Anti-SARS-cov-2 virus protein or mRNA vaccine, preparation method therefor, and use thereof

Abstract

An anti-SARS-CoV-2 virus protein or mRNA vaccine, a preparation method therefor, and a use thereof. The protein has one or more amino acid residues added, deleted, or replaced in the amino acid sequence as shown in SEQ ID NO: 18. The nucleic acid encodes an S protein mutant. Preclinical animal test data shows that the mRNA vaccine has a good protective effect for the current variants of concern (VOC) of the SARS-CoV-2 virus and has broad clinical application prospects.

Description

A protein or mRNA vaccine against SARS-CoV-2 virus and its preparation method and application

[0001] This application claims priority to Chinese Patent Application No. 2023112837656, filed on September 28, 2023. This application incorporates the entirety of the aforementioned Chinese Patent Application. Technical Field

[0002] The present invention belongs to the field of biomedicine, and specifically relates to a protein or mRNA vaccine against SARS-CoV-2 virus, and a preparation method and application thereof. Background Art

[0003] Coronaviruses (CoV) are a class of enveloped, single-stranded, positive-sense RNA viruses with a wide host range, common susceptibility to humans and a variety of animals, and are associated with numerous diseases. Known coronaviruses include HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV, and / or MERS-CoV. Studies have shown that the genetic sequence of the SARS-CoV-2 virus (GenBank accession: MN908947) shares over 85% sequence similarity with the SARS coronavirus.

[0004] Targeting the coronavirus surface spike protein antigen (S protein), recombinant protein vaccines, mRNA vaccines, DNA vaccines, non-replicating viral vector vaccines, replicating viral vector vaccines, peptide vaccines, virus-like particle vaccines, attenuated vaccines, and inactivated vaccines have been developed. However, existing vaccines suffer from low cell transfection rates, low expression efficiency, weak immune responses, and high clinical dose requirements. These issues, on the one hand, lead to high production costs, high medication costs for patients, and a heavy economic burden. On the other hand, high-dose medications introduce more impurities, potentially leading to increased toxic side effects and thus increased clinical risks.

[0005] In the early stages of the COVID-19 pandemic, mRNA vaccines using lipid nanoparticle (LNP) delivery systems achieved unprecedented success. Two mRNA vaccines, encoding the prefusion-stabilized full-length spike protein of SARS-CoV-2, from Moderna (mRNA-1273) and Pfizer-BioNTech (BNT162b2), are currently in widespread use, and several other mRNA vaccine candidates are in clinical trials. However, as the virus continues to evolve and new variants emerge, the efficacy of these two mRNA vaccines is significantly diminishing. Therefore, the development of new broad-spectrum mRNA vaccines is urgently needed.

[0006] Summary of the Invention

[0007] In order to solve the technical problem of the lack of effective or broad-spectrum vaccines for preventing and / or treating SARS-CoV-2 virus infection in the prior art, the present invention provides an anti-SARS-CoV-2 virus protein or mRNA vaccine and its preparation method and application.

[0008] The first aspect of the present invention provides an S protein mutant, which has one or more amino acid residues added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 18.

[0009] In the present invention, the S protein mutant does not contain the amino acid sequence shown in any one of SEQ ID NOs: 6, 7 and 18.

[0010] In some preferred embodiments, the S protein mutant includes one or more of the following in the amino acid sequence shown in SEQ ID NO: 18:

[0011] (i) The Furin protease recognition site was removed and RRAR was mutated to GSAS;

[0012] (ii) the C-terminal 18 amino acids were removed;

[0013] (iii) the substitutions include one or more of: T19I, LPPA24S, V83A, G142D, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K;

[0014] (iv) The deletion is ΔY144.

[0015] In some more preferred embodiments, the S protein mutant comprises the following amino acid sequence as shown in SEQ ID NO: 18:

[0016] (i) The Furin protease recognition site was removed and RRAR was mutated to GSAS;

[0017] (ii) the C-terminal 18 amino acids were removed;

[0018] (iii) the substitutions include: T19I, LPPA24S, V83A, G142D, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K; and

[0019] (iv) The deletion is ΔY144.

[0020] Preferably, the amino acid sequence of the S protein mutant is as shown in SEQ ID NO: 17; or, the S protein mutant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence as shown in SEQ ID NO: 17, and retains the function of the S protein mutant as shown in SEQ ID NO: 17. The above identity and retention of the function of the S protein mutant as shown in SEQ ID NO: 17 are based on satisfying one or more of the above conditions (i), (ii), (iii) and (iv).

[0021] In some preferred embodiments, the S protein mutant further includes a signal peptide.

[0022] Preferably, the signal peptide is located at the N-terminus of the S protein mutant.

[0023] More preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 1.

[0024] The second aspect of the present invention provides a combination of S protein mutants, comprising a first mutant and a second mutant; the first mutant is the S protein mutant as described in the first aspect, and the first mutant is different from the second mutant.

[0025] The third aspect of the present invention provides an isolated nucleic acid encoding the S protein mutant as described in the first aspect or the combination of the S protein mutants as described in the second aspect.

[0026] In some preferred embodiments, in the nucleic acid encoding the combination, the nucleic acid encoding the first mutant and the nucleic acid encoding the second mutant are contained in the same or different nucleic acid molecules.

[0027] In some preferred embodiments, the nucleic acid is RNA, such as mRNA.

[0028] Preferably, when the mRNA encodes the S protein mutant as described in the first aspect, the sequence of the mRNA encoding the S protein mutant is as shown in any one of SEQ ID NOs: 12-15; or, the mRNA has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequence as shown in any one of SEQ ID NOs: 12-15, and retains the function of the mRNA as shown in any one of SEQ ID NOs: 12-15. The mRNA has the above identity and retains the function of the mRNA as shown in any one of SEQ ID NOs: 12-15 based on satisfying one or more conditions (i), (ii), (iii) and (iv) of the above-mentioned S protein mutant.

[0029] In the present invention, the first mRNA and the second mRNA are on the same mRNA chain or on different mRNA chains.

[0030] Preferably, when the first mRNA and the second mRNA are on different mRNA strands, the 5' end of the first mRNA includes a 5'UTR; and / or the 3' end of the first mRNA includes a 3'UTR and / or a 3'poly(A).

[0031] Preferably, when the first mRNA and the second mRNA are on different mRNA strands, the 5' end of the second mRNA includes a 5'UTR; and / or the 3' end of the second mRNA includes a 3'UTR and / or a 3'poly(A).

[0032] Preferably, when the first mRNA and the second mRNA are on the same mRNA chain, the 5' end of the mRNA chain includes a 5' UTR; and / or the 3' end includes a 3' UTR and / or a 3' poly (A).

[0033] Preferably, the nucleotide sequence of the 5'UTR is shown in SEQ ID NO: 10.

[0034] Preferably, the nucleotide sequence of the 3'UTR is shown in SEQ ID NO:11.

[0035] In some preferred embodiments, the nucleic acid comprises a modification at one or more positions selected from the group consisting of: 5'UTR, open reading frame, 3'UTR, and 3'poly(A).

[0036] Preferably, the modification comprises conversion of cytidine (C) to 5-methylcytidine (m5C), conversion of uridine (U) to pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), 5-methoxyuridine (mo5U) or integration of 5-methylcytidine and pseudouridine (m5C / Ψ).

[0037] More preferably, the modification is pseudouridine modification or N1-methylpseudouridine modification.

[0038] In some preferred embodiments, the sequence of the 3'poly(A) is as shown in SEQ ID NO:9.

[0039] In some preferred embodiments, the nucleic acid is DNA.

[0040] Preferably, the nucleotide sequence of the DNA encoding the S protein mutant is as shown in any one of SEQ ID NOs: 2-5; or, the DNA has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in SEQ ID NOs: 2-5, and retains the functions of the DNA shown in SEQ ID NOs: 2-5. The aforementioned identity and retention of the functions of the DNA shown in any one of SEQ ID NOs: 2-5 are based on the fact that the DNA satisfies one or more of the conditions (i), (ii), (iii) and (iv) of the above-mentioned S protein mutant.

[0041] In some embodiments, the DNA sequence encoding the signal peptide is shown in SEQ ID NO:8.

[0042] The fourth aspect of the present invention provides a recombinant expression vector comprising the nucleic acid described in the third aspect.

[0043] Preferably, the backbone of the recombinant expression vector is a plasmid or a virus.

[0044] The plasmid is, for example, pcDNA3.1.

[0045] In some preferred embodiments, the nucleic acid encoding the first mutant and the nucleic acid encoding the second mutant are contained in the same or different recombinant expression vectors.

[0046] The fifth aspect of the present invention provides a transformant comprising the nucleic acid as described above, or the recombinant expression vector as described above.

[0047] In some preferred embodiments, the transformant containing the recombinant expression vector of the nucleic acid encoding the first mutant is the same as or different from the transformant containing the recombinant expression vector of the nucleic acid encoding the second mutant.

[0048] Preferably, the starting cells of the transformant are eukaryotic cells, preferably mammalian cells, such as HEK 293T cells.

[0049] The sixth aspect of the present invention provides a method for preparing an S protein mutant or a combination of S protein mutants, the method comprising culturing the transformant as described in the fifth aspect under conditions suitable for the expression of the S protein mutant.

[0050] The seventh aspect of the present invention provides a composition comprising (1) a nucleic acid encoding as described above, or a recombinant expression vector as described above, or a transformant as described above, and (2) a delivery vector.

[0051] In some preferred embodiments, the delivery vehicle comprises a liposome.

[0052] In some preferred embodiments, the delivery vehicle comprises lipid nanoparticles (LNP).

[0053] Preferably, the lipid nanoparticles comprise cationic lipids and non-cationic lipids.

[0054] More preferably, the cationic lipid comprises RL151 and / or Dlin-MC3.

[0055] The molar ratio of the cationic lipid is, for example, 45% to 55%.

[0056] The molar ratio of the non-cationic lipid is, for example, 55% to 45%.

[0057] Preferably, the non-cationic lipid comprises a phospholipid and / or a lipid conjugate.

[0058] More preferably, the phospholipid comprises distearoylphosphatidylcholine (DSPC).

[0059] More preferably, in the delivery vehicle, the molar ratio of the phospholipid is 9% to 10%.

[0060] More preferably, the lipid conjugate comprises a lipid molecule modified with polyethylene glycol.

[0061] Even more preferably, the polyethylene glycol-modified lipid molecule includes DMG-PEG2000.

[0062] In some preferred embodiments, the molar ratio of the lipid conjugate in the delivery vehicle is 1% to 2%.

[0063] In some preferred embodiments, the delivery vehicle comprises cholesterol.

[0064] Preferably, in the delivery vehicle, the molar ratio of cholesterol is 35% to 40%.

[0065] In some preferred embodiments, the delivery vehicle comprises a cationic lipid, cholesterol, a phospholipid, and a lipid conjugate.

[0066] Preferably, the delivery vehicle comprises RL151, cholesterol, DSPC, and DMG-PEG2000; and the molar ratio of RL151, cholesterol, DSPC, and DMG-PEG2000 is 50:38.5:10:1.5. That is, based on the total molar amount of RL151, cholesterol, DSPC, and DMG-PEG2000 as 100%, the delivery vehicle comprises 50% RL151, 38.5 mol% cholesterol, 10 mol% DSPC, and 1.5 mol% DMG-PEG2000.

[0067] In some preferred embodiments, in the composition, the nucleic acid is mRNA and is contained in the delivery vector.

[0068] The eighth aspect of the present invention provides a pharmaceutical composition comprising the composition as described in the seventh aspect, and optionally a pharmaceutically acceptable carrier and / or excipient.

[0069] The ninth aspect of the present invention provides a vaccine comprising the S protein mutant as described in the first aspect, the combination of the S protein mutants as described in the second aspect, the nucleic acid as described in the third aspect, the recombinant expression vector as described in the fourth aspect, the transformant as described in the fifth aspect, the composition as described in the seventh aspect and / or the pharmaceutical composition as described in the eighth aspect, and pharmaceutically acceptable carriers and / or excipients.

[0070] Preferably, the vaccine is a protein vaccine.

[0071] Preferably, the vaccine is a nucleic acid vaccine.

[0072] Preferably, the vaccine is a DNA vaccine or an mRNA vaccine.

[0073] In the present invention, when the vaccine is an mRNA vaccine, the first mRNA encoding the first mutant and the second mRNA encoding the second mutant are on the same mRNA chain or on different mRNA chains.

[0074] The tenth aspect of the present invention provides a kit or a medicine box, which comprises the S protein mutant as described in the first aspect, the combination of S protein mutants as described in the second aspect, the nucleic acid as described in the third aspect, the recombinant expression vector as described in the fourth aspect, the transformant as described in the fifth aspect, the composition as described in the seventh aspect, the pharmaceutical composition as described in the eighth aspect and / or the vaccine as described in the ninth aspect.

[0075] The eleventh aspect of the present invention provides an S protein mutant as described in the first aspect, a combination of S protein mutants as described in the second aspect, a nucleic acid as described in the third aspect, a recombinant expression vector as described in the fourth aspect, a transformant as described in the fifth aspect, a composition as described in the seventh aspect, a pharmaceutical composition as described in the eighth aspect, a vaccine as described in the ninth aspect and / or a kit or medicine kit as described in the tenth aspect for preparing a medicament for alleviating, preventing and / or treating diseases caused by SARS-CoV-2 virus.

[0076] Preferably, the drug is a vaccine.

[0077] More preferably, the vaccine is a protein vaccine.

[0078] More preferably, the vaccine is a nucleic acid vaccine.

[0079] More preferably, the vaccine is a DNA vaccine or an mRNA vaccine.

[0080] The twelfth aspect of the present invention provides an S protein mutant as described in the first aspect, a combination of S protein mutants as described in the second aspect, a nucleic acid as described in the third aspect, a recombinant expression vector as described in the fourth aspect, a transformant as described in the fifth aspect, a composition as described in the seventh aspect, a vaccine as described in the ninth aspect and / or a kit or medicine kit as described in the tenth aspect for alleviating, preventing and / or treating diseases caused by SARS-CoV-2 virus.

[0081] The thirteenth aspect of the present invention provides a method for alleviating, preventing and / or treating diseases caused by SARS-CoV-2 virus, namely, administering to a subject in need an effective amount of the S protein mutant as described in the first aspect, the combination of S protein mutants as described in the second aspect, the nucleic acid as described in the third aspect, the recombinant expression vector as described in the fourth aspect, the transformant as described in the fifth aspect, the composition as described in the seventh aspect, the pharmaceutical composition as described in the eighth aspect, the vaccine as described in the ninth aspect and / or the kit or medicine box as described in the tenth aspect.

[0082] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0083] The reagents and raw materials used in the present invention are commercially available.

[0084] The positive progress effect of the present invention is:

[0085] The preclinical animal test data of the SARS-CoV-2 virus variant mRNA vaccine of the present invention show that it has a good protective effect against the current mainstream variants of the SARS-CoV-2 virus (variants of concern, VOC) and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 shows the structural characterization of the antigen expressed by Chimera E.

[0087] FIG2 shows the screening of codons and modified nucleotides for mRNA encoding S protein antigen provided by the present invention; wherein Scodon (44) to Scodon (62) indicate that the GC content in the sequence ranges from 44% to 62%.

[0088] FIG3 shows the final optimization scheme of the present invention for the codons after pseudouridine modification; wherein, S(44) to S(62) indicate that the GC content in the sequence is from 44% to 62%.

[0089] FIG4 shows the expression levels of four mRNAs of chimera E.

[0090] Figure 5 shows the content of S protein in cell lysate detected by western blot; NC represents negative control, and M represents marker.

[0091] FIG6 shows the protein expression level of chimera E LNP in HEK293T cells.

[0092] FIG7 shows the IgG antibody titers of chimera E (2 μg and 5 μg doses) against the S protein of the prototype strain and Omicron XBB.1.5 strain in BALB / c mice.

[0093] Figure 8 shows the pseudovirus neutralizing antibody titers of chimera E against Omicron mutant strains (BA.4 / 5, BQ.1.1, XBB.1.5, CH.1.1 and XBB.1.16) in BALB / c mice.

[0094] Figure 9 shows that chimera E (2 μg and 5 μg doses) produced specific IgG antibodies against both the prototype strain and the Omicron XBB.1.5 strain S protein in K18-hACE2 mice.

[0095] The pseudovirus detection results in Figure 10 show that the chimeric E vaccine produced high-titer neutralizing antibodies against different Omicron mutants (XBB.1.5 and XBB.1.16) in K18-hACE2 mice.

[0096] The results of the real virus neutralizing antibody test in Figure 11 show that the chimeric E vaccine produced high titer neutralizing antibodies against the Omicron XBB.1.5 strain in K18-hACE2 mice.

[0097] Figure 12 shows that the chimeric E vaccine (5 μg and 25 μg doses) produced specific IgG antibodies against both the prototype strain S protein and the Omicron XBB.1.5 strain S protein in golden hamsters.

[0098] The pseudovirus test results in Figure 13 show that the chimeric E vaccine produced high-titer neutralizing antibodies against different Omicron mutants (XBB.1.5 and XBB.1.16) in golden hamsters.

[0099] The results of the real virus neutralizing antibody test in Figure 14 show that the chimeric E vaccine produced high titer neutralizing antibodies against the Omicron XBB.1.5 strain in golden hamsters.

[0100] Figure 15 shows the histopathological changes in New Zealand rabbits after immunization with the chimera E vaccine.

[0101] FIG16 shows the serum IgG antibody titers of BALB / c mice after sequential administration.

[0102] FIG17 shows the pseudovirus neutralizing antibody titers in serum of BALB / c mice after sequential administration.

[0103] FIG18 shows the serum true virus neutralizing antibody titers in BALB / c mice after sequential administration.

[0104] FIG19 shows the results of antigen-specific T cell immune responses in the spleen of BALB / c mice 36 and 75 days after the second immunization.

[0105] Figure 20 shows the long-term stability of the Chimera E vaccine under -20°C storage conditions.

[0106] FIG21 shows the results of long-term immunogenicity evaluation of Chimera E vaccine. DETAILED DESCRIPTION

[0107] Unless otherwise specified, the terms of the present invention are defined as follows.

[0108] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to polymers of amino acids of any length. The polymer may include modified amino acids. These terms also include amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a labeling component. For example, polypeptides containing one or more amino acid analogs (e.g., including non-natural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and sarcosine), as well as other modifications known in the art, are also included in the definition.

[0109] The term "polypeptide" refers to proteins and peptides of any size, structure, or function. Polypeptides include encoded polynucleotide products, natural polypeptides, synthetic polypeptides, homologues, orthologues, paralogues, fragments and other equivalents, variants, and analogs of the aforementioned. A polypeptide can be a monomer or a multimolecular complex, such as a dimer, trimer, or tetramer. They can also include single-chain or multi-chain polypeptides. Disulfide linkages are most commonly found in multi-chain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids. In some embodiments, a "polypeptide" can be less than or equal to 50 amino acids in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0110] The term "signal peptide" encompasses the N-terminal 15-60 amino acids of a protein, which are typically required for transmembrane transport in the secretory pathway. Therefore, they universally control the entry of most proteins into the secretory pathway in both eukaryotes and prokaryotes. Signal peptides typically consist of three regions: an N-terminal region of varying length, typically composed of positively charged amino acids; a hydrophobic region; and a short carboxy-terminal peptide region. In eukaryotes, the signal peptide of a nascent precursor protein directs the ribosome to the rough endoplasmic reticulum (ER) membrane and delivers the growing peptide chain to the membrane for processing, producing the mature protein. The signal peptide is then cleaved from the precursor protein. Signal peptides can also facilitate localization of proteins to the cell membrane. However, signal peptides are not responsible for the final destination of the mature protein. Secretory proteins that lack an additional address tag in their sequence are by default secreted into the external environment.

[0111] The term "sequence optimization" refers to a process or series of processes by which bases in a reference nucleic acid sequence are replaced with alternative bases to produce a nucleic acid sequence with improved properties, such as improved protein expression or reduced immunogenicity. Generally, the goal of sequence optimization is to produce a synonymous nucleotide sequence that encodes the same polypeptide sequence as the reference nucleotide sequence. Thus, in a polypeptide encoded by a codon-optimized nucleotide sequence, there are no amino acid substitutions relative to the polypeptide encoded by the reference nucleotide sequence.

[0112] In the context of sequence optimization, the term "codon replacement" refers to replacing a codon in a reference nucleic acid sequence with another codon. A codon can be replaced in a reference nucleic acid sequence, for example, by chemical peptide synthesis or by recombinant methods known in the art. Thus, reference to a "replacement" or "substitution" at a certain position in a nucleic acid sequence (such as mRNA) or in a certain region or subsequence of a nucleic acid sequence (such as mRNA) refers to the replacement of a codon at that position or region. As used herein, the term "coding region" refers to an open reading frame (ORF) in a polynucleotide that produces a polypeptide or protein when expressed.

[0113] As used herein, the term "isolated" refers to a substance or entity that has been separated from at least some of the components with which it is associated (whether in nature or in an experimental setting). Isolated substances (such as polynucleotides or polypeptides) can have varying degrees of purity relative to the substance from which they are isolated. Isolated substances and / or entities can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were originally associated. In some embodiments, the isolated substance is greater than about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components.

[0114] As used herein, the term "isolated" polynucleotide, vector, polypeptide, cell, or any composition refers to a polynucleotide, vector, polypeptide, cell, or composition in a form not found in nature. Isolated polynucleotides, vectors, polypeptides, or compositions include those that have been purified to the point that they no longer exist in a form found in nature. In certain aspects, an isolated polynucleotide, vector, polypeptide, or composition is substantially pure.

[0115] The term "nucleic acid" includes any compound and / or substance consisting of nucleotide polymers. These polymers are generally referred to as polynucleotides. Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), sugar nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), ethylene nucleic acid (ENA), cyclohexyl nucleic acid (CeNA) or a mixture or combination thereof. "Polynucleotide" includes triple-stranded, double-stranded and single-stranded DNA or RNA. It also includes modified, for example, by alkylation, and / or by capping, as well as unmodified forms of polynucleotides. More specifically, the term "polynucleotide" includes polydeoxynucleotides (containing 2-deoxy-D-ribose), polynucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA.

[0116] In certain aspects, " polynucleotide " comprises mRNA. In one aspect, this mRNA is a kind of synthetic mRNA. In some aspects, synthetic mRNA comprises at least one non-natural base. In some aspects, all core bases of a certain class are all replaced by non-natural base (for example, all uridines in polynucleotide disclosed herein can be replaced by non-natural base, for example, 5-methoxyuridine). In some aspects, polynucleotide (for example, synthetic RNA or synthetic DNA) only comprises natural base, i.e., A (adenosine), G (guanosine), C (cytidine) and T (thymidine) in the case of synthetic DNA, or A, C, G and U (uridine) in the case of synthetic RNA.

[0117] It will be understood by those skilled in the art that the T in the codon map disclosed herein is present in DNA, while T will be replaced by U in the corresponding RNA. For example, the codon nucleotide sequence disclosed herein in the form of DNA, such as a vector or in vitro translation (IVT) template, will have its T transcribed as U in its corresponding transcribed mRNA. In this regard, both the codon-optimized DNA sequence (including T) and its corresponding mRNA sequence (including U) are considered to be codon-optimized nucleotide sequences. In addition, equivalent codon maps can also be generated by replacing one or more bases with non-natural bases. Thus, for example, the TTC codon (DNA map) corresponds to the UUC codon (RNA map), which in turn corresponds to the ΨΨC codon (RNA map, in which U is replaced by pseudouridine).

[0118] The term "nucleotide sequence encoding" refers to a nucleic acid (e.g., an mRNA or DNA molecule) coding sequence that encodes a polypeptide. The coding sequence may further include initiation and termination signals operably linked to regulatory elements, including a promoter and a polyadenylation signal, capable of directing expression in cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may further include a sequence encoding a signal peptide.

[0119] In addition, there are untranslated regions at either end of the mRNA ORF (Open Reading Frame), referred to as the 5' UTR and 3' UTR, respectively. The "5' untranslated region" (5' UTR) refers to the region of the mRNA directly upstream (i.e., 5') from the start codon (i.e., the first codon in the mRNA transcript translated by the ribosome), which does not encode a polypeptide. The "3' untranslated region" (3' UTR) refers to the region of the mRNA directly downstream (i.e., 3') from the stop codon (i.e., the codon that signals the end of translation in the mRNA transcript), which does not encode a polypeptide. An "open reading frame" is a continuous stretch of DNA that begins with a start codon (e.g., methionine (ATG)) and ends with a stop codon (e.g., TAA, TAG, or TGA) and encodes a polypeptide. The untranslated region cannot translate amino acids, but can bind RNA-binding proteins, thereby regulating the degradation and translation efficiency of the mRNA product. The 5'-UTR or 3' UTR can be homologous or heterologous to the open reading frame in the polynucleotide. Multiple 5'-UTRs or 3'UTRs may be included in the flanking regions and may be of the same or different sequences.

[0120] "PolyA" or "poly(A)" is a region of an mRNA that is located downstream of, e.g., directly downstream (i.e., 3') of, the 3' UTR and contains multiple consecutive adenosine monophosphates. A polyA tail can contain 10 to 300 adenosine monophosphates. For example, a polyA tail can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, a poly(A) tail contains 50 to 250 adenosine monophosphates. In relevant biological contexts (e.g., in cells, in vivo), the function of the poly(A) tail is to protect mRNA from enzymatic degradation, such as in the cytoplasm, and to aid in transcription termination, mRNA export from the nucleus, and translation.

[0121] The term "cationic lipid" has common meaning in the art and may refer to a lipid comprising one or more positively charged groups. As used herein, "positively charged group" refers to a chemical group having a positive electron charge, for example, a monovalent (+1), a divalent (+2), a trivalent (+3), and the like. Examples of positively charged groups include amine groups, ammonium groups, pyridyl groups, guanidyl groups, and imidazole groups. In certain embodiments, an ionizable lipid molecule may include an amine group and may be referred to as an ionizable amino lipid. In the present invention, cationic lipids include but are not limited to RL151 and / or Dlin-MC3.

[0122] As used herein, the term "PEGylated lipid" or "PEGylated lipid" refers to a lipid modified with polyethylene glycol (PEG) (PEG-lipid). Non-limiting examples of PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (such as PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-dicyanopropane-3-amine. For example, the PEG-lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0123] In some embodiments, PEG-lipids include but are not limited to 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxlpropyl-3amine (PEG-c-DMA).

[0124] In some embodiments, the PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the lipid molecules of the PEG-lipid include those having a length from about C14 to about C22, preferably from about C14 to about C16. In some embodiments, the PEG molecule, such as mPEG-NH2, has a size of about 1000, 2000, 5000, 10000, 15000, or 20000 Daltons. In some embodiments, the PEG-lipid is PEG2000-DMG.

[0125] As used herein, "lipid nanoparticles" or "LNPs" are used for the delivery of mRNA. In some embodiments, the LNPs essentially comprise (i) at least one cationic lipid; (ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE, and SM; (iii) a sterol, such as cholesterol; and (iv) a PEG-lipid, such as PEG2000-DMG, in a molar ratio of 45-55% cationic lipid: 35-40% neutral lipid: 8-12% sterol: 1-2% PEG-lipid.

[0126] As used herein, the term "synthesize" refers to artificial production, preparation, and / or manufacture. The synthesis of polynucleotides or other molecules can be chemical or enzymatic. As used herein, "expression" of a nucleic acid sequence refers to the translation of a polynucleotide (e.g., mRNA) into a polypeptide or protein and / or post-translational modification of the polypeptide or protein. Methods of transfection include, but are not limited to, chemical methods, physical treatments, and cationic lipids or mixtures.

[0127] The term "transcription" refers to the process of producing mRNA (e.g., an mRNA sequence or template) from DNA (e.g., a DNA template or sequence). "Transfection" refers to the introduction of a polynucleotide (e.g., an exogenous nucleic acid) into a cell, wherein the polypeptide encoded by the polynucleotide is expressed (e.g., mRNA) or the polypeptide modulates cellular function (e.g., siRNA, miRNA). For example, transfection can occur in vitro, ex vivo, or in vivo.

[0128] "Modification" refers to any substance, compound, or molecule that is altered in some way. A molecule can undergo a series of modifications, with each modified molecule serving as an "unmodified" starting molecule for subsequent modifications. Herein, the modifications include converting cytidine (C) to 5-methylcytidine (m5C), converting uridine (U) to pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), 5-methoxyuridine (mo5U), or the integration of 5-methylcytidine and pseudouridine (m5C / Ψ).

[0129] The term "variant" or "mutant" includes both natural variants (such as polymorphisms, isomers, etc.) and artificial variants in which at least one amino acid residue in the native or starting sequence (such as the wild-type sequence) has been removed and a different amino acid has been inserted in the same position. These variants may be described as "substitutional variants." The substitution may be single, i.e., only one amino acid is replaced in the molecule, or multiple, i.e., two or more amino acids are replaced in the same molecule. If amino acids are inserted or deleted, the resulting variant will be an "insertional variant" or a "deletional variant," respectively.

[0130] As used herein, the term "immune response" refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the above cells or the liver, resulting in selective damage, destruction, or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancer cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation. In some cases, administration of nanoparticles composed of a lipid component and an encapsulated therapeutic agent can induce an immune response, which can be caused by (i) the encapsulated therapeutic agent (such as mRNA), (ii) the expression product of such encapsulated therapeutic agent (such as a polypeptide encoded by mRNA), (iii) the lipid component of the nanoparticle, or (iv) a combination thereof.

[0131] As used herein, the term "vaccine" is a biological agent that stimulates the immune system to respond to a certain agent or antigen, usually an infectious pathogen or a part thereof, which enters the human or animal body in a non-infectious or non-pathogenic form. Once the immune system is stimulated, the immune system will produce a rapid and powerful immune response when it comes into contact with the pathogen in the future, eliminating the pathogen before it can multiply and infect enough cells in the host organism to cause disease symptoms. The agent or antigen used to stimulate the immune system can be a whole organism with lower infectiousness, i.e., a so-called attenuated organism, or in some cases, a component of an organism, such as a carbohydrate, protein or peptide representing various structural components of the organism. Therefore, according to the present invention, the vaccine can be a protein vaccine or a nucleic acid vaccine. The nucleic acid vaccine can be a DNA vaccine or an mRNA vaccine. When it is a protein vaccine, the vaccine can be encoded by a polynucleotide or mRNA of the present invention.

[0132] The immune response elicited by a single dose of vaccine (primary immunization) is often insufficient to provide effective and / or sustained protection. Repeated administration (boosting) can significantly enhance humoral and cellular responses to vaccine antigens (e.g., Estcoat et al., 2002). "Boosting" as used herein refers to administering a second immunization to the corresponding animal one day after the first immunization using the exact same sample as the first immunization.

[0133] The "combination" used in the present invention refers to any material composition comprising the ingredients of the present invention, i.e., S protein mutants and / or nucleic acids encoding S protein mutants, DNA and / or mRNA encoding S protein mutants. It should be understood that the combination can be formulated as a single component, or it can be prepared as separate preparations, which can then be combined for combined administration. The combination can be used as the effective active ingredient (or effective ingredient, active ingredient) of the composition. When the combination of the present invention is used to prepare a pharmaceutical composition, the pharmaceutical composition may further contain pharmaceutically acceptable excipients and / or carriers well known to those skilled in the art. The combination or pharmaceutical composition of the present invention can also be provided in the form of a complete kit, wherein each component is formulated separately but packaged in a single container.

[0134] As used herein, the term "treat" refers to partially or completely alleviating, ameliorating, improving, relieving, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms or features of a particular infection, disease, disorder, and / or condition. For example, "treating" cancer may refer to inhibiting the survival, growth, and / or spread of a tumor. Treatment may be performed on subjects who do not exhibit signs of a disease, disorder, and / or condition and / or subjects who exhibit only early signs of a disease, disorder, and / or condition to reduce the risk of developing pathology associated with the disease, disorder, and / or condition.

[0135] The term "therapeutic agent" or "prophylactic agent" refers to any agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. Therapeutic agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

[0136] In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). Exemplary polynucleotides used according to the present disclosure include, but are not limited to, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), including messenger mRNA (mRNA) or its hybrids, RNAi inducers, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, and carriers, etc. One or more. In some embodiments, therapeutic and / or preventive drugs are RNA. RNA useful in the compositions and methods described herein includes, but is not limited to, ribozymes, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In certain embodiments, the RNA is mRNA.

[0137] In certain embodiments, the therapeutic and / or preventative measure is an mRNA. The mRNA can encode any polypeptide of interest, including any naturally occurring or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA can be of any size and can have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA can have a therapeutic effect when expressed in a cell.

[0138] As used herein, “and / or” should be considered to specifically disclose each of the two specific features or components, whether or not the other is present. Thus, the term “and / or” as used herein in phrases such as “A and / or B” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, the term “and / or” or “one or more” as used in phrases such as “A, B and / or C” or “one or more of A, B, and C” is intended to include the following: A, B, and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0139] It should also be noted that the term "comprising" is intended to be open-ended and allow, but not require, the inclusion of additional elements or steps. When the term "comprising" is used, the terms "consisting essentially of" and "consisting of" are also inclusive. When a composition is described as having, including, or comprising specific ingredients, it is contemplated that the composition also consists essentially of, or consists of, the described ingredients. Similarly, when a method or process is described as having, including, or comprising specific steps, the method or process also consists essentially of, or consists of, the described steps. Furthermore, it should be understood that the order of the steps or the order in which certain actions are performed is unimportant so long as the invention remains operable. Furthermore, two or more steps or actions may be performed simultaneously.

[0140] The term "identity" refers to the similarity or consistency between the sequences of two or more polypeptides or polynucleotides, determined by comparing these sequences. "Percent identity" applicable to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in an amino acid or nucleic acid sequence that are identical to those in another amino acid or nucleic acid sequence, by aligning the sequences and introducing gaps (if necessary) to achieve maximum percent identity. Methods and computer programs for comparison are well known in the art. Identity depends on the calculation of percent identity, but due to the gaps and penalties introduced in the calculation, its numerical values ​​may be different. "Identity" is determined by those skilled in the art using alignment tools and specific parameters. Such alignment tools include the tools in the BLAST suite (Stephen F. Altschul, et al. (1997), and specific parameter settings are well known to those skilled in the art.

[0141] If other polypeptides or polynucleotide molecules have a certain degree of identity with the polypeptides or polynucleotide molecules herein, they also fall within the scope of protection of the present invention. Specifically, variants of other polynucleotides or polypeptides have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the specific polynucleotides or polypeptides protected by the present invention, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity based on the same core sequence or amino acid residues, and can be considered to be equivalent or identical to the specific polypeptides or polynucleotide molecules protected by the present invention.

[0142] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0143] Example 1: mRNA sequence design

[0144] In this embodiment, chimera E is used to prepare a single-stranded mRNA chimera vaccine designed for the sequences of Omicron subvariants XBB.1, XBB.1.5 and XBB.1.5.1. As shown in Figure 1, the amino acid sequence of chimera E is based on the prototype strain sequence (SEQ ID NO: 18), with a △Y144 mutation (△ indicates that the site is knocked out) in the NTD region, T19I, LPPA24S, V83A, G142D, H146Q, Q183E, V213E, and G252V mutations in the NTD region, and G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, and R376T in the RBD region. 408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H mutations, D614G, H655Y, N679K, P681H mutations in the SD2 region, and N764K, D796Y, Q954H, and N969K mutations in other regions. In addition, the sequence of chimera E mutated the furin protease recognition site and mutated RRAR to GSAS; the C-terminal 18 amino acids were removed, the transmembrane region was retained, and the antigenic conformation was stabilized; thus maintaining the integrity of the S protein expressed by the mRNA vaccine in vivo, improving the stability of the antigenic epitope and the immunogenicity of the vaccine.

[0145] Example 2: In-depth optimization and screening of mRNA

[0146] In this example, the mRNA sequence of the S protein used was deeply optimized in terms of codons and modified nucleotides in order to reduce the immunogenicity in vivo and achieve the strongest antigen expression effect in vivo.

[0147] Specifically, the inventors designed S protein coding sequences with 10 different codons and tested 6 different modified nucleotides, including conversion of cytidine (C) to 5-methylcytidine (m5C), conversion of uridine (U) to pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), 5-methoxyuridine (mo5U) or the integration of 5-methylcytidine and pseudouridine (m5C / Ψ).

[0148] Figure 2 shows the expression levels of mRNAs with different GC contents and modifications in HEK293 cells. The numbers in parentheses after "S Codon" represent the encoded GC content (the GC content described in this invention refers to the ratio of guanine to cytosine among the four bases in DNA), WT represents the prototype strain (wild type, without codon optimization and nucleotide modification), and the numbers 1 to 6 represent: 1 for uridine U, 2 for m5C / Ψ, 3 for Ψ, 4 for mo5U, 5 for m1Ψ, and 6 for m5C. The results in Figure 2 indicate that mRNA modified with Ψ and m1Ψ has the highest expression levels.

[0149] Therefore, the pseudouridine modification with a GC content of 44% (i.e., "S(44)" in Figure 3) to 62% (i.e., "S(62)" in Figure 3) was measured. The results in Figure 3 show that regardless of the GC content of the codon sequence used, pseudouridine modification can improve the expression of the S protein; among them, the S protein sequence containing 60% GC content is the optimal sequence and can obtain the best in vitro expression effect. Therefore, the sequence of chimera E was determined to use pseudouridine modification, and the codon of S(60) was basically determined to be the optimal codon sequence. Therefore, the present invention designed multiple mRNA sequences with a 60% GC content for chimera E, such as those shown in SEQ ID NO:12 to SEQ ID NO:15. The western results of the expression of four of the mRNAs are shown in Figure 4: the expression of three of the mRNAs (SEQ ID NO:13-15, corresponding to v2, v3 and v4 with different codons, respectively) met the expression standards of the mRNA vaccine.

[0150] The mRNA of this example also includes a 5'UTR and a 3'UTR, for example, as shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively. The data show that the UTR sequence selected in this example is conducive to improving the expression efficiency of the S protein.

[0151] To improve the translation efficiency and stability of mRNA, the 3' end of the mRNA of this example further has a poly(A) tail, the nucleotide sequence of which is shown in SEQ ID NO:9.

[0152] On this basis, the mRNA molecules of the prototype strain, chimera E and Omicron subvariant XBB.1.5.1 were transcribed in vitro and transfected into 293T cells respectively. The cells were collected after 19 hours, and the content of S protein in the cell lysate was detected by western blot (Figure 5). The results showed that the expression level of chimera E was the highest in two unmodified Omicron subvariant strains (XBB.1 (amino acid sequence shown in SEQ ID NO:16, DNA sequence shown in SEQ ID NO:6), XBB.1.5.1 (DNA sequence shown in SEQ ID NO:7)) and the other three different codons of chimera E (chimera E (v2), chimera E (v3) and chimera E (v4)). Therefore, chimera E (SEQ ID NO:12) was finally selected for further animal experiments to verify its efficacy.

[0153] Example 3: Preparation of lipid nanoparticles

[0154] The mRNA of Chimera E from Example 2 was ionized (cationic) at low pH and coated into nanoparticles using two helper lipids: DSPC (distearoylphosphatidylcholine, Catalog No. B90536, manufactured by Nippon Seika Co., Ltd.) and cholesterol (Catalog No. C00373, manufactured by Nippon Seika Co., Ltd.), as well as a PEGylated lipid (DMG-PEG2000, Catalog No. M-DMG-2000, purchased from JenKem). An aqueous solution of the mRNA was prepared by mixing the mRNA dissolved in ultrapure water with 100 mM (millimoles per liter, or mmol / L) citrate buffer at pH 4.0 at a volume ratio of 1:1. The ratio of the four lipid components (e.g., cationic lipid RL151 (Cat. No. W211-YB211202, purchased from Zhejiang Shenzhou Pharmaceuticals): cholesterol: DSPC: DMG-PEG2000 = 50:38.5:10:1.5) was adjusted and dissolved in 99.5% ethanol to form a lipid solution. The mRNA and lipid solution were mixed in a NanoAssemblr (Precision Nanosystems) microfluidic mixing system at a volume ratio of HO:EtOH = 3:1 and a constant total flow rate of 12 mL / min to produce lipid nanoparticles (LNPs) containing the mRNA. The lipid nanoparticles were dialyzed, concentrated, filtered, and stored to obtain the chimeric E vaccine.

[0155] Example 4: LNP protein expression and animal experiments

[0156] 1. LNP expression detection

[0157] The protein expression level of chimeric E LNP was tested in HEK293T cells (Figure 6). 5 μg of LNP was incubated with HEK293T cells in one well of a 12-well plate for 18 hours, and the cells were harvested for protein expression analysis by Western blotting. Briefly, cells were harvested and washed once with PBS (phosphate-balanced saline), and lysed with lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 3 mM MgCl2, 1% Triton X-100) containing protease inhibitors. All samples were mixed with SDS (sodium dodecyl sulfate) loading buffer, separated on a 4-20% gradient SDS gel, and transferred to a PVDF membrane (polyvinylidene fluoride membrane) using the Trans-Blot Turbo transfer system (universal protein transfer system, purchased from BioRad). The PVDF membrane was blocked with PBST (phosphate-buffered saline) containing 5% skim milk powder and then incubated with the primary antibody (Manufacturer: Sino Biological; Cat. No. 40589-T62). Signals were detected using an HRP-conjugated secondary antibody and an enhanced chemiluminescence (ECL) detection system.

[0158] 2. Animal Vaccination and Serum Collection

[0159] BALB / c mice: For mouse vaccination, 6- to 8-week-old female BALB / c mice (purchased from Sibeifu (Suzhou) Biotechnology Co., Ltd.) were intramuscularly immunized with the chimeric E vaccine (hereinafter referred to as "immunization"). A second dose was administered on day 21 after vaccination to boost the immune response. Ten BALB / c mice were administered in each group (n = 10).

[0160] K18-hACE2 mice: For mouse vaccination, 6- to 8-week-old female K18-hACE2 mice (purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.) were intramuscularly immunized with the chimeric E vaccine (hereinafter referred to as "immunization"), and a second dose was administered on day 21 after vaccination to boost the immune response. Each group of K18-hACE2 mice consisted of 10 (n=10).

[0161] Golden Hamsters: Female golden hamsters aged 6 to 8 weeks (purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were immunized intramuscularly with either the Chimera E vaccine or a placebo (PBS) at week 0 (within 7 days of the first immunization). All hamsters received a booster at week 3. Each group consisted of 10 hamsters (n=10).

[0162] New Zealand rabbits: For vaccination of New Zealand rabbits (purchased from Qingdao Kangda Aibo Biotechnology Co., Ltd.), eight male or female New Zealand rabbits aged 7-8 months (half male and half female, four rabbits per sex) were administered a placebo (sodium chloride injection) on the left side and Chimera E vaccine on the right side, injected at a single site, twice, two weeks apart. The total number of New Zealand rabbits was 8 (n=8).

[0163] Sera from the immunized mice and golden hamsters were collected and inactivated at 56°C for 0.5 h to detect SARS-CoV-2 S protein-specific IgG and neutralizing antibodies as described below.

[0164] 3. Specific IgG enzyme-linked immunosorbent assay

[0165] SARS-CoV-2 S protein-specific antibody responses in immune sera were measured by enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well plates were coated with 50 μL of coating buffer containing 100 ng (nanograms) / well of recombinant SARS-CoV-2 spike or RBD antigen (manufacturer: Sino Biological) at 4°C overnight. The plates were blocked with PBST solution containing 2% bovine serum albumin for 1 hour at room temperature. The immune mouse sera were diluted 10,000-fold as the initial concentration and then serially diluted 2-fold in PBS buffer for a total of 11 dilutions. The plates were washed with PBST and incubated with the serially diluted sera at 37°C for 1 hour. To determine S-specific antibody reactions, the plates were incubated with goat anti-mouse IgG HRP (for mouse serum, Catalog No. 115-035-003, purchased from Proteintech) or goat anti-Syrian hamster IgG HRP (for hamster serum, Catalog No. ab6892, purchased from abcam) for 1 hour at 37°C, followed by color development using the substrate tetramethylbenzidine (TMB) solution (Catalog No. 00-4201-56, purchased from Invitrogen). After approximately 10 minutes, the color development reaction was terminated with 1 M sulfuric acid, and the absorbance was measured at a wavelength of 450 nm using a microplate reader (VARIOSKAN LUX, purchased from Thermo Fisher Scientific).

[0166] 4. Pseudovirus Neutralization Test

[0167] Serum samples collected from the immunized animals described in Item 3 of this Example were serially diluted with cell culture medium. The diluted serum was mixed with the pseudovirus suspension in a 96-well plate at a ratio of 1:1, and then Opti-HEK293 / ACE2 cells were added to the mixture of serum and pseudovirus in the aforementioned 96-well plate, and the plate was cultured at 37°C in an incubator containing 5% CO2 (i.e., the incubator contained 5% CO2 and 95% air). After 48 hours, the luciferase activity reflecting the degree of SARS-CoV-2 pseudovirus transfection was measured using a luciferase assay kit. NT 50 It is defined as the dilution factor that inhibits pseudovirus infection by more than 50% compared with the control group.

[0168] 5. Real virus neutralization test

[0169] The serum samples collected from the immunized animals described in Item 3 of this Example were tested for true virus neutralizing antibodies using a focus forming assay (FFA). The antibody was diluted in a two-fold series starting from 1:8 using DMEM without FBS, and then the true virus dilution solution was added and evenly mixed, and incubated in a 37°C 5% CO2 incubator. The antibody-virus incubation mixture was then transferred to the pre-laid Vero E6 cell plate and incubated at 37°C and 5% CO2. PFA was then added to inactivate and fix the cells. After blocking with BSA, the cells were washed, and then the diluted primary antibody was added and incubated at 37°C. After washing the cells, the diluted secondary antibody was added and incubated at 37°C. The cells were washed, KPL True-blue colorimetric solution was added, and incubated at room temperature in the dark. The cells were rinsed with water and dried, and the spots were counted using an enzyme-linked immunosorbent assay (ELISA) analyzer. Calculate the inhibition rate = 100% × (1-number of sample spots / number of positive well spots). FRNT 50 = logarithm of virus dilution with higher than 50% inhibition rate + ratio distance × (logarithm of virus dilution with lower than 50% inhibition rate - logarithm of virus dilution with higher than 50% inhibition rate). Where: ratio distance = (number of spots in control positive wells with 50% inhibition rate - number of spots in virus dilution with higher than 50% inhibition rate) / number of spots in virus dilution with lower than 50% inhibition rate - number of spots in virus dilution with higher than 50% inhibition rate). Find the corresponding antilogarithm, which is FRNT. 50 .

[0170] 6. Vaccine safety evaluation

[0171] The safety of the Chimera E vaccine was evaluated in New Zealand rabbits (purchased from Qingdao Kangda Aibo Biotechnology Co., Ltd.). Eight New Zealand rabbits were half male and half female, with 4 rabbits per sex per group. Using the left and right side self-comparison method, each animal was administered intramuscularly in the left and right quadriceps muscles. All animals were injected with a negative control on the left side and a test substance (Chimera E vaccine) on the right side. The test substance dose was 30 μg per animal, injected at a single point. Two immunizations were performed at an interval of 14 days. The first two animals of each sex were euthanized 48 hours after the last dose (D17), and the remaining animals were euthanized 14 days after the last dose (D29). The administration sites of all dosed animals were collected for histopathological analysis and safety assessment.

[0172] 7. Statistical Analysis

[0173] All statistical data were generated and graphed using GraphPad Prism 8.0. EC50 (half-maximal effect concentration, concentration for 50% of maximal effect) values ​​were calculated by nonlinear regression. Statistical analysis was performed using the Student's t test when two groups were analyzed and analysis of variance when two or more groups were analyzed.

[0174] Example 5: Evaluation of the immunogenicity of chimeric E vaccine in BALB / c mice

[0175] This example evaluates the immunogenicity of the chimeric E vaccine in BALB / c mice. The mice were divided into three groups, each with 10 mice, and were administered intramuscularly with 2 μg or 5 μg of the chimeric E vaccine or PBS on day 0. All groups received a booster immunization on day 21. No local inflammation or other adverse reactions were observed throughout the experiment. The levels of binding antibodies and neutralizing antibodies (Nab) in the serum collected on day 28 were evaluated. The results showed that high titers of binding antibodies against the prototype strain S protein and the XBB.1.5 mutant strain S protein were produced in the serum of mice immunized with the chimeric E vaccine (Figure 7). Pseudovirus neutralization experiments showed that high titers of NAbs against Omicron mutant strains (BA.4 / 5, BQ.1.1, XBB.1.5, CH.1.1, and XBB.1.16) could be produced in vaccinated BALB / c mice (Figure 8).

[0176] Example 6: Evaluation of the immunogenicity of chimeric E vaccine in K18-hACE2 mice

[0177] This example evaluates the immunogenicity of the chimeric E vaccine in K18-hACE2 mice. The mice were divided into three groups, each with 10 mice, and were administered 2 μg or 5 μg of the chimeric E vaccine or PBS by intramuscular injection on day 0. All groups were boosted on day 21. The levels of binding antibodies to the prototype strain S protein and the Omicron XBB.1.5 strain S protein in the mouse serum were evaluated. As shown in Figure 9, mice immunized with the chimeric E vaccine produced high-titer binding antibodies to the S protein. The neutralizing antibody levels of the serum collected on day 28 were evaluated using a pseudovirus neutralization experiment. The results showed that mice vaccinated with the chimeric E vaccine produced high-titer neutralizing antibodies to each variant strain of Omicron (XBB.1.5 and XBB.1.16) (Figure 10). Serum was tested for true virus neutralization three weeks after the second immunization. Results (Figure 11) showed that the low-dose Chimera E vaccine elicited effective true virus neutralizing antibodies against the XBB.1.5 strain, with a dose-response effect. These results demonstrate that the Chimera E vaccine offers significant protection against the currently circulating Omicron variant.

[0178] Example 7: Evaluation of the immunogenicity of chimeric E vaccine in golden hamsters

[0179] Golden hamsters with ACE2 receptors are highly sensitive to SARS-CoV-2 and develop pneumonia similar to that of COVID-19 patients, making them a suitable model for evaluating vaccines. Three groups of golden hamsters were vaccinated with PBS (placebo), 5μg chimeric E vaccine, or 25μg chimeric E vaccine on days 0 and 21, respectively. Serum was collected after the second immunization and immunogenicity was evaluated. The results showed that the experimental groups produced high titers of binding antibodies against the prototype strain S protein and the Omicron XBB.1.5 strain S protein (Figure 12). Pseudovirus neutralization experiments showed that high titer NAbs against various Omicron variants (XBB.1.5 and XBB.1.16) could be produced in vaccinated golden hamsters, with a dose-dependent effect (Figure 13). Three weeks after the second immunization, the serum was tested for true virus neutralization. The results showed (Figure 14) that the low-dose group of chimeric E vaccine could produce effective true virus neutralizing antibodies against the XBB.1.5 strain, indicating that the chimeric E vaccine has a good protective effect against the currently prevalent Omicron variant strain.

[0180] Example 8: Safety evaluation of chimera E vaccine in New Zealand rabbits

[0181] This example evaluated the muscle stimulation response of the Chimera E vaccine in New Zealand rabbits. Eight New Zealand rabbits, half male and half female, were enrolled, with four rabbits per sex per group. Using the same-body left-right comparison method, each animal received intramuscular injections into the quadriceps femoris muscles on both sides. All animals received a negative control injection on the left side and the Chimera E vaccine on the right side. The test sample dose was 30 μg per animal, administered at a single site. Two immunizations were performed 14 days apart. The first two animals of each sex were euthanized 48 hours after the last dose (D16), and the remaining animals were euthanized 14 days after the last dose (D28). The administration site of all dosed animals was collected for histopathological analysis and safety assessment. Gross observation of the administration site of animals euthanized 48 hours (±2 hours) (D16) and 14 days (D28) after the last dose revealed no abnormal pathological changes. These data demonstrate the safety of Chimera E vaccine when administered intramuscularly in New Zealand rabbits, providing support for clinical trials (Figure 15).

[0182] Example 9: IgG antibody titers in serum after sequential immunization with placebo and chimera E vaccine in BALB / c mice

[0183] Taking into account the background of widespread completion of basic immunization with inactivated vaccines and other COVID-19 vaccines (the examples of the present invention mainly consider the case where two doses of inactivated vaccines have been administered), a sequential study of inactivated vaccine basic immunization was conducted in a BALB / c mouse model. The levels of binding antibodies to the prototype strain S protein and the Omicron XBB.1.5 strain S protein in mouse serum were evaluated according to Example 5. As shown in Figure 16, mice immunized with the chimeric E vaccine produced high titers of binding antibodies to the S protein.

[0184] Example 10: Neutralizing Antibody Titers in Serum After Sequential Immunization with Placebo and Chimera E Vaccine in BALB / c Mice

[0185] As described in Example 5, the levels of pseudovirus and true virus neutralizing antibodies in mouse serum were assessed against various Omicron subtype mutants. As shown in Figure 17 , mice immunized with the Chimera E vaccine produced high titers of pseudovirus neutralizing antibodies against each variant, including high titers against the currently prevalent Omicron subtypes BA.4 / 5, XBB.1.5, and XBB.1.16. Furthermore, true virus neutralization assays ( Figure 18 ) showed that mouse sera from the experimental group also produced high titers of true virus neutralizing antibodies against the Omicron XBB.1.5 strain. These results demonstrate that the Chimera E vaccine has excellent neutralizing ability against various prevalent Omicron variants.

[0186] Example 11: Evaluation of the timeliness of the cellular immune response stimulated by the chimera E vaccine

[0187] This example evaluated the cellular immune response of BALB / c mice 36 and 75 days after secondary immunization with the Chimera E vaccine. Thirty-six mice were randomly divided into three groups, each containing 12 mice. The three groups were vaccinated with either a placebo or the Chimera E vaccine on days 0 and 21, respectively. Splenic antigen-specific T cell responses were measured in six mice from each group on days 36 and 75 after the second immunization. The results showed that the Chimera E vaccine produced high levels of CD4 T cells at both 36 and 75 days after the second immunization. + and CD8 + The above results indicate that the chimera E vaccine can stimulate a high level of cellular immune response and has a long-term effect.

[0188] Example 12: Long-term stability evaluation of chimera E vaccine

[0189] This example evaluates the long-term stability of the Chimera E vaccine under -20°C storage conditions (PBS buffer: sodium chloride, potassium dihydrogen phosphate, sodium hydrogen phosphate dodecahydrate, potassium chloride, sucrose, water for injection). The particle size, encapsulation efficiency, and antigen expression of the Chimera E vaccine were evaluated after 0, 1, and 3 months of storage at -20°C. The results showed that the encapsulation efficiency did not change significantly between 0 and 3 months, and the encapsulation efficiency remained at 90% after 3 months of storage; there was no significant change in antigen expression between 0 and 3 months (Figure 20, the horizontal axis M represents the natural month). The above results indicate that the Chimera E vaccine has long-term stability under -20°C storage conditions.

[0190] Example 13: Evaluation of long-term immunogenicity of chimera E vaccine

[0191] This example monitored the long-term neutralizing antibody and binding antibody levels against XBB.1.5 in BALB / c mice after immunization with the Chimera E vaccine. The results showed that neutralizing antibody levels remained high 6-14 weeks after immunization in BALB / c mice. In the high-dose group, the neutralizing antibody titer against the XBB.1.5 pseudovirus was still as high as 34,330 at 14 weeks post-immunization. Binding antibody titers decreased slightly 6-10 weeks post-immunization and remained stable at 10-14 weeks. At 14 weeks post-immunization, the binding antibody titers for the high- and low-dose Chimera E vaccines were 242,515 and 160,000, respectively, demonstrating the long-term protective effect of the Chimera E vaccine ( FIG. 21 ).

[0192] The following are the sequences involved in the present invention:

[0193] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A S protein mutant, characterized in that: The S protein mutant has one or more amino acid residues added, deleted or replaced in the amino acid sequence shown in SEQ ID NO:

18.

2. The S protein mutant according to claim 1, characterized in that The S protein mutant includes one or more of the following in the amino acid sequence as shown in SEQ ID NO: 18: (i) The Furin protease recognition site was removed and RRAR was mutated to GSAS; (ii) the C-terminal 18 amino acids were removed; (iii) the substitutions include one or more of the following: T19I, LPPA24S, V83A, G142D, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K; (iv) The deletion is ΔY144.

3. The S protein mutant according to claim 1 or 2, characterized in that The amino acid sequence of the S protein mutant is as shown in SEQ ID NO:17; or, the S protein mutant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in SEQ ID NO:17, and retains the function of the S protein mutant as shown in SEQ ID NO:

17.

4. The S protein mutant according to any one of claims 1 to 3, characterized in that The S protein mutant also includes a signal peptide; Preferably, the signal peptide is located at the N-terminus of the S protein in the variant; and / or, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:

1.

5. A combination of S protein mutants, characterized in that: The combination includes a first mutant and a second mutant; the first mutant is the S protein mutant as described in any one of claims 1-4, and the first mutant is different from the second mutant.

6. An isolated nucleic acid, characterized in that The nucleic acid encodes the S protein mutant according to any one of claims 1 to 4; Preferably, in the nucleic acid encoding the combination, the nucleic acid encoding the first mutant and the nucleic acid encoding the second mutant are contained in the same or different nucleic acid molecules.

7. The nucleic acid according to claim 6, wherein The nucleic acid is RNA, such as mRNA.

8. The nucleic acid according to claim 7, wherein The sequence of the mRNA encoding the S protein mutant or the first mutant is as shown in any one of SEQ ID NOs: 12-15; or, the mRNA has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequence shown in any one of SEQ ID NOs: 12-15, and retains the function of the mRNA shown in any one of SEQ ID NOs: 12-15.

9. The nucleic acid according to claim 7 or 8, characterized in that The 5' end of the mRNA includes a 5'UTR; and / or, the 3' end of the mRNA includes a 3'UTR and / or a 3'poly(A); Preferably, the nucleotide sequence of the 5'UTR is shown as SEQ ID NO:10; and / or, the nucleotide sequence of the 3'UTR is shown as SEQ ID NO:11; and / or, the sequence of the 3'poly(A) is shown as SEQ ID NO:

9.

10. The nucleic acid according to any one of claims 6 to 9, characterized in that It comprises modifications at one or more positions selected from the group consisting of: 5'UTR, open reading frame, 3'UTR and 3'poly(A); Preferably, the modification includes conversion of cytosine nucleoside to 5-methylcytidine, conversion of uridine nucleoside to pseudouridine, N1-methylpseudouridine, 5-methoxyuridine or a combination of 5-methylcytidine and pseudouridine; and / or, the modification is pseudouridine modification or N1-methylpseudouridine modification.

11. The nucleic acid according to claim 6, wherein The nucleic acid is DNA; Preferably, the nucleotide sequence of the DNA encoding the S protein mutant or the first mutant is as shown in any one of SEQ ID NOs: 2-5; or, the DNA has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequence shown in any one of SEQ ID NOs: 2-5, and retains the function of the DNA shown in any one of SEQ ID NOs: 2-5.

12. A recombinant expression vector, characterized in that: It comprises the nucleic acid according to any one of claims 6 to 11; Preferably, the backbone of the recombinant expression vector is a plasmid or a virus, and the plasmid is, for example, pcDNA3.

1.

13. A transformant, characterized in that: It comprises the nucleic acid according to any one of claims 6 to 11, or the recombinant expression vector according to claim 12; Preferably, the starting cell of the transformant is a eukaryotic cell, preferably a mammalian cell such as HEK293 T cell.

14. A method for preparing an S protein mutant or a combination of S protein mutants, characterized in that: The method comprises culturing the transformant according to claim 13 under conditions suitable for the expression of the S protein mutant.

15. A composition, characterized in that It comprises (1) the nucleic acid according to any one of claims 6 to 11, or the recombinant expression vector according to claim 12, or the transformant according to claim 13, and (2) a delivery vector.

16. The composition according to claim 15, characterized in that The delivery vehicle comprises a liposome; and / or, the delivery vehicle comprises a lipid nanoparticle (LNP); Preferably, the lipid nanoparticles include cationic lipids and non-cationic lipids; More preferably, the cationic lipid comprises RL151 and / or Dlin-MC3, and the molar ratio of the cationic lipid is, for example, 45% to 55%; and / or, the non-cationic lipid comprises phospholipid and / or lipid conjugate, and the molar ratio of the non-cationic lipid is, for example, 8% to 12%; Further preferably, the phospholipid includes distearoylphosphatidylcholine (DSPC); and / or, in the delivery vector, the molar ratio of the phospholipid is 9% to 10%; and / or, the lipid conjugate includes a lipid molecule modified with polyethylene glycol, such as DMG-PEG2000; and / or, in the delivery vector, the molar ratio of the lipid conjugate is 1% to 2%.

17. The composition according to claim 15 or 16, characterized in that The delivery vehicle also includes cholesterol; Preferably, in the delivery vector, the molar ratio of cholesterol is 35% to 40%.

18. The composition according to any one of claims 15 to 17, characterized in that The delivery vehicle comprises a cationic lipid, cholesterol, a phospholipid, and a lipid conjugate; And / or, the delivery vector comprises RL151, cholesterol, DSPC and DMG-PEG2000; and the molar ratio of RL151, cholesterol, DSPC and DMG-PEG2000 is 50:38.5:10:1.

5.

19. The composition according to any one of claims 15 to 18, characterized in that The nucleic acid is mRNA and is contained in the delivery vector.

20. A pharmaceutical composition, characterized in that It comprises the composition according to any one of claims 15 to 19, and Optionally, a pharmaceutically acceptable carrier and / or excipient.

21. A vaccine, characterized in that It comprises the S protein mutant according to any one of claims 1 to 4, the combination of S protein mutants according to claim 5, the nucleic acid according to any one of claims 6 to 11, the recombinant expression vector according to claim 12, the transformant according to claim 13, the composition according to any one of claims 15 to 19 and / or the pharmaceutical composition according to claim 20, and a pharmaceutically acceptable adjuvant.

22. The vaccine according to claim 21, characterized in that The vaccine is a protein vaccine; and / or, the vaccine is a nucleic acid vaccine; And / or, the vaccine is a DNA vaccine or an mRNA vaccine.

23. A test kit or a medicine box, characterized in that: The kit or medicine box comprises the S protein mutant according to any one of claims 1 to 4, the combination of S protein mutants according to claim 5, the nucleic acid according to any one of claims 6 to 11, the recombinant expression vector according to claim 12, the transformant according to claim 13, the composition according to any one of claims 15 to 19, the pharmaceutical composition according to claim 20 and / or the vaccine according to claim 21 or 22.

24. Use of an S protein mutant as described in any one of claims 1 to 4, a combination of S protein mutants as described in claim 5, a nucleic acid as described in any one of claims 6 to 11, a recombinant expression vector as described in claim 12, a transformant as described in claim 13, a composition as described in any one of claims 15 to 19, a pharmaceutical composition as described in claim 20, a vaccine as described in claim 21 or 22, and / or a kit or a medicine kit as described in claim 23 in the preparation of a medicament for alleviating, preventing and / or treating diseases caused by SARS-CoV-2 virus.

25. The use according to claim 24, characterized in that The drug is a vaccine; Preferably, the vaccine is a protein vaccine; and / or, the vaccine is a nucleic acid vaccine; and / or, the vaccine is a DNA vaccine or an mRNA vaccine.

26. An S protein mutant according to any one of claims 1 to 4, a combination of S protein mutants according to claim 5, a nucleic acid according to any one of claims 6 to 11, a recombinant expression vector according to claim 12, a transformant according to claim 13, a composition according to any one of claims 15 to 19, a pharmaceutical composition according to claim 20, a vaccine according to claim 21 or 22, and / or a kit or a medicine box according to claim 23 for alleviating, preventing and / or treating diseases caused by SARS-CoV-2 virus.

27. A method for alleviating, preventing and / or treating diseases caused by SARS-CoV-2 virus, characterized in that: The method comprises administering to a subject in need thereof an effective amount of an S protein mutant as described in any one of claims 1-4, a combination of S protein mutants as described in claim 5, a nucleic acid as described in any one of claims 6-11, a recombinant expression vector as described in claim 12, a transformant as described in claim 13, a composition as described in any one of claims 15-19, a pharmaceutical composition as described in claim 20, a vaccine as described in claim 21 or 22, and / or a kit or drug kit as described in claim 23.

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