WO2024055429A1

WO2024055429A1 - Sars-cov-2 antigen polypeptide, and recombinant adeno-associated virus thereof and use thereof in preparation of vaccine

Info

Publication number: WO2024055429A1
Application number: PCT/CN2022/133918
Authority: WO
Inventors: 李展如
Original assignee: 广东珩达生物医药科技有限公司; 广州国家实验室
Priority date: 2022-09-16
Filing date: 2022-11-24
Publication date: 2024-03-21
Also published as: CN116063411A

Abstract

Disclosed in the present invention are an SARS-COV-2 antigen polypeptide, a recombinant adeno-associated virus and the use thereof. The present invention uses an S protein or RBD of the SARS-COV-2 antigen polypeptide as an antigen. A truncated domain has a shorter sequence length, a smaller molecular weight and a clearer structure, and has specificity. The recombinant adeno-associated virus, which contains a nucleic acid sequence encoding the antigen polypeptide achieves gene delivery via intramuscular injection and can effectively and continuously express the antigen polypeptide. The antigen polypeptide, which is expressed by the recombinant adeno-associated virus of the present invention in vivo, can induce a specific immune response against the SARS-COV-2 antigen polypeptide, and an antibody serum produced thereby can not only specifically recognize the SARS-COV-2 RBD antigen polypeptide, but also has a neutralizing titer, and can inhibit the replication and transmission of variant strains Delta and Omicron or prevent the variant strains Delta and Omicron from settling in a host, thereby effectively preventing and/or treating COVID-19 caused by SARS-CoV-2.

Description

Application of novel coronavirus antigen polypeptide and its recombinant adeno-associated virus and vaccine preparation

Technical field

The present invention relates to the field of biotechnology, and more specifically, to novel coronavirus antigen polypeptides and their recombinant adeno-associated viruses and their application in preparing vaccines.

Background technique

The novel coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2) belongs to the genus β-coronavirus and is a single-stranded RNA virus with a genome size of approximately 30kDa. SARS-CoV-2 is highly insidious and contagious. As SARS-CoV-2 spreads across countries, it continues to change or genetically mutate. New viral mutations have increased mortality, spread, and vaccine evasion. high, making it more destructive. Immune escape caused by the mutational dynamics of SARS-CoV-2 mutations poses an unforeseen danger to the entire world, so the development of vaccines against variant strains must be accelerated.

The efficacy of SARS-CoV-2 vaccines in the existing technology is basically greatly reduced against mutant strains such as Delta. The Chinese patent discloses a fusion protein, nasal spray vaccine and its preparation for the new coronavirus mutant strain Delta. Method, the fusion protein is formed by the fusion of protein S, protein M and nucleocapsid protein N. However, the vaccine needs to include the coding genes of the three proteins used. The gene sequence is long, the expressed protein is relatively complex, and the vaccine effect is single. In order to enrich the response to SARS-COV-2 variant strains Delta and cope with the mutations of SARS-COV-2, it is necessary to develop more simple, stable and effective against multiple SARS-COV-2 variant strains Delta and Omicron. New vaccines with high transmissibility and site mutations.

Contents of the invention

In order to overcome the shortcomings of the above-mentioned prior art, the present invention has developed and designed a novel SARS-COV-2 antigen polypeptide, and provides an expression vector for the antigen polypeptide, a method for preparing the antigen polypeptide recombinant adeno-associated virus, and a method for preparing the recombinant adeno-associated virus. Application in vaccines to prevent COVID-19.

The purpose of the present invention is to provide novel antigenic polypeptides of SARS-COV-2 virus.

Another object of the present invention is to provide a recombinant adeno-associated virus expression vector containing the above-mentioned antigen polypeptide.

Another object of the present invention is to provide the application of the above-mentioned antigen polypeptide and its recombinant adeno-associated virus in preparing a vaccine for COVID-19.

Another object of the present invention is to provide a SARS-COV-2 virus vaccine.

Another object of the present invention is to provide a preparation method of SARS-COV-2 virus vaccine.

In order to achieve the above objects, the present invention adopts the following technical solutions:

The invention provides a SARS-COV-2 antigen polypeptide, the amino acid sequence of which is one of the following amino acid sequences:

(1) The sequence shown in SEQ ID NO.1;

(2) The sequence shown in SEQ ID NO.2;

(3) The sequence shown in SEQ ID NO.3;

(4) The sequence shown in SEQ ID NO.4.

Among them, SEQ ID NO.1 is obtained by mutating the R ₆₈₂ RAR ₆₈₅ site of the S protein of SARS-CoV-2 variant strain Delta to G ₆₈₂ SAS ₆₈₅ , and the K ₉₈₆ V ₉₈₇ site to P ₉₈₆ P ₉₈₇ The 13-1208th amino acid sequence;

SEQ ID NO.2 is the amino acid sequence 13-1208 obtained by mutating the K ₉₈₆ V ₉₈₇ site of the S protein of the SARS-CoV-2 variant strain Delta to P ₉₈₆ P ₉₈₇ ;

SEQ ID NO.3 is the R ₆₈₂ RAR ₆₈₅ site of the S protein of SARS-CoV-2 variant strain Delta mutated to S ₆₈₂ RAG ₆₈₅ , and the K ₉₈₆ V ₉₈₇ site mutated to P ₉₈₆ P ₉₈₇ . 13-1208 amino acid sequence;

SEQ ID NO.4 is the 319-593rd amino acid sequence of the gene sequence of SARS-CoV-2 variant strain Delta, which is the spike protein receptor RBD domain of the S protein.

This proposal also claims protection for the nucleotide sequence encoding the above-mentioned antigen polypeptide.

Further, the nucleotide sequence encoding the antigen polypeptide has been optimized by human codons;

This proposal also claims protection for a recombinant adenovirus expression vector containing the above-mentioned antigen polypeptide.

Further, the expression vector contains an adeno-associated virus inverted terminal repeat sequence, a nucleotide sequence encoding a signal peptide, and a nucleotide sequence encoding the above-mentioned antigen polypeptide.

Preferably, the adeno-associated virus inverted terminal repeat sequence is selected from the following sequences: SEQ ID NO.8-10.

Furthermore, the expression vector also contains necessary expression control elements, such as promoter sequences, upstream regulatory regions, coding regions, transcription control elements, terminators, etc.

Further, the above-mentioned signal peptide is one of the following signal peptides:

(1) tPA signal peptide: the amino acid sequence is shown in SEQ ID NO.5;

(2) New tPA signal peptide: the amino acid sequence is shown in SEQ ID NO.6;

(3) Natural signal peptide: The amino acid sequence is shown in SEQ ID NO.7.

The tPA signal peptide is the signal peptide of human tissue plasminogen activator; the new tPA signal peptide is the mutation of the 22nd amino acid of the tPA signal peptide from proline (P) to alanine (tPA22P/A); the natural signal peptide It is the signal peptide of the S spike protein of the new coronavirus.

Further, the adeno-associated virus inverted terminal repeat sequence comes from serotype adeno-associated virus type 2.

This plan also claims the use of the above-mentioned antigen polypeptide, the nucleotide sequence encoding the above-mentioned antigen polypeptide, the expression vector containing the above-mentioned antigen polypeptide, or the above-mentioned recombinant adeno-associated virus in the preparation of a vaccine to prevent new coronavirus pneumonia.

Preferably, the new coronavirus pneumonia is caused by mutant strain Delta and/or mutant strain Omicron.

Further, the vaccine also contains pharmaceutically acceptable diluents and/or excipients.

This plan also claims protection for the SARS-COV-2 virus vaccine prepared using the above-mentioned antigen polypeptide as an antigen.

This proposal also claims protection for the SARS-COV-2 virus vaccine expressed by an expression vector containing a nucleotide sequence encoding the above-mentioned antigen polypeptide.

Further, the expression vector is a recombinant adeno-associated virus expressing the above antigen polypeptide.

Furthermore, the dosage form of the vaccine is injection.

More preferably, the dosage form of the vaccine is intramuscular injection.

This plan also requests protection for a preparation method of SARS-COV-2 virus vaccine, which is prepared through the following steps:

pHelper, pRep2Cap5 and the expression vector expressing the above antigen polypeptide are co-incubated; the cells are transfected in the presence of the transfection reagent polyethylenimine; after culturing the cells, the cells are collected by centrifugation, lysed and purified, and SARS-COV-2 is obtained Virus vaccine.

Compared with the existing technology, the beneficial effects of the present invention are reflected in:

(1) The present invention uses the antigenic polypeptide S protein or RBD domain of the new coronavirus SARS-COV-2 virus as the antigen. The truncated domain sequence is short, the molecular weight is smaller and has specificity; the nucleic acid sequence of this type of antigenic polypeptide is in the recombinant adenoid-related Gene delivery on the virus is achieved through intramuscular injection, which can effectively and continuously express antigen polypeptides.

(2) The antigen polypeptide expressed by the recombinant adeno-associated virus of the present invention in vivo can induce the

Specific immune response to SARS-COV-2 variant strain Delta and Omicron antigen polypeptides. The antibody serum produced can not only specifically recognize the RBD antigen polypeptide of SARS-COV-2, but also has a specific immune response to variant strains Delta and Omicron. Neutralizing potency, the vaccine can inhibit the replication and spread of SARS-CoV-2 variant strains Delta and Omicron or prevent them from colonizing the host body, thereby effectively preventing and/or treating SARS-CoV-2 variant strains. This vaccine is effective against multiple strains of SARS-CoV-2 against new coronavirus pneumonia caused by Delta and Omicron.

Description of drawings

Figure 1 is a schematic diagram of the expression vector map of vaccine 1. In the figure, the key components are described as follows: ITR refers to the inverted terminal repeat sequence of AAV2; tPA signal is the signal peptide, which refers to the SEQ ID NO.5 sequence; Delta ( B.1.617.2)-spike refers to the SEQ ID NO.1 sequence; WPRE refers to the woodchuck hepatitis virus post-transcriptional regulatory element; SV40polyA refers to the monkey vacuolating virus 40 (SV40) polyadenylation sequence.

Figure 2 is a schematic diagram of the expression vector map of vaccine 2; in the figure, tPA signal is the signal peptide, which refers to the SEQ ID NO.5 sequence; Delta(B.1.617.2)-spike refers to the SEQ ID NO.2 sequence; Other key components are the same as Figure 1.

Figure 3 is a schematic diagram of the expression vector map of vaccine 3; in the figure, tPA signal is the signal peptide, which refers to the SEQ ID NO.6 sequence; Delta(B.1.617.2)-spike refers to the SEQ ID NO.3 sequence; Other key components are the same as Figure 1.

Figure 4 is a schematic diagram of the expression vector map of vaccine 4; in the figure, tPA signal is the signal peptide, which refers to the SEQ ID NO.5 sequence; Delta(B.1.617.2)-spike refers to the SEQ ID NO.3 sequence; Other key components are the same as Figure 1.

Figure 5 is a schematic diagram of the expression vector map of vaccine 5; in the figure, tPA signal is the signal peptide, which refers to the SEQ ID NO.7 sequence; Delta(B.1.617.2)-spike refers to the SEQ ID NO.3 sequence; Other key components are the same as Figure 1.

Figure 6 is a schematic diagram of the expression vector map of vaccine 6; in the figure, CMV is the cytomegalovirus enhancer, chickenβ-actin is the promoter splicing acceptor; tPA signal is the signal peptide, which refers to the SEQ ID NO.5 sequence; Delta (B.1.617.2)-spike-RBD refers to SEQ ID NO.4 sequence; WPRE refers to woodchuck hepatitis virus post-transcriptional regulatory element; SV40polyA refers to monkey vacuolating virus 40 (SV40) polyadenylation sequence .

Figure 7 shows the effect of ELISA on the expression of IgG antibodies in antibody serum on day 40 after immunizing C57 mice with recombinant adeno-associated virus vaccine 1-6.

Figure 8 shows the effect of recombinant adeno-associated virus vaccine 4 on the expression of IgG antibodies in the antibody serum on the 40th day after immunizing C57 mice with different immunization methods.

Figure 9 shows the results of the serum neutralization test against the SARS-CoV-2 variant strain Delta virus on the 40th day after immunizing C57 mice with the recombinant adeno-associated virus vaccine 1-6.

Figure 10 shows the results of the serum neutralization test against the SARS-CoV-2 variant strain Omicron virus on the 60th day after immunizing C57 mice with the recombinant adeno-associated virus vaccine 1-6.

Detailed ways

The invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

In the description of the present invention, the following terms will be used, with brief descriptions and definitions as follows:

Vector refers to the delivery tool or element of genetic material, such as plasmids, viruses, virus-like particles, phages, etc. The term is also often referred to as cloning vectors, expression vectors or backbone vectors, as well as viral vectors depending on the type of application or scenario.

rAAV vector refers to a recombinant non-replicating adeno-associated virus. The rAAV vector includes a serotype protein capsid and encapsulates the recombinant genome. The genome includes functional 5' and 3' inverted terminal repeats (ITR sequences), and The flanking exogenous nucleotide sequence replaces the rep or cap gene of wild-type AAV. The ITR sequence provides functional rescue, replication, and packaging of rAAV. In some embodiments, the ITR sequence is from AAV2. Foreign nucleotide sequences usually consist of a series of expression regulatory elements and coding regions.

AAV serotype plasmid pREPCAP includes two open reading frames (ORFs) encoding Rep and Cap expression products. Cap refers to the capsid protein of AAV that is familiar to those skilled in the art, encoding capsid proteins VP1, VP2, VP3 and AAP and other functional proteins. Different serotypes of AAV have different capsid protein sequences.

AAV helper plasmid pHelper usually includes adenovirus VA RNA, E4ORF6, E2A, E1B and other coding regions to provide necessary functions for AAV replication.

Expression regulatory elements are usually a collection of promoter sequences, upstream regulatory regions, coding regions, transcriptional regulatory elements, etc., which together achieve the replication, transcription and translation of coding region sequences in recipient cells. A promoter is a DNA sequence that RNA polymerase recognizes, binds to, and starts transcription. It contains conserved sequences required for specific binding of RNA polymerase and initiation of transcription. Most of them are located upstream of the transcription start point of structural genes. The promoter itself is not affected by Transcription. In some cases, the promoter sequence is a CAG promoter. Suitable promoters also include promoters known to those skilled in the art, such as human cytomegalovirus (CMV) promoter, ubiquitin C promoter (UbC), EF1α A promoter, etc., optionally, may be selected as an expression control sequence for the transcription of the mRNA. In some cases SV40 polyA was chosen as the transcription terminator. Suitable polyA sequences include, but are not limited to, SV40, BGH, synthetic polyA, etc. known in the art. Some cases also include the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), a regulatory element that enhances transcription, and a sequence that enhances translation efficiency (Kozak sequence).

Unless otherwise stated, the reagents and materials used in the following examples were all commercially available.

Experimental methods without specifying specific conditions in the following examples usually follow conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing The manufacturer provides instructions and conditions.

Example 1 Construction of AAV expression vector plasmid

1. Obtaining antigen peptides

The gene sequence of SARS-COV-2 strain Delta (GenBank accession no.OM471068.1) was obtained through the NCBI database (https://www.ncbi.nlm.nih.gov/), and Uniprot blast (https://www .uniprot.org/blast/) Use the uniprotkb_refprotswissprot database to predict the conserved domains within the S protein sequence of variant strain Delta, as well as the RBD domain.

And the S protein and RBD domain of the variant strain Delta are processed as follows:

(1) Mutation obtains the sequence shown in SEQ ID NO.1;

Specifically, the R ₆₈₂ RAR ₆₈₅ site of the S protein of the SARS-CoV-2 variant strain Delta was mutated to G ₆₈₂ SAS ₆₈₅ , and the K ₉₈₆ V ₉₈₇ site was mutated to P ₉₈₆ P ₉₈₇ to obtain amino acids 13-1208 sequence.

(2) Mutation obtains the sequence shown in SEQ ID NO.2;

Specifically, it is the amino acid sequence 13-1208 obtained by mutating the K ₉₈₆ V ₉₈₇ site of the S protein of the SARS-CoV-2 variant strain Delta to P ₉₈₆ P ₉₈₇ .

(3) Mutation obtains the sequence shown in SEQ ID NO.3;

Specifically, the R ₆₈₂ RAR ₆₈₅ site of the S protein of the SARS-CoV-2 variant strain Delta was mutated to S ₆₈₂ RAG ₆₈₅ , and the K ₉₈₆ V ₉₈₇ site was mutated to P ₉₈₆ P ₉₈₇ to obtain amino acids 13-1208 sequence.

(4) Obtain the sequence shown in SEQ ID NO.4.

Specifically, it is the 319-593rd amino acid sequence of the gene sequence of the SARS-CoV-2 variant strain Delta, which is the spike protein receptor RBD domain of the S protein.

2. Construct expression vector

The obtained sequence was spliced after the tPA signal peptide, human codon optimization was performed through GenSmart Optimization (Version Beta 1.0), and DNAWorks (v3.2.4) (https://hpcwebapps.cit.nih.gov/dnaworks/) was used Design primers to use

HS DNA Polymerase (Takarabio) amplifies the synthesized sequence, and uses the endonucleases FastDigest ^TM EcoRI and FastDigest ^TM HindIII (ThermoFisher) to digest the adeno-associated virus backbone vector pAAV-CAG-MCS-WPRE-SV40polyA, using the ClonExpress MultiS One Step Cloning Kit The recombination kit (Nanjing Novozymes) performs a homologous recombination ligation reaction, transforms E. coli DH5ɑ, and spreads it on an ampicillin-resistant petri dish. After 16 hours, the colonies are picked for detection, and the positive clones are sent to a sequencing company (Suzhou Genewise ) was sequenced, and the plasmid with the correct sequencing result was named expression vector 1. The complete sequence of expression vector 1 is shown in SEQ ID NO. 15, and the composition and sequence of its components are shown in Figure 1. In the figure, the AAV2ITR (alternate) corresponding adeno-associated virus inverted terminal repeat sequence is shown in the sequence SEQ ID NO.8 in Table 1; tPA signal is the signal peptide and refers to the SEQ ID NO.5 sequence; Delta (B.1.617.2)- spike refers to SEQ ID NO.1 sequence; WPRE3 refers to woodchuck hepatitis virus post-transcriptional regulatory element, see sequence SEQ ID NO.13; SV40 late poly(A) signal refers to monkey vacuolating virus 40 (SV40) poly For the adenylate sequence, see sequence SEQ ID NO. 14.

Expression vector 2-6 was constructed according to the above method, and the sequences of its elements are shown in Table 1 below.

The schematic diagram of the completed vector map of expression vector 2 is shown in Figure 2. Among them, tPA signal is the signal peptide and refers to the SEQ ID NO.5 sequence; Delta(B.1.617.2)-spike refers to the SEQ ID NO.2 sequence; others The key components are the same as in Figure 1, and the complete sequence of expression vector 2 is shown in SEQ ID NO.16.

The schematic diagram of the completed vector map of expression vector 3 is shown in Figure 3, in which tPA signal is the signal peptide and refers to the SEQ ID NO.6 sequence; Delta(B.1.617.2)-spike refers to the SEQ ID NO.3 sequence; others The key components are the same as in Figure 1, and the complete sequence of expression vector 3 is shown in SEQ ID NO.17.

The schematic diagram of the completed vector map of expression vector 4 is shown in Figure 4, in which tPA signal is the signal peptide and refers to the SEQ ID NO.5 sequence; Delta(B.1.617.2)-spike refers to the SEQ ID NO.3 sequence; others The key components are the same as in Figure 1, and the complete sequence of expression vector 4 is shown in SEQ ID NO.18.

The schematic diagram of the completed vector map of expression vector 5 is shown in Figure 5, in which tPA signal is the signal peptide and refers to the SEQ ID NO.7 sequence; Delta(B.1.617.2)-spike refers to the SEQ ID NO.3 sequence; others The key components are the same as in Figure 1, and the complete sequence of expression vector 5 is shown in SEQ ID NO.19.

The schematic diagram of the completed vector map of expression vector 6 is shown in Figure 6. Among them, CMV Enhancer-SC40intron is the cytomegalovirus enhancer, see sequence SEQ ID NO.12, chickenβ-actin is the promoter splicing acceptor; tPA signal is the signal peptide. refers to the SEQ ID NO.5 sequence; Delta(B.1.617.2)-spike-RBD refers to the SEQ ID NO.4 sequence; ITR refers to the sequence SEQ ID NO.9, and WPRE3 refers to the woodchuck hepatitis virus post-transcriptional regulation The elements are the same as expression vector 1; SV40 late poly(A)signal is the polyadenylic acid sequence of monkey vacuolating virus 40 (SV40) and expression vector 1. The complete sequence of expression vector 6 is shown in SEQ ID NO.20.

Table 1 Elements and gene sequences in expression vectors 1-6

Among them, the element AAV2ITR (alternate) in expression vectors 1-5 corresponds to the -5' end of the adeno-associated virus inverted terminal repeat sequence; the element ITR in expression vector 6 corresponds to the -5' end of the adeno-associated virus inverted terminal repeat sequence; expression The element AAV2ITR in vector 1-6 corresponds to the 3' end of the adeno-associated virus inverted terminal repeat; the element SV40Promoter in expression vector 1-5 corresponds to the promoter of expression vector 1-5; the element CMV Enhancer- in expression vector 6 SC40intron corresponds to the promoter of expression vector 6; the element WPRE3 in expression vectors 1-6 corresponds to the post-transcriptional regulatory sequence; the element SV40 late poly(A)signal in expression vectors 1-6 corresponds to the terminator.

Example 2 Preparation of recombinant adeno-associated virus vaccine

293T cells were seeded in a 150 mm culture dish at a density of 1 × ¹⁰ cells per culture dish 24 hours before transfection, and 12 μg pHelper, 8 μg pRep2Cap5, 5 μg expression vector 1, and 10 μg transfection reagent polyethylenimine (25 kD ) for incubation for transfection. After 72 hours of transfection, cells were harvested by centrifugation at 4°C. Cells were resuspended in lysis buffer containing 50mM Tris-HCl (pH 8.0), 150mM NaCl. Harvest lysates were subjected to three freeze-thaw cycles in dry ice/ethanol and a 37°C water bath. Then 1 unit/mL nuclease and 0.5% sodium deoxycholate were added, the cell suspension was incubated at 37°C for 1 hour, centrifuged for 20 minutes, and the rAAV supernatant crude lysate was collected at 4°C. The crude lysate was diluted to a final volume of 10 mL with 10 mM Tris-HCl (pH 8.0) buffer, and then added to an ultracentrifuge tube to load iodixanol according to a gradient of 15%, 25%, 40%, and 60% mass to volume ratio. . Centrifuge at 350,000 × g for 1 hour at 18°C. Collect 3 mL of the 40% lower layer component and 0.5 mL of the 60% upper layer component as the purified solution. Use a 100 kDa cut-off ultrafiltration tube (Millipore) for ultrafiltration replacement. Virus preservation solution. The recombinant virus storage solution is PBS phosphate buffer (pH 7.4) and 0.05% Prosamer 188. The purified rAAV was labeled as vaccine 1, and SYBRGreenI qPCR was used to determine the virus titer. Store in -80°C refrigerator before use.

The preparation method of vaccine 2-6 is the same as vaccine 1, only the expression vector 2-6 and the above corresponding elements need to be modified.

Example 3 Immunization of C57 mice

(1) C57 mice aged 6-8 weeks were randomly divided into 7 groups, with 5 mice in each group. Vaccine groups 1-6 were injected intramuscularly into the right hind limbs with the recombinant adeno-associated virus vaccine 1-6 in Example 2 (the concentration was 2.5 ×10 ¹² vg/ml, injection volume 40 μL); the negative control group was injected with the same dose of recombinant green fluorescent protein adeno-associated virus blank vector AAV-GFP (concentration 2.5×10 ¹² vg/ml, injection volume 40 μL).

(2) C57 mice aged 6-8 weeks were randomly divided into 2 groups, with 5 mice in each group. In the vaccine 4-two hindlimb group, the mice were injected into the muscles of the hindlimbs with a volume of 20 μL of the recombinant adenoid-related protein in Example 2. Virus Vaccine 4 (concentration: 2.5×10 ¹² vg/ml); Vaccine 4-nasal drip group administered 20 μL of recombinant adeno-associated virus vaccine 4 in Example 2 (concentration: 5×10 ¹² vg/ml) intranasally to the mice. ml).

Adopt a one-time immunization protocol and inject on day 0; collect blood on days 40 and 60 respectively. Take 0.1mL-0.2mL of blood from each mouse, place it at 0°C for 60 minutes, and centrifuge at 4000 rpm for 15 minutes. , take the upper serum for ELISA detection analysis and antibody neutralization experiment.

Example 4 ELISA immunoassay

(1) Use 0.1M carbonate buffer (pH 9.6) to prepare the protein RBD319-541 (wild-type strain) into a solution with a concentration of 0.5μg/ml, add 100μL per well to a 96-well plate, and place Incubate overnight at 4°C; incubate in a 37°C incubator for 1 hour the next day; wash the plate three times with PBST (PBS+0.1% Tween 20), add 300 μL washing solution to each well; add 2% skimmed milk powder after washing the plate. Add 250 μL to each well and incubate at 37°C for 1 hour; wash the plate three times with PBST (PBS+0.1% Tween 20). Add the antibody serum produced by each group of mice in Example 5 to each well (dilution factor 1:300), add 100 μL to each well, and place it in a 37°C incubator for 1 hour; wash the plate three times; For the mouse serum group, HRP (horseradish peroxidase)-labeled IgG was added, 100 μL was added to each well, and incubated at room temperature for 1 hour; the plate was washed three times. Add TMB solution, 100 μL to each well, and develop color for 15 minutes at room temperature in the dark. Add 0.5M H ₂ SO ₄ solution to stop the color development, add 100 μL to each well, and detect the absorbance with a microplate reader. The detection wavelength is 450 nm and 570 nm is used as the background wavelength.

(2)Absorbance result analysis

1) Plot the absorbance (OD) value against the recombinant adeno-associated virus vaccine in Example 2. As shown in Figure 7, the recombinant adeno-associated virus vaccine in Example 2 of the present invention induced mice to produce 1-6 groups. The IgG antibody titer was significantly higher than the AAV-GFP blank control group, indicating that the vaccine induced a strong immune response in mice; the antibody titer result was 4>2>1, and the site mutation to S ₆₈₂ SAG ₆₈₅ increased the immunity of the vaccine activity but the site mutation is G ₆₈₂ SAS ₆₈₅ , the antibody titer is reduced, indicating that the mutation of the R ₆₈₂ RAR ₆₈₅ site has an impact on the immune activity of the vaccine; the vaccine 4 with the signal peptide tPA and the vaccine 3 with the signal peptide tPA new The antibody titer was significantly higher than the natural signal peptide, indicating that different signal peptides have an impact on the activity of the vaccine.

2) Plot the absorbance (OD) value against different administration methods, as shown in Figure 8, 4-two hind limbs>4-right hind limb>4-nasal instillation, the antibody titer after intramuscular injection administration is higher than that of instillation Nasal administration, especially intramuscular injection of the two hind limbs, is more effective.

Example 5 Neutralizing Antibody Experiment

Vero E6 cells (2 × 10 cells/ ^well ) were seeded into a 96-well plate and cultured overnight at 37°C and 5% CO _until a monolayer formed. Mix 100 TCID50 of SARS-CoV-2 variant strain Delta and SARS-CoV-2 variant strain Omicron with mouse antiserum serially diluted 4 times, and incubate at 37°C for one hour. Serum samples are then incubated at 56°C. Heat for 30 minutes, then add to Vero E6 cell culture wells and mix. In each assay, cells infected with 100 TCID50 of SARS-CoV-2 variant strain Delta and SARS-CoV-2 variant strain Omicron were respectively used as positive controls, while cells without virus were used as negative controls, and then CPE (cytopathic effect) was recorded on day 3 post-infection. The Reed-Muench method was used to count the number of Vero E6 cells (CPE) infected by SARS-CoV-2 variant strain Delta and SARS-CoV-2 variant strain Omicron, respectively, which can cause 50% of the inoculated cells to produce cytopathic effects in mice. The maximum dilution factor of the serum is the neutralizing antibody titer (NA) of the vaccine.

The higher the neutralizing antibody titer, the lower the level of virus replication and the stronger the protection against viral infection. The neutralizing antibody (NA) values were plotted against the recombinant adeno-associated virus vaccines 1-6 in Example 2. As shown in Figure 9, the serum neutralizing antibody titers induced by the vaccine groups 1-6 were significantly higher than those of AAV- The GFP blank control group shows that the recombinant adeno-associated virus vaccine 1-6 produces a specific humoral immune response in mice and protects cells from infection by the SARS-CoV-2 variant strain Delta. The vaccine has an obvious prevention and treatment effect.

As shown in Figure 10, in the experimental results of neutralizing antibodies against the SARS-CoV-2 variant strain Omicron, the serum neutralizing antibody titers induced by vaccine groups 1-6 were higher than those in the AAV-GFP blank control group, and The vaccines in vaccine groups 1, 4 and 5 were significantly higher than those in other groups, indicating that recombinant adeno-associated virus vaccines 1-6 produced specific humoral immune responses in mice and protected cells from SARS-CoV-2 variant strain Omicron infection, the vaccine has obvious preventive and therapeutic effects.

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, etc. may be made without departing from the spirit and principles of the present invention. All simplifications should be equivalent substitutions, and are all included in the protection scope of the present invention.

Claims

SARS-COV-2 virus antigen polypeptide is characterized in that its amino acid sequence is one of the following amino acid sequences:

(1) The sequence shown in SEQ ID NO.1;

(2) The sequence shown in SEQ ID NO.2;

(3) The sequence shown in SEQ ID NO.3;

(4) The sequence shown in SEQ ID NO.4.
A nucleotide sequence encoding the antigen polypeptide of claim 1.
A recombinant adeno-associated virus expression vector, characterized by containing the nucleotide sequence of claim 2.
The recombinant adeno-associated virus expression vector according to claim 3, characterized in that it contains an adeno-associated virus inverted terminal repeat sequence, a nucleotide sequence encoding a signal peptide, and a nucleotide encoding the antigen polypeptide of claim 1 sequence.
The recombinant adeno-associated virus expression vector according to claim 4, wherein the signal peptide is one of the following:

(1) tPA signal peptide: the amino acid sequence is shown in SEQ ID NO.5;

(2) New tPA signal peptide: the amino acid sequence is shown in SEQ ID NO.6;

(3) Natural signal peptide: The amino acid sequence is shown in SEQ ID NO.7.
Application of the antigen polypeptide of claim 1, the DNA molecule of claim 2, and the expression vector of any one of claims 3 to 5 in the preparation of a vaccine to prevent new coronavirus pneumonia.
A SARS-COV-2 virus vaccine, characterized in that it is prepared using the antigen polypeptide of claim 1 as an antigen.
A SARS-COV-2 virus vaccine, characterized in that it is expressed by an expression vector including any one of claims 3-5.
The SARS-COV-2 virus vaccine according to claim 8, further comprising a pharmaceutically acceptable diluent and/or excipient.
A method for preparing a SARS-COV-2 virus vaccine, which is characterized in that it is prepared through the following steps:

pHelper, pRep2Cap5 and the expression vector described in any one of claims 3 to 5 are co-incubated; the cells are transfected in the presence of the transfection reagent polyethylenimine; after culturing the cells, the cells are collected by centrifugation and lysed and purified to obtain SARS-COV-2 virus vaccine.