WO2021233213A1 - mRNA或mRNA组合物及其制备方法和应用 - Google Patents

mRNA或mRNA组合物及其制备方法和应用 Download PDF

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WO2021233213A1
WO2021233213A1 PCT/CN2021/093741 CN2021093741W WO2021233213A1 WO 2021233213 A1 WO2021233213 A1 WO 2021233213A1 CN 2021093741 W CN2021093741 W CN 2021093741W WO 2021233213 A1 WO2021233213 A1 WO 2021233213A1
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mrna
protein
variants
cov
rbd
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PCT/CN2021/093741
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French (fr)
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朱涛
王浩猛
李荩
晏巧玲
莘春林
邵忠琦
李军强
宇学峰
巢守柏
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康希诺生物股份公司
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Priority to CA3179412A priority Critical patent/CA3179412A1/en
Priority to KR1020227043901A priority patent/KR20230011369A/ko
Priority to US17/926,444 priority patent/US20230312659A1/en
Priority to JP2022571198A priority patent/JP2023525936A/ja
Priority to EP21807863.2A priority patent/EP4155406A4/en
Publication of WO2021233213A1 publication Critical patent/WO2021233213A1/zh

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Definitions

  • the present invention relates to the technical field of vaccine development, in particular to the mRNA sequence containing the spike protein (S protein) or its variants encoding the new coronavirus SARS-CoV-2 and the receptor binding domain (RBD) or the receptor binding domain (RBD) encoding the S protein
  • the mRNA sequence of the variant relates to a composition containing one or two mRNAs. And the application of the mRNA or the composition in the preparation of medicines (especially vaccines) for preventing and/or treating SARS-CoV-2 infection of the novel coronavirus.
  • Coronavirus is an unsegmented single-stranded positive-stranded RNA virus, belonging to the Orthocoronavirinae family of Nidovirales, Coronavirus (Coronaviridae), and according to the serotype and genome characteristics, the Coronavirus subfamily is divided into ⁇ , Four genera of ⁇ , ⁇ and ⁇ . So far, there are 7 types of coronaviruses that can infect humans: including 229E and NL63 of the ⁇ genera, OC43 and HKU1 of the ⁇ genera, Middle East Respiratory Syndrome-related Coronavirus (MERSr-CoV), and Severe Acute Respiratory Syndrome-related Coronavirus (SARSr). -CoV) and the new coronavirus (SARS-CoV-2). Only the latter three can cause serious human diseases and even death.
  • MERSr-CoV Middle East Respiratory Syndrome-related Coronavirus
  • SARSr Severe Acute Respiratory Syndrome-related Coronavirus
  • SARS-CoV Severe Acute Respiratory Syndrome
  • Coronavirus has an envelope, the particles are round or oval, often pleomorphic, and the diameter is usually 50 to 200 nm.
  • the S protein is located on the surface of the virus to form a rod-like structure. As one of the main antigen proteins of the virus, it is the main gene used for typing.
  • the N protein wraps the viral genome and can be used as a diagnostic antigen. The understanding of the physical and chemical properties of coronavirus mostly comes from the study of SARS-CoV and MERS-CoV.
  • Vaccines designed based on the full-length S protein of SARS-CoV have been reported to induce a large number of non-neutralizing antibodies, fail to challenge the virus in animal models and cause serious side effects, such as increased morbidity, strong inflammation in liver tissues and liver Damage (see literature: "Evaluation of modified vaccine virus ankara based recombinant SARS vaccine in ferrets", Vaccine 23, 2273-2279.). Therefore, avoiding exposure to non-neutralizing epitopes with immunological advantages in vaccine design is the basis for ensuring vaccine safety.
  • the RBD of SARS-CoV and MERS-CoV is composed of two parts: a highly similar core structure and a very different receptor binding motif (RBM).
  • RBM receptor binding motif
  • SARS-CoV and MERS-CoV recognize different receptors: SARS-CoV recognizes angiotensin converting enzyme 2 (ACE2), and MERS-CoV recognizes dipeptidyl peptidase 4 (DPP4).
  • ACE2 angiotensin converting enzyme 2
  • DPP4 dipeptidyl peptidase 4
  • the vaccine platforms involved in the development of SARS-CoV and MERS-CoV vaccines include: viral vector vaccines, DNA vaccines, subunit vaccines, viroid particle (VLP) vaccines, whole virus inactivated vaccines and attenuated vaccines.
  • viral vector vaccines viral vector vaccines
  • DNA vaccines DNA vaccines
  • subunit vaccines subunit vaccines
  • viroid particle (VLP) vaccines whole virus inactivated vaccines and attenuated vaccines.
  • SARS-CoV and MERS-CoV whole virus inactivated vaccines under development have been reported to have allergic pathological phenomena in the lungs after challenge in mouse models (see literature: "Immunization with inactivated middle east respiratory syndrome leads to lung immunopathology on challenge with live virus", Hum.Vaccin.Immunother.12, 2351-2356.), so the form of whole virus inactivated vaccine is not developed for use in COVID-19 vaccine The best choice.
  • the vaccine prepared by the live attenuated vaccine platform is not suitable for the elderly and individuals with low immunity, so it is not suitable for use in the COVID-19 vaccine. Research and development.
  • mRNA vaccines have achieved certain research results in influenza virus, Ebola virus and Zika virus and other infectious diseases.
  • mRNA vaccines deliver mRNA to cells, express and produce proteins. So that the body obtains immune protection.
  • inactivated vaccines and attenuated vaccines mRNA vaccine preparation steps are simple, which is of great significance for the control of infectious diseases.
  • mRNA vaccines are more resistant to high temperatures and more stable than traditional recombinant vaccines.
  • mRNA vaccines can cause a strong CD4+ or CD8+ T cell response.
  • mRNA vaccines in animals can produce antibodies through one or two low-dose inoculations.
  • the present invention provides a safe and reliable mRNA vaccine, avoiding the defects of other vaccine platforms.
  • an mRNA or mRNA composition comprising an mRNA sequence encoding the S protein (spike protein) of the novel coronavirus SARS-CoV-2 or a variant thereof and the encoding The mRNA sequence of the RBD (Receptor Binding Domain) or its variants in the S protein.
  • S protein spike protein
  • RBD Receptor Binding Domain
  • the mRNA sequence encoding the S protein of the new coronavirus SARS-CoV-2 or its variants and the mRNA sequence encoding the RBD or its variants in the S protein are derived from the same SARS-CoV-2 mutant strain or Different SARS-CoV-2 mutant strains.
  • the S protein or a variant thereof comprises a wild-type full-length S protein or a full-length S protein fixed in a pre-fusion conformation.
  • the full-length S protein fixed in the pre-fusion conformation contains the 682RRAR 685 mutation and/or the 986KV987 mutation, so that the S protein is fixed in the pre-fusion conformation.
  • the full-length S protein fixed in the pre-fusion conformation is the mutation of 682RRAR685 of the wild-type full-length S protein to GSAG and/or the mutation of 986KV987 to PP.
  • part of the content disclosed in the existing patents also supports the technical solutions of the present invention, such as replacing one or two amino acids with proline near the first segment of the heptapeptide repeat region or in the first segment of the heptapeptide repeat region. It can stabilize the conformation very efficiently (see US Patent, Application No. 20200061185, PREFUSION CORONAVIRUS SPIKE PROTEINS and THEIR USE).
  • the amino acid sequence of the wild-type full-length S protein is such as SEQ ID NO:1 or 70%, 75%, 80%, 85%, 90% of SEQ ID NO:1. , 95%, 99% identical amino acid sequence.
  • the amino acid sequence of the full-length S protein fixed in the pre-fusion conformation is as SEQ ID NO: 2, SEQ ID NO: 15 or 70% with SEQ ID NO: 2 or 15 , 75%, 80%, 85%, 90%, 95%, 99% identical amino acid sequences.
  • the S protein or its variant does not contain a signal peptide, a signal peptide containing a wild-type S protein or a signal peptide containing a wild-type S protein and a strong signal peptide is added before it.
  • the strong signal peptide is preferably It is the signal peptide of tissue-type plasminogen activator (tPA) or the signal peptide of serum immunoglobulin E (lgE).
  • nucleotide sequence encoding the wild-type 2019-nCoV S protein without a signal peptide is shown in SEQ ID NO: 6.
  • the amino acid sequence of the RBD is such as SEQ ID NO: 3, SEQ ID NO: 13, or 70%, 75%, 80%, 85% with SEQ ID NO: 3 or 13 %, 90%, 95%, 99% identical amino acid sequences.
  • the RBD or its variant does not contain a signal peptide, a signal peptide containing a wild-type S protein or a signal peptide containing a wild-type S protein and a strong signal peptide is added before it, and the strong signal peptide is preferably The signal peptide of tissue-type plasminogen activator (tPA) or the signal peptide of serum immunoglobulin E (lgE).
  • tissue-type plasminogen activator tPA
  • LgE serum immunoglobulin E
  • nucleotide sequence of the RBD encoding wild-type SARS-CoV-2 containing the IgE signal peptide is shown in any one of SEQ ID NO: 7, 9-11.
  • the mRNA is monocistronic, bicistronic or polycistronic mRNA.
  • the bicistronic or polycistronic mRNA is an mRNA containing two or more coding regions.
  • the mRNA sequence encoding the S protein or its variant of the new coronavirus SARS-CoV-2 and the mRNA sequence encoding the RBD or its variant in the S protein are two separate mRNA sequences or connected into one mRNA sequence.
  • connection sequence of a mRNA sequence from 5'to 3' is: the mRNA sequence encoding the S protein of the new coronavirus SARS-CoV-2 or its variant first, and then encoding the RBD or its variants in the S protein.
  • the mRNA sequence of the variant is either the mRNA sequence encoding the RBD or its variants in the S protein first, and then the mRNA sequence encoding the S protein or its variants of the new coronavirus SARS-CoV-2.
  • the mRNA sequence encoding the S protein of the new coronavirus SARS-CoV-2 or its variants and the mRNA sequence encoding the RBD or its variants in the S protein pass through the internal ribosome entry site (IRES) connect.
  • IRS internal ribosome entry site
  • the IRES can be used to separate two coding regions.
  • the IRES sequence includes but is not limited to picornavirus (e.g. FMDV), pestivirus (e.g. CFFV), polio virus (e.g. PV), encephalomyocarditis virus (e.g. ECMV) , Foot-and-mouth disease virus (e.g. FMDV), hepatitis C virus (e.g. HCV), classical swine fever virus (e.g. CSFV), mouse corneal leukoplakia virus (e.g. MLV), simian immunodeficiency virus (e.g. SIV) or cricket paralysis virus (e.g. CrPV) .
  • picornavirus e.g. FMDV
  • pestivirus e.g. CFFV
  • polio virus e.g. PV
  • encephalomyocarditis virus e.g. ECMV
  • ECMV Foot-and-mouth disease virus
  • HCV hepatitis C virus
  • classical swine fever virus
  • the mRNA or mRNA composition further comprises a 5'cap structure, a 5'non-coding region and a polyadenylic acid tail.
  • the mRNA or mRNA composition further comprises one or more of 5'conserved sequence elements, RNA replicase coding regions, subgenomic promoters, 3'conserved sequence elements or 3'non-coding regions The combination.
  • the mRNA is traditional mRNA, autonomously amplified mRNA or trans-amplified mRNA.
  • the mRNA is traditional mRNA, that is, the mRNA includes the mRNA sequence encoding the S protein of the new coronavirus SARS-CoV-2 or its variants and the mRNA sequence encoding the S protein.
  • the mRNA sequence of the RBD or its variants it also contains an mRNA sequence of a 5'cap structure, a 5'non-coding region, a 3'non-coding region and/or a polyadenylic acid tail.
  • the mRNA is an autonomously amplified mRNA, that is, the mRNA includes the mRNA sequence encoding the S protein of the new coronavirus SARS-CoV-2 or its variants and the mRNA sequence encoding the S protein.
  • the mRNA sequence of the RBD or its variants in the protein it also includes 5'cap structure, 5'conserved sequence elements, RNA replicase coding region, subgenomic promoter, 3'conserved sequence elements and polyadenylic acid tail composition .
  • the RNA replicase coding region that can be used includes but is not limited to alphavirus (e.g. SFV), picornavirus (e.g. FMDV), flavivirus (e.g. DENV), paramyxovirus (e.g. HMPV) or calicivirus (e.g. NV ).
  • the mRNA is trans-amplified mRNA, that is, the mRNA encoding the target gene except for those containing the S protein encoding the new coronavirus SARS-CoV-2 or its variants
  • the mRNA sequence and the mRNA sequence encoding the RBD or its variants in the S protein it also includes a 5'cap structure, a 5'conservative sequence element, a subgenomic promoter, a 3'conservative sequence element and a polyadenylic acid tail; RNA replicase is encoded by a single traditional mRNA.
  • the mRNA or mRNA composition includes an mRNA sequence encoding the S protein of the new coronavirus SARS-CoV-2 or its variants and an mRNA sequence encoding the RBD or its variants in the S protein.
  • An mRNA sequence formed by the connection of mRNA sequences which is selected from any of the following groups:
  • the 5'cap structure, the 5'non-coding region, the mRNA sequence encoding the S protein of the new coronavirus SARS-CoV-2 or its variants, the mRNA sequence encoding the RBD or its variants in the S protein, 3' Non-coding region and polyadenylic acid tail constitute;
  • IRS internal ribosome entry site
  • D consisting of 5'cap structure, 5'non-coding region, mRNA sequence encoding RBD or its variants in S protein, IRES, mRNA sequence encoding S protein of new coronavirus SARS-CoV-2 or its variants, 3'non-coding region and polyadenylic acid tail;
  • G Consists of 5'cap structure, 5'conservative sequence elements, RNA replicase coding region, subgenomic promoter, mRNA sequence encoding RBD or its variants in S protein, IRES, encoding new coronavirus SARS-CoV-2
  • H Composed of 5'cap structure, 5'conservative sequence elements, RNA replicase coding region, subgenomic promoter, mRNA sequence encoding the S protein of the new coronavirus SARS-CoV-2 or its variants, IRES, encoding the S protein The mRNA sequence of the RBD or its variants, 3'conserved sequence elements and polyadenylic acid tail.
  • the mRNA or mRNA composition includes a combination of two mRNA sequences, which is selected from any of the following groups:
  • mRNA consisting of 5'cap structure, 5'non-coding region, mRNA sequence encoding the S protein of the new coronavirus SARS-CoV-2 or its variants, 3'non-coding region and polyadenylic acid tail Combines mRNA consisting of 5'cap structure, 5'non-coding region, mRNA sequence encoding RBD or its variants in S protein, 3'non-coding region and polyadenylic acid tail;
  • mRNA composed of the tail is composed of 5'cap structure, 5'conserved sequence elements, RNA replicase coding region, subgenomic promoter, mRNA sequence encoding the S protein of the new coronavirus SARS-CoV-2 or its variants, 3 'MRNA composed of conserved sequence elements and polyadenylic acid tail;
  • the mRNA sequence of the body, the mRNA composed of 3'conserved sequence elements and polyadenylic acid tail, the combination consists of 5'cap structure, 5'non-coding region, RNA replicase coding region, 3'non-coding region and polyadenosine MRNA composed of acid tail;
  • d) It consists of 5'cap structure, 5'conservative sequence elements, subgenomic promoter, mRNA sequence encoding RBD or its variants in S protein, IRES, S protein encoding new coronavirus SARS-CoV-2 or its variants
  • the mRNA sequence of the body, the mRNA composed of 3'conserved sequence elements and polyadenylic acid tail, the combination consists of 5'cap structure, 5'non-coding region, RNA replicase coding region, 3'non-coding region and polyadenosine MRNA composed of acid tail;
  • 5'cap structure 5'conserved sequence elements, subgenomic promoter, mRNA sequence encoding RBD or variants of S protein, S protein or variants of new coronavirus SARS-CoV-2 mRNA sequence, 3'conservative sequence elements and polyadenylic acid tail composed of mRNA, a combination of 5'cap structure, 5'non-coding region, RNA replicase coding region, 3'non-coding region and polyadenylic acid tail Composition of mRNA.
  • the mRNA sequence contained in the mRNA or mRNA composition includes any one or a combination of two or more of SEQ ID NOs: 16-19.
  • the mRNA or mRNA composition further includes a cationic or polycationic compound.
  • the cationic or polycationic compound is free or binds to mRNA.
  • a form in which a cationic or polycationic compound binds to the mRNA is selected.
  • the mRNA or mRNA composition also contains lipids.
  • the lipids include, but are not limited to, liposomes that can promote self-assembly to form virus-sized particles ( ⁇ 100nm), liposomes that release mRNA from endosomes into cells, and support phospholipid bimolecules Layered liposomes or liposomes used as stabilizers.
  • the lipid may also include PEGylated lipid.
  • the lipids comprise cationic lipids, PEGylated lipids, cholesterol and/or phospholipids.
  • the mRNA or mRNA composition of the present invention can be liposomes, lipid complexes or lipid nanoparticles.
  • the liposomes can be liposomes prepared in the following forms: 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, 1,2-diamine liposomes Oleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxane Pentane (DLin-KC2-DMA) liposomes.
  • DODMA 1,2-dioleyloxy-N,N-dimethylaminopropane
  • DLin-DMA 1,2-diamine liposomes Oleyloxy-3-dimethylaminopropane
  • DLin-KC2-DMA 2,2-Dilinoleyl-4-(2-dimethylamin
  • the lipid complex or lipid nanoparticle may be formed by a lipid selected from the group consisting of DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA , DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids.
  • the mRNA or mRNA composition of the present invention may also include pharmaceutically acceptable excipients.
  • the pharmaceutically acceptable excipient may be a carrier, a diluent, an adjuvant or a nucleotide sequence encoding an adjuvant, a solubilizer, a binder, a lubricant, a suspending agent, a transfection promoter, and the like.
  • the transfection promoters include, but are not limited to, surfactants such as immunostimulatory complexes, Freunds incomplete adjuvant, LPS analogs (e.g.
  • monophosphoryl ester A cell wall peptides, benzoquinone analogs, Squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, cations, polycations (e.g. poly-L-glutamic acid (LGS)) or nanoparticles or other known transfection promoters .
  • LGS poly-L-glutamic acid
  • the nucleotide sequence encoding the adjuvant is a nucleotide sequence encoding at least one of the following adjuvants: GM-CSF, IL-17, IFNg, IL-15, IL-21, anti-PD1/2, lactoferrin Protein, protamine, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF- ⁇ , INF- ⁇ , Lymphotoxin- ⁇ , hGH, MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM -1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, CD40, CD40L , Vascular growth factor, fibro
  • an mRNA vaccine comprising any one of the mRNA or mRNA composition of the present invention.
  • the S protein or variants of the new coronavirus SARS-CoV-2 encoded in the mRNA vaccine and the sequences encoding the RBD or its variants in the S protein are from different SARS-CoV-2 mutant strains. , So that immunization produces cross-protection against different SARS-CoV-2 mutant strains.
  • the RBD encoding wild-type SARS-CoV-2 containing the IgE signal peptide in the mRNA vaccine contains the K417N, E484K and N501Y mutations of the 501Y.V2 lineage, preferably its amino acid sequence is as SEQ ID NO: 13; the sequence encoding the full-length S protein fixed in the pre-fusion conformation with 682RRAR685 mutated to GSAG and 986KV987 mutated to PP is from Wuhan-Hu-1 isolate, which contains L18F, D80A, and L18F of the 501Y.V2 lineage. D215G, L242-L244 deletion mutation (L242-244del), R246I, K417N, E484K, N501Y and A701V preferably have an amino acid sequence as shown in SEQ ID NO: 15.
  • the mRNA vaccine comprises an mRNA sequence encoding the S protein of the new coronavirus SARS-CoV-2 or its variants and an mRNA sequence encoding the RBD or its variants in the S protein, Among them, the amino acid sequence of the S protein of the new coronavirus SARS-CoV-2 or its variants is shown in SEQ ID NO: 15, and the RBD or its variants in the S protein is shown in SEQ ID NO: 13.
  • the mRNA vaccine comprises an mRNA sequence encoding the S protein of the new coronavirus SARS-CoV-2 or its variants and an mRNA sequence encoding the RBD or its variants in the S protein , wherein the amino acid sequence of the S protein of the new coronavirus SARS-CoV-2 or its variants is shown in SEQ ID NO: 2, and the RBD or its variants in the S protein is shown in SEQ ID NO: 13.
  • the mRNA vaccine comprises an mRNA sequence encoding the S protein of the new coronavirus SARS-CoV-2 or its variants and an mRNA sequence encoding the RBD or its variants in the S protein ,
  • the amino acid sequence of the S protein of the novel coronavirus SARS-CoV-2 or its variants is shown in SEQ ID NO: 2
  • the RBD or its variants in the S protein is shown in SEQ ID NO: 3.
  • the mass ratio of the mRNA encoding the S protein of the new coronavirus SARS-CoV-2 or its variants to the mRNA encoding the RBD or its variants in the S protein is (1-5): (1-5).
  • the mRNA vaccine encoding the wild-type SARS-CoV-2 RBD mRNA containing the IgE signal peptide and the encoding 682RRAR685 mutated to GSAG and 986KV987 mutated to PP are fixed in the pre-fusion conformation
  • the mass ratio of the mRNA of the full-length S protein is (1-2): (1-2).
  • the mRNA vaccine also includes a cationic or polycationic compound.
  • the cationic or polycationic compound is free or binds to mRNA.
  • a form in which a cationic or polycationic compound binds to the mRNA is selected.
  • the mRNA vaccine also contains lipids.
  • the lipids include, but are not limited to, liposomes that can promote self-assembly to form virus-sized particles ( ⁇ 100nm), liposomes that release mRNA from endosomes into cells, and support phospholipid bimolecules Layered liposomes or liposomes used as stabilizers.
  • the lipids may also include PEGylated lipids.
  • the lipids comprise cationic lipids, PEGylated lipids, cholesterol and/or phospholipids.
  • the mRNA vaccines of the present invention can be liposomes, lipid complexes or lipid nanoparticles.
  • the liposomes can be liposomes prepared in the following forms: 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, 1,2-diamine liposomes Oleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxane Pentane (DLin-KC2-DMA) liposomes.
  • DODMA 1,2-dioleyloxy-N,N-dimethylaminopropane
  • DLin-DMA 1,2-diamine liposomes Oleyloxy-3-dimethylaminopropane
  • DLin-KC2-DMA 2,2-Dilinoleyl-4-(2-dimethylaminoeth
  • the lipid complex or lipid nanoparticle may be formed by a lipid selected from the group consisting of DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA , DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids.
  • the mRNA vaccine of the present invention may also include pharmaceutically acceptable excipients.
  • the pharmaceutically acceptable excipient may be a carrier, a diluent, an adjuvant or a nucleotide sequence encoding an adjuvant, a solubilizer, a binder, a lubricant, a suspending agent, a transfection promoter, and the like.
  • the transfection promoters include, but are not limited to, surfactants such as immunostimulatory complexes, Freunds incomplete adjuvant, LPS analogs (e.g.
  • monophosphoryl ester A cell wall peptides, benzoquinone analogs, Squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, cations, polycations (e.g. poly-L-glutamic acid (LGS)) or nanoparticles or other known transfection promoters .
  • LGS poly-L-glutamic acid
  • the nucleotide sequence encoding the adjuvant is a nucleotide sequence encoding at least one of the following adjuvants: GM-CSF, IL-17, IFNg, IL-15, IL-21, anti-PD1/2, lactoferrin Protein, protamine, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF- ⁇ , INF- ⁇ , Lymphotoxin- ⁇ , hGH, MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM -1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF,, CD40, CD40L, vascular growth factor, fibro
  • a method for preparing mRNA or an mRNA composition which comprises mixing the mRNA with a cationic or polycationic compound and then packaging it with lipids.
  • the lipids include, but are not limited to, liposomes that can promote self-assembly to form virus-sized particles ( ⁇ 100nm), liposomes that release mRNA from endosomes into cells, and support phospholipid bilayers Structured liposomes or liposomes used as stabilizers. More preferably, in order to increase the half-life of LNP, the lipids may also include PEGylated lipids.
  • the lipids comprise cationic lipids, PEGylated lipids, cholesterol and/or phospholipids.
  • a method for preparing an mRNA vaccine includes mixing any one of the mRNA or mRNA composition of the present invention with a cationic or polycationic compound and then packaging it with lipids. Preferably, it is packaged into lipid nanoparticles.
  • the lipids include, but are not limited to, liposomes that can promote self-assembly to form virus-sized particles ( ⁇ 100nm), liposomes that release mRNA from endosomes into cells, and support phospholipid bilayers Structured liposomes or liposomes used as stabilizers. More preferably, in order to increase the half-life of LNP, the lipids may also include PEGylated lipids.
  • the lipids comprise cationic lipids, PEGylated lipids, cholesterol and/or phospholipids.
  • the fifth aspect of the present invention provides an application of the mRNA or mRNA composition or mRNA vaccine of any one of the present invention in preventing and/or treating diseases caused by SARS-CoV-2 infection of the new coronavirus.
  • the diseases caused by the novel coronavirus SARS-CoV-2 infection include but are not limited to COVID-19.
  • the seventh aspect of the present invention provides an application of any one of the mRNA or mRNA composition or mRNA vaccine of the present invention in the preparation of medicines for preventing and/or treating diseases caused by SARS-CoV-2 infection of the new coronavirus .
  • the diseases caused by the novel coronavirus SARS-CoV-2 infection include but are not limited to COVID-19.
  • the eighth aspect of the present invention provides an application of any one of the mRNA or mRNA composition or mRNA vaccine of the present invention in the preparation of medicines against SARS-CoV-2 infection by the novel coronavirus.
  • the ninth aspect of the present invention provides a method for treating and/or preventing a disease caused by a novel coronavirus SARS-CoV-2 infection, which comprises administering to an individual an effective amount of the mRNA or mRNA composition of any one of the present invention , Or mRNA vaccine.
  • the tenth aspect of the present invention provides a method for preventing SARS-CoV-2 infection by the new coronavirus, comprising administering an effective amount of the mRNA vaccine of the present invention to individuals who are not infected with the new coronavirus SARS-CoV-2.
  • the eleventh aspect of the present invention provides a method for treating SARS-CoV-2 infection of the new coronavirus, comprising administering an effective amount of the mRNA of the present invention or containing the mRNA composition or mRNA vaccine to enable individuals to produce neutralizing antibodies against the new coronavirus SARS-CoV-2.
  • the twelfth aspect of the present invention provides a method for antibody screening, the method comprising the step of administering to an individual an effective amount of the mRNA or mRNA composition of any one of the present invention, or an mRNA vaccine.
  • the method of antibody screening is not a method of treatment. This method is used to screen neutralizing antibodies, test and compare the efficacy of antibodies to determine which antibodies can be used as drugs and which cannot be used as drugs, or to compare the sensitivity of different drugs, that is, the therapeutic effect is not inevitable. It's just a possibility.
  • the thirteenth aspect of the present invention provides a method for inducing an individual to neutralize an antigen-specific immune response, the method comprising administering any one of the mRNA or mRNA composition of the present invention, or an mRNA vaccine to the individual.
  • the antigen-specific immune response includes T cell response and/or B cell response.
  • the fourteenth aspect of the present invention provides a protein encoded by the mRNA or mRNA composition of the present invention.
  • the protein is a full-length S protein fixed in a pre-fusion conformation. It is further preferred that the full-length S protein fixed in the pre-fusion conformation contains the 682RRAR 685 mutation and/or the 986KV987 mutation, so that the S protein is fixed in the pre-fusion conformation. Most preferably, the full-length S protein fixed in the pre-fusion conformation is the mutation of 682RRAR685 of the wild-type full-length S protein to GSAG and/or the mutation of 986KV987 to PP.
  • the fifteenth aspect of the present invention provides a nucleotide sequence encoding the protein of the present invention.
  • the sixteenth aspect of the present invention provides a vector containing the nucleotide sequence of the present invention.
  • the seventeenth aspect of the present invention provides a cell comprising the protein of the present invention, the nucleotide sequence and/or the vector.
  • the mRNA or mRNA composition and the mRNA vaccine containing the mRNA or mRNA composition of the present invention have the following advantages: 1. In vitro synthesis, without cell culture, and no risk of contamination from animal sources; 2. Faster development and production, and standardization Production, easy to mass production and quality control, the same production process is suitable for multiple different products; 3. It can be expressed continuously for a period of time, and the antigen exposure time is prolonged to improve the intensity and quality of the immune response; 4. The process of simulating natural infections, Translated and modified in human cells, it can be presented by MHC class I molecules to induce stronger cellular immunity; 5.
  • the expression product of the mRNA or mRNA composition of the present invention includes RBD or its variants and S protein or its variants, wherein the RBD contains the main neutralizing epitope, which can induce high levels of neutralizing antibody titers ; Moreover, there are relatively few non-neutralizing epitopes and high safety.
  • the full-length S protein can induce a high level of specific cellular immunity, and with the RBD that induces neutralizing antibodies, it can produce an extremely excellent immune effect. Further, the examples also confirmed that the expression product of the mRNA or mRNA composition of the present invention can induce the production of high levels of neutralizing antibodies and cytokines in the human body.
  • part of the content disclosed in the prior art also supports the technical solution of the present invention.
  • S protein of SARS-CoV-2 The structural conformation of the interaction between the S protein of SARS-CoV and ACE2 is maintained (see Xintian, X, etc. (2020), Evolution of the novel coronavirus from the ongoing and modeling of its spike protein for risk of human transmission, Science China Life Sci.).
  • protein fragments 377-588 are the key neutralizing domains, which can cause the highest neutralizing antibody titers in mouse and rabbit models; and this key neutralizing domain can still be used
  • Binding to the receptor hDPP4 proves that it maintains a structural conformation, not only can provide linear epitopes, but also provide structural epitopes (see Cuiqing, M et al. (2014), Searching for an ideal vaccine candidate, and different MERS coronavirus receptor-binding fragments) --the importance of immunofocusing in subunit vacuum design, Vaccine.32(46):6170-6176.).
  • the "individual” in the present invention includes mammals and humans.
  • the mammals include, but are not limited to, rodents (such as mice, rats), monkeys, zebrafish, pigs, chickens, rabbits, and so on.
  • prevention in the present invention refers to the use of the product of the present invention before or after the disease starts to develop to avoid symptoms or delay all behaviors of specific symptom stress; preferably, the prevention includes the use of the products described in the present invention mRNA or a composition containing mRNA is used as a vaccine.
  • the "treatment" of the present invention refers to therapeutic intervention to improve the signs, symptoms, etc. of the disease or pathological state after the disease has begun to develop; preferably, the treatment includes screening for the mRNA or the mRNA-containing compound of the present invention.
  • the composition binds the antibody and uses it for therapy.
  • the "effective amount” in the present invention refers to the amount or dose of the product of the present invention that provides the desired treatment or prevention after being administered to a patient or organ in a single or multiple doses.
  • the "S protein” in the present invention is a structural protein that composes the new coronavirus SARS-CoV-2, and the name is spike protein.
  • the "RBD” in the present invention is a structural protein constituting the new coronavirus SARS-CoV-2, and its name is the spike protein receptor binding domain.
  • the "identity" in the present invention refers to the use of amino acid sequence or nucleotide sequence. Those skilled in the art can adjust the sequence according to actual work needs, so that the used sequence is compared with the sequence obtained in the prior art. With (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15 %, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48% , 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%,
  • Figure 1 Sequence sequencing results of RBD encoding wild-type SARS-CoV-2 containing tPA signal peptide.
  • Figure 2 The splicing of Figures 2A and 2B is the sequence sequencing result of the S protein encoding wild-type SARS-CoV-2.
  • Figure 3 Sequence diagram of the basic plasmid template containing T7 promoter, 5'UTR, 3'UTR, and polyA tail.
  • Figure 4 Detection of capped and purified mRNA with formaldehyde denaturing gel, where M is Marker, 1 is the mRNA encoding the RBD of wild-type SARS-CoV-2 containing the tPA signal peptide, and 2 is the mutation encoding 682RRAR685 to GSAG and 986KV987 The mRNA of the full-length S protein mutated to PP fixed in the pre-fusion conformation.
  • Figure 5 WB (Western Blot, Western Blot) detection results, where 1 is the expression supernatant of the RBD mRNA encoding wild-type SARS-CoV-2 containing the tPA signal peptide, and 2 is the negative control.
  • Figure 7 DLS (Dynamic Light Scattering) detection of mRNA-LNP particle size and particle size distribution results, where A represents RBD+S-1-LNP, B represents RBD+S-2-LNP, and C represents RBD+S-3-LNP, D stands for RBD-LNP, E stands for S-LNP.
  • A represents RBD+S-1-LNP
  • B represents RBD+S-2-LNP
  • C represents RBD+S-3-LNP
  • D stands for RBD-LNP
  • E stands for S-LNP.
  • Figure 8 Formaldehyde denaturing glue detects the mRNA integrity results of the packaged sample, where 1 is the mRNA encoding the RBD of wild-type SARS-CoV-2 containing the tPA signal peptide, 2 is RBD+S-1, and 3 is RBD+S -2, 4 are RBD+S-3, and 5 are mRNAs encoding the full-length S protein in the pre-fusion conformation with 682RRAR685 mutated to GSAG and 986KV987 mutated to PP.
  • Figure 9 S protein specific antibody titers after the first and second immunizations, where Negative is the negative control.
  • Figure 10 Results of detection of interferon gamma (IFN- gamma) by ELISpot (enzyme-linked immunospot method), where the ordinate is spot forming unit (SFU) per million spleen cells, and Negative is the negative control .
  • IFN- gamma interferon gamma
  • ELISpot enzyme-linked immunospot method
  • FIG 11 CD4CK intracellular staining (ICS) detection results of IFN- ⁇ , interleukin-2 (IL-2) and tumor necrosis factor- ⁇ (TNF- ⁇ ).
  • FIG 12 CD8CK intracellular staining (ICS) detection results of IFN- ⁇ , interleukin-2 (IL-2) and tumor necrosis factor- ⁇ (TNF- ⁇ ).
  • Figure 13 Detection of capped and purified mRNA with formaldehyde denaturing gel, where M is Marker and 1 is the full-length S protein encoding 682RRAR685 mutated to GSAG and 986KV987 mutated to PP prepared in Example 1 and fixed in the pre-fusion conformation mRNA, 2 is the mRNA encoding the full-length S protein of the 501Y.V2 lineage with 682RRAR685 mutated to GSAG and 986KV987 mutated to PP fixed in the pre-fusion conformation, and 3 is the wild-type SARS- encoding the 501Y.V2 lineage containing the IgE signal peptide
  • M is Marker and 1 is the full-length S protein encoding 682RRAR685 mutated to GSAG and 986KV987 mutated to PP prepared in Example 1 and fixed in the pre-fusion conformation mRNA
  • 2 is the mRNA encoding the full-length
  • Figure 14 WB detection results, where 1 is the expression supernatant of mRNA encoding the RBD sequence of wild-type SARS-CoV-2 of the 501Y.V2 lineage containing the IgE signal peptide, 2 is the mutation encoding 682RRAR685 to GSAG and 986KV987 to PP The mRNA expression supernatant of the full-length S protein of the 501Y.V2 lineage fixed in the pre-fusion conformation, 3 is the cell supernatant of the negative control, and 4 is the wild-type SARS-CoV encoding the 501Y.V2 lineage containing the IgE signal peptide -2 RBD sequence mRNA expression cell precipitation, 5 is encoding 682RRAR685 mutated to GSAG and 986KV987 mutated to PP fixed in the pre-fusion conformation 501Y.V2 lineage full-length S protein mRNA expression cell precipitation, 6 is negative Control cell pellet.
  • Figure 15 DLS (Dynamic Light Scattering, dynamic light scattering) detection of mRNA-LNP particle size and particle size distribution results.
  • Figure 16 S protein-specific antibody titers against 501Y.V2 lineage after the first and second immunizations.
  • Figure 17 S protein-specific antibody titers against Wuhan-Hu-1 isolate after the first and second immunizations.
  • Figure 18 Results of alternative neutralizing antibody titers, the left 4 groups are alternative neutralizing antibody titers against Wuhan-Hu-1 isolate, and the right 4 groups are alternative neutralizing antibody titers against 501Y.V2 lineage.
  • Figure 19 CD4+ T cell Th1 type cellular immune response test results.
  • Figure 20 CD8+ T cell Th1 type cellular immune response test results.
  • Alternative neutralizing antibody detection is to detect neutralizing antibodies in the serum of vaccine-immunized mice, using the competitive binding of ACE2 protein and RBD protein.
  • Specific antibody detection is to detect specific antibodies in the serum of vaccine-immunized mice, using RBD protein.
  • the RBD protein used to detect Wuhan-Hu-1 isolate is a wild-type RBD protein (manufacturer: GenScript, catalog number: Z03483-1).
  • the RBD protein used to detect the 501Y.V2 lineage is the RBD protein containing mutations in the 501Y.V2 lineage (manufacturer: Nearshore, Item No.: DRA125).
  • the primers serve as templates for PCR amplification.
  • step 4 Connect the amplified product of step 3 to the pUC57 vector, and transform and sequence.
  • Figure 1 shows the sequencing results of the RBD encoding wild-type SARS-CoV-2 containing the tPA signal peptide.
  • the nucleotide sequence is shown in SEQ ID NO: 4, and the amino acid sequence is shown in SEQ ID NO: 3.
  • Figure 2 shows the sequencing results of the full-length S protein encoding 682RRAR685 mutated to GSAG and 986KV987 mutated to PP, fixed in the pre-fusion conformation, the nucleotide sequence is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 2 shown.
  • the basic plasmid template is linearized with the restriction endonuclease BsmBI.
  • the PCR products were respectively connected to the basic plasmid template through homologous recombination, respectively transformed into the Xl1-Blue strain, and sequenced to confirm that the sequence was correct and the transcription template was constructed successfully.
  • the strain was fermented in a shake flask, and the transcription template was obtained by purification with an endotoxin-free plasmid large-scale extraction kit.
  • the transcription template was linearized with restriction endonuclease BbsI. T7 in vitro transcription kit was used for transcription, and uncapped mRNA of SEQ ID NO: 4-5 was obtained (the specific mRNA sequence is SEQ ID NO: 16-17, respectively).
  • the transcription template was digested with DNaseI, and mRNA was purified by precipitation method. Use Cap1 capping kit to cap the mRNA, and use the mRNA purification kit to purify the capped mRNA. Dissolve the purified mRNA in acidic sodium citrate buffer and set aside.
  • the capped and purified mRNA was detected with formaldehyde denaturing gel.
  • 1 is the mRNA encoding the wild-type SARS-CoV-2 RBD containing the tPA signal peptide
  • 2 is the mRNA encoding the 682RRAR685 mutation to GSAG and the 986KV987 mutation to
  • the PP is fixed to the mRNA of the full-length S protein in the pre-fusion conformation. The results showed that the size of mRNA was correct and there was basically no degradation.
  • Pave 3 wells of HEK293 cells in a 24-well plate of which wells 1 and 2 were respectively transfected with lipofectamine 2000 with 0.5 ⁇ g capped and purified mRNA encoding wild-type SARS-CoV-2 RBD containing tPA signal peptide, and The mRNA encoding the full-length S protein with 682RRAR685 mutation to GSAG and 986KV987 mutation to PP was fixed in the pre-fusion conformation, and lipofectamine 2000 transfection reagent was added to well 3 as a negative control.
  • lipid mixture mRNA flow rate ratio of 1:3, the RBD+S-1, RBD+S-2, RBD+S-3, and wild-type code containing tPA signal peptide were mixed and packaged in Precision Nanosystems’ nanoparticle preparation instrument Ignite.
  • the packaged mRNA-LNP (LNP is lipid nanoparticles) was dialyzed and ultrafiltered and concentrated into DPBS, and after aseptic filtration, samples for subsequent animal experiments were obtained.
  • DLS was used to detect the particle size and particle size distribution of mRNA-LNP, and the test results are shown in Figure 7.
  • the particle size of the packaged samples are all 70nm-100nm, and the PDI is less than 0.2.
  • RBD+S-1-LNP average particle size 77.15nm, PDI value 0.038, intercept (intercept) 0.958, see Table 1 for details
  • RBD+S-2-LNP average particle size 77.04nm, PDI value 0.055, intercept (intercept) 0.959, see Table 2 for details
  • RBD+S-3-LNP average particle size 91.43nm, PDI value 0.049, intercept (intercept) 0.974, see Table 3 for details
  • RBD-LNP grain The average diameter is 77.92nm, the PDI value is 0.036, and the intercept (intercept) is 0.954, as shown in Table 4
  • S-LNP the average particle size is 76.89nm, the PDI value
  • Peak 1 81.06 100 18.64 Peak 2 0 0 0 0 Peak 3 0 0 0 0
  • Peak 1 82.01 100 19.41 Peak 2 0 0 0 0 Peak 3 0 0 0 0
  • mice about 6 weeks old were randomly divided into 6 groups.
  • the leg muscles were inoculated with 10 ug on the 0th day and the 28th day, respectively, the S protein-specific antibody titers were detected on the 28th day and the 42nd day, and the mice were killed to detect the cytokine on the 42nd day.
  • the S protein specific antibody titers after the first immunization and the second immunization are shown in Figure 9. It can be seen that the specific antibody titers of the single S full-length immunization and the second immunization are significantly lower than the single RBD immunization, while the three groups of S and RBD There is no significant difference in the specific antibody titers between the two groups and the single RBD immunization.
  • RBD+S-1 has a synergistic gain effect, and the specific antibody titers are significantly higher than that of the single RBD immunization.
  • the results of detecting interferon- ⁇ (IFN- ⁇ ) with ELISpot are shown in Figure 10.
  • the response of CD4+ T cells is low, individual differences are large, and the guiding significance is small; while the detection results of CD8+ T cells are basically the same as those of ELISpot.
  • the combination of three groups of S and RBD is immunized, of which two groups are with single S Full-length immunization has no significant difference in the secretion levels of IFN- ⁇ , IL-2 and TNF- ⁇ , while RBD+S-3 has a significant synergistic gain effect. It proves that the combination of S full length and RBD can not only combine the cellular immune advantage of S full length and the humoral immune advantage of RBD, but also achieve synergistic gains, which can achieve more excellent prevention in the prevention of 2019-nCoV coronavirus infection Effect.
  • Example 3 Preparation and detection of mRNA with sequences derived from different SARS-CoV-2 mutant strains
  • sequence of the full-length S protein of the 501Y.V2 lineage fixed in the pre-fusion conformation which encodes the 682RRAR685 mutation to GSAG and the 986KV987 mutation to PP, is synthesized by primer mutual template amplification, which contains the L18F, D80A, and L18F lines of the 501Y.V2 lineage.
  • D215G, L242-L244 deletion mutations (L242-244del), R246I, K417N, E484K, N501Y and A701V have the nucleotide sequence shown in SEQ ID NO: 14, and the amino acid sequence shown in SEQ ID NO: 15.
  • the basic plasmid template is linearized with the restriction endonuclease BsmBI.
  • the PCR products were respectively connected to the basic plasmid template through homologous recombination, respectively transformed into the Xl1-Blue strain, and sequenced to confirm that the sequence was correct and the transcription template was constructed successfully.
  • the strain was fermented with shake flask, and the transcription template was obtained by purification with endotoxin-free plasmid large-scale extraction kit.
  • the transcription template was linearized with restriction endonuclease BbsI.
  • T7 in vitro transcription kit was used for transcription, and the uncapped mRNA of SEQ ID NO: 12 and 14 were obtained respectively (the specific mRNA sequence is SEQ ID NO: 18 and 19, respectively).
  • the transcription template was digested with DNaseI, and mRNA was purified by precipitation method.
  • Use Cap1 capping kit to cap the mRNA, and use the mRNA purification kit to purify the capped mRNA. Dissolve the purified mRNA in acidic sodium citrate buffer and set aside.
  • the capped and purified mRNA was detected with a formaldehyde denaturing gel, as shown in Figure 13, where 1 is the mRNA encoding the full-length S protein in the pre-fusion conformation that encodes 682RRAR685 mutated to GSAG and 986KV987 mutated to PP prepared in Example 1.
  • 2 is the mRNA encoding the full-length S protein of the 501Y.V2 lineage with the 682RRAR685 mutation to GSAG and the 986KV987 mutation to PP fixed in the pre-fusion conformation
  • 3 is the wild-type SARS-CoV- encoding the wild-type SARS-CoV- of the 501Y.V2 lineage containing the IgE signal peptide 2 mRNA of the RBD sequence.
  • the WB detection results are shown in Figure 14, where 1 is the expression supernatant of mRNA encoding the RBD sequence of wild-type SARS-CoV-2 of the 501Y.V2 lineage containing the IgE signal peptide, and 2 is the mutation encoding 682RRAR685 to GSAG and 986KV987 mutations.
  • 3 is the cell supernatant of the negative control
  • 4 is the wild-type SARS of the 501Y.V2 lineage that encodes the IgE signal peptide -CoV-2 RBD sequence mRNA expression cell precipitation
  • 5 is encoding 682RRAR685 mutation to GSAG and 986KV987 mutation to PP fixed in the pre-fusion conformation 501Y.V2 lineage full-length S protein mRNA expression cell precipitation
  • 6 The cell pellet for the negative control. The results showed that the size of the expressed protein was correct.
  • Example 4 Preparation and immunization of mRNA combination vaccines with sequences derived from different SARS-CoV-2 mutant strains
  • Example 2 2) Prepare the mRNA encoding the full-length S protein in the pre-fusion conformation that encodes the 682RRAR685 mutation to GSAG and the 986KV987 mutation to PP prepared in Example 1, and the prepared in Example 3 encodes the 682RRAR685 mutation to GSAG and 986KV987 mutation to PP.
  • the combo A, combo B, and the coding 682RRAR685 prepared in Example 3 were mutated to GSAG and 986KV987 to PP fixed in fusion.
  • the packaged mRNA-LNP was dialyzed and concentrated into DPBS by ultrafiltration. After sterile filtration, samples were obtained for subsequent animal experiments. DLS was used to detect the particle size and particle size distribution of mRNA-LNP, and the results are shown in Figure 15. The particle size of the packaged samples were all 70nm-100nm, and the PDI was all less than 0.2.
  • combo A-LNP average particle size 79.87nm, PDI value 0.132, intercept (intercept) 0.962, see Table 6 for details
  • combo B-LNP average particle size 80.61nm, PDI value 0.123, intercept (intercept) ) 0.958, see Table 7 for details
  • S-SA-LNP average particle size 81.13nm, PDI value 0.159, intercept (intercept) 0.939, see Table 8 for details
  • RBD-SA-LNP average particle size 82.74nm, The PDI value is 0.112 and the intercept is 0.960. See Table 9 for details.
  • Peak 1 91.43 100 32.02 Peak 2 0 0 0 Peak 3 0 0 0 0
  • mice about 6 weeks old were randomly divided into groups of 6 and divided into 5 groups.
  • the leg muscles were inoculated with 5ug on the 0th day and the 14th day, respectively, the S protein-specific antibody titers were detected on the 14th day and the 28th day, and the mice were killed to detect the cytokine on the 28th day.
  • the titers of S protein specific antibodies against 501Y.V2 lineage after the first and second immunizations are shown in Figure 16, and there is no significant difference between the groups.
  • the titers of S protein specific antibodies against Wuhan-Hu-1 isolate after the first and second immunizations are shown in Figure 17.
  • the results show that there is no significant difference in the specific antibody titers caused by the combination of S and RBD between the two groups, but The specific antibody titers of single S full length and single RBD after two immunizations were significantly lower than mRNA combinations derived from different SARS-CoV-2 mutant strains (combo B).
  • the results of Th1 type cellular immune response of CD4+ T cells are shown in Figure 19, and the results of Th1 type of cellular immune response detection of CD8+ T cells are shown in Figure 20.
  • the results show that the combination of S full length and RBD has cells compared with single RBD. Immune advantage. Once again verified the superiority of the combined design of S full length and RBD in vaccine application.

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Abstract

提供了一种mRNA或mRNA组合物以及包含该mRNA或mRNA组合物的mRNA疫苗。该mRNA或mRNA组合物包含编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列以及编码S蛋白中的RBD或其变体的mRNA序列。还提供了该mRNA或mRNA组合物以及包含该mRNA或mRNA组合物的mRNA疫苗在制备预防和/或治疗新型冠状病毒SARS-CoV-2感染导致的疾病的药物中的应用。

Description

mRNA或mRNA组合物及其制备方法和应用 技术领域
本发明涉及疫苗研制技术领域，具体涉及包含编码新型冠状病毒SARS-CoV-2的刺突蛋白(S蛋白)或其变体的mRNA序列以及编码S蛋白中的受体结合域(RBD)或其变体的mRNA序列。本发明涉及包含一种或两种mRNA的组合物。以及所述mRNA或组合物在制备预防和/或治疗新型冠状病毒SARS-CoV-2感染的药物(尤其是疫苗)中的应用。
背景技术
冠状病毒为不分节段的单股正链RNA病毒，属于巢病毒(Nidovirales)冠状病毒(Coronaviridae)正冠状病毒亚科(Orthocoronavirinae)，根据血清型和基因组特点冠状病毒亚科被分为α、β、γ和δ四个属。迄今为止，共有7种冠状病毒可感染人类：包括α属的229E和NL63，β属的OC43和HKU1、中东呼吸综合征相关冠状病毒(MERSr-CoV)、严重急性呼吸综合征相关冠状病毒(SARSr-CoV)和新型冠状病毒(SARS-CoV-2)。其中只有后三种会导致严重的人类疾病甚至死亡。
冠状病毒有包膜，颗粒呈圆形或椭圆形，经常为多形性，直径通常为50～200nm。S蛋白位于病毒表面形成棒状结构，作为病毒的主要抗原蛋白之一，是用于分型的主要基因。N蛋白包裹病毒基因组，可用作诊断抗原。对冠状病毒理化特性的认识多来自对SARS-CoV和MERS-CoV的研究。基于SARS-CoV的全长S蛋白设计的疫苗被报道会诱导大量非中和抗体，在动物模型上攻毒失败且引起严重的副反应，比如增加发病率，引起肝部组织强烈炎症反应及肝损伤(参见文献：“Evaluation of modified vaccinia virus ankara based recombinant SARS vaccine in ferrets”，Vaccine 23，2273-2279.)。因此，在疫苗设计中避免暴露能具有免疫优势的非中和表位是保障疫苗安全性的基础。
SARS-CoV和MERS-CoV的RBD都由两部分组成：一个高度相似的核心结构和一段差异极大的受体结合基序(RBM)。RBM的不同使得SARS-CoV和MERS-CoV分别识别不同的受体：SARS-CoV识别血管紧张素转化酶2(ACE2)，而MERS-CoV识别二肽基肽酶4(DPP4)。
目前SARS-CoV和MERS-CoV疫苗研发所涉及到的疫苗平台有：病毒载体疫苗、DNA疫苗、亚单位疫苗、类病毒颗粒(VLP)疫苗、全病毒灭活疫苗和减毒疫苗。
虽然全病毒灭活疫苗理论上可以快速生产应对新型冠状病毒SARS-CoV-2疫情的爆发，但是一方面病毒的培养需要生物安全三级实验室，疫苗企业一般很难满足其生产要求；另一方面安全性可能也存在问题，研发中的SARS-CoV和MERS-CoV的全病毒灭活疫苗均有报道在小鼠模型上攻毒后会在肺部发现过敏型病理现象(参见文献：“Immunization with inactivated middle east respiratory syndrome  coronavirus vaccine leads to lung immunopathology on challenge with live virus”，Hum.Vaccin.Immunother.12，2351-2356.)，因此全病毒灭活疫苗的形式并不是研发用于COVID-19疫苗的最佳选择。
由于老年人以及免疫力低下的人群，也是SARS-CoV-2感染的宿主，而减毒活苗平台制备得到的疫苗不适合老年人及免疫力低下个体，因此不适合用于COVID-19疫苗的研发。
DNA疫苗和病毒载体疫苗都是将DNA递送至疫苗接种者的细胞内表达，虽然诸如基于腺病毒载体的疫苗目前并没有整合重组进基因组的报导，但是也不能完全排除此种可能，仍有一定安全风险。亚单位疫苗和VLP疫苗则是需要建立优化表达、纯化方法，并且一般需要选择搭配适合的佐剂，需要的研究时间往往以年计算，难以用于应对快速发展的疫情。
近年来RNA分子领域相关技术突破性进展，mRNA疫苗在流感病毒、埃博拉病毒和寨卡病毒等多种传染病上取得了一定的研究成果，mRNA疫苗将mRNA传递至细胞，表达产生蛋白，从而使机体获得免疫保护。与传统的重组蛋白疫苗、灭活疫苗和减毒疫苗相比，mRNA疫苗制备步骤简单，对于传染性疾病的控制有着重大意义。此外，mRNA疫苗比传统重组疫苗更耐高温也更加稳定。同时，mRNA疫苗能够引起强烈的CD4+或CD8+的T细胞应答，与DNA免疫接种不同，在动物体内mRNA疫苗通过一两次低剂量接种就能够产生抗体。
因此，本发明提供了一种安全可靠的mRNA疫苗，避免了其他疫苗平台的缺陷。
发明内容
本发明的第一方面，提供了mRNA或mRNA组合物，所述的mRNA或mRNA组合物包含编码新型冠状病毒SARS-CoV-2的S蛋白(刺突蛋白)或其变体的mRNA序列以及编码S蛋白中的RBD(受体结合域)或其变体的mRNA序列。
其中，所述的编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列与编码S蛋白中的RBD或其变体的mRNA序列来源于相同的SARS-CoV-2突变株或不同的SARS-CoV-2突变株。
优选的，所述的S蛋白或其变体包含野生型全长S蛋白或固定在融合前构象的全长S蛋白。
进一步优选的，为稳定构象，所述的固定在融合前构象的全长S蛋白包含682RRAR685位突变和/或将986KV987位突变，以使S蛋白固定到融合前构象。最为优选的，所述的固定在融合前构象的全长S蛋白为将野生型全长S蛋白的682RRAR685突变为GSAG和/或将986KV987突变为PP。
同时，现有专利中公开的部分内容也支持了本发明的技术方案，例如在接近第一段七肽重复区域或在第一段七肽重复区域中将一个或两个氨基酸替换为脯氨酸可极有效率地稳定构象(见美国专利，申请号20200061185，PREFUSION CORONAVIRUS SPIKE PROTEINS and THEIR USE)。
在本发明的一个具体实施方式中，所述的野生型全长S蛋白的氨基酸序列如SEQ ID NO：1或与SEQ ID NO：1具有70％、75％、80％、85％、90％、95％、99％同一性的氨基酸序列。
在本发明的一个具体实施方式中，所述的固定在融合前构象的全长S蛋白的氨基酸序列如SEQ ID NO：2、SEQ ID NO：15或与SEQ ID NO：2或15具有70％、75％、80％、85％、90％、95％、 99％同一性的氨基酸序列。
优选的，所述的S蛋白或其变体不包含信号肽、包含野生型S蛋白的信号肽或包含野生型S蛋白的信号肽及在其前添加强信号肽，所述的强信号肽优选为组织型纤溶酶原激活剂(tPA)的信号肽或血清免疫球蛋白E(lgE)的信号肽。
在本发明的一个具体实施方式中，编码不含信号肽的野生型2019-nCoV S蛋白的核苷酸序列如SEQ ID NO：6所示。
在本发明的一个具体实施方式中，所述的RBD的氨基酸序列如SEQ ID NO：3、SEQ ID NO：13、或与SEQ ID NO：3或13具有70％、75％、80％、85％、90％、95％、99％同一性的氨基酸序列。
优选的，所述的RBD或其变体不包含信号肽、包含野生型S蛋白的信号肽或包含野生型S蛋白的信号肽及在其前添加强信号肽，所述的强信号肽优选为组织型纤溶酶原激活剂(tPA)的信号肽或血清免疫球蛋白E(lgE)的信号肽。
在本发明的一个具体实施方式中，编码含IgE信号肽的野生型SARS-CoV-2的RBD的核苷酸序列如SEQ ID NO：7、9-11任一所示。
优选的，所述的mRNA为单顺反子、双顺反子或多顺反子mRNA。所述的双顺反子或多顺反子mRNA即含有两个及以上编码区的mRNA。
优选的，所述的编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列与编码S蛋白中的RBD或其变体的mRNA序列为分别的两条mRNA序列或连接为一条mRNA序列。
进一步优选的，连接为一条mRNA序列从5’至3’的连接顺序为：先编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列、后编码S蛋白中的RBD或其变体的mRNA序列，或者为先编码S蛋白中的RBD或其变体的mRNA序列、后编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列。再进一步优选的，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列与编码S蛋白中的RBD或其变体的mRNA序列之间通过内部核糖体进入位点(IRES)连接。
其中，所述的IRES可以用于分隔两个编码区。
在本发明的一个具体实施方式中，所述的IRES序列包括但不限于小RNA病毒(例如FMDV)、瘟病毒(例如CFFV)、脊髓灰质炎病毒(例如PV)、脑心肌炎病毒(例如ECMV)、口蹄疫病毒(例如FMDV)、丙肝病毒(例如HCV)、古典猪瘟病毒(例如CSFV)、小鼠角膜白斑病毒(例如MLV)、猿免疫缺陷病毒(例如SIV)或蟋蟀麻痹病毒(例如CrPV)。
优选的，所述的mRNA或mRNA组合物还包含5’帽子结构、5’非编码区和多聚腺苷酸尾。
进一步优选的，所述的mRNA或mRNA组合物还包含5’保守序列元素、RNA复制酶编码区、亚基因组启动子、3’保守序列元素或3’非编码区中的一种或两种以上的组合。
优选的，所述的mRNA为传统mRNA、自主扩增型mRNA或反式扩增型mRNA。
在本发明的一个具体实施方式中，所述的mRNA为传统mRNA，即所述的mRNA除包含编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列以及编码S蛋白中的RBD或其变体的mRNA序列外，还包含5’帽子结构，5’非编码区，3’非编码区和/或多聚腺苷酸尾的mRNA序列。
在本发明的一个具体实施方式中，所述的mRNA为自主扩增型mRNA，即所述的mRNA除包含编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列以及编码S蛋白中的RBD或其变体的mRNA序列外，还包括5’帽子结构，5’保守序列元素，RNA复制酶编码区，亚基因组启动子，3’保守序列元素和多聚腺苷酸尾构成。其中，可使用的RNA复制酶编码区包括但不限于甲病毒(例如SFV)、小RNA病毒(例如FMDV)、黄病毒(例如DENV)、副粘病毒(例如HMPV)或杯状病毒(例如NV)。
在本发明的一个具体实施方式中，所述的mRNA为反式扩增型mRNA，即所述的编码目的基因 的mRNA除包含编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列以及编码S蛋白中的RBD或其变体的mRNA序列外，还包括5’帽子结构，5’保守序列元素，亚基因组启动子，3’保守序列元素和多聚腺苷酸尾构成；RNA复制酶由单独的传统mRNA编码。
在本发明的一个具体实施方式中，所述的mRNA或mRNA组合物包括编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列与编码S蛋白中的RBD或其变体的mRNA序列连接构成的一条mRNA序列，其选自下列任一组:
A)由5’帽子结构，5’非编码区，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，编码S蛋白中的RBD或其变体的mRNA序列，3’非编码区和多聚腺苷酸尾构成；
B)由5’帽子结构，5’非编码区，编码S蛋白中的RBD或其变体的mRNA序列，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，3’非编码区和多聚腺苷酸尾构成；
C)由5’帽子结构，5’非编码区，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，内部核糖体进入位点(IRES)，编码S蛋白中的RBD或其变体的mRNA序列，3’非编码区和多聚腺苷酸尾构成；
D)由5’帽子结构，5’非编码区，编码S蛋白中的RBD或其变体的mRNA序列，IRES，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，3’非编码区和多聚腺苷酸尾构成；
E)由5’帽子结构，5’保守序列元素，RNA复制酶编码区，亚基因组启动子，编码S蛋白中的RBD或其变体的mRNA序列，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，3’保守序列元素和多聚腺苷酸尾构成；
F)由5’帽子结构，5’保守序列元素，RNA复制酶编码区，亚基因组启动子，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，编码S蛋白中的RBD或其变体的mRNA序列，3’保守序列元素和多聚腺苷酸尾构成；
G)由5’帽子结构，5’保守序列元素，RNA复制酶编码区，亚基因组启动子，编码S蛋白中的RBD或其变体的mRNA序列，IRES，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，3’保守序列元素和多聚腺苷酸尾构成；或
H)由5’帽子结构，5’保守序列元素，RNA复制酶编码区，亚基因组启动子，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，IRES，编码S蛋白中的RBD或其变体的mRNA序列，3’保守序列元素和多聚腺苷酸尾构成。
在本发明的一个具体实施方式中，所述的mRNA或mRNA组合物包括两条mRNA序列的组合，其选自下列任一组：
a)由5’帽子结构，5’非编码区，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序 列，3’非编码区和多聚腺苷酸尾构成的mRNA，组合由5’帽子结构，5’非编码区，编码S蛋白中的RBD或其变体的mRNA序列，3’非编码区和多聚腺苷酸尾构成的mRNA；
b)由5’帽子结构，5’保守序列元素，RNA复制酶编码区，亚基因组启动子，编码S蛋白中的RBD或其变体的mRNA序列，3’保守序列元素和多聚腺苷酸尾构成的mRNA，组合由5’帽子结构，5’保守序列元素，RNA复制酶编码区，亚基因组启动子，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，3’保守序列元素和多聚腺苷酸尾构成的mRNA；
c)由5’帽子结构，5’保守序列元素，亚基因组启动子，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，IRES，编码S蛋白中的RBD或其变体的mRNA序列，3’保守序列元素和多聚腺苷酸尾构成的mRNA，组合由5’帽子结构，5’非编码区，RNA复制酶编码区，3’非编码区和多聚腺苷酸尾构成的mRNA；
d)由5’帽子结构，5’保守序列元素，亚基因组启动子，编码S蛋白中的RBD或其变体的mRNA序列，IRES，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，3’保守序列元素和多聚腺苷酸尾构成的mRNA，组合由5’帽子结构，5’非编码区，RNA复制酶编码区，3’非编码区和多聚腺苷酸尾构成的mRNA；
e)由5’帽子结构，5’保守序列元素，亚基因组启动子，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，编码S蛋白中的RBD或其变体的mRNA序列，3’保守序列元素和多聚腺苷酸尾构成的mRNA，组合由5’帽子结构，5’非编码区，RNA复制酶编码区，3’非编码区和多聚腺苷酸尾构成的mRNA；或
f)由5’帽子结构，5’保守序列元素，亚基因组启动子，编码S蛋白中的RBD或其变体的mRNA序列，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，3’保守序列元素和多聚腺苷酸尾构成的mRNA，组合由5’帽子结构，5’非编码区，RNA复制酶编码区，3’非编码区和多聚腺苷酸尾构成的mRNA。
在本发明的一个具体实施方式中，所述的mRNA或mRNA组合物中包含的mRNA序列包含SEQ ID NO：16-19中的任一个或两个以上的组合。
优选的，所述的mRNA或mRNA组合物还包括阳离子或聚阳离子化合物。
优选的，所述的阳离子或聚阳离子化合物游离或者与mRNA结合。
在本发明的一个具体实施方式中，为了使得所述mRNA或mRNA组合物更稳定选择阳离子或聚阳离子化合物与所述mRNA结合的形式。
优选的，所述的mRNA或mRNA组合物中还包含脂质。
进一步优选的，所述的脂质包括但不限于能够促进自组装形成病毒大小的颗粒(～100nm)的脂质体、使得mRNA从内涵体中释放到胞内的脂质体、支撑磷脂双分子层结构的脂质体或用作稳定剂的脂质体。
更优选的，为了增加LNP(脂质纳米颗粒)的半衰期，所述的脂质还可以包含PEG化脂质。
在本发明的一个具体实施方式中，所述的脂质包含阳离子脂质、PEG化脂质、胆固醇和/或磷脂。
本发明所述的mRNA或mRNA组合物可以为脂质体、脂质复合物或脂质纳米粒子。所述的脂质体可以为以下形式制备得到的脂质体：1，2-二油烯基氧基-N，N-二甲基氨基丙烷(DODMA)脂质体、1，2-二亚油基氧基-3-二甲基氨基丙烷(DLin-DMA)、2，2-二亚油基-4-(2-二甲基氨基乙基)-[1，3]-二氧杂环戊烷(DLin-KC2-DMA)脂质体。所述脂质复合物或脂质纳米粒子可以由选自以下的脂质形成：DLin-DMA、DLin-K-DMA、98N12-5、C12-200、DLin-MC3-DMA、DLin-KC2-DMA、DODMA、PLGA、PEG、PEG-DMG、聚乙二醇化脂质和氨基醇脂质。
本发明所述的mRNA或mRNA组合物还可以包含药学上可接受的赋形剂。所述药学上可接受的赋形剂可以是载体、稀释剂、佐剂或编码佐剂核苷酸序列、增溶剂、粘合剂、润滑剂、助悬剂、转染促进剂等。所述转染促进剂包括但不限于表面活性剂如免疫刺激复合物、费氏(Freunds)不完全佐剂、LPS类似物(例如单磷酰酯A)、胞壁肽、苯醌类似物、角鲨烯、透明质酸、脂质、脂质体、钙离子、病毒蛋白质、阳离子、聚阳离子(例如聚-L-谷氨酸(LGS))或纳米粒子或其他已知的转染促进剂。所述的编码佐剂的核苷酸序列为编码如下至少一种佐剂的核苷酸序列：GM-CSF、IL-17、IFNg、IL-15、IL-21、抗PD1/2、乳铁蛋白、鱼精蛋白、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-12、INF-α、INF-γ、Lymphotoxin-α、hGH、MCP-1、MIP-1a、MIP-1p、IL-8、RANTES、L-选择蛋白、P-选择蛋白、E-选择蛋白、CD34、GlyCAM-1、MadCAM-1、LFA-1、VLA-1、Mac-1、pl50.95、PECAM、ICAM-1、ICAM-2、ICAM-3、CD2、LFA-3、M-CSF、CD40、CD40L、血管生长因子、成纤维细胞生长因子、神经生长因子、血管内皮生长因子、Apo-1、p55、WSL-1、DR3、TRAMP、Apo-3、AIR、LARD、NGRF、DR4、DR5、KILLER、TRAIL-R2、TRICK2、DR6、半胱天冬酶ICE、Fos、c-jun、Sp-1、Ap-1、Ap-2、p38、p65Rel、MyD88、IRAK、TRAF6、IkB、无活性的NIK、SAP K、SAP-1、JNK、NFkB、Bax、TRAIL、TRAILrec、TRAILrecDRC5、TRAIL-R3、TRAIL-R4、RANK、RANK LIGAND、Ox40、Ox40LIGAND、NKG2D、MICA、MICB、NKG2A、NKG2B、NKG2C、NKG2E、NKG2F、TAP1、TAP2以及其功能性片段。
本发明的第二方面，提供了一种mRNA疫苗，所述的mRNA疫苗包括本发明任一所述的mRNA或mRNA组合物。
优选的，所述的mRNA疫苗中编码的新型冠状病毒SARS-CoV-2的S蛋白或其变体与编码S蛋白中的RBD或其变体的序列分别来自于不同SARS-CoV-2突变株，从而免疫产生对不同SARS-CoV-2突变株的交叉保护。在本发明的一个具体实施方式中，所述的mRNA疫苗中编码含IgE信号肽的野生型SARS-CoV-2的RBD中含有501Y.V2谱系的K417N、E484K和N501Y突变，优选其氨基酸序列如SEQ ID NO：13所示；编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的全长S蛋白的序列来自Wuhan-Hu-1 isolate，其中含有501Y.V2谱系的L18F、D80A、D215G、L242-L244删除突变(L242-244del)、R246I、K417N、E484K、N501Y和A701V，优选其氨基酸 序列为SEQ ID NO：15所示。
在本发明的一个具体实施方式中，所述的mRNA疫苗包含编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列以及编码S蛋白中的RBD或其变体的mRNA序列，其中，新型冠状病毒SARS-CoV-2的S蛋白或其变体的氨基酸序列如SEQ ID NO：15所示，S蛋白中的RBD或其变体如SEQ ID NO：13所示。
在本发明的另一个具体实施方式中，所述的mRNA疫苗包含编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列以及编码S蛋白中的RBD或其变体的mRNA序列，其中，新型冠状病毒SARS-CoV-2的S蛋白或其变体的氨基酸序列如SEQ ID NO：2所示，S蛋白中的RBD或其变体如SEQ ID NO：13所示。
在本发明的另一个具体实施方式中，所述的mRNA疫苗包含编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列以及编码S蛋白中的RBD或其变体的mRNA序列，其中，新型冠状病毒SARS-CoV-2的S蛋白或其变体的氨基酸序列如SEQ ID NO：2所示，S蛋白中的RBD或其变体如SEQ ID NO：3所示。优选的，所述的mRNA疫苗中编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA与编码S蛋白中的RBD或其变体的mRNA的质量比为(1-5)：(1-5)。
在本发明的一个具体实施方式中，所述的mRNA疫苗中编码含IgE信号肽的野生型SARS-CoV-2的RBD的mRNA和编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的全长S蛋白的mRNA的质量比为(1-2)：(1-2)。
优选的，所述的mRNA疫苗还包括阳离子或聚阳离子化合物。
优选的，所述的阳离子或聚阳离子化合物游离或者与mRNA结合。
在本发明的一个具体实施方式中，为了使得所述mRNA疫苗更稳定选择阳离子或聚阳离子化合物与所述mRNA结合的形式。
优选的，所述的mRNA疫苗中还包含脂质。
进一步优选的，所述的脂质包括但不限于能够促进自组装形成病毒大小的颗粒(～100nm)的脂质体、使得mRNA从内涵体中释放到胞内的脂质体、支撑磷脂双分子层结构的脂质体或用作稳定剂的脂质体。
更优选的，为了增加LNP的半衰期，所述的脂质还可以包含PEG化脂质。
在本发明的一个具体实施方式中，所述的脂质包含阳离子脂质、PEG化脂质、胆固醇和/或磷脂。
本发明所述的mRNA疫苗可以为脂质体、脂质复合物或脂质纳米粒子。所述的脂质体可以为以下形式制备得到的脂质体：1，2-二油烯基氧基-N，N-二甲基氨基丙烷(DODMA)脂质体、1，2-二亚油基氧基-3-二甲基氨基丙烷(DLin-DMA)、2，2-二亚油基-4-(2-二甲基氨基乙基)-[1，3]-二氧杂环戊烷(DLin-KC2-DMA)脂质体。所述脂质复合物或脂质纳米粒子可以由选自以下的脂质形成：DLin-DMA、DLin-K-DMA、98N12-5、C12-200、DLin-MC3-DMA、DLin-KC2-DMA、DODMA、PLGA、PEG、PEG-DMG、聚乙二醇化脂质和氨基醇脂质。
本发明所述的mRNA疫苗还可以包含药学上可接受的赋形剂。所述药学上可接受的赋形剂可以是载体、稀释剂、佐剂或编码佐剂核苷酸序列、增溶剂、粘合剂、润滑剂、助悬剂、转染促进剂等。所述转染促进剂包括但不限于表面活性剂如免疫刺激复合物、费氏(Freunds)不完全佐剂、LPS类似物(例如单磷酰酯A)、胞壁肽、苯醌类似物、角鲨烯、透明质酸、脂质、脂质体、钙离 子、病毒蛋白质、阳离子、聚阳离子(例如聚-L-谷氨酸(LGS))或纳米粒子或其他已知的转染促进剂。所述的编码佐剂的核苷酸序列为编码如下至少一种佐剂的核苷酸序列：GM-CSF、IL-17、IFNg、IL-15、IL-21、抗PD1/2、乳铁蛋白、鱼精蛋白、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-12、INF-α、INF-γ、Lymphotoxin-α、hGH、MCP-1、MIP-1a、MIP-1p、IL-8、RANTES、L-选择蛋白、P-选择蛋白、E-选择蛋白、CD34、GlyCAM-1、MadCAM-1、LFA-1、VLA-1、Mac-1、pl50.95、PECAM、ICAM-1、ICAM-2、ICAM-3、CD2、LFA-3、M-CSF、、CD40、CD40L、血管生长因子、成纤维细胞生长因子、神经生长因子、血管内皮生长因子、Apo-1、p55、WSL-1、DR3、TRAMP、Apo-3、AIR、LARD、NGRF、DR4、DR5、KILLER、TRAIL-R2、TRICK2、DR6、半胱天冬酶ICE、Fos、c-jun、Sp-1、Ap-1、Ap-2、p38、p65Rel、MyD88、IRAK、TRAF6、IkB、无活性的NIK、SAP K、SAP-1、JNK、NFkB、Bax、TRAIL、TRAILrec、TRAILrecDRC5、TRAIL-R3、TRAIL-R4、RANK、RANK LIGAND、Ox40、Ox40LIGAND、NKG2D、MICA、MICB、NKG2A、NKG2B、NKG2C、NKG2E、NKG2F、TAP1、TAP2以及其功能性片段。
本发明的第三方面，提供了一种mRNA或mRNA组合物的制备方法，包括将mRNA与阳离子或聚阳离子化合物混合后用脂质包装。
优选的，所述的脂质包括但不限于能够促进自组装形成病毒大小的颗粒(～100nm)的脂质体、使得mRNA从内涵体中释放到胞内的脂质体、支撑磷脂双分子层结构的脂质体或用作稳定剂的脂质体。更优选的，为了增加LNP的半衰期，所述的脂质还可以包含PEG化脂质。
在本发明的一个具体实施方式中，所述的脂质包含阳离子脂质、PEG化脂质、胆固醇和/或磷脂。
本发明的第四方面，提供了一种mRNA疫苗的制备方法，所述的制备方法包括将本发明任一所述的mRNA或mRNA组合物与阳离子或聚阳离子化合物混合后用脂质包装。优选的，包装成脂质纳米颗粒。
优选的，所述的脂质包括但不限于能够促进自组装形成病毒大小的颗粒(～100nm)的脂质体、使得mRNA从内涵体中释放到胞内的脂质体、支撑磷脂双分子层结构的脂质体或用作稳定剂的脂质体。更优选的，为了增加LNP的半衰期，所述的脂质还可以包含PEG化脂质。
在本发明的一个具体实施方式中，所述的脂质包含阳离子脂质、PEG化脂质、胆固醇和/或磷脂。
本发明的第五方面，提供了一种本发明任一所述的mRNA或mRNA组合物或mRNA疫苗在预防和/或治疗新型冠状病毒SARS-CoV-2感染导致的疾病的应用。
优选的，所述的新型冠状病毒SARS-CoV-2感染导致的疾病包括但不限于COVID-19。
本发明的第六方面，提供了一种本发明任一所述的mRNA或mRNA组合物或mRNA疫苗在抗新型冠状病毒SARS-CoV-2感染中的应用。
本发明的第七方面，提供了一种本发明任一所述的mRNA或mRNA组合物或mRNA疫苗在制备预防和/或治疗新型冠状病毒SARS-CoV-2感染导致的疾病的药物中的应用。
优选的，所述的新型冠状病毒SARS-CoV-2感染导致的疾病包括但不限于COVID-19。
本发明的第八方面，提供了一种本发明任一所述的mRNA或mRNA组合物或mRNA疫苗在制备抗新型冠状病毒SARS-CoV-2感染的药物中的应用。
本发明的第九方面，提供了一种治疗和/或预防新型冠状病毒SARS-CoV-2感染导致的疾病的方法，包括向个体施加有效量的本发明任一所述的mRNA或mRNA组合物，或者mRNA疫苗。
本发明的第十方面，提供了一种预防新型冠状病毒SARS-CoV-2感染的方法，包括向未感染新型冠状病毒SARS-CoV-2的个体施加有效量的本发明所述的mRNA疫苗。
本发明的第十一方面，提供了一种治疗新型冠状病毒SARS-CoV-2感染的方法，包括向感染新型冠状病毒SARS-CoV-2的个体施加有效量的本发明所述的mRNA或包含mRNA的组合物或mRNA疫苗，以使得个体体内产生中和抗体抵制新型冠状病毒SARS-CoV-2。
本发明的第十二方面，提供了一种抗体筛选的方法，所述的方法包括向个体施加有效量的本发明任一所述的mRNA或mRNA组合物，或者mRNA疫苗的步骤。
其中，所述的抗体筛选的方法不是治疗方法。该方法用来筛选中和抗体，对抗体的药效进行检测和比较，以确定哪些抗体可以作为药物，哪些不能作为药物，或者，比较不同药物的药效敏感程度，即治疗效果不是必然的，只是一种可能性。
本发明的第十三方面，提供了一种诱导个体中和抗原特异性免疫应答的方法，所述的方法包括向个体施加本发明任一所述的mRNA或mRNA组合物，或者mRNA疫苗。
优选的，所述的抗原特异性免疫应答包括T细胞应答和/或B细胞应答。
本发明的第十四方面，提供了本发明所述的mRNA或mRNA组合物编码的蛋白。
优选的，所述的蛋白为固定在融合前构象的全长S蛋白。进一步优选的，所述的固定在融合前构象的全长S蛋白包含682RRAR685位突变和/或将986KV987位突变，以使S蛋白固定到融合前构象。最为优选的，所述的固定在融合前构象的全长S蛋白为将野生型全长S蛋白的682RRAR685突变为GSAG和/或将986KV987突变为PP。
本发明的第十五方面，提供了一种编码本发明所述蛋白的核苷酸序列。
本发明的第十六方面，提供了一种包含本发明所述核苷酸序列的载体。
本发明的第十七方面，提供了一种包含本发明所述的蛋白、所述的核苷酸序列和/或所述的载体的细胞。
本发明所述的mRNA或mRNA组合物以及包含mRNA或mRNA组合物的mRNA疫苗具备的优势在于：1、体外合成，无需细胞培养，无动物源污染的风险；2、研发、生产较快，标准化生产，易于量产和质量控制，同一生产流程适用于多个不同产品；3、可以在一段时间内持续表达，延长抗原暴露时间从而提高免疫反应的强度和质量；4、模拟天然感染的过程，在人体细胞内被翻译、修饰，可以被MHC I类分子呈递，诱发更强的细胞免疫；5、支持多种蛋白形式，包括胞内蛋白、跨膜蛋白、VLP等，且可避开VLP产量低而造成的纯化问题；6、具有自佐剂效应，无需进行佐剂筛选；7、无感染、基因组整合风险；8、无预存免疫，可多次免疫。
本发明所述的mRNA或mRNA组合物的表达产物包含RBD或其变体以及S蛋白或其变体，其中，RBD 中包含了主要的中和表位，可以诱发高水平的中和抗体滴度；而且，非中和表位相对较少，安全性高。全长S蛋白可诱导出高水平的特异性细胞免疫，配合针对性诱导中和抗体的RBD，可以产生极为优异的免疫效果。进一步的，实施例也证实了本发明所述的mRNA或mRNA组合物的表达产物能够在人体中诱发高水平中和抗体和细胞因子的产生。
同时，现有技术中公开的部分内容也支持了本发明的技术方案，例如通过对SARS-CoV-2的RBD的序列分析，以及对其结构的模拟预测，认为SARS-CoV-2的S蛋白维持了SARS-CoV的S蛋白和ACE2相互作用的结构构象(见Xintian,X等(2020)，Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission，Sci China Life Sci.)。通过比对MERS-CoV的RBD的不同片段，确定蛋白片段377-588为关键中和域，即可以在小鼠和兔子模型中引起最高的中和抗体滴度；而且此关键中和域仍可以和受体hDPP4结合，证明其保持了结构构象，不止可以提供线性表位，也可以提供结构表位(见Cuiqing,M等(2014)，Searching for an ideal vaccine candidate among different MERS coronavirus receptor-binding fragments--the importance of immunofocusing in subunit vaccine design，Vaccine.32(46):6170-6176.)。
本发明所述的“个体”包括哺乳动物和人。所述的哺乳动物包括但不限于啮齿类动物(例如小鼠、大鼠)、猴子、斑马鱼、猪、鸡、兔子等等。
本发明所述的“预防”指在疾病开始发展之前或之后通过施用本发明所述的产品来避免症状或者延缓特定症状紧张的所有行为；优选的，所述的预防包括将本发明所述的mRNA或包含mRNA的组合物用作疫苗。
本发明所述的“治疗”指在疾病已开始发展后改善疾病或病理状态的体征、症状等等的治疗干预；优选的，所述的治疗包括筛选与本发明所述的mRNA或包含mRNA的组合物结合的抗体，并将其用于治疗。
本发明所述的“有效量”是指在以单个或多个剂量给予至患者或器官之后提供所希望的治疗或预防的本发明所述产品的量或剂量。
本发明所述的“S蛋白”为组成新型冠状病毒SARS-CoV-2的结构蛋白，名称为刺突蛋白。
本发明所述的“RBD”为组成新型冠状病毒SARS-CoV-2的结构蛋白，名称为刺突蛋白受体结合域。
本发明所述的“同一性”是指在使用氨基酸序列或核苷酸序列的方面，本领域技术人员可以根据实际工作需要对序列进行调整，使使用序列与现有技术获得的序列相比，具有(包括但不限于)1％，2％，3％，4％，5％，6％，7％，8％，9％，10％，11％，12％，13％，14％，15％，16％，17％，18％，19％，20％，21％，22％，23％，24％，25％，26％，27％，28％，29％，30％，31％，32％，33％，34％，35％，36％，37％，38％，39％， 40％，41％，42％，43％，44％，45％，46％，47％，48％，49％，50％，51％，52％，53％，54％，55％，56％，57％，58％，59％，60％，70％，80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，99.1％，99.2％，99.3％，99.4％，99.5％，99.6％，99.7％，99.8％，99.9％的相似度，且依然具有与原氨基酸序列或核苷酸序列相同或类似的功能，例如与原序列具有同一性的氨基酸序列依然具有在体内诱发中和抗体和产生细胞因子的功能，与原序列具有同一性的mRNA序列表达产物依然具有在体内诱发中和抗体和产生细胞因子的功能。
附图说明
以下，结合附图来详细说明本发明的实施例，其中：
图1：编码含tPA信号肽的野生型SARS-CoV-2的RBD的序列测序结果。
图2：图2A和图2B的拼接为编码野生型SARS-CoV-2的S蛋白的序列测序结果。
图3：含有T7启动子，5’UTR，3’UTR，和polyA尾的基础质粒模板序列图。
图4：用甲醛变性胶检测加帽纯化后的mRNA，其中，M为Marker，1为编码含tPA信号肽的野生型SARS-CoV-2的RBD的mRNA，2为编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的全长S蛋白的mRNA。
图5：WB(Western Blot，蛋白质印迹法)检测结果，其中1为编码含tPA信号肽的野生型SARS-CoV-2的RBD的mRNA的表达上清，2为阴性对照。
图6：免疫荧光结果。
图7：DLS(Dynamic Light Scattering，动态光散射)检测mRNA-LNP的粒径和粒径分布结果，其中，A代表RBD+S-1-LNP，B代表RBD+S-2-LNP，C代表RBD+S-3-LNP，D代表RBD-LNP，E代表S-LNP。
图8：甲醛变性胶检测包装后样品的mRNA完整性结果，其中1为编码含tPA信号肽的野生型SARS-CoV-2的RBD的mRNA，2为RBD+S-1，3为RBD+S-2，4为RBD+S-3，5为编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的全长S蛋白的mRNA。
图9：一免、二免后的S蛋白特异性抗体滴度，其中，Negative为阴性对照。
图10：用ELISpot(酶联免疫斑点法)检测干扰素γ(IFN-γ)的结果，其中，纵坐标为每百万脾细胞的点形成单位(spot forming unit，SFU)，Negative为阴性对照。
图11：CD4CK胞内染色法(ICS)检测IFN-γ、白细胞介素-2(IL-2)和肿瘤坏死因子-α(TNF-α)的结果。
图12：CD8CK胞内染色法(ICS)检测IFN-γ、白细胞介素-2(IL-2)和肿瘤坏死因子-α(TNF-α)的结果。
图13：用甲醛变性胶检测加帽纯化后的mRNA，其中，M为Marker，1为实施例1制备的编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的全长S蛋白的mRNA，2为编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的501Y.V2谱系的全长S蛋白的mRNA，3为编码含IgE信号肽的501Y.V2谱系的野生型SARS-CoV-2的RBD序列的mRNA。结果显示，mRNA的大小正确且基本无降解。
图14：WB检测结果，其中1为编码含IgE信号肽的501Y.V2谱系的野生型SARS-CoV-2的RBD序列的mRNA的表达上清，2为编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的501Y.V2谱系的全长S蛋白的mRNA的表达上清，3为阴性对照的细胞上清，4为编码含IgE信号肽的501Y.V2谱系的野生型SARS-CoV-2的RBD序列的mRNA的表达细胞沉淀，5为编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的501Y.V2谱系的全长S蛋白的mRNA的表达细胞沉淀，6为阴性对照的细胞沉淀。
图15：DLS(Dynamic Light Scattering，动态光散射)检测mRNA-LNP的粒径和粒径分布结果。
图16：一免、二免后针对501Y.V2谱系的S蛋白特异性抗体滴度。
图17：一免、二免后针对Wuhan-Hu-1 isolate的S蛋白特异性抗体滴度。
图18：替代性中和抗体滴度结果，左边4组为针对Wuhan-Hu-1 isolate的替代性中和抗体滴度，右边4组为针对501Y.V2谱系的替代性中和抗体滴度。
图19：CD4+T细胞Th1类细胞免疫反应检测结果。
图20：CD8+T细胞Th1类细胞免疫反应检测结果。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的部分实施例，而不是全部。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例中使用的试剂来源：
替代性中和抗体检测是检测的疫苗免疫小鼠血清中的中和抗体，利用的是ACE2蛋白和RBD蛋白的竞争结合。
特异性抗体体检测是检测的疫苗免疫小鼠血清中的特异性抗体，利用的是RBD蛋白。
检测针对Wuhan-Hu-1 isolate用到的RBD蛋白为野生型RBD蛋白(厂家：金斯瑞，货号：Z03483-1)。
检测针对501Y.V2谱系用到的RBD蛋白为含501Y.V2谱系突变的RBD蛋白(厂家：近岸，货号：DRA125)。
实施例1：mRNA的制备与检测
1、分别人工合成抗原设计的基因序列。
2、通过固相亚磷酰胺三酯法合成短核苷酸链(引物)。
3、引物互为模板进行PCR扩增。
4、将步骤3扩增的产物连接到pUC57载体上，转化测序。
5、经测序验证，序列与预期一致，结果见图1-2所示。具体的，图1显示了编码含tPA信号肽的野生型SARS-CoV-2的RBD的序列测序结果，其核苷酸序列如SEQ ID NO：4所示，氨基酸序列如SEQ ID NO：3所示。图2显示编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的全长S蛋白的序列测序结果，其核苷酸序列如SEQ ID NO：5所示，氨基酸序列如SEQ ID NO：2所示。
6、准备含有T7启动子，5’UTR，3’UTR，和polyA尾的基础质粒模板，序列如图3(SEQ ID NO：8)所示。
7、用含有与基础质粒模板同源的引物进行PCR，结果显示正确。
基础质粒模板用限制性核酸内切酶BsmBI线性化。将PCR产物分别通过同源重组的方式分别连到基础质粒模板上，分别转化到Xl1-Blue菌株中，并进行测序确认序列正确，转录模板构建成功。用摇瓶发酵菌株，用无内毒素质粒大提试剂盒纯化获得转录模板。
将转录模板用限制性核酸内切酶BbsI线性化。用T7体外转录试剂盒进行转录，分别获得SEQ ID NO：4-5的未加帽的mRNA(mRNA具体序列分别为SEQ ID NO：16-17)。分别用DNaseI消化转录模板，并用沉淀法纯化mRNA。用Cap1加帽试剂盒给mRNA加帽，并分别用mRNA纯化试剂盒对加帽后的mRNA进行纯化。将纯化后的mRNA溶解于酸性柠檬酸钠缓冲液中，待用。
用甲醛变性胶检测加帽纯化后的mRNA，如图4所示，其中1为编码含tPA信号肽的野生型SARS-CoV-2的RBD的mRNA，2为编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的全长S蛋白的mRNA。结果显示，mRNA的大小正确且基本无降解。
用24孔板铺3个孔的HEK293细胞，其中1、2号孔分别用lipofectamine 2000转染0.5μg加帽纯化后的编码含tPA信号肽的野生型SARS-CoV-2的RBD的mRNA，和编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的全长S蛋白的mRNA，3号孔作为阴性对照加入lipofectamine 2000转染试剂。转染24h后，取1、3号孔的细胞上清进行WB检测，2、3号孔的细胞固定后用抗S蛋白多抗进行免疫荧光检测。WB检测结果如图5所示，其中1为编码含tPA信号肽的野生型SARS-CoV-2的RBD的mRNA的表达上清，2为阴性对照。结果显示表达出来的蛋白大小正确。免疫荧光结果如图6所示，证明编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的全长S蛋白的 mRNA可以正常表达。
实施例2：mRNA疫苗的制备与免疫
1、原料准备
1)将阳离子脂质D-Lin-MC3-DMA、二硬脂酰基磷脂酰胆碱DSPC、胆固醇、PEG化脂质PEG-DMG四个组分按摩尔比50:10:38.5:1.5在乙醇中溶解、混合。
2)实施例1制备的编码含tPA信号肽的野生型SARS-CoV-2的RBD的mRNA和编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的全长S蛋白的mRNA，将这两种mRNA分别按质量比1:1、2:1、1：2进行混合，得到混合后的mRNA，简写为RBD+S-1、RBD+S-2和RBD+S-3。
2、试验步骤
以脂质混合物：mRNA流速比1:3，在Precision Nanosystems的纳米颗粒制备仪器Ignite中分别混合包装RBD+S-1、RBD+S-2、RBD+S-3、编码含tPA信号肽的野生型SARS-CoV-2的RBD的mRNA和编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的全长S蛋白的mRNA。将包装好的mRNA-LNP(LNP为脂质纳米颗粒)透析并超滤浓缩到DPBS中，无菌过滤后获得用于后续动物实验的样品。用DLS检测mRNA-LNP的粒径和粒径分布，检测结果如图7所示，包装后样品的粒径大小均在70nm-100nm，PDI均小于0.2。其中，RBD+S-1-LNP：粒径平均值77.15nm，PDI值0.038，截距(intercept)0.958，具体见表1；RBD+S-2-LNP：粒径平均值77.04nm，PDI值0.055，截距(intercept)0.959，具体见表2；RBD+S-3-LNP：粒径平均值91.43nm，PDI值0.049，截距(intercept)0.974，具体见表3；RBD-LNP：粒径平均值77.92nm，PDI值0.036，截距(intercept)0.954，具体见表4；S-LNP：粒径平均值76.89nm，PDI值0.031，截距(intercept)0.977，具体见表5。
表1：RBD+S-1-LNP
  粒径(nm) 强度(％) 标准差
峰1 81.06 100 18.64
峰2 0 0 0
峰3 0 0 0
表2：RBD+S-2-LNP
  粒径(nm) 强度(％) 标准差
峰1 81.93 100 20.68
峰2 0 0 0
峰3 0 0 0
表3：RBD+S-3-LNP
  粒径(nm) 强度(％) 标准差
峰1 97 100 24.54
峰2 0 0 0
峰3 0 0 0
表4：RBD-LNP
  粒径(nm) 强度(％) 标准差
峰1 82.01 100 19.41
峰2 0 0 0
峰3 0 0 0
表5：S-LNP
  粒径(nm) 强度(％) 标准差
峰1 80.72 100 18.76
峰2 0 0 0
峰3 0 0 0
用甲醛变性胶检测包装后样品的mRNA完整性，结果如图8所示，其中1为编码含tPA信号肽的野生型SARS-CoV-2的RBD的mRNA，2为RBD+S-1，3为RBD+S-2，4为RBD+S-3，5为编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的全长S蛋白的mRNA。可见mRNA基本无降解。
6周龄左右的BALB/c雌性小鼠随机6只一组，分为6组。分别在第0天和第28天免疫后腿肌肉接种10ug，在第28天和第42天检测S蛋白特异性抗体滴度，在第42天杀鼠检测细胞因子。
3、实验结果
一免、二免后的S蛋白特异性抗体滴度如图9所示，可以看到单S全长免疫二免后的特异性抗体滴度显著低于单RBD免疫，而三组S与RBD的组合免疫，其中两组与单RBD免疫的特异性抗体滴度无显著性差异，RBD+S-1出现了协同增益的效果，特异性抗体滴度显著高于单RBD免疫。用ELISpot检测干扰素γ(IFN-γ)的结果如图10所示，可以看到单S全长免疫引起的IFN-γ分泌水平显著高于单RBD免疫，而三组S与RBD的组合免疫，其中两组与单S全长免疫的IFN-γ分泌水平无显著性差异，RBD+S-3出现了协同增益的效果，IFN-γ分泌水平显著高于单S全长免疫。用CK胞内染色法(ICS)检测IFN-γ、白细胞介素-2(IL-2)和肿瘤坏死因子-α(TNF-α)的结果如图11和图12所示。CD4+T 细胞的反应较低，个体差异较大，指导意义较小；而CD8+T细胞的检测结果与ELISpot的检测结果基本一致，三组S与RBD的组合免疫，其中两组与单S全长免疫在IFN-γ、IL-2和TNF-α的分泌水平上无显著性差异，而RBD+S-3出现了显著性的协同增益的效果。证明S全长和RBD的组合不仅可以结合S全长的细胞免疫优势和RBD的体液免疫优势，而且可以达到协同增益的效果，可在2019-nCoV型冠状病毒感染的预防上取得更为优异的效果。
实施例3：序列来源于不同SARS-CoV-2突变株的mRNA的制备与检测
1、通过引物互为模板扩增合成编码含IgE信号肽的的野生型SARS-CoV-2的RBD序列，其中含有501Y.V2谱系的K417N、E484K和N501Y突变，其核苷酸序列如SEQ ID NO：12所示，氨基酸序列如SEQ ID NO：13所示。
2、通过引物互为模板扩增合成编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的501Y.V2谱系的全长S蛋白的序列，其中含有501Y.V2谱系的L18F、D80A、D215G、L242-L244删除突变(L242-244del)、R246I、K417N、E484K、N501Y和A701V，其核苷酸序列如SEQ ID NO：14所示，氨基酸序列如SEQ ID NO：15所示。
3、准备含有T7启动子，5’UTR，3’UTR，和polyA尾的基础质粒模板，序列如图3(SEQ ID NO：8)所示。
4、用含有与基础质粒模板同源的引物进行PCR，结果显示正确。
基础质粒模板用限制性核酸内切酶BsmBI线性化。将PCR产物分别通过同源重组的方式分别连到基础质粒模板上，分别转化到Xl1-Blue菌株中，并进行测序确认序列正确，转录模板构建成功。用摇瓶发酵菌株，用无内毒素质粒大提试剂盒纯化获得转录模板。
将转录模板用限制性核酸内切酶BbsI线性化。用T7体外转录试剂盒进行转录，分别获得SEQ ID NO：12、14的未加帽的mRNA(mRNA具体序列分别为SEQ ID NO：18、19)。分别用DNaseI消化转录模板，并用沉淀法纯化mRNA。用Cap1加帽试剂盒给mRNA加帽，并分别用mRNA纯化试剂盒对加帽后的mRNA进行纯化。将纯化后的mRNA溶解于酸性柠檬酸钠缓冲液中，待用。
用甲醛变性胶检测加帽纯化后的mRNA，如图13所示，其中1为实施例1制备的编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的全长S蛋白的mRNA，2为编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的501Y.V2谱系的全长S蛋白的mRNA，3为编码含IgE信号肽的501Y.V2谱系的野生型SARS-CoV-2的RBD序列的mRNA。结果显示，mRNA的大小正确且基本无降解。
用24孔板铺3个孔的HEK293细胞，其中1、2号孔分别用lipofectamine 2000转染0.5μg加帽 纯化后的编码含IgE信号肽的501Y.V2谱系的野生型SARS-CoV-2的RBD序列的mRNA，和编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的501Y.V2谱系的全长S蛋白的mRNA，3号孔作为阴性对照。转染24h后，离心获得细胞上清和细胞沉淀分别进行WB检测。WB检测结果如图14所示，其中1为编码含IgE信号肽的501Y.V2谱系的野生型SARS-CoV-2的RBD序列的mRNA的表达上清，2为编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的501Y.V2谱系的全长S蛋白的mRNA的表达上清，3为阴性对照的细胞上清，4为编码含IgE信号肽的501Y.V2谱系的野生型SARS-CoV-2的RBD序列的mRNA的表达细胞沉淀，5为编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的501Y.V2谱系的全长S蛋白的mRNA的表达细胞沉淀，6为阴性对照的细胞沉淀。结果显示表达出来的蛋白大小正确。
实施例4：序列来源于不同SARS-CoV-2突变株的mRNA组合疫苗的制备与免疫
1、原料准备
1)将阳离子脂质D-Lin-MC3-DMA、二硬脂酰基磷脂酰胆碱DSPC、胆固醇、PEG化脂质PEG-DMG四个组分按摩尔比50:10:38.5:1.5在乙醇中溶解、混合。
2)准备实施例1制备的编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的全长S蛋白的mRNA，实施例3制备的编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的501Y.V2谱系的全长S蛋白的mRNA，和实施例3制备的编码含IgE信号肽的501Y.V2谱系的野生型SARS-CoV-2的RBD序列的mRNA。
3)将实施例3制备的编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的501Y.V2谱系的全长S蛋白的mRNA和实施例3制备的编码含IgE信号肽的501Y.V2谱系的野生型SARS-CoV-2的RBD序列的mRNA按质量比1：2进行混合，得到混合后的mRNA，简写为combo A。
4)将实施例1制备的编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的全长S蛋白的mRNA和实施例3制备的编码含IgE信号肽的501Y.V2谱系的野生型SARS-CoV-2的RBD序列的mRNA按质量比1：2进行混合，得到混合后的mRNA，简写为combo B。
2、试验步骤
以脂质混合物：mRNA流速比1:3，在Precision Nanosystems的纳米颗粒制备仪器Ignite中分别混合包装combo A、combo B、实施例3制备的编码682RRAR685突变为GSAG和986KV987突变为PP的固定在融合前构象的501Y.V2谱系的全长S蛋白的mRNA，和实施例3制备的编码含IgE信号肽的501Y.V2谱系的野生型SARS-CoV-2的RBD序列的mRNA，得到包装好的mRNA-LNP，分别简写为combo A-LNP，combo B-LNP，S-SA-LNP和RBD-SA-LNP。将包装好的mRNA-LNP透析并超滤浓缩到DPBS中，无 菌过滤后获得用于后续动物实验的样品。用DLS检测mRNA-LNP的粒径和粒径分布，检测结果如图15所示，包装后样品的粒径大小均在70nm-100nm，PDI均小于0.2。其中，combo A-LNP：粒径平均值79.87nm，PDI值0.132，截距(intercept)0.962，具体见表6；combo B-LNP：粒径平均值80.61nm，PDI值0.123，截距(intercept)0.958，具体见表7；S-SA-LNP：粒径平均值81.13nm，PDI值0.159，截距(intercept)0.939，具体见表8；RBD-SA-LNP：粒径平均值82.74nm，PDI值0.112，截距(intercept)0.960，具体见表9。
表6：combo A-LNP
  粒径(nm) 强度(％) 标准差
峰1 91.43 100 32.02
峰2 0 0 0
峰3 0 0 0
表7：comboB-LNP
  粒径(nm) 强度(％) 标准差
峰1 91.05 100 30.06
峰2 0 0 0
峰3 0 0 0
表8：S-SA-LNP
  粒径(nm) 强度(％) 标准差
峰1 93.36 100 33.28
峰2 0 0 0
峰3 0 0 0
表9：RBD-SA-LNP
  粒径(nm) 强度(％) 标准差
峰1 92.91 100 30.56
峰2 0 0 0
峰3 0 0 0
6周龄左右的BALB/c雌性小鼠随机6只一组，分为5组。分别在第0天和第14天免疫后腿肌肉接种5ug，在第14天和第28天检测S蛋白特异性抗体滴度，在第28天杀鼠检测细胞因子。
3、实验结果
一免、二免后针对501Y.V2谱系的S蛋白特异性抗体滴度如图16所示，各组之间没有显著性差异。一免、二免后针对Wuhan-Hu-1 isolate的S蛋白特异性抗体滴度如图17所示，结果显示，两组S与RBD组合免疫引起的特异性抗体滴度无显著性差异，但单S全长和单RBD二免后的特异性抗体滴度均显著低于来源于不同SARS-CoV-2突变株的mRNA组合(combo B)。而替代性中和抗体滴度结果也显示(如图18，左边4组为针对Wuhan-Hu-1 isolate的替代性中和抗体滴度，右边4组为针对501Y.V2谱系的替代性中和抗体滴度)，combo B针对Wuhan-Hu-1 isolate的替代性中和抗体滴度显著高于来源于相同SARS-CoV-2突变株(501Y.V2谱系)的mRNA组合(combo A)、单S全长和单RBD；combo B针对501Y.V2谱系的替代性中和抗体滴度则和combo A及单RBD无显著性差异，显著高于单S全长。说明S全长和RBD的组合相对于单S全长有体液免疫优势，且来源于不同SARS-CoV-2突变株的mRNA组合在体液免疫方面相比于来源于相同SARS-CoV-2突变株的mRNA组合可以提供更好地交叉保护效果。
CD4+T细胞Th1类细胞免疫反应检测结果如图19所示，CD8+T细胞Th1类细胞免疫反应检测结果如图20所示，结果显示，S全长和RBD的组合相对于单RBD有细胞免疫优势。再次验证了S全长和RBD的组合设计在疫苗应用方面的优越性。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。

Claims (24)

  1. mRNA或mRNA组合物，其特征在于，所述的mRNA或mRNA组合物包含编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列以及编码S蛋白中的RBD或其变体的mRNA序列。
  2. 根据权利要求1所述的mRNA或mRNA组合物，其特征在于，所述的编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列与编码S蛋白中的RBD或其变体的mRNA序列来源于相同的SARS-CoV-2突变株或不同的SARS-CoV-2突变株。
  3. 根据权利要求1或2所述的mRNA或mRNA组合物，其特征在于，所述的S蛋白或其变体包含野生型全长S蛋白或固定在融合前构象的全长S蛋白；优选的，所述的固定在融合前构象的全长S蛋白包含682RRAR685位突变和/或将986KV987位突变，以使S蛋白固定到融合前构象；进一步优选的，所述的固定在融合前构象的全长S蛋白为将野生型全长S蛋白的682RRAR685突变为GSAG和/或将986KV987突变为PP。
  4. 根据权利要求3所述的mRNA或mRNA组合物，其特征在于，所述的野生型全长S蛋白的氨基酸序列如SEQ ID NO：1具有70％、75％、80％、85％、90％、95％、99％同一性的氨基酸序列；所述的固定在融合前构象的全长S蛋白的氨基酸序列如SEQ ID NO：2、SEQ ID NO：15或与SEQ ID NO：2或15具有70％、75％、80％、85％、90％、95％、99％同一性的氨基酸序列。
  5. 根据权利要求1-4任一所述的mRNA或mRNA组合物，其特征在于，所述的S蛋白或其变体不包含信号肽、包含野生型S蛋白的信号肽或包含野生型S蛋白的信号肽及在其前添加强信号肽，所述的强信号肽优选为组织型纤溶酶原激活剂的信号肽或血清免疫球蛋白E的信号肽。
  6. 根据权利要求1所述的mRNA或mRNA组合物，其特征在于，所述的RBD的氨基酸序列如SEQ ID NO：3、SEQ ID NO：13、或与SEQ ID NO：3或13具有70％、75％、80％、85％、90％、95％、99％同一性的氨基酸序列。
  7. 根据权利要求1或6所述的mRNA或mRNA组合物，其特征在于，所述的RBD或其变体不包含信号肽、包含野生型S蛋白的信号肽或包含野生型S蛋白的信号肽及在其前添加强信号肽，所述的强信号肽优选为组织型纤溶酶原激活剂的信号肽或血清免疫球蛋白E的信号肽。
  8. 根据权利要求1-7任一所述的mRNA或mRNA组合物，其特征在于，所述的编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列与编码S蛋白中的RBD或其 变体的mRNA序列为分别的两条mRNA序列或连接为一条mRNA序列。
  9. 根据权利要求8所述的mRNA或mRNA组合物，其特征在于，连接为一条mRNA序列从5’至3’的连接顺序为：先编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列、后编码S蛋白中的RBD或其变体的mRNA序列，或者为先编码S蛋白中的RBD或其变体的mRNA序列、后编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列；优选的，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列与编码S蛋白中的RBD或其变体的mRNA序列之间通过内部核糖体进入位点连接；其中，所述的内部核糖体进入位点选自小RNA病毒、瘟病毒、脊髓灰质炎病毒、脑心肌炎病毒、口蹄疫病毒、丙肝病毒、古典猪瘟病毒、小鼠角膜白斑病毒、猿免疫缺陷病毒或蟋蟀麻痹病。
  10. 根据权利要求1-9任一所述的mRNA或mRNA组合物，其特征在于，所述的mRNA或mRNA组合物还包含5’帽子结构、5’非编码区和多聚腺苷酸尾。
  11. 根据权利要求1-10任一所述的mRNA或mRNA组合物，其特征在于，所述的mRNA或mRNA组合物还包含5’保守序列元素、RNA复制酶编码区、亚基因组启动子、3’保守序列元素或3’非编码区中的一种或两种以上的组合。
  12. 根据权利要求1-11任一所述的mRNA或mRNA组合物，其特征在于，所述的mRNA为传统mRNA、自主扩增型mRNA或反式扩增型mRNA。
  13. 根据权利要求1-12任一所述的mRNA或mRNA组合物，其特征在于，所述的mRNA或mRNA组合物包括编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列与编码S蛋白中的RBD或其变体的mRNA序列连接构成的一条mRNA序列，其选自下列任一组:
    A)由5’帽子结构，5’非编码区，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，编码S蛋白中的RBD或其变体的mRNA序列，3’非编码区和多聚腺苷酸尾构成；
    B)由5’帽子结构，5’非编码区，编码S蛋白中的RBD或其变体的mRNA序列，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，3’非编码区和多聚腺苷酸尾构成；
    C)由5’帽子结构，5’非编码区，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体 的mRNA序列，内部核糖体进入位点(IRES)，编码S蛋白中的RBD或其变体的mRNA序列，3’非编码区和多聚腺苷酸尾构成；
    D)由5’帽子结构，5’非编码区，编码S蛋白中的RBD或其变体的mRNA序列，IRES，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，3’非编码区和多聚腺苷酸尾构成；
    E)由5’帽子结构，5’保守序列元素，RNA复制酶编码区，亚基因组启动子，编码S蛋白中的RBD或其变体的mRNA序列，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，3’保守序列元素和多聚腺苷酸尾构成；
    F)由5’帽子结构，5’保守序列元素，RNA复制酶编码区，亚基因组启动子，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，编码S蛋白中的RBD或其变体的mRNA序列，3’保守序列元素和多聚腺苷酸尾构成；
    G)由5’帽子结构，5’保守序列元素，RNA复制酶编码区，亚基因组启动子，编码S蛋白中的RBD或其变体的mRNA序列，IRES，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，3’保守序列元素和多聚腺苷酸尾构成；或
    H)由5’帽子结构，5’保守序列元素，RNA复制酶编码区，亚基因组启动子，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，IRES，编码S蛋白中的RBD或其变体的mRNA序列，3’保守序列元素和多聚腺苷酸尾构成。
  14. 根据权利要求1-12任一所述的mRNA或mRNA组合物，其特征在于，所述的mRNA或mRNA组合物包括两条mRNA序列的组合，其选自下列任一组：
    a)由5’帽子结构，5’非编码区，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，3’非编码区和多聚腺苷酸尾构成的mRNA，组合由5’帽子结构，5’非编码区，编码S蛋白中的RBD或其变体的mRNA序列，3’非编码区和多聚腺苷酸尾构成的mRNA；
    b)由5’帽子结构，5’保守序列元素，RNA复制酶编码区，亚基因组启动子，编 码S蛋白中的RBD或其变体的mRNA序列，3’保守序列元素和多聚腺苷酸尾构成的mRNA，组合由5’帽子结构，5’保守序列元素，RNA复制酶编码区，亚基因组启动子，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，3’保守序列元素和多聚腺苷酸尾构成的mRNA；
    c)由5’帽子结构，5’保守序列元素，亚基因组启动子，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，IRES，编码S蛋白中的RBD或其变体的mRNA序列，3’保守序列元素和多聚腺苷酸尾构成的mRNA，组合由5’帽子结构，5’非编码区，RNA复制酶编码区，3’非编码区和多聚腺苷酸尾构成的mRNA；
    d)由5’帽子结构，5’保守序列元素，亚基因组启动子，编码S蛋白中的RBD或其变体的mRNA序列，IRES，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，3’保守序列元素和多聚腺苷酸尾构成的mRNA，组合由5’帽子结构，5’非编码区，RNA复制酶编码区，3’非编码区和多聚腺苷酸尾构成的mRNA；
    e)由5’帽子结构，5’保守序列元素，亚基因组启动子，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，编码S蛋白中的RBD或其变体的mRNA序列，3’保守序列元素和多聚腺苷酸尾构成的mRNA，组合由5’帽子结构，5’非编码区，RNA复制酶编码区，3’非编码区和多聚腺苷酸尾构成的mRNA；或
    f)由5’帽子结构，5’保守序列元素，亚基因组启动子，编码S蛋白中的RBD或其变体的mRNA序列，编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA序列，3’保守序列元素和多聚腺苷酸尾构成的mRNA，组合由5’帽子结构，5’非编码区，RNA复制酶编码区，3’非编码区和多聚腺苷酸尾构成的mRNA。
  15. 根据权利要求13或14所述的mRNA或mRNA组合物，其特征在于，所述的RNA复制酶编码区选自甲病毒、小RNA病毒、黄病毒、副粘病毒或杯状病毒。
  16. 一种mRNA疫苗，其特征在于，所述的mRNA疫苗包括权利要求1-15任一所述的mRNA或mRNA组合物。
  17. 根据权利要求16所述的mRNA疫苗，其特征在于，所述的mRNA疫苗中编码新型冠状病毒SARS-CoV-2的S蛋白或其变体的mRNA与编码S蛋白中的RBD或其变体的 mRNA的质量比为(1-5)：(1-5)。
  18. 根据权利要求16或17所述的mRNA疫苗，其特征在于，所述的mRNA疫苗还包括阳离子或聚阳离子化合物。
  19. 根据权利要求16-18任一所述的mRNA疫苗，其特征在于，所述的mRNA疫苗还包含脂质。
  20. 根据权利要求16-19任一所述的mRNA疫苗，其特征在于，所述的mRNA疫苗为脂质体、脂质复合物或脂质纳米粒子。
  21. 一种mRNA疫苗的制备方法，其特征在于，所述的制备方法包括将权利要求1-15任一所述的mRNA或mRNA组合物与阳离子或聚阳离子化合物混合后用脂质包装。
  22. 一种权利要求1-15任一所述的mRNA或mRNA组合物或权利要求16-20任一所述的mRNA疫苗的应用，其特征在于，所述的应用包括：
    A)在制备预防和/或治疗新型冠状病毒SARS-CoV-2感染导致的疾病的药物中的应用；或，
    B)在制备抗新型冠状病毒SARS-CoV-2感染的药物中的应用；
    优选的，所述的新型冠状病毒SARS-CoV-2感染导致的疾病为COVID-19。
  23. 一种抗体筛选的方法，其特征在于，所述的方法包括向个体施加权利要求1-15任一所述的mRNA或mRNA组合物，或者权利要求16-20任一所述的mRNA疫苗。
  24. 一种诱导个体中和抗原特异性免疫应答的方法，其特征在于，所述的方法包括向个体施加权利要求1-15任一所述的mRNA或mRNA组合物，或者权利要求16-20任一所述的mRNA疫苗。
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