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

Abstract

The present invention relates to a recombinant protein vaccine for preventing and treating SARS-CoV-2 variants JN.1, BA.2.86, and XBB lineages, a drug for combined use, and use. To solve the problem that Omicron JN.1, BA.2.86, and XBB lineage subvariants exhibit significant immune escape and blotting immunity after previous vaccination and viral infection, an XBB.1.5 recombinant protein vaccine is provided. The vaccine, when administered either intramuscularly or intranasally, induces strong humoral, cellular, and mucosal immune responses against JN.1, BA.2.86, and XBB lineage variants. Compared with homologous vaccination, after inactivated or mRNA vaccines are applied, the vaccine is used for heterologous vaccination to obtain a better immune response. Moreover, the vaccine induces effective protective immunity in vivo against Omicron EG.5.1 live virus attacks. Thus, the XBB.1.5 vaccine has clinical efficacy.

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 with wild-type spike mRNA or inactivated vaccines in humans tends to skew the immune response toward older 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 reevaluate the composition of current vaccines and accelerate the development of next-generation COVID-19 vaccines specifically targeting the evolving XBB lineage. Furthermore, unlike most currently approved COVID-19 vaccines, which are administered via intramuscular injection, intranasal delivery of antigens offers a more promising approach to effectively block novel coronavirus infection and transmission. Subunit recombinant proteins are widely used as a vaccine platform for intranasal vaccine development due to their safety, cost-effectiveness of large-scale production, and ease of transport. However, pure protein antigens are often rapidly cleared through mucociliary clearance, resulting in poor immunogenicity in the respiratory mucosa and insufficient induction of protective immune responses. To address this challenge, protein antigens have been supplemented with various intranasal adjuvants, including polymers, membrane vesicles, and nanoparticles, to enhance the magnitude and durability of the immune response. However, these adjuvants have not yet received widespread clinical approval and their safety is a concern. Therefore, it is crucial to develop a next-generation COVID-19 recombinant protein vaccine that specifically targets the XBB lineage and has high safety and efficacy.

[0004] Summary of the Invention

[0005] Considering that Omicron JN.1, BA.2.86 and XBB lineage subvariants are prevalent around the world, they have shown breakthrough significant immune escape after previous vaccination and viral infection, as well as imprinting immune phenomena, and there is an urgent need to develop a next-generation novel coronavirus vaccine specifically for the emerging subvariants. The present invention successfully constructs and expresses the XBB.1.5 recombinant protein by tandem with a receptor binding domain (RBD) sequence from the XBB.1.5 variant and a partial sequence from the spike S2 ​​subunit, and mixes it with the nationally approved and safe, well-tolerated adjuvant MF59 to prepare an XBB.1.5 recombinant protein vaccine. The vaccine induces stable and lasting humoral and cellular immune responses against the currently prevalent JN.1, BA.2.86 and XBB lineage variants through intramuscular and intranasal immunization, especially intranasal immunization induces high levels of neutralizing antibodies against XBB lineage subvariants, producing an effective mucosal immune response. Moreover, the vaccine is used as a heterologous booster vaccination of an inactivated vaccine or mRNA vaccine, whether it is intramuscular injection or intranasal immunization, and obtains a better immune response and a more effective booster effect. At the same time, after intramuscular injection of the vaccine, the vaccine was immunized again by intranasal immunization, showing excellent systemic and mucosal protective immunity.

[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, EG.5.1, FL.1.5.1, HV.1, HK.3, 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 and / or an XBB.1.5 recombinant protein vaccine against infection with SARS-CoV-2 or its variants, administered separately, simultaneously or sequentially.

[0011] Furthermore, the mRNA vaccine is an mRNA vaccine encoding the full-length BA.5 spike protein or XBB.1.5 of SARS-CoV-2. Preferably, the antigen amino acid sequence of the mRNA vaccine is shown in SEQ ID No. 5. The antigen RNA sequence is shown in SEQ ID No. 7.

[0012] 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 and / or XBB.1.5 recombinant protein vaccine against infection with SARS-CoV-2 or its variants, administered separately, simultaneously or sequentially.

[0013] Furthermore, the inactivated vaccine is an inactivated vaccine prepared based on wild SARS-CoV-2 virus or variant.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] Furthermore, the host cell is at least one of insect cells, mammalian cells, Escherichia coli, and yeast.

[0020] 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.

[0021] 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.

[0022] Preferably, the auxiliary component is an immune adjuvant.

[0023] 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.

[0024] 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.

[0025] Furthermore, the XBB.1.5 recombinant protein vaccine is at least one of an injectable formulation, an intranasal formulation, or an oral formulation. Preferably, the vaccine is an intramuscular injection formulation and / or an intranasal formulation.

[0026] The XBB.1.5 recombinant protein vaccine described herein may be administered intramuscularly and / or intranasally, meaning that different formulations of the XBB.1.5 recombinant protein vaccine can be combined, for example, to provide systemic and mucosal protective immunity via both intramuscular and intranasal administration. Preferably, the vaccine is administered intranasally in at least two doses.

[0027] 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.

[0028] 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.

[0029] 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, EG.5.1, FL.1.5.1, HV.1, HK.3, BA.2.86 or JN.1.

[0030] In the first aspect above, the 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;

[0031] Or the use of a composition or combination of the above-mentioned XBB.1.5 recombinant protein vaccine and mRNA vaccine or inactivated vaccine in the preparation of a drug for preventing and / or treating infection with SARS-CoV-2 and its variants; the dosage form of the drug is at least one of an injection preparation, an intranasal preparation, and an oral preparation; preferably, the drug is an intramuscular injection preparation and / or an intranasal preparation.

[0032] The drug dosage form of the present invention is an intramuscular injection preparation and / or an intranasal administration preparation, which means that drugs in different dosage forms can be combined, for example, the drug can be administered simultaneously through intramuscular and intranasal immunization for systemic and mucosal protective immunity.

[0033] More preferably, when intramuscular and intranasal immunization are used in combination, the recombinant protein vaccine in the medicament is administered intranasally at least twice.

[0034] Sequence information of the novel coronavirus XBB.1.5 recombinant protein vaccine of the present invention:

[0035] Nucleic acid sequence of the novel coronavirus XBB.1.5 recombinant protein: SEQ ID No.1

[0036] Amino acid sequence of the novel coronavirus XBB.1.5 recombinant protein: SEQ ID No. 2

[0037] SARS-CoV-2 XBB.1.5 recombinant protein vaccine antigen nucleic acid sequence: SEQ ID No.3

[0038] SARS-CoV-2 XBB.1.5 recombinant protein vaccine antigen protein sequence: SEQ ID No.4

[0039] 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.

[0040] At the same time, intranasal administration of the vaccine elicited a strong humoral immune response in mice and rats, inducing high levels of neutralizing antibodies against XBB lineage subvariants and generating a potent mucosal immune response characterized by the induction of tissue-resident T cells, local cellular immunity, and germinal center responses in the respiratory tract. Furthermore, combined intramuscular and intranasal delivery of the XBB.1.5 recombinant protein vaccine demonstrated excellent systemic and mucosal protective immunity.

[0041] Furthermore, compared with homologous vaccination, heterologous vaccination with either an intramuscular or intranasal monovalent recombinant vaccine after administration of an inactivated or mRNA-based vaccine resulted in a superior immune response and a more effective booster effect. 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 protection against the currently circulating Omicron variant. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is a molecular sieve and SDS-PAGE identification diagram of the recombinant protein XBB.1.5 in Example 1; M is a protein marker, and 1, 2, and 3 are the molecular weights of the target protein after removing all redundant amino acids;

[0043] FIG2 is a graph showing pseudovirus neutralizing antibody results of serum from mice immunized with the XBB.1.5 recombinant protein vaccine according to Example 4;

[0044] Figure 3 shows the enzyme-linked immunospot (ELISpot) assay for specific cellular immunity following immunization with the XBB.1.5 recombinant protein vaccine in Example 5; ab, Representative images (a) and quantitative analysis (b) of RBD-specific IgG antibody-secreting cells (ASC) in bone marrow and spleen tissues (n = 5 mice / group). cd, Representative images (c) and quantitative analysis (d) of IFN-γ-secreting lymphocytes in the spleen following stimulation with a peptide library of the XBB.1.5 spike protein (n = 5 mice per group).

[0045] Figure 4 is a graph showing the results of flow cytometry detection of specific cellular immunity of the XBB.1.5 recombinant protein vaccine in Example 6; ab, secretion of IFN-γ after stimulation with the spike peptide library + (a) and TNF-α + (b) CD4 + and CD8 + The percentage of memory T cells (n = 5 mice per group). cd, Tfh (CD4 + CXCR5 + PD-1 + ) and GCB cells (CD19 + GL7 + CD95 + ) percentage.

[0046] Figure 5 is a graph showing true virus neutralizing antibodies in the immune serum of mice vaccinated with the XBB.1.5 recombinant protein vaccine according to Example 7;

[0047] Figure 6 is a graph showing the long-term protective immunity induced by the XBB.1.5 recombinant protein vaccine in Example 8; 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 (LLPC) in the bone marrow (n = 5 mice / group). f, Percentage of memory B cells (MBC) in the spleen and lymph nodes (n = 5 mice / group). gh, Secretion of IFN-γ after stimulation with the SARS-CoV-2 XBB.1.5 peptide library + (g) and TNF-α + (h) Percentages of CD4+ and CD8+ memory T cells (n=5 mice per group).

[0048] Figure 7 shows the neutralizing antibody titers of the heterologous sequential inactivated and mRNA vaccines of the XBB.1.5 recombinant protein vaccine in Example 9; a-c, NIH mice were immunized three times with an inactivated virus vaccine, followed by a single dose of a homologous inactivated vaccine (IV×3>IV), or a single heterologous inoculation of the XBB.1.5 recombinant protein vaccine (IV×3>XBB.1.5) (n=6 mice per group). Neutralizing antibodies against the real virus (b) and pseudovirus (c) were measured in serum samples. d, NIH mice were immunized three times with the full-length spike BA.5 mRNA vaccine, followed by a single dose of a homologous mRNA vaccine (mRNA×3>mRNA), or a single dose of a heterologous XBB.1.5 recombinant protein vaccine (mRNA×3>XBB.1.5) (n=6 mice per group). Neutralizing antibody titers against various 0-micron XBB pseudoviruses were measured in immune sera. The immunization doses for mice were: 50 U / mouse of inactivated vaccine, 10 μg / mouse of mRNA vaccine, and 100 μL / mouse of the new coronavirus XBB.1.5 recombinant protein vaccine as shown in Example 3.

[0049] Figure 8 shows the results of the Omicron EG.5.1 live virus challenge in mice immunized with the XBB.1.5 recombinant protein vaccine in Example 10; a, 1×10 6PFU of live EG.5.1 true virus were 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 day 5 after infection, infected mice were euthanized and nasal concha, trachea, and lung tissues were collected, and gRNA levels were then detected by RT-qPCR. d. Histopathological changes and pathological scores of lung tissues of mice challenged with live EG.5.1 true virus. Scale bar in d represents 100 μm.

[0050] Figure 9 is a graph showing the humoral immune response results after immunization with intranasal delivery of the XBB.1.5 recombinant protein vaccine in Example 13; a, Schematic diagram of the immunization and serum collection scheme for mice and rats. Animals were given the XBB.1.5 recombinant protein vaccine intranasally on days 0, 21, and 42, and serum was collected on day 14 after each immunization. b, Endpoint titers of anti-RBD IgG in sera from mice immunized intranasally with low-dose (5 μg) and high-dose (10 μg) XBB.1.5 recombinant protein vaccines (n=6 mice per group). c, Endpoint titers of serum anti-RBD IgG in rats receiving 40 μg intranasal XBB.1.5 recombinant protein vaccine (n=5 rats per group). d, Antibody subtypes of anti-RBD IgG in mouse sera collected on day 56 (n=6 mice / group). e, Neutralizing antibodies against pseudovirus in mouse sera collected on day 56 (n=6 mice / group). fg, Endpoint titers of anti-RBD IgA, IgG (f), and neutralizing antibodies (g) in mouse bronchoalveolar lavage fluid (BALF) samples collected on day 72 (n=6 mice / group).

[0051] Figure 10 is a graph showing the results of local mucosal immune responses induced by intranasal delivery of the XBB.1.5 recombinant protein vaccine in Example 14; a, CD8 + and CD4 + Tissue-resident memory T(T RM ) Absolute number of cells. b, Memory CD8 T cells producing antigen-specific IFN-γ or TNF-α in lung tissue after stimulation with peptide library of SARS-CoV-2 XBB.1.5 spike + and CD4 + The percentage of T cells (n = 6 mice per group). c, T follicular helper cells (CD4 + PD-1 + CXCR5 + ), germinal center B cells (CD19 + GL7 + CD95 + ) and RBD-specific B cells (RBD+ CD19 + ) (n=6 mice per group).

[0052] Figure 11 is a graph showing the mucosal and systemic humoral immune responses elicited by combined intramuscular and intranasal immunization of the XBB.1.5 recombinant protein vaccine in Example 15; ab, NIH mice were injected with two doses of XBB.1.5 recombinant protein vaccine intramuscularly, followed by one intranasal delivery (2×IM+1×IN), or injected with one dose of XBB.1.5 recombinant protein vaccine, followed by two intranasal deliveries (1×IM+2×IN). Mice received three intranasal deliveries of intranasal XBB.1.5 recombinant protein vaccine (3×IN) as a control (n=6 mice per group). cd, endpoint titer of RBD-specific IgG in serum (c), endpoint titer of IgA and IgG in BALF samples (d). ef, neutralization of XBB lineage pseudoviruses in serum (e) and BALF samples (f).

[0053] Figure 12 shows the results of mucosal and systemic cellular immune responses induced by combined intramuscular and intranasal immunization with the XBB.1.5 recombinant protein vaccine in Example 15; a, Percentage of memory T cells producing antigen-specific IFN-γ or TNF-α in lung tissue after stimulation with the peptide library of the XBB.1.5 spike protein (n=6 mice per group). b, Percentage of Tfh (CD4 + PD-1 + CXCR5 + )、GC B(CD19 + GL7 + CD95 + ) and the frequency of antigen-specific B cells (RBD + CD19 + ) (n = 6 mice per group) c, Antigen-specific T cells and antigen-specific memory B cells (RBD) in spleen tissue + CD19 + CD138 + ) percentage.

[0054] Figure 13 is a graph showing the mucosal and systemic immune responses induced by intranasal delivery of the XBB.1.5 recombinant protein vaccine followed by heterologous sequential mRNA vaccines in Example 16; ab, NIH mice were immunized with three doses of full-length BA.5 spike mRNA vaccine, followed by one dose of homologous injection of mRNA vaccine (4×mRNA), or one dose of heterologous intranasal delivery of XBB.1.5 recombinant protein vaccine (3×mRNA+1×IN). Another group of mice received two mRNA vaccine injections and subsequently two doses of intranasal XBB.1.5 recombinant protein vaccine (2×mRNA+2×IN) (n=6 mice per group). cd, endpoint titer of RBD-specific IgG in serum (c), endpoint titer of IgA and IgG in BALF samples (d). ef, neutralization of XBB lineage pseudoviruses in serum (e) and BALF samples (f). gh, GC B (CD19 in mediastinal lymph nodes) + GL7 + CD95 + ), percentage of antigen-specific B cells and Tfh (n=6 mice per group). i, CD8 + and CD4 + Tissue-resident memory T(T RM ) Absolute number of T cells (n=6 mice per group). j, Percentage of memory T cells producing antigen-specific IFN-γ or TNF-α in lung tissue (n=6 mice per group).

[0055] Figure 14 shows the results of intranasal delivery of XBB.1.5 recombinant protein vaccine to mice challenged with Omicron EG.5.1 live virus in Example 17; a, NIH mice were intranasally immunized three times with XBB.1.5 recombinant protein vaccine. Mice treated with adjuvant were used as controls. 6 NIH mice immunized with PFU of live SARS-CoV-2 EG.5.1 Omicron virus were challenged (n = 6 mice per group). Changes in viral load in throat swabs after novel coronavirus infection were monitored daily. b, Mice were euthanized on day 5 after infection, and multiple respiratory tissues including nasal concha, trachea, and lungs were collected to detect gRNA levels. cd, Representative images of histopathological changes (c) and pathological scores (d) in lung tissues of immunized mice infected with EG.5.1 virus. Scale bar in c represents 100 μm. DETAILED DESCRIPTION

[0056] 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.

[0057] Abbreviations:

[0058] 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).

[0059] First, regarding the currently available vaccines against 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 have shown significantly lower neutralizing activity, especially against the XBB family of the current novel coronavirus lineage. Despite the rapid production of bivalent mRNA vaccines incorporating BA.4 / 5 spike protein sequences, the neutralization capacity induced by these vaccines remains limited when faced with the XBB lineage. The emergence of various Omicron subvariants characterized by significant immune evasion properties has led to an increase in breakthrough infections in the community. 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.

[0060] Second, the primary route of administration of approved COVID-19 vaccines (intramuscular injection) has demonstrated limited efficacy in eliciting sufficient local mucosal protective immunity. Given this pressing challenge posed by persistent infection in the human population, there is an urgent need to accelerate the development of next-generation intranasal COVID-19 vaccines capable of preventing upper respiratory tract viral infections. Subunit proteins offer a promising platform for the development of intranasal vaccines; however, intranasal adjuvants must be incorporated to enhance the magnitude and durability of the immune response elicited by the protein antigen. Selecting adjuvants that have been clinically evaluated in humans could significantly accelerate the clinical translation process.

[0061] MF59 is an oil-in-water emulsion containing squalene dissolved in a citric acid buffer containing Tween 80 and Span 85. It has been approved for use in pandemic and seasonal influenza vaccines in many countries and has demonstrated good tolerability and safety in humans. However, little is known about whether this adjuvant can be used with novel coronavirus antigens to formulate an effective intranasal COVID-19 vaccine. Although existing studies have demonstrated the effectiveness of MF59 as an adjuvant for intranasal vaccines against novel coronavirus, the specific immune mechanisms by which MF59-like oil-in-water adjuvants elicit potent mucosal immunity have not been thoroughly investigated and warrant further investigation.

[0062] Finally, the antigenic composition of the vaccine is a key determinant of its effectiveness, particularly in the context of COVID-19 vaccines targeting various variants with enhanced immune evasion capabilities. But most current vaccines utilize antigens from previously circulating strains. Therefore, for the next generation of intranasal vaccines, antigens derived from recently emerged variants that exhibit strong immunogenicity capable of eliciting cross-neutralizing responses must be selected. In addition, repeated exposure to previous antigens tends to bias the immune response toward early lineage variants, thereby reducing the efficacy of immunity against recent Omicron subvariants. Compared with spike proteins from other Omicron subvariants, immunization with antigens from the XBB.1.5 variant can elicit broad-spectrum neutralization capabilities against common Omicron subvariants. However, to date, no subunit protein-based intranasal vaccine has been developed for the XBB lineage.

[0063] Therefore, in light of the above analysis, in one embodiment of the present invention, a recombinant protein of the RBD based on the XBB.1.5 mutation was successfully expressed and mixed with an equal volume of the adjuvant MF59 to prepare the XBB.1.5 recombinant protein vaccine, which is the world's first recombinant protein vaccine targeting the XBB lineage.

[0064] The experimental results show that the XBB.1.5 recombinant protein vaccine with the adjuvant MF59 added to the present invention can induce robust and sustained humoral, cellular and mucosal immune responses against JN.1, BA.2.86 and XBB lineage submutants, whether delivered by intramuscular injection or intranasal delivery, whether as a stand-alone vaccine or as a booster vaccine. In addition, the vaccine immunization can induce effective protective immunity against live EG.5.1 variant infection in the local upper and lower respiratory tracts. It is worth noting that the vaccine showed good safety and tolerability in humans and showed excellent ability to induce strong neutralizing efficacy against the XBB lineage and the recent JN.1 submutants. 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.

[0065] Secondly, although Moderna and Pfizer / BioNTech have updated the sequences of their mRNA vaccines to produce the monovalent XBB.1.5 spike mRNA vaccine, a booster dose of the XBB.1.5 monovalent mRNA vaccine can confer high levels of neutralizing antibodies against both XBB and JN.1 subvariants. However, given the widespread global administration of mRNA-based COVID-19 vaccines, heterologous vaccination using an alternative vaccine platform as a booster injection may elicit a more robust immune response than repeated homologous vaccination with an mRNA-based vaccine. Furthermore, mRNA-based COVID-19 vaccines have not yet been approved for use in mainland China.

[0066] Therefore, in one embodiment of the present invention, after intramuscular immunization with three doses of inactivated virus vaccine or mRNA-based vaccine, intramuscular vaccination with a protein-based recombinant vaccine induced a superior immune response, characterized by elevated serum neutralizing antibody levels in animal models. 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.

[0067] Second, the mixed immune landscape in the population is influenced by various factors, such as the type of vaccine administered (mRNA, inactivated virus, adenovirus), the variant sequences encountered (pre-Omicron, Omicron, XBB lineages), and the time interval between vaccination and infection, posing increasingly complex challenges to the development of next-generation vaccine boosters. Therefore, it is important to explore the potential of intranasal vaccines as booster injections in heterologous vaccination regimens. Although Moderna and Pfizer / BioNTech updated the sequences of their mRNA vaccines to incorporate the XBB.1.5 spike protein for booster injections, considering the widespread use of mRNA-based COVID-19 vaccines worldwide, heterologous vaccination with alternative vaccine platforms and incorporating mucosal delivery may enhance systemic and mucosal immune responses.

[0068] Therefore, in another embodiment of the present invention, heterologous immunization with two intranasal administrations of the XBB.1.5 recombinant protein vaccine following mRNA vaccine injection can achieve superior antibody responses, accompanied by robust local mucosal immune responses. Compared with homologous vaccination, a single single-dose intranasal XBB.1.5 recombinant protein vaccine did not significantly increase serum neutralizing antibody levels. However, at least one intranasal delivery of the XBB.1.5 recombinant protein vaccine did provide additional mucosal protective immunity.

[0069] Thirdly, a disadvantage of intranasal administration is its tendency to induce relatively low systemic and cellular immune responses compared to intramuscular administration. Three intranasal vaccine administrations cannot fully stimulate antigen-specific T cells and memory B cells in spleen tissue. However, a strategy combining intramuscular and intranasal vaccination routes appears to provide a viable solution to this challenge. In a specific embodiment of the present invention, at least one intramuscular injection is included before intranasal administration to elicit a systemic cellular immune response (e.g., 2×IM+1×IN, 1×IM+2×IN). Given that a large proportion of the population has already received at least one dose of intramuscular vaccine, this problem may not be a substantial issue. In addition, compared with other groups, two intranasal administrations of the XBB.1.5 recombinant protein vaccine after a single intramuscular injection (1×IM+2×IN) resulted in the highest levels of systemic and mucosal antibodies. And at least one intranasal delivery can stimulate local mucosal responses, as demonstrated by the generation of cellular immune responses in the lungs and germinal center reactions in the mediastinal lymph nodes. These findings suggest that in order to optimally induce humoral and cellular immune responses in systemic and local mucosal immunity, the XBB.1.5 recombinant protein vaccine should be administered intranasally at least twice.

[0070] 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.

[0071] Experimental data are presented as geometric mean ± SD or mean ± SEM, and statistical analysis was performed using Prism 9.0 (GraphPad Software). P values ​​were determined using Student's t-test for comparisons between two groups or one-way analysis of variance among multiple groups. ****P ≤ 0.0001; ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05; ns, not significant.

[0072] The mRNA vaccine preparation method used in the embodiment is as follows:

[0073] The amino acid sequence of the mRNA vaccine antigen protein is shown in SEQ ID No. 5:

[0074] The mRNA vaccine antigen DNA sequence is shown in SEQ ID No. 6:

[0075] The mRNA vaccine antigen RNA sequence is shown in SEQ ID No. 7:

[0076] The preparation method of mRNA vaccine is as follows:

[0077] According to conventional methods, the template DNA was inserted into the pUC57 vector to obtain a plasmid containing the above antigen DNA sequence. The plasmid template was linearized by enzyme digestion and electrophoresis was performed to confirm whether the linearization was complete.

[0078] Prepare mRNA using RNA polymerase, NTPs, and cap analogs. Add the relevant reactants to a 15 mL centrifuge tube in the order and dosage shown in Table 1. Mix by pipetting three times or tapping the bottom of the tube. Briefly centrifuge to collect the reaction mixture at the bottom of the tube.

[0079] Table 1 Components and amounts for preparing mRNA

[0080] After incubating in a 37°C water bath for 4 hours, add 400 μL of DNase I and incubate at 37°C for 30 minutes. Then, add 4 mL of a phenol / chloroform / isoamyl alcohol (25:24:1) solution to extract mRNA. After mixing, centrifuge at 10,000 rpm for 5 minutes, and remove the aqueous phase. Then, add 4 mL of chloroform solution to extract mRNA. After mixing, centrifuge at 10,000 rpm for 5 minutes, and remove the aqueous phase. Then, add 4 mL of 5 M ammonium acetate solution and incubate at 4°C overnight to precipitate mRNA. Then, remove the pellet and centrifuge at 10,000 rpm for 2 minutes at 4°C. Carefully remove the supernatant. Wash the pellet with 4 mL of 70% ethanol and centrifuge at 10,000 rpm for 2 minutes at 4°C. Carefully remove the supernatant and air-dry the pellet. Reconstitute the pellet with 20 mL of nuclease-free water to obtain the mRNA solution. The purity of mRNA was detected by agarose gel electrophoresis or nucleic acid fragment analyzer, and the concentration was 1.08 mg / mL and the integrity was 75.6%.

[0081] Preparation of mRNA-LNP (mRNA vaccine):

[0082] The ionizable lipid Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 were dissolved in anhydrous ethanol in a molar ratio of 45:10:43.5:1.5 to form a certain volume of organic phase. At the same time, the above-mentioned mRNA solution (mRNA sequence such as SEQ ID No.7) was used as the aqueous phase, wherein the volume ratio of the aqueous phase to the organic phase was 3:1. They were mixed through a microfluidic chip to self-assemble into mRNA-lipid nanoparticles, and then the ethanol was removed through a tangential flow system to obtain the mRNA vaccine. The prepared mRNA vaccine had a particle size of 61.63 nm, a PDI of 0.154, an mRNA content of 0.1068 mg / mL, an encapsulation efficiency of 97.30% and an mRNA integrity of 68.7%, indicating that the mRNA lipid nanoparticles had a small and uniform particle size and a high encapsulation efficiency.

[0083] Example 1: Expression of the novel coronavirus XBB.1.5 recombinant protein using the insect baculovirus system

[0084] 1. Construction and design of the 2019-nCoV XBB.1.5 recombinant protein

[0085] 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. Furthermore, the recombinant protein was 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. Furthermore, 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.

[0086] 2. Preparation, Identification, and Purification of the SARS-CoV-2 XBB.1.5 Recombinant Protein

[0087] 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).

[0088] Example 2: Preparation of the novel coronavirus XBB.1.5 recombinant protein vaccine

[0089] 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.

[0090] The experimental operation and effect of intramuscular injection of the novel coronavirus XBB.1.5 recombinant protein vaccine prepared above are shown in Examples 3-10:

[0091] Example 3: Vaccination of mice

[0092] 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 200 μg / 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 100 μL 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.

[0093] Example 4: Detection of neutralizing antibodies in serum from XBB.1.5 recombinant protein vaccine by pseudovirus neutralization assay

[0094] 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 4 HEK-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%.

[0095] 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.

[0096] Example 5: ELISpot assay to detect specific cellular immunity after immunization with the novel coronavirus XBB.1.5 recombinant protein vaccine

[0097] 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.

[0098] 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.

[0099] 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, followed by incubation 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 using 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).

[0100] Example 6: Detection of specific cellular immunity by flow cytometry

[0101] 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 / Cyanine5.5-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.

[0102] 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).

[0103] 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).

[0104] Example 7: Neutralization test of live novel coronavirus

[0105] 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.

[0106] 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.

[0107] Example 8: Long-term protective immunity induced by the SARS-CoV-2 XBB.1.5 recombinant protein vaccine

[0108] 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.

[0109] 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.

[0110] 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.

[0111] Example 9: Heterologous sequential inactivated vaccine and mRNA vaccine of the novel coronavirus XBB.1.5 recombinant protein vaccine

[0112] 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.

[0113] The present invention first immunized NIH mice three times with either an inactivated vaccine based on wild-type virus (provided by the Institute of Medical Biology, Chinese Academy of Medical Sciences; for detailed preparation methods, see Clinical Infectious Diseases, Volume 73, Issue 11, December 2021, Pages e3949–e3955, https: / / doi.org / 10.1093 / cid / ciaa1703.) or a full-length spike protein mRNA vaccine based on the Omicron BA.5 variant sequence. The mice were then sequentially immunized 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 containing XBB exhibited significant immune evasion of neutralization elicited by the inactivated vaccine, even with the mRNA vaccine designed based on the BA.5 sequence (Figures 7b-d). Heterologous vaccination with the new coronavirus XBB.1.5 recombinant protein vaccine significantly restored neutralizing activity against all variants. The neutralizing antibody titer after heterologous sequential inactivated vaccination of the vaccine was detected by 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 vaccination 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, respectively.

[0114] 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.

[0115] Example 10: Novel Coronavirus EG.5.1 Variant Challenge Protection Test

[0116] 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 the 5th day 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.8 forward), 5'-TCTGGTTACTGCCAGTTGAATCTG-3' (SEQ ID No.9 reverse), 5'-FAM-ACNGCCGCATTACGTTGGTGGACC-BHQ1-3' (SEQ ID No.10 probe sequence) were used to detect gRNA levels.

[0117] 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.

[0118] Daily monitoring of the body 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).

[0119] 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.

[0120] The experimental procedures and effects of intranasal immunization with the novel coronavirus XBB.1.5 recombinant protein vaccine prepared above are shown in Examples 11-17:

[0121] Example 11: Animal vaccination

[0122] Experiments involving mice and rats were performed in accordance with the guidelines established by the Institutional Animal Care and Use Committee of Sichuan University (Chengdu, Sichuan, China). Specific pathogen-free (SPF) female NIH mice aged 6-8 weeks were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. (China). They were housed in the SPF animal facility of the Animal Center of the State Key Laboratory of Biotherapy and acclimated for 1 week before the start of the experiment. NIH mice were randomly divided into groups and intranasally administered with 50 mL PBS, XBB.1.5 recombinant protein antigen prepared in Example 1, MF59, or an XBB.1.5 recombinant protein vaccine formulation containing XBB.1.5 recombinant protein antigen and an equal volume of MF59 adjuvant on days 0, 21, and 42. Mice in the low-dose group received 5 μg of XBB.1.5 recombinant protein vaccine, while mice in the high-dose group received 10 μg of XBB.1.5 recombinant protein vaccine. Following the same prime-boost regimen, rats were immunized with 200 μL PBS or 40 μg of XBB.1.5 recombinant protein antigen in combination with adjuvant at 21-day intervals. Blood samples were collected from mice and rats 2, 5, or 8 weeks after the primary immunization.

[0123] To evaluate the immune response generated by a combination of intranasal (IN) and intramuscular (IM) immunization, NIH mice were administered three doses of 10 μg of XBB.1.5 recombinant protein vaccine by IM or IN injection on days 0, 21, and 42. Mice in the 1×IM+2×IN group received one dose by IM injection and then two doses by IN injection. Mice in the 2×IM+1×IN group received two doses by IM injection and then one dose by IN injection. Mice in the 3×IN group received three doses of vaccine delivered only by IN.

[0124] Example 12: Method for detecting the immune effect of intranasal immunization with the novel coronavirus XBB.1.5 recombinant protein vaccine

[0125] (1) Enzyme-linked immunosorbent assay (ELISA)

[0126] ELISA was used to measure RBD-specific IgG and IgA levels. Briefly, XBB.1.5 recombinant protein (1 μg / mL) was coated onto 96-well plates (NUNC-MaxiSorp, Thermo Fisher Scientific) by incubation overnight at 4°C or for 2 hours at room temperature. After washing three times with PBST (PBS containing 0.1% Tween 20), 100 μL of diluted serum or BALF was added to each well. After incubation at 37°C for 1 hour, the plates were washed again with PBST. Subsequently, 100 μL of HRP-conjugated antibody (anti-mouse IgG, IgG1, IgG3, IgG2a, IgG2b, IgG2c, IgA, or anti-rat IgG, diluted 1:10,000) was added to each well and incubated at 37°C for 1 hour. After washing five times, 100 μL of TMB (3,3',5,5'-tetramethylbenzyldiamine) substrate was added to each well. After reacting at room temperature for 10 minutes, color development was stopped by adding 50 μL of stop buffer (H 2 SO 4 ) to each well, and the absorbance at 450 nm was measured using a microplate reader (Spectramax ABS, Molecular Devices).

[0127] (2) SARS-CoV-2 pseudovirus neutralization test

[0128] The pseudovirus neutralization test is a widely used method to assess the presence of neutralizing antibodies in serum or BALF. SARS-CoV-2 pseudoviruses XBB, XBB.1.5, XBB.1.6, XBB.1.9.1, XBB.1.16, XBB.2.3, EG.5.1, and JN.1 pseudoviruses were provided by Genomeditech. Serum or BALF samples were first heat inactivated in a 56°C water bath for 30 minutes and then diluted in a 3-fold gradient in a 96-well plate (Catalog number: WHB-96-03, Shanghai Wohong Biotechnology Co., Ltd.). Subsequently, 50 μL of diluted pseudovirus was added to each well of the plate and incubated at 37°C for 1 hour. Then, 293T / ACE2 cells (1.5×10 4 / well) were added to the plate and incubated in a cell culture incubator (37 ° C, 0.5% CO2) for two days to allow luciferase expression. On the last day, the supernatant was removed, luciferase substrate (100 μL / well) was added, and luminescence was measured using a multi-mode microplate reader with Kaleido3.0 software (PerkinElmer, USA).

[0129] (3) Flow cytometry

[0130] To detect tissue-resident memory (T RM ) cells, collected and stained with PerCP / Cyanine5.5-conjugated anti-mouse CD3 (BioLegend, catalog number 100218), Brilliant Violet 421-conjugated anti-mouse CD4 (BioLegend, catalog number 100438), Brilliant Violet 510-conjugated anti-mouse CD8 (BioLegend, catalog number 100751), PE-conjugated anti-mouse CD69 (BioLegend, catalog number 164204) and APC-conjugated anti-mouse CD103 antibodies (BioLegend, catalog number 121414).

[0131] To measure T follicular helper cells (Tfh) in mediastinal lymph nodes (MLN), cells were incubated with the following antibodies: PerCP / Blue 5.5-conjugated anti-mouse CD3, Brilliant Violet 421-conjugated anti-mouse CD4, PE / Blue 7 anti-mouse / human B220 (BioLegend, catalog number 103222), APC anti-mouse CD185 (CXCR5, BioLegend, catalog number 145506) and FITC anti-mouse CD279 (PD-1, BioLegend, catalog number 135214). To detect germinal center (GC) and RBD-specific B cells, cells in MLN were treated with biotinylated RBD protein (1 μg / mL, SPD-C82Q3, ACROBiosystems) for 30 minutes at room temperature. After washing with PBS, cells were incubated with PerCP / Cyanine 5.5-conjugated anti-mouse CD3, Pacific Blue 7 anti-mouse CD4, PE / Blue 7 anti-mouse / human B220 (BioLegend, catalog number 103222), APC anti-mouse CD185 (CXCR5, BioLegend, catalog number 145506) and FITC anti-mouse CD279 (PD-1, BioLegend, catalog number 135214). TM Anti-mouse CD19 (BioLegend, catalog number 152416), APC anti-mouse CD95 (BioLegend, catalog number 152604), Alexa Cells were stained with 647 anti-mouse / human GL7 (BioLegend, catalog number 144606) and streptavidin-labeled PE (BioLegend).

[0132] Lung tissue was collected aseptically and minced. Subsequently, in a buffer containing DMEM culture medium (Gibco, USA) supplemented with collagenase I (1 mg / mL, Gibco, USA), collagenase IV (0.5 mg / mL, Gibco) and DNase I (40 U / mL, KeyGen biotech), tissue was enzymatically digested for 1 hour at 37°C. After digestion, the tissue homogenate was filtered through a 70-mesh sieve, erythrocyte lysis was performed, and washed to obtain a single cell suspension. Lymphocytes in the spleen were separated using mouse lymphocyte separation fluid. T cells from lung tissue or lymphocytes from the spleen were cultured overnight in 1640 complete culture media with a spike peptide pool (1 μg / mL). Brefeldin A (BFA, Invitrogen, catalog number 00-4506-51) was added to the culture 4 hours before the cells were collected to block intracellular cytokine secretion. Subsequently, the cells were stained with PerCP / Blue 5.5-labeled anti-mouse CD3, APC anti-mouse CD4 (BioLegend, catalog number 100412), and FITC anti-mouse CD8 (BioLegend, catalog number 100705) antibodies at 4°C. The cells were then fixed and incubated with PE / Blue 7 anti-mouse IFN-γ (BioLegend, catalog number 505826) and Brilliant Violet 510. TMAnti-mouse TNF-α (BioLegend, catalog number 506339) antibody treatment. In splenocytes, RBD+ plasma cells were detected by first treating the cells with biotinylated RBD protein for 30 minutes and then washing with PBS. Brilliant Violet 510 was then used to detect RBD+ plasma cells. TM Anti-mouse CD4 (BioLegend, catalog number 116025), PE / Blue 5 anti-mouse CD19 (BioLegend, catalog number 115510), APC anti-mouse / human GL7 (BioLegend, catalog number 144618), FITC anti-mouse IgD (BioLegend, catalog number 405704), Brilliant Violet 421 TM Cells were stained with anti-mouse C138 (BioLegend, catalog number 144618) and streptavidin-PE to detect RBD+ plasma cells.

[0133] Example 13: Intranasal delivery of XBB.1.5 recombinant protein vaccine elicits broad neutralizing activity against the XBB lineage

[0134] In previous studies, intranasal delivery of recombinant protein antigens alone had poor immunogenicity and could hardly induce a visible immune response. Therefore, it was investigated whether MF59-like water-in-oil adjuvants could enhance the antigenicity of XBB.1.5 recombinant protein in the respiratory mucosa to induce substantial protective immunity. After a 21-day prime-boost regimen, 5 μg (low dose) or 10 μg (high dose) of XBB.1.5 recombinant protein vaccine were administered intranasally to mice (Figure 9a). Mice received PBS or XBB.1.5 recombinant protein alone as a control group. Antigen-specific binding antibody assays showed that XBB.1.5 recombinant protein alone could hardly elicit serum (Figure 9b) and mucosal (Figure 9f) RBD-specific antibodies, while all animals in the group immunized with adjuvanted protein showed significant improvement in the endpoint titers of antigen-specific IgG and IgA. Similar improvements were observed in rats to which the XBB.1.5 recombinant protein vaccine was delivered intranasally (Figure 9c). In addition, it was found that both helper T cell type 1 (Th1) and Th2-biased immune responses could be elicited by the adjuvanted XBB.1.5 recombinant protein vaccine, as evidenced by the production of various antibody subtypes, including antigen-specific IgG1, IgG2a, IgG2b, IgG2c, and IgG3 antibodies (Figure 9d). High-dose XBB.1.5 recombinant protein vaccine induced stronger Th1-biased immunity, as determined by a higher ratio of IgG2a / IgG1 antibodies.

[0135] Neutralizing antibody responses are a key indicator of vaccine protective efficacy. Therefore, neutralizing activity against XBB lineage variants was assessed after intranasal immunization. Although neutralizing antibody (NAb) levels showed only modest improvement on day 35 after immunization, both doses resulted in an order of magnitude increase in neutralizing antibodies after the third dose. Specifically, the group receiving low-dose adjuvanted XBB.1.5 recombinant protein vaccine showed 50% neutralization geometric mean titers (GMTs) against several pseudoviruses, including XBB (2890), XBB.1.5 (17489), XBB.1.6 (5062), XBB.1.9.1 (18195), XBB.1.16 (3691), XBB.2.3 (23886), EG.5.1 (23899) and JN.1 (5516), while the GMTs in the group receiving high-dose vaccine were determined to be 2457, 13216, 3989, 17764, 4695, 14099, 15384 and 6618 (Figure 9e). Furthermore, such robust neutralizing antibody protection was also evident in bronchoalveolar lavage fluid (BALF) in addition to serum samples (Figure 9g), indicating that intranasal delivery of the XBB.1.5 recombinant protein vaccine was able to induce systemic and mucosal humoral immune responses, characterized by high levels of neutralizing antibodies against XBB lineage variants.

[0136] Example 14: Intranasal delivery of XBB.1.5 recombinant protein vaccine induces upper airway cellular immune response

[0137] Tissue-resident memory cells (T RM ) are considered essential components of mucosal immune responses in host defense because they can rapidly respond to pathogens at the site of infection. We then quantified CD8 + and CD4 + T RM cells (CD44 + CD103 + CD69 + Both doses of intranasal XBB.1.5 recombinant protein vaccine produced a high number of CD4 + T RM (Figure 10a). Although there was no significant difference between the control group and the low-dose vaccine group in CD8 + T RM There was no statistically significant difference in the number of mucosal CD8 cells, but an increase in frequency was observed, and the high-dose vaccine further enhanced the + T RM Induction.

[0138] Intracellular cytokine staining (ICS) was used to further evaluate the lung antigen-specific cellular immune response. Lung tissue from vaccinated mice was isolated and processed into single-cell suspensions, which were then stimulated ex vivo with a full-length spike peptide library to detect the expression of intracellular cytokines. In the absence of the stimulating peptide library, T cells in the lungs appeared to be in a resting state, whereas we noted that both doses of the intranasal XBB.1.5 recombinant protein vaccine resulted in a significant increase in the percentage of IFN-γ secreted after stimulation, including CD8 + T cells and CD4 T cells secreting IFN-γ and TNF-α + T cells, and high-dose immunization further enhanced these improvements (Figure 10b). Therefore, the induction of multiple cytokine secretion in antigen-specific T cells suggests the presence of multifunctional cellular immune responses in the respiratory mucosa.

[0139] We also evaluated the germinal center (GC) responses induced by the intranasal XBB.1.5 recombinant protein vaccine in the mediastinal lymph nodes (MLN), as GC B and T follicular helper (Tfh) cells are crucial for long-term protective immune responses. As expected, GC B cells (CD19 ) were significantly increased in immunized mice compared to mice that received PBS and XBB.1.5 recombinant protein antigen alone. + GL7 + CD95 + ) and Tfh cells (CD4 + CXCR5 + PD-1 + ) were significantly more frequent in the MLN (Figure 10c). In addition, more RBD-specific B cells were detected in the MLN, and more cells were involved in antibody production (Figure 10c). These findings strongly support the view that intranasal XBB.1.5 recombinant protein vaccine can induce robust and long-lasting mucosal immune responses.

[0140] Example 15: Protective immunity elicited by combined vaccination with intramuscular and intranasal delivery of XBB.1.5 recombinant protein vaccines

[0141] To further explore the potential of combined intramuscular and intranasal delivery of XBB.1.5 recombinant protein vaccines and determine whether this combined approach can elicit superior immune responses, immunization experiments were performed in mice using different vaccination schedules (Figures 11a-b). NIH mice were initially injected intramuscularly with 10 μg of XBB.1.5 recombinant protein vaccine on days 0 and 21, and three doses (2×IM+1×IN) were administered intranasally on day 42. In addition, mice were vaccinated intramuscularly and subsequently received two booster doses (1×IM+2×IN) by intranasal delivery. The control group consisted of mice treated with PBS or three doses of intranasal XBB.1.5 recombinant protein vaccine administered by intranasal delivery (3×IN).

[0142] No differences in serum RBD-specific IgG antibody levels were observed between the three vaccination regimens (Figure 11c). However, it is noteworthy that the 1×IM+2×IN immunization regimen elicited the highest levels of neutralizing antibodies in all tested groups (Figure 11e). Although all regimens involving at least one intranasal vaccine delivery induced significant antibody responses in the respiratory tract, the 1×IM+2×IN regimen produced stronger binding and neutralizing antibody responses compared to the 2×IM+1×IN group (Figures 11d and 11f).

[0143] To comprehensively evaluate the systemic and mucosal cellular immune responses induced by different vaccination regimens, we analyzed the generation of effector immune responses in spleen, lung and lymph node tissues. All regimens that included at least one intranasal protein vaccine delivery demonstrated the ability to elicit substantial respiratory mucosal cellular immune responses (Figure 12a). In addition, intranasal immunization induced germinal center responses in mediastinal lymph nodes, as demonstrated by increased percentages of Tfh, GC B and RBD-specific B cells (Figure 12b). However, three intranasal deliveries of the XBB.1.5 recombinant protein vaccine resulted in negligible production of antigen-specific T cells and memory B cells (MBC) in spleen tissue, indicating that intranasal immunization elicited a weak systemic cellular immune response (Figure 12c). Importantly, both the 2×IM+1×IN and 1×IM+2×IN immunization regimens overcame this limitation by increasing the frequency of IFN-γ-secreting T cells and MBC in the spleen. Therefore, the combination of intramuscular and intranasal immunization, especially one intramuscular dose followed by two intranasal deliveries of XBB.1.5 recombinant protein vaccine (1×IM+2×IN), can elicit mucosal and systemic immune responses to protect against infection with the new coronavirus mutant strains.

[0144] Example 16: Mucosal and systemic immune responses elicited by intranasal delivery of XBB.1.5 recombinant protein vaccine followed by heterologous sequential mRNA vaccine

[0145] Messenger RNA (mRNA)-based vaccines for the novel coronavirus have been widely deployed globally. However, repeated administration of the same type of vaccine may reduce the ability of heterologous vaccination to elicit enhanced immunity. mRNA vaccines are primarily administered via the intramuscular route, limiting the potential and utilization of mucosal immunity. Therefore, we evaluated the characteristics of intranasal XBB.1.5 recombinant protein as an additional heterologous booster injection in a sequential immunization schedule.

[0146] NIH mice were immunized intramuscularly with an mRNA vaccine based on the full-length BA.5 spike protein on days 0, 21, and 42, followed by either a fourth dose of the homologous mRNA vaccine (4×mRNA) or a heterologous intranasal delivery of an XBB.1.5 recombinant protein vaccine (3×mRNA+1×XBB.1.5 recombinant protein) on day 63 (Figures 13a–b). Additionally, another group of mice received two doses of the mRNA vaccine, followed by two intranasal administrations of the XBB.1.5 recombinant protein vaccine (2×mRNA+2×XBB.1.5 recombinant protein) (Figures 13a–b), with both mRNA and XBB.1.5 recombinant protein vaccines administered at a dose of 10 μg each time. Notably, a single intranasal dose of the XBB.1.5 recombinant protein vaccine did not appear to induce a stronger systemic humoral immune response, as evidenced by similar serum binding and neutralizing antibody levels compared to the 4×mRNA group (Figures 13c and 13e). However, two injections of mRNA followed by two intranasal delivery of XBB.1.5 recombinant protein (2×mRNA+2×XBB.1.5 recombinant protein) induced a better whole-body humoral immune response, with the highest levels of serum antigen-specific IgG (Figure 13c) and neutralizing antibodies (Figure 13e). Compared with the homologous mRNA vaccination group (4×mRNA), the 2×mRNA+2×IN XBB.1.5 recombinant protein group had GMTs of 10.69, 52.05, 12.81, 11.39, 28.10, 20.89, 32.43, and 105.42 times for XBB, XBB.1.5, XBB.1.6, XBB.1.9.1, XBB.1.16, XBB.2.3, EG.5.1, and JN.1 pseudoviruses, respectively. Consistent with the antibody assay, the memory B cells in the lung tissue were significantly improved by two intranasal deliveries rather than a single dose of the XBB.1.5 recombinant protein vaccine. However, we observed enhanced mucosal antibody responses induced by at least one intranasal delivery of the XBB.1.5 recombinant protein vaccine (Figures 13d and 13f).

[0147] Local mucosal cellular immune responses were also assessed. Consistent with the findings from the respiratory antibody assays, intranasal delivery of the XBB.1.5 recombinant protein vaccine resulted in mucosal T RM Heterologous vaccination consisted of two pre-injections of mRNA followed by two doses of XBB.1.5 recombinant protein vaccine (2×mRNA+2×IN XBB.1.5 recombinant protein), resulting in CD8 + and CD4 + T RM The cell counts were the highest (Figure 13i), and the CD4 secretion of IFN-γ and TNF-α + The proportion of T cells was the highest (Figure 13j). RMThe number of cells increased without statistical significance, but it was observed that a single booster dose of XBB.1.5 recombinant protein vaccine (3×mRNA+1×IN XBB.1.5 recombinant protein) may not be sufficient to induce better mucosal T cell immune responses compared with homologous mRNA vaccination (4×mRNA). However, both single and double intranasal administration of XBB.1.5 recombinant protein vaccine induced a large number of GC B cells, Tfh cells, and antigen-specific B cell responses in mediastinal lymph nodes (Figure 13g-h). These findings suggest that intranasal XBB.1.5 recombinant protein vaccine, especially when administered in two doses, represents a promising candidate for boosting in heterologous immunization strategies to elicit superior mucosal and systemic immune responses.

[0148] Example 17: Intranasal XBB.1.5 recombinant protein vaccine protection against EG.5.1 variant challenge

[0149] Mice were challenged with Omicron EG.5.1 live virus: NIH mice (6-8 weeks old) in the vaccine group were given three doses of 10 μg XBB.1.5 recombinant protein vaccine intranasally on days 0, 21, and 42, while mice in the control group received three doses of adjuvant. 21 days after the last immunization, live EG.5.1 virus (1×10 6 All mice were challenged intranasally with 100 μg / mL of 50 μg / mL of 1 μg / mL of 1 μg / mL of 2 ...1 μg / mL of 2 μg / mL of 1 μg / mL of 1 μg / mL of 2 μg / mL of 1 μg / mL of 1 μg / mL of 1 μg / mL of 2 μg / mL of 1 μg / mL of 1 μg / mL of 1 μg / mL of 2 μg / mL of 1 μg / mL of 1 μg / mL of 1 μg / mL of 2 μg / mL of 1 μg / mL of 1 μg / mL of 1 μg / mL of 1 μg / mL of 1 μg / mL of 1 μg / mL of 1 μg / mL of 1 μg / mL of 1 μg / mL of 1 μg / mL of

[0150] The efficacy of intranasal XBB.1.5 recombinant protein vaccine against circulating EG.5.1 virus was evaluated. NIH mice were immunized intranasally with three doses of high-dose XBB.1.5 recombinant protein vaccine (10 μg per mouse) and then immunized intranasally with 1×10 6 Plaque forming units (PFU) of live EG.5.1 virus challenge. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay was used to monitor changes in viral load in throat swabs every day. Throughout the experiment, samples from mice administered PBS showed high levels of viral load (Figure 14a). In contrast, intranasal administration of the XBB.1.5 recombinant protein vaccine significantly reduced viral genomic RNA (gRNA) on day 1 after infection (dpi) and demonstrated rapid clearance of the virus. By day 5 dpi, negligible viral load was observed in the throat swab samples of the group receiving the intranasal XBB.1.5 recombinant protein vaccine.

[0151] On day 5 dpi, the mice were then euthanized for tissue collection. Intranasal immunization with the XBB.1.5 recombinant protein vaccine resulted in a significant decrease in viral gRNA levels in nasal turbinates and lung tissues compared to administration of PBS (Figure 14b). In addition, no detectable gRNA was found in the tracheal tissue of the vaccinated group. These findings strongly suggest that the intranasal XBB.1.5 recombinant protein vaccine provides effective protection against live Omicron virus in both the upper and lower respiratory tracts.

[0152] In addition to changes in viral load, the effective protection conferred by intranasal XBB.1.5 recombinant protein vaccination was also associated with significantly reduced histopathological changes. In the lung tissues of mice treated with PBS, mild pathological changes were evident, including multifocal areas of consolidation, mild thickening of the alveolar septa, alveolar congestion, and small patches of inflammation composed of lymphocytes, neutrophils, and macrophages (Figures 14c–d). In contrast, lung tissues from the group receiving intranasal XBB.1.5 recombinant protein vaccine exhibited normal histological structure with intact alveolar architecture and no obvious inflammation. Therefore, intranasal administration of XBB.1.5 recombinant protein vaccination effectively protected the upper and lower respiratory tracts from challenge with live EG.5.1 virus.

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, EG.5.1, FL.1.5.1, HV.1, HK.3, 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 / or XBB.1.5 recombinant protein vaccine against 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 inactivated vaccine and / or XBB.1.5 recombinant protein vaccine against SARS-CoV-2 or its variants infection, 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 and pharmaceutically acceptable 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 at least one of an injection preparation, an intranasal preparation, and an oral preparation; preferably, the vaccine is an intramuscular injection preparation and / or an intranasal preparation; more preferably, the vaccine is administered at least twice when administered intranasally.

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, EG.5.1, FL.1.5.1, HV.1, HK.3, BA.2.86 or JN.

1.

20. The use according to any one of claims 1 to 3 or any one of claims 17 to 19, characterized in that: The dosage form of the drug is at least one of an injection preparation, an intranasal preparation, and an oral preparation; preferably, the drug is an intramuscular injection preparation and / or an intranasal preparation; more preferably, the recombinant protein vaccine in the drug is administered at least twice when administered intranasally.

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