Recombinant protein vaccine for preventing and treating SARS-CoV-2 variants JN.1, BA.2. 86 and XBB pedigree, combined medicine and application

Abstract

The invention belongs to the field of medicines, and particularly relates to a recombinant protein vaccine for preventing and treating SARS-CoV-2 variants JN.1, BA.2. 86 and XBB pedigree, a combined medicine and application. In order to solve the problem that Aumitron JN.1, BA.2. 86 and XBB pedigree variants show significant immune escape and blot immune phenomena after previous vaccination and virus infection, the invention provides an XBB.1. 5 recombinant protein vaccine, which induces powerful body fluid, cell and mucous membrane immune responses to JN.1, BA.2. 86 and XBB pedigree variants by intramuscular and intranasal vaccination. Compared with homologous vaccine inoculation, the inactivated or mRNA vaccine is applied and then heterologous vaccine inoculation is carried out by using the vaccine, so that better immune response is obtained. And the vaccine induces effective protective immunity against the attack of the live Omitron EG.5.1 virus in vivo. Therefore, the XBB.1.5 vaccine disclosed by the invention has clinical use value.

Description

Technical Field

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

[0002] Since the first discovery of the Omicron (B.1.1.529) BA.1 variant of severe acute respiratory coronavirus-2 (SARS-CoV-2), the evolutionary trajectory of the virus has deviated significantly. Since then, Omicron has quickly become the dominant variant, spreading around the world and producing a variety of descendant subvariants, including well-known subvariants such as BA.2, BA.5 and BQ.1.1, leading to widespread infection around the world. With the prevalence of these subvariants, 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 subvariants. It is worth noting that a rare amino acid mutation (F486P) occurred in the spike protein of some XBB lineage strains, such as XBB.1.5 and XBB.1.16, which spread rapidly in the past few months and became 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. For example, a subvariant outside the XBB lineage, BA.2.86, was discovered. 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 that 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 continued threat of COVID-19 reinfection highlights the critical importance of widespread vaccination to reduce the risks associated with symptomatic infection, severe disease, and death. However, on the one hand, emerging JN.1 and XBB lineage subvariants have demonstrated significant immune evasion capabilities, which significantly impairs neutralization in individuals who receive booster shots 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) has relatively high efficacy against several Omicron subvariants, neutralization activity against XBB subvariants is still significantly reduced. On the other hand, the phenomenon of immunological imprinting must be considered during vaccine development, and this repeated prior antigen exposure may hinder the response of neutralizing antibodies to new Omicron variants. Experimental studies in animal models have confirmed vaccination-induced imprinting immunity, highlighting its relevance in human vaccine development. In addition, studies have shown that repeated vaccination with wild-type spike mRNA vaccines or inactivated vaccines in humans tends to bias immune responses toward earlier lineage variants, thereby reducing immune responses to newer Omicron subvariants. Similarly, bivalent vaccines only enhanced immune responses to Omicron subvariants to a limited extent. These findings highlight the urgent need to re-evaluate the composition of current vaccines and accelerate the development of next-generation novel coronavirus vaccines specifically targeting the evolving XBB lineage. Furthermore, unlike most currently approved novel coronavirus vaccines that are administered by 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 vaccine platforms for intranasal vaccine development due to their safety, cost-effectiveness of large-scale production, and ease of transportation. However, pure protein antigens are often rapidly cleared by 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 added with various intranasal adjuvants to improve the immune effect, including polymers, membrane vesicles, nanoparticles, etc., to increase the magnitude and durability of the immune response. However, these adjuvants have not yet been widely clinically approved, and their safety is a concern. Therefore, it is crucial to develop a next-generation SARS-CoV-2 recombinant protein vaccine that specifically targets the XBB lineage and has high safety and efficacy. Summary of the invention

[0004] Considering that Omicron JN.1, BA.2.86 and XBB lineage subvariants are prevalent around the world, they show breakthrough and significant immune escape after previous vaccination and viral infection, as well as imprinted immune phenomena, and there is an urgent need to develop a next-generation novel coronavirus vaccine specifically for emerging subvariants. The present invention successfully constructs and expresses the XBB.1.5 recombinant protein by connecting the receptor binding domain (RBD) sequence from the XBB.1.5 variant with a partial sequence from the spike S2 ​​subunit in series, and mixes it with the nationally approved and safe, well-tolerated adjuvant MF59 to prepare the XBB.1.5 recombinant protein vaccine, which induces stable and lasting humoral and cellular immune responses against the currently popular JN.1, BA.2.86 and XBB lineage variants for intramuscular and intranasal immunization, especially intranasal immunization induces high levels of neutralizing antibodies against XBB lineage subvariants, producing effective mucosal immune responses. 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 better immune responses and more effective booster effects are obtained. 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.

[0005] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0006] In the 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.

[0007] Further, 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.

[0008] 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.

[0009] 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, which are administered separately, simultaneously or sequentially.

[0010] 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.

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

[0012] Furthermore, the inactivated vaccine is an inactivated vaccine prepared based on wild SARS-CoV-2 virus or variant.

[0013] 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.

[0014] 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.

[0015] Furthermore, the method for preparing the XBB.1.5 recombinant protein comprises the following steps: culturing a host cell containing the XBB.1.5 recombinant protein or a protein precursor to express the protein or precursor, and then recovering the protein.

[0016] Preferably, 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 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.

[0017] 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.

[0018] Furthermore, the host cell is at least one of insect cells, mammalian cells, Escherichia coli, and yeast.

[0019] 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.

[0020] Furthermore, the XBB.1.5 recombinant protein vaccine is obtained by mixing the 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.

[0021] Preferably, the auxiliary component is an immune adjuvant.

[0022] 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, monophosphoryl lipid A, muramyl dipeptide, muramyl tripeptide, bacterial toxin, GM-CSF cytokine, lipid, cationic liposome material.

[0023] 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-dioleyl chloride)propyl]-N,N,N-trimethylammonium chloride, cationic cholesterol, trifluoroacetic acid dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, dimethyldioctadecylammonium bromide, CpG ODN.

[0024] Furthermore, the XBB.1.5 recombinant protein vaccine is at least one of an injection preparation, an intranasal preparation or an oral preparation. Preferably, the vaccine is an intramuscular injection preparation and / or an intranasal preparation.

[0025] The dosage form of the XBB.1.5 recombinant protein vaccine of the present invention is an intramuscular injection preparation and / or an intranasal administration preparation, which means that different dosage forms of the XBB.1.5 recombinant protein vaccine can be combined, such as simultaneous intramuscular and intranasal immunization of the XBB.1.5 recombinant protein vaccine for systemic and mucosal protective immunity. Preferably, the vaccine is administered intranasally at least twice.

[0026] 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.

[0027] Further, 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.

[0028] 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.

[0029] In the first aspect above, the use of XBB.1.5 recombinant protein or vaccine in the preparation of a drug for preventing and / or treating infection with SARS-CoV-2 and its variants;

[0030] 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 medicament for preventing and / or treating infection by SARS-CoV-2 and its variants; the dosage form of the medicament is at least one of an injection preparation, an intranasal preparation, and an oral preparation; preferably, the medicament is an intramuscular injection preparation and / or an intranasal preparation.

[0031] 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 immunized simultaneously through muscle and intranasal immunization for systemic and mucosal protective immunity.

[0032] More preferably, when intramuscular and intranasal immunization are used in combination, the recombinant protein vaccine in the medicament is administered intranasally at least twice.

[0033] Sequence information of the novel coronavirus XBB.1.5 recombinant protein vaccine of the present invention:

[0034] Novel coronavirus XBB.1.5 recombinant protein construction nucleic acid sequence: SEQ ID No.1

[0035]

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

[0037] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYA DSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0038] Novel coronavirus XBB.1.5 recombinant protein vaccine antigen nucleic acid sequence: SEQ ID No.3

[0039] GTGCAGCCAACCGAATCAATCGTCCGCTTCCCAAACATCACTAACCTGTGCCCATTCCACGAGGTGTTCAACGCCACCACTTTCGCCTCTGTCTACGCTTGGAACCGCAAGCGCATCTCCAACTGTGTCGCCGACTACAGCGTGATATACAACTTCGCTCCTTTCTTCGCATTCAAGTGTTACGGAGTGTCCCCTACCAAGCTCAACGATTTGTGCTTCACAAACGTGTACGCTGACAGCTTCGTGATCCGTGGTAACGAGGTTTCTCAAATCGCCCCTGGTCAGACAGGTAACATCGCTGACTACAACTACAAGCTCCCAGACGACTTCACTGGCTGTGTTATCGCTTGGAACTCAAACAAGCTGGACTCTAAGCCGTCTGGTAACTACAACTACCTCTACCGTCTGTTCCGCAAGAGCAAGCTCAAGCCATTCGAACGTGACATCAGCACAGAGATCTACCAAGCCGGTAACAAGCCCTGCAACGGCGTTGCCGGACCTAACTGCTACTCTCCCCTCCAGTCCTACGGATTCCGCCCAACCTACGGCGTGGGTCACCAGCCATACCGCGTGGTTGTGCTGAGCTTCGAACTGCTGCACGCCCCCGCTACCGTGTGTGGCCCTAAGAAGAGCACTAACCTCGTTAAGAACAAGTCCGTGAACTTCAACTTCAACGGTCTCTACGAAAACCAGAAGCTGATCGCTAACCAGTTCAACTCCGCCATCGGCAAGATCCAGGACTCCCTGTCCTCCACAGCTTCCGCTCTGGGCAAGCTGCAGGACGTTGTGAACCAAAACGCTCAGGCCCTGAACACACTGGTGAAGCAACTGAAGAACCACACCAGCCCCGACGTTGACCTGGGTGACATCAGCGGTATCAACGCTAGCGTGGTGAACATCCAGAAGGAGATCGACAGGCTGAACGAAGTGGCTAAGAACTTGAACGAGAGCCTCATCGACCTGCAGGAACTGTAA

[0040] Novel coronavirus XBB.1.5 recombinant protein vaccine antigen protein sequence: SEQ ID No.4

[0041] VQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCN GVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0042] 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, and develops a novel coronavirus XBB.1.5 recombinant protein vaccine, which is a recombinant protein (whose amino acid sequence is shown in SEQ ID No.4) expressing the XBB.1.5RBD and S2 sequences on an insect cell (Sf9) protein expression platform, and a water-in-oil adjuvant similar to MF59 is incorporated to prepare the XBB.1.5 monovalent protein vaccine. Experiments have shown that the vaccine (with SEQ ID No.4 as the antigen protein sequence) induces stable and persistent humoral and cellular immune responses against the currently popular JN.1, BA.2.86 and XBB lineage variants by intramuscular immunization, especially improving the level of neutralizing antibodies against subvariant lineages including JN.1, and generating a large number of antigen-specific T memory cell populations. Vaccination with 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.

[0043] Meanwhile, the vaccine elicited a strong humoral immune response in mice and rats by intranasal administration, induced high levels of neutralizing antibodies against XBB lineage subvariants, and produced an effective mucosal immune response characterized by the induction of tissue-resident T cells, local cellular immunity, and germinal center responses in the respiratory tract. Moreover, the combined intramuscular and intranasal delivery of the XBB.1.5 recombinant protein vaccine showed excellent systemic and mucosal protective immunity.

[0044] In addition, heterologous vaccination with either intramuscular or intranasal monovalent recombinant vaccines after administration of inactivated or mRNA-based vaccines resulted in better immune responses and more effective boosting effects compared with homologous vaccination. In vivo challenge experiments demonstrated that the novel coronavirus XBB.1.5 recombinant protein vaccine effectively protected the body from infection with live virus of the EG.5.1 strain. These findings suggest that it has good clinical potential in preventing the currently circulating Omicron variant. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The molecular sieve and SDS-PAGE identification diagram of the recombinant protein of Example 1XBB.1.5; M is the protein marker, 1, 2, 3 are the molecular weights of the target protein after removing all redundant amino acids;

[0046] Figure 2 This is a graph showing the pseudovirus neutralizing antibody results of the XBB.1.5 recombinant protein vaccine mice immune serum in Example 4;

[0047] Figure 3 This is an ELISpot assay for specific cellular immunity after 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 after stimulation with the peptide library of the XBB.1.5 spike protein (n = 5 mice per group).

[0048] Figure 4 The results of flow cytometry detection of XBB.1.5 recombinant protein vaccine-specific cellular immunity in Example 6; ab, secretion of IFN-γ after stimulation with spike peptide library + (a) and TNF-α + (b) CD4 + and CD8 + Percentage of memory T cells (n = 5 mice per group). cd, Tfh (CD4 + CXCR5 + PD-1 + ) and GC B cells (CD19 + GL7 + CD95 + ) percentage.

[0049] Figure 5 This is a graph of true virus neutralizing antibodies in the immune serum of mice vaccinated with the XBB.1.5 recombinant protein vaccine in Example 7;

[0050] Figure 6 The results of long-term protective immunity induced by the XBB.1.5 recombinant protein vaccine in Example 8 are shown in Figure 2; 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 at 43 weeks after the first immunization and the 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).

[0051] Figure 7 The neutralizing antibody results of the heterologous sequential inactivated vaccine and mRNA vaccine of XBB.1.5 recombinant protein vaccine in Example 9 are shown in Figure 4; ac, NIH mice were immunized three times with inactivated virus vaccine, followed by one dose of homologous vaccine inactivated vaccine (IV×3>IV), or one dose of heterologous inoculation of XBB.1.5 recombinant protein vaccine (IV×3>XBB.1.5) (n=6 mice per group) to measure the neutralizing antibodies against real virus (b) and pseudovirus (c) in serum samples. d, NIH mice were immunized with three doses of full-length spike BA.5 mRNA vaccine, followed by one dose of homologous inoculation of mRNA vaccine (mRNA×3>mRNA), or one dose of heterologous inoculation of XBB.1.5 recombinant protein vaccine (mRNA×3>XBB.1.5) (n=6 mice per group) to measure the neutralizing antibody titers of immune serum against various Omicron XBB pseudoviruses. 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.

[0052] Figure 8 The results of the challenge with Omicron EG.5.1 live virus in mice immunized with the recombinant protein vaccine of Example 10 are shown in Figure 10; a, 1×10 6PFU live EG.5.1 true virus was instilled into NIH mice immunized with 100 μL of the new coronavirus XBB.1.5 recombinant protein vaccine prepared in Example 3. Weight loss (a) and changes in viral genomic RNA (gRNA) in throat swabs (b) were monitored daily. c. On the 5th day after infection, the infected mice were euthanized to collect nasal concha, trachea and lung tissues, and then gRNA levels were detected by RT-qPCR. d. Histopathological changes and pathological scores of lung tissues of mice attacked by live EG.5.1 true virus. The scale bar represents 100 μm in d.

[0053] Fig. 9 This is a diagram of the humoral immune response results after immunization with intranasal delivery of 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 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 titer of anti-RBD IgG in sera from mice immunized intranasally with low-dose (5μg) and high-dose (10μg) XBB.1.5 recombinant protein vaccine (n=6 mice per group). c, Endpoint titer of serum anti-RBD IgG in rats receiving 40μg 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).

[0054] Fig.10 The results of the local mucosal immune response induced by intranasal delivery of XBB.1.5 recombinant protein vaccine in Example 14 are shown in Figure 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 pool 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).

[0055] Fig.11 The results of mucosal and systemic humoral immune responses induced by combined intramuscular and intranasal immunization of XBB.1.5 recombinant protein vaccine in Example 15 are shown in Figure 1; 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 against XBB lineage pseudovirus in serum (e) and BALF samples (f).

[0056] Fig.12 The results of mucosal and systemic cellular immune responses induced by combined intramuscular and intranasal immunization with XBB.1.5 recombinant protein vaccine in Example 15 are shown in Figure 15; a, the percentage of memory T cells producing antigen-specific IFN-γ or TNF-α in lung tissue after stimulation with peptide library of XBB.1.5 spike protein (n=6 mice per group). b, 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.

[0057] Fig.13The results of mucosal and systemic immune responses induced by heterologous sequential mRNA vaccines of intranasally delivered XBB.1.5 recombinant protein vaccine in Example 16 are shown in the figure; ab, NIH mice were immunized with three doses of full-length BA.5 spike mRNA vaccine, followed by one dose of homologous mRNA vaccine (4×mRNA), or one dose of heterologous intranasally delivered XBB.1.5 recombinant protein vaccine (3×mRNA+1×IN). Another group of mice received two mRNA vaccine injections and subsequent 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 against XBB lineage pseudovirus in serum (e) and BALF samples (f). gh, GCB in mediastinal lymph nodes (CD19 + 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).

[0058] Fig.14 The results of intranasal delivery of XBB.1.5 recombinant protein vaccine to immunize mice 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

[0059] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clear, the present application is further described in detail below in combination with the implementation methods. Unless otherwise defined, all scientific and technological terms used herein have the same meanings as understood by ordinary technicians in the field.

[0060] Abbreviations:

[0061] Monophosphoryl lipid A (MPL), squalane 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-dioleyl 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 (nucleotide sequence containing non-methylated cytosine and guanine dinucleotides as the core sequence, artificially synthesized CpG).

[0062] On the first hand, 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 showed 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 escape 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.

[0063] Second, the primary route of administration of approved SARS-CoV-2 vaccines (via intramuscular injection) has shown limited efficacy in eliciting sufficient local mucosal protective immunity. Given this pressing challenge posed by persistent infection in the population, there is an urgent need to accelerate the development of next-generation intranasal SARS-CoV-2 vaccines that can prevent 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 antigens. Selecting adjuvants that have been clinically evaluated in humans could significantly accelerate the clinical translation process.

[0064] 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 shown good tolerability and safety in humans. However, little is known about whether this adjuvant can be used with SARS-CoV-2 antigens to formulate an effective intranasal SARS-CoV-2 vaccine. Although existing studies have demonstrated the effectiveness of MF59 as an adjuvant for intranasal SARS-CoV-2 vaccines, the specific immune mechanisms by which MF59-like oil-in-water adjuvants induce strong mucosal immunity have not been thoroughly studied and deserve further investigation.

[0065] Finally, the antigenic composition of the vaccine is a key determinant of its effectiveness, particularly in the context of novel coronavirus 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 universal Omicron subvariants. However, to date, no subunit protein-based intranasal vaccine has been developed for the XBB lineage.

[0066] Therefore, in view of the above analysis, in one embodiment of the present invention, the present invention successfully expressed the RBD recombinant protein based on the XBB.1.5 mutation, and mixed it with the adjuvant MF59 in equal volumes to prepare the XBB.1.5 recombinant protein vaccine, which is the world's first recombinant protein vaccine targeting the XBB lineage.

[0067] The experimental results show that the XBB.1.5 recombinant protein vaccine with 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 shows good safety and tolerability in humans, and shows excellent ability to induce strong neutralizing efficacy against the XBB lineage and the recent JN.1 subvariants. Emphasize 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 shows excellent immunogenicity in animal models, thus showing good clinical prospects.

[0068] Second, first, although Moderna and Pfizer / BioNTech updated the sequence of their mRNA vaccines to produce monovalent XBB.1.5 spike mRNA vaccines, XBB.1.5 monovalent mRNA vaccine boosters can confer high levels of neutralizing antibodies against XBB and JN.1 subvariants. However, considering the widespread administration of mRNA-based new coronavirus vaccines worldwide, choosing heterologous vaccination with alternative vaccine platforms as booster injections may induce a more robust immune response than repeated homologous vaccination with mRNA-based vaccines.

[0069] 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 an excellent immune response, characterized by increased serum neutralizing antibody levels in animal models. It can be seen that the new coronavirus XBB.1.5 recombinant protein vaccine of the present invention can be used as a safe and advantageous subunit protein-based booster vaccine to provide immune protection in this hybrid immune context.

[0070] Second, the hybrid immune landscape in a population is influenced by 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 for 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, given the widespread use of mRNA-based novel coronavirus vaccines worldwide, heterologous vaccination with alternative vaccine platforms and incorporating mucosal delivery may enhance systemic and mucosal immune responses.

[0071] Therefore, in another embodiment of the present invention, after mRNA vaccine injection, heterologous immunization by two intranasal administrations of XBB.1.5 recombinant protein vaccine can achieve excellent antibody response, accompanied by a strong local mucosal immune response. Compared with homologous vaccination, a single single dose of intranasal XBB.1.5 recombinant protein vaccine does not significantly increase the level of neutralizing antibodies in serum. However, at least one intranasal delivery of XBB.1.5 recombinant protein vaccine does provide additional mucosal protective immunity.

[0072] On the third hand, one disadvantage of intranasal administration is that it tends to cause 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, the strategy of combining intramuscular and intranasal vaccination routes seems to provide a feasible solution to this challenge. In a specific embodiment of the present invention, at least one intramuscular injection before intranasal administration can induce a systemic cellular immune response (e.g., 2×IM+1×IN, 1×IM+2×IN). Given that a large part of the population has received at least one dose of intramuscular vaccine, this problem may not be a substantial problem. In addition, compared with other groups, two intranasal administrations of XBB.1.5 recombinant protein vaccines 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 responses in 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.

[0073] Specific examples will be listed below to explain the scheme of the present invention. It will be appreciated by those skilled in the art 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 indicated in the examples, the techniques or conditions described in the literature in this area or the product instructions are used. Reagents or instruments used that do not indicate manufacturers are all conventional products that can be obtained commercially.

[0074] Experimental data are presented as geometric mean ± SD or mean ± SEM, and statistical analysis was performed using Prism 9.0 (GraphPad software). When analyzing comparisons between two groups or one-way ANOVA between multiple groups, Student's t test was used to determine P values. ****P ≤ 0.0001; ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05; ns, not significant.

[0075] The mRNA vaccine preparation method used in the embodiment is as follows:

[0076] The amino acid sequence of the mRNA vaccine antigen protein is shown in SEQ ID No.5:

[0077] MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTW

[0078] FHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIK

[0079] VCEFQFCNDPFLDVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNL

[0080] REFVFKNIDGYFKIYSKHTPINLGRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGD

[0081] SSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQ

[0082] TSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAF

[0083] KCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNS

[0084] NKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRP

[0085] TYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFL

[0086] PFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVA

[0087] IHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHRRAR

[0088] SVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS

[0089] NLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKYFGGFNFSQILPDPSKPS

[0090] KRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTS

[0091] ALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQD

[0092] SLSSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLNDILSRLDPPEAEVQIDRLITG

[0093] RLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHG

[0094] VVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF

[0095] VSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEI

[0096] DRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCL

[0097] KGCCSCGSCCKFDEDDSEPVLKGVKLHYT

[0098] The DNA sequence of the mRNA vaccine antigen is shown in SEQ ID No.6:

[0099] ATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCCAGTGCGTGAACCTGATCA

[0100] CCAGGACCCAGAGCTACACCAACAGCTTCACCAGGGGCGTGTACTACCCCGACAAGGT

[0101] GTTCAGGAGCAGCGTGCTGCACAGCACCCAGGACCTGTTCCTGCCCTTCTTCAGCAACG

[0102] TGACCTGGTTCCACGCCATCAGCGGCACCAACGGCACCAAGAGGTTCGACAACCCCGT

[0103] GCTGCCCTTCAACGACGGCGTGTACTTCGCCAGCACCGAGAAGAGCAACATCATCAGG

[0104] GGCTGGATCTTCGGCACCACCCTGGACAGCAAGACCCAGAGCCTGCTGATCGTGAACA

[0105] ACGCCACCAACGTGGTGATCAAGGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTG

[0106] GACGTGTACTACCACAAGAACAACAAGAGCTGGATGGAGAGCGAGTTCAGGGTGTACA

[0107] GCAGCGCCAACAACTGCACCTTCGAGTACGTGAGCCAGCCCTTCCTGATGGACCTGGA

[0108] GGGCAAGCAGGGCAACTTCAAGAACCTGAGGGAGTTCGTGTTCAAGAACATCGACGGC

[0109] TACTTCAAGATCTACAGCAAGCACACCCCCATCAACCTGGGCAGGGACCTGCCCCAGGG

[0110] CTTCAGCGCCCTGGAGCCCCTGGTGGACCTGCCCATCGGCATCAACATCACCAGGTTCC

[0111] AGACCCTGCTGGCCCTGCACAGGAGCTACCTGACCCCCGGCGACAGCAGCAGCGGCTG

[0112] GACCGCCGGCGCCGCCGCCTACTACGTGGGCTACCTCCAGCCCAGGACCTTCCTGCTGA

[0113] AGTACAACGAGAACGGCACCATCACCGACGCCGTGGACTGCGCCCTGGACCCCCTGAG

[0114] CGAAACCAAGTGCACCCTGAAGAGCTTCACCGTGGAGAAGGGCATCTACCAGACCAGC

[0115] AACTTCAGGGTGCAGCCCACCGAGAGCATCGTGAGGTTCCCCAACATCACCAACCTGT

[0116] GCCCCTTCGACGAGGTGTTCAACGCCACCAGGTTCGCCAGCGTGTACGCCTGGAACAG

[0117] GAAGAGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACTTCGCCCCCTTCT

[0118] TCGCCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAAC

[0119] GTGTACGCCGACAGCTTCGTGATCAGGGGCAACGAGGTGAGCCAGATCGCCCCCGGCC

[0120] AGACCGGCAACATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGT

[0121] GATCGCCTGGAACAGCAACAAGCTGGACAGCAAGGTGGGCGGCAACTACAACTACAGG

[0122] TACAGGCTGTTCAGGAAGAGCAACCTGAAGCCCTTCGAGAGGGACATCAGCACCGAGA

[0123] TCTACCAGGCCGGCAACAAGCCCTGCAACGGCGTGGCCGGCGTGAACTGCTACTTCCC

[0124] CCTGCAGAGCTACGGCTTCAGGCCCACCTACGGCGTGGGCCACCAGCCCTACAGGGTG

[0125] GTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACCGTGTGCGGCCCCAAGAAGA

[0126] GCACCAACCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGACCGGCAC

[0127] CGGCGTGCTGACCGAGAGCAACAAGAAGTTCCTGCCCTTCCAGCAGTTCGGCAGGGAC

[0128] ATCGCCGACACCACCGACGCCGTGAGGGACCCCCAGACCCTGGAGATCCTGGACATCA

[0129] CCCCATGCAGCTTCGGCGGCGTGAGCGTGATCACCCCCGGCACCAACACCAGCAACCA

[0130] GGTGGCCGTGCTGTACCAGGGCGTGAACTGCACCGAGGTGCCCGTGGCCATCCACGCC

[0131] GACCAGCTGACCCCCACCTGGAGGGTGTACAGCACCGGCAGCAACGTGTTCCAGACCA

[0132] GGGCCGGCTGCCTGATCGGCGCCGAGTACGTGAACAACAGCTACGAGTGCGACATCCC

[0133] CATCGGCGCCGGCATCTGCGCCAGCTACCAGACCCAGACCAAGAGCCACAGGAGGGCC

[0134] AGGAGCGTGGCCAGCCAGAGCATCATCGCCTACACCATGAGCCTGGGCGCCGAGAACA

[0135] GCGTGGCCTACAGCAACAACAGCATCGCCATCCCCACCAACTTCACCATCAGCGTGACC

[0136] ACCGAGATCCTGCCCGTGAGCATGACCAAGACCAGCGTGGACTGCACCATGTACATCTG

[0137] CGGCGACAGCACCGAGTGCAGCAACCTGCTGCTCCAGTACGGCAGCTTCTGCACCCAG

[0138] CTGAAGAGGGCCCTGACCGGCATCGCCGTGGAGCAGGACAAGAACACCCAGGAGGTG

[0139] TTCGCCCAGGTGAAGCAGATCTACAAGACCCCCCCCATCAAGTACTTCGGCGGCTTCAA

[0140] CTTCAGCCAGATCCTGCCCGACCCCAGCAAGCCCAGCAAGAGGAGCTTCATCGAGGAC

[0141] CTGCTGTTCAACAAGGTGACCCTGGCCGACGCCGGCTTCATCAAGCAGTACGGCGACTG

[0142] CCTGGGCGACATCGCCGCCAGGGACCTGATCTGCGCCCAGAAGTTCAACGGCCTGACC

[0143] GTGCTGCCCCCCCTGCTGACCGACGAGATGATCGCCCAGTACACCAGCGCCCTGCTGGC

[0144] CGGCACCATCACCAGCGGCTGGACCTTCGGCGCCGGCGCCGCCCTCCAGATCCCCTTCG

[0145] CCATGCAGATGGCCTACAGGTTCAACGGCATCGGCGTGACCCAGAACGTGCTGTACGAG

[0146] AACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCAAGATCCAGGACAGCC

[0147] TGAGCAGCACCGCCAGCGCCCTGGGCAAGCTCCAGGACGTGGTGAACCACAACGCCCA

[0148] GGCCCTGAACACCCTGGTGAAGCAGCTGAGCAGCAAGTTCGGCGCCATCAGCAGCGTG

[0149] CTGAACGACATCCTGAGCAGGCTGGACCCCCCCGAGGCCGAGGTGCAGATCGACAGGC

[0150] TGATCACCGGCAGGCTCCAGAGCCTCCAGACCTACGTGACCCAGCAGCTGATCAGGGC

[0151] CGCCGAGATCAGGGCCAGCGCCAACCTGGCCGCCACCAAGATGAGCGAGTGCGTGCTG

[0152] GGCCAGAGCAAGAGGGTGGACTTCTGCGGCAAGGGCTACCACCTGATGAGCTTCCCCC

[0153] AGAGCGCCCCCCACGGCGTGGTGTTCCTGCACGTGACCTACGTGCCCGCCCAGGAGAA

[0154] GAACTTCACCACCGCCCCCGCCATCTGCCACGACGGCAAGGCCCACTTCCCCAGGGAG

[0155] GGCGTGTTCGTGAGCAACGGCACCCACTGGTTCGTGACCCAGAGGAACTTCTACGAGC

[0156] CCCAGATCATCACCACCGACAACACCTTCGTGAGCGGCAACTGCGACGTGGTGATCGGC

[0157] ATCGTGAACAACACCGTGTACGACCCCCTCCAGCCCGAGCTGGACAGCTTCAAGGAGG

[0158] AGCTGGACAAGTACTTCAAGAACCACACCAGCCCCGACGTGGACCTGGGCGACATCAG

[0159] CGGCATCAACGCCAGCGTGGTGAACATCCAGAAGGAGATCGACAGGCTGAACGAGGTG

[0160] GCCAAGAACCTGAACGAGAGCCTGATCGACCTCCAGGAGCTGGGCAAGTACGAGCAGT

[0161] ACATCAAGTGGCCCTGGTACATCTGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGA

[0162] TGGTGACCATCATGCTGTGCTGCATGACCAGCTGCTGTAGCTGCCTGAAGGGCTGCTGT

[0163] AGCTGCGGCAGCTGCTGCAAGTTCGACGAGGACGACAGCGAGCCCGTGCTGAAGGGC

[0164] GTGAAGCTGCACTACACC

[0165] The antigen RNA sequence of the mRNA vaccine is shown in SEQ ID No.7:

[0166] AUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCCAGUGCGUGAACCUGA

[0167] UCACCAGGACCCAGAGCUACACCAACAGCUUCACCAGGGGCGUGUACUACCCCGACA

[0168] AGGUGUUCAGGAGCAGCGUGCUGCACAGCACCCAGGACCUGUUCCUGCCCUUCUUCA

[0169] GCAACGUGACCUGGUUCCACGCCAUCAGCGGCACCAACGGCACCAAGAGGUUCGACA

[0170] ACCCCGUGCUGCCCUUCAACGACGGCGUGUACUUCGCCAGCACCGAGAAGAGCAACA

[0171] UCAUCAGGGGCUGGAUCUUCGGCACCACCCUGGACAGCAAGACCCAGAGCCUGCUGA

[0172] UCGUGAACAACGCCACCAACGUGGUGAUCAAGGUGUGCGAGUUCCAGUUCUGCAAC

[0173] GACCCCUUCCUGGACGUGUACUACCACAAGAACAACAAGAGCUGGAUGGAGAGCGA

[0174] GUUCAGGGUGUACAGCAGCGCCAACAACUGCACCUUCGAGUACGUGAGCCAGCCCU

[0175] UCCUGAUGGACCUGGAGGGCAAGCAGGGCAACUUCAAGAACCUGAGGGAGUUCGUG

[0176] UUCAAGAACAUCGACGGCUACUUCAAGAUCUACAGCAAGCACACCCCCAUCAACCUG

[0177] GGCAGGGACCUGCCCCAGGGCUUCAGCGCCCUGGAGCCCCUGGUGGACCUGCCCAUC

[0178] GGCAUCAACAUCACCAGGUUCCAGACCCUGCUGGCCCUGCACAGGAGCUACCUGACC

[0179] CCCGGCGACAGCAGCAGCGGCUGGACCGCCGGCGCCGCCGCCUACUACGUGGGCUAC

[0180] CUCCAGCCCAGGACCUUCCUGCUGAAGUACAACGAGAACGGCACCAUCACCGACGCC

[0181] GUGGACUGCGCCCUGGACCCCCUGAGCGAAACCAAGUGCACCCUGAAGAGCUUCACC

[0182] GUGGAGAAGGGCAUCUACCAGACCAGCAACUUCAGGGUGCAGCCCACCGAGAGCAU

[0183] CGUGAGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGACGAGGUGUUCAACGCCAC

[0184] CAGGUUCGCCAGCGUGUACGCCCUGGAACAGGAAGAGGAUCAGCAACUGCGUGGCCG

[0185] ACUACAGCGUGCUGACAACUUCGCCCCCUUCUUCGCCUUCAAGUGCUACGGCGUGA

[0186] GCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGA

[0187] UCAGGGGCAACGAGGUGAGCCAGAUCGCCCCCGGCCGACCGGCAACAUCGCCGACU

[0188] ACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACA

[0189] AGCUGGACAGCAAGGUGGGCGGCCAACUACAACUACAGGUACAGGCUGUUCAGGAAG

[0190] AGCAACCUGAAGCCCUUCGAGAGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAAC

[0191] AAGCCCUGCAACGGCGUGGCCGGCGUGAACUGCUACUUCCCCUGCAGAGCUACGGC

[0192] UUCAGGGCCACCUACGGCGUGGGCCACCAGCCUACAGGGUGGUGGUGCUGAGCUUC

[0193] GAGCUGCUGCACGCCCCCGCCACCGUGUGCGGCCCAAGAAAGAGCACCAACCUGGUG

[0194] AAGAACAAGUGCGUGAACUUCAACUUCAACGGCCUGACCGGCACCGGCGUGCUGAC

[0195] CGAGAGCAACAAGAAGUUCCUGCCCUUCCAGCAGUUCGGCAGGGACAUCGCCGACAC

[0196] CACCGACGCCGUGAGGGACCCCCAGACCCUGGAGAUCCUGGACAUCACCCAUGCAG

[0197] CUUCGGCGGCGUGAGCGUGAUCACCCCCGGCACCAACACCAGCAACCAGGUGGCCGU

[0198] GCUGUACCAGGGCGUGAACUGCACCGAGGUGCCCGUGGCCAUCCACGCCGACCAGCU

[0199] GACCCCCACCUGGAGGGUGUACAGCACCGGCAGCAACGUGUUCCAGACCAGGGCCGG

[0200] CUGCCUGAUCGGCGCCGAGUACGUGAACAACAGCUACGAGUGCGACAUCCCCAUCGG

[0201] CGCCGGCAUCUGCGCCAGCUACCAGACCCAGACCAAGAGCCACAGGAGGGCCAGGAG

[0202] CGUGGCCAGCCAGAGCAUCAUCGCCUACACCAUGAGCCUGGGCGCCGAGAACAGCGU

[0203] GGCCUACAGCAACAACAGCAUCGCCAUCCCCACCAACUUCACCAUCAGCGUGACCAC

[0204] CGAGAUCCUGCCCGUGAGCAUGACCAAGACCAGCGUGGACUGCACCAUGUACAUCU

[0205] GCGGCGACAGCACCGAGUGCAGCAACCUGCUGCUCCAGUACGGCAGCUUCUGCACCC

[0206] AGCUGAAGAGGGCCCUGACCGGCAUCGCCGUGGAGCAGGACAAGAACACCCAGGAG

[0207] GUGUUCGCCCAGGUGAAGCAGAUCUACAAGACCCCCCCCAUCAAGUACUUCGGCGGC

[0208] UUCAACUUCAGCCAGAUCCUGCCCGACCCCAGCAAGCCCAGCAAGAGGAGCUUCAUC

[0209] GAGGACCUGCUGUUCAACAAGGUGACCCUGGCCGACGCCGGCUUCAUCAAGCAGUA

[0210] CGGCGACUGCCUGGGCGACAUCGCCGCCAGGGACCUGAUCUGCGCCCAGAAGUUCAA

[0211] CGGCCUGACCGUGCUGCCCCCCCUGCUGACCGACGAGAUGAUCGCCCAGUACACCAG

[0212] CGCCCUGCUGGCCGGCACCAUCACCAGCGGCUGGACCUUCGGCGCCGGCGCCGCCCU

[0213] CCAGAUCCCCUUCGCCAUGCAGAUGGCCUACAGGUUCAACGGCAUCGGCGUGACCCA

[0214] GAACGUGCUGUACGAGAACCAGAAGCUGAUCGCCAACCAGUUCAACAGCGCCAUCG

[0215] GCAAGAUCCAGGACAGCCUGAGCAGCACCGCCAGCGCCCUGGGCAAGCUCCAGGACG

[0216] UGGUGAACCACAACGCCCAGGCCCUGAACACCCUGGUGAAGCAGCUGAGCAGCAAG

[0217] UUCGGCGCCAUCAGCAGCGUGCUGAACGACAUCCUGAGCAGGCUGGACCCCCCCGAG

[0218] GCCGAGGUGCAGAUCGACAGGCUGAUCACCGGCAGGCUCCAGAGCCUCCAGACCUAC

[0219] GUGACCCAGCAGCUGAUCAGGGCCGCCGAGAUCAGGGCCAGCGCCAACCUGGCCGCC

[0220] ACCAAGAUGAGCGAGUGCGUGCUGGGCCAGAGCAAGAGGGUGGACUUCUGCGGCAA

[0221] GGGCUACCACCUGAUGAGCUUCCCCCAGAGCGCCCCCCACGGCGUGGUGUUCCUGCA

[0222] CGUGACCUACGUGCCCGCCCAGGAGAAGAACUUCACCACCGCCCCCGCCAUCUGCCA

[0223] CGACGGCAAGGCCCACUUCCCCAGGGAGGGCGUGUUCGUGAGCAACGGCACCCACUG

[0224] GUUCGUGACCCAGAGGAACUUCUACGAGCCCCAGAUCAUCACCACCGACAACACCUU

[0225] CGUGAGCGGCAACUGCGACGUGGUGAUCGGCAUCGUGAACAACACCGUGUACGACC

[0226] CCCUCCAGCCCGAGCUGGACAGCUUCAAGGAGGAGCUGGACAAGUACUUCAAGAAC

[0227] CACACCAGCCCCGACGUGGACCUGGGCGACAUCAGCGGCAUCAACGCCAGCGUGGUG

[0228] AACAUCCAGAAGGAGAUCGACAGGCUGAACGAGGUGGCCAAGAACCUGAACGAGAG

[0229] CCUGAUCGACCUCCAGGAGCUGGGCAAGUACGAGCAGUACAUCAAGUGGCCCUGGU

[0230] ACAUCUGGCUGGGCUUCAUCGCCGGCCUGAUCGCCAUCGUGAUGGUGACCAUCAUG

[0231] CUGUGCUGCAUGACCAGCUGCUGUAGCUGCCUGAAGGGCUGCUGUAGCUGCGGCAG

[0232] CUGCUGCAAGUUCGACGAGGACGACAGCGAGCCCGUGCUGAAGGGCGUGAAGCUGC

[0233] ACUACACC

[0234] The preparation method of mRNA vaccine is as follows:

[0235] According to conventional methods, the template DNA is inserted into the pUC57 vector to obtain a plasmid containing the above antigen DNA sequence. The plasmid template is linearized by enzyme digestion, and electrophoresis is used to verify whether the linearization is complete.

[0236] Select RNA polymerase, NTP and cap analogs to synthesize and prepare mRNA. Add relevant reactants to a 15 mL centrifuge tube according to the dosage and order shown in Table 1 below, pipette and release 3 times or tap the bottom of the tube with your fingers to mix, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0237] Table 1 Components and amounts for preparing mRNA

[0238] Components Addition amount ATP(100mM) 400μL GTP (100 mM) 400μL CTP (100 mM) 400μL N1-Me-pUTP (100mM) 400μL Cap analog (100mM) 200μL Linearized DNA template 200 μg 10× Transcription Buffer 400μL Murine RNase Inhibitor(40U / μL) 400μL T7 RNA Polymerase (50U / μL) 400μL Pyrophosphatase,Inorganic(yeast)(0.1U / μL) 100μL RNase-free water Add to 4 mL

[0239] Then, after incubation in a 37°C water bath for 4 hours, add 400μL DNase I and incubate at 37°C for 30 minutes; then add 4mL of phenol / chloroform / isoamyl alcohol (25:24:1) solution to extract mRNA. After mixing, centrifuge at 10,000rpm for 5 minutes, and then take the aqueous phase. Then add 4mL of chloroform solution to extract mRNA. After mixing, centrifuge at 10,000rpm for 5 minutes, and then take the aqueous phase. Then add 4mL of 5M ammonium acetate solution and place it at 4°C overnight to precipitate mRNA. Then take it out and centrifuge it at 4°C and 10,000rpm for 2 minutes, and carefully remove the supernatant. Add 4mL of 70% ethanol to wash the precipitate, centrifuge it at 4°C and 10,000rpm for 2 minutes, carefully remove the supernatant, and air-dry the precipitate. Then add 20mL of nuclease-free water to reconstitute 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%.

[0240] Preparation of mRNA-LNP (mRNA vaccine):

[0241] The ionizable lipids Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 are 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 SEQID No.7) is used as the aqueous phase, wherein the volume ratio of the aqueous phase to the organic phase is 3:1. They are mixed through a microfluidic chip to self-assemble into mRNA-lipid nanoparticles, and then the ethanol is removed through a tangential flow system to obtain an mRNA vaccine. The prepared mRNA vaccine has 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 have a small and uniform particle size and a high encapsulation efficiency.

[0242] Example 1: Preparation of novel coronavirus XBB.1.5 recombinant protein using insect baculovirus system

[0243] 1. Construction and design of the novel coronavirus XBB.1.5 recombinant protein

[0244] 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. In addition, the recombinant protein was expressed using the conventional Bac-to-Bac baculovirus expression system. Because the S protein of the new coronavirus SARS-CoV-2 is a membrane-localized protein, in order to ensure its normal secretion, when constructing the new coronavirus protein, the GP67 signal peptide sequence was added to the N-terminus of the protein to assist the secretory expression of the protein. This signal peptide will be spontaneously removed by insect cells during the protein secretion process; at the same time, the thioredoxin (Trx) tag of Spodoptera frugiperda (S. frugiperda) was added after the GP67 signal peptide to assist protein folding, a 6×his tag was added to assist in subsequent purification, and an EK enzyme cleavage site was added to remove the Trx and 6×his tags. Based on the construction design of this protein, all redundant amino acids that are not the target protein can be removed by EK enzyme cleavage.

[0245] 2. Preparation, identification and purification of the novel coronavirus XBB.1.5 recombinant protein

[0246] The above gene (nucleotide sequence is shown in SEQ ID No.1, amino acid sequence is shown in SEQ ID No.2) was amplified and integrated into the pFastBac1 vector. The verified plasmid was then transformed into E. coli DH10b cells to produce recombinant bacmids. Next, the recombinant bacmids were transferred to Sf9 insect cells to express the protein. The protein was then cleaved by EK protease, purified by Superdex 200Increase 10 / 300GL column, and determined by SDS-Page, Coomassie Brilliant Blue staining and Western blotting. Figure 1 Shown are the results of molecular screening and SDS-PAGE identification of the recombinant protein of the new coronavirus XBB.1.5, which obtained a protein antigen with good purity that can be used for subsequent immune protection research (the nucleotide sequence is shown in SEQ ID No. 3, and the amino acid sequence is shown in SEQ ID No. 4).

[0247] Example 2: Preparation of the novel coronavirus XBB.1.5 recombinant protein vaccine

[0248] The new coronavirus XBB.1.5 recombinant protein antigen (nucleotide sequence as shown in SEQ ID No. 3, amino acid sequence as shown in SEQ ID No. 4) prepared by the method of Example 1 was mixed with equal volumes of squalene oil-in-water emulsion MF59-like adjuvant to prepare a new coronavirus XBB.1.5 recombinant protein vaccine, which was stored at 2-8°C.

[0249] 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:

[0250] Example 3: Vaccination of mice

[0251] SPF female NIH mice (6-8 weeks) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (China) and were 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 preparation method of the vaccine for mouse immunogenicity test is as follows: the new coronavirus XBB.1.5 recombinant protein prepared in Example 1 was diluted to 200μg / mL with PBS, and an equal volume of MF59-like water-in-oil adjuvant was added and mixed thoroughly. The mice were randomly divided into groups, immunized on days 0, 21 and 42, and 100μL of the above-prepared XBB.1.5 recombinant protein vaccine was injected intramuscularly, and serum and tissues were collected 8 weeks or 43 weeks after the last immunization.

[0252] Example 4: Detection of neutralizing antibodies in serum from XBB.1.5 recombinant protein vaccine by pseudovirus neutralization method

[0253] 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 a three-fold factor ranging from 30 to 65610 and then incubated with an equal volume of diluted pseudoviruses at 37°C for 1 hour. Then, 1.2 × 10 4 HEK-293T cells expressing human ACE2 receptors (293T / ACE2) were added and incubated at 37 ° C for 48 hours to express luciferase. Finally, the supernatant was removed, and a lysis reagent (Beyotime, RG005) with a luciferase substrate 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%.

[0254] 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 ( Figure 2). The vaccine showed high neutralizing 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 determined to be 21184, 56336, 39656, 20117, 22905 and 57620, respectively. The vaccine also showed effective neutralization against mutant pseudoviruses such as EG.5.1, FL.1.5.1 and HV.1, which represent the XBB lineage currently circulating globally. 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, which shows that antigens from the XBB lineage induce a broad universal neutralizing response. It is worth noting that the 50% neutralization GMT of this recombinant protein vaccine against BA.2.86 and JN.1 subvariants are 7546 and 8045, respectively, indicating that the new coronavirus XBB.1.5 recombinant protein vaccine can still effectively protect against their infection.

[0255] Example 5: ELISpot detection of specific cellular immunity after immunization with the novel coronavirus XBB.1.5 recombinant protein vaccine

[0256] In view of the strong antibody response induced by the new coronavirus XBB.1.5 recombinant protein vaccine, 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 killed and serum and tissues were collected at week 8 after the first immunization.

[0257] 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 of the new 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 overnight at 37°C. The plates were washed with PBS the next day and incubated with HRP-conjugated goat anti-mouse IgG (1:10000) secondary antibodies (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.

[0258] 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 appear, rinse with water to stop development. Finally, the IFN-γ ELISpot plate was scanned using an auxiliary ELISpot microplate reader. Consistent with the results of the neutralization test, a large number of antigen-specific IgG ASCs ( Figure 3 ab). In addition, in vitro stimulation of spleen lymphocytes with a peptide library of the full-length spike protein of the XBB.1.5 variant revealed that spleen lymphocytes of mice in the vaccine immunization group induced antigen-specific T cells ( Figure 3 cd).

[0259] Example 6: Detection of specific cellular immunity by flow cytometry

[0260] NIH mice were immunized as described in Example 3. Mice were killed 8 weeks after the first immunization, and lymphocytes from spleen tissue were isolated under sterile conditions and inoculated in 12-well plates (1×10 6 / well), the culture medium was 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 with 1 μg / mL spike protein peptide pool from XBB.1.5 variant for 12 hours. The cells were then treated with Brefeldin A (BD Biosciences) for 6 hours before cell collection. The cells were stained 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) at 4°C for 30 minutes. After washing 3 times with PBS, the cells were fixed and permeabilized with a fixation / permeabilization kit (BD, catalog number: 554715) at room temperature for 20 minutes. Then, the perforated cells were stained with PE-conjugated anti-mouse IFN-γ (catalog number: 505808) and BV421 anti-mouse TNF-α (catalog number: 506328) at room temperature for 1 hour. Cells were detected using a NovoCyte flow cytometer (ACEA Biosciences) and NovoExpress 1.4.1 software.

[0261] 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 percentage of Tfh and GC B cells was detected and analyzed using a NovoCyte Advanteon flow cytometer equipped with NovoExpress 1.5.6 (Agilent).

[0262] like Figure 4 As shown, the XBB.1.5 recombinant protein vaccine of the present invention can produce a strong cellular immune response, secreting IFN-γ antigen memory CD8 + and CD4 + T cells ( Figure 4 a) and secretion of TNF-α antigen memory CD8 + and CD4+ T cells ( Figure 4 b) The percentage was significantly increased. At the same time, a significant increase in GC B and Tfh cells was observed in the spleen and lymph nodes ( Figure 4 cd).

[0263] Example 7: Neutralization test of live novel coronavirus

[0264] Neutralizing antibodies against live ancestral and mutated novel coronaviruses in serum samples from mice vaccinated with the novel coronavirus XBB.1.5 recombinant protein vaccine described in Example 3 were measured by real virus neutralization assay. Each group of diluted serum was mixed with live novel coronavirus that can infect 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 ( / well) for 72 hours. The cytopathic effect (CPE) was measured under a microscope to evaluate the true virus neutralizing antibody titer of the serum.

[0265] Serum neutralizing antibody levels against several major circulating SARS-CoV-2 variants were measured. The monovalent vaccine induced strong neutralizing antibody responses against the XBB lineage, particularly against XBB.1.5 and EG.5.1 ( Figure 5 ). The geometric mean titers (GMTs) against live XBB.1.5, XBB.1.16 and EG.5.1 reached 2580, 912 and 2580, respectively. The GMTs against the pre-XBB variants Delta and BA.5.2.48 were determined to be 256 and 1290, respectively.

[0266] Example 8: Long-term protective immunity induced by the novel coronavirus XBB.1.5 recombinant protein vaccine

[0267] Long-lasting antibody responses are essential to prevent repeated infection with SARS-CoV-2. Therefore, neutralizing antibodies induced by the novel coronavirus XBB.1.5 recombinant protein vaccine were further evaluated by pseudovirus neutralization. In addition, antibody-secreting cells (ASC) in the spleen and bone marrow of long-lasting immune mice were further observed by ELISpot, and long-lived plasma cells (LLPC) 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.

[0268] To detect LLPCs and MBCs, flow staining was performed using the following antibodies. LLPCs in the bone marrow were stained with 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 / Cyanine5.5 anti-mouse CD3, BV605 anti-mouse CD19, PE / Cyanine7 anti-mouse CD38 (Catalog No. 165612), and BV421 anti-mouse / human GL7 antibodies.

[0269] Depend on Figure 6 It can be seen that the neutralizing antibodies induced by the recombinant protein vaccine of the present invention can last up to 43 weeks, and the serum neutralizing antibody titer against the XBB lineage subvariants is high ( Figure 6 b). In addition, antigen-specific ASCs were detected in bone marrow and spleen tissues ( Figure 6 cd). The vaccine significantly induced LLPC in the bone marrow ( Figure 6 e) and MBC in spleen tissue and lymph nodes ( Figure 6 f), which is consistent with the results of persistent serum neutralizing antibodies. Similar to the humoral immune response, the vaccine conferred a persistent cellular immune response, as evidenced by antigen-specific CD4 + and CD8 + The presence of T cells ( Figure 6 gh). These results suggest that our SARS-CoV-2 XBB.1.5 recombinant protein vaccine can provide long-term protection against circulating SARS-CoV-2 variants.

[0270] Example 9: Novel coronavirus XBB.1.5 recombinant protein vaccine heterologous sequential inactivated vaccine and mRNA vaccine

[0271] 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 lineages 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 using recombinant protein-based boosters may induce better immune responses than homologous vaccination using inactivated or mRNA vaccines. Therefore, the present invention attempts to evaluate the potential of this vaccine as a booster for mRNA and inactivated vaccines.

[0272] The present invention first immunizes NIH mice three times with an inactivated vaccine based on wild virus (provided by the Institute of Medical Biology, Chinese Academy of Medical Sciences, and the preparation method is detailed in Clinical Infectious Diseases, Volume 73, Issue 11, 1 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, and then sequentially immunizes the mice with inactivated vaccine (inactivated vaccine × 3> inactivated vaccine), mRNA vaccine (3 injections of mRNA>mRNA) or monovalent recombinant vaccine (3 injections of inactivated vaccine>protein vaccine and 3 injections of mRNA>protein vaccine) ( Figure 7 a). Omicron mutants containing XBB showed significant immune evasion from neutralization elicited by inactivated vaccines, even mRNA vaccines designed based on the BA.5 sequence ( Figure 7 bd). Heterologous vaccination with the novel coronavirus XBB.1.5 recombinant protein vaccine significantly restored neutralizing activity against all variants. Neutralizing antibody titers after heterologous sequential inactivated vaccination of this vaccine were tested by real virus neutralization assay to determine whether this vaccine can be used as a fourth booster injection after three injections of inactivated virus vaccine ( Figure 7 b). The GMTs in the heterologous vaccine-vaccinated groups against Delta, BA.2, BA.5.2.48, and XBB.1.16 viruses reached 891, 776, 891, and 1552, respectively, which were 4.00, 8.00, 111.43, and 256.00-fold higher than those in the groups receiving the homologous inactivated vaccines.

[0273] Then, the neutralization reaction of the novel coronavirus XBB.1.5 recombinant protein vaccine heterologously inoculated with inactivated vaccine and mRNA vaccine was comprehensively evaluated by pseudovirus neutralization test ( Figure 7cd). All Omicron variants, especially XBB mutants, significantly escaped neutralizing antibodies induced by inactivated and mRNA vaccines. Notably, the antibody group induced by heterologous immunization showed high levels of neutralization against all XBB lineages, BA.2.86 and the derived submutation JN.1. In the heterologous vaccination group after inactivated vaccination (inactivated vaccine × 3> protein vaccine), 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 JN.1 submutants 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 7 d). These findings strongly confirm that the novel coronavirus XBB.1.5 recombinant protein vaccine can serve as a candidate for booster vaccination, providing immune protection against variants including JN.1, BA.2.86 and XBB lineages.

[0274] Example 10: Novel coronavirus EG.5.1 variant challenge protection test

[0275] 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 globally prevalent 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 prepared in Example 3 or MF59-like adjuvant on days 0, 21, and 42. Then, on day 63, 1×10 6PFU EG.5.1 live virus challenged mice. Changes in body weight and throat swab viral load were monitored daily. On day 5 after infection, 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 evaluated 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.

[0276] 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. In addition, these experiments were performed in the ABSL-4 facility of the Kunming National High-Level Biosafety Primate Research Center.

[0277] Daily monitoring of the weight of challenged mice revealed that the MF59-like adjuvant control group showed significant weight loss on the first day after infection, reaching a maximum on the fourth day. In contrast, vaccine-immunized mice showed transient weight loss, but quickly rebounded on the second day ( Figure 8 a). In addition, high levels of genomic RNA (gRNA) were detected in the throat swab samples of the control group, while the viral load in the throat swabs of mice immunized with the vaccine showed a significant downward trend throughout the experiment ( Figure 8 b).

[0278] Subsequently, mice were euthanized on day 5 post-infection to collect tissues for assessment of viral load and histopathology ( Figure 8 cd). In the control group that received adjuvant, high levels of gRNA were detected in the nasal turbinates, trachea, and lung tissues. The viral load in the nasal turbinates, trachea, and lung tissues of vaccinated mice was significantly reduced. ( Figure 8 c). In addition, mild pathological changes were observed in the lung tissues of the adjuvant control group, characterized by multifocal areas of consolidation, thickening of alveolar septa, alveolar congestion, and small plaque inflammation ( Figure 8 d). As expected, the lung tissue of vaccinated mice showed normal histological structure, with intact alveoli and no obvious inflammation. Therefore, the pathological score of the lung tissue of vaccine-immunized mice was significantly lower than that of the adjuvant control group ( Figure 8d). These results indicate that the novel coronavirus XBB.1.5 recombinant protein vaccine can induce protective immune responses in both the upper and lower respiratory tracts and effectively prevent infection with the live virus.

[0279] The experimental operation and effect of intranasal immunization of the novel coronavirus XBB.1.5 recombinant protein vaccine prepared above are shown in Examples 11-17:

[0280] Example 11: Animal Vaccination

[0281] 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 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. After 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 intervals of 21 days. Blood samples were collected from mice and rats 2, 5 or 8 weeks after the primary immunization.

[0282] To evaluate the immune response generated by a combination of intranasal (IN) and intramuscular (IM) immunizations, NIH mice were administered three doses of 10 μg 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 were dosed twice by IM injection and then once by IN injection. Mice in the 3×IN group received three doses of vaccine delivered only by IN.

[0283] Example 12: Method for detecting the immune effect of intranasal immunization with the novel coronavirus XBB.1.5 recombinant protein vaccine

[0284] (1) Enzyme-linked immunosorbent assay (ELISA)

[0285] ELISA was used to measure RBD-specific IgG and IgA levels. Briefly, XBB.1.5 recombinant protein (1 μg / mL) was coated on 96-well plates (NUNC-MaxiSorp, Thermo Fisher Scientific) by overnight incubation at 4°C or 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 plate was washed again with PBST. Subsequently, 100 μL of HRP-conjugated antibody (anti-mouse IgG, IgG1, IgG3, IgG2a, IgG2b, IgG2c, IgA or anti-rat IgG, 1:10,000 dilution) was added to each well and incubated at 37°C for 1 hour. After washing 5 times, 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) substrate was added to each well. After reacting at room temperature for 10 minutes, 50 μL of stop buffer (H 2 SO 4 ) was used to stop the color development, and the absorbance at 450 nm was measured using a microplate reader (Spectramax ABS, Molecular Devices).

[0286] (2) SARS-CoV-2 pseudovirus neutralization test

[0287] 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 min 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% CO 2 ) 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 Kaleido 3.0 software (PerkinElmer, USA).

[0288] (3) Flow cytometry

[0289] To detect tissue-resident memory (TRM ) cells, collected the cells 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).

[0290] 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 / Cyanine5.5-conjugated anti-mouse CD3, Pacific Blue 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).

[0291] Lung tissue was collected aseptically and minced. Subsequently, the tissue was enzymatically digested for 1 hour at 37°C in a buffer containing DMEM 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). After digestion, the tissue homogenate was filtered through a 70-mesh sieve, erythrocytes were lysed, 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 medium 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 stained with PE / Blue 7 anti-mouse IFN-γ (BioLegend, catalog number 505826) and Brilliant Violet 510. TM Anti-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 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.

[0292] Example 13: Broad neutralization activity against the XBB lineage elicited by intranasally delivered XBB.1.5 recombinant protein vaccine

[0293] In previous studies, intranasal delivery of recombinant protein antigens alone had poor immunogenicity and could hardly induce a visible immune response. Therefore, we 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, mice were intranasally administered 5 μg (low dose) or 10 μg (high dose) of XBB.1.5 recombinant protein vaccine ( Fig. 9 a). Mice received PBS or XBB.1.5 recombinant protein alone as control groups. Antigen-specific binding antibody assay showed that XBB.1.5 recombinant protein alone could hardly elicit serum ( Fig. 9 b) and mucous membranes ( Fig. 9 f) RBD-specific antibodies, while all animals in the group receiving adjuvanted protein immunization showed significantly improved endpoint titers of antigen-specific IgG and IgA. Similar improvements were observed in rats that received intranasal delivery of the XBB.1.5 recombinant protein vaccine ( Fig. 9 c). 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, manifested by the production of various antibody subtypes, including antigen-specific IgG1, IgG2a, IgG2b, IgG2c, and IgG3 antibodies ( Fig. 9 d). High doses of XBB.1.5 recombinant protein vaccine could induce stronger Th1-biased immunity, as determined by a higher ratio of IgG2a / IgG1 antibodies.

[0294] Neutralizing antibody responses are a key indicator of vaccine protective efficacy. Therefore, neutralizing activity against XBB lineage variants after intranasal immunization was assessed. Although the levels of neutralizing antibodies (NAbs) showed only modest improvements at 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 vaccination 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 ( 1371–1364 ). Fig. 9 e). In addition, such robust neutralizing antibody protection was also evident in bronchoalveolar lavage fluid (BALF) in addition to serum samples ( Fig. 9g), it can be seen that intranasal delivery of the XBB.1.5 recombinant protein vaccine is able to induce systemic and mucosal humoral immune responses, characterized by high levels of neutralizing antibodies against XBB lineage variants.

[0295] Example 14: Intranasal delivery of XBB.1.5 recombinant protein vaccine induces upper airway cellular immune response

[0296] 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 ( Fig.10 a). Although there was a significant difference in CD8 + T RM There was no statistically significant difference in the number of , but an increase in frequency was observed, and the high-dose vaccine further enhanced mucosal CD8 + T RM induction.

[0297] Lung antigen-specific cellular immune responses were further assessed using intracellular cytokine staining (ICS). Lung tissues from vaccinated mice were isolated and processed into single-cell suspensions, which were then stimulated ex vivo with the full-length spike peptide library to detect intracellular cytokine expression. In the absence of the stimulatory peptide library, T cells in the lungs appeared to be quiescent, whereas we noted that both doses of intranasal XBB.1.5 recombinant protein vaccine resulted in a significant increase in the percentage of IFN-γ secretion after stimulation, including CD8 + T cells and CD4 T cells secreting IFN-γ and TNF-α + T cells, high-dose immunization further enhanced these improvements ( Fig.10 b) Therefore, the induction of multiple cytokine secretion in antigen-specific T cells suggests the presence of a multifunctional cellular immune response in the respiratory mucosa.

[0298] We also evaluated the germinal center (GC) responses induced by the intranasal XBB.1.5 recombinant protein vaccine in the mediastinal lymph nodes (MLNs), as GC B and T follicular helper (Tfh) cells are essential for long-term protective immune responses. As expected, GC B cells (CD19 + GL7 + CD95 + ) and Tfh cells (CD4 + CXCR5 + PD-1 + ) is significantly more frequent ( Fig.10 c). In addition, more RBD-specific B cells were detected in the MLN, and more cells were involved in antibody production ( Fig.10 c) These findings strongly support the notion that intranasal XBB.1.5 recombinant protein vaccine is capable of eliciting robust and durable mucosal immune responses.

[0299] Example 15: Protective immunity elicited by combined vaccination with intramuscular and intranasal delivery of XBB.1.5 recombinant protein vaccines

[0300] 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 ( Fig.11 ab). NIH mice were initially vaccinated with 10 μg of XBB.1.5 recombinant protein vaccine intramuscularly 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) via intranasal delivery. The control group consisted of mice treated with PBS or three doses of intranasal XBB.1.5 recombinant protein vaccine administered via intranasal delivery (3×IN).

[0301] No differences in serum RBD-specific IgG antibody levels were observed between the three vaccination regimens ( Fig.11 c). However, it is noteworthy that the 1×IM+2×IN immunization regimen elicited the highest level of neutralizing antibodies among all groups tested ( Fig.11 e). 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 with the 2×IM+1×IN group ( Fig.11 d and 11f).

[0302] To comprehensively evaluate the systemic and mucosal cellular immune responses induced by the 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 ( Fig.12 a). In addition, intranasal immunization induced germinal center responses in the mediastinal lymph nodes, as demonstrated by increased percentages of Tfh, GC B, and RBD-specific B cells ( Fig.12 b). 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 (MBCs) in spleen tissue, indicating that intranasal immunization induced a weak systemic cellular immune response ( Fig.12 c). Importantly, both the 2×IM+1×IN and 1×IM+2×IN immunization regimens overcame this limitation by increasing the frequencies of IFN-γ-secreting T cells and MBCs in the spleen. Therefore, the combination of intramuscular and intranasal immunization, especially one intramuscular dose followed by two intranasal deliveries of the XBB.1.5 recombinant protein vaccine (1×IM+2×IN), can elicit mucosal and systemic immune responses to protect against infection with the novel coronavirus mutant strain.

[0303] Example 16: Mucosal and systemic immune responses elicited by intranasal delivery of XBB.1.5 recombinant protein vaccine followed by heterologous sequential mRNA vaccine

[0304] Messenger RNA (mRNA)-based vaccines for the novel coronavirus have been widely deployed worldwide. However, repeated administration of the same type of vaccine may reduce the ability of heterologous vaccination to elicit enhanced immunity. mRNA vaccines are mainly 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.

[0305] mRNA vaccines based on the full-length BA.5 spike protein, and NIH mice were immunized intramuscularly on days 0, 21, and 42, followed by a fourth dose of homologous mRNA vaccine injection (4×mRNA), or heterologous intranasal delivery of XBB.1.5 recombinant protein vaccine (3×mRNA+1×XBB.1.5 recombinant protein) on day 63 ( Fig.13 a–b). In addition, another group of mice received two doses of mRNA vaccine followed by two intranasal administrations of XBB.1.5 recombinant protein vaccine (2×mRNA+2×XBB.1.5 recombinant protein) ( Fig.13ab), mRNA and XBB.1.5 recombinant protein vaccines were administered at a dose of 10 μg each time. Notably, a single dose of intranasal 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 with the 4× mRNA group ( Fig.13 c and 13e). However, two injections of mRNA followed by two intranasal deliveries of XBB.1.5 recombinant protein (2×mRNA+2×XBB.1.5 recombinant protein) induced a better systemic humoral immune response, with the highest levels of serum antigen-specific IgG ( Fig.13 c) and neutralizing antibodies ( Fig.13 e). Compared with the homologous mRNA vaccination group (4×mRNA), the 2×mRNA+2×IN XBB.1.5 recombinant protein group increased the GMT of XBB, XBB.1.5, XBB.1.6, XBB.1.9.1, XBB.1.16, XBB.2.3, EG.5.1 and JN.1 pseudoviruses by 10.69, 52.05, 12.81, 11.39, 28.10, 20.89, 32.43 and 105.42 times, respectively. Consistent with the antibody assay, two intranasal deliveries of XBB.1.5 recombinant protein vaccine rather than a single dose significantly improved memory B cells in lung tissue. However, we observed enhanced mucosal antibody responses induced by at least one intranasal delivery of XBB.1.5 recombinant protein vaccine ( Fig.13 d and 13f).

[0306] 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 group (2×mRNA+2×IN XBB.1.5 recombinant protein), resulting in CD8 + and CD4 + T RM The cell count was the highest ( Fig.13 i) and CD4 secretion of IFN-γ and TNF-α + The highest proportion of T cells ( Fig.13 j) Although T 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 the mediastinal lymph nodes ( Fig.13 These findings suggest that the 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.

[0307] Example 17: Intranasal XBB.1.5 recombinant protein vaccine protection test against EG.5.1 variant

[0308] Mice challenged with live Omicron EG.5.1 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 2 μg / mL of 4 μg / mL of 1 μg / mL of 2 μg / mL of 1 ...

[0309] 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 high-dose XBB.1.5 recombinant protein vaccines (10 μg per mouse) and subsequently treated with 1×10 6 The mice were challenged with 1000 plaque-forming units (PFU) of live EG.5.1 virus. The changes in viral load in throat swabs were monitored daily using a reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay. Samples from mice administered PBS showed high levels of viral load ( Fig.14a). In contrast, intranasal administration of the XBB.1.5 recombinant protein vaccine significantly reduced viral genomic RNA (gRNA) at day 1 post-infection (dpi) and demonstrated rapid clearance of the virus. By day 5 dpi, negligible viral loads were observed in throat swab samples in the group that received the intranasal XBB.1.5 recombinant protein vaccine.

[0310] On day 5 dpi, 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 with administration of PBS ( Fig.14 b). 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.

[0311] In addition to changes in viral load, the effective protection conferred by the intranasal XBB.1.5 recombinant protein vaccine 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 plaque inflammation composed of lymphocytes, neutrophils, and macrophages ( Fig.14 c–d). In contrast, lung tissue from the group that received intranasal XBB.1.5 recombinant protein vaccine showed normal histology with intact alveolar architecture and no obvious inflammation. Therefore, intranasal vaccination with XBB.1.5 recombinant protein vaccine effectively protected both the upper and lower respiratory tracts from challenge with live EG.5.1 virus.

Claims

1. Use of XBB.1.5 recombinant protein or vaccine in the preparation of a drug 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.

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 drug 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 for protection against SARS-CoV-2 or its variants infection, which are administered separately, simultaneously or sequentially.

5. A composition or combination drug for preventing and / or treating infection with SARS-CoV-2 and its variants, characterized by: Contains inactivated vaccines and / or XBB.1.5 recombinant protein vaccines against SARS-CoV-2 or its variants for separate, simultaneous or sequential administration.

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 an amino acid sequence as 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 combined drug according to claim 4 or 5, characterized in that: 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.

8. The use according to any one of claims 1-3, 6-7, the composition or the combined drug according to any one of claims 4-7, characterized in that: The method for preparing the XBB.1.5 recombinant protein comprises the following steps: culturing a host cell containing the XBB.1.5 recombinant protein or a protein precursor to express the protein or the precursor, and then recovering the protein.

9. The use, composition or combination according to claim 8, characterized in that: In the method for preparing 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 uses 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 cell is selected from at least one of sf9 cells, sf21 cells and Hi5 cells; the mammalian cell is a CHO cell.

13. The use according to any one of claims 1 to 3, 6 to 12, the composition or the combined drug 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, monophosphoryl 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: At least one of the following is met: 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, 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 drug 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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