCA
V6(SEQ ID NO：6)：TGCTCTTTCTAAACAGCCGGTACAGATAATTGTAG
重组质粒的构建以及共转染同实施例1。
最终得到的SARS-CoV-2疫苗命名为Ad/S1CR454A疫苗。
实施例10 SARS-CoV-2冠状病毒疫苗Ad/S1CR466A的构建
Ad/S1CR466A疫苗与Ad/S1C疫苗的区别在于S基因对应的S蛋白的第466个氨基酸由R突变为A；通过PCR来实现突变，使用的PCR引物为：
V7(SEQ ID NO：7)：CTGAAGCCCTTCGAGAGGGACATCTCTACAGAA
V8(SEQ ID NO：8)：TTCTGTAGAGATGTCCCTCTCGAAGGGCTTCAG重组质粒的构建以及共转染同实施例1。
最终得到的SARS-CoV-2疫苗命名为Ad/S1CR466A疫苗。
实施例11 SARS-CoV-2冠状病毒疫苗Ad/S1MR454A的构建
Ad/S1MR454A疫苗与Ad/S1M疫苗的区别在于S基因对应的S蛋白的第454个氨基酸由R突变为A；通过PCR来实现突变，使用的PCR引物为：
V5(SEQ ID NO：5)：CTACAATTATCTGTACCGGCTGTTTAGAAAGAGCA
V6(SEQ ID NO：6)：TGCTCTTTCTAAACAGCCGGTACAGATAATTGTAG
重组质粒的构建以及共转染同实施例1。
最终得到的SARS-CoV-2疫苗命名为Ad/S1MR454A疫苗。
实施例12 SARS-CoV-2冠状病毒疫苗Ad/S1MR466A的构建
Ad/S1MR466A疫苗与Ad/S1M疫苗的区别在于S基因对应的S蛋白的第466个氨基酸由R突变为A；通过PCR来实现突变，使用的PCR引物为：
V7(SEQ ID NO：7)：CTGAAGCCCTTCGAGAGGGACATCTCTACAGAA
V8(SEQ ID NO：8)：TTCTGTAGAGATGTCCCTCTCGAAGGGCTTCAG重组质粒的构建以及共转染同实施例1。
最终得到的SARS-CoV-2疫苗命名为Ad/S1MR466A疫苗。
试验例1 SARS-CoV-2疫苗的体外表达验证
将实施例1-12纯化获得的各个SARS-CoV-2疫苗分别以1×10 7pfu/mL感染A549细胞2h后，弃去病毒悬液，加入细胞培养液，37℃、5％CO 2条件下培养。感染后48h收集细胞，提取RNA，并用RT-PCR法检测各插入基因mRNA的表达水平，设置空白对照(不加模板)和阴性对照(模板为不接种疫苗的细胞提取的RNA)。结果见图7。
结果表明：SARS-CoV-2疫苗以1×10 7pfu/mL感染A549细胞后，能够检测到相应S蛋白抗原的mRNA(图7)。
试验例2 SARS-CoV疫苗携带抗原序列的免疫原性检测
将实施例1-4纯化获得的各个SARS-CoV-2疫苗分别以1×10 7pfu/mL感染A549细胞2h后，弃去病毒悬液，加入细胞培养液，37℃、5％CO 2条件下培养。感染后48h收集细胞裂解液，用间接ELISA检测各个SARS-CoV-2疫苗表达的抗原与COVID-19患者恢复期血清的交叉反应，具体结果见图8。
结果表明：SARS-CoV-2疫苗以1×10 7pfu/mL感染A549细胞后，表达的抗原能够与COVID-19患者恢复期的血清发生交叉反应(图8)。这表明，本申请中的SARS-CoV-2疫苗具有良好的免疫原性。
试验例3 SARS-CoV疫苗携带突变体抗原序列的免疫原性检测
将实施例5-12纯化获得的各个突变体SARS-CoV-2疫苗分别以1×10 7pfu/mL感染A549细胞2h后，弃去病毒悬液，加入细胞培养液，37℃、5％CO 2条件下培养。感染后48h收集细胞裂解液，用间接ELISA检测各个SARS-CoV-2疫苗表达的抗原与COVID-19患者恢复期血清的交叉反应，具体结果见图9。
结果表明：SARS-CoV-2疫苗以1×10 7pfu/mL感染A549细胞后，表达的突变体抗原仍能够与COVID-19患者恢复期的血清发生交叉反应(图9)。这表明，本申请中突变体SARS-CoV-2疫苗，在保证更高安全性的基础上，仍具有良好的免疫原性。
试验例4 SARS-CoV-2疫苗诱导的中和抗体效价
将实施例1-13纯化获得的各个SARS-CoV-2疫苗进行验证。
将试验对象Balb/C小鼠按照下述方式分组：
疫苗组与生理盐水对照组。
给药方式：大腿内侧肌肉注射。
疫苗原液浓度：1×10 11vp/mL。
小鼠剂量：1×10 9vp/只。
免疫程序：每6天免疫一次，共3次；分别取第12天，第18天、第24天小鼠血清。
安乐死取血程序：分别取第12天，第18天、第24天小鼠血清。
中和抗体效价检测方法：SARS-CoV-2假病毒中和法。
结果见图10。
该结果为实施例3得到的SARS-CoV-2疫苗的实验结果，该结果表明：将小鼠免疫血清稀释25倍后，小鼠免疫12天的血清能够中和59％的SARS-CoV-2假病毒，免疫18天的血清能够中和65％的SARS-CoV-2假病毒，免疫24天的血清能够中和91％的SARS-CoV-2假病毒。总体而言，Ad/S1C疫苗能够在小鼠体内诱导出高效价的SARS-CoV-2中和抗体，且第12天即能够检测到中和抗体的产生。
试验例5 SARS-CoV-2疫苗抗体效价试验
本实施例选用实施例3得到的SARS-CoV-2疫苗。
将试验对象Balb/C小鼠按照下述方式分组：
低剂量疫苗+胸腺五肽组：疫苗原液2×10 8vp/只+胸腺五肽；
高剂量疫苗+胸腺五肽组：疫苗原液1×10 9vp/只+胸腺五肽；
低剂量疫苗组：疫苗原液2×10 8vp/只；
高剂量疫苗组：疫苗原液1×10 9vp/只；
生理盐水对照组：注射与其他组别的试剂同等体积的生理盐水。
疫苗原液浓度：1×10 11vp/mL。
胸腺五肽在疫苗注射液中浓度为1.6mg/mL。
给药方式：大腿内侧肌肉注射。
免疫程序：每6天免疫一次，共3次；分别取第18天、第24天小鼠血清。
安乐死取血程序：分别取第18天、第24天小鼠血清。
免疫原性检测方法：用ELISA法测定小鼠针对S蛋白RBD区域的特异结合抗体效价，结果见图11。
该结果表明：
(1)针对新冠Spike RBD的血清效价：免疫后18天血清效价约为2500-3400。免疫后24天血清效价约为8000-32000。
(2)胸腺五肽的添加大大增强了小鼠体内抗体的滴度。免疫后18天，小鼠血清效价从2500增至3400；免疫后24天，血清效价从8000增至32000。

Claims (10)

  1. 一种SARS-CoV-2冠状病毒疫苗，其特征在于，包含SARS-CoV-2冠状病毒S基因的片段和复制缺陷型腺病毒的基因序列。
  2. 根据权利要求1所述的SARS-CoV-2冠状病毒疫苗，其特征在于，所述的复制缺陷型腺病毒为E1和/或E3区完全缺失和/或部分缺失的C亚类的5型腺病毒。
  3. 根据权利要求1所述的SARS-CoV-2冠状病毒疫苗，其特征在于，所述的SARS-CoV-2冠状病毒S基因的片段为野生型或密码子优化后的S基因全长序列、截短体序列或它们的突变序列；所述的SARS-CoV-2冠状病毒S基因的全长序列为SEQ ID NO：9。
  4. 根据权利要求3所述的SARS-CoV-2冠状病毒疫苗，其特征在于，所述的SARS-CoV-2冠状病毒S基因的截短体序列中包括S基因的S1和/或S2结构域的全长序列或部分序列。
  5. 根据权利要求4所述的SARS-CoV-2冠状病毒疫苗，其特征在于，所述的SARS-CoV-2冠状病毒S基因的截短体序列包括S1基因的N端序列、S1基因C端序列、S1基因中间片段序列、S2基因的N端序列、S2基因C端序列、S2基因中间片段序列中的一种或多种。
  6. 根据权利要求5所述的SARS-CoV-2冠状病毒疫苗，其特征在于，所述的SARS-CoV-2冠状病毒S基因的截短体序列为S1基因的C端序列。
  7. 根据根据权利要求4所述的SARS-CoV-2冠状病毒疫苗，其特征在于，所述的SARS-CoV-2冠状病毒S基因的片段为SARS-CoV-2冠状病毒S基因全长序列的第1-2055位基因片段、第1-1827位基因片段、第955-2055位基因片段、第955-1827位基因片段或1827-2057位基因片段；或针对以上基因片段的S基因的S蛋白的第454个氨基酸由R突变为A和/或第466个氨基酸由R突变为A的基因序列。
  8. 根据权利要求1-7任一项所述的SARS-CoV-2冠状病毒疫苗，其特征在于，所述的疫苗中还包括胸腺五肽，胸腺五肽在疫苗中的添加量为1-2mg/mL。
  9. 一种SARS-CoV-2冠状病毒疫苗的制备方法，其特征在于，包括以下步骤：
    (1)取得来自SARS-CoV-2冠状病毒的S基因的序列；
    (2)将来自SARS-CoV-2冠状病毒的S基因的序列与缺陷型腺病毒重组结合；
    (3)转染包装细胞；
    (4)经扩增、分离、纯化，制成制剂。
  10. 根据权利要求9所述的制备方法，其特征在于，所述的步骤(1)中来自SARS-CoV-2冠状病毒的S基因的序列为野生型或密码子优化后的S基因的全长序列或部分序列或它们的突变体；所述的来自SARS-CoV-2冠状病毒的S基因的序列通过PCR获得，所述的PCR引物为V1-V8，其中V1的序列为SEQ ID NO：1，V2的序列为SEQ ID NO：2，V3的序列为SEQ ID NO：3，V4的序列为SEQ ID NO：4，V5的序列为SEQ ID NO：5，V6的序列为SEQ ID NO：6，V7的序列为SEQ ID NO：7，V8的序列为SEQ ID NO：8；所述的PCR引物中V1和V4为一对，扩增S1基因；V1和V3为一对，扩增S1基因的N端片段；V2和V4为一对，扩增S1基因C端片段；V2与V3为一对，扩增S1基因中间片段；V5和V6为一对，能将S基因的第454个氨基酸由R突变为A；V7和V8为一对，能将S基因的第466个氨基酸由R突变为A。
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