CN114989308A - Novel coronavirus chimeric nucleic acid vaccine and use thereof - Google Patents

Novel coronavirus chimeric nucleic acid vaccine and use thereof Download PDF

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Publication number
CN114989308A
CN114989308A CN202210515599.7A CN202210515599A CN114989308A CN 114989308 A CN114989308 A CN 114989308A CN 202210515599 A CN202210515599 A CN 202210515599A CN 114989308 A CN114989308 A CN 114989308A
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amino acid
acid sequence
vaccine
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asn
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CN114989308B (en
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高福
王奇慧
戴连攀
杜沛
陈茜
马雪慧
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Institute of Microbiology of CAS
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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

Abstract

The invention relates to a polynucleotide, related products and application thereof in preparing a novel corona vaccine, and a chimeric nucleic acid vaccine or an immunogenic composition based on the polynucleotide; the polynucleotide encodes a recombinant chimeric antigen formed by direct series connection or connection through a linker of an RBD structure domain of an S protein of a prototype strain of the neocoronaviruse and an RBD structure domain of an S protein of a Beta variant strain, or an RBD structure domain of an S protein of a Delta variant strain and an RBD structure domain of an S protein of an Omicron variant strain; the chimeric nucleic acid vaccine based on the polynucleotide can provide stronger immune protective efficacy against various new coronavirus strains, and can induce remarkably increased immune response levels against various new coronavirus strains (namely, broad spectrum) when being sequentially immunized with other types of vaccines.

Description

Novel coronavirus chimeric nucleic acid vaccine and application thereof
Technical Field
The invention relates to the field of biomedicine, in particular to a novel coronavirus chimeric nucleic acid vaccine and application thereof.
Background
The novel coronavirus pneumonia (also called COVID-19) is an acute respiratory infectious disease caused by infection of a novel coronavirus (also called a novel coronavirus, SARS-CoV-2). The new coronavirus belongs to the genus beta-coronavirus of the family Coronaviridae, has an envelope, and is a single-stranded positive-strand RNA virus. The spike protein (also called S protein) on the surface of the new coronavirus is responsible for the binding and membrane fusion of the virus and a host cell membrane receptor, and a Receptor Binding Domain (RBD) exists on the S protein, so that the S protein is an important vaccine target, can stimulate the generation of neutralizing antibodies, and has the advantage of immune focusing.
At present, the epidemic situation of new coronary pneumonia is still severe in the global scope, new coronary virus variant strains are continuously appeared and prevail, and particularly, Deltay (Delta) and Omicronron (Omicron) variant strains are rolled around the world in turn and become dominant epidemic strains. In particular, there are also a number of subtypes of the novel variant coronavirus Omicron (e.g., subtypes ba.1, ba.2, ba.1.1, ba.3), which has a transmission rate exceeding Delta and has become the dominant strain worldwide; among them, the strain of BA.2 subtype has a higher transmission rate than other Omicron subtypes, and now occupies the largest proportion. The S protein of the Omicron variant has more than 50 amino acid mutations, and the amino acid mutations are greatly increased compared with the former Delta variant.
Due to the existence of many mutations in the sequence of the S protein or RBD in these new emergent crown virus variants, the effectiveness of the immune response triggered by the existing vaccine designed and developed based on the new crown virus Prototype strain (Prototype) is greatly reduced when facing the variants (such as Omicron variants), and the phenomenon that the variants break through the vaccine and protect the antibody is called immune escape. The phenomenon of immune escape is particularly obvious on various subtypes of Omicron. In order to solve the problem of immune escape of new coronavirus variant strains, a novel vaccine needs to be developed to adapt to newly-appeared variant strains (such as an Omicron variant strain and various subtypes thereof) so as to have a strong protection effect on current epidemic strains; meanwhile, as a phenomenon that a plurality of variant strains (particularly a plurality of Omicron subtypes) are epidemic at the same time exists at present, the novel vaccine needs to be capable of inducing broad-spectrum immune response so as to prevent a plurality of new coronavirus strains as far as possible at the same time, and the novel vaccine can play a vital role in preventing and controlling new coronavirus strains.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a polynucleotide, related products and the use thereof in the preparation of vaccines for preventing and/or treating new coronavirus, a chimeric nucleic acid vaccine or an immunogenic composition based on the polynucleotide or related products, and a kit comprising the chimeric nucleic acid vaccine or the immunogenic composition; the polynucleotide encodes a recombinant chimeric antigenic peptide formed by (1) the RBD domain of the S protein of the novel coronavirus prototype strain and the RBD domain of the S protein of the novel coronavirus Beta variant (or a part thereof), or (2) the RBD domain of the S protein of the novel coronavirus Delta variant (or a part thereof) and the RBD domain of the S protein of the novel coronavirus Beta variant (or a part thereof), or (3) the RBD domain of the S protein of the novel coronavirus Delta variant (or a part thereof) and the RBD domain of the S protein of the novel coronavirus Omicron variant (or a part thereof) connected in series directly or through a linker; the chimeric nucleic acid vaccine can provide strong immune protective efficacy against various strains of the new coronavirus, and can induce remarkably increased immune response levels against various strains of the new coronavirus (namely, broad spectrum) when sequentially immunized with other types of vaccines (such as inactivated vaccines).
Specifically, the invention provides the following technical scheme:
in a first aspect, the present invention provides a polynucleotide encoding a recombinant chimeric antigen peptide having the structure according to formula (I):
(A-B)-C-(A-B’)
(I)
in formula (I):
option 1: A-B represents the amino acid sequence of the RBD domain of the S protein of a prototype strain of a novel coronavirus, or a portion thereof, or an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and having the same or substantially the same immunogenicity as it;
A-B' represents the amino acid sequence of the RBD domain of the S protein of a novel variant of coronavirus Beta or a part thereof, or an amino acid sequence which is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and has the same or substantially the same immunogenicity as it; or
A-B represents the amino acid sequence of the RBD domain of the S protein of the novel coronavirus Delta variant or a portion thereof, or an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and having the same or substantially the same immunogenicity as it;
A-B' represents the amino acid sequence of the RBD domain of the S protein of a novel variant of coronavirus Beta or a part thereof, or an amino acid sequence which is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and has the same or substantially the same immunogenicity as it; or alternatively
A-B represents the amino acid sequence of the RBD domain of the S protein of the novel coronavirus Delta variant or a portion thereof, or an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and having the same or substantially the same immunogenicity as it;
A-B' represents the amino acid sequence of the RBD domain of S protein of the novel variant S protein of coronavirus, or a part thereof, or an amino acid sequence which is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and has the same or substantially the same immunogenicity as it;
c represents a linker (GGS) n (ii) a Wherein n is 0,1,2,3,4 or 5.
Preferably, for the above polynucleotides, the portion of the RBD domain of the S protein of the novel coronavirus prototype strain is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% of its entire amino acid sequence;
and/or, a portion of the RBD domain of the S protein of the novel variant coronavirus Beta is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% of its entire amino acid sequence;
and/or, a portion of the RBD domain of the S protein of the novel coronavirus Delta variant is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% of its entire amino acid sequence;
and/or, a portion of the RBD domain of the S protein of the novel variant strain of coronavirus which is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% of its entire amino acid sequence;
and/or, n ═ 0,1,2, or 3.
In some preferred embodiments, the amino acid sequence of the RBD domain or a part thereof of the S protein of the novel coronavirus prototype strain is shown as SEQ ID NO. 1, or an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown as SEQ ID NO. 1 and has the same or basically same immunogenicity with the amino acid sequence;
and/or the amino acid sequence of the RBD structural domain of the S protein of the novel coronavirus Beta variant or a part thereof is shown as SEQ ID NO. 2, or the amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids and has the same or basically the same immunogenicity as the amino acid sequence shown as the SEQ ID NO. 2;
and/or the amino acid sequence of the RBD structural domain of the S protein of the novel coronavirus Delta variant or a part of the RBD structural domain is shown as SEQ ID NO. 3, or the amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids and has the same or basically the same immunogenicity as the amino acid sequence shown as SEQ ID NO. 3;
and/or, the amino acid sequence of the RBD structural domain or a part thereof of the S protein of the novel coronavirus Omicron variant strain is shown as SEQ ID NO. 4, or the amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids and has the same or basically the same immunogenicity as the amino acid sequence shown as SEQ ID NO. 4;
and/or, n is 0,1 or 2.
In a further preferred embodiment, when formula (I) is Option 1, the recombinant chimeric antigen peptide having the structure shown in formula (I) has an amino acid sequence shown in SEQ ID NO. 5;
when the formula (I) is Option 2, the recombinant chimeric antigen peptide with the structure shown in the formula (I) has an amino acid sequence shown in SEQ ID NO. 6;
when the formula (I) is Option 3, the recombinant chimeric antigen peptide with the structure shown in the formula (I) has an amino acid sequence shown in SEQ ID NO. 7.
In some preferred embodiments, the polynucleotide is a DNA molecule;
further preferably, when formula (I) is Option 1, the DNA molecule has the DNA sequence shown in SEQ ID NO. 8;
further preferably, when formula (I) is Option 2, the DNA molecule has the DNA sequence shown in SEQ ID NO. 9;
further preferably, when formula (I) is Option 3, the DNA molecule has the DNA sequence shown in SEQ ID NO 10.
In other preferred embodiments, the polynucleotide is an mRNA molecule;
further preferably, when formula (I) is Option 1, the mRNA molecule has the mRNA sequence shown as SEQ ID NO. 11;
further preferably, when formula (I) is Option 2, the mRNA molecule has the mRNA sequence shown as SEQ ID NO 12;
further preferably, when formula (I) is Option 3, the mRNA molecule has the mRNA sequence shown in SEQ ID NO 13.
In a second aspect, the present invention provides a nucleic acid construct comprising a polynucleotide as described in the first aspect above, and optionally, at least one expression control element operably linked to the polynucleotide.
In a third aspect, the present invention provides an expression vector comprising a nucleic acid construct as described above in the second aspect.
In a fourth aspect, the present invention provides a host cell transformed or transfected with a polynucleotide as described in the first aspect above, a nucleic acid construct as described in the second aspect above or an expression vector as described in the third aspect above.
In a fifth aspect, the present invention provides the use of a polynucleotide as defined in the first aspect above, a nucleic acid construct as defined in the second aspect above, an expression vector as defined in the third aspect above or a host cell as defined in the fourth aspect above for the preparation of a vaccine for the prevention and/or treatment of a novel coronavirus;
preferably, the vaccine is used for immunization alone or in sequential immunization with other types of novel coronavirus vaccines; further preferably, the other types of novel coronavirus vaccines include inactivated vaccines.
In a sixth aspect, the present invention provides a chimeric nucleic acid vaccine or immunogenic composition comprising a polynucleotide as described in the first aspect above, a nucleic acid construct as described in the second aspect above, an expression vector as described in the third aspect above or a host cell as described in the fourth aspect above, and a physiologically acceptable vehicle, adjuvant, excipient, carrier and/or diluent.
In a specific embodiment, the chimeric nucleic acid vaccine or immunogenic composition is a novel coronavirus DNA vaccine comprising:
(i) a eukaryotic expression vector; and
(ii) a DNA sequence which is constructed into the eukaryotic expression vector and encodes the recombinant chimeric antigen peptide with the structure shown in the formula (I) as defined in the first aspect, preferably a DNA sequence shown in SEQ ID NO 8, 9 or 10;
preferably, the eukaryotic expression vector is selected from the group consisting of pGX0001, pVAX1, pCAGGS and pcDNA series vectors.
In another specific embodiment, the chimeric nucleic acid vaccine or immunogenic composition is a novel coronavirus mRNA vaccine comprising:
(I) an mRNA sequence encoding a recombinant chimeric antigen peptide having a structure represented by the formula (I) as defined in the above first aspect, preferably an mRNA sequence represented by SEQ ID NO 11, 12 or 13; and
(II) lipid nanoparticles.
In another specific embodiment, the chimeric nucleic acid vaccine or immunogenic composition is a novel coronavirus-viral vector vaccine comprising:
(1) a viral backbone vector; and
(2) a DNA sequence, preferably a DNA sequence shown in SEQ ID NO 8, 9 or 10, constructed into the viral backbone vector encoding a recombinant chimeric antigen peptide having the structure shown in formula (I) as defined in the first aspect above;
optionally, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vectors, poxvirus vectors, influenza virus vectors, adeno-associated virus vectors.
In a possible implementation, the chimeric nucleic acid vaccine or immunogenic composition is in the form of a nasal spray, oral formulation, suppository, or parenteral formulation;
preferably, the nasal spray is selected from the group consisting of an aerosol, a spray and a powder spray;
preferably, the oral formulation is selected from the group consisting of tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film coatings, pellets, sublingual tablets and ointments;
preferably, the parenteral formulation is a transdermal agent, ointment, plaster, topical liquid, injectable or bolus formulation.
In a seventh aspect, the present invention provides a kit comprising a chimeric nucleic acid vaccine or immunogenic composition as described in the above sixth aspect, and optionally a further type of novel coronavirus vaccine, said chimeric nucleic acid vaccine or immunogenic composition being packaged separately from said further type of novel coronavirus vaccine;
preferably, the other type of novel coronavirus vaccine is a novel inactivated coronavirus vaccine.
Advantageous effects
The present inventors designed a polynucleotide encoding a recombinant chimeric antigen peptide formed by (1) the RBD domain (or a part thereof) of the S protein of a novel coronavirus prototype strain and the RBD domain (or a part thereof) of the S protein of a novel coronavirus Beta variant strain, or (2) the RBD domain (or a part thereof) of the S protein of a novel coronavirus Delta variant strain and the RBD domain (or a part thereof) of the S protein of a novel coronavirus Beta variant strain, or (3) the RBD domain (or a part thereof) of the S protein of a novel coronavirus Delta variant strain and the RBD domain (or a part thereof) of the S protein of a novel coronavirus Omicron variant strain in direct tandem connection or in connection via a linker; the chimeric nucleic acid vaccine based on the polynucleotide can provide stronger immune protection efficacy against various new coronavirus strains, can induce remarkably-increased immune response levels (namely, broad spectrum) against various new coronavirus strains when being sequentially immunized with other vaccines, is very suitable for current complex epidemic situation prevention and control, and has potential clinical application value and prospect.
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One or more embodiments are illustrated by the corresponding figures in the drawings, which are not meant to be limiting. The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.
FIG. 1 is a schematic structural diagram of a prototype strain RBD dimer mRNA vaccine of a new coronavirus (abbreviated as PP mRNA vaccine as a control vaccine), a chimeric RBD dimer mRNA vaccine formed by connecting the prototype strain RBD of the new coronavirus and a Beta variant RBD (abbreviated as PB mRNA vaccine), a chimeric RBD dimer mRNA vaccine formed by connecting a Delta variant RBD and a Beta variant RBD (abbreviated as DB mRNA vaccine), and a chimeric RBD dimer mRNA vaccine formed by connecting a Delta variant RBD and an Omicron variant RBD (abbreviated as DO mRNA vaccine) constructed in example 1 of the present invention; the respective segments of the mRNA vaccine are labeled on the figure, wherein 5 'UTR represents a 5' untranslated region, 3 'UTR represents a 3' untranslated region, SP represents a signal peptide sequence, Poly (A) represents a poly A tail, type RBD represents an RBD sequence of a prototype strain, Beta RBD represents an RBD sequence of a Beta variant, Delta RBD represents an RBD sequence of a Delta variant, and Omicron (BA.1) RBD represents an RBD sequence of an Omicron variant, wherein the respective RBDs are also labeled with amino acid mutations relative to the RBD of the prototype strain.
FIG. 2 shows the humoral immunity level elicited by the PP, PB, DB, DO mRNA vaccines constructed in example 1 of the present invention, as tested in examples 3 and 4, wherein LNP represents the negative control group immunized with lipid nanoparticles; wherein, FIG. 2a is a schematic diagram of mRNA vaccine immunized mice and sampling procedure; FIGS. 2b and 2c are the binding antibody titers of sera collected at 14 th and 28 th days after immunization of mice with the mRNA vaccine against RBD antigens of the new coronavirus prototype strain, Delta variant strain, Beta variant strain and Omicron variant strains BA.1, BA.1.1, BA.2, BA.3 subtypes, respectively, in the column diagrams of FIGS. 2b and 2c, the number above each column indicates the ratio of the antibody titer represented by the column to the antibody titer represented by the corresponding column in the LNP group, and, as indicated by the arrows in the diagrams, the heatmaps on the right side of FIGS. 2b and 2c are prepared based on these numbers; FIG. 2d is a serum collected on day 28 after immunization of mice with mRNA vaccine neutralizing NT strains of pseudoviruses of neocoronaviruses prototype strain, Delta variant strain, Omicron variant strain BA.1, BA.1.1, BA.2, BA.3 subtypes in a pseudovirus neutralization experiment 50 Values, the number above each bar represents the Geometric Mean (GMT) of all samples of the experimental group, as indicated by the arrows in the figure, the heatmap on the right being made based on these numbers; all data are presented as GMT ± 95% CI (confidence interval).
FIG. 3 shows the cellular immunity level elicited by the PP, PB, DB, DO mRNA vaccines constructed in example 1 of the present invention, LNP representing the negative control group immunized with lipid nanoparticles as tested in example 5; wherein, FIG. 3a is a schematic diagram of mRNA vaccine immunized mice and sampling procedure; FIG. 3b is a bar graph showing the numbers of IFN γ + cells produced by spleen cells collected on day 21 after immunization of mice with ELISpot assay mRNA vaccine after stimulation with 4 peptide pools (peptide pools constructed by the prototype strain of New coronavirus, the Delta variant strain, the Beta variant strain, and the Omicron variant strain subtype BA.1 RBD), respectively, the numbers above the bar graph indicating the ratio of the number of IFN γ + cells represented by the bar to the number of IFN γ + cells represented by the corresponding bar in the LNP group; all data are presented as Mean ± SEM.
FIG. 4 shows the fold increase in binding antibody titer after sequential immunization of mice with inactivated vaccine and PP, PB, DB, DO mRNA vaccines constructed in example 1 of the invention, compared to pre-sequential immunization (i.e., the group not boosted with mRNA vaccine); wherein, FIG. 4a is a schematic diagram of a mouse sequential immunization and serum sampling procedure, and IV represents inactivated vaccine; fig. 4b shows the binding antibody titer levels of the sera collected at day 35 (shown by open circles) and day 49 (shown by closed circles) for the antigens of the new coronavirus prototype strain, Delta variant strain, Beta variant strain, omitron variant strain subtypes ba.1, ba.1.1, ba.2, and ba.3rbd, and the fold increase of the antibody titer of the latter (i.e., day 49) relative to the former (i.e., day 35) (the "figure x" above each figure represents the fold increase), wherein fig. 4b (i) - (v) represent the two inactivated vaccines + PP mRNA vaccine immunization groups, the two inactivated vaccines + PB mRNA vaccine immunization groups, the two inactivated vaccines + DB mRNA vaccine immunization groups, the two inactivated vaccines + DO mRNA vaccine immunization groups, and the three inactivated vaccine immunization groups, respectively.
FIG. 5 shows the immune response level of mice immunized sequentially with inactivated vaccine and PP, PB, DB, DO mRNA vaccine constructed in example 1 of the present invention, the immunization program is shown in FIG. 4 a; in each bar chart, "PP" represents the two-time inactivated vaccine + PP mRNA vaccine immunization group, "PB" represents the two-time inactivated vaccine + PB mRNA vaccine immunization group, and "DB" represents the two-time inactivated vaccine + DB mRNA vaccineA vaccine immunization group, "DO" represents a two-time inactivated vaccine + DO mRNA vaccine immunization group, "IV" represents a three-time inactivated vaccine immunization group, and "LNP" represents a two-time inactivated vaccine adjuvant (i.e., Al adjuvant) + Lipid Nanoparticle (LNP) immunization group as a negative control; wherein FIG. 5a is the binding antibody titer of the serum collected on day 49 against the antigen of the prototype strain of the new coronavirus, the Delta variant strain, the Beta variant strain, the Omicron variant strain subtypes BA.1, BA.1.1, BA.2, BA.3, in the column chart of FIG. 5a, the number of the first row above each column represents the ratio of the antibody titer represented by the column to the antibody titer represented by the corresponding column in the Inactivated Vaccine (IV) group, the number of the second row represents the ratio of the antibody titer represented by the column to the antibody titer represented by the corresponding column in the LNP group, and as shown by the arrow in the chart, the heat map on the right side of FIG. 5a is prepared based on the second row number; FIG. 5b is NT representation of sera collected at day 49 neutralizing 6 pseudoviruses (prototype, Delta variant, Omicron variant subtypes BA.1, BA.1.1, BA.2, BA.3) in a pseudovirus neutralization experiment 50 Values, in the bar chart of FIG. 5b, the first row of numbers above each bar represents the NT represented by that bar 50 Titer values and NT represented by the corresponding bars in the Inactivated Vaccine (IV) group 50 The ratio of titer values, the second row number above each bar represents the Geometric Mean (GMT) of all samples of the experimental group, and the heatmap on the right is made based on the second row number as indicated by the arrow in the figure; the data in FIGS. 5a and 5b are presented as GMT + -95% CI (confidence interval); FIG. 5c shows the proportion of IFN γ + cells produced by spleen CD8+ and CD4+ cells harvested on day 49 of the ICS assay after stimulation with 4 peptide pools (peptide pools constructed from prototype strain, Delta, Beta, Omicron subtype BA.1 variant strain RBD), respectively, the data in FIG. 5c being presented as Mean + -SEM; statistical differences were calculated by the Mann-Whitney test method (, p)<0.05；**,p<0.01)。
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In some embodiments, materials, elements, methods, means, and the like that are well known to those skilled in the art are not described in detail in order to not unnecessarily obscure the present invention.
Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations such as "comprises" or "comprising", etc., will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.
Example 1: construction, in vitro preparation and packaging of prototype strain RBD dimer mRNA vaccine of new coronavirus (PP mRNA vaccine for short) as a control, chimeric RBD dimer mRNA vaccine of prototype strain and Beta variant (PB mRNA vaccine for short), chimeric RBD dimer mRNA vaccine of Delta variant strain and Beta variant (DB mRNA vaccine for short) and chimeric RBD dimer mRNA vaccine of Delta variant strain and Omicron variant (DO mRNA vaccine for short)
According to the structural schematic diagram of PP, PB, DB and DO mRNA vaccines shown in figure 1, the construction, in vitro preparation and packaging of the mRNA vaccines are carried out according to the following procedures:
1) in vitro transcription and capping of mRNA vaccines
In this example, the basic plasmid for the in vitro transcription of mRNA vaccine was pUC57, provided by Nanjing Kingsrei Biotech, Inc.
The DNA expression elements of the mRNA vaccine were introduced by conventional molecular biology means on the base plasmid pUC57, including: (1) t7 promoter, (2) DNA coding region of mRNA vaccine (PB, DB, DO mRNA vaccine DNA coding sequence is shown as SEQ ID NO:8, 9, 10 respectively, PP mRNA vaccine DNA coding sequence as comparison is shown as SEQ ID NO: 14), (3) upstream of coding region 5 'end UTR sequence (several mRNA vaccines 5' end UTR sequence is the same, all shown as SEQ ID NO: 15), (4) signal peptide sequence (SP, shown as SEQ ID NO: 16), and (5) downstream 3 'end UTR sequence (several mRNA vaccines 3' end UTR sequence is the same, all shown as SEQ ID NO: 17), and Poly A tail (Poly-A-tail).
Firstly, the in vitro transcription plasmid is subjected to enzyme digestion by using a restriction enzyme BamHI, and the in vitro transcription plasmid is linearized; purifying by using a conventional DNA purification method to obtain a template of in vitro transcription; then, based on the template, in vitro transcription was performed using a T7RNA in vitro transcription kit (E131-01A, shoal protein science and technology ltd), to obtain in vitro transcribed mRNA; finally, the mRNA was purified by lithium chloride precipitation using a lithium chloride recovery kit (S125, suzhou near shore protein science, ltd) to obtain purified in vitro transcribed mRNA.
Then, using a capping enzyme kit Cap1 capping enzyme kit (M082-01B, Suzhou near shore protein science and technology Co., Ltd.), capping the 5' end Cap1 of the purified in vitro transcription mRNA to satisfy the condition of being translated in eukaryotic cells; thereafter, the mRNA was purified again by the same lithium chloride precipitation method as described above to obtain purified mRNA with the 5' -end being capped.
2) Lipid Nanoparticles (LNP) package mRNA
Mixing cationic lipid, phosphatidylcholine, cholesterol and PEG lipid according to a ratio of 50: 10: 38.5: 1.5, then mixing with the 5' end-capped mRNA by using a Nanolasembler Nanohhtop nanoliposome packager manufactured by Precision Nano Systems, and packaging. After packaging, the buffer solution is replaced by PBS by centrifugation or dialysis. After packaging is completed, the mRNA packaging efficiency is identified by using a Quan-iT Ribogreen RNA reagent kit of Thermo Fisher company, and the packaging efficiency meets the standard of mRNA vaccine.
Example 2: experimental animal immunization and sample Collection
In this example, female mice of BALB/c strain (purchased from Wintolite, Inc.) 6-8 weeks old were used for animal experiments; the experimental components comprise an mRNA vaccine immunization group and a negative control group, wherein the mRNA vaccine immunization group comprises a PP mRNA vaccine immunization group, a PB mRNA vaccine immunization group, a DB mRNA vaccine immunization group and a DO mRNA vaccine immunization group, and the negative control group is an LNP immunization group. All 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 day 0 and day 14, respectively, and mice in the negative control group were injected with the same amount of empty LNP at the same time. The vaccination methods were intramuscular injections at a dose of 5 μ g mRNA vaccine or empty LNP per mouse. Mouse serum samples were taken at day 14 and day 28, respectively, for testing the immune sera for binding antibody titers and pseudovirus neutralizing antibody titers. In addition, mouse spleen samples were also collected on day 21 for testing T cell immunity.
mRNA vaccine immunized mice and sampling procedure are shown in FIG. 2 a.
Example 3: examination of mouse serum antibody titer
Respectively coating the ELISA plates with a new coronavirus prototype strain (SEQ ID NO:1), a Delta variant strain (SEQ ID NO:3), a Beta variant strain (SEQ ID NO:2) and Omicron variant strains BA.1(SEQ ID NO:4), BA.1.1(SEQ ID NO:18), BA.2(SEQ ID NO:19) and BA.3(SEQ ID NO:20) subtype RBD antigen protein (0.2 mu g/ml), and sealing the coated ELISA plates in 5% skim milk for 1 hour; then, the sera collected from the mice of each experimental group in example 2 were incubated at 56 ℃ for 30 minutes for inactivation; the inactivated serum samples were diluted in a three-fold gradient starting from 1:200 or 1:1000, and then the dilutions were added to each well, followed by incubation of the ELISA plate for 1 hour at 37 ℃; goat anti-mouse IgG-HRP antibody (purchased from beaconigen (EASYBio)) was added to the plate as a secondary antibody and incubated at 37 ℃ for another 1 hour; finally, color development was performed using a 3,3',5,5' -Tetramethylbenzidine (TMB) substrate, and after completion of color development, the reaction was terminated with 2M hydrochloric acid, and absorbances at 450nm and 630nm were measured using a microplate reader (PerkinElmer). The absorbance value was calculated by subtracting the absorbance at 630nm from the absorbance at 450nm of the same well. Endpoint titers were defined as: the serum produced an absorbance (as described above, absorbance at 450nm minus absorbance at 630 nm) that was 2.1 times greater than the background value for the corresponding dilution of the serum. Antibody titers below the detection limit were defined as one third of the detection limit.
The serum collected at day 14 and day 28 of the immunization schedule for each experimental group of mice was shown in fig. 2b and fig. 2c for the binding antibody titers against the RBD antigens of the seven novel coronaviruses, respectively, in fig. 2b and fig. 2c, the left side is a bar graph of the end titer vs vaccine species and the right side is a corresponding heat map prepared based on the ratio of the end antibody titer of each mRNA vaccine to the end antibody titer of the LNP group, as illustrated in the figure.
Figure 2c is the result of day 28 where antibody levels were more stable, as can be seen in figure 2 c:
(1) PB mRNA vaccine
The PB mRNA vaccine can induce higher level of the combined antibody aiming at the tested new coronavirus prototype strain and each variant strain; and, it induces levels of bound antibody titers comparable to, or higher than, PP mRNA vaccines (e.g., 2-3 fold higher for ba.1.1 and ba.2, respectively);
(2) DB mRNA vaccine
For the tested new coronavirus prototype strain and each variant strain, the DB mRNA vaccine can induce higher level of the combined antibody; moreover, the titer level of the combined antibody induced by the strain aiming at various new coronavirus strains is far higher than that of a PP mRNA vaccine, and the titer level is up to more than 3 times;
(3) DO mRNA vaccine
The DO mRNA vaccine can induce higher level of the combined antibody aiming at the tested new coronavirus prototype strain and each variant strain; particularly, the serum antibody titer level induced by the vaccine against all subtypes of the Omicron variant strain is far higher than that of a PP mRNA vaccine; for example, the antibody titer level induced by DO mRNA vaccine is more than 2 times higher than PP mRNA vaccine against ba.1 subtype, the antibody titer level induced by DO mRNA vaccine is more than 5 times higher than PP mRNA vaccine against ba.1.1 subtype, the antibody titer level induced by ba.2 and ba.3 subtype is nearly 6 times higher than PP mRNA vaccine, the antibody titer level induced by ba.3 subtype is more than 3 times higher than PP mRNA vaccine; this suggests that the DO mRNA vaccine of the present invention can induce significantly higher antibody titer levels against various subtypes of the Omicron variant, indicating that it will have significantly higher immunoprotection efficacy against various types of Omicron variant; furthermore, DO mRNA also induced higher antibody titer levels against the prototype strain of the new coronavirus and other variants, suggesting a very broad spectrum.
Example 4: packaging and serum neutralization of pseudoviruses of a novel coronavirus strain
In this example, the sera of immunized mice collected in example 2 were examined for 50% of the titer of neutralizing pseudoviruses (pVNT) against pseudoviruses of the subtypes New coronavirus prototype strain, Delta variant strain, and Omicron variant strain BA.1, BA.1.1, BA.2, and BA.3 (pVNT) 50 ) (ii) a The specific detection method comprises the following steps:
expression plasmid for preparing truncated new coronavirus S protein
The nucleotide of the rear 18 amino acids of the S protein of the new coronavirus prototype strain, the Delta variant strain and the Omicron variant strains BA.1, BA.1.1, BA.2 and BA.3 subtype is removed, the obtained nucleotides are named as WT-S-del18, Delta-S-del18, BA.1-S-del18, BA.1.1-S-del18, BA.2-S-del18 and BA.3-S-del18 respectively, the nucleotide sequences are shown as SEQ ID NO: 21-26 respectively, and the new coronavirus mutant strains are synthesized by Jinzhi company, Suzhou province; then, these nucleotide sequences were cloned into pCAGGS expression vectors to obtain expression plasmids pCAGGS-WT-S-del18, pCAGGS-Delta-S-del18, pCAGGS-BA.1-S-del18, pCAGGS-BA.1.1-S-del18, pCAGGS-BA.2-S-del18 and pCAGGS-BA.3-S-del18, respectively.
Second, packaging of pseudoviruses of prototype strain and variant strain of new coronavirus
1) HEK293T cells were plated in 10cm cell culture dishes to reach a cell density of around 80% the next day. The culture solution is DMEM medium containing 10% FBS.
2) The S protein expression plasmids of the truncated, novel strains of coronavirus prepared above were used to transfect cells in petri dishes (30. mu.g/10 cm cell culture dishes) with PEI. The target plasmid and PEI are mixed evenly according to the proportion of 1:3 and then transfected, the culture solution (DMEM medium containing 10% FBS) is replaced for 4-6h, and the culture is carried out for 24h at 37 ℃.
3) Pseudovirus packaging frame virus G.VSV-delG (Wuhan Shu Ministry of encyclopedia science and technology Co., Ltd.) was added to the above transfected HEK293T cells, incubated at 37 ℃ for 2 hours, the culture medium (DMEM medium containing 10% FBS) was changed, and VSV-G antibody (hybridoma cells expressing the antibody were purchased from ATCC cell bank) was added and the culture was continued in the incubator for 30 hours.
4) Collecting supernatant, centrifuging at 3000rpm for 10min, filtering in 0.45 μm sterile filter in ultra-clean bench, removing cell debris, packaging, and freezing at-80 deg.C.
Through the steps, the pseudoviruses of new coronavirus prototype strains, Delta variant strains and Omicron variant strains BA.1, BA.1.1, BA.2 and BA.3 subtypes are respectively obtained.
Third, evaluation of the inhibitory Effect of the serum of the immunized mouse on the pseudovirus
The sera of the experimental groups of mice collected on day 28 in example 2 were inactivated by incubation at 56 ℃ for 30 minutes; inactivated serum samples were diluted in a 2-fold gradient starting at 1: 80. Then, each pseudovirus was mixed with an equal volume of diluted serum and incubated at 37 ℃ for 1 hour. 100 μ l of the virus-serum mixture was added to Vero cells pre-plated in 96-well plates. After 15 hours of incubation, the number of Transduction Units (TU) was measured using CQ1 confocal imaging cytometry to calculate the neutralizing capacity of the sera of immunized mice against pseudoviruses of the above subtypes neocoronaviruses prototype strain, Delta variant strain and Omicron variant strain ba.1, ba.1.1, ba.2 and ba.3.
The results are shown in FIG. 2 d; as depicted in the FIG. 2d legend, the left hand histogram of FIG. 2d shows pVNT of pseudoviruses of the subtypes BA.1, BA.1.1, BA.2, BA.3 of the seroneutralized prototype strain, Delta variant strain and Omicron variant strain of each immunization group 50 (i.e., 50% pseudovirus neutralization titer), right heatmap shows pVNT for each mRNA vaccine 50 pVNT with LNP group 50 The ratio of (a) to (b).
As can be seen from fig. 2 d:
(1) the serum neutralizing antibody titer level of the PB mRNA vaccine induced by each subtype of the Omicron variant is far higher than that of the PP mRNA vaccine; particularly, the titer level of the neutralizing antibody induced by the PB mRNA vaccine against the BA.1 subtype is more than 7 times higher than that of the PP mRNA vaccine, the titer level of the neutralizing antibody induced by the BA.1.1 subtype is more than 6 times higher than that of the PP mRNA vaccine, and the titer level of the neutralizing antibody induced by the BA.2 subtype and the BA.3 subtype is about 6 times higher than that of the PP mRNA vaccine; this suggests that the PB mRNA vaccine of the present invention can induce significantly higher neutralizing antibody titer levels against each subtype of Omicron variant strain, indicating that it will have significantly higher immunoprotective efficacy against each type of Omicron variant strain; in addition, PB mRNA also has higher neutralizing antibody titer levels for the new corona virus prototype strain and Delta variant strain, suggesting a very broad spectrum.
(2) The serum neutralizing antibody titer level of the DB mRNA vaccine induced by the prototype strain, the Delta variant strain and the Omicron variant strain is far higher than that of the PP mRNA vaccine; specifically, the DB mRNA vaccine has a neutralizing antibody titer level more than 3 times higher than that of a PP mRNA vaccine against a prototype strain, has a neutralizing antibody titer level more than 5 times higher than that of the PP mRNA vaccine against a Delta variant strain, has a neutralizing antibody titer level more than 45 times higher than that of the PP mRNA vaccine against an Omicron variant strain BA.1 subtype, has a neutralizing antibody titer level more than 30 times higher than that of the PP mRNA vaccine against an Omicron variant strain BA.1.1 subtype, has a neutralizing antibody titer level more than 48 times higher than that of the PP mRNA vaccine against an Omicron variant strain BA.2 subtype, and has a neutralizing antibody titer level more than that of the PP mRNA vaccine against an Omicron variant strain BA.3 subtype, and has a neutralizing antibody titer level more than that of the PP mRNA vaccine against an Omicron variant strain BA.69 times higher; this suggests that the DB mRNA vaccines of the present invention can induce significantly higher neutralizing antibody titer levels against each strain of the novel coronavirus, indicating that they will have significantly higher immunoprotection potency against each strain of the novel coronavirus.
(3) The DO mRNA vaccine is far higher than the PP mRNA vaccine in the serum neutralizing antibody titer level induced by the prototype strain, the Delta variant strain and the Omicron variant strain; specifically, the DO mRNA vaccine induces a neutralizing antibody titer level which is approximately 13 times higher than that of the PP mRNA vaccine against the prototype strain, a neutralizing antibody titer level which is approximately 5 times higher than that of the PP mRNA vaccine against the Delta variant strain, a neutralizing antibody titer level which is approximately 200 times higher than that of the PP mRNA vaccine against the Omicron variant strain BA.1 subtype, a neutralizing antibody titer level which is approximately 163 times higher than that of the PP mRNA vaccine against the Omicron variant strain BA.1.1 subtype, a neutralizing antibody titer level which is approximately 230 times higher than that of the PP mRNA vaccine against the Omicron variant strain BA.2 subtype, and a neutralizing antibody titer level which is approximately 407 times higher than that of the PP mRNA vaccine against the Omicron variant strain BA.3 subtype; this suggests that the DO mRNA vaccine of the present invention can induce significantly higher neutralizing antibody titer levels against various strains of the novel coronavirus, indicating that it will have significantly higher immunoprotection efficacy against various strains of the novel coronavirus.
Example 5: evaluation of mRNA vaccine-induced cellular immunity level
In this example, spleen samples of mice of each experimental group collected on day 21 in example 2 (schematic diagram of mouse immunization and sampling procedure is shown in FIG. 3 a) were used to measure the cellular immunity level induced by the mRNA vaccine. The specific method comprises the following steps:
1) mouse spleen sample treatment
Preparing mouse spleen cells into single-cell homogenate in 1ml of serum-free DMEM by using a cell homogenizer, filtering by using a 40-micron cell filter, and lysing erythrocytes by using an erythrocyte lysis buffer (R1010, Beijing Solebao scientific Co., Ltd.); then, the cells were washed with a washing solution (PBS + 0.5% FBS), stained with a 0.4% trypan blue solution (Gibco, 15250061), and counted using a Cell drop FL automatic Cell counter.
2) ELISpot test
Mu.g/ml anti-mouse IFN-. gamma.antibody (purchased from BD company) was incubated overnight at 4 ℃ in flat-bottom 96-well plates to coat the flat-bottom 96-well plates, and blocked for 2 hours at room temperature the next day. Fresh mouse spleen single cell suspension (4X 10) 5 Hole) is added into a 96-hole plate coated by the antibody, and peptide libraries (each polypeptide is 2 mu g/ml) constructed by RBD of new corona virus prototype strains, Delta, Beta and Omicron variant strains BA.1 subtype are respectively used for stimulating for 20 hours; the peptide library adopts a website https:// www.hiv.lanl.gov/content/sequenceDesign is carried out by a software PeptGen Peptide Generator on e/PEPTGEN/PEPTGEN. The length of the short peptide is 18-20 amino acids, the action of the overlapping amino acid segment is 10 amino acids, and the like; the designed peptide library was synthesized by Zhongke Sudoku Biotech Co., Ltd. The positive control wells were stimulated with Phytohemagglutinin (PMA) to generate non-specific cellular immune responses, and the negative control wells were not stimulated with peptide pools. Then, the cells were discarded and the 96-well plate was incubated with biotinylated IFN γ antibody, streptavidin-HRP antibody, and chromogenic substrate in sequence. When spots appear on the bottom of the plate, the sample is rinsed thoroughly with deionized water and the color development is stopped. Finally, pictures were taken using the Immuno Capture 6.5.0 and the number of spots was counted.
As shown in FIG. 3b, it can be seen from FIG. 3b that spleen cells of mice immunized with PB, DB and DO mRNA vaccines produced IFN-. gamma. + cells in the same amount as those of mice immunized with PP mRNA vaccine after stimulation with the above four novel coronavirus RBD peptide libraries, which are much higher than those of LNP control group, indicating that: the PB, DB and DO mRNA vaccines can effectively stimulate cellular immune response, and the level of the stimulated cellular immunity is equivalent to that of the PP mRNA vaccine.
Example 6: sequential immunization of mRNA vaccines and inactivated vaccines
In this example, female mice of BALB/c strain (purchased from Wintolite, Inc.) 6-8 weeks old were used for animal experiments, and the inactivated vaccine used was derived from the Chinese medicine, Zhongsheng BBBIP-CorV.
The experimental groups are: the vaccine comprises a three-time inactivated vaccine immunization group (namely, an 'IV' group), a two-time inactivated vaccine + PP mRNA vaccine immunization group (called as a 'PP' group for short), a two-time inactivated vaccine + PB mRNA vaccine immunization group (called as a 'PB' group for short), a two-time inactivated vaccine + DB mRNA vaccine immunization group (called as a 'DB' group for short), a two-time inactivated vaccine + DO mRNA vaccine immunization group (called as a 'DO' group for short) and an inactivated vaccine adjuvant + LNP immunization group (called as a 'LNP' group for short, and used as a negative control group).
Group "IV": all mice were vaccinated with one dose of inactivated vaccine on day 0, day 21 and day 35, respectively;
"PB" group, "DB" group, "DO" group: all mice were vaccinated with one dose of inactivated vaccine on days 0 and 21, respectively, and then with one dose of each mRNA vaccine on day 35;
group "LNP": all mice were vaccinated with the adjuvant for inactivated vaccine, Al adjuvant, on days 0, 21 and empty LNP on day 35.
The vaccination method of each vaccine is intramuscular injection, wherein the vaccination dose of the inactivated vaccine is 2.6U (0.4 dose of human dose) per mouse, and the vaccination dose of each mRNA vaccine or empty LNP is 5 mug per mouse.
Mouse serum samples were taken at day 35 and day 49, respectively, for testing the immune sera for binding antibody titers and pseudovirus neutralizing antibody titers. In addition, mouse spleen samples were also collected on day 49 for testing T cell immunity.
A schematic of the sequential immunization and serum sampling procedure for mice is shown in figure 4 a.
Example 7: detection of titer level of binding antibody of serum of sequentially immunized mouse to RBD antigen of each strain of new coronavirus
In this example, the binding antibody titers of the sera of the immunized mice collected in example 6 against the RBD antigens of the subtypes New coronavirus prototype strain, Delta variant strain, Beta variant strain, and Omicron variant strain BA.1, BA.1.1, BA.2, and BA.3 were examined by the method described in example 3.
The results are shown in FIG. 4 b. Wherein, fig. 4b (i) to (v) respectively show groups of PP, PB, DB, DO and inactivated vaccine for the third booster immunization, the first four groups are sequential immunizations, and the last group is control; as described in the figure legend, it shows the level of bound antibody titers of sera collected at day 35 (shown as open circles), day 49 (shown as filled circles) for each immunization program against the neocoronavirus prototype strain, Delta variant, Beta variant, Omicron variant subtype ba.1, ba.1.1, ba.2, ba.3rbd antigen, and the fold increase in antibody titer of the latter (i.e., day 49) relative to the former (i.e., day 35) (the "figure x" on each figure represents the fold increase), which reflects the increase in antibody titer levels after and before sequential immunization with mRNA vaccine; from these results, it can be seen that: after the mRNA vaccines are adopted for sequential immunization, the serum antibody titer level is obviously improved compared with that before the sequential immunization, which shows that: the mRNA vaccines of the present application can be used for sequential immunization to boost the level of immune response.
Furthermore, compared to the PP mRNA vaccine sequential immunization group:
1) sequential immunization group of PB mRNA vaccine
Aiming at the seven new coronavirus strains, the times of improving the serum antibody titer at the 49 th day relative to the serum antibody titer at the 35 th day are all far higher than that of a PP mRNA vaccine sequential immunization group and can be up to 10 times;
2) sequential immunization group of DB mRNA vaccines
Aiming at a new coronavirus prototype strain, a Delta variant strain, a Beta variant strain and an Omicron subtype BA.2 variant strain, the fold improvement of the serum antibody titer at the 49 th day relative to the serum antibody titer at the 35 th day is far higher than that of a PP mRNA vaccine sequential immunization group, and the maximum fold is nearly 5 times;
3) DO mRNA vaccine sequential immunization group
Aiming at the seven new coronavirus strains, the serum antibody titer at the 49 th day is improved by more than 5 times compared with the serum antibody titer at the 35 th day in the sequential immunization group of the PP mRNA vaccine.
Furthermore, the results of the antibody titers bound to the antigens of the prototype strain, Delta variant strain, Beta variant strain, and Omicron variant subtypes BA.1, BA.1.1, BA.2, and BA.3 of the new coronavirus, the Delta variant strain, and the Omicron variant strain in each immunization group on day 49 are shown in FIG. 5 a.
Figure 5a shows, compared to the PP mRNA vaccine sequential immunization group:
1) sequential immunization group of PB mRNA vaccine
Aiming at the seven new coronavirus strains, the titer levels of the induced combined antibodies are far higher than that of a PP mRNA vaccine and can reach 3 times at most;
2) sequential immunization group of DB mRNA vaccines
Aiming at a new coronavirus prototype strain, a Delta variant strain, a Beta variant strain and an Omicron subtype BA.2 variant strain, the titer level of the induced combined antibody is far higher than that of a PP mRNA vaccine;
3) DO mRNA vaccine sequential immunization group
Aiming at the seven new coronavirus strains, the titer levels of the induced combined antibodies are far higher than that of a PP mRNA vaccine.
Example 8: evaluation of inhibitory Effect of serum of sequentially immunized mouse on pseudovirus of Each Strain of New coronavirus
In this example, the neutralizing antibody titers against pseudoviruses of the subtypes neocoronaviruse prototype strain, Delta variant strain, Beta variant strain, Omicron variant strain BA.1, BA.1.1, BA.2, and BA.3 were examined with respect to the sera of each of the immunized mice collected on day 49 in example 6 by the method described in example 4.
The results are shown in FIG. 5 b. Fig. 5b shows: compared with the PP mRNA vaccine sequential immunization group:
1) sequential immunization group of PB mRNA vaccine
Aiming at the pseudo viruses of the new coronavirus prototype strain, Delta and Omicron BA.1.1, the induced neutralizing antibody titer level is higher than or equal to that of a PP mRNA vaccine;
however, the induced neutralizing antibody titer level of the pseudoviruses aiming at the new coronavirus Omicron BA.1, BA.2 and BA.3 is more than two times and up to about 3 times higher than that of the PP mRNA vaccine;
2) sequential immunization group of DB mRNA vaccines
Except for the new coronavirus Omicron BA.1.1, the neutralizing antibody titer level induced by the pseudovirus aiming at all other types of strains is obviously higher than that of the PP mRNA vaccine;
3) DO mRNA vaccine sequential immunization group
The pseudovirus, which is directed against each subtype strain of the novel coronavirus Omicron variant, induces neutralizing antibody titer levels higher than, or comparable to, PP mRNA vaccines.
Example 9: evaluation of cellular immune levels induced by sequential immunization
In this example, the level of cellular immunity induced by sequential immunization was measured using the method described in example 5 and spleen samples of each immunized group of mice collected on day 49 in example 6.
As shown in FIG. 5c, it can be seen from FIG. 5c that spleen cells of mice sequentially immunized with PB, DB and DO mRNA vaccines produced IFN-. gamma. + CD4+ and IFN-. gamma. + CD8+ cells in the same number as that of mice sequentially immunized with PP mRNA vaccine, which are much higher than those of LNP control group, after stimulation with the above four new coronavirus RBD peptide libraries, which indicates that: the sequential immunization with PB, DB and DO mRNA vaccines can effectively stimulate cellular immune response, and the level of the stimulated cellular immunity is equivalent to that of the sequential immunization group of the PP mRNA vaccine.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
SEQUENCE LISTING
<110> institute of microbiology of Chinese academy of sciences
<120> novel coronavirus chimeric nucleic acid vaccine and use thereof
<130> 1087-220106F
<160> 26
<170> PatentIn version 3.5
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165 170 175
Ser Phe Arg Pro Thr Tyr Gly Val Gly His Gln Pro Tyr Arg Val Val
180 185 190
Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro
195 200 205
Lys Lys Ser Thr Asn Leu Val Lys Asn Lys
210 215
<210> 5
<211> 434
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthesis of polypeptide
<220>
<221> recombinant chimeric antigen peptide encoded by PB nucleic acid vaccine
<222> (1)..(434)
<400> 5
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Val Gln Pro Thr Glu Ser Ile Val
210 215 220
Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn
225 230 235 240
Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser
245 250 255
Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser
260 265 270
Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys
275 280 285
Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val
290 295 300
Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr
305 310 315 320
Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn
325 330 335
Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu
340 345 350
Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu
355 360 365
Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Lys Gly Phe Asn
370 375 380
Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val
385 390 395 400
Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His
405 410 415
Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys
420 425 430
Asn Lys
<210> 6
<211> 434
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthesis of polypeptide
<220>
<221> recombinant chimeric antigen peptide encoded by DB nucleic acid vaccine
<222> (1)..(434)
<400> 6
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Val Gln Pro Thr Glu Ser Ile Val
210 215 220
Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn
225 230 235 240
Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser
245 250 255
Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser
260 265 270
Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys
275 280 285
Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val
290 295 300
Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr
305 310 315 320
Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn
325 330 335
Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu
340 345 350
Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu
355 360 365
Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Lys Gly Phe Asn
370 375 380
Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val
385 390 395 400
Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His
405 410 415
Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys
420 425 430
Asn Lys
<210> 7
<211> 434
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic Polypeptides
<220>
<221> recombinant chimeric antigen peptide encoded by DO nucleic acid vaccine
<222> (1)..(434)
<400> 7
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Val Gln Pro Thr Glu Ser Ile Val
210 215 220
Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp Glu Val Phe Asn
225 230 235 240
Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser
245 250 255
Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Leu Ala Pro Phe Phe
260 265 270
Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys
275 280 285
Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val
290 295 300
Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr
305 310 315 320
Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn
325 330 335
Lys Leu Asp Ser Lys Val Ser Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu
340 345 350
Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu
355 360 365
Ile Tyr Gln Ala Gly Asn Lys Pro Cys Asn Gly Val Ala Gly Phe Asn
370 375 380
Cys Tyr Phe Pro Leu Arg Ser Tyr Ser Phe Arg Pro Thr Tyr Gly Val
385 390 395 400
Gly His Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His
405 410 415
Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys
420 425 430
Asn Lys
<210> 8
<211> 1302
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic polynucleotide
<220>
<221> DNA coding sequence of PB mRNA vaccine
<222> (1)..(1302)
<400> 8
cgagtgcagc ctaccgaaag catcgtccgt ttcccgaata ttactaatct ctgtccattc 60
ggagaagtct tcaatgccac ccgattcgct tccgtttacg cgtggaaccg taaacgaata 120
tctaattgtg ttgcggacta ttccgtgttg tacaactcag catcattctc tacttttaaa 180
tgctatggag tgtcgccgac taaactcaac gacttgtgtt tcactaatgt ttatgctgac 240
tctttcgtta ttcgtggaga cgaagttcgt caaatcgcac cagggcaaac tggcaagatt 300
gcggactata attataagct gccagatgac tttaccggat gtgtaatagc ctggaactca 360
aataatctcg acagtaaagt gggaggcaac tataattatc tttatcgact cttcagaaag 420
tctaacctta agccatttga acgtgacatt tctacagaaa tttaccaagc cggctctaca 480
ccttgcaatg gcgtggaagg gtttaactgt tatttcccat tacagtctta tggtttccag 540
ccaactaatg gtgtgggata ccaaccttac cgcgtcgttg tcctgtcgtt tgaattgctt 600
cacgcaccag ccaccgtttg tgggccaaag aagagcacta atctcgtagt tcagcctact 660
gaatcgatcg tgaggttccc aaatattacc aatctgtgtc cgttcggaga ggtcttcaat 720
gcgactcgat tcgcgtctgt ttacgcctgg aacaggaaac ggattagcaa ttgtgtcgct 780
gactattcgg tcttatacaa ctctgcatca ttctcaacct tcaagtgtta tggtgtcagc 840
cctacaaagc tgaatgactt atgtttcacc aatgtttatg cggacagttt cgtaatacga 900
ggtgatgaag tccgccaaat tgcacccgga caaaccggca acatagccga ctataattat 960
aagctccctg atgactttac gggctgtgtc atagcttgga atagtaataa tttggactcg 1020
aaagtgggag gtaattataa ttatctctat agactgttcc ggaaatcaaa tctcaagccc 1080
tttgaacggg acataagtac agaaatctac caagctggtt ccacgccgtg taatggagtc 1140
aaggggttta actgttattt cccgctccag tcgtatgggt tccagccaac gtatggcgtc 1200
ggataccaac cttaccgcgt tgtagtatta agctttgaac tgttgcacgc gcccgcgact 1260
gtttgtggcc cgaagaagtc gactaatcta gtaaagaata ag 1302
<210> 9
<211> 1302
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic polynucleotide
<220>
<221> DNA coding sequence of DB mRNA vaccine
<222> (1)..(1302)
<400> 9
cgagtgcagc ctaccgaaag catcgtccgt ttcccgaata ttactaatct ctgtccattc 60
ggagaagtct tcaatgccac ccgattcgct tccgtttacg cgtggaaccg taaacgaata 120
tctaattgtg ttgcggacta ttccgtgttg tacaactcag catcattctc tacttttaaa 180
tgctatggag tgtcgccgac taaactcaac gacttgtgtt tcactaatgt ttatgctgac 240
tctttcgtta ttcgtggaga cgaagttcgt caaatcgcac cagggcaaac tggcaagatt 300
gcggactata attataagct gccagatgac tttaccggat gtgtaatagc ctggaactca 360
aataatctcg acagtaaagt gggaggcaac tataattatc gttatcgact cttcagaaag 420
tctaacctta agccatttga acgtgacatt tctacagaaa tttaccaagc cggctctaag 480
ccttgcaatg gcgtggaagg gtttaactgt tatttcccat tacagtctta tggtttccag 540
ccaactaatg gtgtgggata ccaaccttac cgcgtcgttg tcctgtcgtt tgaattgctt 600
cacgcaccag ccaccgtttg tgggccaaag aagagcacta atctcgtagt tcagcctact 660
gaatcgatcg tgaggttccc aaatattacc aatctgtgtc cgttcggaga ggtcttcaat 720
gcgactcgat tcgcgtctgt ttacgcctgg aacaggaaac ggattagcaa ttgtgtcgct 780
gactattcgg tcttatacaa ctctgcatca ttctcaacct tcaagtgtta tggtgtcagc 840
cctacaaagc tgaatgactt atgtttcacc aatgtttatg cggacagttt cgtaatacga 900
ggtgatgaag tccgccaaat tgcacccgga caaaccggca acatagccga ctataattat 960
aagctccctg atgactttac gggctgtgtc atagcttgga atagtaataa tttggactcg 1020
aaagtgggag gtaattataa ttatctctat agactgttcc ggaaatcaaa tctcaagccc 1080
tttgaacggg acataagtac agaaatctac caagctggtt ccacgccgtg taatggagtc 1140
aaggggttta actgttattt cccgctccag tcgtatgggt tccagccaac gtatggcgtc 1200
ggataccaac cttaccgcgt tgtagtatta agctttgaac tgttgcacgc gcccgcgact 1260
gtttgtggcc cgaagaagtc gactaatcta gtaaagaata ag 1302
<210> 10
<211> 1302
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic polynucleotide
<220>
<221> DNA coding sequence of DO mRNA vaccine
<222> (1)..(1302)
<400> 10
cgagtgcagc ctaccgaaag catcgtccgt ttcccgaata ttactaatct ctgtccattc 60
ggagaagtct tcaatgccac ccgattcgct tccgtttacg cgtggaaccg taaacgaata 120
tctaattgtg ttgcggacta ttccgtgttg tacaactcag catcattctc tacttttaaa 180
tgctatggag tgtcgccgac taaactcaac gacttgtgtt tcactaatgt ttatgctgac 240
tctttcgtta ttcgtggaga cgaagttcgt caaatcgcac cagggcaaac tggcaagatt 300
gcggactata attataagct gccagatgac tttaccggat gtgtaatagc ctggaactca 360
aataatctcg acagtaaagt gggaggcaac tataattatc gttatcgact cttcagaaag 420
tctaacctta agccatttga acgtgacatt tctacagaaa tttaccaagc cggctctaag 480
ccttgcaatg gcgtggaagg gtttaactgt tatttcccat tacagtctta tggtttccag 540
ccaactaatg gtgtgggata ccaaccttac cgcgtcgttg tcctgtcgtt tgaattgctt 600
cacgcaccag ccaccgtttg tgggccaaag aagagcacta atctcgtagt tcagcctact 660
gaatcgatcg tgaggttccc aaatattacc aatctgtgtc cgttcgacga ggtcttcaat 720
gcgactcgat tcgcgtctgt ttacgcctgg aacaggaaac ggattagcaa ttgtgtcgct 780
gactattcgg tcttatacaa cttggcacca ttcttcacct tcaagtgtta tggtgtcagc 840
cctacaaagc tgaatgactt atgtttcacc aatgtttatg cggacagttt cgtaatacga 900
ggtgatgaag tccgccaaat tgcacccgga caaaccggca acatagccga ctataattat 960
aagctccctg atgactttac gggctgtgtc atagcttgga atagtaataa gttggactcg 1020
aaagtgtcag gtaattataa ttatctctat agactgttcc ggaaatcaaa tctcaagccc 1080
tttgaacggg acataagtac agaaatctac caagctggta acaagccgtg taatggagtc 1140
gcagggttta actgttattt cccgctccgg tcgtattcct tccggccaac gtatggcgtc 1200
ggacaccaac cttaccgcgt tgtagtatta agctttgaac tgttgcacgc gcccgcgact 1260
gtttgtggcc cgaagaagtc gactaatcta gtaaagaata ag 1302
<210> 11
<211> 1302
<212> RNA
<213> Artificial Sequence
<220>
<223> synthetic polynucleotide
<220>
<221> mRNA coding sequence of PB mRNA vaccine
<222> (1)..(1302)
<400> 11
cgagugcagc cuaccgaaag caucguccgu uucccgaaua uuacuaaucu cuguccauuc 60
ggagaagucu ucaaugccac ccgauucgcu uccguuuacg cguggaaccg uaaacgaaua 120
ucuaauugug uugcggacua uuccguguug uacaacucag caucauucuc uacuuuuaaa 180
ugcuauggag ugucgccgac uaaacucaac gacuuguguu ucacuaaugu uuaugcugac 240
ucuuucguua uucguggaga cgaaguucgu caaaucgcac cagggcaaac uggcaagauu 300
gcggacuaua auuauaagcu gccagaugac uuuaccggau guguaauagc cuggaacuca 360
aauaaucucg acaguaaagu gggaggcaac uauaauuauc uuuaucgacu cuucagaaag 420
ucuaaccuua agccauuuga acgugacauu ucuacagaaa uuuaccaagc cggcucuaca 480
ccuugcaaug gcguggaagg guuuaacugu uauuucccau uacagucuua ugguuuccag 540
ccaacuaaug gugugggaua ccaaccuuac cgcgucguug uccugucguu ugaauugcuu 600
cacgcaccag ccaccguuug ugggccaaag aagagcacua aucucguagu ucagccuacu 660
gaaucgaucg ugagguuccc aaauauuacc aaucuguguc cguucggaga ggucuucaau 720
gcgacucgau ucgcgucugu uuacgccugg aacaggaaac ggauuagcaa uugugucgcu 780
gacuauucgg ucuuauacaa cucugcauca uucucaaccu ucaaguguua uggugucagc 840
ccuacaaagc ugaaugacuu auguuucacc aauguuuaug cggacaguuu cguaauacga 900
ggugaugaag uccgccaaau ugcacccgga caaaccggca acauagccga cuauaauuau 960
aagcucccug augacuuuac gggcuguguc auagcuugga auaguaauaa uuuggacucg 1020
aaagugggag guaauuauaa uuaucucuau agacuguucc ggaaaucaaa ucucaagccc 1080
uuugaacggg acauaaguac agaaaucuac caagcugguu ccacgccgug uaauggaguc 1140
aagggguuua acuguuauuu cccgcuccag ucguaugggu uccagccaac guauggcguc 1200
ggauaccaac cuuaccgcgu uguaguauua agcuuugaac uguugcacgc gcccgcgacu 1260
guuuguggcc cgaagaaguc gacuaaucua guaaagaaua ag 1302
<210> 12
<211> 1302
<212> RNA
<213> Artificial Sequence
<220>
<223> synthetic polynucleotide
<220>
<221> mRNA coding sequences for DB mRNA vaccines
<222> (1)..(1302)
<400> 12
cgagugcagc cuaccgaaag caucguccgu uucccgaaua uuacuaaucu cuguccauuc 60
ggagaagucu ucaaugccac ccgauucgcu uccguuuacg cguggaaccg uaaacgaaua 120
ucuaauugug uugcggacua uuccguguug uacaacucag caucauucuc uacuuuuaaa 180
ugcuauggag ugucgccgac uaaacucaac gacuuguguu ucacuaaugu uuaugcugac 240
ucuuucguua uucguggaga cgaaguucgu caaaucgcac cagggcaaac uggcaagauu 300
gcggacuaua auuauaagcu gccagaugac uuuaccggau guguaauagc cuggaacuca 360
aauaaucucg acaguaaagu gggaggcaac uauaauuauc guuaucgacu cuucagaaag 420
ucuaaccuua agccauuuga acgugacauu ucuacagaaa uuuaccaagc cggcucuaag 480
ccuugcaaug gcguggaagg guuuaacugu uauuucccau uacagucuua ugguuuccag 540
ccaacuaaug gugugggaua ccaaccuuac cgcgucguug uccugucguu ugaauugcuu 600
cacgcaccag ccaccguuug ugggccaaag aagagcacua aucucguagu ucagccuacu 660
gaaucgaucg ugagguuccc aaauauuacc aaucuguguc cguucggaga ggucuucaau 720
gcgacucgau ucgcgucugu uuacgccugg aacaggaaac ggauuagcaa uugugucgcu 780
gacuauucgg ucuuauacaa cucugcauca uucucaaccu ucaaguguua uggugucagc 840
ccuacaaagc ugaaugacuu auguuucacc aauguuuaug cggacaguuu cguaauacga 900
ggugaugaag uccgccaaau ugcacccgga caaaccggca acauagccga cuauaauuau 960
aagcucccug augacuuuac gggcuguguc auagcuugga auaguaauaa uuuggacucg 1020
aaagugggag guaauuauaa uuaucucuau agacuguucc ggaaaucaaa ucucaagccc 1080
uuugaacggg acauaaguac agaaaucuac caagcugguu ccacgccgug uaauggaguc 1140
aagggguuua acuguuauuu cccgcuccag ucguaugggu uccagccaac guauggcguc 1200
ggauaccaac cuuaccgcgu uguaguauua agcuuugaac uguugcacgc gcccgcgacu 1260
guuuguggcc cgaagaaguc gacuaaucua guaaagaaua ag 1302
<210> 13
<211> 1302
<212> RNA
<213> Artificial Sequence
<220>
<223> synthetic polynucleotide
<220>
<221> mRNA coding sequence of DO mRNA vaccine
<222> (1)..(1302)
<400> 13
cgagugcagc cuaccgaaag caucguccgu uucccgaaua uuacuaaucu cuguccauuc 60
ggagaagucu ucaaugccac ccgauucgcu uccguuuacg cguggaaccg uaaacgaaua 120
ucuaauugug uugcggacua uuccguguug uacaacucag caucauucuc uacuuuuaaa 180
ugcuauggag ugucgccgac uaaacucaac gacuuguguu ucacuaaugu uuaugcugac 240
ucuuucguua uucguggaga cgaaguucgu caaaucgcac cagggcaaac uggcaagauu 300
gcggacuaua auuauaagcu gccagaugac uuuaccggau guguaauagc cuggaacuca 360
aauaaucucg acaguaaagu gggaggcaac uauaauuauc guuaucgacu cuucagaaag 420
ucuaaccuua agccauuuga acgugacauu ucuacagaaa uuuaccaagc cggcucuaag 480
ccuugcaaug gcguggaagg guuuaacugu uauuucccau uacagucuua ugguuuccag 540
ccaacuaaug gugugggaua ccaaccuuac cgcgucguug uccugucguu ugaauugcuu 600
cacgcaccag ccaccguuug ugggccaaag aagagcacua aucucguagu ucagccuacu 660
gaaucgaucg ugagguuccc aaauauuacc aaucuguguc cguucgacga ggucuucaau 720
gcgacucgau ucgcgucugu uuacgccugg aacaggaaac ggauuagcaa uugugucgcu 780
gacuauucgg ucuuauacaa cuuggcacca uucuucaccu ucaaguguua uggugucagc 840
ccuacaaagc ugaaugacuu auguuucacc aauguuuaug cggacaguuu cguaauacga 900
ggugaugaag uccgccaaau ugcacccgga caaaccggca acauagccga cuauaauuau 960
aagcucccug augacuuuac gggcuguguc auagcuugga auaguaauaa guuggacucg 1020
aaagugucag guaauuauaa uuaucucuau agacuguucc ggaaaucaaa ucucaagccc 1080
uuugaacggg acauaaguac agaaaucuac caagcuggua acaagccgug uaauggaguc 1140
gcaggguuua acuguuauuu cccgcuccgg ucguauuccu uccggccaac guauggcguc 1200
ggacaccaac cuuaccgcgu uguaguauua agcuuugaac uguugcacgc gcccgcgacu 1260
guuuguggcc cgaagaaguc gacuaaucua guaaagaaua ag 1302
<210> 14
<211> 1302
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic polynucleotide
<220>
<221> DNA coding sequence of PP mRNA vaccine
<222> (1)..(1302)
<400> 14
cgagtgcagc ctaccgaaag catcgtccgt ttcccgaata ttactaatct ctgtccattc 60
ggagaagtct tcaatgccac ccgattcgct tccgtttacg cgtggaaccg taaacgaata 120
tctaattgtg ttgcggacta ttccgtgttg tacaactcag catcattctc tacttttaaa 180
tgctatggag tgtcgccgac taaactcaac gacttgtgtt tcactaatgt ttatgctgac 240
tctttcgtta ttcgtggaga cgaagttcgt caaatcgcac cagggcaaac tggcaagatt 300
gcggactata attataagct gccagatgac tttaccggat gtgtaatagc ctggaactca 360
aataatctcg acagtaaagt gggaggcaac tataattatc tttatcgact cttcagaaag 420
tctaacctta agccatttga acgtgacatt tctacagaaa tttaccaagc cggctctaca 480
ccttgcaatg gcgtggaagg gtttaactgt tatttcccat tacagtctta tggtttccag 540
ccaactaatg gtgtgggata ccaaccttac cgcgtcgttg tcctgtcgtt tgaattgctt 600
cacgcaccag ccaccgtttg tgggccaaag aagagcacta atctcgtagt tcagcctact 660
gaatcgatcg tgaggttccc aaatattacc aatctgtgtc cgttcggaga ggtcttcaat 720
gcgactcgat tcgcgtctgt ttacgcctgg aacaggaaac ggattagcaa ttgtgtcgct 780
gactattcgg tcttatacaa ctctgcatca ttctcaacct tcaagtgtta tggtgtcagc 840
cctacaaagc tgaatgactt atgtttcacc aatgtttatg cggacagttt cgtaatacga 900
ggtgatgaag tccgccaaat tgcacccgga caaaccggca agatagccga ctataattat 960
aagctccctg atgactttac gggctgtgtc atagcttgga atagtaataa tttggactcg 1020
aaagtgggag gtaattataa ttatctctat agactgttcc ggaaatcaaa tctcaagccc 1080
tttgaacggg acataagtac agaaatctac caagctggtt ccacgccgtg taatggagtc 1140
gaggggttta actgttattt cccgctccag tcgtatgggt tccagccaac gaatggcgtc 1200
ggataccaac cttaccgcgt tgtagtatta agctttgaac tgttgcacgc gcccgcgact 1260
gtttgtggcc cgaagaagtc gactaatcta gtaaagaata ag 1302
<210> 15
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic polynucleotide
<220>
<221> 5' end UTR
<222> (1)..(47)
<400> 15
gggaaataag agagaaaaga agagtaagaa gaaatataag agccacc 47
<210> 16
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic polynucleotide
<220>
<221> DNA coding sequence for signal peptide
<222> (1)..(45)
<400> 16
atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgc 45
<210> 17
<211> 110
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic polynucleotide
<220>
<221> 3' end UTR
<222> (1)..(110)
<400> 17
tgataatagg ctggagcctc ggtggccatg cttcttgccc cttgggcctc cccccagccc 60
ctcctcccct tcctgcaccc gtacccccgt ggtctttgaa taaagtctga 110
<210> 18
<211> 223
<212> PRT
<213> SARS-CoV-2 Omicron variant BA.1.1 subtype
<220>
<221> SARS-CoV-2 Omicron variant BA.1.1 subtype RBD antigen protein
<222> (1)..(223)
<400> 18
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Asp Glu Val Phe Asn Ala Thr Lys Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Leu Ala Pro Phe Phe Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Lys Leu Asp Ser Lys Val Ser
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Lys
145 150 155 160
Pro Cys Asn Gly Val Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser
165 170 175
Tyr Ser Phe Arg Pro Thr Tyr Gly Val Gly His Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
210 215 220
<210> 19
<211> 223
<212> PRT
<213> SARS-CoV-2 Omicron variant BA.2 subtype
<220>
<221> SARS-CoV-2 Omicron variant BA.2 subtype RBD antigen protein
<222> (1)..(223)
<400> 19
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Asp Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Phe Ala Pro Phe Phe Ala Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asn Glu Val Ser Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Lys Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Lys
145 150 155 160
Pro Cys Asn Gly Val Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser
165 170 175
Tyr Gly Phe Arg Pro Thr Tyr Gly Val Gly His Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
210 215 220
<210> 20
<211> 223
<212> PRT
<213> SARS-CoV-2 Omicron variant BA.3 subtype
<220>
<221> SARS-CoV-2 Omicron variant BA.3 subtype RBD antigen protein
<222> (1)..(223)
<400> 20
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Asp Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Phe Ala Pro Phe Phe Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asn Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Lys Leu Asp Ser Lys Val Ser
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Lys
145 150 155 160
Pro Cys Asn Gly Val Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser
165 170 175
Tyr Gly Phe Arg Pro Thr Tyr Gly Val Gly His Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
210 215 220
<210> 21
<211> 3765
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic polynucleotide
<220>
<221> nucleotide sequence of WT-S-del18
<222> (1)..(3765)
<400> 21
atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgaccaca 60
cggacccagc tccctcccgc ctacacaaac tctttcaccc ggggcgtgta ctaccccgac 120
aaggtgttcc ggtctagcgt gctccactct acacaggacc tgttcctccc tttcttcagc 180
aacgtgacat ggttccacgc catccacgtg tctggcacaa acggcacaaa gcggttcgac 240
aaccccgtgc tccctttcaa cgacggcgtg tacttcgcca gcaccgagaa gtctaacatt 300
atccggggct ggattttcgg caccacactc gactctaaga cacagtccct cctgattgtg 360
aacaacgcca caaacgtggt gattaaggtg tgcgagttcc agttctgcaa cgaccctttc 420
ctgggcgtgt actaccacaa gaacaacaag tcttggatgg agtctgagtt cagagtgtac 480
tctagcgcca acaactgcac cttcgagtac gtgtcccagc ctttcctcat ggacctggag 540
ggcaagcagg gcaacttcaa gaacctgaga gagttcgtgt tcaagaacat tgacggctac 600
ttcaagattt actctaagca caccccaatt aacctcgtga gggacctccc tcagggcttc 660
tccgccttag aaccactggt ggacctccct attggcatta acatcacacg cttccagaca 720
ctgctcgccc tccaccggtc ttacctgacc ccaggcgact ctagctctgg ctggacagcc 780
ggcgccgccg cctactacgt gggctacctg cagcctagga ccttcctcct gaagtacaac 840
gagaacggca caattaccga cgccgtggac tgcgccctgg acccactgtc cgagacaaag 900
tgcacactga agtccttcac agtggagaag ggcatttacc agacatctaa cttccgggtg 960
cagcctacag agtctattgt gcggttccca aacatcacaa acctgtgccc tttcggcgag 1020
gtgttcaacg ccacccggtt cgcctctgtg tacgcctgga accggaagcg gatctctaac 1080
tgcgtggccg actactccgt gctgtacaac tccgcctctt tctctacatt caagtgctac 1140
ggcgtgtccc ctacaaagct gaacgacctg tgcttcacca acgtgtacgc cgactctttc 1200
gtgattagag gcgacgaggt gaggcagatt gcccccggcc agacaggcaa gatcgccgac 1260
tacaactaca agctgcccga cgacttcaca ggctgcgtga tcgcctggaa ctctaacaac 1320
ctggactcta aggtgggcgg caactacaac tacctgtaca gactgttccg gaagtctaac 1380
ctgaagccat tcgagaggga cattagcacc gagatttacc aggccggctc taccccatgc 1440
aacggcgtgg agggcttcaa ctgctacttc ccactgcagt cctacggctt ccagcctaca 1500
aacggcgtgg gctaccagcc ttaccgggtg gtggtgctgt ctttcgagct gctccacgcc 1560
cccgccacag tgtgcggccc aaagaagagc acaaacctcg tgaagaacaa gtgcgtgaac 1620
ttcaacttca acggcctcac aggcacaggc gtgctcaccg agtctaacaa gaagttcctc 1680
cctttccagc agttcggccg cgacattgcc gacaccaccg acgccgtgcg ggaccctcag 1740
acactggaaa ttctcgacat caccccttgc agcttcggcg gcgtgtccgt gatcacccca 1800
ggcacaaaca catctaacca ggtggccgtg ctgtaccagg acgtgaactg caccgaggtg 1860
ccagtggcca tccacgccga ccagctcacc ccaacatgga gggtgtacag cacaggctct 1920
aacgtgttcc agacccgggc cggctgcctc attggcgccg agcacgtgaa caactcttac 1980
gagtgcgaca tccctattgg cgccggcatt tgcgcctctt accagaccca gacaaactct 2040
ccacggagag cccggtctgt ggcctctcag agcattattg cctacaccat gtctctgggc 2100
gccgagaact ctgtggccta ctctaacaac tctattgcca tccctacaaa cttcacaatt 2160
tctgtgacca ccgagattct cccagtgtct atgaccaaga catctgtgga ctgcaccatg 2220
tacatttgcg gcgactccac cgagtgctct aacctcctgc tccagtacgg ctctttctgc 2280
acccagctca accgcgccct gacaggcatc gccgtggagc aggacaagaa cacccaggag 2340
gtgttcgccc aggtgaagca gatttacaag acccccccaa ttaaggactt cggcggcttc 2400
aacttctctc agattctccc cgacccatcc aagcctagca agcggtcctt cattgaggac 2460
ctcctgttca acaaggtgac actggccgac gccggcttca ttaagcagta cggcgactgc 2520
ctgggcgaca ttgccgcccg ggacctgatt tgcgcccaga agttcaacgg cctcacagtg 2580
ctccccccac tgctcaccga cgagatgatt gcccagtaca catctgccct cctggccggc 2640
acaattacat ctggctggac cttcggcgcc ggcgccgccc tgcagatccc tttcgccatg 2700
cagatggcct accgcttcaa cggcatcggc gtgacacaga acgtgctgta cgagaaccag 2760
aagctgatcg ccaaccagtt caacagcgcc attggcaaga ttcaggactc tctgagcagc 2820
acagccagcg ccctgggcaa gctgcaggac gtggtgaacc agaacgccca ggccctgaac 2880
acactggtga agcagctgtc ttctaacttc ggcgccattt ctagcgtgct gaacgacatt 2940
ctgtcgcggc tggacaaggt ggaggccgag gtgcagattg acaggctcat cacaggcaga 3000
ctgcagtctc tgcagacata cgtgacccag cagctgatta gagccgccga gattagagcc 3060
tccgccaacc tggccgccac caagatgagc gagtgcgtgc tcggccagtc taagcgggtg 3120
gacttctgcg gcaagggcta ccacctcatg tctttccctc agtccgcccc tcacggcgtg 3180
gtgttcctcc acgtgacata cgtgcccgcc caggagaaga acttcaccac agcccccgcc 3240
atttgccacg acggcaaggc ccacttccct agggagggcg tgttcgtgtc taacggcacc 3300
cactggttcg tgacccagcg gaacttctac gagcctcaga ttattaccac agacaacaca 3360
ttcgtgagcg gcaactgcga cgtggtgatt ggcattgtga acaacacagt gtacgaccca 3420
ctgcagcctg agttggactc tttcaaggag gaactcgaca agtacttcaa gaaccacaca 3480
tctcctgacg tggacctggg cgacattagc ggcattaacg cctctgtggt gaacattcag 3540
aaggagattg acagactgaa cgaggtggcc aagaacctga acgagtctct cattgacctg 3600
caggagctgg gcaagtacga gcagtacatt aagtggcctt ggtacatttg gctgggcttc 3660
attgccggcc tgatcgccat tgtgatggtg accatcatgc tgtgctgcat gacatcttgc 3720
tgcagctgcc tgaagggctg ctgctcttgc ggctcttgct gcaag 3765
<210> 22
<211> 3759
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic polynucleotide
<220>
<221> nucleotide sequence of Delta-S-del18
<222> (1)..(3759)
<400> 22
atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgagaaca 60
cggacccagc tccctcccgc ctacacaaac tctttcaccc ggggcgtgta ctaccccgac 120
aaggtgttcc ggtctagcgt gctccactct acacaggacc tgttcctccc tttcttcagc 180
aacgtgacat ggttccacgc catccacgtg tctggcacaa acggcacaaa gcggttcgac 240
aaccccgtgc tccctttcaa cgacggcgtg tacttcgcca gcaccgagaa gtctaacatt 300
atccggggct ggattttcgg caccacactc gactctaaga cacagtccct cctgattgtg 360
aacaacgcca caaacgtggt gattaaggtg tgcgagttcc agttctgcaa cgaccctttc 420
ctggacgtgt actaccacaa gaacaacaag tcttggatgg agtctggcgt gtactctagc 480
gccaacaact gcaccttcga gtacgtgtcc cagcctttcc tcatggacct ggagggcaag 540
cagggcaact tcaagaacct gagagagttc gtgttcaaga acattgacgg ctacttcaag 600
atttactcta agcacacccc aattaacctc gtgagggacc tccctcaggg cttctccgtg 660
ttagaaccac tggtggacct ccctattggc attaacatca cacgcttcca gacactgctc 720
gccctccacc ggtcttacct gaccccaggc gactctagct ctggctggac agccggcgcc 780
gccgcctact acgtgggcta cctgcagcct aggaccttcc tcctgaagta caacgagaac 840
ggcacaatta ccgacgccgt ggactgcgcc ctggacccac tgtccgagac aaagtgcaca 900
ctgaagtcct tcacagtgga gaagggcatt taccagacat ctaacttccg ggtgcagcct 960
acagagtcta ttgtgcggtt cccaaacatc acaaacctgt gccctttcgg cgaggtgttc 1020
aacgccaccc ggttcgcctc tgtgtacgcc tggaaccgga agcggatctc taactgcgtg 1080
gccgactact ccgtgctgta caactccgcc tctttctcta cattcaagtg ctacggcgtg 1140
tcccctacaa agctgaacga cctgtgcttc accaacgtgt acgccgactc tttcgtgatt 1200
agaggcgacg aggtgaggca gattgccccc ggccagacag gcaagatcgc cgactacaac 1260
tacaagctgc ccgacgactt cacaggctgc gtgatcgcct ggaactctaa caacctggac 1320
tctaaggtgg gcggcaacta caactacaga tacagactgt tccggaagtc taacctgaag 1380
ccattcgaga gggacattag caccgagatt taccaggccg gctctaagcc atgcaacggc 1440
gtggagggct tcaactgcta cttcccactg cagtcctacg gcttccagcc tacaaacggc 1500
gtgggctacc agccttaccg ggtggtggtg ctgtctttcg agctgctcca cgcccccgcc 1560
acagtgtgcg gcccaaagaa gagcacaaac ctcgtgaaga acaagtgcgt gaacttcaac 1620
ttcaacggcc tcacaggcac aggcgtgctc accgagtcta acaagaagtt cctccctttc 1680
cagcagttcg gccgcgacat tgccgacacc accgacgccg tgcgggaccc tcagacactg 1740
gaaattctcg acatcacccc ttgcagcttc ggcggcgtgt ccgtgatcac cccaggcaca 1800
aacacatcta accaggtggc cgtgctgtac cagggcgtga actgcaccga ggtgccagtg 1860
gccatccacg ccgaccagct caccccaaca tggagggtgt acagcacagg ctctaacgtg 1920
ttccagaccc gggccggctg cctcattggc gccgagcacg tgaacaactc ttacgagtgc 1980
gacatcccta ttggcgccgg catttgcgcc tcttaccaga cccagacaaa ctctagacgg 2040
agagcccggt ctgtggcctc tcagagcatt attgcctaca ccatgtctct gggcgccgag 2100
aactctgtgg cctactctaa caactctatt gccatcccta caaacttcac aatttctgtg 2160
accaccgaga ttctcccagt gtctatgacc aagacatctg tggactgcac catgtacatt 2220
tgcggcgact ccaccgagtg ctctaacctc ctgctccagt acggctcttt ctgcacccag 2280
ctcaaccgcg ccctgacagg catcgccgtg gagcaggaca agaacaccca ggaggtgttc 2340
gcccaggtga agcagattta caagaccccc ccaattaagg acttcggcgg cttcaacttc 2400
tctcagattc tccccgaccc atccaagcct agcaagcggt ccttcattga ggacctcctg 2460
ttcaacaagg tgacactggc cgacgccggc ttcattaagc agtacggcga ctgcctgggc 2520
gacattgccg cccgggacct gatttgcgcc cagaagttca acggcctcac agtgctcccc 2580
ccactgctca ccgacgagat gattgcccag tacacatctg ccctcctggc cggcacaatt 2640
acatctggct ggaccttcgg cgccggcgcc gccctgcaga tccctttcgc catgcagatg 2700
gcctaccgct tcaacggcat cggcgtgaca cagaacgtgc tgtacgagaa ccagaagctg 2760
atcgccaacc agttcaacag cgccattggc aagattcagg actctctgag cagcacagcc 2820
agcgccctgg gcaagctgca gaacgtggtg aaccagaacg cccaggccct gaacacactg 2880
gtgaagcagc tgtcttctaa cttcggcgcc atttctagcg tgctgaacga cattctgtcg 2940
cggctggaca aggtggaggc cgaggtgcag attgacaggc tcatcacagg cagactgcag 3000
tctctgcaga catacgtgac ccagcagctg attagagccg ccgagattag agcctccgcc 3060
aacctggccg ccaccaagat gagcgagtgc gtgctcggcc agtctaagcg ggtggacttc 3120
tgcggcaagg gctaccacct catgtctttc cctcagtccg cccctcacgg cgtggtgttc 3180
ctccacgtga catacgtgcc cgcccaggag aagaacttca ccacagcccc cgccatttgc 3240
cacgacggca aggcccactt ccctagggag ggcgtgttcg tgtctaacgg cacccactgg 3300
ttcgtgaccc agcggaactt ctacgagcct cagattatta ccacagacaa cacattcgtg 3360
agcggcaact gcgacgtggt gattggcatt gtgaacaaca cagtgtacga cccactgcag 3420
cctgagttgg actctttcaa ggaggaactc gacaagtact tcaagaacca cacatctcct 3480
gacgtggacc tgggcgacat tagcggcatt aacgcctctg tggtgaacat tcagaaggag 3540
attgacagac tgaacgaggt ggccaagaac ctgaacgagt ctctcattga cctgcaggag 3600
ctgggcaagt acgagcagta cattaagtgg ccttggtaca tttggctggg cttcattgcc 3660
ggcctgatcg ccattgtgat ggtgaccatc atgctgtgct gcatgacatc ttgctgcagc 3720
tgcctgaagg gctgctgctc ttgcggctct tgctgcaag 3759
<210> 23
<211> 3756
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic polynucleotide
<220>
<221> nucleotide sequence of BA.1-S-del18
<222> (1)..(3756)
<400> 23
atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgaccaca 60
cggacccagc tccctcccgc ctacacaaac tctttcaccc ggggcgtgta ctaccccgac 120
aaggtgttcc ggtctagcgt gctccactct acacaggacc tgttcctccc tttcttcagc 180
aacgtgacat ggttccacgt gatctctggc acaaacggca caaagcggtt cgacaacccc 240
gtgctccctt tcaacgacgg cgtgtacttc gccagcattg agaagtctaa cattatccgg 300
ggctggattt tcggcaccac actcgactct aagacacagt ccctcctgat tgtgaacaac 360
gccacaaacg tggtgattaa ggtgtgcgag ttccagttct gcaacgaccc tttcctggac 420
cacaagaaca acaagtcttg gatggagtct gagttcagag tgtactctag cgccaacaac 480
tgcaccttcg agtacgtgtc ccagcctttc ctcatggacc tggagggcaa gcagggcaac 540
ttcaagaacc tgagagagtt cgtgttcaag aacattgacg gctacttcaa gatttactct 600
aagcacaccc caattattgt gagggaacca gaagacctcc ctcagggctt ctccgcctta 660
gaaccactgg tggacctccc tattggcatt aacatcacac gcttccagac actgctcgcc 720
ctccaccggt cttacctgac cccaggcgac tctagctctg gctggacagc cggcgccgcc 780
gcctactacg tgggctacct gcagcctagg accttcctcc tgaagtacaa cgagaacggc 840
acaattaccg acgccgtgga ctgcgccctg gacccactgt ccgagacaaa gtgcacactg 900
aagtccttca cagtggagaa gggcatttac cagacatcta acttccgggt gcagcctaca 960
gagtctattg tgcggttccc aaacatcaca aacctgtgcc ctttcgacga ggtgttcaac 1020
gccacccggt tcgcctctgt gtacgcctgg aaccggaagc ggatctctaa ctgcgtggcc 1080
gactactccg tgctgtacaa cctggcccct ttcttcacat tcaagtgcta cggcgtgtcc 1140
cctacaaagc tgaacgacct gtgcttcacc aacgtgtacg ccgactcttt cgtgattaga 1200
ggcgacgagg tgaggcagat tgcccccggc cagacaggca acatcgccga ctacaactac 1260
aagctgcccg acgacttcac aggctgcgtg atcgcctgga actctaacaa gctggactct 1320
aaggtgtctg gcaactacaa ctacctgtac agactgttcc ggaagtctaa cctgaagcca 1380
ttcgagaggg acattagcac cgagatttac caggccggca acaagccatg caacggcgtg 1440
gccggcttca actgctactt cccactgcgc tcctactcct tccggcctac atacggcgtg 1500
ggccaccagc cttaccgggt ggtggtgctg tctttcgagc tgctccacgc ccccgccaca 1560
gtgtgcggcc caaagaagag cacaaacctc gtgaagaaca agtgcgtgaa cttcaacttc 1620
aacggcctca agggcacagg cgtgctcacc gagtctaaca agaagttcct ccctttccag 1680
cagttcggcc gcgacattgc cgacaccacc gacgccgtgc gggaccctca gacactggaa 1740
attctcgaca tcaccccttg cagcttcggc ggcgtgtccg tgatcacccc aggcacaaac 1800
acatctaacc aggtggccgt gctgtaccag ggcgtgaact gcaccgaggt gccagtggcc 1860
atccacgccg accagctcac cccaacatgg agggtgtaca gcacaggctc taacgtgttc 1920
caaacccggg ccggctgcct cattggcgcc gagtacgtga acaactctta cgagtgcgac 1980
atccctattg gcgccggcat ttgcgcctct taccagaccc agacaaagtc tcaccggaga 2040
gcccggtctg tggcctctca gagcattatt gcctacacca tgtctctggg cgccgagaac 2100
tctgtggcct actctaacaa ctctattgcc atccctacaa acttcacaat ttctgtgacc 2160
accgagattc tcccagtgtc tatgaccaag acatctgtgg actgcaccat gtacatttgc 2220
ggcgactcca ccgagtgctc taacctcctg ctccagtacg gctctttctg cacccagctc 2280
aagcgcgccc tgacaggcat cgccgtggag caggacaaga acacccagga ggtgttcgcc 2340
caggtgaagc agatttacaa gaccccccca attaagtact tcggcggctt caacttctct 2400
cagattctcc ccgacccatc caagcctagc aagcggtcct tcattgagga cctcctgttc 2460
aacaaggtga cactggccga cgccggcttc attaagcagt acggcgactg cctgggcgac 2520
attgccgccc gggacctgat ttgcgcccag aagttcaagg gcctcacagt gctcccccca 2580
ctgctcaccg acgagatgat tgcccagtac acatctgccc tcctggccgg cacaattaca 2640
tctggctgga ccttcggcgc cggcgccgcc ctgcagatcc ctttcgccat gcagatggcc 2700
taccgcttca acggcatcgg cgtgacacag aacgtgctgt acgagaacca gaagctgatc 2760
gccaaccagt tcaacagcgc cattggcaag attcaggact ctctgagcag cacagccagc 2820
gccctgggca agctgcagga cgtggtgaac cacaacgccc aggccctgaa cacactggtg 2880
aagcagctgt cttctaagtt cggcgccatt tctagcgtgc tgaacgacat tttctcgcgg 2940
ctggacaagg tggaggccga ggtgcagatt gacaggctca tcacaggcag actgcagtct 3000
ctgcagacat acgtgaccca gcagctgatt agagccgccg agattagagc ctccgccaac 3060
ctggccgcca ccaagatgag cgagtgcgtg ctcggccagt ctaagcgggt ggacttctgc 3120
ggcaagggct accacctcat gtctttccct cagtccgccc ctcacggcgt ggtgttcctc 3180
cacgtgacat acgtgcccgc ccaggagaag aacttcacca cagcccccgc catttgccac 3240
gacggcaagg cccacttccc tagggagggc gtgttcgtgt ctaacggcac ccactggttc 3300
gtgacccagc ggaacttcta cgagcctcag attattacca cagacaacac attcgtgagc 3360
ggcaactgcg acgtggtgat tggcattgtg aacaacacag tgtacgaccc actgcagcct 3420
gagttggact ctttcaagga ggaactcgac aagtacttca agaaccacac atctcctgac 3480
gtggacctgg gcgacattag cggcattaac gcctctgtgg tgaacattca gaaggagatt 3540
gacagactga acgaggtggc caagaacctg aacgagtctc tcattgacct gcaggagctg 3600
ggcaagtacg agcagtacat taagtggcct tggtacattt ggctgggctt cattgccggc 3660
ctgatcgcca ttgtgatggt gaccatcatg ctgtgctgca tgacatcttg ctgcagctgc 3720
ctgaagggct gctgctcttg cggctcttgc tgcaag 3756
<210> 24
<211> 3756
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic polynucleotide
<220>
<221> BA.1.1-S-del18 nucleotide sequence
<222> (1)..(3756)
<400> 24
atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgaccaca 60
cggacccagc tccctcccgc ctacacaaac tctttcaccc ggggcgtgta ctaccccgac 120
aaggtgttcc ggtctagcgt gctccactct acacaggacc tgttcctccc tttcttcagc 180
aacgtgacat ggttccacgt gatctctggc acaaacggca caaagcggtt cgacaacccc 240
gtgctccctt tcaacgacgg cgtgtacttc gccagcattg agaagtctaa cattatccgg 300
ggctggattt tcggcaccac actcgactct aagacacagt ccctcctgat tgtgaacaac 360
gccacaaacg tggtgattaa ggtgtgcgag ttccagttct gcaacgaccc tttcctggac 420
cacaagaaca acaagtcttg gatggagtct gagttcagag tgtactctag cgccaacaac 480
tgcaccttcg agtacgtgtc ccagcctttc ctcatggacc tggagggcaa gcagggcaac 540
ttcaagaacc tgagagagtt cgtgttcaag aacattgacg gctacttcaa gatttactct 600
aagcacaccc caattattgt ggaaccagaa agggacctcc ctcagggctt ctccgcctta 660
gaaccactgg tggacctccc tattggcatt aacatcacac gcttccagac actgctcgcc 720
ctccaccggt cttacctgac cccaggcgac tctagctctg gctggacagc cggcgccgcc 780
gcctactacg tgggctacct gcagcctagg accttcctcc tgaagtacaa cgagaacggc 840
acaattaccg acgccgtgga ctgcgccctg gacccactgt ccgagacaaa gtgcacactg 900
aagtccttca cagtggagaa gggcatttac cagacatcta acttccgggt gcagcctaca 960
gagtctattg tgcggttccc aaacatcaca aacctgtgcc ctttcgacga ggtgttcaac 1020
gccaccaagt tcgcctctgt gtacgcctgg aaccggaagc ggatctctaa ctgcgtggcc 1080
gactactccg tgctgtacaa cctggcccct ttcttcacat tcaagtgcta cggcgtgtcc 1140
cctacaaagc tgaacgacct gtgcttcacc aacgtgtacg ccgactcttt cgtgattaga 1200
ggcgacgagg tgaggcagat tgcccccggc cagacaggca acatcgccga ctacaactac 1260
aagctgcccg acgacttcac aggctgcgtg atcgcctgga actctaacaa gctggactct 1320
aaggtgtctg gcaactacaa ctacctgtac agactgttcc ggaagtctaa cctgaagcca 1380
ttcgagaggg acattagcac cgagatttac caggccggca acaagccatg caacggcgtg 1440
gccggcttca actgctactt cccactgcgc tcctactcct tccggcctac atacggcgtg 1500
ggccaccagc cttaccgggt ggtggtgctg tctttcgagc tgctccacgc ccccgccaca 1560
gtgtgcggcc caaagaagag cacaaacctc gtgaagaaca agtgcgtgaa cttcaacttc 1620
aacggcctca agggcacagg cgtgctcacc gagtctaaca agaagttcct ccctttccag 1680
cagttcggcc gcgacattgc cgacaccacc gacgccgtgc gggaccctca gacactggaa 1740
attctcgaca tcaccccttg cagcttcggc ggcgtgtccg tgatcacccc aggcacaaac 1800
acatctaacc aggtggccgt gctgtaccag ggcgtgaact gcaccgaggt gccagtggcc 1860
atccacgccg accagctcac cccaacatgg agggtgtaca gcacaggctc taacgtgttc 1920
caaacccggg ccggctgcct cattggcgcc gagtacgtga acaactctta cgagtgcgac 1980
atccctattg gcgccggcat ttgcgcctct taccagaccc agacaaagtc tcaccggaga 2040
gcccggtctg tggcctctca gagcattatt gcctacacca tgtctctggg cgccgagaac 2100
tctgtggcct actctaacaa ctctattgcc atccctacaa acttcacaat ttctgtgacc 2160
accgagattc tcccagtgtc tatgaccaag acatctgtgg actgcaccat gtacatttgc 2220
ggcgactcca ccgagtgctc taacctcctg ctccagtacg gctctttctg cacccagctc 2280
aagcgcgccc tgacaggcat cgccgtggag caggacaaga acacccagga ggtgttcgcc 2340
caggtgaagc agatttacaa gaccccccca attaagtact tcggcggctt caacttctct 2400
cagattctcc ccgacccatc caagcctagc aagcggtcct tcattgagga cctcctgttc 2460
aacaaggtga cactggccga cgccggcttc attaagcagt acggcgactg cctgggcgac 2520
attgccgccc gggacctgat ttgcgcccag aagttcaagg gcctcacagt gctcccccca 2580
ctgctcaccg acgagatgat tgcccagtac acatctgccc tcctggccgg cacaattaca 2640
tctggctgga ccttcggcgc cggcgccgcc ctgcagatcc ctttcgccat gcagatggcc 2700
taccgcttca acggcatcgg cgtgacacag aacgtgctgt acgagaacca gaagctgatc 2760
gccaaccagt tcaacagcgc cattggcaag attcaggact ctctgagcag cacagccagc 2820
gccctgggca agctgcagga cgtggtgaac cacaacgccc aggccctgaa cacactggtg 2880
aagcagctgt cttctaagtt cggcgccatt tctagcgtgc tgaacgacat tttctcgcgg 2940
ctggacaagg tggaggccga ggtgcagatt gacaggctca tcacaggcag actgcagtct 3000
ctgcagacat acgtgaccca gcagctgatt agagccgccg agattagagc ctccgccaac 3060
ctggccgcca ccaagatgag cgagtgcgtg ctcggccagt ctaagcgggt ggacttctgc 3120
ggcaagggct accacctcat gtctttccct cagtccgccc ctcacggcgt ggtgttcctc 3180
cacgtgacat acgtgcccgc ccaggagaag aacttcacca cagcccccgc catttgccac 3240
gacggcaagg cccacttccc tagggagggc gtgttcgtgt ctaacggcac ccactggttc 3300
gtgacccagc ggaacttcta cgagcctcag attattacca cagacaacac attcgtgagc 3360
ggcaactgcg acgtggtgat tggcattgtg aacaacacag tgtacgaccc actgcagcct 3420
gagttggact ctttcaagga ggaactcgac aagtacttca agaaccacac atctcctgac 3480
gtggacctgg gcgacattag cggcattaac gcctctgtgg tgaacattca gaaggagatt 3540
gacagactga acgaggtggc caagaacctg aacgagtctc tcattgacct gcaggagctg 3600
ggcaagtacg agcagtacat taagtggcct tggtacattt ggctgggctt cattgccggc 3660
ctgatcgcca ttgtgatggt gaccatcatg ctgtgctgca tgacatcttg ctgcagctgc 3720
ctgaagggct gctgctcttg cggctcttgc tgcaag 3756
<210> 25
<211> 3756
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic polynucleotide
<220>
<221> nucleotide sequence of BA.2-S-del18
<222> (1)..(3756)
<400> 25
atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgatcaca 60
cggacccaga gctacacaaa ctctttcacc cggggcgtgt actaccccga caaggtgttc 120
cggtctagcg tgctccactc tacacaggac ctgttcctcc ctttcttcag caacgtgaca 180
tggttccacg ccatccacgt gtctggcaca aacggcacaa agcggttcga caaccccgtg 240
ctccctttca acgacggcgt gtacttcgcc agcaccgaga agtctaacat tatccggggc 300
tggattttcg gcaccacact cgactctaag acacagtccc tcctgattgt gaacaacgcc 360
acaaacgtgg tgattaaggt gtgcgagttc cagttctgca acgacccttt cctggacgtg 420
tactaccaca agaacaacaa gtcttggatg gagtctgagt tcagagtgta ctctagcgcc 480
aacaactgca ccttcgagta cgtgtcccag cctttcctca tggacctgga gggcaagcag 540
ggcaacttca agaacctgag agagttcgtg ttcaagaaca ttgacggcta cttcaagatt 600
tactctaagc acaccccaat taacctcggc agggacctcc ctcagggctt ctccgcctta 660
gaaccactgg tggacctccc tattggcatt aacatcacac gcttccagac actgctcgcc 720
ctccaccggt cttacctgac cccaggcgac tctagctctg gctggacagc cggcgccgcc 780
gcctactacg tgggctacct gcagcctagg accttcctcc tgaagtacaa cgagaacggc 840
acaattaccg acgccgtgga ctgcgccctg gacccactgt ccgagacaaa gtgcacactg 900
aagtccttca cagtggagaa gggcatttac cagacatcta acttccgggt gcagcctaca 960
gagtctattg tgcggttccc aaacatcaca aacctgtgcc ctttcgacga ggtgttcaac 1020
gccacccggt tcgcctctgt gtacgcctgg aaccggaagc ggatctctaa ctgcgtggcc 1080
gactactccg tgctgtacaa cttcgccccc ttcttcgcct tcaagtgcta cggcgtgtcc 1140
cctacaaagc tgaacgacct gtgcttcacc aacgtgtacg ccgactcttt cgtgattaga 1200
ggcaacgagg tgagccagat tgcccccggc cagacaggca acatcgccga ctacaactac 1260
aagctgcccg acgacttcac aggctgcgtg atcgcctgga actctaacaa gctggactct 1320
aaggtgggcg gcaactacaa ctacctgtac agactgttcc ggaagtctaa cctgaagcca 1380
ttcgagaggg acattagcac cgagatttac caggccggca acaagccatg caacggcgtg 1440
gccggcttca actgctactt cccactgcgg tcctacggct tccggcctac atacggcgtg 1500
ggccaccagc cttaccgggt ggtggtgctg tctttcgagc tgctccacgc ccccgccaca 1560
gtgtgcggcc caaagaagag cacaaacctc gtgaagaaca agtgcgtgaa cttcaacttc 1620
aacggcctca caggcacagg cgtgctcacc gagtctaaca agaagttcct ccctttccag 1680
cagttcggcc gcgacattgc cgacaccacc gacgccgtgc gggaccctca gacactggaa 1740
attctcgaca tcaccccttg cagcttcggc ggcgtgtccg tgatcacccc aggcacaaac 1800
acatctaacc aggtggccgt gctgtaccag ggcgtgaact gcaccgaggt gccagtggcc 1860
atccacgccg accagctcac cccaacatgg agggtgtaca gcacaggctc taacgtgttc 1920
cagacccggg ccggctgcct cattggcgcc gagtacgtga acaactctta cgagtgcgac 1980
atccctattg gcgccggcat ttgcgcctct taccagaccc agacaaagtc tcaccggaga 2040
gcccggtctg tggcctctca gagcattatt gcctacacca tgtctctggg cgccgagaac 2100
tctgtggcct actctaacaa ctctattgcc atccctacaa acttcacaat ttctgtgacc 2160
accgagattc tcccagtgtc tatgaccaag acatctgtgg actgcaccat gtacatttgc 2220
ggcgactcca ccgagtgctc taacctcctg ctccagtacg gctctttctg cacccagctc 2280
aagcgcgccc tgacaggcat cgccgtggag caggacaaga acacccagga ggtgttcgcc 2340
caggtgaagc agatttacaa gaccccccca attaagtact tcggcggctt caacttctct 2400
cagattctcc ccgacccatc caagcctagc aagcggtcct tcattgagga cctcctgttc 2460
aacaaggtga cactggccga cgccggcttc attaagcagt acggcgactg cctgggcgac 2520
attgccgccc gggacctgat ttgcgcccag aagttcaacg gcctcacagt gctcccccca 2580
ctgctcaccg acgagatgat tgcccagtac acatctgccc tcctggccgg cacaattaca 2640
tctggctgga ccttcggcgc cggcgccgcc ctgcagatcc ctttcgccat gcagatggcc 2700
taccgcttca acggcatcgg cgtgacacag aacgtgctgt acgagaacca gaagctgatc 2760
gccaaccagt tcaacagcgc cattggcaag attcaggact ctctgagcag cacagccagc 2820
gccctgggca agctgcagga cgtggtgaac cacaacgccc aggccctgaa cacactggtg 2880
aagcagctgt cttctaagtt cggcgccatt agcagcgtgc tgaacgacat tctgtcgcgg 2940
ctggacaagg tggaggccga ggtgcagatt gacaggctca tcacaggcag actgcagtct 3000
ctgcagacat acgtgaccca gcagctgatt agagccgccg agattagagc ctccgccaac 3060
ctggccgcca ccaagatgag cgagtgcgtg ctcggccagt ctaagcgggt ggacttctgc 3120
ggcaagggct accacctcat gtctttccct cagtccgccc ctcacggcgt ggtgttcctc 3180
cacgtgacat acgtgcccgc ccaggagaag aacttcacca cagcccccgc catttgccac 3240
gacggcaagg cccacttccc tagggagggc gtgttcgtgt ctaacggcac ccactggttc 3300
gtgacccagc ggaacttcta cgagcctcag attattacca cagacaacac attcgtgagc 3360
ggcaactgcg acgtggtgat tggcattgtg aacaacacag tgtacgaccc actgcagcct 3420
gagttggact ctttcaagga ggaactcgac aagtacttca agaaccacac atctcctgac 3480
gtggacctgg gcgacattag cggcattaac gcctctgtgg tgaacattca gaaggagatt 3540
gacagactga acgaggtggc caagaacctg aacgagtctc tcattgacct gcaggagctg 3600
ggcaagtacg agcagtacat taagtggcct tggtacattt ggctgggctt cattgccggc 3660
ctgatcgcca ttgtgatggt gaccatcatg ctgtgctgca tgacatcttg ctgcagctgc 3720
ctgaagggct gctgctcttg cggctcttgc tgcaag 3756
<210> 26
<211> 3747
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic polynucleotide
<220>
<221> nucleotide sequence of BA.3-S-del18
<222> (1)..(3747)
<400> 26
atgttcgtgt tcctggtgct cctgcccctc gtgagctctc agtgcgtgaa cctgaccacc 60
cggacacagc tgccacctgc ctacaccaac tctttcacaa gaggcgtgta ctaccccgac 120
aaggtgttcc ggagcagcgt gctgcacagc acacaggatc tgttcctgcc cttcttcagc 180
aacgtgacct ggttccacgt gatcagcggc accaacggaa caaaaagatt tgacaacccc 240
gtgctgcctt ttaacgatgg cgtctacttc gcctccaccg agaagagcaa catcatccgc 300
ggctggatct tcggtacaac cctggattcc aagacccaga gcctgctgat cgtgaacaat 360
gccacaaacg tggtgatcaa ggtgtgtgag ttccagttct gtaacgaccc ttttctggga 420
cacaagaata acaagagctg gatggaaagc gagttccgag tgtactccag cgccaacaac 480
tgcactttcg agtacgtgag ccagcctttc ctgatggacc tggaaggcaa gcagggaaat 540
ttcaagaacc tgcgggagtt cgtgtttaag aacattgatg gctactttaa gatctacagc 600
aagcacaccc caatcatcgt gcgggacctg cctcaaggct tcagcgccct cgaacctctg 660
gtggacctgc ccatcggaat caacatcaca cggtttcaga ccctgctggc cctgcatagg 720
agctacctga cacctggcga cagcagctcc ggctggacag ccggagctgc cgcttattac 780
gttggctacc tgcagcctcg tacattcctg cttaagtata atgagaatgg cacaatcacc 840
gacgccgtgg actgcgccct ggaccccctg tctgagacaa aatgcaccct gaagtctttc 900
accgtggaaa agggcatcta ccagacctct aacttccgcg tccagcctac cgagtccatc 960
gtccggttcc ctaacataac caacctgtgc cctttcgacg aggtgtttaa cgccaccaga 1020
ttcgcttctg tgtacgcctg gaacagaaag agaatcagca attgtgtggc tgactacagc 1080
gtgctctaca actttgcccc ttttttcaca ttcaagtgct acggagtgag ccctacaaag 1140
ctgaacgacc tgtgcttcac caacgtgtac gccgacagct ttgttatccg gggcaatgag 1200
gtgagacaga tcgcccctgg acagaccgga aacatcgccg attacaacta caaactgcca 1260
gatgacttca ccggctgcgt gatcgcctgg aactccaaca agctggactc taaggtgagc 1320
ggcaattaca actacctgta cagactgttt cggaagagca acctgaagcc tttcgagaga 1380
gatataagca ccgagatcta ccaggctggc aataaacctt gcaacggcgt tgccggcttc 1440
aactgctact tccctctgag aagctacggc tttaggccca cctacggcgt gggccaccag 1500
ccctaccggg tggtggtgct gagcttcgag ctgctgcacg cccccgcaac cgtgtgcggc 1560
cctaagaaat ctacaaatct cgtgaaaaat aagtgcgtca acttcaactt caatggcctg 1620
accggcacgg gtgtactgac cgagtctaac aagaaattcc tgcccttcca acagttcggc 1680
agagacatcg ccgacaccac cgatgccgtg cgggacccac aaacccttga gatcctggat 1740
atcacacctt gtagttttgg cggcgtgtct gtcatcaccc ctggcaccaa cacctctaac 1800
caagtggccg tcctctacca gggcgttaat tgcaccgagg tccctgtggc aatccacgcc 1860
gaccagctga cccccacatg gagagtgtac agcacaggca gcaacgtgtt ccaaacaaga 1920
gccggctgcc tgatcggcgc tgaatacgtg aataacagct acgagtgcga catccccatc 1980
ggggctggga tctgcgccag ctaccagacc cagaccaaaa gccacagaag agcccggagc 2040
gttgccagcc agtcaatcat cgcctacacc atgagcctcg gcgctgagaa cagcgtggcc 2100
tattccaaca atagtatcgc catccctacc aatttcacca tctcggtgac caccgaaatc 2160
ctgcctgtga gtatgaccaa aacatcagtg gactgcacca tgtacatctg cggcgatagc 2220
accgagtgca gcaacctgct gctgcagtac gggagcttct gcacccaact gaagcgcgct 2280
ctgaccggca tcgctgtgga acaggataag aacacacagg aggtgttcgc ccaggtgaag 2340
cagatctaca agacgcctcc tatcaagtac ttcggcggct tcaacttttc tcagatcctg 2400
cctgacccct caaagcccag caagcggtcc ttcatcgagg acctgctctt taacaaggtg 2460
acgctggccg acgctggctt catcaaacag tatggggatt gcctgggcga catcgccgcc 2520
agagacctga tttgtgccca gaagttcaac ggcctgaccg tcctccctcc tctgctgaca 2580
gacgaaatga tcgcccagta cacaagcgct ctgctggccg gcacaatcac tagcggctgg 2640
accttcggcg ccggagccgc tctgcaaatc cctttcgcca tgcagatggc ctacagattc 2700
aacggcattg gtgttaccca gaacgtgctg tatgagaacc agaagctgat cgccaaccag 2760
tttaatagcg ccatcggaaa gatccaagac tctctgagca gcaccgccag cgccttagga 2820
aagctgcagg acgtggtgaa ccacaacgcc caggccctga atacactggt gaagcagctg 2880
agctccaagt tcggcgccat ctcatctgtc cttaacgaca ttctgagtag actggacaag 2940
gtggaagccg aagtgcagat cgacagactg atcaccggca gactgcaaag cctgcagaca 3000
tatgtgaccc agcagctgat cagagcggcc gagatcagag ccagcgctaa tctggctgcc 3060
acaaagatgt ccgaatgcgt gctcggccag tccaagagag tggatttctg cggcaaaggc 3120
taccacctga tgagcttccc ccagagcgcc cctcacggcg tggtgtttct gcatgtgacc 3180
tacgtgcctg ctcaggaaaa gaacttcaca acagctcctg ccatctgtca cgacggcaag 3240
gcccacttcc ccagagaggg cgtattcgtg tctaacggca cccactggtt cgtgacccag 3300
agaaacttct acgagcctca gatcatcaca accgacaaca ccttcgtgag cggcaactgt 3360
gatgtggtga tcggcatcgt gaacaacacc gtttacgacc ccttacagcc tgagctggat 3420
tctttcaagg aagaactgga taaatacttc aagaatcaca caagtcccga cgtggaccta 3480
ggggacatct ctggcataaa cgcctccgtc gtgaacatcc agaaagaaat cgatagactg 3540
aacgaagtgg ccaagaacct gaacgagagc ctgatcgacc tgcaggagct gggcaaatac 3600
gagcagtaca tcaagtggcc ttggtacatc tggctgggct tcatcgccgg actgatcgcc 3660
atcgtgatgg tgaccatcat gctgtgttgc atgaccagct gctgcagctg cctgaaggga 3720
tgttgctctt gtggctcatg ctgtaaa 3747

Claims (16)

1. A polynucleotide encoding a recombinant chimeric antigen peptide having the structure shown in formula (I):
(A-B)-C-(A-B’)
(I)
in formula (I):
option 1: A-B represents the amino acid sequence of the RBD domain of the S protein of a prototype strain of a novel coronavirus, or a portion thereof, or an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and having the same or substantially the same immunogenicity as it;
A-B' represents the amino acid sequence of the RBD domain of the S protein of a novel variant of coronavirus Beta or a part thereof, or an amino acid sequence which is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and has the same or substantially the same immunogenicity as it; or
A-B represents the amino acid sequence of the RBD domain of the S protein of the novel coronavirus Delta variant or a part thereof, or an amino acid sequence which has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and which has the same or substantially the same immunogenicity as it;
A-B' represents the amino acid sequence of the RBD domain of the S protein of a novel variant of coronavirus Beta or a part thereof, or an amino acid sequence which is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and has the same or substantially the same immunogenicity as it; or
A-B represents the amino acid sequence of the RBD domain of the S protein of the novel coronavirus Delta variant or a portion thereof, or an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and having the same or substantially the same immunogenicity as it;
A-B' represents the amino acid sequence of the RBD domain of the S protein of the novel variant strain of coronavirus, or a part thereof, or an amino acid sequence which has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and which has the same or substantially the same immunogenicity as it;
c represents a linker (GGS) n (ii) a Wherein n is 0,1,2,3,4 or 5.
2. The polynucleotide of claim 1, wherein a portion of the RBD domain of the S protein of said novel coronavirus prototype strain is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% of its entire amino acid sequence;
and/or, a portion of the RBD domain of the S protein of the novel variant coronavirus Beta is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% of its entire amino acid sequence;
and/or, a portion of the RBD domain of the S protein of the novel coronavirus Delta variant is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% of its entire amino acid sequence;
and/or, a portion of the RBD domain of the S protein of the novel variant strain of coronavirus which is at least 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% of its entire amino acid sequence;
and/or, n is 0,1,2 or 3.
3. The polynucleotide according to claim 1 or 2, wherein the amino acid sequence of the RBD domain of the S protein of the novel coronavirus prototype strain or a part thereof is shown in SEQ ID NO. 1, or an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 1 and has the same or substantially the same immunogenicity as the amino acid sequence;
and/or the amino acid sequence of the RBD structural domain of the S protein of the novel coronavirus Beta variant or a part thereof is shown as SEQ ID NO. 2, or the amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids and has the same or basically the same immunogenicity as the amino acid sequence shown as the SEQ ID NO. 2;
and/or the amino acid sequence of the RBD structural domain of the S protein of the novel coronavirus Delta variant or a part of the RBD structural domain is shown as SEQ ID NO. 3, or the amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids and has the same or basically the same immunogenicity as the amino acid sequence shown as SEQ ID NO. 3;
and/or, the amino acid sequence of the RBD structural domain or a part thereof of the S protein of the novel coronavirus Omicron variant strain is shown as SEQ ID NO. 4, or the amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids and has the same or basically the same immunogenicity as the amino acid sequence shown as SEQ ID NO. 4;
and/or, n ═ 0,1, or 2.
4. The polynucleotide of claim 3, wherein when formula (I) is Option 1, the recombinant chimeric antigenic peptide having the structure shown in formula (I) has the amino acid sequence shown in SEQ ID NO. 5;
or when the formula (I) is Option 2, the recombinant chimeric antigen peptide with the structure shown in the formula (I) has an amino acid sequence shown in SEQ ID NO. 6;
or, when the formula (I) is Option 3, the recombinant chimeric antigen peptide with the structure shown in the formula (I) has an amino acid sequence shown in SEQ ID NO. 7.
5. The polynucleotide of any one of claims 1-4, wherein the polynucleotide is a DNA molecule;
preferably, when formula (I) is Option 1, the DNA molecule has the DNA sequence shown in SEQ ID NO. 8;
preferably, when formula (I) is Option 2, the DNA molecule has the DNA sequence shown in SEQ ID NO. 9;
preferably, when formula (I) is Option 3, the DNA molecule has the DNA sequence shown in SEQ ID NO 10.
6. The polynucleotide of any one of claims 1-4, wherein the polynucleotide is an mRNA molecule;
preferably, when formula (I) is Option 1, the mRNA molecule has the mRNA sequence shown as SEQ ID NO. 11;
preferably, when formula (I) is Option 2, the mRNA molecule has the mRNA sequence shown as SEQ ID NO. 12;
preferably, when formula (I) is Option 3, the mRNA molecule has the mRNA sequence shown in SEQ ID NO 13.
7. A nucleic acid construct comprising the polynucleotide of any one of claims 1-6, and optionally, at least one expression control element operably linked to the polynucleotide.
8. An expression vector comprising the nucleic acid construct of claim 7.
9. A host cell into which a polynucleotide according to any one of claims 1 to 6, a nucleic acid construct according to claim 7 or an expression vector according to claim 8 has been transformed or transfected.
10. Use of a polynucleotide according to any one of claims 1 to 6, a nucleic acid construct according to claim 7, an expression vector according to claim 8 or a host cell according to claim 9 for the preparation of a vaccine for the prevention and/or treatment of a novel coronavirus;
preferably, the vaccine is used for immunization alone or in sequential immunization with other types of novel coronavirus vaccines; further preferably, the other types of novel coronavirus vaccines include inactivated vaccines.
11. A chimeric nucleic acid vaccine or immunogenic composition comprising the polynucleotide of any one of claims 1-6, the nucleic acid construct of claim 7, the expression vector of claim 8, or the host cell of claim 9, and a physiologically acceptable vehicle, adjuvant, excipient, carrier, and/or diluent.
12. The chimeric nucleic acid vaccine or immunogenic composition of claim 11, which is a novel coronavirus DNA vaccine comprising:
(i) a eukaryotic expression vector; and
(ii) a DNA sequence which is constructed into the eukaryotic expression vector and codes the recombinant chimeric antigen peptide with the structure shown in the formula (I), preferably a DNA sequence shown in SEQ ID NO 8, 9 or 10;
preferably, the eukaryotic expression vector is selected from the group consisting of pGX0001, pVAX1, pCAGGS and pcDNA series vectors.
13. The chimeric nucleic acid vaccine or immunogenic composition of claim 11, which is a novel coronavirus mRNA vaccine comprising:
(I) the mRNA sequence of the recombinant chimeric antigen peptide with the structure shown in the formula (I) is preferably the mRNA sequence shown in SEQ ID NO 11, 12 or 13; and
(II) lipid nanoparticles.
14. The chimeric nucleic acid vaccine or immunogenic composition of claim 11, which is a novel coronavirus-viral vector vaccine comprising:
(1) a viral backbone vector; and
(2) the DNA sequence of the recombinant chimeric antigen peptide which is constructed into the virus skeleton vector and has the structure shown in the formula (I) is preferably the DNA sequence shown in SEQ ID NO. 8, 9 or 10;
preferably, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vectors, poxvirus vectors, influenza virus vectors, adeno-associated virus vectors.
15. A chimeric nucleic acid vaccine or immunogenic composition according to any of claims 11-14, characterized in that the vaccine or immunogenic composition is in the form of a nasal spray, oral preparation, suppository or parenteral preparation;
preferably, the nasal spray is selected from the group consisting of an aerosol, a spray and a powder spray;
preferably, the oral formulation is selected from the group consisting of tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film coatings, pellets, sublingual tablets and ointments;
preferably, the parenteral formulation is a transdermal agent, an ointment, a plaster, a topical liquid, an injectable or a bolus formulation.
16. A kit comprising a chimeric nucleic acid vaccine or immunogenic composition according to any one of claims 11-14, and optionally a novel coronavirus vaccine of a further type, said chimeric nucleic acid vaccine or immunogenic composition being packaged separately from said novel coronavirus vaccine of a further type;
preferably, the other type of novel coronavirus vaccine is a novel inactivated coronavirus vaccine.
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