Recombinant protein vaccine for preventing and treating SARS-cov-2 variant JN.1, ba.2.86, and XBB lineages, combination drug and use

Abstract

The present invention relates to a recombinant protein vaccine for preventing and treating SARS-CoV-2 variant JN.1, BA.2.86, and XBB lineages, a combination drug, and a use. To address significant immune escape and immune imprinting phenomena observed with Omicron JN.1, BA.2.86 and XBB lineage subvariants following prior vaccination or virus infection, an XBB.1.5 recombinant protein vaccine is provided. This vaccine elicits potent humoral and cellular immune responses against currently circulating JN.1, BA.2.86, and XBB lineage variants. Compared with homologous vaccination, heterologous vaccination with this vaccine following administration of an inactivated or mRNA-based vaccine achieves superior immune responses. Moreover, the vaccine induces effective protective immunity against live Omicron EG.5.1 virus attack in vivo. Therefore, the monovalent XBB.1.5 vaccine has clinical use value.

Description

Recombinant protein vaccines, combination drugs and uses for preventing and treating SARS-CoV-2 variants JN.1, BA.2.86 and XBB lineages Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to recombinant protein vaccines, combined drugs and uses for preventing and treating SARS-CoV-2 variants JN.1, BA.2.86 and XBB lineages. Background Art

[0002] Since the Omicron (B.1.1.529) BA.1 variant of severe acute respiratory coronavirus-2 (SARS-CoV-2) was first discovered in November 2021, the evolutionary trajectory of the virus has deviated significantly. Since then, Omicron has quickly become the dominant variant, spreading globally and producing a variety of descendant sub-variants, including well-known sub-variants such as BA.2, BA.5, and BQ.1.1, leading to widespread infection worldwide. With the prevalence of these sub-variants, the new BA.2.10.1-BA.2.75 recombinant XBB lineage with stronger immune escape ability appeared in early 2023, further leading to the emergence of various descendant sub-variants. It is worth noting that some XBB lineage strains, such as XBB.1.5 and XBB.1.16, have a rare amino acid mutation in the spike protein (F486P), which has spread rapidly in the past few months and become the dominant strain. Subsequently, two subvariants of XBB.1.5, EG.5 (F456L mutation) and EG.5.1 (F456L and Q52H mutations), spread rapidly around the world. In addition, other XBB series, such as FL.1.5.1, HK.3 and HV.1, have also sprung up around the world. In August 2023, a subvariant outside the XBB lineage, BA.2.86, was discovered for the first time. Compared with BA.2 and XBB.1.5, it has undergone more significant spike mutations, and its potential immune escape ability is worrying. Recently, scientists discovered a subtype evolved from BA.2.86, named JN.1, which has only one additional mutation (L455S) in the RBD.

[0003] Despite the World Health Organization (WHO) declaring the emergency phase of the COVID-19 pandemic over, the ongoing threat of reinfection with the novel coronavirus highlights the critical importance of widespread vaccination to reduce the associated risks of symptomatic infection, severe disease, and death. However, emerging JN.1 and XBB lineage subvariants have demonstrated significant immune evasion, significantly impairing neutralization in individuals who receive a booster dose or experience breakthrough infection with a previous XBB lineage subvariant. Although the bivalent vaccine (an approved mRNA vaccine containing spike protein sequences targeting the ancestral virus and Omicron BA.4 / 5) exhibits relatively high efficacy against several Omicron subvariants, neutralizing activity against XBB subvariants remains significantly reduced. Furthermore, the phenomenon of immunological imprinting must be considered during vaccine development; repeated prior antigen exposure may hinder neutralizing antibody responses to new Omicron variants. Experimental studies in animal models have confirmed the induction of vaccination-induced imprinting immunity, highlighting its relevance in human vaccine development. Furthermore, studies have shown that repeated vaccination in humans with wild-type spike mRNA or inactivated vaccines tends to bias the immune response toward earlier lineage variants, thereby reducing the immune response to newer Omicron subvariants. Similarly, bivalent vaccines only enhance the immune response to Omicron subvariants to a limited extent. These findings highlight the urgent need to reassess the composition of current vaccines and accelerate the development of next-generation COVID-19 vaccines that specifically target the evolving XBB lineage. However, to date, no recombinant protein vaccine has been developed that specifically targets the XBB lineage.

[0004] Summary of the Invention

[0005] Considering the global prevalence of Omicron JN.1, BA.2.86 and XBB lineage subvariants, which show breakthrough and significant immune escape after previous vaccination and viral infection, as well as the phenomenon of imprinting immunity, there is an urgent need to develop the next-generation novel coronavirus vaccine specifically targeting emerging subvariants.

[0006] To achieve the above application objectives, the technical solutions adopted in this application are as follows:

[0007] In a first aspect, the present invention provides the use of XBB.1.5 recombinant protein or vaccine in the preparation of a drug for preventing and / or treating infection by SARS-CoV-2 and its variants; the SARS-CoV-2 variants include Delta, Omicron variants and subtypes thereof; the Omicron variants and subtypes thereof are selected from JN.1, BA.2.86 and XBB lineages.

[0008] Furthermore, the XBB lineage includes at least one of XBB, XBB.1.5, XBB.1.6, XBB.1.9.1, XBB.1.16, XBB.1.16.6, XBB.2.3, EG.5.1, FL.1.5.1 or HV.1.

[0009] More preferably, the SARS-CoV-2 variants include at least one of Delta, BA.2.75, BA.5, BA.2, BA.5.2.48, BF.7, BQ.1, BQ.1.1, XBB, XBB.1.5, XBB.1.6, XBB.1.9.1, XBB.1.16, XBB.1.16.6, XBB.2.3, EG.5.1, FL.1.5.1, HV.1, BA.2.86 or JN.1.

[0010] In a second aspect, the present invention provides a composition or combination drug for preventing and / or treating infection with SARS-CoV-2 and its variants, which contains an mRNA vaccine against infection with SARS-CoV-2 or its variants and an XBB.1.5 recombinant protein vaccine administered separately, simultaneously or sequentially.

[0011] In a third aspect, the present invention provides a composition or combination drug for preventing and / or treating infection with SARS-CoV-2 and its variants, which contains an inactivated vaccine against infection with SARS-CoV-2 or its variants and an XBB.1.5 recombinant protein vaccine, which are administered separately, simultaneously or sequentially.

[0012] Furthermore, the XBB.1.5 recombinant protein or vaccine contains an amino acid sequence as shown in any one of SEQ ID No.2 and SEQ ID No.4.

[0013] Furthermore, the XBB.1.5 recombinant protein or vaccine contains a nucleotide sequence as shown in any one of SEQ ID No.1 and SEQ ID No.3.

[0014] Furthermore, the preparation method of the XBB.1.5 recombinant protein comprises the following steps: culturing host cells containing the XBB.1.5 recombinant protein or protein precursor to express the protein or precursor, and then recovering the protein.

[0015] Preferably, the host cell contains a recombinant vector containing a gene for translating the XBB.1.5 recombinant protein or precursor, and the recombinant vector is at least one of an insect baculovirus expression vector, a mammalian cell expression vector, an Escherichia coli expression vector, and a yeast expression vector.

[0016] Preferably, the insect baculovirus expression vector is pFastBac1. Preferably, the Escherichia coli expression vector is pET32a. Preferably, the yeast expression vector is pPICZaA. Preferably, the mammalian cell expression vector is a CHO cell expression vector. Further preferably, the CHO cell expression vector is pTT5 or FTP-002.

[0017] Furthermore, the host cell is at least one of insect cells, mammalian cells, Escherichia coli, and yeast.

[0018] Preferably, the insect cell is selected from at least one of sf9 cells, sf21 cells, and Hi5 cells. Preferably, the mammalian cell is a CHO cell.

[0019] Furthermore, the XBB.1.5 recombinant protein vaccine is obtained by mixing XBB.1.5 recombinant protein and pharmaceutically acceptable excipients or auxiliary ingredients; the amino acid sequence of the XBB.1.5 recombinant protein is shown in any one of SEQ ID No.2 and SEQ ID No.4.

[0020] Preferably, the auxiliary component is an immune adjuvant.

[0021] Further preferably, the immune adjuvant is selected from at least one of the following: squalene oil-in-water emulsion, aluminum salt, calcium salt, plant saponin, plant polysaccharide, monophosphoric acid lipid A, muramyl dipeptide, muramyl tripeptide, bacterial toxin, GM-CSF cytokine, lipid, cationic liposome material.

[0022] More preferably, the squalene oil-in-water emulsion is MF59; the aluminum salt is selected from at least one of aluminum hydroxide and alum; the calcium salt is tricalcium phosphate; the plant saponin is QS-21 or ISCOM; the plant polysaccharide is astragalus polysaccharide; the bacterial toxin is selected from at least one of recombinant cholera toxin and diphtheria toxin; the lipid is selected from at least one of the following: phosphatidylethanolamine, phosphatidylcholine, cholesterol, dioleoylphosphatidylethanolamine; the cationic liposome material is selected from at least one of the following: (2,3-dioleyloxypropyl)trimethylammonium chloride, N-[1-(2,3-dioleoyl chloride)propyl]-N,N,N-trimethylammonium chloride, cationic cholesterol, dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, dimethyldioctadecylammonium bromide, CpG ODN.

[0023] Furthermore, the XBB.1.5 recombinant protein vaccine is an injection preparation, a nasal spray preparation, or an oral preparation. Preferably, the vaccine is an intramuscular injection preparation.

[0024] In a fourth aspect, the present invention provides the use of the above-mentioned composition or combination drug in the preparation of a drug for preventing and / or treating infection by SARS-CoV-2 and its variants; the SARS-CoV-2 variants include Delta, Omicron variants and subtypes thereof; the Omicron variants and subtypes thereof are selected from JN.1, BA.2.86 and XBB lineages.

[0025] Furthermore, the XBB lineage includes at least one of XBB, XBB.1.5, XBB.1.6, XBB.1.9.1, XBB.1.16, XBB.1.16.6, XBB.2.3, EG.5.1, FL.1.5.1 or HV.1.

[0026] More preferably, the SARS-CoV-2 variants include at least one of Delta, BA.2.75, BA.5, BA.2, BA.5.2.48, BF.7, BQ.1, BQ.1.1, XBB, XBB.1.5, XBB.1.6, XBB.1.9.1, XBB.1.16, XBB.1.16.6, XBB.2.3, EG.5.1, FL.1.5.1, HV.1, BA.2.86 or JN.1.

[0027] Sequence information of the novel coronavirus XBB.1.5 recombinant protein vaccine of the present invention:

[0028] Nucleic acid sequence of the novel coronavirus XBB.1.5 recombinant protein: SEQ ID No.1

[0029] Amino acid sequence of the novel coronavirus XBB.1.5 recombinant protein: SEQ ID No. 2

[0030] SARS-CoV-2 XBB.1.5 recombinant protein vaccine antigen nucleic acid sequence: SEQ ID No.3

[0031] SARS-CoV-2 XBB.1.5 recombinant protein vaccine antigen protein sequence: SEQ ID No.4

[0032] Beneficial Effects: The present invention concatenates the receptor binding domain (RBD) sequence from the XBB.1.5 variant with a partial sequence from the spike S2 ​​subunit to develop a novel coronavirus XBB.1.5 recombinant protein vaccine. The recombinant protein (whose amino acid sequence is shown in SEQ ID No. 4) expressing the XBB.1.5 RBD and S2 sequences is expressed on an insect cell (Sf9) protein expression platform. An oil-in-water adjuvant similar to MF59 is incorporated to formulate the XBB.1.5 monovalent protein vaccine. Experiments have shown that intramuscular immunization with this vaccine (using SEQ ID No. 4 as the antigenic protein sequence) induces stable and sustained humoral and cellular immune responses against the currently prevalent JN.1, BA.2.86, and XBB lineage variants, particularly increasing the level of neutralizing antibodies against subvariant lineages including JN.1, and generating a large population of antigen-specific T memory cells. After vaccination, this vaccine can activate long-lasting immune efficacy, promote germinal center B cells (GCB) and follicular helper T cells (Tfh), increase memory B cells (MBC) and long-lived plasma cells (LLPC), and thus produce long-lasting virus neutralization ability.

[0033] Furthermore, heterologous vaccination with a monovalent recombinant vaccine following administration of an inactivated or mRNA-based vaccine resulted in a superior immune response compared to homologous vaccination. In vivo challenge experiments demonstrated that the SARS-CoV-2 XBB.1.5 recombinant protein vaccine effectively protected against infection with the live EG.5.1 strain. These findings suggest its promising clinical potential for preventing the currently circulating Omicron variant. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a molecular sieve and SDS-PAGE identification diagram of the recombinant protein of the new coronavirus XBB.1.5 in Example 1; M is the protein marker, and 1, 2, and 3 are the molecular weights of the target protein after removing all redundant amino acids;

[0035] Figure 2 is a graph showing the results of pseudovirus neutralizing antibodies in mice immune serum from the novel coronavirus XBB.1.5 recombinant protein vaccine in Example 4;

[0036] FIG3 is a diagram of specific cellular immunity detected by ELISpot after vaccine immunization in Example 5;

[0037] FIG4 is a graph showing the results of flow cytometry detection of specific cellular immunity in Example 6;

[0038] Figure 5 is a graph of true virus neutralizing antibodies in the immune serum of mice vaccinated with the novel coronavirus XBB.1.5 recombinant protein vaccine according to Example 7;

[0039] Figure 6 is a graph showing the long-term protective immunity induced by the novel coronavirus XBB.1.5 recombinant protein vaccine in Example 8;

[0040] Figure 7 is a graph showing the neutralizing antibody results of the heterologous sequential inactivated vaccine and mRNA vaccine of the novel coronavirus XBB.1.5 recombinant protein vaccine in Example 9;

[0041] Figure 8 is a graph showing the results of immunizing mice with the novel coronavirus XBB.1.5 recombinant protein vaccine in Example 10 with the live virus Omicron EG.5.1. DETAILED DESCRIPTION

[0042] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clear, the application is further described in detail below in conjunction with the embodiments. Unless otherwise defined, all scientific and technical terms used herein have the same meanings as understood by ordinary technicians in this field.

[0043] Abbreviations:

[0044] Monophosphoryl lipid A (MPL), squalene oil-in-water emulsion (MF59), recombinant cholera toxin (rCTB), astragalus polysaccharide (APS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), cholesterol (Chol), dioleoylphosphatidylethanolamine (DOPE), (2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP), N-[1-(2,3-dioleoyl chloride)propyl]-N,N,N-trimethylammonium chloride (DOTMA), cationic cholesterol (DC-Chol), dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate (DOSPA), trimethyldodecylammonium bromide (DTAB), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), dimethyldioctadecylammonium bromide (DDAB), CpG ODN (a nucleotide sequence containing unmethylated cytosine and guanine dinucleotides as the core sequence, a synthetic CpG).

[0045] First, regarding the currently available vaccines for the prevention and treatment of SARS-CoV-2 variants, including mRNA and inactivated vaccines, these vaccines have been shown to be effective in preventing symptomatic and severe infections. However, individuals with a history of vaccination and breakthrough infection showed significantly lower neutralizing activity, especially against the XBB family of the current novel coronavirus lineage. Although bivalent mRNA vaccines incorporating BA.4 / 5 spike protein sequences were rapidly produced, the neutralization capacity induced by these vaccines remains limited when faced with the XBB lineage. In addition, the phenomenon of immunoblotting introduces a risk that vaccines containing components of previous mutant strains may hinder neutralization responses against recent XBB subvariants.

[0046] Therefore, in one embodiment of the present invention, the present invention successfully expressed an RBD recombinant protein based on the XBB.1.5 mutation. This is the world's first recombinant protein vaccine targeting the XBB lineage. The results of the study showed that the XBB.1.5 recombinant protein vaccine with an adjuvant added to the present invention can be used as a stand-alone vaccine or as a booster vaccine to induce robust and sustained humoral and cellular immune responses against JN.1, BA.2.86 and XBB lineage submutants. In addition, the vaccine immunization can induce effective protective immunity against local infection with live EG.5.1 variants in the upper and lower respiratory tract. It is worth noting that the vaccine showed good safety and tolerability in humans and showed an excellent ability to induce strong neutralizing efficacy against the XBB lineage and the recent JN.1 submutant. It emphasizes its potential for emergency use as a booster injection against the Omicron variant in the clinic. It can be seen that the new coronavirus XBB.1.5 recombinant protein vaccine developed by the present invention showed excellent immunogenicity in animal models, thus showing good clinical prospects.

[0047] Secondly, although Moderna and Pfizer / BioNTech have updated the sequences of their mRNA vaccines to produce the monovalent XBB.1.5 spike mRNA vaccine, the XBB.1.5 monovalent mRNA vaccine booster can confer high levels of neutralizing antibodies against XBB and JN.1 subvariants. However, given the widespread global administration of mRNA-based COVID-19 vaccines, choosing heterologous vaccination with an alternative vaccine platform as a booster injection may elicit a more robust immune response than repeated homologous vaccination with an mRNA-based vaccine. In addition, mRNA-based COVID-19 vaccines have not yet been approved for use in mainland China.

[0048] Therefore, in one embodiment of the present invention, heterologous vaccination with a protein-based recombinant vaccine after three doses of an inactivated virus vaccine or an mRNA-based vaccine induced a superior immune response, characterized by elevated serum neutralizing antibody levels in an animal model. This suggests that the novel coronavirus XBB.1.5 recombinant protein vaccine of the present invention can serve as a safe and advantageous subunit protein-based booster vaccine, providing immune protection in this hybrid immune setting.

[0049] Specific examples will be listed below to explain the scheme of the present invention. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product specifications. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0050] Example 1: Expression of novel coronavirus XBB.1.5 recombinant protein using an insect baculovirus system

[0051] 1. Construction and design of the 2019-nCoV XBB.1.5 recombinant protein

[0052] The recombinant protein was constructed by concatenating the receptor binding domain (RBD) sequence from the XBB.1.5 variant with a partial sequence from the spike S2 ​​subunit, and expressed using a conventional Bac-to-Bac baculovirus expression system. Because the S protein of the SARS-CoV-2 coronavirus is membrane-localized, to ensure proper secretion, a GP67 signal peptide sequence was added to the N-terminus of the protein to facilitate secretory expression. This signal peptide is spontaneously cleaved by insect cells during secretion. A thioredoxin (Trx) tag from the fall armyworm (Spodoptera frugiperda, S. frugiperda) was added after the GP67 signal peptide to aid protein folding. A 6×his tag was added to facilitate subsequent purification, and an EK enzyme cleavage site was added to remove both the Trx and 6×his tags. Based on this protein design, EK enzyme cleavage will remove all redundant amino acids not relevant to the protein of interest.

[0053] 2. Preparation, Identification, and Purification of the SARS-CoV-2 XBB.1.5 Recombinant Protein

[0054] The above-mentioned gene (nucleotide sequence is shown in SEQ ID No.1, and amino acid sequence is shown in SEQ ID No.2) is amplified and integrated into the pFastBac1 vector. The verified plasmid is then transformed into Escherichia coli DH10b cells to produce recombinant bacmids. Next, the recombinant bacmids are transferred to Sf9 insect cells to express the protein. The protein is then cut by EK protease, purified by Superdex 200Increase 10 / 300GL column, and determined by SDS-Page, Coomassie Brilliant Blue staining and Western blotting. Figure 1 shows the molecular sieve and SDS-PAGE identification results of the new coronavirus XBB.1.5 recombinant protein, and a protein antigen with good purity that can be used for subsequent immune protection studies is obtained (nucleotide sequence is shown in SEQ ID No.3, and amino acid sequence is shown in SEQ ID No.4).

[0055] Example 2: Preparation of the novel coronavirus XBB.1.5 recombinant protein vaccine

[0056] The new coronavirus XBB.1.5 recombinant protein antigen prepared by the method of Example 1 (the nucleotide sequence is shown in SEQ ID No. 3, and the amino acid sequence is shown in SEQ ID No. 4) is mixed with equal volumes of squalene oil-in-water emulsion MF59-like adjuvant to prepare the new coronavirus XBB.1.5 recombinant protein vaccine, which is stored at 2-8°C.

[0057] Example 3: Vaccination of mice

[0058] SPF female NIH mice (6-8 weeks) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (China) and housed in the SPF animal facility of the National Key Laboratory of Biotherapy Animal Center (temperature: 21-25°C; humidity: 30-70%; dark / light cycle: 12h / 12h). All mouse experiments have been approved by the Institutional Animal Care and Use Committee of Sichuan University (Chengdu, Sichuan, China). The vaccine for mouse immunogenicity test was prepared as follows: the new coronavirus XBB.1.5 recombinant protein prepared in Example 1 was diluted to 200μg / ml with PBS, an equal volume of MF59-like water-in-oil adjuvant was added and mixed thoroughly. The mice were randomly divided into groups and immunized on days 0, 21 and 42 with an intramuscular injection of 100μl of the above-prepared XBB.1.5 recombinant protein vaccine, and serum and tissues were collected 8 weeks or 43 weeks after the last immunization.

[0059] Example 4: Detection of Neutralizing Antibodies in Serum Immunized with the XBB.1.5 Recombinant Protein Vaccine Using Pseudovirus Neutralization Method

[0060] SARS-CoV-2 pseudoviruses, including Prototype, Delta, BA.2.75, BA.5, BF.7, BQ.1, BQ.1.1, XBB, XBB.1.5, XBB.1.9.1, XBB.1.16, XBB.1.16.6, XBB.2.3, EG.5.1, FL.1.5.1, HV.1, BA.2.86, and JN.1, were provided by Genomeditech. Briefly, inactivated serum samples (56°C for 30 minutes) were diluted in threefold factors ranging from 30 to 65610 and then incubated with an equal volume of diluted pseudovirus at 37°C for 1 hour. Then, 1.2 × 10 4HEK-293T cells expressing human ACE2 receptors (293T / ACE2) were cultured and incubated at 37 ° C for 48 hours to express luciferase. Finally, the supernatant was removed, and a lysis reagent with luciferase substrate (Beyotime, RG005) was added, and the luminescence in 293T / ACE2 cells was measured using a multimode microplate reader (PerkinElmer, USA). The 50% neutralization rate of the pseudovirus was determined and calculated using GraphPad Prism 8.0.2. The positive control group contained only cells and viruses, the negative control group contained only cells, and the sample group contained cells, samples, and viruses. The neutralization percentage was calculated using the following formula: Neutralization (%) = (positive sample - sample to be tested / positive sample - negative sample) × 100%.

[0061] In order to evaluate the immunogenicity of the vaccine, NIH mice were immunized as described in Example 3 and samples were collected at week 8 to evaluate the immune response induced by vaccination. The immunogenicity of the monovalent vaccine was studied by pseudovirus neutralization assay (Fig. 2). The vaccine showed high neutralization efficacy against all XBB lineage subvariants. The 50% neutralization GMTs for XBB, XBB.1.5, XBB.1.9.1, XBB.1.16, XBB.1.16.6 and XBB.2.3 pseudoviruses were measured to be 21184, 56336, 39656, 20117, 22905 and 57620, respectively. The vaccine also showed effective neutralization against mutant pseudoviruses of the XBB lineage currently spreading globally, such as EG.5.1, FL.1.5.1 and HV.1. In addition, the vaccine induced cross-neutralization against other Omicron variants, including BA.2.75, BA.5, BF.7, BQ.1, and BQ.1.1, indicating that antigens from the XBB lineage induce a broad universal neutralizing response. Of particular note, the 50% neutralization GMTs of the recombinant protein vaccine against the BA.2.86 and JN.1 subvariants were 7546 and 8045, respectively, indicating that the SARS-CoV-2 XBB.1.5 recombinant protein vaccine can still effectively protect against infection with these subvariants.

[0062] Example 5: ELISpot assay to detect specific cellular immunity after immunization with the novel coronavirus XBB.1.5 recombinant protein vaccine

[0063] Given that the novel coronavirus XBB.1.5 recombinant protein vaccine induced a strong antibody response, the percentage of antibody-secreting cells (ASCs) and antigen-specific T cells in the bone marrow and spleen tissues of mice at week 8 after immunization were detected by ELISpot. NIH mice were immunized as described in Example 3, and mice were sacrificed and serum and tissues were collected at week 8 after the first immunization.

[0064] To detect the number of antibody-secreting cells (ASCs) in tissues, sterile 96-well ELISpot filter plates (Sigma, USA) were initially coated with 3 μg / mL novel coronavirus RBD protein at 4°C overnight and blocked with culture medium at 37°C for 2 hours. Subsequently, isolated lymphocytes from bone marrow and spleen tissues were added to the plates and incubated at 37°C overnight. The plates were washed with PBS the next day and incubated with HRP-conjugated goat anti-mouse IgG (1:10000) secondary antibody (Invitrogen, USA) at room temperature for 2 hours. After further washing, TMB ELISpot substrate solution (Mabtech, Sweden) was added to form spots. The reaction was terminated with rinse water. The spots were captured and counted using an auxiliary ELISpot reader.

[0065] To detect IFN-γ-secreting lymphocytes, a 96-well IFN-γ ELISpot plate (Catalog No.: 3420-4APT-2, MABTECH) was washed 4 times with PBS. Subsequently, 1640 complete medium (100 μl / well) was added to each well and incubated at room temperature for 1 hour. After removing the medium, the isolated spleen lymphocytes (2×10 5 / well) was added to the wells and stimulated with the Omicron (XBB.1.5) spike protein peptide library in a 37°C incubator overnight. After removing the cells, the plate was washed 5 times with PBS, and 100 μl of detection antibody (7-B6-1-biotin, 1 μg / ml) was added to each well and incubated at room temperature for 2 hours. After washing 5 times with PBS, the plate was treated with streptavidin-ALP (1:1000) and incubated at room temperature for 1 hour. The plate was washed 5 times and developed with a ready-to-use substrate solution (BCIP / NBT-plus). After obvious spots appeared, rinse with water to stop development. Finally, the IFN-γ ELISpot plate was scanned using an auxiliary ELISpot enzyme reader. Consistent with the results of the neutralization test, a large number of antigen-specific IgG ASCs were detected in the spleen and bone marrow of mice immunized with the vaccine (Figures 3a-b). In addition, when splenic lymphocytes were stimulated in vitro with a peptide library of the full-length spike protein of the XBB.1.5 variant, it was found that splenic lymphocytes of the vaccine-immunized group mice induced antigen-specific T cells (Figure 3c-d).

[0066] Example 6: Detection of specific cellular immunity by flow cytometry

[0067] NIH mice were immunized as described in Example 3. Eight weeks after the first immunization, mice were killed and lymphocytes from spleen tissue were isolated under sterile conditions and inoculated into 12-well plates (1×10 6The cells were plated in 1640 complete medium (10% FBS, 100 μg / ml streptomycin, 100 U / ml penicillin, 1 mM pyruvate, 50 μM β-mercaptoethanol, and 20 U / ml IL-2) and stimulated for 12 hours with a 1 μg / ml spike protein peptide pool from an XBB.1.5 variant. The cells were then treated with brefeldin A (BD Biosciences) for 6 hours before cell collection. The cells were stained for 30 minutes at 4°C with PerCP / Cyanine 5.5-labeled anti-mouse CD3 (Catalog No. 100218), FITC-labeled anti-mouse CD4 (Catalog No. 100406), APC-labeled anti-mouse CD8a (Catalog No. 100712), and BV510 anti-mouse / human CD44 (Catalog No. 103044). After washing three times with PBS, cells were fixed and permeabilized using a fixation / permeabilization kit (BD, catalog number: 554715) for 20 minutes at room temperature. Permeabilized cells were then stained with PE-conjugated anti-mouse IFN-γ (catalog number: 505808) and BV421 anti-mouse TNF-α (catalog number: 506328) for 1 hour at room temperature. Cells were analyzed using a NovoCyte flow cytometer (ACEA Biosciences) and NovoExpress 1.4.1 software.

[0068] To determine the GC B cells in the spleen and inguinal lymph nodes, cells were stained with PerCP / Cyanine5.5-conjugated anti-mouse CD3 (Catalog No.: 100218), BV605 anti-mouse CD19 (Catalog No.: 115540), PE / Cyanine7 anti-mouse CD95 (Catalog No.: 152618) and BV421 anti-mouse / human GL7 (Catalog No.: 144614) antibodies. To determine the percentage of Tfh cells in the spleen and lymph nodes, staining was performed using BV605 anti-mouse CD19, PerCP / Cyanine5.5-conjugated anti-mouse CD3, FITC-conjugated anti-mouse CD4, BV421 anti-mouse CXCR5 (Catalog No.: 356920) and PE anti-mouse PD-1 antibodies. The percentages of Tfh and GC B cells were detected and analyzed using a NovoCyte Advanteon flow cytometer equipped with NovoExpress 1.5.6 (Agilent).

[0069] As shown in Figure 4, the XBB.1.5 recombinant protein vaccine of the present invention can generate a strong cellular immune response, secreting IFN-γ antigen memory CD8 + and CD4 + T cells (Figure 4a) and secreted TNF-α antigen memory CD8 + and CD4 +The percentage of T cells (Figure 4b) increased significantly. Concurrently, a significant increase in GC B and Tfh cells was observed in the spleen and lymph nodes (Figures 4c-d).

[0070] Example 7: Neutralization test of live novel coronavirus

[0071] Neutralizing antibodies against live ancestral and mutant novel coronaviruses in serum samples from mice vaccinated with the novel coronavirus XBB.1.5 recombinant protein vaccine described in Example 3 were detected by real virus neutralization assay. Each diluted serum group was mixed with live novel coronavirus capable of infecting half of the cell culture (TCID50). After incubation at 37°C for 1 hour, the mixture was added to a plate covered with Vero E6 cells (5×10 4 The cells were incubated in 96-well microplates (100 μg / well) for 72 hours. The cytopathic effect (CPE) was measured under a microscope to evaluate the true virus neutralizing antibody titer of the serum.

[0072] Serum neutralizing antibody levels against several major circulating SARS-CoV-2 variants were measured. The monovalent vaccine induced robust neutralizing antibody responses against the XBB lineage, particularly against XBB.1.5 and EG.5.1 (Figure 5). Geometric mean titers (GMTs) against live XBB.1.5, XBB.1.16, and EG.5.1 reached 2580, 912, and 2580, respectively. GMTs against pre-XBB variants Delta and BA.5.2.48 were determined to be 256 and 1290, respectively.

[0073] Example 8: Long-term protective immunity induced by the SARS-CoV-2 XBB.1.5 recombinant protein vaccine

[0074] Long-lasting antibody responses are crucial for preventing repeated SARS-CoV-2 infection. Therefore, neutralizing antibodies induced by the SARS-CoV-2 XBB.1.5 recombinant protein vaccine were further evaluated using pseudovirus neutralization. Furthermore, antibody-secreting cells (ASCs) in the spleen and bone marrow of long-lasting immunized mice were further observed using ELISpot assays, and long-lived plasma cells (LLPCs) in the bone marrow, memory B cells (GC B) in the spleen and lymph nodes, and antibody-specific T cells in the spleen were detected by flow cytometry.

[0075] To detect LLPCs and MBCs, flow cytometry staining was performed using the following antibodies. LLPCs in the bone marrow were stained using FITC-conjugated anti-mouse CD44 (Catalog No. 147710), PE-conjugated anti-mouse IgD (Catalog No. 147710), BV605 anti-mouse CD138 (Catalog No. 142518), and BV605 anti-mouse CD19 (Catalog No. 115546). MBCs in the spleen and lymph nodes were detected using PerC P / Cyanine 5.5 anti-mouse CD3, BV605 anti-mouse CD19, PE / Cyanine 7 anti-mouse CD38 (Catalog No. 165612), and BV421 anti-mouse / human GL7 antibodies.

[0076] The results of the pseudovirus neutralization test are shown in Figure 6 (a, schematic diagram of the immunization procedure. NIH mice were intramuscularly injected with 100 μl of the recombinant XBB.1.5 protein vaccine prepared in Example 3 three times at 0, 3, and 6 weeks, respectively. The mice were killed 43 weeks after the first immunization and serum and tissues were collected. b, Serum neutralizing antibodies against multiple pseudoviruses (n = 6 mice per group). cd, representative images (c) and quantitative analysis (d) of RBD-specific IgG antibody-secreting cells (ASC) in bone marrow and spleen tissues (n = 5 mice / group). e, Percentage of long-lived plasma cells (LLPCs) in the bone marrow (n = 5 mice / group). f, Percentage of memory B cells (MBCs) in the spleen and lymph nodes (n = 5 mice / group). gh, Percentage of CD4+ and CD8+ memory T cells secreting IFN-γ-(g) and TNF-α-(h) after stimulation with the spike peptide pool (n = 5 mice per group). Data are presented as geometric mean and SD, b, and mean and SEM, dh. P values ​​in dh were performed by unpaired Student's t-test. ns, not significant. ****P≤0.0001; ***P≤0.001; **P≤0.01; *P≤0.05. ).

[0077] As shown in Figure 6, the neutralizing antibodies induced by the recombinant protein vaccine of the present invention can last for up to 43 weeks, and the serum neutralizing antibody titer against the XBB lineage subvariants is high (Figure 6b). In addition, antigen-specific ASCs can be detected in bone marrow and spleen tissue (Figure 6c-d). The vaccine significantly induced LLPCs in the bone marrow (Figure 6e) and MBCs in spleen tissue and lymph nodes (Figure 6f), which is consistent with the results of persistent serum neutralizing antibodies. Similar to the humoral immune response, the vaccine confers persistence of the cellular immune response, which is manifested as antigen-specific CD4 in the spleen 43 weeks after initial immunization. + and CD8 +The presence of T cells (Figure 6g-h). These results indicate that our SARS-CoV-2 XBB.1.5 recombinant protein vaccine can provide long-term protection against the circulating SARS-CoV-2 variants.

[0078] Example 9: Heterologous sequential inactivated vaccine and mRNA vaccine of the novel coronavirus XBB.1.5 recombinant protein vaccine

[0079] Given that XBB lineage mutants exhibit significant immune escape from immunity induced by previous vaccination and breakthrough infection, as well as adverse effects on immunoblotting, the FDA recommends the use of monovalent XBB lineage as vaccine antigens. Currently, Moderna and Pfizer / BioNTech have obtained marketing authorization for monovalent XBB.1.5 spike mRNA vaccines. However, some studies have shown that heterologous immunization with recombinant protein-based boosters may induce better immune responses than homologous vaccination with inactivated or mRNA vaccines. In addition, the main COVID-19 vaccines administered in mainland China are inactivated vaccines, and there are no mRNA vaccines yet. Therefore, the present invention attempts to evaluate the potential of this vaccine as a booster for mRNA and inactivated vaccines.

[0080] The present invention first immunized NIH mice three times with an inactivated vaccine based on a wild virus or a full-length spike protein mRNA vaccine based on the BA.5 variant sequence of Omicron, and then sequentially immunized the mice with an inactivated vaccine (inactivated vaccine × 3> inactivated vaccine), an mRNA vaccine (3 injections of mRNA> mRNA) or a monovalent recombinant vaccine (3 injections of inactivated vaccine> protein vaccine and 3 injections of mRNA> protein vaccine) (Figure 7a). The Omicron mutant strain containing XBB showed significant immune escape from the neutralization induced by the inactivated vaccine, even the mRNA vaccine designed based on the BA.5 sequence (Figure 7b-d). Heterologous vaccination with the new coronavirus XBB.1.5 recombinant protein vaccine significantly restored the neutralizing activity against all variants. The neutralizing antibody titer after the heterologous sequential inactivated vaccine of the vaccine was detected by a real virus neutralization test to determine whether the vaccine can be used as a fourth booster injection after three injections of inactivated virus vaccine (Figure 7b). The GMTs in the heterologous vaccine-vaccinated groups against Delta, BA.2, BA.5.2.48 and XBB.1.16 viruses reached 891, 776, 891 and 1552, respectively, which were 4.00, 8.00, 111.43 and 256.00 times higher than those in the groups receiving homologous inactivated vaccines.

[0081] The neutralization responses of the SARS-CoV-2 XBB.1.5 recombinant protein vaccine to the inactivated and mRNA vaccines were then comprehensively evaluated using pseudovirus neutralization assays (Figures 7c-d). All Omicron variants, particularly the XBB variant, significantly escaped neutralizing antibodies induced by both the inactivated and mRNA vaccines. Notably, the antibody group induced by heterologous immunization exhibited high levels of neutralization against all XBB lineages, BA.2.86, and the derived submutant JN.1. In the heterologous vaccination group (inactivated vaccine × 3 > protein vaccine) after inactivated vaccination, the GMTs against XBB.1.5, XBB.1.6, XBB.1.9.1, XBB.1.16, XBB.2.3, EG.5.1, BA.2.86, and the JN.1 submutant were determined to be 48923, 29880, 54896, 25842, 56241, 37829, 15129, and 14471, respectively. Compared with the homologous inactivated group (inactivated vaccine × 3 > inactivated vaccine), these values ​​increased by 1168.18, 4139.36, 1740.76, 2472.39, 5028.81, 727.48, 64.65, and 59.07 times, respectively ( Figure 7 c). In addition, in the group that received three doses of mRNA vaccine followed by one dose of the vaccine (mRNA×3>protein vaccine), the GMTs for XBB.1.5, XBB.1.16.6, EG.5.1, FL.1.5.1, HV.1, BA.2.86, and JN.1 were 32838, 25671, 36691, 7837, 10330, 15546, and 11464, respectively, which were 13.88-, 15.27-, 14.18-, 340.02-, 10.95-, 16.24-, and 17.27-fold higher than those for the homologous mRNA vaccine sequence (mRNA×3>mRNA) (Figure 7d). These findings strongly confirm that the SARS-CoV-2 XBB.1.5 recombinant protein vaccine can serve as a booster vaccine candidate, providing immune protection against variants including JN.1, BA.2.86, and XBB lineages.

[0082] Among them, Figure 7a-c, NIH mice were immunized three times with an inactivated virus vaccine, followed by a single dose of homologous inactivated vaccine (IV×3>IV), or a single heterologous inoculation of XBB.1.5 recombinant protein vaccine (IV×3>XBB.1.5) (n=6 mice per group). The immunization doses of mice were: 50U / mouse of inactivated vaccine, 10μg / mouse of mRNA vaccine, and 100μl / mouse of the new coronavirus XBB.1.5 recombinant protein vaccine as described in Example 3. Neutralizing antibodies against the real virus (b) and pseudovirus (c) were measured in serum samples. d, NIH mice were immunized with three doses of full-length spike BA.5 mRNA vaccine, followed by a single dose of homologous mRNA vaccine (mRNA×3>mRNA), or a single dose of heterologous XBB.1.5 recombinant protein vaccine (mRNA×3>XBB.1.5) (n=6 mice per group). The neutralizing antibody titers of immune sera against various Omicron XBB pseudoviruses were measured.

[0083] Example 10: Novel Coronavirus EG.5.1 Variant Challenge Protection Test

[0084] Prevention of viral infection is one of the most important indicators reflecting the protective efficiency of respiratory vaccines. Therefore, it was investigated whether the new coronavirus XBB.1.5 recombinant protein vaccine can provide a protective immune response against the epidemic strain. The EG.5.1 subvariant was selected, which is a global epidemic strain that accounted for 20.2% of the circulating strains in the United States as of November 20, 2023. NIH mice (6-8 weeks) were divided into two groups and injected intramuscularly with 100 μl of the new coronavirus XBB.1.5 recombinant protein vaccine or MF59-like adjuvant prepared in Example 3 on days 0, 21, and 42. Then, on day 63, 1×10 6 PFU EG.5.1 live virus challenged mice. Changes in body weight and throat swab viral load were monitored daily. On day 5 after infection, the mice were euthanized and tissues were collected to detect viral load and pathological changes. Viral genomic RNA (gRNA) in nasal concha, trachea and lung tissue samples was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay, and pathological changes in lung tissue were assessed by hematoxylin and eosin staining (H&E). The following primer and probe sequences, including 5'-GACCCCAAAATCAGCGAAAT-3' (SEQ ID No.5 forward), 5'-TCTGGTTACTGCCAGTTGAATCTG-3' (SEQ ID No.6 reverse), 5'-FAM-ACNGCCGCATTACGTTGGTGGACC-BHQ1-3' (SEQ ID No.7 probe sequence) were used to detect gRNA levels.

[0085] All experiments involving animals challenged with live SARS-CoV-2 virus were thoroughly reviewed and approved by the Institutional Animal Care and Use Committee of the Institute of Medical Biology, Chinese Academy of Medical Sciences. Furthermore, these experiments were conducted in the ABSL-4 facility of the Kunming National High-Level Biosafety Primate Research Center.

[0086] Daily monitoring of the weight of challenged mice revealed that the MF59-like adjuvant control group exhibited significant weight loss on day 1 after infection, reaching a maximum on day 4. In contrast, mice immunized with the vaccine exhibited transient weight loss, which quickly rebounded on day 2 (Figure 8a). In addition, high levels of genomic RNA (gRNA) were detected in throat swab samples from the control group, while the viral load in throat swabs from mice immunized with the vaccine showed a significant downward trend throughout the experiment (Figure 8b).

[0087] Subsequently, the mice were euthanized on day 5 after infection to collect tissues for assessment of viral load and histopathology (Figure 8c-d). In the control group receiving adjuvant, high levels of gRNA were detected in nasal concha, trachea and lung tissues. The viral load in the nasal concha, trachea and lung tissues of vaccinated mice decreased significantly. (Figure 8c). In addition, mild pathological changes were observed in the lung tissue of the adjuvant control group, characterized by multifocal consolidation areas, thickening of alveolar septa, alveolar congestion and small plaque inflammation (Figure 8d). As expected, the lung tissue of the vaccinated mice showed normal histological structure, with intact alveoli and no obvious inflammation. Therefore, the pathological scores of the lung tissues of vaccine-immunized mice were significantly lower than those of the adjuvant control group (Figure 8d). These results show that the new coronavirus XBB.1.5 recombinant protein vaccine can induce protective immune responses in the upper and lower respiratory tracts and effectively prevent live virus infection.

[0088] In Figure 8a, 1×10 6 PFU of live EG.5.1 true virus was instilled into NIH mice immunized with 100 μl of the new coronavirus XBB.1.5 recombinant protein vaccine prepared in Example 3. Weight loss (a) and changes in viral genomic RNA (gRNA) in throat swabs (b) were monitored daily. c. On the 5th day after infection, the infected mice were euthanized and nasal concha, trachea and lung tissues were collected, and then gRNA levels were detected by RT-qPCR. d. Histopathological changes and pathological scores of lung tissues of mice challenged with live EG.5.1 true virus. The scale bar in d represents 100 μm. Data are presented as mean and SEM in ad. P values ​​in bc were performed by two-way ANOVA followed by Sidak multiple comparison test, and P values ​​in d were performed by unpaired Student t test. ****P≤0.0001; ***P≤0.001.

Claims

1. Use of the XBB.1.5 recombinant protein or vaccine in the preparation of a medicament for preventing and / or treating infection with SARS-CoV-2 and its variants; characterized by: The SARS-CoV-2 variants include Delta, Omicron variants and their subtypes; the Omicron variants and their subtypes are selected from JN.1, BA.2.86 and XBB lineages.

2. The use according to claim 1, characterized in that: The XBB lineage includes at least one of XBB, XBB.1.5, XBB.1.6, XBB.1.9.1, XBB.1.16, XBB.1.16.6, XBB.2.3, EG.5.1, FL.1.5.1 or HV.

1.

3. The use according to claim 1 or 2, characterized in that: The SARS-CoV-2 variants include at least one of Delta, BA.2.75, BA.5, BA.2, BA.5.2.48, BF.7, BQ.1, BQ.1.1, XBB, XBB.1.5, XBB.1.6, XBB.1.9.1, XBB.1.16, XBB.1.16.6, XBB.2.3, EG.5.1, FL.1.5.1, HV.1, BA.2.86 or JN.

1.

4. A composition or combination for preventing and / or treating infection with SARS-CoV-2 and its variants, characterized by: Contains mRNA vaccine and XBB.1.5 recombinant protein vaccine for anti-SARS-CoV-2 or its variants infection, which are administered separately, simultaneously or sequentially.

5. A composition or combination for preventing and / or treating infection with SARS-CoV-2 and its variants, characterized by: Contains an inactivated vaccine and XBB.1.5 recombinant protein vaccine against SARS-CoV-2 or its variants, which are administered separately, simultaneously or sequentially.

6. The use according to any one of claims 1 to 3, the composition or the combined drug according to claim 4 or 5, characterized in that: The XBB.1.5 recombinant protein or vaccine contains the amino acid sequence shown in any one of SEQ ID No. 2 and SEQ ID No.

4.

7. The use according to any one of claims 1 to 3, the composition or the combination according to claim 4 or 5, characterized in that: The XBB.1.5 recombinant protein or vaccine contains the nucleotide sequence shown in any one of SEQ ID No.1 and SEQ ID No.

3.

8. The use according to any one of claims 1 to 3, 6 to 7, or the composition or combination according to any one of claims 4 to 7, characterized in that: The preparation method of the XBB.1.5 recombinant protein comprises the following steps: culturing host cells containing the XBB.1.5 recombinant protein or protein precursor to express the protein or precursor, and then recovering the protein.

9. The use, composition or combination according to claim 8, characterized in that: In the preparation method of the XBB.1.5 recombinant protein, the host cell contains a recombinant vector, the recombinant vector contains a gene for translating the XBB.1.5 recombinant protein or precursor, and the recombinant vector adopts at least one of an insect baculovirus expression vector, a mammalian cell expression vector, an Escherichia coli expression vector, and a yeast expression vector.

10. The use, composition or combination according to claim 9, characterized in that: The insect baculovirus expression vector is pFastBac1; the Escherichia coli expression vector is pET32a; the yeast expression vector is pPICZaA; the mammalian cell expression vector is a CHO cell expression vector; preferably, the CHO cell expression vector is pTT5 or FTP-002.

11. The use, composition or combination according to any one of claims 8 to 10, characterized in that: The host cell is at least one of insect cells, mammalian cells, Escherichia coli, and yeast.

12. The use, composition or combination according to claim 11, characterized in that: The insect cells are selected from at least one of sf9 cells, sf21 cells, and Hi5 cells; and the mammalian cells are CHO cells.

13. The use according to any one of claims 1 to 3, 6 to 12, or the composition or combination according to any one of claims 4 to 12, characterized in that: The XBB.1.5 recombinant protein vaccine is obtained by mixing the XBB.1.5 recombinant protein with pharmaceutically acceptable excipients or auxiliary components; the amino acid sequence of the XBB.1.5 recombinant protein is shown in any one of SEQ ID No.2 and SEQ ID No.

4.

14. The use, composition or combination according to claim 13, characterized in that: The auxiliary component is an immune adjuvant; preferably, the immune adjuvant is selected from at least one of the following: squalene oil-in-water emulsion, aluminum salt, calcium salt, plant saponin, plant polysaccharide, monophosphoric acid lipid A, muramyl dipeptide, muramyl tripeptide, bacterial toxin, GM-CSF cytokine, lipid, cationic liposome material.

15. The use, composition or combination according to claim 14, characterized in that: Meet at least one of the following: The squalene oil-in-water emulsion is MF59; The aluminum salt is selected from at least one of aluminum hydroxide and alum; The calcium salt is tricalcium phosphate; The plant saponin is QS-21 or ISCOM; The plant polysaccharide is astragalus polysaccharide; The bacterial toxin is selected from at least one of recombinant cholera toxin and diphtheria toxin; The lipid is selected from at least one of the following: phosphatidylethanolamine, phosphatidylcholine, cholesterol, dioleate Acylphosphatidylethanolamine; The cationic liposome material is selected from at least one of the following: (2,3-dioleyloxypropyl)trimethylammonium chloride, N-[1-(2,3-dioleoyl chloride)propyl]-N,N,N-trimethylammonium chloride, cationic cholesterol, dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, dimethyldioctadecylammonium bromide, and CpG ODN.

16. The use, composition or combination according to any one of claims 13 to 15, characterized in that: The XBB.1.5 recombinant protein vaccine is an injection preparation, a nasal spray preparation, or an oral preparation; preferably, the vaccine is an intramuscular injection preparation.

17. Use of the composition or combination of any one of claims 4 to 16 in the preparation of a medicament for preventing and / or treating infection with SARS-CoV-2 and its variants, characterized in that: The SARS-CoV-2 variants include Delta, Omicron variants and their subtypes; the Omicron variants and their subtypes are selected from JN.1, BA.2.86 and XBB lineages.

18. The use according to claim 17, characterized in that: The XBB lineage includes at least one of XBB, XBB.1.5, XBB.1.6, XBB.1.9.1, XBB.1.16, XBB.1.16.6, XBB.2.3, EG.5.1, FL.1.5.1 or HV.

1.

19. The use according to claim 17 or 18, characterized in that: The SARS-CoV-2 variants include at least one of Delta, BA.2.75, BA.5, BA.2, BA.5.2.48, BF.7, BQ.1, BQ.1.1, XBB, XBB.1.5, XBB.1.6, XBB.1.9.1, XBB.1.16, XBB.1.16.6, XBB.2.3, EG.5.1, FL.1.5.1, HV.1, BA.2.86 or JN.1.

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