WO2021155760A1 - 用于2019-nCoV型冠状病毒mRNA疫苗、制备方法及其应用 - Google Patents

用于2019-nCoV型冠状病毒mRNA疫苗、制备方法及其应用 Download PDF

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WO2021155760A1
WO2021155760A1 PCT/CN2021/074206 CN2021074206W WO2021155760A1 WO 2021155760 A1 WO2021155760 A1 WO 2021155760A1 CN 2021074206 W CN2021074206 W CN 2021074206W WO 2021155760 A1 WO2021155760 A1 WO 2021155760A1
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mrna
protein
ncov
rbd
encoding
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French (fr)
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朱涛
李荩
王浩猛
莘春林
蒋剑豪
李军强
巢守柏
邱东旭
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康希诺生物股份公司
苏州吉玛基因股份有限公司
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
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    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the technical field of vaccine development, and in particular to an mRNA sequence containing a coding region encoding at least one antigenic peptide or protein of the 2019-nCoV coronavirus or a fragment, variant or derivative thereof.
  • the present invention relates to containing one or more mRNAs. Compositions. And the application of the mRNA or composition in the preparation of drugs (especially vaccines) for preventing and/or treating 2019-nCoV type coronavirus infection.
  • 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 new coronavirus (2019-nCoV). 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
  • 2019-nCoV new coronavirus Only the latter three can cause
  • 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 injury ("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.
  • 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.
  • the first aspect of the present invention provides an mRNA comprising at least one mRNA sequence encoding the coding region of at least one antigenic peptide or protein of the 2019-nCoV coronavirus or a fragment, variant or derivative thereof .
  • the antigen peptide or protein or fragments, variants or derivatives thereof can induce an immune response in the human body and produce neutralizing antibodies.
  • the mRNA includes an mRNA sequence encoding the S protein of the 2019-nCoV coronavirus. Further preferably, the mRNA includes an mRNA sequence encoding the RBD of the 2019-nCoV coronavirus.
  • the amino acid sequence of the RBD includes a single site amino acid mutation and N-linked glycosylation is introduced.
  • the mutation site is the mutation of proline at position 190 to aspartic acid.
  • the mRNA includes an mRNA sequence encoding the RBM of the 2019-nCoV coronavirus.
  • the mRNA contains an mRNA sequence encoding dS protein. Further preferably, the mRNA contains an mRNA sequence encoding a fusion protein of dS protein and RBD or RBM.
  • the mRNA includes an mRNA sequence encoding M protein and/or E protein.
  • the antigen peptide or protein encoded by the mRNA or a fragment, variant or derivative thereof is selected from any one of the following:
  • S protein of 2019-nCoV maintains SARS- The structural conformation of the interaction between the S protein of CoV and ACE2 (see Xintian, X, etc. (2020), Evolution of the novel coronavirus from theongoing Wuhan outbreak 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.).
  • S protein of 2019-nCoV coronavirus preferably, it also includes M protein and/or E protein of 2019-nCoV coronavirus; specifically (1) S protein and M protein of 2019-nCoV coronavirus (2) The combination of the S protein and E protein of the 2019-nCoV coronavirus; or (3) the combination of the S protein, M protein and E protein of the 2019-nCoV coronavirus.
  • the molar ratio of mRNA of S protein, M protein and E protein is 1:1:1.
  • the baculovirus expression system is used to simultaneously express the three structural proteins of MERS-CoV S, M, and E in insect cells, and the expressed protein will automatically form and Coronavirus, an enveloped virus, is similar to virus-like particles with a diameter of about 100 nm (see Wang.C et al. (2017), MERS-CoV virus-like particles produced in insect cells produce specific humoural and cellular imminity in rhesus macaques, Oncotarget. 8 (8):12686-12694.).
  • MERS-CoV virus-like particles prepared by this method have good performance, they cannot be purified, and cannot be uniform in nature, and are not feasible for vaccine production; and if the mRNA vaccine is used to achieve the three types
  • the simultaneous expression of antigens can completely avoid the defects of the baculovirus expression system.
  • the molar ratio of the dS protein to the mRNA of the 2019-nCoV RBD-dS fusion protein containing the signal peptide is 1:1.
  • the molar ratio of the dS protein to the mRNA of the 2019-nCoV RBM-dS fusion protein containing the signal peptide is 1:1.
  • the signal peptide encoded by the mRNA is a signal peptide that can be recognized by a human cell, such as a signal peptide of tissue plasminogen activator (tPA) or a signal of serum immunoglobulin E (lgE). Peptides and so on. More preferably, it is tPA.
  • tissue plasminogen activator tPA
  • LgE serum immunoglobulin E
  • the 190th amino acid of the RBD is a site that is not easily accessible in the full-length S protein, but there is indeed a prominent site in the RBD structure, which is very easy to contact.
  • the proline at this site is mutated to aspartic acid Acid, can introduce N-linked glycosylation motif: aspartic acid-non-proline-threonine.
  • the proline at position 190 at the mutation site in B) is mutated to aspartic acid.
  • the amino acid mutation in B) is introduced into the N-linked glycosylated 2019-nCoV coronavirus.
  • the amino acid sequence of the RBD of the 2019-nCoV coronavirus is as shown in SEQ ID NO:1 or with SEQ ID NO:1 Amino acid sequences with 70%, 75%, 80%, 85%, 90%, 95%, 99% identity.
  • the mRNA encoding the S protein and M protein in C) can have the following forms: I) two mRNAs, one encoding the S protein and the other encoding the M protein; II) one mRNA, There are two coding regions encoding S protein and M protein respectively.
  • the mRNA encoding the S protein and E protein in C) can have the following forms: i) two mRNAs, one encoding the S protein and the other encoding the E protein; ii) one mRNA, There are two coding regions encoding S protein and E protein respectively.
  • the mRNA encoding the three proteins of S protein, M protein and E protein in C) can have the following forms: a) Three mRNAs, respectively encoding S protein, M protein and E protein; b) Two mRNAs, one encodes S protein, and the second has two coding regions, which encode M protein and E protein, respectively; c) One mRNA has three coding regions that encode S protein, M protein and E protein, respectively.
  • the amino acid sequence of the dS protein is as SEQ ID NO: 2 or with SEQ ID NO: 2 having 70%, 75%, 80%, 85%, 90%, 95%, Amino acid sequence with 99% identity.
  • the mRNA encoding the signal peptide, dS protein and the fusion protein of dS protein and RBD in D) can have the following forms: (1) Two mRNAs, one encoding the dS protein and the other encoding the signal Peptide and fusion protein of dS protein and RBD; (2) An mRNA with two coding regions respectively encoding dS protein, signal peptide and fusion protein of dS protein and RBD.
  • the amino acid sequence of the fusion protein of dS protein and RBD is as SEQ ID NO: 3 or 70%, 75%, 80%, 85%, 90% with SEQ ID NO: 3 %, 95%, 99% identical amino acid sequences.
  • the mRNA encoding the signal peptide, dS protein and the fusion protein of dS protein and RBM in E) can have the following forms: 1) Two mRNAs, one encoding the dS protein and the other encoding the signal peptide And the fusion protein of dS protein and RBM; 2) an mRNA with two coding regions respectively encoding dS protein, signal peptide and fusion protein of dS protein and RBM.
  • the amino acid sequence of the fusion protein of the dS protein and RBM is as SEQ ID NO: 4 or 70%, 75%, 80%, 85%, 90% with SEQ ID NO: 4 %, 95%, 99% identical amino acid sequences.
  • the amino acid sequence of the S protein is such as SEQ ID NO: 5 or with SEQ ID NO: 5 having 70%, 75%, 80%, 85%, 90%, 95%, Amino acid sequence with 99% identity.
  • the amino acid sequence of the M protein is such as SEQ ID NO: 6 or with SEQ ID NO: 6 having 70%, 75%, 80%, 85%, 90%, 95%, Amino acid sequence with 99% identity.
  • the amino acid sequence of the E protein is as SEQ ID NO: 7 or with SEQ ID NO: 7 having 70%, 75%, 80%, 85%, 90%, 95%, Amino acid sequence with 99% identity.
  • the mRNA is monocistronic, bicistronic or polycistronic mRNA.
  • the bicistronic or polycistronic mRNA is an mRNA containing two or more coding regions.
  • the coding region of the bicistronic or polycistronic mRNA can be separated by at least one internal ribosome entry site (IRES) sequence
  • IRES sequence includes but 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 Classical swine fever virus (e.g. CSFV), mouse white spot virus (e.g. MLV), simian immunodeficiency virus (e.g. SIV) or cricket paralysis virus (e.g. CrPV).
  • picornavirus e.g. FMDV
  • CFFV pestivirus
  • polio virus e.g. PV
  • encephalomyocarditis virus e.g. ECMV
  • the mRNA is composed of several structural elements, that is, in addition to the coding region, the mRNA also includes a 5'cap structure, a 5'non-coding region, a 3'non-coding region and/or polyadenosine The acid tail mRNA sequence.
  • the length of the mRNA sequence is 200-10000 nucleotides. Further preferably, the length of the mRNA sequence is 500-8000 nucleotides.
  • composition comprising mRNA, said mRNA comprising mRNA encoding the coding region of at least one antigenic peptide or protein of the 2019-nCoV coronavirus or a fragment, variant or derivative thereof sequence.
  • the antigen peptide or protein or fragment, variant or derivative thereof is selected from any one of the following:
  • S protein of 2019-nCoV coronavirus preferably, it also includes M protein and/or E protein of 2019-nCoV coronavirus; specifically (1) S protein and M protein of 2019-nCoV coronavirus (2) The combination of S protein and E protein of 2019-nCoV coronavirus; (3) The combination of S protein, M protein and E protein of 2019-nCoV coronavirus;
  • the signal peptide encoded by the mRNA encodes a human cell recognizable secretion signal peptide, such as the signal peptide of tissue plasminogen activator (tPA) or the signal peptide of serum immunoglobulin E (lgE), etc. . More preferably, it is tPA.
  • the 190th amino acid of the RBD is a site that is not easily accessible in the full-length S protein, but there is indeed a prominent site in the RBD structure, which is very easy to contact.
  • the proline at this site is mutated to aspartic acid Acid, can introduce N-linked glycosylation motif: aspartic acid-non-proline-threonine.
  • the proline at position 190 in B) is mutated to aspartic acid.
  • the amino acid sequence of the RBD of the N-linked glycosylated 2019-nCoV coronavirus introduced in B) has an amino acid sequence such as SEQ ID NO:1 or 70% or 75% of SEQ ID NO:1. , 80%, 85%, 90%, 95%, 99% identical amino acid sequences.
  • the mRNA encoding the S protein and M protein in C) can have the following forms: I) two mRNAs, one encoding the S protein and the other encoding the M protein; II) one mRNA, There are two coding regions encoding S protein and M protein respectively.
  • the mRNA encoding the S protein and E protein in C) can have the following forms: i) two mRNAs, one encoding the S protein and the other encoding the E protein; ii) one mRNA, There are two coding regions encoding S protein and E protein respectively.
  • the mRNA encoding the three proteins of S protein, M protein and E protein in C) can have the following forms: a) Three mRNAs, respectively encoding S protein, M protein and E protein; b) Two mRNAs, one encodes S protein, and the second has two coding regions, which encode M protein and E protein, respectively; c) One mRNA has three coding regions that encode S protein, M protein and E protein, respectively.
  • amino acid sequence of the dS protein is SEQ ID NO: 2 or an amino acid having 70%, 75%, 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 2 sequence.
  • the mRNA encoding the signal peptide, dS protein and the fusion protein of dS protein and RBD in D) can have the following forms: (1) Two mRNAs, one encoding the dS protein and the other encoding the signal Peptide and fusion protein of dS protein and RBD; (2) An mRNA with two coding regions respectively encoding dS protein, signal peptide and fusion protein of dS protein and RBD.
  • amino acid sequence of the fusion protein of the dS protein and RBD is as SEQ ID NO: 3 or 70%, 75%, 80%, 85%, 90%, 95%, 99% with SEQ ID NO: 3 % Identity of the amino acid sequence.
  • the mRNA encoding the signal peptide, dS protein and the fusion protein of dS protein and RBM in E) can have the following forms: 1) Two mRNAs, one encoding the dS protein and the other encoding the signal peptide And the fusion protein of dS protein and RBM; 2) an mRNA with two coding regions respectively encoding dS protein, signal peptide and fusion protein of dS protein and RBM.
  • amino acid sequence of the fusion protein of the dS protein and RBM is as SEQ ID NO: 4 or 70%, 75%, 80%, 85%, 90%, 95%, 99% with SEQ ID NO: 4 % Identity of the amino acid sequence.
  • amino acid sequence of the S protein is SEQ ID NO: 5 or an amino acid that is 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 5 sequence.
  • the amino acid sequence of the M protein is SEQ ID NO: 6 or an amino acid that is 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 6 sequence.
  • the amino acid sequence of the E protein is SEQ ID NO: 7 or an amino acid that is 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO: 7 sequence.
  • the mRNA is monocistronic, bicistronic or polycistronic mRNA.
  • the bicistronic or polycistronic mRNA is an mRNA containing two or more coding regions.
  • the coding region of the bicistronic or polycistronic mRNA can be separated by at least one internal ribosome entry site (IRES) sequence
  • IRES sequence includes but 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 Classical swine fever virus (e.g. CSFV), mouse white spot virus (e.g. MLV), simian immunodeficiency virus (e.g. SIV) or cricket paralysis virus (e.g. CrPV).
  • picornavirus e.g. FMDV
  • CFFV pestivirus
  • polio virus e.g. PV
  • encephalomyocarditis virus e.g. ECMV
  • the mRNA is composed of several structural elements, that is, in addition to the coding region, the mRNA also includes a 5'cap structure, a 5'non-coding region, a 3'non-coding region and/or polyadenosine The acid tail mRNA sequence.
  • the length of the mRNA sequence is 200-10000 nucleotides. Further preferably, the length of the mRNA sequence is 500-8000 nucleotides.
  • the composition 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 composition also contains lipids.
  • the lipids include, but are not limited to, lipids that can promote self-assembly to form virus-sized particles ( ⁇ 100nm), lipids that enable mRNA to be released from endosomes into cells, and support phospholipid bilayer structure Lipids or lipids used as stabilizers.
  • the lipids may also include PEGylated lipids.
  • the lipids comprise cationic lipids, PEGylated lipids, cholesterol and/or phospholipids.
  • the mRNA or composition of the present invention may also include pharmaceutically acceptable excipients.
  • the pharmaceutically acceptable excipients may be carriers, diluents, adjuvants or nucleotide sequences encoding adjuvants, solubilizers, binders, lubricants, suspending agents, transfection promoters, 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, lipids, calcium ions, viral proteins, cations, polycations (such as 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
  • the composition of the present invention can be a lipid, a lipid complex or a lipid nanoparticle.
  • the lipid may be a lipid prepared in the following form: 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) lipid, 1,2-dilinoleyloxy Benzyl-3-dimethylaminopropane (DLin-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane ( DLin-KC2-DMA) lipids.
  • DODMA 1,2-dioleyloxy-N,N-dimethylaminopropane
  • DLin-DMA 1,2-dilinoleyloxy Benzyl-3-dimethylaminopropane
  • DLin-KC2-DMA 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-di
  • 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 third aspect of the present invention provides a protein encoded by the mRNA or composition of the present invention.
  • the fourth aspect of the present invention provides an amino acid mutation introduced into the N-linked glycosylated 2019-nCoV type coronavirus RBD protein.
  • the RBD of the 2019-nCoV coronavirus does not contain a signal peptide region.
  • the fifth aspect of the present invention provides a fusion protein of two or three of the S protein, M protein or E protein of the 2019-nCoV coronavirus.
  • the sixth aspect of the present invention provides a fusion protein of dS protein and RBD or RBM.
  • the RBD and RBM do not contain a signal peptide region.
  • the seventh aspect of the present invention provides a nucleic acid encoding the protein or fusion protein of the third to sixth aspects.
  • the sequence of the nucleic acid is the nucleotide sequence of the epitope antigen gene of the protein or fusion protein.
  • the eighth aspect of the present invention provides a vector containing the nucleic acid of the seventh aspect of the present invention.
  • the ninth aspect of the present invention provides a cell containing the mRNA, the protein or fusion protein, the nucleic acid and/or the vector of the present invention.
  • the tenth aspect of the present invention provides a method for preparing an mRNA-containing composition, which includes mixing the mRNA with a cationic or polycationic compound and then packaging it with lipids.
  • the lipids include, but are not limited to, lipids that can promote self-assembly to form virus-sized particles ( ⁇ 100nm), lipids that enable mRNA to be released from endosomes into cells, and support phospholipid bilayer structure Lipids or lipids 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 eleventh aspect of the present invention provides the application of the mRNA or the composition containing mRNA in the prevention and/or treatment of 2019-nCoV type coronavirus infection.
  • the twelfth aspect of the present invention provides the application of the mRNA or the composition containing the mRNA in the preparation of a medicine for preventing and/or treating 2019-nCoV type coronavirus infection.
  • the prevention includes using the mRNA or the composition containing the mRNA of the present invention as a vaccine.
  • the treatment includes screening for antibodies that bind to the mRNA or mRNA-containing composition of the present invention, and using it for treatment.
  • the thirteenth aspect of the present invention provides a method for preventing 2019-nCoV type coronavirus infection, comprising administering an effective amount of the mRNA of the present invention or a combination comprising mRNA to an individual who is not infected with the 2019-nCoV type coronavirus Things.
  • the fourteenth aspect of the present invention provides a method for treating 2019-nCoV type coronavirus infection, comprising applying an effective amount of the mRNA of the present invention or a composition containing mRNA to an individual infected with 2019-nCoV type coronavirus . This allows individuals to produce neutralizing antibodies against the 2019-nCoV coronavirus.
  • the fifteenth aspect of the present invention provides a method for antibody screening, which includes the step of administering to an individual an effective amount of the mRNA or a composition containing mRNA of the present invention.
  • 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 sixteenth aspect of the present invention provides a method for inducing an individual to neutralize an antigen-specific immune response, which comprises administering the mRNA of the present invention or a composition comprising mRNA to the individual.
  • the antigen-specific immune response includes T cell response and/or B cell response.
  • the mRNA or the composition containing mRNA of the present invention has the following advantages: 1. In vitro synthesis, no cell culture, no risk of animal source contamination; 2. Faster R&D and production, standardized production, easy 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, extending the exposure time of the antigen to improve the intensity and quality of the immune response; 4. Simulating the process of natural infection, it is translated and modified in human cells , Can be presented by MHC class I molecules to induce stronger cellular immunity; 5.
  • the "S protein” in the present invention is a structural protein that composes the 2019-nCoV type coronavirus, and is named as a spike protein.
  • the “RBD” in the present invention is a structural protein constituting the 2019-nCoV type coronavirus, and its name is the spike protein receptor binding domain.
  • the RBD includes a core structure and a spike protein receptor binding motif, and the spike protein receptor binding motif is the "RBM" described in the present invention.
  • the "M protein” in the present invention is a structural protein that composes the 2019-nCoV type coronavirus, and the name is envelope protein.
  • the "E protein” in the present invention is a structural protein constituting the 2019-nCoV type coronavirus, and the name is small envelope protein.
  • the "dS protein" of the present invention is a small surface protein of duck hepatitis B virus.
  • the "LNP” in the present invention is a lipid nanoparticle.
  • 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%,
  • 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 all behaviors of avoiding symptoms or delaying the tension of specific symptoms by administering the product of the present invention before or after the disease begins to develop.
  • treatment refers to therapeutic intervention to improve the signs, symptoms, etc. of the disease or pathological state after the disease has begun to develop.
  • 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.
  • Figure 1 The RBD part and S full-length protein of 2019-nCoV were simulated by the protein structure prediction of RaptorX. The figure shows the target mutation position of the amino acid in the predicted protein structure.
  • Figure 2 Sequence diagram of the basic plasmid template containing T7 promoter, 5'UTR, 3'UTR, and polyA tail.
  • Figure 3 Sequence sequencing results of RBD encoding wild-type 2019-nCoV containing tPA signal peptide.
  • Figure 4 Sequence sequencing results of the RBD encoding 2019-nCoV with the 190 mutation of the tPA signal peptide.
  • Figure 5 The sequencing results of the sequence encoding the wild-type 2019-nCoV M protein.
  • Figure 6 Sequencing results of the sequence encoding wild-type 2019-nCoV E protein.
  • Fig. 7 The splicing of Fig. 7A and Fig. 7B is the sequencing result of the sequence encoding the wild-type 2019-nCoV S protein.
  • Figure 8 Sequencing results of the 2019-nCoV RBD-dS fusion protein encoding the tPA signal peptide.
  • Figure 9 Sequencing results of the 2019-nCoV RBM-dS fusion protein encoding the tPA signal peptide.
  • Figure 10 The sequencing results of the sequence encoding the dS protein.
  • Figure 11 The results of PCR amplification of the mRNA prepared in the example with primers containing homology to the basic plasmid template, where RBD-M is the mRNA encoded by the N-linked glycosylation after the 190 mutation is introduced, and SF is The mRNA encoding the full-length S protein.
  • Figure 12 Detection of capped and purified mRNA with formaldehyde denaturing gel
  • M is Marker, where 1 is mRNA encoding 2019-nCoV RBD-dS fusion protein containing tPA signal peptide, and 2 is 2019-nCoV encoding 2019-nCoV containing tPA signal peptide
  • the mRNA of the RBM-dS fusion protein 3 is the mRNA encoding the wild-type 2019-nCoV S protein
  • 4 is the mRNA encoding the wild-type 2019-nCoV M protein
  • 5 is the mRNA encoding the wild-type 2019-nCoV E protein
  • 6 is the encoding
  • 7 is the mRNA encoding the wild-type 2019-nCoV RBD containing the tPA signal peptide
  • 8 is the mRNA encoding the dS protein
  • Figure 13 WB (Western Blot, Western Blot) expression detection results, where M is Marker, 1 is the expression supernatant of mRNA encoding the wild-type 2019-nCoV RBD containing the tPA signal peptide, and 2 is the mRNA encoding the tPA signal Mutation of peptide at position 190 and introduction of N-linked glycosylated RBD mRNA expression supernatant of 2019-nCoV, 3 is a negative control.
  • M Marker
  • 1 is the expression supernatant of mRNA encoding the wild-type 2019-nCoV RBD containing the tPA signal peptide
  • 2 is the mRNA encoding the tPA signal
  • 3 is a negative control.
  • Figure 14 Results of immunofluorescence detection of S protein expression by anti-S protein polyclonal antibodies.
  • FIG. 15 DLS (Dynamic Light Scattering, dynamic light scattering) detects the particle size and particle size distribution of mRNA-LNP (marked by the polymer dispersion index PDI (Polymer dispersion index)), where A is the encoding wild-type 2019- nCOV S protein mRNA-LNP, B is the mRNA-LNP encoding the wild-type 2019-nCOV RBD containing the tPA signal peptide, and C is the 2019-LNP encoding the mutation at position 190 containing the tPA signal peptide and introducing N-linked glycosylation.
  • PDI Polymer dispersion index
  • Figure 16 S protein specific antibody titers after the first and second immunizations, where Negative is the negative control, SF is the mRNA-LNP encoding the wild-type 2019-nCOV S protein, and RBD is the wild-type encoding the tPA signal peptide
  • the mRNA-LNP of the RBD of 2019-nCOV, RBD-M encodes the mRNA-LNP of the RBD of 2019-nCOV that contains a mutation at position 190 of the tPA signal peptide and introduces N-linked glycosylation.
  • FIG. 17 ELISpot (enzyme-linked immunospot method) detection results of interferon gamma, where Negative is the negative control, SF is the mRNA-LNP encoding wild-type 2019-nCOV S protein, and RBD is the wild-type encoding the tPA signal peptide
  • the mRNA-LNP of the RBD of 2019-nCOV, RBD-M encodes the 190 mutation of the tPA signal peptide and the introduction of N-linked glycosylation of the mRNA-LNP of the RBD of 2019-nCOV, the ordinate is per million spleen cells Spot forming unit (SFU).
  • Figure 18 Neutralizing antibody titer after the second immunization, where SF is mRNA-LNP encoding wild-type 2019-nCOV S protein, and RBD is mRNA-LNP, RBD encoding wild-type 2019-nCOV RBD containing tPA signal peptide -M encodes mRNA-LNP containing the mutation at position 190 of the tPA signal peptide and introducing N-linked glycosylated 2019-nCOV RBD, sVNT stands for alternative virus neutralization test.
  • Figure 19 WB detection of cell supernatant, where 1 is the negative control, 2 is the expression supernatant of VLP-1, 3 is the expression supernatant of VLP-2, and 4 is the mRNA encoding the wild-type 2019-nCoV S protein Express the supernatant.
  • Figure 20 DLS detects the particle size and particle size distribution of mRNA-LNP, where A represents VLP-1-LNP, B represents VLP-2-LNP, and C represents mRNA-LNP encoding wild-type 2019-nCOV S protein.
  • Figure 21 Formaldehyde denaturing glue detects the mRNA integrity of the packaged sample, where M is Marker, 1 is VLP-1-LNP, 2 is VLP-2-LNP, and 3 is mRNA encoding wild-type 2019-nCOV S protein- LNP.
  • Figure 22 S protein-specific antibody titers after the first and second immunizations, where Negative is the negative control, and S-F is the mRNA-LNP encoding the wild-type 2019-nCOV S protein.
  • Figure 23 Results of interferon gamma detection by ELISpot, where Negative is a negative control, SF is mRNA-LNP encoding wild-type 2019-nCOV S protein, and the ordinate is spot forming unit per million spleen cells. SFU).
  • Figure 24 Cross-validation results of intracellular CK staining method, where Negative is a negative control, and S-F is mRNA-LNP encoding wild-type 2019-nCOV S protein.
  • Figure 25 The particle size and size distribution of mRNA-LNP detected by DLS.
  • the left image represents VLP-3-LNP
  • the middle image represents VLP-4-LNP
  • the right image represents mRNA encoding wild-type 2019-nCOV S protein. LNP.
  • Figure 26 S protein specific antibody titers after the first and second immunizations, where Negative is the negative control, and S-F is the mRNA-LNP encoding the wild-type 2019-nCOV S protein.
  • Figure 27 Results of interferon gamma detection by ELISpot, where Negative is a negative control, SF is mRNA-LNP encoding wild-type 2019-nCOV S protein, and the ordinate is spot forming unit per million spleen cells. SFU).
  • Figure 28 Cross-validation results of intracellular CK staining method, where Negative is a negative control, and S-F is mRNA-LNP encoding wild-type 2019-nCOV S protein.
  • 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.
  • FIG. 3 shows the sequencing result of the RBD encoding the wild-type 2019-nCoV containing the tPA signal peptide.
  • the nucleotide sequence is shown in SEQ ID NO: 9 and the amino acid sequence is shown in SEQ ID NO: 17.
  • Figure 4 shows the sequencing results of the RBD of 2019-nCoV (see Figure 1 for the location) encoding the 190-mutated 2019-nCoV containing the tPA signal peptide.
  • the nucleotide sequence is shown in SEQ ID NO: 10
  • the amino acid sequence is shown in SEQ ID NO: 18 shown.
  • Figure 5 shows the sequencing results of the sequence encoding the wild-type 2019-nCoV M protein.
  • the nucleotide sequence is shown in SEQ ID NO: 11, and the amino acid sequence is shown in SEQ ID NO: 6.
  • Figure 6 shows the sequencing results of the sequence encoding the wild-type 2019-nCoV E protein, the nucleotide sequence is shown in SEQ ID NO: 12, and the amino acid sequence is shown in SEQ ID NO: 7.
  • Fig. 7 shows the sequencing result of the sequence encoding the wild-type 2019-nCoV S protein.
  • the nucleotide sequence is shown in SEQ ID NO: 13, and the amino acid sequence is shown in SEQ ID NO: 5.
  • Figure 8 shows the sequencing results of the 2019-nCoV RBD-dS fusion protein encoding the tPA signal peptide.
  • the nucleotide sequence is shown in SEQ ID NO: 14 and the amino acid sequence of the RBD-dS fusion protein without the signal peptide As shown in SEQ ID NO: 3.
  • Figure 9 shows the sequencing results of the 2019-nCoV RBM-dS fusion protein encoding the tPA signal peptide.
  • the nucleotide sequence is shown in SEQ ID NO: 15, and the 2019-nCoV RBM-dS fusion without the signal peptide
  • the amino acid sequence of the protein is shown in SEQ ID NO: 4.
  • Fig. 10 shows the sequencing result of the sequence encoding the dS protein. Its amino acid sequence is shown in SEQ ID NO: 2 and its nucleotide sequence is shown in SEQ ID NO: 16.
  • 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 to obtain the uncapped mRNA transcribed from SEQ ID NO: 9-16.
  • 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.
  • Pave 4 wells of HEK293 cells in a 24-well plate of which wells 1, 2, and 3 were transfected with lipofectamine 2000, respectively, with 0.5 ⁇ g capped and purified mRNA encoding wild-type 2019-nCoV RBD containing tPA signal peptide, encoding Containing the mutation at position 190 of the tPA signal peptide and introducing N-linked glycosylated RBD mRNA of 2019-nCoV and mRNA encoding wild-type 2019-nCoV S protein, lipofectamine 2000 transfection reagent was added to well 4 as a negative control.
  • mRNA flow rate ratio of 1:3 the above three types of mRNA were mixed and packaged in Precision Nanosystems' Nanoparticle Preparation Instrument Benchtop.
  • the packaged mRNA-LNP was dialyzed and concentrated into DPBS by ultrafiltration, and then aseptically filtered to obtain samples for animal experiments.
  • DLS was used to detect the particle size and particle size distribution of mRNA-LNP (marked by the polymer dispersibility index PDI).
  • the test results are shown in Figure 15.
  • the particle size of the packaged samples are all around 80nm, and the PDI is less than 0.2.
  • mRNA-LNP encoding wild-type 2019-nCOV S protein average particle size 78.83nm, PDI value 0.028, intercept (intercept) 0.953, see Table 1 for details; encoding wild-type 2019-nCOV containing tPA signal peptide RBD mRNA-LNP: average particle size 83.13nm, PDI value 0.013, intercept (intercept) 0.978, see Table 2 for details; encoding the 190 mutation containing tPA signal peptide and introducing N-linked glycosylation 2019-nCOV MRNA-LNP of RBD: average particle size of 83.41nm, PDI value of 0.031, intercept (intercept) 0.978, see Table 3 for details.
  • Table 1 mRNA-LNP encoding wild-type 2019-nCOV S protein
  • BALB/c female mice about 6 weeks old were randomly divided into groups of 6 and divided into 4 groups. After immunization on day 0 and day 28, the leg muscles were inoculated with 10ug, the S protein-specific antibody titer was detected on day 28 and day 42, cytokine was detected on day 42 and tested by competitive ELISA method. And antibody titers.
  • the S protein specific antibody titers after the first immunization and the second immunization are shown in Figure 16. It can be seen that both RBD designs can quickly stimulate the specificity that is significantly higher than that of the wild-type S full-length protein design during the first immunization. After the second immunization, there was no significant difference in the specific antibody titers of the three.
  • the results of detecting interferon ⁇ with ELISpot are shown in Figure 17. It can be seen that the cellular immunity level caused by the two RBD designs is significantly higher than that of the wild-type S full-length protein design, while the cellular immunity level of the RBD design with the introduction of glycosylation is significantly higher. Higher than wild-type RBD design.
  • the neutralizing antibody titer after the second immunization is shown in Figure 18, which shows that the neutralizing antibody level of the two RBD designs is significantly higher than that of the wild-type S when there is no significant difference in the specific antibody titer of the three designs.
  • the full-length protein design, and the introduction of glycosylation RBD design, the level of neutralizing antibody was significantly higher than that of wild-type RBD design.
  • the RBD design contains the main neutralizing epitopes, and the non-neutralizing epitopes are relatively small, resulting in a high proportion of neutralizing antibodies and higher safety; and the RBD design with N-linked glycosylation introduced by amino acid mutations Masking the exposed non-neutralizing epitopes that may produce immunodominance, making the proportion of neutralizing antibodies higher, can achieve better results in the prevention of 2019-nCoV coronavirus infection.
  • Example 2 2) mRNA encoding wild-type 2019-nCoV S, E and M proteins prepared in Example 1. These three kinds of mRNA were mixed at a molar ratio of 1:1:1 and a mass ratio of 1:1:1 to obtain the mixed mRNA, which is abbreviated as VLP-1 and VLP-2.
  • mRNA flow rate ratio of 1:3, VLP-1, VLP-2 and mRNA encoding wild-type 2019-nCoV S protein were mixed and packaged in Precision Nanosystems' Nanoparticle Preparation Instrument Benchtop.
  • the packaged mRNA-LNP was dialyzed and concentrated by ultrafiltration into DPBS (Dulbecco's Phosphate Buffered Saline), and after aseptic filtration, samples for animal experiments were obtained. DLS was used to detect the particle size and particle size distribution of mRNA-LNP. The test results are shown in Figure 20.
  • the particle size of the packaged samples are all around 80nm, and the PDI is less than 0.2.
  • VLP-1-LNP average particle size 81.44nm, PDI value 0.023, intercept (intercept) 0.978, see Table 4 for details
  • VLP-2-LNP average particle size 82.59nm, PDI value 0.038, intercept (intercept) 0.977, see Table 5 for details
  • mRNA-LNP encoding wild-type 2019-nCOV S protein average particle size 84.77nm, PDI value 0.030, intercept (intercept) 0.957, see Table 6 for details.
  • mice about 6 weeks old were randomly divided into groups of 6 and divided into 4 groups.
  • the leg muscles were inoculated with 10ug 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 and second immunizations are shown in Figure 22. It can be seen that VLP-2 failed to cause high levels of specific antibody titers, but the VLP-1 design and the S full-length protein design There was no significant difference in specific antibody titers during the first and second immunizations.
  • the results of detecting interferon ⁇ with ELISpot are shown in Figure 23. It can be seen that the Th1 cellular immune level caused by the VLP-1 design is significantly higher than that of the wild-type S full-length protein design, and this result is also performed by the intracellular CK staining method. Cross-validation is implemented, as shown in Figure 24.
  • the mRNA encoding wild-type 2019-nCoV S, E, and M proteins can be immunized at a molar ratio of 1:1:1 and can obtain no less than wild-type S full-length protein designed humoral immune response and significantly higher than wild-type
  • the cellular immune response designed by the S full-length protein can achieve better results in the prevention of 2019-nCoV coronavirus infection.
  • the protein expressed by the mRNA vaccine in the animal body will automatically form a virus-like particle similar to the 2019-nCoV coronavirus. At the same time, it avoids the problem of uneven nature of the expression products of the in vitro expression system, and is suitable for mass production as a vaccine.
  • VLP-4 The mRNA encoding the dS protein and the 2019-nCoV RBM-dS fusion protein containing the tPA signal peptide was mixed at a molar ratio of 1:1 to obtain the mixed mRNA, which is abbreviated as VLP-4.
  • mRNA flow rate ratio of 1:3, VLP-3, VLP-4 and mRNA encoding wild-type 2019-nCoV S protein were mixed and packaged in Precision Nanosystems' Nanoparticle Preparation Instrument Benchtop.
  • the packaged mRNA-LNP was dialyzed and concentrated into DPBS by ultrafiltration, and then aseptically filtered to obtain samples for animal experiments. DLS was used to detect the particle size and particle size distribution of mRNA-LNP.
  • the test results are shown in Figure 25.
  • the particle size of the packaged samples are all around 85nm, and the PDI is less than 0.2.
  • VLP-3-LNP average particle size 84.19nm, PDI value 0.018, intercept (intercept) 0.954, see Table 7 for details
  • VLP-4-LNP average particle size 82.16nm, PDI value 0.018, intercept (intercept) 0.959, see Table 8 for details
  • mRNA-LNP encoding wild-type 2019-nCOV S protein average particle size 84.77nm, PDI value 0.030, intercept (intercept) 0.957, see Table 6 for details.
  • the S protein specific antibody titers after the first and second immunizations are shown in Figure 26. It can be seen that VLP-4 failed to cause high levels of specific antibody titers, but the VLP-3 design and the S full-length protein design There was no significant difference in specific antibody titers during the first and second immunizations.
  • the results of detecting interferon ⁇ with ELISpot are shown in Figure 27. It can be seen that the Th1 cellular immunity level caused by the VLP-3 design is significantly higher than that of the wild-type S full-length protein design, and this result is also performed by the intracellular CK staining method. Cross-validation is implemented, as shown in Figure 28.
  • the mRNA encoding the dS protein and the 2019-nCoV RBD-dS fusion protein containing the tPA signal peptide can be mixed and packaged at a molar ratio of 1:1 to obtain a humoral immune response that is not lower than the wild-type S full-length protein design and is highly significant
  • the cellular immune response designed on the wild-type S full-length protein can achieve better results in the prevention of 2019-nCoV coronavirus infection.
  • the protein expressed by the mRNA vaccine in the animal body will automatically form virus-like particles. At the same time, it avoids the difficulty of purification of the expression product of the in vitro expression system, and is suitable for mass production as a vaccine.
  • the mRNA vaccine prepared in Example 2-4 can be used for the prevention of 2019-nCoV type coronavirus infection, and the protein expressed by the mRNA vaccine in animals will automatically form a protein similar to 2019-nCoV type coronavirus.
  • Virus-like particles induce stronger cellular immunity, and there are few or no non-neutralizing antibodies in the body.
  • the production cycle is short, and it can be purified to achieve uniform properties, which is suitable for mass production of vaccines.

Abstract

本发明提供了一种包含编码2019-nCoV型冠状病毒的至少一个抗原肽或蛋白或其片段、变体或衍生物的编码区的mRNA序列及其组合物。本发明还提供了该mRNA或组合物在制备预防和/或治疗2019-nCoV型冠状病毒感染的药物，尤其是疫苗中的应用。

Description

用于2019-nCoV型冠状病毒mRNA疫苗、制备方法及其应用 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2019-nCoV型冠状病毒的至少一个抗原肽或蛋白或其片段、变体或衍生物的编码区的mRNA序列，本发明涉及包含一个或多个mRNA的组合物。以及所述mRNA或组合物在制备预防和/或治疗2019-nCoV型冠状病毒感染的药物(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(2019-nCoV)。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等严重的副反应，比如增加发病率，引起肝部组织强烈炎症反应及肝损伤(“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2019-nCoV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(“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2019-nCoV疫苗的最佳选择。
目前受2019-nCoV感染的患者的年龄绝大部分超过25岁，18岁以下的患者较少，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2019-nCoV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，提供了一种mRNA，所述的mRNA包含至少一种编码2019-nCoV型冠状病毒的至少一个抗原肽或蛋白或其片段、变体或衍生物的编码区的mRNA序列。其中，所述的抗原肽或蛋白或其片段、变体或衍生物能够在人体中诱发免疫反应，产生中和抗体。
优选的，所述的mRNA包含编码2019-nCoV型冠状病毒S蛋白的mRNA序列。进一步优选的，所述的mRNA包含编码2019-nCoV型冠状病毒的RBD的mRNA序列。
在本发明的一个具体实施方式中，所述的RBD的氨基酸序列中包含单个位点氨基酸突变并引入N-连接糖基化。优选的，所述的突变位点为第190位的脯氨酸突变为天冬氨酸。
优选的，所述的mRNA包含编码2019-nCoV型冠状病毒的RBM的mRNA序列。
优选的，所述的mRNA包含编码dS蛋白的mRNA序列。进一步优选的，所述的mRNA包含编码dS蛋白与RBD或RBM的融合蛋白的mRNA序列。
优选的，所述的mRNA包含编码M蛋白和/或E蛋白的mRNA序列。
进一步优选的，所述的mRNA编码的抗原肽或蛋白或其片段、变体或衍生物选自下列任一种：
A)信号肽和2019-nCoV型冠状病毒的RBD，其中，所述的2019-nCoV型冠状病毒的RBD不包含信号肽区域；RBD中包含了主要的中和表位，非中和表位相对较少，安全性高。
B)信号肽和氨基酸突变引入N-连接糖基化的2019-nCoV型冠状病毒的RBD，其中，所述的2019-nCoV型冠状病毒的RBD不包含信号肽区域；为避免脱离全长S蛋白暴露出原本埋在全长S蛋白 中的氨基酸序列，出现免疫优势的非中和表位，则通过氨基酸突变引入N-连接糖基化，以遮蔽非中和表位，并提高免疫原性。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2019-nCoV的RBD的序列分析，以及对其结构的模拟预测，认为2019-nCoV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.)。
C)2019-nCoV型冠状病毒的S蛋白；优选的，还包括2019-nCoV型冠状病毒的M蛋白和/或E蛋白；具体的为(1)2019-nCoV型冠状病毒的S蛋白和M蛋白的组合；(2)2019-nCoV型冠状病毒的S蛋白和E蛋白的组合；或(3)2019-nCoV型冠状病毒的S蛋白、M蛋白和E蛋白的组合。其中，在本发明的一个具体实施方式中，S蛋白、M蛋白与E蛋白的mRNA摩尔比为1：1：1。
同时，现有技术中也有类似的做法支持本技术方案，例如利用杆状病毒表达系统在昆虫细胞内同时表达MERS-CoV的S、M、E三个结构蛋白，表达出的蛋白会自动形成与冠状病毒这种包膜病毒类似的约100nm直径的类病毒颗粒(见Wang.C等(2017)，MERS-CoV virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques，Oncotarget.8(8):12686-12694.)。然而，利用此种方法制备的MERS-CoV病毒样颗粒虽然有很好的表现，但是无法纯化，且无法做到性质均一，不具备疫苗生产的可行性；而如果通过mRNA疫苗来实现对三种抗原的同时表达，则可以完全避开杆状病毒表达系统的缺陷。
D)信号肽、dS蛋白以及dS蛋白与RBD的融合蛋白的组合，其中，所述的RBD不包含信号肽区域。其中，在本发明的一个具体实施方式中，dS蛋白与包含信号肽的2019-nCoV RBD-dS融合蛋白的mRNA的摩尔比为1:1。
同时，现有技术中也有类似的做法支持本技术方案，例如已有研究在酵母中共表达dS和dS融合蛋白，可形成直径约20nm的类包膜病毒颗粒(见Wetzel.C等(2018)，Establishment of a yeast-based VLP platform for antigen presentation，Microb Cell Fact.5；17(1):17.)。然而，利用此方法制备的病毒颗粒无法纯化，一致性难以保证，但是本发明技术方案采用mRNA疫苗的形式，有效避免了这种情况的发生。
或E)信号肽、dS蛋白以及dS蛋白与RBM的融合蛋白的组合，其中，所述的RBM不包含信号肽区域。其中，在本发明的一个具体实施方式中，dS蛋白与包含信号肽的2019-nCoV RBM-dS融合蛋 白的mRNA的摩尔比为1:1。
更进一步优选的，所述的mRNA编码的信号肽为编码一个人类细胞可识别分泌信号肽，如组织型纤溶酶原激活剂(tPA)的信号肽或者血清免疫球蛋白E(lgE)的信号肽等。进一步优选为tPA。
所述的RBD第190位氨基酸为在全长S蛋白中不易接触的位点，但在RBD结构中确实一个突出的位点，极易接触，将该位点的脯氨酸突变为天冬氨酸，可以引入N-连接糖基化基序：天冬氨酸-非脯氨酸-苏氨酸。最为优选的，所述B)中突变位点为第190位的脯氨酸突变为天冬氨酸。
在本发明的一个具体实施方式中，所述的B)中氨基酸突变引入N-连接糖基化的2019-nCoV型冠状病毒的RBD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。
更进一步优选的，所述的编码C)中S蛋白、M蛋白两种蛋白的mRNA可以有以下几种形式：I)两条mRNA，一条编码S蛋白，一条编码M蛋白；II)一条mRNA，有两个编码区分别编码S蛋白和M蛋白。
更进一步优选的，所述的编码C)中S蛋白、E蛋白两种蛋白的mRNA可以有以下几种形式：i)两条mRNA，一条编码S蛋白，一条编码E蛋白；ii)一条mRNA，有两个编码区分别编码S蛋白和E蛋白。
更进一步优选的，所述的编码C)中S蛋白、M蛋白和E蛋白三种蛋白的mRNA可以有以下几种形式：a)三条mRNA，分别编码S蛋白、M蛋白及E蛋白；b)两条mRNA，一条编码S蛋白，第二条有两个编码区，分别编码M蛋白和E蛋白；c)一条mRNA，有三个编码区分别编码S蛋白，M蛋白和E蛋白。
在本发明的一个具体实施方式中，所述的dS蛋白的氨基酸序列如SEQ ID NO：2或与SEQ ID NO：2具有70％、75％、80％、85％、90％、95％、99％同一性的氨基酸序列。
更进一步优选的，所述的编码D)中信号肽、dS蛋白以及dS蛋白与RBD的融合蛋白的mRNA可以有以下几种形式：(1)两条mRNA，一条编码dS蛋白，另一条编码信号肽和dS蛋白与RBD的融合蛋白；(2)一条mRNA，有两个编码区分别编码dS蛋白、信号肽和dS蛋白与RBD的融合蛋白。
在本发明的一个具体实施方式中，所述的dS蛋白与RBD的融合蛋白的氨基酸序列如SEQ ID NO：3或与SEQ ID NO：3具有70％、75％、80％、85％、90％、95％、99％同一性的氨基酸序列。
更进一步优选的，所述的编码E)中信号肽、dS蛋白以及dS蛋白与RBM的融合蛋白的mRNA可以有以下几种形式：1)两条mRNA，一条编码dS蛋白，另一条编码信号肽和dS蛋白与RBM的融合蛋白；2)一条mRNA，有两个编码区分别编码dS蛋白、信号肽和dS蛋白与RBM的融合蛋白。
在本发明的一个具体实施方式中，所述的dS蛋白与RBM的融合蛋白的氨基酸序列如SEQ ID NO： 4或与SEQ ID NO：4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S蛋白的氨基酸序列如SEQ ID NO：5或与SEQ ID NO：5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M蛋白的氨基酸序列如SEQ ID NO：6或与SEQ ID NO：6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E蛋白的氨基酸序列如SEQ ID NO：7或与SEQ ID NO：7具有70％、75％、80％、85％、90％、95％、99％同一性的氨基酸序列。
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。
在本发明的一个具体实施方式中，所述的双顺反子或多顺反子mRNA的编码区可通过至少一种内部核糖体进入位点(IRES)序列分开，可使用的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由若干个结构元件组成，即所述的mRNA除包含上述编码区外还包括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。
优选的，所述的mRNA序列的长度为200-10000个核苷酸。进一步优选的，所述的mRNA序列的长度为500-8000个核苷酸。
本发明的第二方面，提供了一种包含mRNA的组合物，所述的mRNA包含编码2019-nCoV型冠状病毒的至少一个抗原肽或蛋白或其片段、变体或衍生物的编码区的mRNA序列。
优选的，所述抗原肽或蛋白或其片段、变体或衍生物选自下列任一种：
A)信号肽和2019-nCoV型冠状病毒的RBD，其中，所述的2019-nCoV型冠状病毒的RBD不包含信号肽区域；RBD中包含了主要的中和表位，非中和表位相对较少，安全性高。
B)信号肽和氨基酸突变引入N-连接糖基化的2019-nCoV型冠状病毒的RBD，其中，所述的2019-nCoV型冠状病毒的RBD不包含信号肽区域；为避免脱离全长S蛋白暴露出原本埋在全长S蛋白中的氨基酸序列，出现免疫优势的非中和表位，则通过氨基酸突变引入N-连接糖基化，以遮蔽非中和表位，并提高免疫原性。
C)2019-nCoV型冠状病毒的S蛋白；优选的，还包括2019-nCoV型冠状病毒的M蛋白和/或E蛋白；具体的为(1)2019-nCoV型冠状病毒的S蛋白和M蛋白的组合；(2)2019-nCoV型冠状病毒的S蛋白和E蛋白的组合；(3)2019-nCoV型冠状病毒的S蛋白、M蛋白和E蛋白的组合；
D)信号肽、dS蛋白以及dS蛋白与RBD的融合蛋白的组合，其中，所述的RBD不包含信号肽区域；或
E)信号肽、dS蛋白以及dS蛋白与RBM的融合蛋白的组合，其中，所述的RBM不包含信号肽区域。
优选的，所述的mRNA编码的信号肽为编码一个人类细胞可识别分泌信号肽，如组织型纤溶酶原激活剂(tPA)的信号肽或者血清免疫球蛋白E(lgE)的信号肽等。进一步优选为tPA。
所述的RBD第190位氨基酸为在全长S蛋白中不易接触的位点，但在RBD结构中确实一个突出的位点，极易接触，将该位点的脯氨酸突变为天冬氨酸，可以引入N-连接糖基化基序：天冬氨酸-非脯氨酸-苏氨酸。
优选的，所述B)中突变位点为第190位的脯氨酸突变为天冬氨酸。进一步优选的，所述的B)中氨基酸突变引入N-连接糖基化的2019-nCoV型冠状病毒的RBD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。
更进一步优选的，所述的编码C)中S蛋白、M蛋白两种蛋白的mRNA可以有以下几种形式：I)两条mRNA，一条编码S蛋白，一条编码M蛋白；II)一条mRNA，有两个编码区分别编码S蛋白和M蛋白。
更进一步优选的，所述的编码C)中S蛋白、E蛋白两种蛋白的mRNA可以有以下几种形式：i)两条mRNA，一条编码S蛋白，一条编码E蛋白；ii)一条mRNA，有两个编码区分别编码S蛋白和E蛋白。
更进一步优选的，所述的编码C)中S蛋白、M蛋白和E蛋白三种蛋白的mRNA可以有以下几种形式：a)三条mRNA，分别编码S蛋白、M蛋白及E蛋白；b)两条mRNA，一条编码S蛋白，第二条有两个编码区，分别编码M蛋白和E蛋白；c)一条mRNA，有三个编码区分别编码S蛋白，M蛋白和E蛋白。
进一步优选的，所述的dS蛋白的氨基酸序列如SEQ ID NO：2或与SEQ ID NO：2具有70％、75％、80％、85％、90％、95％、99％同一性的氨基酸序列。
更进一步优选的，所述的编码D)中信号肽、dS蛋白以及dS蛋白与RBD的融合蛋白的mRNA可以有以下几种形式：(1)两条mRNA，一条编码dS蛋白，另一条编码信号肽和dS蛋白与RBD的融合蛋白；(2)一条mRNA，有两个编码区分别编码dS蛋白、信号肽和dS蛋白与RBD的融合蛋白。
进一步优选的，所述的dS蛋白与RBD的融合蛋白的氨基酸序列如SEQ ID NO：3或与SEQ ID NO：3具有70％、75％、80％、85％、90％、95％、99％同一性的氨基酸序列。
更进一步优选的，所述的编码E)中信号肽、dS蛋白以及dS蛋白与RBM的融合蛋白的mRNA可 以有以下几种形式：1)两条mRNA，一条编码dS蛋白，另一条编码信号肽和dS蛋白与RBM的融合蛋白；2)一条mRNA，有两个编码区分别编码dS蛋白、信号肽和dS蛋白与RBM的融合蛋白。
进一步优选的，所述的dS蛋白与RBM的融合蛋白的氨基酸序列如SEQ ID NO：4或与SEQ ID NO：4具有70％、75％、80％、85％、90％、95％、99％同一性的氨基酸序列。
进一步优选的，所述的S蛋白的氨基酸序列如SEQ ID NO：5或与SEQ ID NO：5具有70％、75％、80％、85％、90％、95％、99％同一性的氨基酸序列。
进一步优选的，所述的M蛋白的氨基酸序列如SEQ ID NO：6或与SEQ ID NO：6具有70％、75％、80％、85％、90％、95％、99％同一性的氨基酸序列。
进一步优选的，所述的E蛋白的氨基酸序列如SEQ ID NO：7或与SEQ ID NO：7具有70％、75％、80％、85％、90％、95％、99％同一性的氨基酸序列。
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。
在本发明的一个具体实施方式中，所述的双顺反子或多顺反子mRNA的编码区可通过至少一种内部核糖体进入位点(IRES)序列分开，可使用的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由若干个结构元件组成，即所述的mRNA除包含上述编码区外还包括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。
优选的，所述的mRNA序列的长度为200-10000个核苷酸。进一步优选的，所述的mRNA序列的长度为500-8000个核苷酸。
优选的，所述的组合物中还包括阳离子或聚阳离子化合物。其中所述的阳离子或聚阳离子化合物游离或者与mRNA结合。为了使得所述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，所述的组合物中还包含脂质。
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、使得mRNA从内涵体中释放到胞内的脂质、支撑磷脂双分子层结构的脂质或用作稳定剂的脂质。
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或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、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。
本发明所述的组合物可以为脂质、脂质复合物或脂质纳米粒子。所述的脂质可以为以下形式制备得到的脂质：1，2-二油烯基氧基-N，N-二甲基氨基丙烷(DODMA)脂质、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。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。
本发明的第三方面，提供了一种本发明所述的mRNA或组合物编码的蛋白。
本发明的第四方面，提供了一种氨基酸突变引入N-连接糖基化的2019-nCoV型冠状病毒的RBD的蛋白。优选的，所述的2019-nCoV型冠状病毒的RBD不包含信号肽区域。
本发明的第五方面，提供了一种2019-nCoV型冠状病毒的S蛋白、M蛋白或E蛋白中的两种或三种蛋白的融合蛋白。
本发明的第六方面，提供了一种dS蛋白与RBD或RBM的融合蛋白。优选的，所述的RBD和RBM不包含信号肽区域。
本发明的第七方面，提供了一种编码第三至六方面所述蛋白或融合蛋白的核酸。优选的，所述的核酸的序列为所述蛋白或融合蛋白的表位抗原基因的核苷酸序列。
本发明的第八方面，提供了一种包含本发明第七方面所述核酸的载体。
本发明的第九方面，提供了一种包含本发明所述的mRNA、所述的蛋白或融合蛋白、所述的核酸和/或所述的载体的细胞。
本发明的第十方面，提供了一种包含mRNA的组合物的制备方法，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、使得mRNA从内涵体中释放到胞内的脂质、支撑磷脂双分子层结构的脂质或用作稳定剂的脂质。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。
本发明的第十一方面，提供了所述mRNA或包含mRNA的组合物在预防和/或治疗2019-nCoV型冠状病毒感染中的应用。
本发明的第十二方面，提供了所述mRNA或包含mRNA的组合物在制备预防和/或治疗2019-nCoV型冠状病毒感染的药物中的应用。
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。
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。
本发明的第十三方面，提供了一种预防2019-nCoV型冠状病毒感染的方法，包括向未感染2019-nCoV型冠状病毒的个体施加有效量的本发明所述的mRNA或包含mRNA的组合物。
本发明的第十四方面，提供了一种治疗2019-nCoV型冠状病毒感染的方法，包括向感染2019-nCoV型冠状病毒的个体施加有效量的本发明所述的mRNA或包含mRNA的组合物。以使得个体体内产生中和抗体抵制2019-nCoV型冠状病毒。
本发明的第十五方面，提供了一种抗体筛选的方法，包括向个体施加有效量的本发明所述的mRNA或包含mRNA的组合物的步骤。
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。
本发明的第十六方面，提供了一种诱导个体中和抗原特异性免疫应答的方法，包括向个体施加本发明所述的mRNA或包含mRNA的组合物。
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。
本发明所述的mRNA或包含mRNA的组合物具备的优势在于：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“S蛋白”为组成2019-nCoV型冠状病毒的结构蛋白，名称为纤突蛋白。
本发明所述的“RBD”为组成2019-nCoV型冠状病毒的结构蛋白，名称为纤突蛋白受体结合域。所述的RBD包含核心结构和纤突蛋白受体结合基序，所述的纤突蛋白受体结合基序即为本发明所述的“RBM”。
本发明所述的“M蛋白”为组成2019-nCoV型冠状病毒的结构蛋白，名称为包膜蛋白。
本发明所述的“E蛋白”为组成2019-nCoV型冠状病毒的结构蛋白，名称为小包膜蛋白。
本发明所述的“dS蛋白”为鸭乙肝病毒小表面蛋白。
本发明所述的“LNP”为脂质纳米颗粒。
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的功能。
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。
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。
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。
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：通过RaptorX的蛋白结构预测分别对2019-nCoV的RBD部分和S全长蛋白进行模拟，图中显示预测的蛋白结构中氨基酸的目标突变突变位置。
图2：含有T7启动子，5’UTR，3’UTR，和polyA尾的基础质粒模板序列图。
图3：编码含tPA信号肽的野生型2019-nCoV的RBD的序列测序结果。
图4：编码含tPA信号肽的190位突变的2019-nCoV的RBD的序列测序结果。
图5：编码野生型2019-nCoV M蛋白的序列测序结果。
图6：编码野生型2019-nCoV E蛋白的序列测序结果。
图7：图7A和图7B的拼接为编码野生型2019-nCoV S蛋白的序列测序结果。
图8：编码含tPA信号肽的2019-nCoV RBD-dS融合蛋白的序列测序结果。
图9：编码含tPA信号肽的2019-nCoV RBM-dS融合蛋白的序列测序结果。
图10：编码dS蛋白的序列测序结果。
图11：用含有与基础质粒模板同源的引物对实施例制备的mRNA进行PCR扩增的结果图，其中，RBD-M为编码190位突变引入N-连接糖基化后的mRNA，S-F为编码全长S蛋白的mRNA。
图12：用甲醛变性胶检测加帽纯化后的mRNA，M为Marker，其中1为编码含tPA信号肽的2019-nCoV RBD-dS融合蛋白的mRNA，2为编码含tPA信号肽的2019-nCoV RBM-dS融合蛋白的mRNA，3为编码野生型2019-nCoV S蛋白的mRNA，4为编码野生型2019-nCoV M蛋白的mRNA，5为编码野生型2019-nCoV E蛋白的mRNA，6为编码含tPA信号肽的190位突变的2019-nCoV的RBD的mRNA，7为编码含tPA信号肽的野生型2019-nCoV的RBD的mRNA，8为编码dS蛋白的mRNA。
图13：WB(Western Blot，蛋白质印迹法)表达检测结果，其中，M为Marker，1为编码含tPA信号肽的野生型2019-nCoV的RBD的mRNA的表达上清，2为编码含tPA信号肽的190位突变并引入N-连接糖基化的2019-nCoV的RBD的mRNA的表达上清，3为阴性对照。
图14：抗S蛋白多抗免疫荧光检测S蛋白表达的结果。
图15：DLS(Dynamic Light Scattering，动态光散射)检测mRNA-LNP的粒径和粒径分布(用聚合物分散性指数PDI(Polymer dispersity index)标征)，其中，A为编码野生型2019-nCOV S蛋白的mRNA-LNP，B为编码含tPA信号肽的野生型2019-nCOV的RBD的mRNA-LNP，C为编码含tPA信号肽的190位突变并引入N-连接糖基化的2019-nCOV的RBD的mRNA-LNP。
图16：一免、二免后的S蛋白特异性抗体滴度，其中，Negative为阴性对照，S-F为编码野生型2019-nCOV S蛋白的mRNA-LNP，RBD为编码含tPA信号肽的野生型2019-nCOV的RBD的mRNA-LNP，RBD-M编码含tPA信号肽的190位突变并引入N-连接糖基化的2019-nCOV的RBD的 mRNA-LNP。
图17：ELISpot(酶联免疫斑点法)检测干扰素γ的结果，其中，Negative为阴性对照，S-F为编码野生型2019-nCOV S蛋白的mRNA-LNP，RBD为编码含tPA信号肽的野生型2019-nCOV的RBD的mRNA-LNP，RBD-M编码含tPA信号肽的190位突变并引入N-连接糖基化的2019-nCOV的RBD的mRNA-LNP，纵坐标为每百万脾细胞的点形成单位(spot forming unit，SFU)。
图18：二免后中和抗体滴度，其中，S-F为编码野生型2019-nCOV S蛋白的mRNA-LNP，RBD为编码含tPA信号肽的野生型2019-nCOV的RBD的mRNA-LNP，RBD-M编码含tPA信号肽的190位突变并引入N-连接糖基化的2019-nCOV的RBD的mRNA-LNP，sVNT代表替代性病毒中和试验。
图19：对细胞上清进行WB检测，其中1为阴性对照，2为VLP-1的表达上清，3为VLP-2的表达上清，4为编码野生型2019-nCoV S蛋白的mRNA的表达上清。
图20：DLS检测mRNA-LNP的粒径和粒径分布，其中，A代表VLP-1-LNP，B代表VLP-2-LNP，C代表编码野生型2019-nCOV S蛋白的mRNA-LNP。
图21：甲醛变性胶检测包装后样品的mRNA完整性，其中，M为Marker，1为VLP-1-LNP，2为VLP-2-LNP，3为编码野生型2019-nCOV S蛋白的mRNA-LNP。
图22：一免、二免后的S蛋白特异性抗体滴度，其中，Negative为阴性对照，S-F为编码野生型2019-nCOV S蛋白的mRNA-LNP。
图23：ELISpot检测干扰素γ的结果，其中，Negative为阴性对照，S-F为编码野生型2019-nCOV S蛋白的mRNA-LNP，纵坐标为每百万脾细胞的点形成单位(spot forming unit，SFU)。
图24：胞内CK染色法进行了交叉验证结果，其中，Negative为阴性对照，S-F为编码野生型2019-nCOV S蛋白的mRNA-LNP。
图25：DLS检测mRNA-LNP的粒径和粒径分布，其中，左图代表VLP-3-LNP，中图代表VLP-4-LNP，右图代表编码野生型2019-nCOV S蛋白的mRNA-LNP。
图26：一免、二免后的S蛋白特异性抗体滴度，其中，Negative为阴性对照，S-F为编码野生型2019-nCOV S蛋白的mRNA-LNP。
图27：ELISpot检测干扰素γ的结果，其中，Negative为阴性对照，S-F为编码野生型2019-nCOV S蛋白的mRNA-LNP，纵坐标为每百万脾细胞的点形成单位(spot forming unit，SFU)。
图28：胞内CK染色法进行了交叉验证结果，其中，Negative为阴性对照，S-F为编码野生型2019-nCOV S蛋白的mRNA-LNP。
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1 mRNA的制备与检测
1、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3-10所示。具体的，图3显示了编码含tPA信号肽的野生型2019-nCoV的RBD的序列测序结果，其核苷酸序列如SEQ ID NO：9所示，氨基酸序列如SEQ ID NO：17所示。图4显示编码含tPA信号肽的190位突变的2019-nCoV的RBD(位置参见图1)的序列测序结果，其核苷酸序列如SEQ ID NO：10所示，氨基酸序列如SEQ ID NO：18所示。图5显示编码野生型2019-nCoV M蛋白的序列测序结果，其核苷酸序列如SEQ ID NO：11所示，其氨基酸序列如SEQ ID NO：6所示。图6显示编码野生型2019-nCoV E蛋白的序列测序结果，其核苷酸序列如SEQ ID NO：12所示，其氨基酸序列如SEQ ID NO：7所示。图7显示编码野生型2019-nCoV S蛋白的序列测序结果，其核苷酸序列如SEQ ID NO：13所示，其氨基酸序列如SEQ ID NO：5所示。图8显示编码含tPA信号肽的2019-nCoV RBD-dS融合蛋白的序列测序结果，其核苷酸序列如SEQ ID NO：14所示，且不含信号肽的RBD-dS融合蛋白的氨基酸序列如SEQ ID NO：3所示。图9显示编码含tPA信号肽的2019-nCoV RBM-dS融合蛋白的序列测序结果，其核苷酸序列如SEQ ID NO：15所示，且其不含信号肽的2019-nCoV RBM-dS融合蛋白的氨基酸序列如SEQ ID NO：4所示。图10显示编码dS蛋白的序列测序结果，其氨基酸序列如SEQ ID NO：2所示，其核苷酸序列如SEQ ID NO：16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2(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，结果如图11所示。
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，获得SEQ ID NO：9-16转录的未加帽的mRNA。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12所示，其中1为编码含tPA信号肽的2019-nCoV RBD-dS融合蛋白的mRNA，2为编码含tPA信号肽的2019-nCoV RBM-dS融合蛋白的mRNA，3为编码野生型2019-nCoV S蛋白的mRNA，4为编码野生型2019-nCoV M蛋白的mRNA，5为编码野生型2019-nCoV E蛋白的mRNA，6为编码含tPA信号肽的190位突变的2019-nCoV的RBD的mRNA，7为编码含tPA信号肽的野生型2019-nCoV的RBD的mRNA，8为编码dS蛋白的mRNA。可以看到mRNA的大小正确且基本无降解。
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2019-nCoV的RBD的mRNA，编码含tPA信号肽的190位突变并引入N-连接糖基化的2019-nCoV的RBD的mRNA，和编码野生型2019-nCoV S蛋白的mRNA。
2、表达检测
用24孔板铺4个孔的HEK293细胞，其中1、2、3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2019-nCoV的RBD的mRNA，编码含tPA信号肽的190位突变并引入N-连接糖基化的2019-nCoV的RBD的mRNA，和编码野生型2019-nCoV S蛋白的mRNA，4号孔作为阴性对照加入lipofectamine 2000转染试剂。转染24h后，取1、2、4号孔的细胞上清进行WB检测，3、4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13所示，其中1为编码含tPA信号肽的野生型2019-nCoV的RBD的mRNA的表达上清，2为编码含tPA信号肽的190位突变并引入N-连接糖基化的2019-nCoV的RBD的mRNA的表达上清，3为阴性对照。可以看到表达出来的蛋白大小正确，引入糖基化的设计比野生型约大5kDa，证明糖基化位点成功引入。免疫荧光结果如图14所示，证明野生型2019-nCoV S蛋白的mRNA可以正常表达。
3、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Benchtop中分别混合包装上述三种mRNA。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动物实验的样品。用DLS检测mRNA-LNP的粒径和粒径分布(用聚合物分散性指数PDI标征)，检测结果如图15所示，包装后样品的粒径大小均在80nm左右，PDI均小于0.2，其中，编码野生型2019-nCOV S蛋白的mRNA-LNP：粒径平均值78.83nm，PDI值0.028，截距(intercept)0.953，具体见表1；编码含tPA信号肽的野生型2019-nCOV的RBD的mRNA-LNP：粒径平均值83.13nm，PDI值 0.013，截距(intercept)0.978，具体见表2；编码含tPA信号肽的190位突变并引入N-连接糖基化的2019-nCOV的RBD的mRNA-LNP：粒径平均值83.41nm，PDI值0.031，截距(intercept)0.978，具体见表3。
表1：编码野生型2019-nCOV S蛋白的mRNA-LNP
  粒径(nm) 强度(％) 标准差
峰1 82.74 100 19.35
峰2 0 0 0
峰3 0 0 0
表2：编码含tPA信号肽的野生型2019-nCOV的RBD的mRNA-LNP
  粒径(nm) 强度(％) 标准差
峰1 86.64 100 19.17
峰2 0 0 0
峰3 0 0 0
表3：编码含tPA信号肽的190位突变并引入N-连接糖基化的2019-nCOV的RBD的mRNA-LNP
  粒径(nm) 强度(％) 标准差
峰1 87.63 100 20.5
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4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，并用竞争性ELISA法检测中和抗体滴度。
4、实验结果
一免、二免后的S蛋白特异性抗体滴度如图16所示，可以看到两种RBD设计均可以在一免时就快速刺激出显著性高于野生型S全长蛋白设计的特异性抗体，二免后三者的特异性抗体滴度没有显著性差异。用ELISpot检测干扰素γ的结果如图17所示，可以看到两种RBD设计引起的细胞免疫水平显著高于野生型S全长蛋白设计，而引入糖基化的RBD设计的细胞免疫水平显著高于野生型RBD设计。二免后中和抗体滴度如图18所示，显示在三种设计的特异性抗体滴度无显著性差异的情况下，两种RBD设计的中和抗体水平均显著性高于野生型S全长蛋白设计，而引入糖基化的RBD设计的中和抗体水平显著高于野生型RBD设计。说明RBD设计中包含了主要的中和表位，非中和表位相对较少，引起的 中和抗体占比高，安全性更高；而通过氨基酸突变引入N-连接糖基化的RBD设计遮蔽了暴露出来的可能产生免疫优势的非中和表位，使得中和抗体占比更高，可在2019-nCoV型冠状病毒感染的预防上取得更好的效果。
实施例3 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制备的编码野生型2019-nCoV S、E和M蛋白的mRNA。将这三种mRNA分别按摩尔比1:1:1和质量比1:1:1进行混合，得到混合后的mRNA，简写为VLP-1和VLP-2。
2、表达检测
用24孔板铺4个孔的HEK293细胞，其中1、2、3号孔分别用lipofectamine 2000转染0.5μg VLP-1、VLP-2和编码野生型2019-nCoV S蛋白的mRNA，4号孔作为阴性对照加入lipofectamine 2000转染试剂。转染24h后，取细胞上清进行WB检测。WB检测结果如图19所示，其中1为阴性对照，2为VLP-1的表达上清，3为VLP-2的表达上清，4为编码野生型2019-nCoV S蛋白的mRNA的表达上清。可以看到2号泳道有清晰条带，3号泳道有微弱条带，1号和4号泳道无条带。证明编码野生型2019-nCoV S、E和M三种蛋白的mRNA按一定比例混合后可以形成类病毒颗粒表达到细胞上清；而单一的编码野生型2019-nCoV S蛋白的mRNA表达后，S蛋白会锚定在细胞膜上，不会进入细胞上清。
3、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Benchtop中分别混合包装VLP-1、VLP-2和编码野生型2019-nCoV S蛋白的mRNA。将包装好的mRNA-LNP透析并超滤浓缩到DPBS(杜氏磷酸盐缓冲液)中，无菌过滤后获得用于动物实验的样品。用DLS检测mRNA-LNP的粒径和粒径分布，检测结果如图20所示，包装后样品的粒径大小均在80nm左右，PDI均小于0.2。其中，VLP-1-LNP：粒径平均值81.44nm，PDI值0.023，截距(intercept)0.978，具体见表4；VLP-2-LNP：粒径平均值82.59nm，PDI值0.038，截距(intercept)0.977，具体见表5；编码野生型2019-nCOV S蛋白的mRNA-LNP：粒径平均值84.77nm，PDI值0.030，截距(intercept)0.957，具体见表6。
表4：VLP-1-LNP
  粒径(nm) 强度(％) 标准差
峰1 85.14 100 19.10
峰2 0 0 0
峰3 0 0 0
表5：VLP-2-LNP
  粒径(nm) 强度(％) 标准差
峰1 87.01 100 20.52
峰2 0 0 0
峰3 0 0 0
表6：编码野生型2019-nCOV S蛋白的mRNA-LNP
  粒径(nm) 强度(％) 标准差
峰1 89.19 100 21.37
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21所示，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4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。
4、结果显示
一免、二免后的S蛋白特异性抗体滴度如图22所示，可以看到VLP-2没能引起高水平的特异性抗体滴度，但VLP-1设计和S全长蛋白设计的特异性抗体滴度在一免、二免时均没有显著性差异。用ELISpot检测干扰素γ的结果如图23所示，可以看到VLP-1设计引起的Th1类细胞免疫水平显著高于野生型S全长蛋白设计，而此结果也用胞内CK染色法进行了交叉验证，如图24所示。证明编码野生型2019-nCoV S、E和M蛋白的mRNA按摩尔比1:1:1混合包装后免疫可以获得不低于野生型S全长蛋白设计的体液免疫反应和显著性高于野生型S全长蛋白设计的细胞免疫反应，可在2019-nCoV型冠状病毒感染的预防上取得更好的效果。且该mRNA疫苗在动物体内表达出来的蛋白会自动形成与2019-nCoV型冠状病毒类似的类病毒颗粒，同时，避免了体外表达系统表达产品的性质不均一问题，适合大批量作为疫苗生产。
实施例-4 mRNA疫苗的制备
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dS蛋白、含tPA信号肽的2019-nCoV RBD-dS融合蛋白的mRNA、含tPA 信号肽的2019-nCoV RBM-dS融合蛋白的mRNA和编码野生型2019-nCoV S蛋白的mRNA。将编码dS蛋白、含tPA信号肽的2019-nCoV RBD-dS融合蛋白的mRNA按摩尔比1:1进行混合，得到混合后的mRNA，简写为VLP-3。将编码dS蛋白、含tPA信号肽的2019-nCoV RBM-dS融合蛋白的mRNA按摩尔比1:1进行混合，得到混合后的mRNA，简写为VLP-4。
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Benchtop中分别混合包装VLP-3、VLP-4和编码野生型2019-nCoV S蛋白的mRNA。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25所示，包装后样品的粒径大小均在85nm左右，PDI均小于0.2。其中，VLP-3-LNP：粒径平均值84.19nm，PDI值0.018，截距(intercept)0.954，具体见表7；VLP-4-LNP：粒径平均值82.16nm，PDI值0.018，截距(intercept)0.959，具体见表8；编码野生型2019-nCOV S蛋白的mRNA-LNP：粒径平均值84.77nm，PDI值0.030，截距(intercept)0.957，具体见表6。
表7：VLP-3-LNP
  粒径(nm) 强度(％) 标准差
峰1 87.98 100 19.99
峰2 0 0 0
峰3 0 0 0
表8：VLP-4-LNP
  粒径(nm) 强度(％) 标准差
峰1 85.95 100 19.84
峰2 0 0 0
峰3 0 0 0
3、实验结果
一免、二免后的S蛋白特异性抗体滴度如图26所示，可以看到VLP-4没能引起高水平的特异性抗体滴度，但VLP-3设计和S全长蛋白设计的特异性抗体滴度在一免、二免时均没有显著性差异。用ELISpot检测干扰素γ的结果如图27所示，可以看到VLP-3设计引起的Th1类细胞免疫水平显著高于野生型S全长蛋白设计，而此结果也用胞内CK染色法进行了交叉验证，如图28所示。证明编码dS蛋白、含tPA信号肽的2019-nCoV RBD-dS融合蛋白的mRNA按摩尔比1:1混合包装后免疫可以获得不低于野生型S全长蛋白设计的体液免疫反应和显著性高于野生型S全长蛋白设计的细胞免疫反应，可在2019-nCoV型冠状病毒感染的预防上取得更好的效果。且该mRNA疫苗在动物体内表达出来的蛋 白会自动形成类病毒颗粒，同时，避免了体外表达系统表达产品的纯化困难问题，适合大批量作为疫苗生产。
综上所述，实施例2-4制备的mRNA疫苗可以用于2019-nCoV型冠状病毒感染的预防，且该mRNA疫苗在动物体内表达出来的蛋白会自动形成与2019-nCoV型冠状病毒类似的类病毒颗粒，诱发更强的细胞免疫，在体内出现的非中和抗体极少或基本没有。同时，生产周期短，可以纯化做到性质均一，适合大批量作为疫苗生产。
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 (16)

  1. 一种mRNA，其特征在于，所述的mRNA包含编码2019-nCoV型冠状病毒的至少一个抗原肽或其片段、变体或衍生物的编码区的mRNA序列；优选的，所述抗原肽或其片段、变体或衍生物选自下列任一种：
    A)信号肽和2019-nCoV型冠状病毒的RBD，其中，所述的2019-nCoV型冠状病毒的RBD不包含信号肽区域；
    B)信号肽和氨基酸突变引入N-连接糖基化的2019-nCoV型冠状病毒的RBD，其中，所述的2019-nCoV型冠状病毒的RBD不包含信号肽区域；
    C)2019-nCoV型冠状病毒的S蛋白；优选的，还包括2019-nCoV型冠状病毒的M蛋白和E蛋白；或，
    D)信号肽、dS蛋白以及dS蛋白与RBD的融合蛋白的组合，其中，所述的RBD不包含信号肽区域。
  2. 根据权利要求1所述的mRNA，其特征在于，所述B)中突变位点为第190位的脯氨酸突变为天冬氨酸，优选的，所述的B)中氨基酸突变引入N-连接糖基化的2019-nCoV型冠状病毒的RBD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。
  3. 根据权利要求1所述的mRNA，其特征在于，所述的dS蛋白的氨基酸序列如SEQ ID NO：2或与SEQ ID NO：2具有70％、75％、80％、85％、90％、95％、99％同一性的氨基酸序列。
  4. 根据权利要求1所述的mRNA，其特征在于，所述的dS蛋白与RBD的融合蛋白的氨基酸序列如SEQ ID NO：3或与SEQ ID NO：3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所述的mRNA，其特征在于，所述C)中S蛋白、M蛋白与E蛋白的mRNA摩尔比为1：1：1。
  6. 根据权利要求1-5任一所述的mRNA，其特征在于，所述的mRNA为单顺反子、双顺反子或多顺反子mRNA。
  7. 根据权利要求1-6任一所述的mRNA，其特征在于，所述的信号肽为tPA或lgE，优选的，所述的mRNA还包括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。
  8. 一种包含mRNA的组合物，其特征在于，所述的mRNA包含编码2019-nCoV型冠状病毒的至少一个抗原肽或其片段、变体或衍生物的编码区的mRNA序列；优选的，所述的mRNA选自权利要求1-7任一所述的mRNA。
  9. 根据权利要求8所述的组合物，其特征在于，所述的组合物中还包括阳离子或聚阳离子化合物，优选的，所述的组合物中还包含脂质。
  10. 一种权利要求1-7任一所述的mRNA编码的蛋白。
  11. 一种编码权利要求10所述蛋白的核酸。
  12. 一种包含权利要求11所述核酸的载体。
  13. 一种包含权利要求10所述的蛋白、权利要求11所述的核酸和/或权利要求12所述的载体的细胞。
  14. 一种包含mRNA的组合物的制备方法，其特征在于，所述的制备方法包括将权利要求1-7任一所述的mRNA与阳离子或聚阳离子化合物混合后用脂质包装。
  15. 一种权利要求1-7任一所述的mRNA或权利要求8-9任一所述的包含mRNA的组合物在制备预防和/或治疗2019-nCoV型冠状病毒感染的药物中的应用。
  16. 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1-7任一所述的mRNA或权利要求8-9任一所述的包含mRNA的组合物。
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