WO2023125974A1 - mRNA疫苗 - Google Patents

mRNA疫苗 Download PDF

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WO2023125974A1
WO2023125974A1 PCT/CN2022/144148 CN2022144148W WO2023125974A1 WO 2023125974 A1 WO2023125974 A1 WO 2023125974A1 CN 2022144148 W CN2022144148 W CN 2022144148W WO 2023125974 A1 WO2023125974 A1 WO 2023125974A1
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utr
antigen
poly
cap
linker
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PCT/CN2022/144148
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French (fr)
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彭华
曹学智
王修业
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广州国家实验室
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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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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
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    • C07K2319/00—Fusion polypeptide
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    • C12N2760/16011—Orthomyxoviridae
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Definitions

  • the invention belongs to the field of vaccines, in particular to mRNA vaccines and applications thereof.
  • mRNA messenger ribonucleic acid
  • mRNA is a single-stranded RNA molecule corresponding to the genetic sequence of a gene, which is translated by ribosomes during the process of protein synthesis in organisms to produce proteins.
  • Combining T7 bacteriophage RNA polymerase with plasmid DNA can carry out mRNA transcription.
  • In vitro transcribed mRNA has the same structural components as natural mRNA in eukaryotic cells: a 5'cap (5'cap), a 5'untranslated region (5'UTR), an open reading frame (ORF) encoding the relevant antigen , a 3' untranslated region (3'UTR), and a 3' poly A tail [3'Poly(A)].
  • the use of synthetic 5'-cap analogs can improve mRNA stability and increase protein translation.
  • mRNA nucleotides can be modified to reduce innate immune activation and increase mRNA half-life in host cells.
  • An mRNA vaccine is a vaccine that utilizes mRNA molecules encapsulated in lipid nanoparticles (LNP) to generate an immune response.
  • LNP lipid nanoparticles
  • the mRNA can be delivered to immune cells, but also to other cells such as muscle cells or epithelial cells.
  • the proteins translated by these cells can be expressed inside the cell, on the surface of the cell membrane, or secreted outside the cell, and finally targeted to immune cells.
  • the host cells Once the host cells have produced the antigens, the normal adaptive immune system processes begin.
  • the antigen is broken down by the proteasome, and MHC class I and II molecules attach to the antigen and transport it to the cell membrane to activate dendritic cells. Once activated, dendritic cells migrate to lymph nodes where they present antigens to T and B cells, ultimately leading to an immune response.
  • mRNA vaccines The advantage of mRNA vaccines is that it is easy to design, can present multiple antigens, has the potential to develop multi-linked and multivalent vaccines, has a short production cycle, and is easier to expand production capacity. It will not integrate with human DNA, and there is no risk of exogenous virus infection. .
  • mRNA vaccines are the third-generation vaccines after inactivated vaccines, live attenuated vaccines, subunit vaccines, and viral vector vaccines. They have the characteristics of fast response to pathogen mutation, simple production process, and easy scale-up. However, the toxic and side effects of mRNA vaccines are also very obvious, such as sore reaction at the injection site, fatigue, headache, and even facial paralysis. The side effects are even greater after the second injection. There are still many areas for optimization and improvement in the existing mRNA vaccine technology, and there are still many technical barriers that need to be broken through.
  • the research on the improvement of mRNA vaccines mainly focuses on LNP delivery technology, production of plasmids, synthesis and purification of mRNA, and optimization of mRNA structure and sequence itself mostly focuses on the modification of nucleotides encoding antigens. Reports that additionally contain coding sequences encoding immune cell targeting molecules in the open reading frame encoding the antigen of interest.
  • the present invention provides an mRNA vaccine with enhanced immune effect.
  • the mRNA vaccine includes an open reading frame encoding an antigen and an immune cell targeting molecule.
  • the mRNA vaccine with enhanced immune effect of the present invention has the effects of enhancing the immune effect, improving the protection rate of the mRNA vaccine, reducing the dosage of the mRNA vaccine, enlarging the production capacity of the mRNA vaccine and reducing the toxic and side effects of the mRNA vaccine.
  • the present invention provides mRNA comprising an open reading frame (ORF) encoding an antigen (Antigen) and an immune cell targeting molecule.
  • ORF open reading frame
  • Antigen an antigen
  • the antigen and the immune cell targeting molecule encoded by the open reading frame are fusion proteins of the antigen and the immune cell targeting molecule.
  • the immune cell targeting molecule is one or more selected from the following:
  • A Antibodies or polypeptides that can bind to immune cell surface proteins
  • PADRE Pan epitope capable of activating immune cells
  • the open reading frame encodes a fusion protein of an antigen with A; a fusion protein of an antigen with A and B; a fusion protein of an antigen with A, B, and C; a fusion protein of an antigen with A, B, C, and D protein; fusion protein of antigen with B; fusion protein of antigen with B and C; fusion protein of antigen with B, C and D; fusion protein of antigen with C; fusion protein of antigen with C and D; or fusion protein of antigen with D fusion protein.
  • the fusion protein can be any arrangement of the antigen and A and/or B and/or C and/or D, for example, in the fusion protein, the antigen and A and/or B and/or C and/or D are relative to are located at the C-terminal or N-terminal of each other.
  • the fusion protein is a homodimer or a heterodimer.
  • the mRNA provided by the present invention can express secreted fusion protein or membrane fusion protein.
  • the mRNA expressed secreted fusion protein can be efficiently targeted to the surface of immune cells because it contains immune cell targeting molecules.
  • the antibody or polypeptide A capable of binding to immune cell surface proteins includes but is not limited to CD274 (PDL1), PDCD1LG2 (PDL2), CLEC9A, LY75 (DEC205), CD40, TNFSF9 (4-1BB-L) and/or TNFSF4 ( OX4OL), etc., or active fragments of their ligands.
  • the cytokine B capable of activating immune cells includes but not limited to interleukin (interleukin, IL), colony-stimulating factor (Colony-stimulating factor, CSF) and interferon (interferon, IFN).
  • interleukin interleukin
  • CSF colony-stimulating factor
  • IFN interferon
  • the interleukins include IL2, IL12, IL15 and IL21, or active fragments thereof.
  • IL2 or its active fragment or homologue is from human, bovine, sheep, cat, dog, horse, rabbit, monkey, mouse, rat, alpaca or camel.
  • the colony-stimulating factors include CSF1, CSF2 and CSF3, or active fragments thereof.
  • the interferon includes type I interferon, type II interferon and type III interferon, or active fragments thereof, wherein type I interferon includes IFN ⁇ , IFN ⁇ , IFN ⁇ , IFN ⁇ and IFN ⁇ , or active fragments thereof, II Type III interferon includes IFN- ⁇ , or an active fragment thereof, and type III interferon includes IFN- ⁇ 1, IFN- ⁇ 2, IFN- ⁇ 3 and IFN- ⁇ 4, or an active fragment thereof.
  • the Pan epitope (PADRE) C capable of activating immune cells has the amino acid sequence shown in AKFVAAWTLKAAA (SEQ ID NO: 1).
  • Said immunoglobulin Fc(D) may be Fc from IgG, IgM, IgA, IgE or IgD or a mutant thereof, said Fc is mutant to form a heterodimeric protein.
  • the mRNA further comprises a 5'UTR (untranslated region) sequence and a 3'UTR (untranslated region).
  • the 5'UTR and 3'UTR can be the 5'UTR and 3'UTR of any highly expressed gene.
  • the mRNA further comprises a 5' Cap.
  • the mRNA further comprises a 3' Poly(A).
  • the antigen may be an immunogenic protein or an immunogenic fragment thereof capable of inducing an immune response against pathogenic microorganisms.
  • the pathogenic microorganisms include but are not limited to SARS-Cov-2, SARS, cytomegalovirus CMV, herpes virus, RSV, influenza virus, Ebola virus, Epstein-Barr virus (EBV), dengue fever virus, Zike virus, HIV virus , Rabies virus, Plasmodium gametophyte, herpes zoster virus (HZV), hepatitis B virus HBV, hepatitis C virus (HCV), hepatitis D virus HDV, HPV, Mycobacterium tuberculosis, Helicobacter pylori, etc.
  • the antigen may be an immunogenic protein or an immunogenic fragment thereof capable of inducing an immune response against cancer cells.
  • the antigen may be a tumor antigen, such as MelanA/MART1, cancer-germline antigen, gp100, tyrosinase, CEA, PSA, Her-2/neu, survivin, telomerase, or its immunogenic fragments.
  • a tumor antigen such as MelanA/MART1, cancer-germline antigen, gp100, tyrosinase, CEA, PSA, Her-2/neu, survivin, telomerase, or its immunogenic fragments.
  • the open reading frame (ORF) further comprises a region encoding a junctional segment.
  • the antigen and the immune cell targeting molecule, and/or the immune cell targeting molecule can be connected through a linking fragment.
  • the junctional fragment can be a flexible junctional fragment, a rigid junctional fragment, or an in vivo cleavage junctional fragment.
  • the amino acid sequence of the flexible linking fragment can be but not limited to (G) N , (GS) N , (GGS) N , (GGGS) N , or (GGGGS) N .
  • the mRNA has a structure selected from:
  • the above domains expressing antigens and immune cell targeting molecules in the open reading frame of mRNA can be arranged and combined in any form.
  • the domains expressing antigens and immune cell targeting molecules A, B, C and D in the open reading frame can be arranged and combined in any form.
  • the open reading frame in the mRNA is an open reading frame for expressing a fusion protein formed by an immune cell targeting molecule and an antigen.
  • the antigen and immune cell targeting molecule A can be arranged and combined in any form.
  • the antigen and immune cell targeting molecule B can be arranged and combined in any form.
  • the antigen and immune cell targeting molecule C can be arranged and combined in any form.
  • the open reading frame in the mRNA is an open reading frame for expressing a fusion protein formed by two immune cell targeting molecules and an antigen.
  • Antigens and immune cell targeting molecules A and B can be arranged and combined in any form.
  • Antigens and immune cell targeting molecules A and C can be arranged and combined in any form.
  • Antigens and immune cell targeting molecules A and D can be arranged and combined in any form.
  • Antigens and immune cell targeting molecules B and C can be arranged and combined in any form.
  • Antigens and immune cell targeting molecules B and D can be arranged and combined in any form.
  • Antigens and immune cell targeting molecules C and D can be arranged and combined in any form.
  • the open reading frame in the mRNA is an open reading frame for expressing a fusion protein formed by three immune cell targeting molecules and an antigen.
  • Antigens and immune cell targeting molecules A, B, and C can be arranged and combined in any form.
  • Antigens and immune cell targeting molecules A, B, and D can be arranged and combined in any form.
  • Antigens and immune cell targeting molecules A, C, and D can be arranged and combined in any form.
  • Antigens and immune cell targeting molecules B, C, and D can be arranged and combined in any form.
  • the open reading frame in the mRNA is an open reading frame for expressing a fusion protein formed by four immune cell targeting molecules and an antigen.
  • Antigens and immune cell targeting molecules A, B, C, D can be arranged and combined in any form.
  • the antigen is the S protein of SARS-Cov-2 or a fragment thereof.
  • the S protein is a pre-fusion stable S protein.
  • the prefusion stabilized S protein comprises a double proline (S2P) mutation or a hexaproline (S6P) mutation.
  • the antigen is the RBD domain of the S protein of SARS-Cov-2.
  • the present invention provides a composition comprising the mRNA of the first aspect.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier can be lipid nanoparticle (LNP), polymer material and inorganic nanoparticle.
  • LNP lipid nanoparticle
  • LPX cationic lipoplex
  • LPP lipid polyplex
  • PNP polymer nanoparticle
  • INP inorganic nanoparticle
  • CNE cationic nanoemulsion
  • the lipid nanoparticle can be used for mRNA vaccines for infectious diseases or for mRNA tumor vaccines.
  • the cationic lipoplex (LPX) can be used for mRNA tumor vaccines or mRNA vaccines for infectious diseases.
  • the present invention provides a method for preventing or treating infection with pathogenic microorganisms or tumors, the method comprising the step of administering the mRNA of the first aspect or the composition of the second aspect to a subject.
  • the subject is a human or an animal.
  • the animals include but are not limited to cattle, sheep, cats, dogs, horses, rabbits, monkeys, mice, rats, alpacas, camels and the like.
  • the subject is an immunocompromised human or animal.
  • the subject has a chronic lung disease, such as chronic obstructive pulmonary disease or asthma.
  • the patient has an underlying disease, such as heart disease, diabetes, or lung disease.
  • an underlying disease such as heart disease, diabetes, or lung disease.
  • the present invention provides the use of the mRNA of the first aspect or the composition of the second aspect in the preparation of a medicament for preventing or treating a subject related to pathogenic microorganism infection or tumor.
  • the subject is a human or an animal.
  • the animals include, but are not limited to, cows, sheep, cats, dogs, horses, rabbits, monkeys, mice, rats, alpacas, camels, chickens, ducks, geese, and the like.
  • the subject is an immunocompromised human or animal.
  • the subject has a chronic lung disease, such as chronic obstructive pulmonary disease or asthma.
  • the patient has an underlying disease, such as heart disease, diabetes, or lung disease.
  • an underlying disease such as heart disease, diabetes, or lung disease.
  • the mRNA can be delivered to immune cells, but also to other cells such as muscle cells or epithelial cells.
  • the proteins translated by these cells can be secreted and targeted to immune cells.
  • the mRNA provided by the present invention can express secreted fusion protein or membrane fusion protein.
  • the mRNA expressed secreted fusion protein can be efficiently targeted to the surface of immune cells because it contains immune cell targeting molecules.
  • the mRNA vaccine of the present invention has enhanced immune effect, enhanced protection rate, reduced mRNA vaccination dose, enlarged mRNA vaccine production capacity, and reduced mRNA vaccine toxic side effects.
  • the design of antigen fusion expression in the mRNA vaccine of the present invention can also be applied to other types of vaccines, including but not limited to nucleic acid vaccines (such as DNA vaccines and circular RNA vaccines, etc.), viral vector vaccines (such as adenovirus vector vaccines and influenza virus vaccines) Vector vaccines, etc.), recombinant protein vaccines and nanoparticle vaccines, etc.
  • nucleic acid vaccines such as DNA vaccines and circular RNA vaccines, etc.
  • viral vector vaccines such as adenovirus vector vaccines and influenza virus vaccines
  • Vector vaccines etc.
  • recombinant protein vaccines and nanoparticle vaccines etc.
  • UTRs untranslated regions
  • Untranslated regions are capable of being transcribed but not translated.
  • the 5'UTR starts from the transcription start site to but not including the start codon; while the 3'UTR follows the stop codon until the transcription termination signal.
  • UTR affects transcriptional regulation and stability of mRNA, and affects translation efficiency.
  • 5'UTRs are usually selected from naturally occurring stable/highly expressed sequences, or engineered from them.
  • the 3'UTR is usually related to mRNA stability, and similar to the 5'UTR, it is selected from a naturally occurring sequence, or is modified to some extent.
  • 5'Cap is a very important structure for mRNA vaccines. It is usually combined with eukaryotic translation initiation factors to initiate translation. Capping can help mRNA be smoothly transported out of the nucleus. In addition, 5'Cap can also protect mRNA from exonucleases The degradation of mRNA can also promote the circularization of mRNA to form space and structure, and enhance the stability. Usually co-transcriptional capping with capping analogs or post-transcriptional capping with capping enzymes alone.
  • the 3'UTR tail is a PolyA sequence, which is similar to the cap structure.
  • the PolyA tail also protects mRNA from being degraded by exonucleases.
  • PolyA also participates in translation and regulation processes, increasing stability, extending half-life in vivo and improving mRNA translation efficiency.
  • PolyA-binding protein (PABP) can be connected to the 5' cap through translation initiation factors (such as eIF4G and eIF4E) to form a closed-loop structure, and cooperate to participate in the regulation of mRNA stability and translation activity.
  • LNPs lipid nanoparticles LNP in recent years.
  • LNPs currently represent the state-of-the-art RNA delivery technology, the mechanism by which LNPs deliver RNA is not yet fully understood, but it is generally believed that LNPs bind to cell membranes through non-covalent affinity and are endocytosed through a mechanism mediated by clathrin.
  • mRNA escapes endocytic vesicles and is released into the cytoplasm to express the target protein.
  • LNP can also be excreted out of the cell through the opposite exocytosis, which may reduce the delivery efficiency of the drug.
  • the cancer may be prostate cancer, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, brain cancer, melanoma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, squamous cell carcinoma of the head and neck, squamous cell carcinoma of the skin Cystoid cell carcinoma, adenoid cystic carcinoma, glioblastoma, breast cancer, mesothelioma, ovarian cancer, glioma, bladder cancer, liver cancer, bone cancer, bone marrow cancer, stomach cancer, thyroid cancer, lymphoma, Cervical cancer, endometrial cancer, laryngeal cancer, acute lymphoblastic leukemia, etc.
  • the antigen is a tumor antigen, including but not limited to 5T4, AIM2, AKAP4 2, Art-4, AuraA1 (AURKA), Aura B1 (AURKB), BAGE, BCAN, B-cycle BSG, CCND1, CD133, CDC45L, CDCA1 (TTK), CEA, CHI3L2 (chitinase 3-like 2), CSPG4, EpCAM4, Epha2, EPHX1, Ezh2, FABP7, Fosl1 (Fra-1), GAGE, Galt -3, G250(CA9), gBK, glast, GnT-V, gp100, HB-EGF, HER2, HNPRL, HO-1, hTERT, IGF2BP3, IL13-Ra2, IMP-3, IQGAP1, ITGAV, KIF1C, KIF20A, KIF21B, KIFC3, KK-LC-1, LAGE-1, Lck, LRRC8A, MAGE
  • the antigens include, but are not limited to, antigens associated with any tumor/cancer, such as lung cancer (MTFR2 D326Y, CHTF18 L769V, MYADM R30W, HERC1 P3278S, FAM3C K193E, CSMD1G3446E, SLC26A7 R117Q, PGAP1 Y903F, HELB P987S , ANKRD K603T); melanoma (TMEM48F169L, TKT R438W, SEC24A P469L, AKAP13 Q285K, EXOC8 Q656P, PABPC1 R520Q, MRPS5P59L, ABCC2 S1342F, SEC23A P52L, SYTL4 S363F , MAP3K9 E689K, AKAP6 M1482I, RPBMP42L, HCAPG2 P333L, H3F3C T4I, GABPA E16
  • lung cancer
  • the diseases related to the pathogenic microorganisms include but are not limited to: Acquired Immunodeficiency Syndrome (AIDS) (Human Immunodeficiency Virus (HIV)); Argentine Teagan fever (Argentine Teagan fever) (Junin virus); Astrovirus infection (Astrovirus) Viridae); BK virus infection (BK virus); Venezuelan hemorrhagic fever (Machupo virus); Brazilian hemorrhagic fever (Sabiá virus); chickenpox (varicella zoster virus (VZV)); A virus); Colorado tick fever (CTF) (Colorado tick fever virus (CTFV)); common cold, acute viral nasopharyngitis, acute rhinitis (usually rhinovirus and coronavirus); cytomegalovirus infection (CMV); dengue fever (Dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4) and other flaviviruses, including but not limited to West Nile virus (West Nile fever), yellow fever virus (
  • the present invention shows:
  • the immunogenicity of the free RBD mRNA vaccine is weak, and its immunogenicity is improved when the Fc part is added on the basis of RBD, and its immunogenicity is improved when the Pan part is added on the basis of RBD-Fc It has been greatly improved.
  • the cytokine IL12 or CSF2 is added to the Pan-RBD-Fc, its immunogenicity has been further improved.
  • the Pan-RBD-Fc is combined with the immune
  • the immunogenicity of the cell surface protein PDL1 PD1 or aPDL1 antibody has been further improved;
  • the immunogenicity of the free RBD mRNA vaccine is weak, and the antibody titer can be increased after being immunized with Pan alone, and the antibody titer can be further increased when immunized with Pan fusion expression;
  • the antibody titer was not increased after immunization.
  • RBD and ⁇ PDL1-Fc were fused and immunized, the antibody titer could be increased.
  • Fc was added to RBD, the antibody titer could be increased.
  • Pan when adding Pan on the basis of RBD-Fc, the antibody titer can be increased;
  • the immunogenicity of the free HA2mRNA vaccine is weak, and the antibody titer can be increased when it is immunized with Pan alone, and the antibody titer can be further increased when it is immunized with Pan fusion expression.
  • the antibody titer when adding Fc part on the basis of HA2, the antibody titer can be increased, and adding Pan on the basis of HA2-Fc can further increase the antibody titer;
  • HA2 and ⁇ PDL1-Fc mixed immunization did not increase the antibody titer, but when HA2 and ⁇ PDL1-Fc were fused and immunized, the antibody titer could be increased; adding Pan on the basis of ⁇ PDL1-HA2-Fc could further improve the antibody titer. Increase antibody titers;
  • Figure 1 shows the Urea-PAGE electrophoresis identification map of the RBD-related mRNA of the novel coronavirus SARS-CoV-2 spike protein.
  • Figure 2 shows the antibody response induced by the immune cell-targeted mRNA vaccine against the RBD of the SARS-CoV-2 spike protein.
  • Figure 3 shows the Urea-PAGE electrophoresis identification map of the novel coronavirus SARS-CoV-2 spike protein RBD-related mRNA vaccine.
  • Figure 4 shows that immune cells targeting the new coronavirus SARS-CoV-2 spike protein RBD-related mRNA vaccine can elicit stronger antibody responses in mice than RBD mRNA vaccine alone.
  • Figure 5 shows that immune cells targeting the SARS-CoV-2 spike protein RBD-related mRNA vaccine can elicit a stronger antibody response in mice than RBD mRNA vaccine alone.
  • Figure 6 shows the Urea-PAGE electrophoresis identification diagram of influenza virus Influenza A virus hemagglutinin protein HA-related mRNA vaccine.
  • Figure 7 shows that immune cells targeting influenza virus Influenza A virus hemagglutinin protein HA-related mRNA vaccine can elicit a stronger antibody response in mice than pure HA mRNA vaccine.
  • Immune cell targeting mRNA vaccine consists of four structural units: 5'cap, 5'UTR, ORF, 3'UTR and 3'Poly(A).
  • the ORF in immune cell targeting mRNA vaccines is fused with immune cell targeting molecules on the antigen, including the following four components: A. Antibodies or polypeptides that can bind to immune cell surface proteins; B. Cytokines that can activate immune cells; C. . Pan epitope capable of activating immune cells (PADRE); D. Immunoglobulin Fc capable of binding immune cells.
  • the antigen can be arranged and combined with one, two, three or four of these four components in any form to form an open reading frame for expressing fusion protein.
  • the antigen is connected with the immune cell targeting molecule and the immune cell targeting molecule through a connecting fragment.
  • the construction and production of the vaccine platform are described by taking the spike protein RBD of the new coronavirus SARS-CoV-2 as an example.
  • the mRNA vaccine structural unit was constructed downstream of the T7 RNA polymerase promoter by molecular cloning, so as to obtain a plasmid capable of transcribing mRNA.
  • KOZAK is the Kozak sequence
  • SP signal peptide
  • the antigen is connected with the immune cell targeting molecule and the immune cell targeting molecule through a connecting fragment.
  • the amino acid sequences of linker fragments are as follows: linker 1 (Linker 1) is (G) 3 , linker 2 (Linker 2) is (GS) 3 , and linker 3 (Linker 3) is (GGGGS) 3 .
  • the Cleanup Purification Kit was used to purify mRNA samples.
  • the electrophoresis buffer is 0.5 ⁇ TBE, run the gel at 250V for 2 hours, and image it with a gel imager to check the integrity and purity of mRNA.
  • C57BL/6 male mice (6-8) weeks were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd.
  • HRP horseradish peroxidase
  • HRP horseradish peroxidase
  • IgG1, IgG2b and IgG2c horseradish peroxidase
  • 96-well ELISA assay plate was purchased from Bioland Company
  • ELISA chromogenic solution Purchased from Shanghai Biyuntian Biotechnology Co., Ltd.
  • ELISA stop solution was purchased from Beijing Suolaibao Technology Co., Ltd.
  • microplate reader Multiskan FC was purchased from Thermo Fisher Scientific.
  • mice were immunized with mRNA vaccine. After the mRNA was mixed with the transfection reagent Lipofectamine Messenger MAX 3 times the mass of the mRNA, it was allowed to stand at room temperature for 5 minutes. Each mouse was inoculated with 0.5 ⁇ g mRNA or other mRNA with the same molar amount, and each mouse was injected intramuscularly with 50 ⁇ l. Mice were immunized on days 0 and 21 using a two-immunization schedule. On the 14th day after each immunization, mouse serum was collected by cheek blood collection for antibody detection.
  • the immunogenicity of the free RBD mRNA vaccine was weak, and its immunogenicity was improved when the Fc part was added on the basis of RBD, and its immunogenicity was extremely improved when the Pan part was added on the basis of RBD-Fc.
  • Great improvement when the cytokine IL12 or CSF2 is added on the basis of Pan-RBD-Fc, its immunogenicity is further improved, and when Pan-RBD-Fc is added on the basis of immune cell surface protein
  • the immunogenicity of PDL1 PD1 or aPDL1 antibody was further improved, and the antibody level after the second immunization was generally higher than that after the first immunization, as shown in Figure 2(a).
  • IL12, CSF2, PD1 or aPDL1 induced higher levels of IgG1 representing Th2 immune responses on the basis of Pan-RBD-Fc, as shown in Figure 2(b).
  • IL12, PD1 or aPDL1 induced higher levels of IgG2b and IgG2c representing Th1 immune responses on the basis of Pan-RBD-Fc, as shown in Figure 2(c) and Figure 2(d).
  • the ratio of (IgG2b+IgG2c)/IgG1 showed that Pan-RBD-Fc, CSF2-Pan-RBD-Fc and PD1-Pan-RBD-Fc induced relatively balanced Th1 and Th2 immune responses, while IL12-Pan-RBD-Fc and aPDL1-Pan-RBD-Fc induced a Th1-biased immune response, as shown in Figure 2(e).
  • Example 4 Construction, production and identification of mRNA vaccines related to SARS-CoV-2 spike protein RBD
  • the mRNA vaccine structural unit was constructed downstream of the T7 RNA polymerase promoter by molecular cloning, so as to obtain a plasmid capable of transcribing mRNA.
  • KOZAK is the Kozak sequence
  • SP signal peptide
  • the antigen is connected with the immune cell targeting molecule and the immune cell targeting molecule through a connecting fragment.
  • the amino acid sequences of linker fragments are as follows: linker 1 (Linker 1) is (G) 3 , linker 2 (Linker 2) is (GS) 3 , and linker 3 (Linker 3) is (GGGGS) 3 .
  • the Cleanup Purification Kit was used to purify mRNA samples.
  • the electrophoresis buffer is 0.5 ⁇ TBE, run the gel at 250V for 2 hours, and image it with a gel imager to check the integrity and purity of mRNA.
  • Example 5 Immune cells targeting the new coronavirus SARS-CoV-2 spike protein RBD-related mRNA vaccine can elicit a stronger antibody response in mice than a simple RBD mRNA vaccine
  • C57BL/6 male mice (6-8) weeks were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd. Co., Ltd.; 96-well ELISA assay plate was purchased from Bioland; ELISA chromogenic solution was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; ELISA stop solution was purchased from Beijing Solaibao Technology Co., Ltd.; Microplate reader Multiskan FC was purchased from Thermo Fisher Scientific company.
  • HRP horseradish peroxidase
  • mice were immunized with mRNA vaccine. After the mRNA was mixed with the transfection reagent Lipofectamine Messenger MAX 3 times the mass of the mRNA, it was allowed to stand at room temperature for 5 minutes. Each mouse was inoculated with 0.5 ⁇ g mRNA or other mRNA with the same molar amount, and each mouse was injected intramuscularly with 50 ⁇ l. Mice were immunized on days 0 and 21 using a two-immunization schedule. On the 14th day after each immunization, mouse serum was collected by cheek blood collection for antibody detection.
  • Example 6 Immune cells targeting the new coronavirus SARS-CoV-2 spike protein RBD-related mRNA vaccine can elicit a stronger antibody response in mice than a simple RBD mRNA vaccine
  • C57BL/6 male mice (6-8) weeks were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd. Co., Ltd.; 96-well ELISA assay plate was purchased from Bioland; ELISA chromogenic solution was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; ELISA stop solution was purchased from Beijing Solaibao Technology Co., Ltd.; Microplate reader Multiskan FC was purchased from Thermo Fisher Scientific company.
  • HRP horseradish peroxidase
  • mice were immunized with mRNA vaccine.
  • Dissolve SM-102, DSPC, Cholesterol and DMG-PEG2000 in ethanol so that the molar ratio is 50:10:38.5:1.5, dissolve mRNA in 20mM sodium acetate (pH 4), and mix mRNA and lipid
  • the mixed immunization of RBD and ⁇ PDL1-Fc does not increase the antibody titer, but the antibody titer can be increased when RBD is fused with ⁇ PDL1-Fc and immunized, when IFN ⁇ 4 is added on the basis of ⁇ PDL1-RBD-Fc Instead, the antibody titer was reduced.
  • the antibody titer can be increased, when adding Pan on the basis of RBD-Fc, the antibody titer can be increased, and when adding IL12 on the basis of RBD-Fc, the antibody titer can not be improved Spend.
  • Example 7 Construction, production and identification of influenza virus Influenza A virus hemagglutinin protein HA-related mRNA vaccine
  • HA in influenza A There are at least 18 different subtypes of HA in influenza A.
  • H1 in human influenza virus as the research object, and each HA is cleaved into HA1 and HA2 subunits by cellular protease.
  • HA1 forming the globular head while containing the predominant neutralizing epitope, is highly variable between subtypes.
  • the HA2 subunit constitutes most of the stalk domain, is fairly conserved among subtypes, and contains few but cross-reactive neutralizing epitopes.
  • the mRNA vaccine structural unit was constructed downstream of the T7 RNA polymerase promoter by molecular cloning, so as to obtain a plasmid capable of transcribing mRNA.
  • KOZAK is the Kozak sequence
  • SP signal peptide
  • the antigen is connected with the immune cell targeting molecule and the immune cell targeting molecule through a connecting fragment.
  • the amino acid sequences of linker fragments are as follows: linker 1 (Linker 1) is (G) 3 , linker 2 (Linker 2) is (GS) 3 , and linker 3 (Linker 3) is (GGGGS) 3 .
  • the Cleanup Purification Kit was used to purify mRNA samples.
  • the electrophoresis buffer is 0.5 ⁇ TBE, run the gel at 250V for 2 hours, and image it with a gel imager to check the integrity and purity of mRNA.
  • Example 8 Immune cells target influenza virus Influenza A virus hemagglutinin protein HA-related mRNA vaccine can cause stronger antibody response in mice than simple HA mRNA vaccine
  • C57BL/6 male mice (6-8) weeks were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd. Co., Ltd.; 96-well ELISA assay plate was purchased from Bioland; ELISA chromogenic solution was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; ELISA stop solution was purchased from Beijing Solaibao Technology Co., Ltd.; Microplate reader Multiskan FC was purchased from Thermo Fisher Scientific company.
  • HRP horseradish peroxidase
  • the immunogenicity of the free HA2mRNA vaccine is weak, and the antibody titer can be increased when it is immunized with Pan alone, and the antibody titer can be further increased when it is immunized with Pan fusion expression.
  • the antibody titer when adding Fc part on the basis of HA2, the antibody titer can be improved, when adding Pan on the basis of HA2-Fc, the antibody titer can be further improved, and when adding IFN ⁇ 4 on the basis of Pan-HA2-Fc Instead, the antibody titer was reduced, as shown in Figure 7.

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Abstract

提供了一种mRNA疫苗，所述mRNA疫苗包含与抗原融合表达并增强mRNA疫苗免疫效果的免疫细胞靶向分子。

Description

mRNA疫苗 技术领域
本发明属于疫苗领域，具体地涉及mRNA疫苗及其应用。
背景技术
信使核糖核酸(mRNA)是与基因的遗传序列相对应的单链RNA分子，在生物体内合成蛋白质的过程中被核糖体翻译产生蛋白质。将T7噬菌体RNA聚合酶与质粒DNA结合可进行mRNA的转录。体外转录的mRNA具有与真核细胞中天然mRNA相同的结构成分：一个5'帽(5'cap)，一个5'非翻译区(5'UTR)，一个编码相关抗原的开放阅读框(ORF)，一个3'非翻译区(3'UTR)，以及一个3'多聚A尾巴[3'Poly(A)]。利用合成的5'-cap类似物可以提高mRNA的稳定性，增加蛋白质的翻译。mRNA核苷酸可以被修改以降低先天免疫激活以及增加mRNA在宿主细胞中的半衰期。
mRNA疫苗是一种利用包裹在脂质纳米颗粒(LNP)中的mRNA分子来产生免疫应答的疫苗。mRNA可以递送到免疫细胞，也可以是其他细胞，如肌肉细胞或上皮细胞等。这些细胞翻译产生的蛋白可以表达在细胞内部，细胞膜表面，也可以分泌到细胞外面，最终靶向到免疫细胞。一旦宿主细胞产生了抗原，正常的适应性免疫系统过程就开始了。抗原被蛋白酶体分解，I类和II类MHC分子附着在抗原上，并将其运输到细胞膜上，激活树突状细胞。一旦被激活，树突状细胞迁移到淋巴结，将抗原呈递给T细胞和B细胞，最终导致免疫应答。mRNA疫苗优点在于设计简便，可呈递多种抗原，有多联、多价疫苗研发潜力，生产周期短，扩产能也更容易，不会与人体DNA整合，也没有外源性病毒感染的风险。
因此，mRNA疫苗是继灭活疫苗、减毒活疫苗、亚单位疫苗和病毒载体疫苗后的第三代疫苗，具有针对病原体变异反应速度快、生产工艺简单、易规模化扩大等特点。然而，mRNA疫苗的毒副作用也非常明显，比如注射部位酸疼反应、疲倦、头疼，甚至面瘫，第二针接种后副反应更大。现有mRNA疫苗技术还有很多可以优化和改进的地方，而且仍有较多的技术壁垒尚需突破。目前对于mRNA疫苗的改进研究主要集中在LNP递送技术，质粒的生产，mRNA的合成和纯化，对于mRNA结构及序列本身的优化也多集中在编码抗原的核苷酸本身的修饰，尚未有关于在编码相关抗原的开放阅读框中另外包含编码免疫细胞靶向 分子的编码序列的报道。
发明内容
本发明提供了免疫效果增强的mRNA疫苗，所述mRNA疫苗包括开放阅读框，所述开放阅读框编码抗原和免疫细胞靶向分子。本发明的免疫效果增强的mRNA疫苗具有增强免疫效果，提高mRNA疫苗保护率，降低mRNA疫苗接种剂量，放大mRNA疫苗产能以及降低mRNA疫苗毒副作用的效果。
第一方面，本发明提供了含有开放阅读框(ORF)的mRNA，所述开放阅读框编码抗原(Antigen)和免疫细胞靶向分子。所述开放阅读框编码的抗原和免疫细胞靶向分子为抗原和免疫细胞靶向分子融合蛋白。
在一些实施方案中，所述免疫细胞靶向分子为选自如下的一种或多种：
A：能够结合免疫细胞表面蛋白的抗体或多肽；
B：能够激活免疫细胞的细胞因子；
C：能够激活免疫细胞的Pan表位(PADRE)；
D：能够结合免疫细胞的免疫球蛋白Fc。
在一些实施方案中，所述开放阅读框编码抗原与A的融合蛋白；抗原与A和B的融合蛋白；抗原与A，B和C的融合蛋白；抗原与A，B，C和D的融合蛋白；抗原与B的融合蛋白；抗原与B和C的融合蛋白；抗原与B，C和D的融合蛋白；抗原与C的融合蛋白；抗原与C和D的融合蛋白；或抗原与D的融合蛋白。
所述融合蛋白可以是抗原与A和/或B和/或C和/或D的任意排列方式，例如，所述融合蛋白中抗原和A和/或B和/或C和/或D相对于彼此位于C端或者N端。
在一些实施方案中，所述融合蛋白是同源二聚体或异源二聚体。
本发明提供的mRNA可以表达分泌型的融合蛋白或膜融合蛋白。在一些实施方案中，所述mRNA表达分泌型的融合蛋白因为带有免疫细胞靶向分子，因此能被高效地靶向到免疫细胞表面。
所述能够结合免疫细胞表面蛋白的抗体或多肽A包括但不限于针对CD274(PDL1)，PDCD1LG2(PDL2)，CLEC9A，LY75(DEC205)，CD40，TNFSF9(4-1BB-L)和/或TNFSF4(OX4OL)等的抗体，或其配体的活性片段。
所述能够激活免疫细胞的细胞因子B包括但不限于白介素(interleukin，IL)，集落刺激因子(Colony-stimulating factor，CSF)和干扰素(interferon，IFN)。
优选地，所述白介素包括IL2，IL12，IL15和IL21，或其活性片段。IL2或其活性片段或同源物来自人，牛，羊，猫，犬，马，兔，猴，小鼠，大鼠，羊驼或骆驼。
优选地，所述集落刺激因子包括CSF1，CSF2和CSF3，或其活性片段。
优选地，所述干扰素包括I型干扰素，II型干扰素和III型干扰素，或其活性片段，其中I型干扰素包括IFNα，IFNω，IFNε，IFNк和IFNβ，或其活性片段，II型干扰素包括IFN-γ，或其活性片段，III型干扰素包括IFN-λ1，IFN-λ2，IFN-λ3和IFN-λ4，或其活性片段。
优选地，所述能够激活免疫细胞的Pan表位(PADRE)C具有AKFVAAWTLKAAA(SEQ ID NO：1)所示的氨基酸序列。
所述免疫球蛋白Fc(D)可以为来自IgG，IgM，IgA，IgE或IgD的Fc或其突变体，所述Fc为突变体以形成异源二聚体蛋白。
在一些实施方案中，所述mRNA还包含5'UTR(非翻译区)序列和3'UTR(非翻译区)。所述5'UTR和3'UTR可以为任何高表达基因的5'UTR和3'UTR。
在一些实施方案中，所述mRNA还包含5'Cap。
在一些实施方案中，所述mRNA还包含3'Poly(A)。
所述抗原(Antigen)可以为能够诱导针对病原微生物的免疫反应的免疫原性蛋白或其免疫原性片段。
所述病原微生物包括但不限于SARS-Cov-2，SARS，巨细胞病毒CMV，疱疹病毒，RSV，流感病毒，Ebola病毒，爱泼斯坦-巴尔病毒(EBV)，登革热病毒，Zike病毒，HIV病毒，狂犬病毒，疟原虫配子体，带状疱疹病毒(HZV)，乙肝病毒HBV，丙型肝炎病毒(HCV)，丁肝病毒HDV，HPV，结核分枝杆菌，幽门螺旋杆菌等。
所述抗原可以为能够诱导针对癌症细胞的免疫反应的免疫原性蛋白或其免疫原性片段。
在一些实施方案中，所述抗原可以为肿瘤抗原，例如MelanA/MART1、癌-种系抗原、gp100、酪氨酸酶、CEA、PSA、Her-2/neu、存活蛋白、端粒酶，或其免疫原性片段。
在一些实施方案中，所述开放阅读框(ORF)还包含编码连接片段的区域。
抗原与免疫细胞靶向分子之间，和/或免疫细胞靶向分子之间可以通过连接片段进行连接。
在一些实施方案中，所述连接片段可以是柔性连接片段、刚性连接片段或体内剪切连接片段。其中柔性连接片段的氨基酸序列可以是但不限于(G) N，(GS) N，(GGS) N，(GGGS) N，或(GGGGS) N。
在一些实施方案中，所述mRNA具有选自如下的结构：
5'Cap–5'UTR–A–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–CLEC9A binding peptide–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αDEC205–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–A–Linker–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–Linker–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–(GGGGS) 3–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–CLEC9A binding peptide–Linker–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–CLEC9A binding peptide–(GGGGS) 3–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αDEC205–Linker–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αDEC205–(GGGGS) 3–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–A–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–αPDL1–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–CLEC9A binding peptide–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–CD40L–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–4-1BB–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–OX40–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–Linker–A–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–Linker–αPDL1–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–(GGGGS) 3–αPDL1–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–Linker–CLEC9A binding peptide–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–(GGGGS) 3–CLEC9A binding peptide–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–Linker–CD40L–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–(GGGGS) 3–CD40L–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–Linker–4-1BB–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–(GGGGS) 3–4-1BB–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–Linker–OX40–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–(GGGGS) 3–OX40–3'UTR–3'Poly(A)；
5'Cap–5'UTR–B–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IL2–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IL12–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IL15–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IL21–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–CSF2–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IFNα–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–B–Linker–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IL2–Linker–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IL2–(GGGGS) 3–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IL12–Linker–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IL12–(GGGGS) 3–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IL15–Linker–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IL15–(GGGGS) 3–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IL21–Linker–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IL21–(GGGGS) 3–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–CSF2–Linker–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–CSF2–(GGGGS) 3–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IFNα–Linker–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IFNα–(GGGGS) 3–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–B–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–Linker–B–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–Linker–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–(GGGGS) 3–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–C–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Pan–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–C–Linker–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Pan–Linker–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Pan–(GS) 3–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–C–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–Pan–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–Linker–C–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–Linker–Pan–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–(GS) 3–Pan–3'UTR–3'Poly(A)；
5'Cap–5'UTR–A–Antigen–B–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–Antigen–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–A–Linker–Antigen–Linker–B–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–Linker–Antigen–Linker–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–(GGGGS) 3–Antigen–(GS) 3–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–A–C–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–Pan–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–A–Linker–C–Linker–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–Linker–Pan–Linker–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–(GGGGS) 3–Pan–(GS) 3–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–A–Antigen–D–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–Antigen–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–A–Linker–Antigen–Linker–D–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–(GGGGS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–B–C–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–CSF2–Pan–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–B–Linker–C–Linker–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–CSF2–Linker–Pan–Linker–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–CSF2–(GGGGS) 3–Pan–(GS) 3–Antigen–3'UTR–3'Poly(A)；
5'Cap–5'UTR–B–Antigen–D–3'UTR–3'Poly(A)；
5'Cap–5'UTR–CSF2–Antigen–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–B–Linker–Antigen–Linker–D–3'UTR–3'Poly(A)；
5'Cap–5'UTR–CSF2–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–CSF2–(GGGGS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–D–B–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–Fc–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–Linker–D–Linker–B–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–Linker–Fc–Linker–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Antigen–(G) 3–Fc–(GS) 3–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–C–Antigen–D–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–C–Linker–Antigen–Linker–D–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–A–C–Antigen–B–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–Pan–Antigen–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–A–Linker–C–Linker–Antigen–Linker–B–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–Linker–Pan–Linker–Antigen–Linker–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–(GGGGS) 3–Pan–(GS) 3–Antigen–(GS) 3–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–A–Antigen–D–B–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–Antigen–Fc–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–A–Linker–Antigen–Linker–D–Linker–B–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–Linker–Antigen–Linker–Fc–Linker–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–(GGGGS) 3–Antigen–(G) 3–Fc–(GS) 3–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–A–C–Antigen–D–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–PD1–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–CLEC9A binding peptide–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αDEC205–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–A–Linker–C–Linker–Antigen–Linker–D–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–Linker–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–PD1–Linker–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–PD1–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–CLEC9A binding peptide–Linker–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–CLEC9A binding peptide–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αDEC205–Linker–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αDEC205–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–B–C–Antigen–D–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IL2–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IL12–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IL21–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–CSF2–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IFNα–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–B–Linker–C–Linker–Antigen–Linker–D–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IL2–Linker–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IL2–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IL12–Linker–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IL12–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IL21–Linker–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IL21–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–CSF2–Linker–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–CSF2–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IFNα–Linker–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–IFNα–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–C–Antigen–D–B–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Pan–Antigen–Fc–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–C–Linker–Antigen–Linker–D–Linker–B–3'UTR–3'Poly(A)；
5'Cap–5'UTR–Pan–(GS) 3–Antigen–(G) 3–Fc–(GS) 3–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–A–C–Antigen–D–B–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–Pan–Antigen–Fc–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–CLEC9A binding peptide–Pan–Antigen–Fc–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–Pan–Antigen–Fc–IFNα–3'UTR–3'Poly(A)；
5'Cap–5'UTR–A–Linker–C–Linker–Antigen–Linker–D–Linker–B–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–Linker–Pan–Linker–Antigen–Linker–Fc–Linker–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–(GS) 3–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–CLEC9A binding peptide–Linker–Pan–Linker–Antigen–Linker–Fc–Linker–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–CLEC9A binding peptide–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–(GS) 3–CSF2–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–Linker–Pan–Linker–Antigen–Linker–Fc–Linker–IFNα–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–(GS) 3–IFNα–3'UTR–3'Poly(A)；
5'Cap–5'UTR–A–B–C–Antigen–D–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–CSF2–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–A–Linker–B–Linker–C–Linker–Antigen–Linker–D–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–Linker–CSF2–Linker–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
5'Cap–5'UTR–αPDL1–(GGGGS) 3–CSF2–(GS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)。
上述其中mRNA中开放阅读框中表达抗原与免疫细胞靶向分子的结构域可以用任意形式排列组合。开放阅读框中表达抗原与免疫细胞靶向分子A，B，C，D的结构域可以任意形式排列组合。
在一些实施方案中，所述mRNA中开放阅读框为一种免疫细胞靶向分子与 抗原形成表达融合蛋白的开放阅读框。分别按照A-抗原，B-抗原，C-抗原的顺序排列组合的示意图。抗原与免疫细胞靶向分子A可以用任意形式排列组合。抗原与免疫细胞靶向分子B可以用任意形式排列组合。抗原与免疫细胞靶向分子C可以用任意形式排列组合。
在一些实施方案中，所述mRNA中开放阅读框为两种免疫细胞靶向分子与抗原形成表达融合蛋白的开放阅读框。分别按照A-B-抗原，A-C-抗原，A-D-抗原，B-C-抗原，B-D-抗原，C-D-抗原的顺序排列组合的示意图。抗原与免疫细胞靶向分子A，B可以用任意形式排列组合。抗原与免疫细胞靶向分子A，C可以用任意形式排列组合。抗原与免疫细胞靶向分子A，D可以用任意形式排列组合。抗原与免疫细胞靶向分子B，C可以用任意形式排列组合。抗原与免疫细胞靶向分子B，D可以用任意形式排列组合。抗原与免疫细胞靶向分子C，D可以用任意形式排列组合。
在一些实施方案中，所述mRNA中开放阅读框为三种免疫细胞靶向分子与抗原形成表达融合蛋白的开放阅读框。分别按照A-B-C-抗原，A-B-D-抗原，A-C-D-抗原，B-C-D-抗原的顺序排列组合的示意图。抗原与免疫细胞靶向分子A，B，C可以用任意形式排列组合。抗原与免疫细胞靶向分子A，B，D可以用任意形式排列组合。抗原与免疫细胞靶向分子A，C，D可以用任意形式排列组合。抗原与免疫细胞靶向分子B，C，D可以用任意形式排列组合。
在一些实施方案中，所述mRNA中开放阅读框为四种免疫细胞靶向分子与抗原形成表达融合蛋白的开放阅读框。按照A-B-C-D-抗原的顺序排列组合的示意图。抗原与免疫细胞靶向分子A，B，C，D可以用任意形式排列组合。
在一些实施方案中，所述抗原为SARS-Cov-2的S蛋白或其片段。优选地，所述S蛋白为预融合稳定的S蛋白。在一些实施方案中，所述预融合稳定的S蛋白包含双脯氨酸(S2P)突变或六脯氨酸(S6P)突变。
在一些实施方案中，所述抗原为SARS-Cov-2的S蛋白的RBD结构域。
第二方面，本发明提供了含有第一方面的mRNA的组合物。
在一些实施方案中，所述组合物还包含可药用载体。所述可药用载体可以为脂质纳米颗粒(LNP)、高分子材料和无机纳米颗粒。例如，还可以为阳离子脂质复合物(LPX)，脂质多聚复合物(LPP)，聚合物纳米颗粒(PNP)，无机纳米颗 粒(INP)，阳离子纳米乳(CNE)等。
优选地，所述脂质纳米颗粒(LNP)可用于感染性疾病的mRNA疫苗或用于mRNA肿瘤疫苗。优选地，所述阳离子脂质复合物(LPX)可用于mRNA肿瘤疫苗或用于感染性疾病的mRNA疫苗。
第三方面，本发明提供了用于预防或治疗与病原微生物感染或肿瘤的方法，所述方法包括向对象给药第一方面的mRNA或第二方面的组合物的步骤。
所述对象为人类或动物。所述动物包括但不限于牛，羊，猫，犬，马，兔，猴，小鼠，大鼠，羊驼，骆驼等。
在一些实施方案中，所述对象为免疫功能低下的人或动物。
在一些实施方案中，所述对象患有慢性肺病，例如慢性阻塞性肺疾病或哮喘。
在一些实施方案中，所述患者患有基础疾病，例如心脏病，糖尿病或肺病。
第四方面，本发明提供了第一方面的mRNA或第二方面的组合物在制备用于预防或治疗对象与病原微生物感染或肿瘤相关的药物中的应用。
所述对象为人类或动物。所述动物包括但不限于牛，羊，猫，犬，马，兔，猴，小鼠，大鼠，羊驼，骆驼，鸡，鸭，鹅，等。
在一些实施方案中，所述对象为免疫功能低下的人或动物。
在一些实施方案中，所述对象患有慢性肺病，例如慢性阻塞性肺疾病或哮喘。
在一些实施方案中，所述患者患有基础疾病，例如心脏病，糖尿病或肺病。
mRNA可以递送到免疫细胞，也可以是其他细胞，如肌肉细胞或上皮细胞。这些细胞翻译产生的蛋白可以分泌出来，靶向到免疫细胞。
本发明提供的mRNA可以表达分泌型的融合蛋白或膜融合蛋白。在一些实施方案中，所述mRNA表达分泌型的融合蛋白因为带有免疫细胞靶向分子，因此能被高效地靶向到免疫细胞表面。
因此，本发明的mRNA疫苗具有增强的免疫效果，增强的保护率，降低的mRNA疫苗接种剂量，放大的mRNA疫苗产能，降低的mRNA疫苗毒副作用。
本发明中mRNA疫苗中抗原融合表达的设计同样可以应用于其它类型的疫苗，包括但不限于核酸疫苗(如DNA疫苗和环状RNA疫苗等)，病毒载体疫苗(如腺病毒载体疫苗和流感病毒载体疫苗等)，重组蛋白疫苗以及纳米颗粒疫苗等。
名词解释：
侧翼区：非翻译区(UTR)
非翻译区(UTR)能够被转录但不被翻译。5'UTR开始于转录起始位点至起始密码子但不包括起始密码子；而3'UTR紧随终止密码子直到转录终止信号。UTR影响转录调控和mRNA的稳定性，并且影响翻译效率。5'UTR通常筛选自天然存在的稳定/高表达序列，或由其改造而来。3'UTR通常与mRNA稳定性有关，与5'UTR类似选自天然存在的序列，或进行一定修饰。
5'Cap是mRNA疫苗非常重要的结构，通常与真核生物翻译起始因子结合来启动翻译，加帽能够帮助mRNA被顺利转运出细胞核，另外5'Cap也可以使mRNA免于核酸外切酶的降解，还可促使mRNA环化形成空间及结构，增强稳定性。通常用帽结构类似物共转录加帽或单独使用加帽酶进行转录后加帽。
3'UTR尾部是一段PolyA序列，与帽子结构类似，PolyA尾也起到保护mRNA防止被核酸外切酶降解的作用，PolyA也参与到翻译及调控过程，增加稳定性，延长体内半衰期并提高mRNA的翻译效率。PolyA结合蛋白(PABP)可通过翻译起始因子(如elF4G和elF4E)与5'帽连接形成闭环结构，协同参与对mRNA的稳定性及翻译活性的调节。
药物递送一直是mRNA药物发展所面临的较大的技术障碍。大多数真核细胞能够通过胞吞作用主动将裸露的mRNA摄入胞体内，但其效率极低，10,000个分子中通常仅有1个能够通过细胞膜。mRNA进入体内后，首先面临周围组织液或血液中核酸酶的降解，此外还需要通过负电性的磷脂双分子生物膜。因此，其制剂系统需要保护mRNA不被核酸酶降解，同时还需要促进其被细胞摄取。截至目前，对于mRNA药物的递送系统已经经历了3代技术演变。从20世纪90年代出现的以鱼精蛋白、阳离子高分子PEI、阳离子脂质体为代表的第1代递送系统，到90年代末出现的以可降解、可离子化高分子为代表的第2代递送系统，再到近些年出现的以可离子化脂质纳米粒LNP为代表的第3代递送系统。尽管LNPs目前代表着最先进的RNA输送技术，LNP递送RNA的原理目前还不完全清楚，但通常认为，LNP通过非共价亲和力和细胞膜结合并通过由网格蛋白clatherin介导的机制内吞入细胞，mRNA逃离内吞小泡，被释放到细胞质中表达目标蛋白，然而LNP还可以通过相反的胞吐作用被排出细胞外，可能降 低药物的递送效率。
所述癌症可以为前列腺癌，非小细胞肺癌，小细胞肺癌，肾细胞癌，脑癌，黑色素瘤，急性髓细胞性白血病，胰腺癌，结直肠癌，头颈部鳞状细胞癌，皮肤鳞状细胞癌，腺样囊性癌，胶质母细胞瘤，乳腺癌，间皮瘤，卵巢癌，神经胶质瘤，膀胱癌，肝癌，骨癌，骨髓癌，胃癌，甲状腺癌，淋巴癌，宫颈癌，子宫内膜癌，喉癌，急性淋巴细胞性白血病，等。
在一些实施方案中，所述抗原为肿瘤抗原，所述肿瘤抗原包括但不限于5T4,AIM2,AKAP4 2,Art-4,AuraA1(AURKA),Aura B1(AURKB),BAGE,BCAN,B-周期素,BSG,CCND1,CD133,CDC45L,CDCA1(TTK),CEA,CHI3L2(几丁质酶3样2),CSPG4,EpCAM4,Epha2,EPHX1,Ezh2,FABP7,Fosl1(Fra-1),GAGE,Galt-3,G250(CA9),gBK,glast,GnT-V,gp100,HB-EGF,HER2,HNPRL,HO-1,hTERT,IGF2BP3,IL13-Ra2,IMP-3,IQGAP1,ITGAV,KIF1C,KIF20A,KIF21B,KIFC3,KK-LC-1,LAGE-1,Lck,LRRC8A,MAGE-1(MAGEA1),MAGE-2(MAGEA2B),MAGE-3,MAGE-4,MAGE-6,MAGE-10,MAGE-12,MAGE-C1(CT7),MAGE-C2,MAGE-C3,Mart-1,MELK,MRP3,MUC1,NAPSA,NLGN4X,Nrcam,NY-ESO-1(CTAG1B),NY-SAR-35,OFA/iLRP,PCNA,PIK3R1,Prame,PRKDC,PTH-rP,PTPRZ1,PTTG1 2,PRKDC,RAN,RGS1,RGS5,RHAMM(RHAMM-3R),RPL19,Sart-1,Sart-2,Sart-3,SEC61G,SGT-1,SOX2,Sox10,Sox11,SP17,SPANX-B,SQSTM1,S.S.X-2,STAT1,STAT3,生存素,TARA,TNC,Trag-3,TRP-1,TRP2,酪氨酸酶,URLC10(LY6K),Ube2V,WT1,XAGE-1b(GAGED2a),YKL-40(CHI3L1),ACRBP,SCP-1,S.S.X-1,S.S.X-4,NY-TLU-57,CAIX,Brachyury,NY-BR-1,ErbB,间皮素,EGFRvIII,IL-13Ra2,MSLN,GPC3,FR,PSMA,GD2,L1-CAM,VEGFR1,VEGFR2,KOC1,OFA,SL-701,突变体P53,DEPDC1,MPHOSPH1,ONT-10,GD2L,GD3L,TF,PAP,BRCA1DLC1,XPO1,HIF1A,ADAM2,CALR3,SAGE1,SCP-1,ppMAPkkk,WHSC,突变体Ras,COX1,COX2,FOXP3,IDO1,IDO2,TDO,PDL1,PDL2和PGE2。
在一些实施方案中，所述抗原包括但不限于与任何肿瘤/癌症有关的抗原，例如肺癌(MTFR2 D326Y,CHTF18 L769V,MYADM R30W,HERC1 P3278S,FAM3C K193E,CSMD1G3446E,SLC26A7 R117Q,PGAP1 Y903F,HELB P987S, ANKRD K603T)；黑色素瘤(TMEM48F169L,TKT R438W,SEC24A P469L,AKAP13 Q285K,EXOC8 Q656P,PABPC1 R520Q,MRPS5P59L,ABCC2 S1342F,SEC23A P52L,SYTL4 S363F,MAP3K9 E689K,AKAP6 M1482I,RPBMP42L,HCAPG2 P333L,H3F3C T4I,GABPA E161K,SEPT2 Q125R,SRPX P55L,WDR46 T300I,PRDX3 P101L,HELZ2 D614N,GCN1L1 P769L,AFMID A52V,PLSCR4 R247C,CENPL P79L,TPX2H458Y,SEC22C H218Y,POLA2 L420F,SLC24A5 mut)；间皮瘤(NOTCH2 G703D,PDE4DIPL288M,BAP1 V523fs,ATP10B E210K,NSD1 K2482T)；胶质瘤/胶质母细胞瘤(IDH1 R132H,POLE L424V)；乳腺癌(mPALB2,mROBO3,mZDHHC16,mPTPRS,RBPJ H204L)；胆管癌((ERBB2IPE805G)；和宫颈癌(MAPK1 E322K,PIK3CA E545K,PIK3CA E542K,EP300 D1399N,ERBB2S310F,ERBB3 V104M,KRAS G12D)。
所述病原微生物相关的疾病包括但不限于：获得性免疫缺陷综合症(AIDS)(人类免疫缺陷病毒(HIV))；阿根廷提根热(ArgentineTeagan fever)(Junin病毒)；星状病毒感染(星状病毒科)；BK病毒感染(BK病毒)；玻利维亚出血热(Machupo病毒)；巴西出血热(Sabiá病毒)；水痘(水痘带状疱疹病毒(VZV))；基孔肯雅热(Chikungunya)(甲病毒)；科罗拉多蜱传热(Colorado tick fever)(CTF)(科罗拉多蜱传热病毒(Colorado tick fever virus)(CTFV))；普通感冒，急性病毒性鼻咽炎，急性鼻炎(通常是鼻病毒和冠状病毒)；巨细胞病毒感染(巨细胞病毒)；登革热(登革热病毒(DEN-1，DEN-2，DEN-3和DEN-4)和其他黄病毒，包括但不限于西尼罗河病毒(西尼罗河热)，黄热病病毒(黄热病)；寨卡病毒(寨卡热)和蜱传脑炎病毒；埃博拉出血热(埃博拉病毒(EBOV))；肠病毒感染(肠病毒种)；感染性红斑病(第五种病)(细小病毒B19)；幼儿急疹(Exanthemsubitum)(第六种病)(人类疱疹病毒6(HHV-6)和人类疱疹病毒7(HHV-7))；手足口病(HFMD)(肠病毒，主要是柯萨奇A病毒和肠病毒71(EV71))；汉坦病毒肺综合征(HantavirusPulmonary Syndrome，HPS)(Sin Nombre病毒)；甲型肝炎(甲型肝炎病毒)；乙型肝炎(乙型肝炎病毒)；丙型肝炎(丙型肝炎病毒)；丁型肝炎(丁型肝炎病毒)；戊型肝炎(戊型肝炎病毒)；单纯疱疹(单纯疱疹病毒1和2(HSV-1和HSV-2))；人博卡病毒感染(人博卡病毒(HBoV))；人间质性肺炎病毒感染(人间质性肺炎病毒(hMPV))；人乳头瘤病毒(HPV)感染(人乳头瘤病毒(HPV))；人副流感病毒感染(人副流感病毒(HPIV))；爱 泼斯坦-巴尔病毒传染性单核细胞增多症(Mono)(爱泼斯坦-巴尔病毒(EBV))；人流感病毒(甲型流感，包括但不限于H1N1，H2N2，H3N2，H5N1，H7N9，乙型流感和正黏病毒科的其他成员)；拉沙热(拉沙病毒)；淋巴细胞性脉络丛脑膜炎(淋巴细胞性脉络丛脑膜炎病毒(LCMV))；马尔堡出血热(MHF)(马尔堡病毒)；麻疹(麻疹病毒)；中东呼吸综合征(MERS)(中东呼吸综合征冠状病毒)；传染性软疣(MC)(传染性软疣病毒(MCV))；猴痘(猴痘病毒)；流行性腮腺炎(流行性腮腺炎病毒)；诺如病毒(Norovirus)(儿童和婴儿)(诺如病毒)；脊髓灰质炎(脊髓灰质炎病毒)；进行性多灶性白质脑病(JC病毒)；狂犬病(狂犬病病毒)；呼吸道合胞病毒感染(呼吸道合胞病毒(RSV))；鼻病毒感染(鼻病毒)；裂谷热(RVF)(裂谷热病毒)；轮状病毒感染(轮状病毒)；风疹(风疹病毒)；带状疱疹(Shingles)(带状疱疹(Herpes zoster))(水痘带状疱疹病毒(VZV))；天花(Smallpox)(天花(Variola))(大天花或小天花)；亚急性硬化性全脑炎(麻疹病毒)；委内瑞拉马脑炎(委内瑞拉马脑炎病毒)；委内瑞拉出血热(瓜纳里托病毒)；病毒性肺炎。
本发明显示了：
(1)游离的RBD mRNA疫苗免疫原性较弱，当在RBD的基础上加上Fc部分时其免疫原性得到了提升，当在RBD-Fc的基础上加上Pan部分时其免疫原性得到了极大的提升，当在Pan-RBD-Fc的基础上加上细胞因子IL12或CSF2时其免疫原性得到了进一步的提升，当在Pan-RBD-Fc的基础上加上能够结合免疫细胞表面蛋白PDL1的PD1或aPDL1抗体时其免疫原性得到了进一步的提升；
(2)游离的RBD mRNA疫苗免疫原性较弱，当与Pan单纯混合免疫后可提高抗体滴度，当与Pan融合表达免疫后，可进一步提高抗体滴度；
(3)RBD与αPDL1-Fc单纯混合免疫后没有提高抗体滴度，当RBD与αPDL1-Fc融合表达免疫后，可提高抗体滴度，当在RBD的基础上加上Fc时可提高抗体滴度，当在RBD-Fc的基础上加上Pan时可提高抗体滴度；
(4)游离的HA2mRNA疫苗免疫原性较弱，当与Pan单纯混合免疫后可提高抗体滴度，当与Pan融合表达免疫后，可进一步提高抗体滴度。同时，当在HA2的基础上加上Fc部分时可提高抗体滴度，在HA2-Fc的基础上加上Pan时可进一步提高抗体滴度；
(5)HA2与αPDL1-Fc单纯混合免疫后没有提高抗体滴度，当HA2与 αPDL1-Fc融合表达免疫后，可提高抗体滴度；当在αPDL1-HA2-Fc的基础上加上Pan可进一步提高抗体滴度；
(6)当在HA2-Fc的基础上加上IL12时相对于HA2抗体滴度有所提高，当在IL12-HA2-Fc的基础上加上Pan时可提高抗体滴度。
附图说明
图1显示了新冠病毒SARS-CoV-2刺突蛋白RBD相关mRNA的Urea-PAGE电泳鉴定图。
图2显示了免疫细胞靶向mRNA疫苗诱导的针对新冠病毒SARS-CoV-2刺突蛋白RBD的抗体反应。
图3显示了新冠病毒SARS-CoV-2刺突蛋白RBD相关mRNA疫苗的Urea-PAGE电泳鉴定图。
图4显示了免疫细胞靶向新冠病毒SARS-CoV-2刺突蛋白RBD相关mRNA疫苗相较于单纯的RBD mRNA疫苗在小鼠体内可以引起更强的抗体反应。
图5显示了免疫细胞靶向新冠病毒SARS-CoV-2刺突蛋白RBD相关mRNA疫苗相较于单纯的RBD mRNA疫苗在小鼠体内可以引起更强的抗体反应。
图6显示流感病毒Influenza A virus血凝素蛋白HA相关mRNA疫苗的Urea-PAGE电泳鉴定图。
图7显示免疫细胞靶向流感病毒Influenza A virus血凝素蛋白HA相关mRNA疫苗相较于单纯的HA mRNA疫苗在小鼠体内可以引起更强的抗体反应。
以下所述的是本发明的优选实施方式，本发明所保护的不限于以下优选实施方式。应当指出，对于本领域的技术人员来说在此发明创造构思的基础上，做出的若干变形和改进，都属于本发明的保护范围。所用试剂未注明生产商者，均为可以通过市购获得的常规产品。
序列：
Figure PCTCN2022144148-appb-000001
Figure PCTCN2022144148-appb-000002
Figure PCTCN2022144148-appb-000003
Figure PCTCN2022144148-appb-000004
Figure PCTCN2022144148-appb-000005
Figure PCTCN2022144148-appb-000006
Figure PCTCN2022144148-appb-000007
Ψ＝1-methyl-3'-pseudouridylyl
实施例
实施例1.mRNA疫苗平台的设计
免疫细胞靶向mRNA疫苗由四个结构单元构成：5'cap，5'UTR，ORF，3'UTR 和3'Poly(A)。免疫细胞靶向mRNA疫苗中的ORF在抗原上融合了免疫细胞靶向分子，包括以下四种成分：A.能够结合免疫细胞表面蛋白的抗体或多肽；B.能够激活免疫细胞的细胞因子；C.能够激活免疫细胞的Pan表位(PADRE)；D.能够结合免疫细胞的免疫球蛋白Fc。抗原可以和这四种成分中的一种，两种，三种或四种以任意形式排列组合形成表达融合蛋白的开放阅读框。抗原与免疫细胞靶向分子以及免疫细胞靶向分子之间通过连接片段进行连接。
实施例2.mRNA疫苗平台的构建，生产和鉴定
以新冠病毒SARS-CoV-2刺突蛋白RBD为例对该疫苗平台的构建和生产进行描述。
1、载体构建
以pBluescript II KS(+)为载体通过分子克隆的方式将mRNA疫苗结构单元构建到T7RNA聚合酶启动子下游，从而得到可以转录出mRNA的质粒。其中KOZAK为科扎克序列，SP(signal peptide)为信号肽。抗原与免疫细胞靶向分子以及免疫细胞靶向分子之间通过连接片段进行连接。连接片段氨基酸序列如下：连接片段1(Linker 1)为(G) 3，连接片段2(Linker 2)为(GS) 3，连接片段3(Linker 3)为(GGGGS) 3。
RBD：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-RBD-XhoI-3’UTR-Poly(A)-NsiI
RBD-Fc：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-RBD-EcoRI-Linker1-Fc-XhoI-3’UTR-Poly(A)-NsiI
Pan-RBD-Fc：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-Pan-Linker2-HindIII-RBD-EcoRI-Linker1-Fc-XhoI-3’UTR-Poly(A)-NsiI
IL4-Pan-RBD-Fc：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-IL4-SfuI-Linker3-Pan-Linker2-XbaI-RBD-XhoI-Linker 1-Fc-EcoRI-3’UTR-Poly(A)-NsiI
IL10-Pan-RBD-Fc：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-IL10-Linker3-SfuI-Pan-Linker2-XbaI-RBD-XhoI-Linker1-Fc-EcoRI-3’UTR-Poly(A)-NsiI
IL12-Pan-RBD-Fc：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-IL12-Linker3-SfuI-Pan-Linker2-XbaI-RBD-XhoI-Linker1-Fc-EcoRI-3’UTR-Poly(A)-NsiI
CSF2-Pan-RBD-Fc：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-CSF2-Linker3-SfuI-Pan-Linker2-XbaI-RBD-XhoI-Linker1-Fc-EcoRI-3’UTR-Poly(A)-NsiI
PD1-Pan-RBD-Fc：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-PD1-Linker3-SfuI-Pan-Linker2-XbaI-RBD-XhoI-Linker1-Fc-EcoRI-3’UTR-Poly(A)-NsiI
αPDL1-Pan-RBD-Fc：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-αPDL1-Linker3-SfuI-Pan-Linker2-XbaI-RBD-XhoI-Linker1-Fc-EcoRI-3’UTR-Poly(A)-NsiI
IFNε-Pan-RBD-Fc：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-IFNε-Linker3-SfuI-Pan-Linker2-XbaI-RBD-XhoI-Linker 1-Fc-EcoRI-3’UTR-Poly(A)-NsiI
IFNα4-Pan-RBD-Fc：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-IFNα4-Linker3-SfuI-Pan-Linker2-XbaI-RBD-XhoI-Linker 1-Fc-EcoRI-3’UTR-Poly(A)-NsiI
2、双链DNA模板的制备
(1)利用内切酶Sca I和Nsi I按照如下体系双酶切构建好的质粒：4μg质粒，3μl Sca I，3μl Nsi I，6μl 10x酶切缓冲液，加水补齐至60μl，37℃反应1小时；(2)制作1％琼脂糖胶，将酶切产物跑胶，胶回收目的条带，用32μl水洗脱，洗脱后的溶液含有双链DNA模板；
3、体外转录mRNA
(1)按照如下体系准备20μl的体外转录反应混合液：7μl DNA模板，2μl 100mM DTT，1μl 20mM ATP，1μl 20mM CTP，1μl 20mM GTP，1μl 20mM N1-UTP，2μl 40mM ARCA，2μl 10 x Transcription buffer(NEB)，2μl T7 RNA polymerase(NEB)，总共20μl。37℃反应2.5小时。
(2)把样品放到冰上，加入1μl DNase I，37℃反应15分钟。
(3)取0.5μl上样到1.5％的琼脂糖胶上，电泳检测有无mRNA条带。
4、mRNA的纯化
利用
Figure PCTCN2022144148-appb-000008
Cleanup纯化试剂盒对mRNA样品进行纯化。
(1)在20μl体外转录体系中加入80μl水，总共100μl。
(2)加入350μl RLT溶液，用移液器混匀。
(3)加入250μl无水乙醇，混匀后转移溶液到离心柱里，8000g室温离心15秒。
(4)将离心柱转移到新的2ml离心管中，加入500μl RPE溶液，8000g室温离心15秒。
(5)加入500μl 80％乙醇，8000g室温离心2分钟。
(6)把离心柱转移到新的2ml离心管中，最高速离心5分钟。
(7)将离心柱转移到1.5ml离心管中，加入20μl水，溶解mRNA，高速离心1分钟。
5、mRNA的浓度和纯度鉴定
取100ng mRNA上样到5％尿素-聚丙烯酰胺胶上，电泳缓冲液为0.5×TBE，250V跑胶2小时，用凝胶成像仪成像，查看mRNA的完整性和纯度。
6、所述mRNA Urea-PAGE电泳鉴定图如图1所示。
实施例3.免疫细胞靶向新冠病毒SARS-CoV-2刺突蛋白RBD mRNA疫苗相较于单纯的RBD mRNA疫苗在小鼠体内可以引起更强的抗体反应
1、材料
C57BL/6雄性小鼠(6-8)周购买于江苏集萃药康生物科技股份有限公司；辣根过氧化物氧化酶(HRP)标记的山羊抗小鼠IgG购自江苏康为世纪生物科技股份有限公司；辣根过氧化物氧化酶(HRP)标记的山羊抗小鼠IgG1，IgG2b和IgG2c购自江苏康为世纪生物科技股份有限公司；96孔ELISA测定板购自Bioland公司；ELISA显色液购自上海碧云天生物技术有限公司；ELISA终止液购自北京索莱宝科技有限公司；酶标仪Multiskan FC购自Thermo Fisher Scientific公司。
2、方法
(1)mRNA疫苗免疫小鼠。将mRNA与3倍于mRNA质量的转染试剂Lipofectamine Messenger MAX混和后，室温静置5分钟。每只小鼠接种0.5μg mRNA或者相同摩尔数的其它mRNA，每只小鼠肌肉注射50μl。采用两次免疫 程序在第0天与21天免疫小鼠。每次免疫后第14天通过脸颊取血的方式收集小鼠血清，进行抗体检测。
(2)ELISA检测血清中RBD特异性抗体。将RBD(2μg/ml)包被液以每孔100μl的体系加入到Elisa板中，4℃包被过夜。5％的封闭液(5％FBS的PBS)37℃封闭1小时。PBST洗3次，血清样品按照10倍梯度稀释，每孔加100μl到封闭好的ELISA板中，37℃孵育1小时。PBST洗3次，每孔加入100μl酶标二抗(1:5000)，37℃孵育1小时。用PBST洗5次，加底物TMB 100μl/孔，室温避光孵育，等待底物显色；每孔加50μl ELISA终止液终止显色，酶标仪读板，OD450-620。
3、结果
游离的RBD mRNA疫苗免疫原性较弱，当在RBD的基础上加上Fc部分时其免疫原性得到了提升，当在RBD-Fc的基础上加上Pan部分时其免疫原性得到了极大的提升，当在Pan-RBD-Fc的基础上加上细胞因子IL12或CSF2时其免疫原性得到了进一步的提升，当在Pan-RBD-Fc的基础上加上能够结合免疫细胞表面蛋白PDL1的PD1或aPDL1抗体时其免疫原性得到了进一步的提升，二免后抗体的水平普遍高于一免后的抗体水平，如图2(a)所示。IL12，CSF2，PD1或aPDL1在Pan-RBD-Fc基础上诱导了更高水平的代表Th2免疫反应的IgG1，如图2(b)所示。IL12，PD1或aPDL1在Pan-RBD-Fc基础上诱导了更高水平的代表Th1免疫反应的IgG2b和IgG2c，如图2(c)和图2(d)所示。(IgG2b+IgG2c)/IgG1的比例显示Pan-RBD-Fc，CSF2-Pan-RBD-Fc和PD1-Pan-RBD-Fc诱导了比较平衡的Th1和Th2免疫反应，而IL12-Pan-RBD-Fc和aPDL1-Pan-RBD-Fc诱导了偏向Th1的免疫反应，如图2(e)所示。
实施例4.新冠病毒SARS-CoV-2刺突蛋白RBD相关mRNA疫苗的构建，生产和鉴定
1、载体构建
以pBluescript II KS(+)为载体通过分子克隆的方式将mRNA疫苗结构单元构建到T7RNA聚合酶启动子下游，从而得到可以转录出mRNA的质粒。其中KOZAK为科扎克序列，SP(signal peptide)为信号肽。抗原与免疫细胞靶向分子以及免疫细胞靶向分子之间通过连接片段进行连接。连接片段氨基酸序列如下：连接片 段1(Linker 1)为(G) 3，连接片段2(Linker 2)为(GS) 3，连接片段3(Linker 3)为(GGGGS) 3。
Pan：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-Pan-XhoI-3’UTR-Poly(A)-NsiI
Pan-RBD：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-Pan-Linker2-HindIII-RBD-XhoI-3’UTR-Poly(A)-NsiI
αPDL1-Fc：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-αPDL1-SfuI-Linker1-Fc-EcoRI-3’UTR-Poly(A)-NsiI
αPDL1-RBD-Fc：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-αPDL1-SfuI-Linker3-XbaI-RBD-XhoI-Linker1-Fc-EcoRI-3’UTR-Poly(A)-NsiI
αPDL1-RBD-Fc-IFNα4：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-αPDL1-SfuI-Linker3-XbaI-RBD-XhoI-Linker1-Fc-Linker3-IFNα4-EcoRI-3’UTR-Poly(A)-NsiI
IL12-RBD-Fc：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-IL12-SfuI-Linker3-XbaI-RBD-XhoI-Linker1-Fc-EcoRI-3’UTR-Poly(A)-NsiI
2、双链DNA模板的制备
(1)利用内切酶Sca I和Nsi I按照如下体系双酶切构建好的质粒：4μg质粒，3μl Sca I，3μl Nsi I，6μl 10×酶切缓冲液，加水补齐至60μl，37℃反应1小时；
(2)制作1％琼脂糖胶，将酶切产物跑胶，胶回收目的条带，用32μl水洗脱，洗脱后的溶液含有双链DNA模板；
3、体外转录mRNA
(1)按照如下体系准备20μl的体外转录反应混合液：7μl DNA模板，2μl 100mM DTT，1μl 20mM ATP，1μl 20mM CTP，1μl 20mM GTP，1μl 20mM N1-UTP，2μl 40mM ARCA，2μ10×Transcription buffer(NEB)，2μl T7RNA polymerase(NEB)，总共20μl。37℃反应2.5小时。
(2)把样品放到冰上，加入1μl DNase I，37℃反应15分钟。
(3)取0.5μl上样到1.5％的琼脂糖胶上，电泳检测有无mRNA条带。
4、mRNA的纯化
利用
Figure PCTCN2022144148-appb-000009
Cleanup纯化试剂盒对mRNA样品进行纯化。
(1)在20μl体外转录体系中加入80μl水，总共100μl。
(2)加入350μl RLT溶液，用移液器混匀。
(3)加入250μl无水乙醇，混匀后转移溶液到离心柱里，8000g室温离心15秒。
(4)将离心柱转移到新的2ml离心管中，加入500μl RPE溶液，8000g室温离心15秒。
(5)加入500μl 80％乙醇，8000g室温离心2分钟。
(6)把离心柱转移到新的2ml离心管中，最高速离心5分钟。
(7)将离心柱转移到1.5ml离心管中，加入20μl水，溶解mRNA，高速离心1分钟。
5、mRNA的浓度和纯度鉴定
取100ng mRNA上样到5％尿素-聚丙烯酰胺胶上，电泳缓冲液为0.5×TBE，250V跑胶2小时，用凝胶成像仪成像，查看mRNA的完整性和纯度。
6、所述mRNA Urea-PAGE电泳鉴定图如图3所示。
实施例5.免疫细胞靶向新冠病毒SARS-CoV-2刺突蛋白RBD相关mRNA疫苗相较于单纯的RBD mRNA疫苗在小鼠体内可以引起更强的抗体反应
1、材料
C57BL/6雄性小鼠(6-8)周购买于江苏集萃药康生物科技股份有限公司；辣根过氧化物氧化酶(HRP)标记的山羊抗小鼠IgG购自江苏康为世纪生物科技股份有限公司；96孔ELISA测定板购自Bioland公司；ELISA显色液购自上海碧云天生物技术有限公司；ELISA终止液购自北京索莱宝科技有限公司；酶标仪Multiskan FC购自Thermo Fisher Scientific公司。
2、方法
(1)mRNA疫苗免疫小鼠。将mRNA与3倍于mRNA质量的转染试剂Lipofectamine Messenger MAX混和后，室温静置5分钟。每只小鼠接种0.5μg mRNA或者相同摩尔数的其它mRNA，每只小鼠肌肉注射50μl。采用两次免疫 程序在第0天与21天免疫小鼠。每次免疫后第14天通过脸颊取血的方式收集小鼠血清，进行抗体检测。
(2)ELISA检测血清中RBD特异性抗体。将RBD(2μg/ml)包被液以每孔100μl的体系加入到Elisa板中，4℃包被过夜。5％的封闭液(5％FBS的PBS)37℃封闭1小时。PBST洗3次，血清样品按照10倍梯度稀释，每孔加100μl到封闭好的ELISA板中，37℃孵育1小时。PBST洗3次，每孔加入100μl酶标二抗(1:5000)，37℃孵育1小时。用PBST洗5次，加底物TMB 100μl/孔，室温避光孵育，等待底物显色；每孔加50μl ELISA终止液终止显色，酶标仪读板，OD450-620。
3、结果
参见图4，游离的RBD mRNA疫苗免疫原性较弱，当与Pan单纯混合免疫后可提高抗体滴度，当与Pan融合表达免疫后，可进一步提高抗体滴度。这些结果说明Pan-RBD的免疫原性高于RBD和Pan与RBD单纯混合。
实施例6.免疫细胞靶向新冠病毒SARS-CoV-2刺突蛋白RBD相关mRNA疫苗相较于单纯的RBD mRNA疫苗在小鼠体内可以引起更强的抗体反应
1、材料
C57BL/6雄性小鼠(6-8)周购买于江苏集萃药康生物科技股份有限公司；辣根过氧化物氧化酶(HRP)标记的山羊抗小鼠IgG购自江苏康为世纪生物科技股份有限公司；96孔ELISA测定板购自Bioland公司；ELISA显色液购自上海碧云天生物技术有限公司；ELISA终止液购自北京索莱宝科技有限公司；酶标仪Multiskan FC购自Thermo Fisher Scientific公司。
2、方法
(1)mRNA疫苗免疫小鼠。
溶解SM-102、DSPC、Cholesterol和DMG-PEG2000四种成分于乙醇中，使其摩尔比为50:10:38.5:1.5，溶解mRNA于20mM醋酸钠(pH＝4)中，将mRNA和脂质体按照质量比1:40和体积比1:3混匀，形成mRNA/脂质体纳米颗粒，超滤去除乙醇并过滤除菌，用PBS(pH＝7.4)将mRNA/脂质体纳米颗粒浓度稀释为1μg mRNA/100μl，取100μl肌肉注射小鼠进行免疫。采用两次免疫程序在第0天与21天免疫小鼠。每次免疫后第14天通过脸颊取血的方式收集小鼠血清，进 行抗体检测。
(2)ELISA检测血清中RBD特异性抗体。将RBD(2μg/ml)包被液以每孔100μl的体系加入到Elisa板中，4℃包被过夜。5％的封闭液(5％FBS的PBS)37℃封闭1小时。PBST洗3次，血清样品按照10倍梯度稀释，每孔加100μl到封闭好的ELISA板中，37℃孵育1小时。PBST洗3次，每孔加入100μl酶标二抗(1:5000)，37℃孵育1小时。用PBST洗5次，加底物TMB 100μl/孔，室温避光孵育，等待底物显色；每孔加50μl ELISA终止液终止显色，酶标仪读板，OD450-620。
3、结果
参见图5，RBD与αPDL1-Fc单纯混合免疫后没有提高抗体滴度，当RBD与αPDL1-Fc融合表达免疫后，可提高抗体滴度，当在αPDL1-RBD-Fc的基础上加上IFNα4时反而降低了抗体滴度。当在RBD的基础上加上Fc时可提高抗体滴度，当在RBD-Fc的基础上加上Pan时可提高抗体滴度，当在RBD-Fc的基础上加上IL12时没有提高抗体滴度。
实施例7.流感病毒Influenza A virus血凝素蛋白HA相关mRNA疫苗的构建，生产和鉴定
甲型流感中的HA至少有18种不同的亚型，我们选择人流感病毒中的H1作为研究对象，每个HA经过细胞蛋白酶裂解为HA1和HA2亚基。形成球状头部的HA1虽然包含主要的中和表位，但是在亚型之间高度可变。HA2亚基构成大部分茎结构域，在亚型中相当保守，包含很少但交叉反应的中和表位。我们选择保守的HA2亚基作为我们的研究对象。
1、载体构建
以pBluescript II KS(+)为载体通过分子克隆的方式将mRNA疫苗结构单元构建到T7RNA聚合酶启动子下游，从而得到可以转录出mRNA的质粒。其中KOZAK为科扎克序列，SP(signal peptide)为信号肽。抗原与免疫细胞靶向分子以及免疫细胞靶向分子之间通过连接片段进行连接。连接片段氨基酸序列如下：连接片段1(Linker 1)为(G) 3，连接片段2(Linker 2)为(GS) 3，连接片段3(Linker 3)为(GGGGS) 3。
HA2：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-HA2-EcoRI-3’UTR-Poly(A)-NsiI
Pan-HA2：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-Pan-Linker2-HindIII-HA2-XhoI-3’UTR-Poly(A)-NsiI
HA2-Fc：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-HA2-XhoI-Linker1-Fc-EcoRI-3’UTR-Poly(A)-NsiI
Pan-HA2-Fc：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-Pan-Linker2-HindIII-HA2-EcoRI-
Linker1-Fc-XhoI-3’UTR-Poly(A)-NsiI
IFNα4-Pan-HA2-Fc：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-IFNα4-SfuI-Linker3-Pan-Linker2-XbaI-HA2-XhoI-Linker1-Fc-EcoRI-3’UTR-Poly(A)-NsiI
αPDL1-HA2-Fc：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-αPDL1-SfuI-Linker3-XbaI-HA2-XhoI-Linker1-Fc-EcoRI-3’UTR-Poly(A)-NsiI
αPDL1-HA2-Fc-IFNα4：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-αPDL1-SfuI-Linker3-XbaI-HA2-XhoI-Linker1-Fc-Linker3-IFNα4-EcoRI-3’UTR-Poly(A)-NsiI
αPDL1-Pan-HA2-Fc：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-αPDL1-SfuI-Linker3-Pan-Linker2-XbaI-HA2-XhoI-Linker1-Fc-EcoRI-3’UTR-Poly(A)-NsiI
αPDL1-Pan-HA2-Fc-IFNα4：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-αPDL1-SfuI-Linker3-Pan-Linker2-XbaI-HA2-XhoI-Linker1-Fc-Linker3-IFNα4-EcoRI-3’UTR-Poly(A)-NsiI
IL12-HA2-Fc：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-IL12-Linker3-XbaI-HA2-XhoI-Linker1-Fc-EcoRI-3’UTR-Poly(A)-NsiI
IL12-Pan-HA2-Fc：
SacI-5’UTR-XbaI-KOZAK-Start-SP-BsiwI-IL12-SfuI-Linker3-Pan-Linker2-XbaI-HA2-XhoI-Linker1-Fc-EcoRI-3’UTR-Poly(A)-NsiI
2、双链DNA模板的制备
(1)利用内切酶Sca I和Nsi I按照如下体系双酶切构建好的质粒：4μg质粒，3μl Sca I，3μl Nsi I，6μl 10×酶切缓冲液，加水补齐至60μl，37℃反应1小时；(2)制作1％琼脂糖胶，将酶切产物跑胶，胶回收目的条带，用32μl水洗脱，洗脱后的溶液含有双链DNA模板；
3、体外转录mRNA
(1)按照如下体系准备20μl的体外转录反应混合液：7μl DNA模板，2μl 100mM DTT，1μl 20mM ATP，1μl 20mM CTP，1μl 20mM GTP，1μl 20mM N1-UTP，2μl 40mM ARCA，2μl 10 x Transcription buffer(NEB)，2μl T7 RNA polymerase(NEB)，总共20μl。37℃反应2.5小时。
(2)把样品放到冰上，加入1μl DNase I，37℃反应15分钟。
(3)取0.5μl上样到1.5％的琼脂糖胶上，电泳检测有无mRNA条带。
4、mRNA的纯化
利用
Figure PCTCN2022144148-appb-000010
Cleanup纯化试剂盒对mRNA样品进行纯化。
(1)在20μl体外转录体系中加入80μl水，总共100μl。
(2)加入350μl RLT溶液，用移液器混匀。
(3)加入250μl无水乙醇，混匀后转移溶液到离心柱里，8000g室温离心15秒。
(4)将离心柱转移到新的2ml离心管中，加入500μl RPE溶液，8000g室温离心15秒。
(5)加入500μl 80％乙醇，8000g室温离心2分钟。
(6)把离心柱转移到新的2ml离心管中，最高速离心5分钟。
(7)将离心柱转移到1.5ml离心管中，加入20μl水，溶解mRNA，高速离心1分钟。
5、mRNA的浓度和纯度鉴定
取100ng mRNA上样到5％尿素-聚丙烯酰胺胶上，电泳缓冲液为0.5×TBE，250V跑胶2小时，用凝胶成像仪成像，查看mRNA的完整性和纯度。
6、所述mRNA Urea-PAGE电泳鉴定图如图6所示。
实施例8.免疫细胞靶向流感病毒Influenza A virus血凝素蛋白HA相关mRNA疫苗相较于单纯的HA mRNA疫苗在小鼠体内可以引起更强的抗体反应
1、材料
C57BL/6雄性小鼠(6-8)周购买于江苏集萃药康生物科技股份有限公司；辣根过氧化物氧化酶(HRP)标记的山羊抗小鼠IgG购自江苏康为世纪生物科技股份有限公司；96孔ELISA测定板购自Bioland公司；ELISA显色液购自上海碧云天生物技术有限公司；ELISA终止液购自北京索莱宝科技有限公司；酶标仪Multiskan FC购自Thermo Fisher Scientific公司。
2、方法
(1)溶解SM-102、DSPC、Cholesterol和DMG-PEG2000四种成分于乙醇中，使其摩尔比为50:10:38.5:1.5，溶解mRNA于20mM醋酸钠(pH＝4)中，将mRNA和脂质体按照质量比1:40和体积比1:3混匀，形成mRNA/脂质体纳米颗粒，超滤去除乙醇并过滤除菌，用PBS(pH＝7.4)将mRNA/脂质体纳米颗粒浓度稀释为1μg mRNA/100μl，取100μl肌肉注射小鼠进行免疫。采用两次免疫程序在第0天与21天免疫小鼠。每次免疫后第14天通过脸颊取血的方式收集小鼠血清，进行抗体检测。
(2)ELISA检测血清中RBD特异性抗体。将RBD(2μg/ml)包被液以每孔100μl的体系加入到Elisa板中，4℃包被过夜。5％的封闭液(5％FBS的PBS)37℃封闭1小时。PBST洗3次，血清样品按照10倍梯度稀释，每孔加100μl到封闭好的ELISA板中，37℃孵育1小时。PBST洗3次，每孔加入100μl酶标二抗(1:5000)，37℃孵育1小时。用PBST洗5次，加底物TMB 100μl/孔，室温避光孵育，等待底物显色；每孔加50μl ELISA终止液终止显色，酶标仪读板，OD450-620。
3、结果参见图7：
(1)游离的HA2mRNA疫苗免疫原性较弱，当与Pan单纯混合免疫后可提高抗体滴度，当与Pan融合表达免疫后，可进一步提高抗体滴度。同时，当在HA2的基础上加上Fc部分时可提高抗体滴度，在HA2-Fc的基础上加上Pan时可进一步提高抗体滴度，在Pan-HA2-Fc的基础上加上IFNα4时反而降低了抗体滴度，如图7所示。这些结果说明Pan-HA2的免疫原性高于HA2和Pan与HA2单纯混合，Pan-HA2-Fc的免疫原性高于HA2，HA2-Fc和IFNα4-Pan-HA2-Fc。
(2)HA2与αPDL1-Fc单纯混合免疫后没有提高抗体滴度，当HA2与αPDL1-Fc 融合表达免疫后，可提高抗体滴度，当在αPDL1-HA2-Fc的基础上加上IFNα4时反而降低了抗体滴度。当在αPDL1-HA2-Fc的基础上加上Pan可进一步提高抗体滴度，当在αPDL1-Pan-HA2-Fc的基础上加上IFNα4时反而降低了抗体滴度，如图7所示。这些结果说明αPDL1-HA2-Fc的免疫原性高于HA2，αPDL1-Fc与HA2单纯混合，HA2-Fc和αPDL1-HA2-Fc-IFNα4。αPDL1-Pan-HA2-Fc的免疫原性高于HA2，HA2-Fc，αPDL1-HA2-Fc和αPDL1-Pan-HA2-Fc-IFNα4。
(3)当在HA2-Fc的基础上加上IL12没有提高抗体滴度，但相对于HA2有所提高，当在IL12-HA2-Fc的基础上加上Pan时可提高抗体滴度，如图7所示。这些结果说明IL12-HA2-Fc的免疫原性高于HA2。IL12-Pan-HA2-Fc的免疫原性高于HA2，HA2-Fc和IL12-HA2-Fc。

Claims (16)

  1. 含有开放阅读框(ORF)的mRNA，所述开放阅读框编码抗原(Antigen)和免疫细胞靶向分子。
  2. 根据权利要求1所述的mRNA，所述开放阅读框编码的抗原和免疫细胞靶向分子为抗原和免疫细胞靶向分子融合蛋白。
  3. 根据权利要求1所述的mRNA，所述免疫细胞靶向分子选自如下的一种或多种：
    A：能够结合免疫细胞表面蛋白的抗体或多肽；
    B：能够激活免疫细胞的细胞因子；
    C：能够激活免疫细胞的Pan表位(PADRE)；
    D：能够结合免疫细胞的免疫球蛋白Fc；
    优选地，所述A选自针对CD274(PDL1)，PDCD1LG2(PDL2)，CLEC9A，LY75(DEC205)，CD40，TNFSF9(4-1BB-L)和/或TNFSF4(OX4OL)的抗体，或其配体的活性片段；
    优选地，所述B选自白介素(interleukin，IL)，集落刺激因子(Colony-stimulating factor，CSF)和/或干扰素(interferon，IFN)，或其活性片段，更优选地，所述白介素选自IL2，IL12，IL15和/或IL21或其活性片段；
    优选地，所述集落刺激因子选自CSF1，CSF2和/或CSF3，或其活性片段；
    优选地，所述干扰素选自I型干扰素，II型干扰素和/或III型干扰素，或其活性片段，更优选地，所述I型干扰素选自IFNα，IFNω，IFNε，IFNк和/或IFNβ，或其活性片段；更优选地，所述II型干扰素选自IFN-γ，或其活性片段；更优选地，所述III型干扰素选自IFN-λ1，IFN-λ2，IFN-λ3和/或IFN-λ4，或其活性片段；
    优选地，所述C具有AKFVAAWTLKAAA(SEQ ID NO：1)所示的氨基酸序列；
    优选地，所述D选自来自IgG，IgM，IgA，IgE或IgD的Fc或其突变体。
  4. 根据权利要求1-3任一项所述的mRNA，所述开放阅读框编码抗原与A的融合蛋白；抗原与A和B的融合蛋白；抗原与A，B和C的融合蛋白；抗原与A，B，C和D的融合蛋白；抗原与A和C的融合蛋白；抗原与A和D的融合蛋白；抗原与A，C和D的融合蛋白；抗原与B的融合蛋白；抗原与B和C的融合蛋白；抗原与B和D 的融合蛋白；抗原与B，C和D的融合蛋白；抗原与C的融合蛋白；抗原与C和D的融合蛋白；或抗原与D的融合蛋白；
    所述融合蛋白是抗原与A和/或B和/或C和/或D的任意排列方式；或者
    所述融合蛋白中抗原和A和/或B和/或C和/或D相对于彼此位于C端或者N端；
    优选地，所述融合蛋白是同源二聚体或异源二聚体，
    优选地，所述融合蛋白为分泌型的融合蛋白或膜融合蛋白，
    更优选地，所述融合蛋白为分泌型的融合蛋白；
    所述能够结合免疫细胞表面蛋白的抗体或多肽A选自针对CD274(PDL1)，PDCD1LG2(PDL2)，CLEC9A，LY75(DEC205)，CD40，TNFSF9(4-1BB-L)，和/或TNFSF4(OX4OL)的抗体，或其配体的活性片段。
  5. 根据权利要求1-4任一项所述的mRNA，所述mRNA还包含5'UTR序列和3'UTR序列。
  6. 根据权利要求1-5任一项所述的mRNA，所述mRNA还包含5'Cap和3'Poly(A)。
  7. 根据权利要求1-6任一项所述的mRNA，所述开放阅读框(ORF)还包含编码连接片段的区域，
    优选地，抗原与免疫细胞靶向分子之间，和/或免疫细胞靶向分子之间通过连接片段进行连接；
    优选地，所述连接片段是柔性连接片段、刚性连接片段或体内剪切连接片段，更优选地，所述柔性连接片段的氨基酸序列是(G) N，(GS) N，(GGS) N，(GGGS) N，或(GGGGS) N。
  8. 根据权利要求1-7任一项所述的mRNA，所述mRNA具有选自如下的结构：
    5'Cap–5'UTR–A–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–CLEC9A binding peptide–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αDEC205–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–A–Linker–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–Linker–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–(GGGGS) 3–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–CLEC9A binding peptide–Linker–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–CLEC9A binding peptide–(GGGGS) 3–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αDEC205–Linker–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αDEC205–(GGGGS) 3–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–A–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–αPDL1–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–CLEC9A binding peptide–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–CD40L–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–4-1BB–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–OX40–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–Linker–A–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–Linker–αPDL1–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–(GGGGS) 3–αPDL1–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–Linker–CLEC9A binding peptide–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–(GGGGS) 3–CLEC9A binding peptide–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–Linker–CD40L–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–(GGGGS) 3–CD40L–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–Linker–4-1BB–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–(GGGGS) 3–4-1BB–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–Linker–OX40–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–(GGGGS) 3–OX40–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–B–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IL2–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IL12–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IL15–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IL21–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–CSF2–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IFNα–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–B–Linker–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IL2–Linker–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IL2–(GGGGS) 3–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IL12–Linker–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IL12–(GGGGS) 3–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IL15–Linker–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IL15–(GGGGS) 3–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IL21–Linker–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IL21–(GGGGS) 3–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–CSF2–Linker–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–CSF2–(GGGGS) 3–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IFNα–Linker–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IFNα–(GGGGS) 3–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–B–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–Linker–B–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–Linker–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–(GGGGS) 3–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–C–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Pan–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–C–Linker–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Pan–Linker–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Pan–(GS) 3–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–C–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–Pan–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–Linker–C–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–Linker–Pan–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–(GS) 3–Pan–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–A–Antigen–B–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–Antigen–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–A–Linker–Antigen–Linker–B–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–Linker–Antigen–Linker–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–(GGGGS) 3–Antigen–(GS) 3–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–A–C–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–Pan–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–A–Linker–C–Linker–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–Linker–Pan–Linker–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–(GGGGS) 3–Pan–(GS) 3–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–A–Antigen–D–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–Antigen–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–A–Linker–Antigen–Linker–D–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–(GGGGS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–B–C–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–CSF2–Pan–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–B–Linker–C–Linker–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–CSF2–Linker–Pan–Linker–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–CSF2–(GGGGS) 3–Pan–(GS) 3–Antigen–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–B–Antigen–D–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–CSF2–Antigen–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–B–Linker–Antigen–Linker–D–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–CSF2–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–CSF2–(GGGGS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–D–B–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–Fc–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–Linker–D–Linker–B–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–Linker–Fc–Linker–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Antigen–(G) 3–Fc–(GS) 3–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–C–Antigen–D–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–C–Linker–Antigen–Linker–D–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–A–C–Antigen–B–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–Pan–Antigen–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–A–Linker–C–Linker–Antigen–Linker–B–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–Linker–Pan–Linker–Antigen–Linker–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–(GGGGS) 3–Pan–(GS) 3–Antigen–(GS) 3–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–A–Antigen–D–B–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–Antigen–Fc–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–A–Linker–Antigen–Linker–D–Linker–B–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–Linker–Antigen–Linker–Fc–Linker–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–(GGGGS) 3–Antigen–(G) 3–Fc–(GS) 3–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–A–C–Antigen–D–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–PD1–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–CLEC9A binding peptide–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αDEC205–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–A–Linker–C–Linker–Antigen–Linker–D–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–Linker–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–PD1–Linker–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–PD1–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–CLEC9A binding peptide–Linker–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–CLEC9A binding peptide–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αDEC205–Linker–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αDEC205–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–B–C–Antigen–D–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IL2–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IL12–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IL21–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–CSF2–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IFNα–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–B–Linker–C–Linker–Antigen–Linker–D–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IL2–Linker–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IL2–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IL12–Linker–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IL12–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IL21–Linker–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IL21–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–CSF2–Linker–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–CSF2–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IFNα–Linker–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–IFNα–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–C–Antigen–D–B–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Pan–Antigen–Fc–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–C–Linker–Antigen–Linker–D–Linker–B–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–Pan–(GS) 3–Antigen–(G) 3–Fc–(GS) 3–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–A–C–Antigen–D–B–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–Pan–Antigen–Fc–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–CLEC9A binding peptide–Pan–Antigen–Fc–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–Pan–Antigen–Fc–IFNα–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–A–Linker–C–Linker–Antigen–Linker–D–Linker–B–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–Linker–Pan–Linker–Antigen–Linker–Fc–Linker–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–(GS) 3–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–CLEC9A binding peptide–Linker–Pan–Linker–Antigen–Linker–Fc–Linker–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–CLEC9A binding peptide–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–(GS) 3–CSF2–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–Linker–Pan–Linker–Antigen–Linker–Fc–Linker–IFNα–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–(GGGGS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–(GS) 3–IFNα–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–A–B–C–Antigen–D–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–CSF2–Pan–Antigen–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–A–Linker–B–Linker–C–Linker–Antigen–Linker–D–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–Linker–CSF2–Linker–Pan–Linker–Antigen–Linker–Fc–3'UTR–3'Poly(A)；
    5'Cap–5'UTR–αPDL1–(GGGGS) 3–CSF2–(GS) 3–Pan–(GS) 3–Antigen–(G) 3–Fc–3'UTR–3'Poly(A)。
  9. 根据权利要求1-8任一项所述的mRNA，所述抗原(Antigen)为能够诱导针对病原微生物的免疫反应的免疫原性蛋白或其免疫原性片段；
    优选地，所述病原微生物为SARS-Cov-2，SARS，巨细胞病毒CMV，疱疹病毒，RSV，流感病毒，Ebola病毒，爱泼斯坦-巴尔病毒(EBV)，登革热病毒，Zike病毒，HIV病毒，狂犬病毒，疟原虫配子体，带状疱疹病毒(HZV)，乙肝病毒HBV，丙型肝炎病毒(HCV)，丁肝病毒HDV，HPV，结核分枝杆菌，或幽门螺旋杆菌。
  10. 根据权利要求9所述的mRNA，所述抗原为SARS-Cov-2的S蛋白或其片段，优选地，所述S蛋白为预融合稳定的S蛋白，更优选地，所述预融合稳定的S蛋白包含双脯氨酸(S2P)突变；或者所述抗原为SARS-Cov-2的S蛋白的RBD结构域。
  11. 根据权利要求1-8任一项所述的mRNA，所述抗原为能够诱导针对癌症细胞的免疫反应的免疫原性蛋白或其免疫原性片段；
    优选地，所述抗原为选自如下的肿瘤抗原或其免疫原性片段：MelanA/MART1、癌-种系抗原、gp100、酪氨酸酶、CEA、PSA、Her-2/neu、存活蛋白、端粒酶，或其免疫原性片段；
    优选地，所述癌症可以为前列腺癌，非小细胞肺癌，小细胞肺癌，肾细胞癌，脑癌，黑色素瘤，急性髓细胞性白血病，胰腺癌，结直肠癌，头颈部鳞状细胞癌，皮肤鳞状细胞癌，腺样囊性癌，胶质母细胞瘤，乳腺癌，间皮瘤，卵巢癌，神经 胶质瘤，膀胱癌，肝癌，骨癌，骨髓癌，胃癌，甲状腺癌，淋巴癌，宫颈癌，子宫内膜癌，喉癌，急性淋巴细胞性白血病。
  12. 包含权利要求1-11任一项的mRNA的组合物。
  13. 根据权利要求12所述的组合物，所述组合物还包含可药用载体，
    优选地，所述可药用载体为脂质纳米颗粒(LNP)、高分子材料或无机纳米颗粒；
    优选地，所述可药用载体为阳离子脂质复合物(LPX)，脂质多聚复合物(LPP)，聚合物纳米颗粒(PNP)，无机纳米颗粒(INP)或阳离子纳米乳(CNE)。
  14. 用于预防或治疗与病原微生物感染或肿瘤的方法，所述方法包括向对象给药权利要求1-11任一项的mRNA或权利要求12或13的组合物的步骤。
  15. 根据权利要求14的方法，所述对象为人类或动物，
    优选地，所述动物为牛，羊，猫，犬，马，兔，猴，小鼠，大鼠，羊驼或骆驼；
    优选地，所述对象为免疫功能低下的人或动物；
    优选地，所述对象患有慢性肺病，慢性阻塞性肺疾病或哮喘；
    优选地，所述患者患有选自心脏病，糖尿病或肺病的基础疾病。
  16. 权利要求1-11任一项的mRNA或权利要求12或13的组合物在制备用于诊断，预防或治疗对象与病原微生物感染或肿瘤相关的药物或试剂盒中的应用，
    所述对象为人类或动物，
    优选地，所述动物为牛，羊，猫，犬，马，兔，猴，小鼠，大鼠，羊驼或骆驼；
    优选地，所述对象为免疫功能低下的人或动物；
    优选地，所述对象患有慢性肺病，慢性阻塞性肺疾病或哮喘；
    优选地，所述患者患有选自心脏病，糖尿病或肺病的基础疾病。
PCT/CN2022/144148 2021-12-31 2022-12-30 mRNA疫苗 WO2023125974A1 (zh)

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