CN113633764B

CN113633764B - New crown DNA vaccine containing adjuvant

Info

Publication number: CN113633764B
Application number: CN202111026912.2A
Authority: CN
Inventors: 黄维金; 赵爱华; 徐苗; 王佑春; 周泽华; 付丽丽; 聂建辉; 张黎; 赵晨燕
Original assignee: National Institutes for Food and Drug Control
Current assignee: National Institutes for Food and Drug Control
Priority date: 2021-09-02
Filing date: 2021-09-02
Publication date: 2024-05-14
Anticipated expiration: 2041-09-02
Also published as: CN113633764A

Abstract

The invention relates to a DNA vaccine composition and application thereof. In particular, the invention relates to DNA vaccine compositions directed against coronaviruses, and pharmaceutical compositions comprising said DNA vaccine compositions. Furthermore, the invention relates to the use of said DNA vaccine composition. The DNA vaccine compositions of the invention are useful for the prevention and/or treatment of coronavirus infections and/or diseases caused by said infections.

Description

New crown DNA vaccine containing adjuvant

Technical Field

The present invention relates to the fields of immunology and molecular virology, in particular to the fields of diagnosis, prevention and treatment of coronaviruses. In particular, the invention relates to DNA vaccine compositions directed against novel coronaviruses, and pharmaceutical compositions comprising said DNA vaccine compositions. Furthermore, the invention relates to the use of said DNA vaccine composition. The DNA vaccine compositions of the invention are useful for the prevention and/or treatment of coronavirus infections and/or diseases caused by said infections.

Background

Coronavirus-caused disease in 2019 (COVID-19) has caused a health crisis worldwide. Severe acute respiratory syndrome 2 (SARS-CoV-2) is a single stranded positive sense ribonucleic acid (RNA) virus whose Spike protein (Spike protein) determines the infectivity of the virus and its ability to spread in a host. The Spike protein of the virus is also very unstable and mutations in the Spike protein may result in increased infectivity of the virus. Since there is currently no effective treatment for the public, there is an urgent need for effective prophylactic methods, especially vaccines.

Currently, many studies have found that the Spike protein gene of SARS-CoV-2 has a variety of mutations, of which D614G is the most important mutation and combinations of mutations, for example D614G+I472V(Zhou,B.,et al.,SARS-CoV-2 spike D614G variant confers enhanced replication and transmissibility.bioRxiv,2020;Plante,J.A.,et al.,Spike mutation D614G alters SARS-CoV-2 fitness.Nature,2020;Li,Q.,et al., The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity.Cell,2020.182(5):p.1284-1294e9.). other studies have found that certain mutation sites can reduce viral infectivity, such as L452R and V483A, but that these sites can tolerate some neutralizing antibodies (Li,Q.,et al.,The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity.Cell,2020. 182(5):p.1284-1294e9.). and that the B.1.1.7 variant, as well as many genetically altered variants, increases rapidly in the United kingdom, and that the B.1.1.7 variant, due to mutation of N501Y, may increase viral binding to human angiotensin converting enzyme 2 (ACE 2) in the south African strain (501Y.V2) unique to (Leung,K.,et al.,Early transmissibility assessment of the N501Y mutant strains of SARS-CoV-2 in the United Kingdom,October to November 2020.Euro Surveill,2021.26(1).). also includes a number of non-synonymous Spike mutations that may have functional significance, such as K417N, E K and K in the Spike receptor binding domain N501Y(Tegally,H.,et al.,Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2(SARS-CoV-2)lineage with multiple spike mutations in South Africa.medRxiv,2020:p. 2020.12.21.20248640.).

Previously, DNA vaccines have been successful in preventing a variety of different infectious diseases and have great advantages over conventional vaccines because they are very simple in design and require only one step to clone into a plasmid vector. Furthermore, the expression of the antigen gene in vivo can maintain the natural structure of the protein, ensuring proper processing and immune presentation. Notably, the greatest challenge in practical use of DNA vaccines is whether sufficient immunogenicity can be elicited.

To address this problem, a number of different strategies have been applied in preclinical models, including the formulation of DNA vaccines with molecular adjuvants. Adjuvants are immunomodulators and have been used for decades to treat various clinical conditions.

For example, cpG ODN has been recognized as an immunoadjuvant for a variety of vaccines, as it can aid in activating both innate and adaptive immune responses in animals and humans. CPG-ODN also stimulates the secretion of various cytokines such as TNF- α and GM-CSF by monocytes, macrophages and dendritic cells directly, which in turn stimulates the production of an immune response by helper T cells.

Disclosure of Invention

The inventors of the present application have made extensive experiments and repeated experiments to construct a DNA vaccine expressing SARS-Cov-2 Spike protein, and used BC01 as an adjuvant. It has surprisingly been found that such a combination can elicit efficient production of neutralizing antibodies against the neocrowns and produce cell-mediated immune responses to prevent and/or treat infection or disease caused by SRAS-CoV-2 virus.

In a first aspect, the present application provides a vaccine composition comprising or consisting of: a first nucleic acid molecule as an adjuvant component and a second nucleic acid molecule as an immunogen component; the first nucleic acid molecule comprises a nucleotide sequence encoding a BCG unmethylated CpG motif and the second nucleic acid molecule comprises a nucleotide sequence encoding a SARS-CoV-2 Spike protein.

In certain embodiments, the BCG unmethylated CpG motif is obtained by lysing the bacillus calmette guerin cells and extracting the nucleic acid from the lysate.

In certain embodiments, the SARS-CoV-2S protein has a sequence as set forth below: genBank: mn_908947.

In certain embodiments, the first nucleic acid molecule and the second nucleic acid molecule are contained in the same vector (e.g., a pSV10 vector), or are contained in separate vectors.

The adjuvant component and the immunogenic component of the vaccine composition may be in the form of a mixture of the two components in a single pharmaceutical formulation or in separate form of the individual components in a kit. Also, the two components may be administered separately, sequentially or simultaneously. In certain embodiments, the two components are administered substantially simultaneously.

In certain embodiments, the first nucleic acid molecule is prepared by the method described in chinese application patent ZL200410033878.1, the disclosure of which is incorporated herein in its entirety. In certain embodiments, the method of making the first nucleic acid molecule comprises: inoculating the strain into a culture medium suitable for the growth of mycobacterium, and culturing until the strain reaches a logarithmic phase, and collecting thalli; crushing thalli, and centrifugally collecting supernatant; dissolving the supernatant with CTAB precipitate in NaCl solution, extracting with organic solvent to collect protein-free layer, treating the supernatant with ethanol to collect precipitate, and post-treating the precipitate.

In certain embodiments, the first nucleic acid molecule is prepared by the method described in chinese application patent zl201310586057.X, the disclosure of which is incorporated herein in its entirety. In certain embodiments, the method of making the first nucleic acid molecule comprises: and separating the BCG CpG-DNA from the BCG lysate by using a Q Sepharose HP ion exchange column, wherein the ion exchange column separation of the BCG CpG-DNA lysate can be performed in a TE buffer system or a sodium phosphate buffer system. In certain preferred embodiments, wherein the ion exchange column separation of the bcg CpG-DNA lysate is performed in a sodium phosphate buffer system, the elution buffer is 1M sodium chloride +50mM sodium phosphate buffer, ph7.5-8.5. In certain preferred embodiments, wherein the elution buffer is ph7.5. In certain preferred embodiments, a loading buffer is used at the time of loading, the loading buffer being 0-0.5M sodium chloride+50 mM sodium phosphate buffer, pH7.5-8.5. In certain preferred embodiments, the loading buffer is 0.5M sodium chloride+50 mM sodium phosphate buffer, pH7.5. In certain preferred embodiments, wherein the elution after loading employs a gradient elution, the gradient elution is performed by eluting from 50% to 65% of the elution buffer, continuing 100% of the elution buffer. In certain preferred embodiments, wherein the elution after loading employs a gradient elution, the gradient elution is performed by eluting from 50% to 65% of the elution buffer, continuing 100% of the elution buffer. In certain preferred embodiments, wherein the ion exchange column separation of the bcg CpG-DNA lysate is performed in a TE buffer system, the elution buffer is 1M NaCl+50mM Tris+1mM EDTA pH7.5-8.5. In certain preferred embodiments, wherein the elution buffer is ph7.5. In certain preferred embodiments, the following loading buffers are used in the loading, and the loading buffers are in the range of 0-0.5MNaCl+50mM Tris+1mM EDTA pH7.5-8.5. In certain preferred embodiments, the loading buffer is 0.5M NaCl+50mM Tris+1mM EDTA pH7.5. In certain preferred embodiments, wherein the elution after loading employs a gradient elution, the gradient elution is performed by eluting 25-65% of the elution buffer, continuing 100% of the elution buffer. In certain preferred embodiments, the BCG lysate is further clarified prior to separation and purification on a Q Sepharose HP ion exchange column. In certain preferred embodiments, wherein said BCG lysate clarification treatment is by diluting the disrupted cell lysate to a concentration of 200mg/ml, centrifuging at a high speed refrigerated centrifuge at 4℃at 8000-12000rpm/min, 10-20min X2 times, collecting the supernatant, and/or filtering through a 1.0-1.2 μm filter.

The first nucleic acid molecule may also be prepared by modifications of the above-described methods, as known to those skilled in the art. The first nucleic acid molecules obtained by these exemplary methods are useful in the vaccine compositions of the present invention and produce good immunoadjuvant effects.

In certain embodiments, the CpG content of the first nucleic acid molecule can be obtained by high performance liquid detection. For example, cpG can be quantified by modifying cytosine (dC) of CpG dinucleotides to 5-methylcytosine (m 5-dC) by reverse phase-high performance liquid chromatography (RP-HPLC) using SsssI, which is described in ZL200410033878.1, hydrolyzing DNA to single deoxynucleosides using nuclease P1 and Bacterial Alkaline Phosphatase (BAP), and quantifying the difference in the amount of m5-dC detected in the hydrolyzed sample of modified and unmodified DNA by reverse phase-high performance liquid chromatography (RP-HPLC).

In certain embodiments, the bcg seed strain is the bcg seed strain D2PB302 for chinese bcg preparation provided by the vaccine house of the chinese pharmaceutical biologicals institute.

In certain embodiments, the first nucleic acid molecule is contained or not contained in a vector (e.g., a pSV10 vector).

In certain embodiments, the second nucleic acid molecule is contained or not contained in a vector. In certain embodiments, the second nucleic acid molecule is contained in a vector capable of expressing the SARS-CoV-2S protein (e.g., a pSV10 vector).

In certain embodiments, the DNA vaccine is an aqueous solution or reconstituted lyophilized powder for injection that can be administered via injection or via mucous membrane.

In certain embodiments, the ratio of the content of the first nucleic acid molecule to the content of the second nucleic acid molecule is 1:2 to 1:10 (e.g., 1:2,1:3,1:4,1:5,1:6,1:7, 1:8,1:9, or 1:10).

In certain embodiments, the ratio of the first nucleic acid molecule to the second nucleic acid molecule is 1:5.

In certain embodiments, the first nucleic acid molecule is present in an amount of 1 μg to 100 μg (e.g., 1 μg,10 μg,20 μg,40 μg,60 μg,80 μg,100 μg). In certain embodiments, the first nucleic acid molecule is present in an amount of 10 μg.

In certain embodiments, the second nucleic acid molecule is present in an amount of 5 μg to 500 μg (e.g., 5 μg,50 μg,100 μg,200 μg,300 μg,400 μg,500 μg). In certain embodiments, the second nucleic acid molecule is present in an amount of 50 μg.

In another aspect, the application provides a host cell comprising a nucleic acid molecule or vector in a vaccine composition as described above.

In certain embodiments, the host cell comprises a first nucleic acid molecule and a second nucleic acid molecule, or the host cell comprises a first nucleic acid molecule and the pSV10 vector.

In another aspect, the application provides a combination of host cells, wherein a first host cell comprises a first nucleic acid molecule or vector in a vaccine composition as described above; the second host cell comprises a second nucleic acid molecule or vector in a vaccine composition as described previously.

In another aspect, the application provides a pharmaceutical composition comprising a vaccine composition as described above, and a pharmaceutically acceptable carrier and/or excipient.

In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent, such as an additional antiviral agent (e.g., interferon, lopinavir, ritonavir, chloroquine phosphate, fepima Weirui darunavir, etc.).

In another aspect, the application provides a method for neutralizing the virulence of a coronavirus in a sample comprising contacting a sample comprising the coronavirus with a vaccine composition as described above.

In certain embodiments, the coronavirus is SARS-CoV-2.

In certain embodiments, the first nucleic acid molecule or vector comprising the same is contacted with the sample simultaneously with the second nucleic acid molecule or vector comprising the same, or separately.

In another aspect, the application provides a method for preventing and/or treating a coronavirus infection or a disease associated with a coronavirus infection in a subject (e.g., a human), comprising: administering (e.g., injecting) an effective amount of a vaccine composition as described above or a pharmaceutical composition as described above to a subject in need thereof.

In certain embodiments, the first nucleic acid molecule or vector comprising the same is administered to a subject simultaneously with the second nucleic acid molecule or vector comprising the same, or separately.

In certain embodiments, the coronavirus is SARS-CoV-2.

In certain embodiments, the disease associated with coronavirus infection is COVID-19 and/or SARS.

In another aspect, the application provides a method of inducing an immune response (e.g., production of neutralizing antibodies) against a coronavirus in a subject (e.g., a human) comprising: administering (e.g., injecting) an effective amount of a vaccine composition as described above or a pharmaceutical composition as described above to the subject.

In certain embodiments, the injection is intradermal or intramuscular.

In certain embodiments, the method further comprises the step of electroporating the tissue with an electroporation amount of electrical energy.

In certain embodiments, the coronavirus is SARS-CoV-2.

In another aspect, the application provides the use of a vaccine composition as described above for the prevention and/or treatment of a coronavirus infection, or for the prevention and/or treatment of a disease caused by a coronavirus infection, or for inducing or generating an immune response against a coronavirus (e.g. generating neutralizing antibodies) in a subject.

In certain embodiments, the coronavirus is SARS-CoV-2.

In another aspect, the application provides a method of preparing a vaccine composition as described above, comprising synthesizing a first nucleic acid molecule and a second nucleic acid molecule, respectively, by an organic synthesis reaction or an enzymatic synthesis reaction. Optionally, the first nucleic acid molecule and/or the second nucleic acid synthesized as described above are separately constructed into a vector.

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Further, the procedures of molecular genetics, nucleic acid chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA, etc., as used herein, are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, "Severe acute respiratory syndrome coronavirus 2 (severe acute respiratory syndrome coronavirus, SARS-CoV-2)", which is known as "novel coronavirus" or "2019-nCov", belongs to the genus beta coronavirus, which is a enveloped single-stranded plus sense RNA virus. The genomic sequence of SARS-CoV-2 is known to those skilled in the art and can be found in, for example, genBank: MN908947.SARS-CoV-2 contains at least three membrane proteins, including surface spike protein (S), integral membrane protein (M) and membrane protein (E). The receptor of SARS-CoV-2 is, like SARS-CoV, specifically combined with angiotensin transferase 2 (ACE 2) on host cell by receptor binding domain (Receptor binding domain, RBD) on S protein, and then grafted with membrane fusion and cell entry of virus, which plays a vital role in the process of virus infection of cells.

As used herein, the term "COVID-19" refers to pneumonia caused by SARS-CoV-2 infection.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as e.g. escherichia coli or bacillus subtilis, a fungal cell such as e.g. yeast cells or aspergillus, an insect cell such as e.g. S2 drosophila cells or Sf9, or an animal cell such as e.g. fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells or human cells.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and includes, but is not limited to: for example, pH modifiers include, but are not limited to, phosphate buffers, surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80, ionic strength enhancers include, but are not limited to, sodium chloride, preservatives include, but are not limited to, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and the like, agents that maintain osmotic pressure include, but are not limited to, sugar, naCl, and the like, agents that delay absorption include, but are not limited to, monostearates and gelatin, diluents include, but are not limited to, water, preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like, stabilizers have the meaning commonly understood by those skilled in the art that stabilizes the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin or casein) or degradation products thereof (e.g., lactalbumin hydrolysate), and the like. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), dextrose solutions (e.g., 5% dextrose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), ringer's solution, and any combination thereof.

As used herein, the term "preventing" refers to a method performed in order to prevent or delay the occurrence of a disease or disorder or symptom (e.g., SARS-CoV-2 infection) in a subject. As used herein, the term "treatment" refers to a method that is performed in order to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., no longer worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and diminishment of symptoms (whether partial or total), whether detectable or undetectable. Furthermore, "treatment" may also refer to an extension of survival compared to the expected survival (if not treated).

As used herein, the term "subject" refers to a mammal, such as a human. In certain embodiments, the subject (e.g., human) has, or is at risk of having, a SARS-CoV-2 infection or a disease associated with a SARS-CoV-2 infection (e.g., COVID-19).

As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, a prophylactically effective amount of a disease (e.g., SARS-CoV-2 infection) refers to an amount sufficient to prevent, arrest, or delay the onset of the disease (e.g., SARS-CoV-2 infection); a therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determination of such effective amounts is well within the ability of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.

As used herein, the term "neutralizing activity" refers to the functional activity of an antibody or antibody fragment that binds to an antigenic protein on a virus, thereby preventing the maturation of virus-infected cells and/or virus progeny and/or the release of virus progeny, and an antibody or antibody fragment having neutralizing activity may prevent the amplification of a virus, thereby inhibiting or eliminating the infection by a virus.

As used herein, the term "CpG motif" is fully referred to as an unmethylated cytosine-guanine motif, which refers to unmethylated CpG dinucleotides. Usually there are more unmethylated CpG dinucleotides in the bacterial genome. The immune system of vertebrates regards CpG motifs as recognition molecules for foreign substances, which in turn can excite the body to produce a protective immune response. The sequences of CpG motifs can be obtained by the person skilled in the art from existing databases.

In general, methylation refers to the substitution of hydrogen in a nucleic acid molecule with methyl (-CH 3). The term "unmethylated CpG motif" refers to CpG motifs that do not undergo the methylation reaction.

As used herein, the term "BCG" (Bacillus Calmette-Guerin), also known as "bacillus calmette-Guerin", is a live mycobacterium tuberculosis (Mycobacterium bovis) attenuated but capable of producing immunity to humans, and is useful as a vaccination against tuberculosis in children, which may be followed by a particular resistance to tuberculosis.

As used herein, the term "BCG-CpG-DNA" refers to nucleic acids (e.g., double-stranded DNA fragments) extracted from BCG, containing a large number of unmethylated CpG motifs.

As used herein, the term "immunogen" refers to a protein or peptide sequence capable of inducing an immune response in a host.

As used herein, the term "adjuvant" also known as immunomodulator or immunopotentiator refers to a substance that is injected into a mammal prior to/after or simultaneously with an antigen, and that can nonspecifically alter or enhance the specific immune response of the body to the antigen, thereby acting as an adjunct.

The DNA vaccine of the invention can be water solution or freeze-dried powder injection for re-dissolution which can be injected or applied through mucous membrane. In certain embodiments, the aqueous solution or reconstituted lyophilized powder for injection is prepared by aseptic procedures known in the art. In certain embodiments, they are sterile during storage, shipping, and use.

According to the present invention, the pharmaceutical composition may be in any pharmaceutical form known in the art for administration, including pharmaceutical compositions, pharmaceutical preparations, kits, and the like. Although the DNA vaccines involved in the use of the present invention may be administered by injection, mucosal or the like, and these modes of administration are also part of the present invention. It will be clear to those skilled in the art that the most preferred route of administration suitable for the use described herein is parenteral or by injection. For the practice of the present invention, the pharmaceutical compositions are preferably formulations for parenteral administration, including but not limited to topical and systemic injection formulations, and specific dosage forms include but are not limited to injectable solutions and injectable powders. In certain embodiments, the medicament is a sterile injectable aqueous solution or a sterile injectable powder prepared by reconstitution with injectable water prior to clinical use, particularly a lyophilized powder. In the preparation of lyophilized powder for injection, pharmaceutically acceptable excipient such as mannitol can be also contained.

The DNA vaccine of the present invention may be introduced into an organism by methods well known in the art. Such methods of introduction include, but are not limited to, intramuscular injection, gene gun introduction, ③ mucosal immunization, intravenous injection, intraperitoneal injection, and the like, as disclosed in more detail in Alpar HO, et al Expert Opin Drug Deliv,2005,2:829-842, which is incorporated herein by reference in its entirety.

In addition, based on the results of the studies provided below, one skilled in the art can readily determine an effective dose of the vaccine composition of the present application when used in mammals, particularly when used in humans. The dosage of one day may be administered to the subject all at once throughout the day, or the desired dosage may be divided into two, three, four or more small doses administered at appropriate intervals throughout the day. The small doses may be formulated in unit dosage forms, for example, each unit dosage form containing a corresponding amount of the total daily dose divided an appropriate number of times. Of course, administration may also be performed at a certain time period, such as once a day, once two days, once a week, once a month, once february, once a trimester, once a six month, once a year, once two years, etc.

As used herein, the term "ID50" refers to a 50% inhibition dose, which is typically used to evaluate the potency of neutralizing antibodies.

Advantageous effects of the invention

Compared with the prior art, the vaccine composition prepared by the application can early generate the anti-neocrown antibody, improve the titer of the generated neutralizing antibody and enhance the immune response of cells. The DNA vaccine compositions of the application are useful for the prevention and/or treatment of coronavirus infections and/or diseases caused by said infections.

Further, the BC01 adjuvant and the vaccine composition composed of the pSV10-SARS-CoV-2 vaccine are superior to the following: vaccine composition composed of other adjuvants and pSV10-SARS-CoV-2 vaccine; and, a composition of a BC01 adjuvant and the remainder of the vaccine. For example, the present application has been experimentally confirmed to be able to produce only neutralizing antibodies against the D614G, D614G+I472V and V483A strains, and not against the L452R strain, using the pSV10-SARS-CoV2 vaccine alone. Whereas the use of pSV10-SARS-CoV2 vaccine with BC01 is capable of producing neutralizing antibodies against the L452R, D614G, D G+I472V and V483A strains, and also induces higher titers of neutralizing antibodies.

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

Drawings

FIG. 1 shows a schematic design and expression results of SARS-Cov-2 DNA vaccine construct. In which FIG. 1A shows a schematic representation of a DNA vaccine, i.e., pSV10-SARS-CoV-2 contains the inserted SARS-CoV-2 Spike protein. FIG. 1B shows Western blot analysis of Spike proteins in vitro after transfection of pSV10-SARS-CoV-2 and MOCK plasmids into 293T cells.

Figure 2 shows the humoral immune response of mice vaccinated with different types of vaccines. Among them, fig. 2A shows the results of different sets of neutralizing antibodies ID 50. FIG. 2B shows the binding of IgG to SARS-CoV-2S protein antigen in serial dilutions of mice at weeks 4 and 6.

FIG. 3 shows the effect of neutralizing antibodies on different variants produced by inoculating different sets of reagents. Wherein, fig. 3A shows neutralization for the D614G variant, fig. 3B shows neutralization for the d614g+i472V variant, fig. 3C shows neutralization for the V483A variant, and fig. 3D shows neutralization for the L452R variant.

FIG. 4 shows the T cell epitope pattern of BALB/c mice vaccinated with pSV10-SARS-CoV-2 vaccine with BC 01.

FIG. 5 shows the T cell response of mice after inoculation with pSV10-SARS-CoV-2 vaccine with BC 01. Wherein, FIG. 5A shows IFN-. Gamma.ELISPOT produced at week 4; FIG. 5B shows IFN-. Gamma.ELISPOT at week 6, FIG. 5C shows IL-2 ELISPOT at week 4, and FIG. 5D shows IL-2 ELISPOT at week 6.

Sequence information

The information of the partial sequences to which the present invention relates is provided in table 1 below.

Table 1: description of the sequence

/>

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

The experiments and methods described in the examples were performed substantially in accordance with conventional methods well known in the art and described in various references unless specifically indicated. For example, for the conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention, reference may be made to Sambrook (Sambrook), friech (Fritsch) and manitis (Maniatis), molecular cloning: laboratory Manual (MOLECULAR CLONING: A LABORATORY MANUAL), edit 2 (1989); the handbook of contemporary molecular biology (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) (edited by f.m. ausubel et al, (1987)); the enzyme methods series (METHODS IN ENZYMOLOGY) (academic publishing Co): PCR 2: practical methods (PCR 2:A PRACTICAL APPROACH) (M.J. MaxFrson (M.J. MacPherson), B.D. Hemsl (B.D. Hames) and G.R. Taylor (G.R. Taylor) editions (1995)), and animal cell CULTURE (ANIMAL CELL CULTURE) (R.I. Fu Lei Xieni (R.I. Freshney) editions (1987)).

In addition, the specific conditions are not specified in the examples, and the process is carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

EXAMPLE 1 preparation and validation of DNA vaccine

Preparation of DNA vaccine

DNA vaccine was designed based on the sequence of SARS-CoV-2 spike (S) protein Wuhan-1 (GenBank: MN_ 908947). Nucleotide sequences encoding the full length of the S protein were synthesized. The nucleotide sequence synthesized was cloned into a mammalian expression plasmid pSV10 (obtained from the center for prevention and control of AIDS, center for prevention and control of disease in China), the DNA vaccine was obtained and designated pSV10-SARS-CoV-2, and the sequence of the DNA vaccine was verified using Sanger sequencing. The synthesized plasmid was extracted and dissolved in sterile water for use.

Preparation of BC01 adjuvant

The preparation method of the BC01 adjuvant used in the embodiment is shown in the Chinese patent application ZL201310586057.X, and the BC01 adjuvant used in the embodiment is BCG-CpG-DNA in the patent. Briefly, bacillus calmette-guerin strain (bacillus calmette-guerin strain D2PB302 for preparing Chinese bacillus calmette-guerin, provided by a bacillus calmette-guerin laboratory of Chinese medicine biological product detection) is subjected to cell culture and then is split, and the split solution is purified by an ion exchange purification method to remove polysaccharide, protein, RNA and other impurities. Finally, BCG vaccine (BCG) CpG-DNA is separated from the lysate through an ion exchange column and is dissolved in sterile water, so that the BC01 adjuvant is obtained.

Western Blot validation

293T cells were seeded into 6-well plates and at about 70% density, pSV10-SARS-CoV-2 plasmid (4 μg) was transfected with Lipofectamine 3000 (Invitrogen). Cell lysates were collected 48 hours after transfection, heated at 95℃for 5 minutes, spotted on a pre-prepared 10% SDS-PAGE gel (Bio-Rad) and electrophoresis was started. Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and the PVDF membranes were membrane blocked in Phosphate Buffer (PBST) containing 0.2% tween 20 (Sigma) (V/V) and 5% (W/V) skimmed milk powder at 4 ℃. After blocking overnight, PVDF membranes were incubated for one hour in PBST with 5% (W/V) skimmed milk powder containing a 1:1000 dilution of mouse anti-SARS-CoV-2 Spike S1 mab. After incubation, PVDF membranes were washed 5 times with 5% (W/V) skimmed milk powder in PBST followed by incubation with 1:10,000 goat anti-mouse secondary antibodies in 5% (W/V) skimmed milk powder in PBST. Then, the PVDF membrane was washed again 5 times with 5% (W/V) of skimmed milk powder in PBST and detected by the Touch Imager XLI system (e-BLOT).

The vector map of pSV10 after insertion into SARS-CoV-2 Spike protein is shown in FIG. 1A. The Spike protein has a molecular weight of 140-142kDa, and slight changes in molecular weight are due to the 22 potential N-linked glycans in the S protein. Western blot analysis of Spike protein is shown in fig. 1B, which demonstrates that Spike protein can be expressed after transfection of vaccine vectors.

EXAMPLE 2 mouse humoral immune response

Treatment of animal samples

39 Female mice, 4 to 6 weeks old, were divided into four groups and injected with the following reagents, respectively: the first group was pSV10-SARS-CoV-2 (sample number 10) with BC01, the second group was pSV10-SARS-CoV-2 (sample number 10), the third group was BC01 (sample number 10), and the fourth group was PBS (sample number 9). Mice received 50ug of DNA vaccine at week 0, week 2 and week 4, respectively. The four groups of agents were injected into mice of the respective experimental groups at week 0, week 2 and week 4 with needles and syringes, respectively, and then subjected to in vivo electroporation. Serum was collected at weeks 4 and 6 post immunization.

Antigen binding ELISA

ELISA was used to detect serum antibody binding titers. ELISA plates were coated overnight at 4℃with 1. Mu.g/ml SARS-CoV-2 Spike protein in 1 Xphosphate buffer (DPBS). ELISA plates were washed 3 times with wash buffer and blocked with 3% Bovine Serum Albumin (BSA) in DPBS (0.05% Tween) for two to three hours at room temperature. The blocking solution was discarded and the ELISA plate incubated with serially diluted heat-inactivated mouse serum for one hour at room temperature. And incubated with anti-mouse IgG horseradish peroxidase (HRP) at 1:4000 dilution in the dark for one hour at room temperature. The plates were then washed 5 times with wash buffer. 100. Mu.L of TMB solution was added to each well, and after 5min, the reaction was stopped by adding a stop solution. Absorbance at 450nm and 630nm was recorded.

Statistical analysis

Analysis was performed using GRAPHPAD PRISM 8.4.2 (GraphPad Software). Comparison of data between groups was performed using one-way anova and multiple comparison tests of Holm-Sidak. A P value of less than 0.05 is considered significant.

Experimental results

39 Mice (4-6 weeks old) were divided into 4 test groups, the first group was injected with 50. Mu.g of pSV-10-SARS-CoV-2 vaccine (10 vaccinations), the second group was injected with 50. Mu.g of pSV-10-SARS-CoV-2 vaccine with 10. Mu.g of BC01 adjuvant (10 vaccinations), the third group was injected with 10. Mu.g of BC01 adjuvant (10 vaccinations), and the fourth group was injected with 60. Mu.g of PBS (9 vaccinations). The average titer of ID50 of neutralizing antibodies observed in the first group of vaccinated mice was 87 and the average titer of ID50 of neutralizing antibodies detected in the second group of vaccinated mice was 141 at the fourth week after injection. No neutralizing antibodies were detected in the test groups of the third and fourth groups. At week 6 post injection, the average titer of neutralizing ID50 for the first group was 262, the average titer of neutralizing ID50 for the second group was 309, and no neutralizing antibodies were detected in the control group of both the third and fourth groups (fig. 2A). The positive conversion rate was 80% in the second group, significantly higher than 50% in the first group (i.e. the experimental group injected with pSV-10-SARS-CoV-2 vaccine only) at the fourth week after injection. These results indicate that BC01 adjuvant can promote early production of neutralizing antibodies in mice (table 2).

TABLE 2 neutralization Activity of serum after BC01 administration of pSV10-SARS-CoV-2 to mice

At week 4, a titer of bound antibody was detected in both the first and second groups. No bound antibodies were detected in the third and fourth groups. At week 6, a titer of bound antibody was detected in both the first and second groups. No binding antibodies were detected in the third and fourth groups (fig. 2B).

EXAMPLE 3 pseudovirus neutralization experiments

Production and titration of pseudoviruses

The production of SARS-CoV-2 pseudovirus is described in literature (Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2.Emerg Microbes Infect,2020.9(1):p.680-686.). Briefly, the nucleotide sequences of SEQ ID NOS 1-4 were inserted into the multiple cloning site of pSV10, respectively, plasmids expressing the different SARS-CoV-2 Spike proteins were inserted into 293T cells, and these cells were then infected with G.DELTA.G-VSV (VSV G pseudotyped virus) at a concentration of 7.0X10. 10 ⁴ TCID 50/ml.

Cells were incubated at 37℃for 6-8 hours, and then the supernatant was removed. 15ml of fresh cell culture broth was added to the flask and cultured for 24 hours. Finally, the culture supernatant containing SARS-CoV-2 pseudovirus was collected, filtered through a filter membrane (0.45 mm pore size, millipore, cat# SLHP033 RB) and stored at-80℃to prepare pseudoviruses D614G, D G+I472V, L452R and V483A. 24 hours after infection, the prepared SARS-CoV-2 pseudovirus was titrated to produce a Relative Luminescence Unit (RLU) more than ten times that of the negative control (cell only). The grouping and inoculation of the mice were the same as described in example 2.

Experimental results

The level of neutralization of antibodies produced by the vaccine against currently circulating pseudoviruses D614G, D g+i472V, L452R and V483A was assessed. Week 6 sera from 5 vaccinated mice in each group were used for neutralization experiments. Mouse serum from different vaccinated groups was heat-inactivated at 56℃for 30 min, with each assay of mouse serum serially diluted three times starting at 1:30 dilution. Serum was mixed with 50 μl pseudovirus for 60 minutes. After 60 minutes Huh-7 cells stably expressing ACE2 were added and then incubated at 37℃for 24 hours. Cells were then lysed using britelite plus luminescent reporter gene analysis system (PERKIN ELMER catalog No. 6066769) and RLU was measured. Virus neutralization titers (ID 50) were calculated using the Reed-Muench method.

The second group injected with the DNA vaccine with BC01 could neutralize the d614G, D g+i472V, L452R and V483A variants, while the first group injected with the DNA vaccine only could neutralize the d614G, D g+i472V and V483A variants, but not the L452R variants. Mice injected with the third and fourth groups were unable to neutralize these variants (fig. 3).

EXAMPLE 4 cellular immune response

ELISPOT detection

Spleens of mice were collected and ground to a single cell suspension in RPMI1640 medium containing 1% penicillin/streptomycin (R0). The cell pellet was resuspended in 5mL ACK lysis buffer for 5 minutes, then 8mL PBS was added to stop the reaction. The sample was centrifuged at 1500g for 5 min and the cells were resuspended in RPMI1640 medium containing 10% peptide bovine serum (FBS (R10)). Mouse IFN-. Gamma. ELISpotPLUS plates and mouse IL-2ELISpotPLUS plates (MABTECH) were activated for 30 min at 200. Mu. L R10/well. 5x10 ⁵ mouse spleen cells were added to each well and the cells were stimulated with a pool of 18-mer peptides overlapping with 9 amino acids from the SARS-CoV-2 Spike protein. In addition, localization (matrix mapping) was performed using peptide libraries in the matrix to identify immunodominant reactions. The spleen cells of the mice were stimulated in R10 with a final concentration of 5 μg/ml of each peptide per well. R10 and the cell stimulator PAM+ ION (Invitrogen) served as negative and positive controls, respectively. Spots were counted by an ImmunoSpot CTL reader. The grouping and inoculation of the mice were the same as described in example 2.

Experimental results

IFN-. Gamma.ELISPOT was used to detect cellular immune responses. Spleen cells of BALB/c mice receiving 50. Mu.g of pSV-10-SARS-CoV-2 vaccine were epitope mapped. Spike proteins were covered by 20 peptide libraries. Each peptide pool contains 7 peptide fragments, each peptide fragment has 18 amino acids, and adjacent peptide fragments overlap 9 amino acids. Cellular immune responses were detected in multiple peptide pools, but the strongest response was in peptide pool 5 (FIG. 4), the region located at amino acids 234-297 of the spike protein. In subsequent cellular immune experiments, peptide pool 5 was used as a peptide stimulator. BALB/c mice were sacrificed at weeks 4 and 6 after vaccine injection and spleen cells were harvested. Single cell suspensions were stimulated with peptide pool 5 for 20 hours. And detected using the IFN-. Gamma.ELISPOT kit.

Vaccine-induced cellular immune response

The results of the cellular immune response at week 4 showed that the average IFN- γ of the first group was 154 Spot Forming Units (SFU) per 5X10 ⁵ spleen cells, while the results of the second group were 179.1 SFU per 5X10 ⁵ spleen cells. At week 6, the average IFN- γ for the first group was ⁵ splenocytes 253 SFU per 5x10, while the average IFN- γ for the second group was 389.9 SFU per 5x10 ⁵ splenocytes (FIG. 5A). The pSV-10-SARS-CoV-2 vaccine with BC01 enhances the cellular immune response and increases the number of IFN-gamma spots.

A cellular immune response was also detected with IL-2 ELISPOT. The average number of IL-2 spots was 29.3 SFU per 5X10 ⁵ spleen cells in the first group and 37.7 SFU per 5X10 ⁵ spleen cells in the second group. At week 6, the results of the cellular immune response showed that the average number of IL-2 spots was 39.4 SFU per 5X10 ⁵ spleen cells in the first group and 60.4SFU per 5X10 ⁵ spleen cells in the second group (FIG. 5B). The results show that the pSV-10-SARS-CoV-2 vaccine with BC01 increases the number of IL-2 secreting cells.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

SEQUENCE LISTING

<110> National food and drug verification institute

<120> A novel coronal DNA vaccine containing adjuvant

<130> IDC210273

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1273

<212> PRT

<213> artificial

<220>

<223> Insertion fragment of D614G plasmid

<400> 1

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

1 5 10 15

Asn Leu 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 Asp

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 Asp 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 Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

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

355 360 365

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

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

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

405 410 415

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

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

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

450 455 460

Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

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

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

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

545 550 555 560

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

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

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

595 600 605

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

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

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

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

945 950 955 960

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

965 970 975

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

980 985 990

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

995 1000 1005

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

1010 1015 1020

Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys

1025 1030 1035

Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1040 1045 1050

Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val

1055 1060 1065

Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His

1070 1075 1080

Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn

1085 1090 1095

Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln

1100 1105 1110

Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val

1115 1120 1125

Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro

1130 1135 1140

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

1145 1150 1155

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

1160 1165 1170

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

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

Val Leu Lys Gly Val Lys Leu His Tyr Thr

1265 1270

<210> 2

<211> 1273

<212> PRT

<213> artificial

<220>

<223> Insertion of D614G+I472V plasmid

<400> 2

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

1 5 10 15

Asn Leu 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 Asp

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 Asp 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 Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

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

355 360 365

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

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

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

405 410 415

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

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

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

450 455 460

Glu Arg Asp Ile Ser Thr Glu Val Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

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

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

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

545 550 555 560

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

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

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

595 600 605

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

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

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

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

945 950 955 960

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

965 970 975

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

980 985 990

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

995 1000 1005

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

1010 1015 1020

Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys

1025 1030 1035

Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1040 1045 1050

Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val

1055 1060 1065

Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His

1070 1075 1080

Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn

1085 1090 1095

Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln

1100 1105 1110

Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val

1115 1120 1125

Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro

1130 1135 1140

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

1145 1150 1155

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

1160 1165 1170

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

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

Val Leu Lys Gly Val Lys Leu His Tyr Thr

1265 1270

<210> 3

<211> 1273

<212> PRT

<213> artificial

<220>

<223> Insertion fragment of V483A plasmid

<400> 3

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

1 5 10 15

Asn Leu 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 Asp

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 Asp 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 Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

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

355 360 365

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

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

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

405 410 415

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

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

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

450 455 460

Glu Arg Asp Ile Ser Thr Glu Val Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Ala Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

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

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

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

545 550 555 560

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

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

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

595 600 605

Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

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

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

945 950 955 960

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

965 970 975

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

980 985 990

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

995 1000 1005

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

1010 1015 1020

Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys

1025 1030 1035

Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1040 1045 1050

Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val

1055 1060 1065

Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His

1070 1075 1080

Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn

1085 1090 1095

Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln

1100 1105 1110

Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val

1115 1120 1125

Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro

1130 1135 1140

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

1145 1150 1155

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

1160 1165 1170

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

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

Val Leu Lys Gly Val Lys Leu His Tyr Thr

1265 1270

<210> 4

<211> 1273

<212> PRT

<213> artificial

<220>

<223> Insertion fragment of L452R plasmid

<400> 4

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

1 5 10 15

Asn Leu 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 Asp

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 Asp 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 Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

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

355 360 365

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

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

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

405 410 415

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

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

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

450 455 460

Glu Arg Asp Ile Ser Thr Glu Val Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Ala Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

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

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

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

545 550 555 560

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

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

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

595 600 605

Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

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

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

945 950 955 960

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

965 970 975

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

980 985 990

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

995 1000 1005

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

1010 1015 1020

Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys

1025 1030 1035

Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1040 1045 1050

Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val

1055 1060 1065

Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His

1070 1075 1080

Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn

1085 1090 1095

Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln

1100 1105 1110

Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val

1115 1120 1125

Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro

1130 1135 1140

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

1145 1150 1155

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

1160 1165 1170

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

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

Val Leu Lys Gly Val Lys Leu His Tyr Thr

1265 1270

Claims

1. A vaccine composition comprising or consisting of: a first nucleic acid molecule as an adjuvant component and a second nucleic acid molecule as an immunogen component; the first nucleic acid molecule comprises a nucleotide sequence encoding a bcg unmethylated CpG motif and the second nucleic acid molecule comprises a nucleotide sequence encoding a SARS-CoV-2Spike protein.

2. The vaccine composition of claim 1, wherein said bcg non-methylated CpG motif is obtained by lysing bacillus calmette guerin and extracting nucleic acid from the lysate.

3. The vaccine composition of claim 1, having one or more characteristics selected from the group consisting of:

(1) The first nucleic acid molecule is contained or not contained in a vector;

(2) The second nucleic acid molecule is contained or not contained in a vector;

(3) The first nucleic acid molecule and the second nucleic acid molecule are comprised in the same vector or are comprised in separate vectors.

4. The vaccine composition of claim 1, wherein the second nucleic acid molecule is contained in a vector capable of expressing SARS-CoV-2Spike protein.

5. The vaccine composition of claim 1, wherein the ratio of the first nucleic acid molecule to the second nucleic acid molecule is 1:2 to 1:10.

6. The vaccine composition of claim 5, wherein the ratio of the first nucleic acid molecule to the second nucleic acid molecule is 1:2,1:3,1:4,1:5,1:6,1:7,1:8,1:9 or 1:10.

7. The vaccine composition of claim 5, wherein the first nucleic acid molecule is present in an amount of 1 μg to 100 μg.

8. The vaccine composition of claim 5, wherein the first nucleic acid molecule is present in an amount of 1 μg,10 μg,20 μg,40 μg,60 μg,80 μg or 100 μg.

9. The vaccine composition of claim 5, wherein the second nucleic acid molecule is present in an amount of 5 μg to 500 μg.

10. The vaccine composition of claim 5, wherein the second nucleic acid molecule is present in an amount of 5 μg,50 μg,100 μg,200 μg,300 μg,400 μg or 500 μg.

11. A combination of host cells, wherein a first host cell comprises a first nucleic acid molecule or a vector comprising the same in the vaccine composition of any one of claims 1-10; a second host cell comprising a second nucleic acid molecule or a vector comprising the same in a vaccine composition according to any one of claims 1-10.

12. A pharmaceutical composition comprising the vaccine composition of any one of claims 1-10, and a pharmaceutically acceptable excipient.

13. The pharmaceutical composition of claim 12, further comprising an additional antiviral agent.

14. The pharmaceutical composition of claim 13, further comprising an interferon, lopinavir, ritonavir, chloroquine phosphate, fepima Weirui darcy, or any combination thereof.

15. Use of the vaccine composition according to any one of claims 1-10 for the preparation of a kit for the prevention and/or treatment of a coronavirus infection, or for the prevention and/or treatment of a disease caused by a coronavirus infection, or for inducing or generating an immune response against a coronavirus in a subject.

16. The use of claim 15, wherein the coronavirus is SARS-CoV-2.

17. The use according to claim 15, wherein the disease caused by coronavirus infection is COVID-19 and/or SARS.

18. A method of preparing the vaccine composition of any one of claims 1-10, comprising synthesizing the first nucleic acid molecule and the second nucleic acid molecule, respectively, by an organic synthesis reaction or an enzymatic synthesis reaction; optionally, the first nucleic acid molecule and/or the second nucleic acid synthesized as described above are separately constructed into a vector.