CN113398260B

CN113398260B - New corona vaccine using vaccinia virus as vector

Info

Publication number: CN113398260B
Application number: CN202110877781.2A
Authority: CN
Inventors: 袁明
Original assignee: Shenzhen Huayao Kangming Biopharmaceutical Co ltd
Current assignee: Shenzhen Huayao Kangming Biopharmaceutical Co ltd
Priority date: 2021-08-02
Filing date: 2021-08-02
Publication date: 2021-12-24
Anticipated expiration: 2041-08-02
Also published as: CN113398260A

Abstract

The application relates to a novel coronavirus south Africa mutant vaccine based on a genetically engineered vaccinia virus as a vector. The vaccine takes A46R-deficient vaccinia virus as a vector to carry a new corona virus south Africa mutant strain S gene nucleic acid sequence, the vaccinia virus vector can also carry IL-21, and the vaccine can generate antibodies aiming at the new corona virus south Africa mutant strain after mice are immunized.

Description

New corona vaccine using vaccinia virus as vector

Technical Field

The application relates to the field of biomedicine, in particular to a new corona vaccine of a new corona virus south Africa variant strain with vaccinia virus as a vector.

Background

The new coronavirus is a coronavirus which can infect upper respiratory tract to cause common cold symptoms such as fever, cough, laryngitis and the like, and can infect lower respiratory tract to cause acute respiratory tract symptoms such as bronchitis, pneumonia and the like. The novel coronavirus is an enveloped single-stranded positive-strand RNA virus, and spike protein S embodied on the surface of the virus is a specific tissue structure on the virus envelope, and plays an important role in invasion of a target cell by the virus and recognition of the virus and the cell. In order to combat pneumonia caused by new coronavirus, development of various vaccines capable of effectively preventing infection with new coronavirus is urgently needed.

Disclosure of Invention

The application provides a vaccinia virus vector vaccine which comprises a modified new coronavirus south Africa variant spike protein S gene and/or interleukin 21 (IL-21) gene. The vaccinia virus vector vaccine can effectively improve the level of in vivo antibody production, the produced antibody can effectively neutralize the new coronavirus and the variant thereof, and the vaccinia virus vector vaccine can effectively prevent the infection of the south Africa variant of the new coronavirus.

In one aspect, the present application provides a vaccinia virus vector vaccine comprising an altered new coronavirus south african variant spike protein S gene, wherein the vaccinia virus vector has all of the genes encoding a46R deleted, the altered new coronavirus south african variant spike protein S having the following mutations compared to the amino acid sequence of wild-type new coronavirus spike protein S: L18F, D80A, D215G, Delta 241-Across 243, R246I, K417N, E484K, N501Y, D614G, D663G, P681H, A701V, K986P and V987P.

In certain embodiments, the vaccinia virus vector is derived from a vaccinia virus listeria strain.

In certain embodiments, the altered mutant south african coronavirus spike protein S gene is located in the a46R region of a vaccinia virus vector.

In certain embodiments, the a46R gene of the vaccinia virus vector is replaced with the spike protein S gene of the altered new coronavirus south african variant.

In some embodiments, the amino acid sequence of the spike protein S of the modified novel south African variant coronavirus is shown as SEQ ID NO. 2.

In some embodiments, the nucleic acid sequence of the spike protein S of the modified novel south African variant coronavirus is shown as SEQ ID NO. 1.

In certain embodiments, the vaccinia virus vector further comprises an exogenous interleukin 21 (IL-21) gene.

In certain embodiments, the IL-21 is human IL-21.

In certain embodiments, the IL-21 gene is located in the a46R region of a vaccinia virus vector.

In certain embodiments, the a46R gene of the vaccinia virus vector is replaced with the altered south african variant spike protein S gene and the IL-21 gene.

In certain embodiments, the amino acid sequence of IL-21 is set forth in SEQ ID NO 3.

In another aspect, the present application provides nucleic acid molecules encoding the vaccinia virus vector vaccines.

In another aspect, the present application also provides pharmaceutical compositions comprising the vaccinia virus vector vaccine, and optionally a pharmaceutically acceptable carrier.

In another aspect, the present application also provides methods of making the vaccinia virus vector vaccines.

In some embodiments, the method for preparing a vaccinia virus vector vaccine comprises recombining the shuttle vector with vaccinia virus to obtain vaccinia virus with a deleted gene in the A46R region, and inserting the S gene and the IL-21 gene of the south Africa mutant strain of the New crown virus into the A46R region of the vaccinia virus.

In another aspect, the present application also provides a virus-producing cell capable of producing the vaccinia virus vector vaccine.

In certain embodiments, the virus-producing cell comprises a CV-1 cell.

In certain embodiments, the virus-producing cell comprises a HEK293 cell.

In another aspect, the application further provides the use of the vaccinia virus vector vaccine in the preparation of a medicament for preventing and/or treating new coronary pneumonia.

Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.

Drawings

The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The drawings are briefly described as follows:

FIG. 1 shows the mutation sites of S protein of the modified south African variant of the novel coronavirus as described in the present application.

FIG. 2 shows a schematic diagram of a pmSA shuttle vector constructed from the novel vaccine of south African variant coronavirus, the recombination process and its location.

FIG. 3 shows a schematic structural diagram of a novel south African variant vaccine against coronavirus according to the present application.

FIG. 4 shows the identification of Vac-351 vector as a mutant vaccine of New Guannan Africa.

FIG. 5 shows the detection of the titer of specific anti-new coronavirus S protein antibody produced by Vac-351 vaccine mutant strain of New Guannan Africa in the present application.

FIG. 6 shows the effect of neutralizing wild-type neocoronavirus in mice immunized with south African variant vaccine of the new coronavirus.

FIG. 7 shows the effect of neutralizing the south African variant of New coronavirus produced by mice immunized with the south African variant vaccine of New coronavirus described herein.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure of the present specification.

Definition of terms

In the present application, the term "vaccinia virus vector vaccine" generally refers to a vaccine in which vaccinia virus is used as a vector. In certain embodiments, the vaccine can be constructed by loading a gene of interest into a vaccinia virus vector. In certain embodiments, the vaccinia virus comprises a listeria strain (Lister). In certain embodiments, the vaccinia virus vector comprises one or more mutations. For example, the a46R gene of the vaccinia virus vector is deleted.

In the present application, the term "SARS-CoV-2 (Severe acid Respiratory Syndrome Virus 2)" generally refers to Severe Acute Respiratory Syndrome Coronavirus type 2. In the present application, the term encompasses any form of SAR-CoV-2 and homologues, derivatives, mutants and functionally active fragments thereof.

In the present application, the term "S protein" generally refers to the Spike protein of the coronary protein (Spike protein). The S protein may typically contain two subunits (subbunit), S1 and S2. S1 mainly contains a receptor binding domain (receptor binding domain), which can be responsible for recognizing a receptor of a cell. S2 contains the essential elements required for the membrane fusion process. In the present application, the S protein may be the S protein of SAR-Cov-2. In the present application, the term encompasses homologues, derivatives, mutants and functionally active fragments of the S protein.

In the present application, the term "mutant" generally refers to an amino acid sequence having a deletion, insertion and/or substitution of one or more amino acids. For example, the mutant may comprise a protein or polypeptide that has been altered by at least 1, such as 1-30, 1-20 or 1-10, and further such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions and/or insertions. The mutant may substantially retain the biological properties of the protein or the polypeptide prior to the alteration (e.g., substitution, deletion, or addition). In the present application, the mutant may comprise a sequence homology of at least about 80% (e.g., at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) as compared to the amino acid sequence of the protein.

In this application, the term "a 46R" generally refers to the viral protein of vaccinia virus that contains the TIR domain. In the present application, the term also encompasses homologues, derivatives, mutants and functionally active fragments thereof. In the present application, the left and right arms of the a46R region are generally defined as being located upstream and downstream of the S gene. For example, the left arm of the a46R region may be located upstream of the S gene. For example, the right arm of the a46R region can be located downstream of the S gene.

In this application, the term "IL-21" also referred to as "interleukin-21", which term also encompasses homologues, derivatives, mutants and functionally active fragments of IL-21. For example, the IL-21 may comprise human IL-21. For example, the cDNA and amino acid sequences of human IL-21 can be found, for example, in GenBank accession numbers BC066260.1 and AAH 69124.1. For example, the IL-21 may comprise the amino acid sequence shown in SEQ ID NO. 3.

In the present application, the terms "include" and "comprise" generally mean including, summarizing, containing or including. In some cases, the meaning of "is", "consisting of … …" may also be indicated.

In the present application, the term "about" generally means varying from 0.5% to 10% above or below the stated value, for example, varying from 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the stated value.

In the present application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to the cells or animals to which they are exposed at the dosages and concentrations employed. Physiologically acceptable carriers can include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides, and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions, such as sodium, and/or nonionic surfactants.

In the present application, the term "pharmaceutical composition" generally refers to a formulation in a form that allows the biological activity of the active ingredient to be effective, and which does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is to be administered. These formulations may be sterile.

In one aspect, the present application provides a vaccinia virus vector vaccine comprising an altered new coronavirus south african variant spike protein S gene, wherein the vaccinia virus vector has all of the genes encoding a46R deleted, the altered new coronavirus south african variant spike protein S having the following mutations compared to the amino acid sequence of a wild-type new coronavirus spike protein S: L18F, D80A, D215G, Delta 241-Across 243, R246I, K417N, E484K, N501Y, D614G, D663G, P681H, A701V, K986P and V987P.

In the present application, a vaccinia virus expression vector is provided having a deletion of the a46R gene. For example, the vaccinia virus expression vector has a deletion of all of the a46R gene. In the present application, the vaccinia virus vector may express a gene of interest. For example, the gene of interest may comprise an exogenous gene of interest. For example, the gene of interest may include one or more exogenous genes of interest.

In the present application, the vaccinia virus vector comprises a deletion in the a46R gene. In the present application, a shuttle plasmid carrying the modified S gene can be constructed, and homologous recombinant vaccinia virus carrying the modified S gene can be obtained by transfection using Cas9 as a mediator. In the present application, the vaccinia virus vector vaccine may comprise an altered spike protein S gene of a new variant of south africa of corona virus. In the present application, the altered south african variant spike protein S of the novel coronavirus may comprise one or more amino acid mutations compared to the amino acid sequence of the wild-type novel coronavirus. For example, the S protein may comprise a L18F mutation. For example, the S protein may comprise a D80A mutation. For example, the S protein may comprise a D215G mutation. For example, the S protein may comprise the Δ 241-243 mutation. For example, the S protein may comprise a R246I mutation. For example, the S protein may comprise a K417N mutation. For example, the S protein may comprise an E484K mutation. For example, the S protein may comprise the N501Y mutation. For example, the S protein may comprise a D614G mutation. For example, the S protein may comprise a D663G mutation. For example, the S protein may comprise a P681H mutation. For example, the S protein may comprise a701V mutation. For example, the S protein may further comprise a stability-increasing mutation, e.g., the stability-increasing mutation may comprise a proline mutation. For example, the S protein may comprise a proline mutation at position 986. For example, the S protein may comprise a proline mutation at position 987.

In the present application, the altered south african variant spike protein S of the new coronavirus may comprise a mutation at one or more of the following positions compared to the amino acid sequence of the wild-type new coronavirus: 18 th, 80 th, 215 th, 241 th, 242 th, 243 th, 246 th, 417 th, 484 th, 501 th, 614 th, 663 th, 681 th, 701 th, 986 th, and 987 th bits.

In the present application, the amino acid position of the spike protein S of the modified south african variant of the new coronavirus is determined by the amino acid sequence of the spike protein S of the wild-type new coronavirus.

In the present application, the modified south african variant spike protein S of the new coronavirus may comprise the following mutations compared to the amino acid sequence of the wild-type new coronavirus: L18F, D80A, D215G, Delta 241-Across 243, R246I, K417N, E484K, N501Y, D614G, D663G, P681H, A701V, K986P and V987P. In the application, the modified south African variant of the novel coronavirus spike protein S can comprise an amino acid sequence shown as SEQ ID NO. 2. In the application, the spike protein S of the modified south African variant strain of the novel coronavirus can comprise a nucleic acid sequence shown as SEQ ID NO. 1. In the present application, the altered south african variant spike protein S of the novel coronavirus may comprise an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% homology to the amino acid sequence of the wild-type novel coronavirus.

In the present application, the a46R gene of the vaccinia virus can be replaced with the spike protein S gene of the modified south african variant of the new coronavirus.

In the present application, the engineered novel south african variant spike protein S gene of coronavirus may be located between the left and right arms of the a46R region.

In the present application, the vaccinia virus vector vaccine may also express IL-21. In the present application, the IL-21 may comprise human IL-21. In the present application, the IL-21 may comprise the amino acid sequence shown in SEQ ID NO. 3. In the present application, the vaccinia virus vector may comprise an IL-21 gene. In the present application, the vaccinia virus may comprise both the spike protein S gene and the IL-21 gene of the altered south African variant of the novel coronavirus.

In the present application, the IL-21 gene is located between the left and right arms of the A46R region of the vaccinia virus vector.

In the present application, the 5 'end of the IL-21 gene may be directly or indirectly linked to the 3' end of the spike protein S gene of the modified south African variant of the novel coronavirus.

In the present application, the 3 'end of the IL-21 gene may be directly or indirectly linked to the 5' end of the spike protein S gene of the modified south African variant of the novel coronavirus.

In the present application, the IL-21 may comprise the amino acid sequence shown in SEQ ID NO. 3. In the present application, the IL-21 may comprise an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% homology to the amino acid sequence set forth in SEQ ID NO. 3.

In another aspect, the present application also provides a nucleic acid molecule encoding the vaccinia virus vector vaccine.

In another aspect, the present application also provides a pharmaceutical composition comprising a vaccinia virus vector vaccine as described herein, and optionally a pharmaceutically acceptable carrier.

In certain embodiments, the vaccinia virus vector vaccines described herein can be prepared as injectable formulations. For example, the injection may be subcutaneous. For example, the injection may be intramuscular. For example, the injection may be an intradermal injection.

In another aspect, the present application also provides methods of making the vaccinia virus vector vaccines. In this application, the preparation method may comprise constructing a shuttle plasmid carrying the engineered S gene, co-transfecting a46RgRNA vector (targeting region) with Cas9 into CV-1 cells, transfecting the shuttle plasmid into cells after viral infection, and purifying the virus.

In another aspect, the present application also provides a virus-producing cell capable of producing the vaccinia virus vector vaccine. For example, the virus-producing cells can comprise CV-1 cells. For example, the virus-producing cell may comprise a HEK293 cell.

In another aspect, the present application also provides the vaccinia virus vector vaccine for use in preventing and/or treating a new coronary pneumonia drug.

In another aspect, the present application also provides a method of preventing and/or treating a new coronary pneumonia drug comprising administering to a subject in need thereof a vaccinia virus vector vaccine as described herein.

In certain embodiments, the new coronary pneumonia may comprise new coronary pneumonia caused by a new variant of the south africa of the new coronary virus.

Without wishing to be bound by any theory, the following examples are only intended to illustrate various aspects of the invention of the present application and are not intended to limit the scope of the invention of the present application.

EXAMPLE 1 Synthesis of vaccinia Virus vector vaccine

1. Construction of shuttle plasmid

Designing a mutant S gene according to a genome sequence of a south African mutant strain B.1.351/501Y.V2 of the novel crown virus, wherein the designed S gene sequence contains a first part and a second part of the S gene of a mutation site of the south African mutant strain of the novel crown virus, and the specific mutation site is shown in figure 1. On the basis, recombinant sequences including the A46R arm and the green fluorescent protein gene, the S gene of the mutant site of the new south Africa mutant strain and the human interleukin 21 (IL-21) gene sequence are designed, a promoter H5 is added in front of each gene, and a schematic diagram of a recombinant region is shown as a shuttle plasmid pmSA recombinant region in FIG. 2. In FIG. 2, H5 is a promoter, GFP is green fluorescent protein, mSA is a mutant S gene designed based on a mutant strain of south Africa New crown, and IL-21 is human interleukin 12 gene. And (3) carrying out recombinant complete sequence whole gene synthesis, adding EcoRV enzyme cutting sites at two ends of the gene, obtaining the synthesized gene, and cloning the synthesized gene into the EcoRV/EcoRV enzyme cutting sites of a pUC57 vector, thereby obtaining the shuttle plasmid pmSA. Nanjing Kingsrey Biotechnology, Inc. provides for the cloning of gene synthesis and cloning vector pUC57 and shuttle plasmid pmSA.

The shuttle plasmid pmSA was confirmed to carry the correct S gene mutation sequence by EcoRV cleavage and sequencing.

2. Preparation of vaccinia virus vector vaccine

1) Homologous recombination vaccinia virus recombination mediated by Cas9

3X 10 cells were transfected the day before transfection⁵One CV-1 cell was seeded into one well of a six-well plate. The a46RgRNA vector (targeting region) was co-transfected with Cas9 into CV-1 cells in six-well plates using Lipofectamine3000 (Thermo Fisher Scientific). The next day, wells transfected with a46RgRNA vector and Cas9 gene were infected with 0.01 PFU/cell of the backbone virus. Shuttle vectors for homologous recombination were transfected into infected wells 2 hours after virus infection. Cells were harvested after 24 hours and frozen at-80 ℃ for recombinant virus purification. The resulting recombinant vaccinia virus was designated Vac-351.

The insertion of the gene of interest and the deletion of vaccinia virus A46R gene were identified. CV1 cells were infected with purified recombinant vaccinia virus, DNA was extracted from the cells after 1 to 2 days of infection, and recombinant virus was identified by PCR under 94 ℃ annealing for 2 min, followed by 36 PCR cycles: 94 ℃ 20 seconds, 52 ℃ 20 seconds, 72 ℃ 30 seconds. The PCR products were analyzed by electrophoresis in 1% agarose.

PCR primers to identify deletion of vaccinia virus a46R gene:

A46R Forward primer 5'-TTGGCTATTAAACAGTATGGA-3' (SEQ ID NO: 4)

A46R reverse primer 5'-GGATCCCGATAACAAATG-3' (SEQ ID NO: 5)

Primers for identifying S gene inserted vaccinia virus:

s Gene forward primer 5'-CGTGGTGTTTATTACCCTGA-3' (SEQ ID NO: 6)

S Gene reverse primer 5'-ACAATAAGTAGGGACTGGGT-3' (SEQ ID NO: 7)

The results are shown in FIG. 4, in which C is the DNA of the control virus and B is the DNA of Vac-351 constructed in the present application. C19 is amplified new coronavirus S gene, A46R is amplified A46R gene, and M is DNA molecular weight marker. The result shows that the A46R gene is deleted and the S gene of the new south-coronal Africa mutant vaccine is inserted in the new south-coronal Africa mutant vaccine.

2) Purification of recombinant viruses

Collected cell lysates of Cas 9-mediated homologous recombination were released of virus by freeze-thawing and 0.5 μ l of the lysates were used to infect all 6 wells of a six-well plate containing CV1 cells grown to 80-90% confluence. 48 hours after infection, each well was examined carefully under a fluorescent microscope for green-fluorescent virus. After identifying the positive lesions, their positions were marked on the lower surface of the plate with a marker pen. The virus plaques were then carefully picked with a 20 μ l tip after aspirating the medium from the wells in a super clean bench where the cells were cultured. The tip was then submerged in a cryovial containing 200 microliters of cell culture fluid. After one freeze-thaw cycle, 5-20 μ l of the virus solution was added to each well of a new 6-well plate containing CV1 cells. This process was repeated for 3 to 5 cycles until each viral plaque fluoresced green, i.e., all viral plaques were caused by the recombinant virus. After confirming that the virus was purified, the infected cells were scraped off and centrifuged to obtain cell pellets. Then, a part of the cells was taken to extract viral DNA. The purity of the virus was confirmed by PCR amplification of the target gene from the extracted viral DNA.

3) Amplification of viruses

After confirming that the recombinant virus was the desired recombinant virus by PCR, 100 μ l of the virus lysate was added to a T175 flask containing CV1 cells and grown to 90-100% confluence in a cell culture medium containing about 30 ml. After 48 hours, the cells and medium were scraped and the "primary virus amplification" was preserved to obtain seed viruses.

4) Production and purification of viruses

CV-1 cells were cultured and expanded to 36 flasks using T175 cell culture flasks. When the confluency of the cells in the flask was observed to be 100%, infection with the virus started. Taking out the frozen seed virus from a refrigerator at-80 deg.C, thawing at 37 deg.C, vortex shaking for 30s, and adding 0.25-0.3 ml seed virus per bottle. The flask was gently shaken to shake the mixture and placed back into the cell incubator for culture.

After 48 hours of infection, GFP is observed under a fluorescence microscope, and the virus can be harvested when the infection amount reaches more than 100%.

The infected cells were harvested with a scraper along with the cell sap and dispensed into 50ml centrifuge tubes. Centrifuging at 4 deg.C for 5 min at 2000 rpm, removing supernatant, adding the above infected cell mixture into the tube, and centrifuging repeatedly until the infected cell mixture is completely removed.

Finally, 20mL of cold 10mM Tris-HCl (pH =9.0) was added to the pellet, the pellet was blown up and mixed well, and frozen in a freezer at-80 ℃ for use.

3. Virus purification

The frozen virus solution was removed from the-80 ℃ freezer and thawed at 37 ℃ to release virus particles. The virus solution was then added to the mill and repeatedly milled 30-60 times before transferring to a 50mL centrifuge tube. The mill was rinsed with the appropriate amount of Tris-HCl (PH =9.0) and the liquids were combined into the above 50mL centrifuge tube, which was placed on ice. 2000 rpm, 5 min, centrifugation at 4 ℃, the supernatant (containing released virus particles) was collected and diluted with 10mM Tris-HCl (PH =9.0) buffer to a total volume of 30 mL.

23 mL of 36% sucrose (w/v) was added to a SW28Beckman centrifuge tube (typically, the SW28Beckman centrifuge tube has a volume of 38.5mL, and during ultracentrifugation, the volume of the solution should be larger than 2/3 of the volume of the centrifuge tube), and 10 mL of the virus mixture was added slowly to form two layers. General sucrose: virus fluid =2:1-2.5:1, weighed, trimmed, and replated with 10mM Tris-HCl (PH =9.0) to a weight error of less than 0.5 g.

General numbers

1 and 4, 2 and

5, 3 and 6 baskets were symmetrically trimmed, 1 mL liquid =1 g. Centrifuge at 13500 rpm for 80 min at 4 ℃. The supernatant was carefully blotted dry and the pellet resuspended with 2-2.5 mL of resuspension buffer (PBS +10% glycerol +138mM NaCl).

Packaging the virus solution into 200 μ L/tube, and freezing at-80 deg.C in ultra-low temperature refrigerator for storage.

Example 2 in vivo antibody titer detection in mice after immunization

C57BL/6 mice, 4-6 weeks old, were harvested, the right dorsal coat removed, and injected intradermally with a needle. The injection dose is 10⁶PFU/mouse. 20. mu.l of blood was taken through the tail vein on days 14, 28, 35, 42 and 56 after inoculation of the vaccinia virus vector neocoronary vaccine, respectively, and serum was isolated and antibody detection against the SARS-CoV-2S protein of the neocoronary virus was performed.

The ELISA method detects antibodies against the novel coronavirus spike protein in the immunized mice. The antibody titer was detected by using the anti-novel coronavirus antibody kit (Shenzhen, New Productivity Biotech), and the specific procedures were described in the kit.

The results are shown in fig. 5, C01, C02, C03, C04 are four immunized mice; the control was mice not immunized with the new corona vaccine. D14, D28, D35, D42, D56 refer to days 14, 28, 35, 42 and 56 after immunization. Antibody detection specific anti-neocoronavirus S protein antibodies were determined by ELISA. The Y-axis values are calculated for the antibody dilution Log 10. Antibody detection results it was found that 4 immunized animals detected specific antibodies against the S protein of the novel coronavirus at each detection time point. The experimental result shows that the new south China African mutant vaccine Vac-351 obtained in the invention can generate antibodies against S protein of new corona virus, so that the vaccine can be further developed for preventing the new south China African mutant.

Example 3 neutralization of New coronavirus by antibodies generated from south Africa mutant vaccine against New coronavirus

Neutralizing antibody experiments were performed using pseudoviruses. The neutralizing effect of the antibody produced by the south Africa mutant vaccine of the new coronavirus on the pseudovirus of the wild type of the new coronavirus (SARS-CoV-2) and the pseudovirus of the south Africa mutant of the new coronavirus (B.1.351/501Y V2) is respectively tested.

HeLa cells expressing human ACE2 were used as target cells for pseudovirus infection. Diluting the immune mouse serum to obtain antibodies with different dilutions, and respectively adding the antibodies with different dilutions to test the ability of the antibodies to neutralize pseudoviruses and prevent the entering pseudoviruses from entering target cells. The amount of the pseudovirus entering the target cell is judged by the luciferase activity in the target cell. Higher enzyme activity indicates more pseudovirus entering the target cell, and less neutralization. In this experiment, the percentage of the ability to inhibit infection by the new coronavirus (at a viral load of 2000 TCID) after neutralization at different dilutions was tested using serum from non-immunized mice as a control.

The results of the experiments are shown in fig. 6 and 7, with the Y-axis being the percentage of virus infected cells and the X-axis being the different dilutions of the antibody. The results show that the antibody produced by the south African mutant vaccine of the new coronavirus can neutralize wild-type new coronavirus and south African mutant of the new coronavirus.

The foregoing detailed description is provided by way of illustration and example, and is not intended to limit the scope of the appended claims. Various modifications of the presently recited embodiments will be apparent to those of ordinary skill in the art and are intended to be within the scope of the appended claims and their equivalents. .

Sequence listing

<110> Shenzhen Hua Yao kang Ming biological medicine industry Limited liability company

<120> novel corona vaccine using vaccinia virus as vector

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<170> PatentIn version 3.5

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gattattctg tcctatataa ttccgcatca ttttccactt ttaagtgtta tggagtgtct 1140

cctactaaat taaatgatct ctgctttact aatgtctatg cagattcatt tgtaattaga 1200

ggtgatgaag tcagacaaat cgctccaggg caaactggaa acattgctga ttataattat 1260

aaattaccag atgattttac aggctgcgtt atagcttgga attctaacaa tcttgattct 1320

aaggttggtg gtaattataa ttacctgtat agattgttta ggaagtctaa tctcaaacct 1380

tttgagagag atatttcaac tgaaatctat caggccggta gcacaccttg taatggtgtt 1440

aaaggtttta attgttactt tcctttacaa tcatatggtt tccaacccac ttatggtgtt 1500

ggttaccaac catacagagt agtagtactt tcttttgaac ttctacatgc accagcaact 1560

gtttgtggac ctaaaaagtc tactaatttg gttaaaaaca aatgtgtcaa tttcaacttc 1620

aatggtttaa caggcacagg tgttcttact gagtctaaca aaaagtttct gcctttccaa 1680

caatttggca gagacattgc tgacactact gatgctgtcc gtgatccaca gacacttgag 1740

attcttgaca ttacaccatg ttcttttggt ggtgtcagtg ttataacacc aggaacaaat 1800

acttctaacc aggttgctgt tctttatcag ggtgttaact gcacagaagt ccctgttgct 1860

attcatgcag atcaacttac tcctacttgg cgtgtttatt ctacaggttc taatgttttt 1920

caaacacgtg caggctgttt aataggggct gaacatgtca acaactcata tgagtgtggc 1980

atacccattg gtgcaggtat atgcgctagt tatcagactc agactaattc tcatcggcgg 2040

gcacgtagtg tagctagtca atccatcatt gcctacacta tgtcacttgg tgcagaaaat 2100

tcagttgctt actctaataa ctctattgcc atacccacaa attttactat tagtgttacc 2160

acagaaattc taccagtgtc tatgaccaag acatcagtag attgtacaat gtacatttgt 2220

ggtgattcaa ctgaatgcag caatcttttg ttgcaatatg gcagtttttg tacacaatta 2280

aaccgtgctt taactggaat agctgttgaa caagacaaaa acacccaaga agtttttgca 2340

caagtcaaac aaatttacaa aacaccacca attaaagatt ttggtggttt taatttttca 2400

caaatattac cagatccatc aaaaccaagc aagaggtcat ttattgaaga tctacttttc 2460

aacaaagtga cacttgcaga tgctggcttc atcaaacaat atggtgattg ccttggtgat 2520

attgctgcta gagacctcat ttgtgcacaa aagtttaacg gccttactgt tttgccacct 2580

ttgctcacag atgaaatgat tgctcaatac acttctgcac tgttagcggg tacaatcact 2640

tctggttgga cctttggtgc aggtgctgca ttacaaatac catttgctat gcaaatggct 2700

tataggttta atggtattgg agttacacag aatgttctct atgagaacca aaaattgatt 2760

gccaaccaat ttaatagtgc tattggcaaa attcaagact cactttcttc cacagcaagt 2820

gcacttggaa aacttcaaga tgtggtcaac caaaatgcac aagctttaaa cacgcttgtt 2880

aaacaactta gctccaattt tggtgcaatt tcaagtgttt taaatgatat cctttcacgt 2940

cttgaccccc cggaggctga agtgcaaatt gataggttga tcacaggcag acttcaaagt 3000

ttgcagacat atgtgactca acaattaatt agagctgcag aaatcagagc ttctgctaat 3060

cttgctgcta ctaaaatgtc agagtgtgta cttggacaat caaaaagagt tgatttttgt 3120

ggaaagggct atcatcttat gtccttccct cagtcagcac ctcatggtgt agtcttcttg 3180

catgtgactt atgtccctgc acaagaaaag aacttcacaa ctgctcctgc catttgtcat 3240

gatggaaaag cacactttcc tcgtgaaggt gtctttgttt caaatggcac acactggttt 3300

gtaacacaaa ggaattttta tgaaccacaa atcattacta cagacaacac atttgtgtct 3360

ggtaactgtg atgttgtaat aggaattgtc aacaacacag tttatgatcc tttgcaacct 3420

gaattagact cattcaagga ggagttagat aaatatttta agaatcatac atcaccagat 3480

gttgatttag gtgacatctc tggcattaat gcttcagttg taaacattca aaaagaaatt 3540

gaccgcctca atgaggttgc caagaattta aatgaatctc tcatcgatct ccaagaactt 3600

ggaaagtatg agcagtatat aaaatggcca tggtacattt ggctaggttt tatagctggc 3660

ttgattgcca tagtaatggt gacaattatg ctttgctgta tgaccagttg ctgtagttgt 3720

ctcaagggct gttgttcttg tggatcctgc tgcaaatttg atgaagacga ctctgagcca 3780

gtgctcaaag gagtcaaatt acattacaca taa 3813

<210> 2

<211> 1270

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> modified amino acid sequence of S protein

<400> 2

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Phe Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Ala

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

115 120 125

Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro Ile Asn Leu Val Arg Gly Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu His Ile Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr

245 250 255

Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe

260 265 270

Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys

275 280 285

Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr

290 295 300

Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr

305 310 315 320

Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly

325 330 335

Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg

340 345 350

Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser

355 360 365

Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu

370 375 380

Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg

385 390 395 400

Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala

405 410 415

Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala

420 425 430

Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr

435 440 445

Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp

450 455 460

Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val

465 470 475 480

Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro

485 490 495

Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe

500 505 510

Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr

515 520 525

Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr

530 535 540

Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln

545 550 555 560

Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro

565 570 575

Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val

580 585 590

Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu

595 600 605

Tyr Gln Gly Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp

610 615 620

Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val Phe

625 630 635 640

Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val Asn Asn Ser

645 650 655

Tyr Glu Cys Gly Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln

660 665 670

Thr Gln Thr Asn Ser His Arg Arg Ala Arg Ser Val Ala Ser Gln Ser

675 680 685

Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr

690 695 700

Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr

705 710 715 720

Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr

725 730 735

Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln

740 745 750

Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala

755 760 765

Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln

770 775 780

Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe Ser

785 790 795 800

Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu

805 810 815

Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys

820 825 830

Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys

835 840 845

Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp

850 855 860

Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr

865 870 875 880

Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala

885 890 895

Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val

900 905 910

Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile

915 920 925

Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys

930 935 940

Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val

945 950 955 960

Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp

965 970 975

Ile Leu Ser Arg Leu Asp Pro Pro Glu Ala Glu Val Gln Ile Asp Arg

980 985 990

Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln

995 1000 1005

Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr

1010 1015 1020

Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys

1025 1030 1035 1040

Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly

1045 1050 1055

Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe

1060 1065 1070

Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His Phe Pro Arg

1075 1080 1085

Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg

1090 1095 1100

Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser

1105 1110 1115 1120

Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp

1125 1130 1135

Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr

1140 1145 1150

Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly

1155 1160 1165

Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn

1170 1175 1180

Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu

1185 1190 1195 1200

Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly

1205 1210 1215

Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu Cys

1220 1225 1230

Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly

1235 1240 1245

Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly

1250 1255 1260

Val Lys Leu His Tyr Thr

1265 1270

<210> 3

<211> 162

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IL-21

<400> 3

Met Arg Ser Ser Pro Gly Asn Met Glu Arg Ile Val Ile Cys Leu Met

1 5 10 15

Val Ile Phe Leu Gly Thr Leu Val His Lys Ser Ser Ser Gln Gly Gln

20 25 30

Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln

35 40 45

Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro

50 55 60

Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln

65 70 75 80

Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile

85 90 95

Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala

100 105 110

Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr

115 120 125

Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu

130 135 140

Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu

145 150 155 160

Asp Ser

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> A46R Forward primer

<400> 4

ttggctatta aacagtatgg a 21

<210> 5

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> A46R reverse primer

<400> 5

ggatcccgat aacaaatg 18

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> S Gene Forward primer

<400> 6

cgtggtgttt attaccctga 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> S Gene reverse primer

<400> 7

acaataagta gggactgggt 20

Claims

1. A vaccinia virus vector vaccine comprising an altered new coronavirus south african variant spike protein S gene, wherein the gene encoding a46R is deleted in its entirety in the vaccinia virus vector derived from a vaccinia virus listeria, wherein the altered new coronavirus south african variant spike protein S has the following mutations compared to the amino acid sequence of a wild-type new coronavirus spike protein S: L18F, D80A, D215G, delta 241-243, R246I, K417N, E484K, N501Y, D614G, D663G, P681H, A701V, K986P and V987P, wherein the amino acid sequence of the spike protein S of the south African variant of the engineered new coronavirus is shown as SEQ ID NO: 2.

2. The vaccinia virus vector vaccine of claim 1 wherein the altered mutant south African variant spike protein S gene of the new coronavirus is located in region A46R of the vaccinia virus vector.

3. The vaccinia virus vector vaccine of claim 1, wherein a46R gene of the vaccinia virus vector is replaced by the spike protein S gene of the altered south african variant of new corona virus.

4. The vaccinia virus vector vaccine of claim 1, wherein the nucleic acid sequence of the spike protein S gene of the altered south african variant of new coronavirus is set forth in SEQ ID No. 1.

5. The vaccinia virus vector vaccine of claim 1, wherein the vaccinia virus vector further comprises an exogenous interleukin 21 (IL-21) gene.

6. The vaccinia virus vector vaccine of claim 5, wherein the IL-21 is human IL-21.

7. The vaccinia virus vector vaccine of claim 5, wherein the IL-21 gene is located in the A46R region of vaccinia virus vector.

8. The vaccinia virus vector vaccine of claim 7, wherein A46R gene of the vaccinia virus vector is replaced with the modified south African variant spike protein S gene and the IL-21 gene.

9. The vaccinia virus vector vaccine of claim 5 wherein the amino acid sequence of IL-21 is set forth in SEQ ID NO 3.

10. A pharmaceutical composition comprising a vaccinia virus vector vaccine of any of claims 1-9, and optionally a pharmaceutically acceptable carrier.

11. The method for preparing the vaccinia virus vector vaccine of any of claims 1-9, comprising recombining a shuttle vector with vaccinia virus to obtain vaccinia virus with a deleted gene in a46R region, and inserting the S gene and the IL-21 gene of the modified south african mutant strain of new corona virus into a46R region of vaccinia virus to obtain recombinant vaccinia virus.

12. The method of claim 11, comprising infecting a virus-producing cell with the recombinant vaccinia virus.

13. The production method according to claim 12, wherein the virus-producing cell is a CV-1 cell.

14. The preparation method of claim 12, wherein the virus-producing cell is a HEK293 cell.

15. Use of a vaccinia virus vector vaccine of any of claims 1-9 in the preparation of a medicament for preventing new coronary pneumonia.