WO2023142283A1 - 一种新型冠状病毒mRNA疫苗及其制备方法与应用 - Google Patents

一种新型冠状病毒mRNA疫苗及其制备方法与应用 Download PDF

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WO2023142283A1
WO2023142283A1 PCT/CN2022/090196 CN2022090196W WO2023142283A1 WO 2023142283 A1 WO2023142283 A1 WO 2023142283A1 CN 2022090196 W CN2022090196 W CN 2022090196W WO 2023142283 A1 WO2023142283 A1 WO 2023142283A1
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seq
mrna
mrna molecule
rbd
amino acid
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PCT/CN2022/090196
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English (en)
French (fr)
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胡勇
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深圳市瑞吉生物科技有限公司
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Priority to US18/260,497 priority Critical patent/US11931410B1/en
Priority to EP22891168.1A priority patent/EP4242308A1/en
Priority to CN202280001268.XA priority patent/CN114729373B/zh
Priority to AU2022369834A priority patent/AU2022369834A1/en
Priority to TW112102868A priority patent/TW202330924A/zh
Publication of WO2023142283A1 publication Critical patent/WO2023142283A1/zh

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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
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • 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

Definitions

  • the present invention relates to an mRNA vaccine and its preparation method and application, in particular to an mRNA vaccine against novel coronavirus (including original strain and/or mutant strain), its preparation method and related applications.
  • mRNA vaccine technology can be used as a fast and flexible technology platform to effectively deal with the threats of various emerging viruses. Therefore, mRNA vaccine is considered to be the most potential vaccine against novel coronavirus (SARS-CoV-2).
  • SARS-CoV-2 novel coronavirus
  • the target antigen epitope sequence (including B cell epitope and T cell epitope) directly determines the type of immune response. Given that both B-cell immunity and T-cell immunity are indispensable in the clearance of SARS-CoV-2 virus, selecting the appropriate immunogenic fragment as much as possible is a key step in the design of a new coronavirus vaccine.
  • selecting the appropriate immunogenic fragment as much as possible is a key step in the design of a new coronavirus vaccine.
  • NTD fragment and RBD fragment of the S protein contain multiple effective epitopes of B cells and T cells, and can trigger strong protective antiviral immunity, which further confirms the design of the novel coronavirus.
  • epitope analysis in vaccines is crucial to the production of B cells and T cells.
  • WO2021159040A9 discloses an mRNA vaccine encoding the NTD and RBD regions of the novel coronavirus S protein.
  • a glycine-serine linker (glycine-serine linker) is used to connect the NTD and RBD regions.
  • One object of the present invention is to provide an mRNA vaccine against novel coronavirus.
  • Another object of the present invention is to provide a method for preparing an mRNA vaccine against novel coronavirus.
  • Another object of the present invention is to provide a DNA template for an mRNA vaccine against a novel coronavirus.
  • Another object of the present invention is to provide the application of the mRNA vaccine against novel coronavirus.
  • the present invention provides an mRNA molecule capable of encoding a target polypeptide, wherein the target polypeptide includes the NTD-RBD natural domain in the SARS-CoV-2 spike protein S, and the NTD-RBD natural domain
  • the target polypeptide includes the NTD-RBD natural domain in the SARS-CoV-2 spike protein S, and the NTD-RBD natural domain
  • the NTD fragment and the RBD fragment are included, and the natural amino acid sequence derived from the S protein is used as a linker to connect the NTD fragment and the RBD fragment.
  • the mRNA molecule provided by the present invention also encodes a signal peptide at the N-terminal of the NTD-RBD natural domain.
  • the amino acid sequence encoded by the mRNA molecule provided by the present invention sequentially includes a signal peptide, an NTD fragment, a linker, and an RBD fragment from the N-terminus to the C-terminus.
  • the signal peptide includes, but is not limited to: a sequence consisting of amino acids 1-12 of SEQ ID NO: 1 (MFVFLVLLPLVS).
  • the amino acid sequence of the linker is SEQ ID NO:50.
  • the amino acid sequence of the NTD fragment is selected from:
  • the amino acid sequence of the RBD fragment is selected from:
  • the "same function" refers to having the same immunogenicity.
  • the amino acid sequence of the encoded NTD-RBD natural domain is:
  • the amino acid sequence of the encoded NTD-RBD natural domain is SEQ ID NO: 49, SEQ ID NO: 48, SEQ ID NO: 47, SEQ ID NO A kind of in :46, SEQ ID NO:45, SEQ ID NO:44.
  • the mRNA molecule provided by the present invention has an encoded amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 9, SEQ ID NO: 5 or SEQ ID NO:1.
  • the mRNA molecule provided by the present invention its protein coding region sequence comprises the sequence that as SEQ ID NO:25 the 37th-1623rd nucleotide composition, the 37th-1623rd of SEQ ID NO:26
  • the sequence consisting of nucleotides, the sequence consisting of 37-1623 nucleotides of SEQ ID NO: 27, the sequence consisting of 37-1614 nucleotides of SEQ ID NO: 28, the sequence consisting of nucleotides 37-1614 of SEQ ID NO: 29 The sequence consisting of nucleotides 37-1614, the sequence consisting of nucleotides 37-1614 of SEQ ID NO:30, the sequence consisting of nucleotides 37-1617 of SEQ ID NO:31, SEQ ID
  • the mRNA molecules provided by the present invention have protein coding region sequences such as SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 , SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ Shown in ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:42.
  • the mRNA molecule provided by the present invention is modified with 1-methylpseudouridine.
  • the mRNA molecule provided by the present invention further includes a 5'-UTR sequence and/or a 3'-UTR sequence.
  • the 5'-UTR sequence may or may not contain a Kozak sequence.
  • the 5'-UTR has the sequence shown in SEQ ID NO:51.
  • the 3'-UTR sequence has the sequence shown in SEQ ID NO:52.
  • the mRNA molecule provided by the present invention is further modified by 3' tailing and/or at least one 5' capping modification.
  • the 3' tailing modification comprises, for example, a poly-A tail, which may be polyadenylation with or without a linker inserted in the middle.
  • the cap structure of at least one 5'capping modification can be selected from Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8- Oxo-guanosine, 2-amino-guanosine, LNA-guanosine or 2-azido-guanosine.
  • the mRNA molecule provided by the invention is an isolated mRNA
  • the mRNA molecule provided by the present invention is purified.
  • the purification includes but not limited to chromatography, lithium chloride or ethanol precipitation, spin column, chlorine extraction, ethanol precipitation or gel purification.
  • the present invention also provides a DNA molecule encoding any one of the aforementioned mRNA molecules of the present invention.
  • the present invention also provides a recombinant plasmid containing the aforementioned DNA molecule of the present invention.
  • the present invention also provides a lipid nanoparticle loaded with any one of the aforementioned mRNA molecules of the present invention.
  • the present invention also provides a pharmaceutical composition, which comprises: any one of the aforementioned mRNA molecules of the present invention, and a pharmaceutically acceptable excipient.
  • a pharmaceutically acceptable excipient can be selected from solvent, water solvent, non-aqueous solvent, dispersion medium, diluent, dispersant, suspending aid, surfactant, isotonicity agent, thickener or emulsifier, preservative, fat Substances, lipidoid liposomes, lipid nanoparticles, core-shell nanoparticles, polymers, lipoplexes (lipoplexes) peptides, proteins, cells, hyaluronidase, and mixtures thereof.
  • the present invention also provides the mRNA molecule of the present invention, the DNA molecule, the recombinant plasmid, the lipid nanoparticle or the pharmaceutical composition in the preparation of the novel coronavirus mRNA vaccine in the application.
  • the present invention also provides a novel coronavirus mRNA vaccine comprising the mRNA molecule of the present invention.
  • the vaccine is in the form of lipid nanoparticles.
  • the particle size of the lipid nanoparticles is 50nm-200nm, preferably 50nm-150nm.
  • the novel coronavirus mRNA vaccine of the present invention wherein the lipid nanoparticles include mRNA and lipids, wherein the lipids include:
  • the novel coronavirus mRNA vaccine of the present invention wherein, the nitrogen-phosphorus molar ratio of described positively charged lipid and/or ionizable lipid to mRNA is 5:1 ⁇ 20: 1.
  • the molar ratio of each lipid component is:
  • PEG-modified lipids 0-3% PEG-modified lipids 0-3%.
  • the ionizable lipids include but are not limited to: 4-(N,N-dimethyl (Dilinoleyl)methyl butyrate ((Dlin-MC3-DMA), SM-102, ((4-hydroxybutyl) azadialkyl)bis(hexane-6,1-di One or more of two (2-hexyldecanoate) (ALC-0315), the positively charged lipids include but not limited to: one or more of DOTMA, DOTAP; the Neutral helper lipids include, but are not limited to: one or more of DSPC, DOPE, DSPE; the PEG-modified lipids include, but are not limited to: methoxypolyethylene glycol ditetradecylacetamide One or more of (ALC-0159), DMG-PEG.
  • the novel coronavirus mRNA vaccine described in the present invention is in a freeze-dried dosage form or a frozen dosage form.
  • the present invention also provides a method for preparing a novel coronavirus mRNA vaccine, which includes the process of preparing the mRNA molecule. Specifically, the following steps are included:
  • the DNA fragment encoding the NTD-RBD natural domain peptide was synthesized, cloned into a plasmid as a template, and transcribed to prepare the target mRNA molecule.
  • the target mRNA molecule is any one of the aforementioned mRNA molecules of the present invention.
  • the present invention provides a method for preparing the mRNA molecule, wherein the process of synthesizing the DNA fragment encoding the NTD-RBD native domain peptide can be self-synthesized or commissioned.
  • the DNA fragment is cloned into a plasmid as a template, and the target mRNA molecule can be prepared according to the following reaction:
  • Two-step method in vitro transcription to obtain tailed and uncapped mRNA; under the catalysis of capping enzyme, to add a cap structure to the 5' end of uncapped mRNA to obtain capped and tailed mRNA molecules;
  • One-step method perform in vitro transcription and capping to obtain capped and tailed mRNA molecules.
  • the method for preparing the mRNA molecule also includes the purification process of mRNA: the mRNA is subjected to lithium chloride/ethanol precipitation, spin column, chlorine extraction/ethanol precipitation or gel purification, to Obtain purified mRNA.
  • the preparation method of novel coronavirus mRNA vaccine of the present invention also includes:
  • the prepared mRNA molecule is dissolved in the aqueous phase composed of citrate buffer, and mixed with the lipid component dissolved in the ethanol phase by microfluidic or impact jet method to prepare the lipid nanoparticle loaded with mRNA.
  • the lipid component may include ionizable cationic phospholipids (ionizable lipids), neutral auxiliary phospholipids, cholesterol, PEGylated lipids, etc. as mentioned above.
  • the preparation method of the novel coronavirus mRNA vaccine of the present invention also includes the process of making the prepared lipid nanoparticles into a frozen preparation or a freeze-dried preparation.
  • the preparation method of the novel coronavirus mRNA vaccine of the present invention includes:
  • the freeze-dried protective agent includes but is not limited to one or more of the following protective agents 1 to 3:
  • Protective agent 1 sucrose
  • Protective agent 2 sucrose and nonionic surfactant
  • Protective agent 3 sucrose and trehalose.
  • the mass volume concentration of sucrose is 10% to 20%. (that is, 10-20g/100mL), preferably 12-18%; the mass volume concentration of trehalose is 0%-20%, preferably 0%-5%, more preferably 0.5%-3%; non-ionic surface
  • the mass volume concentration of the active agent is 0%-2%.
  • the non-ionic surfactant includes but is not limited to poloxamer, such as Pluronic F-68.
  • the mass volume concentration of the poloxamer in the prepared buffer containing the lipid nanoparticles and the freeze-drying protective agent is preferably 0% to 1%.
  • SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, and SEQ ID NO: 21 are the original strain of novel coronavirus, Alpha strain, Amino acid sequences of NTD-RBD antigens of Beta strain, Gamma strain, Delta strain and Omicron strain.
  • SEQ ID NO: 2 SEQ ID NO: 3, and SEQ ID NO: 4 are the codon-optimized DNA fragment sequences of human, mouse, and rat codon-optimized DNA fragments of the original strain of the new coronavirus respectively;
  • SEQ ID NO: 27 is its corresponding mRNA coding region sequence.
  • SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 are the human, mouse, and rat codon-optimized DNA fragment sequences of the new coronavirus Alpha strain, respectively;
  • SEQ ID NO: 28, SEQ ID NO : 29, SEQ ID NO: 30 is its corresponding mRNA coding region sequence.
  • SEQ ID NO: 10 SEQ ID NO: 11, and SEQ ID NO: 12 are the human, mouse, and rat codon-optimized DNA fragment sequences of the new coronavirus Beta strain, respectively;
  • SEQ ID NO: 31, SEQ ID NO : 32, SEQ ID NO: 33 is its corresponding mRNA coding region sequence.
  • SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16 are the codon-optimized DNA fragment designs of human, mouse, and rat codons of the new coronavirus Gamma strain, respectively;
  • SEQ ID NO: 34, SEQ ID NO : 35, SEQ ID NO: 36 are their corresponding mRNA coding region sequences.
  • SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20 are the codon-optimized DNA fragment designs of the human, mouse, and rat codon-optimized DNA fragments of the new coronavirus Delta strain, respectively;
  • SEQ ID NO: 37, SEQ ID NO : 38, SEQ ID NO: 39 are their corresponding mRNA coding region sequences.
  • SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24 are the human, mouse, and rat codon-optimized DNA fragment designs of the new coronavirus Omicron strain, respectively;
  • SEQ ID NO: 40, SEQ ID NO : 41, SEQ ID NO: 42 is its corresponding mRNA coding region sequence.
  • SEQ ID NO: 43 is the mRNA sequence of the RBD antigen of the new coronavirus Delta strain.
  • SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49 are original strain, Alpha strain, Beta strain, Gamma strain, Delta strain respectively The amino acid sequence of the NTD-RBD native domain of strain and Omicron strain.
  • SEQ ID NO:50 is the amino acid sequence of the linker in the NTD-RBD native domain.
  • novel coronavirus described includes original strains and/or mutant strains.
  • the present invention first analyzes the epitope of the novel coronavirus (SARS-CoV-2), and determines that NTD-RBD is the antigen target, LNP is the delivery carrier, and the immune mode is muscle Injected mRNA vaccine dosage form, at the same time through UTR and codon optimization, and in vitro transcription methods to synthesize mRNA encoding viral antigen fragments, and finally achieve high-efficiency expression in human cells, and have protective effects on the main mutant strains currently circulating; the entire mRNA vaccine production
  • the cycle is short, the process operation is simple, the production cost is low, the storage time is long, no cold chain is required, and the transportation is convenient.
  • Traditional vaccines cannot respond quickly to public health events caused by many new viruses, but mRNA vaccines have wider applicability, and their sequences can be flexibly designed to deal with different pathogens. It is extremely important to develop rapidly compiled acute infectious disease vaccines role.
  • the mRNA vaccine with NTD-RBD as antigen designed by the present invention can induce stronger neutralizing antibody effect under the condition of the same inoculation amount.
  • Figure 1 shows the particle size and PDI test results of mRNA-LNP prepared in a specific embodiment of the present invention.
  • Figure 2 shows the test results of the encapsulation efficiency of the mRNA-LNP prepared in a specific embodiment of the present invention.
  • Fig. 3 shows the appearance of the mRNA freeze-dried vaccine prepared in a specific embodiment of the present invention.
  • Fig. 4 shows the effect of in vitro activity of the mRNA freeze-dried vaccine prepared in a specific embodiment of the present invention.
  • Fig. 5 shows the in vivo activity effect of the mRNA freeze-dried vaccine prepared in a specific embodiment of the present invention.
  • Fig. 6 shows the specific antibody titer test results of the mRNA vaccine of the present invention.
  • Figure 7 shows the comparison of immunogenicity of RBD and NTD-RBD antigens.
  • Fig. 8 shows the cross-protection effect of NTD-RBD mRNA vaccine of the present invention to different strains.
  • Figure 9 shows that the NTD-RBD vaccine of the present invention induces strong cellular immunity.
  • Figure 10 shows the results of the NTD-RBD vaccine of the present invention in vivo challenge experiments on ACE2 mice.
  • Figure 11 shows the experimental results of the NTD-RBD vaccine of the present invention as a booster.
  • Fig. 12 is the experimental results of the relative expression of protein induced by mRNA in some specific embodiments of the present invention.
  • the present invention can be further described by the following examples, however, the scope of the present invention is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made in the present invention without departing from the spirit and scope of the present invention.
  • the present invention provides general and/or specific descriptions of the materials and test methods used in the tests. While many of the materials and methods of manipulation which are employed for the purposes of the invention are well known in the art, the invention has been described here in as much detail as possible. For methods not specified in detail, perform the conventional operations in the art or the operations suggested by the manufacturer's instructions.
  • Embodiment 1 mRNA vaccine and preparation method thereof
  • This embodiment provides an mRNA vaccine, the preparation method of which is mainly carried out according to the following operations.
  • the above enzyme digestion system was placed at 50 °C for 1 h. After the reaction, 1 ⁇ l of the system before and after the enzyme digestion reaction was taken for DNA agarose gel electrophoresis (1.5% agarose gel, 5 V/min, 40 min). According to the comparison of electrophoresis results, whether the recombinant plasmid is digested completely.
  • Eligibility criteria A single band appears in the electrophoresis test; compared with the supercoiled plasmid before digestion, the band is located above the supercoiled plasmid; the size meets the expected requirements.
  • Measurement results a single band; the size is in line with expectations and the band is located above the supercoiled plasmid.
  • the DNA templates obtained above were concentrated using Millipore 30Kd ultrafiltration tubes.
  • NanoDrop to detect the concentration of the purified template and the ratios of 260/280 and 260/230. Samples were taken for DNA agarose gel electrophoresis detection (1.5% agarose, 5V/min, 40min).
  • Eligibility criteria 260/280 between 1.8 and 2.1, 260/230 between 1.6 and 2.2.
  • the DNA template purified by FPLC was concentrated by Millipore 30Kd ultrafiltration tube, and eluted with RNase-free water to dissolve. Use NanoDrop to detect the concentration of the template after ultrafiltration, and the ratios of 260/280 and 260/230. Finally, it was diluted to 150ng/ ⁇ l with RNase-free water.
  • Reaction volume 1600 ⁇ l (placed in a 2ml RNase-free Tube tube, which is the reaction volume of a single tube, multiple tubes can be reacted at the same time): RNA-free water 440 ⁇ l, 7.5mM ATP 160 ⁇ l, 7.5mM N1-methyl-pseudo Uridine 160 ⁇ l, 7.5mM CTP 160 ⁇ l, 7.5mM GTP 160 ⁇ l, 7.5mM M7G (2'OMeA)pG 160 ⁇ l, 150ng/ ⁇ l DNA template 40 ⁇ l, 10 ⁇ Buffer 160 ⁇ l and Enzyme Mix 160 ⁇ l.
  • RNA synthesis in vitro is 37° C. for 10 h.
  • the target mRNA molecule is obtained.
  • the target mRNA molecule in addition to the coding sequence, also includes 5'UTR (AGGGAGAUAAGAGAGAAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCGCCACC, SEQ ID NO: 51) and 3'UTR (GCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCU GAGUAGGAAG, SEQ ID NO:52), 5'CAP is m7G+- 5'-ppp-5'-Am2'-3'-p-(cap1), 3' poly-A tail (SEQ ID NO:53).
  • the reaction solution was combined into an RNase-free 50ml Tube tube to detect the residue of DNA fragments.
  • the three measurements were 0.013ng, 0.016ng, 0.017ng per 100 ⁇ g mRNA.
  • the recovered mRNA concentration detected by NanoDrop was 5 ⁇ g/ ⁇ l, A260/A280 was 1.90, and A260/A230 was 2.0.
  • the detection result is: the band meets the size and the fragment is complete.
  • the purified mRNA was diluted to 2 ⁇ g/ ⁇ l with 0.1 M citric acid.
  • phase A mRNA buffer
  • phase B lipid compound dissolved in absolute ethanol
  • Encapsulation efficiency detection Take 64 ⁇ L of the sample in step (6) and step (7) and dilute it 5 times respectively, as the LNP RNA sample before and after lysis; measure the RNA concentration; divide the concentration difference before and after lysis by the concentration after lysis , to obtain the encapsulation rate;
  • Fig. 1 and Fig. 2 The detection results of the particle size, PDI and encapsulation efficiency of the mRNA-loaded LNP prepared in this example are shown in Fig. 1 and Fig. 2 .
  • the abscissa numbers correspond to the sequence numbers of each mRNA respectively, that is, the sample corresponding to 25 in the figure is the mRNA sample of SEQ ID NO: 25, and the sample corresponding to 26 in the figure is the mRNA of SEQ ID NO: 26 Sample, the sample corresponding to 27 in the figure is the mRNA sample of SEQ ID NO:27, and so on, the sample corresponding to 42 in the figure is the mRNA sample of SEQ ID NO:42.
  • the lyoprotectant added to the LNP solution is put into a freeze dryer, and the mass volume fraction (w/v%) is 15% of sucrose and 2% of trehalose.
  • Xinzhi Scientz-10N After the cold trap was pre-frozen for 4 hours, the vacuum was turned on for 48 hours, and then the sample was transferred to the upper layer of the freeze dryer for secondary drying for 16 hours. When drying in the cold trap, the sample temperature probe shows about -30°C, and when drying in the upper layer, the sample temperature probe shows about 4°C. After drying, collect the product (i.e. LNP freeze-dried powder), reconstitute with the same volume of ultrapure water as before freeze-drying, and the freeze-dried powder dissolves rapidly after adding ultrapure water.
  • the whole process does not exceed 20s. Sealing rate, European and American NS-90Z nanoparticle size and potential analyzer to measure the particle size and zeta potential of nanoparticles.
  • the mRNA freeze-dried vaccine of this embodiment was prepared. The finished product picture and the appearance after reconstitution of the prepared vaccine are shown in FIG. 3 .
  • 100 ⁇ g mRNA-LNP powders of 25# to 42# (that is, mRNA samples corresponding to SEQ ID NO: 25 to SEQ ID NO: 42 respectively) were reconstituted with 200 ⁇ l of water for injection before injection inoculation, and the first 6-week-old balb/c mice were inoculated twice on day 1 and day 14, and the serum of the mice was collected on day 35 to detect the titer of anti-S protein-specific antibody in the serum. Specifically, proceed as follows:
  • Plate washing Pour out the coating liquid on the coated 96-well plate, put it on the buckle absorbent paper, and buckle the plate hard until there is no residue in the well. Prepare the eluent, dilute 50x Washing buffer with deionized water, add it to the liquid inlet bottle of the plate washer, set the program, set the washing volume of each well to 300 ⁇ l, and repeat the wash 4 times.
  • Serum incubation Dilute the mouse serum to 40x, 400x, 4000x, 40000x, 400000x, 4000000x, 40000000x with dilution buffer, add to the washed 96-well plate according to the volume of 100 ⁇ l per well, and then seal the plate at room temperature Incubate for 1.5h.
  • Plate washing Complete the plate washing according to step 2, and the number of plate washing is increased to 6 times.
  • Plate washing Complete plate washing according to step 2. In this step, be sure to wash the plate and dry the solution.
  • Termination Add 100 ⁇ l of Stop buffer, read on the microplate within 10 minutes, and set the absorption wavelength to 450nm.
  • the mRNA vaccine (43#, i.e. corresponding to the sample of SEQ ID NO:43) and the encoding NTD-RBD vaccine (37#, i.e. the sample corresponding to SEQ ID NO:37) of encoding RBD were investigated by experiment.
  • Antibody titer Specifically, proceed as follows:
  • mice aged 6 to 8 weeks were inoculated with 5 ⁇ g of novel coronavirus mRNA vaccine (dissolved in PBS, 200 ⁇ l, injected intramuscularly) at 0 d and 14 d respectively; the peripheral blood of the mice was collected at 28 d.
  • novel coronavirus mRNA vaccine dissolved in PBS, 200 ⁇ l, injected intramuscularly
  • Vero E6 cells 24-well plate
  • 100 ⁇ l serum and 100 ⁇ l virus stock solution 100 PFU
  • 100 ⁇ l DMEM containing 2% FBS
  • 100 ⁇ l virus solution was mixed in equal volume with 100 ⁇ l virus solution as a negative control. Incubate at 37°C for 1h.
  • the above serum-virus mixture (200 ⁇ l in total) was transferred to Vero E6 cells in a 24-well plate, and adsorbed for 1 hour. Gently mix 3-4 times during this period. Remove the adsorption solution from the previous step, replace the methylcellulose medium, and culture for 3 days. Fixed with paraformaldehyde, stained with crystal violet, and counted plaques. Calculate the serum neutralization percentage based on the negative control. Curve fitting was carried out with the JMP analysis software Probit method, and the PRNT 50 value was calculated.
  • Vaccination 6-8 week-old female mice were inoculated with 5 ⁇ g of novel coronavirus mRNA vaccine (dissolved in PBS, 200 ⁇ l, intramuscular injection) at 0 d and 14 d respectively; at 28 d, the peripheral blood of the mice was taken, and microneutralization was used to The heat-inactivated serum was tested in the experiment to detect the level of antibodies neutralizing the monolayer cells expressing ACE2 infected by the new coronavirus pseudovirus; dilute 4 wells on a 96-well plate, and detect the virus in the cells on the 3rd and 4th day Sexual lesion effect (cpe), the serum dilution of the serum that completely inhibits cpe in the 50% endpoint method was calculated by Reed Muench formula; statistical analysis was carried out by non-parametric two-tailed t test (Mann-Whiteny).
  • the PVDF membrane in the 96-well plate was soaked with 70% ethanol for 30s.
  • Vaccination 6-8 weeks old female mice were inoculated with 5 ⁇ g novel coronavirus mRNA vaccine RH109 (40#) (dissolved in PBS, 200 ⁇ l, intramuscular injection) at 0d and 14d respectively; peripheral blood of the mice was taken at 28 days , PBMC were isolated from fresh blood with Ficoll and counted, and the cells were diluted with culture medium and added to a 96-well plate. Usually the number of cells used is 1-2 ⁇ 10 5 /well.
  • the reaction was terminated by washing with distilled water.
  • CD4 and CD8 cells were further analyzed by flow cytometry intracellular staining experiments, CD4 + cells of IL2 + /TNF- ⁇ + /IFN- ⁇ + , and CD8 + cells of TNF- ⁇ + /IFN- ⁇ + Significantly increased, indicating that the RH109 vaccine activated a strong Th1 type and CD8 killer T cell immune response.
  • Vaccination when 0d and 21d, female mice aged 6 to 8 weeks were inoculated with 5 ⁇ g or 10ug novel coronavirus mRNA vaccine (37#) (dissolved in PBS, 200 ⁇ l, intramuscular injection); ivD was the challenge period, ivD0 is the day of the challenge, the new coronavirus (Delta strain) was instilled into the hACE2 transgenic mice, and the infection dose was initially 105PFU. D42 is the attack time. After challenge ivD3 and ivD5, the animals were euthanized in batches.
  • mice aged 6-8 weeks were inoculated with two injections of Kexing inactivated vaccine on 0d and 21d respectively, and 5 ⁇ g of the new coronavirus mRNA vaccine RH109 (dissolved in PBS, 200 ⁇ l, intramuscular injection) on 42 days; On the 56th day, the peripheral blood of the mice was taken, and the heat-inactivated serum was measured by a micro-neutralization experiment to detect the level of antibody neutralization and the level of monolayer cells expressing ACE2 infected by the new coronavirus pseudovirus; dilute 4 wells on a 96-well plate, On the 3rd day and the 4th day, the viral pathological effect (cpe) of the cells was detected, and the serum dilution of the serum that completely inhibited the cpe was calculated in the 50% endpoint method by the Reed Muench formula; Mann-Whiteny) for statistical analysis.
  • RH109 dissolved in PBS, 200 ⁇ l, intramuscular injection
  • the sera after RH109 immunization produced high neutralizing antibody levels against Omicron, Delta, and the original strain, and the geometric mean titers were 7970, 9091, and 13998, respectively, and Omicron increased by 39% compared with the second injection.
  • the times of Omicron, Detla and the original strain are 35, 6.2, and 3.4 times of the latter respectively, indicating that RH109 is very suitable as an inactivated vaccine.
  • the strengthening needle is used.
  • the present invention also compares different doses of different mRNAs of the present invention to induce protein expression experiments, respectively incubating mRNA-LNP solutions of SEQ ID NO: 28, SEQ ID NO: 54, and SEQ ID NO: 55 with 2 ⁇ g and 4 ⁇ g
  • the cell pellet was taken after 24 hours, the cells were lysed, and the target protein bands obtained by parallel detection were used for grayscale analysis of the relative expression of intracellular proteins within 24 hours.
  • the results are shown in Figure 12 (the number 28-1 in the figure corresponds to SEQ ID NO:28, the number 30-1 corresponds to SEQ ID NO:54, and the number 30-3 corresponds to SEQ ID NO:55).
  • the mRNA of the present invention has good protein expression efficiency.

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Abstract

一种能编码目标多肽的mRNA分子，其中，目标多肽包括SARS-CoV-2刺突蛋白中NTD-RBD天然结构域，该NTD-RBD天然结构域包括NTD片段与RBD片段以及两者之间的连接子，该连接子是来源于刺突蛋白的天然氨基酸序列。

Description

一种新型冠状病毒mRNA疫苗及其制备方法与应用 技术领域
本发明是关于一种mRNA疫苗及其制备方法与应用，具体是一种针对新型冠状病毒(包括原始株和/或突变株)的mRNA疫苗、其制备方法与相关应用。
背景技术
mRNA疫苗技术可以作为快速灵活的技术平台，有效应对各种新兴病毒的威胁。因此mRNA疫苗被认为是对抗新型冠状病毒(SARS-CoV-2)最有潜力的一种疫苗。随着化学修饰mRNA和纳米脂质载体技术的发展和成熟，mRNA在多种病毒疫苗研发中的优势愈发凸显。
由于免疫反应的特异性，靶抗原表位序列(包括B细胞表位和T细胞表位)直接决定了免疫反应的种类。鉴于B细胞免疫和T细胞免疫都在SARS-CoV-2病毒的清除中不可或缺，尽可能去选择合适的免疫原性片段，是新型冠状病毒疫苗设计的关键一步。在设计针对SARS-CoV-2突变株的靶抗原时，需要考虑到有些抗原表位会诱导出非中和抗体，这类抗体不能起到有效的保护作用，反而有概率引起ADE效应，加重病毒的感染程度。因此在这种情况下，全长S蛋白能否继续作为疫苗抗原值得深入研究探讨。经过研究COVID-19病人体内的血清发现S蛋白的NTD片段和RBD片段含有多个B细胞和T细胞有效表位，并能够引发强烈的保护性抗病毒免疫，这也进一步证实了设计新型冠状病毒疫苗时进行抗原表位分析的重要性。
WO2021159040A9公开了一种编码包含新型冠状病毒S蛋白NTD和RBD区域的mRNA疫苗，在氨基酸序列设计中，使用了甘氨酸-丝氨酸连接子(glycine-serine linker)对NTD和RBD区域进行连接。
另一方面，现已发现新型冠状病毒新型变异株，并且世界范围内的流行毒株发生了明显变化，其不同程度的免疫逃逸对现有新型冠状病毒疫苗和后续疫苗研发工作带来了挑战。
发明内容
本发明的一个目的在于提供一种针对新型冠状病毒的mRNA疫苗。
本发明的另一个目的在于提供针对新型冠状病毒的mRNA疫苗的制备方法。
本发明的另一个目的在于提供针对新型冠状病毒的mRNA疫苗的DNA模板。
本发明的另一个目的在于提供针对新型冠状病毒的mRNA疫苗的应用。
一方面，本发明提供了一种能编码目标多肽的mRNA分子，其中，所述的目标多肽包括SARS-CoV-2刺突蛋白S中NTD-RBD天然结构域，所述NTD-RBD天然结构域包括NTD片段与RBD片段，NTD片段与RBD片段之间以来源于S蛋白的天然氨基酸序列作为连接子连接。
根据本发明的具体实施方案，本发明提供的mRNA分子，还编码NTD-RBD天然结构域N端的信号肽。
根据本发明的具体实施方案，本发明提供的mRNA分子编码的氨基酸序列从N端到C端依次包括信号肽、NTD片段、连接子、RBD片段。
在本发明的一些具体实施方案中，所述信号肽包括但不限于：如SEQ ID NO:1第1-12位氨基酸组成的序列(MFVFLVLLPLVS)。
根据本发明的具体实施方案，本发明提供的mRNA分子，所编码的NTD-RBD天然结构域中，所述的连接子的氨基酸序列为SEQ ID NO:50。
根据本发明的具体实施方案，本发明提供的mRNA分子，所编码的NTD-RBD天然结构域中，NTD片段的氨基酸序列选自：
(a)SEQ ID NO:49的第1至第289位组成的氨基酸序列；
(b)由(a)的氨基酸序列经过替换、增加和/或缺失一个或几个氨基酸且具有与(a)相同功能的衍生序列。
根据本发明的具体实施方案，本发明提供的mRNA分子，所编码的NTD-RBD天然结构域中，RBD片段的氨基酸序列选自：
(c)SEQ ID NO:49的第304至第526位组成的氨基酸序列；
(d)由(c)的氨基酸序列经过替换、增加和/或缺失一个或几个氨基酸且具有与(a)相同功能的衍生序列。
本发明中，所述的“相同功能”是指同样具有免疫原性。
根据本发明的具体实施方案，本发明提供的mRNA分子，所编码的NTD-RBD天然结构域的氨基酸序列为：
SEQ ID NO:44至SEQ ID NO:49中任一序列所示氨基酸序列；或
与SEQ ID NO:44至SEQ ID NO:49中任一序列同一性在92.78％以上的衍生的氨基酸序列。
根据本发明的一些具体实施方案，本发明提供的mRNA分子，所编码的NTD-RBD 天然结构域的氨基酸序列为SEQ ID NO:49、SEQ ID NO:48、SEQ ID NO:47、SEQ ID NO:46、SEQ ID NO:45、SEQ ID NO:44中的一种。
根据本发明的具体实施方案，本发明提供的mRNA分子，所编码的氨基酸序列为SEQ ID NO:21、SEQ ID NO:17、SEQ ID NO:13、SEQ ID NO:9、SEQ ID NO:5或SEQ ID NO:1。
根据本发明的具体实施方案，本发明提供的mRNA分子，其蛋白质编码区序列包括如SEQ ID NO:25的第37-1623位核苷酸组成的序列、SEQ ID NO:26的第37-1623位核苷酸组成的序列、SEQ ID NO:27的第37-1623位核苷酸组成的序列、SEQ ID NO:28的第37-1614位核苷酸组成的序列、SEQ ID NO:29的第37-1614位核苷酸组成的序列、SEQ ID NO:30的第37-1614位核苷酸组成的序列、SEQ ID NO:31的第37-1617位核苷酸组成的序列、SEQ ID NO:32的第37-1617位核苷酸组成的序列、SEQ ID NO:33的第37-1617位核苷酸组成的序列、SEQ ID NO:34的第37-1623位核苷酸组成的序列、SEQ ID NO:35的第37-1623位核苷酸组成的序列、SEQ ID NO:36的第37-1623位核苷酸组成的序列、SEQ ID NO:37的第37-1623位核苷酸组成的序列、SEQ ID NO:38的第37-1623位核苷酸组成的序列、SEQ ID NO:39的第37-1623位核苷酸组成的序列、SEQ ID NO:40的第37-1614位核苷酸组成的序列、SEQ ID NO:41的第37-1614位核苷酸组成的序列或SEQ ID NO:42的第37-1614位核苷酸组成的序列。
根据本发明的具体实施方案，本发明提供的mRNA分子，其蛋白质编码区序列如SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41或SEQ ID NO:42所示。
根据本发明的具体实施方案，本发明提供的mRNA分子，经过1-甲基假尿苷修饰。
根据本发明的具体实施方案，本发明提供的mRNA分子，还包括5’-UTR序列和/或3’-UTR序列。
具体地，5’-UTR序列可包含或不包含Kozak序列。在本发明的一些优选实施方案中，所述5'-UTR具有SEQ ID NO:51所示序列。
在本发明的一些优选实施方案中，3’-UTR序列具有SEQ ID NO:52所示序列。
根据本发明的具体实施方案，本发明提供的mRNA分子，还经过3'加尾修饰和/或至少一个5'加帽修饰。
具体地，3'加尾修饰例如包含聚-A尾，所述聚A尾可以是中间插入或不插入连接子的多聚腺苷酸。
具体地，至少一个5'加帽修饰的帽结构可选自Cap0、Cap1、ARCA、肌苷、N1-甲基-鸟苷、2'氟-鸟苷、7-脱氮-鸟苷、8-氧代-鸟苷、2-氨基-鸟苷、LNA-鸟苷或2-叠氮基-鸟苷。
根据本发明的具体实施方案，本发明提供的mRNA分子，其是分离的mRNA；
根据本发明的具体实施方案，本发明提供的mRNA分子，是经纯化的。所述的纯化包括但不限于经过层析、氯化锂或乙醇沉淀、离心柱、氯萃取、乙醇沉淀或凝胶纯化。
另一方面，本发明还提供了一种DNA分子，其编码本发明前述任意一种mRNA分子。
另一方面，本发明还提供了一种重组质粒，其含有本发明前述的DNA分子。
另一方面，本发明还提供了一种脂质纳米颗粒，其负载有本发明前述任意一种mRNA分子。
另一方面，本发明还提供了一种药物组合物，其包含：本发明前述任意一种mRNA分子，以及药学上可接受的赋形剂。所述的赋形剂可选自溶剂、水溶剂、非水溶剂、分散介质、稀释剂、分散剂、悬浮助剂、表面活性剂、等渗剂、增稠剂或乳化剂、防腐剂、脂质、类脂质(lipidoid)脂质体、脂质纳米颗粒、核-壳纳米颗粒、聚合物、脂复合物(lipoplexe)肽、蛋白、细胞、透明质酸酶、及它们的混合物。
另一方面，本发明还提供了本发明所述的mRNA分子、所述的DNA分子、所述的重组质粒、所述的脂质纳米颗粒或所述的药物组合物在制备新型冠状病毒mRNA疫苗中的应用。
另一方面，本发明还提供了一种新型冠状病毒mRNA疫苗，其包含本发明所述的mRNA分子。在本发明的一些具体实施方案中，所述的疫苗为脂质纳米颗粒剂型。
根据本发明的具体实施方案，本发明所述的新型冠状病毒mRNA疫苗，其中，脂质纳米颗粒的粒径为50nm～200nm，优选为50nm～150nm。
根据本发明的具体实施方案，本发明所述的新型冠状病毒mRNA疫苗，其中，所述的脂质纳米颗粒包括mRNA和脂质，其中，所述脂质包括：
a)正电荷脂质和/或可离子化脂质中的一种或多种；
b)中性辅助脂质；
c)胆固醇；
d)PEG修饰的脂质。
根据本发明的具体实施方案，本发明所述的新型冠状病毒mRNA疫苗，其中，所述的正电荷脂质和/或可离子化脂质与mRNA的氮磷摩尔比为5:1～20:1。
根据本发明的具体实施方案，本发明所述的新型冠状病毒mRNA疫苗，所述的脂质纳米颗粒中，以脂质的总摩尔量为100％计，各脂质成分的摩尔比为：
正电荷脂质或可离子化脂质  46％～50％；
中性辅助脂质  5％～10％；
胆固醇  38.5％～48％；
PEG修饰的脂质  0～3％。
根据本发明的具体实施方案，本发明所述的新型冠状病毒mRNA疫苗，所述的脂质纳米颗粒中，所述的可离子化脂质包括但不限于：4-(N,N-二甲基氨基)丁酸(二亚油基)甲酯((Dlin-MC3-DMA)、SM-102、((4-羟基丁基)氮杂二烷基)双(己烷-6,1-二基)双(2-己基癸酸酯)(ALC-0315)中的一种或多种，所述的正电荷脂质包括但不限于：DOTMA、DOTAP中的一种或多种；所述的中性辅助脂质包括但不限于：DSPC、DOPE、DSPE中的一种或多种；所述的PEG修饰的脂质包括但不限于：甲氧基聚乙二醇双十四烷基乙酰胺(ALC-0159)、DMG-PEG中的一种或多种。
根据本发明的具体实施方案，本发明所述的新型冠状病毒mRNA疫苗，为冷冻干燥剂型或冷冻剂型。
另一方面，本发明还提供了一种新型冠状病毒mRNA疫苗的制备方法，其包括制备所述mRNA分子的过程。具体地，包括以下步骤：
合成编码NTD-RBD天然结构域肽段的DNA片段，并克隆到质粒作为模板，转录制备得到目标mRNA分子。
优选地，所述目标mRNA分子为本发明前述的任意一项mRNA分子。
在本发明的一些具体实施方案，本发明提供了制备所述mRNA分子的方法，其中，合成编码NTD-RBD天然结构域肽段的DNA片段的过程可自行合成或委托合成。将所述DNA片段克隆到质粒作为模板进行，可按照如下反应制备目标mRNA分子：
a.三步法：体外转录得到未加帽未加尾的mRNA，在RNA聚合酶和ATP的条件下，在mRNA尾部加上polyA结构(polyA结构中间可插入连接子或不插入连接子)，在加帽酶催化下，以给未加帽的mRNA的5'端加上帽子结构，得到加帽加尾的mRNA分子；
b.两步法：体外转录得到加尾未加帽的mRNA；在加帽酶催化下，以给未加帽的 mRNA的5'端加上帽子结构，得到加帽加尾的mRNA分子；
c.一步法：进行体外转录和加帽，得到加帽加尾的mRNA分子。
在本发明的一些具体实施方案，制备所述mRNA分子的方法还包括mRNA的纯化过程：将所述的mRNA进行氯化锂/乙醇沉淀、离心柱、氯萃取/乙醇沉淀或凝胶纯化，以获得纯化后的mRNA。
根据本发明的具体实施方案，本发明的新型冠状病毒mRNA疫苗的制备方法，还包括：
将所制备的mRNA分子溶解在柠檬酸缓冲液组成的水相中，采用微流控或冲击式射流的方法与溶解在乙醇相中的脂质成分混合，制备负载有mRNA的脂质纳米颗粒。其中，所述的脂质成分如前所述可包括可电离的阳离子磷脂(ionizable lipids)，中性辅助磷脂，胆固醇，聚乙二醇修饰的磷脂(PEGylated lipid)等。
根据本发明的具体实施方案，本发明的新型冠状病毒mRNA疫苗的制备方法，还包括将所制备的脂质纳米颗粒制成冷冻制剂或冷冻干燥制剂的过程。
根据本发明的具体实施方案，本发明的新型冠状病毒mRNA疫苗的制备方法，将脂质纳米颗粒制成冷冻干燥制剂的过程包括：
a)配制含有脂质纳米颗粒和冷冻干燥保护剂的缓冲液；
b)降温进行预冻；
c)在真空条件下升温进行干燥，使体系含水量在3％以下，制备得到脂质纳米颗粒的干燥制剂。
根据本发明的一些具体实施方案，本发明的新型冠状病毒mRNA疫苗的制备方法中，所述的冷冻干燥保护剂包括但不限于以下保护剂1至保护剂3中的一种或多种：
保护剂1：蔗糖；
保护剂2：蔗糖和非离子表面活性剂；
保护剂3：蔗糖和海藻糖。
根据本发明的具体实施方案，本发明的新型冠状病毒mRNA疫苗的制备方法中，所配制的含有脂质纳米颗粒和冷冻干燥保护剂的缓冲液中，蔗糖的质量体积浓度为10％～20％(即，10～20g/100mL)，优选为12～18％；海藻糖的质量体积浓度为0％～20％，优选为0％～5％，更优选为0.5％～3％；非离子表面活性剂的质量体积浓度为0％～2％。在本发明的一些具体实施方案中，所述非离子表面活性剂包括但不限于泊洛沙姆，例如可以是Pluronic F-68。泊洛沙姆在所配制的含有脂质纳米颗粒和冷冻干燥保护 剂的缓冲液中的质量体积浓度优选为0％～1％。
本发明中，SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:9、SEQ ID NO:13、SEQ ID NO:17、SEQ ID NO:21分别为新型冠状病毒原始株、Alpha株、Beta株、Gamma株、Delta株和Omicron株的NTD-RBD抗原的氨基酸序列。
SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4分别为新型冠状病毒原始株的人源、小鼠、大鼠密码子优化的DNA片段序列；SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27为其对应的mRNA编码区序列。
SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8分别为新型冠状病毒Alpha株的人源、小鼠、大鼠密码子优化的DNA片段序列；SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30为其对应的mRNA编码区序列。
SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12分别为新型冠状病毒Beta株的人源、小鼠、大鼠密码子优化的DNA片段序列；SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33为其对应的mRNA编码区序列。
SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16分别为新型冠状病毒Gamma株的人源、小鼠、大鼠密码子优化的DNA片段设计；SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36为其对应的mRNA编码区序列。
SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20分别为新型冠状病毒Delta株的人源、小鼠、大鼠密码子优化的DNA片段设计；SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39为其对应的mRNA编码区序列。
SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24分别为新型冠状病毒Omicron株的人源、小鼠、大鼠密码子优化的DNA片段设计；SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42为其对应的mRNA编码区序列。
SEQ ID NO:43为新型冠状病毒Delta株RBD抗原的mRNA序列。
SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49分别为原始株、Alpha株、Beta株、Gamma株、Delta株和Omicron株的NTD-RBD天然结构域的氨基酸序列。
SEQ ID NO:50是NTD-RBD天然结构域中连接子的氨基酸序列。
本发明中，除特别注明以及结合上下文能明确确定外，所描述的新型冠状病毒包括原始株和/或突变株。
在本发明的一些具体实施方案中，本发明先通过对新型冠状病毒(SARS-CoV-2) 进行抗原表位分析，确定以NTD-RBD为抗原靶点，LNP为递送载体，免疫方式为肌肉注射的mRNA疫苗剂型，同时通过UTR和密码子优化、体外转录方法合成编码病毒抗原片段的mRNA，最终实现人源细胞高效表达，并且对目前流行的主要突变株均具有保护作用；整个mRNA疫苗生产周期短、工艺操作简单、生产成本低，保存时间久，不需要冷链，便于运输。传统疫苗对很多新型病毒引起的公共卫生事件无法做到快速响应，而mRNA疫苗适用性更广，序列可以灵活设计，以应对不同的病原体，针对快速编译的急性传染病疫苗快速研发有及其重要的作用。
本发明设计的以NTD-RBD为抗原的mRNA疫苗，能在相同接种量的条件下，引起更强的中和抗体效应。
附图说明
图1显示本发明一具体实施例中所制备得到的mRNA-LNP的粒径和PDI测试结果。
图2显示本发明一具体实施例中所制备得到的mRNA-LNP的包封率测试结果。
图3显示本发明一具体实施例中所制备得到的mRNA冻干疫苗外观。
图4显示本发明一具体实施例中所制备得到的mRNA冻干疫苗体外活性效果。
图5显示本发明一具体实施例中所制备得到的mRNA冻干疫苗体内活性效果。
图6显示本发明的mRNA疫苗的特异性抗体滴度测试结果。
图7显示RBD和NTD-RBD抗原的免疫原性对比。
图8显示本发明的NTD-RBD mRNA疫苗对不同毒株的交叉保护效果。
图9显示本发明的NTD-RBD疫苗诱导强烈的细胞免疫。
图10显示本发明的NTD-RBD疫苗对ACE2小鼠体内攻毒实验结果。
图11显示本发明的NTD-RBD疫苗作为加强针的实验结果。
图12为本发明一些具体实施例中的mRNA诱导表达的蛋白相对表达量实验结果。
具体实施方式
通过下面的实施例可以对本发明进行进一步的描述，然而，本发明的范围并不限于下述实施例。本领域的专业人员能够理解，在不背离本发明的精神和范围的前提下，可以对本发明进行各种变化和修饰。本发明对试验中所使用到的材料以及试验方法进行一般性和/或具体的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的，但是本发明仍然在此作尽可能详细描述。未详细注明的方法操作，按照所属领域现有技术的常规操作或商厂说明书建议的操作进行。
实施例1：mRNA疫苗及其制备方法
本实施例提供了mRNA疫苗，其制备方法主要按照以下操作进行。
1、根据待合成的mRNA合成对应的DNA片段，并将所述DNA片段克隆至表达质粒获得重组质粒，将所述重组质粒转入宿主细胞获得重组细胞，从扩繁后的重组细胞中提取质粒。
对构建好的质粒进行酶切线性化，酶切反应体系如下(以酶切2μg的重组质粒为例)：
10×Digestion Buffer I 2μl
BspQI(10U/μl)(购买自诺唯赞)  1μl
Plasmid DNA 2μg
RNase-free Water  补水至20μl
将上述酶切体系置于50℃反应1h。待反应结束后，取酶切反应前后体系各1μl进行DNA琼脂糖凝胶电泳(1.5％琼脂糖凝胶，5V/min，40min)。根据电泳结果比较显示，重组质粒是否酶切完全。
合格标准：电泳检测出现单一条带；与酶切前的超螺旋质粒比较，条带位于超螺旋质粒上方；大小符合预期要求。
测定结果：条带单一；大小符合预期且条带位于超螺旋质粒上方。
2、DNA模板超滤
利用Millipore 30Kd超滤管浓缩上述获得的DNA模板。
3、DNA模板FPLC纯化
将上述超滤得到的DNA浓缩液，加入等体积的苯酚/氯仿/异戊醇混合液(苯酚/氯仿/异戊醇体积比＝25/24/1)，充分震荡后，12000g离心15min。
去掉沉淀，转移上清至新的离心管中，加入上清体积的1/10 3M NaAc(pH5.2)，混匀，然后加入2倍体积的无水乙醇，混匀，至于-20℃静置30min。
4℃，12000g离心10min，弃上清。
用70％乙醇洗涤沉淀，12000g离心5min，取上清，于超净台晾干5min。
用适当的RNase-free水溶解纯化后的DNA模板。
用NanoDrop检测纯化后模板的浓度，以及260/280、260/230的比值。取样进行DNA琼脂糖凝胶电泳检测(1.5％琼脂糖，5V/min，40min)。
合格标准：260/280介于1.8至2.1之间，260/230在1.6至2.2之间。
测定结果：浓度为500ng/μl，260/280＝1.90，260/230＝1.7。
4、FPLC纯化后模板超滤
Millipore 30Kd超滤管浓缩FPLC纯化后的DNA模板，用RNase-free水洗脱溶解。用NanoDrop检测超滤后模板的浓度，以及260/280、260/230的比值。最终用RNase-free水稀释至150ng/μl。
测定结果：浓度为150ng/μl，260/280＝1.95，260/230＝1.85。
5、mRNA的体外合成
在恒温反应器中，进行mRNA的体外合成。
按照如下合成体系进行(反应试剂按照先后顺序添加)：
反应体积，1600μl(置于2ml RNase-free Tube管中，为单个管的反应体积，一次同时反应多管)：RNA-free水440μl、7.5mM的ATP 160μl、7.5mM的N1-甲基-假尿苷160μl、7.5mM的CTP 160μl、7.5mM的GTP 160μl、7.5mM的M7G(2’OMeA)pG160μl、150ng/μl的DNA模板40μl、10×Buffer 160μl和Enzyme Mix 160μl。
所述RNA体外合成的程序为37℃，10h。
经过体外转录和加帽，得到目标mRNA分子。
本发明各具体实验例中，目标mRNA分子除编码序列外，还包括5’UTR(AGGGAGAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCGCCACC，SEQ ID NO:51)和3’UTR(GCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAG，SEQ ID NO:52)，5’CAP为m7G+-5'-ppp-5'-Am2'-3'-p-(cap1)，3’加聚-A尾(SEQ ID NO:53)。
6、DNase I消化去除DNA模板
向mRNA体外合成后的每个Tube管中各加入120μl DNase I。
上下颠倒10次混匀，1000rpm离心10s。
重新置于恒温反应器中，37℃，1h。
反应结束后，将反应液合并到RNase-free 50ml Tube管中，检测DNA片段的残留。三次测量结果为0.013ng，0.016ng，0.017ng每100μg mRNA。
7、mRNA沉淀回收
向上一步骤中的每个50ml Tube管中，加入等体积的醋酸铵溶液。
上下颠倒10次混匀。
置于-20℃2h，沉淀。
17000g，4℃离心，30min。
去掉上清，用70％乙醇洗涤沉淀。
17000g，4℃离心，10min。
去掉70％乙醇，于超净台中蒸干，每管加入RNase-free水20ml。
静置10min后，用枪头轻吹混匀。
用NanoDrop检测回收后的mRNA浓度为5μg/μl，A260/A280为1.90、A260/A230为2.0。
取1μl，稀释10倍，进行RNA ScreenTape assay以及琼脂糖凝胶电泳检测其片段完整性。
检测结果为：条带符合大小，片段完整。
8、LiCl沉淀纯化mRNA
将上一步骤中回收的mRNA按照其1.5倍体积加入Rnase-free水，混匀。
加入原mRNA 1.5倍体积-20预冷的LiCl溶液，混匀。
然后于-20℃静至2h。
16000g离心20min。
弃上清，用70％乙醇洗涤沉淀，16000g离心15min。
取上清，于超净台晾干5min。
用适当的RNase-free水溶解纯化后的mRNA。
纯化后的mRNA用0.1M柠檬酸稀释至2μg/μl。
9、LNP制备
配制水相：将mRNA按照2μg/ul的终浓度稀释于柠檬酸buffer；
按照表1配制乙醇相溶液；
表1
组分 配制1mL所需组分/μL 配制0.5mL所需组分/μL
ALC-0315 559.7 279.8
ALC-0159 117.2 58.6
DSPC 260.5 130.2
Cholesterol 62.7 31.3
准备好PBS作为LNP稀释液；
注射泵仪器操作步骤：
(1).将A相(mRNA缓冲液)装入5mL注射器，B相(脂质化合物溶于无水乙醇) 装入5mL注射器，安装于注射泵，夹紧；
(2).将芯片连接到注射器，设定注射泵流速；
(3).点击注射泵的开始按键，将料液注入芯片；
(4).观察芯片出口的产品颜色，弃去前5滴乳白色液滴(约为100ul)后，开始收集到60mL PBS中；
(5).收集完成的产品，轻柔地上下颠倒混匀，保存于4℃；
(6).取0.3mL作为包封率检测样品；
(7).裂解：取步骤(6)的0.1mL，加入2μL 10％Triton X-100，混匀，室温孵育10min；
(8).包封率检测：分别取64μL步骤(6)和步骤(7)的样品稀释5倍，作为裂解前后的LNP RNA样品；测定RNA浓度；用裂解前后浓度差值除以裂解后浓度，得出包封率；
(9).于马尔文公司的Zetasizer nano仪器上使用标准检测方法进行粒径与PDI检测、Zeta电位分析，上样体积为600μl，样品池为DTS1070，检测温度为25℃。
本实施例所制备得到的负载mRNA的LNP的粒径、PDI和包封率的检测结果如图1、图2所示。图1、图2中，横坐标编号分别对应各mRNA的序列编号，即，图中25对应的样品为SEQ ID NO:25的mRNA样品，图中26对应的样品为SEQ ID NO:26的mRNA样品，图中27对应的样品为SEQ ID NO:27的mRNA样品，以此类推，图中42对应的样品为SEQ ID NO:42的mRNA样品。
10.LNP冻干
LNP溶液加入的冻干保护剂，放入冷冻干燥机，质量体积分数(w/v％)为蔗糖15％、海藻糖2％。(新芝Scientz-10N)冷阱预冻4小时后，开启真空抽真空48小时，随后样品转移到冷冻干燥机上层，二次干燥16小时。在冷阱中干燥时样品温度探头显示-30℃左右，在上层干燥时样品温度探头显示为4℃左右。干燥结束后收集产物(即LNP冻干粉)，使用与冻干前等体积的超纯水复溶，加入超纯水后冻干粉迅速溶解，整个过程不超过20s，用RiboGreenTM测定纳米颗粒包封率，欧美克NS-90Z纳米粒度及电位分析仪测定纳米颗粒粒径及zeta电位。制备得到本实施例的mRNA冻干疫苗。所制备得到的疫苗的成品图及复溶后外观参见图3所示。分别将37#(对应SEQ ID NO:37的mRNA样品)2μg mRNA-LNP溶液、冻干复溶液和GFP mRNA-LNP孵育培养的293T细胞，24小时后取细胞沉淀，裂解细胞，收获可溶性蛋白，进行Westernblot(蛋白免疫印迹)，检测蛋白表达效率，以及将37#(对应SEQ ID NO:37的mRNA样品)100μg mRNA-LNP粉末在注射接种前用200μl注射用水复溶，在第1天和第14天两次注射接种6周龄balb/c 小鼠，在第35天取小鼠血清后检测血清中抗S蛋白特异性抗体的滴度，结果如图4和图5所示，冻干工艺基本不影响mRNA脂质颗粒的生物学活性。
实施例2
本实施例中，分别将25#～42#(即分别对应SEQ ID NO:25～SEQ ID NO:42的mRNA样本)100μg mRNA-LNP粉末在注射接种前用200μl注射用水复溶，在第1天和第14天两次注射接种6周龄balb/c小鼠，在第35天取小鼠血清，检测血清中抗S蛋白特异性抗体的滴度。具体按照以下操作进行：
1、包被：将S1蛋白(义翘神州，40591-MM43)用coating buffer稀释成200ng/ml的溶液，加入到酶标板中，每孔加入的体积为100μl，每个稀释度重复3个孔，盖上封板膜，4℃过夜包被。
2、洗板：包被好的96孔板，倒出包被液，到扣吸水纸上，使劲扣板，直到孔中无残留为止。配制洗脱液，用去离子水稀释50x的Washing buffer，加入到洗板机的进液瓶中，设置程序，每孔洗板体积设为300μl，重复洗4次。
3、封闭：洗好的板，扣干里面的溶液，按每孔250μl的体积加入Blocking buffer，随后封上封板膜，室温封闭2h。
4、洗板：封闭好的酶标板按照步骤2完成洗板。
5、血清孵育：用dilution buffer将小鼠血清稀释成40x，400x，4000x，40000x，400000x，4000000x，40000000x，按照每孔100μl的体积加入洗好的96孔板中，随后封上封板膜室温下孵育1.5h。
6、洗板：按照步骤2完成洗板，其中洗板次数增加为6次。
7、加入二抗：用Dilution buffer稀释HRP标记山羊抗小鼠IgG，稀释倍数为10000x，稀释好的抗体按照每孔100μl的体积加入酶标板中，封上封板膜，室温下避光孵育1h。
8、洗板：按照步骤2完成洗板，此步骤务必洗干净板，扣干溶液。
9、显色：加入TMB buffer 100μl，避光显色20-30分钟，此时阳性样品显蓝色。
10、终止：加入Stop buffer 100μl，10分钟内在酶标板上读数，设置吸收波长450nm。
根据标准品孔光吸收值绘制标准曲线，线性回归的相关系数应大于0.0995。根据供试品吸光度的大小计算样品中S蛋白特异性抗体的残留含量。
特异性抗体滴度测试结果参见图6所示。结果显示，编码NTD-RBD的mRNA候选疫苗免疫效果具有良好的免疫原性，诱导产生了针对S蛋白的特异性抗体，对照组注射生理盐水的小鼠不能产生针对S蛋白的特异性抗体。
实施例3
本实施例中，通过实验考察了编码RBD的mRNA疫苗(43#，即对应SEQ ID NO:43的样本)和编码NTD-RBD疫苗(37#，即对应SEQ ID NO:37的样本)的中抗体滴度。具体按照以下操作进行：
接种疫苗，分别在0d、14d时，对6～8周龄的雌性小鼠接种5μg新型冠状病毒mRNA疫苗(溶解于PBS，200μl，肌肉注射)；28d时取小鼠外周血。
准备Vero E6细胞(24孔板)，血清热灭活，连续3倍梯度稀释。100μl血清与100μl病毒储存液(100PFU)等体积混匀，同时100μl DMEM(含2％FBS)与100μl病毒液等体积混匀，作为阴性对照。37℃孵育1h。
将上述血清-病毒混合液(共200μl)转移至24孔板Vero E6细胞，吸附1h。期间可轻柔混匀3-4次。移除上一步的吸附液，更换甲基纤维素培养基，培养3天。多聚甲醛固定、结晶紫染色，空斑计数。以阴性对照为基准，计算血清中和百分比。以JMP分析软件Probit方法进行拟合曲线，计算PRNT 50值。
实验结果如图7所示，NTD-RBD诱导的中和抗体滴度高于RBD，因此NTD-RBD的免疫原性由于RBD。
实施例4
本实施例中，通过实验考察了NTD-RBD mRNA疫苗对2019-nCoV假病毒的交叉免疫保护。具体按照以下操作进行：
接种疫苗，分别在0d、14d时，对6～8周龄的雌性小鼠接种5μg新型冠状病毒mRNA疫苗(溶解于PBS，200μl，肌肉注射)；28d时取小鼠外周血，采用微量中和实验对热灭活血清进行测定，以检测抗体中和新型冠状假病毒侵染表达ACE2单层细胞的水平高低；于96孔板上稀释4孔，在第3天和第4天时检测细胞的病毒性病变作用(cpe)，通过Reed Muench公式计算出完全抑制cpe的血清在50％终点法中测定的血清稀释度；采用非参数双尾t检测(Mann-Whiteny)进行统计分析。
实验结果如图8所示(图中各图案的数字编号分别为对应相同SEQ ID NO编号的样本)，本发明所构建的mRNA疫苗对新型冠状病毒突变株均有不同程度的交叉保护效果。
实施例5：NTD-RBD新型冠状疫苗诱导强烈的细胞免疫
本实施例中，考察了NTD-RBD新型冠状疫苗诱导细胞免疫的作用。具体按照以下操作进行：
一、包被96孔板
用70％乙醇浸润96孔板中的PVDF膜30s。
加入捕获抗体(PBS稀释)，4℃过夜。
倒空板中包被液，轻轻在纸上拍干，用PBS洗涤，禁用洗板机。
加入100ul 2％的脱脂奶粉(或者BSA)室温孵育2小时，封闭板中空白位点。
PBS洗涤1次。
二、细胞刺激和细胞因子捕获
接种疫苗，分别在0d、14d时，对6～8周龄的雌性小鼠接种5μg新型冠状病毒mRNA疫苗RH109(40#)(溶解于PBS，200μl，肌肉注射)；28d时取小鼠外周血，从新鲜血液中用Ficoll分离PBMC并进行计后，将细胞用培养液稀释后加入96孔板中。通常所用细胞数量为1-2×10 5/孔。
将96孔板在37℃CO 2孵箱中孵育过夜。禁止移动或晃动板子。
培养板中加入S蛋白pepmix刺激物孵育8小时；
用含0.1％Tween 20的PBS孵育10分钟，去除细胞和未结合的细胞因子。然后用含0.1％Tween 20的PBS洗涤3次。
三、加入检测抗体检测
加入标记的检测抗体(用含1％BSA的PBS稀释)，室温孵育1-2小时。
加入底物显色，在加入底物之前用蒸馏水洗涤板子膜的两侧，以防止部分溶液漏出后导致的背景。监测斑点的形成，适时终止反应。
用蒸馏水洗涤终止反应。
四、结果分析
将96孔板干燥。(将板子放在4℃避光过夜，可使斑点边缘锐化，更易分辨)
使用读板仪进行结果分析，结果如图9所示，ELISPot实验所示，多肽刺激后，疫苗组小鼠能够产生大量的IFN-γ斑点，达到3000spots/100万细胞，而生理盐水组几乎没有斑点产生，说明RH109激发了强细胞免疫反应。
用流式细胞术胞内染色实验对CD4和CD8细胞进行进一步分析，发现IL2 +/TNF-α +/IFN-γ +的CD4 +细胞，以及TNF-α +/IFN-γ +的CD8 +细胞明显升高，说明RH109疫苗激活了强Th1型和CD8杀伤性T细胞免疫反应。
实施例6
接种疫苗，分别在0d、21d时，对6～8周龄的雌性小鼠接种5μg或10ug新型冠状 病毒mRNA疫苗(37#)(溶解于PBS，200μl，肌肉注射)；ivD为攻毒期，ivD0为攻毒当天，新型冠状病毒(Delta株)滴鼻感染hACE2转基因小鼠，感染剂量初步为105PFU。D42为攻毒时间。攻毒后ivD3和ivD5，分批将动物安乐死，动物安乐死后取肺(右肺)等组织器官，提取RNA，用Q-PCR法测定病毒载量或检测活病毒滴度。期间每天两次观察，每周称量一次体重。病毒载量及生存曲线如图10所示。由图10可知，接种疫苗后的小鼠在进行真病毒攻毒实验后，肺部病毒载量显著低于对照组。佐剂对照组小鼠于攻毒后第7天均达到安乐死标准。与佐剂对照组相比，疫苗低剂量组和疫苗高剂量组的小鼠全部存活，表明疫苗对SARS-CoV-2病毒Delta毒株感染小鼠有显著的保护作用。
实施例7
接种疫苗，分别在0d、21d时，对6～8周龄的雌性小鼠接种两针科兴灭活疫苗，42d时接种5μg新型冠状病毒mRNA疫苗RH109(溶解于PBS，200μl，肌肉注射)；56d时取小鼠外周血，采用微量中和实验对热灭活血清进行测定，以检测抗体中和新型冠状假病毒侵染表达ACE2单层细胞的水平高低；于96孔板上稀释4孔，在第3天和第4天时检测细胞的病毒性病变作用(cpe)，通过Reed Muench公式计算出完全抑制cpe的血清在50％终点法中测定的血清稀释度；采用非参数双尾t检测(Mann-Whiteny)进行统计分析。如图11所示，RH109免疫后的血清对Omicron，Delta,原始毒株均产生了高中和抗体水平，几何平均滴度分别是7970、9091、13998，其中针对Omicron比第二针后提高了39倍数；同时，相比于用科兴灭活苗作为第三针加强，对Omicron、Detla和原始毒株分别是后者的倍35、6.2、3.4倍，说明RH109非常适合作为灭活苗基础上的加强针使用。另，本发明还比较了本发明的不同mRNA不同剂量诱导表达蛋白实验，分别将SEQ ID NO:28、SEQ ID NO:54、SEQ ID NO:55的mRNA-LNP溶液以2μg、4μg孵育培养的293T细胞，24小时后取细胞沉淀，裂解细胞，平行检测得到的目的蛋白条带进行灰度分析24h胞内蛋白相对表达量。结果参见图12(图中编号28-1对应SEQ ID NO:28、编号30-1对应SEQ ID NO:54、编号30-3对应SEQ ID NO:55)。本发明的mRNA具有良好的蛋白表达效率。
应当指出的是，以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (31)

  1. 一种能编码目标多肽的mRNA分子，其中，所述的目标多肽包括SARS-CoV-2刺突蛋白S中NTD-RBD天然结构域，所述NTD-RBD天然结构域包括NTD片段与RBD片段，NTD片段与RBD片段之间以来源于S蛋白的天然氨基酸序列作为连接子连接。
  2. 根据权利要求1所述的mRNA分子，该mRNA还编码NTD-RBD天然结构域N端的信号肽。
  3. 根据权利要求1或2所述的mRNA分子，该mRNA分子编码的氨基酸序列从N端到C端依次包括信号肽、NTD片段、连接子、RBD片段。
  4. 根据权利要求1至3任意一项所述的mRNA分子，其中，所述的连接子的氨基酸序列为SEQ ID NO:50。
  5. 根据权利要求1至4中任意一项所述的mRNA分子，其中，NTD片段的氨基酸序列选自：
    (a)SEQ ID NO:49的第1至第289位组成的氨基酸序列；
    (b)由(a)的氨基酸序列经过替换、增加和/或缺失一个或几个氨基酸且具有与
    (a)相同功能的衍生序列。
  6. 根据权利要求1至5中任意一项所述的mRNA分子，其中，RBD片段的氨基酸序列选自：
    (c)SEQ ID NO:49的第304至第526位组成的氨基酸序列；
    (d)由(c)的氨基酸序列经过替换、增加和/或缺失一个或几个氨基酸且具有与
    (a)相同功能的衍生序列。
  7. 根据权利要求1至6中任意一项所述的mRNA分子，其中，所述的NTD-RBD天然结构域的氨基酸序列为：
    SEQ ID NO:44至SEQ ID NO:49中任一序列所示氨基酸序列；或
    与SEQ ID NO:44至SEQ ID NO:49中任一序列同一性在94.3％以上的衍生的氨基酸序列。
  8. 根据权利要求7所述的mRNA分子，其中，所述的NTD-RBD天然结构域的氨基酸序列为SEQ ID NO:49、SEQ ID NO:48、SEQ ID NO:47、SEQ ID NO:46、SEQ ID NO:45、SEQ ID NO:44中的一种。
  9. 根据权利要求3至7中任意一项所述的mRNA分子，其中，所述的mRNA编码的氨基酸序列为SEQ ID NO:21、SEQ ID NO:17、SEQ ID NO:13、SEQ ID NO:9、SEQ  ID NO:5或SEQ ID NO:1。
  10. 根据权利要求1至9中任意一项所述的mRNA分子，其蛋白质编码区序列如SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41或SEQ ID NO:42所示。
  11. 根据权利要求1至10中任意一项所述的mRNA分子，其中，该mRNA经过1-甲基假尿苷修饰。
  12. 根据权利要求1至11中任意一项所述的mRNA分子，其中，该mRNA还包括5’-UTR序列和/或3’-UTR序列。
  13. 根据权利要求12所述的mRNA分子，其中，5’-UTR序列包含或不包含Kozak序列；
    优选地，所述5'-UTR具有SEQ ID NO:51所示序列。
  14. 根据权利要求12所述的mRNA分子，其中，3’-UTR序列具有SEQ ID NO:52所示序列。
  15. 根据权利要求1至14中任意一项所述的mRNA分子，其中，该mRNA还经过3'加尾修饰和/或至少一个5'加帽修饰；
    优选地，3'加尾修饰包含聚-A尾，所述聚-A尾为中间插入或不插入连接子的多聚腺苷酸；
    优选地，至少一个5'加帽修饰的帽结构选自Cap0、Cap1、ARCA、肌苷、N1-甲基-鸟苷、2'氟-鸟苷、7-脱氮-鸟苷、8-氧代-鸟苷、2-氨基-鸟苷、LNA-鸟苷或2-叠氮基-鸟苷。
  16. 根据权利要求1至15中任一项所述的mRNA分子，其是分离的mRNA；
    优选地，所述mRNA分子是经纯化的。
  17. 一种DNA分子，其编码权利要求1至16中任意一项所述的mRNA分子。
  18. 一种重组质粒，其含有权利要求17所述的DNA分子。
  19. 一种脂质纳米颗粒，其负载有权利要求1至16中任意一项所述的mRNA分子。
  20. 一种药物组合物，其包含：权利要求1至16中任一项所述的mRNA分子，以及药学上可接受的赋形剂。
  21. 权利要求1至16中任意一项所述的mRNA分子、权利要求17所述的DNA分 子、权利要求18所述的重组质粒、权利要求19所述的脂质纳米颗粒或权利要求20所述的药物组合物在制备新型冠状病毒mRNA疫苗中的应用。
  22. 一种新型冠状病毒mRNA疫苗，其包含权利要求1至16中任意一项所述的mRNA分子，所述的疫苗为脂质纳米颗粒剂型。
  23. 根据权利要求22所述的新型冠状病毒mRNA疫苗，其中，所述的脂质纳米颗粒包括mRNA和脂质，其中，所述脂质包括：
    a)正电荷脂质和/或可离子化脂质中的一种或多种；
    b)中性辅助脂质；
    c)胆固醇；
    d)PEG修饰的脂质。
  24. 根据权利要求23所述的新型冠状病毒mRNA疫苗，其中，所述的正电荷脂质和/或可离子化脂质与mRNA的氮磷摩尔比为5:1～20:1。
  25. 根据权利要求23或24所述的新型冠状病毒mRNA疫苗，其中，所述的脂质纳米颗粒中，以脂质的总摩尔量为100％计，各脂质成分的摩尔比为：
    正电荷脂质或可离子化脂质  46％～50％；
    中性辅助脂质  5％～10％；
    胆固醇  38.5％～48％；
    PEG修饰的脂质  0～3％。
  26. 根据权利要求22至25中任意一项所述的新型冠状病毒mRNA疫苗，其中，所述的可离子化脂质包括但不限于：4-(N,N-二甲基氨基)丁酸(二亚油基)甲酯、SM-102、((4-羟基丁基)氮杂二烷基)双(己烷-6,1-二基)双(2-己基癸酸酯)中的一种或多种，所述的正电荷脂质包括但不限于：DOTMA、DOTAP中的一种或多种；所述的中性辅助脂质包括但不限于：DSPC、DOPE、DSPE中的一种或多种；所述的PEG修饰的脂质包括但不限于：甲氧基聚乙二醇双十四烷基乙酰胺、DMG-PEG中的一种或多种。
  27. 根据权利要求22至25中任意一项所述的新型冠状病毒mRNA疫苗，其中，疫苗剂型为冷冻干燥剂型或冷冻剂型。
  28. 一种新型冠状病毒mRNA疫苗的制备方法，其包括以下步骤：
    合成编码NTD-RBD天然结构域肽段的DNA片段，并克隆到质粒作为模板，转录制备得到目标mRNA分子；
    优选地，所述目标mRNA分子为权利要求1至16中任意一项所述的mRNA分子。
  29. 根据权利要求28所述的制备方法，该方法还包括：
    将所制备的mRNA分子溶解在柠檬酸缓冲液组成的水相中，采用冲击式射流或微流控等方法与溶解在乙醇相中的脂质成分混合，制备负载有mRNA的脂质纳米颗粒。
  30. 根据权利要求29所述的制备方法，该方法还包括将所制备的脂质纳米颗粒制成冷冻制剂或冷冻干燥制剂的过程。
  31. 根据权利要求30所述的制备方法，其中，将脂质纳米颗粒制成冷冻干燥制剂的过程包括：
    a)配制含有脂质纳米颗粒和冷冻干燥保护剂的缓冲液；
    b)降温进行预冻；
    c)在真空条件下升温进行干燥，使体系含水量在3％以下，制备得到脂质纳米颗粒的干燥制剂。
PCT/CN2022/090196 2022-01-27 2022-04-29 一种新型冠状病毒mRNA疫苗及其制备方法与应用 WO2023142283A1 (zh)

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