Coronavirus S protein mutant and application thereof

Abstract

The invention discloses a coronavirus S protein mutant and application thereof. The invention provides a coronavirus S protein mutant, the S protein mutant is a mutant having mutation in a conserved region in a cytoplasm tail region of a wild type S protein, and the mutation is that the fourth amino acid in the conserved region is mutated into leucine. According to the present invention, the fourth amino acid in the conserved region in the cytoplasm tail region of the S protein is mutated into the leucine, and the mutation is introduced into the vaccine of SARS-CoV-2, SARS-CoV, MERS-CoV and other viruses so as to significantly enhance the immunogenicity.

Description

TECHNICAL FIELD

[0001] The present application relates to a coronavirus S protein mutant and application thereof, and belongs to the technical field of biological medicine and vaccine. BACKGROUND

[0002] Coronaviridae virus is an enveloped single-stranded RNA virus, which has the largest genome among RNA viruses, up to 27-32 kb. Coronaviridae is divided into four genera: alpha, beta, gamma and delta. Among them, alpha and beta genera are susceptible to mammals, while gamma and delta genera mainly infect birds. So far, seven human coronaviruses have been discovered, including human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU1 (HCoV-HKU1), SARS coronavirus (SARS-CoV, which causes severe acute respiratory syndrome), Middle East respiratory syndrome coronavirus (MERS-CoV, which causes Middle East respiratory syndrome) and novel coronavirus (SARS-CoV-2).

[0003] SARS-CoV-2 has significantly enhanced its transmission ability and immune escape ability due to its extensive mutation characteristics. SARS-CoV-2 contains four structural proteins, namely spike protein (Spike, S), envelope protein (Envelope, E), membrane protein (Membrane, M) and nucleocapsid protein (Nucleocapsid, N). The virus enters the cell by binding to the receptor through the S protein and mediating cell membrane fusion. The E protein forms an ion channel on the surface of the virus to participate in regulating the lysis process of the virus and the subsequent release of the viral genome; the M protein plays a crucial role in virus assembly by interacting with other structural proteins; the main function of the N protein is to protect the genomic RNA by packaging the genomic RNA. The four structural proteins can self-assemble to form virus-like particles (VLP).

[0004] As a key surface antigen of SARS-CoV-2, the S protein not only mediates the process of virus entering host cells, but also plays a crucial role in immunogenicity and vaccine development. It can stimulate the body to produce specific antibodies and T cell immune response. At present, various VLP vaccines use S protein or its specific fragments as the main antigen component in order to achieve effective immune prevention.

[0005] VLPs have made significant progress in vaccine development over the past three decades. VLP vaccines not only have similar spatial structures and compositions to natural virus particles, but also effectively mimic the structural characteristics of natural viruses, allowing antigen-presenting cells to efficiently uptake and process these particles. In addition, since VLP vaccines do not contain viral nucleic acids, they lack replication ability, thereby greatly reducing the safety risks that may occur after vaccination. Currently, there are six SARS-CoV-2 VLP-based candidate vaccines in the clinical trial stage. Although the Canadian government has approved a plant-derived VLP vaccine, the high mutation rate of the virus still poses challenges to the production and application of vaccines. Therefore, we emphasize the importance of optimizing the immunogenicity of vaccines during the development process, aiming to stimulate a stronger immune response and provide long-term immune protection for the host. Variants of SARS-CoV-2 continue to emerge, such as the Omicron variant, which has stronger transmissibility than the original strain. In addition, as a virus that spreads through the respiratory tract, several members of the coronavirus family have acquired the ability to infect human hosts during evolution and have rapidly spread from nature to humans and caused disease. Therefore, developing a universal strategy to enhance vaccine protection can quickly respond to future infectious disease outbreaks caused by new coronavirus variants or other coronaviruses. SUMMARY

[0006] Problem to be solved by the invention

[0007] The present application aims to address the technical problem of the current vaccine's slow response to mutant viruses and reduced effectiveness. As viruses continue to mutate, the development and production cycle of traditional vaccines has become difficult to meet the demand for rapid response. Therefore, the present application is committed to developing a vaccine technology that can quickly adapt to viral mutations, aiming to ensure that when faced with future coronavirus mutations, the vaccine formula can be quickly adjusted to improve the effectiveness and protection range of the vaccine, thereby better protecting public health.

[0008] Solution for solving the problem

[0009] In a first aspect of the present application, a coronavirus S protein mutant is provided, wherein the S protein mutant is a mutant having a mutation in a conserved region in the cytoplasmic tail region of a wild-type S protein.

[0010] In some embodiments, the conserved region in the cytoplasmic tail region of the wild-type S protein has the sequence X1X2X3X4X5X6X7, wherein,

[0011] X1 is D, E, A, or P;

[0012] X2 is any amino acid;

[0013] X3 is E;

[0014] X4 is any amino acid;

[0015] X5 is V, I, or absent;

[0016] X6 is L, H, or absent;

[0017] X7 is K, V, I, or H.

[0018] In some embodiments, the mutation is mutation of amino acid X4 in the conserved region to leucine (L).

[0019] In some preferred embodiments, the conserved region in the cytoplasmic tail of the wild type S protein has the sequence X1X2X3X4X5X6X7, wherein:

[0020] X1 is D or E;

[0021] X2 is any amino acid;

[0022] X3 is E;

[0023] X4 is any amino acid;

[0024] X5 is V, I, or absent;

[0025] X6 is L, H, or absent;

[0026] X7 is K, V, I, or H.

[0027] In some embodiments, the coronavirus comprises a human coronavirus, a porcine epidemic diarrhea virus, or a porcine transmissible gastroenteritis virus.

[0028] In some optional embodiments, the coronavirus is selected from SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-229E, HCoV-NL63, a porcine epidemic diarrhea virus, or a porcine transmissible gastroenteritis virus.

[0029] In some optional embodiments, the amino acid sequence of the conserved region in the cytoplasmic tail of the S protein is set forth in any one of SEQ ID NOs: 23-29.

[0030] In some preferred embodiments, the coronavirus is selected from SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-229E, or HCoV-NL63;

[0031] In some more preferred embodiments, the coronavirus is selected from SARS-CoV, MERS-CoV, or SARS-CoV-2.

[0032] In some specific embodiments, the S protein mutant is selected from any one of the following:

[0033] (a1) comprises an amino acid sequence as set forth in SEQ ID NO: 2, and comprises a mutation from proline (P) to leucine (L) at position 1260 of the amino acid sequence as set forth in SEQ ID NO: 2;

[0034] (a2) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in (a1), and retains the activity of the amino acid sequence as set forth in (a1);

[0035] (b1) comprises an amino acid sequence as set forth in SEQ ID NO: 3, and comprises a mutation from proline (P) to leucine (L) at position 1259 of the amino acid sequence as set forth in SEQ ID NO: 3;

[0036] (b2) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in (b1), and retains the activity of the amino acid sequence as set forth in (b1);

[0037] (c1) comprises an amino acid sequence as set forth in SEQ ID NO: 4, and comprises a mutation from proline (P) to leucine (L) at position 1347 of the amino acid sequence as set forth in SEQ ID NO: 4;

[0038] (c2) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in (c1), and retains the activity of the amino acid sequence as set forth in (c1);

[0039] (d1) comprises an amino acid sequence as set forth in SEQ ID NO: 5, and comprises a mutation from proline (P) to leucine (L) at position 1245 of the amino acid sequence as set forth in SEQ ID NO: 5;

[0040] (d2) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in (d1), and retains the activity of the amino acid sequence as set forth in (d1).

[0041] A second aspect of the present application provides an isolated polynucleotide encoding the coronavirus S protein mutant as described in the first aspect of the present application.

[0042] In some optional embodiments, the polynucleotide is DNA or RNA.

[0043] In some preferred embodiments, the RNA is mRNA.

[0044] A third aspect of the application provides an expression cassette comprising the polynucleotide as described in the second aspect of the application.

[0045] A fourth aspect of the application provides a recombinant vector comprising the polynucleotide as described in the second aspect of the application or the expression cassette as described in the third aspect of the application.

[0046] A fifth aspect of the application provides a host cell comprising the coronavirus S protein mutant as described in the first aspect of the application, the polynucleotide as described in the second aspect of the application, the expression cassette as described in the third aspect of the application or the recombinant vector as described in the fourth aspect of the application.

[0047] A sixth aspect of the application provides a composition comprising the coronavirus S protein mutant as described in the first aspect of the application, the polynucleotide as described in the second aspect of the application, the expression cassette as described in the third aspect of the application, the recombinant vector as described in the fourth aspect of the application or the host cell as described in the fifth aspect of the application.

[0048] In some embodiments, the composition further comprises a delivery vehicle.

[0049] In some optional embodiments, the delivery vehicle comprises protamine, a lipid nanoparticle, a polymeric material or an inorganic nanoparticle.

[0050] In some preferred embodiments, the delivery vehicle comprises a lipid nanoparticle.

[0051] In some embodiments, the composition is a vaccine composition.

[0052] In some optional embodiments, the vaccine composition further comprises one or more adjuvants.

[0053] In some embodiments, the vaccine composition is a virus-like particle vaccine.

[0054] In some optional embodiments, the vaccine composition further comprises at least one of:

[0055] (i) at least one of an envelope protein, a membrane protein and a nucleocapsid protein from a coronavirus;

[0056] (ii) at least one of a polynucleotide encoding the envelope protein, a polynucleotide encoding the membrane protein, and a polynucleotide encoding the nucleocapsid protein;

[0057] (iii) an expression cassette comprising the polynucleotide in (ii);

[0058] (iv) a recombinant vector comprising the polynucleotide in (ii) or the expression cassette in (iii);

[0059] (v) a host cell comprising the polynucleotide in (ii), the expression cassette in (iii), the recombinant vector in (iv), or the recombinant vector in (iv).

[0060] The seventh aspect of the present application provides use of the coronavirus S protein mutant as described in the first aspect of the present application, the polynucleotide as described in the second aspect of the present application, the expression cassette as described in the third aspect of the present application, the recombinant vector as described in the fourth aspect of the present application, or the host cell as described in the fifth aspect of the present application, or the composition as described in the sixth aspect of the present application, in the preparation of a medicament for preventing and / or treating a disease or disorder associated with coronavirus infection.

[0061] In some alternative embodiments, the medicament is a vaccine.

[0062] In some preferred embodiments, the vaccine is a virus-like particle vaccine.

[0063] Effects of the invention

[0064] In some embodiments of the present application, a new mutation site P1263L of the novel coronavirus is found. Specifically, the site is at position 1263 of the S protein of the novel coronavirus, and the mutation from proline (P) to leucine (L) is P1263L.

[0065] In some embodiments of the present application, the novel coronavirus sequence and other coronavirus sequences are aligned, and it is found that P1263L has a conserved region with other coronaviruses, i.e., the fourth amino acid in the conserved region is mutated to L. When applied to the SARS-CoV-2 Omicron BA.1 sublineage strain, it can enhance the immunogenicity of the novel coronavirus VLP vaccine and improve the effectiveness and protection of the vaccine.

[0066] In some embodiments of the present application, in view of the poor protection of existing vaccines against novel coronavirus variants, the mutation of the fourth amino acid in the conserved region to L is introduced into different variants of the novel coronavirus (such as the Omicron XBB strain), which can significantly enhance the immunogenicity.

[0067] In some embodiments of the present application, the mutation of the fourth amino acid in the conserved region to L can also significantly enhance the immunogenicity of the VLP vaccine of SARS virus, MERS virus and other coronaviruses. BRIEF DESCRIPTION OF DRAWINGS

[0068] Figure 1 The results of the mouse humoral immune response experiment are shown in which:

[0069] Figure 1 A in the above is a schematic diagram of the mouse immunization process in the embodiments of the present application;

[0070] Figure 1 B in the above is a statistical diagram of the percentage of germinal center B cells in lymphocytes;

[0071] Figure 1 C in the above is a statistical diagram of the percentage of follicular helper T cells in lymphocytes;

[0072] Figure 1 D in the above is a representative flow cytogram of the percentage of germinal center B cells and follicular helper T cells;

[0073] Figure 1 E in the above is a schematic diagram of the results of the detection of IgG levels in mice after immunization by ELISA method;

[0074] Figure 1 F in the above is a schematic diagram of the results of the detection of neutralizing antibodies in mice after immunization by pseudovirus neutralization experiment.

[0075] Figure 2 The results of the mouse cellular immune response experiment are shown in which:

[0076] Figure 2 A in the above is a schematic diagram of the results of the level of TNF-α produced by CD8 + T cells;

[0077] Figure 2 B in the above is a schematic diagram of the results of the level of IFN-γ produced by CD8 + T cells;

[0078] Figure 2 C in the above is a schematic diagram of the results of the level of TNF-α produced by CD4 + T cells;

[0079] Figure 2 D in the above is a schematic diagram of the results of the level of IFN-γ produced by CD4 + T cells;

[0080] Figure 2 E in the above is a schematic diagram of the results of the secretion of IFN-γ detected by ELISpot experiment;

[0081] Figure 2 Figure 2 is a schematic diagram showing the results of ELISpot assay for detecting the secretion of TNF-α in the mice immunized with the P1263L mutant.

[0082] Figure 3 Figure 3 shows the conserved region of the P1263L mutation site in the coronavirus.

[0083] Figure 4 Figure 4 shows the immune level against other coronavirus vaccines, wherein:

[0084] Figure 4 Figure 5 is a schematic diagram showing the results of detecting the IgG antibody specific to the spike protein in the mice after immunization.

[0085] Figure 4 Figure 6 is a schematic diagram showing the results of detecting the neutralizing antibody in the mice after immunization. DETAILED DESCRIPTION

[0086] Various illustrative embodiments, features and aspects of the present application are described below in detail. The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any implementation described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations.

[0087] In addition, for the purpose of convenience and brevity, detailed descriptions of well-known functions and structures incorporated in the present application can be omitted. It will be appreciated that those skilled in the art will be able to devise many alternative embodiments that will be suited to the particular necessities of a given situation without benefit of additional teachings or guidance from this detailed description.

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The meaning of "a", "an", and "the" includes plural references. The meaning of "in" includes "in" and "on." The use of "including", "comprising", "having" and "with" are not limiting. The use of "or" means "and / or". The use of parentheses means "and". The use of "based on" does not mean "based only on" and is intended to allow for being based on indicated items as well as items being "independent of" indicated items. Numeric ranges are inclusive of the numbers defining the range. Numeric ranges include the end points.

[0089] In this specification, the use of "may" includes both negligent and intentional aspects of possibilities.

[0090] In this specification, references to "some embodiments", "other embodiments", "exemplary embodiments", etc. indicate that the described feature is included in at least one embodiment. Such phrases do not necessarily refer to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the purview of those skilled in the art to effect such feature, structure, or characteristic in connection with other embodiments whether or not explicit reference to that particular feature, structure, or characteristic is made. Thus, it is intended that such a particular feature, structure, or characteristic can be included in any of the embodiments.

[0091] In the present specification, a numerical range indicated using "numerical value A to numerical value B" means a range including the end point numerical values A and B.

[0092] In the present specification, the terms "polypeptide", "peptide", and "protein" are used interchangeably herein and refer to an amino acid polymer of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The term also encompasses an amino acid polymer that has been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0093] In the present specification, the term "amino acid" can include natural amino acids, unnatural amino acids, amino acid analogs, and all their D and L stereoisomers. The amino acids and abbreviations and English abbreviations in the present invention are shown as follows: histidine (His, H); serine (Ser, S); glutamic acid (Glu, E); glutamine (Gln, Q); glycine (Gly, G); threonine (Thr, T); phenylalanine (Phe, F); aspartic acid (Asp, D); tyrosine (Tyr, Y); leucine (Leu, L); isoleucine (lie, I); arginine (Arg, R); alanine (Ala, A); valine (Val, V); tryptophan (Trp, W); methionine (Met, M); asparagine (Asn, N); cysteine (Cys, C); lysine (Lys, K); proline (Pro, P).

[0094] In the present specification, the term "mutant" refers to a polynucleotide or polypeptide comprising an alteration (i.e., substitution, insertion, and / or deletion) at one or more (e.g., several) positions relative to a "wild type", or "compared" polynucleotide or polypeptide, wherein substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. Deletion refers to removing a nucleotide or amino acid occupying a position. Insertion refers to adding a nucleotide or amino acid after a nucleotide or amino acid occupying a position, which is adjacent and immediately follows.

[0095] In the present specification, the term "polynucleotide" refers to a polymer composed of nucleotides. A polynucleotide can be in the form of a separate fragment or can be a component of a larger nucleotide sequence structure derived from a nucleotide sequence isolated at least once in quantity or concentration, capable of being recognized, manipulated and the sequence and its component nucleotides recovered by standard molecular biology methods (e.g., using cloning vectors). When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" is substituted for "T". In other words, "polynucleotide" refers to a nucleotide polymer removed from other nucleotides (either a separate fragment or an entire fragment) or can be a component or ingredient of a larger nucleotide structure or composition, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA and cDNA sequences.

[0096] In the present specification, the terms "sequence identity" and "percent identity" refer to the percentage of nucleotides or amino acids that are the same (i.e., identical) between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be determined by aligning the nucleotide or amino acid sequences of the polynucleotides or polypeptides and scoring the number of positions in the aligned polynucleotides or polypeptides that contain the same nucleotide or amino acid residue, and comparing it to the number of positions in the aligned polynucleotides or polypeptides that contain different nucleotides or amino acid residues. A polynucleotide can differ at a position, for example, by containing a different nucleotide (i.e., substitution or mutation) or a missing nucleotide (i.e., an insertion or a deletion of a nucleotide in one or both polynucleotides). A polypeptide can differ at a position, for example, by containing a different amino acid (i.e., substitution or mutation) or a missing amino acid (i.e., an insertion or a deletion of an amino acid in one or both polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0097] In the present specification, two or more sequences or subsequences are said to have "sequence identity" or "percent identity" when the sequences or subsequences are compared and aligned for maximum correspondence, using a sequence comparison algorithm or by visual inspection, and have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotides. In certain embodiments, the sequences are substantially identical over the entire length of either or both of the biological polymers (e.g., polynucleotides) being compared.

[0098] In the present specification, the term "corresponding to" has the meaning generally understood by one of ordinary skill in the art. Specifically, "corresponding to" means that, after homology or sequence identity alignment, a position in one sequence corresponds to a specified position in another sequence.

[0099] In the present specification, the term "recombinant vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the recombinant vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. In another embodiment, the recombinant vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.

[0100] In the present specification, the terms "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. The term "transformant" and "transformed cell" includes the primary subject cell and cultures derived from it, regardless of the number of transfers. It is also understood that all progeny can not necessarily be identical to the parental cell since there can be deliberate or inadvertent mutations that occur upon multiplication. Mutant progeny that have the same function or biological activity as the screened original transfected cell are included.

[0101] In the present specification, the mRNA, also known as messenger RNA, is generally a single-stranded ribonucleic acid that is transcribed from one strand of DNA as a template, carrying genetic information that can guide protein synthesis. After transcription of mRNA from the gene in the cell as a template according to the principle of base complementary pairing, the mRNA contains the base sequence corresponding to some functional fragments in the DNA molecule, which serves as a direct template for protein biosynthesis.

[0102] In the present specification, the antigen (abbreviation Ag) generally refers to a substance that can induce the production of antibodies, and is any substance that can induce an immune response.

[0103] In the present specification, the antibody generally refers to the immunoglobulin produced by the plasma cells differentiated from the B cells under the stimulation of the antigenic substance, which can have specific binding reaction with the corresponding antigen.

[0104] In the present specification, the neutralizing antibody generally refers to the antibodies produced after the invasion of microorganisms into the human body, but only part of the antibodies can rapidly recognize the microorganisms and "catch" them before they invade the human cells, thereby protecting the human body from infection. This process is called neutralization, and the antibody that plays a role is the neutralizing antibody.

[0105] In the present specification, a lipid nanoparticle (LNP) is a nanometer-sized carrier composed of a lipid bilayer, which can encapsulate and deliver hydrophobic or hydrophilic drug molecules, including small molecule drugs, proteins, nucleic acids, etc.

[0106] In the present specification, the virus-like particle (VLP) generally refers to a hollow particle containing one or more structural proteins of a certain virus, without viral nucleic acid, and cannot replicate autonomously, and is identical or similar in morphology to a real virus particle, commonly known as a pseudo-virus.

[0107] In the present specification, the term "wild type" refers to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism, can be isolated from a source in nature and has not been intentionally modified by humans in the laboratory is naturally occurring. DETAILED DESCRIPTION

[0109] <Coronavirus S protein mutant>

[0110] In some aspects of the present application, a coronavirus S protein mutant is provided, wherein the S protein mutant is a mutant having a mutation in a conserved region in the cytoplasmic tail region of a wild type S protein.

[0111] In some embodiments, the conserved region in the cytoplasmic tail region of the wild type S protein has the sequence: X1X2X3X4X5X6X7;

[0112] X1is D, E, A or P;

[0113] X2is any amino acid;

[0114] X3is E;

[0115] X4is any amino acid;

[0116] X5is V, I or deletion;

[0117] X6is L, H or deletion;

[0118] X7is K, V, I or H.

[0119] In some exemplary embodiments, X2is S, L, V, F or I.

[0120] In some exemplary embodiments, X4is P or K.

[0121] In some preferred embodiments, the conserved region in the cytoplasmic tail region of the wild type S protein has the sequence: X1X2X3X4X5X6X7, wherein:

[0122] X1is D or E;

[0123] X2is any amino acid;

[0124] X3is E;

[0125] X4is any amino acid;

[0126] X5is V, I or absent;

[0127] X6is L, H or absent;

[0128] X7is K, V, I or H.

[0129] In some exemplary embodiments, X2is S, L, V or F.

[0130] In some exemplary embodiments, X4is P or K.

[0131] In some specific embodiments, the mutation is a mutation of the fourth amino acid X4in the conserved region to leucine (L).

[0132] The S protein comprises one large ectodomain, one transmembrane domain (TMD) and one short cytoplasmic tail (CT, also known as cytoplasmic tail region). The cytoplasmic tail region of most coronaviruses comprises about 40 amino acids, and this tail region can regulate intracellular transport and subcellular localization of the S protein through one or two classical intracellular targeting motifs.

[0133] The position of the conserved region in the cytoplasmic tail region of the S protein in different coronaviruses can be determined by the method of homology alignment well known to those skilled in the art. For example, in the examples of the present application, the Mega software is used for alignment, the "Align by muscle" parameter is selected, and after multiple sequence alignment of coronaviruses, the position of the conserved region of various coronaviruses is determined, and then the position of the fourth amino acid X4in the conserved region is determined.

[0134] In some embodiments, the coronavirus comprises a virus of the alpha genus and / or the beta genus in the Coronaviridae family.

[0135] In some embodiments, the coronavirus comprises a human coronavirus selected from HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV or SARS-CoV-2.

[0136] In some embodiments, the coronavirus further comprises Porcine epidemic diarrhea virus (PEDV) or Transmissible gastroenteritis virus (TGEV).

[0137] In some specific embodiments, the coronavirus comprises SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-229E or HCoV-NL63.

[0138] In some preferred embodiments, the coronavirus comprises SARS-CoV, MERS-CoV or SARS-CoV-2.

[0139] In some specific embodiments, the sequence of the conserved region in the cytoplasmic tail of the S protein of SARS-CoV-2 is: DSE P VLK (SEQ ID NO: 23; X4 is underlined).

[0140] In some specific embodiments, the sequence of the conserved region in the cytoplasmic tail of the S protein of SARS-CoV is: DSE P VLK (SEQ ID NO: 24; X4 is underlined).

[0141] In some specific embodiments, the sequence of the conserved region in the cytoplasmic tail of the S protein of MERS-CoV is: DLE P -H (SEQ ID NO: 25; X4 is underlined; "-" indicates a deletion).

[0142] In some specific embodiments, the sequence of the conserved region in the cytoplasmic tail of the S protein of HCoV-229E is: DVE K IHI (SEQ ID NO: 26; X4 is underlined).

[0143] In some specific embodiments, the sequence of the conserved region in the cytoplasmic tail of the S protein of HCoV-NL63 is: EFE K VHV (SEQ ID NO: 27; X4 is underlined).

[0144] In some specific embodiments, the sequence of the conserved region in the cytoplasmic tail of the S protein of Porcine epidemic diarrhea virus is: AFE K VHV (SEQ ID NO: 28; X4 is underlined).

[0145] In some specific embodiments, the sequence of the conserved region in the cytoplasmic tail of the S protein of the porcine transmissible gastroenteritis virus is: PI E K VHV (SEQ ID NO: 29; X4 is underlined).

[0146] As demonstrated in the examples of the present application, after alignment of the S protein amino acid sequence of SARS-CoV-2 and the S protein amino acid sequences of other coronaviruses, it was found that position 1263 and its vicinity have a conserved region in the cytoplasmic tail of the S protein of other coronaviruses, and thus the P1263L mutation point (i.e., mutation of amino acid X4 of the conserved region amino acid sequence as defined above to L) can be constructed in the S protein of other coronaviruses, such as other variant strains, sub-type strains of SARS-CoV-2, MERS-CoV, and SARS-CoV, and it was confirmed that the vaccine immunization effect is also improved.

[0147] Therefore, it can be understood that for coronaviruses, as long as the conserved region in the cytoplasmic tail of the S protein has a sequence as defined above after alignment and analysis of the sequence, mutation of the fourth amino acid to leucine (L) is expected to significantly enhance immunogenicity.

[0148] In some exemplary embodiments, the amino acid sequence as shown in SEQ ID NO: 1 is the amino acid sequence of the wild-type S protein of SARS-CoV-2, and the mutation of X4 in the conserved region in the cytoplasmic tail of the S protein to leucine (L) means that corresponding to the amino acid sequence as shown in SEQ ID NO: 1, the mutation from proline (P) to leucine (L) at position 1263.

[0149] Similarly, in some exemplary embodiments, the human coronavirus includes the SARS-CoV-2 Omicron variant BA.1 sub-type strain, the amino acid sequence of the wild-type S protein of which is as shown in SEQ ID NO: 2, and the S protein mutant is the mutation from proline (P) to leucine (L) at position 1260 of the amino acid sequence as shown in SEQ ID NO: 2.

[0150] Similarly, in some exemplary embodiments, the human coronavirus includes the SARS-CoV-2 Omicron variant XBB sub-type strain, the amino acid sequence of the wild-type S protein of which is as shown in SEQ ID NO: 3, and the S protein mutant is the mutation from proline (P) to leucine (L) at position 1259 of the amino acid sequence as shown in SEQ ID NO: 3.

[0151] Similarly, in some exemplary embodiments, the human coronavirus comprises MERS-CoV, the amino acid sequence of the wild-type S protein of which is shown as SEQ ID NO: 4, and the S protein mutant is a mutation from proline (P) to leucine (L) at position 1347 of the amino acid sequence shown as SEQ ID NO: 4.

[0152] Similarly, in some exemplary embodiments, the human coronavirus comprises SARS-CoV, the amino acid sequence of the wild-type S protein of which is shown as SEQ ID NO: 5, and the S protein mutant is a mutation from proline (P) to leucine (L) at position 1245 of the amino acid sequence shown as SEQ ID NO: 5.

[0153] <Isolated polynucleotide>

[0154] The present application provides an isolated polynucleotide encoding the coronavirus S protein mutant described above.

[0155] In some embodiments, the polynucleotide can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded, preferably double-stranded DNA. The DNA can be a coding strand or a non-coding strand. The RNA form includes mRNA or hnRNA, etc. Unless otherwise specified, a specific polynucleotide sequence also implicitly covers conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.

[0156] In some embodiments, the polynucleotide comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 6-9, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleotide sequence shown in any one of SEQ ID NOs: 6-9.

[0157] <Expression cassette, recombinant vector, host cell>

[0158] Based on the polynucleotide obtained by the present application, an expression cassette comprising the polynucleotide as described in the present application is provided.

[0159] In other embodiments, the present application provides a recombinant vector comprising the polynucleotide or expression cassette as described above.

[0160] In some embodiments, the recombinant vector can be in the form of a plasmid vector or a viral vector. In some embodiments, the recombinant vector can be used to prepare the polynucleotide of the present application, such as mRNA, in vitro by transcription.

[0161] In other embodiments, the present application provides a host cell comprising a coronavirus S protein mutant, a polynucleotide, an expression cassette or a recombinant vector as described herein.

[0162] In some specific embodiments, the host cell is a bacterium; illustratively, the host cell is Escherichia coli, Bacillus subtilis, but not limited thereto.

[0163] In other specific embodiments, the host cell is a Saccharomyces; illustratively, the host cell is Pichia Pastoris, Saccharomyces cerevisiae, but not limited thereto.

[0164] In other specific embodiments, the host cell is a mammalian cell; illustratively, the host cell is a mammalian cell line, such as HEK293T cells, HEK293F cells, HEK293 cells, CHO cells, but not limited thereto.

[0165] <Composition>

[0166] The present application provides a composition comprising a coronavirus S protein mutant, a polynucleotide, an expression cassette, a recombinant vector or a host cell as described herein.

[0167] In some embodiments, the composition further comprises a delivery vehicle.

[0168] In some optional embodiments, the delivery vehicle comprises protamine, a lipid nanoparticle, a polymeric material and an inorganic nanoparticle.

[0169] In some preferred embodiments, the delivery vehicle comprises a lipid nanoparticle.

[0170] In some more preferred embodiments, the polynucleotide is an mRNA and is encapsulated in the delivery vehicle, such as a lipid nanoparticle.

[0171] In some embodiments, the composition of the present application further comprises one or more pharmaceutically acceptable carriers, excipients or diluents.

[0172] Exemplary carriers for use in the compositions of the present application include saline, buffered saline, dextrose, and water. Exemplary excipients for use in the compositions of the present application include fillers, binders, disintegrants, coating agents, adsorbents, antiadherents, glidants, preservatives, antioxidants, flavoring agents, coloring agents, sweetening agents, solvents, cosolvents, buffers, chelating agents, viscosity conferring agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers, and tonicity adjusting agents. One of skill in the art would know to select appropriate excipients to make the compositions of the present application. Generally, the selection of appropriate excipients depends, inter alia, on the active agent used, the disease to be treated, and the desired dosage form of the composition.

[0173] Depending on the active agent employed (e.g., mRNA), the compositions of the present disclosure can be prepared in various forms, such as solid, liquid, gaseous, or lyophilized forms, particularly in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, infusions, tinctures, or flowable extracts, or in a form particularly suitable for the desired method of administration. Processes known to the art for the manufacture of pharmaceuticals are shown in Remington’s Pharmaceutical Sciences, 22ndEd. (Ed. Maack Publishing Co, Easton, Pa., 2012), and can include, for example, conventional mixing, dissolving, granulation, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0174] In some embodiments, the composition is a vaccine composition, optionally the vaccine composition further comprises one or more adjuvants.

[0175] As used herein, the term "vaccine composition" refers to a biological agent that induces or improves immunity to a particular disease. The immune system of an individual is primed with a vaccine composition to induce formation and / or proliferation of immune cells that specifically recognize a compound comprised by the vaccine. At least a portion of the immune cells remain viable for a period of time, which can extend up to 10, 20, or 30 years after vaccination. If the immune system of the individual encounters a pathogen that is derived from the compound that elicits an immune response within the aforementioned time period, then the immune cells generated by vaccination are reactivated and the immune response against the pathogen is enhanced compared to the immune response of an individual who has not been primed with the vaccine and first encounters the immunogenic compound of the pathogen.

[0176] In the present context, "vaccination", "immunization", "immunizing" or "inoculation" means the administration of a vaccine to a subject with the aim of preventing the subject from developing one or more symptoms of a disease. In principle, vaccination comprises a primary vaccination and optionally one or more booster vaccinations. A primary vaccination or primary immunization is defined as the initial administration schedule of a composition or unit dose disclosed herein to establish a protective immune response. A booster vaccination or booster immunization refers to an administration or administration schedule that is performed after a primary vaccination, e.g. at least one week, at least 2 weeks, at least one month, at least 6 months, at least 1 year or even 5 or 10 years after the last administration of a primary vaccination schedule. A booster administration seeks to enhance or re-establish the immune response of a primary vaccination.

[0177] An immune response to a composition or vaccine composition of the present application is the development in a subject of a humoral and / or cellular immune response to the antigenic proteins present in the composition. For the purposes of the present application, a "humoral immune response" refers to an immune response mediated by antibody molecules, including secretory (IgA) or IgG molecules, while a "cellular immune response" refers to an immune response mediated by T-lymphocytes and / or other leukocytes. An important aspect of cellular immunity involves the antigen-specific response of cytolytic T cells ("CTLs"). CTLs are specific for peptide antigens presented in conjunction with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surface of cells. CTLs help induce and promote the destruction of intracellular microorganisms, or the lysis of cells infected by such microorganisms. Another aspect of cellular immunity involves the antigen-specific response of helper T cells. Helper T cells act to help stimulate functions, and focus the activity of non-specific effector cells against cells displaying peptide antigens in conjunction with MHC molecules on their surface. Cellular immune responses also involve the production of cytokines, chemokines and other such molecules by activated T cells and / or other leukocytes, including those derived from CD4+ and CD8+ T cells.

[0178] Thus, an immune response can be a response that stimulates CTLs, and / or helper T cells to produce or become activated. The production of chemokines and / or cytokines can also be stimulated. A composition or vaccine composition of the present application can also elicit an antibody-mediated immune response. Thus, an immune response can include one or more of the following effects: antibody (e.g. IgA or IgG) production by B cells; and / or the activation of suppressor, cytotoxic or helper T cells and / or T cells specific for a protein present in the vaccine.

[0179] These responses can serve to neutralize infectivity, and / or mediate antibody-complement, or antibody-dependent cellular cytotoxicity (ADCC) to provide protection to the immunized individual. Such responses can be determined using standard immunization assays and neutralization assays known in the art.

[0180] As used herein, the term "adjuvant" refers to an agent that increases, stimulates, activates, potentiates, or modulates an immune response at the cellular or humoral level to an active ingredient of a composition, e.g., an immunological adjuvant stimulates the immune system's response to the actual antigen but has no immunological effect itself. Examples of such adjuvants include, but are not limited to, inorganic adjuvants (e.g., inorganic metal salts such as aluminum phosphate or aluminum hydroxide), organic adjuvants (e.g., saponins or squalene), oil-based adjuvants (such as Freund's complete and incomplete adjuvants), cytokines (such as IL-1 beta, IL-2, IL-7, IL-12, IL-18, GM-CFS, and INF-gamma), particulate adjuvants (e.g., immunostimulatory complexes (ISCOMS), liposomes, or biodegradable microspheres), viral particles, bacterial adjuvants (e.g., monophosphoryl lipid A or muramyl peptide), synthetic adjuvants (e.g., non-ionic block copolymers, muramyl peptide analogs, or synthetic lipid A), or synthetic polynucleotide adjuvants (e.g., polyarginine or polylysine). Preferably, the adjuvant is selected from the group consisting of an aluminum adjuvant (e.g., aluminum hydroxide, aluminum phosphate, aluminum sulfate, alum), MF59, AS03, virosomes (e.g., hepatitis virus virosomes and influenza virus virosomes), AS04, heat-reversible oil-in-water emulsions, ISA51, Freund's adjuvant, IL-12, CpG motifs, mannose, or any combination thereof.

[0181] In some embodiments, the vaccine composition is a virus-like particle vaccine.

[0182] In some more particular embodiments, the vaccine composition further comprises at least one of:

[0183] (i) at least one of a membrane protein, a nucleocapsid protein, and an envelope protein from a coronavirus;

[0184] (ii) at least one of a polynucleotide encoding the membrane protein, a polynucleotide encoding the nucleocapsid protein, and a polynucleotide encoding the envelope protein;

[0185] (iii) an expression cassette comprising the polynucleotide of (ii);

[0186] (iv) a recombinant vector comprising the polynucleotide of (ii) or the expression cassette of (iii);

[0187] (v) a host cell comprising the polynucleotide of (ii), the expression cassette of (iii), or the recombinant vector of (iv).

[0188] In some embodiments, the composition or vaccine composition further comprises one or more additional therapeutic agents. For example, the therapeutic agent can be selected from the group consisting of an additional antigenic protein or polypeptide, an antibody, a hormone or hormone analog, and a small molecule drug.

[0189] <Uses and Methods>

[0190] The present application provides methods of preventing and / or treating a disease or condition associated with a coronavirus infection in a subject comprising administering to the subject an effective amount of a coronavirus S protein mutant, an isolated polynucleotide, an expression cassette, a recombinant vector, a host cell, and / or a composition of the present application.

[0191] The terms "prevent," "preventing," and "prevention," as used herein, refer to a reduction in risk of acquiring or developing a disease or condition, i.e., causing the clinical symptoms of a disease not to develop in a subject who is predisposed to the disease, or not exposed to the causative agent of the disease. For example, treatment can include: (i) preventing the disease, disorder, and / or condition from occurring in a patient that can be predisposed to the disease, disorder, and / or condition but has not yet been diagnosed as having it; (ii) inhibiting the disease, disorder, and / or condition, i.e., arresting its development; or (iii) relieving the disease, disorder, and / or condition, i.e., causing regression of the disease, disorder, and / or condition.

[0192] The term "effective amount," as used herein, means the amount of a compound that, when administered to a subject for treating or preventing a disease, is sufficient to effect such treatment or prevention. The "effective amount" can vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated. A "therapeutically effective amount" refers to an amount effective, when administered to a subject, for the treatment of a disease. A "prophylactically effective amount" refers to an amount effective, when administered to a subject, for the prevention of a disease.

[0193] The term "administering" as used herein means physically introducing a pharmaceutical agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, e.g., by injection or infusion.

[0194] As used herein, the terms "subject," "individual," and "patient" are art-recognized and used interchangeably herein, and refer to any subject for which treatment is desired, particularly a mammalian subject. Examples include, but are not limited to, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species. The terms individual, subject, and patient do not by themselves indicate a particular age, sex, race, etc.

[0195] In embodiments of the methods of the present application, the disease or condition is a disease or condition caused by a coronavirus infection, e.g., SARS-CoV, SARS-CoV-2, MERS-CoV, and variants thereof, e.g., a SARS-CoV-2 variant, such as the Delta variant, the Omicron variant, or the Lambda variant, e.g., severe acute respiratory syndrome, Middle East respiratory syndrome, novel coronavirus infection.

[0196] The present application provides use of the coronavirus S protein mutant, the isolated polynucleotide, the expression cassette, the recombinant vector, the host cell, and / or the composition of the present application in the preparation of a medicament (e.g., a vaccine, in particular a virus-like particle vaccine) for preventing and / or treating a disease or disorder associated with coronavirus infection (in a subject).

[0197] The present application provides a coronavirus S protein mutant, an isolated polynucleotide, an expression cassette, a recombinant vector, a host cell, and / or a composition of the present application for use in preventing and / or treating a disease or disorder associated with coronavirus infection in a subject.

[0198] In an embodiment of the use of the present application, the disease or disorder is a disease or disorder caused by coronavirus infection, e.g., SARS-CoV, SARS-CoV-2, MERS-CoV, and variants thereof, e.g., SARS-CoV-2 variants, such as Delta variant, Omicron variant, or Lambda variant, e.g., severe acute respiratory syndrome, Middle East respiratory syndrome, novel coronavirus infection.

[0199] Examples

[0200] The embodiments of the present application will be described in detail below with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. The specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, which are all conventional products that can be obtained by purchase.

[0201] I. Construction of protein cloning

[0202] The cloning involved in the embodiments of the present application is constructed by plasmid vector pCAGGS, including T7 promoter and PolyA tail.

[0203] Taking pCAGGS-BA.1-P1263L as an example, the construction method of plasmid vector pCAGGS is briefly described as follows:

[0204] pCAGGS is a commercialized vector, which is purchased from Addgene. In the multiple cloning site of pCAGGS, the elements include, in order: T7 promoter, 5'UTR, Kozak sequence, SEQ ID NO: 6, 3'UTR, PolyA.

[0205] T7 promoter (SEQ ID NO: 19):

[0206] taatacgactcactatagg

[0207] 5' UTR (SEQ ID NO: 20):

[0208] GAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCGCTAGCCTCGAG

[0209] Kozak sequence:

[0210] GCCACC

[0211] 3' UTR (SEQ ID NO: 21):

[0212] gatatctgataataggctggagcctcggtggccatgcttcttgccccttgggcctccccccagcccctcctccccttcctgcacccgtacccccgtggtctttgaataaagtctga

[0213] PolyA (SEQ ID NO: 22):

[0214] aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

[0215] All sequences built into the vector are codon-optimized.

[0216] The nucleotide sequence of the SARS-CoV-2 Omicron variant BA.1 sublineage S protein mutant (BA.1-P1263L) is shown below (SEQ ID NO: 6):

[0217]

[0218]

[0219]

[0220] wherein the bold and underlined positions are mutated relative to the wild-type S protein.

[0221] The nucleotide sequence of the SARS-CoV-2 Omicron variant XBB sublineage S protein mutant (XBB-P1263L) is shown below (SEQ ID NO: 7):

[0222]

[0223]

[0224]

[0225] wherein the bold and underlined shows the site of mutation relative to the wild type S protein.

[0226] The nucleotide sequence encoding the MERS-CoV S protein mutant (MERS-CoV-P1263L) is shown as follows (SEQ ID NO: 8):

[0227]

[0228]

[0229]

[0230] wherein the bold and underlined shows the site of mutation relative to the wild type S protein.

[0231] The nucleotide sequence encoding the SARS-CoV S protein mutant (SARS-CoV-P1263L) is shown as follows (SEQ ID NO: 9):

[0232]

[0233]

[0234]

[0235] wherein the bold and underlined shows the site of mutation relative to the wild type S protein. The SARS-CoV-2 wild type S protein amino acid sequence (SEQ ID NO: 1):

[0236]

[0237]

[0238] wherein, represents a conserved region, wherein the bold and underlined shows the fourth amino acid X4 in the conserved region of the wild type S protein.

[0239] The SARS-CoV-2 Omicron (Omicron) variant BA.1 subtype strain (referred to as BA.1) wild type S protein amino acid sequence (SEQ ID NO: 2):

[0240]

[0241] wherein, represents a conserved region, wherein the fourth amino acid X4 in the conserved region in the BA.1 wild-type S protein is shown in bold underlined. The BA.1-P1263L mutant, i.e., mutating P at this position to L, is actually mutating P at position 1260 to L for the BA.1 wild-type S protein.

[0242] BA.1 M protein amino acid sequence (SEQ ID NO: 10):

[0243] MADSNGTITVEELKKLLEQWNLVIGFLFLTWICLLQFAYANRNRFLYIIKLIFLWLLWPVTLACFVLAAVYRINWITGGIAIAMACLVGLMWLSYFIASFRLFARTRSMWSFNPETNILLNVPLHGTILTRPLLESELVIGAVILRGHLRIAGHHLGRCDIKDLPKEITVATSRTLSYYKLGASQRVAGDSGFAAYSRYRIGNYKLNTDHSSSSDNIALLVQ

[0244] BA.1 E protein amino acid sequence (SEQ ID NO: 11):

[0245] MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPSFYVYSRVKNLNSSRVPDLLV

[0246] BA.1 N protein amino acid sequence (SEQ ID NO: 12):

[0247] MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA

[0248] SARS-CoV-2 Omicron variant XBB sublineage strain (referred to as XBB) wild-type S protein amino acid sequence (SEQ ID NO: 3):

[0249]

[0250]

[0251] wherein, represents a conserved region, wherein the fourth amino acid X4 in the conserved region in the XBB wild-type S protein is shown in bold and underlined. The XBB-P1263L mutant, i.e., mutating P at this position to L, is actually mutating P at position 1259 to L for the XBB wild-type S protein.

[0252] XBB M protein amino acid sequence:

[0253] Same as BA.1 M amino acid sequence

[0254] XBB E protein amino acid sequence:

[0255] Same as BA.1 E amino acid sequence

[0256] XBB N protein amino acid sequence:

[0257] Same as BA.1 N amino acid sequence

[0258] MERS-CoV wild-type S protein amino acid sequence (SEQ ID NO: 4):

[0259]

[0260]

[0261] wherein, represents a conserved region, wherein the fourth amino acid X4 in the conserved region in the MERS-CoV wild-type S protein is shown in bold underlined. The MERS-CoV S protein mutant, i.e. mutating P at this position to L, is actually mutating P at position 1347 to L for the MERS-CoV wild-type S protein.

[0262] MERS-CoV M protein amino acid sequence (SEQ ID NO: 13):

[0263] MSNMTQLTEAQIIAIIKDWNFAWSLIFLLITIVLQYGYPSRSMTVYVFKMFVLWLLWPSSMALSIFSAVYPIDLASQIISGIVAAVSAMMWISYFVQSIRLFMRTGSWWSFNPETNCLLNVPFGGTTVVRPLVEDSTSVTAVVTNGHLKMAGMHFGACDYDRLPNEVTVAKPNVLIALKMVKRQSYGTNSGVAIYHRYKAGNYRSPPITADIELALLRA

[0264] MERS-CoV E protein amino acid sequence (SEQ ID NO: 14):

[0265] MLPFVQERIGLFIVNFFIFTVVCAITLLVCMAFLTATRLCVQCMTGFNTLLVQPALYLYNTGRSVYVKFQDSKPPLPPDEWV

[0266] MERS-CoV N protein amino acid sequence (SEQ ID NO: 15):

[0267] MASPAAPRAVSFADNNDITNTNLSRGRGRNPKPRAAPNNTVSWYTGLTQHGKVPLTFPPGQGVPLNANSTPAQNAGYWRRQDRKINTGNGIKQLAPRWYFYYTGTGPEAALPFRAVKDGIVWVHEDGATDAPSTFGTRNPNNDSAIVTQFAPGTKLPKNFHIEGTGGNSQSSSRASSLSSNSSRSSSQGSRSGNSTRGTSPGPSGIGAVGGDLLYLDLLNRLQALESGKVKQSQPKVITKKDAAAAKNKMRHKRTSTKSFNMVQAFGLRGPGDLQGNFGDLQLNKLGTEDPRWPQIAELAPTASAFMGMSQFKLTHQNNDDHGNPVYFLRYSGAIKLDPKNPNYNKWLELLEQNIDAYKTFPKKEKKQKAPKEESTDQMSEPPKEQRVQGSITQRTRTRPSVQPGPMIDVNTD

[0268] SARS-CoV wild-type S protein amino acid sequence (SEQ ID NO: 5):

[0269]

[0270]

[0271] wherein, represents a conserved region, wherein the fourth amino acid X4 in the conserved region in the SARS-CoV wild-type S protein is shown in bold and underlined. The SARS-CoV S protein mutant, i.e. mutating P at this position to L, is actually mutating P at position 1245 to L for the SARS-CoV S protein.

[0272] SARS-CoV M protein amino acid sequence (SEQ ID NO: 16):

[0273] MADNGTITVEELKQLLEQWNLVIGFLFLAWIMLLQFAYSNRNRFLYIIKLVFLWLLWPVTLACFVLAAVYRINWVTGGIAIAMACIVGLMWLSYFVASFRLFARTRSMWSFNPETNILLNVPLRGTIVTRPLMESELVIGAVIIRGHLRMAGHSLGRCDIKDLPKEITVATSRTLSYYKLGASQRVGTDSGFAAYNRYRIGNYKLNTDHAGSNDNIALLVQ

[0274] SARS-CoV E protein amino acid sequence (SEQ ID NO: 17):

[0275] MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPTVYVYSRVKNLNSSEGVPDLLV

[0276] SARS-CoV N protein amino acid sequence (SEQ ID NO: 18):

[0277] MSDNGPQSNQRSAPRITFGGPTDSTDNNQNGGRNGARPKQRRPQGLPNNTASWFTALTQHGKEELRFPRGQGVPINTNSGPDDQIGYYRRATRRVRGGDGKMKELSPRWYFYYLGTGPEASLPYGANKEGIVWVATEGALNTPKDHIGTRNPNNNAATVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRGNSRNSTPGSSRGNSPARMASGGGETALALLLLDRLNQLESKVSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKQYNVTQAFGRRGPEQTQGNFGDQDLIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYHGAIKLDDKDPQFKDNVILLNKHIDAYKTFPPTEPKKDKKKKTDEAQPLPQRQKKQPTVTLLPAADMDDFSRQLQNSMSGASADSTQA

[0278] For each plasmid vector of wild-type S protein, M protein, E protein and N protein of each virus, pCAGGS is also used for construction, and the nucleotide sequence (CDS region) encoding S protein in the vector is replaced with the nucleotide sequence (CDS region) encoding the corresponding wild-type S protein, M protein, E protein or N protein.

[0279] II. Experimental process

[0280] 1. Mouse immunization

[0281] First, mRNA is synthesized and lipid nanoparticles are prepared, and then the prepared lipid nanoparticles are immunized into C57BL / 6 mice.

[0282] Specifically, it includes the following steps:

[0283] (1) The wild-type / mutant S, M, E and N clones of BA.1 constructed in “I. Construction of mutant clones” are transcribed in vitro to synthesize the corresponding mRNA in the pCAGGS vector;

[0284] (2) The mRNA of S (wild-type or mutant), M, E, N synthesized in vitro in step (1) is used to prepare lipid nanoparticles (LNP) using a microfluidic mixing device (Micro & Nano Technologies), and the aqueous phase is composed of mRNA and 100 mM citric acid buffer with a pH of 5.0. LNP is composed of ionizable lipids, cholesterol, distearoyl phosphatidylcholine (DSPC) and lipid-anchored polyethylene glycol (PEG) with a molar ratio of 45:10:42:3. In the microfluidic system, the aqueous phase and the ethanol phase are mixed at a ratio of 3:1. Subsequently, the mRNA-LNP is dialyzed (molecular weight cut off (MWCO) = 3.5 kDa) in PBS for four hours to prepare lipid nanoparticles (LNP), and the corresponding mRNA LNP is obtained, and then the corresponding mRNA LNP is mixed in a mass ratio of S (wild-type or mutant): M: E: N = 5: 1.25: 0.75: 3, and the obtained lipid nanoparticles using wild-type S protein are called Omicron BA.1 VLP mRNA, and the obtained lipid nanoparticles using mutant S protein are called Omicron BA.1 P1263L VLP mRNA, which are abbreviated as WT VLP in Figure 1 and Figure 2 , and P1263L VLP in Figure 1 and Figure 2 .

[0285] (3) 6-8 weeks old C57BL / 6 mice were randomly divided into three groups (n=6 for each group). Prime and boost immunization with 10 μg mRNA LNP per dose, with S:M:E:N=5:1.25:0.75:3, the different mRNA LNP synthesized in step (2) were diluted in PBS at the ratio, 100 μl per mouse, the injection site was located in the thigh muscle of the mouse, 50 μl per leg. PBS as a control, wild type (WT VLP) represents Omicron BA.1, and mutant represents P1263L mutant (P1263L VLP) vaccine. Mice were sacrificed 7 days or 14 days after the boost immunization to collect serum, isolate lymphocytes and spleen cells to analyze germinal center B cells, follicular helper T cells and cytokines.

[0286] 2. Humoral immune response in mice:

[0287] As described in step 1, 18 C57BL / 6 mice were divided into 3 groups, 6 mice in each group, and immunized with PBS, 10 μg Omicron BA.1 VLP mRNA and 10 μg Omicron BA.1 P1263L VLP mRNA respectively, each 100 μl, which were abbreviated as PBS, WT VLP and P1263L VLP respectively. Three weeks later, the same dose of vaccine was boosted, and the inguinal lymph nodes of mice were collected 7 days after the boost immunization (A in Figure 1 ). Flow cytometry was used to analyze germinal center (GC) B cells by labeling B220 + CD95 + GL-7 + CD4 + CD185 + PD-1+ to identify follicular helper T (T follicular helper, Tfh) cells. The proportion of GC B cells in the lymph nodes of mice inoculated with P1263L VLP increased significantly, indicating that the vaccine could significantly induce germinal center response in mice (B in Figure 1 and D in Figure 1 ). At the same time, the proportion of Tfh cells also increased significantly, indicating that these cells were significantly activated. The results showed that the vaccine after P1263L mutation could more effectively promote B cell antibody-mediated humoral immune response by activating Tfh cells (C in Figure 1 and D in Figure 1 ).

[0288] In addition, the serum of mice was collected at the second week after the booster immunization, and the specific IgG antibody titer was detected by ELISA method, and the neutralizing antibody titer of mice was detected by using the pseudovirus neutralization experiment. By measuring the IgG titer in the serum of immunized mice against S protein by ELISA method, we found that the binding antibody of P1263L VLP immunized mice was significantly higher than that of WT VLP immunized mice (E) in the Figure 1 Meanwhile, it can be found that the neutralizing antibody titer of the animal group inoculated with P1263L VLP vaccine is significantly higher than that of the animal group inoculated with WT VLP vaccine (F) in the Figure 2

[0289] The specific steps of ELISA are as follows:

[0290] The 96-well plate was coated with BA.1 recombinant protein (Sino Biological) at 4°C overnight. After coating, the plate was washed once with 1xPBS and 0.05% Tween-20 (PBST). Subsequently, the blocking solution containing 5% skim milk was added to the plate and incubated at 37°C for 2h. The serum of immunized mice was initially diluted 30 times, and then serially diluted by 3 times in PBST+0.5% BSA buffer, a total of 11 gradients, and incubated at 37°C for 2h. In order to determine the specific antibody response against S, after washing three times with PBST, goat anti-mouse IgG HRP (Proteintech, SA00001-1) was added at a ratio of 1:5,000 and incubated at 37°C for 1h. The enzyme reaction was initiated by adding substrate 3,3',5,5'-tetramethylbenzidine, and terminated by adding 1M H2SO4. The absorbance was measured at 450nm using a microplate reader.

[0291] 3. Mouse cellular immune response

[0292] On day 14 after the booster vaccination, the spleen was harvested from the post-immunized mice (n=5 per group). The spleen was briefly lysed with ammonium chloride-potassium buffer to lyse the red blood cells. After washing the spleen cells with PBS, they were resuspended in RPMI 1640 medium containing 10% fetal bovine serum. The cells were first stained with Ghost Dye TM Red 780 to label dead cells. Then stained with the following mixture of fluorescently labeled antibodies: anti-CD45-Alexa Fluor TM ​700, anti-CD4-FITC, anti-CD8-PerCP-Cyanine5.5. All antibodies were diluted in cell staining buffer and incubated at room temperature for 20 min in the dark. For intracellular cytokine staining, cells also needed to be fixed and permeabilized, Cytofix / Cytoperm reagent was used and the procedure was performed according to the manufacturer’s instruction. Subsequently, cells were stained with anti-IFN-γ-PE and / or anti-TNF-α-BV421 antibodies. After washing, cells were resuspended in cell staining buffer and ready for flow cytometry analysis.

[0293] Splenocytes were isolated from immunized and control mice (n=5) and found that splenocytes from mice inoculated with P1263L VLPs were able to release higher levels of interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) than mice inoculated with WT VLPs or mice injected with PBS only (A-D in FIG. 1). Figure 2 Figure 2 ELISpot assays were performed according to the manufacturer’s instruction using Mouse IFN-γ (Mabtech, Cat. No# 3321-4APT-10) and IL-4 ELISpot kit (Mabtech, Cat. No# 3311-4HPW-10). A total of 1 x 105splenocytes (for IFN-γ and IL-4) were restimulated in vitro with Spike 15 amino acid overlapping polypeptide library (2 μg / ml) or DMSO control. Spot numbers were calculated using ELISpot reader (iSpot). By ELISpot experiment, it was observed that P1263L VLP immunized mice produced a significant increase in the number of IFN-γ positive specific splenocytes upon stimulation with S protein peptide library (E in FIG. 1). However, no significant difference was found in the number of interleukin-4 (IL-4) producing splenocytes between the two groups of immunized mice (F in FIG. 1). 6 Figure 2 Figure 3

[0294] 4. P1263L mutation site in conserved region of coronavirus

[0295] The novel coronavirus sequence and other coronavirus sequences were aligned using Mega software with the parameter of “Align by muscle”. After alignment, it was found that P1263L had a conserved region X1X2X3X4X5X6X7 with other coronaviruses; wherein X1was D, E, A or P; X2was S, L, V, F or I; X3was E; X4was P or K; X5was V, I or absent; X6was L, H or absent; X7was K, V, I or H (FIG. 2). Figure 4 ​​​​). The region displayed by the new coronavirus is "DSEPVLK", the conserved amino acid of SARS-CoV is "DSEPVLK", and the conserved amino acid of MERS-CoV is "DLEP--H". Therefore, the P1263L mutation point was constructed in the Spike plasmid of MERS-CoV and SARS-CoV (the specific method is described in "I. Construction of protein cloning"), which was then applied to the subsequent immune experiment.

[0296] 5. Immune level against other coronavirus vaccines

[0297] The same method as in "1. Mouse immunization" was used to prepare lipid nanoparticles and immunize mice. Briefly, the P1263L mutation was introduced into the vaccines against XBB, MERS-CoV and SARS-CoV, and 6-8 week old C57BL / 6 mice were randomly assigned to three groups (n = 6 per group). The initial and booster immunization was 10 μg per dose, S:M:E:N = 5:1.25:0.75:3, with a three-week interval, and the synthesized mRNA LNP was diluted in PBS according to the proportion, 100 μl per mouse, and the injection site was located in the thigh muscle of the mouse, 50 μl per leg. The mouse serum was collected 14 days after the booster immunization. The same method as in "2. Mouse humoral immune response" and "3. Mouse cellular immune response" was used for detection, and the experimental results showed that the P1263L mutation could induce higher IgG titers in XBB, MERS-CoV and SARS-CoV, indicating that the mutation had a universal significance in improving the immune effect of the vaccine Figure 4 A) in the specification. Subsequently, a pseudovirus neutralization experiment was performed, and it was found that the P1263L mutation could significantly enhance the level of pseudovirus neutralizing antibodies ​ B) in the specification.

[0298] It should be noted that although the technical solutions of the present application are described with specific examples, those skilled in the art can understand that the present application should not be limited thereto.

[0299] The above has described various embodiments of the present application, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes are obvious to those skilled in the art without departing from the scope and spirit of the described embodiments. The choice of terms used herein is intended to best explain the principles, practical applications, or technical improvements in the market of the embodiments, or to enable other ordinary skilled persons in the art to understand the embodiments disclosed herein.

Claims

1. A coronavirus S protein mutant, wherein, the S protein mutant is a mutant having a mutation in a conserved region in the cytoplasmic tail of a wild-type S protein, the conserved region in the cytoplasmic tail of the wild-type S protein has the sequence X1X2X3X4X5X6X7, wherein, X1is D, E, A or P; X2is any amino acid; X3is E; X4is any amino acid; X5is V, I or deletion; X6is L, H or deletion; X7is K, V, I or H; the mutation is that the amino acid X4in the conserved region is mutated to leucine (L); Preferably, the conserved region in the cytoplasmic tail of the wild-type S protein has the sequence X1X2X3X4X5X6X7, wherein: X1is D or E; X2is any amino acid; X3is E; X4is any amino acid; X5is V, I or deletion; X6is L, H or deletion; X7is K, V, I or H.

2. The coronavirus S protein mutant of claim 1, wherein, the coronavirus comprises a human coronavirus, a porcine epidemic diarrhea virus or a porcine transmissible gastroenteritis virus; Optionally, the coronavirus is selected from SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-229E, HCoV-NL63, a porcine epidemic diarrhea virus or a porcine transmissible gastroenteritis virus, and / or the amino acid sequence of the conserved region in the cytoplasmic tail of the S protein is set forth in any one of SEQ ID NOs: 23-29; Preferably, the coronavirus is selected from SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-229E or HCoV-NL63; More preferably, the coronavirus is selected from SARS-CoV, MERS-CoV or SARS-CoV-2.

3. The coronavirus S protein mutant of claim 1 or 2, wherein, the S protein mutant is selected from any one of: (a1) comprises an amino acid sequence set forth in SEQ ID NO: 2, and comprises a mutation from proline (P) to leucine (L) at position 1260 of the amino acid sequence set forth in SEQ ID NO: 2; (a2) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in (a1), and retains the activity of the amino acid sequence set forth in (a1); (b1) comprises an amino acid sequence set forth in SEQ ID NO: 3, and comprises a mutation from proline (P) to leucine (L) at position 1259 of the amino acid sequence set forth in SEQ ID NO: 3; (b2) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in (b1), and retains the activity of the amino acid sequence set forth in (b1); (c1) comprises an amino acid sequence set forth in SEQ ID NO: 4, and comprises a mutation from proline (P) to leucine (L) at position 1347 of the amino acid sequence set forth in SEQ ID NO: 4; (c2) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in (c1), and retains the activity of the amino acid sequence set forth in (c1). (c2) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in (c1), and retains the activity of the amino acid sequence set forth in (c1); (d1) comprises an amino acid sequence as set forth in SEQ ID NO: 5, and comprises a mutation from proline (P) to leucine (L) at position 1245 of the amino acid sequence set forth in SEQ ID NO: 5; (d2) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in (d1), and retains the activity of the amino acid sequence set forth in (d1).

4. An isolated polynucleotide encoding the coronavirus S protein mutant of any one of claims 1-3. Optionally, the polynucleotide is DNA or RNA; preferably, the RNA is mRNA.

5. An expression cassette comprising the polynucleotide of claim 4.

6. A recombinant vector comprising the polynucleotide of claim 4 or the expression cassette of claim 5.

7. A host cell comprising the coronavirus S protein mutant of any one of claims 1-3, the polynucleotide of claim 4, the expression cassette of claim 5, or the recombinant vector of claim 6.

8. A composition comprising the coronavirus S protein mutant of any one of claims 1-3, the polynucleotide of claim 4, the expression cassette of claim 5, the recombinant vector of claim 6, or the host cell of claim 7.

9. The composition of claim 8, wherein, The composition further comprises a delivery vehicle; Optionally, the delivery vehicle comprises protamine, a lipid nanoparticle, a polymeric material, or an inorganic nanoparticle; Preferably, the delivery vehicle comprises a lipid nanoparticle.

10. The composition according to claim 8 or 9, wherein, The composition is a vaccine composition, optionally, the vaccine composition further comprises one or more adjuvants.

11. The composition of claim 10, wherein, The vaccine composition is a virus-like particle vaccine; Optionally, the vaccine composition further comprises at least one of: (i) at least one of an envelope protein, a membrane protein, and a nucleocapsid protein from a coronavirus; (ii) at least one of a polynucleotide encoding the envelope protein, a polynucleotide encoding the membrane protein, and a polynucleotide encoding the nucleocapsid protein; (iii) an expression cassette comprising the polynucleotide of (ii); (iv) a recombinant vector comprising the polynucleotide of (ii) or the expression cassette of (iii); (v) a host cell comprising the polynucleotide of (ii), the expression cassette of (iii), the recombinant vector of (iv), or the recombinant vector of (iv).

12. Use of the coronavirus S protein mutant of any one of claims 1-3, the polynucleotide of claim 4, the expression cassette of claim 5, the recombinant vector of claim 6, the host cell of claim 7, or the composition of any one of claims 8-11 in the manufacture of a medicament for preventing and / or treating a disease or disorder associated with coronavirus infection. Optionally, the medicament is a vaccine; preferably, the vaccine is a virus-like particle vaccine.

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