MRNA vaccines and recombinant protein vaccines for infection by SAR-COV-2 or its variants, and their application.

Abstract

The present invention relates to mRNA vaccines and recombinant protein vaccines against infection by SARS-CoV-2 or its variants, and their application, and belongs to the field of pharmaceuticals. In order to solve the problem of drugs for preventing and treating infection by SARS-CoV-2 or its variant viruses, the present invention provides recombinant protein vaccines and / or mRNA vaccines for preventing and / or treating infection by SARS-CoV-2 or its variants, and in particular provides methods for applying mRNA vaccines and recombinant protein vaccines, the application of which can induce a strong antibody and cellular immune response in vivo and block the binding of the SARS-CoV-2 S protein to the ACE2 receptor on host cells, thereby enabling the host to resist coronavirus infection.

Description

Technical Field

[0001] The present invention relates to drugs and combination drugs for infection by SARS-CoV-2 or its variants, as well as their combination administration methods. In particular, it relates to mRNA vaccines and / or recombinant protein vaccines for infection by SARS-CoV-2 or its variants, as well as their administration methods, and belongs to the field of medicine.

Background Art

[0002] SARS-Cov-2 is a novel beta coronavirus named by the World Health Organization. This virus has an envelope, its particles are circular or oval, often polymorphic, and have a diameter of 60 nm to 140 nm. Its genetic characteristics are significantly different from those of SARS-CoV and MERS-CoV, so this virus is a branch of novel coronaviruses that have never been discovered in humans before. Currently, there are five main types of SARS-CoV-2 variants: alpha, beta, gamma, delta, and omicron, and the omicron variant is further classified into several sub-lineages such as BA.1, BA.2, BA.2.12.1, BA.4, and BA.5.

[0003] The main structural proteins of SARS-CoV-2 include the spike (S) protein, envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein. Of these, the S protein plays a crucial role in viral infection and pathogenicity and is often used as a vaccine antigen. SARS-CoV-2 mutants possess multiple mutation sites, and the virus's S protein also possesses multiple mutation sites. As a result, mutants can evade to some extent antibodies induced by SARS-CoV-2 precursor vaccines, thereby reducing the effectiveness of SARS-CoV-2 vaccines or diminishing protective immunity, posing a significant challenge to epidemic control efforts. Therefore, the development of vaccines against various variants of the SARS-CoV-2 virus, especially broad-spectrum vaccines against various variants of the SARS-CoV-2 virus, is crucial for the prevention and control of SARS-CoV-2.

[0004] Currently, more than 10 vaccines based on adenovirus vectors, mRNA, and recombinant protein subunits are approved worldwide. Some typical vaccines include Pfizer Inc.'s BNT162B2 (mRNA), AstraZeneca's ChAdOx1 nCoV-19 (adenovirus vector), Moderna's mRNA-1273 (mRNA), Novavax's NVX-CoV2373 (protein subunit), and AstraZeneca's AZD1222 (viral vector). These SARS-CoV-2 vaccines effectively prevent disease and fatal consequences caused by SARS-CoV-2 and its variants.

[0005] However, the continued evolution of SARS-CoV-2 has led to the persistent emergence of new variants, posing new challenges to vaccine development. In particular, the dominant variant, Omicron (e.g., B.1.1.529), possesses numerous spike protein variants, is highly transmissible, and weakens the herd protection provided by currently available vaccines. For example, neutralization ability against the Omicron variant decreases 30-fold compared to the WA1 / 2020 bacterial strain 2-4 weeks after vaccination with BNT162B2. Furthermore, another study revealed a 22-fold difference in the geometric mean titer (GMT) of 50% neutralizing antibodies between the WA1 / 2020 strain and the Omicron variant (1953 vs. 89). Recently, a series of new Omicron variants have emerged, including BA.2, BA.3, BA.2.12.1, BA.4, and BA.5 (BA.4 / 5). Omicron BA.4 / 5 is dominant in South Africa and the United States. While the spike protein mutations in BA.4 / 5 are equivalent to those in BA.2, the significantly enhanced neutralization evasion ability of BA.4 / 5 poses a more significant obstacle to vaccine development.

[0006] Given the continued spread and successive mutations of SARS-CoV-2, administering a third booster dose or developing a new generation vaccine against SARS-CoV-2 is a crucial strategy. Existing researchers have demonstrated that a third dose of BNT162B2 can significantly increase its neutralizing ability against Omicron. Similarly, homologous mRNA-1273 boosters can improve the efficacy of a two-dose mRNA-1273 vaccine against the Omicron variant. While a third booster dose can enhance its neutralizing ability, its efficacy against the BA.4 / 5 Omicron variant remains significantly lower. [Overview of the project] [Means for solving the problem]

[0007] The present invention aims to solve one of the technical problems that exist in the prior art. Therefore, the object of the present invention is to provide recombinant protein vaccines and mRNA vaccines against infection by SARS-CoV-2 or its variants, as well as methods for administering them in combination.

[0008] The present invention provides mRNA vaccines and recombinant protein vaccines for preventing and / or treating infection by SARS-CoV-2 or its variants, the two vaccines being preparations containing either an mRNA vaccine or a recombinant protein vaccine as an active ingredient, respectively, against infection by SARS-CoV-2 or its variants.

[0009] The present invention further provides a composition for infection by SARS-CoV-2 or a variant thereof, the composition comprising a preparation having an mRNA vaccine and a recombinant protein vaccine as active ingredients, wherein the mRNA vaccine comprises mRNA constructed based on the nucleotide sequence shown in Sequence ID No. 1 or Sequence ID No. 2, and the recombinant protein vaccine comprises a protein constructed based on the amino acid sequence shown in Sequence ID No. 4.

[0010] The present invention further provides a combination drug for preventing and / or treating infection by SARS-CoV-2 or a variant thereof, the combination drug comprising an mRNA vaccine and a recombinant protein vaccine against SARS-CoV-2 or a variant thereof, the mRNA vaccine and the recombinant protein vaccine being administered simultaneously or sequentially, the mRNA vaccine comprising mRNA constructed based on the nucleotide sequence shown in Sequence ID No. 1, and the recombinant protein vaccine comprising a protein constructed based on the amino acid sequence shown in Sequence ID No. 4.

[0011] The mRNA vaccine contains the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or the nucleotide sequence is homologous to SEQ ID NO: 1 or SEQ ID NO: 2, exhibits the same or similar biological activity, and is a variant obtained by substitution and / or deletion and / or insertion of at least one amino acid in the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Preferably, the variant sequence is a variant obtained by substitution and / or deletion and / or insertion of 1 to 400 amino acids in the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and more preferably, the variant sequence is a variant obtained by substitution and / or deletion and / or insertion of 5 to 30 amino acids in the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0012] The amino acid sequence of the recombinant protein vaccine protein precursor is the sequence shown in SEQ ID NO: 4, or the amino acid sequence is homologous and identical or similar to the sequence of SEQ ID NO: 4 and is a variant obtained by substitution and / or deletion and / or insertion of at least one amino acid in the sequence of SEQ ID NO: 4. Preferably, the RBD sequence is a variant obtained by substitution and / or deletion and / or insertion of 1 to 400 amino acids in the sequence of SEQ ID NO: 4. Preferably, the RBD sequence is a variant obtained by substitution and / or deletion and / or insertion of 5 to 30 amino acids in the sequence of SEQ ID NO: 4.

[0013] Furthermore, mRNA vaccines, recombinant protein vaccines, or combination drugs are formulated as intramuscular injections, nasal drops, sprays, nasal sprays, or inhalants. Preferably, mRNA vaccines are formulated as intramuscular injections, and recombinant protein vaccines are formulated as intramuscular injections and nasal sprays.

[0014] Furthermore, recombinant protein vaccines contain proteins and / or protein precursors that protect against infection by SARS-CoV-2 or its variants.

[0015] Furthermore, the protein and / or protein precursor comprises a protein having at least one RBD sequence and at least one HR sequence derived from the SARS-CoV-2 S protein, the sequence of the protein and / or protein precursor is shown in SEQ ID NO: 4.

[0016] Alternatively, the sequence may exhibit homology and identical or similar biological activity to the sequence of SEQ ID NO: 4 and be a variant obtained by substitution and / or deletion and / or insertion of at least one amino acid in the sequence of SEQ ID NO: 4, preferably the RBD sequence is a variant obtained by substitution and / or deletion and / or insertion of 1 to 400 amino acids in the sequence of SEQ ID NO: 4, preferably the RBD sequence is a variant obtained by substitution and / or deletion and / or insertion of 5 to 30 amino acids in the sequence of SEQ ID NO: 4.

[0017] Furthermore, the proteins formed by the RBD and HR sequences of the S protein spontaneously form trimers.

[0018] Furthermore, homologous amino acid sequences are selected from at least one of the alpha, beta, gamma, delta, and omicron RBD sequences.

[0019] Furthermore, the protein precursor is a protein against infection by SARS-CoV-2 or a variant thereof, the protein is linked to a signal peptide and / or a protein tag, preferably the signal peptide comprises a native signal peptide of the S protein or a variant thereof, and / or a human tPA signal peptide further added before the native signal peptide, preferably the protein tag is selected from at least one of a histidine tag, a thioredoxin tag, a glutathione transferase tag, a ubiquitin-like modifier protein tag, a maltose-binding protein tag, a c-Myc protein tag, and an Avi tag protein tag, more preferably the protein tag is a Trx tag and / or a 6His tag.

[0020] Furthermore, a protease recognition site for cleaving the protein tag is further linked to the protein against infection by SARS-CoV-_{2} or its variant, and preferably, the protease is selected from at least one of enterokinase, TEV protease, thrombin, coagulation factor Xa, carboxypeptidase A and rhinovirus 3c protease.

[0021] Furthermore, the amino acid sequence of the protein and / or the protein precursor is selected from the sequence of SEQ ID NO: 4.

[0022] Furthermore, the nucleotide sequence encoding the amino acid sequence is shown in the sequence of SEQ ID NO: 5.

[0023] Furthermore, the recombinant protein vaccine and / or mRNA vaccine contain nucleic acids against infection by SARS-CoV-_{2} or its variant.

[0024] Furthermore, the nucleotide sequence of the nucleic acid is shown in the sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 5.

[0025] The present invention further provides a recombinant vector containing a polynucleotide sequence, and the polynucleotide sequence is selected from at least one of the sequences of SEQ ID NO: 1 and SEQ ID NO: 5.

[0026] Furthermore, 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. Preferably, the insect baculovirus expression vector is pFastBac1. Preferably, the mammalian cell expression vector is a CHO cell expression vector. More preferably, the CHO cell expression vector is pTT5 or FTP-002. Preferably, the Escherichia coli expression vector is pET32a. Preferably, the yeast expression vector is pPICZaA.

[0027] The present invention further provides a host cell containing the above recombinant vector.

[0028] Furthermore, the host cell is selected from at least one of insect cells, mammalian cells, Escherichia coli, and yeast. Preferably, the insect cell is selected from at least one of sf9 cells, sf21 cells, and Hi5 cells. Preferably, the mammalian cell is either a CHO cell or a HEK293 cell.

[0029] The recombinant protein vaccine and / or mRNA vaccine further contains a pharmaceutically acceptable excipient or adjuvant component.

[0030] Furthermore, the adjuvant component is an immune adjuvant. Preferably, the immune adjuvant is selected from at least one of squalene-based oil-in-water emulsions, aluminum salts, calcium salts, plant saponins, plant polysaccharides, monophosphoryl lipid A, muramyl dipeptide, muramyl tripeptide, bacterial toxins, GM-CSF cytokines, lipids, and cationic liposome materials.

[0031] Furthermore, the immunoadjuvant satisfies at least one of the following: the squalene-based 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 either 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, and the lipid is at least phosphatidylethanolamine, phosphatidylcholine, cholesterol, and dioleoylphosphatidylethanolamine. The cationic liposome material, selected from one of the following, is chosen from at least one of (2,3-dioleoyloxypropyl)trimethylammonium chloride, N-[1-(2,3-dioleoylchloride)propyl]-N,N,N-trimethylamine chloride, cationic cholesterol, dimethyl-2,3-dioleyloxypropyl-2-(2-spermidinecarboxamide)ethylammonium trifluoroacetate, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, dimethyldioctadecylammonium bromide, and CpG ODN.

[0032] The present invention further provides the use of the above-mentioned drugs, administered alone or in combination with other drugs, in the preparation of drugs for preventing and / or treating infection by SARS-CoV-2 or its variants.

[0033] Furthermore, SARS-CoV-2 mutants include at least one of the alpha, beta, gamma, delta, and omicron forms.

[0034] The present invention further provides a method for preparing a protein, the method comprising the steps of culturing host cells to express a required protein or precursor, and then recovering the required protein.

[0035] The present invention also provides a method for preparing recombinant proteins, the method comprising the steps of constructing a recombinant vector containing polynucleotides and immunizing a human body to produce the protein.

[0036] Furthermore, the vector is selected from at least one of mRNA, DNA vaccines, adenoviruses, vaccinia ankara viruses, and adeno-associated viruses.

[0037] Beneficial Effects: The present invention provides proteins and vaccines against infection by SARS-CoV-2 or its variants, particularly mRNA vaccines and recombinant protein vaccines against infection by SARS-CoV-2 or its variants, wherein the nucleotide sequences encoding both vaccines are selected from at least one of SEQ ID NOs: 1, SEQ ID NOs: 2, and SEQ ID NOs: 5. According to the present invention, experiments show that the two vaccines combined (i.e., the recombinant protein vaccine combined with two mRNA vaccines as a third heterologous booster) can enhance humoral and cellular immune responses, showing higher anti-RBD IgG levels, stronger neutralizing antibodies, and an increase in IFN-γ secreting T cells. These experiments provide robust evidence for a heterologous boosting strategy against SARS-CoV-2 and demonstrate that a third heterologous protein subunit booster (RBD-HR / trimeric vaccine) is a very suitable candidate after two doses of mRNA vaccine.

[0038] Sequence ID 1: Nucleotide sequence of the S antigen in the SARS-CoV-2 delta mRNA vaccine:

[0039] Sequence ID 2: mRNA sequence of the S antigen in the SARS-CoV-2 delta mRNA vaccine:

[0040] Sequence ID 3: Amino acid sequence of the S antigen in the SARS-CoV-2 delta mRNA vaccine:

[0041] Sequence ID 4: SARS-CoV-2 Delta RBD-HR / Trimeric Protein Precursor Sequence (Signal Peptide - Trx Tag - 6His Tag - EK Enzyme Digestion Site - RBD Sequence - HR1 Sequence - HR2 Sequence): MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYA DSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0042] Sequence ID 5: Nucleotide sequence encoding Sequence ID 4

[0043] Terms and abbreviations: Monophosphate peptide a (MPL); squalene-based oil-in-water emulsion (MF59), recombinant cholera toxin (rCTB), astragalus polysaccharide (APS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), cholesterol (Chol), dioleoyl phosphatidylethanolamine (DOPE), (2,3-dioleoxypropyl)trimethylammonium chloride (DOTAP), N-[N-trimethylamine chloride (DOTMA), cationic cholesterol (DC-Chol), dimethyltrifluoroacetate-2,3-dioleoxypropyl-2-(2-speramide)ethylammonium (DOSPA), trimethyldodecylammonium bromide (DDAB), trimethyltetradecylammonium bromide (TTAB), trimethylcetylammonium bromide (CTAB), dimethyldidoctadecylammonium bromide (DDAB), CpG ODN (nucleotide sequence with unmethylated cytosine and guanine dinucleotide as core elements, artificially synthesized CpG). [Brief explanation of the drawing]

[0044] [Figure 1] This is a structural diagram of the mRNA sequence in Embodiment 1. [Figure 2] This is a schematic diagram of the preparation of the mRNA vaccine in Embodiment 1. [Figure 3] This is a schematic diagram of the recombinant protein in Embodiment 1. [Figure 4] This is a schematic diagram of the immunization program and grouping in Test Example 1. [Figure 5] This figure shows the test results for anti-RBD-specific antibodies in mice at 0, 21, and 42 days after vaccination in Test Example 2. [Figure 6] This figure shows the test results for anti-RBD-specific antibodies in mice at 0, 21, and 111 days after vaccination in Test Example 2. [Figure 7]This figure shows the levels of pseudovirus neutralizing antibodies in mice 0, 21, and 42 days after vaccination in Test Example 3. [Figure 8] This figure shows the levels of pseudovirus neutralizing antibodies in mice at 0, 21, and 111 days after vaccination in Test Example 3. [Figure 9] This figure shows the levels of pseudovirus neutralizing antibodies against Omicron and its variants in mice 0, 21, and 111 days after vaccination in Test Example 3. [Figure 10] This is a flow cytometry plot of serum blocking assays in mice at 0, 21, and 42 days post-vaccination in Test Example 4. [Figure 11] This figure shows the statistical results of serum blocking assays in mice at 0, 21, and 42 days post-vaccination in Test Example 4. [Figure 12] This is a flow cytometry plot of serum blocking assays in mice at 0, 21, and 111 days post-vaccination in Test Example 4. [Figure 13] This figure shows the statistical results of serum blocking assays in mice at 0, 21, and 111 days post-vaccination in Test Example 4. [Figure 14] These are typical flow cytometry plots of CD4+ memory T cells (top figure) and CD8+ memory T cells (bottom figure) generated by RBD-specific IL-4 in Test Example 5. [Figure 15] This graph shows the IL-4 level results in the cell supernatant analyzed by ELISA in Test Example 5. [Figure 16] These are typical flow cytometry plots of CD4+ memory T cells (top figure) and CD8+ memory T cells (bottom figure) generated by RBD-specific IFN-γ in Test Example 5. [Figure 17] This is a graph of IFN-γ levels in the cell supernatant analyzed by ELISA in Test Example 5. [Figure 18]These are typical flow cytometry plots of CD4+ memory T cells (top figure) and CD8+ memory T cells (bottom figure) generated by RBD-specific IL-2 in Test Example 5. [Figure 19] This is a flow cytometry plot of CD8+CD44+IL-2 cells in Test Example 5. [Figure 20] This is a flow cytometry plot of CD4+CD44+IL-2 cells in Test Example 5. [Figure 21] This is a flow cytometry plot of CD4+CD44+CD62L- cells in Test Example 5. [Figure 22] This is a flow cytometry plot of CD8+CD44+CD62L- cells in Test Example 5. [Figure 23] This is a typical flow cytometry plot of six follicular helper T cells in Test Example 6. [Figure 24] This is a flow cytometry plot of T follicular helper cells in Test Example 6. [Figure 25] This is a typical flow cytometry plot of plasmablasts in Test Example 6. [Figure 26] This is a flow cytometry plot of plasmablasts in Test Example 6. [Figure 27] This is a flow cytometry plot of germinal center B cells in Test Example 6. [Figure 28] This figure shows the viral load in the nasal turbinate tissue of the experimental mice in Test Example 7. [Figure 29] This figure shows the viral load in the tracheal tissue of experimental mice in Test Example 7. [Figure 30] This figure shows the viral load in the lung tissue of the experimental mice used in Test Example 7. [Modes for carrying out the invention]

[0045] Currently, research into serial immunization using mRNA and protein vaccines remains largely blank.

[0046] The present invention provides recombinant protein vaccines and mRNA vaccines against infection by SARS-CoV-2 or its variants, the two vaccines being used in combination or together. In a specific embodiment of the present invention, the protein vaccine RBD-HR / trimer is used as a third dose heterologous booster after two doses of the mRNA vaccine have been administered.

[0047] Compared to recombinant protein vaccines prepared with MF59-like adjuvants, single-dose mRNA vaccines induce a superior germinal center (GC) response against SARS-CoV-2 infection. However, this study found no significant difference in germinal center response between three-dose allogeneic RBD-HR / trimeric vaccines and allogeneic mRNA vaccines. The researchers also found that mRNA-omicron boosters failed to induce a stronger humoral or cellular immune response compared to those inoculated with mRNA-1273. Furthermore, this study also showed that the thermal stability and antigenicity of recombinant omicron RBD were impaired. Therefore, in this study, recombinant RBD proteins based on the delta mutant were designed instead of the omicron mutant to effectively achieve specific and broad mutant neutralization against the delta mutant. Experimental validation demonstrated that heterologous recombinant protein vaccine boosters provide stronger immunity and protection than allogeneic mRNA vaccines.

[0048] The solutions of the present invention will be described with reference to embodiments. Those skilled in the art will understand that the following embodiments are not intended to limit the scope of the invention, but merely to illustrate it. Where no specific technical or condition is specified in an embodiment, the technical or condition described in the relevant art literature or product specifications shall prevail. Where the manufacturer of the reagents or equipment used is not indicated, they are all conventional products available by market purchase. [Examples]

[0049] Embodiment 1: Preparation of mRNA vaccine and recombinant protein vaccine As shown in Figure 1, an mRNA vaccine capable of expressing a full-length spike protein having the amino acid sequence shown in SEQ ID NO: 3 and the mRNA sequence shown in SEQ ID NO: 2 was prepared based on the full-length S protein of the delta strain of the SARS-CoV-2 mutant (Figure 1). The mRNA vaccine was prepared by mixing an oil phase (E1-1 lipid, DOPE, cholesterol, DMG-PEG2000, and ethanol) and an aqueous phase (mRNA sodium citrate solution) in appropriate proportions (Figure 2). The endotoxin levels of the mRNA vaccine were less than 15 EU / dose or less than 30 EU / mL.

[0050] We developed a recombinant trimer vaccine containing delta-derived receptor-binding domain (RBD) proteins (including L452R and T478K). RBD / HR trimer proteins were prepared and mixed with an equal volume of MF59-like adjuvant to prepare the RBD-HR / trimer recombinant protein vaccine (Figure 3). Considering the well-established efficacy of MF59-like adjuvants in the antibody-T cell response, MF59-like adjuvants were selected as adjuvants to enhance the immunogenicity of RBD-HR / trimer proteins. The specific preparation process includes the following:

[0051] (1) Preparation of RBD / HR trimer recombinant protein vaccine: 1. Construction and design of RBD / HR trimer proteins The SARS-CoV-2 S protein was a membrane-located protein. Therefore, to mimic its secretion process, the GP67 signal peptide sequence was added to the N-terminus of the protein to assist in protein secretion and expression during the construction of S-RBD-HR protein expression in SARS-CoV-2, although this signal peptide would be spontaneously removed by insect cells during the protein secretion process. On the other hand, the thioredoxin (Trx) tag from Spodoptera fulgiperda (S. fulgiperda) was added after the GP67 signal peptide to assist in S-RBD-HR folding, a 6×his tag was added to facilitate subsequent purification, and an EK enzyme digestion site was added to remove both the Trx and 6×his tags. Protein expression construction and design can be performed by removing all non-S-RBD-HR redundant amino acids by EK enzyme digestion. The amino acid design sequence of the protein is shown in SEQ ID NO: 4.

[0052] An S protein expression vector was constructed based on the pFastBac1 vector (ampicillin resistant), inserted into the pFast-bacI vector using BamHI and HindIII enzyme digestion sites, and optimized according to the preferred codons of insect cells.

[0053] 2. Amplification of recombinant baculoviruses Based on the principle of bacterial transposons, site-directed transposition was induced via the Tn7 transposon element through a BAC-to-BAC expression system, completing the construction of recombinant bacmid in E. coli (including DH10Bac, bacmid (kanamycin-resistant), and helper plasmid (tetracycline-resistant)). Recombinant bacmid was extracted and transfected into sf9 insect cells using Beyotime's Lipoinsect® transfection reagent to produce recombinant baculovirus capable of expressing target genes. 72 hours after transfection, the first generation virus was collected, P2-P4 generation viruses were amplified, and proteins were expressed using P3 or P4 generation viruses.

[0054] 3. Protein expression Hi5 insect cells (sf9 cells) were infected with P3 or P4 generation viruses at different infection multiplicities (MOI 0.5-10). After 48-72 hours of culture, the supernatant was collected. The optimal collection time may vary depending on the viral load and cell condition, but generally, it is appropriate to examine approximately 50% of the cellular lesions under a microscope.

[0055] 4. Protein purification The recovered culture supernatant was rapidly centrifuged at 4°C and filtered through a 0.22 μm filtration membrane, and the recombinant protein was pre-purified by affinity purification (HisTrap nickel column). Subsequently, the recombinant protein was purified using a MonoQ ion column and Superdex 200 10 / 300GL molecular sieve, and the protein purity was identified by SDS-PAGE, which showed that the required purity was over 95%. After purification by molecular exclusion chromatography, a good purity RBD-HR / trimeric protein antigen was obtained, which could be used in subsequent immunodefence studies. The purified recombinant RBD-HR / trimeric protein antigen was diluted in PBS at 120 μg / ml.

[0056] An RBD-HR / trimeric recombinant protein vaccine was prepared by mixing an equal volume of MF59-like adjuvant with RBD / HR trimer protein.

[0057] (2) Preparation of mRNA vaccine An mRNA vaccine capable of expressing the full-length spike protein was prepared using microfluidic technology. E1-1 lipids, DOPE, cholesterol, and DMG-PEG2000 were dissolved in anhydrous ethanol to prepare a predetermined volume of E1-1 organic phase solution with a concentration of 18 mg / mL in a molar ratio of 50:10:38.5:1.5. Meanwhile, mRNA expressing the full-length spike protein (SEQ ID NO: 2) was prepared to a predetermined volume of 0.6 mg / mL using citrate buffer. The mRNA-LNP complex was prepared using a microfluidic instrument with a flow rate ratio of 3:1 between the aqueous and organic phases and a total flow rate of 9 mL / min. After removing ethanol by ultrafiltration with phosphate buffer (PBS), an mRNA vaccine capable of expressing the full-length spike protein was obtained.

[0058] The following experiment illustrates the beneficial effects of the present invention.

[0059] Test Example 1: Mouse Immunotherapy Experiment Six-to-eight-week-old female NIH mice were purchased from Charles River Laboratories and raised in an environment free of specific pathogens. As shown in Figure 4, the NIH female mice were randomly divided into four groups: a PBS group, a homologous mRNA group (mRNA), a homologous RBD-HR / trimer group (RBD-HR), and a heterologous group (mRNA+RBD-HR).

[0060] Mice were administered according to either homologous or heterologous vaccination sequences. NIH mice were immunized with 1 μg, 5 μg, and 10 μg mRNA vaccines on days 0 and 21, respectively, and then immunized with the corresponding doses of mRNA or 10 μg RBD-HR vaccine on day 42, respectively. Control mice were administered three doses of the same type and dose of PBS, mRNA, and RBD-HR / trimer vaccines, respectively. In other words, each mouse in the homologous mRNA group received three doses of 1 μg, 5 μg, or 10 μg mRNA vaccine, and each mouse in the homologous RBD-HR group received three doses of 10 μg RBD-HR vaccine. Heterologous groups received the same dose of mRNA vaccine for the first two doses, followed by a 10 μg RBD-HR vaccine for the third dose.

[0061] To test the efficacy of the RBD-HR vaccine on the long-term immune response after two doses of mRNA vaccine, mice were immunized with a third booster dose on day 111. Blood samples were collected via the orbital vein on days 56, 84, 125, and 153 and centrifuged at 6000 rpm for 10 minutes at 4°C. Serum samples were stored at -20°C before use. Seven days after the third immunization, mice were sacrificed and their spleens and lymph nodes were removed. Spleen tissue and lymph nodes were homogenized, and single-cell suspensions were prepared using lymphocyte isolation medium.

[0062] Test Example 2: Detection of specific antibodies against RBD RBD-specific antibodies in serum were detected using enzyme-linked immunosorbent assay (ELISA). 96-well NUNC-MaxiSorp plates (Thermo Fisher Scientific, USA) were coated with recombinant RBD protein (0.1 μg / well) at 4°C for 12 hours. The plates were washed three times with 1×PBST (1×PBS + 0.1% Tween-20) and then blocked with 1% bovine serum albumin (BSA) at 37°C for 1 hour. The plates were incubated with a 2-fold dilution of serum sample at room temperature for 1 hour and washed three times with 1×PBST. Horseradish peroxidase (HRP)-goat anti-mouse IgG antibody, diluted 1:10000, was added to the plates, incubated at room temperature for 1 hour, and washed five times. 3,3',5,5'-tetramethylbiphenyldiamine (TMB) was added to the plates and incubated in the dark for 10 minutes. The incubation was stopped with 1M H2SO4 (100 μl / well), and the absorbance at 450 nm was measured.

[0063] As shown in Figures 5 and 6, in immunization programs with short time intervals (immunization on days 0, 21, and 42) (Figure 5) and long time intervals (immunization on days 0, 21, and 111) (Figure 6), heterologous immunization regimens induced higher levels of anti-RBD binding antibodies than homologous immunization with three doses of mRNA vaccine.

[0064] Test Example 3: Pseudovirus Neutralization Test To evaluate whether heterologous vaccine boosters have superior protective effects compared to homologous vaccine boosters, mice from the homologous mRNA group were injected with 1 μg of mRNA vaccine each time, and serum was collected 84 and 153 days after the first immunization (long-term immunization) to perform pseudovirus neutralization tests.

[0065] SARS-CoV-2 pseudoviruses (GFP-luciferase), BA.3 and BA.4 / 5 pseudoviruses, and other SARS-CoV-2 variant pseudoviruses were purchased from Vazyme Biotech Co., Ltd. (China) and Genomeditech (China). mRNA vaccines were intramuscularly injected into NIH mice. After inactivation at 60°C for 30 minutes, serum was diluted in 3-fold gradient complete dimethyl ether medium. Diluted serum and luciferase pseudoviruses (wild type, B.1.1.7, B.1.351, P.1, B.1.617, C.37, BA.1, BA.2, BA.2.12.1, BA.3, BA.4 / 5) were incubated at 37°C for 1 hour. 1.2 × 10⁴ 293T / ACE2 cells were added to each well to express the reporter gene. After 48 hours, the supernatant of the infected cells was removed, and then 100 μL of fluorescein substrate (50 μL of PBS + 50 μL of fluorescein substrate), which had been pre-mixed with PBS 1:1 and equilibrated at room temperature, was added to each well and incubated for approximately 2 minutes. Finally, chemiluminescence was detected using a fluorescence analyzer.

[0066] As shown in Figure 7, in short-interval immunization programs, the heterologous sequential immunization group induced higher levels of broad-spectrum neutralizing antibodies compared to the homologous mRNA vaccine immunization group.

[0067] As shown in Figure 8, in long-interval immunization programs, the heterologous immunization group also induced higher broad-spectrum neutralizing antibody titers compared to the allologous mRNA vaccine group.

[0068] As shown in Figure 9, in long-interval immunization programs, heterologous immunization groups were able to induce higher neutralizing antibody levels against Omicron and its variants, including the BA.4 / 5 variant.

[0069] Test Example 4: Blocking assay of RBD protein binding to ACE2 receptor Binding to the ACE2 receptor was detected by the RBD-Fc (omicron) protein. RBD-Fc protein (0.3 μg / mL) was serially diluted in immunoserum (immunoimmun dose for mRNA vaccine was 1 μg / dose / animal, and immunoimmunoimmun dose for protein was 10 μg / dose / animal) and incubated at 37°C for 30 minutes. Then, 293T / ACE2 cells (5 × 10⁴) were added and incubated at room temperature for 30 minutes. The cells were washed three times with BPBS (PBS with 1% BSA added). Anti-human IgG-Fc antibody labeled with pe was added to the cells and incubated in the dark at 4°C for 30 minutes. Binding concentrations were measured by flow cytometry, and the results were analyzed using a NovoCyte flow cytometer (ACEA Biosciences).

[0070] As shown in Figures 10 and 11, in an immunization program with 21-day intervals, the third dose of the heterologous RBD / HR trimer booster injection effectively enhanced the binding ability of mouse serum to block hACE2, improving the blocking rate.

[0071] As shown in Figures 12 and 13, in an immunization program immunized with a third-dose booster injection 90 days after the second-dose immunization, the third-dose booster injection of the heterologous RBD / HR trimer vaccine also effectively enhanced the binding ability of mouse serum to block hACE2 and improved the blocking rate compared to the third-dose allologous mRNA vaccine.

[0072] In summary, to investigate the potential mechanism of antiviral neutralization, flow cytometry was used to detect blocking of binding between the ACE2 receptor on the cell surface and RBD-Fc(omicron). Consistent with the results of pseudoviral assays from two immunization regimens using different time intervals, heterologous immunization also demonstrated a higher inhibition rate between RBD-Fc(omicron) and the human ACE2 receptor than that of allogeneic mRNA immunization. In particular, shorter regimens showed higher inhibition rates, highlighting the importance of receiving co-immunization at the appropriate time. These results suggest that heterologous vaccines containing protein subunits may be a better strategy for inducing stronger humoral immunity than allogeneic mRNA vaccines.

[0073] Test Example 5: Flow cytometry detection of splenic lymphocytes Splenic lymphocytes were cultured in 1640 medium for 72 hours and subjected to intracellular cytokine staining (ICS). 10% fetal bovine serum, 100 μg / mL streptomycin, 100 U / mL penicillin, 1 mM pyruvate (all purchased from Gibco, USA), 50 μM β-mercaptoethanol, and 20 U / mL IL-2 (all purchased from Sigma-Aldrich) were added to the 1640 medium. In addition, RBD (10 μg / well) was added to the live cells. To block intracellular cytokine secretion, the cells were incubated with brefeldin A (BFA, BD Biosciences) for 6 hours and then stained. Cells were washed with 1× chilled PBS and stained with CD4 (Brilliant Violet 421), CD8 (FITC), CD44 (Brilliant Violet 510), B220 (PerCP / Cyanine 5.5), and MHC-II (BV711) (all purchased from BioLegend) at 4°C for 30 minutes. Cells were then fixed and permeabilized, and intracellular staining with anti-IFN-γ (PE), anti-IL-4 (Brilliant Violet 421), and IL-2 (APC) (all flow antibodies from BioLegend) was accelerated at room temperature for 2 hours. Cells were washed with 1× cold PBS and then detected by flow cytometry.

[0074] T cell response is a major determinant of clinical outcomes and plays a crucial role in SARS-CoV-2 prevention. Homologous mRNA mice were administered 5 μg of mRNA intramuscularly each time. CD4 response to SARS-CoV-2 RBD + and CD8 + T cell responses were evaluated. Single-cell suspensions of splenic lymphocytes were prepared to assess the boosting effect of the RBD-HR / trimeric heterologous vaccine on cellular immune responses. After stimulation with RBD protein at 37°C for 72 hours, secreted cytokines (IL-4 and IFN-γ) were detected by flow cytometry and ELISA. Higher levels of IL-4 (Figures 14 and 15) and IFN-γ (Figures 16 and 17) were observed in the heterologous vaccine group and the allogeneic protein vaccine group, indicating that the RBD-HR / trimeric vaccine can induce a strong T cell response.

[0075] It has been reported that mRNA vaccines were able to elicit a clear T cell response related to IL-2, while protein vaccines were not. In particular, both heterologous and homologous mRNA vaccines elicited equivalent IL-2-related CD8 responses. + This induced a T cell response (Figures 18-20). Memory T cell responses are crucial for accelerating and enhancing the immune response, becoming even more important during reinfection by the same pathogen. Furthermore, immune T cell memory responses can also provide protective immunity against various variants. On day 49, splenic effector memory (CD44) was detected. + CD62L - We evaluated a subset of T cells. The number of effector memory T cells in both CD4+ and CD8+ was greater than in the allogeneic mRNA and RBD-HR / trimer vaccine groups (Figures 21 and 22). In summary, cellular immunity induced by xenogeneic RBD-HR / trimer vaccination is stronger than that induced by allogeneic mRNA vaccination.

[0076] Test Example 6: Detection of spleen memory T cells After preparing a single-cell suspension of splenic lymphocytes, Tfh cells, GC B cells, plasmablasts, and effector memory T cells were stained using CD3 (PerCP / cyanine 5.5), CD62L (Brilliant Violet 421), CD4 (APC), CD8 (FITC), CD69 (PE-cy7), and anti-mouse CD44 (Brilliant Violet 510), CD95 (FITC), B220 (PE-cy7), GL7 (PE), CD4 (Brilliant Violet 510), CD8 (Brilliant Violet 510), CD19 (Brilliant Violet 421), CD138 (APC), CXCR5 (APC), PD1 (Brilliant Violet 421), and anti-mouse CD19 (PE) (all flow antibodies from BioLegend). Cells were stained in the dark at 4°C. After 30 minutes, the cells were washed with 1×PBS and detected by flow cytometry.

[0077] The long-term memory response provides rapid and effective protective immunity upon reinfection. Homologous mRNA mice were intramuscularly injected with 5 μg of mRNA each time. To assess whether xenogeneic vaccination could induce a further memory response with RBD-HR / trimer, the formation of follicular T helper (Tfh) cell effects, plasmablast responses, and germinal center (GC) effects, which are crucial for the long-term memory response, was analyzed. Furthermore, GC B cells and Tfh cells are closely associated with the neutralizing antibody response. The results showed that xenogeneic vaccination with RBD-HR / trimer could enhance the responses of Tfh (Figures 23 and 24), plasmablasts (Figures 25 and 26), and GC-B (Figure 27). In particular, the xenogeneic vaccination group showed the highest frequencies of Tfh and GC B cells. Therefore, these results indicate that xenogeneic RBD-HR / trimer vaccines can induce a greater long-term memory immune response than mRNA vaccine boosters.

[0078] Test Example 7: Live Virus Challenge Assay Mice were administered vaccines according to either homologous or heterologous vaccination sequences. Mice were immunized with 5 μg of mRNA vaccine on days 0 and 21, and then immunized again on day 42 with the corresponding doses of 5 μg of mRNA or 10 μg of RBD-HR vaccine, respectively. The PBS group served as the control group.

[0079] To test the protective effect of the RBD-HR vaccine on mice pre-stimulated with two doses of mRNA vaccine, live virus challenge was performed on day 56 by intranasal vaccination. All experimental animals were challenged with Omicron (BA.1) by intranasal vaccination, with a challenge dose of 1 × 10⁶ per mouse. 6 The TCID50 count was 1.45. Four days after the challenge, the right lung lobe, nasal turbinate, and tracheal tissue of mice were excised for viral load detection. The tissue was cut into small pieces, mixed, and weighed. Approximately 100 mg of tissue was weighed and homogenized with 800 μL of Trizol. RNA was extracted using 400 μL of tissue homogenate, and finally the RNA was dissolved in 50 μL of nuclease-free solution and stored at -80°C for future use. The RNA was used for subsequent viral load detection by qRT-PCR (one-step method). Viral genomic RNA (gRNA) was detected by real-time fluorescence quantitative PCR (qRT-PCR). The primers and probes used targeted the N gene of SARS-CoV-2. Positive: SEQ ID NO: 6 5'-GACCCCAAAATCAGCGAAAT-3', Reverse: SEQ ID NO: 7 5'-TCTGGTTACTGCCAGTTGAATCTG-3', Probe: SEQ ID NO: 8 5'-FAM-ACNCCGCATTACGTTTGGTGGACC-BHQ 1-3'.

[0080] The results showed high viral loads in the nasal turbinates, trachea, and lung tissue of experimental mice in the PBS group (Figures 28-30). The mRNA vaccine group showed a significant reduction in viral load, with only low levels of viral residue. No viral load was detected in any tissue of the RBD-HR serial mRNA vaccine group. This demonstrates that the RBD-HR serial mRNA vaccine can better inhibit viral replication and eliminate residual virus in mice.

[0081] In summary, in this invention, a third dose xenobolic booster of the self-assembling recombinant RBD-HR vaccine and a second dose of the mRNA vaccine can significantly enhance humoral and cellular immune responses, resulting in higher anti-RBD IgG levels, stronger neutralizing antibodies, and IFN-γ secreting T cells. Furthermore, xenobolic vaccination can induce stronger effector memory responses than allogeneic RBD-HR and mRNA vaccination. Moreover, the lymphoblastic response induced by xenobolic vaccination is higher than that of the allogeneic mRNA group, and the induced GC-B cell and Tfh responses are higher than those of both groups. This indicates that xenobolic vaccination can induce higher long-term memory responses. Compared to mRNA homologous sequencing, RBD-HR xenobolic sequencing provides a better protective effect. Therefore, this invention provides a novel strategy for enhanced xenobolic immunity. A third dose xenobolic protein subunit booster (RBD-HR / trimeric vaccine) is a very suitable candidate after a second dose of mRNA vaccination.

Claims

1. An mRNA vaccine for the prevention and / or treatment of SARS-CoV-2 or its variant infections, characterized by containing an mRNA vaccine against SARS-CoV-2 variant infection as an active ingredient.

2. A recombinant protein vaccine for preventing and / or treating infection by SARS-CoV-2 or its variants, characterized by containing a recombinant protein vaccine as an active ingredient against infection by SARS-CoV-2 variants.

3. The mRNA vaccine according to claim 1, wherein the mRNA sequence comprises a full-length nucleic acid sequence encoding the SARS-CoV-2 mutant S protein.

4. The mRNA vaccine according to claim 1, wherein the full-length nucleic acid sequence encoding the SARS-CoV-2 mutant S protein is shown in the sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2, or exhibits homology and identical or similar biological activity to the sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2, and is a mutant obtained by substitution and / or deletion and / or insertion of at least one amino acid in the sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2, preferably the mutant sequence is a mutant obtained by substitution and / or deletion and / or insertion of 1 to 400 amino acids in the sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2, and preferably the mutant sequence is a mutant obtained by substitution and / or deletion and / or insertion of 5 to 30 amino acids in the sequence of SEQ ID NO:

1.

5. The recombinant protein vaccine according to claim 2, wherein the protein and / or protein precursor comprises a protein having at least one RBD sequence and at least one HR sequence derived from the S protein of SARS-CoV-2 or a mutant thereof.

6. The recombinant protein vaccine according to claim 2, wherein the protein and / or protein precursor comprises an amino acid sequence shown in the sequence of SEQ ID NO: 4, or a mutant that exhibits homology and identical or similar biological activity to the sequence of SEQ ID NO: 4 and is obtained by substitution and / or deletion and / or insertion of at least one amino acid in the sequence of SEQ ID NO: 4, preferably the RBD sequence is a mutant obtained by substitution and / or deletion and / or insertion of 1 to 400 amino acids in the sequence of SEQ ID NO: 4, and preferably the RBD sequence is a mutant obtained by substitution and / or deletion and / or insertion of 5 to 30 amino acids in the sequence of SEQ ID NO:

4.

7. A composition for infection by SARS-CoV-2 or a variant thereof, comprising a preparation containing an mRNA vaccine and a recombinant protein vaccine as active ingredients, wherein the mRNA vaccine is the mRNA vaccine described in claim 1, 3, or 4, and the recombinant protein vaccine is the recombinant protein vaccine described in claim 2, 5, or 6.

8. A combination drug for infection by SARS-CoV-2 or a variant thereof, comprising an mRNA vaccine and a recombinant protein vaccine for SARS-CoV-2 or a variant thereof, wherein the mRNA vaccine and the recombinant protein vaccine are administered simultaneously or sequentially, the mRNA vaccine being the mRNA vaccine according to claim 1, 3, or 4, and the recombinant protein vaccine being the recombinant protein vaccine according to claim 2, 5, or 6.

9. The mRNA vaccine, the recombinant protein vaccine, or the combination drug according to claim 8 is formulated as an intramuscular injection, nasal spray, spray, nasal spray, or inhalant.

10. The combination drug according to claim 8, wherein the protein formed by the RBD sequence and HR sequence of the recombinant S protein spontaneously forms a trimer.

11. The combination drug according to claim 8, wherein the homologous amino acid sequence is selected from at least one of the alpha, beta, gamma, delta, and omicron RBD sequences.

12. The combination drug according to claim 10, wherein the protein precursor is linked to a signal peptide and / or protein tag on a protein resistant to SARS-CoV-2 or its variant infection, preferably the signal peptide comprises a native signal peptide of the S protein or a variant thereof, and / or a human tPA signal peptide further added before the native signal peptide, preferably the protein tag is selected from at least one of a histidine tag, a thioredoxin tag, a glutathione transferase tag, a ubiquitin-like modifier protein tag, a maltose-binding protein tag, a c-Myc protein tag, and an Avi-tag protein tag, and more preferably the protein tag is a Trx tag and / or a 6His tag.

13. The combination drug according to claim 8, wherein the protease recognition site for cleaving the protein tag is further linked to a protein against infection by SARS-CoV-2 or a variant thereof, and preferably the protease is selected from at least one of enterokinase, TEV protease, thrombin, coagulation factor Xa, carboxypeptidase A, and rhinovirus 3c protease.

14. Host cells used in recombinant protein vaccines according to claim 2, wherein the host cells are selected from at least one of insect cells, mammalian cells, Escherichia coli, and yeast, preferably the insect cells are selected from at least one of sf9 cells, sf21 cells, and Hi5 cells, and preferably the mammalian cells are either CHO cells or HEK293 cells.

15. The mRNA vaccine and / or recombinant protein vaccine according to claim 1, wherein the mRNA vaccine and / or recombinant protein vaccine further comprises a pharmaceutically acceptable excipient or adjuvant component.

16. The recombinant protein vaccine further comprises a pharmaceutically acceptable excipient or adjuvant component, wherein the adjuvant component is an immunoadjuvant, preferably selected from at least one of a squalene-based oil-in-water emulsion, an aluminum salt, a calcium salt, a plant saponin, a plant polysaccharide, monophosphate tripide A, muramyl dipeptide, muramyl tripeptide, a bacterial toxin, a GM-CSF cytokine, a lipid, and a cationic liposome material, further wherein the immunoadjuvant is MF59 as the squalene-based oil-in-water emulsion, selected from at least one of aluminum hydroxide and alum as the aluminum salt, tricalcium phosphate as the calcium salt, QS-21 or ISCOM as the plant saponin, and astragalus polysaccharide as the plant polysaccharide. The combination drug according to claim 13, wherein 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 phosphatidylethanolamine, phosphatidylcholine, cholesterol, and dioleoylphosphatidylethanolamine; and the cationic liposome material is selected from at least one of (2,3-dioleoyloxypropyl)trimethylammonium chloride, N-[1-(2,3-dioleoylchloride)propyl]-N,N,N-trimethylamine chloride, cationic cholesterol, dimethyl-2,3-dioleyloxypropyl-2-(2-spermidinecarboxamide)ethylammonium trifluoroacetate, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, dimethyldioctadecylammonium bromide, and CpG ODN.

17. Use of the mRNA vaccine according to any one of claims 1, 3, 4, and 9, the recombinant protein vaccine according to any one of claims 2, 5, 6, and 9, the composition according to claim 7, or the combination drug according to any one of claims 8, 9 to 13, in preparing a drug for preventing and / or treating infection by SARS-CoV-2 or a variant thereof.

18. Use of an mRNA vaccine according to any one of claims 1, 3, 4, and 9, a recombinant protein vaccine according to any one of claims 2, 5, 6, and 9, the composition according to claim 7, or a combination drug according to any one of claims 8, 9 to 13, in preparing a drug for treating and / or preventing infection or pathogenicity caused by a SARS-CoV-2 mutant strain, wherein the SARS-CoV-2 mutant strain comprises at least one of alpha, beta, gamma, delta, and omicron.

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