Nucleotide chain for expressing Omicro BA.1 spike protein, virus and vaccine

Abstract

The invention provides a nucleotide chain for expressing Omicro BA.1 spike protein, a virus and a vaccine, and belongs to the technical field of viral vaccines, the nucleotide chain or the virus comprises a nucleotide sequence as shown in SEQ ID NO.1, on the premise that an expression protein main body is still novel coronavirus BA.1 variant spike protein, the nucleotide sequence is a nucleotide sequence as shown in SEQ ID NO.1, and the nucleotide sequence is a nucleotide sequence as shown in SEQ ID NO.2. A recombinant virus vector vaccine prepared from a nucleotide sequence after experience optimization can effectively stimulate an organism to generate a binding antibody, a neutralizing antibody and cellular immune response aiming at BA.1 variant virus after immunization, and has good immunogenicity.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus vaccines, and in particular relates to a nucleotide chain, a virus and a vaccine expressing an Omicron BA.1 spike protein. Background Art

[0002] The new coronavirus is an RNA virus that is very prone to mutation during its transmission, and through natural selection, dominant variants with stronger transmission ability are selected. Omicron BA.1 is one of the variants. Compared with other Omicron subtypes, the basic transmission number (R0) of Omicron BA.1 is slightly higher than that of other subtypes, which means that under similar environments, cases infected with BA.1 may spread to more people. In addition, the incubation period of BA.1 is relatively short, and the infected person is highly contagious in the early stages, which makes it possible for it to spread rapidly.

[0003] Mutations in the receptor binding domain (RBD) region of Omicron BA.1 (e.g., Q493R, N501Y, S371L, S373P, S375F, Q498R, and T478K) enhance the binding of the virus to angiotensin-converting enzyme 2 (ACE2), making it more contagious, and the efficacy of the inactivated vaccine based on the original strain sequence against Omicron BA.1 is only 56.0%, indicating that Omicron BA.1 has a strong potential for immune escape relative to existing inactivated vaccines. Summary of the invention

[0004] Based on the above problems in the prior art, the present invention provides a nucleotide chain for expressing Omicron BA.1 spike protein, which comprises a nucleotide sequence as shown in SEQ ID NO.1.

[0005] The present invention also provides a virus expressing Omicron BA.1 spike protein, wherein the genomic nucleotide sequence of the virus comprises the nucleotide sequence shown in SEQ ID NO.1.

[0006] Preferably, the virus expressing the Omicron BA.1 spike protein can express the spike protein encoded by the nucleotide sequence shown in SEQ ID NO.1.

[0007] Preferably, the virus is a recombinant human adenovirus type 5 replication-deficient.

[0008] Preferably, the virus is a human type 5 replication-deficient adenovirus in which the E1 and E3 genes are jointly deleted.

[0009] The present invention also discloses the use of the above-mentioned virus expressing Omicron BA.1 spike protein in the preparation of a vaccine for a new coronavirus.

[0010] The present invention also provides a vaccine expressing the Omicron BA.1 spike protein, which comprises the virus expressing the Omicron BA.1 spike protein as described above.

[0011] Preferably, the vaccine is in the form of an injection, nasal drops, spray or inhaler.

[0012] The present invention also provides a method for preparing a virus expressing the Omicron BA.1 spike protein as described above, comprising the following steps: Step S1, constructing a shuttle plasmid vector comprising the nucleotide chain as described above; Step S2, the shuttle plasmid vector and the backbone plasmid are co-transfected into host cells to package into a recombinant replication-deficient adenovirus.

[0013] Preferably, the host cell in step S2 is HEK293 cell.

[0014] The beneficial effects of the present invention are as follows: the recombinant adenovirus (nucleotide sequence) capable of expressing a mutant of the spike protein of the novel coronavirus BA.1 variant provided by the present invention is used as a novel coronavirus vaccine. On the premise that the main body of the expressed protein is still the spike protein of the novel coronavirus BA.1 variant, the recombinant virus vector vaccine prepared by the empirically optimized nucleic acid sequence can effectively stimulate the body to produce binding antibodies, neutralizing antibodies and cellular immune responses against the BA.1 variant virus after immunization, and has good immunogenicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the map of plasmid pADV-mCMV-Ad5-BA.1-S.

[0016] Figure 2 This is the electrophoresis diagram of the S protein expression level of the recombinant new coronavirus BA.1 variant vaccine.

[0017] Figure 3 A single immunization 2 and 4 weeks later induced IgG binding antibody levels.

[0018] Figure 4 Two immunizations at 2 and 4 weeks induced IgG binding antibody levels.

[0019] Figure 5 A single immunization induced neutralizing antibody levels against the original live virus at 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks and 8 weeks.

[0020] Figure 6A single immunization induced the production of neutralizing antibodies against the BA.1 variant live virus at 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks and 8 weeks.

[0021] Figure 7 The levels of neutralizing antibodies against the live virus of the XBB.1.5 variant were induced by a single immunization at 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks and 8 weeks.

[0022] Figure 8 Two immunizations at 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks and 8 weeks induced the production of neutralizing antibody levels against the original live virus.

[0023] Fig. 9 Two immunizations at 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks and 8 weeks induced the production of neutralizing antibody levels against the BA.1 variant live virus.

[0024] Fig.10 Two immunizations at 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks and 8 weeks induced the production of neutralizing antibody levels against the live virus of the XBB.1.5 variant.

[0025] Fig.11 It is the statistical result of flow cytometry IFN-γ detection in the cellular immune response level of BA.1 vaccine.

[0026] Fig.12 Representative results of flow cytometry.

[0027] Fig.13 It is the statistical result of IFN-γ detection by ELISPOT method in the cellular immune response level of BA.1 vaccine.

[0028] Fig.14 Representative results of ELISPOT.

[0029] Fig.15 This is a flow chart for the preparation and detection of viruses expressing the Omicron BA.1 spike protein. DETAILED DESCRIPTION

[0030] The present invention is described in detail below with reference to the accompanying drawings and specific examples. Unless otherwise specified, the starting plasmids, enzymes and related reagents used in the following examples can all be purchased from commercial companies.

[0031] Example 1: A plasmid expressing Omicron BA.1 spike protein.

[0032] As attached Figure 1A plasmid expressing Omicron BA.1 spike protein is shown, which contains the nucleotide sequence shown in SEQ ID NO.1. The nucleotide sequence shown in SEQ ID NO.1 is first edited and synthesized, and then constructed into a plasmid vector. The nucleotide sequence encodes the S protein of the new coronavirus BA.1 variant.

[0033] Example 2: A virus expressing Omicron BA.1 spike protein.

[0034] A virus expressing Omicron BA.1 spike protein, which is a human type 5 replication-deficient adenovirus with a combined deletion of E1 and E3 genes, whose genomic nucleotide sequence contains the nucleotide sequence shown in SEQ ID NO.1, and can express the spike protein encoded by the nucleotide sequence shown in SEQ ID NO.1. The virus can be used in the preparation of a vaccine for the new coronavirus.

[0035] Example 3: A vaccine expressing the Omicron BA.1 spike protein.

[0036] A vaccine expressing Omicron BA.1 spike protein, comprising the virus as described in Example 2, wherein the vaccine is in the form of an injection, nasal drops, spray or inhaler, and in this example is prepared as an intramuscular injection.

[0037] Example 4: A method for preparing a virus expressing Omicron BA.1 spike protein.

[0038] As attached Fig.15 A method for preparing a virus expressing Omicron BA.1 spike protein is shown, comprising the following steps: Step S1, constructing a shuttle plasmid vector comprising the nucleotide sequence shown in SEQ ID NO.1; Step S2, the shuttle plasmid vector and the backbone plasmid are co-transfected into a host cell, the host cell is a HEK293 cell, and packaged to form a recombinant replication-deficient adenovirus.

[0039] Example 5: Obtaining an optimized nucleotide sequence of the novel coronavirus Omicron BA.1 variant antigen.

[0040] The BA.1 variant (SARS-CoV-2 / E6 / RYS / 2023 / ZJ16) was selected as a template to obtain the nucleotide sequence encoding the S protein of the BA.1 variant, and the nucleotide sequence shown in SEQ ID NO.1 was optimized, with a total length of 3879 bp.

[0041] Example 6: Packaging and in vitro expression identification of recombinant virus vaccine.

[0042] In the nucleotide chain synthesis process of Example 5, the synthesized product was cloned into the pDC316 vector (Microbix Biosystems Inc.), and the map of the resulting plasmid (pAdeno-CMV-BA.1) is shown in Figure 1 As shown. Subsequently, the pAdeno-CMV-BA.1 plasmid and the adenovirus backbone plasmid pBHGlox(delta)E1, 3Cre were co-transfected into HEK293 cells and cultured in DMEM medium containing 5% FBS until the cells showed pathological changes. In this process, the pDC316 vector contains the left inverted repeat sequence, packaging signal and partial sequence of the antigen gene of the adenovirus. With the help of Cre / Loxp site-specific recombination, it is connected to the viral backbone sequence to form a complete viral genome, thereby initiating the synthesis and assembly of the virus.

[0043] With the continuous synthesis of progeny viruses, the cytopathic effect gradually increased. When more than 90% of the cells were significantly diseased and fell off the bottom of the culture dish, the cells were collected by centrifugation at 1000 g for 10 minutes.

[0044] After repeated freezing and thawing and centrifugation, the native virus solution was obtained, followed by plaque purification and virus amplification. After determining and verifying the virus titer, HEK293 cells were infected with MOI = 1. After 24 hours, cell samples were collected for Western Blot analysis. The results are shown in Figure 2 As shown, the SARS-CoV-2 Spike protein is approximately 142 KDa, and the experimental results show that protein bands were detected in the range of Marker100-180 KDa.

[0045] Example 7: Evaluation of the immune response to the novel coronavirus Omicron BA.1 variant vaccine.

[0046] Mice were immunized by intramuscular injection, and 5×108VP doses of Ad5-nCoV-BA.1 variant vaccine were respectively inoculated, and Ad5-nCoV prototype vaccine was used as a control, with 10 mice in each group. Blood was collected at 1, 2, 3, 4, 6 and 8 weeks after immunization, and serum was separated for the detection of binding antibodies and neutralizing antibodies, respectively. At the same time, 4 identical immunization groups were set up, and mice were killed 14 days after immunization, and spleen lymphocytes were separated for cellular immunity detection.

[0047] (A) The COVID-19 Omicron BA.1 variant vaccine induces a higher level of variant-specific binding antibody response.

[0048] The S protein of the BA.1 variant was used as the antigen, and the specific IgG and IgA antibody titers in the serum were detected by ELISA. The test results were as follows: Figure 3 and4 As shown (*P<0.05; **P<0.01; ***P<0 .001). Two weeks after intramuscular injection, the Ad5-nCoV-BA.1 vaccine can effectively stimulate mice to produce specific IgG antibody responses. In the experiment, the SARS-CoV-2 S protein was coated on a 96-well plate and treated with 0.05 M bicarbonate buffer overnight. Subsequently, it was washed 5 times with PBST, and the blocking solution was added and incubated at 37°C for 1 hour. Then, the diluted serum samples were added to each well and incubated at 37°C for 1 hour, and then washed 5 times with PBST. Then the primary antibody was added for incubation for 1 hour, washed again with PBST 5 times, and then incubated with horseradish peroxidase-conjugated secondary antibody at 37°C for 1 hour and washed 5 times with PBST. Finally, 3,3',5,5'-tetramethylbenzidine was added, and the reaction was terminated with 2 M sulfuric acid after 5 min. The optical density was measured at 450 nm and 630 nm by an enzyme labeling instrument, and the data were fitted to the standard curve.

[0049] (2) The new coronavirus Omicron BA.1 variant vaccine induces a higher level of variant-specific neutralizing antibody response.

[0050] Neutralizing antibodies (NAB) in mouse sera 4 weeks after immunization were evaluated using the BA.1 variant virus. The specific method is as follows: The neutralizing activity of mouse sera was detected by live neutralization test. The diluted serum was mixed with SARS-CoV-2 virus and incubated at 37°C for 1 hour. Subsequently, the mixture was added to a 96-well plate to infect Vero E6 cells. After 72 hours of incubation at 37°C, the virus-induced cytopathic effect (CPE) was observed using ×40 magnification, and the neutralization titer (the reciprocal of the 50% serum dilution required to neutralize virus infection) was calculated.

[0051] The above experiments were all conducted in a biosafety level 3 environment. The results of the neutralizing antibody test were as follows: Figure 5-10 As shown, the Ad5-nCoV-BA.1 variant vaccine can effectively stimulate mice to produce high-titer specific serum neutralizing antibodies against the Omicron variant. This conclusion confirms the good application potential of using Ad5-nCoV-BA.1 vaccine to prevent the Omicron variant.

[0052] (3) A single administration of the new coronavirus Omicron BA.1 variant vaccine can effectively stimulate the cellular immune response in mice.

[0053] IFN-γ and related cytokines were measured by flow cytometry and Elispot method.

[0054] First, spleen cells and peripheral blood PBMCs were isolated aseptically and diluted to 5.0 × 10 in RPMI1640 medium containing 2% FBS. 6 cells / mL, 500 μL (2×10 6 cells / well), 100 μL of stimulator, the final peptide concentration was 2 μg / mL, and 1 μL of Brilliant Violet 421TM anti-mouse CD107a mixed peptide stimulator and stimulation control was added.

[0055] Cell culture was performed in a 24-well plate, with one well for peptide stimulation and one well for non-stimulation control for each mouse. 100 μL of mixed peptide stimulation, non-stimulation control or positive stimulation was first added to each well, followed by the corresponding mouse spleen cells, with a total of 500 μL (2.5×10 6 cells / well), cultured in a 37°C / CO2 cell culture incubator for 6 hours, determined the appropriate intracellular cytokine staining scheme, and tested on the machine after staining. The results were as follows Figure 11-14 shown.

[0056] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A nucleotide chain expressing Omicron BA.1 spike protein, characterized in that It comprises the nucleotide sequence shown in SEQ ID NO.

1.

2. A virus expressing Omicron BA.1 spike protein, characterized in that: Its genomic nucleotide sequence includes the nucleotide sequence shown in SEQ ID NO.

1.

3. A virus expressing Omicron BA.1 spike protein according to claim 2, characterized in that: It can express the spike protein encoded by the nucleotide sequence shown in SEQ ID NO.

1.

4. A virus expressing Omicron BA.1 spike protein according to claim 3, characterized in that: The virus is a recombinant human adenovirus type 5 replication-deficient.

5. A virus expressing Omicron BA.1 spike protein according to claim 4, characterized in that: The virus is a human type 5 replication-deficient adenovirus in which the E1 and E3 genes are jointly deleted.

6. Use of a virus expressing the Omicron BA.1 spike protein as described in any one of claims 2 to 5 in the preparation of a vaccine for a new coronavirus.

7. A vaccine expressing Omicron BA.1 spike protein, characterized in that: It comprises the virus according to any one of claims 2 to 5.

8. A vaccine expressing Omicron BA.1 spike protein according to claim 7, characterized in that The vaccine is in the form of injection, nasal drops, spray or inhalation.

9. A method for preparing a virus according to any one of claims 2 to 5, characterized in that: It includes the following steps: Step S1, constructing a shuttle plasmid vector comprising the nucleotide chain as claimed in claim 1; Step S2, the shuttle plasmid vector and the backbone plasmid are co-transfected into host cells to package into a recombinant replication-deficient adenovirus.

10. The preparation method according to claim 9, characterized in that: The host cells in step S2 are HEK293 cells.

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