Nucleotide chain for expressing Omicro BF.7 spike protein, virus and vaccine

Abstract

The invention provides a nucleotide chain for expressing Omicron BF.7 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 shown as SEQ ID NO.1. On the premise that an expression protein main body is still novel coronavirus BF.7 variant spike protein, the nucleotide sequence is a nucleotide sequence shown as SEQ ID NO.1. The nucleotide sequence is a nucleotide sequence shown as SEQ ID NO.2. The nucleotide sequence is a nucleotide sequence shown as SEQ ID NO.1. The nucleotide sequence is a nucleotide sequence shown as SEQ ID NO.1. The recombinant virus vector prepared from the optimized nucleic acid sequence is used as a vaccine, can effectively stimulate an organism to generate a binding antibody, a neutralizing antibody and cellular immune response aiming at the BF.7 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 the Omicron BF.7 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, and Omicron BF.7 is one of the variants. Compared with other Omicron subtypes, the basic reproduction number (R0) of Omicron BF.7 is slightly higher than that of other subtypes, which means that under the same environment, cases infected with BF.7 may spread to more people. In addition, the incubation period of BF.7 is relatively short, and the infected person can be highly contagious at an early stage, which makes it possible for it to spread rapidly.

[0003] Omicron BF.7 has a series of spike protein mutations, especially the R346T mutation, which significantly enhances its resistance to neutralizing antibodies, making BF.7 about 4.4 times more resistant to neutralizing antibodies than the original Omicron subtype. Due to this mutation, BF.7 is able to partially escape the immune response generated by previous infection or vaccination, reducing the protective effect of early vaccines (based on prototype strains or early variants). Summary of the invention

[0004] Based on the above problems in the prior art, the present invention provides a nucleotide chain expressing Omicron BF.7 spike protein, which comprises a nucleotide sequence as shown in SEQ ID NO.1.

[0005] The present invention also provides a virus expressing Omicron BF.7 spike protein, whose genome nucleotide sequence contains the nucleotide sequence shown in SEQ ID NO.1.

[0006] Preferably, the virus expressing the Omicron BF.7 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 replication-deficient human adenovirus type 5.

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

[0009] The present invention also discloses the use of the above-mentioned virus expressing Omicron BF.7 spike protein in the preparation of a vaccine for a new coronavirus.

[0010] The present invention also provides a vaccine expressing the Omicron BF.7 spike protein, which comprises the virus expressing the Omicron BF.7 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 BF.7 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 BF.7 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 BF.7 variant, the recombinant virus vector prepared with the optimized nucleic acid sequence is used as a vaccine. After immunization, it can effectively stimulate the body to produce binding antibodies, neutralizing antibodies and cellular immune responses against the BF.7 variant virus, and has good immunogenicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a map of plasmid pADV-mCMV-Ad5-BF.7-S.

[0016] Figure 2 This is the electrophoresis diagram of the S protein expression level of the recombinant new coronavirus BF.7 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 6 The levels of neutralizing antibodies against the BF.7.14 variant live virus induced by a single immunization 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 BF.7.14 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 These are the statistical results of flow cytometry IFN-γ detection in the cellular immune response level of BF.7-S vaccine.

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

[0027] Fig.13 These are the statistical results of IFN-γ detection using the ELISPOT method in the cellular immune response level of the BF.7-S 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 BF.7 spike protein.

[0030] in Fig.12 and Fig.14 This is a graph of experimental results. The colors in the graph are part of the results. A color graph is used to clearly express the results. DETAILED DESCRIPTION

[0031] 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.

[0032] Example 1: A plasmid expressing Omicron BF.7 spike protein.

[0033] As attached Figure 1A plasmid expressing Omicron BF.7 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 BF.7 variant.

[0034] Example 2: A virus expressing the Omicron BF.7 spike protein.

[0035] A virus expressing Omicron BF.7 spike protein, which is a replication-deficient human adenovirus type 5 in which both E1 and E3 genes are deleted, and 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.

[0036] Example 3: A vaccine expressing the Omicron BF.7 spike protein.

[0037] A vaccine expressing Omicron BF.7 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.

[0038] Example 4: A method for preparing a virus expressing Omicron BF.7 spike protein.

[0039] As attached Fig.15 A method for preparing a virus expressing Omicron BF.7 spike protein is shown, comprising 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 a host cell, the host cell is a HEK293 cell, and packaged to form a recombinant replication-deficient adenovirus.

[0040] Example 5: Obtaining an optimized nucleotide sequence of the novel coronavirus Omicron BF.7 variant antigen.

[0041] The BF.7 variant (SARS-CoV-2 / E6 / RYS / 2023 / ZJ16) was selected as a template to obtain the nucleotide sequence encoding the S protein of the BF.7 variant, and the nucleotide sequence shown in SEQ ID NO.1 was optimized, with a total length of 3873 bp.

[0042] Example 6: Packaging and in vitro expression identification of recombinant virus vaccine.

[0043] In the nucleotide chain synthesis process of Example 5, the synthesized product was cloned into the pDC316 vector. The map of the resulting plasmid (pAdeno-CMV-BF.7) is shown in Figure 1 As shown. Subsequently, the pAdeno-CMV-BF.7 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. Relying on Cre / Loxp site-specific recombination, it is spliced ​​with the viral backbone sequence to form a complete viral genome, initiating the synthesis and assembly of the virus.

[0044] With the continuous synthesis of progeny viruses, the cytopathic effect worsened. When more than 90% of the cells showed obvious lesions and fell off the bottom of the culture dish, the cells were collected by centrifugation at 1000 g for 10 minutes.

[0045] After repeated freezing and thawing and centrifugation, the native virus solution was obtained. The virus was amplified and cultured after plaque purification. 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 the figure. Figure 2 As shown, the SARS-CoV-2 Spike protein is approximately 142 KDa, and the experimental results show that protein bands were detected between Marker 100-180KDa.

[0046] Example 7: Evaluation of the immune response to the new coronavirus Omicron BF.7 variant vaccine.

[0047] The mice were immunized with 5x10°VP doses of Ad5-nCoV-BF.7 variant vaccine by intramuscular injection, and the 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 binding antibody and neutralizing antibody detection. At the same time, the same 4 immunization groups were set up, and the mice were killed 14 days after immunization, and the spleen lymphocytes of the mice were separated for cellular immunity detection.

[0048] (A) The new coronavirus Omicron BF.7 variant vaccine induces a higher level of variant-specific binding antibody response.

[0049] The S protein of the BF.7 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 and 4As shown (*P<0.05; **P<0.01; ***P<0 .001). Ad5-nCoV-BF.7 vaccine can effectively stimulate serum-specific IgG antibody response in mice 2 weeks after intramuscular immunization. SARS-CoV-2 S protein was coated on 96-well plates and 0.05 M bicarbonate buffer overnight. After washing with PBST five times, blocking solution was added and incubated at 37°C for 1 h, and serum samples were diluted and added to each well. After incubation at 37°C for 1 h, the samples were washed five times with PBST. Primary antibody was added and incubated for 1 h, and then the samples were washed five times with PBST, and then incubated with horseradish peroxidase-conjugated secondary antibody at 37°C for 1 h at 37°C and washed five times with PBST. After adding 3,30,5,50-tetramethylbiphenyl anhydride for 5 min, the reaction was terminated with 2 M sulfuric acid. The optical density was measured at 450 nm and 630 nm by an enzyme labeling instrument and then fitted to the standard curve.

[0050] (II) The COVID-19 Omicron BF.7 variant vaccine induces a higher level of variant-specific neutralizing antibody response Neutralizing antibody (NAb) in mouse serum 4 weeks after immunization was determined using the BF.7 variant virus. The specific method is as follows: The neutralizing activity of mouse serum was evaluated by a live neutralization test. The diluted serum was mixed with SARS-CoV-2 and incubated at 37°C for 1 hour. The mixture was added to a 96-well plate to infect Vero E6 cells. After incubation at 37°C for 72 hours, the viral cytopathic effect (CPE) was observed at ×40 magnification, and the neutralization titer (the reciprocal of the 50% serum dilution required to neutralize viral infection) was calculated.

[0051] The above procedures were all carried out in a biosafety level 3 environment. The results of the neutralizing antibody test are shown in Figure 5-10 The results showed that the Ad5-nCoV-BF.7 variant vaccine can effectively stimulate mice to produce high titers of specific serum true virus neutralizing antibodies against the Omicron variant. This conclusion confirms that the use of Ad5-nCoV-BF.7 vaccine to prevent the Omicron variant has a high application potential.

[0052] (3) A single administration of the new coronavirus Omicron BF.7 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] Aseptically isolate mouse spleen cells from peripheral blood PBMCs and dilute the cells to 5.0 × 10 using RPMI1640 medium containing 2% FBS. 6cells / mL, 500 μL (2×10 6 Peptide stimulators and stimulation controls were prepared with 100 μL of stimulator, 2 μg / mL peptide final concentration, and 1 μL of Brilliant Violet 421TM anti-mouse CD107a per well.

[0055] Cell culture: 24-well plate, 1 well for each mouse with peptide stimulation and 1 well without stimulation. First, add 100 μL of mixed peptide stimulation or non-stimulated control or positive stimulation to each well, and then add spleen cells from each mouse to the corresponding wells, 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 BF.7 spike protein, characterized in that: It comprises the nucleotide sequence shown in SEQ ID NO.

1.

2. A virus expressing Omicron BF.7 spike protein, characterized in that: Its genomic nucleotide sequence includes the nucleotide sequence shown in SEQ ID NO.

1.

3. A virus expressing Omicron BF.7 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 BF.7 spike protein according to claim 3, characterized in that: The virus is a recombinant replication-deficient human adenovirus type 5.

5. A virus expressing Omicron BF.7 spike protein according to claim 4, characterized in that: The virus is a replication-deficient human adenovirus type 5 in which both E1 and E3 genes are deleted.

6. Use of a virus expressing the Omicron BF.7 spike protein as described in any one of claims 2-5 in the preparation of a vaccine for a new coronavirus.

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

8. A vaccine expressing Omicron BF.7 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.

Images