CN113633764A - Novel corona DNA vaccine containing adjuvant - Google Patents

Novel corona DNA vaccine containing adjuvant Download PDF

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CN113633764A
CN113633764A CN202111026912.2A CN202111026912A CN113633764A CN 113633764 A CN113633764 A CN 113633764A CN 202111026912 A CN202111026912 A CN 202111026912A CN 113633764 A CN113633764 A CN 113633764A
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CN113633764B (en
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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
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National Institutes for Food and Drug Control
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Abstract

The invention relates to a DNA vaccine composition and application thereof. In particular, the present invention relates to DNA vaccine compositions against coronaviruses, and pharmaceutical compositions comprising the DNA vaccine compositions. Furthermore, the invention relates to the use of said DNA vaccine composition. The DNA vaccine composition of the present invention can be used for preventing and/or treating coronavirus infection and/or diseases caused by the infection.

Description

Novel corona DNA vaccine containing adjuvant
Technical Field
The present invention relates to the fields of immunology and molecular virology, in particular the fields of diagnosis, prevention and treatment of coronaviruses. In particular, the present invention relates to DNA vaccine compositions against novel coronaviruses, and pharmaceutical compositions comprising the DNA vaccine compositions. Furthermore, the invention relates to the use of said DNA vaccine composition. The DNA vaccine composition of the present invention can be used for preventing and/or treating coronavirus infection and/or diseases caused by the infection.
Background
Coronavirus-induced disease (COVID-19) has caused a health crisis globally in 2019. Severe acute respiratory syndrome 2(SARS-CoV-2) is a single-stranded positive-sense ribonucleic acid (RNA) virus, the Spike protein (Spike protein) of which determines the infectivity and the ability to spread in the host. The Spike protein of the virus is also very unstable and mutations in the Spike protein may lead to 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.
At present, 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, and combinations of Mutations, such as D614G + I472V (Zhou, B., et al, SARS-CoV-2Spike D614G variant enhanced reproduction and transmission. bioRxiv, 2020; plant, J.A., et al, Spike repetition D614G variants SARS-CoV-2 fixed. Nature, 2020; Li, Q., et al, The Impact of Mutations in SARS-CoV-2Spike visual infection and immunity. cell,2020.182(5): p.4-1294 e 9). Other studies have found that certain mutation sites can reduce The Infectivity of The virus, such as L452R and V483A, but these sites can tolerate some neutralizing antibodies (Li, Q., et al., The Impact of Mutations in SARS-CoV-2Spike on Viral infection and immunity. cell,2020.182(5): p.1284-1294e 9.). In addition, the b.1.1.7 variant and many genetically altered variants are rapidly increasing in the uk, and the b.1.1.7 variant may increase the binding of the virus to human angiotensin converting enzyme 2(ACE2) due to mutation of N501Y (Leung, k., et al., Early transmission assessment of the N501Y mutant strains of SARS-CoV-2 in the United Kingdom, October to November 2020.Euro surfille, 2021.26 (1)). The unique South African strain (501Y.V2) also includes multiple nonsynonymous spike mutations that may be of functional significance, such as K417N, E484K, and N501Y (Tegality, H., et al, Emergene and Rapid spread of a new segment acid regulated syndrome-related polynucleotide vaccine 2(SARS-CoV-2) line with multiple spike mutations in South African. medRxiv,2020: p. 2020.12.21.20248640.) in the spike receptor binding domain.
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, expression of antigenic genes in vivo can preserve the native structure of the protein, ensuring its proper processing and immune presentation. It is noteworthy that 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 a variety of clinical conditions.
For example, CpG ODN has been recognized as an immunological adjuvant for a variety of vaccines because it can help activate both innate and adaptive immune responses in animals and humans. CPG-ODN can also directly stimulate monocytes, macrophages and dendritic cells to secrete various cytokines, such as TNF-alpha and GM-CSF, which in turn stimulate helper T cells to generate immune responses.
Disclosure of Invention
Through a large number of experiments and repeated groping, the inventor constructs a DNA vaccine expressing SARS-Cov-2Spike protein, and uses BC01 as adjuvant. It has been surprisingly found that such a combination can elicit an effective production of neutralizing antibodies against neocorona and generate a cell-mediated immune response to prevent and/or treat infections or diseases 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 immunogenic component; the first nucleic acid molecule contains a nucleotide sequence encoding a BCG motif that is unmethylated by BCG, and the second nucleic acid molecule contains a nucleotide sequence encoding a SARS-CoV-2Spike protein.
In certain embodiments, the BCG-unmethylated CpG motifs are obtained by lysing the cells of BCG and extracting the nucleic acids from the lysate.
In certain embodiments, the SARS-CoV-2S protein has a sequence as shown 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., the pSV10 vector), or are contained separately in different 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 forms 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 a method described in chinese invention patent ZL200410033878.1, the entire contents of which are incorporated herein by reference. In certain embodiments, the first nucleic acid molecule is prepared by a method comprising: inoculating the strain into a culture medium suitable for growth of mycobacteria, and collecting the thallus when the strain is cultured to a logarithmic phase; crushing the thalli, centrifuging and collecting supernate; dissolving the precipitate of the supernatant with NaCl solution, extracting with organic solvent to collect protein-free layer, treating the re-extracted supernatant with ethanol to collect precipitate, and post-treating the precipitate.
In certain embodiments, the first nucleic acid molecule is prepared by a method described in chinese invention patent zl201310586057.x, the contents of which are incorporated herein in its entirety. In certain embodiments, the first nucleic acid molecule is prepared by a method comprising: and (2) separating the BCG-DNA from the BCG lysate by using a Q Sepharose HP ion exchange column, wherein the ion exchange column separation of the BCG-DNA lysate can be carried out in a TE buffer system or a sodium phosphate buffer system. In certain preferred embodiments, wherein the ion exchange column separation of BCG-DNA lysates is performed in a sodium phosphate buffer system, the elution buffer is 1M sodium chloride +50mM sodium phosphate buffer, pH 7.5-8.5. In certain preferred embodiments, wherein the elution buffer is at pH 7.5. In certain preferred embodiments, a loading buffer is used for loading, the loading buffer being 0-0.5M sodium chloride +50mM sodium phosphate buffer, pH 7.5-8.5. In certain preferred embodiments, the loading buffer is 0.5M sodium chloride +50mM sodium phosphate buffer, pH 7.5. In certain preferred embodiments, wherein the elution after loading is performed using a gradient elution, the gradient elution is performed by eluting from 50% to 65% of the elution buffer, and continuing with elution with 100% of the elution buffer. In certain preferred embodiments, wherein the elution after loading is performed using a gradient elution, the gradient elution is performed by eluting from 50% to 65% of the elution buffer, and continuing with elution with 100% of the elution buffer. In certain preferred embodiments, wherein the ion exchange column separation of BCG-DNA lysates is performed in a TE buffer system, the elution buffer is 1M NaCl +50mM Tris +1mM EDTA pH 7.5-8.5. In certain preferred embodiments, wherein the elution buffer is at pH 7.5. In certain preferred embodiments, the loading buffer is 0-0.5M NaCl +50mM Tris +1mM EDTA pH 7.5-8.5. In certain preferred embodiments, the loading buffer is 0.5M NaCl +50mM Tris +1mM EDTA pH 7.5. In certain preferred embodiments, wherein the elution after loading is performed using a gradient elution, the gradient elution is performed by eluting from 25% to 65% of the elution buffer, and continuing with elution with 100% of the elution buffer. In certain preferred embodiments, wherein the bcg lysate is further clarified prior to Q Sepharose HP ion exchange column separation purification. In some preferred embodiments, the method for clarifying BCG lysate comprises diluting the disrupted lysate to a concentration of 200mg/ml, centrifuging at 8000-.
As known to those skilled in the art, the first nucleic acid molecule may also be prepared by a modification of the above-described method. The first nucleic acid molecules obtained by these exemplary methods can be used in the vaccine composition 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 chromatography. For example, CpG can be quantified by modifying cytosine (dC) of CpG dinucleotides to 5-methylcytosine (m5-dC) using specific methylase SssI by reverse phase-high performance liquid chromatography (RP-HPLC) as described in ZL200410033878.1, hydrolyzing DNA to single deoxynucleosides using nuclease P1 and Bacterial Alkaline Phosphatase (BAP), and detecting the difference in the amount of m5-dC in the modified and unmodified DNA hydrolysis samples by reverse phase-high performance liquid chromatography (RP-HPLC).
In certain embodiments, the bcg strain is bcg strain D2PB302 for chinese bcg preparation, provided by the chinese pharmaceutical biologies certification institute bacterin chamber.
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 lyophilized powder for reconstitution that can be injected or can be administered via the mucosa.
In certain embodiments, the ratio of the amount of the first nucleic acid molecule to the amount 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 and the second nucleic acid molecule is 1: 5.
in certain embodiments, the first nucleic acid molecule is present in an amount of 1 μ g to 100 μ g (e.g., 1 μ g, 10 μ g, 20 μ g, 40 μ g, 60 μ g, 80 μ g, 100 μ g). In certain embodiments, the first nucleic acid molecule is present in an amount of 10 μ g.
In certain embodiments, the second nucleic acid molecule is present in an amount of 5 μ g to 500 μ g (e.g., 5 μ g, 50 μ g, 100 μ g, 200 μ g, 300 μ g, 400 μ g, 500 μ g). In certain embodiments, the second nucleic acid molecule is present in an amount of 50 μ g.
In another aspect, the present application provides a host cell comprising a nucleic acid molecule or vector as in the vaccine composition as described above.
In certain embodiments, the host cell comprises the first nucleic acid molecule and the second nucleic acid molecule, or alternatively, the host cell comprises the first nucleic acid molecule and the pSV10 vector.
In another aspect, the present application provides a combination of host cells, wherein a first host cell comprises a first nucleic acid molecule or vector as in the vaccine composition as described above; the second host cell comprises a second nucleic acid molecule or vector as in the vaccine composition described previously.
In another aspect, the present application provides a pharmaceutical composition comprising a vaccine composition as described above, together with 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, fabiravir, ridciclovir, and the like).
In another aspect, the present application provides a method for neutralizing the virulence of a coronavirus in a sample comprising contacting a sample comprising a 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 present 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 previously or a pharmaceutical composition as described previously to a subject in need thereof.
In certain embodiments, the first nucleic acid molecule or vector comprising the same is administered to the 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 present 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 previously or a pharmaceutical composition as described previously to the subject.
In certain embodiments, the injection is an intradermal injection or an intramuscular injection.
In certain embodiments, the method further comprises the step of electroporating the tissue with an electroporating amount of electrical energy.
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 present application provides the use of a vaccine composition as described previously 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 (e.g., generating neutralizing antibodies) against a coronavirus in a subject.
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 present application provides a method of preparing a vaccine composition as described above, comprising synthesizing the first nucleic acid molecule and the second nucleic acid molecule by organic synthesis reaction or enzymatic synthesis reaction, respectively. Optionally, the first nucleic acid molecule and/or the second nucleic acid synthesized above are constructed into a vector separately.
Definition of terms
In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the procedures of molecular genetics, nucleic acid chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, etc., used herein, are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.
As used herein, "Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)", formerly known as "novel coronavirus" or "2019-nCov", belongs to its genus β -coronavirus, and is an enveloped, single-stranded, positive-sense RNA virus. The genomic sequence of SARS-CoV-2 is known to those skilled in the art and can be found, for example, in GenBank: MN 908947. SARS-CoV-2 contains at least three membrane proteins, including surface spike protein (S), integral membrane protein (M) and membrane protein (E). Like SARS-CoV, the SARS-CoV-2 Receptor is specifically bound to angiotensin transferase 2(ACE2) on host cells via Receptor Binding Domain (RBD) on S protein, and then is connected to viral membrane fusion and cell entry, and plays a crucial role in the process of viral infection of cells.
As used herein, the terms "novel coronavirus pneumonia" and "COVID-19" refer to pneumonia resulting from SARS-CoV-2 infection, which have the same meaning and are used interchangeably.
As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing 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, and 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 Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (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 contain a replication initiation site.
As used herein, the term "host cell" refers to a cell that can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, 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 carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical sciences. edited by geno AR,19th ed. pennsylvania: mach Publishing Company,1995), and include, but are not limited to: pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusting agents 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, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate salts and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, for example, thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning generally understood by those skilled in the art to be capable of stabilizing the desired activity of the active ingredient in a medicament, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or degradation products thereof (such as lactalbumin hydrolysate), and the like. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), glucose solutions (e.g., 5% glucose), 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 to prevent or delay the onset 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 performed in order to obtain a beneficial or desired clinical result. For 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., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Furthermore, "treatment" may also refer to prolonging survival as compared to 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 SARS-CoV-2 infection or a disease associated with 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, a desired effect. For example, an amount effective to prevent a disease (e.g., SARS-CoV-2 infection) is an amount sufficient to prevent, or delay the onset of a disease (e.g., SARS-CoV-2 infection); a therapeutically effective amount for a disease is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such effective amounts. For example, an 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, e.g., age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, and the like.
As used herein, the term "neutralizing activity" means that the antibody or antibody fragment has a functional activity of binding to an antigenic protein on the virus, thereby preventing the virus from infecting cells and/or maturation of viral progeny and/or release of viral progeny, and the antibody or antibody fragment having neutralizing activity can prevent amplification of the virus, thereby inhibiting or eliminating infection by the virus.
As used herein, the term "CpG motif, collectively referred to as an unmethylated cytosine guanine motif, refers to an unmethylated CpG dinucleotide. Typically, the bacterial genome contains a large proportion of unmethylated CpG dinucleotides. The immune system of vertebrates uses CpG motifs as recognition molecules for foreign substances, which in turn can stimulate the body to produce a protective immune response. The sequences of CpG motifs can be obtained from existing databases by those skilled in the art.
Generally, methylation refers to a reaction in which a hydrogen in a nucleic acid molecule is replaced with a methyl group (-CH 3). The term "unmethylated CpG motif" refers to a CpG motif that is not subject to the methylation reaction.
As used herein, the term "Bacillus Calmette-Guerin (BCG)" also known as "Bacillus Calmette-Guerin", a live Mycobacterium bovis (Bacillus bovis) bacterium that is attenuated but provides immunity to the organism, is used as a prophylactic vaccine for tuberculosis in children, and provides the organism with a particular resistance to tuberculosis after vaccination.
As used herein, the term "BCG-CpG-DNA" refers to nucleic acids (e.g., double stranded DNA fragments) extracted from Bacillus Calmette-Guerin (BCG) that contain a significant amount 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 an immunomodulator or immunopotentiator, refers to a substance that is injected into a mammal before or after an antigen or mixed with an antigen, and that can nonspecifically alter or enhance the body's specific immune response to the antigen, thereby aiding in its action.
The DNA vaccine of the invention can be an aqueous solution or a lyophilized powder for reconstitution which can be injected or can be administered through mucosa. In certain embodiments, the aqueous solution or reconstituting lyophilized powder for injection is prepared by aseptic processing techniques well known in the art. In certain embodiments, they are sterile during storage, transport, 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 formulations, kits, and the like. Although the DNA vaccine involved in the use of the present invention may be administered by injection, mucosal, etc., and these modes of administration are also part of the present invention. However, it will be clear to the skilled person that the most preferred route of administration suitable for the use according to the invention is parenteral or by injection. For the practice of the present invention, the pharmaceutical composition is preferably a formulation for parenteral administration, including but not limited to topical injection formulations and systemic injection formulations, and specific dosage forms include but are not limited to injectable solutions and injectable powder injections. In certain embodiments, the medicament is a sterile injectable aqueous solution or a sterile injectable powder, particularly a lyophilized powder, for reconstitution with injectable water prior to clinical use. When the freeze-dried powder injection is prepared, the freeze-dried powder injection can also contain pharmaceutically acceptable excipient, such as mannitol.
The DNA vaccine of the present invention can be introduced into an organism by a method known in the art. Such introduction methods include, but are not limited to, intramuscular injection, gene gun introduction, ③ mucosal immunization, intravenous injection, intraperitoneal injection, and the like, and are 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 effective dosages of the vaccine compositions of the present application for use in mammals, particularly in humans. The daily dose may be administered to a subject all at once during the day, or the required dose may be divided into two, three, four or more small doses for administration at appropriate intervals throughout the day. The small doses may be formulated as unit dosage forms, e.g., each unit dosage form containing the respective amount divided into a total daily dose an appropriate number of times. Of course, administration may also be performed over a period of time, such as once a day, once every two days, once a week, once a month of february, once a month of march, once a month of june, once a year of two years, and the like.
As used herein, the term "ID 50" refers to a 50% inhibitory dose, which is commonly 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 be used for preventing the generation of new crown antibodies in advance, improving the titer of generated neutralizing antibodies and enhancing the immune response of cells. The DNA vaccine composition of the present invention can be used for preventing and/or treating coronavirus infection and/or diseases caused by the infection.
Furthermore, the vaccine composition consisting of the BC01 adjuvant and the pSV10-SARS-CoV-2 vaccine of the application is superior to: the vaccine composition consisting of the rest adjuvant and pSV10-SARS-CoV-2 vaccine; and, a combination of BC01 adjuvant with the remaining vaccine. For example, the application proves through experiments that only the neutralizing antibodies aiming at D614G, D614G + I472V and V483A strains can be generated by using the pSV10-SARS-CoV2 vaccine alone, and the neutralizing antibodies aiming at the L452R strain cannot be generated. While the use of pSV10-SARS-CoV2 vaccine with BC01 enabled the production of neutralizing antibodies against the L452R, D614G, D614G + I472V and V483A strains, and also induced higher titers of neutralizing antibodies.
Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit 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 accompanying drawings and the following detailed description of the preferred embodiments.
Drawings
FIG. 1 shows the design scheme and expression results of SARS-Cov-2 DNA vaccine construct. Among them, FIG. 1A shows a schematic diagram of the DNA vaccine, i.e., pSV10-SARS-CoV-2 contains an inserted SARS-CoV-2Spike protein. FIG. 1B shows the results of Western blot analysis of Spike protein in vitro after transfection of 293T cells with pSV10-SARS-CoV-2 and MOCK plasmids.
Figure 2 shows the humoral immune response of mice vaccinated with different types of vaccines. Among them, fig. 2A shows the results of different groups of neutralizing antibody ID 50. FIG. 2B shows the results of IgG binding to SARS-CoV-2S protein antigen in serial mouse dilution sera at weeks 4 and 6.
FIG. 3 shows the effect of neutralizing antibodies generated by inoculation of different groups of reagents on different variants. Wherein fig. 3A shows neutralization against the D614G variant, fig. 3B shows neutralization against the D614G + I472V variant, fig. 3C shows neutralization against the V483A variant, and fig. 3D shows neutralization against the L452R variant.
FIG. 4 shows the T cell epitope map after BALB/c mice were inoculated with pSV10-SARS-CoV-2 vaccine with BC 01.
FIG. 5 shows the T cell response of mice after vaccination 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-2ELISPOT at week 4, and FIG. 5D shows IL-2ELISPOT at week 6.
Sequence information
Information on the partial sequences to which the present invention relates is provided in table 1 below.
Table 1: description of the sequences
Figure BDA0003243626520000141
Figure BDA0003243626520000151
Figure BDA0003243626520000161
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.
Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. For example, conventional techniques in immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in the present invention can be found in Sambrook (Sambrook), friesch (Fritsch), and manitis (manitis), molecular cloning: a LABORATORY Manual (Molecular CLONING: A Laboratory Manual), 2 nd edition (1989); a Current Manual of MOLECULAR BIOLOGY experiments (Current PROTOCOLS IN MOLECULAR BIOLOGY BIOLOGY) (edited by F.M. Otsubel et al, (1987)); METHODS IN ENZYMOLOGY (METHODS IN Enzymology) series (academic Press): PCR 2: practical methods (PCR 2: A PRACTICAL APPROACH) (M.J. Mefferson, B.D. Hemsh (B.D. Hames) and G.R. Taylor (edited by G.R. Taylor) (1995)), and animal cell CULTURE (ANIMAL CELL CURTURE) (edited by R.I. Fresherni (R.I. Freshney) (1987)).
In addition, those whose specific conditions are not specified in the examples are conducted under the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. The examples are given by way of illustration 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 vaccines
Preparation of DNA vaccine
The DNA vaccine is designed based on the sequence of SARS-CoV-2spike (S) protein Wuhan-1 (GenBank: MN-908947). The nucleotide sequence encoding the full length of the S protein was synthesized. The nucleotide sequence synthesized was cloned into a mammalian expression plasmid pSV10 (obtained from the AIDS prevention and control center, China disease prevention and control center), the DNA vaccine was obtained and named 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 this example refers to chinese patent application zl201310586057.x, and the BC01 adjuvant used in this example is BCG-CpG-DNA in this patent. In brief, bacillus calmette-guerin strains (bacillus calmette-guerin strain D2PB302 for preparing Chinese bacillus calmette-guerin, provided by the bacterin chamber of China pharmaceutical and biological products institute) are subjected to thalli culture and then are cracked, and the cracked liquid is purified by an ion exchange purification method to remove impurities such as polysaccharide, protein, RNA and the like in the cracked liquid. Finally, BCG vaccine (BCG) CpG-DNA is separated from the lysate by an ion exchange column and dissolved in sterile water, and the BC01 adjuvant is obtained.
Western Blot validation
293T cells were seeded into 6-well plates and at approximately 70% density, the cells were transfected with pSV10-SARS-CoV-2 plasmid (4. mu.g) using 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 run. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes and PVDF membranes were membrane blocked at 4 ℃ in Phosphate Buffered Saline (PBST) containing 0.2% Tween 20(Sigma) (V/V) and 5% (W/V) skimmed milk powder. After overnight blocking, PVDF membrane in 1:1000 dilution of mouse anti SARS-CoV-2Spike S1 monoclonal antibody 5% (W/V) skimmed milk powder PBST were incubated for one hour. After incubation, the PVDF membrane was washed 5 times with 5% (W/V) nonfat dry milk in PBST and then incubated with 1:10,000 goat anti-mouse secondary antibody in 5% (W/V) nonfat dry milk in PBST. Then, the PVDF membrane was washed again 5 times with 5% (W/V) non-fat dry milk in PBST and detected by Touch Imager XLI system (e-BLOT).
The vector map of pSV10 after insertion into SARS-CoV-2Spike protein is shown in FIG. 1A. The molecular weight of the Spike protein was 140-142kDa, with slight variations in molecular weight due to the 22 potential N-linked glycans in the S protein. Western blot analysis of Spike proteins is shown in fig. 1B, which demonstrates that Spike proteins can be expressed after transfection of a vaccine vector.
Example 2 humoral immune response in mice
Treatment of animal samples
39 female mice 4 to 6 weeks old were divided into four groups and injected with the following agents: the first group was pSV10-SARS-CoV-2 (sample size 10) with BC01, the second group was pSV10-SARS-CoV-2 (sample size 10), the third group was BC01 (sample size 10), and the fourth group was PBS (sample size 9). Mice received 50ug of DNA vaccine at week 0, week 2 and week 4, respectively. The above four groups of reagents were injected into mice of the respective experimental groups with needles and syringes at weeks 0, 2 and 4, respectively, and then subjected to in vivo electroporation. Sera were collected at weeks 4 and 6 after 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-2Spike protein in 1 Xphosphate buffer (DPBS). The ELISA plate was washed 3 times with wash buffer and blocked with 3% Bovine Serum Albumin (BSA) in DPBS (containing 0.05% Tween) for two to three hours at room temperature. The blocking solution was discarded and the ELISA plate was incubated with serial dilutions of heat-inactivated mouse serum for one hour at room temperature. And incubated with anti-mouse IgG horseradish peroxidase (HRP) diluted 1:4000 for one hour at room temperature in the dark. The plates were then washed 5 times with wash buffer. 100 μ L of TMB solution was added to each well, and after 5min, stop solution was added to terminate the reaction. The absorbance at 450nm and 630nm was recorded.
Statistical analysis
Analysis was performed using GraphPad Prism 8.4.2(GraphPad Software). Comparisons of data between groups were performed using one-way analysis of variance and multiple comparison tests of Holm-Sidak. Differences were considered significant with P values less than 0.05.
Results of the experiment
The 39 mice (4-6 weeks old) were divided into 4 experimental groups, the first group injected with 50. mu.g of pSV-10-SARS-CoV-2 vaccine (vaccination number 10), the second group injected with 50. mu.g of pSV-10-SARS-CoV-2 vaccine with 10. mu.g of BC01 adjuvant (vaccination number 10), the third group injected with 10. mu.g of BC01 adjuvant (vaccination number 10), and the fourth group injected with 60. mu.g of PBS (vaccination number 9). At the fourth week post-injection, the average titer of ID50 for neutralizing antibodies observed in the first group of vaccinated mice was 87 and the average titer of ID50 for neutralizing antibodies detected in the second group of vaccinated mice was 141. No neutralizing antibodies were detected in the test groups of the third and fourth groups. At week 6 post-injection, the mean titer of neutralizing ID50 was 262 for the first group, 309 for the second group, and no neutralizing antibody was detected in the control group of both the third and fourth groups (fig. 2A). At the fourth week after injection, the positive conversion rate of the second group was 80%, which was significantly higher than 50% of the first group (i.e., the test group injected with pSV-10-SARS-CoV-2 vaccine only). These results indicate that BC01 adjuvant can promote early production of neutralizing antibodies in mice (table 2).
TABLE 2 neutralizing Activity of serum after administration of pSV10-SARS-CoV-2 to mice with BC01
Figure BDA0003243626520000201
At week 4, a certain titer of bound antibody was detected in both the first and second groups. No bound antibody was detected in the third and fourth groups. At week 6, a certain titer of bound antibody was detected in both the first and second groups. No bound antibody was detected in the third and fourth groups (fig. 2B).
Example 3 neutralization of pseudoviruses
Production and titration of pseudoviruses
The production of SARS-CoV-2 pseudovirus is described in the literature (Establishment and validation of a pseudo viral replication assay for SARS-CoV-2. emery Microbes infection, 2020.9(1): p.680-686.). Briefly, the nucleotide sequences of SEQ ID NOS: 1-4 were inserted into the multiple cloning sites of pSV10, respectively, plasmids expressing different SARS-CoV-2Spike proteins were inserted into 293T cells, and then used at a concentration of 7.0X 104 TCID 50/ml G.DELTA.G-VSV (VSV G-pseudotype)Virus) to infect these cells.
The cells were cultured 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, culture supernatants containing SARS-CoV-2 pseudovirus were collected, filtered through a filter (0.45 mm pore size, Millipore, Cat # SLHP033RB) and stored at-80 ℃ to produce pseudoviruses D614G, D614G + I472V, L452R and V483A. 24 hours after infection, the prepared SARS-CoV-2 pseudovirus was titrated to produce more than ten times the Relative Luminescence Units (RLU) of the negative control (cells only). The grouping of mice and the manner of inoculation were the same as described in example 2.
Results of the experiment
The level of neutralization of the antibodies produced by the vaccine against the currently circulating pseudoviruses D614G, D614G + I472V, L452R and V483A was evaluated. Week 6 sera from 5 vaccinated mice each were taken from each group for neutralization. Mouse sera from different vaccinated groups were heat-inactivated at 56 ℃ for 30 minutes, with three serial dilutions starting at 1:30 dilution for each assay. Serum was mixed with 50 μ L of pseudovirus for 60 min. After 60 minutes Huh-7 cells stably expressing ACE2 were added and incubated at 37 ℃ for 24 hours. Cells were then lysed and RLU measured using the britelite plus luminescent reporter assay system (Perkin Elmer catalog No. 6066769). Virus neutralization titers were calculated using the Reed-Muench method (ID 50).
The second group injected with DNA vaccine with BC01 could neutralize D614G, D614G + I472V, L452R and V483A variants, while the first group injected with DNA vaccine alone could neutralize D614G, D614G + I472V and V483A variants, but not neutralize L452R variants. Mice injected with the third and fourth groups were unable to neutralize these variants (figure 3).
Example 4 cellular immune response
ELISPOT detection
Mouse spleens were harvested and ground to single cell suspensions in RPMI1640 medium containing 1% penicillin/streptomycin (R0). The cell pellet was resuspended in 5mL of ACK lysis buffer for 5 minutes, and then 8mL of PBS was added to stop the reaction. The sample was centrifuged at 1500g for 5minCells 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 from 200. mu. L R10/well for 30 min. Add 5X10 to each well5Mouse splenocytes and stimulated with an 18-mer peptide library (these peptides overlap with 9 amino acids from SARS-CoV-2Spike protein). In addition, mapping (matrix mapping) was performed using a peptide library in the matrix to identify immunodominant reactions. Mouse splenocytes were stimulated in R10 with a final concentration of 5. mu.g/ml of each peptide per well. R10 and cell stimulator PAM + ion (invitrogen) were used as negative and positive controls, respectively. Spots were counted by an ImmunoSpot CTL reader. The grouping of mice and the manner of inoculation were the same as described in example 2.
Results of the experiment
IFN-gamma ELISPOT was used to detect cellular immune responses. Splenocytes from BALB/c mice receiving 50. mu.g of pSV-10-SARS-CoV-2 vaccine were epitopically mapped. The Spike protein was covered by a library of 20 peptides. Each peptide pool comprises 7 peptide fragments, each peptide fragment has 18 amino acids, and adjacent peptide fragments are overlapped by 9 amino acids. Cellular immune responses were detected in the peptide pools, but the strongest response was in peptide pool 5 (FIG. 4), which was located at amino acid 234-297 of spike proteins. In subsequent cellular immunity experiments, peptide library 5 was used as a peptide stimulator. BALB/c mice were sacrificed at 4 and 6 weeks post vaccine injection and splenocytes harvested. Single cell suspensions were stimulated with peptide library 5 for 20 hours. And detecting by using an IFN-gamma ELISPOT kit.
Vaccine-induced cellular immune response
The results of the cellular immune response at week 4 showed that the mean IFN-. gamma.of the first group was every 5X105Individual splenocytes 154 Spot Formation Units (SFU), while the second group resulted in every 5x105179.1 SFU per splenocyte. At week 6, the average IFN-. gamma.of the first group was every 5X105Individual splenocytes 253 SFU, and the average IFN-. gamma.of the second group was every 5X105Individual splenocytes 389.9 SFU (fig. 5A). The pSV-10-SARS-CoV-2 vaccine with BC01 enhanced the cellular immune response and increased the number of IFN-. gamma.spots.
Return checkCellular immune responses to IL-2ELISPOT were measured. In the first group, the average number of IL-2 spots was 10 per 5X529.3 SFU of individual splenocytes, every 5X10 in the second group5Individual splenocytes 37.7 SFU. At week 6, the cellular immune response results showed that the average number of IL-2 spots in the first group was every 5X105Individual splenocytes 39.4 SFU, the average number of IL-2 spots in the second group was every 5X105Individual splenocytes 60.4SFU (fig. 5B). The results showed that pSV-10-SARS-CoV-2 vaccine with BC01 increased the number of IL-2 secreting cells.
While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.
SEQUENCE LISTING
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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 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 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 (10)

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 immunogenic component; the first nucleic acid molecule comprises a nucleotide sequence encoding a BCG motif that is unmethylated by BCG, and the second nucleic acid molecule comprises a nucleotide sequence encoding a SARS-CoV-2Spike protein;
preferably, the BCG motif that is unmethylated by BCG is obtained by lysing the cells of BCG and extracting the nucleic acid from the lysate.
2. 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 (e.g., a pSV10 vector);
(2) the second nucleic acid molecule is contained or not contained in a vector; preferably, the second nucleic acid molecule is contained in a vector capable of expressing the SARS-CoV-2S protein (e.g., a pSV10 vector);
(3) the DNA vaccine is an aqueous solution or a freeze-dried powder injection for reconstitution which can be injected or can be applied through mucosa;
(4) the first nucleic acid molecule and the second nucleic acid molecule are contained in the same vector (e.g., the pSV10 vector), or are contained in separate vectors.
3. The vaccine composition of claim 1 or 2, wherein the ratio of the content of the first nucleic acid molecule and 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); preferably, the ratio of the first nucleic acid molecule to the second nucleic acid molecule is 1: 5;
preferably, the first nucleic acid molecule is present in an amount of 1 μ g to 100 μ g (e.g., 1 μ g, 10 μ g, 20 μ g, 40 μ g, 60 μ g, 80 μ g, 100 μ g); more preferably, the first nucleic acid molecule is present in an amount of 10 μ g;
preferably, the second nucleic acid molecule is present in an amount of 5 μ g to 500 μ g (e.g., 5 μ g, 50 μ g, 100 μ g, 200 μ g, 300 μ g, 400 μ g, 500 μ g); more preferably, the second nucleic acid molecule is present in an amount of 50. mu.g.
4. A host cell comprising the nucleic acid molecule or vector in the vaccine composition of any one of claims 1-3;
preferably, the host cell comprises the first nucleic acid molecule and the second nucleic acid molecule, or alternatively, the host cell comprises the first nucleic acid molecule and the pSV10 vector.
5. A combination of host cells, wherein a first host cell comprises a first nucleic acid molecule or vector in a vaccine composition according to any one of claims 1-3; the second host cell comprises the second nucleic acid molecule or vector of the vaccine composition of any one of claims 1-3.
6. A pharmaceutical composition comprising the vaccine composition of any one of claims 1-3, and a pharmaceutically acceptable carrier and/or excipient;
optionally, the pharmaceutical composition further comprises an additional pharmaceutically active agent, such as an additional antiviral agent (e.g., interferon, lopinavir, ritonavir, chloroquine phosphate, fabiravir, ridciclovir, and the like).
7. Use of the vaccine composition of any one of claims 1-3 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 (e.g., generating neutralizing antibodies) against a coronavirus in a subject;
preferably, the coronavirus is SARS-CoV-2;
preferably, the disease associated with coronavirus infection is COVID-19 and/or SARS.
8. A method of preparing the vaccine composition of any one of claims 1-3, 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 above are constructed into a vector separately.
9. A method for neutralizing the virulence of a coronavirus in a sample comprising contacting a sample comprising a coronavirus with a vaccine composition according to any one of claims 1-3;
preferably, the first nucleic acid molecule or a vector comprising the same is contacted with the sample simultaneously with the second nucleic acid molecule or a vector comprising the same, or separately;
preferably, the coronavirus is SARS-CoV-2.
10. A method for inducing an immune response (e.g., production of neutralizing antibodies) against a coronavirus in a subject (e.g., a human), or 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 the vaccine composition of any one of claims 1-3 or the pharmaceutical composition of claim 5 to a subject in need thereof;
preferably, the first nucleic acid molecule or a vector comprising the same is administered to the subject simultaneously with the second nucleic acid molecule or a vector comprising the same, or separately;
preferably, the injection is intradermal or intramuscular;
preferably, the method further comprises the step of electroporating the tissue with an electroporating amount of electrical energy;
preferably, the coronavirus is SARS-CoV-2;
preferably, the disease associated with coronavirus infection is COVID-19 and/or SARS.
CN202111026912.2A 2021-09-02 2021-09-02 New crown DNA vaccine containing adjuvant Active CN113633764B (en)

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CN113633764B CN113633764B (en) 2024-05-14

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