WO2023236468A1

WO2023236468A1 - Coronavirus s protein variant and use thereof

Info

Publication number: WO2023236468A1
Application number: PCT/CN2022/136551
Authority: WO
Inventors: 林金钟; 卢静; 谭舒丹
Original assignee: 复旦大学; 上海蓝鹊生物医药有限公司
Priority date: 2021-07-09
Filing date: 2022-12-05
Publication date: 2023-12-14
Also published as: CN115594742A; WO2023280220A9; WO2023280220A1

Abstract

Provided is an S protein variant, which does not contain a complete cytoplasmic tail domain compared to the S protein of a wild-type coronavirus. Further provided are a nucleic acid molecule encoding the S protein variant, and the use of the S protein variant and the nucleic acid molecule in the preparation of a vaccine.

Description

A coronavirus S protein variant and its application

Technical field

This application relates to the field of biomedicine, specifically to a coronavirus S protein variant and its application in preparing vaccines.

Background technique

Coronavirus (CoV) is a type of enveloped single-stranded positive-sense RNA virus with a wide host range. Humans and various animals are generally susceptible to it and are associated with many diseases. Known coronaviruses include HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV and/or MERS-CoV. Research shows that the genetic sequence of the SARS-CoV-2 virus (GenBank accession: MN908947) It has more than 85% sequence similarity compared with SARS coronavirus.

For the coronavirus surface spike protein antigen (S protein), we have mainly developed recombinant protein vaccines, mRNA vaccines, DNA vaccines, non-replicating viral vector vaccines, replicating viral vector vaccines, peptide vaccines, virus-like particle vaccines, and attenuated vaccines. and inactivated vaccines. However, there are problems such as low cell transfection rate, low expression efficiency, weak stimulated immune response, and high clinical dosage. On the one hand, it leads to high production costs, high medication costs, and heavy patient burden. On the other hand, high-dose medication makes Introducing more impurities may lead to increased toxic and side effects, thereby increasing clinical risks. Therefore, there is an urgent need to prepare new vaccines.

Contents of the invention

The present application provides a coronavirus S protein variant, and nucleic acid molecules encoding it (including DNA and/or RNA), which can stimulate the body to produce an immune response. Compared with the S protein of wild-type coronavirus, the S protein variant of the present application has a higher expression level, and when used to stimulate an immune response, it can produce higher total antibodies, while also having protective antibodies/neutralizing antibodies. A huge improvement. The S protein variant and its nucleic acid molecule of the present application can be prepared as a vaccine. For example, mRNA encoding an S protein variant and lipid nanoparticles containing it can be prepared as a vaccine. Compared with the S protein vaccine of wild-type coronavirus, the vaccine of the present application can greatly improve the immune response, reduce the dosage and reduce side effects.

In one aspect, the present application provides an S protein variant that does not contain a complete cytoplasmic tail domain compared with the S protein of wild-type coronavirus.

In certain embodiments, the S protein variant comprises a truncated cytoplasmic tail domain compared to the S protein of a wild-type coronavirus.

In certain embodiments, the S protein variant has 18-20 amino acids truncated at the C-terminus compared to the S protein of wild-type coronavirus.

In certain embodiments, the S protein variant is truncated by 18 amino acids at the C-terminus compared to the S protein of wild-type coronavirus.

In certain embodiments, the S protein variant does not comprise a cytoplasmic tail domain compared to the S protein of wild-type coronavirus.

In certain embodiments, the S protein variant does not comprise a transmembrane domain compared to the S protein of wild-type coronavirus.

In certain embodiments, the S protein variant has 22-64 amino acids truncated at the C-terminus compared to the S protein of wild-type coronavirus.

In certain embodiments, the coronavirus is a MERS coronavirus and/or a SARS-CoV-like virus.

In certain embodiments, the coronavirus is SARS-CoV-2 virus.

In certain embodiments, the cytoplasmic tail domain of the S protein of the wild-type coronavirus comprises the amino acid sequence shown in any one of SEQ ID NO: 48, 49, 86 and 87.

In certain embodiments, the transmembrane domain of the S protein of the wild-type coronavirus comprises the amino acid sequence shown in any one of SEQ ID NO: 50, 51, 88 and 89.

In certain embodiments, the S protein of the wild-type coronavirus comprises the amino acid sequence shown in any one of SEQ ID NO: 5, 34 and 44.

In certain embodiments, the S protein variant comprises any one of SEQ ID NOs: 8, 21, 24, 27, 30, 33, 35, 38, 41, 45, 59, 61 and 94. Amino acid sequence.

In certain embodiments, the S protein variant comprises the amino acid sequence set forth in SEQ ID NO:8.

In certain embodiments, the S protein variant further includes a signal peptide, and the signal peptide is located at the N-terminus of the S protein variant.

In certain embodiments, the signal peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, and 68.

In another aspect, the present application provides an isolated nucleic acid molecule comprising a polynucleotide encoding an S protein variant described herein.

In certain embodiments, the nucleic acid molecule is DNA.

In certain embodiments, the nucleic acid molecule comprises SEQ ID NO: 6, 9-19, 22, 25, 28, 31, 36, 39, 42, 46, 60, 62, 63, 71, 72, 74, The nucleotide sequence of any one of 76, 78, 80, 82, 84, 95, 96 and 97.

In certain embodiments, the nucleic acid molecule is mRNA.

In certain embodiments, the nucleic acid molecule comprises a modification at one or more positions selected from the group consisting of: a 5' cap, a 5' untranslated region, an open reading frame, a 3' untranslated region, and a 3' polygon glycosides.

In certain embodiments, the nucleic acid molecule comprises at least one modified nucleotide.

In certain embodiments, the nucleic acid molecule is codon optimized.

In certain embodiments, the nucleic acid molecule comprises SEQ ID NOs: 7, 20, 23, 26, 29, 32, 37, 40, 43, 47, 73, 75, 77, 79, 81, 83, and 85 The nucleotide sequence shown in any one.

In another aspect, the present application provides vectors comprising the nucleic acid molecules described in the present application.

In another aspect, the application provides cells comprising said nucleic acid molecule, and/or said vector.

On the other hand, the present application provides a method for preparing the S protein variant described in the present application, the method comprising culturing the cell under conditions such that the S protein variant is expressed.

In another aspect, the present application provides a composition comprising (1) an mRNA encoding an S protein variant described herein, and (2) a delivery vector.

In certain embodiments, the mRNA comprises modifications at one or more positions selected from the group consisting of: a 5' cap, a 5' untranslated region, an open reading frame, a 3' untranslated region, and a poly A tail.

In certain embodiments, the mRNA includes at least one modified nucleotide.

In certain embodiments, the mRNA comprises a modified nucleotide that is pseudouridine (Ψ).

In certain embodiments, the mRNA is codon optimized.

In certain embodiments, the mRNA comprises as in SEQ ID NO: 7, 20, 23, 26, 29, 32, 37, 40, 43, 47, 73, 75, 77, 79, 81, 83 and 85 The nucleotide sequence shown in any one.

In certain embodiments, the S protein variant is truncated by 18 amino acids at the C-terminus compared to the S protein of a wild-type coronavirus (e.g., SARS CoV-2 virus).

In certain embodiments, the mRNA comprises a nucleotide sequence set forth in SEQ ID NO: 7 or 67.

In certain embodiments, the delivery vehicle includes liposomes.

In certain embodiments, the delivery vehicle includes lipid nanoparticles (LNP).

In certain embodiments, the delivery vehicle includes a cationic lipid.

In certain embodiments, the molar ratio of the cationic lipid in the delivery vehicle is from about 45% to about 55%.

In certain embodiments, the cationic lipid includes SM102 and/or Dlin-MC3-DMA. For example, the cationic lipid may be SM102. For example, the chemical structural formula of SM102 can be:

For example, the cationic lipid can be Dlin-MC3-DMA. For example, the chemical structural formula of Dlin-MC3-DMA can be:

In certain embodiments, the delivery vehicle includes non-cationic lipids.

In certain embodiments, the noncationic lipids include phospholipids and/or lipid conjugates.

In certain embodiments, the phospholipid includes distearoylphosphatidylcholine (DSPC).

In certain embodiments, in the delivery vehicle, the molar ratio of the phospholipids is from about 35% to about 40%.

In certain embodiments, the lipid conjugates comprise polyethylene glycol modified lipid molecules.

In certain embodiments, in the delivery vehicle, the molar ratio of the lipid conjugate is from about 1% to about 2%.

In certain embodiments, the polyethylene glycol modified lipid molecule includes DMG-PEG2000.

In certain embodiments, the delivery vehicle includes cholesterol.

In certain embodiments, in the delivery vehicle, the molar ratio of cholesterol is from about 8% to about 12%.

In certain embodiments, the delivery vehicle comprises cationic lipids, cholesterol, phospholipids and lipid conjugates, and the mass ratio of the cationic lipids, cholesterol, phospholipids and lipid conjugates is 50:10: 38.5:1.5.

In certain embodiments, the delivery vehicle includes SM102, cholesterol, DSPC and DMG-PEG2000, and the mass ratio of SM102, cholesterol, DSPC and DMG-PEG2000 is 50:10:38.5:1.5.

In certain embodiments, the mRNA is encapsulated in the delivery vector.

In another aspect, the present application provides a pharmaceutical composition comprising the S protein variant of the present application, the nucleic acid molecule, the vector, the cell and/or the composition, and optionally a pharmaceutically acceptable carrier.

In another aspect, the present application provides a vaccine comprising the S protein variant, the nucleic acid molecule, the vector, the cell, the composition and/or the pharmaceutical composition of the present application, and Pharmaceutically acceptable adjuvants.

In certain embodiments, the vaccine is a protein vaccine.

In certain embodiments, the vaccine is a nucleic acid vaccine.

In certain embodiments, the vaccine is a DNA vaccine.

In certain embodiments, the vaccine is an mRNA vaccine.

On the other hand, the present application provides a kit comprising the S protein variant of the present application, the nucleic acid molecule, the vector, the cell, the compound, the pharmaceutical composition and/or or said vaccine.

On the other hand, the present application provides the S protein variant, the nucleic acid molecule, the vector, the cell, the composition, the pharmaceutical composition and/or the vaccine described in the present application in the preparation of medicines. The medicine is used to alleviate, prevent and/or treat diseases caused by coronavirus.

In certain embodiments, the disease or condition is novel coronavirus pneumonia (COVID-19) and/or severe acute respiratory syndrome (SARS).

On the other hand, the present application provides methods for alleviating, preventing and/or treating diseases or conditions caused by coronavirus, the method comprising administering the S protein variant, the nucleic acid molecule described in the present application to a subject in need. , the vector, the cell, the composition, the pharmaceutical composition and/or the vaccine.

On the other hand, the present application provides a method for producing antibodies against coronavirus, the method comprising administering the S protein variant, the nucleic acid molecule, the vector, the cell, the composition, the the pharmaceutical composition and/or the vaccine.

In another aspect, the application provides a method of activating immunity, the method comprising administering the S protein variant, the nucleic acid molecule, the vector, the cell, the composition, the pharmaceutical composition and/or or said vaccine.

Those skilled in the art will readily appreciate other aspects and advantages of the present application from the detailed description below. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will realize, the contents of this application enable those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention covered by this application. Accordingly, the drawings and descriptions of the present application are illustrative only and not restrictive.

Description of the drawings

The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates can be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

Figure 1A shows the in vitro expression results of 293T cells transfected with the mRNA of the S protein variant described in this application.

Figure 1B shows the in vitro expression results of cells transfected with the mRNA expressing the S protein of SARS virus described in this application.

Figure 1C shows the results of Western Blot detection of protein expression after the mRNA of the S protein variant described in the present application was transfected with liposomes into 293A cells, where, S: wild-type S protein mRNA; SDC60 Nat: band Nat (original virus) signal peptide, codon-optimized wild-type S protein mRNA (codon sequence code SDC60, in which the GC content in the sequence is 60%); SDC60 Alb: SDC60 codon with Alb (serum albumin) signal peptide The sequence of S protein mRNA; FL: full-length S protein; DC18: S protein mutant with 18 amino acids truncated at the C terminus; 2P: S protein mutation containing 2 proline (Proline) sequence mutations (K986P and V987P) body.

Figure 2 shows a schematic diagram of immunizing mice using lipid nanoparticles of the present application.

Figure 3 shows the S protein-binding antibody titers in the first week after the second immunization of the mRNA vaccine. Among them, S: wild-type S protein mRNA; SDC60 Alb-2P: with Alb signal peptide and containing 2 proline mutations (K986P , V987P), S protein mRNA of SDC60 codon sequence; SDC60 Alb-DC18: with Alb signal peptide, S protein mRNA with 18 amino acids truncated at the C-terminus based on the SDC60 codon sequence.

Detailed ways

The implementation of the invention of the present application will be described below with specific examples. Those skilled in the art can easily understand other advantages and effects of the invention of the present application from the contents disclosed in this specification.

Definition of Terms

In this application, the term "delivery vector" generally refers to a transfer vehicle capable of delivering an agent (eg, mRNA) to a target cell. Delivery vehicles can deliver agents (eg, mRNA) to specific cell subtypes. For example, by means of inherent characteristics of the delivery vehicle or by means of a moiety coupled to, contained within (or a moiety bound to the carrier) such that the moiety and the delivery vehicle are maintained together, thereby rendering the moiety sufficient to target the target. Targeting the delivery vector to certain types of cells. The delivery vehicle may also increase the in vivo half-life of the agent to be delivered (eg, mRNA) and/or the bioavailability of the agent to be delivered. Delivery vectors may include viral vectors, virus-like particles, polycationic vectors, peptide vectors, liposomes, and/or hybrid vectors. For example, if the target cell is a hepatocyte, the delivery vector has properties (e.g., size, charge, and/or pH) that are effective in delivering the delivery vector and/or the molecule entrapped therein (e.g., mRNA) to the target. cells, reduce immune clearance and/or promote retention in that target cell.

In this application, the term "polynucleotide" generally includes DNA molecules (eg, cDNA or genomic DNA), RNA molecules (eg, mRNA), nucleotide analogs (eg, peptide nucleic acids, and non-naturally occurring nucleotide analogs). ), and hybrids thereof. Nucleic acid molecules can be single-stranded or double-stranded.

In this application, the term "isolated nucleic acid molecule" generally refers to a single- or double-stranded polymer or analog thereof of deoxyribonucleotide or ribonucleotide bases, read from the 5' to the 3' end, which has been separated from at least about 50% of the polypeptides, peptides, lipids, carbohydrates, polynucleotides or other materials that are naturally found with the nucleic acid molecules when the total nucleic acid is isolated from the source cell. For example, an isolated nucleic acid molecule is substantially free of any other contaminating nucleic acid molecules or other molecules found in the nucleic acid's natural environment that could interfere with its use or its therapeutic, diagnostic, prophylactic, or research uses.

In this application, the term "mRNA" generally refers to an RNA transcript that has been processed to remove introns and capable of being translated into a polypeptide. mRNA usually includes a 5' cap, a 5' untranslated region (5'UTR), a coding region (also known as an open reading frame), a 3' untranslated region (3'UTR), and a poly(A) tail.

In this application, the term "modification" when applied to a nucleic acid, such as RNA or DNA, generally means that the nucleic acid has a different nucleotide molecule, a different nucleotide sequence, compared to the corresponding wild type, consisting of Different bond compositions and/or the incorporation of unnatural moieties into their structure. For example, the modification may include modification of a nucleotide, for example, the nucleotide may include a modified base, sugar, or phosphate group. For example, the modifications may include polypeptides or proteins with different nucleotide sequences but encoding the same amino acid sequence, or the same function. The modification may be chemical modification and/or biological modification. "Chemical modification" may include modifications that introduce chemicals different from those found in wild-type or naturally occurring nucleic acids, e.g., covalent modifications, e.g., the introduction of modified nucleotides (e.g., nucleotide analogs , or introduce side groups not naturally found in these nucleic acid molecules). The term "modified nucleotide" generally refers to units in nucleic acid polymers that contain modified bases, sugars, or phosphate groups, or that incorporate non-natural moieties into their structure.

In this application, the term "transmembrane domain" generally refers to the region of a protein sequence that spans the cell membrane.

As used herein, the term "codon optimization", when applied to nucleic acids, generally means the replacement of one, at least one, of the parent polypeptide-encoding nucleic acid with a codon encoding the same amino acid residue that has a different relative frequency of use in the cell. , or a nucleic acid encoding a polypeptide in which one or more codons have been modified to have improved expression in a cell, such as a mammalian cell or a bacterial cell.

In this application, the term "signal peptide" generally refers to a propeptide present as an N-terminal peptide on the precursor form of a protein. The function of the signal peptide is to facilitate the translocation of the expressed polypeptide to the endoplasmic reticulum. The signal peptide is usually cleaved during this process. A signal peptide may be heterologous or homologous to the organism used to produce the polypeptide.

In this application, the term "pharmaceutical composition" generally refers to a preparation in a form that is effective to allow the biological activity of the active ingredient (e.g., S protein variant, nucleic acid molecule of the present application), and which does not contain ingredients that are harmful to the preparation. Additional ingredients that have unacceptable toxicity to the subject are to be administered. These preparations can be sterile.

In this application, the term "S protein", which may also be called "Spike protein" or "Spike protein", generally refers to the membrane protein on the surface of coronavirus, which can form protruding homotrimers on the surface of the virus. The term may also include glycosylated forms of S protein. S protein usually contains two functional subunits, S1 and S2. The S1 subunit is responsible for binding to host cell receptors, and the S2 subunit is responsible for the fusion of viral membranes and cell membranes. The S protein can mediate coronavirus entry into host cells. Different coronaviruses use different domains within the S1 subunit to recognize attachment receptors on the surface of target cells and enter target cells through the receptors. For SARS-CoV and/or SARS-CoV-2, it can enter target cells through direct interaction between domain B of the S1 subunit of the S protein and angiotensin-converting enzyme 2 (ACE2). In addition, virus entry into the host requires priming of the S protein, which requires cleavage of the S protein at the S1/S2 and S2' sites by the host's protease, and the S2 subunit drives viral membrane fusion. The S1 subunit contains the N-terminal domain and the receptor binding domain (RBD), and the S2 subunit contains the fusion peptide (FP), heptapeptide repeat 1 (HR1), heptapeptide repeat 2 (HR2), and the transmembrane domain (TM). domain) and cytoplasmic tail domain. "S protein" encompasses S protein of any viral origin, for example, MERS CoV, SARS-CoV and/or SARS-CoV-2. The exemplary amino acid sequence of the S protein of SARS-CoV-2 can be found in the NCBI database accession number YP_009724390.1. The exemplary amino acid sequence of the S protein of SARS-CoV can be found in the NCBI database accession number YP009825051.1. The amino acid sequence of the exemplary S protein of MERS-CoV can be found in the NCBI database accession number NC019843.

In this application, the term "S protein of wild-type coronavirus" generally refers to the S protein of a coronavirus that contains the complete sequence of the cytoplasmic tail domain and/or the transmembrane domain. The S protein of wild-type coronavirus can be the S protein of the full-length sequence of coronavirus obtained from nature. The full-length amino acid sequence of the exemplary S protein of SARS-CoV-2 can be found in the NCBI database accession number YP_009724390.1 or the Uniprot database accession number PODTC2. The S protein of wild-type SARS-CoV-2 can contain 1273 amino acids (aa), from the N-terminus to the C-terminus, usually including a 13aa signal peptide in sequence, followed by the S1 subunit (amino acid residues 14-685) and S2 Subunit (amino acid residues 686-1273). Within the S1 subunit, it contains the N-terminal domain (amino acid residues 14-305) and a receptor binding domain (RBD; amino acid residues 319-541) from the N terminus to the C terminus. In the S2 subunit From N-terminus to C-terminus, it contains fusion peptide (FP; amino acid residues 788-806), heptapeptide repeat sequences 1 and 2 (HR1; amino acid residues 912-984 and HR2; amino acid residues 1163-1213) , transmembrane domain (TMD; amino acid residues 1213-1237) and cytoplasmic tail domain (cytoplasmic tail; amino acid residues 1237-1273). The full-length amino acid sequence of the exemplary SARS-CoV S protein can be found in NCBI database accession number YP_009825051.1 or Uniprot database accession number P59594. The wild-type SARS-CoV S protein can contain 1255 amino acids (aa), and its cytoplasmic tail The amino acid residues of the domain are 1217-1255). "Position" when used herein means the location within the sequence of a protein. Positions can be numbered consecutively or according to a defined format. , for example, K986P or V987P is relative to the wild-type S protein with amino acids K and V at positions 986 and 987 both mutated to P.

In this application, the term "cytoplasmic tail domain" generally refers to the domain of the wild-type S protein that is located inside the cytoplasm. The cytoplasmic tail domain of coronaviruses is usually Cys-rich and palmitoylated.

In this application, the term "truncated cytoplasmic tail domain" generally refers to a cytoplasmic tail domain with an amino acid deletion relative to an intact cytoplasmic tail domain.

As used herein, the term "vaccine" generally refers to an agent or composition containing an active component effective to induce a therapeutic degree of immunity in a subject against a particular pathogen or disease.

In this application, the term "lipid nanoparticle" generally refers to particles that contain multiple (ie, more than one) lipid molecules physically bound to each other (eg, covalently or non-covalently) by intermolecular forces. Lipid nanoparticles can be, for example, microspheres (including unilamellar and multilamellar vesicles, such as liposomes), the dispersed phase in emulsions, micelles or the internal phase in suspensions. Lipid nanoparticles can include one or more lipids (eg, cationic lipids, noncationic lipids, and PEG-modified lipids).

In this application, the term "liposome" generally refers to a vesicle having an internal space separated from an external medium by a membrane of one or more bilayers. For example, the membrane of the bilayer can be formed by amphipathic molecules, such as synthetic or naturally derived lipids containing spatially separated hydrophilic and hydrophobic domains; as another example, the membrane of the bilayer can be formed by amphiphilic molecules. Polymer and surfactant formation.

In this application, the term "SARS-CoV-2" generally refers to severe acute respiratory syndrome coronavirus 2, the full English name is Severe Acute Respiratory Syndrome Coronavirus 2. SARS-CoV-2 belongs to the Betacoronavirus subgenus of the Coronaviridae family and the Sarbecovirus subgenus. SARS-CoV-2 is an enveloped, non-segmented, positive-stranded single-stranded RNA virus.

In this application, the term "SARS-CoV-like" generally refers to Severe acute respiratory syndrome-related coronavirus, a species of the genus Betacoronavirus in the family Coronaviridae. SARS-CoV-like viruses may also be called SARS coronavirus or SARS-related coronavirus.

In this application, the term "SARS-CoV" generally refers to SARS coronavirus, that is, severe acute respiratory syndrome coronavirus (full name in English: Severe acute respiratory syndrome coronavirus), which belongs to the genus Betacoronavirus in the family Coronaviridae. (Betacoronavirus) Subgenus Sarbecovirus.

In this application, the term "novel coronavirus pneumonia (COVID-19)" generally refers to coronavirus disease 2019 (full English name is coronavirus disease 2019), which is a disease caused by SARS-CoV-2. Most COVID-19 patients mainly present with lower respiratory tract symptoms, and common clinical manifestations include fever, limb weakness, dry cough and other symptoms.

In this application, the term "severe acute respiratory syndrome (SARS)" generally refers to the disease caused by SARS-CoV. SARS is usually characterized by systemic symptoms of muscle pain, headache, and fever, with respiratory symptoms such as cough, dyspnea, and pneumonia appearing within 2 to 14 days.

In this application, the term "pharmaceutically acceptable adjuvant" generally refers to any adjuvant, excipient such as solvents, dispersion media, coatings, isotonic and absorption delaying agents that are compatible with the active ingredient being administered or other pharmaceutical carriers.

In this application, the term "SM102" generally refers to an ionizable amino lipid that has been combined with other lipids to form lipid nanoparticles. The molecular structure of SM102 can be found in CAS 2089251-47-6.

Detailed description of the invention

S protein variants

In one aspect, the present application provides an S protein variant, which may not include a complete cytoplasmic tail domain compared with the S protein of wild-type coronavirus. In the present application, the S protein variant may comprise a truncated cytoplasmic tail domain compared to the S protein of wild-type coronavirus.

In the present application, compared with the S protein of wild-type coronavirus, the S protein variant may not include a cytoplasmic tail domain.

In this application, compared with the S protein of wild-type coronavirus, the cytoplasmic tail domain of the S protein variant may be deleted at the C-terminus of about 14, about 15, about 16, about 17, or about 18 , about 19 or about 20 amino acids. In the present application, compared with the S protein of wild-type coronavirus, the cytoplasmic tail domain of the S protein variant may be deleted at the C terminus of about 10, about 20, about 30, about 40, or about 50 or about 60 amino acids.

In the present application, compared with the S protein of wild-type coronavirus, the cytoplasmic tail domain of the S protein variant may be deleted from about 22 to about 64 amino acids at the C terminus. In the present application, compared with the S protein of wild-type coronavirus, the cytoplasmic tail domain of the S protein variant may be deleted from about 22 to about 60 amino acids at the C terminus. In the present application, compared with the S protein of wild-type coronavirus, the cytoplasmic tail domain of the S protein variant may be deleted from about 30 to about 60 amino acids at the C terminus. In the present application, compared with the S protein of wild-type coronavirus, the cytoplasmic tail domain of the S protein variant may be deleted from about 40 to about 60 amino acids at the C terminus. In the present application, compared with the S protein of wild-type coronavirus, the cytoplasmic tail domain of the S protein variant may be deleted from about 30 to about 40 amino acids at the C terminus. In the present application, compared with the S protein of wild-type coronavirus, the cytoplasmic tail domain of the S protein variant may be deleted from about 20 to about 30 amino acids at the C terminus.

In the present application, compared with the S protein of wild-type coronavirus, the cytoplasmic tail domain of the S protein variant may lack 18-20 amino acids at the C-terminus. In the present application, compared with the S protein of wild-type coronavirus, the cytoplasmic tail domain of the S protein variant may have 18 amino acids deleted at the C-terminus.

In the present application, compared with the S protein of wild-type coronavirus, the S protein variant may not include a transmembrane domain. In this application, compared with the S protein of wild-type coronavirus, the S protein variant may not include a complete transmembrane domain. In the present application, the S protein variant may comprise a truncated transmembrane domain compared to the S protein of wild-type coronavirus.

In this application, the coronavirus may be MERS coronavirus and/or SARS-CoV-like viruses. The SARS-CoV-like virus may be SARS-CoV or SARS-CoV-2 virus.

In this application, the coronavirus may be SARS-CoV-2 virus. In the present application, compared with the S protein of the wild-type SARS-CoV-2 virus, the cytoplasmic tail domain of the S protein variant may be deleted at the C-terminus of about 14, about 15, about 16, or about 17 , about 18, about 19 or about 20 amino acids. In this application, compared with the S protein of the wild-type SARS-CoV-2 virus, the cytoplasmic tail domain of the S protein variant may be deleted at the C-terminus of about 10, about 20, about 30, or about 40 , about 50 or about 60 amino acids.

In the present application, compared with the S protein of wild-type SARS-CoV-2 virus, the cytoplasmic tail domain of the S protein variant may be deleted from about 22 to about 64 amino acids at the C terminus. In the present application, compared with the S protein of wild-type SARS-CoV-2 virus, the cytoplasmic tail domain of the S protein variant may be deleted from about 22 to about 60 amino acids at the C terminus. In the present application, compared with the S protein of wild-type SARS-CoV-2 virus, the cytoplasmic tail domain of the S protein variant may be deleted from about 30 to about 60 amino acids at the C terminus. In the present application, compared with the S protein of wild-type SARS-CoV-2 virus, the cytoplasmic tail domain of the S protein variant may be deleted from about 40 to about 60 amino acids at the C terminus. In the present application, compared with the S protein of wild-type SARS-CoV-2 virus, the cytoplasmic tail domain of the S protein variant may be deleted from about 30 to about 40 amino acids at the C terminus. In the present application, compared with the S protein of wild-type SARS-CoV-2 virus, the cytoplasmic tail domain of the S protein variant may be deleted from about 20 to about 30 amino acids at the C terminus.

In the present application, compared with the S protein of the wild-type SARS-CoV-2 virus, the cytoplasmic tail domain of the S protein variant may have 18-20 amino acids deleted at the C-terminus. In this application, compared with the S protein of the wild-type SARS-CoV-2 virus, the cytoplasmic tail domain of the S protein variant may have 18 amino acids deleted at the C-terminus.

In this application, the S protein variant may comprise the amino acid sequence shown in any one of SEQ ID NO: 8, 21, 24, 27, 30, 33, 38, 41, 45, 59 and 61. For example, the S protein variant may comprise the amino acid sequence shown in SEQ ID NO:8.

In this application, the coronavirus may be SARS-CoV virus. In the present application, compared with the S protein of the wild-type SARS-CoV virus, the cytoplasmic tail domain of the S protein variant may be deleted at the C terminus of about 14, about 15, about 16, about 17, About 18, about 19 or about 20 amino acids. In the present application, compared with the S protein of the wild-type SARS-CoV virus, the cytoplasmic tail domain of the S protein variant may be deleted at the C-terminus of about 10, about 20, about 30, about 40, About 50 or about 60 amino acids.

In the present application, compared with the S protein of wild-type SARS-CoV virus, the cytoplasmic tail domain of the S protein variant may be deleted from about 22 to about 64 amino acids at the C terminus. In the present application, compared with the S protein of wild-type SARS-CoV virus, the cytoplasmic tail domain of the S protein variant may be deleted from about 22 to about 60 amino acids at the C terminus. In the present application, compared with the S protein of wild-type SARS-CoV virus, the cytoplasmic tail domain of the S protein variant may be deleted from about 30 to about 60 amino acids at the C terminus. In the present application, compared with the S protein of wild-type SARS-CoV virus, the cytoplasmic tail domain of the S protein variant may be deleted from about 40 to about 60 amino acids at the C terminus. In the present application, compared with the S protein of wild-type SARS-CoV virus, the cytoplasmic tail domain of the S protein variant may be deleted from about 30 to about 40 amino acids at the C terminus. In the present application, compared with the S protein of wild-type SARS-CoV virus, the cytoplasmic tail domain of the S protein variant may be deleted from about 20 to about 30 amino acids at the C terminus.

In the present application, compared with the S protein of the wild-type SARS-CoV virus, the cytoplasmic tail domain of the S protein variant may have 18-20 amino acids deleted at the C terminus. In the present application, compared with the S protein of the wild-type SARS-CoV virus, the cytoplasmic tail domain of the S protein variant may have 18 amino acids deleted at the C-terminus.

In this application, the S protein variant may comprise the amino acid sequence shown in SEQ ID NO: 35.

In this application, the coronavirus may be MERS-CoV virus. In the present application, compared with the S protein of the wild-type MERS-CoV virus, the cytoplasmic tail domain of the S protein variant may be deleted at the C terminus of about 14, about 15, about 16, about 17, About 18, about 19 or about 20 amino acids. In the present application, compared with the S protein of the wild-type MERS-CoV virus, the cytoplasmic tail domain of the S protein variant may be deleted at the C terminus of about 10, about 20, about 30, about 40, About 50 or about 60 amino acids.

In the present application, compared with the S protein of wild-type MERS-CoV virus, the cytoplasmic tail domain of the S protein variant may be deleted from about 22 to about 64 amino acids at the C terminus. In the present application, compared with the S protein of wild-type MERS-CoV virus, the cytoplasmic tail domain of the S protein variant may be deleted from about 22 to about 60 amino acids at the C terminus. In the present application, compared with the S protein of wild-type MERS-CoV virus, the cytoplasmic tail domain of the S protein variant may be deleted from about 30 to about 60 amino acids at the C terminus. In the present application, compared with the S protein of wild-type MERS-CoV virus, the cytoplasmic tail domain of the S protein variant may be deleted from about 40 to about 60 amino acids at the C terminus. In the present application, compared with the S protein of wild-type MERS-CoV virus, the cytoplasmic tail domain of the S protein variant may be deleted from about 30 to about 40 amino acids at the C terminus. In the present application, compared with the S protein of wild-type MERS-CoV virus, the cytoplasmic tail domain of the S protein variant may be deleted from about 20 to about 30 amino acids at the C terminus.

In the present application, compared with the S protein of the wild-type MERS-CoV virus, the cytoplasmic tail domain of the S protein variant may have 18-20 amino acids deleted at the C terminus. In the present application, compared with the S protein of the wild-type MERS-CoV virus, the cytoplasmic tail domain of the S protein variant may have 18 amino acids deleted at the C-terminus.

In this application, the S protein variant may comprise the amino acid sequence shown in SEQ ID NO: 45.

In this application, the S protein variant may include a signal peptide, and the signal peptide is located at the N-terminus of the S protein variant. In this application, the signal peptide may comprise the amino acid sequence shown in SEQ ID NO: 1 or 2.

nucleic acid molecules

In another aspect, the application provides an isolated nucleic acid molecule, which may comprise a polynucleotide encoding the S protein variant.

In this application, the nucleic acid molecule may be DNA. For example, the DNA may comprise the nucleotide sequence shown in any one of SEQ ID NOs: 6, 9-19, 22, 25, 28, 31, 36, 39, 42, 46, 60, 62 and 63. In this application, the DNA may comprise SEQ ID NOs: 6, 9-19, 22, 25, 28, 31, 36, 39, 42, 46, 60, 62, 63, 71, 72, 74, 76 , 78, 80, 82, 84, 95, 96 and 97 have at least 80% (for example, 82%, 85%, 88%, 90%, 95%, 98%, 99% or above) sequence identity.

For example, the DNA may comprise the nucleotide sequence shown in any one of SEQ ID NOs: 6, 9-19, 22, 25, 28 and 31.

For example, the DNA may comprise the nucleotide sequence described in any one of SEQ ID NOs: 6, 9-18, 60, 62 and 63.

For example, the DNA may comprise the nucleotide sequence described in any one of SEQ ID NOs: 71, 72, 74, 76, 78, 80, 82, 84, 95, 96 and 97.

In this application, the DNA may comprise modifications at the 5' cap. In this application, the DNA may comprise modifications at the 5' untranslated region. In this application, the DNA may comprise modifications at the open reading frame. In this application, the DNA may comprise modifications at the 3' untranslated region. In this application, the DNA may comprise modifications at the 3' poly(A). In this application, the DNA contains at least one modified nucleotide.

In this application, the DNA may be codon optimized. For example, the DNA may comprise the nucleotide sequence described in any one of SEQ ID NOs: 9-18.

In this application, the nucleic acid molecule may be RNA. In this application, the nucleic acid molecule may be mRNA. The mRNA may include a 5' cap, a 5' untranslated region (5' UTR), an open reading frame, a 3' untranslated region (3' UTR), and a poly A tail.

For example, the 5' UTR may comprise the nucleotide sequence shown in SEQ ID NO: 64.

For example, the 5' UTR may comprise the nucleotide sequence shown in SEQ ID NO: 65.

For example, the poly A may comprise the nucleotide sequence shown in SEQ ID NO: 66.

For example, the open reading frame may comprise the nucleotide sequence shown in any one of SEQ ID NO: 7, 20, 23, 26, 29, 32, 37, 40, 43 and 47. In this application, the open reading frame may contain the same sequence as SEQ ID NO: 7, 20, 23, 26, 29, 32, 37, 40, 43, 47, 73, 75, 77, 79, 81, 83 and 85 A nucleotide sequence having a sequence identity of at least 80% (e.g., 82%, 85%, 88%, 90%, 95%, 98%, 99% or more) .

For example, the open reading frame may comprise the nucleotide sequence shown in any one of SEQ ID NO: 7, 20, 23, 26, 29 and 32.

For example, the open reading frame may comprise the nucleotide sequence shown in SEQ ID NO:7.

For another example, the mRNA may include a 5' cap, a 5' untranslated region (5'UTR), an open reading frame, a 3' untranslated region (3'UTR) and a poly A tail; wherein the 5' UTR can Containing the nucleotide sequence shown in SEQ ID NO:64, the 5'UTR may include the nucleotide sequence shown in SEQ ID NO:65, and the poly A may include the nucleoside shown in SEQ ID NO:66 acid sequence, the open reading frame may include the nucleotide sequence shown in SEQ ID NO:7; the mRNA may include the nucleotide sequence shown in SEQ ID NO:67.

In this application, the RNA (e.g., mRNA) may comprise modifications at the 5' cap. In this application, the RNA (e.g., mRNA) may comprise modifications at the 5' untranslated region. In this application, the RNA (eg, mRNA) may comprise modifications at the open reading frame. In this application, the RNA (e.g., mRNA) may comprise modifications at the 3' untranslated region. In this application, the RNA (e.g., mRNA) may comprise modifications at the 3' poly(A). In this application, the RNA (eg, mRNA) includes at least one modified nucleotide.

In this application, the mRNA may be codon optimized.

The modification of the nucleic acid molecule (eg DNA or RNA) may be a chemical modification or a biological modification. In this application, the nucleic acid molecule may comprise one or more modified nucleobases, nucleosides or nucleotides. Modified nucleic acid molecules can have useful properties compared to an unmodified reference sequence (e.g., a naturally occurring or wild-type nucleic acid molecule), including, for example, enhanced stability, increased intracellular retention, enhanced translation effect and/or reduced immunogenicity. Therefore, the use of modified nucleic acid molecules can increase the efficiency of protein production, improve nucleic acid retention within cells, and reduce immunogenicity.

Codon optimization methods for nucleic acid molecules (e.g., DNA or RNA) are known in the art and can be used for a variety of purposes: matching codon frequencies in the host organism to ensure correct folding; biasing GC content to increase nucleic acid molecule stability; or Reduce secondary structure; minimize tandem repeat codons that may be repeated or base sequences that may impair gene construction or expression; customize transcription and translation control regions; insert or remove protein transport sequences; remove/add post-translational sequences in encoded proteins Modify sites (such as glycosylation sites); add, remove, or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and nucleic acid molecule degradation sites; regulate translation rates to allow individual The domain folds correctly; or, problematic secondary structure within the polynucleotide is reduced or eliminated.

lipid nanoparticles

On the other hand, the present application provides a composition, which may include the mRNA described in the present application and may also include a delivery carrier.

The mRNAs of the present application may be formulated in nanoparticles or other delivery vehicles, for example, to avoid their degradation upon delivery to a subject. In this application, the mRNA can be encapsulated within nanoparticles. In specific embodiments, the nanoparticles include lipids. Lipid nanoparticles may include, but are not limited to, liposomes and micelles. In this application, the lipid nanoparticles may include cationic and/or ionizable lipids, anionic lipids, neutral lipids, amphipathic lipids, pegylated lipids and/or structural Lipid, or a combination of the above. In certain embodiments, lipid nanoparticles comprise one or more RNAs described herein, such as mRNA, and for example, mRNA encoding an S protein variant.

The delivery vehicle in the compositions described herein may be lipid nanoparticles. The nanolipid particles may comprise one or more (

eg

1, 2, 3, 4, 5, 6, 7 or 8) cationic and/or ionizable lipids. "Cationic lipid" generally refers to a lipid that carries any number of net positive charges at a certain pH (eg, physiological pH). The cationic lipids may include, but are not limited to, SM102, 3-(didodecylamino)-N1,N1,4-triacontyl-1-piperazineethylamine (KL10), N1-[2- (Docosylamino)ethyl]-N1,N4,N4-tricosyl-1,4-piperazinediethylamine (KL22), 14,25-tricosyl-15,18 ,21,24-tetraazaoctaporanane (KL25), DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, Octyl-CLinDMA, octyl-CLinDMA (2S), DODAC, DOTMA, DDAB, DOTAP, DOTAP.C1, DC-Choi, DOSPA, DOGS, DODAP, DODMA and DMRIE. For example, the cationic lipid can be SM102

In certain embodiments, the molar proportion of the cationic lipid in the lipid nanoparticle is about 40-70%, for example, about 40-65%, about 40-60%, about 45-55%, or about 48 -53%. In certain embodiments, the molar proportion of the cationic lipid (eg, SM102) in the lipid nanoparticle is about 50%.

In this application, the nanolipid particles may comprise one or more (

eg

1, 2, 3, 4, 5, 6, 7 or 8) non-cationic lipids. The non-cationic lipids may include anionic lipids. Anionic lipids suitable for lipid nanoparticles of the present application may include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-lauroylphosphatidylethanolamine, N-succinylphosphatidylethanolamine , N-glutarylphosphatidylphosphoethanol group, and other neutral lipids to which anionic groups are attached.

The non-cationic lipids may include neutral lipids. Neutral lipids suitable for lipid nanoparticles of the present application may include phospholipids, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine ( DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl- Phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-trans Formula PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or mixtures thereof. Additionally, lipids having a mixture of saturated and unsaturated fatty acid chains can be used. For example, the neutral lipids described herein may be selected from DOPE, DSPC, DPPC, POPC or any related phosphatidylcholine.

In certain embodiments, the molar proportion of the phospholipid in the lipid nanoparticle is about 30-45%, for example, about 33-42%, about 35-40%, or about 38-39%. In certain embodiments, the molar proportion of the phospholipid (eg, DSPC) in the lipid nanoparticle is about 38.5%.

In the present application, the nanolipid particles may comprise lipid conjugates, for example, polyethylene glycol (PEG) modified lipids and derivatized lipids. PEG-modified lipids may include, but are not limited to, polyethylene glycol chains up to 5 kDa in length covalently linked to lipids having alkyl chains of C6-C20 length. The addition of these components can prevent lipid aggregation, increase circulation duration, facilitate delivery of lipid-nucleic acid compositions to target cells, or rapidly release nucleic acids. For example, the polyethylene glycol (PEG) modified lipid molecule can be a PEG-ceramide with a shorter acyl chain (eg, C14 or C18). For example, the nanolipid particles may comprise PEG2000-DMG.

In certain embodiments, the molar proportion of polyethylene glycol (PEG)-modified lipid molecules in the lipid nanoparticles is about 0.5-2%, for example, about 1-2%, about 1.2-1.8%, or About 1.4-1.6%. In certain embodiments, the molar proportion of polyethylene glycol (PEG)-modified lipid molecules (eg, PEG2000-DMG) in the lipid nanoparticle is about 1.5%.

In this application, the nanolipid particles may also contain cholesterol. In certain embodiments, the molar proportion of cholesterol in the lipid nanoparticle is about 5-15%, for example, about 6-14%, about 7-13%, about 8-12%, or about 9-11% %. In certain embodiments, the molar proportion of cholesterol in the lipid nanoparticle can be about 10%.

In this application, the nanolipid particles may include cationic lipids, cholesterol, phospholipids, and polyethylene glycol-modified lipid molecules. In certain embodiments, the molar ratio of the cationic lipid, cholesterol, phospholipid and polyethylene glycol-modified lipid molecules may be 45-55:5-15:35-45:0.5-2. In certain embodiments, the molar ratio of the cationic lipid, cholesterol, phospholipid, and polyethylene glycol-modified lipid molecules may be 50:10:38.5:1.5.

In another aspect, the application provides a vector comprising the nucleic acid molecule. For example, the vector may be a viral vector, such as an adenoviral vector, an adeno-associated viral vector, and/or a lentiviral vector.

In another aspect, the application provides cells comprising said nucleic acid molecule, and/or said vector. In this application, the cell may be a prokaryotic cell, for example, E. coli. In this application, the cells may be eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells. In this application, the cells may be mammalian cells, such as mouse cells, human cells, etc.

In another aspect, the present application provides a method of preparing the S protein variant, the method comprising culturing the cell under conditions such that the S protein variant is expressed.

In another aspect, the application provides a pharmaceutical composition comprising the S protein variant, the nucleic acid molecule, the vector, the cell and/or the composition, and optionally a pharmaceutical acceptable carrier.

On the other hand, the present application provides a vaccine comprising the S protein variant, the nucleic acid molecule, the vector, the cell, the composition and/or the pharmaceutical composition, and a pharmaceutical Acceptable adjuvants.

In this application, the vaccine may be a protein vaccine, which may comprise the S protein variant.

In this application, the vaccine may be a nucleic acid vaccine, which may include the nucleic acid molecule. In this application, the vaccine may be a DNA vaccine. In this application, the vaccine may be an mRNA vaccine.

On the other hand, the present application provides a kit comprising the S protein variant, the nucleic acid molecule, the vector, the cell, the compound, the pharmaceutical composition and/or the Describe vaccines.

On the other hand, the application provides the use of the S protein variant, the nucleic acid molecule, the vector, the cell, the composition, the pharmaceutical composition and/or the vaccine in the preparation of medicines , the drug is used to alleviate, prevent and/or treat diseases caused by coronavirus.

In another aspect, the present application provides a method of alleviating, preventing and/or treating a disease or condition caused by coronavirus, the method comprising administering the S protein variant, the nucleic acid molecule, the the vector, the cell, the composition, the pharmaceutical composition and/or the vaccine.

In this application, the disease or condition may be novel coronavirus pneumonia (COVID-19) and/or severe acute respiratory syndrome (SARS).

On the other hand, the present application provides a method for producing antibodies against coronavirus, the method comprising administering the S protein variant, the nucleic acid molecule, the vector, the cell, the composition, the drug compositions and/or said vaccines.

In this application, the S protein variant, the nucleic acid molecule, the vector, the cell, the composition, the pharmaceutical composition and/or the vaccine can be administered once or multiple times. or continuous administration, and both are safe. After the initial administration, subsequent administration of the composition and/or pharmaceutical composition may shorten the onset of action. In this application, the S protein variant, the nucleic acid molecule, the vector, the cell, the composition, the pharmaceutical composition and/or the vaccine can be combined with other active substances or treatment/prevention Ingredients are co-administered. In this application, the S protein variant, the nucleic acid molecule, the vector, the cell, the composition, the pharmaceutical composition and/or the vaccine can be used in other active substances or treatments/preventions. before or after the ingredients are applied.

This application also discloses the following specific implementation modes:

1. S protein variant, compared with the S protein of wild-type coronavirus, it does not contain a complete cytoplasmic tail domain.

2. The S protein variant according to embodiment 1, which contains a truncated cytoplasmic tail domain compared with the S protein of wild-type coronavirus.

3. The S protein variant according to any one of embodiments 1-2, which has 18-20 amino acids truncated at the C-terminus compared with the S protein of wild-type coronavirus.

4. The S protein variant according to embodiment 3, which has 18 amino acids truncated at the C-terminus compared with the S protein of wild-type coronavirus.

5. The S protein variant according to embodiment 1, which does not include a cytoplasmic tail domain compared with the S protein of wild-type coronavirus.

6. The S protein variant according to embodiment 5, which does not include a transmembrane domain compared with the S protein of wild-type coronavirus.

7. The S protein variant according to any one of embodiments 5-6, which has 22-64 amino acids truncated at the C-terminus compared with the S protein of wild-type coronavirus.

8. The S protein variant according to any one of embodiments 1-7, wherein the coronavirus is a MERS coronavirus and/or a SARS-CoV-like virus.

9. The S protein variant according to any one of embodiments 1-8, wherein the coronavirus is a SARS-CoV-2 virus.

10. The S protein variant according to any one of embodiments 1-9, wherein the cytoplasmic tail domain of the S protein of the wild-type coronavirus comprises the S protein variant shown in any one of SEQ ID NO: 48-49 Amino acid sequence.

11. The S protein variant according to any one of embodiments 6-10, wherein the transmembrane domain of the S protein of the wild-type coronavirus comprises the amino acid shown in any one of SEQ ID NO: 50-51 sequence.

12. The S protein variant according to any one of embodiments 1-11, wherein the S protein of the wild-type coronavirus comprises the amino acid sequence shown in any one of SEQ ID NO: 5, 34 and 44.

13. The S protein variant according to any one of embodiments 1-12, comprising SEQ ID NO:8, 21, 24, 27, 30, 33, 35, 38, 41, 45, 59 and 61 The amino acid sequence shown in any one.

14. The S protein variant according to any one of embodiments 1-13, which comprises the amino acid sequence shown in SEQ ID NO:8.

15. The S protein variant according to any one of embodiments 1-14, further comprising a signal peptide, and the signal peptide is located at the N-terminus of the S protein variant.

16. The S protein variant according to embodiment 15, wherein the signal peptide comprises the amino acid sequence shown in any one of SEQ ID NO: 1-2.

17. An isolated nucleic acid molecule comprising a polynucleotide encoding the S protein variant of any one of embodiments 1-16.

18. The nucleic acid molecule of embodiment 17, which is DNA.

19. The nucleic acid molecule according to any one of embodiments 17-18, comprising SEQ ID NOs: 6, 9-19, 22, 25, 28, 31, 36, 39, 42, 46, 60, 62 and The nucleotide sequence described in any one of 63.

20. The nucleic acid molecule of embodiment 17, which is mRNA.

21. The nucleic acid molecule of embodiment 20, comprising modifications at one or more positions selected from the group consisting of: 5' cap, 5' untranslated region, open reading frame, 3' untranslated region, and 3' poly(A).

22. The nucleic acid molecule of any one of embodiments 20-21, comprising at least one modified nucleotide.

23. The nucleic acid molecule of any one of embodiments 20-22, which is codon optimized.

24. The nucleic acid molecule according to any one of embodiments 20-23, which comprises SEQ ID NO:7, 20, 23, 26, 29, 32, 37, 40, 43 and 47. Nucleotide sequence.

25. A vector comprising the nucleic acid molecule of any one of embodiments 17-24.

26. A cell comprising the nucleic acid molecule of any one of embodiments 17-24, and/or the vector of embodiment 25.

27. A method for preparing the S protein variant according to any one of embodiments 1-16, the method comprising culturing under conditions such that the S protein variant according to any one of embodiments 1-16 is expressed. The cell according to embodiment 26.

28. A composition comprising (1) an mRNA encoding the S protein variant of any one of embodiments 1-16, and (2) a delivery vector.

29. The composition of embodiment 28, wherein the mRNA comprises modifications at one or more positions selected from the group consisting of: 5' cap, 5' untranslated region, open reading frame, 3' untranslated region and poly A tail.

30. The composition of any one of embodiments 28-29, wherein the mRNA comprises at least one modified nucleotide.

31. The composition of any one of embodiments 28-30, wherein the mRNA comprises a modified nucleotide that is pseudouridine (Ψ).

32. The composition of any one of embodiments 28-31, wherein the mRNA is codon optimized.

33. The composition according to any one of embodiments 28-32, wherein the mRNA comprises any one of SEQ ID NOs: 7, 20, 23, 26, 29, 32, 37, 40, 43 and 47 The nucleotide sequence shown in the item.

34. The composition of any one of embodiments 28-32, wherein the S protein variant is derived from the SARS CoV-2 virus.

35. The composition according to any one of embodiments 28-32, wherein the mRNA comprises the nucleotide sequence shown in SEQ ID NO:7.

36. The composition of any one of embodiments 28-35, wherein the delivery vehicle comprises liposomes.

37. The composition of any one of embodiments 28-36, wherein the delivery vehicle comprises lipid nanoparticles (LNP).

38. The composition of any one of embodiments 28-37, wherein the delivery vehicle comprises a cationic lipid.

39. The composition of embodiment 38, wherein the molar ratio of the cationic lipid in the delivery vehicle is from about 45% to about 55%.

40. The composition of embodiment 38 or 39, wherein the cationic lipid comprises SM102 and/or Dlin-MC3.

41. The composition of any one of embodiments 28-40, wherein the delivery vehicle comprises a non-cationic lipid.

42. The composition of embodiment 41, wherein the non-cationic lipids comprise phospholipids and/or lipid conjugates.

43. The composition of embodiment 42, wherein the phospholipid comprises distearoylphosphatidylcholine (DSPC).

44. The composition of embodiment 42 or 43, in the delivery vehicle, wherein the molar ratio of the phospholipids is from about 35% to about 40%.

45. The composition of any one of embodiments 42-44, wherein the lipid conjugate comprises a polyethylene glycol modified lipid molecule.

46. The composition of any one of embodiments 42-45, in the delivery vehicle, wherein the molar ratio of the lipid conjugate is from about 1% to about 2%.

47. The composition of any one of embodiments 45-46, wherein the polyethylene glycol modified lipid molecule comprises DMPE-PEG2000.

48. The composition of any one of embodiments 28-47, wherein the delivery vehicle includes cholesterol.

49. The composition of embodiment 48, wherein the molar ratio of cholesterol in the delivery vehicle is from about 8% to about 12%.

50. The composition of any one of embodiments 28-49, wherein the delivery vehicle comprises a cationic lipid, cholesterol, phospholipid, and lipid conjugate, and the cationic lipid, cholesterol, phospholipid, and lipid conjugate The mass ratio of the conjugate is 50:10:38.5:1.5.

51. The composition according to any one of embodiments 28-50, wherein the delivery vehicle comprises SM102, cholesterol, DSPC and DMPE-PEG2000, and the mass ratio of SM102, cholesterol, DSPC and DMPE-PEG2000 is 50:10:38.5:1.5.

52. The composition of any one of embodiments 28-51, wherein said mRNA is encapsulated in said delivery vehicle.

53. A pharmaceutical composition comprising the S protein variant according to any one of embodiments 1-16, the nucleic acid molecule according to any one of embodiments 17-24, the vector according to embodiment 25, and the embodiment 26 and/or the composition of any one of embodiments 28-52, and optionally a pharmaceutically acceptable carrier.

54. A vaccine comprising the S protein variant according to any one of embodiments 1-16, the nucleic acid molecule according to any one of embodiments 17-24, the vector according to embodiment 25, and the vector according to embodiment 26. The cells described above, the composition described in any one of embodiments 28-52 and/or the pharmaceutical composition described in embodiment 53, and a pharmaceutically acceptable adjuvant.

55. The vaccine of embodiment 54, which is a protein vaccine.

56. The vaccine according to embodiment 54, which is a nucleic acid vaccine.

57. The vaccine of embodiment 56, which is a DNA vaccine.

58. The vaccine of embodiment 56, which is an mRNA vaccine.

59. Kit, comprising the S protein variant according to any one of embodiments 1-16, the nucleic acid molecule according to any one of embodiments 17-24, the vector according to embodiment 25, and the vector according to embodiment 26. The cells described above, the composition described in any one of embodiments 28-52, the pharmaceutical composition described in embodiment 53, and/or the vaccine described in any one of embodiments 54-58.

60. The S protein variant according to any one of embodiments 1-16, the nucleic acid molecule according to any one of embodiments 17-24, the vector according to embodiment 25, the cell according to embodiment 26, The use of the composition described in any one of embodiments 28-52, the pharmaceutical composition described in embodiment 53, and/or the vaccine described in any one of embodiments 54-58 in the preparation of a medicine, the medicine For use in mitigating, preventing, and/or treating illness caused by coronavirus.

61. Use according to embodiment 60, wherein the disease or condition is novel coronavirus pneumonia (COVID-19) and/or severe acute respiratory syndrome (SARS).

62. A method of alleviating, preventing and/or treating a disease or condition caused by coronavirus, the method comprising administering to a subject in need thereof the S protein variant of any one of embodiments 1-16, embodiment The nucleic acid molecule described in any one of 17-24, the vector described in embodiment 25, the cell described in embodiment 26, the composition described in any one of embodiments 28-52, the vector described in embodiment 53 Pharmaceutical compositions and/or vaccines according to any of embodiments 54-58.

63. Use according to embodiment 62, wherein the disease or condition is novel coronavirus pneumonia (COVID-19) and/or severe acute respiratory syndrome (SARS).

64. A method of producing antibodies against coronavirus, comprising administering the S protein variant described in any one of embodiments 1-16, the nucleic acid molecule described in any one of embodiments 17-24, or the method described in embodiment 25 The vector, the cell described in Embodiment 26, the composition described in any one of Embodiments 28-52, the pharmaceutical composition described in Embodiment 53, and/or the method described in any one of Embodiments 54-58 vaccine.

65. A method of activating immunity, comprising administering the S protein variant of any one of embodiments 1-16, the nucleic acid molecule of any one of embodiments 17-24, the vector of embodiment 25, The cell of embodiment 26, the composition of any one of embodiments 28-52, the pharmaceutical composition of embodiment 53, and/or the vaccine of any one of embodiments 54-58.

This application also discloses the following specific implementation modes:

13. The S protein variant according to any one of embodiments 1-12, which comprises the amino acid sequence shown in any one of SEQ ID NO: 38, 41 and 59.

16. The S protein variant according to embodiment 15, wherein the signal peptide comprises the amino acid sequence shown in any one of SEQ ID NO: 1, 2 and 68.

18. The nucleic acid molecule of embodiment 17, which is DNA.

19. The nucleic acid molecule according to any one of embodiments 17-18, comprising SEQ ID NO: 17, 19, 22, 25, 28, 31, 36, 39, 42, 46, 60, 62 and 63 The nucleotide sequence of any one.

20. The nucleic acid molecule of embodiment 17, which is mRNA.

47. The composition of any one of embodiments 45-46, wherein the polyethylene glycol modified lipid molecule comprises DMG-PEG2000.

51. The composition according to any one of embodiments 28-50, wherein the delivery vehicle comprises SM102, cholesterol, DSPC and DMG-PEG2000, and the mass ratio of SM102, cholesterol, DSPC and DMG-PEG2000 is 50:10:38.5:1.5.

55. The vaccine of embodiment 54, which is a protein vaccine.

56. The vaccine according to embodiment 54, which is a nucleic acid vaccine.

57. The vaccine of embodiment 56, which is a DNA vaccine.

58. The vaccine of embodiment 56, which is an mRNA vaccine.

Without intending to be limited by any theory, the following examples are only for illustrating various technical solutions of the invention of the present application, and are not intended to limit the scope of the invention of the present application.

Example

Example 1

Example 1.1 Preparation of mRNA

The mRNA was obtained by in vitro transcription using a linearized plasmid as a template through a one-step capping method. The cap1 cap analog (m7G(5')ppp(5')(2'OMeA)pG (APExBIO Company) was used in the transcription system. Product EZ Cap#B8176, cap1), ATP, GTP, CTP, Pseudo-UTP (referred to as pseudoU, ApexBio, #B7972), to produce pseudouridine (Ψ)-modified mRNA of the cap1 structure, which is purified to high purity through two-step chromatography mRNA.

The following mRNA samples were prepared: SARS-Cov-2 virus S protein containing Alb signal peptide (code: SDC60 Alb) and SARS virus S protein (SARS S). Among them, the SARS-Cov-2 virus SDC60 Alb protein has the following variants: full-length protein (FL), full-length protein containing K986P and V987P mutations (2P), and the C-terminus of the full-length protein is truncated by 10, 18, respectively. Proteins of 20, 40, 60, and 67 amino acids (DC10, DC18, DC20, DC40, DC60, DC67); SARS S protein variants of SARS virus include: full-length protein (FL) and full-length C-terminus truncated by 10 respectively , 18, 40 amino acid proteins (DC10, DC18, DC40).

Example 1.2 Preparation of lipid nanoparticles

The mRNA of Example 1.1 is ionizable (cationic) at low pH, and coated into nanoparticles with two auxiliary lipids (DSPC and cholesterol) and a pegylated lipid (DMG-PEG2000). An aqueous solution of mRNA was prepared by mixing the mRNA dissolved in ultrapure water 1:1 (v/v) with 100 μmM citrate buffer, pH 4.0. The ratio of the four lipid components (cationic lipid SM102: cholesterol: DSPC: DMG-PEG2000) was adjusted (50:10:38.5:1.5) and dissolved in ethanol (99.5%) to form a lipid solution. The mRNA and lipid solutions were mixed in a NanoAssemblr (Precision Nanosystems) microfluidic mixing system with a volume mixing ratio of Aq:EtOH=3:1 and a constant total flow rate of 12mL/min to obtain liposome nanoparticles containing mRNA ( LNP). Lipid nanoparticles are stored after dialysis, concentration, and filtration.

Example 2 Determination of S protein content in cells transfected with mRNA using enzyme-linked immunosorbent assay

The mRNA prepared in Example 1 was transfected into 293T cells respectively (via the standard operating procedure of ThermoFisher transfection reagent Lipofectamine 2000). The cells were collected after 19 hours, and the total protein amount of the cell lysis sample was detected using the Micro BCA ^TM protein detection kit. The enzyme-linked immunosorbent assay was then used to determine the S protein content in the cell lysate.

Use 2 μg/mL capture antibody (antibody stock solution is diluted 500 times in 1×PBS), coat a 96-well ELISA plate, 50 μL/well, a total of 100ng per well, and store overnight in the dark for 4 days. For the S protein of SARS-Cov-2 virus (SDC60 Alb) and the S protein of SARS virus (SARS S), 40150-D006 was used as the capture antibody, and the antibody was purchased from Sino Biological. Wash the plate three times with 1×PBST (0.05% Tween20), 200 μL/well, invert the ELISA plate each time and tap to clean. Add 100 μL/well 2% BSA (dissolved in 1×PBST) to block, and incubate at room temperature for 1 hour. Wash the plate three times with 1×PBST (0.05% Tween20), 200 μL/well, invert the ELISA plate each time and tap to clean. SARS-CoV-2 and SARS proteins were used as standards for quantification. For SDC60 Alb protein and its variants, protein 40589-V08B1 was used as the standard. For SARS S protein and its variants, 40150-V08B1 was used as the standard. The proteins were all purchased from Sino Biological. According to the previous BCA quantification results, the cell lysates of the samples to be tested were diluted with 1×PBS at corresponding concentrations to keep the total protein amount added to the sample wells consistent. After mixing evenly, pipet 100 μL and add to the ELISA plate. Finally, add the total protein in each well of the ELISA plate, that is, the total protein amount of S mRNA transfected with SARS-Cov-2 virus is 10 μg, and the total protein amount of S mRNA transfected with SARS virus is 10 μg. The total protein amount was 20 μg and incubated at room temperature for 2 hours. Wash the plate 5 times with 1×PBST (0.05% Tween20), 200 μL/well, invert the ELISA plate each time and tap to clean. Dilute the detection antibody to the working concentration with 1×PBS, mix well, add 100 μL to each well, and incubate at room temperature for 1 hour. The detection antibody used for SDC60 Alb protein is 40591-MM43, with a working concentration of 0.609 μg/mL. The detection antibody used for SARS S protein is 40150-MM10, with a working concentration of 1 μg/mL. The antibodies were purchased from Sino Biological. Wash the plate three times with 1×PBST (0.05% Tween20), 200 μL/well, invert the ELISA plate each time and tap to clean. Dilute Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) 5000 times with 1×PBS, add 100μL to each well, and incubate at room temperature for 1 hour. Wash 3 times with 1×PBST (0.05% Tween20), 200 μL/well, invert the ELISA plate each time and tap lightly to clean. Add TMB substrate respectively, 50μL/well, wait at room temperature for 5-15 minutes (protect from light), blue will appear. Add 1M sulfuric acid respectively to stop the reaction, 150μL/well, the blue will turn yellow. Read the OD450 with a microplate reader.

As shown in Figure 1A, the in vitro expression results of cells transfected with the SARS-Cov-2 S protein variant mRNA of the present application show that for the S protein (SDC60 Alb) of the SARS-Cov-2 virus, the C-terminus is truncated by 10, The expression level of the protein after 18, 20, 40, 60 and 67 amino acids is higher than that of the full-length protein, and the expression level of the full-length protein is the same as the expression level of the protein after the C-terminal truncation of 10, 18 and 40 amino acids. There is a significant difference between them.

As shown in Figure 1B, the in vitro expression results of cells transfected with mRNA expressing the S protein of SARS virus (SARS S) show that for the SARS S protein, the protein expression after the C-terminal truncation of 18 or 40 amino acids is lower than that of the full-length protein. The expression level is high, and there is a significant difference between the expression level of the full-length protein and the protein expression level after C-terminal truncation of 18 or 40 amino acids. (The test method is Ordinary one-way ANOVA, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns: not significant.)

Example 3 Transfection of Cells with Lipid Nanoparticles

The lipid nanoparticles obtained in Example 2 were transfected into 293A cells respectively. After 24 hours, the cells were collected and Western Blot was used to detect protein expression. The results obtained are shown in Figure 1C. The results showed that for the wild-type S protein, the wild-type S protein with the SDC60 codon, and the wild-type S protein with the SDC60 codon of the Alb signal peptide, the protein expression levels after the C-terminal truncation of 18 amino acids were all higher.

Example 4 Mouse immunization experiment

The coated lipid nanoparticles expressing the SARS-CoV-2 Spike protein or the mRNA of each mutant (SDC60 Alb, SDC60 Alb-2P, SDC60 Alb-DC18) were injected intramuscularly (i.m.) into Balb/c mice, specifically The information is shown in Table 2 below, and the mouse immunization schematic is shown in Figure 2. Blood samples were collected on days 1, 14, 28, 42, and 70, and the sera were analyzed in a fluorescent antibody virus neutralization assay, as detailed in the following examples described.

Table 1 Administration Information

Example 5 Determination of antibody titer in serum using enzyme-linked immunosorbent assay

Coat 96-well ELISA plate with 2 μg/ml antigen protein (dissolved in PBS), 100 ng, 50 μl/well, and keep overnight for 4 days in the dark. The S protein antigen was purchased from Sino Biological, product number 40589-V08B1; the S protein RBD domain was purchased from Novoprotein, product number DRA36. Wash 3 times with PBST (0.05% Tween), 200 μl/well, invert the ELISA plate each time and tap to clean. Add 100 μl/well 2% BSA (dissolved in PBST) to block, and incubate at room temperature for 1 hr. Wash 3 times with PBST (0.05% Tween), 200 μl/well, invert the ELISA plate each time and tap to clean. Add mouse serum (diluted 100 times as the starting concentration, followed by 5-fold gradient dilution, a total of 11 gradients) in PBS, mix evenly, add 100 μl to the ELISA plate, and incubate at room temperature for 2 hours. From the mice in Example 4, 100 μl of approximately 40 μl of serum was collected around the eyes. After washing the plate, add HRP-anti-mouse IgG (1:5000 diluted in PBS), 100 μl/well, and incubate at room temperature for 1 hr. After washing the plate, add TMB substrate (Thermo Fisher, Cat. No. 34022), 50 μl/well, and wait at room temperature for 5-15 minutes (protect from light), and it will appear blue. Add 1M sulfuric acid respectively to stop the reaction, 150μl/well, the blue will turn yellow. Read the OD450 with a microplate reader.

Figure 3 shows that the S protein variant mRNA vaccines of the present application can effectively activate the mouse immune system and produce antibodies in the serum, and are highly safe and effective. One week after the second immunization of mice, at the same immunization dose (1 μg), the mRNA of the S protein variant truncated to 18 amino acids produced the highest antibody titer.

The foregoing detailed description is provided by way of explanation and example, and is not intended to limit the scope of the appended claims. Various modifications to the embodiments described herein will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.

Claims

S protein variant, which does not contain a complete cytoplasmic tail domain compared to the S protein of wild-type coronavirus.
The S protein variant according to claim 1, compared with the S protein of wild-type coronavirus, it contains a truncated cytoplasmic tail domain.
According to the S protein variant according to any one of claims 1-2, compared with the S protein of wild-type coronavirus, 18-20 amino acids are truncated at the C terminus.
According to the S protein variant of claim 3, compared with the S protein of wild-type coronavirus, 18 amino acids are truncated at the C terminus.
The S protein variant according to claim 1, compared with the S protein of wild-type coronavirus, it does not contain a cytoplasmic tail domain.
The S protein variant according to claim 5, compared with the S protein of wild-type coronavirus, it does not contain a transmembrane domain.
According to the S protein variant according to any one of claims 5-6, compared with the S protein of wild-type coronavirus, 22-64 amino acids are truncated at the C terminus.
The S protein variant according to any one of claims 1-7, wherein the coronavirus is a MERS coronavirus and/or a SARS-CoV-like virus.
The S protein variant according to any one of claims 1-8, wherein the coronavirus is a SARS-CoV-2 virus.
The S protein variant according to any one of claims 1-9, wherein the cytoplasmic tail domain of the S protein of the wild-type coronavirus comprises any one of SEQ ID NOs: 48, 49, 86 and 87. The amino acid sequence shown.
The S protein variant according to any one of claims 6-10, wherein the transmembrane domain of the S protein of the wild-type coronavirus includes any one of SEQ ID NO: 50, 51, 88 and 89 amino acid sequence.
The S protein variant according to any one of claims 1-11, wherein the S protein of the wild-type coronavirus comprises the amino acid sequence shown in any one of SEQ ID NO: 5, 34 and 44.
The S protein variant according to any one of claims 1-12, which includes SEQ ID NO: 8, 21, 24, 27, 30, 33, 35, 38, 41, 45, 59, 61 and 94 The amino acid sequence shown in any one.
The S protein variant according to any one of claims 1-13, which comprises the amino acid sequence shown in SEQ ID NO:8.
The S protein variant according to any one of claims 1 to 14, further comprising a signal peptide, and the signal peptide is located at the N-terminus of the S protein variant.
The S protein variant according to claim 15, wherein the signal peptide comprises the amino acid sequence shown in any one of SEQ ID NO: 1, 2 and 68.
An isolated nucleic acid molecule comprising a polynucleotide encoding the S protein variant of any one of claims 1-16.
The nucleic acid molecule of claim 17, which is DNA.
The nucleic acid molecule according to any one of claims 17-18, comprising SEQ ID NO: 6, 9-19, 22, 25, 28, 31, 36, 39, 42, 46, 60, 62, 63, The nucleotide sequence of any one of 71, 72, 74, 76, 78, 80, 82, 84, 95, 96 and 97.
The nucleic acid molecule of claim 17, which is mRNA.
The nucleic acid molecule of claim 20, comprising modifications at one or more positions selected from the group consisting of: 5' cap, 5' untranslated region, open reading frame, 3' untranslated region and 3' polygland glycosides.
The nucleic acid molecule of any one of claims 20-21, comprising at least one modified nucleotide.
The nucleic acid molecule according to any one of claims 20-22, which is codon optimized.
The nucleic acid molecule according to any one of claims 20-23, comprising SEQ ID NO: 7, 20, 23, 26, 29, 32, 37, 40, 43, 47, 73, 75, 77, 79, The nucleotide sequence shown in any one of 81, 83 and 85.
A vector comprising the nucleic acid molecule of any one of claims 17-24.
Cells comprising the nucleic acid molecule of any one of claims 17-24, and/or the vector of claim 25.
A method for preparing the S protein variant according to any one of claims 1 to 16, the method comprising culturing the S protein variant according to any one of claims 1 to 16 under conditions allowing expression of the S protein variant according to any one of claims 1 to 16. The cell of claim 26.
A composition comprising (1) an mRNA encoding the S protein variant of any one of claims 1-16, and (2) a delivery vector.
The composition of claim 28, wherein the mRNA comprises modifications at one or more positions selected from the group consisting of: 5' cap, 5' untranslated region, open reading frame, 3' untranslated region and poly A tail.
The composition of any one of claims 28-29, wherein said mRNA comprises at least one modified nucleotide.
The composition of any one of claims 28-30, wherein the modified nucleotide comprised by the mRNA is pseudouridine (Ψ).
The composition of any one of claims 28-31, wherein said mRNA is codon optimized.
The composition according to any one of claims 28-32, wherein the mRNA comprises SEQ ID NO: 7, 20, 23, 26, 29, 32, 37, 40, 43, 47, 73, 75, The nucleotide sequence shown in any one of 77, 79, 81, 83 and 85.
The composition of any one of claims 28-32, wherein the S protein variant is derived from SARS CoV-2 virus.
The composition of any one of claims 28-32, wherein the mRNA comprises a nucleotide sequence as shown in SEQ ID NO: 7 or 67.
The composition of any one of claims 28-35, wherein the delivery vehicle comprises liposomes.
The composition of any one of claims 28-36, wherein the delivery vehicle comprises lipid nanoparticles (LNP).
The composition of any one of claims 28-37, wherein the delivery vehicle comprises a cationic lipid.
The composition of claim 38, wherein the molar ratio of the cationic lipid in the delivery vehicle is from about 45% to about 55%.
The composition of claim 38 or 39, wherein the cationic lipid comprises SM102 and/or Dlin-MC3.
The composition of any one of claims 28-40, wherein the delivery vehicle comprises a non-cationic lipid.
The composition of claim 41, wherein the non-cationic lipids comprise phospholipids and/or lipid conjugates.
The composition of claim 42, wherein the phospholipid includes distearoylphosphatidylcholine (DSPC).
The composition of claim 42 or 43, in the delivery vehicle, wherein the molar ratio of the phospholipids is from about 35% to about 40%.
The composition of any one of claims 42-44, wherein the lipid conjugate comprises a polyethylene glycol modified lipid molecule.
The composition of any one of claims 42-45, wherein the molar ratio of the lipid conjugate in the delivery vehicle is from about 1% to about 2%.
The composition of any one of claims 45-46, wherein the polyethylene glycol modified lipid molecule comprises DMG-PEG2000.
The composition of any one of claims 28-47, wherein the delivery vehicle includes cholesterol.
The composition of claim 48, wherein the molar ratio of cholesterol in the delivery vehicle is from about 8% to about 12%.
The composition of any one of claims 28-49, wherein the delivery vehicle comprises cationic lipids, cholesterol, phospholipids and lipid conjugates, and the cationic lipids, cholesterol, phospholipids and lipid conjugates The mass ratio of the compound is 50:10:38.5:1.5.
The composition according to any one of claims 28-50, wherein the delivery carrier comprises SM102, cholesterol, DSPC and DMG-PEG2000, and the mass ratio of SM102, cholesterol, DSPC and DMG-PEG2000 is 50: 10:38.5:1.5.
The composition of any one of claims 28-51, wherein said mRNA is entrapped in said delivery vehicle.
A pharmaceutical composition comprising the S protein variant of any one of claims 1-16, the nucleic acid molecule of any one of claims 17-24, the vector of claim 25, the vector of claim 26 The cells and/or the composition of any one of claims 28-52, and optionally a pharmaceutically acceptable carrier.
A vaccine comprising the S protein variant according to any one of claims 1 to 16, the nucleic acid molecule according to any one of claims 17 to 24, the vector according to claim 25, or the vector according to claim 26. Cells, the composition of any one of claims 28-52 and/or the pharmaceutical composition of claim 53, and a pharmaceutically acceptable adjuvant.
The vaccine according to claim 54, which is a protein vaccine.
The vaccine according to claim 54, which is a nucleic acid vaccine.
The vaccine according to claim 56, which is a DNA vaccine.
The vaccine according to claim 56, which is an mRNA vaccine.
A kit comprising the S protein variant according to any one of claims 1 to 16, the nucleic acid molecule according to any one of claims 17 to 24, the vector according to claim 25, and the nucleic acid molecule according to any one of claims 17 to 24. Cells, the composition of any one of claims 28-52, the pharmaceutical composition of claim 53, and/or the vaccine of any one of claims 54-58.
The S protein variant of any one of claims 1-16, the nucleic acid molecule of any one of claims 17-24, the vector of claim 25, the cell of claim 26, the The use of the composition according to any one of 28-52, the pharmaceutical composition according to claim 53 and/or the vaccine according to any one of claims 54-58 in the preparation of a medicine, the medicine is used for Mitigation, prevention and/or treatment of illness caused by coronavirus.
The use according to claim 60, wherein the disease or condition is novel coronavirus pneumonia (COVID-19) and/or severe acute respiratory syndrome (SARS).
A method of alleviating, preventing and/or treating diseases or conditions caused by coronavirus, the method comprising administering to a subject in need the S protein variant of any one of claims 1-16, claim 17- The nucleic acid molecule according to any one of 24, the vector according to claim 25, the cell according to claim 26, the composition according to any one of claims 28-52, and the pharmaceutical combination according to claim 53 and/or the vaccine according to any one of claims 54-58.
The use according to claim 62, wherein the disease or condition is novel coronavirus pneumonia (COVID-19) and/or severe acute respiratory syndrome (SARS).
A method of producing antibodies against coronavirus, comprising administering the S protein variant of any one of claims 1-16, the nucleic acid molecule of any one of claims 17-24, and the vector of claim 25 , the cell of claim 26, the composition of any one of claims 28-52, the pharmaceutical composition of claim 53, and/or the vaccine of any one of claims 54-58.
A method of activating immunity, comprising administering the S protein variant of any one of claims 1-16, the nucleic acid molecule of any one of claims 17-24, the vector of claim 25, 26, the composition of any one of claims 28-52, the pharmaceutical composition of claim 53, and/or the vaccine of any one of claims 54-58.