Novel coronavirus SARS-CoV-2 mutant vaccine and application

Abstract

The invention relates to a novel coronavirus SARS-CoV-2 mutant vaccine and application, the vaccine comprises a fusion protein, and the fusion protein comprises: (1) interferon or a functional fragment thereof; (2) a novel coronavirus SARS-CoV-2Omicron mutant strain S protein or a functional fragment of the novel coronavirus SARS-CoV-2Omicron mutant strain S protein; and (3) an immunoglobulin Fc region. Molecule and process optimization of the fusion protein comprises improvement of process developability and immunogenicity, and the vaccine can improve immunogenicity and antibody titer of a mutant new crown antigen through convergence mutation design, efficiently generate a neutralizing antibody, significantly improve defense ability to a variant strain, and can be massively and industrially produced.

Description

Technical Field

[0001] The present disclosure belongs to the field of biotechnology, and specifically relates to a novel coronavirus SARS-CoV-2 mutant vaccine and application. Background Art

[0002] The new coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, 2019-nCoV, SARS-CoV-2) belongs to the subgenus Rotavirus of the Coronaviridae family and is the seventh known coronavirus that can infect humans. Coronaviruses are a class of enveloped RNA viruses. Animal coronaviruses rarely spread to humans, but some can cause serious illness in humans, such as the new coronavirus (SARS-CoV) and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Infection with the virus can cause patients to experience symptoms such as fever, dry cough, and fatigue; some patients will develop acute respiratory distress syndrome, septic shock, coagulation disorders, and multiple organ failure, and even death. The new coronavirus is composed of four structural proteins (spike protein, envelope protein, membrane protein, and nucleocapsid protein) and RNA nucleic acid chains. Among them, the spike protein (SpikeGlycoprotein, S protein) is located on the surface of the new coronavirus membrane and mainly acts on cell adhesion and cell membrane fusion. The S protein is composed of two subunits, S1 and S2. The S1 subunit contains the receptor binding domain (RBD), which is responsible for recognizing the host cell receptor ACE2. It is a key factor in the interaction between the virus and the receptor and the invasion of the virus into the cell, and is also a key target for vaccine design. The S2 subunit contains the basic elements required for the membrane fusion process and can promote the fusion of the virus and the host cell membrane.

[0003] Although the RNA polymerase of SARS-CoV-2 has a certain proofreading ability, the rapid mutation rate of the virus and its pandemic scale have further led to a large number of SARS-CoV-2 single-point random / escape evolution mutations. In particular, the S protein has undergone thousands of mutations, and variants containing multiple mutations have begun to appear. As of now, more than 3,120 variant sublineages have been derived worldwide (GISAID data). Since the outbreak of the Omicron variant on November 26, 2021, a large number of variants have appeared rapidly and simultaneously. Its early sublineage BA.1 replaced all previous VOC / VOI variants in just 1-2 months, but BA.2 evolved almost at the same time, and then was replaced by the BA.5 / BF.7 sublineage in just 2 months, while BQ.1.1, CH.1.1, and the newly generated XBB series of recombinant variant sublines were continuously generated. These variants show advantages over BA.5 / BF.7, and the rapid and simultaneous emergence of multiple variants with huge growth advantages is unprecedented. Although the evolutionary process of each variant is different, due to the influence of immunoblotting, the mutations on their RBD are concentrated on several common sites, including: R346, K356, K444, V445, G446, N450, L452, N460, F486, F490, R493 and S494. The combination of these different sites forms a variable "convergent mutation". The above high-frequency mutations are all generated to escape neutralizing antibodies in the body. After convergence, they will further obtain optimization capabilities such as improving ACE2 receptor affinity and enhancing S protein structural stability, which is crucial to the effect of vaccines and antibodies. Many studies have confirmed that the neutralizing efficacy of the main new crown therapeutic antibodies currently in clinical use or under development, such as Etesevimab, Bamlanivimab, REGEN-COV (casirivimab and imdevimab), Evusheld (cilgavimab and tixagevimab), Bebtelovimab and Xevudy (sotrovimab) is greatly reduced. There are signs that the XBB series of recombinant variants are antigenically more distant than SARS-CoV or some Sarbecoviruses in animals, so people with a three-shot inactivated immune background are simply not able to defend against Omicron.

[0004] Domestic and foreign regulations have also been adjusted simultaneously, and it is recommended to develop vaccines that can effectively deal with new variants. However, most of the commercially available / under-development vaccines are designed with wild-type full-length S protein antigens, which include NTD structures. Because the NTD domain is prone to produce enhanced NTD antibodies, it increases the infectivity of the new coronavirus, making it easy to infect other variants after vaccination with wild-type vaccines. With the implementation of the booster immunization strategy, the enhanced NTD antibodies will increase as the total antibody titer increases. In addition, breakthrough infections in early Omicron sublineages, especially BA.5, reduced the diversity of neutralizing antibody binding sites and increased the proportion of non-neutralizing antibodies, which in turn concentrated humoral immune pressure and promoted Omicron convergent evolution. Therefore, the current herd immunity and BA.4 / 5 vaccine booster shots may not be able to effectively prevent infection with Omicron recombinant variants. In this regard, using RBD as an antigen and taking into account the antigen design of convergent mutations may be a better strategy for vaccine design.

[0005] Based on the high variability of the Omicron variant and the fact that vaccine development requires a certain R&D and production cycle, it will be difficult to meet future needs by designing a vaccine targeting a single strain. Vaccines not only need to be effective against the current mainstream sub-lineages, such as BQ.1.1, CH.1.1, and XBB series recombinant variants, but also need to be effective against future new variants. Therefore, it is urgent to redesign a vaccine that can effectively fight against the new coronavirus variants. Summary of the invention

[0006] In order to avoid the limitations of existing vaccines, the present disclosure provides a fusion protein vaccine comprising interferon, a new coronavirus antigen and an immunoglobulin Fc region. The vaccine can enhance the immunogenicity of mutant new coronavirus antigens and the titer of neutralizing antibodies through the fusion-expressed IFN, thereby ensuring the efficient production of neutralizing antibodies, and can significantly enhance the defense capability against mutant strains (especially Omicron variants).

[0007] In one aspect, the present disclosure provides an antigen for a new coronavirus vaccine, which has at least one or more of the following amino acid substitutions relative to the RBD of the wild-type new coronavirus S protein shown in SEQ ID NO: 1: R28T, L50I, D87N, R90S, K99N, N122K, K126T, G128S, L134R, N142K, S159N, T160K, E166A, F168P or F168V, F172S, Q180R, N183Y and Y187H.

[0008] In one aspect, the present disclosure provides a fusion protein comprising:

[0009] (1) interferon or its functional fragments;

[0010] (2) Antigens for novel coronavirus vaccines; and

[0011] (3) Immunoglobulin Fc.

[0012] In another aspect, the present disclosure provides a nucleic acid encoding the above antigen or fusion protein.

[0013] In another aspect, the present disclosure provides a vector comprising the above nucleic acid.

[0014] On the other hand, the present disclosure provides a host cell expressing the above-mentioned antigen, fusion protein, comprising the above-mentioned nucleic acid and / or the above-mentioned vector.

[0015] In another aspect, the present disclosure provides a vaccine for treating and / or preventing infection by the novel coronavirus SARS-CoV-2 or the novel coronavirus disease COVID-19.

[0016] In another aspect, the present disclosure provides a vaccine combination product for treating or preventing diseases or conditions associated with SARS-CoV-2, wherein the vaccine combination product comprises a component selected from the following (1)-(3):

[0017] (1) Primer vaccines and booster vaccines, wherein the prime vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; the booster vaccine comprises the above-mentioned antigen, fusion protein, nucleic acid, vector, host cell, vaccine and / or vaccine combination product; optionally, the prime vaccine and booster vaccine independently further comprise one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants;

[0018] (2) a priming vaccine, a first boosting vaccine, and a second boosting vaccine, wherein the priming vaccine or the first boosting vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; the second boosting vaccine comprises the above-mentioned antigen, fusion protein, nucleic acid, vector, host cell, vaccine and / or vaccine combination product; optionally, the priming vaccine, the first boosting vaccine and / or the second boosting vaccine independently further comprise one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants; and

[0019] (3) a priming vaccine, a first booster vaccine, a second booster vaccine and a third booster vaccine, wherein the priming vaccine, the first booster vaccine and / or the second booster vaccine comprise any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; the third booster vaccine comprises the above-mentioned antigen, fusion protein, nucleic acid, vector, host cell, vaccine and / or vaccine combination product; optionally, the priming vaccine, the first booster vaccine, the second booster vaccine and / or the third booster vaccine independently further comprise one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants.

[0020] In some embodiments, the vaccine against wild-type SARS-CoV-2 or the vaccine against mutant SARS-CoV-2 is an inactivated vaccine, an mRNA vaccine, an adenovirus vaccine, a parental strain vaccine, or a mutant strain vaccine.

[0021] In one aspect, the present disclosure provides a method for preparing a vaccine or a vaccine combination product for preventing and / or treating novel coronavirus SARS-CoV-2 infection or coronavirus disease COVID-19.

[0022] In one aspect, the present disclosure provides a use of the above-mentioned fusion protein, nucleic acid, vector, host cell, vaccine and / or vaccine combination product in the preparation of a drug or product for preventing and / or treating novel coronavirus SARS-CoV-2 infection and / or novel coronavirus disease COVID-19.

[0023] In one aspect, the present disclosure provides a method of inducing a neutralizing antigen-specific immune response in an individual.

[0024] In one aspect, the present disclosure provides a method for rapid screening of engineered cell lines.

[0025] The Omicron broad-spectrum vaccine disclosed herein has the following advantages:

[0026] (1) Immunogenicity: The high immunogenicity of the variant vaccine comes from the selection of vaccine antigens. The RBD antigen design with a combination of highly immunogenic mutations can effectively stimulate the production of neutralizing antibodies to defend against viral invasion of the host.

[0027] (2) Broad-spectrum effectiveness: Combining the law of convergent evolution, an efficient convergent mutation combination is used as the antigen design, which simultaneously covers the different mutation site combinations of multiple mutant strains in one molecular structure, achieving broad-spectrum effectiveness against multiple Omicron strains.

[0028] (3) Large-scale production achievable: Omicron’s newly added mutation sites increase the polarity and hydrophobicity of the protein to varying degrees. The optimized combination of mutation sites can ensure high immunogenicity while achieving large-scale production achievable.

[0029] (4) Large-scale production platform process: After testing multiple variant vaccine strains, the optimized Omicron vaccine expression process can complete the screening of engineered cell lines in an extremely short time and achieve stable large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a summary of the mutation sites in the RBD region of representative SARS-CoV-2 strains.

[0031] Figure 2 This is a diagram showing the charge and hydrophobic surface analysis of the RBD domain of the parental strain and the mainstream subline of Omicron.

[0032] Figure 3 The structure and site diagram of the parental strain vaccine (V-01) and the Omicron strain vaccine (B5, QX, CX). Figure 3 a is a schematic diagram of the differential sites of the parental strain and Omicron strain vaccine; Figure 3 b is a diagram showing the charge and hydrophobic surface analysis of the RBD domain of the parental strain and Omicron vaccine.

[0033] Figure 4 The expression plasmid maps of the parent strain vaccine (V-01) and the Omicron strain vaccine (B5, QX, CX). Figure 4 a is the map of the parental strain vaccine (V-01); Figure 4 b is the map of Omicron strain vaccine (B5); Figure 4 c is the map of Omicron strain vaccine (QX); Figure 4 d is the map of Omicron strain vaccine (CX).

[0034] Figure 5 The results of identification of plasmid enzymes expressed by parental vaccine (V-01) and Omicron vaccine (B5, QX, CX). Among them, M1: DL15000 nucleic acid molecule marker; 1. Parental vaccine (V-01) HindIII single digestion; 2. Parental vaccine (V-01) HindIII / PacI double digestion; 3. Omicron vaccine (B5) HindIII single digestion; 4. Omicron vaccine (B5) HindIII / PacI double digestion; 5. Omicron vaccine (QX) HindIII single digestion; 6. Omicron vaccine (QX) HindIII / PacI double digestion; 7. Omicron vaccine (CX) HindIII single digestion; 8. Omicron vaccine (CX) HindIII / PacI double digestion; M2: DL10000 nucleic acid molecule marker.

[0035] Figure 6 The reactor results of the parent strain vaccine (V-01) and the Omicron strain vaccine (B5, QX, CX) expressed by the final process in Example 2 are shown in FIG. Figure 6 a is the growth trend expressed in tank culture (the left ordinate represents the density of living cells, and the right ordinate represents the cell viability); Figure 6 b shows the changing trend of the metabolite lactic acid in tank culture.

[0036] Figure 7 The expression levels of the parent strain vaccine (V-01) and the Omicron strain vaccine (B5, QX, CX) expressed by the final process in Example 2 and the purity of the purified monomers. Among them, 7a is the SEC-HPLC purity of the parent strain vaccine (V-01); 7b is the SEC-HPLC purity of the Omicron strain vaccine (B5); 7c is the SEC-HPLC purity of the Omicron strain vaccine (QX); 7d is the SEC-HPLC purity of the Omicron strain vaccine (CX)

[0037] Figure 8 The results of the binding activity of the parental strain vaccine (V-01) and the Omicron strain vaccine (B5, QX, CX) with ACE2 / IFNAR2 were determined based on the ELISA method. Figure 8 a Fitting curve of the binding activity of parental strain vaccine (V-01), Omicron strain vaccine (B5, QX, CX) and ACE2; Figure 8 b, Fitting curve of the binding activity of parental strain vaccine (V-01), Omicron strain vaccine (B5, QX, CX) and IFNAR2.

[0038] Fig. 9 The graph shows the biological activity of the IFNRα domain of the parental strain vaccine (V-01) and Omicron strain vaccine (B5, QX, CX) measured based on the indicator cell method.

[0039] Fig.10 To evaluate the in vivo efficacy of parental strain vaccine (V-01) and Omicron strain vaccine (B5, QX, CX) in mice.

[0040] Fig.11 To evaluate the neutralizing potency of the bivalent parental strain vaccine (V-01) and Omicron strain vaccine (B5, QX, CX) against the currently prevalent Omicron subtype mutant pseudovirus.

[0041] Fig.12 The neutralization potency of pseudovirus of bivalent vaccines was evaluated after two injections of inactivated vaccine followed by sequential immunization with parental strain vaccine (V-01) and Omicron strain vaccine (B5, QX, CX).

[0042] Fig.13 The neutralization potency of pseudovirus of bivalent vaccines of parental strain vaccine (V-01) and Omicron strain vaccine (QX, CX) was evaluated after two injections of inactivated vaccine followed by inoculation of B5 bivalent vaccine (simulating BA.5 infection) and then booster immunization.

[0043] Fig.14 To evaluate the immunogenic pseudovirus neutralization titer of the bivalent vaccine consisting of the parental strain vaccine (V-01) and the Omicron strain vaccine (B5, QX) in booster immunization of people aged 18 years and older who have completed 3 doses of inactivated vaccine and recovered for 3 months or more. DETAILED DESCRIPTION

[0044] I. Definitions

[0045] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0046] As used herein and unless otherwise specified, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0047] As used herein and unless otherwise specified, the terms "comprising", "including", "having", "containing", including grammatical equivalents thereof, should generally be understood as open and non-limiting, for example, not excluding other unlisted elements or steps.

[0048] The term "fusion protein" refers to a natural or synthetic molecule composed of one or more molecules, in which two or more peptide- or protein-based (including glycoprotein) molecules with different specificities are optionally fused together through chemical or amino acid-based linker molecules. The connection can be achieved by CN fusion or NC fusion (in 5'→3' direction), preferably CN fusion.

[0049] The term "interferon" (IFN) refers to a class of cytokines that are produced by the cell genome under the control of viral infection or other interferon inducers and have multiple biological activities such as antiviral, anti-tumor and immunomodulatory activities. Interferons can be divided into three major categories according to their biological and physical properties: type I, type II and type III interferons.

[0050] Type I interferons constitute a structurally related family (IFN-α (α), IFN-β (β), IFN-κ (κ), IFN-δ (δ), IFN-ε (ε), IFN-τ (τ), IFN-ω (ω), and IFN-ζ (ζ)), of which IFN-δ and IFN-τ do not occur in humans. Human type I interferon (IFN) genes are clustered on human chromosome 9p21, while mouse genes are located in a conserved colinear region on mouse chromosome 4. To date, 14 IFN-α genes and 3 pseudogenes have been identified in mice. In humans, 13 IFN-α (or IFNA) genes (IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17 and IFNA21) and 1 pseudogene have been identified, of which two human IFN-α genes (IFNA1 / IFN-α1 and IFNA13 / IFN-α13) encode for the same protein. All human type I interferons bind to cell surface receptors (IFNα receptors, IFNARs) composed of two transmembrane proteins (IFNAR-1 and IFNAR-2), which cause JAK-STAT activation, ISGF3 formation and subsequent gene expression. Interferon gamma (IFN-γ) is the only known type II interferon, which is mainly involved in inducing antibacterial and anti-tumor mechanisms through macrophage stimulation. The IFN-γ receptor (IFNGR) is a heterodimeric receptor composed of two ligand-binding IFNGR1 chains associated with two signal transducing IFNGR2 chains. Type III interferons consist of three subtypes and are also known as IFNλ (IFNλ1 or IL-29, IFNλ2 or IL-28A and IFNλ3 or IL-28B) and have antiviral, antitumor and immunomodulatory activities. The IFN-λ receptor is also a heterodimeric complex composed of the only ligand-binding chain IFN-λR1 (also designated IL-28Rα) and the side chain IL-10R2 shared with the receptor for the IL-10 related cytokine.

[0051] The term "coronavirus" belongs to the Coronaviridae family, Coronavirus genus, which can infect mammals and poultry, causing various diseases of the respiratory system, digestive system and central nervous system. Coronaviruses can be divided into four different genera based on genomic and serological differences: α, β, γ and δ. Currently, only α and β coronaviruses infect humans. So far, six human coronaviruses (HCoV) from two genera (α and β) have been identified. The α coronaviruses include NL63 and 229E, and the β coronaviruses include OC43, HKU1, acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and new coronavirus (SARS-CoV-2).

[0052] The term "antibody" or "immunoglobulin" has the broadest meaning, specifically including intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) composed of at least two intact antibodies, and antibody fragments, as long as they show the desired biological activity. This term generally includes hybrid antibodies composed of two or more antibodies or antibody fragments with different binding specificities linked together.

[0053] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including a native sequence Fc region and a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from position Cys226, or from an amino acid residue at Pro230 to the carboxyl end of the heavy chain. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody. Therefore, the composition of the complete antibody can include an antibody population from which all K447 residues have been removed, an antibody population from which no K447 residues have been removed, and an antibody population with a mixture of antibodies with and without the K447 residue.

[0054] Sequence "identity" or "identity" has a meaning recognized in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule. Although there are many methods for measuring the identity between two polynucleotides or polypeptides, the term "identity" is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).

[0055] The term "Th cell helper epitope" refers to all epitopes that activate helper T cells, including PADRE.

[0056] PADRE is a short peptide sequence of 13 amino acids that can bind to different DR molecules in humans and various animals, present on the cell surface, and then activate CD4+T helper cells to play an immunomodulatory role. PADRE's ability to induce T cell responses is more than 1,000 times that of natural epitopes, so PADRE has some characteristics as an immune adjuvant (PMID: 7895164). PADRE peptides can immunologically activate helper T cells (Th1) in the body to assist in the activation of CTLs, and can activate helper T cells (Th2) to assist B cells in secreting specific antibodies, thereby further enhancing the antigen immune response caused by the recombinant protein.

[0057] The term "treat" refers to any sign of success in treating or ameliorating an injury, disease, lesion or condition, including any objective or subjective parameter, such as alleviation; remission; attenuation of symptoms or making the injury, lesion or condition more tolerable to the patient; slowing the rate of degeneration or decline; making the degenerative endpoint less debilitating; improving the patient's physical or mental health. Treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of physical examinations, neuropsychiatric testing, and / or psychiatric assessments.

[0058] The term "effective amount" is an amount sufficient to achieve the stated purpose (e.g., to achieve the effect for which it is administered, to treat a disease, to reduce enzyme activity, to increase enzyme activity, to reduce protein function, to alleviate one or more symptoms of a disease or condition). An example of an "effective amount" is an amount sufficient to promote the treatment, prevention, or alleviation of one or more symptoms of a disease, which may also be referred to as a "therapeutically effective amount."

[0059] II. Detailed description of specific implementation scheme

[0060] In one aspect, the present disclosure provides an antigen for a new coronavirus vaccine, which has at least one or more of the following amino acid substitutions relative to the RBD of the wild-type new coronavirus S protein shown in SEQ ID NO: 1: R28T, L50I, D87N, R90S, K99N, N122K, K126T, G128S, L134R, N142K, S159N, T160K, E166A, F168P or F168V, F172S, Q180R, N183Y and Y187H.

[0061] In some embodiments, the above-mentioned antigen has at least the following mutations relative to the RBD of the wild-type novel coronavirus S protein shown in SEQ ID NO: 1: R28T, D87N, R90S, K99N, N122K, K126T, L134R, N142K, S159N, T160K, E166A, F168P or F168V, F172S, Q180R, N183Y and Y187H.

[0062] In some embodiments, the above-mentioned antigen has at least the following mutations relative to the RBD of the wild-type novel coronavirus S protein shown in SEQ ID NO: 1: L50I and G128S.

[0063] In some embodiments, the antigen has the sequence shown in SEQ ID NO:7 or 8.

[0064] In another aspect, the present disclosure provides a fusion protein comprising:

[0065] (1) interferon or its functional fragments;

[0066] (2) antigens of the above-mentioned novel coronavirus vaccines; and

[0067] (3) Immunoglobulin Fc region;

[0068] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: the interferon or a functional fragment thereof, the antigen, and the immunoglobulin Fc region;

[0069] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: the antigen, the interferon or a functional fragment thereof, and the immunoglobulin Fc region.

[0070] In some embodiments, the interferon is selected from type I interferon, type II interferon and / or type III interferon.

[0071] In some embodiments, the interferon mentioned above may be of human or mouse origin.

[0072] In some embodiments, the above-mentioned type I interferon is selected from IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω and IFN-ζ.

[0073] In some embodiments, the above-mentioned type II interferon is interferon gamma.

[0074] In some embodiments, the above-mentioned type III interferon is selected from IFN-λ1 (IL-29), IFN-λ2 (IL-28a) and IFN-λ (IL-28b).

[0075] In some embodiments, the interferon is selected from human IFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α13, IFN-α14, IFN-α16, IFN-α17 and IFN-α21;

[0076] More preferably, the above-mentioned interferon is IFN-α2a; preferably, the amino acid sequence of the above-mentioned IFN-α2a comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 18, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity; more preferably, the amino acid sequence of the above-mentioned IFN-α2a is as shown in SEQ ID NO: 18.

[0077] In some embodiments, the above-mentioned antigen comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO:1, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; more preferably, the amino acid sequence of the above-mentioned antigen is as shown in SEQ ID NO:7 or 8.

[0078] In some embodiments, the immunoglobulin Fc region is selected from the constant region amino acid sequences of IgG1, IgG2, IgG3 and / or IgG4.

[0079] In some embodiments, the immunoglobulin Fc region is an IgG1 Fc region; preferably, the IgG1 Fc region comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 19, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; more preferably, the amino acid sequence of the IgG1 Fc region is as shown in SEQ ID NO: 19.

[0080] In some embodiments, the above fusion protein further comprises one or more Th cell helper epitopes and / or connecting fragments.

[0081] In some embodiments, the Th cell helper epitope is PADRE or a derivative thereof; the amino acid sequence of the PADRE or a derivative thereof is selected from SEQ ID NO: 20, 21, 22 and 23; preferably, the amino acid sequence of the PADRE or a derivative thereof is as shown in SEQ ID NO: 20.

[0082] In some embodiments, the connecting fragment is a flexible polypeptide sequence; preferably, the amino acid sequence of the flexible polypeptide is selected from SEQ ID NO: 24 and SEQ ID NO: 25.

[0083] In some embodiments, the fusion protein comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO:14 or SEQ ID NO:16, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; more preferably, the amino acid sequence of the fusion protein is selected from the amino acid sequence shown in SEQ ID NO:14 or SEQ ID NO:16.

[0084] In another aspect, the present disclosure provides a nucleic acid encoding the above antigen or fusion protein.

[0085] In some embodiments, the nucleic acid comprises a nucleotide sequence selected from the group consisting of a nucleotide sequence having 80% or more identity with the nucleotide sequence shown in SEQ ID NO: 15 or SEQ ID NO: 17, preferably a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably a nucleotide sequence having 98% or 99% or more identity; more preferably, the nucleic acid is selected from the group consisting of a nucleic acid shown in SEQ ID NO: 15 or SEQ ID NO: 17.

[0086] In another aspect, the present disclosure provides a vector comprising the above nucleic acid.

[0087] On the other hand, the present disclosure provides a host cell expressing the above antigen or fusion protein, comprising the above nucleic acid and / or comprising the above vector.

[0088] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0089] In some embodiments, the prokaryotic cell is a bacterial cell; preferably, the prokaryotic cell is an Escherichia coli cell.

[0090] In some embodiments, the eukaryotic cells are selected from yeast cells, insect cells and mammalian cells; preferably, the mammalian cells are selected from CHO, HEK293, SP2 / 0, BHK, C127, etc.; more preferably, the eukaryotic cells are CHO cells.

[0091] On the other hand, the present disclosure provides a vaccine for treating and / or preventing infection by the novel coronavirus SARS-CoV-2 or the novel coronavirus disease COVID-19, comprising the above-mentioned fusion protein, nucleic acid, vector and / or host cell, and optionally, one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants.

[0092] In some embodiments, the vaccine is in the form of an inactivated vaccine, an mRNA vaccine or an adenovirus vaccine; preferably, it is a recombinant protein subunit vaccine, a recombinant protein mRNA vaccine or a recombinant protein viral vector vaccine, and preferably, the viral vector vaccine is an adenovirus vector vaccine.

[0093] On the other hand, the present disclosure provides a vaccine combination product for treating or preventing diseases or conditions associated with SARS-CoV-2, wherein the vaccine combination product comprises at least the above-mentioned antigen, fusion protein, nucleic acid, vector, host cell and / or vaccine.

[0094] In some embodiments, the above vaccine combination product comprises a component selected from the following (1)-(3):

[0095] (1) Primer vaccines and booster vaccines, wherein the prime vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; the booster vaccine comprises the above-mentioned antigen, fusion protein, nucleic acid, vector, host cell, vaccine and / or vaccine combination product; optionally, the prime vaccine and booster vaccine independently further comprise one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants;

[0096] (2) a priming vaccine, a first boosting vaccine, and a second boosting vaccine, wherein the priming vaccine or the first boosting vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; the second boosting vaccine comprises the above-mentioned antigen, fusion protein, nucleic acid, vector, host cell, vaccine and / or vaccine combination product; optionally, the priming vaccine, the first boosting vaccine and / or the second boosting vaccine independently further comprise one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants; and

[0097] (3) a priming vaccine, a first booster vaccine, a second booster vaccine and a third booster vaccine, wherein the priming vaccine, the first booster vaccine and / or the second booster vaccine comprise any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; the third booster vaccine comprises the above-mentioned antigen, fusion protein, nucleic acid, vector, host cell, vaccine and / or vaccine combination product; optionally, the priming vaccine, the first booster vaccine, the second booster vaccine and / or the third booster vaccine independently further comprise one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants.

[0098] In some embodiments, the above-mentioned priming vaccine, first booster vaccine and / or second booster vaccine comprise a monovalent vaccine against wild-type SARS-CoV-2 or a vaccine against mutant SARS-CoV-2.

[0099] In some embodiments, the above-mentioned priming vaccine, first booster vaccine and / or second booster vaccine comprise a bivalent vaccine consisting of a vaccine against wild-type SARS-CoV-2 and a vaccine against mutant SARS-CoV-2.

[0100] In some embodiments, the above-mentioned priming vaccine, the first booster vaccine, and the second booster vaccine all comprise monovalent vaccines against wild-type SARS-CoV-2.

[0101] In some embodiments, the monovalent vaccine of the above-mentioned vaccine against wild-type SARS-CoV-2 is an inactivated new coronavirus vaccine; more preferably, it is an inactivated new coronavirus vaccine from Beijing Sinovac Biotech Co., Ltd. or an inactivated new coronavirus vaccine from Sinopharm Beijing Institute of Biological Products.

[0102] In some embodiments, the third booster vaccine comprises a bivalent vaccine consisting of a vaccine against wild-type SARS-CoV-2 and a vaccine against mutant SARS-CoV-2.

[0103] In some embodiments, the active ingredient of the above-mentioned vaccine against wild-type SARS-CoV-2 comprises a parent strain vaccine having an amino acid sequence as shown in SEQ ID NO:10.

[0104] In some embodiments, the active ingredients of the above-mentioned vaccine against mutant SARS-CoV-2 include the above-mentioned antigens, fusion proteins, nucleic acids, vectors, host cells, vaccines and / or vaccine combination products;

[0105] In some embodiments, the ratio of the vaccine against mutant SARS-CoV-2 and the vaccine against mutant SARS-CoV-2 in the above-mentioned bivalent vaccine is 1:1-3, preferably 1:1.

[0106] In some embodiments, the above-mentioned vaccine against wild-type SARS-CoV-2 or the vaccine against mutant SARS-CoV-2 is an inactivated vaccine, an mRNA vaccine or an adenovirus vaccine.

[0107] In some embodiments, the adjuvant is selected from aluminum hydroxide, aluminum phosphate, saponin such as Quil A, QS-21, GPI-0100, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion.

[0108] In some embodiments, the priming vaccine and / or boosting vaccine described above is in the form of a liquid, emulsion, solid, aerosol, mist or gas.

[0109] In some embodiments, the above-mentioned priming vaccine, first boosting vaccine, second boosting vaccine and / or third boosting vaccine are in the form of liquid, emulsion, solid, aerosol, mist or gas.

[0110] In another aspect, the present disclosure provides a kit of parts, which comprises components selected from the following (1)-(3), wherein the above components are provided in the form of separate administration:

[0111] (1) (i) a first container comprising a priming vaccine and one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants, wherein the priming vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; and (ii) a second container comprising a boosting vaccine and one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants; the boosting vaccine comprises the above-mentioned antigen, fusion protein, nucleic acid, vector, host cell, vaccine and / or vaccine combination product;

[0112] (2) (i) a first container comprising a priming vaccine and one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants, wherein the priming vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; (ii) a second container comprising a first boosting vaccine and one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants, wherein the first boosting vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; and (iii) a third container comprising a second boosting vaccine and a pharmaceutically acceptable carrier, diluent or excipient, wherein the second boosting vaccine comprises the above-mentioned antigen, fusion protein, nucleic acid, vector, host cell, vaccine and / or vaccine combination product; and

[0113] (3) (i) a first container comprising a priming vaccine and one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants, wherein the priming vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; (ii) a second container comprising a first boosting vaccine and one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants, wherein the first boosting vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; (iii) a third container comprising a second boosting vaccine and one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants, wherein the second boosting vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; (iv) a fourth container comprising a third boosting vaccine and one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants, wherein the third boosting vaccine comprises the above-mentioned antigen, fusion protein, nucleic acid, vector, host cell, vaccine and / or vaccine combination product.

[0114] On the other hand, the present disclosure provides a method for preparing a vaccine or a vaccine combination product for preventing and / or treating infection by the new coronavirus SARS-CoV-2 or the coronavirus disease COVID-19, the method comprising expressing the above-mentioned antigen or fusion protein.

[0115] On the other hand, the present disclosure provides a method for inducing an immune response against SARS-CoV-2 or preventing and / or treating a disease or condition associated with SARS-CoV-2 in a subject in need thereof, the method comprising at least administering to the subject a composition comprising the above-mentioned antigen, fusion protein, nucleic acid, vector, host cell, vaccine and / or vaccine combination product and / or complete kit; optionally, the above-mentioned fusion protein, nucleic acid, vector, host cell and / or vaccine further comprises a pharmaceutically acceptable carrier and / or excipient.

[0116] In some embodiments, the above method comprises a method selected from the following (1)-(3):

[0117] (1) (i) administering a priming vaccine to a subject; and (ii) administering a boosting vaccine to a subject, wherein the priming vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; the boosting vaccine comprises the above-mentioned antigen, fusion protein, nucleic acid, vector, host cell, vaccine and / or vaccine combination product and / or complete kit; optionally, the priming vaccine and / or the boosting vaccine independently further comprise one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants;

[0118] (2) (i) administering a priming vaccine to a subject; (ii) administering a first boosting vaccine to a subject; and (iii) administering a second boosting vaccine to a subject, wherein the priming vaccine and / or the first boosting vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; the second boosting vaccine comprises the above-mentioned antigen, fusion protein, nucleic acid, vector, host cell, vaccine and / or vaccine combination product and / or complete kit; optionally, the priming vaccine, the first boosting vaccine and / or the second boosting vaccine independently further comprise one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants; and

[0119] (3) (i) administering a priming vaccine to a subject; (ii) administering a first boosting vaccine to a subject; (iii) administering a second boosting vaccine to a subject, and (iv) administering a third boosting vaccine to a subject, wherein the priming vaccine, the first boosting vaccine and / or the second boosting vaccine comprise any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; the third boosting vaccine comprises the above-mentioned antigen, fusion protein, nucleic acid, vector, host cell, vaccine and / or vaccine combination product and / or complete set of kits; optionally, the priming vaccine, the first boosting vaccine, the second boosting vaccine and / or the third boosting vaccine independently further comprise one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants.

[0120] In some embodiments, the subject is a mammal or a bird.

[0121] In some embodiments, the subject is a human, cow, dog, cat, goat, sheep, pig, horse, turkey, duck, or chicken.

[0122] In some embodiments, in the method of (1), the booster vaccine is administered at an interval after the initial administration of the priming vaccine for immunization.

[0123] In some embodiments, the booster vaccine is administered approximately 1-10 weeks after the initial administration of the priming vaccine, preferably 2-3, 2-8, 3-8, 1-3, 2-5, 3-5, 5-8 weeks, and more preferably 2-3, 2-8, 2-5, 3-5 weeks.

[0124] In some embodiments, in the method (2), the first booster vaccine is first administered about 1-10 weeks after the initial administration of the priming vaccine, preferably 2-3, 2-8, 3-8, 1-3, 2-5, 3-5, 5-8 weeks, more preferably 2-3, 2-8, 2-5, 3-5 weeks; the second booster vaccine is administered again about 4-72 weeks after the initial administration of the priming vaccine, preferably 4-12, 6-52, 6-45, 6-30, 6-25, 8-52, 8-30, 8-25 weeks, more preferably 4-12, 6-25, 8-25 weeks. In some embodiments, the first booster vaccine is administered about 2-3 weeks after the initial administration of the priming vaccine, and the second booster vaccine is administered about 4-12 weeks after the initial administration of the priming vaccine.

[0125] In some embodiments, in method (3), the first booster vaccine is first administered about 1-10 weeks after the initial administration of the priming vaccine, preferably 2-3, 2-8, 3-8, 1-3, 2-5, 3-5, 5-8 weeks, more preferably 2-3, 2-8, 2-5, 3-5 weeks; the second booster vaccine is administered again about 4-72 weeks after the initial administration of the priming vaccine, preferably 4-12, 6-52, 6-45, 6-30, 6-25, 8-52, 8-30, 8-25 weeks, more preferably 4-12, 6-25, 8-25 weeks; the third booster vaccine is administered about 24-52 weeks after the second booster vaccine, preferably 24-32 weeks.

[0126] On the other hand, the present disclosure provides the use of the above-mentioned antigens, fusion proteins, nucleic acids, vectors, host cells, vaccines, vaccine combination products and / or complete kits in the preparation of a pharmaceutical composition for inducing an immune response against the new coronavirus SARS-CoV-2 in a subject in need thereof, or in the preparation of a pharmaceutical composition for preventing and / or treating a disease or condition associated with SARS-CoV-2.

[0127] In some embodiments, the above-mentioned disease or condition associated with SARS-CoV-2 is SARS-CoV-2 infection or COVID-19.

[0128] In another aspect, the present disclosure provides a culture medium for culturing cells expressing the above fusion protein, comprising Dynamis Medium, FeNa-EDTA, methionine sulfoximine (MSX) and NaCl.

[0129] In some embodiments, the concentration of FeNa-EDTA is 0.005-0.06 mM; preferably 0.02 mM.

[0130] In some embodiments, the concentration of methionine sulfoximine is 6-25 μM; preferably 6.25-12.5 μM.

[0131] In some embodiments, the concentration of sodium chloride is 25-45 mM; preferably 25-30 mM.

[0132] In another aspect, the present disclosure provides a method for prolonging cell culture time, comprising the following steps:

[0133] (1) Cultivating cells in a basal medium, wherein the basal medium comprises Dynamis Medium, FeNa-EDTA, methionine sulfoximine (MSX) and NaCl.

[0134] In some embodiments, the concentration of FeNa-EDTA is 0.005-0.06 mM; preferably 0.02 mM.

[0135] In some embodiments, the concentration of methionine sulfoximine is 6-25 μM; preferably 6.25-12.5 μM.

[0136] In some embodiments, the concentration of sodium chloride is 25-45 mM; preferably 25-30 mM;

[0137] (2) adding 3-8% / 3-8% / 5-10% / 5-10% / 3-8% feed medium on culture day 3 / 5 / 7 / 9 / 11, respectively, preferably 5% / 5% / 8% / 8% / 5% feed medium, wherein the feed medium is Advanced CHOFeed1; and

[0138] (3) On the fifth day of culture, the temperature is lowered to 25-35°C, preferably 31°C, until the end of the fed-batch culture.

[0139] In another aspect, the present disclosure provides a cell culture method for increasing the percentage of product protein monomers in cells, comprising the following steps:

[0140] (1) Cultivating cells in a basal medium, wherein the basal medium comprises the above-mentioned Dynamis Medium, FeNa-EDTA, methionine sulfoximine (MSX) and NaCl.

[0141] In some embodiments, the concentration of FeNa-EDTA is 0.005-0.06 mM; preferably 0.02 mM.

[0142] In some embodiments, the concentration of methionine sulfoximine is 6-25 μM; preferably 6.25-12.5 μM.

[0143] In some embodiments, the concentration of sodium chloride is 25-45 mM; preferably 25-30 mM;

[0144] (2) adding 3-8% / 3-8% / 5-10% / 5-10% / 3-8% feed medium on culture day 3 / 5 / 7 / 9 / 11, respectively, preferably 5% / 5% / 8% / 8% / 5% feed medium, wherein the feed medium is Advanced CHOFeed1; and

[0145] (3) On the fifth day of culture, the temperature is lowered to 25-35°C, preferably 31°C, until the end of the fed-batch culture.

[0146] In another aspect, the present disclosure provides a cell culture method for increasing the production of a product protein in cells, comprising the following steps:

[0147] (1) Sodium chloride is added to a basal medium, and cells are cultured in the basal medium supplemented with sodium chloride, wherein the basal medium comprises Dynamis Medium, FeNa-EDTA, methionine sulfoximine (MSX), and NaCl.

[0148] In some embodiments, the concentration of FeNa-EDTA is 0.005-0.06 mM; preferably 0.02 mM.

[0149] In some embodiments, the concentration of methionine sulfoximine is 6-25 μM; preferably 6.25-12.5 μM.

[0150] In some embodiments, the concentration of sodium chloride is 25-45 mM; preferably 25-30 mM;

[0151] (2) adding 3-8% / 3-8% / 5-10% / 5-10% / 3-8% feed medium on culture day 3 / 5 / 7 / 9 / 11, respectively, preferably 5% / 5% / 8% / 8% / 5% feed medium, wherein the feed medium is Advanced CHOFeed1; and

[0152] (3) On the fifth day of culture, the temperature is lowered to 25-35°C, preferably 31°C, until the end of the fed-batch culture.

[0153] In another aspect, the present disclosure provides a cell culture method comprising the following steps:

[0154] (1) Sodium chloride is added to a basal medium, and cells are cultured in the basal medium supplemented with sodium chloride, wherein the basal medium comprises Dynamis Medium, FeNa-EDTA, methionine sulfoximine (MSX), and NaCl.

[0155] In some embodiments, the concentration of FeNa-EDTA is 0.005-0.06 mM; preferably 0.02 mM.

[0156] In some embodiments, the concentration of methionine sulfoximine is 6-25 μM; preferably 6.25-12.5 μM.

[0157] In some embodiments, the concentration of sodium chloride is 25-45 mM; preferably 25-30 mM;

[0158] (2) on culture day 3 / 5 / 7 / 9 / 11, adding 3-8% / 3-8% / 5-10% / 5-10% / 3-8% feed medium, preferably 5% / 5% / 8% / 8% / 5% feed medium, wherein the feed medium is Advanced CHOFeed1;

[0159] (3) on the 5th day of culture, lowering the temperature to 25-35°C, preferably 31°C, until the 11th to 13th day or the cell viability is <60%; and

[0160] (4) When the sugar concentration is lower than 3 g / L, replenish it to at least 6 g / L;

[0161] In some embodiments, during the cell culture process, the parameters of the bioreactor are controlled: temperature is controlled at 25-40°C, carbon dioxide is controlled at 3%-8%, pH is controlled at 6.7-7.3, dissolved oxygen is controlled at 20-80%, and the rotation speed is controlled at 50-200 rpm.

[0162] In some preferred embodiments, the temperature is controlled at 37° C., the carbon dioxide is controlled at 5%, the pH is controlled at pH 7.0, the dissolved oxygen is controlled at 40%, and the rotation speed is controlled at 100 rpm.

[0163] In this article, "basic immunization vaccine" includes "priming vaccine" or "priming vaccine". In the "priming-boosting immunization" vaccination strategy, the process of completing the priming is regarded as completing the basic immunization. The basic immunization vaccine may include one or more doses of vaccine, for example, the basic immunization vaccine may be one dose, two doses or three doses. In the booster immunization after completing the basic immunization or the primary immunization, the "boosting vaccine" used in this article may include one or more doses of booster vaccine, for example, the booster vaccine may be one dose, two doses or more doses. In the complete "priming-boosting immunization" vaccination strategy, it may include, for example, (1) one dose of vaccine is given as a basic immunization vaccine or a primary immunization vaccine, followed by one dose of vaccine as a booster vaccine; (2) two doses of vaccine are given as a basic immunization vaccine or a primary immunization vaccine, followed by one dose of vaccine as a booster vaccine; or (3) one dose of vaccine is given as a basic immunization vaccine or a primary immunization vaccine, followed by two doses of vaccine as a booster vaccine.

[0164] The experiments disclosed in this paper prove that the above-mentioned vaccine can enhance the immunogenicity and titer of neutralizing antibodies against mutant new coronavirus antigens through the fusion-expressed IFN, ensure the efficient production of neutralizing antibodies, and significantly enhance the defense capability against mutant strains.

[0165] For purposes of clarity and concise description, features are described herein as part of the same or separate embodiments, however, it will be understood that the scope of the present disclosure may include embodiments having a combination of all or some of the described features.

[0166] It should be noted that the following examples are intended to facilitate understanding of the present invention and have no limiting effect. In the examples, if specific conditions are not specified, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer, and if the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0167] Example

[0168] Example 1: Omicron protein vaccine design based on protein structure calculation assistance

[0169] First, the RBD mutation sites of VOC / VOI (Variant of Interest (VOI) / Variant of Concem (VOC)) variants with strong escape ability were compared (such as Figure 1). The amino acid sequence of the wild-type novel coronavirus S protein is shown in SEQ ID NO: 9. Compared with the wild-type novel coronavirus S protein, the A1pha mutant strain has only one mutation, N501Y; the Beta / Gamma mutant strain has three mutations, K417N / T, E484K, and N501Y; the Delta mutant strain has two mutations, L452R and T478K; and the Mu mutant strain has three mutations, R346K, E484K, and N501Y. Omicron includes almost all VOC / VOIs in the above early stages, as well as other newly added immune escape sites. The main mutations are concentrated in G339D / H, R346K / T, L368I, S371L / F, S373P, S375F, T376A, D405H / N, R408S, K417N / T, N440K, K444T, V445P, G446S, and L45 2R / Q, N460K, S477N, T478K, E484A / K, F486V / S / P, F490S, Q493R / * (* indicates reverse mutation), G496S, Q498R, N501Y, Y505H and other sites are all immune escape sites, which can reduce the neutralizing antibody titer to varying degrees, resulting in a significant decrease or ineffectiveness of the efficacy of most neutralizing antibodies on the market / under development against Omicron. After high-frequency recombination and "convergent mutation" of the above sites, new strains with stronger escape ability are derived, such as: BA.4 / 5 is equivalent to BA.2+L452R+F486V+R493Q, BA.2.75 is equivalent to BA.2+D339H+G446S+N460K+R493Q, BQ.1.1 is equivalent to BA.4 / 5+D405N+K444T+N460K+S477N, CH.1.1 is equivalent to BA.2.75+K444T+L452R+F486S, BJ.1 and BM1.1.1 are recombined into XBB.1.5, etc. (as shown in Table 1). Due to the convergent evolution of Omicron, the convergent variants after convergent mutation show stronger escape ability, such as XBB.1.9.1 and XBB.1.16, but their mutation sites are highly overlapping. Therefore, using efficient convergent mutation combinations as antigen design and simultaneously covering different mutation site combinations of multiple mutant strains in one molecular structure is of great significance for constructing a broad-spectrum new coronavirus vaccine.

[0170] Table 1 Summary of high escape Omicron sublines and their origins

[0171] Strain name Source strain Date of discovery BA.1 BA.1 (B.1.1.529.1) November 2021 BA.2 BA.2 (B.1.1.529.2) January 2022 BA.2.75 BA.2 August 2022 BA.4.1.9 BA.4 August 2022 BA.5.1 BA.5 August 2022 BA.4.6 BA.4 September 2022 BJ.1 BA.2 September 2022 BA.2.3.20 BA.2 September 2022 BA.2.75.2 BA.2.75 September 2022 BA.2.75.6 BA.2.75 September 2022 BM.1.1.1 BA.2 September 2022 BF.7 BA.4 September 2022 BQ.1.1 BQ.1 October 2022 XBB BJ.1&BM1.1.1 October 2022 CH.1.1 BA.2.75 October 2022 XBB.1 XBB[BJ.1 / BM1.1.1] November 2022 BN.1 BA.2.75 November 2022 BF.11 BA.5.2 November 2022 XBF BA.5.2&CJ.1 December 2022 BR.2.1 BA.2.75 December 2022 XBB.1.5 XBB[BJ.1 / BM1.1.1] December 2022 BA.5.2.48 BA.5.2 January 2023 BF.7.14 BF.7 January 2023

[0172] According to the analysis of the GISAID big database, the strains with the strongest growth rate and escape ability are BQ.1.1, CH.1.1, and XBB.1.5. Based on the law of Omicron convergent evolution, the newly recombinant variants are very likely to be derived or recombined from the above high escape strains. Protein vaccine design should take into account both immunogenicity and process development. Therefore, this embodiment analyzes the charge polarity and hydrophobicity of each mutation point (as shown in Table 2) by using UCSF Chimera software to analyze the RBD domain of the preferred strain one by one. Among them, the hydrophobic coefficients of sites such as L368I, S371L / F, S375F, T376A, E484A, F486V, and N501Y are relatively high, especially S371L / F, S373P, S375F, and T376A sites are combined into large clusters of hydrophobic epitopes, which will significantly increase the aggregation after protein expression. Mutations of G339D / H, N440K, L452R, N460K, T478K, E484K, Q498R, and Y505H increase molecular polarity and have a greater impact on molecular aggregation. If the aggregation risk site is directly introduced for protein vaccine expression, the product protein aggregation leads to a decrease in the yield of industrial large-scale production. In addition, it will be difficult to meet the needs of future immune barrier establishment by designing a vaccine against a single strain. Therefore, the vaccine not only needs to be effective against the current mainstream subsystems, such as variants such as BQ.1.1, CH.1.1, and XBB.1.5, but it is key to be effective against new foreign mutants. The immunogenicity of each amino acid, the mutation rate of each site, and the effect on the spatial conformation should be combined to design a suitable site combination to simultaneously meet the requirements of high immunogenicity and industrial large-scale production of protein vaccines. According to the one-by-one comparison and analysis of high-frequency mutation sites, it was found that the evolution trend of the mutation site Omicron is to increase hydrophobicity and molecular polarity to stabilize the intermolecular stability of its RBD, so the selection of mutation sites should consider the influence of the site itself.

[0173] Table 2 Analysis of high-frequency mutation sites of representative strains with high Omicon escape

[0174]

[0175] "-" indicates no impact, "+" indicates risk

[0176] The epitope region (Receptor binding motif, RBM) where RBD binds to ACE2, namely RBD aa436-aa507, is the main stimulating region for the formation of receptor-directed neutralizing antibodies (RBM-mAb), and is also the central region of the important escape site of Omicron. The RBM epitope region is not suitable for modification. Therefore, all combination designs retain mutations in the RBM region to stimulate a wider spectrum of neutralizing antibodies. In addition, according to the serum test reported in the literature, it was found that BA.5+N460K escaped most of the neutralizing antibodies after combining with F486V, and it was found that BA.5+R346T+K444T+N460K was highly similar to BA.5+R346T+N417T+K444N+N460K, showing the strongest escape ability. Combined with the above conclusions, using efficient convergent mutation combinations as antigen design, and covering different mutation site combinations of multiple mutant strains in one molecular structure at the same time, is of great significance for the construction of a broad-spectrum new crown vaccine. Therefore, based on the influence of molecular conformational changes, receptor epitope clustering, improved immunogenicity after combination and other factors, differentiated candidate mutation combinations 2 to 8 of the wild-type S protein RBD (combination-1) and wild-type S protein RBD variants in the new crown vaccine were designed, as shown in Table 3 for details.

[0177] Table 3 Combinations of mutation sites of RBD or its variants of wild-type S protein in COVID-19 vaccines:

[0178]

[0179]

[0180] Example 2: Preparation and investigation of candidate mutation combinations

[0181] 2.1 Construction of recombinant protein vaccine expression vector

[0182] The vaccine of this embodiment is based on the structure of the parent strain vaccine (V-01), and the antigen is optimized to improve the immunogenicity and process developability of the Omicron strain. Therefore, the Omicron strain vaccine referred to in this embodiment is a fusion protein of IFN, the RBD domain of the S protein of the new coronavirus, and Fc, which comprises genetically fusing the C-terminus of IFN with RBD-Fc via a flexible linker (GGGGS)3-PADRE-flexible linker GSGSGS.

[0183] According to the differentially designed antigen mutation site combinations 1 to 8, full gene synthesis was performed respectively, the difference being the different point mutation combinations of the RBD antigen. The double restriction enzyme digestion was connected to the mammalian cell transient transfection expression vector pcDNA3.3 (purchased from: Invitrogen TM ) to express.

[0184] 2.2 Transient expression of recombinant protein vaccine

[0185] This embodiment uses a large-volume transient transfection scheme for expression, specifically using the ExpiCHO expression system (purchased from: Life technologies, item number: A29133), referring to the 7-day transient transfection process of the kit, and performing transient expression on a 1L scale for combinations 1 to 8. Under the same expression process, the same signal peptide and expression vector conditions, the parental strain vaccine (V-01) has an advantage over other combination vaccine protein expression levels. After clarification, the cell culture fluid is directly captured on a Protein A affinity chromatography column, and the fusion protein is eluted under pH 3.6 conditions to obtain a preliminary purified (hereinafter referred to as primary pure) fusion protein. After SEC-HPLC analysis, the results are shown in Table 4. The primary pure monomer content of combination-1 (parental strain vaccine (V-01)) is close to 80% and meets the requirements, while the purity of other vaccine proteins is low. Considering the purity results of combination-5, combination-7, and combination-8 that affect aggregation due to conformational changes is significantly better than combination-2, combination-3, combination-4, and combination-6 that do not consider the effect of conformational changes on aggregation.

[0186] Table 4 Effects of different candidate mutation combinations on expression level and purity

[0187] combination Transfection plasmid (μg) Expression level (mg / L) monomer(%) Polymer(%) Fragment (%) Combination-1 800 63.1 80.2 14.5 5.3 Combination-2 800 25.4 14.2 84.3 1.5 Combination-3 800 32.2 10.7 88.7 0.6 Combination-4 800 27.7 15.6 81.3 3.1 Combination-5 800 31.3 23.6 72.3 4.1 Combination 6 800 23.9 9.8 90.2 ND Combination-7 800 26.0 34.1 63.1 2.7 Combination-8 800 30.8 27.4 68.1 4.5

[0188] The design of protein vaccines should ensure that the product protein vaccine can stimulate higher and broader immunogenicity, while avoiding the aggregation risk introduced by the molecule itself and reducing the difficulty of process preparation. Therefore, combination-5, combination-7, and combination-8, which consider the effect of conformational changes on aggregation and have better transient expression purity results, were selected as more preferred Omicron strain vaccine combination molecules, named Omicron vaccines (B5, QX, CX), respectively. The molecular structure and mutation site combination schematic diagram are shown in the figure. Figure 3 As shown in a. The mutations in the RBD region of the B5 vaccine include: N440K, L452R, S477N, T478K, E484A, F486V, Q493R, Q498R, N501Y, Y505H; the mutations in the RBD region of the QX and CX vaccines include K444T, N460K, (R493Q), and F490S added on the basis of B5. Non-RBM, namely RBD aa319-aa435, among which the mutations in the non-RBM region of the B5 vaccine include: R346K, D405N, R408S, and K417N; the non-RBM mutations of the QX vaccine include R346T added on the basis of B5; the non-RBM mutations of the CX vaccine include L368I added on the basis of QX. The newly added epitopes formed by these mutations constitute the potential escape site region of Omicron, and the hydrophobicity of the relevant amino acids has not changed significantly ( Figure 3b). The high-frequency mutation sites in the non-RBM region of the Omicron mutant strain include G339D, S371L / F, S373P, S375F and T376A mutations. These mutations are outside the RBM epitope region and do not participate in the binding of RBD to ACE2. In addition, due to the addition of charges (G339D) and large hydrophobic regions (S371L / F, S373P, S375F and T376A) to the mutations, the aggregation risk introduced by the molecule itself. Therefore, in order to reduce the aggregation risk introduced by the molecule, the Omicron vaccine (B5, QX, CX) was designed with G339D, S371L / F, S373P, S375F and T376A reverse mutations, and it was found that a better monomer rate was shown in transient expression. Since the changes in the clustering of antigenic epitopes displayed after different combinations of mutation sites have an impact on antigenicity and process developability, this example compares the process developability and immunogenicity of three Omicron vaccines (B5, QX, and CX) with different site combinations based on the above differences in structure and antigenicity.

[0189] Example 3: Screening of cell processes for extending culture period and reducing protein aggregation

[0190] The inventors systematically compared and studied a series of practical issues that may affect the expression and preparation of Omicron vaccines, such as lactic acid production, protein aggregation, and yield reduction, which can control the expression of Omicron variant vaccines. Combining cell density and viability, and taking protein yield and purity as evaluation indicators, the inventors determined the optimal expression process using a pressurized culture process with the addition of FeNa-EDTA and NaCl.

[0191] 3.1 Construction and preparation of expression plasmid

[0192] The expression plasmids of the parent strain vaccine (V-01) and the Omicron strain vaccine (B5, QX, CX) were constructed, and the difference was the different point mutation combinations of the RBD antigen. All genes involved in this example were synthesized by whole gene synthesis, and then ligated to the mammalian cell stable expression vector pCGS3 (purchased from Sigma-Aldrich) for expression (such as Figure 4 , Figure 5 The plasmid was extracted using Omega's endotoxin-free plasmid extraction kit (Cat. No.: D6926-03B; purchased from Omega Bio-tek), linearized with endonuclease PvuI, and then extracted and recovered using an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) solution and 2 times the volume of anhydrous ethanol, and the concentration was adjusted to 800 ng / μl for stable cell line construction.

[0193] in:

[0194] The amino acid sequence of the parent strain vaccine (V-01) is shown in SEQ ID NO: 10, and the encoding nucleic acid sequence thereof is shown in SEQ ID NO: 11;

[0195] The amino acid sequence of the Omicron strain vaccine (B5) is shown in SEQ ID NO: 12, and the encoding nucleic acid sequence thereof is shown in SEQ ID NO: 13;

[0196] The amino acid sequence of the Omicron strain vaccine (QX) is shown in SEQ ID NO: 14, and the encoding nucleic acid sequence thereof is shown in SEQ ID NO: 15;

[0197] The amino acid sequence of the Omicron strain vaccine (CX) is shown in SEQ ID NO:16, and its encoding nucleic acid sequence is shown in SEQ ID NO:17.

[0198] The expression system is GS system. GS enzyme is an enzyme in the body that uses glutamate and ammonia as substrates to synthesize glutamine. The activity of GS enzyme can be selectively inhibited by methionine sulfoximine (MSX). CHO cells cannot grow in a medium without glutamine. The GS gene can be used as a screening marker to provide a selection pressure for cell growth under the action of methionine sulfoximine (MSX) at a high level sufficient to inhibit the synthesis of GS enzyme.

[0199] 3.2 Construction of stable expression cell lines

[0200] This embodiment uses LIFE's Freedom TM CHO-S TM (cGMP Banked, referred to as CHO-S) was used as the host to construct stable cell lines of the parental strain vaccine (V-01), Omicron strain vaccine (B5), Omicron strain vaccine (QX), and Omicron strain vaccine (CX).

[0201] (1) Electrotransfection:

[0202] Before transfection, adjust the density of CHO-S cells to 1.35 × 10 7cells / mL, take 3.75mL CHO-S cells and 250μL linearized vector and mix thoroughly, then carefully add to 4 electroporation cups, 800μL / electroporation cup, and incubate on ice for 5 minutes. Electroporation conditions: 0.4cm electroporation cup, square wave (voltage 280V; electroporation 20ms; pulse 2 times; interval 0.1 seconds), parallel electroporation of 4 systems. After electroporation, carefully transfer the cells of each system to 4mL Dynamis Medium (8mM L-Glutamine) culture medium, and culture at 37℃, 5.0% CO2 for 1-2 days to confirm whether the cells are expressed and whether the viability is normal.

[0203] (2)Planning:

[0204] According to the counting results of electroporated cells cultured for 1 to 2 days, the concentration of 0.5 to 0.7 × 10 6 Cell suspension was prepared at 10000 cells / mL, and incubated with anti-human IgG-Fc FITC antibody (purchased from Biolegend, catalog number 409310) for more than 30 minutes. Microfluidic single-cell sorting equipment was used to sort >10,000 positive cells / well, and plated on 96-well plates. The culture medium was: 50% CDCHO Medium + 50% Cloning Medium + 12.5μM MSX, 250μL / well, 12 plates, and cultured at 37°C and 5.0% CO2. Among them: CD CHO Medium was purchased from Gibco, catalog number 10743029; Cloning Medium was purchased from Sigma, catalog number C6366; methionine sulfoximine (MSX) was purchased from Aladdin, catalog number M111096.

[0205] (3) Minipool (MP) screening:

[0206] On the 15th to 20th day after plating, the confluence at the bottom of the plate was observed with the naked eye, and MPs with a confluence of 30% to 50% were selected and screened for yield in batches using the Protein A probe of ForteBio Octet (purchased from Sartorius, catalog number 185010). Top-96 MPs were selected and subcultured to 24-well plates for culture. After MPs were subcultured to 24-well plates for 3 days, the yield was measured, and the top-10 MPs were selected for direct monoclonal screening.

[0207] (4) Monoclonal screening:

[0208] Monoclonal isolation was performed using a microfluidic single cell sorting device. The Top-10 MP mixed suspension was plated at 1 cell / well in a 96-well plate. The plating medium was QuaMonoTM Plus CHO Cloning Medium (A+B), 200μL. In order to verify the monoclonal origin, a single-cell imager was used to track and scan the plate to capture the division process of the monoclonal "1→2→3 / 4" (i.e., the division process of 1 to 2 to 3 or 4 cells). Cells that met the trend within 2 times were marked as monoclonal. On the 14th day after plating, the confluence at the bottom of the plate was observed with the naked eye, and monoclones with a confluence of 30-50% were selected. The yield was screened in batches using the Protein A probe of ForteBio Octet (purchased from Sartorius, catalog number 185010), and the Top-200 monoclones were selected and subcultured to 24-well plates for culture. After 3 days of culture in 24-well plates, the culture was expanded to 6-well plates. After 3 days of culture in 6-well plates, the monoclones were subcultured to shake tubes for continued culture. After the cells were cultured in shake tubes for 2 to 3 generations and the cell viability was ≥ 95%, at least Top-96 monoclones were selected for cell cryopreservation.

[0209] (5) Clone yield and quality screening:

[0210] Fed-batch yield screening was performed on frozen monoclonal clones at the same time. The basic culture medium was Dynamis Medium + 1% anti-clumping agent, and the initial VCD (viable cell density) was 5×10 5 cells / mL, the initial culture volume was 20mL, and on the 3rd / 5th / 7th / 9th / 11th day after inoculation, 5% / 5% / 8% / 8% / 5% feed (Advanced CHO Feed1) was added, and the sugar concentration was adjusted to 6g / L. Among them: Dynamis Medium was purchased from Gibco, catalog number A2661501; anti-clumping agent was purchased from Gibco, catalog number 0010057DG; Advanced CHO Feed1 was purchased from Sigma, catalog number 24368C. Among the clones that completed the Fed-batch evaluation, the Top-48 clones with the best growth and expression were selected for quality evaluation. Finally, the parent strain vaccine (V-01) and Omicron strain vaccine (B5), Omicron strain vaccine (QX), and Omicron strain vaccine (CX) high-expressing cell line clones that met the requirements of growth, expression level, and quality were selected for vaccine production.

[0211] 3.3 Process screening for extending the culture cycle

[0212] This embodiment provides a cell culture process that can extend the production cycle of Omicron strain vaccine (B5), Omicron strain vaccine (QX), and Omicron strain vaccine (CX). The use of 12.5μM MSX pressurized culture process can directly solve the lactic acid problem introduced by the Omicron+L452R molecule, thereby optimizing the growth, maintenance, and product protein expression effects of CHO cells.

[0213] (1) Using the parent strain vaccine (V-01) cell process, it was found that Omicron strain vaccines (B5, QX, CX) also have lactic acid problems:

[0214] The frozen monoclones were selected for Fed-batch yield screening at the same time. The basic culture medium was: Dynamis Medium + 1% anti-clumping agent, and the initial VCD was 5×10 5 cells / ml, the initial culture volume was 20ml, and on the 3rd / 5th / 7th / 9th day after inoculation, 5% / 5% / 8% / 8% feed (Advanced CHO Feed1) were added, and the sugar concentration was adjusted to 6g / L. The parent strain vaccine (V-01) used the same process, and no abnormal lactate metabolism was found. The lactate concentration was always lower than 15mM during the culture period (RAPIDPoint 500 blood gas analyzer, Siemens), and the lactate was consumed after the temperature was lowered on Day-5. The lactate concentration dropped to 0 when the cell culture was completed on Day-13. In comparison, it was found that the culture system of Omicron strain vaccine (B5, QX, CX) all had abnormal lactate problems, and the concentration was far beyond the normal lactate range of 10-25mM during the culture process. After the temperature was lowered on Day-5, the cells could not metabolize lactate normally, resulting in a sharp drop in cell viability after Day-7 and incomplete expression (Table 5-7).

[0215] Table 5 Cell viability change trend (%)

[0216] Time (day) V-01 B5 QX CX 0 98.7 96.8 98.8 98.1 3 97.9 98 98.8 98.4 5 97.4 98.3 99.3 99 7 98.1 89 76.7 82.3 8 97.2 57.3 - - 9 94.2 - - - 11 92.1 - - -

[0217] Note: “-” indicates that the sample has been received but not tested

[0218] Table 6 Trend of viable cell density (×10 6 cells / ml)

[0219] Time (day) V-01 B5 QX CX 0 0.55 0.55 0.55 0.55 3 4.39 4.04 6.29 3.92 5 8.94 8.11 9.95 8.15 7 9.31 8.39 8.89 8.17 8 9.30 5.23 - - 9 10.8 - - - 11 10 - - -

[0220] Note: “-” indicates that the sample has been received but not tested

[0221] Table 7 Trend of lactic acid content in culture medium (mM)

[0222] Time (day) V-01 B5 QX CX 0 1.4 0.9 1.3 1.6 3 14.6 3.5 4.7 3.2 5 15.2 3.9 3.2 8.1 7 10.1 32.3 32.8 64.4 8 5.3 57.2 - - 9 2.5 - - - 11 1.4 - - -

[0223] Note: “-” indicates that the sample has been received but not tested

[0224] The only difference that causes the increase in lactic acid is the difference in the mutation site of the antigen RBD part, and some reports have been made, such as Zhang Y, Zhang T, Fang Y, et al. SARS-CoV-2 spike L452R mutation increases Omicron variant fusogenicity and infectivity as well as host glycolysis. Signal transduction and targeted therapy, 2022, 7(1): 1-3, which found that new coronavirus variants such as Delta, B.1, Omicron, and Omicron+L452R can cause cells infected with the new coronavirus (SARS-CoV-2) to produce more lactic acid, among which Omicron+L452R is the most significant, and further proposed that the mechanism of lactic acid production may be triggered by glycolysis. Therefore, the Omicron strain vaccine (B5, QX, CX) contains the L452R site, which is the key to causing the lactic acid problem. Since L452R is located in the RBM region, it is the main stimulating area for the formation of receptor-directed neutralizing antibodies (RBM-mAb) and is also the central area of ​​the important escape site of Omicron. This epitope area is not easy to modify, and the lactic acid problem needs to be solved through cell process optimization.

[0225] (2) Using pressurized process to control lactic acid metabolism:

[0226] Lactic acid is the main metabolite of CHO cell technology, mainly through glycolysis pathway, or other amino acid metabolism, such as glutamine. The production of lactic acid will reduce the pH value of the culture medium. In addition, since one glucose molecule can produce two lactic acid molecules, the osmotic pressure (osmolarity) in the culture medium will increase. Lactic acid accumulation itself will also affect the metabolism of cells. Specifically, if the lactic acid in the culture medium is higher than 30mM and cannot be metabolized to below 15mM after cooling, the cell process will not be able to be scaled up.

[0227] When the Omicron strain vaccine (B5, QX, CX) adopted the V-01 platform cell process, it was found that there were problems with lactic acid accumulation and consumption (Table 4), and lactic acid should be controlled in the initial stage. The development of variant vaccines should be based on the parent strain vaccine (V-01) process, and there should be no major process changes, and the product protein quality properties (mainly referring to the glycosylation level) should not change significantly. Considering the above points, the Omicron strain vaccine (B5, QX, CX) process can only select simple and easy methods, and should not interfere with the protein glycoform. This embodiment adopts the MSX pressurization method to successfully solve the lactic acid problem in the cell culture process stage of the Omicron strain vaccine (B5, QX, CX). V-01 adopts the original process, and the specific experimental steps and methods for expressing the Omicron strain vaccine (B5, QX, CX) are as follows:

[0228] (a) Resuscitate the cell lines with high expression of Omicron strain vaccine (B5, QX, CX), and amplify them 2-3 times until the viability is restored to more than 95%;

[0229] (b) 0.5×10 6 Cells / ml were inoculated, and Fed-batch expression was carried out at the same time. The basic culture medium was: DynamisMedium+12.5μM MSX+1% anti-clumping agent, and the initial VCD was 5×10 5 cells / ml, the initial culture volume was 20 ml, and on days 3 / 5 / 7 / 9 / 11 after inoculation, 5% / 5% / 8% / 8% / 5% feed (AdvancedCHO Feed 1) were added, respectively, and the sugar concentration was adjusted to the range of 6 g / L.

[0230] According to the steps shown above, 20 ml shake flask culture was carried out, and V-01 was expressed using the original process. From the actual results in Tables 8 to 10, it can be seen that the cell viability, viable cell density, and lactate metabolism level under MSX pressurized conditions were good, which is consistent with the performance of V-01 cell line using the platform process and can meet the requirements for scale-up.

[0231] Table 8 Cell viability change trend (%)

[0232] Time (day) V-01 B5 QX CX 0 98.7 96.8 98.8 98.1 3 98.5 98.5 98.6 97.8 5 97.2 98.4 98.3 98.4 7 98 98.7 98.6 98.4 9 95.9 97.6 98.1 97.8 11 94.7 96.1 97 97.8 13 93.1 94.3 95.8 96.4

[0233] Table 9 Trend of viable cell density changes (×10 6 cells / ml)

[0234] Time (day) V-01 B5 QX CX 0 0.55 0.55 0.55 0.55 3 5.32 3.77 3.13 2.87 5 8.48 7.1 5.75 6.07 7 8.14 7.15 5.91 6.85 9 8.03 7.05 5.57 6.04 11 9.37 8.26 6.94 7.56 13 8.36 7.57 6.06 6.90

[0235] Table 10 Trend of lactic acid content in culture medium (mM)

[0236] Time (day) V-01 B5 QX CX 0 - - - - 3 1.2 3.4 1.7 1.7 5 2.9 7.2 4.7 6.3 7 6.2 9.0 9.7 6.8 9 3.9 4.9 8.5 5.6 11 2.0 4.2 3.5 1.6 13 1.81 3.4 1.8 0.0

[0237] Therefore, under the MSX (12.5 μM) pressure condition, the Omicron strain vaccine (B5, QX, CX) cells can maintain a low lactate level, maintain a good cell state, and significantly prolong the culture time. Therefore, the 12.5 μM MSX pressure condition was selected as the basic culture process for the Omicron strain vaccine (B5, QX, CX).

[0238] (3) Preliminary protein purification and purity comparison

[0239] The cell culture fluid of the V-01 control and Omicron strain vaccines (B5, QX, CX) was directly applied to the Protein A affinity column for capture after clarification, and the primary pure fusion protein was eluted under pH 3.6 conditions. After SEC-HPLC analysis, the results are shown in Table 11. The primary pure monomer content of the parent strain vaccine (V-01) was close to 80%, which met the requirements, while the purity of the Omicron strain vaccines (B5, QX, CX) was low.

[0240] Table 11 Protein purity of primary pure product

[0241] sample monomer(%) Polymer(%) Fragment (%) V-01 79.9 19.6 0.5 B5 38.7 53.6 7.7 QX 35.3 59.4 5.3 CX 34.2 64.6 1.2

[0242] 3.4 Process screening for increasing monomer content

[0243] The present disclosure also provides a cell culture process that can increase the monomer content of the Omicron strain vaccine. The use of FeNa-EDTA and increased osmotic pressure culture process can further solve the protein aggregation problem introduced by the Omicron molecules, thereby optimizing the growth, maintenance, expression ability and product protein monomers of CHO cells.

[0244] (1) Cell technology suitable for Omicron strain vaccines

[0245] By comparing the various mutations, the hydrophobicity and polarity of different Omicron variants after convergent mutations continue to increase, resulting in a significant increase in molecular aggregation. Optimization should be based on the parental strain vaccine (V-01) process, and no major process changes should occur. First, the impact of the substances in the culture medium components should be analyzed. The cell anti-clumping agent (ACA) used in the basic culture process of Omicron strain vaccines (B5, QX, CX) is sodium dextran sulfate (DSS), a polyanionic derivative of dextran, which can significantly reduce the primary pure SEC-HPLC monomer content of Omicron strain vaccine product proteins. DDS can reduce cell aggregation, promote CHO cell growth, increase viability, and the purity and yield of recombinant proteins. It is the most widely used anti-clumping agent in CHO cell suspension culture. If DSS is removed, additional substitutes or culture medium adaptation steps are required to reduce other problems introduced by DSS dependence. Studies have found that the optimized process using FeNa-EDTA as a substitute and adding the N-1 adaptation step can significantly improve the monomer purity of different Omicron strain vaccines.

[0246] The revived Omicron strain vaccine (B5) high-expressing cell line was passaged 2-3 times and cultured at 0.5×10 6 Cells / ml were inoculated, and Fed-batch expression studies were carried out at the same time. The basal culture medium was: Dynamis Medium+12.5μM MSX, the initial culture volume was 20ml, and the conditions were changed from the N-1 stage to adapt to the changed process (as shown in Table 12). On the 3rd / 5th / 7th / 9th / 11th day after inoculation, 5% / 5% / 8% / 8% / 5% feed (Advanced CHO Feed1) were added respectively, and the sugar concentration was adjusted to the range of 6g / L, and the goods were collected on Day-13. In this example, FeNa-EDTA (purchased from Sigma, item number E6760) was added to the basal culture medium, and a comparative test was carried out in parallel with the original process conditions. The cell culture fluid was captured by a Protein A affinity chromatography column after clarification, and the primary pure fusion protein was obtained by elution under pH3.6 conditions, and SEC-HPLC analysis was performed. The results are shown in Table 12.

[0247] Table 12 Process growth and product quality of different anti-caking agent additions (B5)

[0248]

[0249] (2) Expression enhancement and process optimization

[0250] Since the basic process should not be changed significantly, optimization should be based on the basic culture medium composition. Studies have found that increasing the osmotic pressure (+30mM NaCl) can further increase the expression of Omicron strain vaccines (B5, QX, CX) without affecting the cell state and protein quality.

[0251] Resuscitation of Omicron strain vaccine (B5, QX, CX) high expression cell lines, after 2-3 passages, at 0.5×10 6 The cells / ml were inoculated, and the process listed in Condition-4 of Table 12 was used to conduct a small-scale shake flask study of the 30ml system. The initial culture volume was 20ml. On the 3rd / 5th / 7th / 9th / 11th day after inoculation, 5% / 5% / 8% / 8% / 5% of feed (Advanced CHOFeed1) was added respectively, and the sugar concentration was adjusted to the range of 6g / L, and the goods were received on Day-13. In this embodiment, 30mM sodium chloride NaCl (purchased from Sigma, item number F5879) was added on the basis of the full pressure basal medium, and comparative tests were performed respectively. The cell culture fluid was captured by a Protein A affinity chromatography column after clarification, and the primary pure fusion protein was eluted under pH3.6 conditions, and SEC-HPLC was performed for analysis, and the results are shown in Table 13. After detection and analysis, the addition of NaCl had no effect on the monomer content, and there was no obvious change in the product protein glycoform (not listed). The addition of 30mM NaCl to vaccine cells of different Omicron strains can increase the expression of product protein.

[0252] Table 13 Process growth and product quality at different osmotic pressures

[0253]

[0254] “-” means not added.

[0255] 3.5 Protein expression

[0256] For the expression of the parental strain vaccine (V-01) and the Omicron strain vaccine (B5, QX, CX), this example uses a 200 L tank culture scheme for protein expression.

[0257] (1) Resuscitation of V-01 and Omicron strain vaccine (B5, QX, CX) high-expressing cell lines, with 0.5×10 6 Cells / ml were inoculated and cultured 2-3 times until the viable cell density reached 4×10 6 More than cells / ml.

[0258] (2) 0.5×10 6The control parameters are set to be consistent with those of later reactors of 200L / 2000L or above, so that the quality of the expression product can be as consistent as possible with the commercial scale.

[0259] (3) The basal medium was Dynamis Medium + 12.5 μM MSX + 0.02 mM FeNa-EDTA + 30 mM-NaCl. On days 3 / 5 / 7 / 9 / 11 after inoculation, 5% / 5% / 8% / 8% / 5% feed (Advanced CHO Feed 1) was added respectively. The sugar concentration was lower than 3 g / L and supplemented to 6 g / L. The bioreactor was cultured under the following culture parameter conditions: 37°C, 5% CO2, pH 7.0, dissolved oxygen 40%, and rotation speed 100 rpm.

[0260] (4) On Day 5, the temperature was lowered to 31°C. On Days 11 to 13, or when the cell viability was <60%, the supernatant was collected for protein quantification and the next step of purification.

[0261] According to the above steps, the parent strain vaccine (V-01) and Omicron strain vaccine (B5, QX, CX) high expression cell lines were cultured in 200L tanks. The change trend of cell viability and viable cell density during expression culture was similar, and the high cell density and cell viability were always maintained ( Figure 6 a), lactate metabolism is normal during the culture process ( Figure 6 b) In this example, the reaction conditions are precisely controlled by a bioreactor, which is basically consistent with the control of later commercial-scale production, making the product quality more representative.

[0262] 3.6 Protein purification

[0263] The sample of the cell culture fluid was clarified and captured by Protein A affinity chromatography column. The primary pure fusion protein was eluted under pH 3.6 conditions, and then separated by mixed mode chromatography Diamond Mix-A. After ultrafiltration and dialysis with a 30kDa cutoff pore size, the pure product protein was obtained. The concentration was adjusted to 1 mg / ml for binding activity, animal experiments and preparation of bivalent vaccines. The results of SEC-HPLC analysis are as follows: Figure 7 As shown, the parental strain vaccine (V-01) ( Figure 7 a) With Omicron strain vaccines (B5, QX, CX) Figure 7 b, 7c, 7d) purity is ≥95%.

[0264] Example 4: Binding to ACE2 and IFNAR2

[0265] The enzyme-linked immunosorbent assay (ELISA) was used to test the binding ability of the parental strain vaccine (V-01) and the Omicron strain vaccine (B5, QX, CX), including the binding of the RBD domain of the SARS-CoV-2 S protein to the ACE2 receptor, and the binding of the IFNα domain to the IFNAR2 receptor. The details are as follows:

[0266] 4.1 ACE2 binding assay

[0267] Research method: Take recombinant human ACE2 protein, prepare it into 1.5μg / mL with PBS (10mM pH 7.4), add 100μL / well to the ELISA plate (Costar, Cat#:9018), seal it with plastic wrap, and place it at 5±3℃ overnight; wash the plate twice with PBST, 300μL / well, add blocking solution (PBST containing 1% skimmed milk powder) to the ELISA plate, and incubate it at room temperature for 1.5 hours; dilute the parent strain vaccine (V-01) and Omicron strain vaccine (B5, QX, CX) prepared in Example 2 to 200ng / mL, respectively, and perform a 2-fold gradient dilution, with a concentration range of 0.098ng / mL to 200ng / mL. Add 100 μL / well of the pre-diluted test solution to the sealed ELISA plate, shake and incubate at room temperature for 2 hours; wash the plate 4 times with detergent, dilute the goat anti-human IgGFc labeled with horseradish peroxidase to an appropriate concentration, add 100 μL / well to the ELISA plate, shake and incubate at room temperature for 1 hour; wash the plate with detergent, add TMB substrate solution at 100 μL / well, and place it at room temperature away from light for 30 minutes; add 50 μL / well of stop solution to terminate the reaction. Use an ELISA reader at 620 nm as the reference wavelength, measure the absorbance at a wavelength of 450 nm, record the measurement results, and use four-parameter fitting to obtain the half effective concentration EC 50 .

[0268] Results and Conclusions: The results are shown in Table 14 and Figure 8 a. Both the parent strain vaccine (V-01) and the Omicron strain vaccine (B5, QX, CX) can bind to ACE2, EC 50 They are 1.93ng / mL, 5.51ng / mL, 1.92ng / mL and 1.98ng / mL respectively, which are basically consistent with the binding ability of ACE2.

[0269] Table 14 Binding activity of parental strain vaccine (V-01) and Omicron strain vaccine (QX, CX) with ACE2

[0270] sample <![CDATA[EC 50 (ng / mL)]]> Parent strain vaccine (V-01) 1.93 Omicron strain vaccine (B5) 5.51 Omicron strain vaccine (QX) 1.92 Omicron strain vaccine (CX) 1.98

[0271] 4.2 IFNAR2 binding assay

[0272] Research method: Take recombinant human IFNAR2 protein, prepare it into 1.5μg / mL with PBS (10mM pH 7.4), add 100μL / well to an ELISA plate (Costar, Cat#:9018), seal it with plastic wrap, and place it at 5±3℃ overnight; wash the plate twice with PBST, 300μL / well, add blocking solution (PBST containing 1% skimmed milk powder) to the ELISA plate, and incubate it at room temperature for 1.5 hours; dilute the parent strain vaccine (V-01) and Omicron strain vaccine (B5, QX, CX) prepared in Example 2 to 500ng / mL respectively, and perform 2.3-fold gradient dilution, with a concentration range of 0.052ng / mL to 500ng / mL. Add 100 μL / well of the pre-diluted test solution to the sealed ELISA plate, shake and incubate at room temperature for 2 hours; wash the plate 4 times with detergent, dilute the goat anti-human IgG Fc labeled with horseradish peroxidase to an appropriate concentration, add 100 μL / well to the ELISA plate, shake and incubate at room temperature for 1 hour; wash the plate with detergent, add TMB substrate solution at 100 μL / well, and place it at room temperature away from light for 30 minutes; add 50 μL / well of stop solution to terminate the reaction. Use an ELISA reader at 620 nm as the reference wavelength, measure the absorbance at a wavelength of 450 nm, record the measurement results, and use four-parameter fitting to obtain the half effective concentration EC 50 .

[0273] Results and Conclusions: The results are shown in Tables 15 and Figure 8 b, Parental strain vaccine (V-01) and Omicron strain vaccine (B5, QX, CX) can bind to IFNAR2, EC 50 They were 10.50 ng / mL, 10.50 ng / mL, 7.86 ng / mL, and 7.57 ng / mL, respectively, which were basically consistent with the binding ability of IFNAR2.

[0274] Table 15 Binding activity of parental strain vaccine (V-01), Omicron strain vaccine (B5, QX, CX) and IFNAR2

[0275] sample <![CDATA[EC 50 (ng / mL)]]> Parent strain vaccine (V-01) 10.50 Omicron strain vaccine (B5) 10.50 Omicron strain vaccine (QX) 7.86 Omicron strain vaccine (CX) 7.57

[0276] Example 5: IFNα Cell Biological Activity

[0277] After the IFNα-2b in the structure of the parental strain vaccine (V-01) and the Omicron strain vaccine (B5, QX, CX) binds to the endogenous receptors IFNAR2 and IFNAR1 on the cell membrane, the interferon-stimulated response element can be activated through signal transduction to start the expression of luciferase. The expression amount is positively correlated with the biological activity of interferon. After adding cell lysate and luciferase substrate, the luminescence intensity is measured to measure its biological activity. Therefore, the cell activity of the IFNα domain in the structure of the Omicron strain vaccine (B5, QX, CX) and the parental strain vaccine (V-01) prepared in Example 2 was detected using interferon recombinant cells (reporter gene method). Specifically as follows:

[0278] Research methods: HEK293puroISRE-Luc was used to measure the culture medium (containing 2mmol / L L-glutamine, 1mmol / L sodium pyruvate, 1% Pen Strep, 10% FBS MEM (containing NEAA (Non-Essential Amino Acids): non-essential amino acids) Dilute the parent strain vaccine (V-01) and Omicron strain vaccine (QX, CX) to 500ng / mL (2×, final concentration is 250ng / mL), add to the second column of the dilution plate as the starting concentration, and dilute to the 11th concentration gradient according to 3.5 times in columns 3-8 and 6 times in columns 9-12. The concentrations are 500, 143, 40.8, 11.7, 3.33, 0.95, 0.27, 0.045, 0.0076, 0.0013, and 0.00021ng / mL, respectively. Add the test solution of each dilution gradient to a 96-well all-leukocyte plate at 50μL / well, with 3 replicates in each well. Collect HEK293puroISRE-Luc cells and adjust the cell density to 8×10 5 50 μL / well was added to the 96-well full leukocyte plate sample, i.e. 4×10 cells / mL. 4 100 μL / well of Bio-Glo luciferase reagent was added, and the reaction was carried out at 200-500 rpm at room temperature in the dark for 10-30 minutes. The chemiluminescence unit RLU value was detected by an enzyme reader, and the half effective concentration EC was obtained by four-parameter fitting analysis. 50 .

[0279] Results and Conclusions: The results are shown in Table 16 and Fig. 9The IFNα domains of the parental vaccine (V-01) and Omicron vaccine (B5, QX, CX) can activate the IFNα signaling pathway and have the expected cell biological activity. The EC 50 The biological activities of IFNα domains of Omicron strain vaccines (B5, QX, CX) were basically the same as those of the parent strain vaccine (V-01).

[0280] Table 16 Biological activity of IFNα domain of parental strain vaccine (V-01) and Omicron strain vaccine (B5, QX, CX)

[0281] sample <![CDATA[EC 50 (ng / mL)]]> Parent strain vaccine (V-01) 0.240 Omicron strain vaccine (B5) 0.621 Omicron strain vaccine (QX) 0.233 Omicron strain vaccine (CX) 0.261

[0282] Example 6: Evaluation of Basic Immunogenicity of Monovalent Vaccines

[0283] The in vivo efficacy evaluation of vaccines in animals is crucial, as it can directly reflect the immunogenicity of the vaccine and is directly related to the protection it produces. Therefore, C57BL / 6 mice were used to evaluate the in vivo efficacy of the parental strain vaccine (V-01) and the Omicron strain vaccine (B5, QX, CX). Due to the danger of the new coronavirus, it is very difficult to evaluate the attack with a real virus. Therefore, a pseudovirus method using a recombinant new coronavirus S protein, a VSV G-based skeleton for packaging, and a luciferase reporter gene was used for evaluation.

[0284] Research method: The parent strain vaccine (V-01) and Omicron strain vaccine (B5, QX, CX) prepared in Example 2 were used to immunize 6-8 week old C57BL / 6 mice. The vaccine concentration was 20 μg / ml. 0.1 mL was injected into the thigh muscle of each mouse each time. 10 mice were given to each group. Each mouse was immunized twice (on the 0th day and once on the 14th day). Blood was collected from the eye sockets to obtain serum 14 days after the second immunization, i.e. on the 28th day. The blood was allowed to stand at room temperature for coagulation, and then centrifuged at 4000 rpm, 2-8°C for 10 min. The supernatant was taken and inactivated in a 56°C water bath for 30 min.

[0285] Detection method: Take Omicron neutralizing antibody (purchased from Novozyme Biotechnology Co., Ltd., catalog number DD1604-01), first dilute it to 25μg / mL with detection culture medium (10% FBS DMEM) as a positive quality control (PC). Take the serum sample to be tested, dilute it with detection culture medium in a 96-well all-leukocyte plate to a concentration of 5% (i.e. 1:20) in the first well, and then dilute it with the positive quality control product at a ratio of 1:3, for a total of 8 dilution gradients, and the serum sample for each dilution gradient is 100μL / well. Thaw the pseudovirus at 4°C in advance, and dilute the BQ.1.1, XBB.1.5, and CH.1.1 pseudoviruses (purchased from Beijing Yunling Biology, catalog numbers 80126, 80135, and 80139, respectively) to 13000TCID with detection culture medium. 50 / mL, add 50μL / well of pseudovirus dilution to the serum dilution plate, the pseudovirus amount is 650TCID 50 / well, the initial serum dilution was 1:30. Simultaneously, a pseudovirus control (VC, without serum) and a cell control (CC, without serum and pseudovirus) were set up, and then the whole white blood cell plate was placed in a 37°C, 5% CO2 incubator for 1 to 2 hours. HEK293T-ACE2 cells (purchased from Jiman Bio, catalog number GM-C012968) were collected, resuspended and counted in the detection culture medium, and the cell concentration was adjusted to 2.5×10 5 Cells were added at 100 μL / well, i.e., 2.5×10 cells per well. 4 The whole white blood cell plate was placed in a 37°C, 5% CO2 incubator for 20-28 hours. Before the test, 150 μL / well supernatant was discarded, and then 100 μL / well luciferase detection reagent was added, and the liquid in the reaction well was repeatedly blown and aspirated 6-8 times with a multichannel pipette, and reacted at room temperature in the dark for 5 minutes. The chemiluminescence unit RLU value was read by an enzyme reader, and the inhibition rate was calculated according to the following formula: Inhibition rate (%) = [1-(RLU value of sample group-mean value of cell control CC) / (mean value of pseudovirus control VC-mean value of cell control CC)] × 100%.

[0286] The inhibition rate data results were imported into GraphPad Prism software (version 8.4.3), with the horizontal axis representing the logarithm of the dilution multiple (Log Titer) and the vertical axis representing the inhibition rate percentage (% Inhibition). Four-parameter fitting analysis was performed, and the software automatically calculated pNT 50 The value is rounded to the integer value, which is the pseudovirus neutralization titer.

[0287] Results and Conclusions: The results are shown in Tables 17 and Fig.10The immunogenicity of the parental strain vaccine (V-01) and the Omicron strain vaccine (B5) against BQ.1.1, XBB.1.5, and CH.1.1 strains were weak, but the neutralization titer of the serum after immunization with the Omicron strain vaccine (QX) against BQ.1.1 and XBB.1.5 was significantly improved. At the same time, the neutralization titer of the serum after immunization with the Omicron strain vaccine (CX) against XBB.1.5 and CH.1.1 was also significantly improved, which is consistent with the molecular theoretical design, indicating that the Omicron strain vaccine (QX) and the Omicron strain vaccine (CX) are the preferred vaccines for dealing with the current epidemic strains.

[0288] Table 17 Neutralization titer of monovalent vaccine basic immune pseudovirus

[0289]

[0290] Example 7: Evaluation of basic immunogenicity of bivalent vaccine

[0291] Since the neutralization titers of the monovalent Omicron strain vaccines (QX, CX) were relatively weak, the pseudovirus protection of the bivalent vaccines prepared from the parent strain vaccine (V-01) prepared in Example 2 and the Omicron strain vaccines (B5, QX, CX) was evaluated.

[0292] Research method: The parent strain vaccine (V-01) prepared in Example 2 and the Omicron strain vaccine (B5, QX, CX) were made into bivalent vaccines at a ratio of 1:1, and 6-8 week old C57BL / 6 mice were immunized. The vaccine concentration was 40 μg / ml, and 0.1 mL was injected into the thigh muscle of each mouse each time. 10 mice / group, each mouse was immunized twice (on the 0th day and the 14th day respectively), and the bivalent vaccine (V-01+B5) was injected in the same way and dose as a control. Blood was collected from the eye sockets on the 28th day after the second immunization for serum. After the blood was allowed to stand at room temperature for coagulation, it was centrifuged at 4000 rpm, 2-8°C for 10 minutes, and the supernatant was taken and inactivated in a 56°C water bath for 30 minutes.

[0293] The detection method is the same as in Example 6.

[0294] Results and Conclusions: The results are shown in Table 18 and Fig.11 Compared with the monovalent vaccine, the neutralization titer of XBB.1.5 was significantly improved after the bivalent vaccine (V-01+QX) and the bivalent vaccine (V-01+CX) were administered to mice twice, and the former also showed a high neutralizing activity against BQ.1.1, and the latter showed a high neutralizing activity against CH.1.1. For the current epidemic mutant strains, the bivalent vaccines (V-01+QX) and (V-01+CX) both showed advantages over the bivalent vaccine (V-01+B5).

[0295] Table 18 Bivalent vaccine basic immune pseudovirus neutralization titer

[0296]

[0297] Example 8: Sequential Immunogenicity Evaluation of Bivalent Vaccines

[0298] The immunogenicity of sequential booster shots of bivalent vaccines prepared from the parental strain vaccine (V-01) prepared in Example 2 and the Omicron strain vaccines (B5, QX, CX) were evaluated.

[0299] Research method: The parent strain vaccine (V-01) prepared in Example 2 and the Omicron strain vaccine (B5, QX, CX) were made into bivalent vaccines in a 1:1 ratio for sequential immunization studies. BALB / C mice aged 6 to 8 weeks were used and randomly divided into groups of 10 mice / group. Each mouse was given an intramuscular injection of inactivated vaccine twice on the 0th day and the 21st day, 1 μg / mouse each time. On the 91st day (i.e., 70 days between the three immunizations), the parent strain vaccine (V-01) and the bivalent vaccine were sequentially boosted. The V-01 immunization dose was 2 μg / mouse, and the bivalent vaccine immunization dose was 4 μg / mouse. Blood was collected from the eye sockets 14 days after the three immunizations, i.e., on the 105th day, serum was obtained. After the blood was allowed to stand at room temperature for coagulation, it was centrifuged at 4000 rpm, 2 to 8°C for 10 minutes, the supernatant was taken, and it was inactivated in a 56°C water bath for 30 minutes.

[0300] The detection method is the same as in Example 6.

[0301] Results and Conclusions: The results are shown in Tables 19 and Fig.12 The immunogenicity of the bivalent vaccine (V-01+QX) and the bivalent vaccine (V-01+CX) were significantly improved after sequential immunization. The neutralization titers of the serum after sequential immunization against XBB.1.5, BQ.1.1, and CH.1.1 were higher than those of the bivalent vaccine (V-01+B5), indicating that the bivalent vaccines (V-01+QX) and (V-01+CX) have broad-spectrum neutralizing activity.

[0302] Table 19 Bivalent vaccine sequential immunization pseudovirus neutralization titer

[0303]

[0304] Example 9: Evaluation of the immunogenicity of bivalent vaccines after sequential boosting simulating BA.5 infection

[0305] The pseudovirus protective capacity of bivalent vaccines prepared from the parental strain vaccine (V-01) prepared in Example 2 and Omicron strain vaccines (B5, QX, CX) was evaluated for sequential booster immunization after simulating BA.5 infection.

[0306] Research method: The parent strain vaccine (V-01) prepared in Example 2 and the Omicron strain vaccine (B5, QX, CX) were prepared into bivalent vaccines at a 1:1 ratio for sequential booster immunization studies after simulating BA.5 infection. BALB / C mice aged 6 to 8 weeks were used and randomly divided into groups of 10 mice. Each mouse was immunized twice with inactivated vaccine by intramuscular injection on day 0 and day 21, respectively, at 1 μg / mouse each time. On day 91 (i.e., 70 days between three immunizations), three immunizations of bivalent vaccine (V-01+B5) were sequentially boosted (simulating BA.5 infection). On day 162 (i.e., 71 days between four immunizations), bivalent vaccine (V-01+QX) or bivalent vaccine (V-01+CX) was given after simulating BA.5 infection and sequentially boosted. The immunization dose of bivalent vaccine was 4 μg / mouse. Blood was collected from the eye sockets for serum 14 days after the four immunizations, i.e., on day 176. The blood was allowed to stand at room temperature for coagulation, and then centrifuged at 4000 rpm, 2 to 8°C for 10 min. The supernatant was taken and inactivated in a 56°C water bath for 30 min.

[0307] The detection method is the same as in Example 6.

[0308] Results and Conclusions: The results are shown in Table 20 and Fig.13 After two injections of inactivated vaccine plus one injection of B5 bivalent vaccine (simulating BA.5 infection), sequential booster therapy with bivalent vaccine (V-01+QX) or bivalent vaccine (V-01+CX) greatly improved the neutralizing titers of BQ.1.1 and XBB.1.5, indicating that based on the current status of the immune population, there is great advantage in adding bivalent vaccines (V-01+QX) and (V-01+CX).

[0309] Table 20 Neutralization titer of pseudoviruses after sequential boosting of immunogenicity of bivalent vaccines simulating BA.5 infection

[0310]

[0311] Example 10: Evaluation of the immunogenicity of the booster bivalent vaccine in people aged 18 years and over who have completed 3 doses of inactivated vaccine and recovered 3 months or more

[0312] According to the domestic real-world COVID-19 vaccination statistics released by the China Centers for Disease Control and Prevention, the proportion of people who have completed 2 basic immunizations in China reached 96.7%, and the proportion of people who have completed 3 injections reached 92.6%. In order to evaluate the true effectiveness of this implementation vaccine, an evaluation of the effectiveness of the booster will be conducted based on the domestic immune background. The bivalent vaccine prepared by the parental strain vaccine (V-01) and the Omicron strain vaccine (B5, QX) prepared in Example 2 was evaluated for the neutralization efficacy of the enhanced immunogenic pseudovirus in people aged 18 years and over who have completed 3 doses of inactivated vaccination and are 3 months or more after Yangkang.

[0313] Research method: The parental strain vaccine (V-01) and the Omicron strain vaccine (B5, QX) prepared in Example 2 were prepared into bivalent vaccines in a 1:1 ratio for clinical research. 20 subjects aged 18 years and above who had completed 3 doses of inactivated vaccine and were 3 months or more after being healthy were screened and enrolled. The subjects were randomly vaccinated with 1 dose of the parental strain vaccine (V-01) and Omicron strain vaccine (QX) bivalent vaccine or the parental strain vaccine (V-01) and Omicron strain vaccine (B5) bivalent vaccine in a 1:1 ratio, with 10 cases in each group. 14 days after vaccination (D14), blood was collected and serum was separated for pseudovirus neutralizing antibody detection to evaluate the immunogenicity of the booster bivalent vaccine in people aged 18 years and above who had completed 3 doses of inactivated vaccine and were 3 months or more after being healthy.

[0314] The method of immunization with three doses of inactivated vaccine: the first immunization time is D0, the interval between the second immunization and the first immunization is about 1 month, and the interval between the third immunization and the second immunization is about 7 months; all three immunizations are injected with one dose of inactivated vaccine (Beijing Kexing or Sinopharm Group) into the deltoid muscle of the upper arm, with a dose of 0.5mL; the interval between completing the three doses of inactivated vaccine and infection is about 1 year, and then people aged 18 years and above who have completed the three doses of inactivated vaccine and have been healthy for 3 months or more will be screened, and divided into the experimental group and the control group with 10 cases in each group for the immunogenicity evaluation of the booster immunization bivalent vaccine. The method of this booster immunization is as follows:

[0315] ﹒ Experimental group (n=10): One dose of parental vaccine (V-01) and Omicron vaccine (QX) bivalent vaccine was injected into the deltoid muscle of the upper arm on D0, with a dose of 0.5mL (containing 10μg each of parental vaccine (V-01) / Omicron vaccine (QX)).

[0316] ﹒ Control group (n=10): One dose of parental vaccine (V-01) and Omicron vaccine (B5) bivalent vaccine was injected into the deltoid muscle of the upper arm on D0, with a dose of 0.5 mL (containing 10 μg each of parental vaccine (V-01) / Omicron vaccine (B5)).

[0317] Detection method: The serum was first inactivated in a 56°C water bath for 30 minutes. Take Omicron neutralizing antibody (purchased from Novozyme Biotechnology Co., Ltd., catalog number DD1604-01), first dilute it to 20μg / mL with detection culture medium, then dilute the inactivated human negative mixed serum with detection culture medium to a serum concentration of 30%, and finally mix the diluted Omicron neutralizing antibody and inactivated human negative mixed serum in equal volumes, then the neutralizing antibody concentration is 10μg / mL, and the serum concentration is 15%, as a positive quality control (PC). Take the serum sample to be tested, dilute it with detection culture medium in a 96-well all-leukocyte plate to a concentration of 15% in the first well, and then dilute it with the positive quality control product at a ratio of 1:3, with a total of 7 dilution gradients, and the serum sample of each dilution gradient is 100μL / well. Thaw the pseudovirus at 4°C in advance, and dilute the XBB.1.16 and XBB.1.5 pseudoviruses (purchased from Novozyme Biotechnology Co., Ltd., catalog numbers DD15105-03 and DD1597-03, respectively) to 13,000 TCID using the test culture medium. 50 / mL, add 50μL / well of pseudovirus dilution to the serum dilution plate, the pseudovirus amount is 650TCID 50 / well, the initial serum dilution was 1:10. Simultaneously, a pseudovirus control (VC, without serum) and a cell control (CC, without serum and pseudovirus) were set up, and then the whole white blood cell plate was placed in a 37°C, 5% CO2 incubator for 1 to 2 hours. HEK293T-ACE2 cells (purchased from Jiman Bio, catalog number GM-C012968) were collected, resuspended and counted in the detection culture medium, and the cell concentration was adjusted to 2.5×10 5 Cells were added at 100 μL / well, i.e., 2.5×10 cells per well. 4 The whole white blood cell plate was placed in a 37°C, 5% CO2 incubator for 20-28 hours. Before the test, 150 μL / well supernatant was discarded, and then 100 μL / well luciferase detection reagent was added, and the liquid in the reaction well was repeatedly blown and aspirated 6-8 times with a multichannel pipette, and reacted at room temperature in the dark for 5 minutes. The chemiluminescence unit RLU value was read by an enzyme reader, and the inhibition rate was calculated according to the following formula: Inhibition rate (%) = [1-(RLU value of sample group-mean value of cell control CC) / (mean value of pseudovirus control VC-mean value of cell control CC)] × 100%.

[0318] The inhibition rate data results were imported into GraphPad Prism software (version 8.4.3), with the horizontal axis representing the logarithm of the dilution multiple (Log Titer) and the vertical axis representing the inhibition rate percentage (% Inhibition). Four-parameter fitting analysis was performed, and the software automatically calculated pNT 50 The value is rounded to the integer value, which is the pseudovirus neutralization titer.

[0319] Results and Conclusions: The results are shown in Table 21 and Fig.14 Among people aged 18 years and above who have completed three doses of inactivated vaccination and have been healthy for 3 months or more, the sequential booster bivalent vaccine (V-01+QX) had a higher neutralizing titer against XBB.1.16 and XBB.1.5, which was significantly better than the booster bivalent vaccine (V-01+B5) (p<0.05).

[0320] Table 21 Immunogenicity of the pseudovirus neutralization titer of the booster bivalent vaccine in people aged 18 years and above who have received 3 doses of inactivated vaccine and have been healthy for 3 months or more

[0321] sample XBB.1.16 pseudovirus neutralizing antibody titer XBB.1.5 pseudovirus neutralizing antibody titer Bivalent vaccine (V-01+B5) plus 105 93 Bivalent vaccine (V-01+QX) plus 407 393 T-test p-value 0.0003 0.0022

Claims

1. An antigen for a new coronavirus vaccine, which antigen has at least one or more of the following amino acid substitutions relative to the RBD of the wild-type new coronavirus S protein shown in SEQ ID NO: 1: R28T, L50I, D87N, R90S, K99N, N122K, K126T, G128S, L134R, N142K, S159N, T160K, E166A, F168P or F168V, F172S, Q180R, N183Y and Y187H.

2. The antigen of claim 1, wherein the antigen has at least the following mutations relative to the RBD of the wild-type novel coronavirus S protein shown in SEQ ID NO: 1: R28T, D87N, R90S, K99N, N122K, K126T, L134R, N142K, S159N, T160K, E166A, F168P or F168V, F172S, Q180R, N183Y and Y187H.

3. The antigen of claim 2, wherein the antigen has at least the following mutations relative to the RBD of the wild-type novel coronavirus S protein shown in SEQ ID NO: 1: L50I and G128S; Preferably, the antigen has the sequence shown in SEQ ID NO: 7 or 8.

4. A fusion protein comprising: (1) interferon or its functional fragments; (2) an antigen of the novel coronavirus vaccine according to any one of claims 1 to 3; and (3) Immunoglobulin Fc region; Preferably, the fusion protein comprises, from N-terminus to C-terminus: the interferon or a functional fragment thereof, the antigen and the immunoglobulin Fc region; Preferably, the fusion protein comprises, from N-terminus to C-terminus: the antigen, the interferon or a functional fragment thereof and the immunoglobulin Fc region.

5. The fusion protein according to claim 4, wherein The interferon is selected from type I interferon, type II interferon and / or type III interferon; Preferably, the interferon may be of human or mouse origin; Preferably, the type I interferon is selected from IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω and IFN-ζ; Preferably, the type II interferon is interferon gamma; Preferably, the type III interferon is selected from IFN-λ1 (IL-29), IFN-λ2 (IL-28a) and IFN-λ (IL-28b); Preferably, the interferon is selected from human IFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α13, IFN-α14, IFN-α16, IFN-α17 and IFN-α21; More preferably, the interferon is IFN-α2a; preferably, the amino acid sequence of the IFN-α2a comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 18, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity; more preferably, the amino acid sequence of the IFN-α2a is as shown in SEQ ID NO:

18.

6. The fusion protein according to claim 4 or 5, wherein The antigen comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 1, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; more preferably, the amino acid sequence of the antigen is as shown in SEQ ID NO: 7 or 8.

7. The fusion protein according to any one of claims 4 to 6, wherein The immunoglobulin Fc region is selected from the constant region amino acid sequence of IgG1, IgG2, IgG3 and / or IgG4; Preferably, the immunoglobulin Fc region is the Fc region of IgG1; preferably, the IgG1 Fc region comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 19, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, more preferably an amino acid sequence that is 98% or more identical; more preferably, the amino acid sequence of the IgG1 Fc region is as shown in SEQ ID NO:

19.

8. The fusion protein according to any one of claims 4 to 7, wherein The fusion protein further comprises one or more Th cell helper epitopes and / or linker fragments; Preferably, the Th cell helper epitope is PADRE or a derivative thereof; the amino acid sequence of the PADRE or a derivative thereof is selected from SEQ ID NO: 20, 21, 22 and 23; preferably, the amino acid sequence of the PADRE or a derivative thereof is as shown in SEQ ID NO: 20; Preferably, the connecting fragment is a flexible polypeptide sequence; preferably, the amino acid sequence of the flexible polypeptide is selected from SEQ ID NO: 24 and SEQ ID NO:

25.

9. According to any one of claims 4 to 8, wherein The fusion protein comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO:14 or SEQ ID NO:16, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; more preferably, the amino acid sequence of the fusion protein is selected from the amino acid sequence shown in SEQ ID NO:14 or SEQ ID NO:

16.

10. A nucleic acid encoding the antigen according to any one of claims 1 to 3 or the fusion protein according to any one of claims 4 to 9; Preferably, the nucleic acid comprises a nucleotide sequence selected from the group consisting of nucleotides having 80% or more identity with the nucleotide sequence shown in SEQ ID NO: 15 or SEQ ID NO: 17, preferably a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably a nucleotide sequence having 98% or 99% or more identity; more preferably, the nucleic acid is selected from the group consisting of the nucleic acids shown in SEQ ID NO: 15 or SEQ ID NO:

17.

11. A vector comprising the nucleic acid of claim 10.

12. A host cell expressing the antigen according to any one of claims 1 to 3 or the fusion protein according to any one of claims 4 to 9, comprising the nucleic acid according to claim 10 and / or comprising the vector according to claim 11; Preferably, the host cell is a prokaryotic cell or a eukaryotic cell; Preferably, the prokaryotic cell is a bacterial cell; preferably, the prokaryotic cell is an Escherichia coli cell; Preferably, the eukaryotic cell is selected from yeast cells, insect cells and mammalian cells; preferably, the mammalian cell is selected from CHO, HEK293, SP2 / 0, BHK, C127, etc.; more preferably, the eukaryotic cell is a CHO cell.

13. A vaccine for treating and / or preventing infection by the novel coronavirus SARS-CoV-2 or the novel coronavirus disease COVID-19, comprising an antigen according to any one of claims 1 to 3, a fusion protein according to any one of claims 4 to 9, a nucleic acid according to claim 10, a vector according to claim 11 and / or a host cell according to claim 12, and optionally, one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants; Preferably, the vaccine is in the form of an inactivated vaccine, an mRNA vaccine or an adenovirus vaccine; preferably, it is a recombinant protein subunit vaccine, a recombinant protein mRNA vaccine or a recombinant protein viral vector vaccine; preferably, the viral vector vaccine is an adenovirus vector vaccine.

14. A vaccine combination product for treating or preventing a disease or condition associated with SARS-CoV-2, the vaccine combination product comprising at least the antigen according to any one of claims 1 to 3, the fusion protein according to any one of claims 4 to 9, the nucleic acid according to claim 10, the vector according to claim 11 and / or the host cell according to claim 12 and / or the vaccine according to claim 13; Preferably, the vaccine combination product comprises components selected from the following (1)-(3): (1) Primer and booster vaccines, where: The priming vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; the boosting vaccine comprises the antigen according to any one of claims 1 to 3, the fusion protein according to any one of claims 4 to 9, the nucleic acid according to claim 10, the vector according to claim 11 and / or the host cell according to claim 12 and / or the vaccine according to claim 13; optionally, the priming vaccine and the boosting vaccine independently further comprise one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants; (2) a priming vaccine, a first boosting vaccine, and a second boosting vaccine, wherein the priming vaccine or the first boosting vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; the second boosting vaccine comprises the antigen according to any one of claims 1 to 3, the fusion protein according to any one of claims 4 to 9, the nucleic acid according to claim 10, the vector according to claim 11 and / or the host cell according to claim 12 and / or the vaccine according to claim 13; optionally, the priming vaccine, the first boosting vaccine and / or the second boosting vaccine independently further comprise one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants; and (3) a priming vaccine, a first boosting vaccine, a second boosting vaccine and a third boosting vaccine, wherein the priming vaccine, the first boosting vaccine and / or the second boosting vaccine comprise any one of a vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; the third boosting vaccine comprises the antigen according to any one of claims 1 to 3, the fusion protein according to any one of claims 4 to 9, the nucleic acid according to claim 10, the vector according to claim 11 and / or the host cell according to claim 12 and / or the vaccine according to claim 13; optionally, the priming vaccine, the first boosting vaccine, the second boosting vaccine and / or the third boosting vaccine independently further comprise one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants; Preferably, the priming vaccine, the first booster vaccine and / or the second booster vaccine comprise a monovalent vaccine against wild-type SARS-CoV-2 or a vaccine against mutant SARS-CoV-2; Preferably, the priming vaccine, the first booster vaccine and / or the second booster vaccine comprise a bivalent vaccine consisting of a vaccine against wild-type SARS-CoV-2 and a vaccine against mutant SARS-CoV-2; Preferably, the priming vaccine, the first booster vaccine and the second booster vaccine all comprise monovalent vaccines against wild-type SARS-CoV-2; Preferably, the monovalent vaccine against wild-type SARS-CoV-2 is an inactivated novel coronavirus vaccine; Preferably, the third booster vaccine comprises a bivalent vaccine consisting of a vaccine against wild-type SARS-CoV-2 and a vaccine against mutant SARS-CoV-2; Preferably, the active ingredient of the vaccine against wild-type SARS-CoV-2 comprises a parent strain vaccine having an amino acid sequence as shown in SEQ ID NO: 10; Preferably, the active ingredients of the vaccine against mutant SARS-CoV-2 comprise the antigen according to any one of claims 1 to 3, the fusion protein according to any one of claims 4 to 9, the nucleic acid according to claim 10, the vector according to claim 11 and / or the host cell according to claim 12 and / or the vaccine according to claim 13; Preferably, the ratio of the vaccine against wild-type SARS-CoV-2 and the vaccine against mutant SARS-CoV-2 in the bivalent vaccine is 1:1-3, preferably 1:1; Preferably, the vaccine against wild-type SARS-CoV-2 or the vaccine against mutant SARS-CoV-2 is an inactivated vaccine, an mRNA vaccine or an adenovirus vaccine; Preferably, the adjuvant is selected from aluminum hydroxide, aluminum phosphate, saponin such as Quil A, QS-21, GPI-0100, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; Preferably, the priming vaccine and / or booster vaccine is in the form of a liquid, emulsion, solid, aerosol, mist or gas; Preferably, the priming vaccine, the first boosting vaccine, the second boosting vaccine and / or the third boosting vaccine are in the form of a liquid, an emulsion, a solid, an aerosol, a mist or a gas.

15. A kit of parts comprising components selected from the following (1) to (3), wherein the components are provided in the form of separate administration: (1) (i) a first container comprising a priming vaccine and one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants, wherein the priming vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; and (ii) a second container comprising a boosting vaccine and one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants; the boosting vaccine comprises the antigen according to any one of claims 1 to 3, the fusion protein according to any one of claims 4 to 9, the nucleic acid according to claim 10, the vector according to claim 11 and / or the host cell according to claim 12 and / or the vaccine according to claim 13; (2) (i) a first container comprising a priming vaccine and one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants, wherein the priming vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; (ii) a second container comprising a first booster vaccine and one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants, wherein the first booster vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; and (iii) a third container comprising a second booster vaccine and a pharmaceutically acceptable carrier, diluent or excipient, wherein the second booster vaccine comprises the antigen according to any one of claims 1 to 3, the fusion protein according to any one of claims 4 to 9, the nucleic acid according to claim 10, the vector according to claim 11 and / or the host cell according to claim 12 and / or the vaccine according to claim 13; and (3) (i) a first container comprising a priming vaccine and one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants, wherein the priming vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; (ii) a second container comprising a first boosting vaccine and one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants, wherein the first boosting vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; (iii) a third container comprising a second boosting vaccine and one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants. More pharmaceutically acceptable carriers, excipients, diluents or adjuvants, the second booster vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; (iv) a fourth container comprising a third booster vaccine and one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants, the third booster vaccine comprising the antigen according to any one of claims 1 to 3, the fusion protein according to any one of claims 4 to 9, the nucleic acid according to claim 10, the vector according to claim 11 and / or the host cell according to claim 12 and / or the vaccine according to claim 13; Preferably, the priming vaccine, the first booster vaccine and / or the second booster vaccine comprise a monovalent vaccine against wild-type SARS-CoV-2 or a vaccine against mutant SARS-CoV-2; Preferably, the priming vaccine, the first booster vaccine and / or the second booster vaccine comprise a bivalent vaccine consisting of a vaccine against wild-type SARS-CoV-2 and a vaccine against mutant SARS-CoV-2; Preferably, the priming vaccine, the first booster vaccine and the second booster vaccine all comprise monovalent vaccines against wild-type SARS-CoV-2; Preferably, the monovalent vaccine against wild-type SARS-CoV-2 is an inactivated novel coronavirus vaccine; Preferably, the third booster vaccine comprises a bivalent vaccine consisting of a vaccine against wild-type SARS-CoV-2 and a vaccine against mutant SARS-CoV-2; Preferably, the active ingredient of the vaccine against wild-type SARS-CoV-2 comprises a parent strain vaccine having an amino acid sequence as shown in SEQ ID NO: 10; Preferably, the active ingredients of the vaccine against mutant SARS-CoV-2 comprise the antigen according to any one of claims 1 to 3, the fusion protein according to any one of claims 4 to 9, the nucleic acid according to claim 10, the vector according to claim 11 and / or the host cell according to claim 12 and / or the vaccine according to claim 13; Preferably, the ratio of the vaccine against wild-type SARS-CoV-2 and the vaccine against mutant SARS-CoV-2 in the bivalent vaccine is 1:1-3, preferably 1:

1.

16. A method for preparing a vaccine or a vaccine combination product for preventing and / or treating infection with the new coronavirus SARS-CoV-2 or coronavirus disease COVID-19, the method comprising expressing an antigen as described in any one of claims 1 to 3 or a fusion protein as described in any one of claims 4 to 9.

17. A method for inducing an immune response against SARS-CoV-2 in a subject in need thereof or preventing and / or treating a disease or condition associated with SARS-CoV-2, the method comprising administering to the subject at least an antigen according to any one of claims 1 to 3, a fusion protein according to any one of claims 4 to 9, a nucleic acid according to claim 10, a vector according to claim 11, a host cell according to claim 12, a vaccine according to claim 13, a vaccine combination product according to claim 14, and / or a kit according to claim 15; optionally, the fusion protein, nucleic acid, vector, host cell, and / or vaccine further comprises a pharmaceutically acceptable carrier and / or excipient; Preferably, the method comprises a method selected from the following (1)-(3): (1) (i) administering a priming vaccine to a subject; and (ii) administering a boosting vaccine to a subject, wherein The priming vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; the boosting vaccine comprises the antigen according to any one of claims 1 to 3, the fusion protein according to any one of claims 4 to 9, the nucleic acid according to claim 10, the vector according to claim 11, the host cell according to claim 12, the vaccine according to claim 13, the vaccine combination product according to claim 14 and / or the complete kit according to claim 15; optionally, the priming vaccine and / or the boosting vaccine independently further comprise one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants; (2) (i) administering a priming vaccine to a subject; (ii) administering a first boosting vaccine to a subject; and (iii) administering a second boosting vaccine to a subject, wherein the priming vaccine and / or the first boosting vaccine comprises any vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; the second boosting vaccine comprises the antigen of any one of claims 1 to 3, the fusion protein of any one of claims 4 to 9, the nucleic acid of claim 10, the vector of claim 11, the host cell of claim 12, the vaccine of claim 13, the vaccine combination product of claim 14 and / or the kit of parts of claim 15; optionally, the priming vaccine, the first boosting vaccine and / or the second boosting vaccine independently further comprise one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants; and (3) (i) administering a priming vaccine to a subject; (ii) administering a first boosting vaccine to a subject; (iii) administering a second boosting vaccine to a subject, and (iv) administering a third boosting vaccine to a subject, wherein the priming vaccine, the first boosting vaccine and / or the second boosting vaccine comprise any one of a vaccine against wild-type SARS-CoV-2 and / or a vaccine against mutant SARS-CoV-2; the third boosting vaccine comprises an antigen according to any one of claims 1 to 3, a fusion protein according to any one of claims 4 to 9, a nucleic acid according to claim 10, a vector according to claim 11, a host cell according to claim 12, a vaccine according to claim 13, a vaccine combination product according to claim 14, and / or a kit of parts according to claim 15; optionally, the priming vaccine, the first boosting vaccine, the second boosting vaccine and / or the third boosting vaccine independently further comprise one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants; Preferably, the priming vaccine, the first booster vaccine and / or the second booster vaccine comprise a monovalent vaccine against wild-type SARS-CoV-2 or a vaccine against mutant SARS-CoV-2; Preferably, the priming vaccine, the first booster vaccine and / or the second booster vaccine comprise a bivalent vaccine consisting of a vaccine against wild-type SARS-CoV-2 and a vaccine against mutant SARS-CoV-2; Preferably, the priming vaccine, the first booster vaccine and the second booster vaccine all comprise monovalent vaccines against wild-type SARS-CoV-2; Preferably, the monovalent vaccine against wild-type SARS-CoV-2 is an inactivated novel coronavirus vaccine; Preferably, the third booster vaccine comprises a bivalent vaccine consisting of a vaccine against wild-type SARS-CoV-2 and a vaccine against mutant SARS-CoV-2; Preferably, the active ingredient of the vaccine against wild-type SARS-CoV-2 comprises a parent strain vaccine having an amino acid sequence as shown in SEQ ID NO: 10; Preferably, the active ingredients of the vaccine against mutant SARS-CoV-2 comprise the antigen according to any one of claims 1 to 3, the fusion protein according to any one of claims 4 to 9, the nucleic acid according to claim 10, the vector according to claim 11 and / or the host cell according to claim 12 and / or the vaccine according to claim 13; Preferably, the ratio of the vaccine against wild-type SARS-CoV-2 and the vaccine against mutant SARS-CoV-2 in the bivalent vaccine is 1:1-3, preferably 1:1; Preferably, the vaccine against wild-type SARS-CoV-2 or the vaccine against mutant SARS-CoV-2 is an inactivated vaccine, an mRNA vaccine or an adenovirus vaccine; Preferably, the subject is a mammal or a bird; Preferably, the subject is a human, bovine, canine, feline, goat, sheep, pig, horse, turkey, duck or chicken.

18. The method of claim 14, wherein in the method of (1), the booster vaccine is administered at an interval after the initial administration of the priming vaccine for immunization; Preferably, the booster vaccine is administered approximately 1-10 weeks after the initial administration of the priming vaccine, preferably 2-3, 2-8, 3-8, 1-3, 2-5, 3-5, 5-8 weeks, more preferably 2-3, 2-8, 2-5, 3-5 weeks.

19. The method of claim 14, wherein in the method described in (2), the first booster vaccine is first administered about 1-10 weeks after the initial administration of the priming vaccine, preferably 2-3, 2-8, 3-8, 1-3, 2-5, 3-5, 5-8 weeks, more preferably 2-3, 2-8, 2-5, 3-5 weeks; the second booster vaccine is administered again about 4-72 weeks after the initial administration of the priming vaccine, preferably 4-12, 6-52, 6-45, 6-30, 6-25, 8-52, 8-30, 8-25 weeks, more preferably 4-12, 6-25, 8-25 weeks; More preferably, the first booster vaccine is administered approximately 2-3 weeks after the initial administration of the priming vaccine, and the second booster vaccine is administered approximately 4-12 weeks after the initial administration of the priming vaccine.

20. Use of the antigen according to any one of claims 1 to 3, the fusion protein according to any one of claims 4 to 9, the nucleic acid according to claim 10, the vector according to claim 11, the host cell according to claim 12, the vaccine according to claim 13, the vaccine combination product according to claim 14 and / or the kit according to claim 15 in the preparation of a pharmaceutical composition for inducing an immune response against the new coronavirus SARS-CoV-2 in a subject in need thereof or in the preparation of a pharmaceutical composition for preventing and / or treating a disease or condition associated with SARS-CoV-2; Preferably, the disease or condition associated with SARS-CoV-2 is SARS-CoV-2 infection or COVID-19.

21. A culture medium for culturing cells expressing the fusion protein according to any one of claims 4 to 9, comprising Dynamis Medium, FeNa-EDTA, methionine sulfoximine (MSX) and NaCl; Preferably, the concentration of FeNa-EDTA is 0.005-0.06 mM; preferably 0.02 mM; Preferably, the concentration of methionine sulfoximine is 6-25 μM; preferably 6.25-12.5 μM; Preferably, the concentration of sodium chloride is 25-45 mM; preferably 25-30 mM.

22. A method for prolonging cell culture time, comprising the following steps: (1) culturing cells in a basal medium, wherein the basal medium comprises Dynamis Medium, FeNa-EDTA, methionine sulfoximine (MSX) and NaCl; Preferably, the concentration of FeNa-EDTA is 0.005-0.06 mM; preferably 0.02 mM; Preferably, the concentration of methionine sulfoximine is 6-25 μM; preferably 6.25-12.5 μM; Preferably, the concentration of sodium chloride is 25-45 mM; preferably 25-30 mM; (2) adding 3-8% / 3-8% / 5-10% / 5-10% / 3-8% feed medium on culture day 3 / 5 / 7 / 9 / 11, respectively, preferably 5% / 5% / 8% / 8% / 5% feed medium, wherein the feed medium is Advanced CHO Feed 1; and (3) On the fifth day of culture, the temperature is lowered to 25-35°C, preferably 31°C, until the end of the fed-batch culture.

23. A cell culture method for increasing the percentage of product protein monomers in cells, comprising the following steps: (1) culturing cells in a basal medium, wherein the basal medium comprises Dynamis Medium, FeNa-EDTA, methionine sulfoximine (MSX) and NaCl; Preferably, the concentration of FeNa-EDTA is 0.005-0.06 mM; preferably 0.02 mM; Preferably, the concentration of methionine sulfoximine is 6-25 μM; preferably 6.25-12.5 μM; Preferably, the concentration of sodium chloride is 25-45 mM; preferably 25-30 mM; (2) adding 3-8% / 3-8% / 5-10% / 5-10% / 3-8% feed medium on culture day 3 / 5 / 7 / 9 / 11, respectively, preferably 5% / 5% / 8% / 8% / 5% feed medium, wherein the feed medium is Advanced CHO Feed 1; and (3) On the fifth day of culture, the temperature is lowered to 25-35°C, preferably 31°C, until the end of the fed-batch culture.

24. A cell culture method for increasing the yield of a cell product protein, comprising the following steps: (1) adding sodium chloride to a basal medium, and culturing the cells in the basal medium supplemented with sodium chloride, wherein the basal medium comprises Dynamis Medium, FeNa-EDTA, methionine sulfoximine (MSX), and NaCl; Preferably, the concentration of FeNa-EDTA is 0.005-0.06 mM; preferably 0.02 mM; Preferably, the concentration of methionine sulfoximine is 6-25 μM; preferably 6.25-12.5 μM; Preferably, the concentration of sodium chloride is 25-45 mM; preferably 25-30 mM; (2) adding 3-8% / 3-8% / 5-10% / 5-10% / 3-8% feed medium on culture day 3 / 5 / 7 / 9 / 11, respectively, preferably 5% / 5% / 8% / 8% / 5% feed medium, wherein the feed medium is Advanced CHO Feed 1; and (3) On the fifth day of culture, the temperature is lowered to 25-35°C, preferably 31°C, until the end of the fed-batch culture.

25. A cell culture method comprising the following steps: (1) adding sodium chloride to a basal medium, and culturing the cells in the basal medium supplemented with sodium chloride, wherein the basal medium comprises Dynamis Medium, FeNa-EDTA, methionine sulfoximine (MSX), and NaCl; Preferably, the concentration of FeNa-EDTA is 0.005-0.06 mM; preferably 0.02 mM; Preferably, the concentration of methionine sulfoximine is 6-25 μM; preferably 6.25-12.5 μM; Preferably, the concentration of sodium chloride is 25-45 mM; preferably 25-30 mM; (2) on culture day 3 / 5 / 7 / 9 / 11, adding 3-8% / 3-8% / 5-10% / 5-10% / 3-8% of feed medium, preferably 5% / 5% / 8% / 8% / 5% of feed medium, respectively, wherein the feed medium is Advanced CHO Feed 1; (3) on the 5th day of culture, lowering the temperature to 25-35°C, preferably 31°C, until the 11th to 13th day or the cell viability is <60%; and (4) When the sugar concentration is lower than 3 g / L, replenish it to at least 6 g / L; Preferably, during the cell culture process, the parameters of the bioreactor are controlled as follows: temperature is controlled at 25-40°C, carbon dioxide is controlled at 3%-8%, pH is controlled at 6.7-7.3, dissolved oxygen is controlled at 20-80%, and the rotation speed is controlled at 50-200 rpm; More preferably, the temperature is controlled at 37° C., the carbon dioxide is controlled at 5%, the pH is controlled at pH 7.0, the dissolved oxygen is controlled at 40%, and the rotation speed is controlled at 100 rpm.

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