WO2023217206A1 - 新冠病毒嵌合核酸疫苗及其用途 - Google Patents

新冠病毒嵌合核酸疫苗及其用途 Download PDF

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WO2023217206A1
WO2023217206A1 PCT/CN2023/093373 CN2023093373W WO2023217206A1 WO 2023217206 A1 WO2023217206 A1 WO 2023217206A1 CN 2023093373 W CN2023093373 W CN 2023093373W WO 2023217206 A1 WO2023217206 A1 WO 2023217206A1
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amino acid
acid sequence
vaccine
seq
new coronavirus
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PCT/CN2023/093373
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English (en)
French (fr)
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高福
王奇慧
戴连攀
杜沛
陈茜
马雪慧
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中国科学院微生物研究所
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This application relates to the field of biomedicine, specifically a novel coronavirus chimeric nucleic acid vaccine and its use.
  • Novel coronavirus pneumonia is an acute respiratory infectious disease caused by infection with a new coronavirus (also known as new coronavirus, SARS-CoV-2).
  • the new coronavirus belongs to the genus ⁇ -coronavirus of the family Coronaviridae. It has an envelope and is a single-stranded positive-strand RNA virus.
  • the spike protein also known as S protein
  • S protein on the surface of the new coronavirus is responsible for the binding and membrane fusion between the virus and the host cell membrane receptor.
  • RBD receptor binding domain
  • the COVID-19 epidemic is still severe around the world, and new coronavirus mutant strains continue to emerge and spread.
  • the Delta and Omicron mutant strains have successively swept the world and become the dominant epidemic strains.
  • the Omicron variant of the new coronavirus also exists in multiple subtypes (for example, BA.1, BA.2, BA.1.1, BA.3 subtypes), has a transmission speed exceeding Delta, and has become the dominant strain in the world. ;
  • the BA.2 subtype strain spreads faster than other Omicron subtypes and now accounts for the largest proportion.
  • the S protein of the Omicron mutant strain has more than 50 amino acid mutations, which is much more than the previous Delta mutant strain.
  • this application provides a polynucleotide, its related products and its use in preparing a vaccine for preventing and/or treating the new coronavirus, based on the polynucleotide or its related products.
  • the S protein RBD domain (or a part thereof), or (3) the new coronavirus Delta variant S protein RBD domain (or a part thereof) is directly connected in series with the new coronavirus Omicron variant S protein RBD domain (or a part thereof) Or recombinant chimeric antigen peptides formed through linkers; the chimeric nucleic acid vaccine can provide strong immune protection against a variety of new coronavirus strains, and can be sequenced with other types of vaccines
  • the present application provides a polynucleotide encoding a recombinant chimeric antigen peptide with a structure shown in formula (I): (AB)-C-(A-B') (I)
  • A-B represents the amino acid sequence of the RBD domain of the S protein of the new coronavirus prototype strain or a part thereof, or is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to it and is identical to it Amino acid sequences with the same or substantially the same immunogenicity;
  • A-B' represents the amino acid sequence of the RBD domain of the S protein of the new coronavirus Beta variant strain or a part thereof, or has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with it and An amino acid sequence that has the same or substantially the same immunogenicity; or
  • A-B represents the amino acid sequence of the RBD domain of the S protein of the new coronavirus Delta variant strain or a part thereof, or has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with it and An amino acid sequence that has the same or substantially the same immunogenicity;
  • A-B' represents the amino acid sequence of the RBD domain of the S protein of the new coronavirus Beta variant strain or a part thereof, or has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with it and An amino acid sequence that has the same or substantially the same immunogenicity; or
  • AB represents the amino acid sequence of the RBD domain of the S protein of the new coronavirus Delta variant strain or a part thereof, or has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with it and An amino acid sequence that has the same or substantially the same immunogenicity;
  • A-B' represents the amino acid sequence of the RBD domain of the S protein of the new coronavirus Omicron variant strain or a part thereof, or has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with it and An amino acid sequence that has the same or substantially the same immunogenicity;
  • a part of the RBD domain of the S protein of the new coronavirus prototype strain is at least 70%, 80%, 85%, 90%, 92%, 95%, 96% of its entire amino acid sequence. , 97%, 98% or 99%;
  • a part of the RBD domain of the S protein of the new coronavirus Beta variant strain is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97% of its entire amino acid sequence. 98% or 99%;
  • a part of the RBD domain of the S protein of the new coronavirus Delta variant strain is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97% of its entire amino acid sequence. 98% or 99%;
  • a part of the RBD domain of the S protein of the new coronavirus Omicron variant is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97% of its entire amino acid sequence. 98% or 99%;
  • n 0,1,2 or 3.
  • the amino acid sequence of the S protein RBD domain of the new coronavirus prototype strain or a part thereof is as shown in SEQ ID NO: 1, or the amino acid sequence as shown in SEQ ID NO: 1 is substituted , an amino acid sequence obtained by deleting or adding one or several amino acids and having the same or substantially the same immunogenicity;
  • amino acid sequence of the S protein RBD domain of the new coronavirus Beta variant strain or a part thereof is as shown in SEQ ID NO: 2, or the amino acid sequence as shown in SEQ ID NO: 2 is substituted, deleted or added
  • amino acid sequence derived from one or several amino acids and having the same or substantially the same immunogenicity is substituted, deleted or added.
  • amino acid sequence of the S protein RBD domain of the new coronavirus Delta variant strain or a part thereof is as shown in SEQ ID NO:3, or the amino acid sequence as shown in SEQ ID NO:3 is substituted, deleted or added
  • amino acid sequence derived from one or several amino acids and having the same or substantially the same immunogenicity is substituted, deleted or added.
  • amino acid sequence of the S protein RBD domain of the new coronavirus Omicron variant strain or a part thereof is as shown in SEQ ID NO: 4, or the amino acid sequence as shown in SEQ ID NO: 4 is substituted, deleted or added
  • amino acid sequence derived from one or several amino acids and having the same or substantially the same immunogenicity is substituted, deleted or added.
  • n 0, 1 or 2.
  • the recombinant chimeric antigen peptide with the structure shown in formula (I) has the amino acid sequence shown in SEQ ID NO: 5;
  • the recombinant chimeric antigen peptide with the structure shown in formula (I) has the amino acid sequence shown in SEQ ID NO: 6;
  • the recombinant chimeric antigen peptide with the structure shown in formula (I) has the structure shown in SEQ ID NO:7 amino acid sequence.
  • the polynucleotide is a DNA molecule
  • the DNA molecule has the DNA sequence shown in SEQ ID NO: 8;
  • the DNA molecule has the DNA sequence shown in SEQ ID NO: 9;
  • the DNA molecule has the DNA sequence shown in SEQ ID NO: 10.
  • the polynucleotide is an mRNA molecule
  • the mRNA molecule has the mRNA sequence shown in SEQ ID NO: 11;
  • the mRNA molecule has the mRNA sequence shown in SEQ ID NO: 12;
  • the mRNA molecule has the mRNA sequence shown in SEQ ID NO: 13.
  • the present application provides a nucleic acid construct comprising the polynucleotide as described in the first aspect above, and optionally, at least one expression control element operably linked to the polynucleotide.
  • the present application provides an expression vector comprising the nucleic acid construct as described in the above second aspect.
  • the present application provides a host cell, which is transformed or transfected with the polynucleotide as described in the first aspect, the nucleic acid construct as described in the second aspect, or the third aspect as described above.
  • Expression vector
  • the present application provides the polynucleotide as described in the first aspect, the nucleic acid construct as described in the second aspect, the expression vector as described in the third aspect, or the fourth aspect as described above.
  • the vaccine is used for separate immunization or sequential immunization with other types of new coronavirus vaccines; further preferably, the other types of new coronavirus vaccines include inactivated vaccines.
  • the present application provides a chimeric nucleic acid vaccine or immunogenic composition, which includes the polynucleotide as described in the first aspect, the nucleic acid construct as described in the second aspect, and the polynucleotide as described in the first aspect.
  • the chimeric nucleic acid vaccine or immunogenic composition is a new coronavirus DNA vaccine, and the DNA vaccine includes:
  • the DNA sequence of the recombinant chimeric antigen peptide is preferably the DNA sequence shown in SEQ ID NO: 8, 9 or 10;
  • the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors.
  • the chimeric nucleic acid vaccine or immunogenic composition is a new coronavirus mRNA vaccine, and the mRNA vaccine includes:
  • the chimeric nucleic acid vaccine or immunogenic composition is a novel coronavirus-viral vector vaccine, which includes:
  • the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, poxvirus vector, influenza virus vector, and adeno-associated virus vector.
  • the chimeric nucleic acid vaccine or immunogenic composition is in the form of a nasal spray, oral preparation, suppository or parenteral preparation;
  • the nasal spray is selected from aerosols, sprays and powder sprays;
  • the oral preparation is selected from tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film-coated agents, pellets, sublingual tablets and ointments;
  • the parenteral preparation is a transdermal preparation, an ointment, a plaster, a topical liquid, an injectable or a pushable preparation.
  • the present application provides a kit, which includes the chimeric nucleic acid vaccine or immunogenic composition as described in the sixth aspect, and optionally other types of new coronavirus vaccines, the chimeric Nucleic acid vaccines or immunogenic compositions are packaged separately from other types of novel coronavirus vaccines;
  • novel coronavirus vaccines are novel coronavirus inactivated vaccines.
  • the inventor of the present application has designed a polynucleotide encoding a combination of (1) the S protein RBD domain of the new coronavirus prototype strain (or a part thereof) and the S protein RBD domain of the new coronavirus Beta variant strain (or a part thereof), or (2) the novel coronavirus Delta variant S protein RBD domain (or a part thereof) and the novel coronavirus Beta variant strain S protein RBD domain (or a part thereof), or (3) the novel coronavirus A recombinant chimeric antigen peptide formed by directly connecting the S protein RBD domain (or a part thereof) of the virus Delta variant strain and the S protein RBD domain (or a part thereof) of the new coronavirus Omicron variant strain in series or through a linker; based on this polynucleoside
  • the acid-based chimeric nucleic acid vaccine can provide strong immune protection against a variety of new coronavirus strains, and is compatible with other Sequential immunization of various types of vaccines
  • Figure 1 shows the RBD dimer mRNA vaccine of the new coronavirus prototype strain constructed in Example 1 of the present application (referred to as PP mRNA vaccine, as a control vaccine), the chimeric RBD dimer formed by connecting the RBD of the new coronavirus prototype strain and the RBD of the Beta variant strain.
  • Body mRNA vaccine (referred to as PB mRNA vaccine), chimeric RBD dimer mRNA vaccine formed by the connection of Delta variant RBD and Beta variant RBD (referred to as DB mRNA vaccine), Chimeric RBD variant RBD formed by connection of Delta variant RBD and Omicron variant RBD
  • DO mRNA vaccine chimeric RBD dimer mRNA vaccine formed by the connection of Delta variant RBD and Beta variant RBD
  • DO mRNA vaccine chimeric RBD dimer mRNA vaccine formed by the connection of Delta variant RBD and Beta variant RBD
  • DO mRNA vaccine Schematic structural diagram of the combined RBD dimer mRNA vaccine
  • each segment of the mRNA vaccine is marked on the diagram, where 5'UTR represents the 5' untranslated region and 3'UTR represents the 3' untranslated region Region, SP represents the signal peptide sequence, Poly(A) represents the poly(A) tail, Protype RBD represents the RBD sequence of the prototype strain, Beta RBD represents the RBD sequence of the Beta variant strain, Delta
  • Figure 2 shows the humoral immunity levels stimulated by the PP, PB, DB, and DO mRNA vaccines constructed in Example 1 of the present application, as detected in Examples 3 and 4, where LNP represents immunity using lipid nanoparticles
  • the negative control group among them, Figure 2a is a schematic diagram of the mice immunized with the mRNA vaccine and the sampling procedure; Figure 2b and Figure 2c are respectively the serum collected on the 14th and 28th days after immunizing the mice with the mRNA vaccine against the new coronavirus.
  • the binding antibody titers of RBD antigens of the prototype strain, Delta variant strain, Beta variant strain and Omicron variant strain BA.1, BA.1.1, BA.2 and BA.3 subtypes are shown in the bar graphs of Figure 2b and Figure 2c , the number above each bar represents the ratio of the antibody titer represented by that bar to the antibody titer represented by the corresponding bar in the LNP group, and, as indicated by the arrows in the figure, the heat on the right side of Figures 2b and 2c The figure is based on these figures; Figure 2d shows how the serum collected on day 28 after immunizing mice with the mRNA vaccine neutralized the new coronavirus prototype strain, Delta variant strain, Omicron variant strain BA.1, and BA in the pseudovirus neutralization experiment.
  • the number above each column represents the geometric mean (GMT) of all samples in the experimental group, as shown by the arrow in the figure. The one on the right The heat map is based on these numbers; all data are presented as GMT ⁇ 95% CI (confidence interval).
  • Figure 3 shows the level of cellular immunity stimulated by the PP, PB, DB, and DO mRNA vaccines constructed in Example 1 of the present application.
  • LNP represents the negative control group immunized with lipid nanoparticles.
  • Figure 3a is a schematic diagram of the mice immunized with the mRNA vaccine and the sampling procedure
  • Figure 3b shows the ELISpot test of the spleen cells collected on the 21st day after the mice were immunized with the mRNA vaccine.
  • IFN ⁇ + cells produced after stimulation by a peptide library constructed from strains, Beta variant strains, and Omicron variant strain subtypes BA.1RBD
  • a bar chart of the number of cells. The number above the bar chart represents the ratio of the number of IFN ⁇ + cells represented by the bar to the number of IFN ⁇ + cells represented by the corresponding bar in the LNP group; all data are presented as Mean ⁇ SEM.
  • Figure 4 shows that after sequential immunization of mice using inactivated vaccines and the PP, PB, DB, DO mRNA vaccines constructed in Example 1 of the present application, compared with before sequential immunization (i.e., without using the mRNA vaccine Enhanced group), the fold increase in binding antibody titers;
  • Figure 4a is a schematic diagram of the sequential immunization and serum sampling procedures for mice, and IV represents the inactivated vaccine;
  • Figure 4b shows the results of each immunization procedure on day 35 (indicated by open circles) shown), and the serum collected on day 49 (shown as filled circles) was against the new coronavirus prototype strain, Delta variant strain, Beta variant strain, and Omicron variant strain subtypes BA.1, BA.1.1, BA.2, and BA.3RBD
  • the antigen-binding antibody titer level, and the fold improvement of the latter (i.e., day 49) relative to the former (i.e., day 35) antibody titer (the "number
  • Figure 5 shows the immune response level after sequential immunization of mice with inactivated vaccines and the PP, PB, DB, and DO mRNA vaccines constructed in Example 1 of the present application.
  • the immunization program is as shown in Figure 4a; in each column In the graph, "PP” represents the immune group of two inactivated vaccines + PP mRNA vaccine, "PB” represents the immune group of two inactivated vaccines + PB mRNA vaccines, and “DB” represents the immune group of two inactivated vaccines + DB mRNA vaccines.
  • Figure 5a shows the serum collected on day 49 against the new coronavirus prototype strain, Delta variant strain, Beta variant strain, Omicron variant strain subtype BA.1, BA.1.1, The binding antibody titers of BA.2 and BA.3 antigens.
  • the numbers in the first row above each column indicate the antibody titer represented by the column and the inactivated vaccine (IV) group.
  • the ratio of the antibody titer represented by the corresponding column in , the heat map on the right side of Figure 5a is based on the second row of numbers;
  • Figure 5b shows that the serum collected on day 49 neutralized 6 pseudoviruses (prototype strain, Delta variant strain, Omicron variant subtype) in the pseudovirus neutralization experiment.
  • BA.1, BA.1.1, BA.2, BA.3 in the bar chart in Figure 5b, the first row of numbers above each bar indicates the NT 50 titer value represented by the bar .
  • Example 1 New coronavirus prototype strain RBD dimer mRNA vaccine (referred to as PP mRNA vaccine, as a control), prototype strain and Beta variant chimeric RBD dimer mRNA vaccine (referred to as PB mRNA vaccine), Delta variant strain and Beta Construction, in vitro preparation and packaging of chimeric RBD dimer mRNA vaccines of mutant strains (DB mRNA vaccine for short) and chimeric RBD dimer mRNA vaccines of Delta mutant strains and Omicron mutant strains (DO mRNA vaccine for short)
  • the basic plasmid used for in vitro transcription of the mRNA vaccine is pUC57, provided by Nanjing GenScript Biotechnology Co., Ltd.
  • the DNA expression elements of the mRNA vaccine were introduced through conventional molecular biology methods, including: (1) T7 promoter, (2) DNA coding region of the mRNA vaccine (PB, DB, DO DNA coding sequence of the mRNA vaccine As shown in SEQ ID NO:8, 9, and 10 respectively, the DNA coding sequence of the PP mRNA vaccine as a control is shown in SEQ ID NO:14), (3) the 5' end UTR sequence upstream of the coding region (several The 5'-end UTR sequences of the mRNA vaccines are the same, as shown in SEQ ID NO:15), (4) signal peptide sequence (i.e.
  • the capping enzyme kit Cap1 capping enzyme kit (M082-01B, Suzhou Nearshore Protein Technology Co., Ltd.) was used to cap the 5' end of the purified in vitro transcribed mRNA with Cap1 to meet the requirements for eukaryotic expression. Conditions for translation in cells; thereafter, the mRNA was purified again using the same lithium chloride precipitation method as above to obtain purified 5'-end capped mRNA.
  • mice of the BALB/c strain purchased from Viton Lever
  • the experimental groups were divided into an mRNA vaccine immunization group and a negative control group, in which the mRNA vaccine immunization group
  • the groups include PP mRNA vaccine immunization group, PB mRNA vaccine immunization group, DB mRNA vaccine immunization group and DO mRNA vaccine immunization group, and the negative control group is the LNP immunization group.
  • mice in the mRNA vaccine immunization group were immunized with the same designed mRNA vaccine (i.e., PP, PB, DB or DO mRNA vaccine) on days 0 and 14, and mice in the negative control group were immunized at the same time and The same amount of empty LNP was injected.
  • the vaccination method was intramuscular injection, and the vaccination dose was 5 ⁇ g of mRNA vaccine or empty LNP per mouse.
  • Mouse serum samples were collected on days 14 and 28 respectively to test the binding antibody titer and pseudovirus neutralizing antibody titer of immune serum. In addition, mouse spleen samples were collected on day 21 for testing T cell immunity.
  • the coated ELISA plate was blocked in 5% skim milk for 1 hour; then, the serum collected from the mice in each experimental group in Example 2 was incubated at 56°C for 30 minutes for inactivation; the inactivated Serum samples were diluted threefold starting from 1:200 or 1:1000, and then the dilution was added to each well, followed by incubating the ELISA plate at 37°C for 1 hour; goat anti-mouse IgG-HRP antibody (purchased from EASYBio) was added to the plate as a secondary antibody and incubated again at 37°C for 1 hour; finally, 3,3',5,5'-tetramethylbenzidine (TMB) substrate was used for After color development, the reaction was terminated with 2M hydrochloric acid, and the absorbance at 450 nm and 630 nm was measured using a microplate reader (PerkinElmer).
  • TMB 3,3',5,5'-tetramethylbenzidine
  • the endpoint titer is defined as the dilution factor corresponding to the serum dilution when the absorbance produced by the serum (as mentioned above, the absorbance at 450 nm minus the absorbance at 630 nm) is 2.1 times greater than the background value.
  • Antibody titers below the detection limit were defined as one-third of the detection limit.
  • FIG. 2b and Figure 2c The binding antibody titers of the serum collected from mice in each experimental group on the 14th and 28th days of the immunization program against the RBD antigens of the seven new coronaviruses are shown in Figure 2b and Figure 2c respectively.
  • Figure 2b and Figure 2c In the figure, the left side is a bar graph of endpoint titer vs. vaccine type, and the right side is the corresponding heat map.
  • the heat map is based on the ratio of the endpoint antibody titer of each mRNA vaccine to the endpoint antibody titer of the LNP group. , as described in the figure captions.
  • Figure 2c shows the results on day 28 when the antibody level is relatively stable. It can be seen from Figure 2c:
  • the PB mRNA vaccine can induce higher binding antibody levels; and the binding antibody titer level it induces is equivalent to or higher than the PP mRNA vaccine (For example, those for BA.1.1 and BA.2 are 2-3 times higher respectively);
  • the DB mRNA vaccine can induce higher binding antibody levels for the tested new coronavirus prototype strains and various mutant strains; and the binding antibody titer levels it induces against various new coronavirus strains are much higher than PP mRNA vaccines, some are more than 3 times higher;
  • the DO mRNA vaccine can induce higher binding antibody levels; in particular, the serum antibody titer levels it induces against each subtype of the Omicron mutant strain are far higher than Higher than the PP mRNA vaccine; for example, against the BA.1 subtype, the antibody titer level induced by the DO mRNA vaccine is more than 2 times higher than that of the PP mRNA vaccine, and against the BA.1.1 subtype, the antibody titer induced by the DO mRNA vaccine The level is more than 5 times higher than that of PP mRNA vaccine. The antibody titer level induced against BA.2 and BA.3 subtypes is nearly 6 times higher than that of PP mRNA vaccine.
  • the antibody titer level induced against BA.3 subtype is higher than that of PP mRNA vaccine.
  • the PP mRNA vaccine is more than 3 times higher; this suggests that the DO mRNA vaccine of this application can induce significantly higher antibody titer levels against all subtypes of Omicron mutant strains, indicating that it will have significantly higher antibody titer levels against all types of Omicron mutant strains.
  • High immune protection efficacy; and, DO mRNA also induces higher antibody titer levels against the new coronavirus prototype strain and other mutant strains, which suggests that it has a good broad spectrum.
  • Example 4 Packaging and serum neutralization of pseudoviruses of new coronavirus strains
  • the immune mouse sera collected in Example 2 above were tested for their false positives against the new coronavirus prototype strain, Delta variant strain and Omicron variant strain BA.1, BA.1.1, BA.2, and BA.3 subtypes.
  • the 50% pseudovirus neutralizing titer of the virus (pVNT 50 ); the specific detection method is as follows:
  • the culture medium was DMEM medium containing 10% FBS.
  • pseudoviruses of the new coronavirus prototype strain, Delta variant strain and Omicron variant strain BA.1, BA.1.1, BA.2 and BA.3 subtypes were obtained respectively.
  • mice serum of each experimental group collected on the 28th day in Example 2 was incubated at 56°C for 30 minutes for inactivation; the inactivated serum samples were diluted, and a 2-fold gradient dilution was performed starting from 1:80. .
  • each pseudovirus was mixed with an equal volume of diluted serum and incubated at 37°C for 1 hour. Add 100 ⁇ l of the virus-serum mixture to the pre-plated Vero cells in a 96-well plate.
  • the CQ1 confocal image cytometer was used to detect the number of transduction units (TU) to calculate the response of the immune mouse serum to the above-mentioned new coronavirus prototype strain, Delta variant strain and Omicron variant strain BA.1, BA.1.1, Neutralizing ability of pseudoviruses of BA.2 and BA.3 subtypes.
  • TU transduction units
  • FIG. 2d shows the results of Figure 2d; as described in the figure description of Figure 2d, the left column chart of Figure 2d shows the neutralization of the prototype strain, Delta variant strain and Omicron variant strain BA.1 and BA in the serum of each immune group.
  • the pVNT 50 i.e., 50% pseudovirus neutralization titer
  • the heat map on the right shows the ratio of the pVNT 50 of each mRNA vaccine to the pVNT 50 of the LNP group. .
  • the serum neutralizing antibody titers induced by the PB mRNA vaccine against each subtype of the Omicron mutant strain are much higher than those induced by the PP mRNA vaccine; in particular, the neutralizing antibody titers induced by the PB mRNA vaccine against the BA.1 subtype Antibody titer
  • the level is more than 7 times higher than that of the PP mRNA vaccine.
  • the neutralizing antibody titer level induced against the BA.1.1 subtype is more than 6 times higher than that of the PP mRNA vaccine.
  • the neutralizing antibody titer induced against the BA.2 and BA.3 subtypes The titer level is about 6 times higher than that of the PP mRNA vaccine; this suggests that the PB mRNA vaccine of this application can induce significantly higher neutralizing antibody titer levels against all subtypes of Omicron variant strains, indicating that it can target all types of Omicron variant strains. All strains will have significantly higher immune protection efficacy; in addition, PB mRNA also has higher neutralizing antibody titer levels against the new coronavirus prototype strain and Delta variant strain, which suggests that it has a good broad spectrum.
  • the serum neutralizing antibody titer levels induced by the DB mRNA vaccine against the prototype strain, Delta variant strain, and Omicron variant strain subtypes are much higher than those of the PP mRNA vaccine; specifically, the DB mRNA vaccine induces against the prototype strain.
  • the neutralizing antibody titer level is more than 3 times higher than that of the PP mRNA vaccine
  • the neutralizing antibody titer level induced against the Delta variant strain is more than 5 times higher than that of the PP mRNA vaccine
  • the neutralizing antibody titer level induced against the Omicron variant BA.1 subtype The neutralizing antibody titer level is nearly 45 times higher than that of the PP mRNA vaccine.
  • the neutralizing antibody titer level induced against the Omicron variant BA.1.1 subtype is nearly 30 times higher than that of the PP mRNA vaccine.
  • the neutralizing antibody titer level induced against the Omicron variant BA.2 subtype is
  • the neutralizing antibody titer level induced by the Omicron variant strain BA.3 subtype is nearly 48 times higher than that of the PP mRNA vaccine, and the neutralizing antibody titer level induced by the Omicron variant BA.3 subtype is nearly 69 times higher than that of the PP mRNA vaccine; this suggests that this
  • the applied DB mRNA vaccine can induce significantly higher neutralizing antibody titer levels against various strains of the new coronavirus, indicating that it will have significantly higher immune protection efficacy against various strains of the new coronavirus.
  • the serum neutralizing antibody titer levels induced by the DO mRNA vaccine against the prototype strain, Delta variant strain, and Omicron variant strain subtypes are much higher than those of the PP mRNA vaccine; specifically, the DO mRNA vaccine induces against the prototype strain.
  • the neutralizing antibody titer level is nearly 13 times higher than that of the PP mRNA vaccine
  • the neutralizing antibody titer level induced against the Delta variant strain is nearly 5 times higher than that of the PP mRNA vaccine
  • the neutralizing antibody titer level is nearly 200 times higher than that of the PP mRNA vaccine.
  • the neutralizing antibody titer level induced against the Omicron variant BA.1.1 subtype is nearly 163 times higher than that of the PP mRNA vaccine.
  • the neutralizing antibody titer level induced against the Omicron variant BA.2 subtype is nearly 163 times higher than that of the PP mRNA vaccine.
  • the neutralizing antibody titer level induced by the Omicron mutant strain BA.3 subtype is nearly 230 times higher than that of the PP mRNA vaccine, and the neutralizing antibody titer level induced by the Omicron variant BA.3 subtype is nearly 407 times higher than that of the PP mRNA vaccine; this suggests that this
  • the applied DO mRNA vaccine can induce significantly higher neutralizing antibody titer levels against various strains of the new coronavirus, indicating that it will have significantly higher immune protection efficacy against various strains of the new coronavirus.
  • Example 5 Evaluation of the level of cellular immunity induced by mRNA vaccines
  • mice from each experimental group collected on the 21st day in Example 2 were used to detect the level of cellular immunity induced by the mRNA vaccine.
  • the specific method is as follows:
  • a cell homogenizer to prepare mouse spleen cells into single-cell homogenate in 1 ml of serum-free DMEM, filter it with a 40 ⁇ m cell filter, and lyse red blood cells with red blood cell lysis buffer (Beijing Solebao Technology Co., Ltd., R1010) cells; then, the cells were washed with washing solution (PBS+0.5% FBS), stained with 0.4% trypan blue solution (Gibco, 15250061), and counted using a Cell drop FL automatic cell counter.
  • the constructed peptide library (2 ⁇ g/ml of each polypeptide) was stimulated for 20 hours; the peptide library was performed using the software PeptGen Peptide Generator on the website https://www.hiv.lanl.gov/content/sequence/PEPTGEN/peptgen.html Design, the key parameters of the design include: the length of the short peptide is 18-20 amino acids, the overlapping amino acid fragment is 10 amino acids, etc.; the designed peptide library was synthesized by Zhongke Yaguang Biotechnology Co., Ltd.
  • the positive control wells were stimulated with phytohemagglutinin (PMA) to produce non-specific cellular immune responses, and the negative control wells were not stimulated with the peptide library.
  • PMA phytohemagglutinin
  • the experimental groups are: three times inactivated vaccine immunization group (i.e., "IV” group), two times inactivated vaccine + PP mRNA vaccine immunization group (referred to as "PP” group), two times inactivated vaccine + PB mRNA vaccine immunization group ( (referred to as "PB” group), twice inactivated vaccine + DB mRNA vaccine immune group (referred to as “DB” group), twice inactivated vaccine + DO mRNA vaccine immune group (referred to as "DO” group) and inactivated vaccine adjuvant +LNP immune group (referred to as the "LNP” group, serving as the negative control group).
  • IV group three times inactivated vaccine immunization group
  • PP two times inactivated vaccine + PP mRNA vaccine immunization group
  • PB two times inactivated vaccine + PB mRNA vaccine immunization group
  • DB twice inactivated vaccine + DB mRNA vaccine immune group
  • DO twice inactivated vaccine + DO mRNA vaccine immune group
  • IV group All mice were vaccinated with one dose of inactivated vaccine on days 0, 21 and 35;
  • mice All mice were vaccinated with one dose of inactivated vaccine on days 0 and 21, and then one dose of each mRNA vaccine on day 35;
  • LNP LNP group: All mice were vaccinated with Al adjuvant, the adjuvant of the inactivated vaccine, on days 0 and 21, and empty LNP on day 35.
  • the vaccination method for each of the above vaccines is intramuscular injection.
  • the vaccination dose of the inactivated vaccine is 2.6U per mouse each time (0.4 doses of the human dose).
  • the vaccination dose of each mRNA vaccine or empty LNP is 2.6U per mouse. 5 ⁇ g each time for mice.
  • Mouse serum samples were collected on days 35 and 49 respectively to test the binding antibody titer and pseudovirus neutralizing antibody titer of the immune serum. In addition, mouse spleen samples were collected on day 49 to examine T cell immunity.
  • FIG. 4a A schematic diagram of the mouse sequential immunization and serum sampling procedure is shown in Figure 4a.
  • Example 7 Detection of binding antibody titer levels of sequentially immunized mouse sera to RBD antigens of each strain of the new coronavirus
  • Example 3 the method described in Example 3 was used to detect whether the mouse serum of each immune group collected in Example 6 was against the new coronavirus prototype strain, Delta variant strain, Beta variant strain, and Omicron variant strain BA.1 , binding antibody titers of RBD antigens of BA.1.1, BA.2, and BA.3 subtypes.
  • Figure 4b(i) ⁇ (v) shows the groups that received the third booster immunization with PP, PB, DB, DO and inactivated vaccines respectively.
  • the first four groups are sequential immunizations, and the last group is the control; such as As mentioned in the description of the figures, it shows that the serum collected on the 35th day (shown as open circles) and the 49th day (shown as solid circles) of each immunization program is against the new coronavirus prototype strain, Delta variant strain, and Beta variant strain.
  • the multiple increase in titer (the "number Improvement; from these results, it can be seen that after sequential immunization with each mRNA vaccine, the serum antibody titer level is significantly higher than before sequential immunization, indicating that the mRNA vaccine of the present application can be used for sequential immunization to strengthen the immune response. level.
  • the increase in serum antibody titer on day 49 compared to the serum antibody titer on day 35 was much higher than PP mRNA vaccine sequential immunization group can reach up to nearly 5 times;
  • the increase in serum antibody titer on day 49 compared to the serum antibody titer on day 35 was much higher than that of the PP mRNA vaccine sequential immunization group, up to more than 5 times. .
  • the serum collected from each immune group on day 49 had binding antibodies against the new coronavirus prototype strain, Delta variant strain, Beta variant strain, and Omicron variant strain subtypes BA.1, BA.1.1, BA.2, and BA.3 antigens.
  • the titer results are shown in Figure 5a.
  • Figure 5a shows that compared with the PP mRNA vaccine sequential immunization group:
  • the binding antibody titer levels induced by them are much higher than those of the PP mRNA vaccine, up to 3 times;
  • the binding antibody titer levels induced are much higher than those of the PP mRNA vaccine;
  • the binding antibody titer levels induced by them are much higher than those of the PP mRNA vaccine.
  • Example 8 Evaluation of the inhibitory effect of sequentially immunized mouse serum on pseudoviruses of various strains of the new coronavirus
  • Example 4 the method described in Example 4 was used to detect the sensitivity of the mouse sera of each immune group collected on the 49th day in Example 6 against the new coronavirus prototype strain, Delta variant strain, Beta variant strain, and Omicron variant. Neutralizing antibody titers of pseudoviruses of strains BA.1, BA.1.1, BA.2, and BA.3 subtypes.
  • Figure 5b shows: compared with the PP mRNA vaccine sequential immunization group:
  • the neutralizing antibody titer levels induced by pseudoviruses against the prototype strain of the new coronavirus, Delta, and Omicron BA.1.1 are higher than those of the PP mRNA vaccine, or equivalent to those of the PP mRNA vaccine;
  • the neutralizing antibody titer levels induced by pseudoviruses against the new coronavirus Omicron BA.1, BA.2, and BA.3 are more than twice, up to about 3 times higher than those of the PP mRNA vaccine;
  • the neutralizing antibody titers induced by pseudoviruses against various subtypes of the new coronavirus Omicron variant are higher than, or equivalent to, the PP mRNA vaccine.
  • Example 9 Evaluation of cellular immunity levels induced by sequential immunization
  • Example 5 the method described in Example 5 and the spleen samples of mice in each immunized group collected on day 49 in Example 6 were used to detect the level of cellular immunity induced by sequential immunization.
  • novel coronavirus chimeric nucleic acid vaccine provided by this application can provide strong immune protection against multiple strains of the novel coronavirus, and can induce immunity against various strains of the novel coronavirus when used in sequential immunization with other types of vaccines ( That is, broad-spectrum) and significantly higher immune response levels are very suitable for the current complex epidemic prevention and control, and have potential clinical application value and prospects.

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Abstract

一种多核苷酸，其相关产品及其在制备新冠疫苗中的用途，以及基于该多核苷酸的嵌合核酸疫苗或免疫原性组合物；所述多核苷酸编码由新冠病毒原型株S蛋白RBD结构域与Beta变异株S蛋白RBD结构域、或者Delta变异株S蛋白RBD结构域与Beta变异株S蛋白RBD结构域、或者Delta变异株S蛋白RBD结构域与Omicron变异株S蛋白RBD结构域直接串联或通过连接子连接形成的重组嵌合抗原；基于该多核苷酸的嵌合核酸疫苗针对多种新冠病毒毒株均可提供较强的免疫保护效力，并且在与其他类型疫苗进行序贯免疫时可诱导针对新冠病毒各型毒株的(即，广谱的)、显著增高的免疫反应水平。

Description

新冠病毒嵌合核酸疫苗及其用途
交叉引用
本申请要求于2022年5月12日提交的、申请号为202210515599.7、发明名称为“新冠病毒嵌合核酸疫苗及其用途”的中国专利申请的优先权，其全部内容通过引用并入本文。
技术领域
本申请涉及生物医药领域，具体涉及一种新型冠状病毒嵌合核酸疫苗及其用途。
背景技术
新型冠状病毒肺炎(也称COVID-19)是由新型冠状病毒(也称新冠病毒，SARS-CoV-2)感染导致的一种急性呼吸道传染病。新冠病毒属于冠状病毒科β-冠状病毒属，具有囊膜，是单股正链RNA病毒。新冠病毒表面的刺突蛋白(又称S蛋白)负责病毒与宿主细胞膜受体的结合和膜融合，S蛋白上存在受体结合结构域(RBD)，其是一个重要的疫苗靶点，能激发中和抗体的产生，具有免疫聚焦的优势。
目前，新冠肺炎疫情在全球范围内仍很严峻，新冠病毒变异株不断出现和流行，特别是，德尔塔(Delta)和奥密克戎(Omicron)变异株依次席卷全球，成为了优势流行毒株。特别是，新冠病毒Omicron变异株还存在多种亚型(例如，BA.1，BA.2，BA.1.1，BA.3亚型)，具有超过Delta的传播速度，已经成为全球的主导毒株；其中，BA.2亚型毒株的传播速度高于其他的Omicron亚型，现在已经占据了最大的比例。Omicron变异株的S蛋白上具有超过50个氨基酸突变，相比之前的Delta突变株的氨基酸突变大大增多。
由于这些新出现的新冠病毒变异株中S蛋白或RBD的序列存在很多突变，导致现有的基于新冠病毒原型株(Prototype)设计和开发的疫苗所激发的免疫反应在面对变异株(如Omicron变异株)时的效力大大下降，这种变异株突破疫苗、抗体保护的现象称为免疫逃逸。免疫逃逸的现象在Omicron的多种亚型上表现得尤为明显。为了解决新冠病毒变异株的免疫逃逸问题，需要开发新型疫苗以使其适应新出现的变异株(如Omicron变异株及其各种亚型)，使其对当前的流行株具有较强保护效果；同时，由于目前存在多种变异株(特别是多种Omicron亚型)同时流行的现象，新型疫苗需要能够诱导广谱的免疫反应，以尽可能地同时防范多种新冠病毒毒株，这对于新冠疫情的防控可以起到至关重要的作用。
公开于该背景技术部分的信息仅仅旨在增加对本申请的总体背景的理解，而不应当 被视为承认或以任何形式暗示该信息构成已为本领域一般技术人员所公知的现有技术。
发明内容
为克服上述现有技术中存在的问题，本申请提供了一种多核苷酸，其相关产品及其在制备用于预防和/或治疗新冠病毒疫苗中的用途，基于该多核苷酸或其相关产品的嵌合核酸疫苗或免疫原性组合物，以及包括该嵌合核酸疫苗或免疫原性组合物的试剂盒；所述多核苷酸编码由(1)新型冠状病毒原型株S蛋白RBD结构域(或其一部分)与新型冠状病毒Beta变异株S蛋白RBD结构域(或其一部分)，或者(2)新型冠状病毒Delta变异株S蛋白RBD结构域(或其一部分)与新型冠状病毒Beta变异株S蛋白RBD结构域(或其一部分)，或者(3)新型冠状病毒Delta变异株S蛋白RBD结构域(或其一部分)与新型冠状病毒Omicron变异株S蛋白RBD结构域(或其一部分)直接串联或通过连接子连接形成的重组嵌合抗原肽；所述嵌合核酸疫苗针对多种新冠病毒毒株均可提供较强的免疫保护效力，并且在与其他类型疫苗(如灭活疫苗)进行序贯免疫时可诱导针对新冠病毒各型毒株的(即，广谱的)、显著增高的免疫反应水平。
具体地，本申请提供了以下技术方案：
第一方面，本申请提供了一种多核苷酸，其编码如式(I)所示结构的重组嵌合抗原肽：
(A-B)-C-(A-B’)           
(I)
式(I)中：
Option 1:A-B表示新型冠状病毒原型株S蛋白RBD结构域或其一部分的氨基酸序列，或与其具有至少90％，92％，95％，96％，97％，98％或99％同一性且与其具有相同或基本相同的免疫原性的氨基酸序列；
A-B’表示新型冠状病毒Beta变异株S蛋白RBD结构域或其一部分的氨基酸序列，或与其具有至少90％，92％，95％，96％，97％，98％或99％同一性且与其具有相同或基本相同的免疫原性的氨基酸序列；或者
Option 2:A-B表示新型冠状病毒Delta变异株S蛋白RBD结构域或其一部分的氨基酸序列，或与其具有至少90％，92％，95％，96％，97％，98％或99％同一性且与其具有相同或基本相同的免疫原性的氨基酸序列；
A-B’表示新型冠状病毒Beta变异株S蛋白RBD结构域或其一部分的氨基酸序列，或与其具有至少90％，92％，95％，96％，97％，98％或99％同一性且与其具有相同或基本相同的免疫原性的氨基酸序列；或者
Option 3:A-B表示新型冠状病毒Delta变异株S蛋白RBD结构域或其一部分的氨基酸序列，或与其具有至少90％，92％，95％，96％，97％，98％或99％同一性且与其具有相同或基本相同的免疫原性的氨基酸序列；
A-B’表示新型冠状病毒Omicron变异株S蛋白RBD结构域或其一部分的氨基酸序列，或与其具有至少90％，92％，95％，96％，97％，98％或99％同一性且与其具有相同或基本相同的免疫原性的氨基酸序列；
C表示连接子(GGS)n；其中，n＝0,1,2,3,4或5。
对于上述多核苷酸，优选地，所述新型冠状病毒原型株S蛋白RBD结构域的一部分为其全部氨基酸序列的至少70％、80％、85％、90％、92％、95％、96％、97％、98％或99％；
和/或，所述新型冠状病毒Beta变异株S蛋白RBD结构域的一部分为其全部氨基酸序列的至少70％、80％、85％、90％、92％、95％、96％、97％、98％或99％；
和/或，所述新型冠状病毒Delta变异株S蛋白RBD结构域的一部分为其全部氨基酸序列的至少70％、80％、85％、90％、92％、95％、96％、97％、98％或99％；
和/或，所述新型冠状病毒Omicron变异株S蛋白RBD结构域的一部分为其全部氨基酸序列的至少70％、80％、85％、90％、92％、95％、96％、97％、98％或99％；
和/或，n＝0,1,2或3。
在一些优选的具体实施方案中，所述新型冠状病毒原型株S蛋白RBD结构域或其一部分的氨基酸序列如SEQ ID NO:1所示，或者如SEQ ID NO:1所示的氨基酸序列经取代、缺失或添加一个或几个氨基酸获得的、与其具有相同或基本相同的免疫原性的氨基酸序列；
和/或，所述新型冠状病毒Beta变异株S蛋白RBD结构域或其一部分的氨基酸序列如SEQ ID NO:2所示，或者如SEQ ID NO:2所示的氨基酸序列经取代、缺失或添加一个或几个氨基酸获得的、与其具有相同或基本相同的免疫原性的氨基酸序列；
和/或，所述新型冠状病毒Delta变异株S蛋白RBD结构域或其一部分的氨基酸序列如SEQ ID NO:3所示，或者如SEQ ID NO:3所示的氨基酸序列经取代、缺失或添加一个或几个氨基酸获得的、与其具有相同或基本相同的免疫原性的氨基酸序列；
和/或，所述新型冠状病毒Omicron变异株S蛋白RBD结构域或其一部分的氨基酸序列如SEQ ID NO:4所示，或者如SEQ ID NO:4所示的氨基酸序列经取代、缺失或添加一个或几个氨基酸获得的、与其具有相同或基本相同的免疫原性的氨基酸序列；
和/或，n＝0,1或2。
在进一步优选的具体实施方案中，当式(I)为Option 1时，如式(I)所示结构的重组嵌合抗原肽具有如SEQ ID NO:5所示的氨基酸序列；
当式(I)为Option 2时，如式(I)所示结构的重组嵌合抗原肽具有如SEQ ID NO:6所示的氨基酸序列；
当式(I)为Option 3时，如式(I)所示结构的重组嵌合抗原肽具有如SEQ ID NO:7所示 的氨基酸序列。
在一些优选的具体实施方案中，所述多核苷酸为DNA分子；
进一步优选地，当式(I)为Option 1时，所述DNA分子具有如SEQ ID NO:8所示的DNA序列；
进一步优选地，当式(I)为Option 2时，所述DNA分子具有如SEQ ID NO:9所示的DNA序列；
进一步优选地，当式(I)为Option 3时，所述DNA分子具有如SEQ ID NO:10所示的DNA序列。
在另一些优选的具体实施方案中，所述多核苷酸为mRNA分子；
进一步优选地，当式(I)为Option 1时，所述mRNA分子具有如SEQ ID NO:11所示的mRNA序列；
进一步优选地，当式(I)为Option 2时，所述mRNA分子具有如SEQ ID NO:12所示的mRNA序列；
进一步优选地，当式(I)为Option 3时，所述mRNA分子具有如SEQ ID NO:13所示的mRNA序列。
第二方面，本申请提供了一种核酸构建体，其包含如上述第一方面所述的多核苷酸，以及任选地，与所述多核苷酸可操作地连接的至少一个表达调控元件。
第三方面，本申请提供了一种表达载体，其包含如上述第二方面所述的核酸构建体。
第四方面，本申请提供了一种宿主细胞，其中转化或转染有如上述第一方面所述的多核苷酸、如上述第二方面所述的核酸构建体或如上述第三方面所述的表达载体。
第五方面，本申请提供了如上述第一方面所述的多核苷酸、如上述第二方面所述的核酸构建体、如上述第三方面所述的表达载体或如上述第四方面所述的宿主细胞在制备用于预防和/或治疗新型冠状病毒的疫苗中的应用；
优选地，所述疫苗用于单独免疫或者与其他类型的新型冠状病毒疫苗进行序贯免疫；进一步优选地，所述其他类型的新型冠状病毒疫苗包括灭活疫苗。
第六方面，本申请提供了一种嵌合核酸疫苗或免疫原性组合物，其包含如上述第一方面所述的多核苷酸、如上述第二方面所述的核酸构建体、如上述第三方面所述的表达载体或如上述第四方面所述的宿主细胞，以及生理学可接受的媒介物、佐剂、赋形剂、载体和/或稀释剂。
在一个具体实施方案中，所述嵌合核酸疫苗或免疫原性组合物为新型冠状病毒DNA疫苗，所述DNA疫苗包括：
(i)真核表达载体；和
(ii)构建入所述真核表达载体中的、编码如上述第一方面中所定义的如式(I)所示结构 的重组嵌合抗原肽的DNA序列，优选为如SEQ ID NO:8、9或10所示的DNA序列；
优选地，所述真核表达载体选自pGX0001、pVAX1、pCAGGS和pcDNA系列载体。
在另一个具体实施方案中，所述嵌合核酸疫苗或免疫原性组合物为新型冠状病毒mRNA疫苗，所述mRNA疫苗包括：
(I)编码如上述第一方面中所定义的如式(I)所示结构的重组嵌合抗原肽的mRNA序列，优选为如SEQ ID NO:11、12或13所示的mRNA序列；和
(II)脂质纳米颗粒。
在另一个具体实施方案中，所述嵌合核酸疫苗或免疫原性组合物为新型冠状病毒-病毒载体疫苗，其包括：
(1)病毒骨架载体；和
(2)构建入所述病毒骨架载体中的、编码如上述第一方面中所定义的如式(I)所示结构的重组嵌合抗原肽的DNA序列，优选为如SEQ ID NO:8、9或10所示的DNA序列；
可选地，所述病毒骨架载体选自以下病毒载体中的一种或几种：腺病毒载体、痘病毒载体、流感病毒载体、腺相关病毒载体。
在可行的实现方式中，所述嵌合核酸疫苗或免疫原性组合物为鼻喷剂、口服制剂、栓剂或胃肠外制剂的形式；
优选地，所述鼻喷剂选自气雾剂、喷雾剂和粉雾剂；
优选地，所述口服制剂选自片剂、粉末剂、丸剂、散剂、颗粒剂、细粒剂、软/硬胶囊剂、薄膜包衣剂、小丸剂、舌下片和膏剂；
优选地，所述胃肠外制剂为经皮剂、软膏剂、硬膏剂、外用液剂、可注射或可推注制剂。
第七方面，本申请提供了一种试剂盒，其包括如上述第六方面所述的嵌合核酸疫苗或免疫原性组合物，以及任选地其他类型的新型冠状病毒疫苗，所述嵌合核酸疫苗或免疫原性组合物与所述其他类型的新型冠状病毒疫苗单独包装；
优选地，所述其他类型的新型冠状病毒疫苗为新型冠状病毒灭活疫苗。
有益效果
本申请的发明人设计了一种多核苷酸，所述多核苷酸编码由(1)新型冠状病毒原型株S蛋白RBD结构域(或其一部分)与新型冠状病毒Beta变异株S蛋白RBD结构域(或其一部分)，或者(2)新型冠状病毒Delta变异株S蛋白RBD结构域(或其一部分)与新型冠状病毒Beta变异株S蛋白RBD结构域(或其一部分)，或者(3)新型冠状病毒Delta变异株S蛋白RBD结构域(或其一部分)与新型冠状病毒Omicron变异株S蛋白RBD结构域(或其一部分)直接串联或通过连接子连接形成的重组嵌合抗原肽；基于该多核苷酸的嵌合核酸疫苗，针对多种新冠病毒毒株均可提供较强的免疫保护效力，并且在与其他 类型疫苗进行序贯免疫时可诱导针对新冠病毒各型毒株的(即，广谱的)、显著增高的免疫反应水平，非常适用于当前复杂的疫情防控，具有潜在的临床应用价值和前景。
附图说明
一个或多个实施例通过与之对应的附图中的图片进行示例性说明，这些示例性说明并不构成对实施例的限定。在这里专用的词“示例性”意为“用作例子、实施例或说明性”。这里作为“示例性”所说明的任何实施例不必解释为优于或好于其它实施例。
图1是本申请实施例1中构建的新冠病毒原型株RBD二聚体mRNA疫苗(简称PP mRNA疫苗，作为对照疫苗)、新冠病毒原型株RBD与Beta变异株RBD连接形成的嵌合RBD二聚体mRNA疫苗(简称PB mRNA疫苗)、Delta变异株RBD与Beta变异株RBD连接形成的嵌合RBD二聚体mRNA疫苗(简称DB mRNA疫苗)、Delta变异株RBD与Omicron变异株RBD连接形成的嵌合RBD二聚体mRNA疫苗(简称DO mRNA疫苗)的结构示意图；图上标注了mRNA疫苗的各个区段，其中，5’UTR表示5’端非翻译区，3’UTR表示3’端非翻译区，SP表示信号肽序列，Poly(A)表示多聚腺苷酸尾，Protype RBD表示原型株的RBD序列，Beta RBD表示Beta变异株的RBD序列，Delta RBD表示Delta变异株的RBD序列，Omicron(BA.1)RBD表示Omicron变异株的RBD序列，其中，Beta RBD、Delta RBD和Omicron RBD上还标注了其各自相对于原型株RBD的氨基酸突变。
图2显示了本申请实施例1中所构建的PP、PB、DB、DO mRNA疫苗所激发的体液免疫水平，如实施例3和4中所检测的，其中，LNP代表采用脂质纳米颗粒免疫的阴性对照组；其中，图2a为mRNA疫苗免疫小鼠及采样程序示意图；图2b和图2c分别为在用mRNA疫苗免疫小鼠后第14天、第28天所采集的血清分别针对新冠病毒原型株、Delta变异株、Beta变异株和Omicron变异株BA.1、BA.1.1、BA.2、BA.3亚型的RBD抗原的结合抗体滴度，在图2b和图2c的柱形图中，每个柱上方的数字表示该柱所代表的抗体滴度与LNP组中的相应柱所代表的抗体滴度的比值，并且，如图中箭头所示，图2b和2c右侧的热图基于这些数字制作；图2d为在用mRNA疫苗免疫小鼠后第28天所采集的血清在假病毒中和实验中中和新冠病毒原型株、Delta变异株、Omicron变异株BA.1、BA.1.1、BA.2、BA.3亚型的假病毒的NT50值，每个柱上方的数字代表该实验组所有样品的几何平均值(GMT)，如图中箭头所示，右侧的热图基于这些数字制作；所有数据的展示方式为GMT±95％CI(置信区间)。
图3显示了本申请实施例1中所构建的PP、PB、DB、DO mRNA疫苗所激发的细胞免疫水平，如实施例5中所检测的，LNP代表采用脂质纳米颗粒免疫的阴性对照组；其中，图3a为mRNA疫苗免疫小鼠及采样程序示意图；图3b为显示ELISpot检验mRNA疫苗免疫小鼠后第21天采集的脾脏细胞在分别被4种肽库(新冠病毒原型株、Delta变异株、Beta变异株、Omicron变异株亚型BA.1RBD构建的肽库)刺激后产生的IFNγ+细 胞数量的柱形图，柱形图上方的数字表示该柱所代表的IFNγ+细胞数量与LNP组中相应的柱所代表的IFNγ+细胞数量的比值；所有数据的展示方式为Mean±SEM。
图4显示了采用灭活疫苗与本申请实施例1中所构建的PP、PB、DB、DO mRNA疫苗对小鼠进行序贯免疫后，相较于序贯免疫前(即，未用mRNA疫苗加强的组)，结合抗体滴度的提高倍数；其中，图4a为小鼠序贯免疫和血清采样程序示意图，IV代表灭活疫苗；图4b显示了各免疫程序在第35天(以空心圆显示)、第49天(以实心圆显示)所采集的血清针对新冠病毒原型株、Delta变异株、Beta变异株、Omicron变异株亚型BA.1、BA.1.1、BA.2、BA.3RBD抗原的结合抗体滴度水平，以及后者(即，第49天)相对于前者(即，第35天)抗体滴度的提高倍数(每张图上面的“数字×”表示提高倍数)，其中，图4b(i)～(v)分别代表两次灭活疫苗+PP mRNA疫苗免疫组、两次灭活疫苗+PB mRNA疫苗免疫组、两次灭活疫苗+DB mRNA疫苗免疫组、两次灭活疫苗+DO mRNA疫苗免疫组、三次灭活疫苗免疫组。
图5显示了采用灭活疫苗与本申请实施例1中所构建的PP、PB、DB、DO mRNA疫苗对小鼠进行序贯免疫后的免疫反应水平，其免疫程序参照图4a；在各柱形图中，“PP”代表两次灭活疫苗+PP mRNA疫苗免疫组，“PB”代表两次灭活疫苗+PB mRNA疫苗免疫组，“DB”代表两次灭活疫苗+DB mRNA疫苗免疫组，“DO”代表两次灭活疫苗+DO mRNA疫苗免疫组，“IV”代表三次灭活疫苗免疫组，“LNP”代表两次灭活疫苗佐剂(即，Al佐剂)+脂质纳米颗粒(LNP)免疫组，作为阴性对照；其中，图5a为第49天采集的血清针对新冠病毒原型株、Delta变异株、Beta变异株、Omicron变异株亚型BA.1、BA.1.1、BA.2、BA.3抗原的结合抗体滴度，在图5a的柱形图中，每个柱上方的第一行的数字表示该柱所代表的抗体滴度与灭活疫苗(IV)组中的相应柱所代表的抗体滴度的比值，第二行的数字表示该柱所代表的抗体滴度与LNP组中的相应柱所代表的抗体滴度的比值，并且如图中箭头所示，图5a右侧的热图基于第二行数字制作；图5b为第49天采集的血清在假病毒中和实验中中和6种假病毒(原型株、Delta变异株、Omicron变异株亚型BA.1、BA.1.1、BA.2、BA.3)的NT50值，在图5b的柱形图中，每个柱上方的第一行数字表示该柱所代表的NT50滴度值与灭活疫苗(IV)组中的相应柱所代表的NT50滴度值的比值，每个柱上方的第二行数字代表该实验组所有样品的几何平均值(GMT)，并且如图中箭头所示，右侧的热图基于第二行数字制作；图5a和图5b中数据的展示方式为GMT±95％CI(置信区间)；图5c显示ICS检验第49天采集的脾脏的CD8+和CD4+细胞在分别被4种肽库(原型株、Delta、Beta、Omicron亚型BA.1变异株RBD构建的肽库)刺激后产生IFNγ+细胞的比例，图5c中数据的展示方式为Mean±SEM；统计学差异由Mann-Whitney test方法计算(*,p<0.05；**,p<0.01)。
具体实施方式
为使本申请实施例的目的、技术方案和优点更加清楚，下面将对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
另外，为了更好的说明本申请，在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解，没有某些具体细节，本申请同样可以实施。在一些实施例中，对于本领域技术人员熟知的原料、元件、方法、手段等未作详细描述，以便于凸显本申请的主旨。
除非另有其它明确表示，否则在整个说明书和权利要求书中，术语“包括”或其变换如“包含”或“包括有”等等将被理解为包括所陈述的元件或组成部分，而并未排除其它元件或其它组成部分。
实施例1：新冠病毒原型株RBD二聚体mRNA疫苗(简称PP mRNA疫苗，作为对照)、原型株和Beta变异株嵌合RBD二聚体mRNA疫苗(简称PB mRNA疫苗)、Delta变异株和Beta变异株的嵌合RBD二聚体mRNA疫苗(简称DB mRNA疫苗)以及Delta变异株和Omicron变异株的嵌合RBD二聚体mRNA疫苗(简称DO mRNA疫苗)的构建、体外制备与包装
按照图1所示的PP、PB、DB、DO mRNA疫苗的结构示意图，根据下述程序进行mRNA疫苗的构建、体外制备与包装：
1)mRNA疫苗的体外转录和加帽
本实施例中，用于体外转录mRNA疫苗的基础质粒为pUC57，由南京金斯瑞生物科技有限公司提供。
在基础质粒pUC57上，通过常规分子生物学手段引入mRNA疫苗的DNA表达元件，包括：(1)T7启动子，(2)mRNA疫苗的DNA编码区(PB、DB、DO mRNA疫苗的DNA编码序列分别如SEQ ID NO:8、9、10所示，作为对照的PP mRNA疫苗的DNA编码序列如SEQ ID NO:14所示)，(3)在编码区上游的5’端UTR序列(几种mRNA疫苗的5’端UTR序列相同，均为如SEQ ID NO:15所示序列)，(4)信号肽序列(即SP，如SEQ ID NO:16所示)，和(5)下游的3’端UTR序列(几种mRNA疫苗的3’端UTR序列相同，均为如SEQ ID NO:17所示序列)，和多聚腺苷酸尾(Poly-A-tail)。
首先，使用限制性内切酶BamHI对上述体外转录质粒进行酶切，将其线性化；使用常规DNA纯化方法进行纯化，获得体外转录的模板；然后，基于该模板，使用T7RNA体外转录试剂盒(E131-01A，苏州近岸蛋白质科技股份有限公司)进行体外转录，获得体外转录的mRNA；最后，使用氯化锂回收试剂盒(S125，苏州近岸蛋白质科技股份有限公司)，通过氯化锂沉淀法对mRNA进行纯化，获得提纯的体外转录mRNA。
然后，使用加帽酶试剂盒Cap1加帽酶试剂盒(M082-01B，苏州近岸蛋白质科技股份有限公司)，对上述提纯的体外转录mRNA进行5’端Cap1加帽，使其满足在真核细胞内被翻译的条件；其后，使用与上述相同的氯化锂沉淀法对mRNA进行再次纯化，获得纯化的经5’端加帽修饰的mRNA。
2)脂质纳米颗粒(Lipid nanoparticle,LNP)包装mRNA
将阳离子脂质、磷脂酰胆碱、胆固醇和PEG脂质按照50：10：38.5：1.5的比例进行混合，然后使用Precision Nano Systems公司生产的Nanoassemblr Benchtop纳米脂质体包装仪，将其与上述5’端加帽修饰的mRNA进行混合、包装。包装完成后，使用离心或透析的方法更换缓冲溶液为PBS。完成包装后，使用Thermo Fisher公司的Quan-iT Ribogreen RNA reagent试剂盒，对mRNA包装效率进行鉴定，包装效率符合mRNA疫苗的标准。
实施例2：实验动物免疫和样品采集
本实施例中，使用6-8周龄的BALB/c品系的雌性小鼠(购自维通利华)进行动物实验；实验组分为mRNA疫苗免疫组和阴性对照组，其中，mRNA疫苗免疫组包括PP mRNA疫苗免疫组、PB mRNA疫苗免疫组、DB mRNA疫苗免疫组和DO mRNA疫苗免疫组，所述阴性对照组为LNP免疫组。mRNA疫苗免疫组的所有小鼠在第0天和第14天分别免疫接种同一种设计的mRNA疫苗(即，PP、PB、DB或DO mRNA疫苗)，阴性对照组的小鼠在同样的时间、注射相同量的空的LNP。接种方法均为肌肉注射，接种剂量为每只小鼠每次5μg mRNA疫苗或空LNP。分别在第14天和第28天采集小鼠血清样本，用于检验免疫血清的结合抗体滴度和假病毒中和抗体滴度。此外，还在第21天采集小鼠脾脏样本，用于检验T细胞免疫。
mRNA疫苗免疫小鼠及采样程序如图2a所示。
实施例3：小鼠血清抗体滴度的检验
用新冠病毒原型株(SEQ ID NO:1)、Delta变异株(SEQ ID NO:3)、Beta变异株(SEQ ID NO:2)和Omicron变异株BA.1(SEQ ID NO:4)、BA.1.1(SEQ ID NO:18)、BA.2(SEQ ID NO:19)、BA.3(SEQ ID NO:20)亚型的RBD抗原蛋白(0.2μg/ml)分别包被ELISA板，将包被好的ELISA板在5％脱脂牛奶中封闭1小时；然后，将实施例2中的、由各实验组小鼠采集的血清在56℃孵育30分钟，进行灭活；将经过灭活的血清样本从1:200或1:1000开始进行三倍梯度稀释，然后将稀释液加入每个孔，随后将ELISA板在37℃下孵育1小时；将山羊抗小鼠IgG-HRP抗体(购自柏奥易杰(EASYBio))添加到板中作为二抗，并在37℃下再次孵育1小时；最后，用3,3',5,5'-四甲基联苯胺(TMB)底物进行显色，显色完成后用2M盐酸终止反应，使用酶标仪(PerkinElmer)测量450nm和630nm处的吸光度。通过从同一孔的450nm处的吸光度减去630nm处的吸光度，来计算 吸光度值。终点滴度定义为：血清产生的吸光度(如上所述，450nm的吸光度减去630nm的吸光度)大于背景值2.1倍时对应的血清稀释倍数。低于检测限的抗体滴度定义为检测限的三分之一。
各实验组小鼠在免疫程序的第14天、第28天所采集的血清针对上述七种新冠病毒的RBD抗原的结合抗体滴度分别如图2b和图2c所示，在图2b和图2c中，左侧为终点滴度vs疫苗种类的柱形图，右侧为相应的热图，所述热图是基于各mRNA疫苗的终点抗体滴度与LNP组的终点抗体滴度的比值制作的，如附图说明中所述。
图2c为抗体水平较为稳定的第28天的结果，由图2c可以看出：
(1)PB mRNA疫苗
针对所测试的新冠病毒原型株和各变异株，PB mRNA疫苗均能诱导出较高的结合抗体水平；并且，其所诱导的结合抗体滴度水平与PP mRNA疫苗相当，或者高于PP mRNA疫苗(如，针对BA.1.1和针对BA.2的，分别高出2-3倍)；
(2)DB mRNA疫苗
针对所测试的新冠病毒原型株和各变异株，DB mRNA疫苗均能诱导出较高的结合抗体水平；并且，其针对各型新冠病毒毒株所诱导的结合抗体滴度水平均远远高于PP mRNA疫苗，有的高达3倍以上；
(3)DO mRNA疫苗
针对所测试的新冠病毒原型株和各变异株，DO mRNA疫苗均能诱导出较高的结合抗体水平；特别是，其针对Omicron变异株的各亚型所诱导的血清抗体滴度水平均远远高于PP mRNA疫苗；例如，针对BA.1亚型，DO mRNA疫苗所诱导的抗体滴度水平比PP mRNA疫苗高2倍以上，针对BA.1.1亚型，DO mRNA疫苗所诱导的抗体滴度水平比PP mRNA疫苗高5倍以上，针对BA.2和BA.3亚型所诱导的抗体滴度水平比PP mRNA疫苗高将近6倍，针对BA.3亚型所诱导的抗体滴度水平比PP mRNA疫苗高3倍以上；这提示，本申请的DO mRNA疫苗针对Omicron变异株各亚型均可诱导出明显更高的抗体滴度水平，说明其针对Omicron各型变异株都将具有明显更高的免疫保护效力；并且，DO mRNA对于新冠病毒原型株和其他变异株也诱导出了较高的抗体滴度水平，这提示其具有很好的广谱性。
实施例4：新冠病毒毒株假病毒的包装和血清中和
本实施例中，分别检测上述实施例2所采集的免疫小鼠血清对新冠病毒原型株、Delta变异株和Omicron变异株BA.1、BA.1.1、BA.2、BA.3亚型的假病毒的50％假病毒中和滴度(pVNT50)；具体检测方法如下：
一、制备截短的新冠病毒S蛋白的表达质粒
分别将编码新冠病毒原型株、Delta变异株和Omicron变异株BA.1、BA.1.1、BA.2、 BA.3亚型的S蛋白的后18位氨基酸的核苷酸去掉，所得核苷酸分别命名为WT-S-del18、Delta-S-del18、BA.1-S-del18、BA.1.1-S-del18、BA.2-S-del18、BA.3-S-del18，其核苷酸序列分别如SEQ ID NO:21～26所示，由苏州金唯智公司进行合成；然后，将这些核苷酸序列各自克隆到pCAGGS表达载体上，分别得到表达质粒pCAGGS-WT-S-del18、pCAGGS-Delta-S-del18、pCAGGS-BA.1-S-del18、pCAGGS-BA.1.1-S-del18、pCAGGS-BA.2-S-del18、pCAGGS-BA.3-S-del18。
二、新冠病毒原型株和各型变异株的假病毒的包装
1)在10cm细胞培养皿中铺HEK293T细胞，使第二天细胞密度至80％左右。培养液为含10％FBS的DMEM培养基。
2)将上文所制备的截短的、新冠病毒各型株的S蛋白的表达质粒，用PEI转染培养皿中的细胞(30μg/10cm细胞培养皿)。目的质粒与PEI按1:3比例混匀后转染，4-6h换培养液(含10％FBS的DMEM培养基)，37℃培养24h。
3)将假病毒包装骨架病毒G*VSV-delG(武汉枢密脑科学技术有限公司)加入上述转染后的HEK293T细胞，37℃孵育2h，换培养液(含10％FBS的DMEM培养基)，并加入VSV-G抗体(表达该抗体杂交瘤细胞购自ATCC细胞库)，在培养箱中继续培养30h。
4)收上清，3000rpm离心10min，在超净工作台中经0.45μm无菌滤器过滤，去除细胞碎片，分装，-80℃冰箱冻存。
通过上述步骤，分别得到新冠病毒原型株、Delta变异株和Omicron变异株BA.1、BA.1.1、BA.2、BA.3亚型的假病毒。
三、免疫小鼠血清对假病毒抑制效果的评价
将实施例2中的、于第28天采集的各实验组小鼠血清在56℃孵育30分钟，进行灭活；将灭活后的血清样本进行稀释，从1:80开始进行2倍梯度稀释。然后，将每种假病毒与等体积的稀释后血清混合，在37℃下孵育1小时。取100μl病毒-血清混合物加入到96孔板中预铺板的Vero细胞上。孵育15小时后，使用CQ1共聚焦图像细胞仪，检测转导单位(TU)数，从而计算免疫小鼠血清对上述新冠病毒原型株、Delta变异株和Omicron变异株BA.1、BA.1.1、BA.2、BA.3亚型的假病毒的中和能力。
结果如图2d所示；如图2d的附图说明中所述，图2d的左侧柱形图显示了各免疫组血清中和原型株、Delta变异株和Omicron变异株BA.1、BA.1.1、BA.2、BA.3亚型的假病毒的pVNT50(即，50％假病毒中和滴度)，右侧热图显示了各mRNA疫苗的pVNT50与LNP组的pVNT50的比值。
由图2d可以看出：
(1)PB mRNA疫苗针对Omicron变异株的各亚型所诱导的血清中和抗体滴度水平均远远高于PP mRNA疫苗；特别是，PB mRNA疫苗针对BA.1亚型所诱导的中和抗体滴度 水平比PP mRNA疫苗高7倍以上，针对BA.1.1亚型所诱导的中和抗体滴度水平比PP mRNA疫苗高6倍以上，针对BA.2和BA.3亚型所诱导的中和抗体滴度水平比PP mRNA疫苗高6倍左右；这提示，本申请的PB mRNA疫苗针对Omicron变异株各亚型均可诱导出明显更高的中和抗体滴度水平，说明其针对Omicron各型变异株都将具有明显更高的免疫保护效力；此外，PB mRNA对于新冠病毒原型株和Delta变异株也具有较高的中和抗体滴度水平，这提示其具有很好的广谱性。
(2)DB mRNA疫苗针对原型株、Delta变异株、Omicron变异株各亚型所诱导的血清中和抗体滴度水平均远远高于PP mRNA疫苗；具体地，DB mRNA疫苗针对原型株所诱导的中和抗体滴度水平比PP mRNA疫苗高3倍以上，针对Delta变异株所诱导的中和抗体滴度水平比PP mRNA疫苗高5倍以上，针对Omicron变异株BA.1亚型所诱导的中和抗体滴度水平比PP mRNA疫苗高将近45倍，针对Omicron变异株BA.1.1亚型所诱导的中和抗体滴度水平比PP mRNA疫苗高将近30倍，针对Omicron变异株BA.2亚型所诱导的中和抗体滴度水平比PP mRNA疫苗高将近48倍，针对Omicron变异株BA.3亚型所诱导的中和抗体滴度水平比PP mRNA疫苗高将近69倍；这提示，本申请的DB mRNA疫苗针对新冠病毒各型毒株均可诱导出明显更高的中和抗体滴度水平，说明其针对新冠病毒各型毒株都将具有明显更高的免疫保护效力。
(3)DO mRNA疫苗针对原型株、Delta变异株、Omicron变异株各亚型所诱导的血清中和抗体滴度水平均远远高于PP mRNA疫苗；具体地，DO mRNA疫苗针对原型株所诱导的中和抗体滴度水平比PP mRNA疫苗高将近13倍，针对Delta变异株所诱导的中和抗体滴度水平比PP mRNA疫苗高将近5倍，针对Omicron变异株BA.1亚型所诱导的中和抗体滴度水平比PP mRNA疫苗高将近200倍，针对Omicron变异株BA.1.1亚型所诱导的中和抗体滴度水平比PP mRNA疫苗高将近163倍，针对Omicron变异株BA.2亚型所诱导的中和抗体滴度水平比PP mRNA疫苗高将近230倍，针对Omicron变异株BA.3亚型所诱导的中和抗体滴度水平比PP mRNA疫苗高将近407倍；这提示，本申请的DO mRNA疫苗针对新冠病毒各型毒株均可诱导出明显更高的中和抗体滴度水平，说明其针对新冠病毒各型毒株都将具有明显更高的免疫保护效力。
实施例5：mRNA疫苗诱导细胞免疫水平的评价
本实施例中，采用实施例2中的、于第21天采集的各实验组小鼠的脾脏样本(小鼠免疫和采样程序示意图如图3a所示)，检测mRNA疫苗诱导的细胞免疫水平。具体方法如下：
1)小鼠脾脏样品处理
用细胞匀浆器在1ml无血清DMEM中将小鼠脾脏细胞制备成单细胞匀浆，用40μm细胞过滤器过滤，用红细胞裂解缓冲液(北京索莱宝科技有限公司，R1010)裂解红细 胞；然后，细胞经过清洗液(PBS+0.5％FBS)清洗后，用0.4％台盼蓝溶液(Gibco，15250061)染色，使用Cell drop FL自动细胞计数器进行计数。
2)ELISpot检验
将10μg/ml anti-mouse IFN-γ抗体(购自BD公司)在平底96孔板中在4℃过夜孵育，以包被该平底96孔板，第二天，在室温下封闭2小时。将新鲜的小鼠脾脏单细胞悬液(4×105/孔)加入上述抗体包被的96孔板，并分别用新冠病毒原型株、Delta、Beta、Omicron变异株BA.1亚型的RBD构建的肽库(每条多肽2μg/ml)刺激20小时；所述肽库采用网站https://www.hiv.lanl.gov/content/sequence/PEPTGEN/peptgen.html上的软件PeptGen Peptide Generator进行设计，设计的关键参数包括：短肽的长度在18-20个氨基酸，重叠氨基酸片段在10个氨基酸作用等；设计好的肽库由中科亚光生物科技有限公司合成。阳性对照孔用植物血凝素(PMA)刺激以产生非特异细胞免疫反应，阴性对照孔不用肽库刺激。然后，丢弃细胞，并用生物素化的IFNγ抗体、链霉亲和素-HRP抗体和显色底物先后孵育96孔板。当板底显现斑点后，用去离子水彻底冲洗样品，停止显色。最后，使用Immuno Capture 6.5.0拍照并对斑点数量进行计数。
结果如图3b所示，由图3b可知，在使用上述四种新冠病毒RBD肽库刺激后，PB、DB、DO mRNA疫苗免疫的小鼠脾脏细胞所产生的IFN-γ+细胞的数量与PP mRNA疫苗免疫的小鼠相当，它们均远远高于LNP对照组，这表明：PB、DB、DO mRNA疫苗均能有效激发细胞免疫应答，并且其所激发的细胞免疫水平与PP mRNA疫苗相当。
实施例6：mRNA疫苗与灭活疫苗的序贯免疫
本实施例中，使用6-8周龄的BALB/c品系的雌性小鼠(购自维通利华)进行动物实验，所用灭活疫苗来自国药中生BBBIP-CorV。
实验分组为：三次灭活疫苗免疫组(即，“IV”组)、两次灭活疫苗+PP mRNA疫苗免疫组(简称“PP”组)、两次灭活疫苗+PB mRNA疫苗免疫组(简称“PB”组)、两次灭活疫苗+DB mRNA疫苗免疫组(简称“DB”组)、两次灭活疫苗+DO mRNA疫苗免疫组(简称“DO”组)和灭活疫苗佐剂+LNP免疫组(简称“LNP”组，作为阴性对照组)。
“IV”组：所有小鼠在第0天、第21天和第35天分别接种一剂灭活疫苗；
“PB”组、“DB”组、“DO”组：所有小鼠在第0天、第21天分别接种一剂灭活疫苗，然后在第35天接种一剂各mRNA疫苗；
“LNP”组：所有小鼠在第0天、第21天接种灭活疫苗的佐剂——Al佐剂，在第35天接种空的LNP。
上述各疫苗的接种方法均为肌肉注射，其中，灭活疫苗的接种剂量为每只小鼠每次2.6U(为人用剂量的0.4剂)，各mRNA疫苗或空的LNP的接种剂量为每只小鼠每次5μg。
分别在第35天和第49天采集小鼠血清样本，用于检验免疫血清的结合抗体滴度和假病毒中和抗体滴度。此外，在第49天还采集了小鼠脾脏样本，用于检验T细胞免疫。
小鼠序贯免疫和血清采样程序示意图如图4a所示。
实施例7：序贯免疫小鼠血清对新冠病毒各毒株RBD抗原的结合抗体滴度水平检测
本实施例中，采用实施例3中所记载的方法，检测了实施例6中所采集的各免疫组小鼠血清针对新冠病毒原型株、Delta变异株、Beta变异株、Omicron变异株BA.1、BA.1.1、BA.2、BA.3亚型的RBD抗原的结合抗体滴度。
结果如图4b所示。其中，图4b(i)～(v)分别显示了以PP、PB、DB、DO和灭活疫苗进行第三次加强免疫的组，前四组为序贯免疫，最后一组为对照；如附图说明中所述，其显示了各免疫程序在第35天(以空心圆显示)、第49天(以实心圆显示)所采集的血清针对新冠病毒原型株、Delta变异株、Beta变异株、Omicron变异株亚型BA.1、BA.1.1、BA.2、BA.3RBD抗原的结合抗体滴度水平，以及后者(即，第49天)相对于前者(即，第35天)抗体滴度的提高倍数(每张图上面的“数字×”表示提高倍数)，这种血清抗体滴度的提高倍数反映了采用mRNA疫苗进行序贯免疫后、较序贯免疫前的抗体滴度水平的提高；由这些结果可知：采用各mRNA疫苗进行序贯免疫后，其血清抗体滴度水平较序贯免疫前均显著提高，表明：本申请的mRNA疫苗可用于序贯免疫，以加强免疫反应水平。
此外，与PP mRNA疫苗序贯免疫组相比：
1)PB mRNA疫苗序贯免疫组
针对上述七种新冠病毒毒株，在第49天的血清抗体滴度相对于第35天的血清抗体滴度的提高倍数均远远高于PP mRNA疫苗序贯免疫组，最高可达10倍之多；
2)DB mRNA疫苗序贯免疫组
针对新冠病毒原型株、Delta变异株、Beta变异株和Omicron亚型BA.2变异株，在第49天的血清抗体滴度相对于第35天的血清抗体滴度的提高倍数均远远高于PP mRNA疫苗序贯免疫组，最高可达将近5倍；
3)DO mRNA疫苗序贯免疫组
针对上述七种新冠病毒毒株，在第49天的血清抗体滴度相对于第35天的血清抗体滴度的提高倍数均远远高于PP mRNA疫苗序贯免疫组，最高可达5倍以上。
此外，各免疫组在第49天采集的血清针对新冠病毒原型株、Delta变异株、Beta变异株、Omicron变异株亚型BA.1、BA.1.1、BA.2、BA.3抗原的结合抗体滴度结果如图5a所示。
图5a显示，与PP mRNA疫苗序贯免疫组相比：
1)PB mRNA疫苗序贯免疫组
针对上述七种新冠病毒毒株，其所诱导的结合抗体滴度水平均远远高于PP mRNA疫苗，最高可达3倍；
2)DB mRNA疫苗序贯免疫组
针对新冠病毒原型株、Delta变异株、Beta变异株和Omicron亚型BA.2变异株，其所诱导的结合抗体滴度水平均远远高于PP mRNA疫苗；
3)DO mRNA疫苗序贯免疫组
针对上述七种新冠病毒毒株，其所诱导的结合抗体滴度水平均远远高于PP mRNA疫苗。
实施例8：序贯免疫小鼠血清对新冠病毒各毒株的假病毒的抑制效果的评价
本实施例中，采用实施例4中所记载的方法，检测了实施例6中在第49天所采集的各免疫组小鼠血清针对新冠病毒原型株、Delta变异株、Beta变异株、Omicron变异株BA.1、BA.1.1、BA.2、BA.3亚型的假病毒的中和抗体滴度。
结果如图5b所示。图5b显示：与PP mRNA疫苗序贯免疫组相比：
1)PB mRNA疫苗序贯免疫组
针对新冠病毒原型株、Delta、Omicron BA.1.1的假病毒，其所诱导的中和抗体滴度水平高于PP mRNA疫苗，或与PP mRNA疫苗相当；
但是，针对新冠病毒Omicron BA.1、BA.2、BA.3的假病毒，其所诱导的中和抗体滴度水平比PP mRNA疫苗高两倍以上，最高达3倍左右；
2)DB mRNA疫苗序贯免疫组
除了新冠病毒Omicron BA.1.1以外，其针对其他所有类型毒株的假病毒所诱导的中和抗体滴度水平均显著高于PP mRNA疫苗；
3)DO mRNA疫苗序贯免疫组
针对新冠病毒Omicron变异株的各亚型毒株的假病毒，其所诱导的中和抗体滴度水平高于PP mRNA疫苗，或与其相当。
实施例9：序贯免疫所诱导细胞免疫水平的评价
本实施例中，采用实施例5中所记载的方法，和实施例6中在第49天所采集的各免疫组小鼠的脾脏样本，检测序贯免疫所诱导的细胞免疫水平。
结果如图5c所示，由图5c可知，在使用上述四种新冠病毒RBD肽库刺激后，PB、DB、DO mRNA疫苗序贯免疫的小鼠脾脏细胞所产生的IFN-γ+CD4+、IFN-γ+CD8+细胞的数量与PP mRNA疫苗序贯免疫的小鼠相当，它们均远远高于LNP对照组，这表明：采用PB、DB、DO mRNA疫苗进行的序贯免疫均能有效激发细胞免疫应答，并且其所激发的细胞免疫水平与PP mRNA疫苗序贯免疫组相当。
最后应说明的是：以上实施例仅用以说明本申请的技术方案，而非对其限制；尽管参照前述实施例对本申请进行了详细的说明，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申请技术方案的精神和范围。
工业实用性
本申请提供的新型冠状病毒嵌合核酸疫苗针对多种新冠病毒毒株均可提供较强的免疫保护效力，并且在与其他类型疫苗进行序贯免疫时可诱导针对新冠病毒各型毒株的(即，广谱的)、显著增高的免疫反应水平，非常适用于当前复杂的疫情防控，具有潜在的临床应用价值和前景。

Claims (16)

  1. 一种多核苷酸，其编码如式(I)所示结构的重组嵌合抗原肽：
    (A-B)-C-(A-B’)
    (I)
    式(I)中：
    Option 1:A-B表示新型冠状病毒原型株S蛋白RBD结构域或其一部分的氨基酸序列，或与其具有至少90％，92％，95％，96％，97％，98％或99％同一性且与其具有相同或基本相同的免疫原性的氨基酸序列；
    A-B’表示新型冠状病毒Beta变异株S蛋白RBD结构域或其一部分的氨基酸序列，或与其具有至少90％，92％，95％，96％，97％，98％或99％同一性且与其具有相同或基本相同的免疫原性的氨基酸序列；或者
    Option 2:A-B表示新型冠状病毒Delta变异株S蛋白RBD结构域或其一部分的氨基酸序列，或与其具有至少90％，92％，95％，96％，97％，98％或99％同一性且与其具有相同或基本相同的免疫原性的氨基酸序列；
    A-B’表示新型冠状病毒Beta变异株S蛋白RBD结构域或其一部分的氨基酸序列，或与其具有至少90％，92％，95％，96％，97％，98％或99％同一性且与其具有相同或基本相同的免疫原性的氨基酸序列；或者
    Option 3:A-B表示新型冠状病毒Delta变异株S蛋白RBD结构域或其一部分的氨基酸序列，或与其具有至少90％，92％，95％，96％，97％，98％或99％同一性且与其具有相同或基本相同的免疫原性的氨基酸序列；
    A-B’表示新型冠状病毒Omicron变异株S蛋白RBD结构域或其一部分的氨基酸序列，或与其具有至少90％，92％，95％，96％，97％，98％或99％同一性且与其具有相同或基本相同的免疫原性的氨基酸序列；
    C表示连接子(GGS)n；其中，n＝0,1,2,3,4或5。
  2. 根据权利要求1所述的多核苷酸，其特征在于，所述新型冠状病毒原型株S蛋白RBD结构域的一部分为其全部氨基酸序列的至少70％、80％、85％、90％、92％、95％、96％、97％、98％或99％；
    和/或，所述新型冠状病毒Beta变异株S蛋白RBD结构域的一部分为其全部氨基酸序列的至少70％、80％、85％、90％、92％、95％、96％、97％、98％或99％；
    和/或，所述新型冠状病毒Delta变异株S蛋白RBD结构域的一部分为其全部氨基酸序列的至少70％、80％、85％、90％、92％、95％、96％、97％、98％或99％；
    和/或，所述新型冠状病毒Omicron变异株S蛋白RBD结构域的一部分为其全部氨基 酸序列的至少70％、80％、85％、90％、92％、95％、96％、97％、98％或99％；
    和/或，n＝0,1,2或3。
  3. 根据权利要求1或2所述的多核苷酸，其特征在于，所述新型冠状病毒原型株S蛋白RBD结构域或其一部分的氨基酸序列如SEQ ID NO:1所示，或者如SEQ ID NO:1所示的氨基酸序列经取代、缺失或添加一个或几个氨基酸获得的、与其具有相同或基本相同的免疫原性的氨基酸序列；
    和/或，所述新型冠状病毒Beta变异株S蛋白RBD结构域或其一部分的氨基酸序列如SEQ ID NO:2所示，或者如SEQ ID NO:2所示的氨基酸序列经取代、缺失或添加一个或几个氨基酸获得的、与其具有相同或基本相同的免疫原性的氨基酸序列；
    和/或，所述新型冠状病毒Delta变异株S蛋白RBD结构域或其一部分的氨基酸序列如SEQ ID NO:3所示，或者如SEQ ID NO:3所示的氨基酸序列经取代、缺失或添加一个或几个氨基酸获得的、与其具有相同或基本相同的免疫原性的氨基酸序列；
    和/或，所述新型冠状病毒Omicron变异株S蛋白RBD结构域或其一部分的氨基酸序列如SEQ ID NO:4所示，或者如SEQ ID NO:4所示的氨基酸序列经取代、缺失或添加一个或几个氨基酸获得的、与其具有相同或基本相同的免疫原性的氨基酸序列；
    和/或，n＝0,1或2。
  4. 根据权利要求3所述的多核苷酸，其特征在于，当式(I)为Option 1时，如式(I)所示结构的重组嵌合抗原肽具有如SEQ ID NO:5所示的氨基酸序列；
    或者，当式(I)为Option 2时，如式(I)所示结构的重组嵌合抗原肽具有如SEQ ID NO:6所示的氨基酸序列；
    或者，当式(I)为Option 3时，如式(I)所示结构的重组嵌合抗原肽具有如SEQ ID NO:7所示的氨基酸序列。
  5. 根据权利要求1-4任一项所述的多核苷酸，其特征在于，所述多核苷酸为DNA分子；
    优选地，当式(I)为Option 1时，所述DNA分子具有如SEQ ID NO:8所示的DNA序列；
    优选地，当式(I)为Option 2时，所述DNA分子具有如SEQ ID NO:9所示的DNA序列；
    优选地，当式(I)为Option 3时，所述DNA分子具有如SEQ ID NO:10所示的DNA序列。
  6. 根据权利要求1-4任一项所述的多核苷酸，其特征在于，所述多核苷酸为mRNA分子；
    优选地，当式(I)为Option 1时，所述mRNA分子具有如SEQ ID NO:11所示的mRNA序列；
    优选地，当式(I)为Option 2时，所述mRNA分子具有如SEQ ID NO:12所示的mRNA 序列；
    优选地，当式(I)为Option 3时，所述mRNA分子具有如SEQ ID NO:13所示的mRNA序列。
  7. 一种核酸构建体，其包含如权利要求1-6任一项所述的多核苷酸，以及任选地，与所述多核苷酸可操作地连接的至少一个表达调控元件。
  8. 一种表达载体，其包含如权利要求7所述的核酸构建体。
  9. 一种宿主细胞，其中转化或转染有如权利要求1-6任一项所述的多核苷酸、如权利要求7所述的核酸构建体或如权利要求8所述的表达载体。
  10. 如权利要求1-6任一项所述的多核苷酸、如权利要求7所述的核酸构建体、如权利要求8所述的表达载体或如权利要求9所述的宿主细胞在制备用于预防和/或治疗新型冠状病毒的疫苗中的应用；
    优选地，所述疫苗用于单独免疫或者与其他类型的新型冠状病毒疫苗进行序贯免疫；进一步优选地，所述其他类型的新型冠状病毒疫苗包括灭活疫苗。
  11. 一种嵌合核酸疫苗或免疫原性组合物，其包含如权利要求1-6任一项所述的多核苷酸、如权利要求7所述的核酸构建体、如权利要求8所述的表达载体或如权利要求9所述的宿主细胞，以及生理学可接受的媒介物、佐剂、赋形剂、载体和/或稀释剂。
  12. 根据权利要求11所述的嵌合核酸疫苗或免疫原性组合物，其为新型冠状病毒DNA疫苗，所述DNA疫苗包括：
    (i)真核表达载体；和
    (ii)构建入所述真核表达载体中的、编码如式(I)所示结构的重组嵌合抗原肽的DNA序列，优选为如SEQ ID NO:8、9或10所示的DNA序列；
    优选地，所述真核表达载体选自pGX0001、pVAX1、pCAGGS和pcDNA系列载体。
  13. 根据权利要求11所述的嵌合核酸疫苗或免疫原性组合物，其为新型冠状病毒mRNA疫苗，所述mRNA疫苗包括：
    (I)编码如式(I)所示结构的重组嵌合抗原肽的mRNA序列，优选为如SEQ ID NO:11、12或13所示的mRNA序列；和
    (II)脂质纳米颗粒。
  14. 根据权利要求11所述的嵌合核酸疫苗或免疫原性组合物，其为新型冠状病毒-病毒载体疫苗，其包括：
    (1)病毒骨架载体；和
    (2)构建入所述病毒骨架载体中的、编码如式(I)所示结构的重组嵌合抗原肽的DNA序列，优选为如SEQ ID NO:8、9或10所示的DNA序列；
    优选地，所述病毒骨架载体选自以下病毒载体中的一种或几种：腺病毒载体、痘病 毒载体、流感病毒载体、腺相关病毒载体。
  15. 根据权利要求11-14任一项所述的嵌合核酸疫苗或免疫原性组合物，其特征在于，所述疫苗或免疫原性组合物为鼻喷剂、口服制剂、栓剂或胃肠外制剂的形式；
    优选地，所述鼻喷剂选自气雾剂、喷雾剂和粉雾剂；
    优选地，所述口服制剂选自片剂、粉末剂、丸剂、散剂、颗粒剂、细粒剂、软/硬胶囊剂、薄膜包衣剂、小丸剂、舌下片和膏剂；
    优选地，所述胃肠外制剂为经皮剂、软膏剂、硬膏剂、外用液剂、可注射或可推注制剂。
  16. 一种试剂盒，其包括根据权利要求11-14任一项所述的嵌合核酸疫苗或免疫原性组合物，以及任选地其他类型的新型冠状病毒疫苗，所述嵌合核酸疫苗或免疫原性组合物与所述其他类型的新型冠状病毒疫苗单独包装；
    优选地，所述其他类型的新型冠状病毒疫苗为新型冠状病毒灭活疫苗。
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