WO2024021817A1 - 新型冠状病毒疫苗及其制备方法和应用 - Google Patents

新型冠状病毒疫苗及其制备方法和应用 Download PDF

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WO2024021817A1
WO2024021817A1 PCT/CN2023/096148 CN2023096148W WO2024021817A1 WO 2024021817 A1 WO2024021817 A1 WO 2024021817A1 CN 2023096148 W CN2023096148 W CN 2023096148W WO 2024021817 A1 WO2024021817 A1 WO 2024021817A1
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seq
reading frame
open reading
nucleic acid
acid molecule
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PCT/CN2023/096148
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English (en)
French (fr)
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彭育才
刘隽
刘琪
雷奕欣
李爽
罗丽平
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珠海丽凡达生物技术有限公司
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Priority claimed from CN202210892843.1A external-priority patent/CN117467677A/zh
Priority claimed from CN202211207683.9A external-priority patent/CN115716866A/zh
Application filed by 珠海丽凡达生物技术有限公司 filed Critical 珠海丽凡达生物技术有限公司
Publication of WO2024021817A1 publication Critical patent/WO2024021817A1/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
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/295—Polyvalent viral antigens; Mixtures of viral and bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus

Definitions

  • the present disclosure relates to the field of vaccine technology, and in particular to a novel coronavirus vaccine and its preparation method and application.
  • the new coronavirus epidemic has caused great social and economic losses around the world.
  • the new coronavirus is extremely easy to mutate. Since its discovery, the original strain of the new coronavirus, the new coronavirus Alpha strain, the new coronavirus Beta strain, the new coronavirus Gamma mutant strain, the new coronavirus Viruses such as the Kappa strain of the virus, the Delta strain of the new coronavirus, and the Omicron strain of the new coronavirus have appeared one after another.
  • the Omicron variant has at least 60 new mutation points, including more than 35 mutations in the spike protein (S protein), and the spike protein (S protein) It carries 15 mutations in the most critical receptor-binding domain.
  • the Delta variant has only 2 mutations in this region. According to the difference in mutation sites, the Omicron mutant strain can be divided into at least 5 sub-mutant strains, namely BA.1, BA.2.12.1, BA.2, BA.4 and BA.5.
  • a novel coronavirus vaccine comprising a nucleic acid molecule containing a first open reading frame and a second open reading frame;
  • the first open reading frame encodes the S protein of the new coronavirus Delta variant strain; the second open reading frame encodes the BA.5S protein of the new coronavirus Omikrong variant strain.
  • the novel coronavirus vaccine includes: a nucleic acid molecule containing a first open reading frame; and, a nucleic acid molecule containing a second open reading frame;
  • the novel coronavirus vaccine includes: a fusion nucleic acid molecule containing both a first open reading frame and a second open reading frame.
  • the amino acid sequence of the S protein of the new coronavirus Delta variant strain is shown in SEQ ID.NO.8, or includes an amino acid sequence that is at least 80% identical to SEQ ID.NO.8, such as It may be, but is not limited to, an amino acid sequence comprising at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID.NO.8.
  • the amino acid sequence of the BA.5S protein of the new coronavirus Omicron variant is as shown in SEQ ID.NO.26, or contains at least 80% of the same as SEQ ID.NO.26
  • the amino acid sequence of identity may, for example, be, but is not limited to, an amino acid sequence that contains at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID. NO. 26.
  • the nucleic acid molecules include DNA molecules and/or RNA molecules.
  • the DNA molecules include chain DNA molecules and/or circular DNA molecules.
  • the RNA molecule includes mRNA or circular RNA.
  • the nucleic acid molecule is RNA.
  • the total GC% content of the open reading frame in the RNA is 30 to 70%, and the GC% content of any 60 bp fragment of the open reading frame is not less than 40%. .
  • the total GC% content of the portion of the open reading frame in the RNA is 50% to 60%, more preferably 54% to 60%.
  • the RNA further includes one or more of a 5' cap, 5' UTR, 3' UTR, polyA tail, initiation region, termination region, signal sequence region and linker sequence.
  • the nucleotide sequence of the first open reading frame is such as SEQ ID.NO.9, SEQ ID.NO.36, SEQ ID.NO.37, SEQ ID.NO.38, SEQ ID.NO.39, SEQ ID.NO.40, SEQ ID.NO.41, SEQ ID.NO.42 or SEQ ID.NO.29.
  • the nucleotide sequence of the second open reading frame is such as SEQ ID.NO.27, SEQ ID.NO.32, SEQ ID.NO.20, SEQ ID.NO.21, SEQ ID.NO.22, SEQ ID.NO.23, SEQ ID.NO.24, SEQ ID.NO.25, SEQ ID.NO.28 are shown.
  • the combination of the first open reading frame and the second open reading frame is selected from: SEQ ID.NO.9 and SEQ ID.NO.27, SEQ ID.NO.9 and SEQ ID. NO.32; SEQ ID.NO.36 and SEQ ID.NO.32, SEQ ID.NO.37 and SEQ ID.NO.20, SEQ ID.NO.38 and SEQ ID.NO.21, SEQ ID.NO .39 and SEQ ID.NO.22, SEQ ID.NO.40 and SEQ ID.NO.23, SEQ ID.NO.41 and SEQ ID.NO.24, SEQ ID.NO.42 and SEQ ID.NO. 25.
  • the mass ratio of the nucleic acid molecule encoding the first open reading frame to the nucleic acid molecule encoding the second open reading frame is (1:9) to (9:1);
  • the mass ratio of the nucleic acid molecule of the open reading frame to the nucleic acid molecule encoding the second open reading frame is (1:9), (1:8), (1:7), (1:6), (1:5) , (1:4), (1:3), (1:2), (1:1), (2:1), (3:1), (4:1), (5:1), ( 6:1), (7:1), (8:1), (9:1); in one or more embodiments, a nucleic acid molecule encoding a first open reading frame and a nucleic acid molecule encoding a second open reading frame The mass ratio of nucleic acid molecules is 3:1.
  • the nucleic acid molecule is a fusion nucleic acid molecule, and the number of repetitions of the first open reading frame and the second open reading frame in the fusion nucleic acid molecule is (1:9) to (9:1 ); the repeat numbers of the first open reading frame and the second open reading frame in the fusion nucleic acid molecule are (1:9), (1:8), (1:7), (1:6), (1 :5), (1:4), (1:3), (1:2), (1:1), (2:1), (3:1), (4:1), (5:1 ), (6:1), (7:1), (8:1), (9:1); in one or more embodiments, the first open reading frame and the second open reading frame are fused in the The number of repeats in a nucleic acid molecule is 3:1.
  • RNA sequence based on the provided RNA sequence, one of ordinary skill in the art will be able to obtain the corresponding DNA sequence (eg, uracil to thymine conversion). Likewise, based on the provided DNA sequence, one of ordinary skill in the art will derive the corresponding RNA sequence (eg, thymine to uracil). In alternative embodiments, based on the provided RNA or DNA sequence, one of ordinary skill in the art will be able to obtain the corresponding amino acid sequence.
  • the vaccine further includes a delivery formulation.
  • the novel coronavirus vaccine contains nucleic acid lipid nanoparticles composed of the nucleic acid molecules and lipid components.
  • the novel coronavirus vaccine is selected from (a), (b) or (c):
  • the novel coronavirus vaccine includes: nucleic acid lipid nanoparticles wrapped with a nucleic acid molecule containing a first open reading frame, and nucleic acid lipid nanoparticles wrapped with a nucleic acid molecule containing a second open reading frame;
  • the novel coronavirus vaccine includes: nucleic acid lipid nanoparticles wrapped with a nucleic acid molecule containing a first open reading frame and a nucleic acid molecule containing a second open reading frame;
  • the novel coronavirus vaccine includes: nucleic acid lipid nanoparticles wrapped with a fusion nucleic acid molecule containing both a first open reading frame and a second open reading frame.
  • the present disclosure also provides a method for preparing the above-mentioned novel coronavirus vaccine.
  • the preparation method includes mixing the nucleic acid molecule and optional auxiliary materials to obtain the novel coronavirus vaccine.
  • the novel coronavirus vaccine includes nucleic acid lipid nanoparticles, and the preparation method includes:
  • Nucleic acid lipid nanoparticles coated with nucleic acid molecules containing the first open reading frame and nucleic acid lipid nanoparticles coated with nucleic acid molecules containing the second open reading frame are prepared respectively; and then the two nucleic acid lipids are mixed according to the formula amount. Nanoparticle blending;
  • nucleic acid lipid nanoparticles wrapped with the two nucleic acid molecules first mix the nucleic acid molecule containing the first open reading frame and the nucleic acid molecule containing the second open reading frame according to the formula amount, and then prepare nucleic acid lipid nanoparticles wrapped with the two nucleic acid molecules;
  • nucleic acid lipid nanoparticles coated with a nucleic acid molecule containing both a first open reading frame and a second open reading frame are prepared.
  • the present disclosure also provides the above-mentioned novel coronavirus vaccine, or the application of the above-mentioned preparation method in preparing products configured to prevent or treat diseases caused by the novel coronavirus.
  • the present disclosure also provides a product configured to prevent or treat diseases caused by the new coronavirus, the product comprising the above-mentioned new coronavirus vaccine.
  • Figure 1-1 shows the pseudovirus neutralizing activity test results of the serum produced after immunizing cynomolgus monkeys with each vaccine preparation in Example 4 against the new coronavirus.
  • Figure 1-2 shows the pseudovirus neutralizing activity test results of the serum produced after immunizing cynomolgus monkeys with the vaccine preparation in Example 4 against the novel coronavirus.
  • Figure 2 shows the pseudovirus neutralizing activity test results of the serum produced after immunizing C57 mice with each vaccine preparation in Example 5 against the novel coronavirus.
  • Figure 3 shows the test results of the pseudovirus neutralizing activity of the serum produced after immunizing C57 mice with each vaccine preparation in Example 6 against the new coronavirus.
  • Figure 4 shows the pseudovirus neutralizing activity test results of the serum produced after immunizing C57 mice with each vaccine preparation in Example 8 against the novel coronavirus.
  • Figure 5 shows the test results of the pseudovirus neutralizing activity of the serum produced after immunizing C57 mice with each vaccine preparation in Example 9 against the new coronavirus.
  • Figure 6 shows the pseudovirus neutralizing activity test results of the serum produced after immunizing C57 mice with each vaccine preparation in Example 10 against the new coronavirus.
  • Figure 7 shows the test results of the pseudovirus neutralizing activity of the serum produced after immunizing C57 mice with each vaccine preparation in Example 11 against the new coronavirus.
  • Figure 8 shows the pseudovirus neutralizing activity test results of the serum produced after immunizing C57 mice with each vaccine preparation in Example 12 against the new coronavirus.
  • Figure 9 shows the test results of the pseudovirus neutralizing activity of the serum produced after immunizing C57 mice with each vaccine preparation in Example 13 against the new coronavirus.
  • Figure 10 shows the pseudovirus neutralizing activity test results of the serum produced after immunizing C57 mice with each vaccine preparation in Example 14 against the new coronavirus.
  • Figure 11 shows the true virus cross-neutralizing activity test results of SPF mice immunized with each vaccine preparation in Example 15 against the original strain and the currently circulating virus strain.
  • the "range” disclosed in this disclosure is in the form of a lower limit and an upper limit, which may be one or more lower limits and one or more upper limits respectively; unless otherwise stated, the operating steps may be performed in sequence or not in sequence.
  • the present disclosure provides a new coronavirus vaccine, which mainly uses nucleic acid molecules as immunogenic substances, including a first open reading frame and a second open reading frame.
  • Nucleic acid molecule in which the first open reading frame encodes the S protein of the new coronavirus Delta variant strain, and the second open reading frame encodes the BA.5S protein of the new coronavirus Omicron sub-variant strain.
  • the new coronavirus vaccine immunizes the body, it can express the new coronavirus Delta variant strain and Omicron variant BA.5S protein in the body, making it a multivalent vaccine.
  • nucleic acid molecule refers to a polymeric form of nucleotides of any length, including ribonucleotides and/or deoxyribonucleotides.
  • nucleic acids include, but are not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA; vector DNA integrating exogenous genes, such as expression cassettes or plasmids; DNA-RNA hybrids or containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived polymers of nucleotide bases.
  • the first open reading frame and the second open reading frame may exist in the same nucleic acid molecule or in different nucleic acid molecules. Nucleic acid molecules. Therefore, as long as the nucleic acid molecule contained in the new coronavirus vaccine contains the first open reading frame and the second open reading frame, it can produce the new coronavirus Delta variant strain and the Omicron variant BA.5S in the body after administration to the body.
  • the protein belongs to the new coronavirus vaccine provided by this disclosure.
  • the novel coronavirus vaccine includes: a nucleic acid molecule containing a first open reading frame; and, a nucleic acid molecule containing a second open reading frame;
  • the novel coronavirus vaccine includes: a fusion nucleic acid molecule containing both a first open reading frame and a second open reading frame.
  • This disclosure has discovered through experiments that after immunizing animals with nucleic acid molecules encoding the S protein of the new coronavirus Delta variant strain, the antibodies produced in the animal serum are resistant to the original strain, Alpha variant strain, Beta variant strain, Gamma variant strain, and Delta variant strain of the new coronavirus.
  • the new coronavirus vaccine contains nucleic acid molecules that can encode the S protein of the Delta variant strain and the S protein of the Omicron variant strain, and can have both protection against the new coronavirus The preventive effect of various epidemic strains.
  • the S protein of the new coronavirus encoded by the open reading frame can optionally be the S protein obtained by mutation of the Delta mutant strain and/or the Omicron mutant strain under natural conditions; it can also be optional
  • the ground is an S protein that has been artificially mutated and modified.
  • the mutation and modification can be wild-type mutation and modification to obtain an amino acid sequence of the S protein consistent with the Delta variant or the Omicron variant; or, it can also be in the Delta Based on the amino acid sequence of the S protein of the mutant strain and the Omicron mutant strain, the amino acid sequence of the S protein was obtained by further mutation and transformation.
  • amino acid sequence of the S protein of the novel coronavirus encoded by the open reading frame is as follows:
  • the amino acid sequence of the S protein of the new coronavirus Delta variant strain is preferably as shown in SEQ ID. NO. 8, or contains an amino acid sequence that is at least 80% identical to SEQ ID NO. ID NO.8 is an amino acid sequence of at least 80%, 85%, 90%, 95% or 98% identity; the amino acid sequence of the S protein of the new coronavirus Omicron variant is derived from the BA.5 sub-variant strain, BA .2 S protein of the sub-variant strain, BA.3 sub-variant strain or S protein of the BA.5 sub-variant strain; or, the amino acid sequence of the S protein of the Omicron variant strain is optionally derived from wild-type S protein The amino acid sequence obtained by mutating a protein.
  • the novel coronavirus Omicron variant is the S protein of the BA.2 sub-variant strain or the BA.1 sub-variant strain or the BA.5 sub-variant strain; more preferably, it is BA. 5 mutant strains.
  • the amino acid sequence of the S protein of the new coronavirus Omicron variant strain BA.1 sub-variant strain is preferably as shown in SEQ ID.NO.16, or contains an amino acid sequence that is at least 80% identical to SEQ ID NO.16, for example It can be, but is not limited to, an amino acid sequence that contains at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID NO. 16.
  • the amino acid sequence of the S protein of the new coronavirus Omicron variant strain BA.2 sub-variant strain is preferably as shown in SEQ ID.NO.14, or contains an amino acid sequence that is at least 80% identical to SEQ ID NO.14, for example It may be, but is not limited to, an amino acid sequence that contains at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID NO. 14.
  • the amino acid sequence of the S protein of the new coronavirus Omicron variant strain BA.5 sub-variant strain is preferably as shown in SEQ ID.NO.26 or contains an amino acid sequence that is at least 80% identical to SEQ ID NO.26, for example Be, but are not limited to, comprise an amino acid sequence that is at least 80%, 85%, 90%, 95% or 98% identical to SEQ ID NO. 26.
  • Sequence identity between two nucleotide sequences indicates the percentage of identical nucleotides between the sequences.
  • Sequence identity between two amino acid sequences indicates the percentage of identical amino acids between the sequences.
  • % identity refers to the percentage of identical nucleotides or amino acids between the sequences to be compared, under optimal alignment. This percentage is purely statistical, and the differences between the two sequences may, but need not, be randomly distributed over the entire length of the sequences to be compared. Comparison of two sequences is usually performed by comparing the sequences against fragments or "comparison windows" after optimal alignment to identify local regions of corresponding sequences.
  • SEQ ID.NO.26 is the S protein of the Omicron BA.5 strain that has undergone SN mutation (with two proline residue substitutions at positions 981 and 982 of the amino acid sequence of the full-length S protein), which has higher expression quantity.
  • the position of the amino acid sequence described in this disclosure is based on the full-length amino acid sequence of the wild-type S protein of the original strain of the new coronavirus.
  • the novel coronavirus vaccine provided by this disclosure uses nucleic acid molecules as the main functional component. After the novel coronavirus vaccine is administered to the body, it expresses in the body the S protein of the novel coronavirus Delta variant strain and the virus Omicron strain. In order to further improve the immune effect of the vaccine, the present disclosure also optimizes the nucleic acid molecules, the ratio of two nucleic acids encoding S protein, and the vaccine preparation.
  • the nucleic acid molecule can optimize the mRNA sequence through sequence optimization means to improve properties related to expression efficacy after in vivo administration: for example, improving mRNA stability, increasing translation efficacy in target tissues, and reducing expression interception.
  • sequence optimization means to improve properties related to expression efficacy after in vivo administration: for example, improving mRNA stability, increasing translation efficacy in target tissues, and reducing expression interception.
  • Goals of sequence optimization also include: optimizing the formulation and delivery characteristics of nucleic acid-based therapeutics while maintaining structural and functional integrity; overcoming expression thresholds; increasing expression rates; half-life and/or protein concentration; optimizing protein localization; and avoiding Adverse biological responses such as immune responses and/or degradation pathways.
  • Sequence optimization methods include: (1) Codon optimization based on codon frequencies in specific organs and/or host organisms to ensure proper folding and proper expression; (2) Adjustment of G/C content to increase mRNA stability or reduce diodes hierarchical structure; (3) Minimize tandemly repeated codons or baseruns that may impair gene construction or expression; (4) Customize transcription and translation control regions; (5) Reduce or eliminate problems within polynucleotides secondary structure.
  • the nucleic acid molecule contained in the new coronavirus vaccine is preferably RNA, and the new coronavirus vaccine is preferably an RNA vaccine.
  • the total GC% content of the open reading frame part in the RNA is 30-70%, for example, it can be but is not limited to 30%, 35%, 40%, 45%, 50%, 55 %, 60%, 65% or 70%, or the range value between any two points thereof, preferably 50% to 60%, more preferably 54% to 60%.
  • the GC% content of any 60 bp fragment in the open reading frame part is not less than 40%.
  • the RNA also includes one or more of a 5' cap, 5'UTR, 3'UTR, polyA tail, initiation region, termination region, signal sequence region and linker sequence;
  • the preferred structure of RNA in the new coronavirus vaccine is as follows:
  • the RNA includes in sequence from the 5' end to the 3' end: 5' cap - 5' UTR - first open reading frame and/or second open reading frame - 3' UTR - 3' polyA tail; including the expressions in sequence
  • the RNA contains the above-mentioned fragments in sequence from the 5' end to the 3' end, and each fragment may or may not contain at least one ribonucleotide or functional nucleic acid fragment.
  • the RNA contains both the first open reading frame and the second open reading frame, which is a fusion RNA.
  • the structure of the fusion RNA is preferably as follows:
  • An alternative example structure is as follows: 5’ cap-5’UTR-starting region-first open reading frame-linker sequence-second open reading frame-3’UTR-terminating region-3’polyA tail;
  • An optional example structure is as follows: 5' cap-5'UTR-starting region-(first coding region-linker sequence)n-(linker sequence-second coding region)m-3'UTR-termination region- 3'polyA tail;
  • n is: the number of repetitions of the fragment "first coding region-linker sequence”
  • m is: the number of repetitions of the fragment "linker sequence-second coding region”
  • n and m are independently positive integers.
  • the encoding can be adjusted by adjusting the number of repeats of the fragment "first coding region-linker sequence” and the fragment "linker sequence-second coding region", that is, the values of n and m, or adjusting the ratio of n and m.
  • the 5' cap structure is used to increase the stability of mRNA and prevent the mRNA from being degraded by exonucleases.
  • the 5' cap structure modification group is selected from ARCA, m7G(5"")ppp(5"")(2""OMeA)pG , m7G(5"")ppp(5"")(2""OMeG)pG, m7(3""OMeG)(5"")ppp(5"")(2""OMeG)pG, m7(3 ""OMeG)(5"”)ppp(5"")(2"”OMeA)pG, mCAP, dmCAP, tmCAP or dmCAP.
  • An alternative example 5' cap structure is m7G(5')(2'-OMeA)pG.
  • 5’UTR and 3’UTR are used to regulate the translation of mRNA.
  • the 5’UTR sequence is preferably: GGGAGAAAGCUUACC (as shown in SEQ ID.NO.1).
  • the 3'UTR sequence is preferably: GGACUAGUUAUAAGACUGACUAGCCCGAUGGGCCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGAGAUUAAU (as shown in SEQ ID.NO.2).
  • the 3'polyA tail is used to prevent mRNA from being degraded by exonucleases and to terminate transcription.
  • the length of polyA is 50 to 200.
  • the length of optional polyA is 80 to 200.
  • the length of optional polyA is 100 bp.
  • the sequence is as SEQ. Shown as ID.NO.3.
  • RNA sequence based on the provided RNA sequence, one of ordinary skill in the art will be able to obtain the corresponding DNA sequence (eg, uracil to thymine conversion). Likewise, based on the provided DNA sequence, one of ordinary skill in the art will derive the corresponding RNA sequence (eg, thymine to uracil). In alternative embodiments, based on the provided RNA or DNA sequence, one of ordinary skill in the art will be able to obtain the corresponding amino acid sequence.
  • one or more uridines in the mRNA are replaced with modified nucleosides.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m1 ⁇ ), or 5-methyl-uridine (m5U).
  • the Linker sequence contains at least one part encoding a protein cleavage signal.
  • the protein cleavage signal may be, for example, but is not limited to, a cleavage signal for the following substances with cleavage function: protein precursor convertase, hormone pro- In body convertase, thrombin and factor Xa protein.
  • the protein cleavage signal preferably includes a Furin cleavage site (FCS, reference US7374930B2). Furin cleavage sites are widely distributed in most cell types.
  • FCS Furin cleavage site
  • Furin cleavage sites can effectively express the active polypeptides expressed by the above-mentioned fusion RNA in vivo. Cut, so that the first and second open reading frames fused to the same RNA express the Delta variant S protein and the Omicron variant S protein respectively.
  • the DNA sequence of the Furin cleavage site is preferably CGTCAACGTCGT (SEQ ID.NO.6); the RNA sequence is preferably CGUCAACGUCGU (SEQ ID.NO.7).
  • the Linker is a cleavable linker or a protease-sensitive linker.
  • the cleavable linker is preferably 2A peptide.
  • 2A peptides (2A self-cleaving peptides) are a type of peptide fragments with a length of 18 to 22 amino acid residues, which can induce self-cleavage of recombinant proteins containing 2A peptides in cells.
  • viruses use the 2A peptide to produce two proteins from one transcript through ribosome hopping, such that the normal peptide bond is weakened at the 2A peptide sequence, resulting in the production of two discontinuous proteins from one translation event.
  • Examples of 2A peptides can be, for example, but are not limited to, F2A linkers, P2A linkers, E2A linkers, T2A linkers; the amino acid sequence of the F2A linker (foot-and-mouth disease virus (FMDV) 2A peptide) such as SEQ ID.NO.43 or SEQ ID.NO.44 shown; the amino acid sequence of the P2A linker (porcine Jieshen virus-12A peptide) is shown in SEQ ID.NO.45 or SEQ ID.NO.46; the amino acid sequence of the E2A linker (equine rhinitis A virus 2A peptide) is shown in SEQ ID .NO.47 or SEQ ID.NO.48 is shown; the amino acid sequence of the T2A linker (Termovirus 2A peptide) is shown in SEQ ID.NO.49 or SEQ ID.NO.50.
  • F2A linkers foot-and-mouth disease virus (FMDV) 2A peptide
  • FMDV foot
  • GSG Gly-Ser-Gly, glycine, serine, glycine
  • the nucleotide sequence encoding the 2A peptide includes, but is not limited to, the following sequence, or based on the following sequence, the polynucleotide sequence of the 2A peptide is modified or codon optimized by methods described above and/or known in the art. In some alternative embodiments, the nucleotide sequence encoding the 2A peptide is shown in SEQ ID.NO.30 or SEQ ID.NO.31.
  • linker sequence in the fragment "first coding region-linker sequence” and the linker sequence in the fragment “linker sequence-second coding region” may be the same or different.
  • the nucleotide sequence encoding the first open reading frame of the S protein of the new coronavirus Delta variant strain is such as SEQ ID.NO.9, SEQ ID.NO.36, SEQ ID.NO.37 , SEQ ID.NO.38, SEQ ID.NO.39, SEQ ID.NO.40, SEQ ID.NO.41, SEQ ID.NO.42 or SEQ ID.NO.29.
  • the second open reading frame encodes the new coronavirus Omicron variant BA.5 sub-variant
  • the nucleotide sequence of the second open reading frame is such as SEQ ID.NO.27, SEQ ID.NO.32, SEQ ID.NO.20, SEQ ID.NO.21, SEQ ID.NO.22, SEQ ID.NO.23, SEQ ID.NO.24, SEQ ID.NO.25 or SEQ ID .NO.28 is shown.
  • the nucleotide sequence of the first open reading frame is selected from SEQ ID.NO.9, SEQ ID.NO.36, SEQ ID.NO.37, SEQ ID.NO.38, SEQ ID expressing Delta variant S protein.
  • the acid sequence is selected from SEQ ID.NO.27, SEQ ID.NO.32, SEQ ID.NO.20, SEQ ID.NO.21, SEQ ID.NO expressing S protein of Omicron BA.2 sub-variant strain. .22, one of SEQ ID.NO.23, SEQ ID.NO.24, SEQ ID.NO.25 or SEQ ID.NO.28.
  • Specific combinations of the first open reading frame and the second open reading frame may be, for example, but are not limited to: SEQ ID.NO.9 and SEQ ID.NO.27, SEQ ID.NO.9 and SEQ ID.NO. 32; SEQ ID.NO.36 and SEQ ID.NO.32, SEQ ID.NO.37 and SEQ ID.NO.20, SEQ ID.NO.38 and SEQ ID.NO.21, SEQ ID.NO.39 and SEQ ID.NO.22, SEQ ID.NO.40 and SEQ ID.NO.23, SEQ ID.NO.41 and SEQ ID.NO.24, SEQ ID.NO.42 and SEQ ID.NO.25, SEQ ID.NO.29 and SEQ ID.NO.28, SEQ ID.NO.29 and SEQ ID.NO.28, SEQ ID.NO.42 and SEQ ID.NO.25, SEQ ID.NO.41 and SEQ ID.NO.24, SEQ ID.NO.40 and SEQ ID.NO.23, SEQ ID.
  • the two nucleic acids produced in the body after the vaccine immunizes the body are adjusted.
  • the first open reading frame and the second open reading frame are located on different nucleic acid molecules, and the mass ratio of the nucleic acid molecule encoding the first open reading frame to the nucleic acid molecule encoding the second open reading frame is It is (1:9) ⁇ (9:1); for example, it can be but is not limited to 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1: 2. 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or 9:1, preferably (1:1) ⁇ (9:1 ), more preferably 3:1.
  • the nucleic acid molecule is preferably an RNA molecule, and has a sequence including from the 5' end to the 3' end: 5' cap - 5' UTR - first open reading frame and/or second open reading frame -Structure of 3'UTR-3'polyA tail.
  • the nucleic acid molecule is a fusion nucleic acid molecule, that is, the same nucleic acid molecule contains both a first open reading frame and a second open reading frame, where the first open reading frame and the second open reading frame are
  • the number of repeats in the fusion nucleic acid molecule is (1:9) to (9:1); for example, it can be but is not limited to 1:9, 1:8, 1:7, 1:6, 1:5, 1: 4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or 9:1, preferably (1 :1) ⁇ (9:1), more preferably 3:1.
  • novel coronavirus vaccine provided by the present disclosure may also contain other auxiliary materials or functional ingredients acceptable in the art for vaccine preparation, including but not limited to vaccine adjuvants, delivery formulations, solvents, preservatives, stabilizers, At least one or more of agents, pH adjusters, buffer substances and lyoprotectants.
  • the vaccine further includes a delivery preparation, which is preferably a lipid component.
  • the lipid component preferably forms nucleic acid lipid nanoparticles (LNP) with the nucleic acid molecules of the new coronavirus vaccine.
  • LNP is a nanoparticle formed by using lipid components to wrap nucleic acid. LNP can make the nucleic acid wrapped in it more effectively delivered into cells.
  • the vaccine preferably also includes a delivery preparation.
  • the delivery preparation is preferably a lipid component.
  • the lipid component preferably forms nucleic acid lipid nanoparticles (LNP) with the nucleic acid molecules of the new coronavirus vaccine.
  • the LNP is wrapped with a lipid component. Nanoparticles formed from nucleic acids, LNPs, can enable the nucleic acids wrapped in them to be delivered more effectively into cells.
  • novel coronavirus vaccine containing LNP is optionally as follows (a), (b) or (c):
  • the novel coronavirus vaccine includes: LNP wrapped with a nucleic acid molecule containing a first open reading frame, and LNP wrapped with a nucleic acid molecule containing a second open reading frame.
  • the novel coronavirus vaccine includes: LNP wrapped with a nucleic acid molecule containing a first open reading frame and a nucleic acid molecule containing a second open reading frame.
  • the first open reading frame and the second open reading frame exist in the same nucleic acid molecule, and the nucleic acid molecule is wrapped in LNP, that is, the new coronavirus vaccine includes: wrapped with a first open reading frame and a second open reading frame. Two open reading frames of the nucleic acid molecule LNP.
  • the lipid component used to form LNP is preferably as follows: on a molar basis, the lipid component forming LNP includes 20 to 50% protonatable cationic lipids, such as but not limited to 20%, 25%, 30%, 35%, 40%, 45% or 50%; 20-50% structural lipid, for example but not limited to 20%, 25%, 30%, 35%, 40%, 45% or 50%; 5-50% 20% auxiliary lipid, for example, but not limited to 5%, 10%, 15% or 20%; and 1 to 5% surfactant, for example, but not limited to 1%, 2%, 3%, 4% or 5%.
  • the total molar content of protonatable cationic lipids, structural lipids, auxiliary lipids and surfactants is 100%.
  • the protonatable cationic lipid preferably includes at least one of DlinMC3-DMA, DODMA, C12-200 and DlinDMA.
  • the auxiliary lipid preferably includes at least one of DSPC, DOPE, DOPC, DOPG and DOPS.
  • Structural lipids preferably include cholesterol and/or cholesterol derivatives.
  • the surfactant preferably includes at least one of PEG-DMG, PEG-DSPE and TPGS.
  • the lipid component includes Dlin-MC3-DMA 50%, DOPG 10%, cholesterol 38.5%, and PEG-DMG 1.5% on a molar percentage basis. In some preferred embodiments, the lipid composition includes Dlin-MC3-DMA 50%, DOPG 20%, cholesterol 29%, and PEG-DMG 1% on a molar percentage basis.
  • the LNP in the vaccine is prepared as follows: uniformly mix the aqueous phase containing nucleic acid molecules and the organic phase containing the lipid component to obtain a mixed liquid, remove the organic phase and allow the system to
  • concentration of nucleic acid molecules in is 1 to 100 ⁇ g/ml, for example, but is not limited to 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 ⁇ g/ml, and the preferred concentration is 55 ⁇ g/ml.
  • the mixing of the aqueous phase and the organic phase is preferably performed using microfluidic equipment, with the flow rate controlled to >3ml/min.
  • a buffer solution for example but not limited to 50, 60, 70, 80, 90 or 100 times, and use tangential flow filtration (TFF) to remove the solution.
  • THF tangential flow filtration
  • the organic phase is then concentrated to bring the nucleic acid molecules in the system to the target concentration.
  • the aqueous phase is an aqueous phase buffer containing 0.08 to 1.2 mg/L of the nucleic acid molecules.
  • concentration of the nucleic acid molecules in the aqueous phase can be, for example, but not limited to, 0.08, 0.1, 0.2, 0.5, 0.8, 1.0, 1.1 Or 1.2 mg/L; the aqueous buffer is citrate buffer or sodium acetate buffer.
  • the organic phase is anhydrous C1-C4 lower alcohol containing 5-7 mg/L of the lipid component.
  • concentration of the lipid component may be, for example, but is not limited to 5, 5.5, 6, 6.5 or 7 mg/ml;
  • the anhydrous C1-C4 lower alcohol is preferably ethanol.
  • the volume ratio of the aqueous phase and the organic phase is 1:2-4, for example, but is not limited to 1:2, 1:3 or 1:4.
  • LNPs can optionally be prepared in (a), (b) or (c), or other delivery methods can optionally be used; in vaccine preparation optimization
  • different combinations of first open reading frames and second open reading frames are used to prepare a variety of LNPs wrapped with different nucleic acids; when the nucleic acid molecules are other types of molecules, such as expression cassettes integrated with DNA Or a carrier, it can also be prepared into LNP wrapped with DNA using the method (a), (b) or (c) to serve as the main functional component of the vaccine.
  • Specific examples may be, for example, but are not limited to
  • the novel coronavirus vaccine contains two RNA molecules, the two RNA molecules respectively contain a first open reading frame and a second open reading frame, and the nucleotide sequence of the first open reading frame As shown in SEQ ID.NO.9, the nucleotide sequence of the second open reading frame is as shown in SEQ ID.NO.27.
  • the sequence characteristics of the two RNA molecules also include 5' cap (m7G(5')(2'-OMeA)pG), 5'UTR (as shown in SEQ ID.NO.1), 3'UTR (as shown in SEQ ID.NO.
  • the mass ratio of the RNA molecule containing the first open reading frame and the RNA molecule containing the second open reading frame is (1:9) to (9:1); for example, it can be but is not limited to 1:9, 1:8, 1 :7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 3:1, 4:1 or 9:1.
  • the two RNA molecules are first made into LNPs respectively, and then mixed according to the formula amount based on RNA quality to obtain the active ingredients in the new coronavirus vaccine.
  • the nucleotide sequence of the first open reading frame can also be selected from SEQ ID.NO.9, SEQ ID.NO.36, SEQ ID.NO.37, SEQ ID.NO.38, SEQ ID. Any one of NO.39, SEQ ID.NO.40, SEQ ID.NO.41, SEQ ID.NO.42 or SEQ ID.NO.29; the nucleotide sequence of the second open reading frame is also Can be selected from SEQ ID.NO.27, SEQ ID.NO.32, SEQ ID.NO.20, SEQ ID.NO.21, SEQ ID.NO.22, SEQ ID.NO.23, SEQ ID.NO. 24. Any one shown in SEQ ID.NO.25 or SEQ ID.NO.28.
  • the specific combination of the first open reading frame and the second open reading frame can be, for example, but is not limited to: SEQ ID.NO.9 and SEQ ID.NO.27, SEQ ID.NO.9 and SEQ ID.NO.32; SEQ ID.NO.36 and SEQ ID.NO.32, SEQ ID.NO.37 and SEQ ID.NO.20, SEQ ID.NO.38 and SEQ ID.NO.21, SEQ ID.NO.39 and SEQ ID.NO.22, SEQ ID.NO.40 and SEQ ID.NO.23, SEQ ID.NO.41 and SEQ ID.NO.24, SEQ ID.NO.42 and SEQ ID .NO.25, SEQ ID.NO.29 and SEQ ID.NO.28, SEQ ID.NO.29 and SEQ ID.NO.28, SEQ ID.NO.42 and SEQ ID.NO.25, SEQ ID.
  • the novel coronavirus vaccine contains two RNA molecules, the two RNA molecules respectively contain a first open reading frame and a second open reading frame, and the nucleotides of the first open reading frame
  • the sequence is shown in SEQ ID.NO.9
  • the nucleotide sequence of the second open reading frame is shown in SEQ ID.NO.27.
  • the sequence characteristics of the two RNA molecules also include 5' cap (m7G(5')(2'-OMeA)pG), 5'UTR (as shown in SEQ ID.NO.1), 3'UTR (as shown in SEQ ID.NO.
  • the mass ratio of the RNA molecule containing the first open reading frame and the RNA molecule containing the second open reading frame is (1:9) to (9:1); for example, it can be but is not limited to 1:9, 1:8, 1 :7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1 , 8:1 or 9:1.
  • the nucleotide sequence of the first open reading frame can also be selected from SEQ ID.NO.9, SEQ ID.NO.36, SEQ ID.NO.37, SEQ ID.NO.38, SEQ ID. Any one of NO.39, SEQ ID.NO.40, SEQ ID.NO.41, SEQ ID.NO.42 or SEQ ID.NO.29; the nucleotide sequence of the second open reading frame is also Can be selected from SEQ ID.NO.27, SEQ ID.NO.32, SEQ ID.NO.20, SEQ ID.NO.21, SEQ ID.NO.22, SEQ ID.NO.23, SEQ ID.NO. 24. Any one shown in SEQ ID.NO.25 or SEQ ID.NO.28.
  • the specific combination of the first open reading frame and the second open reading frame can be, for example, but not limited to: SEQ ID.NO.9 and SEQ ID.NO.27, SEQ ID.NO.9 and SEQ ID.NO.32; SEQ ID.NO.36 and SEQ ID.NO.32, SEQ ID.NO.37 and SEQ ID.NO.20, SEQ ID.NO.38 and SEQ ID.NO.21, SEQ ID.NO.39 and SEQ ID.NO.22, SEQ ID.NO.40 and SEQ ID.NO.23, SEQ ID.NO.41 and SEQ ID.NO.24, SEQ ID.NO.42 and SEQ ID .NO.25, SEQ ID.NO.29 and SEQ ID.NO.28, SEQ ID.NO.29 and SEQ ID.NO.28, SEQ ID.NO.42 and SEQ ID.NO.25, SEQ ID.
  • the novel coronavirus vaccine contains a fusion RNA molecule, and the fusion RNA molecule contains at least two coding regions containing a first open reading frame and a second open reading frame, wherein the first open reading frame
  • the frame sequence is selected from the nucleotide sequence shown in SEQ ID.NO.9
  • the second open reading frame sequence is selected from the nucleotide sequence shown in SEQ ID.NO.27.
  • the structure of the fusion RNA molecule is 5' cap-5'UTR-(first coding region-linker sequence)n-(linker sequence-second coding region)m-3'UTR-3'polyA tail, n and m are independent is a positive integer.
  • the 5' cap is m7G(5')(2'-OMeA)pG, the 5'UTR is as shown in SEQ ID.NO.1, the 3'UTR is as shown in SEQ ID.NO.2, and the polyA is as shown in SEQ ID.NO.
  • the Linker sequence is shown as SEQ ID.NO.21.
  • the ratio of n and m is (1:9) to (9:1); for example, it can be but is not limited to 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1 :3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or 9:1.
  • the above fusion RNA molecules were prepared to obtain LNP, which was used as an active ingredient in the new coronavirus vaccine.
  • the nucleotide sequence of the first open reading frame can also be selected from the group consisting of SEQ ID.NO.9, SEQ ID.NO.36, SEQ ID. Any of NO.37, SEQ ID.NO.38, SEQ ID.NO.39, SEQ ID.NO.40, SEQ ID.NO.41, SEQ ID.NO.42 or SEQ ID.NO.29 One; the nucleotide sequence of the second open reading frame can also be selected from SEQ ID.NO.27, SEQ ID.NO.32, SEQ ID.NO.20, SEQ ID.NO.21, SEQ ID.NO. 22. Any one of SEQ ID.NO.23, SEQ ID.NO.24, SEQ ID.NO.25 or SEQ ID.NO.28.
  • the specific combination of the first open reading frame and the second open reading frame can be, for example, but is not limited to: SEQ ID.NO.9 and SEQ ID.NO.27, SEQ ID.NO.9 and SEQ ID.NO.32; SEQ ID.NO.36 and SEQ ID.NO.32, SEQ ID.NO.37 and SEQ ID.NO.20, SEQ ID.NO.38 and SEQ ID.NO.21, SEQ ID.NO.39 and SEQ ID.NO.22, SEQ ID.NO.40 and SEQ ID.NO.23, SEQ ID.NO.41 and SEQ ID.NO.24, SEQ ID.NO.42 and SEQ ID .NO.25, SEQ ID.NO.29 and SEQ ID.NO.28, SEQ ID.NO.29 and SEQ ID.NO.28, SEQ ID.NO.42 and SEQ ID.NO.25, SEQ ID.
  • the present disclosure also provides a method for preparing the above-mentioned novel coronavirus vaccine.
  • the preparation method includes mixing the nucleic acid molecule and optional auxiliary materials to obtain the novel coronavirus vaccine.
  • the new coronavirus vaccine includes nucleic acid lipid nanoparticles, and the preparation method includes:
  • Nucleic acid lipid nanoparticles wrapped with a nucleic acid molecule containing a first open reading frame and nucleic acid lipid nanoparticles wrapped with a nucleic acid molecule containing a second open reading frame are respectively prepared to obtain nucleic acid lipids that wrap different open reading frames respectively. Then mix the two nucleic acid lipid nanoparticles according to the formula amount.
  • nucleic acid lipid nanoparticles wrapped with the two nucleic acid molecules first mix the nucleic acid molecule containing the first open reading frame and the nucleic acid molecule containing the second open reading frame according to the formula amount, and then prepare nucleic acid lipid nanoparticles wrapped with the two nucleic acid molecules.
  • nucleic acid lipid nanoparticles encapsulated with a nucleic acid molecule containing both a first open reading frame and a second open reading frame are prepared.
  • the preparation method of the above-mentioned nucleic acid lipid nanoparticles is preferably based on the lipid nanoparticles (LNP) method described in the above-mentioned novel coronavirus vaccine technical solution section, and will not be described in detail here.
  • LNP lipid nanoparticles
  • the present disclosure also provides the application of the above-mentioned novel coronavirus vaccine, or the preparation method of the above-mentioned novel coronavirus vaccine, in the preparation of products configured to prevent or treat diseases caused by the novel coronavirus.
  • the present disclosure also provides a product configured to prevent or treat diseases caused by the new coronavirus, which product includes the above-mentioned new coronavirus vaccine.
  • the above-mentioned product configured to prevent or treat diseases caused by the new coronavirus may be, for example, but not limited to, a kit that also contains a device configured to inoculate the above-mentioned new coronavirus vaccine; a kit that also contains other preventive or therapeutic active ingredients; Or, it also contains a kit for evaluating the effectiveness of the new coronavirus vaccine, etc.
  • This embodiment provides a method for preparing lipid nanoparticles containing RNA, wherein the lipid nanoparticles include in molar percentage: Dlin-MC3-DMA 50%, DOPG 20%, cholesterol 29% and PEG-DMG 1% , the preparation method is as follows:
  • step (c) Mix the aqueous phase of step (a) and the organic phase of step (b) at a volume ratio of 1:3 using a microfluidic device at a flow rate of 12 mL/min.
  • the mixed solution is immediately mixed with PBS at pH 7.4.
  • the solution was diluted 100 times, and tangential flow filtration (TFF) was used to remove the ethanol component in the solution, and then concentrated until the concentration of mRNA in the system was 55 ⁇ g/ml, to obtain lipid nanoparticles containing RNA encoding SARS-CoV-2 viral antigens. Particles.
  • MC3 refers to Dlin-MC3-DMA
  • + after administration, the formulations of different vaccine carriers are studied by in vivo fluorescence imaging of small animals.
  • the phase system detects the efficiency of delivering luciferase gene-encoding mRNA in mice, and detects the physical and chemical indicators of different composite preparations (see Example 1 for the preparation method). The results are shown in Table 1 .
  • MC3 refers to Dlin-MC3-DMA.
  • This example designed a series of mRNA sequences, in which the sequence of the open reading frame is shown in Table 3; in addition to the open reading frame sequence, the sequence characteristics of this series of mRNA also include 5' cap (m7G(5')(2'-OMeA )pG), 5'UTR (as shown in SEQ ID.NO.1), 3'UTR (as shown in SEQ ID.NO.2) and the 3' tail of 100 polyA (as shown in SEQ ID.NO.3 Show).
  • Each of the above mRNAs was prepared into lipid nanoparticles according to the preparation method provided in Example 1.
  • Experiment A Select normal cynomolgus monkeys aged about 5 to 8 years old to evaluate the immunogenicity of the vaccine.
  • the animals were randomly divided into 5 groups, namely the original strain group (SEQ ID.NO.13) (50 ⁇ g/animal), the Delta group (SEQ ID.NO.9) (50 ⁇ g/animal), and the Beta group (SEQ ID.NO. 5) (50 ⁇ g/animal), Omicron group (SEQ ID.NO.11) (50 ⁇ g/animal) and negative control group, each group has 12 animals, half male and half female.
  • the mRNALNP preparation prepared in Example 2 was used in the cynomolgus monkey immunity experiment. Each cynomolgus monkey was injected with 50 micrograms (in terms of mRNA) through the muscle of the hind limb thigh on D0 (the day of administration is D0) and D21 respectively. Intramuscular injection corresponds to the test vaccine and the control vaccine, among which the negative control group was injected with 0.5 mL of PBS. 28 days after the first immunization, cynomolgus monkey serum was extracted and sent to a third-party laboratory for SARS-CoV-2 pseudovirus neutralizing activity testing.
  • the vaccine preparation prepared from four antigens can stimulate cynomolgus monkeys to produce antibodies with neutralizing ability against the target strain pseudovirus.
  • the vaccine preparation encoding the S protein of the new coronavirus Delta variant strain stimulates the antibodies produced by cynomolgus monkeys against the original strain, Alpha variant strain, Beta variant strain, Gamma variant strain and Delta strain of the new coronavirus.
  • the six pseudovirus strains of mutant strains and Omicron mutant strains all have strong neutralizing activity;
  • the vaccine preparation encoding the S protein of the original strain of the new coronavirus stimulates the antibodies produced by cynomolgus monkeys against the original strain, Alpha variant strain, Beta variant strain, Gamma variant strain and Delta variant strain of the new coronavirus.
  • the pseudovirus strains also have strong neutralizing activity, while the neutralizing activity against Omicron mutant strains is weak;
  • the mRNA (SEQ ID.NO.5) vaccine preparation encoding the S protein of the new coronavirus Beta variant strain stimulates the antibodies produced by cynomolgus monkeys to be more effective against the three pseudoviruses of the new coronavirus Beta variant strain, Gamma variant strain and Delta variant strain. Strong neutralizing activity, but weak neutralizing activity against the original strain, Alpha mutant strain and Omicron mutant strain.
  • the antibodies produced by the mRNA vaccine preparation encoding the new coronavirus Delta mutant strain and the S protein of the original new coronavirus strain have broader capabilities. neutralizing activity.
  • Experiment B Select normal cynomolgus monkeys aged about 5 to 8 years old to evaluate the immunogenicity of the vaccine.
  • the animals were randomly divided into 3 groups, namely the original strain group (SEQ ID.NO.13) (50 ⁇ g/animal), the Delta group (SEQ ID.NO.9) (50 ⁇ g/animal), and the negative control group, each group had 36 Only, half male and half female.
  • the mRNA vaccine preparation sample encoding the S protein of the new coronavirus Delta variant strain is recorded as sample 1-1
  • the mRNA vaccine preparation sample encoding the S protein of the original strain of the new coronavirus is recorded as sample 1-2.
  • the mRNA vaccine preparation encoding the S protein of the new coronavirus Delta variant has better neutralizing activity against popular strains such as Beta variant and Omicron variant.
  • This example designed a series of mRNA sequence information, in which the sequence of the open reading frame is shown in Table 4; in addition to the open reading frame sequence, the series of mRNA sequence features also include 5' cap (m7G(5')(2'- OMeA)pG), 5'UTR (as shown in SEQ ID.NO.1), 3'UTR (as shown in SEQ ID.NO.2) and the 3' tail of 100 polyA (as shown in SEQ ID.NO.3 shown).
  • 5' cap m7G(5')(2'- OMeA)pG
  • 5'UTR as shown in SEQ ID.NO.1
  • 3'UTR as shown in SEQ ID.NO.2
  • the 3' tail of 100 polyA as shown in SEQ ID.NO.3 shown.
  • a total of four different antigens in samples 1-4 were made into vaccines according to the method of Example 1, and different groups of mRNA mixtures were prepared into LNP preparations.
  • the encapsulation rate of the LNP preparations was tested to be more than 90%, and the particle size was about 70 nm. about.
  • mice The prepared mRNALNP preparation was used in the C57 mouse immunization experiment. Each mouse was injected with 5 micrograms (in terms of mRNA) through the muscle of the lateral thigh of the hind limb. Secondary immunization was performed after an interval of 7 days, with 3 mice in each group. 14 days after the first immunization, mouse serum was extracted and sent to a third-party laboratory for SARS-CoV-2 pseudovirus neutralizing activity testing. After diluting the mouse serum according to different proportions in a 96-well plate (the initial dilution factor is 30), add the SARS-CoV-2 pseudovirus that can infect it, and set up cell controls and virus controls at the same time.
  • the initial dilution factor is 30
  • the mRNA vaccine open reading frame encodes different S proteins (amino acid sequences such as SEQ ID.NO.14, SEQ ID.NO.16, SEQ ID.NO.18, SEQ ID.NO.27
  • the mRNA vaccine encoding the S protein of the amino acid sequence such as SEQ ID.NO.27 stimulates mice to produce a response to the new coronavirus Omicron.
  • the neutralizing antibody activity of each sub-variant pseudovirus decreased less.
  • This embodiment provides a series of new coronavirus bivalent vaccines, and the preparation method is as follows:
  • the mRNA encoding the S protein of the new coronavirus Omicron variant and Delta variant also includes a 5' cap (m7G(5')(2'-OMeA)pG), 5'UTR (such as SEQ ID. NO.1), 3'UTR (SEQ ID.NO.2) and the 3' tail of 100 polyA (SEQ ID.NO.3).
  • mice The three samples prepared above were used in the C57 mouse immunization experiment. Each mouse was injected with 5 micrograms (in terms of mRNA) through the muscle of the outer thigh of the hind limb. A second immunization was performed after an interval of 7 days. There were 3 mice in each group. Fourteen days after the first immunization, mouse serum was extracted and sent to a third-party laboratory for SARS-CoV-2 pseudovirus neutralizing activity testing. After diluting the mouse serum according to different proportions in a 96-well plate (the initial dilution factor is 30), add the SARS-CoV-2 pseudovirus that can infect it, and set up cell controls and virus controls at the same time.
  • the initial dilution factor is 30
  • the new coronavirus bivalent mRNA vaccine sample 3 (including the mRNA lipid nanoparticles encoding the S protein of the new coronavirus Omicron BA.5 variant strain and the mRNA lipid nanoparticles encoding the S protein of the new coronavirus Delta variant strain)
  • the activity of antibodies produced against each virus variant dropped less.
  • mRNA sequences were designed based on the S protein of the new coronavirus Omicron strain (the amino acid sequence is shown in SEQ ID.NO.26) and the principles of mRNA sequence optimization.
  • the open reading frame information is shown in Table 4 below.
  • sequence characteristics of this series of mRNAs also include 5' cap (m7G(5')(2'-OMeA)pG), 5'UTR (as shown in SEQ ID.NO.1), 3 'UTR (as shown in SEQ ID.NO.2) and the 3' tail of 100 polyA (as shown in SEQ ID.NO.3).
  • the cells were transfected with the mRNA shown in Table 4, and the expression of S full-length protein in the cells was detected.
  • the results are shown in Table 4.
  • the detailed method is as follows: HEK293 cells were lysed 24 hours after transfection of each mRNA. According to the loading amount of 10 ⁇ g total protein, the target protein was specifically detected by SDS-PAGE immunoblotting method. In this example, anti-SARS was used respectively.
  • -S1 protein antibody was used as the primary antibody
  • goat anti-mouse-HRP antibody was used as the secondary antibody for incubation, and then color development was performed.
  • Example 7 Two different antigens of samples 1 and 2 in Example 7 were made into vaccines according to the method of Example 1, and different groups of mRNA mixtures were respectively prepared into LNP preparations. The encapsulation rates of the LNP preparations were tested to be more than 90%. The particle size is approximately 70nm. The obtained two groups of vaccine preparations were used in C57 mouse immunization experiments. The experimental methods are shown in Example 6. The corresponding group numbers and results of serum are shown in Figure 4.
  • This embodiment provides a series of new coronavirus bivalent vaccines, and the preparation method is as follows:
  • the mRNA encoding the S protein of the new coronavirus Omicron BA.5 variant and Delta variant also includes a 5' cap (m7G(5')(2' -OMeA)pG), 5'UTR (as shown in SEQ ID.NO.1), 3'UTR (as shown in SEQ ID.NO.2) and the 3' tail of 100 polyA (as shown in SEQ ID.NO. shown in 3).
  • the bar graph in Figure 5 shows the EC50 value of the pseudovirus strain of samples 2-1 to 2-11; the dotted line is an indicator line, indicating the position where the EC50 value of the pseudovirus strain is 200.
  • This embodiment provides a series of new coronavirus bivalent vaccines, and the preparation method is as follows:
  • the new coronavirus bivalent vaccine provided in this example contains lipid nanoparticles encoding RNA for antigen 1 and antigen 2, wherein the lipid nanoparticles include Dlin-MC3-DMA50%, DOPG10%, cholesterol 38.5% and PEG in molar percentage. -DMG1.5%.
  • the preparation method is as follows:
  • step (c) Mix the aqueous phase of step (a) and the organic phase of step (b) at a volume ratio of 1:3 using a microfluidic device at a flow rate of 12 mL/min.
  • the mixed solution is immediately mixed with PBS at pH 7.4.
  • the solution was diluted 100 times, and tangential flow filtration (TFF) was used to remove the ethanol component in the solution, and then concentrated until the concentration of mRNA in the system was 0.55 mg/ml, to obtain lipid nanoparticles containing RNA encoding antigen 1 and antigen 2. .
  • TMF tangential flow filtration
  • nucleotide open reading frame sequence encoding antigen 1 is SEQ ID.NO.9 (encoding the S protein of the new coronavirus Delta variant strain)
  • nucleotide open reading frame sequence encoding antigen 2 is SEQ ID.NO.27 ( When encoding the S protein of the new coronavirus Omicron BA.5 variant), antigen 1 and antigen 2 are expressed in the order of 1:15, 1:10, 1:9, 1:4, 1:3, 1:1, 3:1, 4 Samples 1-11 were prepared with :1, 9:1, 10:1 and 15:1.
  • nucleotide sequences encoding antigens 1 and 2 include, in addition to the open reading frame, 5' cap (m7G(5')(2'-OMeA)pG), 5'UTR (as shown in SEQ ID.NO.1 ), 3'UTR (as shown in SEQ ID.NO.2) and the 3' tail of 100 polyA (as shown in SEQ ID.NO.3).
  • M1 and M2 respectively include 5' Mass of the cap (m7G(5')(2'-OMeA)pG), 5'UTR, open reading frame, 3'UTR and 3' tail of the mRNA encoding antigen 1 and the mass of the mRNA encoding antigen 2.
  • the bar graph in Figure 6 shows the EC50 value of the pseudovirus strain of samples 1-11; the dotted line is an indicator line, indicating the position where the EC50 value of the pseudovirus strain is 200.
  • Example 9 prepare the new coronavirus mRNA encoding the S protein of the new coronavirus Delta variant strain (SEQ ID.NO.9) and the mRNA encoding the S protein of the new coronavirus OmicronBA.5 variant strain (SEQ ID.NO.27).
  • SEQ ID.NO.9 the S protein of the new coronavirus Delta variant strain
  • SEQ ID.NO.27 the mRNA encoding the S protein of the new coronavirus OmicronBA.5 variant strain.
  • the mass ratios of the two lipid nanoparticles are 1:1 and 3:1 respectively, which are recorded as samples a and b respectively.
  • mRNA sequence characteristics also include 5' cap (m7G(5')(2'-OMeA)pG), 5'UTR (as shown in SEQ ID.NO.1), 3'UTR (as shown in SEQ ID.NO. 2) and the 3' tail of 100 polyA (shown in SEQ ID.NO.3).
  • RNA molecules in the new coronavirus vaccine provided in this embodiment are fusion RNA structures, and the specific structures are as follows:
  • nucleotide sequence of the first coding region is shown in SEQ ID.NO.9; the nucleotide sequence of the second coding region is shown in SEQ ID.NO.27;
  • the 5' cap is m7G(5')(2'-OMeA)pG; the 5'UTR is as shown in SEQ ID.NO.1; the 3'UTR is as shown in SEQ ID.NO.2; the 3' tail of 100 polyA As shown in SEQ ID.NO.3;
  • This example provides a series of bivalent vaccines for the new coronavirus, prepared according to the method described in Example 9, in which the mRNA open reading frame sequence encoding the S protein of the new coronavirus Omicron BA.5 variant strain and the S protein encoding the Delta variant strain of the new coronavirus
  • the mRNA open reading frame sequence and quality ratio are shown in the table below:
  • This example provides a series of bivalent vaccines for the new coronavirus, prepared according to the method described in Example 10, in which the mRNA open reading frame sequence encoding the S protein of the new coronavirus Omicron BA.5 variant strain and the sequence of the mRNA encoding the S protein of the new coronavirus Delta variant strain are provided.
  • the mRNA open reading frame sequence and quality ratio are shown in the table below:
  • This example provides the immune effects of the new coronavirus bivalent vaccine and the new coronavirus original strain vaccine in mice, and uses the CPE method to detect the true virus cross-neutralizing activity against the original strain and the currently circulating virus strain.
  • the bivalent vaccine for the new coronavirus was prepared according to the method described in Example 10, in which the sequence of the mRNA open reading frame encoding the S protein of the new coronavirus Omicron BA.5 variant is shown in SEQ ID.NO.27, encoding the S protein of the new coronavirus Delta variant.
  • the protein's mRNA open reading frame sequence is shown in SEQ ID.NO.9.
  • the mass ratio of the mRNA encoding the S protein of the new coronavirus Omicron BA.5 variant and the mRNA encoding the S protein of the new coronavirus Delta variant is 1:3.
  • the mRNA sequence of the new coronavirus bivalent vaccine also includes 5' cap (m7G(5')(2'-OMeA)pG), 5'UTR (as shown in SEQ ID.NO.1), 3 'UTR (as shown in SEQ ID.NO.2) and the 3' tail of 100 polyA (as shown in SEQ ID.NO.3).
  • 5' cap m7G(5')(2'-OMeA)pG
  • 5'UTR as shown in SEQ ID.NO.1
  • 3 'UTR as shown in SEQ ID.NO.2
  • the 3' tail of 100 polyA as shown in SEQ ID.NO.3
  • the new coronavirus original strain vaccine was prepared according to the method described in Example 1, in which the mRNA open reading frame sequence encoding the S protein of the new coronavirus original strain is shown in SEQ ID.NO.13; in addition to the open reading frame, the new coronavirus bivalent vaccine
  • the mRNA sequence also includes 5' cap (m7G(5')(2'-OMeA)pG), 5'UTR (as shown in SEQ ID.NO.1), 3'UTR (as shown in SEQ ID.NO.2 ) and 100 polyA 3' tails (as shown in SEQ ID.NO.3).
  • 5' cap m7G(5')(2'-OMeA)pG
  • 5'UTR as shown in SEQ ID.NO.1
  • 3'UTR as shown in SEQ ID.NO.2
  • 100 polyA 3' tails as shown in SEQ ID.NO.3
  • Two new coronavirus mRNA vaccine samples 14-1 and 14-2 were used to immunize 6-8-week-old female KM mice by intramuscular injection (i.m.) in the anterolateral area of the hind legs. The dose was 15 ⁇ g/mouse, 10 in each group. Only, 3 groups in total. Both vaccines were immunized on D0 and D28 according to the clinically developed immunization schedule. Retroorbital blood collection was performed on D34 animals, and the serum was separated and the post-immune serum was tested for cross-neutralizing activity against the original strain and the currently prevalent virus strain using the CPE method. By detecting serum neutralizing antibody levels after immunization with two different vaccines, the superiority of the two vaccines, Sample 14-1 and Sample 14-2, was compared.
  • the new coronavirus vaccine uses nucleic acid as the main immunogenic substance of the vaccine, and includes a nucleic acid molecule containing a first open reading frame and a second open reading frame, wherein the first open reading frame encodes the new coronavirus Delta variant strain S Protein, the second open reading frame encodes the BA.5S protein of the new coronavirus Omicron sub-variant strain.
  • the new coronavirus vaccine immunizes the body, it can express the S protein of the new coronavirus Delta variant strain and the Omikron variant strain BA.5 in the body, and the antibody activity produced by the new coronavirus variant strains is less reduced, which is one of the Multivalent vaccine.

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Abstract

本公开提供了一种新型冠状病毒疫苗及其制备方法和应用，涉及疫苗的技术领域。该新型冠状病毒疫苗包含编码新型冠状病毒Delta变异株S蛋白和编码新型冠状病毒奥密克戎子变异株BA.5S蛋白的核酸分子，为一种多价疫苗。

Description

新型冠状病毒疫苗及其制备方法和应用
相关申请的交叉引用
本申请要求申请日为2022年07月27日的中国专利申请(申请号：2022108928431，发明名称：编码SARS-CoV-2病毒抗原的mRNA和疫苗)的优先权，其全部内容通过引用方式并入到本申请；以及申请日为2022年09月30日的中国专利申请(申请号：2022112076839，发明名称：新型冠状病毒疫苗及其制备方法和应用)的优先权，其全部内容通过引用方式整体并入到本申请。
技术领域
本公开涉及疫苗技术领域，尤其是涉及一种新型冠状病毒疫苗及其制备方法和应用。
背景技术
新冠肺炎疫情造成了全世界范围内社会和经济的极大损失，新冠病毒极易变异，从被发现至今，新冠病毒原始株、新冠病毒Alpha株、新冠病毒Beta株、新冠病毒Gamma变异株、新冠病毒Kappa株、新冠病毒Delta株和新冠病毒Omicron株等毒株相继出现。
目前上市和大多数处在临床试验阶段的新冠疫苗均是针对新冠病毒原始毒株进行的抗原设计。与最初的新冠病毒原始株序列相比，奥密克戎(Omicron)变异株至少新增了60个突变点，其中刺突蛋白(S蛋白)超过35个突变，而刺突蛋白(S蛋白)内最关键的受体结合结构域上携带15个突变，相对而言，德尔塔(Delta)变异株在该区域仅有2个突变。根据突变位点的差异，奥密克戎变异株可分为至少5个子变异株，分别是BA.1、BA.2.12.1、BA.2、BA.4和BA.5等。
研究发现，已上市的疫苗对变异株的保护效果出现不同程度的下降，尤其是针对新冠病毒奥密克戎株。因此针对变异株具有更好保护效果的新冠疫苗亟待研发。
发明内容
根据本公开的各种实施例，提供了一种新型冠状病毒疫苗，所述新型冠状病毒疫苗包含含有第一开放阅读框和含有第二开放阅读框的核酸分子；
所述第一开放阅读框编码新型冠状病毒Delta变异株S蛋白；所述第二开放阅读框编码新型冠状病毒奥密克戎子变异株BA.5S蛋白。
在一个或多个实施例中，所述新型冠状病毒疫苗包含：含有第一开放阅读框的核酸分子；和，含有第二开放阅读框的核酸分子；
或，所述新型冠状病毒疫苗包含：同时含有第一开放阅读框和第二开放阅读框的融合核酸分子。
在一个或多个实施例中，新型冠状病毒Delta变异株S蛋白的氨基酸序列如SEQ ID.NO.8所示，或，包含与SEQ ID.NO.8至少80％同一性的氨基酸序列，例如可以为但不限于为包含与SEQ ID.NO.8至少80％、85％、90％、95％或98％同一性的氨基酸序列。
在一个或多个实施例中，所述新型冠状病毒奥密克戎子变异株BA.5S蛋白的氨基酸序列如SEQ ID.NO.26所示，或，包含与SEQ ID.NO.26至少80％同一性的氨基酸序列，例如可以为但不限于为包含与SEQ ID.NO.26至少80％、85％、90％、95％或98％同一性的氨基酸序列。
在一个或多个实施例中，所述核酸分子包括DNA分子和/或RNA分子。
在一个或多个实施例中，所述DNA分子包括链状DNA分子和/或环状DNA分子。
在一个或多个实施例中，所述RNA分子包括mRNA或环形RNA。
在一个或多个实施例中，所述核酸分子为RNA。
在一个或多个实施例中，RNA中开放阅读框的部分总GC％含量为30～70％，所述开放阅读框的片段中任意一长度为60bp的片段中GC％含量不低于40％。
在一个或多个实施例中，RNA中开放阅读框的部分总GC％含量为50％～60％，更优选为54％～60％。
在一个或多个实施例中，所述RNA还包括5’帽子、5’UTR、3’UTR、polyA尾、起始区、终止区、信号序列区和linker序列中的一种或多种。
在一个或多个实施例中，所述第一开放阅读框的核苷酸序列如SEQ ID.NO.9、SEQ ID.NO.36、SEQ ID.NO.37、SEQ ID.NO.38、SEQ ID.NO.39、SEQ ID.NO.40、SEQ ID.NO.41、SEQ ID.NO.42或SEQ ID.NO.29所示。
在一个或多个实施例中，所述第二开放阅读框的核苷酸序列如SEQ ID.NO.27、SEQ ID.NO.32、SEQ ID.NO.20、SEQ ID.NO.21、SEQ ID.NO.22、SEQ ID.NO.23、SEQ ID.NO.24、SEQ ID.NO.25、SEQ ID.NO.28所示。
在一个或多个实施例中，第一开放阅读框和第二开放阅读框的组合方式选自：SEQ ID.NO.9和SEQ ID.NO.27、SEQ ID.NO.9和SEQ ID.NO.32；SEQ ID.NO.36和SEQ ID.NO.32、SEQ ID.NO.37和SEQ ID.NO.20、SEQ ID.NO.38和SEQ ID.NO.21、SEQ ID.NO.39和SEQ ID.NO.22、SEQ ID.NO.40和SEQ ID.NO.23、SEQ ID.NO.41和SEQ ID.NO.24、SEQ ID.NO.42和SEQ ID.NO.25、SEQ ID.NO.29和SEQ ID.NO.28、SEQ ID.NO.29和SEQ ID.NO.28、SEQ ID.NO.42和SEQ ID.NO.25、SEQ ID.NO.41和SEQ ID.NO.24、SEQ ID.NO.40和SEQ ID.NO.23、SEQ ID.NO.39和SEQ ID.NO.32、SEQ ID.NO.38和SEQ ID.NO.22、SEQ ID.NO.37和SEQ ID.NO.21、SEQ ID.NO.36和SEQ ID.NO.20。
在一个或多个实施例中，含有编码第一开放阅读框的核酸分子和含有编码第二开放阅读框的核酸分子的质量比为(1:9)～(9:1)；含有编码第一开放阅读框的核酸分子和含有编码第二开放阅读框的核酸分子的质量比为(1:9)、(1:8)、(1:7)、(1:6)、(1:5)、(1:4)、(1:3)、(1:2)、(1:1)、(2:1)、(3:1)、(4:1)、(5:1)、(6:1)、(7:1)、(8:1)、(9:1)；在一个或多个实施例中，含有编码第一开放阅读框的核酸分子和含有编码第二开放阅读框的核酸分子的质量比为3:1。
在一个或多个实施例中，所述核酸分子为融合核酸分子，第一开放阅读框和第二开放阅读框在所述融合核酸分子中的重复数为(1:9)～(9:1)；第一开放阅读框和第二开放阅读框在所述融合核酸分子中的重复数为(1:9)、(1:8)、(1:7)、(1:6)、(1:5)、(1:4)、(1:3)、(1:2)、(1:1)、(2:1)、(3:1)、(4:1)、(5:1)、(6:1)、(7:1)、(8:1)、(9:1)；在一个或多个实施例中，第一开放阅读框和第二开放阅读框在所述融合核酸分子中的重复数为3:1。
在可选的实施方式中，基于提供的RNA序列，本领域普通技术人员将能得到相应的DNA序列(例如尿嘧啶转换为胸腺嘧啶)。同样地，基于所提供的DNA序列，本领域普通技术人员将得到相应的RNA序列(例如胸腺嘧啶转换为尿嘧啶)。在可选的实施方式中，基于提供的RNA或DNA序列，本领域普通技术人员将能得到相应的氨基酸序列。
在一个或多个实施例中，所述疫苗还包括递送制剂。
在一个或多个实施例中，所述新型冠状病毒疫苗含有由所述核酸分子和脂质成分构成的核酸脂质纳米颗粒。
在一个或多个实施例中，所述新型冠状病毒疫苗选自(a)、(b)或(c)：
(a)、所述新型冠状病毒疫苗包含:包裹有含有第一开放阅读框的核酸分子的核酸脂质纳米颗粒，和，包裹有含有第二开放阅读框的核酸分子的核酸脂质纳米颗粒；
(b)、所述新型冠状病毒疫苗包含:包裹有含有第一开放阅读框的核酸分子，和含有第二开放阅读框的核酸分子的核酸脂质纳米颗粒；
(c)、所述新型冠状病毒疫苗包含：包裹有同时含有第一开放阅读框和第二开放阅读框的融合核酸分子的核酸脂质纳米颗粒。
根据本公开的各种实施例，本公开还提供了一种上述新型冠状病毒疫苗的制备方法，该制备方法包括将所述核酸分子与任选地辅料混合，得到所述新型冠状病毒疫苗。
在一个或多个实施例中，所述新型冠状病毒疫苗包括核酸脂质纳米颗粒，所述制备方法包括：
分别制备包裹有含有第一开放阅读框的核酸分子的核酸脂质纳米颗粒，和，包裹有含有第二开放阅读框的核酸分子的核酸脂质纳米颗粒；然后按照配方量将两种核酸脂质纳米颗粒混合；
或，先将含有第一开放阅读框的核酸分子和含有第二开放阅读框的核酸分子按照配方量混合，再制备得到包裹有含有两种核酸分子的核酸脂质纳米颗粒；
或，制备包裹有同时含有第一开放阅读框和第二开放阅读框的核酸分子的核酸脂质纳米颗粒。
在一个或多个实施例中，本公开还提供了上述新型冠状病毒疫苗，或上述制备方法在制备被配置为预防或治疗新型冠状病毒引发的疾病的产品中的应用。
根据本公开的另一个方面，本公开还提供了一种被配置为预防或治疗新型冠状病毒引发的疾病的产品，所述产品包含上述新型冠状病毒疫苗。
本公开的一个或多个实施例的细节在下面的附图和描述中提出。本公开的其它特征、目的和优点将从说明书、附图以及权利要求书变得明显。
附图说明
为了更清楚地说明本公开具体实施方式或现有技术中的技术方案，可以参考一幅或多幅附图。用于描述附图的附加细书或示例不应当被认为是对所公开的发明、目前描述的实施例和或示例以及目前理解的这些发明的最佳模式中的任何一者的范围的限制。下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本公开的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1-1为实施例4中各疫苗制剂免疫食蟹猴后产生的血清对新型冠状病毒的假病毒中和活性测试结果。
图1-2为实施例4中疫苗制剂免疫食蟹猴后产生的血清对新型冠状病毒的假病毒中和活性测试结果。
图2为实施例5中各疫苗制剂免疫C57小鼠后产生的血清对新型冠状病毒的假病毒中和活性测试结果。
图3为实施例6各疫苗制剂免疫C57小鼠后产生的血清对新型冠状病毒的假病毒中和活性测试结果。
图4为实施例8中各疫苗制剂免疫C57小鼠后产生的血清对新型冠状病毒的假病毒中和活性测试结果。
图5为实施例9中各疫苗制剂免疫C57小鼠后产生的血清对新型冠状病毒的假病毒中和活性测试结果。
图6为实施例10中各疫苗制剂免疫C57小鼠后产生的血清对新型冠状病毒的假病毒中和活性测试结果。
图7为实施例11中各疫苗制剂免疫C57小鼠后产生的血清对新型冠状病毒的假病毒中和活性测试结果。
图8为实施例12中各疫苗制剂免疫C57小鼠后产生的血清对新型冠状病毒的假病毒中和活性测试结果。
图9为实施例13中各疫苗制剂免疫C57小鼠后产生的血清对新型冠状病毒的假病毒中和活性测试结果。
图10为实施例14中各疫苗制剂免疫C57小鼠后产生的血清对新型冠状病毒的假病毒中和活性测试结果。
图11为实施例15中各疫苗制剂免疫SPF小鼠针对原始株和当下流行毒株的真病毒交叉中和活性测试结果。
具体实施方式
下面将结合实施例对本公开的技术方案进行清楚、完整地描述，显然，所描述的实施例是本公开一部分实施例，而不是全部的实施例。基于本公开中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本公开保护的范围。
需要说明的是：本公开中，如果没有特别的说明，本文所提到的所有实施方式以及优选实施方法可以相互组合形成新的技术方案；本文所提到的所有技术特征以及优选特征可以相互组合形成新的技术方案；所涉及的各组分或其优选组分可以相互组合形成新的技术方案。
本公开所公开的“范围”以下限和上限的形式，可以分别为一个或多个下限，和一个或多个上限；除非另有说明，操作步骤可以顺序进行，也可以不按照顺序进行。
除非另有说明，本文中所用的专业与科学术语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法或材料也可应用于本公开中。
根据本公开的各种实施例，本公开提供了一种新型冠状病毒疫苗，所述新型冠状病毒疫苗主要以核酸分子作为免疫原物质，包含含有第一开放阅读框和含有第二开放阅读框的核酸分子，其中第一开放阅读框编码新型冠状病毒Delta变异株S蛋白，第二开放阅读框编码新型冠状病毒奥密克戎(Omicron)子变异株BA.5S蛋白。该新型冠状病毒疫苗免疫机体后，能够在机体内表达新型冠状病毒Delta变异株和奥密克戎子变异株BA.5S蛋白，为一种多价疫苗。
本公开中所述的“核酸分子”指的是任何长度的核苷酸的聚合物形式，核苷酸包括核糖核苷酸和/或脱氧核糖核苷酸。核酸的实例包括但不限于单链、双链或多链DNA或RNA、基因组DNA、cDNA；整合由外源基因的载体DNA，例如表达盒或质粒；DNA-RNA杂合体或者包含嘌呤和嘧啶碱基或其他天然、化学或生化修饰、非天然或衍生的核苷酸碱基的聚合物。
需要说明的是，所述包含含有第一开放阅读框和含有第二开放阅读框的核酸分子中，第一开放阅读框和第二开放阅读框可以存在于同一核酸分子，也可以存在于不同的核酸分子。因此，只要新型冠状病毒疫苗包含的核酸分子中含有第一开放阅读框和第二开放阅读框，施用于机体后能够在机体内产生新型冠状病毒Delta变异株和奥密克戎子变异株BA.5S蛋白，即属于本公开提供的新型冠状病毒疫苗。
在一些可选的实施方式中，所述新型冠状病毒疫苗包含：含有第一开放阅读框的核酸分子；和，含有第二开放阅读框的核酸分子；
或，所述新型冠状病毒疫苗包含：同时含有第一开放阅读框和第二开放阅读框的融合核酸分子。
本公开通过实验发现，含有编码新型冠状病毒Delta变异株S蛋白的核酸分子免疫动物后，动物血清中产生的抗体对新冠病毒的原始株、Alpha变异株、Beta变异株、Gamma变异株、Delta变异株和Omicron变异株6种毒株假病毒都具有较强的中和活性；含有编码新型冠状病毒奥密克戎变异株S蛋白的核酸分子免疫动物后，动物血清中产生的抗体对Omicron变异株具有较强的中和活性；结合新冠病毒世界流行毒株分布，使新型冠状病毒疫苗含有能够编码Delta变异株S蛋白和奥密克戎变异株S蛋白的核酸分子，能兼具对新型冠状病毒各流行株的预防作用。
本公开提供的新型冠状病毒疫苗中，开放阅读框编码的新型冠状病毒的S蛋白可选地为Delta变异株和/或奥密克戎变异株在自然条件下突变得到的S蛋白；也可选地为经人工突变和改造的S蛋白，所述突变和改造可以为经野生型突变和改造得到符合Delta变异株或奥密克戎变异株的S蛋白的氨基酸序列；或，也可以为在Delta变异株和奥密克戎变异株的S蛋白的氨基酸序列的基础上，进一步进行突变和改造而获得的S蛋白的氨基酸序列。
在一个或多个实施例中，开放阅读框编码的新型冠状病毒的S蛋白的氨基酸序列如下：
新型冠状病毒Delta变异株S蛋白的氨基酸序列优选如SEQ ID.NO.8所示，或，包含与SEQ ID NO.8至少80％同一性的氨基酸序列，例如可以为但不限于为包含与SEQ ID NO.8至少80％、85％、90％、95％或98％同一性的氨基酸序列；新型冠状病毒奥密克戎变异株的S蛋白的氨基酸序列来源于BA.5子变异株、BA.2子变异株、BA.3子变异株的S蛋白或BA.5子变异株的S蛋白；或，所述奥密克戎变异株的S蛋白的氨基酸序列可选地来源于野生型S蛋白经突变后得到的氨基酸序列。
在一个或多个实施例中，所述新型冠状病毒奥密克戎变异株为BA.2子变异株或BA.1子变异株或BA.5子变异株的S蛋白；更优选为BA.5子变异株。
新型冠状病毒奥密克戎变异株BA.1子变异株S蛋白的氨基酸序列优选如SEQ ID.NO.16所示，或，包含与SEQ ID NO.16至少80％同一性的氨基酸序列，例如可以为但不限于为包含与SEQ ID NO.16至少80％、85％、90％、95％或98％同一性的氨基酸序列。
新型冠状病毒奥密克戎变异株BA.2子变异株S蛋白的氨基酸序列优选如SEQ ID.NO.14所示，或，包含与SEQ ID NO.14至少80％同一性的氨基酸序列，例如可以为但不限于为包含与SEQ ID NO.14至少80％、85％、90％、95％或98％同一性的氨基酸序列。
新型冠状病毒奥密克戎变异株BA.5子变异株S蛋白的氨基酸序列优选如SEQ ID.NO.26所示或，包含与SEQ ID NO.26至少80％同一性的氨基酸序列，例如可以为但不限于为包含与SEQ ID NO.26至少80％、85％、90％、95％或98％同一性的氨基酸序列。
两个核苷酸序列之间的「序列同一性」指示序列之间相同核苷酸的百分比。两个氨基酸序列之间的「序列同一性」指示序列之间相同氨基酸的百分比。
术语「％同一性」或类似术语是指，在最佳比对下，待比较序列之间相同核苷酸或氨基酸之百分比。该百分比为纯粹统计学的，且两个序列之间的差异可能为(但不一定)随机分布于待比较序列之整个长度上。两个序列之比较通常藉由在最佳比对之后，相对于片段或「比较窗口」比较改等序列而进行，以鉴别对应序列之局部区。
SEQ ID.NO.26为Omicron BA.5毒株的S蛋白经SN突变(在全长S蛋白的氨基酸序列的位置981和982处具有两处脯氨酸残基取代)，具有更高的表达量。
本公开中所述的氨基酸序列的位置，是以新型冠状病毒原始株野生型S蛋白全长氨基酸序列定位。
本公开提供的新型冠状病毒疫苗以核酸分子作为主要功效成分，该新型冠状病毒疫苗施用于机体后在体内表达产生新型冠状病毒Delta变异株和病毒奥密克戎株的S蛋白。为了进一步提高疫苗的免疫效果，本公开还对核酸分子、两种编码S蛋白的核酸比例以及疫苗制剂进行了优化。
在一个或多个实施例中，核酸分子可以通过序列优化手段优化mRNA序列，改善与体内施用后的表达功效相关的特性：例如提高mRNA稳定性、增加靶组织中的翻译功效、减少表达的截短蛋白的数量、改善表达的蛋白质的折叠或防止其错误折叠、降低表达产物的毒性、减少由表达产物引起的细胞死亡、增加和/或减少蛋白质聚集，得到改善特性的mRNA。序列优化的目的还包括：保持结构和功能完整性的同时优化基于核酸的治疗剂的配制和递送的特征；克服表达的阈值；提高表达率；半衰期和/或蛋白质浓度；优化蛋白质定位；以及避免不利的生物响应如免疫响应和/或降解途径。序列优化手段包括：(1)根据特定器官和/或宿主生物体中的密码子频率进行密码子优化以确保恰当折叠和适当表达；(2)调节G/C含量以增加mRNA稳定性或减少二级结构；(3)使可能损害基因构建或表达的串联重复密码子或碱基串(baseruns)最小化；(4)定制转录和翻译控制区；(5)减少或消除多核苷酸内的问题二级结构。
核酸分子的优化：
新型冠状病毒疫苗中含有的核酸分子优选为RNA，所述新型冠状病毒疫苗优选为RNA疫苗。
在一个或多个实施例中，RNA中的开放阅读框部分的总GC％含量为30～70％，例如可以为但不限于为30％、35％、40％、45％、50％、55％、60％、65％或70％，或其任意两点之间的范围值，优选为50％～60％，更优选为54％～60％。同时，开放阅读框部分中任意一长度为60bp的片段中GC％含量不低于40％，本公开通过实验发现，符合上述条件的RNA的新型冠状病毒S蛋白的表达量更高。
在一个或多个实施例中，所述RNA还包括5’帽子、5’UTR、3’UTR、polyA尾、起始区、终止区、信号序列区和linker序列中的一种或多种；新型冠状病毒疫苗中的RNA的结构优选如下：
可选地，RNA从5’端到3’端依次包括：5’帽子-5’UTR-第一开放阅读框和/或第二开放阅读框-3’UTR-3’polyA尾；依次包括表示RNA从5’端到3’依次含有上述片段，各片段之间可以含有也可以不含有至少一个核糖核苷酸或具有功能的核酸片段。
可选地，RNA同时含有第一开放阅读框和第二开放阅读框，为融合RNA，融合RNA的结构优选如下：
一种可选地示例结构如下：5’帽子-5’UTR-起始区-第一开放阅读框-linker序列-第二开放阅读框-3’UTR-终止区-3’polyA尾；
或，5’帽子-5’UTR-第一开放阅读框-linker序列-第二开放阅读框-3’UTR-3’polyA尾；
一种可选地示例结构如下：5’帽子-5’UTR-起始区-(第一编码区-linker序列)n-(linker序列-第二编码区)m-3’UTR-终止区-3’polyA尾；
或，5’帽子-5’UTR-(第一编码区-linker序列)n-(linker序列-第二编码区)m-3’UTR-3’polyA尾；
其中，n为：片段“第一编码区-linker序列”的重复数；m为：片段“linker序列-第二编码区”的重复数；n和m分别独立的为正整数。
在上述示例中，可以通过调节片段“第一编码区-linker序列”和片段“linker序列-第二编码区”的重复数，即n和m的值，或调整n和m的比值来调整编码新型冠状病毒Delta变异株S蛋白的开放阅读框和编码新型冠状病毒奥密克戎变异株S蛋白的开放阅读框的含量，从而实现新型冠状病毒中RNA免疫机体后，能够产生不同含量和比例的Delta变异株S蛋白和奥密克戎变异株S蛋白。
RNA中其他功能片段优选如下：
5’帽子结构用于增加mRNA稳定性，避免mRNA被核酸外切酶降解，5’帽子结构修饰基团选自ARCA、m7G(5"")ppp(5"")(2""OMeA)pG、m7G(5"")ppp(5"")(2""OMeG)pG、m7(3""OMeG)(5"")ppp(5"")(2""OMeG)pG、m7(3""OMeG)(5"")ppp(5"")(2""OMeA)pG、mCAP、dmCAP、tmCAP或dmCAP。一种可选地示例5’帽子结构为m7G(5’)(2’-OMeA)pG。
5’UTR和3’UTR用于调控mRNA的翻译，5’UTR序列优选为：GGGAGAAAGCUUACC(如SEQ ID.NO.1所示)。
3’UTR序列优选为：GGACUAGUUAUAAGACUGACUAGCCCGAUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGAGAUUAAU(如SEQ ID.NO.2所示)。
3’polyA尾用于避免mRNA被核酸外切酶降解，同时终结转录，polyA的长度为50～200，可选的polyA的长度为80～200，可选的polyA的长度为100bp，序列如SEQ ID.NO.3所示。
在一个或多个实施例中，基于提供的RNA序列，本领域普通技术人员将能得到相应的DNA序列(例如尿嘧啶转换为胸腺嘧啶)。同样地，基于所提供的DNA序列，本领域普通技术人员将得到相应的RNA序列(例如胸腺嘧啶转换为尿嘧啶)。在可选的实施方式中，基于提供的RNA或DNA序列，本领域普通技术人员将能得到相应的氨基酸序列。
在一个或多个实施例中，mRNA中的一个或多个尿苷用修饰核苷进行置换。在一些实施方案中，置换尿苷的修饰核苷为假尿苷(ψ)、N1-甲基-假尿苷(m1ψ)或5-甲基-尿苷(m5U)。
在一些可选地实施方式中，Linker序列含有至少一个编码蛋白质切割信号的部分，所述蛋白质切割信号例如可以为但不限于为如下具有切割功能物质的切割信号：蛋白前体转化酶、激素前体转化酶、凝血酶和因子Xa蛋白中。蛋白质切割信号优选包括Furin切割位点(furin cleavage site，FCS，参考US7374930B2)。Furin切割位点广泛分布在大多数细胞类型中，上述融合RNA可以在体内几乎任何类型的细胞中有效表达活性多肽，因此采用Furin切割位点可以使上述融合RNA在体内表达的活性多肽得到有效的切割，使融合于同一RNA上的第一和第二开放阅读框分别表达Delta变异株S蛋白和奥密克戎变异株S蛋白。
Furin切割位点的DNA序列优选为CGTCAACGTCGT(SEQ ID.NO.6)；RNA序列优选为CGUCAACGUCGU(SEQ ID.NO.7)。
在一些可选地实施方式中，Linker为可切割接头或蛋白酶敏感接头。可切割接头优选采用2A肽，2A肽(2Aself-cleavingpeptides)是一类长18～22个氨基酸残基的肽片段，能诱导细胞内含有2A肽的重组蛋白自我剪切。若干病毒使用2A肽通过核糖体跳跃由一种转录物产生两种蛋白质，使得正常肽键在2A肽序列处削弱，导致由一个翻译事件产生两种不连续的蛋白质。
2A肽的实例例如可以为但不限于F2A接头、P2A接头、E2A接头、T2A接头；F2A接头(口蹄疫病毒(FMDV)2A肽)的氨基酸序列如SEQ ID.NO.43或SEQ ID.NO.44所示；P2A接头(猪捷申病毒-12A肽)的氨基酸序列如SEQ ID.NO.45或SEQ ID.NO.46所示；E2A接头(马鼻炎A病毒2A肽)的氨基酸序列如SEQ ID.NO.47或SEQ ID.NO.48所示；T2A接头(明脉扁刺蛾病毒2A肽)的氨基酸序列如SEQ ID.NO.49或SEQ ID.NO.50所示。
在2A肽序列的N端加上一个GSG(Gly-Ser-Gly，甘氨酸、丝氨酸、甘氨酸)序列能提高2A肽诱导剪切的效率。
编码2A肽的核苷酸序列包括但不限于以下序列，或以以下序列为基础，通过本文上述和/或本领域中已知的方法对2A肽的多核苷酸序列进行修饰或密码子优化。在一些可选地实施方式中，编码2A肽的核苷酸序列如SEQ ID.NO.30或SEQ ID.NO.31所示。
片段“第一编码区-linker序列”中的linker序列和片段“linker序列-第二编码区”中的linker序列可以相同也可以不同。
在一些可选的实施方式中，编码新型冠状病毒Delta变异株S蛋白的第一开放阅读框的核苷酸序列如SEQ ID.NO.9、SEQ ID.NO.36、SEQ ID.NO.37、SEQ ID.NO.38、SEQ ID.NO.39、SEQ ID.NO.40、SEQ ID.NO.41、SEQ ID.NO.42或SEQID.NO.29所示。
在一些可选的实施方式中，第二开放阅读框编码新型冠状病毒奥密克戎变异株BA.5子变异株，第二开放阅读框的核苷酸序列如SEQ ID.NO.27、SEQ ID.NO.32、SEQ ID.NO.20、SEQ ID.NO.21、SEQ ID.NO.22、SEQ ID.NO.23、SEQ ID.NO.24、SEQ ID.NO.25或SEQ ID.NO.28所示。
第一开放阅读框和第二开放阅读框可选的组合方式如下：
第一开放阅读框的核苷酸序列选自表达Delta变异株S蛋白的SEQ ID.NO.9、SEQ ID.NO.36、SEQ ID.NO.37、SEQ ID.NO.38、SEQ ID.NO.39、SEQ ID.NO.40、SEQ ID.NO.41、SEQ ID.NO.42或SEQ ID.NO.29所示所示中的一种；且，第二开放阅读框的核苷酸序列选自表达奥密克戎BA.2子变异株S蛋白的SEQ ID.NO.27、SEQ ID.NO.32、SEQ ID.NO.20、SEQ ID.NO.21、SEQ ID.NO.22、SEQ ID.NO.23、SEQ ID.NO.24、SEQ ID.NO.25或SEQ ID.NO.28所示中的一种。
第一开放阅读框和第二开放阅读框的具体的组合方式例如可以为但不限于为：SEQ ID.NO.9和SEQ ID.NO.27、SEQ ID.NO.9和SEQ ID.NO.32；SEQ ID.NO.36和SEQ ID.NO.32、SEQ ID.NO.37和SEQ ID.NO.20、SEQ ID.NO.38和SEQ ID.NO.21、SEQ ID.NO.39和SEQ ID.NO.22、SEQ ID.NO.40和SEQ ID.NO.23、SEQ ID.NO.41和SEQ ID.NO.24、SEQ ID.NO.42和SEQ ID.NO.25、SEQ ID.NO.29和SEQ ID.NO.28、SEQ ID.NO.29和SEQ ID.NO.28、SEQ ID.NO.42和SEQ ID.NO.25、SEQ ID.NO.41和SEQ ID.NO.24、SEQ ID.NO.40和SEQ ID.NO.23、SEQ ID.NO.39和SEQ ID.NO.32、SEQ ID.NO.38和SEQ ID.NO.22、SEQ ID.NO.37和SEQ ID.NO.21、SEQ ID.NO.36和SEQ ID.NO.20。
编码S蛋白的核酸比例：
在一些可选的实施方式中，通过调节新型冠状病毒疫苗中编码Delta变异株S蛋白的核酸含量和编码奥密克戎变异株S蛋白的含量来调节疫苗免疫机体后，在机体中产生的两种S蛋白的比例。
在一些可选的实施方式中，第一开放阅读框和第二开放阅读框位于不同的核酸分子，含有编码第一开放阅读框的核酸分子和含有编码第二开放阅读框的核酸分子的质量比为(1:9)～(9:1)；例如可以为但不限于为1:9、1:8、1:7、1:6、1:5、1:4、1:3、1:2、1:1、2:1、3:1、4:1、5:1、6:1、7:1、8:1或9:1，优选为(1:1)～(9:1)，更优选为3:1。在该实施方式中，所述核酸分子优选为RNA分子，且具有如从5’端到3’端依次包括：5’帽子-5’UTR-第一开放阅读框和/或第二开放阅读框-3’UTR-3’polyA尾的结构。
在一些可选的实施方式中，所述核酸分子为融合核酸分子，即同一核酸分子中同时含有第一开放阅读框和第二开放阅读框，第一开放阅读框和第二开放阅读框在所述融合核酸分子中的重复数为(1:9)～(9:1)；例如可以为但不限于为1:9、1:8、1:7、1:6、1:5、1:4、1:3、1:2、1:1、2:1、3:1、4:1、5:1、6:1、7:1、8:1或9:1，优选为(1:1)～(9:1)，更优选为3:1。
疫苗制剂优化：
可以理解的是，本公开提供的新型冠状病毒疫苗还可以含有本领域可接受的其他用于制备疫苗的辅料或功能性成分，包括但不限于疫苗佐剂、递送制剂、溶剂、防腐剂、稳定剂、pH调节剂、缓冲物质和冻干保护剂中的至少一种或多种。
在一个或多个实施例中，所述疫苗还包括递送制剂，所述递送制剂优选为脂质成分，所述脂质成分优选和新型冠状病毒疫苗的核酸分子构成核酸脂质纳米颗粒(LNP)，LNP是使用脂质成分包裹核酸形成的纳米颗粒，LNP能够使包裹其中的核酸更有效的递送至细胞内。
所述疫苗优选还包括递送制剂，所述递送制剂优选为脂质成分，所述脂质成分优选和新型冠状病毒疫苗的核酸分子构成核酸脂质纳米颗粒(LNP)，LNP是使用脂质成分包裹核酸形成的纳米颗粒，LNP能够使包裹其中的核酸更有效的递送至细胞内。
所述新型冠状病毒疫苗中含有LNP可选地为如下(a)、(b)或(c)：
(a)、第一开放阅读框和第二开放阅读框分别存在与不同的核酸分子，含有第一开放阅读框的核酸分子和含有第二开放阅读框的核酸分子分别包裹于不同的LNP中，即所述新型冠状病毒疫苗包含:包裹有含有第一开放阅读框的核酸分子的LNP，和，包裹有含有第二开放阅读框的核酸分子的LNP。
(b)、第一开放阅读框和第二开放阅读框分别存在与不同的核酸分子，含有第一开放阅读框的核酸分子和含有第二开放阅读框的核酸分子混合后包裹于同一LNP中，即所述新型冠状病毒疫苗包含:包裹有含有第一开放阅读框的核酸分子，和含有第二开放阅读框的核酸分子的LNP。
(c)、第一开放阅读框和第二开放阅读框存在于同一核酸分子中，该核酸分子包裹于LNP中，即所述新型冠状病毒疫苗包含：包裹有同时含有第一开放阅读框和第二开放阅读框的核酸分子的LNP。
用于构成LNP的脂质成分优选如下：按摩尔百分比计，形成LNP的脂质成分包括20～50％可质子化阳离子脂质，例如可以为但不限于为20％、25％、30％、35％、40％、45％或50％；20～50％结构脂质，例如可以为但不限于为20％、25％、30％、35％、40％、45％或50％；5～20％辅助脂质，例如可以为但不限于为5％、10％、15％或20％；和1～5％表面活性剂，例如可以为但不限于为1％、2％、3％、4％或5％。其中，可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂的摩尔含量合计100％。
可质子化阳离子脂质优选包括DlinMC3-DMA、DODMA、C12-200和DlinDMA中的至少一种。辅助脂质优选包括DSPC、DOPE、DOPC、DOPG和DOPS中的至少一种。结构脂质优选包括胆固醇和/或胆固醇衍生物。表面活性剂优选包括PEG-DMG、PEG-DSPE和TPGS中的至少一种。
在一些优选的实施方式中，按照摩尔百分比计，所述脂质成分包括Dlin-MC3-DMA 50％、DOPG 10％、胆固醇38.5％和PEG-DMG 1.5％。在一些优选的实施方式中，按照摩尔百分比计，所述脂质成分包括Dlin-MC3-DMA 50％、DOPG 20％、胆固醇29％和PEG-DMG 1％。
在一些可选的实施方式中，所述疫苗中的LNP按照如下方法制备得到：将含有核酸分子的水相和含有所述脂质成分的有机相混合均匀得到混合液，去除有机相并使体系中的核酸分子浓度为1～100μg/ml，例如可以为但不限于为1、5、10、20、30、40、50、60、70、80、90或100μg/ml，优选浓度为55μg/ml，得到所述核酸脂质纳米颗粒。
水相和有机相的混合优选使用微流控设备进行，流速控制为>3ml/min。
去除有机相优选采用先使用缓冲液将混合液稀释50～100倍，例如可以为但不限于为50、60、70、80、90或100倍，并使用切向流过滤(TFF)去除溶液中有机相，然后浓缩使体系中的核酸分子至目标浓度。
所述水相为含有0.08～1.2mg/L所述核酸分子的水相缓冲液，水相中的核酸分子的浓度例如可以为但不限于为0.08、0.1、0.2、0.5、0.8、1.0、1.1或1.2mg/L；所述水相缓冲液为柠檬酸盐缓冲液或醋酸钠缓冲液。
所述有机相为含有5～7mg/L所述脂质成分的无水C1～C4低碳醇，脂质成分的浓度例如可以为但不限于为5、5.5、6、6.5或7mg/ml；无水C1～C4低碳醇优选为乙醇。
所述水相和所述有机相的体积比为1:2～4，例如可以为但不限于为1:2、1:3或1:4。
需要说明的是，上述对核酸分子的优化、编码S蛋白的核酸比例的优化和疫苗制剂优化中所提到的所有技术特征以及优选特征可以相互组合形成新的技术方案。例如，在优化的编码S蛋白的核酸比例的方案中，可选地采用(a)、(b)或(c)方式制备LNP，也可选地采用或不采用其他递送方式；在疫苗制剂优化的方案中，采用不同的第一开放阅读框和第二开放阅读框的组合，制备得到多种包裹有不同核酸的LNP；当所述核酸分子采用其他类型分子时，如整合有DNA的表达盒或载体，也可以采用(a)、(b)或(c)方式制备成包裹有DNA的LNP，以作为疫苗的主要功效成分。具体的实例例如可以为但不限于为
在一些可选地实施方式中，所述新型冠状病毒疫苗中含有两种RNA分子，两种RNA分子分别含有第一开放阅读框和第二开放阅读框，第一开放阅读框的核苷酸序列如SEQ ID.NO.9所示，第二开放阅读框的核苷酸序列如SEQ ID.NO.27所示。两种RNA分子序列特征还包括5’帽子(m7G(5’)(2’-OMeA)pG)、5’UTR(如SEQ ID.NO.1所示)、3’UTR(如SEQ ID.NO.2所示)、100个polyA的3’尾(如SEQ ID.NO.3所示)、N1-甲基-假尿苷(m1ψ)修饰核苷(置换全部尿苷)。含有第一开放阅读框的RNA分子和含有第二开放阅读框的RNA分子的质量比(1:9)～(9:1)；例如可以为但不限于为1:9、1:8、1:7、1:6、1:5、1:4、1:3、1:2、1:1、3:1、4:1或9:1。两种RNA分子先分别制成LNP，再以RNA质量计，按照配方量混合，得到所述新型冠状病毒疫苗中的有效成分。
该实施方式中，第一开放阅读框的核苷酸序列还可以选自SEQ ID.NO.9、SEQ ID.NO.36、SEQ ID.NO.37、SEQ ID.NO.38、SEQ ID.NO.39、SEQ ID.NO.40、SEQ ID.NO.41、SEQ ID.NO.42或SEQ ID.NO.29所示中的任意一种；第二开放阅读框的核苷酸序列还可以选自SEQ ID.NO.27、SEQ ID.NO.32、SEQ ID.NO.20、SEQ ID.NO.21、SEQ ID.NO.22、SEQ ID.NO.23、SEQ ID.NO.24、SEQ ID.NO.25或SEQ ID.NO.28所示中的任意一种。
上述实施方式中，第一开放阅读框和第二开放阅读框的具体的组合方式例如可以为但不限于为：SEQ ID.NO.9和SEQ ID.NO.27、SEQ ID.NO.9和SEQ ID.NO.32；SEQ ID.NO.36和SEQ ID.NO.32、SEQ ID.NO.37和SEQ ID.NO.20、SEQ ID.NO.38和SEQ ID.NO.21、SEQ ID.NO.39和SEQ ID.NO.22、SEQ ID.NO.40和SEQ ID.NO.23、SEQ ID.NO.41和SEQ ID.NO.24、SEQ ID.NO.42和SEQ ID.NO.25、SEQ ID.NO.29和SEQ ID.NO.28、SEQ ID.NO.29和SEQ ID.NO.28、SEQ ID.NO.42和SEQ ID.NO.25、SEQ ID.NO.41和SEQ ID.NO.24、SEQ ID.NO.40和SEQ ID.NO.23、SEQ ID.NO.39和SEQ ID.NO.32、SEQ ID.NO.38和SEQ ID.NO.22、SEQ ID.NO.37和SEQ ID.NO.21、SEQ ID.NO.36和SEQ ID.NO.20。
在另一些可选地实施方式中，所述新型冠状病毒疫苗中含有两种RNA分子，两种RNA分子分别含有第一开放阅读框和第二开放阅读框，第一开放阅读框的核苷酸序列如SEQ ID.NO.9所示，第二开放阅读框的核苷酸序列如SEQ ID.NO.27所示。两种RNA分子序列特征还包括5’帽子(m7G(5’)(2’-OMeA)pG)、5’UTR(如SEQ ID.NO.1所示)、3’UTR(如SEQ ID.NO.2所示)和100个polyA的3’尾(如SEQ ID.NO.3所示)、N1-甲基-假尿苷(m1ψ)修饰核苷(置换全部尿苷)。含有第一开放阅读框的RNA分子和含有第二开放阅读框的RNA分子的质量比(1:9)～(9:1)；例如可以为但不限于为1:9、1:8、1:7、1:6、1:5、1:4、1:3、1:2、1:1、2:1、3:1、4:1、5:1、6:1、7:1、8:1或9:1。先将两种RNA分子按照配方量混合，然后制备包裹有两种RNA分子的LNP，得到所述新型冠状病毒疫苗中的有效成分。
上述实施方式中，第一开放阅读框的核苷酸序列还可以选自SEQ ID.NO.9、SEQ ID.NO.36、SEQ ID.NO.37、SEQ ID.NO.38、SEQ ID.NO.39、SEQ ID.NO.40、SEQ ID.NO.41、SEQ ID.NO.42或SEQ ID.NO.29所示中的任意一种；第二开放阅读框的核苷酸序列还可以选自SEQ ID.NO.27、SEQ ID.NO.32、SEQ ID.NO.20、SEQ ID.NO.21、SEQ ID.NO.22、SEQ ID.NO.23、SEQ ID.NO.24、SEQ ID.NO.25或SEQ ID.NO.28所示中的任意一种。
上述实施方式中，第一开放阅读框和第二开放阅读框的具体的组合方式例如可以为但不限于为：SEQ ID.NO.9和SEQ ID.NO.27、SEQ ID.NO.9和SEQ ID.NO.32；SEQ ID.NO.36和SEQ ID.NO.32、SEQ ID.NO.37和SEQ ID.NO.20、SEQ ID.NO.38和SEQ ID.NO.21、SEQ ID.NO.39和SEQ ID.NO.22、SEQ ID.NO.40和SEQ ID.NO.23、SEQ ID.NO.41和SEQ ID.NO.24、SEQ ID.NO.42和SEQ ID.NO.25、SEQ ID.NO.29和SEQ ID.NO.28、SEQ ID.NO.29和SEQ ID.NO.28、SEQ ID.NO.42和SEQ ID.NO.25、SEQ ID.NO.41和SEQ ID.NO.24、SEQ ID.NO.40和SEQ ID.NO.23、SEQ ID.NO.39和SEQ ID.NO.32、SEQ ID.NO.38和SEQ ID.NO.22、SEQ ID.NO.37和SEQ ID.NO.21、SEQ ID.NO.36和SEQ ID.NO.20。
在另一些可选地实施方式中，所述新型冠状病毒疫苗中含有融合RNA分子，融合RNA分子含有至少含有第一开放阅读框和第二开放阅读框的两个编码区，其中第一开放阅读框序列选自SEQ ID.NO.9所示核苷酸序列，第二开放阅读框序列选自SEQ ID.NO.27所示核苷酸序列。
融合RNA分子的结构为5’帽子-5’UTR-(第一编码区-linker序列)n-(linker序列-第二编码区)m-3’UTR-3’polyA尾，n和m分别独立的为正整数。其中5’帽子为m7G(5’)(2’-OMeA)pG、5’UTR如SEQ ID.NO.1所示、3’UTR如SEQ ID.NO.2所示和polyA如SEQ ID.NO.3所示，Linker序列均如SEQ ID.NO.21所示。n和m的比为(1:9)～(9:1)；例如可以为但不限于为1:9、1:8、1:7、1:6、1:5、1:4、1:3、1:2、1:1、2:1、3:1、4:1、5:1、6:1、7:1、8:1或9:1。将上述融合RNA分子制备得到LNP，作为新型冠状病毒疫苗中的有效成分。
上述实施方式中，第一开放阅读框的核苷酸序列还可以选自第一开放阅读框的核苷酸序列还可以选自SEQ ID.NO.9、SEQ ID.NO.36、SEQ ID.NO.37、SEQ ID.NO.38、SEQ ID.NO.39、SEQ ID.NO.40、SEQ ID.NO.41、SEQ ID.NO.42或SEQ ID.NO.29所示中的任意一种；第二开放阅读框的核苷酸序列还可以选自SEQ ID.NO.27、SEQ ID.NO.32、SEQ ID.NO.20、SEQ ID.NO.21、SEQ ID.NO.22、SEQ ID.NO.23、SEQ ID.NO.24、SEQ ID.NO.25或SEQ ID.NO.28所示中的任意一种。
上述实施方式中，第一开放阅读框和第二开放阅读框的具体的组合方式例如可以为但不限于为：SEQ ID.NO.9和SEQ ID.NO.27、SEQ ID.NO.9和SEQ ID.NO.32；SEQ ID.NO.36和SEQ ID.NO.32、SEQ ID.NO.37和SEQ ID.NO.20、SEQ ID.NO.38和SEQ ID.NO.21、SEQ ID.NO.39和SEQ ID.NO.22、SEQ ID.NO.40和SEQ ID.NO.23、SEQ ID.NO.41和SEQ ID.NO.24、SEQ ID.NO.42和SEQ ID.NO.25、SEQ ID.NO.29和SEQ ID.NO.28、SEQ ID.NO.29和SEQ ID.NO.28、SEQ ID.NO.42和SEQ ID.NO.25、SEQ ID.NO.41和SEQ ID.NO.24、SEQ ID.NO.40和SEQ ID.NO.23、SEQ ID.NO.39和SEQ ID.NO.32、SEQ ID.NO.38和SEQ ID.NO.22、SEQ ID.NO.37和SEQ ID.NO.21、SEQ ID.NO.36和SEQ ID.NO.20。
根据本公开的另一个方面，本公开还提供了上述新型冠状病毒疫苗的制备方法，所述制备方法包括将所述核酸分子与任选地辅料混合，得到所述新型冠状病毒疫苗。
在一些可选地实施方式中，所述新型冠状病毒疫苗包括核酸脂质纳米颗粒，所述制备方法包括：
分别制备包裹有含有第一开放阅读框的核酸分子的核酸脂质纳米颗粒，和，包裹有含有第二开放阅读框的核酸分子的核酸脂质纳米颗粒，得到分别包裹不同开放阅读框的核酸脂质纳米颗粒，然后按照配方量将两种核酸脂质纳米颗粒混合。
或者，先将含有第一开放阅读框的核酸分子和含有第二开放阅读框的核酸分子按照配方量混合，再制备得到包裹有含有两种核酸分子的核酸脂质纳米颗粒。
或者，制备包裹有同时含有第一开放阅读框和第二开放阅读框的核酸分子的核酸脂质纳米颗粒。
上述核酸脂质纳米颗粒的制备方法优选按照上述新型冠状病毒疫苗技术方案部分记载的脂质纳米颗粒(LNP)的方法，在此不再赘述。
在一些可选的实施方式中，本公开还提供了上述新型冠状病毒疫苗，或上述新型冠状病毒疫苗的制备方法在制备被配置为预防或治疗新型冠状病毒引发的疾病的产品中的应用。
在一些可选的实施方式中，本公开还提供了一种被配置为预防或治疗新型冠状病毒引发的疾病的产品，该产品包含上述新型冠状病毒疫苗。
上述被配置为预防或治疗新型冠状病毒引发的疾病的产品例如可以为但不限于为还含有被配置为接种上述新型冠状病毒疫苗装置的试剂盒；还含有其他预防或治疗活性成分的试剂盒；或，还含有用于评价上述新型冠状病毒疫苗有效性物质的试剂盒等。
下面结合优选实施例进一步说明本公开的技术方案和有益效果。
实施例1
本实施例提供了一种包含RNA的脂质纳米颗粒的制备方法，其中脂质纳米颗粒按照摩尔百分比计包括：Dlin-MC3-DMA 50％、DOPG 20％、胆固醇29％和PEG-DMG 1％，制备方法如下：
(a)将RNA溶解于pH4的柠檬酸盐缓冲液，将浓度调整为0.1mg/ml，得到水相。
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(b)按照配方量将Dlin-MC3-DMA、DOPG、胆固醇和PEG-DMG按照配方量溶解于无水乙醇，将有机相中的脂质成分的浓度调整至6mg/mL，得到有机相。
(c)将步骤(a)的水相和步骤(b)的有机相按照1:3的体积比，使用微流控设备，按照12mL/min的流速混合，混合液立刻用pH7.4的PBS溶液稀释100倍，并使用切向流过滤(TFF)除去溶液中乙醇成分，然后浓缩至体系中的mRNA的浓度为55μg/ml，得到包含编码SARS-CoV-2病毒抗原的RNA的脂质纳米颗粒。
实施例2
利用荧光素酶作为报告基因，通过活体荧光成像技术研究不同疫苗载体的配方(如下表1所示，“MC3”是指Dlin-MC3-DMA，“+”：给药后通过小动物活体荧光成相系统检测到小鼠体内荧光素酶表达)在小鼠体内递送编码荧光素酶基因的mRNA的效率，并检测不同复合制剂(制备方法参见实施例1)的理化指标，结果如表1所示。通过研究发现，提高脂质与mRNA质量比有利于增加mRNA在脂质纳米颗粒中的包封率从而使其具有更高的稳定性，此外适度提高配方中聚乙二醇(PEG)的含量有利于提高mRNA在体内的表达效率。因此综合考虑mRNA包封率以及mRNA体内递送效率等因素，选取了3和4号配方用于后续mRNA疫苗的研究。
表1
实施例3
不同配方的阳离子脂质纳米颗粒包裹编码S蛋白全长的mRNA的能力及形成纳米颗粒的粒度数据如表2所示，几种配方均能将S蛋白mRNA压缩成粒径100nm以下表面电位净中性的纳米颗粒，同时均能包裹至少50％的mRNA，因此均可具备一定的体内递送效果。“MC3”是指Dlin-MC3-DMA。
表2
实施例4
本实施例设计了一系列mRNA序列，其中开放阅读框的序列如表3所示；除开放阅读框序列外，该系列mRNA序列特征还包括5’帽子(m7G(5’)(2’-OMeA)pG)、5’UTR(如SEQ ID.NO.1所示)、3’UTR(如SEQ ID.NO.2所示)和100个polyA的3’尾(如SEQ ID.NO.3所示)。
表3 mRNA序列设计方案
将上述各个mRNA按照实施例1提供的制备方法制备为脂质纳米颗粒。
实验A：选取5～8岁龄左右正常食蟹猴，进行疫苗免疫原性评价。动物被随机分为5组，即原始株组(SEQ ID.NO.13)(50μg/只)、Delta组(SEQ ID.NO.9)(50μg/只)、Beta组(SEQ ID.NO.5)(50μg/只)、Omicron组(SEQ ID.NO.11)(50μg/只)和阴性对照组，每组12只，雌雄各半。
将实施例2制备的mRNALNP制剂用于食蟹猴免疫实验，每只食蟹猴通过后肢大腿肌肉注射50微克(以mRNA计)，分别于D0(给药当天即为D0)和D21通过后肢大腿肌肉注射对应受试疫苗及对照疫苗，其中，阴性对照组注射0.5mL的PBS。首次免疫后28天抽取食蟹猴血清送第三方实验室进行SARS-CoV-2假病毒中和活性测试。在96孔板内将食蟹猴血清按照不同比例稀释后(初始稀释倍数为30)加入可感染SARS-CoV-2假病毒，并同时设置细胞对照和病毒对照，孵育1小时，加入事先准备好的细胞，于细胞培养箱中培养20～28小时，吸弃一部分上清，加入荧光素酶检测试剂，室温避光反应后，反复吹吸孔内细胞，使细胞充分裂解后，置于化学发光检测仪中读取发光值。确保病毒对照和细胞对照成立的前提下，采用Reed-Muench法计算EC50值。血清对应分组编号及结果如图1-1所示。
从图1-1可以看出：
(1)4种抗原制备而成疫苗制剂可刺激食蟹猴产生对目标毒株假病毒具有中和能力的抗体。
(2)编码新冠病毒Delta变异株S蛋白的mRNA(SEQ ID.NO.9)疫苗制剂刺激食蟹猴产生的抗体对新冠病毒的原始株、Alpha变异株、Beta变异株、Gamma变异株、Delta变异株和Omicron变异株6种毒株假病毒都具有较强的中和活性；
编码新冠病毒原始株S蛋白的mRNA(SEQ ID.NO.13)疫苗制剂刺激食蟹猴产生的抗体对新冠病毒的原始株、Alpha变异株、Beta变异株、Gamma变异株和Delta变异株5种毒株假病毒同样都具有较强的中和活性，而对Omicron变异株的中和活性则较弱；
编码新冠病毒Beta变异株S蛋白的mRNA(SEQ ID.NO.5)疫苗制剂刺激食蟹猴产生的抗体对新冠病毒的Beta变异株、Gamma变异株和Delta变异株3种毒株假病毒具有较强的中和活性，而对原始株、Alpha变异株和Omicron变异株的中和活性则较弱。
从刺激食蟹猴产生的抗体对各新冠病毒毒株及变异株的假病毒中和活性来看，编码新冠病毒Delta变异株和编码新冠病毒原始株S蛋白的mRNA疫苗制剂产生的抗体具备更广泛的中和活性。
实验B：选取5～8岁龄左右正常食蟹猴，进行疫苗免疫原性评价。动物被随机分为3组，即原始株组(SEQ ID.NO.13)(50μg/只)、Delta组(SEQ ID.NO.9)(50μg/只)、和阴性对照组，每组36只，雌雄各半。按照实验A所述方法开展SARS-CoV-2假病毒中和活性测试。确保病毒对照和细胞对照成立的前提下，采用Reed-Muench法计算EC50值。血清对应分组编号及结果如图1-2所示。编码新冠病毒Delta变异株S蛋白的mRNA疫苗制剂样品记为样品1-1，编码新冠病毒原始株S蛋白的mRNA疫苗制剂样品记为样品1-2。
编码新冠病毒Delta变异株S蛋白的mRNA疫苗制剂对流行毒株如Beta变异株和Omicron变异株具有更好的中和活性。
实施例5
Omicron毒株亚型毒株S蛋白的筛选：
本实施例设计了一系列mRNA序列信息，其中开放阅读框的序列如表4所示；除开放阅读框序列外，该系列mRNA序列特征还包括5’帽子(m7G(5’)(2’-OMeA)pG)、5’UTR(如SEQ ID.NO.1所示)、3’UTR(如SEQ ID.NO.2所示)和100个polyA的3’尾(如SEQ ID.NO.3所示)。
表4 mRNA序列设计方案
将样品1-4共四种不同抗原分别按照实施例1的方法制成疫苗，将不同组别mRNA混合物分别制备成LNP制剂，LNP制剂经检测包封率均90％以上，粒径约为70nm左右。
将制备的mRNALNP制剂用于C57小鼠免疫实验，每只小鼠通过后肢外侧大腿肌肉注射5微克(以mRNA计)，在间隔7天后进行二次免疫，每组3只小鼠。首次免疫后后14天抽取小鼠血清送第三方实验室进行SARS-CoV-2假病毒中和活性测试。在96孔板内将小鼠血清按照不同比例稀释后(初始稀释倍数为30)加入可感染SARS-CoV-2假病毒，并同时设置细胞对照和病毒对照，孵育1小时，加入事先准备好的细胞，于细胞培养箱中培养20～28小时，吸弃一部分上清，加入荧光素酶检测试剂，室温避光反应后，反复吹吸孔内细胞，使细胞充分裂解后，置于化学发光检测仪中读取发光值。确保病毒对照和细胞对照成立的前提下，采用Reed-Muench法计算EC50值。样品1-4制备的不同抗原的假病毒毒株的EC50值，结果如图2所示。
从图2结果可以看出：在mRNA疫苗开放阅读框编码不同的S蛋白(氨基酸序列如SEQ ID.NO.14、SEQ ID.NO.16、SEQ ID.NO.18、SEQ ID.NO.27所示)制备而成疫苗制剂中，编码氨基酸序列如SEQ ID.NO.27(编码氨基酸为新冠病毒Omicron BA.5毒株的S蛋白)的S蛋白的mRNA疫苗刺激小鼠产生对新冠病毒Omicron各个子变异株假病毒的中和抗体活性降低较少。
实施例6
本实施例提供了一系列新冠病毒二价疫苗，制备方法如下：
(a)按照实施例1的方法制备编码新冠病毒Omicron BA.1变异株S蛋白、编码新冠病毒Omicron BA.2变异株S蛋白和编码新冠病毒Omicron BA.5变异株S蛋白的mRNA(开放阅读框序列分别如SEQ ID.NO.14、SEQ ID.NO.16和SEQ ID.NO.27)脂质纳米颗粒样品1.1、1.2和1.3。
(b)按照实施例1的方法制备编码新冠病毒Delta变异株S蛋白的mRNA(SEQ ID.NO.9)脂质纳米颗粒样品2；
(c)将样品1.1、1.2和1.3分别与样品2以质量比3:1混合制备新冠病毒二价mRNA疫苗样品1、2和3。编码新冠病毒Omicron变异株和Delta变异株S蛋白的mRNA除了上述开放阅读框序列外，还包括5’帽子(m7G(5’)(2’-OMeA)pG)、5’UTR(如SEQ ID.NO.1所示)、3’UTR(如SEQ ID.NO.2所示)和100个polyA的3’尾(如SEQ ID.NO.3所示)。
将上述制备的3个样品用于C57小鼠免疫实验，每只小鼠通过后肢外侧大腿肌肉注射5微克(以mRNA计)，在间隔7天后进行二次免疫，每组3只小鼠。首次免疫后14天抽取小鼠血清送第三方实验室进行SARS-CoV-2假病毒中和活性测试。在96孔板内将小鼠血清按照不同比例稀释后(初始稀释倍数为30)加入可感染SARS-CoV-2假病毒，并同时设置细胞对照和病毒对照，孵育1小时，加入事先准备好的细胞，于细胞培养箱中培养20～28小时，吸弃一部分上清，加入荧光素酶检测试剂，室温避光反应后，反复吹吸孔内细胞，使细胞充分裂解后，置于化学发光检测仪中读取发光值。确保病毒对照和细胞对照成立的前提下，采用Reed-Muench法计算EC50值。血清对应分组编号及结果如图3所示。
从图上可以看到，新冠病毒二价mRNA疫苗样品3(包括编码新冠病毒Omicron BA.5变异株S蛋白的mRNA脂质纳米颗粒和编码新冠病毒Delta变异株S蛋白的mRNA脂质纳米颗粒)在假病毒测试中，针对各病毒变异株所产生的抗体活性下降较少。
实施例7
编码BA.5子变异毒株S蛋白mRNA序列的优化：
本实施例根据新冠病毒Omicron毒株S蛋白(氨基酸序列如SEQ ID.NO.26所示)及mRNA序列优化原则，设计了一系列mRNA序列，其中开放阅读框信息如下表4所示。
除了表4的开放阅读框外，该系列mRNA序列特征还包括5’帽子(m7G(5’)(2’-OMeA)pG)、5’UTR(如SEQ ID.NO.1所示)、3’UTR(如SEQ ID.NO.2所示)和100个polyA的3’尾(如SEQ ID.NO.3所示)。
表5 mRNA序列设计方案及相对表达量
表4中“局部的GC％含量”：从ORF的3’端到5’端，以60bp作为windowsize的局部序列中GC％含量。
将表4所示的mRNA转染细胞，检测S全长蛋白在细胞中的表达，结果如表4所示。详细方法如下：转染各mRNA24小时后的HEK293细胞进行裂解，按10μg总蛋白的上样量，采用SDS-PAGE免疫印记法对目的蛋白进行特异性检测，在本实施例中，分别使用抗SARS-S1蛋白抗体作为一抗、用羊抗鼠-HRP抗体作为二抗进行孵育，然后显色。对蛋白表达量检测结果进行分析时，使用内参β-actin进行标化定量，同时设置未转染mRNA的细胞作为阴性对照，比较不同mRNA转染细胞后表达蛋白量的差异。检测结果显示，均可以检测到S蛋白全长的表达以及S1亚基。各个序列的表达量以相对OD值计量，如表4所示；其中相对OD值的计算方法为：样品OD值/样品1的OD值。
从表4可以看出：当mRNA开放阅读框序列总体的GC％含量为54-60％时，局部的GC％含量不低于40％时，S蛋白的相对表达量较高(见SEQ ID.NO.27和32)；另外相比于mRNA开放阅读框序列SEQ ID.NO.32，mRNA开放阅读框序列为SEQ ID.NO.27的S蛋白表达量更高一些。
实施例8
将实施例7中的样品1和2共两种不同抗原分别按照实施例1的方法制成疫苗，将不同组别mRNA混合物分别制备成LNP制剂，LNP制剂经检测包封率均90％以上，粒径约为70nm。将所得两组疫苗制剂用于C57小鼠免疫实验，实验方法见实施例6所示。血清对应分组编号及结果如图4所示。
从图4中可以看出：由于mRNA序列Seq ID NO.27的表达量较高，由mRNA序列Seq ID NO.27制备的mRNA疫苗制剂产生的抗体中和能力最优。
实施例9
本实施例提供了一系列新冠病毒二价疫苗，制备方法如下：
(a)按照实施例1的方法制备编码新冠病毒Omicron BA.5变异株S蛋白的mRNA(SEQ ID.NO.27)脂质纳米颗粒样品1。
(b)按照实施例1的方法制备编码新冠病毒Delta变异株S蛋白的mRNA(SEQ ID.NO.9)脂质纳米颗粒样品2；
(c)将样品1与样品2以质量比1:15、1:10、1:9、1:4、1:3、1:1、3:1、4:1、9:1、10:1和15:1混合制备新冠病毒二价mRNA疫苗样品2-1～2-11。编码新冠病毒Omicron BA.5变异株和Delta变异株S蛋白的mRNA除了SEQ ID.NO.27和SEQ ID.NO.9开放阅读框外，还包括5’帽子(m7G(5’)(2’-OMeA)pG)、5’UTR(如SEQ ID.NO.1所示)、3’UTR(如SEQ ID.NO.2所示)和100个polyA的3’尾(如SEQ ID.NO.3所示)。
将上述制备的11个样品用于C57小鼠免疫实验，实验方法见实施例6所述。血清对应分组编号及结果如图5所示。
假病毒毒株EC50值不低于200时，认为所测试的疫苗产生的抗体中和活性更佳。图5中的条形图展示了样品2-1～2-11的假病毒毒株EC50值；虚线为指示线，指示假病毒毒株EC50值为200的位置。
实施例10
本实施例提供了一系列新冠病毒二价疫苗，制备方法如下：
本实施例提供的新冠病毒二价疫苗包含编码抗原1和抗原2的RNA的脂质纳米颗粒，其中脂质纳米颗粒按照摩尔百分比计包括Dlin-MC3-DMA50％、DOPG10％、胆固醇38.5％和PEG-DMG1.5％。
制备方法如下：
(a)将编码不同抗原的RNA溶解于pH4的柠檬酸盐缓冲液，将浓度调整为0.1mg/ml，得到水相；
(b)按照配方量将Dlin-MC3-DMA、DOPG、胆固醇和PEG-DMG按照配方量溶解于无水乙醇，将有机相中的脂质成分的浓度调整至6mg/mL，得到有机相。
(c)将步骤(a)的水相和步骤(b)的有机相按照1:3的体积比，使用微流控设备，按照12mL/min的流速混合，混合液立刻用pH7.4的PBS溶液稀释100倍，并使用切向流过滤(TFF)除去溶液中乙醇成分，然后浓缩至体系中的mRNA的浓度为0.55mg/ml，得到包含编码抗原1和抗原2的RNA的脂质纳米颗粒。
当编码抗原1的核苷酸开放阅读框序列为SEQ ID.NO.9(编码新冠病毒Delta变异株的S蛋白)、编码抗原2的核苷酸开放阅读框序列为SEQ ID.NO.27(编码新冠病毒Omicron BA.5变异株的S蛋白)时，抗原1和抗原2以1:15、1:10、1:9、1:4、1:3、1:1、3:1、4:1、9:1、10:1和15:1制备样品1-11。当编码抗原1和2的核苷酸序列除了开放阅读框外，还包括5’帽子(m7G(5’)(2’-OMeA)pG)、5’UTR(如SEQ ID.NO.1所示)、3’UTR(如SEQ ID.NO.2所示)和100个polyA的3’尾(如SEQ ID.NO.3所示)。M1和M2分别为包括5’ 帽子(m7G(5’)(2’-OMeA)pG)、5’UTR、开放阅读框、3’UTR和3’尾的编码抗原1的mRNA质量和编码抗原2的mRNA质量。
将上述制备的11个样品用于C57小鼠免疫实验，实验方法见实施例6所述。血清对应分组编号及结果如图6。
假病毒毒株EC50值不低于200时，认为所测试的疫苗产生的抗体中和活性更佳。图6中的条形图展示了样品1-11的假病毒毒株EC50值；虚线为指示线，指示假病毒毒株EC50值为200的位置。
从图6中可以看出：当M1：M2为(1：9)～(9:1)时(样品3～9)，新冠病毒二价mRNA疫苗刺激小鼠产生的抗体对流行的新冠病毒各变异株的中和活性较好。其中M1：M2为(1：1)～(9:1)时(样品6～9)，抗体的中和活性更优。
实施例11
按照实施例9所述的方法制备编码新冠病毒Delta变异株S蛋白的mRNA(SEQ ID.NO.9)和编码新冠病毒OmicronBA.5变异株S蛋白的mRNA(SEQ ID.NO.27)的新冠二价mRNA脂质纳米颗粒疫苗，2种脂质纳米颗粒的质量比例分别为1:1和3:1，分别记作样品a和b。
按照实施例10所述的方法制备编码新冠病毒Delta变异株S蛋白的mRNA(SEQ ID.NO.9)和编码新冠病毒OmicronBA.5变异株S蛋白的mRNA(SEQ ID.NO.27)的新冠二价mRNA脂质纳米颗粒疫苗，2种mRNA的质量比为1:1和3:1，记作样品c和样品d。
上述的mRNA序列特征还包括5’帽子(m7G(5’)(2’-OMeA)pG)、5’UTR(如SEQ ID.NO.1所示)、3’UTR(如SEQ ID.NO.2所示)和100个polyA的3’尾(如SEQ ID.NO.3所示)。
按照实施例6的方法测试本实施例中制备的新冠病毒二价mRNA疫苗样品a、b、c和d的假病毒毒株EC50值，实验结果如图7所示。
实施例12
本实施例提供的新型冠状病毒疫苗中的RNA分子为融合RNA的结构，具体结构如下：
5’帽子-5’UTR-(第一编码区-linker序列)n-(linker序列-第二编码区)m-3’UTR--3’polyA尾；n＝3，m＝1；
其中，第一编码区的核苷酸序列如SEQ ID.NO.9所示；第二编码区的核苷酸序列如SEQ ID.NO.27所示；
5’帽子为m7G(5’)(2’-OMeA)pG；5’UTR如SEQ ID.NO.1所示；3’UTR如SEQ ID.NO.2所示；100个polyA的3’尾如SEQ ID.NO.3所示；
Linker序列均如SEQ ID.NO.31所示。
将上述融合RNA按照实施例1提供的制备方法制备成LNP，按照实施例6的方法测试本实施例中制备的新冠病毒二价mRNA疫苗样品的假病毒毒株EC50值，实验结果如图8所示。
实施例13
本实施例提供了一系列新冠病毒二价疫苗，按照实施例9所述的方法制备，其中编码新冠病毒Omicron BA.5变异株S蛋白的mRNA开放阅读框序列和编码新冠病毒Delta变异株S蛋白的mRNA开放阅读框序列、以及质量比如下表所示：
表6
按照实施例6的方法测试本实施例中制备的新冠病毒二价mRNA疫苗样品12-1～12-11的假病毒毒株EC50值，实验结果如图9所示。
实施例14
本实施例提供了一系列新冠病毒二价疫苗，按照实施例10所述的方法制备，其中编码新冠病毒Omicron BA.5变异株S蛋白的mRNA开放阅读框序列和编码新冠病毒Delta变异株S蛋白的mRNA开放阅读框序列、以及质量比如下表所示：
表7
按照实施例6的方法测试本实施例中制备的新冠病毒二价mRNA疫苗样品13-1～13-11的假病毒毒株EC50值，实验结果如图10所示。
实施例15
本实施例提供了新冠病毒二价疫苗和新冠原始株疫苗在小鼠体内的免疫效果，利用CPE法进行针对原始株和当下流行毒株的真病毒交叉中和活性检测。
新冠病毒二价疫苗按照实施例10所述的方法制备，其中编码新冠病毒Omicron BA.5变异株S蛋白的mRNA开放阅读框序列为SEQ ID.NO.27所示，编码新冠病毒Delta变异株S蛋白的mRNA开放阅读框序列为SEQ ID.NO.9所示，编码新冠病毒Omicron BA.5变异株S蛋白的mRNA和编码新冠病毒Delta变异株S蛋白的mRNA质量比为1:3。除了开放阅读框外，新冠病毒二价疫苗的mRNA序列还包括5’帽子(m7G(5’)(2’-OMeA)pG)、5’UTR(如SEQ ID.NO.1所示)、3’UTR(如SEQ ID.NO.2所示)和100个polyA的3’尾(如SEQ ID.NO.3所示)。制备为样品14-1。
新冠原始株疫苗按照实施例1所述的方法制备，其中编码新冠病毒原始株S蛋白的mRNA开放阅读框序列为SEQ ID.NO.13所示；除了开放阅读框外，新冠病毒二价疫苗的mRNA序列还包括5’帽子(m7G(5’)(2’-OMeA)pG)、5’UTR(如SEQ ID.NO.1所示)、3’UTR(如SEQ ID.NO.2所示)和100个polyA的3’尾(如SEQ ID.NO.3所示)。制备为样品14-2。
分别使用两种新冠mRNA疫苗样品14-1和样品14-2，分别对6-8周雌性KM小鼠后腿前外侧区肌肉注射给药(i.m.)免疫，剂量为15μg/只，每组10只，共3组。两种疫苗均按照临床拟定的免疫程序进行在D0、D28进行免疫。在D34动物进行眶后采血，分离血清后用CPE法对免后血清进行针对原始株和当下流行毒株的交叉中和活性检测。通过检测两种不同的疫苗免疫后血清中和抗体水平，进行品14-1和样品14-2两种疫苗的优效性比较。
分别比较样品14-2和样品14-1两种疫苗在D0、D28免疫两次后血清对新冠病毒原始株和当下流行毒株新冠病毒变异株XBB.1，新冠病毒变异株BQ1.1和新冠病毒变异株BA.5的交叉中和活性，结果显示，样品14-2对原始株、BA.5、XBB.1和BQ1.1的中和抗体几何平均滴度(GMT)分别为：32、12、12和7；样品14-1对原始株、BA.5、XBB.1和BQ1.1的中和抗体几何平均滴度(GMT)分别为：12、2702、446和676(详见下表)；在针对BA.5、XBB.1和BQ1.1三种当下流行毒株时，与样品14-2相比，样品14-1中和活性分别提高225.2倍、37.2倍和96.6倍，样品14-1的中和活性均显著优于样品14-2(P＜0.001，针对原始株，两者无显著性差异(见图11)。
表8
最后应说明的是：以上各实施例仅用以说明本公开的技术方案，而非对其限制；尽管参照前述各实施例对本公开进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本公开各实施例技术方案的范围。
工业实用性
本公开提供的新型冠状病毒疫苗以核酸作为疫苗的主要免疫原物质，包含含有第一开放阅读框和含有第二开放阅读框的核酸分子，其中第一开放阅读框编码新型冠状病毒Delta变异株S蛋白，第二开放阅读框编码新型冠状病毒奥密克戎(Omicron)子变异株BA.5S蛋白。该新型冠状病毒疫苗免疫机体后，能够在机体内表达新型冠状病毒Delta变异株和奥密克戎子变异株BA.5的S蛋白，对于新冠病毒各变异株产生的抗体活性下降较少，为一种多价疫苗。

Claims (10)

  1. 新型冠状病毒疫苗，其特征在于，所述新型冠状病毒疫苗包含含有第一开放阅读框和含有第二开放阅读框的核酸分子；
    所述第一开放阅读框编码新型冠状病毒Delta变异株S蛋白；所述第二开放阅读框编码新型冠状病毒奥密克戎子变异株BA.5 S蛋白。
  2. 根据权利要求1所述的新型冠状病毒疫苗，其特征在于，所述新型冠状病毒疫苗包含：含有第一开放阅读框的核酸分子；和，含有第二开放阅读框的核酸分子；
    或，所述新型冠状病毒疫苗包含：同时含有第一开放阅读框和第二开放阅读框的融合核酸分子。
  3. 根据权利要求1所述的新型冠状病毒疫苗，其特征在于，新型冠状病毒Delta变异株S蛋白的氨基酸序列如SEQ ID.NO.8所示，或，包含与SEQ ID.NO.8至少80％同一性的氨基酸序列，例如可以为但不限于为包含与SEQ ID.NO.8至少80％、85％、90％、95％或98％同一性的氨基酸序列；
    所述新型冠状病毒奥密克戎子变异株BA.5 S蛋白的氨基酸序列如SEQ ID.NO.26所示，或，包含与SEQ ID.NO.26至少80％同一性的氨基酸序列，例如可以为但不限于为包含与SEQ ID.NO.26至少80％、85％、90％、95％或98％同一性的氨基酸序列。
  4. 根据权利要求1所述的新型冠状病毒疫苗，其特征在于，所述核酸分子为RNA；
    在一个或多个实施例中，RNA中开放阅读框的部分总GC％含量为30～70％，所述开放阅读框的片段中任意一长度为60bp的片段中GC％含量不低于40％；在一个或多个实施例中，RNA中开放阅读框的部分总GC％含量为50％～60％；
    在一个或多个实施例中，所述RNA还包括5’帽子、5’UTR、3’UTR、polyA尾、起始区、终止区、信号序列区和linker序列中的一种或多种；
    在一个或多个实施例中，所述RNA从5’端到3’端依次包括：5’帽子-5’UTR-第一开放阅读框和/或第二开放阅读框-3’UTR-3’polyA尾；在一个或多个实施例中，所述RNA含有第一开放阅读框和第二开放阅读框；
    在一个或多个实施例中，所述RNA从5’端到3’端依次包括：5’帽子-5’UTR-起始区-第一开放阅读框-linker序列-第二开放阅读框-3’UTR-终止区-3’polyA尾；
    在一个或多个实施例中，所述RNA从5’端到3’端依次包括：5’帽子-5’UTR-(第一开放阅读框-linker序列)n-(linker序列-第二开放阅读框)m-3’UTR-3’polyA尾；
    n为片段第一编码区-linker序列的重复数；m为片段linker序列-第二编码区的重复数；n和m分别独立的为正整数；
    在一个或多个实施例中，所述5’帽子为选自ARCA、m7G(5"")ppp(5"")(2""OMeA)pG、m7G(5"")ppp(5"")(2""OMeG)pG、m7(3""OMeG)(5"")ppp(5"")(2""OMeG)pG、m7(3""OMeG)(5"")ppp(5"")(2""OMeA)pG、mCAP、dmCAP、tmCAP或dmCAP；
    在一个或多个实施例中，所述5’UTR的序列如SEQ ID.NO.1所示；在一个或多个实施例中，所述3’UTR的序列如SEQ ID.NO.2所示；在一个或多个实施例中，所述3’polyA尾长度为50～200；在一个或多个实施例中，所述3’polyA尾长度为80～200；在一个或多个实施例中，所述3’polyA尾的序列如SEQ ID.NO.3所示；
    在一个或多个实施例中，所述RNA中的一个或多个尿苷用修饰核苷进行置换；在一些实施方案中，所述修饰核苷选自假尿苷(ψ)、N1-甲基-假尿苷(m1ψ)或5-甲基-尿苷(m5U)中的至少一种；
    在一个或多个实施例中，linker序列中含有蛋白质切割信号；
    在一个或多个实施例中，所述蛋白质切割信号包括蛋白前体转化酶、激素前体转化酶、凝血酶、因子Xa蛋白酶中至少一种的切割信号；在一个或多个实施例中，所述蛋白质切割信号为Furin切割位点；
    在一个或多个实施例中，所述Furin切割位点的RNA核苷酸序列如SEQ ID.NO.7所示；
    在一个或多个实施例中，所述linker序列为可切割接头或蛋白酶敏感接头；
    在一个或多个实施例中，所述可切割接头包括F2A接头、P2A接头、T2A切割接头或E2A切割接头。
  5. 根据权利要求4所述的新型冠状病毒疫苗，其特征在于，所述第一开放阅读框的核苷酸序列如SEQ ID.NO.9、SEQ ID.NO.36、SEQ ID.NO.37、SEQ ID.NO.38、SEQ ID.NO.39、SEQ ID.NO.40、SEQ ID.NO.41、SEQ ID.NO.42或SEQ ID.NO.29所示；
    在一个或多个实施例中，所述第二开放阅读框编码新型冠状病毒奥密克戎变异株为BA.5子变异株；所述第二开放阅读框的核苷酸序列如SEQ ID.NO.27、SEQ ID.NO.32、SEQ ID.NO.20、SEQ ID.NO.21、SEQ ID.NO.22、SEQ ID.NO.23、SEQ ID.NO.24、SEQ ID.NO.25或SEQ ID.NO.28所示；
    在一个或多个实施例中，第一开放阅读框和第二开放阅读框的组合方式选自：SEQ ID.NO.9和SEQ ID.NO.27、SEQ ID.NO.9和SEQ ID.NO.32；SEQ ID.NO.36和SEQ ID.NO.32、SEQ ID.NO.37和SEQ ID.NO.20、SEQ ID.NO.38和SEQ ID.NO.21、SEQ ID.NO.39和SEQ ID.NO.22、SEQ ID.NO.40和SEQ ID.NO.23、SEQ ID.NO.41和SEQ ID.NO.24、
    SEQ ID.NO.42和SEQ ID.NO.25、SEQ ID.NO.29和SEQ ID.NO.28、SEQ ID.NO.29和SEQ ID.NO.28、SEQ ID.NO.42和SEQ ID.NO.25、SEQ ID.NO.41和SEQ ID.NO.24、SEQ ID.NO.40和SEQ ID.NO.23、SEQ ID.NO.39和SEQ ID.NO.32、SEQ ID.NO.38和SEQ ID.NO.22、SEQ ID.NO.37和SEQ ID.NO.21、SEQ ID.NO.36和SEQ ID.NO.20。
  6. 根据权利要求1-5任一项所述的新型冠状病毒疫苗，其特征在于，含有编码第一开放阅读框的核酸分子和含有编码第二开放阅读框的核酸分子的质量比为(1:9)～(9:1)；在一个或多个实施例中，含有编码第一开放阅读框的核酸分子和含有编码第二开放阅读框的核酸分子的质量比为(1:9)、(1:8)、(1:7)、(1:6)、(1:5)、(1:4)、(1:3)、(1:2)、(1:1)、(2:1)、(3:1)、(4:1)、(5:1)、(6:1)、(7:1)、(8:1)、(9:1)；在一个或多个实施例中，含有编码第一开放阅读框的核酸分子和含有编码第二开放阅读框的核酸分子的质量比为3:1；
    在一个或多个实施例中，所述核酸分子为融合核酸分子，第一开放阅读框和第二开放阅读框在所述融合核酸分子中的重复数为(1:9)～(9:1)；在一个或多个实施例中，第一开放阅读框和第二开放阅读框在所述融合核酸分子中的重复数为(1:9)、(1:8)、(1:7)、(1:6)、(1:5)、(1:4)、(1:3)、(1:2)、(1:1)、(2:1)、(3:1)、(4:1)、(5:1)、(6:1)、(7:1)、(8:1)、(9:1)；在一个或多个实施例中，第一开放阅读框和第二开放阅读框在所述融合核酸分子中的重复数为3:1。
  7. 根据权利要求1-5任一项所述的新型冠状病毒疫苗，其特征在于，所述疫苗还包括递送制剂；
    在一个或多个实施例中，所述新型冠状病毒疫苗含有由所述核酸分子和脂质成分构成的核酸脂质纳米颗粒；
    在一个或多个实施例中，所述新型冠状病毒疫苗选自(a)、(b)或(c)：
    (a)、所述新型冠状病毒疫苗包含:包裹有含有第一开放阅读框的核酸分子的核酸脂质纳米颗粒，和，包裹有含有第二开放阅读框的核酸分子的核酸脂质纳米颗粒；
    (b)、所述新型冠状病毒疫苗包含:包裹有含有第一开放阅读框的核酸分子，和含有第二开放阅读框的核酸分子的核酸脂质纳米颗粒；
    (c)、所述新型冠状病毒疫苗包含：包裹有同时含有第一开放阅读框和第二开放阅读框的融合核酸分子的核酸脂质纳米颗粒；
    在一个或多个实施例中，含有编码第一开放阅读框的核酸分子和含有编码第二开放阅读框的核酸分子的质量比为(1:9)～(9:1)；在一个或多个实施例中，含有编码第一开放阅读框的核酸分子和含有编码第二开放阅读框的核酸分子的质量比为(1:9)、(1:8)、(1:7)、(1:6)、(1:5)、(1:4)、(1:3)、(1:2)、(1:1)、(2:1)、(3:1)、(4:1)、(5:1)、(6:1)、(7:1)、(8:1)、(9:1)；在一个或多个实施例中，含有编码第一开放阅读框的核酸分子和含有编码第二开放阅读框的核酸分子的质量比为3:1。
    在一个或多个实施例中，所述核酸分子为融合核酸分子，第一开放阅读框和第二开放阅读框在所述融合核酸分子中的重复数为(1:9)～(9:1)；在一个或多个实施例中，第一开放阅读框和第二开放阅读框在所述融合核酸分子中的重复数为(1:9)、(1:8)、(1:7)、(1:6)、(1:5)、(1:4)、(1:3)、(1:2)、(1:1)、(2:1)、(3:1)、(4:1)、(5:1)、(6:1)、(7:1)、(8:1)、(9:1)；在一个或多个实施例中，第一开放阅读框和第二开放阅读框在所述融合核酸分子中的重复数为3:1；
    在一个或多个实施例中，按摩尔百分比计，所述脂质成分包括20～50％可质子化阳离子脂质、20～50％结构脂质、5～20％辅助脂质和1～5％表面活性剂，其中，可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂的摩尔含量合计100％；
    在一个或多个实施例中，所述可质子化阳离子脂质包括DlinMC3-DMA、DODMA、C12-200和DlinDMA中的至少一种；
    在一个或多个实施例中，所述辅助脂质包括DSPC、DOPE、DOPC、DOPG和DOPS中的至少一种；在一个或多个实施例中，所述结构脂质包括胆固醇和/或胆固醇衍生物；在一个或多个实施例中，所述表面活性剂包括PEG-DMG、PEG-DSPE和TPGS中的至少一种；
    在一个或多个实施例中，按照摩尔百分比计所述脂质成分包括Dlin-MC3-DMA 50％、DOPG 10％、胆固醇38.5％和PEG-DMG 1.5％；
    在一个或多个实施例中，按照摩尔百分比计，所述脂质成分包括Dlin-MC3-DMA 50％、DOPG 20％、胆固醇29％和PEG-DMG 1％；
    在一个或多个实施例中，所述核酸脂质纳米颗粒按照如下方法制备得到：
    将含有核酸分子的水相和含有所述脂质成分的有机相混合均匀得到混合液，去除有机相后使体系中的核酸分子的浓度为1～100μg/ml，得到所述核酸脂质纳米颗粒；
    所述水相为含有0.08～1.2mg/L核酸分子的水相缓冲液，所述水相缓冲液为柠檬酸盐缓冲液或醋酸钠缓冲液；
    所述有机相为含有5～7mg/L所述脂质成分的无水C1～C4低碳醇；所述水相和所述有机相的体积比为1:2～4。
  8. 权利要求1-7任一项所述的新型冠状病毒疫苗的制备方法，其特征在于，包括将所述核酸分子与任选地辅料混合，得到所述新型冠状病毒疫苗；
    在一个或多个实施例中，所述新型冠状病毒疫苗包括核酸脂质纳米颗粒，所述制备方法包括：
    分别制备包裹有含有第一开放阅读框的核酸分子的核酸脂质纳米颗粒，和，包裹有含有第二开放阅读框的核酸分子的核酸脂质纳米颗粒；然后按照配方量将两种核酸脂质纳米颗粒混合；
    或，先将含有第一开放阅读框的核酸分子和含有第二开放阅读框的核酸分子按照配方量混合，再制备得到包裹有含有两种核酸分子的核酸脂质纳米颗粒；
    或，制备包裹有同时含有第一开放阅读框和第二开放阅读框的核酸分子的核酸脂质纳米颗粒；
    在一个或多个实施例中，所述核酸脂质纳米颗粒按照如下方法制备得到：
    将含有核酸分子的水相和含有脂质成分的有机相混合均匀得到混合液，去除有机相后使体系中的RNA的浓度为1～100μg/ml，得到所述核酸脂质纳米颗粒；
    所述水相为含有0.08～1.2mg/L核酸分子的水相缓冲液，所述水相缓冲液为柠檬酸盐缓冲液或醋酸钠缓冲液；
    所述有机相为含有5～7mg/L所述脂质成分的无水C1～C4低碳醇；所述水相和所述有机相的体积比为1:2～4。
  9. 权利要求1-7任一项所述的新型冠状病毒疫苗，或权利要求8所述的制备方法在制备被配置为预防或治疗新型冠状病毒引发的疾病的产品中的应用。
  10. 被配置为预防或治疗新型冠状病毒引发的疾病的产品，其特征在于，包含权利要求1-7任一项所述的新型冠状病毒疫苗。
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