WO2022135563A1 - 同时诱导抗多种病毒的免疫应答的方法 - Google Patents

同时诱导抗多种病毒的免疫应答的方法 Download PDF

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WO2022135563A1
WO2022135563A1 PCT/CN2021/141198 CN2021141198W WO2022135563A1 WO 2022135563 A1 WO2022135563 A1 WO 2022135563A1 CN 2021141198 W CN2021141198 W CN 2021141198W WO 2022135563 A1 WO2022135563 A1 WO 2022135563A1
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virus
seq
region
vaccine
tag
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French (fr)
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徐建青
张晓燕
周东明
曹康丽
王祥
胡杨洋
丁龙飞
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上海市公共卫生临床中心
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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • 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
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to the field of vaccines, in particular to a method for simultaneously inducing immune responses against multiple viruses including influenza (eg, coronaviruses, especially novel coronaviruses).
  • influenza eg, coronaviruses, especially novel coronaviruses.
  • Influenza referred to as influenza
  • influenza virus which is mainly divided into seasonal and pandemic influenza. Since the 16th century, major influenza outbreaks in different countries and regions around the world have become one of the severe challenges faced by the public health field. Influenza is associated with high morbidity and mortality, resulting in 2,000,000 to 5,000,000 severe cases and 250,000 to 500,000 deaths annually. It is estimated that adults account for 5-10% of influenza infections worldwide and children account for 20-30%. Influenza not only causes a severe disease burden, but also a huge direct and indirect economic burden.
  • influenza virus genome is divided into 8 segments, encoding hemagglutinin (HA), neuraminidase (NA), matrix proteins 1 and 2 (M1 and M2), nonstructural proteins 1 and 2 (NS1 and NS2), 10 proteins including nucleocapsid protein (NP) and three polymerase complexes (PB1, PB2 and PA).
  • HA hemagglutinin
  • NA neuraminidase
  • M1 and M2 matrix proteins 1 and 2
  • NS1 and NS2 nonstructural proteins 1 and 2
  • NP nucleocapsid protein
  • PB1, PB2 and PA polymerase complexes
  • influenza viruses can be divided into three types, A, B and C.
  • avian influenza virus (AIV) is influenza A virus (influenza A virus).
  • influenza A can be divided into 18 H subtypes and 9 N subtypes.
  • the 18 H subtypes can be divided into group I (H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, H18) and group II (H3, H4, H7, H10, H14, H15).
  • group I H1, H2, H3, H5, H7, H9 and H10
  • group II H3, H4, H7, H10, H14, H15
  • the subtypes known to directly infect humans are H1, H2, H3, H5, H7, H9 and H10
  • the viruses that cause highly pathogenic avian influenza are both H5 and H7 subtypes.
  • HA protein a trimeric surface glycoprotein composed of HA1 and HA2 domains.
  • Most HA1 chains form the globular crown, which contains the receptor binding site, while HA2 forms the stem, supporting HA1 and forming trimers.
  • HA crown (HA1) has a significant immunological advantage in inducing the production of neutralizing antibodies, it is easily mutated, resulting in antigenic drift and antigenic variation of the virus, forming new influenza virus subtypes.
  • the HA2 region is more conservative than HA1.
  • H7N9HA2 has broad-spectrum protection against influenza A
  • 3SDY has broad-spectrum protection against influenza A group II
  • 4FQV has broad-spectrum protection against influenza A
  • influenza B have broad-spectrum protection. Therefore, HA2 of H7N9 is crucial as a broad-spectrum influenza neutralizing antibody.
  • Coronaviruses contain four structural proteins, including spike protein (S protein), envelope protein, membrane protein, and nucleocapsid protein. Among them, the S protein plays the most important role in the attachment, fusion and entry process of the virus, and is also the main target of antibodies, entry inhibitors and vaccines.
  • S protein mediates viral entry into host cells, first by binding to host receptors through the receptor binding domain (RBD) of the S1 subunit, and then by fusing the virus and host cell membranes through the S2 subunit.
  • RBD receptor binding domain
  • animals immunized with the coronavirus vaccine may develop more severe symptoms when re-exposed to the live virus.
  • Non-neutralizing antibodies or lower antibody levels produced by vaccine immunization may cause antibody-dependent enhancement (ADE), enhancing the pathogenicity of the virus. Therefore, in order to reduce the side effects of ADE, the RBD region in the new coronavirus S protein will be the most effective target for vaccine development.
  • ADE antibody-dependent enhancement
  • immunogenic peptides capable of simultaneously inducing immune responses against multiple viruses, including influenza virus, nucleotide molecules encoding the same, vectors, host cells, vaccines, and uses thereof.
  • an immunogenic peptide comprising the following moieties:
  • immunogenic stem it contains the HA2 region of the influenza virus hemagglutinin HA;
  • an immunogenic corona it comprises a viral membrane protein or an immunogenic fragment thereof, wherein the source virus of the viral membrane protein is different from the source virus of the HA2 region in (a);
  • the source of the HA2 region is selected from the following group: any one of H1 to H18, especially from the widespread human influenza H1, H2, H3 and the multiple-occurring human infection avian influenza H5 and H7, for example, is derived from H1 (H1N1) popular in 2009 and H7 (H7N9) popular in 2013.
  • the amino acid sequence of the HA2 region is set forth in SEQ ID NO:1, or encoded by a nucleotide molecule having the sequence set forth in SEQ ID NO:2.
  • the viral membrane protein is an immunogenic membrane protein.
  • the source of the immunogen crown is selected from: influenza viruses whose sources are different from the HA2 region in (a); other viruses that are not influenza viruses.
  • the source of the immunogen crown is selected from the group consisting of coronavirus (eg, SARS-CoV-2), HIV, influenza virus (eg, the HA1 region of influenza virus hemagglutinin HA), rabies virus, swine fever Viruses, PRRS, measles, Ebola, herpes, arboviruses (eg, Zika, Japanese encephalitis, forest encephalitis, dengue, hantavirus, haemorrhagic heat virus).
  • the source of the immunogen crown is selected from the group consisting of: coronaviruses SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat -CoV, such as the S1 protein comprising the receptor binding domain of coronavirus, or the receptor binding domain (RBD), or an engineered receptor binding domain (eg, the RBD region is modified with a terminal cysteine to form an sRBD region) or its immunogenic fragments.
  • coronaviruses SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat -CoV such as the S1 protein comprising the receptor binding domain of coronavirus, or the receptor binding domain (RBD), or an engineered receptor binding domain (eg, the RBD region is modified
  • the immunogen crown is selected from the S1 of the coronavirus SARS-CoV-2, especially from the RBD region of S1 (e.g., the sequence of which is shown in SEQ ID NO:3 or by SEQ ID NO: 4) or its modified RBD region (such as the sRBD region modified by terminal Cys modification, such as the peptide segment shown in SEQ ID NO: 5 or encoded by the nucleotide molecule shown in SEQ ID NO: 6) , or an immunogenic fragment of the RBD region (for example, a peptide segment shown in SEQ ID NO: 21 or encoded by a nucleotide molecule shown in SEQ ID NO: 22).
  • the RBD region of S1 e.g., the sequence of which is shown in SEQ ID NO:3 or by SEQ ID NO: 4
  • its modified RBD region such as the sRBD region modified by terminal Cys modification, such as the peptide segment shown in SEQ ID NO: 5 or encoded by the nucleotide
  • the other moiety is selected from the group consisting of immunomodulatory sequences such as IL-2, IL-7, IL-12, IL-18, IL-21, GM-CSF, CD40L, CD40 stimulating antibody, PD- 1 with PD-L1 antibody, CTLA4 antibody, chemokines CXCL9, CXCL10, CXCL11, CXCL12, CXCL3, XCL1, CCL4, CCL20, cholera toxin and its subunits, bacterial flagellin, FimH, HIV p24, HIV gp41.
  • immunomodulatory sequences such as IL-2, IL-7, IL-12, IL-18, IL-21, GM-CSF, CD40L, CD40 stimulating antibody, PD- 1 with PD-L1 antibody, CTLA4 antibody, chemokines CXCL9, CXCL10, CXCL11, CXCL12, CXCL3, XCL1, CCL4, CCL20, cholera toxin and its subunits
  • the other moiety is selected from: a moiety that enables an immunogenic peptide to form a nanoparticle, such as transferrin (Fn, eg, the peptide molecule set forth in SEQ ID NO: 7 or a moiety represented by SEQ ID NO: 8 Nucleotide molecules indicated).
  • transferrin Fn, eg, the peptide molecule set forth in SEQ ID NO: 7 or a moiety represented by SEQ ID NO: 8 Nucleotide molecules indicated.
  • the other moiety is selected from the group consisting of: signal peptides, eg, CD33, CD8, CD16, mouse IgGl antibody.
  • the other moiety is selected from: a transmembrane region that enables the expression of an immunogenic peptide on the surface of a viral vector, such as the CD8 transmembrane region (CD8TM, such as the peptide molecule shown in SEQ ID NO: 17 or a The nucleotide molecule shown in SEQ ID NO: 18 encodes), HA2 transmembrane region, CD4 transmembrane region, gp41 transmembrane region.
  • CD8TM such as the peptide molecule shown in SEQ ID NO: 17 or a The nucleotide molecule shown in SEQ ID NO: 18 encodes
  • HA2 transmembrane region such as the peptide molecule shown in SEQ ID NO: 17 or a The nucleotide molecule shown in SEQ ID NO: 18 encodes
  • HA2 transmembrane region such as the peptide molecule shown in SEQ ID NO: 17 or a The nucleotide molecule shown in SEQ ID
  • the other moiety is selected from the group consisting of linking peptides, eg, (G4S) 3 , (G4S) n , GSAGSAAGSGEF, (Gly) 6 , EFPKPSTPPGSSGGAP, KESGSVSSEQLAQFRSLD, (Gly) 8 , EGKSSGSGSESKST.
  • linking peptides eg, (G4S) 3 , (G4S) n , GSAGSAAGSGEF, (Gly) 6 , EFPKPSTPPGSSGGAP, KESGSVSSEQLAQFRSLD, (Gly) 8 , EGKSSGSGSESKST.
  • the other moiety is selected from the group consisting of tags, such as His-tag, AviTag, Calmodulin tag, polyglutamate tag, E-tag, FLAG tag, HA-tag, Myc-tag, S-tag, SBP-tag , Sof-tag 1, Sof-tag3, Strep-tag, TC tag, V5 tag, T7 tag, VSV tag, Xpress tag, 3X FLAG tag, Isopep tag, Spytag, Snoop tag and PNE tag.
  • tags such as His-tag, AviTag, Calmodulin tag, polyglutamate tag, E-tag, FLAG tag, HA-tag, Myc-tag, S-tag, SBP-tag , Sof-tag 1, Sof-tag3, Strep-tag, TC tag, V5 tag, T7 tag, VSV tag, Xpress tag, 3X FLAG tag, Isopep tag, Spytag, Snoop tag and PNE tag.
  • the immunogenic peptide comprises an RBD region or sRBD region or an immunogenic fragment thereof linked to the HA2 region, and optionally other moieties linked to the aforementioned moieties.
  • the immunogenic peptide comprises: an RBD region or sRBD region (eg, the peptide molecule set forth in SEQ ID NO:5 or encoded by the nucleotide molecule set forth in SEQ ID NO:6) linked to the HA2 region ) and the Fn region (e.g. the peptide molecule shown in SEQ ID NO: 7 or encoded by the nucleotide molecule shown in SEQ ID NO: 8); the RBD region or sRBD region linked to the HA2 region and the CD8 transmembrane region (e.g.
  • SEQ ID NO: 8 The peptide molecule shown in ID NO: 17 or encoded by the nucleotide molecule shown in SEQ ID NO: 18); the immunogenic fragment of the RBD region linked to the HA2 region (for example, the peptide molecule shown in SEQ ID NO: 21 or encoded by the nucleotide molecule shown in SEQ ID NO: 18); The nucleotide molecule shown in SEQ ID NO: 22 encodes) and the Fn region.
  • the amino acid sequence of the immunogenic peptide is set forth in SEQ ID NO: 9 or 15 or 25, or the coding sequence of the immunogenic peptide is set forth in SEQ ID NO: 10 or 16 or 26 .
  • the present application adopts the HA2 region of H7N9 virus to connect the RBD region of the new coronavirus or its immunogenic fragment as the immunogen, and further modifies the immunogen at the same time, such as adding a disulfide bond to the RBD region, fusion protein, fusion Cytokines, thereby simultaneously inducing neutralizing antibodies against influenza virus and 2019-nCoV.
  • the present application provides an immunogenic peptide against influenza and novel coronavirus at the same time, which includes the HA2 region of the H7N9 virus hemagglutinin HA and the RBD region of the SARS-CoV-2 virus spike protein S, so The RBD region can be further modified with cysteine to form an sRBD region.
  • the cysteine modification adds a pair of cysteines at the base of the RBD domain to enable disulfide bond formation.
  • nucleotide molecule encoding an immunogenic peptide herein.
  • the coding sequence of the HA2 region comprises the nucleotide sequence shown in SEQ ID NO: 2; the coding sequence of the immunogen crown comprises the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 4 The nucleotide sequence set forth in ID NO: 6 or the nucleotide sequence set forth in SEQ ID NO: 22; and/or the coding sequence of the other portion comprises the nucleotide sequence set forth in SEQ ID NO: 8 or set forth in SEQ ID NO: 18 The nucleotide sequence shown.
  • sequence of the nucleotide molecule is set forth in SEQ ID NO: 10 or 16 or 26.
  • a vector comprising a nucleotide molecule herein.
  • a host cell comprising a nucleotide molecule or vector herein or capable of expressing an immunogenic peptide described herein.
  • the host cell is a mammalian cell or an insect cell, such as HEK293, HeLa, K562, CHO, NSO, SP2/0, PER.C6, Vero, RD, BHK, HT1080, A549, Cos-7 , ARPE-19 and MRC-5 cells; High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5 and Ao38.
  • HEK293, HeLa, K562, CHO, NSO, SP2/0, PER.C6, Vero, RD, BHK, HT1080, A549, Cos-7 , ARPE-19 and MRC-5 cells High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn36
  • a vaccine capable of simultaneously inducing an immune response against an influenza virus and another non-influenza or non-syngeneic influenza virus, comprising the immunogenic peptides, nucleotides described herein Molecules, vectors and/or host cells.
  • the vaccine is a nucleic acid vaccine (DNA or RNA vaccine), recombinant protein subunit vaccine, recombinant viral vector vaccine, recombinant bacterial vector vaccine, virus-like particle vaccine, nanoparticle vaccine, cell vector vaccine.
  • the vaccine is a viral vector vaccine selected from the group consisting of: poxviruses (eg, Tiantan strain, North American vaccine strain, Wyeth-derived strain, Listerial strain, Ankara-derived strain, Copenhagen strain, and New York strain) , Adenovirus (Ad5, Ad11, Ad26, Ad35, AdC68 or modified variants thereof), adeno-associated virus, herpes simplex virus, measles virus, reovirus, rhabdovirus, forest encephalitis virus, influenza virus, respiratory syndrome virus, poliovirus.
  • poxviruses eg, Tiantan strain, North American vaccine strain, Wyeth-derived strain, Listerial strain, Ankara-derived strain, Copenhagen strain, and New York strain
  • Adenovirus Ad5, Ad11, Ad26, Ad35, AdC68 or modified variants thereof
  • AdC68 Ad5
  • adeno-associated virus herpes simplex virus, measles virus, reovirus, rhabdovirus, forest
  • the vaccine comprises or is used in combination with an adjuvant
  • the adjuvant is selected from the group consisting of: aluminum adjuvant, cholera toxin and subunits thereof, oligodeoxynucleotides, manganese ion adjuvant, colloid Manganese adjuvant, Freund's adjuvant, MF59 adjuvant, QS-21 adjuvant, Poly I:C and other TLR ligands, GM-CSF, IL-2, IL-3, IL-7, IL-11, IL -12, IL-18, IL-21.
  • the vaccine is in a form suitable for intramuscular, intradermal, subcutaneous, intranasal, aerosol inhalation, genital tract, rectal, oral, or a combination of the above different inoculation methods (eg, intramuscular injection + intranasal instillation). ).
  • the vaccine is in a form suitable for combined vaccination (eg, co-vaccination or sequential vaccination) of two or more, such as with a coronavirus (eg, SARS-CoV-2, SARS-CoV, MERS-CoV) , HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV) S or S1 vaccines are inoculated sequentially, or with influenza virus HA or HA2 (such as derived from any of H1-H18) HA or HA2) vaccines of different species were vaccinated sequentially.
  • a coronavirus eg, SARS-CoV-2, SARS-CoV, MERS-CoV
  • HCoV-229E HCoV-229E
  • HCoV-OC43 HCoV-NL63
  • influenza virus HA or HA2 such as derived from any of H1-H18
  • an immunogenic peptide, nucleotide molecule, vector and/or host cell described herein in the manufacture of a medicament for the simultaneous prevention or treatment of an influenza virus and another non-influenza virus application.
  • immunogenic peptides for use in the simultaneous prevention or treatment of influenza and novel coronavirus infections.
  • a method for simultaneously preventing or treating influenza and novel coronavirus infection comprising administering to a subject in need the immunogenic peptide, nucleotide molecule, vector, host of the present disclosure cells and/or vaccines.
  • the vaccine is a nucleic acid vaccine (DNA or RNA vaccine), a recombinant protein subunit vaccine, a recombinant viral vector vaccine, a recombinant bacterial vector vaccine, a virus-like particle vaccine, a nanoparticle vaccine, a cell-based vaccine.
  • the vaccine comprises or is used in combination with adjuvants including, but not limited to, aluminum adjuvants, cholera toxin and subunits thereof, oligodeoxynucleotides, manganese ion adjuvants, Colloidal manganese adjuvant, Freund's adjuvant, SAS adjuvant, MF59 adjuvant, QS-21 adjuvant, Poly I:C and other TLR ligands, GM-CSF, IL-2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21, etc.
  • adjuvants including, but not limited to, aluminum adjuvants, cholera toxin and subunits thereof, oligodeoxynucleotides, manganese ion adjuvants, Colloidal manganese adjuvant, Freund's adjuvant, SAS adjuvant, MF59 adjuvant, QS-21 adjuvant, Poly I:C and other TLR ligands, GM-CSF,
  • the vaccine is in a form suitable for the following modes of administration: intramuscular, intradermal, subcutaneous, intranasal, aerosol inhalation, genital, rectal, oral, or any combination thereof.
  • one or more of the vaccines are used for vaccination, eg, in combination or sequentially.
  • one or more of the vaccines are vaccinated with other vaccines against novel coronaviruses or influenza
  • the other vaccines include vaccines against coronavirus S or S1, such as the S or S1 From including but not limited to SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV, etc.
  • the other vaccines include HA against influenza virus or HA2 vaccine, such as said HA or HA2 from including but not limited to H1-H18.
  • the vaccine comprises a nucleic acid vaccine (DNA or RNA vaccine) in combination with an adenovirus vector vaccine.
  • the vaccine comprises pcDNA3.1-S in combination with AdC68-RHAF.
  • the components of the vaccine combination are administered sequentially, preferably the DNA vaccine is administered first.
  • a method for preparing a vaccine against both influenza and novel coronavirus infection comprising:
  • a method of simultaneously inducing an immune response against influenza and other viruses including coronavirus wherein the HA2 region of the influenza virus hemagglutinin HA is used as the immunogenic stem and other viruses Or strain membrane protein as a fusion protein composed of the crown as a vaccine immunogen to simultaneously induce anti-influenza and other virus immune responses.
  • the HA2 immunogen is selected from the group including, but not limited to, H1-H18, especially from the widespread human influenza H1, H2, H3, and the multiple-occurring human infection avian influenza H5 and H7;
  • the HA2 immunogen is derived from H1, which was popular in 2009, and H7, which was popular in 2013.
  • the HA2 immunogen is derived from the HA2 sequence of an early strain of H7 circulating in 2013 and more than 80% full-length or fragment homologous to the sequence.
  • the HA2 immunogen may comprise the amino acid sequence set forth in SEQ ID NO:1 or may be encoded by the coding sequence having SEQ ID NO:2.
  • the coronal immunogen linked to HA2 is from a source including, but not limited to, coronavirus, HIV, influenza, rabies, swine fever, PRRS, measles, Ebola, herpes Viruses, arboviruses (Zika virus, Japanese encephalitis virus, Forest encephalitis virus, Dengue virus, Hantavirus, Hemorrhagic fever virus).
  • the crown immunogen linked to HA2 is selected from coronaviruses including but not limited to SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV- HKU1, bat-CoV, etc., especially the S1 protein comprising the receptor binding domain of coronavirus, or the receptor binding domain (RBD), or the engineered receptor binding domain.
  • coronaviruses including but not limited to SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV- HKU1, bat-CoV, etc.
  • S1 protein comprising the receptor binding domain of coronavirus, or the receptor binding domain (RBD), or the engineered receptor binding domain.
  • the crown immunogen linked to HA2 is selected from S1 of the coronavirus SARS-CoV-2, especially from the RBD region of S1 or full-length and fragments with more than 90% homology thereto.
  • the RBD region immunogen may comprise the amino acid sequence set forth in SEQ ID NO:3 or may be encoded by the coding sequence having SEQ ID NO:4.
  • the RBD region immunogen may comprise the amino acid sequence set forth in SEQ ID NO:5 or may be encoded by the coding sequence having SEQ ID NO:6.
  • a method for simultaneously inducing anti-influenza and other viral immune responses including coronavirus comprising co-expressing the immunogen of the present application with other sequences, and the co-expression manner may be: Fusion expression can also be expressed in a separate reading frame, thereby further increasing antiviral species, or improving the ability of immunogens to induce immune responses, and preventing multiple viral infections.
  • the sequences linked to the immunogens of the present application from different coronaviruses including, but not limited to, influenza virus, HIV, rabies virus, swine fever virus, PRRS virus, measles virus, Ebo Laviruses, herpesviruses, arboviruses (Zika virus, Japanese encephalitis virus, forest encephalitis virus, dengue virus, hantavirus, hemorrhagic fever virus), especially immunogens that respond to activated T cells Co-expression to prepare a composite vaccine capable of simultaneously activating neutralizing antibodies and T cell responses; immunomodulatory sequences, including but not limited to IL-2, IL-7, IL-12, IL-18, IL-21, GM-CSF , CD40L, CD40 stimulating antibodies, PD-1 and PD-L1 antibodies, CTLA4 antibodies, chemokines CXCL9, CXCL10, CXCL11, CXCL12, CXCL3, XCL1, CCL4, CCL20,
  • a method of simultaneously inducing an immune response against influenza and other viruses comprising combining an immunogen described herein, alone, or with a co-expression molecule described herein
  • nucleic acid vaccines DNA or RNA vaccines
  • recombinant protein subunit vaccines for the preparation of nucleic acid vaccines (DNA or RNA vaccines), recombinant protein subunit vaccines, recombinant viral vector vaccines, recombinant bacterial vector vaccines, virus-like particle vaccines, nanoparticle vaccines, cell vector vaccines, etc.
  • a method of simultaneously inducing an immune response against influenza and other viruses comprising administering an immunogenic peptide described herein, a nucleotide molecule encoding it, a vector , host cells or vaccines.
  • the vaccination is performed with a combination of two or more of the vaccines, either in combination or sequentially.
  • the vaccines herein are sequentially vaccinated with vaccines prepared from coronaviruses S or S1 from sources including but not limited to SARS-CoV-2, SARS-CoV, MERS- CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV, etc; From including but not limited to H1-H18.
  • FIG. 1 Construction of S protein eukaryotic expression vector and sRBD-HA2-hFn (RHAF) adenovirus and expression of fusion protein:
  • pcDNA3.1-S protein was successfully expressed in 293T cells, and adenovirus AdC68-RHAF could successfully express RHAF protein.
  • FIG. 1 Immunogenicity of AdC68-RHAF in C57BL/6 mice:
  • mice used in the experiment were 6-8 week old female C57BL/6, and the immunogens were plasmid pcDNA3.1-S and adenovirus AdC68-RHAF.
  • the abscissa is the immunization group, the ordinate is the titer of the binding antibody, ** means p ⁇ 0.01
  • mice The T cell responses of mice were detected by ELISPOT method at the second week after immunization.
  • the abscissa is the immune group, the ordinate is the number of cells secreting IFN- ⁇ per million splenocytes, * means p ⁇ 0.05.
  • mice used in the experiment were hACE2+ transgenic mice aged 6-8 weeks, and the immunogen was adenovirus AdC68-RHAF.
  • the abscissa is the immunization group, the ordinate is the titer of the binding antibody, and **** indicates p ⁇ 0.0001.
  • B 293T-ACE2 cells detected the neutralizing antibody titer in mouse serum at the second week after immunization.
  • the abscissa is the immunization group, the ordinate is the titer of neutralizing antibody (ID 50 ), and **** indicates p ⁇ 0.0001.
  • FIG. 4 Immunogenicity of AdC68-RHAF and AdC68-RBD-HA2-CD8TM in BALB/c mice:
  • mice used in the experiment were 6-8 week old female BALB/c mice, and the immunogens were adenovirus AdC-RHAF and AdC68-RBD-HA2-CD8TM.
  • the abscissa is the immunization group, and the ordinate is the titer of the binding antibody.
  • mice used in the experiment were hACE2+ transgenic mice aged 6-8 weeks, and the immunogen was adenovirus AdC68-RHAF.
  • A Viral load.
  • the horizontal axis is the immune group, and the vertical axis is the viral load.
  • A Changes in body weight of mice after H7N9 challenge.
  • the abscissa is the days after challenge, and the ordinate is the percentage of body weight.
  • mice after H7N9 challenge The abscissa is the days after challenge, the ordinate is the survival percentage, * means p ⁇ 0.05, ** means p ⁇ 0.01.
  • Figure 7 Construction of R545-HA2-IntN-PAB-7XHis eukaryotic expression vector and R545-HA2-IntN-PAB-7XHis protein expression:
  • Baculovirus can successfully express the protein R545-HA2-IntN-PAB-7XHis in the insect cell line Sf9.
  • R545-HA2-IntN-PAB-7XHis is connected to the Intein of gb1-IntC-Fn; as shown in the figure, the number of monomer gb1-IntC-Fn is significantly reduced, and the by-product IntN-PAB (17kDa) can be seen after cleavage ) and gb1-IntC (11 kDa), indicating the formation of nanoparticles R545HAF.
  • mice used in the experiment were hACE2+ transgenic mice aged 6-8 weeks, and the immunogens were plasmid pcDNA3.1-S and nanoparticle R545HAF.
  • the abscissa is the immunization group, the ordinate is the titer of the binding antibody, and * means p ⁇ 0.05.
  • C 293T-ACE2 cells detected the neutralizing antibody titer in mouse serum at the 4th week after immunization.
  • the abscissa is the immunization group, the ordinate is the titer of neutralizing antibody (ID 50 ), and * means p ⁇ 0.05.
  • D Neutralizing antibody titers against SARS-Cov-2 pseudovirus after the first and third doses of immunization.
  • the left picture shows the overall increase, and the right picture shows the corresponding increase of a single mouse. * means p ⁇ 0.05 .
  • the present disclosure relates to the field of vaccines, and in particular, to a method for simultaneously inducing immune responses against influenza and other viruses including coronavirus.
  • the results of animal experiments confirm that the vaccine of the present disclosure is safe, can continuously produce high-titer neutralizing antibodies, and can be used for the prevention and treatment of influenza and new crowns.
  • 0.1-2.5 mg/day includes 0.1 mg/day, 0.2 mg/day, 0.3 mg/day, etc. up to 2.5 mg/day.
  • the term "immunogenic peptide” refers to a peptide capable of simultaneously inducing an immune response against an influenza virus and another non-influenza virus, comprising the following moieties: (a) an immunogenic stem: which comprises the influenza virus hemagglutination The HA2 region of the prime HA; (b) the immunogenic corona: it comprises a viral membrane protein or an immunogenic fragment thereof, wherein the viral membrane protein is derived from a virus different from the source virus of the HA2 region in (a); (c) Optionally, other moieties linked to the aforementioned moieties.
  • attachment has its broad meaning and can include various means such as fusion, co-expression, covalent attachment, coupling, and the like.
  • sRBD-HA2 immunogenic peptide immunogenic peptide
  • immunogenic peptide against influenza virus and novel coronavirus SARS-CoV-2 are used interchangeably and refer to a peptide that includes influenza hemagglutinin HA.
  • the HA2 region is fused to the RBD region of the SARS-CoV-2 virus spike protein S, and has peptides that stimulate binding and neutralizing antibodies against influenza virus and novel coronavirus SARS-CoV-2, as well as corresponding T cell responses.
  • the immunogenic peptide can be: (a) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence set forth in SEQ ID NO: 3 or 5 or 21; (b) in (a) Homologous or sequence-similar polypeptides of the polypeptides with the same or similar immunogenicity, for example, each segment therein is the same as the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence shown in SEQ ID NO: 3 or 5 or 21, respectively with 80% or higher, 85% higher, 90% higher, 95% higher, 96% higher, 97% higher, 98% higher, greater than or equal to 99% homology or sequence identity, or greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95% with the polypeptide in (a) %, greater than or equal to 96%, greater than or equal to 97%, greater than or equal to 98%, greater than or equal to 99% homology or sequence identity; (c)
  • the immunogenic peptide can be: (a') a polypeptide having the amino acid sequence set forth in SEQ ID NO: 9 or 15 or 25; (b') as set forth in SEQ ID NO: 10 or 16 or 26 Polypeptides encoded by nucleotide sequences; polypeptides that are homologous or sequence-similar to the polypeptides described in (c') (a') and (b') and have the same or similar immunogenicity, such as those of (a') and (b) ') of the polypeptides have greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95%, greater than or equal to 96%, greater than or equal to 97%, greater than or equal to 98%, greater than or equal to 99% homology or sequence identity; (d') substitution, deletion or addition in the amino acid sequence defined in (a') or (b') or (c') A protein or polypeptide derived from (a') or (b
  • the immunogenic peptide may include other moieties attached to the immunogenic stem or crown, eg, to enhance the stability of the immunogenic stem or crown or fusion protein, increase neutralizing antibody responses, form polynucleotides Aggregates, increase cellular responses, etc.
  • other moieties may include, but are not limited to, viral or host derived proteins, transferrin (Fn), IL-2, IL-7, IL-12, IL-18, IL-21, GM- CSF, CD40L, CD40 stimulating antibody, PD-1 and PD-L1 antibody, CTLA4 antibody, chemokines CXCL9, CXCL10, CXCL11, CXCL12, CXCL3, XCL1, CCL4, CCL20, cholera toxin and its subunits, bacterial flagellin, FimH et al.
  • transferrin Fn
  • IL-2 transferrin
  • IL-7 transferrin
  • IL-12 IL-18
  • IL-21 GM- CSF
  • CD40L CD40 stimulating antibody
  • PD-1 and PD-L1 antibody CTLA4 antibody
  • chemokines CXCL9, CXCL10, CXCL11, CXCL12, CXCL3, XCL1, CCL4, CCL20, cholera to
  • a signal peptide element can refer to an amino acid sequence that functions to direct secretion, localization, and/or delivery of a protein, and is typically 5-30 amino acids in length.
  • the signal peptide element can be selected from the group consisting of: protein self signal peptide, CD33 protein signal peptide, CD8 protein signal peptide, CD16 protein signal peptide, mouse IgG1 antibody signal peptide, influenza HA protein signal peptide.
  • a linker peptide sequence (or a linker) can refer to a short peptide that serves to connect different elements in the fusion protein herein, and its length is usually 1-50 (eg, 5-50, 5-40, 10-40) amino acid.
  • the linker peptide does not affect or significantly affects the formation of the correct folding and spatial conformation of the peptides of the invention.
  • the linking peptide element may be selected from the group consisting of: (G4S) 3 linker, (G4S) n , GSAGSAAGSGEF, (G1y) 6 , EFPKPSTPPGSSGGAP, KESGSVSSEQLAQFRSLD, (Gly) 8 , EGKSSGSGSESKST.
  • Immunogenic peptides may also include variant forms thereof, such as one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid deletions, insertions and/or substitutions, and additions of one or more (usually within 20) C-terminal and/or N-terminal , preferably within 10, more preferably within 5) amino acids.
  • substitution with amino acids of similar or similar properties generally does not alter the function of a protein or polypeptide.
  • the addition of one or several amino acids to the C-terminus and/or the N-terminus generally does not alter the function of the protein or polypeptide.
  • Immunogenic peptides can be produced by recombinant expression under appropriate circumstances and conditions, such as from the encoding nucleotide molecules, vectors, host cells of the present disclosure; they can also be obtained by chemical synthesis, etc., as long as they have the desired amino acid sequence and immunogenicity and reactivity.
  • immunogenic peptide-encoding molecule As used herein, the terms “immunogenic peptide-encoding molecule,” “coding sequence,” and the like are used interchangeably and refer to nucleotide molecules that encode the immunogenic peptides described in the present disclosure.
  • the nucleotide molecule can be selected from, for example: (i) nucleosides having the nucleotide sequence set forth in SEQ ID NO:2 and the nucleotide sequence set forth in SEQ ID NO:4 or 6 or 22 Acid molecules; (ii) molecules that hybridize to (i) under stringent conditions; (iii) nucleotide molecules that are homologous or sequence-similar to (i) and capable of expressing functional immunogenic peptides, such as those in Each segment and the nucleotide sequence shown in SEQ ID NO: 2 and the nucleotide sequence shown in SEQ ID NO: 4 or 6 or 22 respectively have greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95%, greater than or equal to 96%, greater than or equal to 97%, greater than or equal to 98%, greater than or equal to 99% homology or sequence identity, or with (i ) or (ii) the sequence has a value greater than
  • the nucleotide molecule can be selected from, for example: (i) a nucleotide molecule having the sequence set forth in SEQ ID NO: 10 or 16 or 26; (ii) under stringent conditions with (i) ) Hybrid molecules; (iii) with sequences in (i) or (ii) having greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95%, greater than or equal to Nucleotide molecules equal to 96%, greater than or equal to 97%, greater than or equal to 98%, greater than or equal to 99% homology or sequence identity and capable of expressing functional immunogenic peptides; (iv) A nucleotide molecule capable of expressing a functional immunogenic peptide with substitution, deletion or addition of one or several nucleotides in the nucleotide sequence defined in (i) or (ii).
  • stringent conditions refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 x SSC, 0.1% SDS, 60°C; or (2) hybridization with added With a denaturant, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 50%, preferably 55% % or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, more preferably 95% or more, hybridization occurs.
  • a denaturant such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.
  • the full-length nucleotide sequence of the present disclosure or a fragment thereof can generally be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • primers can be designed according to the relevant nucleotide sequences disclosed in the present disclosure, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art is used as a template to amplify the Get the relevant sequence.
  • a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art is used as a template to amplify the Get the relevant sequence.
  • the present disclosure also relates to vectors comprising nucleotide molecules encoding the immunogenic peptides of the present application, as well as host cells genetically engineered with the vectors.
  • the coding sequences of the present disclosure can be used to express or produce recombinant immunogenic peptides by conventional recombinant DNA techniques (Science, 1984; 224: 1431). Generally there are the following steps:
  • vector and “recombinant expression vector” are used interchangeably and refer to bacterial plasmids, bacteriophages, yeast plasmids, animal cell viruses, mammalian cell viruses or other vectors well known in the art.
  • An important feature of expression vectors is that they typically contain an origin of replication, a promoter, marker genes and translational control elements.
  • Expression vectors containing immunogenic peptide coding sequences and appropriate transcriptional/translational control signals can be constructed using routine methods in the art. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • Expression vectors also include a ribosome binding site for translation initiation and a transcription terminator. Expression systems such as pcDNA3.1 vector, pIRES2-EGFP vector, AdMaxTM, AdC68, etc. can be employed in the present disclosure.
  • the expression vector may contain one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescence protein (GFP), or for tetracycline or ampicillin resistance in E. coli.
  • selectable marker genes such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescence protein (GFP), or for tetracycline or ampicillin resistance in E. coli.
  • Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, can be used to transform appropriate host cells so that they can express proteins or polypeptides.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as animal cells. Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; animal cells, etc.
  • host cells selected from, for example, HEK293, HeLa, CHO, K562, NSO, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE may be employed -19 and MRC-5 cells; High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5, and Ao38 .
  • Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on a promoter to enhance transcription of a gene. It will be clear to those of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.
  • recombinant polypeptide in the above method can be expressed intracellularly or on the cell membrane or secreted outside the cell.
  • recombinant proteins can be isolated and purified by various isolation methods utilizing their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic disruption, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • a vaccine or immune composition, comprising the immunogenic peptide, nucleotide molecule, vector, and/or host cell of the present disclosure.
  • the vaccine comprises a formulation of an immunogenic peptide and/or nucleic acid molecule of the present disclosure in a form that can be administered to a vertebrate, preferably a mammal, and which induces an immunity-enhancing protective immune response to prevent and/or alleviate influenza Viral infections and other non-influenza viral infections (eg, novel coronavirus infection) and/or at least one symptom thereof.
  • protective immune response refers to an immune response mediated by an immunogen against an infectious agent or disease, exhibited by a vertebrate (eg, human), to prevent or alleviate infection or to reduce at least one disease thereof symptom.
  • vertebrate or “subject” or “patient” refers to any member of the subphylum chordate, including but not limited to: humans and other primates, including non-human primates such as chimpanzees and other apes and monkeys species; domestic animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds including domesticated, wild and game birds such as chickens, fire Chickens and other quail birds, ducks, geese.
  • mammals such as cattle, sheep, pigs, goats and horses
  • domestic mammals such as dogs and cats
  • laboratory animals including rodents such as mice, rats and guinea pigs
  • birds including domesticated, wild and game birds such as chickens, fire Chickens and other quail birds, ducks, geese.
  • mamammal and “animal” are included in this definition and are intended to encompass adult, juvenile, and neonatal individuals.
  • the vaccines herein may be recombinant protein vaccines, recombinant DNA vaccines, recombinant viral vector vaccines (eg, adenovirus vectors, poxvirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, cytomegalovirus vectors), recombinant bacterial vector vaccines, recombinant yeast Vector vaccines or recombinant virus-like particle vaccines.
  • the vaccines herein are selected from recombinant DNA vaccines, recombinant adenoviral vectors, or a combination of one or both.
  • one or more vaccines or combinations thereof selected from the group consisting of recombinant plasmid vaccines (DNA), such as DNA vaccines comprising the RBD region of the SARS-CoV2 virus spike protein S coding sequence (e.g. pcDNA3.1-S); recombinant adenovirus vector vaccines, such as AdC68-RHAF; recombinant protein subunit vaccines, such as nanoparticles, R545HAF.
  • DNA recombinant plasmid vaccines
  • DNA vaccines comprising the RBD region of the SARS-CoV2 virus spike protein S coding sequence (e.g. pcDNA3.1-S)
  • recombinant adenovirus vector vaccines such as AdC68-RHAF
  • recombinant protein subunit vaccines such as nanoparticles, R545HAF.
  • an effective amount of the immunogens herein is included in the vaccine compositions herein.
  • the immunogen is included in the vaccine compositions of the present disclosure in an amount sufficient to achieve the desired biological effect.
  • the term "effective amount” generally refers to an amount of an immunogen that can induce a protective immune response sufficient to induce immunity to prevent and/or alleviate an infection or disease and/or to reduce at least one symptom of an infection or disease.
  • Adjuvants may also be included in the vaccines herein.
  • Adjuvants known to those of ordinary skill in the art may be employed, such as those described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients” (2nd Edition) (hereby incorporated by reference in its entirety).
  • adjuvants include, but are not limited to: aluminum adjuvant, cholera toxin and its subunits, oligodeoxynucleotides, manganese ion adjuvant, colloidal manganese adjuvant, Freund's adjuvant, MF59 adjuvant, QS-21 adjuvant Agents, Poly I:C and other TLR ligands, GM-CSF, IL-2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21, etc.
  • the vaccine compositions herein may also include pharmaceutically acceptable carriers, diluents, preservatives, solubilizers, emulsifiers and other adjuvants.
  • pharmaceutically acceptable carriers include, but are not limited to, water for injection, saline solution, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffers, and combinations thereof.
  • Pharmaceutically acceptable carriers, diluents and other excipients can be found, for example, in Remington's Pharmaceutcal Sciences.
  • the form of the vaccine compositions herein may be suitable for systemic or topical (particularly intra-respiratory) administration.
  • Methods of administering the vaccine composition include, but are not limited to, intramuscular, intradermal, subcutaneous, intranasal, aerosol inhalation, genital tract, rectal, oral, or any combination thereof, such as intramuscular injection followed by intranasal instillation.
  • the vaccines herein prevent, eliminate or reduce influenza virus and novel coronavirus infection or at least one symptom thereof, such as respiratory symptoms (eg, nasal congestion, sore throat, hoarseness), headache, cough, phlegm, in a subject , fever, rales, wheezing, dyspnea, pneumonia caused by infection, severe acute respiratory syndrome, renal failure, etc.
  • respiratory symptoms eg, nasal congestion, sore throat, hoarseness
  • headache e.g, cough, phlegm
  • fever, rales, wheezing e.g., wheezing, dyspnea, pneumonia caused by infection, severe acute respiratory syndrome, renal failure, etc.
  • an immunoconjugate also referred to as an immunoconjugate
  • the other substances can be targeting substances (eg, moieties that specifically recognize a specific target), therapeutic substances (eg, drugs, toxins, cytotoxic agents), labeling substances (eg, fluorescent labels, radioisotope labels).
  • a combination product comprising an immunogenic peptide, nucleotide molecule, vector, host cell and/or vaccine of the present disclosure, and may further comprise one or more of the Other substances that play the function of preventing and/or treating influenza virus infection and other non-influenza virus infections (such as novel coronavirus infection) or their symptoms or enhance the stability of the aforementioned substances.
  • other substances may include other vaccines against coronavirus S or S1, such as from sources including but not limited to SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV - S or S1 vaccines of HKU1, bat-CoV; other vaccines against influenza virus HA or HA2 derived from including but not limited to H1-H18; benefit from T cell activation and/or interaction with T cells Other active substances of a disease or disorder of memory immune response.
  • Also provided herein is a method for preventing and/or treating influenza virus infection and other non-influenza virus infections (eg, novel coronavirus infection) and/or symptoms thereof, comprising: administering at least once a prophylactically and/or therapeutically effective amount one or more vaccines of the present disclosure.
  • the available vaccination methods include, but are not limited to: systemic immunization methods, such as intramuscular injection, subcutaneous injection, and intradermal injection, etc.; intra-respiratory immunization methods, such as atomization, intranasal injection, etc.
  • the primary immunization is systemic or intra-respiratory, preferably systemic.
  • the interval between each inoculation is at least 1 week, eg, 2 weeks, 4 weeks, 2 months, 3 months, 6 months or more.
  • the DNA vaccine is used for the primary immunization and the recombinant viral vaccine is used for one or more booster immunizations.
  • the immunization method of the present disclosure may adopt a "prime-boost” or “prime-boost-re-boost” approach, a single systemic system immunization or a respiratory local immunization approach, or a combination of the two immunization approaches.
  • recombinant DNA vaccines are used for systemic priming to establish a systemic immune response, and then other vaccines (such as recombinant adenovirus vaccines or recombinant poxvirus vaccines) are used for one more time or multiple boosts, which may include at least one intra-respiratory boost (eg, with an adenovirus vaccine).
  • the vaccine-specific immune response can be effectively established in the local and systemic systems of the respiratory tract, which helps to enhance the effectiveness of vaccine protection.
  • Combinations herein may be provided in the form of a pharmaceutical pack or kit, for example, one or more vaccine compositions herein or one or more components thereof may be packaged in one or more containers, such as in the indicated combination. amount of substance in a sealed container such as an ampoule or sachet.
  • Vaccine compositions can be provided in the form of liquids, sterile lyophilized powders, or anhydrous concentrates, which can be diluted, reconstituted and/or formulated with appropriate liquids (eg, water, saline, etc.) immediately prior to use for administration appropriate concentration and form to the subject.
  • the combination product of the present disclosure can be used to locally induce high levels of antigen-specific CD8+ T cell responses in the respiratory tract, making it useful in preventing respiratory pathogen infection, reducing the pathogenicity of respiratory pathogens, and preventing and treating respiratory tumors. prospect.
  • mice The left and right hind limbs of mice were injected intramuscularly; or intranasally.
  • the specific dosage is shown in the examples.
  • the HA2 sequence is from Genebank: AGI60292.1, the S and RBD sequences are both from Genebank: NC_045512.2; the hFn sequence (human transferrin) is from Genebank: M97164.1, and the specific sequence is shown in the sequence table.
  • Recombinant plasmid vaccine (DNA): pcDNA3.1-S, pcDNA3.1 (empty);
  • Recombinant adenovirus vector vaccines AdC68, AdC68-sRBD-HA2-hFn (AdC68-RHAF), AdC68-RBD-HA2-CD8TM.
  • Recombinant protein subunit vaccine (nanoparticles, protein): R545HAF
  • immunogen immunization doses employed in the examples are as follows:
  • Recombinant plasmid vaccine 100 ⁇ g/mouse, 100 ⁇ L, dissolved in sterile saline;
  • Recombinant adenovirus vector vaccine 5E10vp/mouse, 100 ⁇ L (intramuscular injection); 5E10vp/mouse, 40 ⁇ L (nasal).
  • Recombinant protein subunit vaccine protein (dissolved in sterile PBS) and aluminum adjuvant (Aluminium, InvivoGen, item number 5200) are immunized after mixing in a volume ratio of 1 : 1, 20 ⁇ g/small Mouse, 100 ⁇ L;
  • the gene recovery product was ligated with the enzyme-cut linearized vector using T4 DNA ligase (Thermo Scientific Company, Item No. 2011A): The ligated product was transformed into E. coli Stable, and grown on a culture plate containing ampicillin overnight . On the second day, a single colony was randomly selected for sequencing, and the mutation sites were corrected. After verifying that the entire sequence was correct, a lentiviral expression plasmid (pHAGE-hACE2-puro) of the hACE2 gene was successfully cloned.
  • mice 2/4 weeks after the last immunization, before the mice were sacrificed by decapitation, the peripheral whole blood of the mice was collected by encapsulating the eyeballs, collected in a 1.5 mL EP tube, and allowed to stand at room temperature for natural coagulation.
  • the mouse serum was centrifuged at 7000g for 15min. Transfer mouse serum to a new 1.5 mL EP tube. Before the experiment, the sample needs to be inactivated at 56°C for 30min to destroy the complement activity in the serum. Briefly centrifuge before inactivation to avoid residual sample on the tube walls and caps. The level of the water bath should be below the sample level, but not over the bottle cap.
  • ELISA sample diluent (0.5% nonfat dry milk, dissolved in PBST), starting from 1 : 100, and perform 2-fold dilution. Unimmunized mouse serum was used as a negative control. Set up blank wells, only add sample diluent, make 2 duplicate wells for each sample, the final volume of each well is 100 ⁇ L, and incubate at room temperature for 3h;
  • the stimulating peptide library was synthesized by Suzhou Qiangyao Biotechnology Co., Ltd., each single peptide was 15 amino acids, covering the RBD sequence, and there was one peptide library for every five single peptides, that is, a total of 13 peptide libraries.
  • the first peptide is MPTESIVRFPNITNL (SEQ ID NO: 19)
  • the second peptide is aa 8-22 in the RBD peptide sequence of SEQ ID NO: 3
  • each subsequent peptide is shifted by four amino acids compared to other peptides position, that is, the 3rd peptide is aa 12-26 in the RBD peptide sequence of SEQ ID NO: 3
  • the 4th peptide is aa 16-30 in the RBD peptide sequence of SEQ ID NO: 3... and so on.
  • Peptide 64 is aa 256-270 in the RBD peptide sequence of SEQ ID NO:3, and peptide 65 is AVRDPQTLEILDITP (SEQ ID NO:20).
  • Example 1 Construction of S protein eukaryotic expression vector and RHAF and RBD-HA2-CD8TM adenovirus expression vector and protein expression
  • the gene recovery product was ligated with the enzyme-cut linearized vector using T4 DNA ligase method (Thermo Scientific Company, Cat. No. 2011A): the ligated product was transformed into E. coli Stable, and grown overnight on a culture plate containing ampicillin . On the second day, a single colony was randomly selected for sequencing, and the mutation sites were corrected. After verifying that all sequences were correct, the eukaryotic expression vector pcDNA3.1-S of the S protein was successfully cloned. The plasmid construction map is shown in Figure 1A.
  • adenovirus expression vector of RHAF ie, sRBD-HA2-hFn fusion protein
  • AdC68-RHAF adenovirus AdC68-RHAF
  • RHAF gene the specific sequence is shown in SEQ ID NO: 10, wherein the HA2 part is based on the sequence shown in SEQ ID NO: 2, the sRBD part is based on the sequence shown in SEQ ID NO: 6, and the hFn part is based on the sequence shown in SEQ ID NO: 6. the sequence shown in SEQ ID NO: 8).
  • sRBD-HA2 is expressed at the N-terminus of hFn, and the two are connected by a G 4 S linker.
  • the gene recovery product was ligated with the enzyme-cut linearized vector using T4 DNA ligase method (Thermo Scientific Company, Cat. No. 2011A): the ligated product was transformed into E. coli Stable, and grown overnight on a culture plate containing ampicillin . On the second day, a single colony was randomly selected for sequencing, and the mutation sites were corrected. After verifying that the entire sequence was correct, the RHAF adenovirus expression vector pAdC68XY3-RHAF was successfully cloned. The plasmid construction map is shown in Figure 1A.
  • the constructed recombinant plasmids pAdC68XY3-RHAF and pAdC68XY3-RBD-HA2-CD8TM were linearized by restriction endonuclease Pac 1 in a water bath at 37 °C for 3.5 h, and the endonuclease was inactivated at 65 °C.
  • the samples were collected at the time of infection, and the cell supernatant was collected together. After repeated freezing and thawing at -80°C for three times, a small amount of infected 293A cells was taken, and the expression of the target protein was verified by Western blot.
  • the adenovirus samples collected above were infected with 293A cells in 1 T175 cell culture flask, collected after 24 hours, placed at -80°C for three times, and then infected with 293A cells in 6 T175 cell culture flasks, and so on. A large amount of amplification was carried out, and when 36 T175 cell culture flasks were expanded, the cell pellet was collected, the supernatant was discarded, and resuspended in about 10 mL of serum-free and non-resistant DMED medium. Adenoviruses were purified by cesium density gradient centrifugation, and stored at -80°C after aliquoting.
  • Nanogrop 2000 was used to measure the OD 260 value of each tube of eluent, and the eluents with an OD 260 greater than 2 were combined, added with 10% sterile glycerol, and packaged;
  • Nanogrop 2000 was used to measure the OD 260 value of the subpackaged adenovirus solution, and the OD 260 value ⁇ 1.1 ⁇ 10 12 /mL was the titer of the purified adenovirus;
  • the final AdC68-RHAF titer was 1.76 ⁇ 10 13 /mL, and the AdC68-RBD-HA2-CD8TM titer was 1.48 ⁇ 10 13 /mL.
  • C57BL/6 mice were immunized with DNA-pcDNA3.1-S and adenovirus AdC68-RHAF, respectively. After 2 weeks of immunization, the immunization combination induced the binding antibody titers against RBD protein and H7 protein and the titers against SARS-CoV- 2 Neutralizing antibody titers of pseudoviruses. The level of T cell response to the RBD peptide pool was also assessed.
  • mice were randomly divided into two groups, named as control group and AdC68-RHAF group according to the immunogen.
  • the specific immunization combination is shown in Table 1, and the immunization method is intramuscular injection.
  • AdC68-RHAF group produced binding antibody titers against RBD protein and H7 protein as shown in Figure 2A: the binding antibody titers against RBD protein were all above 10,000, and the highest could reach 50,000; the average binding antibody titer against H7 was in Around 6,000, the highest can reach 10,000. At the same time, comparing the data of the binding antibody against the RBD protein before and after the second immunization, it can be found that the titer of the binding antibody has been significantly improved (Figure 2B).
  • AdC68-RHAF group produced neutralizing antibody titers against SARS-Cov-2 pseudovirus as shown in Figure 2C: most of the titers were above 1000, with an average value of 1360, and one mouse had a neutralizing antibody titer as high as 12808. There are individual differences among mice. At the same time, comparing the data of the neutralizing antibody titers against the SARS-Cov-2 pseudovirus before and after the second immunization, it can be found that the neutralizing antibody titer has been significantly improved (Figure 2D).
  • the AdC68-RHAF group generated T cell responses against the RBD peptide pool as shown in Figure 2E: the number of IFN- ⁇ secreting cells per million splenocytes was around 3,000.
  • AdC68-RHAF could simultaneously induce antibodies against influenza and 2019-nCoV, and could induce neutralizing antibodies against 2019-nCoV and T cell responses against 2019-nCoV.
  • Example 2 in order to be able to use the new coronavirus to verify the challenge protection of mice, we verified the immunogenicity of AdC68-RHAF in hACE2+ICR mice. After 2 weeks of immunization, we evaluated the The immunization combination induced binding antibody titers against RBD protein, H3 protein and H7 protein and neutralizing antibody titers against SARS-CoV-2 pseudovirus.
  • the hACE2+ICR mouse used in this example is a preclinical model of COVID-19, which is a SARS-CoV-2-susceptible human angiotensin-converting enzyme 2 (hACE2) transgenic mouse constructed under the background of ICR mice. hACE2 is expressed in its lung, heart, kidney and small intestine. This mouse model is an important tool for the development of SARS-CoV-2 therapeutics and vaccines.
  • hACE2 SARS-CoV-2-susceptible human angiotensin-converting enzyme 2
  • mice were randomly divided into two groups, which were named as the control group and the AdC68-RHAF group according to the immunogen.
  • the specific immunization combination is shown in Table 2, and the immunization method is intramuscular injection.
  • AdC68-RHAF combination produces binding antibody titers against RBD protein, H3 protein and H7 protein as shown in Figure 3A: most of the binding antibody titers against RBD protein are above 10,000, and some can reach 100,000; Most of the titers are above 100, and some can reach 1000; most of the binding antibody titers against H7 protein are around 6,400.
  • the AdC68-RHAF group produced neutralizing antibody titers against SARS-CoV-2 pseudovirus as shown in Figure 3B: most of them were around 1000, and the highest could reach 2253.
  • AdC68-RHAF can simultaneously induce binding antibodies against 2019-nCoV and influenza in hACE2+ICR mice, and can induce binding antibodies against influenza H3 and H7 at the same time, verifying the broad-spectrum of HA2 antibodies.
  • AdC68-RHAF can effectively induce neutralizing antibodies against the new crown, and has a good prospect for development as an anti-influenza and new crown vaccine.
  • mice were immunized with adenovirus AdC68-RHAF and AdC68-RBD-HA2-CD8TM respectively. After 2 weeks of immunization, the immunization combination induced the binding antibody titers against RBD protein and H7 protein and the titers against SARS-CoV- 2 Neutralizing antibody titers of pseudoviruses. The level of T cell response to the RBD peptide pool was also assessed.
  • mice were randomly divided into 3 groups, which were named AdC68 group, AdC68-RHAF group and AdC68-RBD-HA2-CD8TM group according to the immunogen.
  • AdC68 group AdC68-RHAF group
  • AdC68-RBD-HA2-CD8TM group AdC68-RBD-HA2-CD8TM group according to the immunogen.
  • the specific immunization combination is shown in Table 3, and the immunization method is intramuscular injection.
  • Neutralizing antibody titers against SARS-CoV-2 pseudovirus were detected one week after immunization, as shown in Figure 4B: the average neutralizing antibody titer in the AdC68-RHAF group was 128, and the highest was 274; AdC68-RBD-HA2 The average titer of neutralizing antibody in the -CD8TM group was 97, and the highest was 348. At this time, there was no significant difference between the AdC68-RHAF group and AdC68-RBD-HA2-CD8TM.
  • AdC68-RHAF and AdC68-RBD-HA2-CD8TM can effectively induce binding antibodies against influenza virus needle H7; can effectively induce neutralizing antibodies against SARS-CoV-2 pseudovirus;
  • T cell responses against the RBD peptide pool can be induced.
  • Example 5 Immunogenicity of AdC68-RHAF in BALB/c mice by different vaccination methods
  • mice were immunized with DNA-pcDNA3.1-S and adenovirus AdC68-RHAF respectively. After 2 weeks of immunization, the titers of binding antibodies induced by the immunization combination against RBD protein and H7 protein and against SARS-CoV- 2 Neutralizing antibody titers of pseudoviruses. The level of T cell response to the RBD peptide pool was also assessed.
  • mice were randomly divided into 4 groups, which were named AdC68 muscle group, AdC68 intranasal group, AdC68-RHAF intramuscular group and AdC68-RHAF intranasal group according to the immunogen and immunization method.
  • AdC68 muscle group AdC68 muscle group
  • AdC68 intranasal group AdC68-RHAF intramuscular group
  • AdC68-RHAF intranasal group The mice were randomly divided into 4 groups, which were named AdC68 muscle group, AdC68 intranasal group, AdC68-RHAF intramuscular group and AdC68-RHAF intranasal group according to the immunogen and immunization method.
  • the specific immune combinations are shown in Table 4.
  • mice Southern Model Organism ACE2-transgenic mice (C57BL/6-Tgtn(CAG-human ACE2-IRES-Luciferase-WPRE-polyA)Smoc), female.
  • Immunization procedure Mice were randomly divided into 2 groups, named as control group and AdC68-RHAF group according to the immunogen.
  • the specific immunization combination is shown in Table 5, and the immunization mode is intramuscular injection.
  • mice were challenged, observed to 5 days after challenge, and body weight changes were recorded; 3 mice in each group were sacrificed on the third day after challenge, and lung tissue was collected , the left half of the lung tissue (one lobe) of each mouse was fixed with 4% paraformaldehyde for 48 hours and then pathological sections (H&E staining and histochemistry) were made; the right half of the lung tissue (4 lobules) of each mouse Grind to measure viral load, qPCR RNAcopies or viral titer PFU/ml; on the fifth day, if there are still mice alive, they will all be sacrificed after weighing.
  • mice hACE2+ICR mice and BALB/c mice, female.
  • H7N9 (A/Shanghai/4664T/2013), H3N2 (A/Hong Kong/8/68).
  • the challenge test was carried out in the P2 and P3 laboratories of the Shanghai Public Health Clinical Center.
  • Immunization procedure Mice were randomly divided into 2 groups, named as control group and AdC68-RHAF group according to the immunogen.
  • the specific immunization combination is shown in Table 6, and the immunization method is intramuscular injection.
  • Example 8 Construction of R545-HA2-IntN-PAB-7XHis eukaryotic expression vector and R545-HA2-IntN-PAB-7XHis protein expression
  • R545-HA2-IntN In order to express the R545-HA2-IntN protein, we constructed a donor plasmid of R545-HA2-IntN (R545HA-IntN), and obtained a recombinant baculovirus that can efficiently express R545-HA2-IntN in the insect cell line Sf9 by transposition , and the mature protein was eukaryotically expressed in the insect cell line Sf9.
  • the P1 generation vAc R545HA-IntN-PAB-7XHis was infected with Sf9 in suspension culture in 600 mL SF-SFM medium (Womei Bio, S F10111-2) at a volume ratio of 1:100
  • the cell line, the cell density was 2 ⁇ 10 6 /mL, and the cell culture was harvested after 5 days, and the protein expression and distribution of R545HA-IntN-PAB-7XHis were detected ( FIG. 7B ).
  • the results showed that the fusion proteins were mostly expressed in cells, so inclusion body purification was used for subsequent purification.
  • VI.PBS pH 7.4, 2mM dTT, 5% glycerol for 4h.
  • the purified protein was stained with WB and Coomassie brilliant blue to see a band of the correct size (about 70 kDa) (Fig. 8A).
  • Intein is a self-cleaving peptide. As previously described, the C-terminus of R545-HA2 was fused to the N-terminus of the intein to form the recombinant protein R545HA-IntN-PAB-7XHis.
  • the N-terminus of ferritin (Fn) is fused with the C-terminus of the linker gbl-intein (gb1 is the B domin of Protein G as a solubilizing tag) to form a recombinant protein gbl-intC-ferrtin, which self-assembles into a 24-mer Nanoparticles; the N-terminus of the intein specifically recognizes and excises the ferritin exposed on the surface of the nanoparticles; the exogenous protein is covalently cross-linked with ferritin to obtain R545-HA2-hFn, which is also a 24-mer Nanoparticle state.
  • BCA Thermo Fisher, product number: 23235
  • R545HA-IntN-PAB-7XHis purified in Example 9
  • R545HA-IntN-PAB can form a ligation product R545HAF (70kDa band in the left second lane of Figure 8B) by connecting Fn to form a ligation product of about 70kDa (67kDa before glycosylation modification), and cleavage to generate by-products IntN-PAB (17kDa) and gb1 -IntC (11 kDa).
  • the hACE2+ICR mouse used in this example is a preclinical model of COVID-19, which is a SARS-CoV-2-susceptible human angiotensin-converting enzyme 2 (hACE2) transgenic mouse constructed under the background of ICR mice. hACE2 is expressed in its lung, heart, kidney and small intestine. This mouse model is an important tool for the development of SARS-CoV-2 therapeutics and vaccines.
  • hACE2 SARS-CoV-2-susceptible human angiotensin-converting enzyme 2
  • mice were randomly divided into 2 groups, which were named as the control group and the nanoparticle group according to the immunogen.
  • the specific immunization combination is shown in Table 7, and the immunization method is intramuscular injection.
  • the nanoparticle group that is, R545HAF produced binding antibody titers against RBD protein as shown in Figure 9A.
  • most of the binding antibody titers against RBD protein were above 400,000, and some could reach 3,000,000;
  • the data of the binding antibody against the RBD protein after the immunization and the third immunization showed that the titer of the binding antibody was significantly improved (Fig. 9B).
  • R545HAF was also shown to be effective in inducing binding antibodies against influenza virus H7.
  • nanoparticle vaccine R545HAF can simultaneously induce binding antibodies against SARS-CoV-2 and influenza in hACE2+ICR mice.
  • the nanoparticle vaccine R545HAF can effectively induce neutralizing antibodies against the new crown, and has a good prospect of being developed as an anti-influenza and new crown vaccine.

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Abstract

提供同时诱导抗多种病毒(例如流感病毒以及新冠病毒)的免疫应答的方法。提供了一种免疫原性肽，其包含(a)免疫原茎部：其包含流感病毒血凝素HA的HA2区域；(b)免疫原冠部：其包含病毒膜蛋白或其免疫原性片段，其中所述病毒膜蛋白的来源病毒不同于(a)中HA2区域的来源病毒；以及(c)可任选地，与前述部分融合的其他部分。

Description

同时诱导抗多种病毒的免疫应答的方法 技术领域
本发明涉及疫苗领域，特别涉及一种同时诱导抗包括流感在内的多种病毒(例如冠状病毒，尤其是新冠病毒)的免疫应答的方法。
背景技术
流行性感冒简称流感，是一种由流感病毒引发的急性呼吸道传染性疾病，主要分为季节性和大流行性(seasonal and pandemic)流感。16世纪以来多次在全世界不同国家和地区爆发的重大流感疫情，已经成为公共卫生领域面临的严峻挑战之一。流感所引起的发病率与死亡率高，每年导致2,000,000到5,000,000的重症病例患者和250,000到500,000的死亡病例。据估计，全球流感感染患者中，成人占5-10％，儿童占20-30％。流感不仅造成了严重的疾病负担，还会造成巨大的直接与间接经济负担。
流感病毒基因组分为8个节段，分别编码血凝素(HA)、神经氨酸酶(NA)、基质蛋白1和2(M1和M2)、非结构蛋白1和2(NS1和NS2)、核衣壳蛋白(NP)以及三个聚合酶复合体(PB1、PB2和PA)等10种蛋白质。根据核蛋白、M1和M2的抗原性差异，可将流感病毒分为甲、乙、丙三型，例如禽流感病毒(AIV)为甲型流感病毒(甲流病毒)。根据HA和NA的抗原性不同，甲型流感可以分为18个H亚型和9个N亚型。这18个H亚型可以分为group I类(H1、H2、H5、H6、H8、H9、H11、H12、H13、H16、H17、H18)和group II类(H3、H4、H7、H10、H14、H15)。在这些亚型中已知可直接感染人的亚型为H1、H2、H3、H5、H7、H9和H10，引发高致病性禽流感的病毒均为H5和H7亚型。
大多数针对流感病毒产生的保护性抗体都以HA蛋白为靶点，HA蛋白是一种由HA1和HA2结构域组成的三聚体表面糖蛋白。大多数HA1链形成球状的冠部，它包含受体结合部位，而HA2则形成茎部，支撑HA1且形成三聚体。虽然，HA冠部(HA1)在诱导中和抗体的产生时具有显著的免疫优势，但其自身极易发生变异导致病毒产生抗原飘移及至抗原变异，形成新的流感病毒亚型。HA2区域相对于HA1更为保守，目前分离出的针对H7N9HA2的中和抗体中3GBN对于甲型流感有广谱性保护，3SDY对于甲型流感group II有广谱性保护，而4FQV则对甲型及乙型流感有广谱性保护。因此，H7N9的HA2作为诱导广谱性的流感中和抗体至关重要。
截至2020年9月24日，在全球共报告了31,664,104例确诊的新冠病毒性肺炎病例，包括972,221例死亡病例，且新冠病毒型肺炎的全球大流行依旧在持续中。目前，虽 然有部分疫苗已经进入临床试验阶段，但疫苗的上市、生产和使用仍然是迫在眉睫的巨大挑战。
冠状病毒包含四种结构蛋白，包括突刺蛋白(S蛋白)、包膜蛋白、膜蛋白和核衣壳蛋白。其中，S蛋白在病毒的附着、融合和进入过程中起着最重要的作用，也是抗体、进入抑制剂和疫苗的主要靶点。S蛋白介导病毒进入宿主细胞，首先通过S1亚基的受体结合域(RBD)与宿主受体结合，然后通过S2亚基融合病毒和宿主细胞膜。据WHO报道：冠状病毒疫苗免疫后的动物，再次暴露于活病毒时，可能发生更严重的症状。疫苗免疫产生的非中和抗体或较低的抗体水平可能会引起抗体依赖性增强效应(antibody-dependent enhancement，ADE)，增强病毒致病性。因此，为了减少ADE副作用，新冠病毒S蛋白中的RBD区将是最为有效的疫苗研发靶点。
秋冬季是呼吸道疾病的高发季节，其中就包括了流感病毒感染，如何防范流感与新冠等呼吸道传染病叠加流行的风险成为重中之重。在感染性疾病的预防中，疫苗自问世以来就是最为有效和经济的措施，而目前常用的预防疫苗大部分都是以活化中和抗体为目标。因此，设计一种免疫原，使其能够同时活化针对多种流感病毒或同时抗流感病毒及其他病毒(诸如新冠病毒)的中和抗体是我们迫切需要探索和开发的方向。
发明内容
本公开中提供了能够同时诱导针对包括流感病毒在内的多种病毒的免疫应答的免疫原性肽、其编码核苷酸分子、载体、宿主细胞、疫苗及其应用。
在本公开的一个方面中，提供了一种免疫原性肽，其包含如下部分：
(a)免疫原茎部：其包含流感病毒血凝素HA的HA2区域；
(b)免疫原冠部：其包含病毒膜蛋白或其免疫原性片段，其中所述病毒膜蛋白的来源病毒不同于(a)中HA2区域的来源病毒；
( c)可任选地，与前述部分连接的其他部分。
在一些实施方式中，所述HA2区域的来源选自下组：H1～H18中任一种，尤其是来自于广泛流行的人流感H1、H2、H3以及多次出现的人感染禽流感H5与H7，例如源自2009年流行的H1(H1N1)、2013年流行的H7(H7N9)。
在一些实施方式中，所述HA2区域的氨基酸序列如SEQ ID NO：1所示，或由具有SEQ ID NO：2所示序列的核苷酸分子编码。
在一些实施方式中，所述病毒膜蛋白是免疫原性膜蛋白。
在一些实施方式中，所述免疫原冠部的来源选自：来源不同于(a)中HA2区域的流感病毒；非流感病毒的其他病毒。
在一些实施方式中，所述免疫原冠部的来源选自下组：冠状病毒(如新冠病毒)、艾滋病毒、流感病毒(例如流感病毒血凝素HA的HA1区域)、狂犬病毒、猪瘟病毒、蓝耳病病毒、麻疹病毒、埃博拉病毒、疱疹病毒、虫媒病毒(例如，寨卡病毒、流行性乙型脑炎病毒、森林脑炎病毒、登革病毒、汉坦病毒、出血热病毒)。
在一些实施方式中，所述免疫原冠部的来源选自：冠状病毒SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV，例如包含冠状病毒的受体结合域的S1蛋白、或受体结合域(RBD)、或经改造的受体结合域(例如RBD区经末段半胱氨酸修饰形成sRBD区)或其免疫原性片段。
在一些实施方式中，所述免疫原冠部选自冠状病毒SARS-CoV-2的S1，尤其是来自于S1的RBD区(例如其序列如SEQ ID NO：3所示或由SEQ ID NO：4的核苷酸分子编码)或其修饰的RBD区(例如经末端Cys修饰改造的sRBD区，如SEQ ID NO：5所示肽段或由SEQ ID NO：6所示核苷酸分子编码)、或者RBD区的免疫原性片段(例如如SEQ ID NO：21所示肽段或由SEQ ID NO：22所示核苷酸分子编码)。
在一些实施方式中，所述其他部分选自：免疫调节序列，例如IL-2、IL-7、IL-12、IL-18、IL-21、GM-CSF、CD40L、CD40刺激抗体、PD-1与PD-L1抗体、CTLA4抗体、趋化因子CXCL9、CXCL10、CXCL11、CXCL12、CXCL3、XCL1、CCL4、CCL20、霍乱毒素及其亚单位、细菌鞭毛蛋白、FimH、HIV p24、HIV gp41。
在一些实施方式中，所述其他部分选自：使免疫原性肽能够形成纳米颗粒的部分，例如转铁蛋白(Fn，例如SEQ ID NO：7所示的肽分子或由SEQ ID NO：8所示核苷酸分子编码)。
在一些实施方式中，所述其他部分选自：信号肽，例如CD33、CD8、CD16、小鼠IgG1抗体。
在一些实施方式中，所述其他部分选自：使免疫原性肽能够表达在病毒载体表面的跨膜区，例如CD8跨膜区(CD8TM，例如SEQ ID NO：17所示的肽分子或由SEQ ID NO：18所示核苷酸分子编码)、HA2跨膜区、CD4跨膜区、gp41跨膜区。
在一些实施方式中，所述其他部分选自：连接肽，例如(G4S) 3、(G4S) n、GSAGSAAGSGEF、(Gly) 6、EFPKPSTPPGSSGGAP、KESGSVSSEQLAQFRSLD、(Gly) 8、EGKSSGSGSESKST。
在一些实施方式中，所述其他部分选自：标签，例如His-tag、AviTag、Calmodulin tag、polyglutamate tag、E-tag、FLAG tag、HA-tag、Myc-tag、S-tag、SBP-tag、Sof-tag 1、Sof-tag3、Strep-tag、TC tag、V5 tag、T7 tag、VSV tag、Xpress tag、3X FLAG tag、Isopep tag、Spytag、Snoop tag和PNE tag。
在一些实施方式中，所述免疫原性肽包含：与HA2区域连接的RBD区或sRBD区或其免疫原性片段，以及可任选的与前述部分连接的其他部分。
在一些实施方式中，所述免疫原性肽包含：与HA2区域连接的RBD区或sRBD区(例如SEQ ID NO：5所示的肽分子或由SEQ ID NO：6所示核苷酸分子编码)以及Fn区(例如SEQ ID NO：7所示的肽分子或由SEQ ID NO：8所示核苷酸分子编码)；与HA2区域连接的RBD区或sRBD区以及CD8跨膜区(例如SEQ ID NO：17所示的肽分子或由SEQ ID NO：18所示核苷酸分子编码)；与HA2区域连接的RBD区免疫原性片段(例如SEQ ID NO：21所示的肽分子或由SEQ ID NO：22所示核苷酸分子编码)以及Fn区。
在一些实施方式中，所述免疫原性肽的氨基酸序列如SEQ ID NO：9或15或25所示，或者所述免疫原性肽的编码序列如SEQ ID NO：10或16或26所示。
在一些实施方式中，本申请采用H7N9病毒的HA2区连接新冠病毒的RBD区或其免疫原性片段作为免疫原，同时对免疫原进行进一步修饰，例如RBD区加入二硫键、融合蛋白、融合细胞因子，进而同时诱导抗流感病毒和新冠病毒的中和抗体。
在一些实施方式中，本申请提供了一种同时针对流感及新冠的免疫原性肽，其包括H7N9病毒血凝素HA的HA2区及SARS-CoV-2病毒刺突蛋白S的RBD区，所述RBD区可进一步经半胱氨酸修饰形成sRBD区。
在一些实施方式中，所述经半胱氨酸修饰为在RBD结构域的根部增加一对半胱氨酸，使之能够形成二硫键。
在本公开的一个方面中，提供了一种核苷酸分子，其编码本文的免疫原性肽。
在一些实施方式中，所述HA2区域的编码序列包含SEQ ID NO：2所示的核苷酸序列；所述免疫原冠部的编码序列包含SEQ ID NO：4所示核苷酸序列或SEQ ID NO：6所示的核苷酸序列或SEQ ID NO：22所述的核苷酸序列；和/或所述其他部分的编码序列包含SEQ ID NO：8所示或SEQ ID NO：18所示的核苷酸序列。
在一些实施方式中，所述核苷酸分子的序列如SEQ ID NO：10或16或26所示。
在本公开的一个方面中，提供了一种载体，其包含本文的核苷酸分子。
在本公开的一个方面中，提供了一种宿主细胞，其包含本文核苷酸分子或载体或能表达本文所述的免疫原性肽。
在一些实施方式中，所述宿主细胞为哺乳动物细胞或昆虫细胞，如HEK293、HeLa、K562、CHO、NS0、SP2/0、PER.C6、Vero、RD、BHK、HT1080、A549、Cos-7、ARPE-19和MRC-5细胞；High Five、Sf9、Se301、SeIZD2109、SeUCR1、Sf9、Sf900+、Sf21、BTI-TN-5B1-4、MG-1、Tn368、HzAm1、BM-N、Ha2302、Hz2E5以及Ao38。
在本公开的一个方面中，提供了一种能同时诱导针对流感病毒和另一非流感病 毒或非同种流感病毒的免疫应答的疫苗，其包含本文所述的免疫原性肽、核苷酸分子、载体和/或宿主细胞。
在一些实施方式中，所述疫苗为核酸疫苗(DNA或RNA疫苗)、重组蛋白亚单位疫苗、重组病毒载体疫苗、重组细菌载体疫苗、病毒样颗粒疫苗、纳米颗粒疫苗、细胞载体疫苗。
在一些实施方式中，所述疫苗为病毒载体疫苗，所述病毒载体选自：痘病毒(例如天坛株、北美疫苗株、惠氏衍生株、李斯特株、安卡拉衍生株、哥本哈根株和纽约株)、腺病毒(Ad5、Ad11、Ad26、Ad35、AdC68或其修饰变体)、腺相关病毒、单纯疱疹病毒、麻疹病毒、呼肠弧病毒、弹状病毒、森林脑炎病毒、流感病毒、呼吸道合胞病毒、脊髓灰质炎病毒。
在一些实施方式中，所述疫苗包含佐剂或与佐剂联合使用，例如所述佐剂选自：铝佐剂、霍乱毒素及其亚单位、寡脱氧核苷酸、锰离子佐剂、胶体锰佐剂、弗氏佐剂、MF59佐剂、QS-21佐剂、Poly I：C及其他TLR配体、GM-CSF、IL-2、IL-3、IL-7、IL-11、IL-12、IL-18、IL-21。
在一些实施方式中，所述疫苗的形式适于肌肉接种、皮内接种、皮下接种、滴鼻、雾化吸入、生殖道、直肠、口服或上述不同接种方式的组合(例如肌肉注射+滴鼻)。
在一些实施方式中，所述疫苗的形式适于进行2种或以上的组合接种(例如联合接种或序贯接种)，如与冠状病毒(例如SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV)的S或S1疫苗进行前后序贯接种，或与流感病毒的HA或HA2(例如源自H1～H18中任一种的HA或HA2)疫苗进行前后序贯接种。
在本公开的一个方面中，提供了本文所述的免疫原性肽、核苷酸分子、载体和/或宿主细胞在制备用于同时预防或治疗流感病毒和另一非流感病毒的药物中的应用。
在本公开的一个方面中，还提供了本公开的免疫原性肽、核苷酸分子、载体和/或宿主细胞，其用于同时预防或治疗流感及新冠病毒感染。
在本公开的一个方面中，还提供了一种同时预防或治疗流感及新冠病毒感染的方法，所述方法包括给予有需要的对象本公开的免疫原性肽、核苷酸分子、载体、宿主细胞和/或疫苗。
在一些实施方式中，所述疫苗为核酸疫苗(DNA或RNA疫苗)、重组蛋白亚单位疫 苗、重组病毒载体疫苗、重组细菌载体疫苗、病毒样颗粒疫苗、纳米颗粒疫苗、细胞载体疫苗。
在一些实施方式中，所述疫苗包含佐剂或与佐剂联用，所述佐剂包括但不限于：铝佐剂、霍乱毒素及其亚单位、寡脱氧核苷酸、锰离子佐剂、胶体锰佐剂、弗氏佐剂、SAS佐剂、MF59佐剂、QS-21佐剂、Poly I：C及其他TLR配体、GM-CSF、IL-2、IL-3、IL-7、IL-11、IL-12、IL-18、IL-21等。
在一些实施方式中，所述疫苗的形式适于如下接种方式：肌肉接种、皮内接种、皮下接种、滴鼻、雾化吸入、生殖道、直肠、口服或其任意组合。
在一些实施方式中，采用一种或多种所述疫苗进行接种，例如联合接种或前后序贯接种。
在一些实施方式中，采用一种或多种所述疫苗与其他针对新型冠状病毒或流感的疫苗进行接种，例如所述其他疫苗包括针对冠状病毒S或S1的疫苗，例如所述的S或S1来自于包括但不限于SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV等；所述其他疫苗包括针对流感病毒HA或HA2的疫苗，例如所述的HA或HA2来自包括但不限于H1-H18。
在一些实施方式中，所述疫苗包含核酸疫苗(DNA或RNA疫苗)与腺病毒载体疫苗的组合。
在一些实施方式中，所述疫苗包含pcDNA3.1-S与AdC68-RHAF的组合。在一些实施方式中，所述疫苗组合中的组分前后序贯接种，优选先接种DNA疫苗。
在本公开的一个方面中，提供了一种制备同时针对流感及新冠病毒感染的疫苗的方法，所述方法包括：
(a)提供本公开的免疫原性肽、核苷酸分子和载体；
(b)将(a)中所提供的活性物质与免疫学上或药学上可接受的载体组合。
在本公开的一个方面中，提供了一种同时诱导抗流感以及包含冠状病毒在内的其他病毒免疫应答的方法，其中，采用流感病毒血凝素HA的HA2区域作为免疫原茎部与其他病毒或毒株膜蛋白作为冠部组成的融合蛋白作为疫苗免疫原，以同时诱导抗流感及其他病毒免疫应答。
在一些实施方式中，HA2免疫原选自包括但不限于H1-H18，尤其是来自于广泛流行的人流感H1、H2、H3以及多次出现的人感染禽流感H5与H7；
在一些实施方式中，HA2免疫原来自于2009年流行的H1、2013年流行的H7。
在一些实施方式中，HA2免疫原来自于2013年流行H7的早期毒株的HA2序列以及与该序列同源性超过80％全长或片段。在一些实施方式中，HA2免疫原可包含SEQ  ID NO：1所示的氨基酸序列或可由具有SEQ ID NO：2的编码序列所编码。
在一些实施方式中，与HA2连接的冠部免疫原来自于包括但不限于冠状病毒、艾滋病毒、流感病毒、狂犬病毒、猪瘟病毒、蓝耳病病毒、麻疹病毒、埃博拉病毒、疱疹病毒、虫媒病毒(寨卡病毒、流行性乙型脑炎病毒、森林脑炎病毒、登革病毒、汉坦病毒、出血热病毒)。
在一些实施方式中，与HA2连接的冠部免疫原选自冠状病毒包括但不限于SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV等，尤其是包含冠状病毒的受体结合域的S1蛋白、或受体结合域(Receptor binding domain，RBD)、或经改造的受体结合域。
在一些实施方式中，与HA2连接的冠部免疫原选自冠状病毒SARS-CoV-2的S1，尤其是来自于S1的RBD区或与其同源性超过90％的全长与片段。在一些实施方式中，RBD区免疫原可包含SEQ ID NO：3所示的氨基酸序列或可由具有SEQ ID NO：4的编码序列所编码。在一些实施方式中，RBD区免疫原可包含SEQ ID NO：5所示的氨基酸序列或可由具有SEQ ID NO：6的编码序列所编码。
在本公开的一个方面中，提供了一种同时诱导抗流感及包含冠状病毒在内的其他病毒免疫应答的方法，其包括，将本申请的免疫原与其他序列共表达，共表达方式可以为融合表达，也可以是单独阅读框架表达，从而进一步增加抗病毒种类，或提高免疫原诱导免疫应答能力，预防多种病毒感染。
在一些实施方式中，与本申请的免疫原连接的序列：来自于包括但不限于不同的冠状病毒、流感病毒、艾滋病毒、狂犬病毒、猪瘟病毒、蓝耳病病毒、麻疹病毒、埃博拉病毒、疱疹病毒、虫媒病毒(寨卡病毒、流行性乙型脑炎病毒、森林脑炎病毒、登革病毒、汉坦病毒、出血热病毒)，尤其是与活化T细胞应答的免疫原共表达，制备能够同时激活中和抗体与T细胞应答的复合疫苗；为免疫调节序列，包括但不限于IL-2、IL-7、IL-12、IL-18、IL-21、GM-CSF、CD40L、CD40刺激抗体、PD-1与PD-L1抗体、CTLA4抗体、趋化因子CXCL9、CXCL10、CXCL11、CXCL12、CXCL3、XCL1、CCL4、CCL20、霍乱毒素及其亚单位、细菌鞭毛蛋白、FimH等；提供形成纳米颗粒的特征，包括但不限于转铁蛋白(Ferritin)。
在本公开的一个方面中，提供了一种同时诱导抗流感及包含冠状病毒在内的其他病毒免疫应答的方法，其包括将本文所述的免疫原单独、或与本文所述的共表达分子联合，用于制备包括但不限于核酸疫苗(DNA或RNA疫苗)、重组蛋白亚单位疫苗、重组病毒载体疫苗、重组细菌载体疫苗、病毒样颗粒疫苗、纳米颗粒疫苗、细胞载体疫苗等。
在本公开的一个方面中，提供了一种同时诱导抗流感及包含冠状病毒在内的其他病毒免疫应答的方法，其包括给予本文所述的免疫原性肽、其编码核苷酸分子、载体、宿主细胞或疫苗。
在一些实施方式中，用2种或以上所述疫苗的组合进行接种，既可以联合接种，也可以前后序贯接种。在一些实施方式中，将本文的疫苗与冠状病毒S或S1所制备的疫苗进行前后序贯接种，所述的S或S1来自于包括但不限于SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV等；或将本文的疫苗与流感病毒HA或HA2所制备的疫苗进行前后序贯接种，所述的HA或HA2来自于包括但不限于H1-H18。
本领域的技术人员可对前述的技术方案和技术特征进行任意组合而不脱离本公开的发明构思和保护范围。本公开的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
下面结合附图对本公开作进一步说明，其中这些显示仅为了图示说明本公开的实施方案，而不是为了局限本公开的范围。
图1：S蛋白真核表达载体及sRBD-HA2-hFn(RHAF)腺病毒的构建及融合蛋白的表达：
A：pcDNA3.1-S及pAdC68XY3-RHAF质粒构建图谱；
B：pcDNA3.1-S蛋白在293T细胞中成功表达，且腺病毒AdC68-RHAF能成功表达RHAF蛋白。
图2：AdC68-RHAF在C57BL/6小鼠体内的免疫原性：
实验所用小鼠为6-8周龄雌性C57BL/6，免疫原为质粒pcDNA3.1-S及腺病毒AdC68-RHAF。
A：ELISA方法检测免疫结束后第2周小鼠血清中结合抗体的滴度。横坐标为免疫组别，纵坐标为结合抗体的滴度，**表示p＜0.01
B：第二针免疫前后针对RBD蛋白的结合抗体数据，**表示p＜0.01。
C：293T-ACE2细胞检测免疫结束后第2周小鼠血清中中和抗体的滴度。横坐标为免疫组别，纵坐标为中和抗体的滴度(ID 50)，**表示p＜0.01。
D：第二针免疫前后针对SARS-Cov-2假病毒的中和抗体滴度。
E：ELISPOT方法检测免疫结束后第2周小鼠的T细胞应答。横坐标为免疫组别，纵坐标为每百万个脾细胞中分泌IFN-γ的细胞数量，*表示p＜0.05。
图3：AdC68-RHAF在hACE2+转基因小鼠体内的免疫原性：
实验所用小鼠为6-8周龄hACE2+转基因小鼠，免疫原为腺病毒AdC68-RHAF。
A：ELISA方法检测免疫结束后第2周小鼠血清中结合抗体的滴度。横坐标为免疫组别，纵坐标为结合抗体的滴度，****表示p＜0.0001。
B：293T-ACE2细胞检测免疫结束后第2周小鼠血清中中和抗体的滴度。横坐标为免疫组别，纵坐标为中和抗体的滴度(ID 50)，****表示p＜0.0001。
图4：AdC68-RHAF和AdC68-RBD-HA2-CD8TM在BALB/c小鼠体内的免疫原性：
实验所用小鼠为6-8周龄雌性BALB/c小鼠，免疫原为腺病毒AdC-RHAF和AdC68-RBD-HA2-CD8TM。
A：ELISA方法检测免疫结束后1周小鼠血清中结合抗体的滴度。横坐标为免疫组别，纵坐标为结合抗体的滴度。
B：免疫结束后一周检测的针对SARS-CoV-2假病毒的中和抗体滴度。
图5：新冠病毒攻毒实验：
实验所用小鼠为6-8周龄hACE2+转基因小鼠，免疫原为腺病毒AdC68-RHAF。
A：病毒载量。横坐标为免疫组别，纵坐标为病毒载量。
B：肺组织H&E染色。
C：小鼠攻毒后体重变化。横坐标为攻毒后的天数，纵坐标为体重百分比。
D：小鼠攻毒后生存情况。横坐标为攻毒后的天数，纵坐标为生存百分比。
图6：不同流感病毒攻毒实验：
A：H7N9攻毒后小鼠的体重变化。横坐标为攻毒后的天数，纵坐标为体重百分比。
B：H7N9攻毒后小鼠的生存情况。横坐标为攻毒后的天数，纵坐标为生存百分比，*表示p＜0.05，**表示p＜0.01。
C：H3N2攻毒后小鼠的体重变化。横坐标为攻毒后的天数，纵坐标为体重百分比，*表示p＜0.05，**表示p＜0.01。
图7：R545-HA2-IntN-PAB-7XHis真核表达载体的构建及R545-HA2-IntN-PAB-7XHis蛋白表达：
A：pFastBac-Dual-R545-HA2-IntN-PAB-7XHis质粒构建图谱；
B：杆状病毒能够在昆虫细胞系Sf9中成功表达出蛋白R545-HA2-IntN-PAB-7XHis。
图8：R545-HA2-IntN-PAB-7XHis蛋白纯化及纳米颗粒R545HAF的组装：
A：R545-HA2-IntN-PAB-7XHis蛋白的纯化；
B：R545-HA2-IntN-PAB-7XHis与gb1-IntC-Fn的Intein连接；如图所示单体gb1-IntC-Fn的数量明显减少，且能看到切割生成副产物IntN-PAB(17kDa)和gb1-IntC(11kDa)，表明形成纳米颗粒R545HAF。
图9：纳米颗粒R545HAF在ICR小鼠体内的免疫原性：
实验所用小鼠为6-8周龄hACE2+转基因小鼠，免疫原为质粒pcDNA3.1-S及纳米颗粒R545HAF。
A：ELISA方法检测免疫结束后第4周小鼠血清中结合抗体的滴度。横坐标为免疫组别，纵坐标为结合抗体的滴度，*表示p＜0.05。
B：第一针免疫后与第三针免疫后针对RBD蛋白的结合抗体数据，左图为整体提升，右图为单只小鼠的相应提升，*表示p＜0.05。
C：293T-ACE2细胞检测免疫结束后第4周小鼠血清中中和抗体的滴度。横坐标为免疫组别，纵坐标为中和抗体的滴度(ID 50)，*表示p＜0.05。
D：第一针免疫后与第三针免疫后针对SARS-Cov-2假病毒的中和抗体滴度，左图为整体提升，右图为单只小鼠的相应提升，*表示p＜0.05。
具体实施方式
本公开涉及疫苗领域，特别涉及一种同时诱导抗流感以及包含冠状病毒在内的其他病毒免疫应答的方法。动物实验结果证实，本公开的疫苗安全，可持续产生高效价中和抗体，可用于流感及新冠的预防和治疗。
本文中提供的所有数值范围旨在清楚地包括落在范围端点之间的所有数值及它们之间的数值范围。可对本公开提到的特征或实施例提到的特征进行组合。本说明书所揭示的所有特征可与任何组合物形式并用，说明书中所揭示的各个特征，可以任何可提供相同、均等或相似目的的替代性特征取代。因此除有特别说明，所揭示的特征仅为均等或相似特征的一般性例子。
如本文所用，在数值或范围上下文中的“约”表示所引用或要求保护的数值或范围的±10％。
应理解，当提供参数范围时，本发明同样提供了在该范围内的所有整数及其十分位小数。例如，“0.1-2.5毫克/天”包括0.1毫克/天、0.2毫克/天、0.3毫克/天等直至2.5毫克/天。
如本文所用，“含有”、“具有”或“包括”包括了“包含”、“主要由......构成”、“基本上由......构成”、和“由......构成”；“主要由......构成”、“基本上由......构成”和“由......构成”属于“含有”、“具有”或“包括”的下位概念。
免疫原性肽及其编码分子
如本文所用，术语“免疫原性肽”是指能够同时诱导针对流感病毒和另一非流感病毒的免疫应答的肽，其包含如下部分：(a)免疫原茎部：其包含流感病毒血凝素HA的HA2区域；(b)免疫原冠部：其包含病毒膜蛋白或其免疫原性片段，其中所述病毒膜蛋白的来源病毒不同于(a)中HA2区域的来源病毒；(c)可任选地，与前述部分连接的其他部分。
如本文所用，术语“连接”具有其广义含义，可包括例如融合、共表达、共价连接、偶联等各种方式。
例如，如本文所用，术语“sRBD-HA2免疫原性肽”、“针对流感病毒及新型冠状病毒SARS-CoV-2的免疫原性肽”可互换使用，是指包括流感血凝素HA的HA2区融合SARS-CoV-2病毒刺突蛋白S的RBD区、且具有激发针对流感病毒和新型冠状病毒SARS-CoV-2的结合抗体和中和抗体作用以及相应T细胞应答的肽。
在一些实施方式中，免疫原性肽可为：(a)具有SEQ ID NO：1所示氨基酸序列和SEQ ID NO：3或5或21所示氨基酸序列的多肽；(b)(a)中所述多肽的同源或序列相似且具有相同或相似免疫原性的多肽，例如其中的各区段与SEQ ID NO：1所示氨基酸序列和SEQ ID NO：3或5或21所示氨基酸序列分别具有高于或等于80％，高于或等于85％，高于或等于90％，高于或等于95％，高于或等于96％，高于或等于97％，高于或等于98％，高于或等于99％的同源性或序列相同性，或者与(a)中的多肽具有高于或等于80％，高于或等于85％，高于或等于90％，高于或等于95％，高于或等于96％，高于或等于97％，高于或等于98％，高于或等于99％的同源性或序列相同性；(c)在(a)或(b)限定的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有相同或相似免疫原性的由(a)或(b)衍生的蛋白质或多肽。
在一些实施方式中，免疫原性肽可为：(a′)具有SEQ ID NO：9或15或25所示氨基酸序列的多肽；(b′)由SEQ ID NO：10或16或26所示核苷酸序列编码的多肽；(c′)(a′)和(b′)中所述多肽同源或序列相似且具有相同或相似免疫原性的多肽，例如与(a′)和(b′)中的多肽具有高于或等于80％，高于或等于85％，高于或等于90％，高于或等于95％，高于或等于96％，高于或等于97％，高于或等于98％，高于或等于99％的同源性或序列相同性；(d′)在(a′)或(b′)或(c′)限定的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有相同或相似免疫原性的由(a′)或(b′)或(c′)衍生的蛋白质或多肽。
在一些实施方式下，免疫原性肽可包括与免疫原茎部或冠部连接的其他部分，以例如增强免疫原茎部或冠部或融合蛋白的稳定性、提高中和抗体应答、形成多聚体、 增加细胞应答等。在一些实施方式中，其他部分可包括但不限于：病毒或宿主来源的蛋白，转铁蛋白(Fn)、IL-2、IL-7、IL-12、IL-18、IL-21、GM-CSF、CD40L、CD40刺激抗体、PD-1与PD-L1抗体、CTLA4抗体、趋化因子CXCL9、CXCL10、CXCL11、CXCL12、CXCL3、XCL1、CCL4、CCL20、霍乱毒素及其亚单位、细菌鞭毛蛋白、FimH等。
在一些实施方式中，免疫原性肽中还可包括信号肽、接头、分子标签等元件。例如，信号肽元件可指具有引导蛋白质分泌、定位和/或输送功能的氨基酸序列，其长度通常为5-30个氨基酸。在一些实施方式中，信号肽元件可选自：蛋白自身信号肽、CD33蛋白信号肽、CD8蛋白信号肽、CD16蛋白信号肽、小鼠IgG1抗体信号肽、流感HA蛋白信号肽。例如，连接肽序列(或称接头)可指在本文的融合蛋白中起到连接不同元件作用的短肽，其长度通常为1～50(如5～50、5～40、10～40)个氨基酸。通常，连接肽不影响或严重影响本发明肽形成正确的折叠和空间构象。在一些实施方式中，连接肽元件可选自：(G4S) 3接头、(G4S) n、GSAGSAAGSGEF、(G1y) 6、EFPKPSTPPGSSGGAP、KESGSVSSEQLAQFRSLD、(Gly) 8、EGKSSGSGSESKST。
免疫原性肽也可包括其变异形式，例如一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个，例如1、2、3、4、5、6、7、8、9或10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质或多肽的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质或多肽的功能。
免疫原性肽可在适当的环境和条件下通过重组表达产生，例如由本公开的编码核苷酸分子、载体、宿主细胞产生；也可通过化学合成等方式获得，只要其具有所需的氨基酸序列和免疫原性和反应性。
如本文所用，术语“免疫原性肽编码分子”、“编码序列”等可互换使用，均是指编码本公开所述的免疫原性肽的核苷酸分子。
在一些实施方式中，核苷酸分子可选自，例如：(i)具有SEQ ID NO：2所示核苷酸序列和SEQ ID NO：4或6或22所示核苷酸序列的核苷酸分子；(ii)在严格条件下与(i)杂交的分子；(iii)与(i)中序列同源或序列相似且能表达功能性免疫原性肽的核苷酸分子，例如其中的各区段与SEQ ID NO：2所示核苷酸序列和SEQ ID NO：4或6或22所示核苷酸序列分别具有高于或等于80％，高于或等于85％，高于或等于90％，高于或等于95％，高于或等于96％，高于或等于97％，高于或等于98％，高于或等于99％的同源性或序列相同性，或者与(i)或(ii)中序列具有高于或等于80％，高于或等于85％，高于 或等于90％，高于或等于95％，高于或等于96％，高于或等于97％，高于或等于98％，高于或等于99％的同源性或序列相同性且能够表达功能性免疫原性肽的核苷酸分子；(iv)在(i)或(ii)限定的核苷酸序列中经过取代、缺失或添加一个或几个核苷酸且能够表达功能性免疫原性肽的核苷酸分子。
在一些实施方式中，所述核苷酸分子可选自，例如：(i)具有SEQ ID NO：10或16或26所示序列的核苷酸分子；(ii)在严格条件下与(i)杂交的分子；(iii)与(i)或(ii)中序列具有高于或等于80％，高于或等于85％，高于或等于90％，高于或等于95％，高于或等于96％，高于或等于97％，高于或等于98％，高于或等于99％的同源性或序列相同性且能够表达功能性免疫原性肽的核苷酸分子；(iv)在(i)或(ii)限定的核苷酸序列中经过取代、缺失或添加一个或几个核苷酸且能够表达功能性免疫原性肽的核苷酸分子。
如本文所用，术语“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在50％，优选55％以上、60％以上、65％以上、70％以上、75％以上、80％以上、85％以上或90％以上，更优选是95％以上时才发生杂交。
本公开的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本公开所公开的有关核苷酸序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
载体和宿主细胞
本公开还涉及包含本申请免疫原性肽编码核苷酸分子的载体，以及用该载体经基因工程产生的宿主细胞。
通过常规的重组DNA技术(Science，1984；224：1431)，可利用本公开的编码序列可用来表达或生产重组的免疫原性肽。一般来说有以下步骤：
(1)将本公开的编码核苷酸分子，或将含有核苷酸分子的重组表达载体转入合适的宿主细胞；
(2)在合适的培养基中培养的宿主细胞；
(3)从培养基或细胞中分离、纯化蛋白质或多肽。
本公开中，术语“载体”与“重组表达载体”可互换使用，指本领域熟知的细菌质粒、 噬菌体、酵母质粒、动物细胞病毒、哺乳动物细胞病毒或其它载体。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
可采用本领域常规方法构建含免疫原性肽编码序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。本公开中可采用诸如pcDNA3.1载体、pIRES2-EGFP载体、AdMaxTM、AdC68等表达系统。
此外，表达载体可包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质或多肽。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如动物细胞。代表性例子有：大肠杆菌，链霉菌属、农杆菌；真菌细胞如酵母；动物细胞等。在本公开中，可采用例如选自下组的宿主细胞：HEK293、HeLa、CHO、K562、NS0、SP2/0、PER.C6、Vero、RD、BHK、HT 1080、A549、Cos-7、ARPE-19和MRC-5细胞；High Five、Sf9、Se301、SeIZD2109、SeUCR1、Sf9、Sf900+、Sf21、BTI-TN-5B1-4、MG-1、Tn368、HzAm1、BM-N、Ha2302、Hz2E5以及Ao38。
本公开的核苷酸分子在高等真核细胞中表达时，如果在载体中插入增强子序列时将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
在上面的方法中的重组多肽可在细胞内或在细胞膜上表达或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
疫苗和免疫偶联物
本文中还提供了一种包含本公开的免疫原性肽、核苷酸分子、载体和/或宿主细胞的疫苗，或免疫组合物。该疫苗包含其形式能够被给予脊椎动物(优选哺乳动物)的 本公开的免疫原性肽和/或核酸分子的配制品，并且其诱导提高免疫力的保护性免疫应答以预防和/或减轻流感病毒感染和其他非流感病毒感染(例如新型冠状病毒感染)和/或其至少一种症状。
术语“保护性免疫应答”或“保护性应答”是指通过免疫原介导的针对传染原或疾病的免疫应答，通过脊椎动物(例如人)展现，预防或减轻感染或减少其至少一种疾病症状。
术语“脊椎动物”或“对象”或“患者”是指脊索动物亚门的任何成员，包括但不限于：人和其他灵长类动物，包括非人灵长类动物诸如黑猩猩和其他猿和猴物种；家畜诸如牛、绵羊、猪、山羊和马；家养哺乳动物诸如狗和猫；实验室动物，包括啮齿动物诸如小鼠、大鼠和豚鼠；鸟包括驯养、野生和猎鸟诸如鸡、火鸡和其他鹑鸡类鸟、鸭、鹅。术语“哺乳动物”和“动物”被包括在这个定义中，旨在涵盖成年、幼年以及新生个体。
本文的疫苗可为重组蛋白疫苗、重组DNA疫苗、重组病毒载体疫苗(例如腺病毒载体、痘病毒载体、腺相关病毒载体、单纯疱疹病毒载体、巨细胞病毒载体)、重组细菌载体疫苗、重组酵母载体疫苗或重组病毒样颗粒疫苗。在一些实施方式中，本文的疫苗选自重组DNA疫苗、重组腺病毒载体或其中一种或两种的组合。
在一些实施方式中，可采用选自下组的一种或多种疫苗或其组合：重组质粒疫苗(DNA)，如包含RBD区的SARS-CoV2病毒刺突蛋白S编码序列的DNA疫苗(例如pcDNA3.1-S)；重组腺病毒载体疫苗，如AdC68-RHAF；重组蛋白亚单位疫苗，例如纳米颗粒，R545HAF。
本文的疫苗组合物中包含有效量的本文免疫原。本公开的疫苗组合物中包含足以实现希望的生物效应的量的免疫原。术语“有效量”通常是指可以诱导足以诱导免疫力的保护性免疫应答以预防和/或减轻感染或疾病和/或以减少感染或疾病的至少一种症状的免疫原的量。
本文的疫苗中还可包含佐剂。可采用本领域普通技术人员已知的佐剂，例如Vogel等人，“A Compendium ofVaccine Adjuvants and Excipients”(第2版)中所记载的佐剂(通过引用以其全文结合在此)。已知佐剂的例子包括但不限于：铝佐剂、霍乱毒素及其亚单位、寡脱氧核苷酸、锰离子佐剂、胶体锰佐剂、弗氏佐剂、MF59佐剂、QS-21佐剂、Poly I：C及其他TLR配体、GM-CSF、IL-2、IL-3、IL-7、IL-11、IL-12、IL-18、IL-21等。
本文的疫苗组合物还可包括药学上可接受的载体、稀释剂、防腐剂、增溶剂、乳化剂等辅料。例如，药学上可接受的载体是已知的，并且包括但不限于注射用水、盐 溶液、缓冲盐水、右旋糖、水、甘油、无菌等渗水缓冲液及其组合。药学上可接受的载体、稀释剂和其他赋形剂可例如参见《雷明顿药物科学》(Remngton′s Pharmaceutcal Sciences)中。
本文疫苗组合物的形式可适于系统性或局部(尤其是呼吸道内)给予。给予疫苗组合物的方法包括但不限于：肌肉接种、皮内接种、皮下接种、滴鼻、雾化吸入、生殖道、直肠、口服或其任意组合，例如先肌肉注射后滴鼻。
在一些实施方式中，本文的疫苗预防、消除或减轻对象中的流感病毒及新型冠状病毒感染或其至少一种症状，例如呼吸道症状(如鼻塞、咽喉痛、声嘶)、头痛、咳嗽、痰、发热、啰音、喘息、呼吸困难、因感染引起的肺炎、严重急性呼吸综合症、肾衰竭等。
本文中还涉及了一种免疫偶联物(也可称免疫缀合物)，其包含本文的免疫原以及与其偶联的其他物质。所述的其他物质可为靶向性物质(如特异性识别特定靶标的部分)、治疗性物质(如药物、毒素、细胞毒剂)、标记性物质(如荧光标记物、放射性同位素标记物)。
在本公开中还提供了一种组合产品，其包括本公开的免疫原性肽、核苷酸分子、载体、宿主细胞和/或疫苗，且还可包含一种或多种有助于更好发挥预防和/或治疗流感病毒感染以及其他非流感病毒感染(例如新型冠状病毒感染)或其症状的功能或增强前述物质稳定性的其他物质。例如，其他物质可包括针对冠状病毒S或S1的其他疫苗，如来自于包括但不限于SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV的S或S1疫苗；针对流感病毒HA或HA2的其他疫苗，所述的HA或HA2来自于包括但不限于H1-H18；受益于T细胞活化和/或与T细胞的记忆性免疫反应的疾病或病症的其他活性物质。
免疫方法
本文还提供了一种用于预防和/或治疗流感病毒感染以及其他非流感病毒感染(例如新型冠状病毒感染)和/或其症状的方法，其包括：至少一次给予预防和/或治疗有效量的本公开的一种或多种疫苗。可采用的接种方式包括但不限于：系统性免疫接种方式，如肌肉注射、皮下注射和皮内注射等；呼吸道内免疫接种方式，如雾化、滴鼻等。在一些实施方式中，初次免疫采用系统性接种或呼吸道内接种，优选系统性接种。
在本公开的一些实施方式中，每两次接种之间的间隔至少为1周，例如2周、4周、2个月、3个月、6个月或更长间隔。
在一些实施方式中，采用DNA疫苗进行初次免疫，并采用重组病毒疫苗进行一 次或多次加强免疫。本公开的免疫方法可采用“初免-加强”或“初免-加强-再加强”的方式，可采用单一的全身系统免疫或呼吸道局部免疫方式，或采用两种免疫方式的组合。
根据不同载体疫苗的特点，在一些优选的实施方式中，采用重组DNA疫苗进行系统性初免，从而建立全身系统免疫应答，再用其他疫苗(例如重组腺病毒疫苗或重组痘病毒疫苗)进行一次或多种免疫加强，所述免疫加强可包括至少一次呼吸道内免疫加强(例如，采用腺病毒疫苗)。
采用本文的免疫方法可在呼吸道局部和全身系统有效建立的疫苗特异性免疫应答，有助于增强疫苗保护的有效性。
以药物包或试剂盒的形式提供本文的组合产品可，例如可将本文的一种或多种疫苗组合物或其一种或多种成分包装在一个或多个容器中，例如包装在指明组合物的量的密封容器诸如安瓿或小药囊中。可以液体、无菌冻干粉或无水浓缩物等形式提供疫苗组合物，可在临用前用适当液体(例如水、盐水等)对其进行稀释、复原和/或配制以获得用于给予至对象的适当浓度和形式。
本公开中的组合产品可用于在呼吸道内局部诱导高水平抗原特异性CD8+T细胞应答的特征，使得其在预防呼吸道病原体感染、降低呼吸道病原体致病力和呼吸道肿瘤的预防治疗中均有应用前景。
实施例
下面结合具体实施例，进一步阐述本公开。应理解，这些实施例仅用于说明本公开而不用于限制本公开的范围。本领域技术人员可对本公开做出适当的修改、变动，这些修改和变动都在本公开的范围之内。
下列实施例中未注明具体条件的实验方法，可采用本领域中的常规方法，例如参考《分子克隆实验指南》(第三版，纽约，冷泉港实验室出版社，New York：Cold Spring Harbor Laboratory Press，1989)或按照供应商所建议的条件。DNA的测序方法为本领域常规的方法，也可由商业公司提供测试。
除非另外说明，否则百分比和份数按重量计算。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本公开方法中。文中所述的较佳实施方法与材料仅作示范之用。
下述实验中涉及到的实验动物、免疫方式、免疫原、假病毒及检测方法如下：
I.实验动物：
6-8周龄雌性C57/BL6及BALB/c小鼠，购自上海吉辉实验动物饲养有限公司)；
6-8周龄雌性hACE2+ICR小鼠，购自北京唯尚立拓科技有限公司)。
II.免疫方式：
对小鼠左、右后肢分别进行肌肉注射；或进行滴鼻。具体剂量见实施例。
III.免疫原的选择：
HA2序列来自于Genebank：AGI60292.1，S及RBD序列均来自Genebank：NC_045512.2；hFn序列(人转铁蛋白)来自于Genebank：M97164.1，具体序列见序列表。
1.重组质粒疫苗(DNA)：pcDNA3.1-S、pcDNA3.1(空载)；
2.重组腺病毒载体疫苗：AdC68、AdC68-sRBD-HA2-hFn(AdC68-RHAF)、AdC68-RBD-HA2-CD8TM。
3.重组蛋白亚单位疫苗(纳米颗粒，protein)：R545HAF
IV.免疫原制备及免疫剂量：
免疫原的制备参见实施例1
在实施例中所采用的免疫原免疫剂量如下：
1.重组质粒疫苗(DNA)：100μg/只小鼠，100μL，溶于无菌生理盐水中；
2.重组腺病毒载体疫苗：5E10vp/只小鼠，100μL(肌肉注射)；5E10vp/只小鼠，40μL(滴鼻)。
3.重组蛋白亚单位疫苗(纳米颗粒，protein)：蛋白质(溶于无菌PBS中)与铝佐剂(Aluminium，InvivoGen，货号5200)按照体积比1 ∶1混合后进行免疫，20μg/只小鼠，100μL；
V.免疫间隔：
具体免疫间隔见下文表格。
VI.SARS-CoV2包膜假病毒(Pseudovirus)包装：
1.转染前一天准备293T细胞，用于包装质粒的转染与表达。用DMEM完全培养基将细胞稀释至5×10 6个/mL细胞，取1mL稀释好的细胞，铺在10cm的皿中，37℃，5％CO 2，培养过夜；
2.吸取SARS-CoV2膜蛋白质粒pcDNA3.1-S 4μg和pNL4-3Δenv骨架质粒8μg (NIH AIDS Reagent Program，3418)加入500μL双无(无血清、无双抗，双抗为青链霉素混合液)的DMEM中，室温孵育5min；
3.用双无DMEM将24μL TurboFect(Thermo Fisher Scientific)稀释，终体积为500μL/样品，室温孵育5min；
4.将2与3两者混匀，1000μL/样品终体积，室温孵育20min，孵育结束后加到10cm培养皿的293T细胞中。6h后更换新鲜的15mL完全培养基，继续在细胞培养箱中培养48h；
5.培养结束后，收集10cm皿的细胞培养上清，于15mL离心管里，然后4000g，4℃，离心10min，用0.45μm的滤器过滤到新的15mL离心管中，冻存于-80℃保存，滴定后备用。
VII.构建稳定表达hACE2受体的293T细胞：
1.人工合成人源ACE2(hACE2)序列(Genebank#NCBI_NP_001358344.1)，如SEQ ID NO：13所示，序列5’端带有Age1酶切位点，3’端带有Xba1酶切位点，合成片段与载体质粒pHAGE-MCS-puro使用Age1酶切(Thermo Scientific公司，货号FD1464)与Xba1酶切(Thermo Scientific公司，FD0685)，并通过凝胶电泳后切胶回收，采用Sanprep柱式DNA胶回收试剂盒(Promega公司，货号A9282)回收酶切片段。
2.基因回收产物与酶切线性化载体用T4DNA连接酶的方法连接(Thermo Scientific公司，货号2011A)：将连接产物转化至大肠杆菌E.coli Stable，在含氨苄霉素的培养板上过夜生长。第2天，随机挑取单菌落进行测序，突变位点校正，验证全部序列正确后，成功克隆出hACE2基因的慢病毒表达质粒(pHAGE-hACE2-puro)。
3.取10cm皿，在每个皿中接种约5×10 6个293T细胞，保证第二天转染时使细胞密度达90％为宜；将pHAGE-hACE2-puro、慢病毒包装质粒psPAX以及VSVG三种质粒，按照质量比1∶2∶1的比例转染293T细胞。
4. 37℃，5％的孵箱培养48小时左右，具体时间根据细胞情况而定，收集细胞上清。将收集的细胞上清用0.45μm的滤器进行过滤，再用PEG 8000进行浓缩，即可得到较为纯化的hACE2慢病毒。
5.提前一天铺5×10 5个的293T细胞于12孔板的一个孔内，次日向铺好的细胞中加入步骤2中浓缩的病毒500μL，1000g，离心2小时。
6.离心感染结束后，继续在37℃，5％的孵箱培养12小时左右，将培养基换 成添加1μg/mL嘌呤霉素(puro)的细胞培养基培养，最后能够存活的细胞便是整合有hACE2基因的293T细胞，并通过流式分选筛选出稳定表达hACE2的293T细胞(能与S蛋白结合)。
VIII.检测方法：
采血：
最后一次免疫结束后第2/4周，将小鼠脱颈处死前，通过摘眼球的方法采集小鼠外周全血，收集于1.5mL EP管中，室温静置使其自然凝血，凝固后的小鼠血清于7000g，离心15min。将小鼠血清转移至新的1.5mL EP管中。实验前需要将样品在56℃灭活30min，来破坏血清内的补体活性。灭活前短暂离心，避免管壁和瓶盖上的样品残存。水浴液面要没过样品液面，但不能超过瓶盖。
ELSIA检测结合抗体：
1.用4℃预冷的ELISA包被液稀释检测的抗原蛋白(S1，购自北京义翘神州科技有限公司；RBD，购自上海近岸生物科技有限公司)，至终浓度为1μg/mL。在ELISA板的每孔加入100μL包被抗原溶液，4℃过夜；
2.第二天，取出ELISA板，弃掉包被液，用0.05％的PBST缓冲液洗板3次，每次220μL；
3.洗涤完毕后，在吸水纸上拍干，每孔用200μL ELISA封闭液(0.5％脱脂奶粉，PBST溶解)进行封闭，室温封闭2h；
4.封闭结束后，用0.05％的PBST洗板3次，每次220μL；
5.对于血清或者血浆，用ELISA样品稀释液(0.5％脱脂奶粉，PBST溶解)稀释，从1 ∶100起始，进行2倍比稀释。用未免疫的小鼠血清设置为阴性对照。设置空白孔，只加样品稀释液，每个样品需做2个复孔，每孔终体积为100μL，室温孵育3h；
6.样品孵育结束后，继续用PBST洗板5次，每次220μL；
7.用ELISA封闭液(0.5％脱脂奶粉，PBST溶解)稀释相对应比例的二抗(山羊抗鼠，购自北京中杉金桥生物技术有限公司，货号ZB-2305)，每孔加入100μL，室温孵育1-1.5h；
8.二抗孵育结束后，用0.05％的PBST洗板5次，每次220μL；
9.取一对金银片OPD底物，溶解于20mL去离子水中，随后每孔加入100μL，避光反应5min；
10.显色结束后，用50μL 2nM H 2SO 4进行终止，在酶标仪上读取OD 492-OD 630值；
11.以最后一个稀释度OD 492大于2倍的(negative mean+SD)值对应的血清稀释比 的倒数作为抗体滴度。
293T-ACE2细胞检测中和抗体：
1.取96孔透明底黑板进行中和实验，第一列设置细胞对照(CC)(150μL)，第二列设置病毒对照(VC)(100μL)，其他均为样品孔，对血清样品进行倍比稀释，最终孔中体积为100μL。
2.除细胞对照组外，每孔加50μL SARS-CoV-2假病毒稀释液，使每孔最终含假病毒为200TCID 50。
3.轻轻震荡混匀，将上述96孔底黑板置于细胞培养箱中，37℃，5％CO 2孵育1h。
4.当孵育时间至20min时，开始准备293T-hACE2靶细胞，并用完全培养基将细胞稀释至10 5个细胞/mL。
5.当孵育时间至1h，向96孔透明底黑板中每孔加100μL靶细胞，使每孔细胞为10 4个。
6.前后左右轻轻晃动96孔透明底黑板，使孔中的细胞均匀分散，再将板子放入细胞培养箱中，37℃，5％CO 2培养48h。
7.培养48h后，从细胞培养箱中取出96孔透明底黑板，吸掉孔中上清，每孔加入100μL PBS清洗一遍，吸去PBS，每孔加入50μL 1×的裂解缓冲液(购自Promega公司Cat#E153A)，室温在水平摇床上孵育30min使细胞充分裂解；
8.加30μL荧光素酶的底物(购自Promega公司，Cat#E1501)于96孔黑板中，用仪器
Figure PCTCN2021141198-appb-000001
96微孔板发光-检测仪检测荧光素酶活性。
9.导出荧光素读值，计算中和抑制率，结合中和抑制率结果，利用Graphpad Prism 5.0软件计算ID 50。
Figure PCTCN2021141198-appb-000002
ELISPOT检测T细胞应答：
小鼠脾脏单细胞分离：
1)将小鼠仰卧，剖开右侧腹部皮肤，打开腹膜，取下小鼠脾脏，放入加有5mL完全RPIM1640培养基的小平皿中；
2)用无菌镊子将脾脏用无菌纱布包裹起来，用小弯镊夹起纱布，轻轻磨碎脾脏，可使脾细胞全部释放到培养基中；
3)随后用5mL移液器将脾细胞悬液经纱布吸到无菌的15mL离心管中，800g，离心5min；
4)弃掉离心后的上清，轻敲15mL离心管重悬细胞沉淀，每个离心管中加入3mL 红细胞裂解液裂解红细胞，颠倒混匀后室温静置5min，使红细胞充分裂解又不会损伤脾细胞；
5)裂红结束后，用5mLRPIM1640培养基终止裂红。800g，离心5min；
6)弃掉离心后的上清，用5mL RPIM1640培养基洗1次，800g离心5min；
7)弃离心后上清，脾细胞放于冻存液(90％FBS和10％DMSO)中进行冻存备用。
ELISpot实验操作按照Mouse IFN-γ/Monkey IFN-γ说明书进行。
1)用纯化IFN-γ抗体包被试剂盒(购自BD，货号551083)提供的Millipore板，比例1∶250，4℃过夜包被；
2)甩掉板中的包被抗体溶液，用200μL RPMI 1640完全培养基洗板一遍，随后用200μL RPMI 1640完全培养基封闭液封闭Millipore板，室温孵育2h；
3)弃掉孔板中的封闭液，根据不同的实验设计，在Millipore板中加入刺激肽库，50μL/孔，每条肽的浓度为5μg/mL。在阴性对照孔加入50μL RPMI 1640完全培养基；阳性对照孔中加入50μL佛波醇酯类多克隆刺激剂(PMA，购自Sigma，货号FXP012)(终浓度100ng/mL)和离子霉素(Ionomycin，终浓度2μg/mL)的RPMI 1640完全培养基；
其中，刺激肽库由苏州强耀生物科技有限公司合成，每条单肽为15个氨基酸，覆盖RBD序列，每五个单肽一个肽库，即总共13个肽库。其中，第1条肽为MPTESIVRFPNITNL(SEQ ID NO：19)，第2条肽为SEQ ID NO：3的RBD肽序列中的aa 8-22，后面每条肽相比于其他肽氨基酸后移四位，即第3条肽为SEQ ID NO：3的RBD肽序列中的aa 12-26，第4条肽为SEQ ID NO：3的RBD肽序列中的aa 16-30……以此类推到第64条肽为SEQ ID NO：3的RBD肽序列中的aa 256-270，第65条肽为AVRDPQTLEILDITP(SEQ ID NO：20)。
4)对小鼠脾细胞进行计数，将细胞调整为4×10 6个细胞/mL，每孔加入50μL细胞，最终使每孔的细胞数为2×10 5个细胞。将Millipore板放入湿盒，在37℃5％CO 2培养箱中孵育20-22h，期间切勿摇动板子，以免造成细胞的偏移；
5)培养孵育结束后，从培养箱中取出Millipore板，将板中的液体弃掉，用预冷的去离子水洗两遍，每次220μL，每次清洗孵育3min；
6)用0.05％的PBST(PBS+0.05％Tween-20)洗板3次，每次200μL；
7)用10％FBS的PBS抗体稀释液稀释生物素化检测抗体(Biotinylated Detection antibody，比例1∶200)，每孔加入100μL，室温孵育2h；
8)孵育结束后，再用0.05％的PBST洗板3次，每次220μL；
9)将链霉亲和素-HRP偶联抗体用抗体稀释液进行稀释(比例1∶100)，每孔加入100μL，室温孵育1h；
10)孵育结束后，用0.05％的PBST洗板4次，每次220μL；
11)再用PBS清洗板子2次，每次220μL；
12)准备底物溶液(1mL的底物缓冲液加1滴底物溶液)，每孔加入100μL底物溶液。反应5-60min，孵育时间根据斑点形成的情况而定。
13)用去离子水冲洗终止反应，室温晾干后进行计数；
14)利用ChampSpot III型酶联斑点图像分析仪进行斑点形成细胞SFC(spot forming cell，SFC)的计数以及QC。
实施例1：S蛋白真核表达载体及RHAF与RBD-HA2-CD8TM腺病毒表达载体的构建及蛋白质表达
为了研究RBD的功能，我们构建了S蛋白的真核表达载体，并在体外细胞系中真核表达出成熟的蛋白质。
首先，我们人工合成了S基因(其对应氨基酸序列如SEQ ID NO：11所示，基因序列如SEQ ID NO：12所示)，合成片段与载体质粒pcDNA3.1(购自优宝生物)使用BamHI酶切(Thermo Scientific公司，FD0054)与Not1酶切(Thermo Scientific公司，FD0596)，并通过凝胶电泳后切胶回收，采用Sanprep柱式DNA胶回收试剂盒(Promega公司，货号A9282)回收酶切片段。
随后，基因回收产物与酶切线性化载体用T4DNA连接酶的方法连接(Thermo Scientific公司，货号2011A)：将连接产物转化至大肠杆菌E.coli Stable，在含氨苄霉素的培养板上过夜生长。第2天，随机挑取单菌落进行测序，突变位点校正，验证全部序列正确后，成功克隆出S蛋白的真核表达载体pcDNA3.1-S，质粒构建图谱如图1A。
我们进一步检测了S蛋白能否在真核细胞系293T中表达。
首先，取6孔板，在每个孔中接种约6×10 5个293T细胞，保证第二天转染时使细胞密度达90％为宜；用pcDNA3.1及pcDNA3.1-S分别转染293T细胞(转染试剂TurboFect)。37℃，5％的孵箱培养48小时左右，具体时间根据细胞情况而定，收集细胞，进行蛋白质免疫印迹(WB)鉴定，发现S蛋白在细胞中表达，大小为200kD左右；而pcDNA3.1对照转染的细胞中不能检测到S蛋白的表达(图1B左图)。
为了研究HA2的功能，我们构建了RHAF(即sRBD-HA2-hFn融合蛋白)的腺病毒表达载体，并构建了腺病毒AdC68-RHAF，且验证了腺病毒中RHAF的表达。
首先，我们人工合成了RHAF基因(具体序列如SEQ ID NO：10所示，其中HA2部分基于如SEQ ID NO：2所示，sRBD部分基于如SEQ ID NO：6所示的序列，hFn部分基于如SEQ ID NO：8所示的序列)。sRBD-HA2表达在hFn的N端，两者之间采用G 4S接头 连接。合成片段与载体质粒pAdC68XY3(根据专利申请号201910777937.2中所记载方法构建，其中E1/E3缺失的复制缺陷型AdC68载体的E4部分替换为AdHu5的对应E4部分，该改造可增加重组黑猩猩腺病毒的产量而不改变腺病毒的血清学特性)，使用SrfI酶切(NEB公司，#R0629S)，并通过凝胶电泳后切胶回收，采用Sanprep柱式DNA胶回收试剂盒(Promega公司，货号A9282)回收酶切片段。
随后，基因回收产物与酶切线性化载体用T4DNA连接酶的方法连接(Thermo Scientific公司，货号2011A)：将连接产物转化至大肠杆菌E.coli Stable，在含氨苄霉素的培养板上过夜生长。第2天，随机挑取单菌落进行测序，突变位点校正，验证全部序列正确后，成功克隆出RHAF的腺病毒表达载体pAdC68XY3-RHAF，质粒构建图谱如图1A。
为了研究究竟是将RBD-HA2与hFn融合分泌出去还是将RBD-HA2通过CD8跨膜区(CD8TM)表达在腺病毒表面的效果更优越，我们构建了RBD-HA2-CD8TM的腺病毒表达载体，并构建了腺病毒AdC68-RBD-HA2-CD8TM。其构建方法与AdC68-RHAF一致，其氨基酸序列如SEQ ID NO：15所示，编码序列如SEQ ID NO：16所示。质粒构建图谱如图1A。
我们进一步对重组腺病毒进行了包装及扩增。
构建好的重组质粒pAdC68XY3-RHAF与pAdC68XY3-RBD-HA2-CD8TM分别经限制性内切酶Pac 1在37℃水浴中线性化3.5h，65℃灭活内切酶。用293A细胞铺六孔板，将线性化后的重组质粒分别以2.5μg、2μg、1.5μg/孔的量转染至293A细胞，培养11天左右噬斑出现，14天左右至细胞均被病毒感染时收样，细胞上清一起收集，置于-80℃反复冻融三次后，取少量感染293A细胞，Western blot验证目的蛋白的表达，发现有正确大小的蛋白表达(图1B)。
将上述收集的腺病毒样品感染至1个T175细胞培养瓶的293A细胞，24h后收样，置于-80℃反复冻融三次后，感染至6个T175细胞培养瓶的293A细胞，以此类推进行大量扩增，扩增至36个T175细胞培养瓶时，收集细胞沉淀，弃上清，用10mL左右无血清无抗性的DMED培养基重悬，-80℃反复冻融三次后，采用氯化铯密度梯度离心法纯化腺病毒，分装后，-80℃保存。
氯化铯密度梯度纯化腺病毒步骤：
(1)将扩增冻融后的腺病毒4℃，4000g，离心30min，取上清，用0.8μm的滤膜过滤；
(2)向超速离心管中加入3mL 1.2M CsC溶液，将3mL 1.4CsC溶液用注射器从底部轻轻缓慢打入，铺好氯化铯梯度；
(3)将过滤后的腺病毒溶液轻轻加入超速离心管中，用PBS将液面补满，配平后放入SW41套筒中；
(4)选取SW41转子，4℃，25000rpm，离心2.5h，降速调为no brake自然降速；
(5)待离心结束后，轻轻取出超速离心管，可见两条白色病毒条带，用注射器从侧面戳如超立管中，将下层条带吸出，注意避免吸到上层条带；
(6)将Bio-Gel P-6PG gel脱盐胶注入液相色谱柱(Liquid chromatography columns)中，待液体落完，留下2-3cm左右的胶为宜，加入PBS洗涤两遍；
(7)将上述吸出的病毒液加入脱盐胶，待液体落完后，加入2mL左右PBS洗脱，每个1.5mL EP管收集3-4滴洗脱液；
(8)Nanogrop 2000测定每管洗脱液的OD 260值，将OD 260大于2的洗脱液合并，加入10％无菌甘油，分装；
(9)Nanogrop 2000测定分装后腺病毒溶液的OD 260值，OD 260值×1.1×10 12/mL即为纯化后腺病毒的滴度；
(10)最终AdC68-RHAF滴度为1.76×10 13/mL，AdC68-RBD-HA2-CD8TM滴度为1.48×10 13/mL。
实施例2：AdC68-RHAF在C57BL/6小鼠体内的免疫原性
分别采用DNA-pcDNA3.1-S及腺病毒AdC68-RHAF免疫C57BL/6小鼠，在完成免疫2周后，评价免疫组合诱导针对RBD蛋白及H7蛋白的结合抗体滴度及针对SARS-CoV-2假病毒的中和抗体滴度。同时评价针对RBD肽库的T细胞应答水平。
将小鼠随机分为2组，根据免疫原分别命名为对照组、AdC68-RHAF组。具体免疫组合如表1所示，免疫方式为肌肉注射。
AdC68-RHAF组产生针对RBD蛋白及H7蛋白的结合抗体滴度如图2A所示：针对RBD蛋白的结合抗体滴度均在10,000以上，最高的能够到50,000；针对H7的结合抗体滴度均值在6,000左右，最高的能够到达10,000。同时比较第二针免疫前后针对RBD蛋白的结合抗体数据可以发现，结合抗体滴度有了明显的提升(图2B)。
AdC68-RHAF组产生针对SARS-Cov-2假病毒的中和抗体滴度如图2C所示：大部分滴度都在1000以上，均值在1360，有一小鼠中和抗体滴度高达12808，小鼠间存在个体差异性。同时比较第二针免疫前后针对SARS-Cov-2假病毒的中和抗体滴度的数据可以发现，中和抗体滴度有了明显的提升(图2D)。
AdC68-RHAF组产生针对RBD肽库的T细胞应答如图2E所示：每百万个脾细胞里面分泌IFN-γ的细胞数在3,000左右。
该实验证实，在C57BL/6小鼠体内，AdC68-RHAF能够同时诱导针对流感及新冠的抗体，且能诱导针对新冠病毒的中和抗体及针对新冠病毒的T细胞应答。
表1.AdC68-RHAF在C57BL/6小鼠体内免疫原性测试的免疫方案
Figure PCTCN2021141198-appb-000003
实施例3：AdC68-RHAF在hACE2+ICR小鼠体内的免疫原性
根据实施例2得出的结论，为了后续能够采用新冠病毒对小鼠进行攻毒保护验证，我们在hACE2+ICR小鼠体内验证了AdC68-RHAF的免疫原性，在完成免疫2周后，评价免疫组合诱导针对RBD蛋白、H3蛋白及H7蛋白的结合抗体滴度和针对SARS-CoV-2假病毒的中和抗体滴度。
本实施例中所用hACE2+ICR小鼠是一种COVID-19临床前模型，其为ICR小鼠背景下构建的SARS-CoV-2易感人血管紧张素转换酶2(hACE2)转基因小鼠，主要在其肺、心、肾和小肠中表达hACE2。该小鼠模型是SARS-CoV-2治疗药物和疫苗研发的重要工具。
实验步骤如下：将小鼠随机分为2组，根据免疫原分别命名为对照组和AdC68-RHAF组。具体免疫组合如表2所示，免疫方式为肌肉注射。
AdC68-RHAF组合产生针对RBD蛋白、H3蛋白及H7蛋白的结合抗体滴度如图3A所示：针对RBD蛋白的结合抗体滴度大多在10,000以上，部分能够达到100,000；针对H3蛋白的结合抗体滴度大多在100以上，部分能够达到1000；针对H7蛋白的结合抗体滴度大多在6,400左右。
第二针免疫结束后2周，AdC68-RHAF组产生针对SARS-CoV-2假病毒的中和抗体滴度如图3B所示：大部分都在1000左右，最高能到达2253。
该实验证实，AdC68-RHAF能够在hACE2+ICR小鼠体内同时诱导针对新冠及流感的结合抗体，且能够同时诱导针对流感H3及H7的结合抗体，验证了HA2抗体的广谱性。同时AdC68-RHAF能够有效诱导针对新冠的中和抗体，具有开发为抗流感及新冠疫苗的良好前景。
表2.AdC68-RHAF在hACE2+ICR小鼠体内免疫原性测试的免疫方案
Figure PCTCN2021141198-appb-000004
Figure PCTCN2021141198-appb-000005
实施例4：AdC68-RHAF和AdC68-RBD-HA2-CD8TM在BALB/c小鼠体内的免疫原性
分别采用腺病毒AdC68-RHAF及AdC68-RBD-HA2-CD8TM免疫BALB/c小鼠，在完成免疫2周后，评价免疫组合诱导针对RBD蛋白及H7蛋白的结合抗体滴度及针对SARS-CoV-2假病毒的中和抗体滴度。同时评价针对RBD肽库的T细胞应答水平。
将小鼠随机分为3组，根据免疫原分别命名为AdC68组、AdC68-RHAF组和AdC68-RBD-HA2-CD8TM组。具体免疫组合如表3所示，免疫方式为肌肉注射。
免疫结束后一周采集血清检测了针对RBD蛋白的结合抗体滴度，如图4A所示：AdC68-RHAF组针对RBD蛋白的结合抗体滴度均值为4031，最高能到12,800；AdC68-RBD-HA2-CD8TM组针对RBD蛋白的结合抗体滴度均值为10，160，最高的能够到25,600，此时AdC68-RHAF组与AdC68-RBD-HA2-CD8TM组之间没有显著差别。
免疫结束后一周检测了针对SARS-CoV-2假病毒的中和抗体滴度，如图4B所示：AdC68-RHAF组中和抗体滴度均值为128，最高能到274；AdC68-RBD-HA2-CD8TM组中和抗体滴度均值为97，最高的能够到348，此时AdC68-RHAF组与AdC68-RBD-HA2-CD8TM之间没有显著差别。
随后持续检测免疫结束后两周针对RBD的结合抗体滴度及针对SARS-CoV-2假病毒的中和抗体滴度。
同时，试验进一步证明两种腺病毒AdC68-RHAF及AdC68-RBD-HA2-CD8TM：可有效诱导产生针对流感病毒针H7的结合抗体；可有效诱导针对SARS-CoV-2假病毒的中和抗体；同时，可诱导针对RBD肽库的T细胞应答。
表3.AdC68-RHAF和AdC68-RBD-HA2-CD8TM
在BALB/c小鼠体内的免疫原性测试方案
Figure PCTCN2021141198-appb-000006
实施例5：AdC68-RHAF在BALB/c小鼠体内不同接种方式产生的免疫原性
分别采用DNA-pcDNA3.1-S及腺病毒AdC68-RHAF免疫BALB/c小鼠，在完成免疫2周后，评价免疫组合诱导针对RBD蛋白及H7蛋白的结合抗体滴度及针对SARS-CoV-2假病毒的中和抗体滴度。同时评价针对RBD肽库的T细胞应答水平。
将小鼠随机分为4组，根据免疫原及免疫方式分别命名为AdC68肌肉组、AdC68滴鼻组、AdC68-RHAF肌肉组和AdC68-RHAF滴鼻组。具体免疫组合如表4所示。
免疫结束后两周采集血清及肺灌洗液检测针对RBD蛋白和针对流感病毒H7蛋白的结合抗体，包括IgG和IgA；同时检测针对SARS-CoV-2假病毒的中和抗体及针对RBD肽库的T细胞应答。
表4.AdC68-RHAF在BALB/c小鼠体内不同接种方式产生的免疫原性测试方案
Figure PCTCN2021141198-appb-000007
实施例6.新冠病毒攻毒试验
小鼠：南模生物ACE2-转基因小鼠(C57BL/6-Tgtn(CAG-human ACE2-IRES-Luciferase-WPRE-polyA)Smoc)，雌性。
毒株：SARS-CoV-2/human/CHN/Shanghai_CH-02/2020。
委托第二军医大学进行攻毒试验
免疫程序：将小鼠随机分为2组，根据免疫原分别命名为对照组和AdC68-RHAF组。具体免疫组合如表5所示，免疫方式为肌肉注射。
攻毒程序：免疫结束后1-2周对小鼠进行攻毒试验，攻毒后观察至5天，记录体重变化；攻毒后第三天每组各取3只小鼠处死，取肺组织，每只小鼠的左半边肺组织(一个大叶)用4％多聚甲醛固定48小时后做病理切片(H&E染色和组化)；每只小鼠的右半边肺组织(4个小叶)研磨测病毒载量qPCR RNAcopies或者病毒滴度PFU/ml；第五天，若还有小鼠存活，则称完体重后全部处死。
结果显示：从病毒载量来看，AdC68-RHAF组相比于对照组降低了3.7个Log，其中AdC68-RHAF三只中有两只检测不到病毒载量(图5A)。同时，从H&E染色的结果来看，AdC68-RHAF组肺组织病理要明显好于对照组，损伤更小且炎性浸润更少(图5B)。从体重来看，AdC68-RHAF组没有改变，而对照组在第三天出现急剧下降(图5C)。从生存来看，AdC68-RHAF组百分之百存活，而对照组全部死亡(图5D)。总体而言，所有数据均证明了AdC68-RHAF疫苗对新冠有极佳的保护作用。
表5.AdC68-RHAF在hACE2+C57BL/6小鼠体内免疫方案
Figure PCTCN2021141198-appb-000008
实施例7.小鼠流感病毒攻毒试验
小鼠：hACE2+ICR小鼠和BALB/c小鼠，雌性。
毒株：H7N9(A/Shanghai/4664T/2013)、H3N2(A/Hong Kong/8/68)。
在上海市公共卫生临床中心P2及P3实验室进行攻毒试验。
免疫程序：将小鼠随机分为2组，根据免疫原分别命名为对照组和AdC68-RHAF组。具体免疫组合如表6所示，免疫方式为肌肉注射。
攻毒程序：免疫结束后对小鼠进行滴鼻攻毒，攻毒后持续观察14天，每日称重，记录体重变化，并得出生存曲线。
结果显示，对于H7N9攻毒，AdC68-RHAF组小鼠体重在第七天就出现回升，而对照组则一直在下降(图6A)。而从生存来看，AdC68-RHAF组完全存活，而对照组则在第十天全部死亡(图6B)。对于H3N2的攻毒，该剂量对于小鼠是不致死的，因此只看体重变化，可以发现，AdC68-RHAF组的体重下降明显少于对照组(图6C)。总的来说，AdC68-RHAF可有效针对各试验毒株产生保护效果，具有广谱性。
表6.AdC68-RHAF在hACE2+C57BL/6小鼠体内免疫方案
Figure PCTCN2021141198-appb-000009
实施例8.R545-HA2-IntN-PAB-7XHis真核表达载体的构建及R545-HA2-IntN-PAB-7XHis蛋白表达
从前面的实验中，我们发现腺病毒载体疫苗AdC68-RHAF能够诱导针对新冠和流感的抗体，且均有保护效果。在后续的实验中，我们尝试更换载体，即直接采用纯化出的纳米颗粒来进行测试。为了提升蛋白的溶解度和复性率，我们选用了一个更短的RBD肽段(R545，其对应氨基酸序列如SEQ ID NO：21所示，基因序列如SEQ ID NO：22所示)，比前文的RBD更利于用于蛋白质的包涵体纯化。
为了表达R545-HA2-IntN蛋白，我们构建了R545-HA2-IntN(R545HA-IntN)的供体质粒，通过转座获得能在昆虫细胞系Sf9中高效表达R545-HA2-IntN的重组杆 状病毒，并在昆虫细胞系Sf9中真核表达出了成熟的蛋白质。
首先，我们对R545-HA2-IntN基因进行了昆虫Sf9细胞密码子优化并加入了促溶标签PAB(Protein A B domin)和7XHis标签，人工合成了R545-HA2-IntN-PAB-7XHis基因片段(擎科，其对应氨基酸序列如SEQ ID NO：23所示，基因序列如SEQ ID NO：24所示)，并插入至pFastBac-Dual质粒(赛默飞Thermo，10712024)PH启动子下游的酶切位点BamH I(Thermo Scientific公司，FD0054)和Hind III(Thermo Scientific公司，FD0504)之间，获得供体质粒pFBD-R545HA-IntN-PAB-7XHIS(图7A)。
随后我们用供体质粒pFBD-R545HA-IntN-PAB-7XHis转化至含有Bacmid的DH10Bac感受态(唯地)，涂含有X-gal、IPTG、卡那霉素和庆大霉素抗性的平板，37℃培养2天后，挑取白色菌落获得并用M13-F/R引物进行PCR验证，挑取阳性克隆获得bR545HA-IntN-PAB-7XHis。
接着我们用bR545HA-IntN-PAB-7XHis转染贴壁Sf9(Spodoptera frugiperda clone 9，ATCC)细胞，具体步骤见Cellfectin II Reagent(Invitrogen公司，货号10362100)转染步骤，获得P1代重按组杆状病毒vAc R545HA-IntN-PAB-7XHis，将P1代vAc R545HA-IntN-PAB-7XHis以1∶100的体积比感染600mL SF-SFM培养基(沃美生物，S F10111-2)中悬浮培养的Sf9细胞系，细胞密度2×10 6/mL，5天后收获细胞培养物，对R545HA-IntN-PAB-7XHis蛋白表达情况和分布进行检测(图7B)。结果显示，融合蛋白多在细胞内表达，因此后续采用包涵体纯化的方式进行纯化。
实施例9.R545-HA2-IntN-PAB-7XHis蛋白纯化
为了获取展示R545-HA2的纳米颗粒，我们首先对R545HA-IntN-PAB-7XHis蛋白进行了纯化，纯化步骤如下：
(1)将vAc R545HA-IntN-PAB-7XHis感染后5天的600mL Sf9细胞2000g，10min离心，收集细胞沉淀；
(2)将500μL 100X蛋白酶复合物抑制剂(生工生物，货号：600387)加入50mL细胞裂解液(50mM Tris-HCl PH＝8，0.5M NaCl，1％NP40)，超声波细胞粉碎机(SCIENTZ-IID)，15s打，20s停，20min，显微镜镜检至细胞完全破碎。
(3)8000g 4℃离心20min，收集沉淀，用50mL pH＝8.5的50mM Tris-HCl重悬沉淀，加入50μL超级核酸酶(天地人和，PE001B)，室温孵育20min。
(4)8000g 4℃离心20min，收集沉淀，用50mL 2M尿素(50mM Tris-HCl pH＝8，0.5M NaCl，0.5％NP40，0.05％Tween)重悬沉淀，冰置5min。
(5)8000g 4℃离心20min，收集沉淀，重复步骤(4)。
(6)8000g 4℃离心20min，用50mL 8M尿素(50mM Tris-HCl pH＝8，0.5M NaCl)重悬沉淀，冷库旋转过夜，充分溶解沉淀。
(7)8000g 4℃离心20min，收集上清，过0.22μm的滤膜。
(8)取4mL High Affinity Ni-Charged Resin FF(金斯瑞，L00666)，用10mL平衡液(8M尿素，50mM Tris-HCl PH＝8，0.5M NaCl)混匀，800g，离心5min，收集Beads，反复清洗3次。
(9)用2mL平衡液重悬Beads，加入至8M尿素溶解物中，4℃旋转4h，2000g 4℃离心10min，收集Beads。
(10)用10mL的平衡液完全收集Beads上重力柱，收集部分流传液检测挂柱效率。
(11)加入20mL平衡液洗柱，至完全流尽。
(12)加入20mL洗柱液(80mM咪唑，8M尿素，50mM Tris-HCl pH＝8，0.5M NaCl)，至完全流尽。
(13)加入11mL洗脱液(300mM咪唑，8M尿素，50mM Tris-HCl pH＝8，0.5M NaCl)，弃最开始1mL的流传液，收集10mL洗脱液。
(14)加入dTT至终浓度20mM，4℃旋转过夜。
(15)将洗脱液稀释10倍(蛋白浓度小于50μg/mL)，如下进行梯度复性：
I.4M尿素，50mM Tris-HCl PH＝8，0.5M NaCl，0.4mM还原型谷胱甘肽，0.2mM氧化型谷胱甘肽，0.1％PEG6000，5％甘油，4h。
II.2M尿素，50mM Tris-HCl PH＝8，0.5M NaCl，0.4mM还原型谷胱甘肽，0.2mM氧化型谷胱甘肽，0.1％PEG6000，5％甘油，4h。
III.1M尿素，50mM Tris-HCl PH＝8，0.5M NaCl，0.4mM还原型谷胱甘肽，0.2mM氧化型谷胱甘肽，0.1％PEG6000，5％甘油，4h。
IV.50mM Tris-HCl PH＝8，0.5M NaCl，2mM dTT，0.1％PEG6000，5％甘油，4h。
V.50mM Tris-HCl PH＝8，0.5M NaCl，2mM dTT，5％甘油，4h。
VI.PBS PH＝7.4，2mM dTT，5％甘油4h。
(16)透析复性后的蛋白质用BCA(赛默飞，货号：23235)法检测浓度。分装，存于4℃备用(长存于-80℃)。
纯化后的蛋白质用WB和考马斯亮蓝染色均可看到正确大小(约70kDa)的条带(图8A)。
实施例10.纳米颗粒R545HAF的组装
内含肽(Intein)是一种可以自我剪切的肽段。如前所述，R545-HA2的C端融合内含肽的N端形成重组蛋白R545HA-IntN-PAB-7XHis。铁蛋白(Ferritin，Fn)的N端与接头gbl-intein(gb1为Protein G的B domin在此作为促溶标签)的C端融合形成重组蛋白gbl-intC-ferrtin后自装配成24聚体构成纳米颗粒；内含肽的N端特异性识别并切除暴露在所述纳米颗粒表面的铁蛋白；外源蛋白与铁蛋白共价交联而得R545-HA2-hFn，其也呈24聚体的纳米颗粒状态。
为了探究这一连接反应的有效性，我们先纯化了7XHIS-GB1-IntC-Fn(按照专利申请号201910421408.9进行)，具体纯化方式如下：
(1)将pET28a 7XHIS-GB1-IntC-Fn转化至BL21(擎科生物，货号：CD901-02)表达菌株。
(2)挑取单克隆菌株，接种至20mL的卡纳氯霉素双抗LB培养基中，37℃摇床过夜培养。
(3)第二天1∶25接种至500mL的卡纳氯霉素双抗的LB培养基，37℃、250rpm。
(4)培养至OD为0.8-1.0时，加入0.2mM IPTG进行诱导。25℃、250rpm诱导5h后，4000rpm、4℃、30分钟离心收集菌体，弃上清。
(5)用30mL的裂解液(50mM Tris，pH 7.5，150mM NaCl，1％Triton X-100)重悬500mL的LB培养细菌，而后超声破碎。
(6)超声结束后，12000rpm、4℃、30分钟离心，收集上清。
(7)每30mL的溶液加入4.5g的硫酸铵，4℃混匀15min。
(8)12000rpm、4℃、30分钟离心，收集沉淀。
(9)加入30mL的Tris-HCl溶液(0mM Tris，pH 7.5，150mM NaCl)溶解4℃过夜。
(10)BCA(赛默飞，货号：23235)测7XHIS-GB1-IntC-Fn蛋白浓度，分装，存于4℃备用(长存于-80℃)。
将纯化的7XHIS-GB1-IntC-Fn与纯化的R545HA-IntN-PAB-7XHis(实施例9中所纯化)在dTT浓度2mM的条件下进行连接，结果如图8B。可见R545HA-IntN-PAB能通过连接Fn形成大小约70kDa(糖基化修饰前67kDa)的连接产物R545HAF(图8B左二泳道70kDa条带)，并切割生成副产物IntN-PAB(17kDa)和gb1-IntC(11kDa)。
实施例10：纳米颗粒R545HAF在hACE2+小鼠体内的免疫原性
我们在hACE2+ICR小鼠体内验证了纳米颗粒疫苗R545HAF的免疫原性，在完成免疫后4周，评价免疫组合诱导针对RBD蛋白及H7蛋白的结合抗体滴度和针对SARS-CoV-2假病毒的中和抗体滴度。
本实施例中所用hACE2+ICR小鼠是一种COVID-19临床前模型，其为ICR小鼠背景下构建的SARS-CoV-2易感人血管紧张素转换酶2(hACE2)转基因小鼠，主要在其肺、心、肾和小肠中表达hACE2。该小鼠模型是SARS-CoV-2治疗药物和疫苗研发的重要工具。
实验步骤如下：将小鼠随机分为2组，根据免疫原分别命名为对照组和纳米颗粒组。具体免疫组合如表7所示，免疫方式为肌肉注射。
纳米颗粒组，即R545HAF产生针对RBD蛋白的结合抗体滴度如图9A所示，免疫结束后第四周，针对RBD蛋白的结合抗体滴度大多在400,000以上，部分能够达到3,000,000；同时比较第一针免疫后与第三针免疫后针对RBD蛋白的结合抗体数据可以发现，结合抗体滴度有了明显的提升(图9B)。同时，R545HAF也显示可有效诱导针对流感病毒H7的结合抗体。
免疫结束后4周，纳米颗粒组产生针对SARS-CoV-2假病毒的中和抗体滴度如图9C所示：大部分都在10,000左右，最高能到达92,172；同时比较第一针免疫后与第三针免疫后针对RBD蛋白的结合抗体数据可以发现，结合抗体滴度有了明显的提升(图9D)。
该实验证实，纳米颗粒疫苗R545HAF能够在hACE2+ICR小鼠体内同时诱导针对新冠及流感的结合抗体。同时纳米颗粒疫苗R545HAF能够有效诱导针对新冠的中和抗体，具有开发为抗流感及新冠疫苗的良好前景。
表7.R545HAF在hACE2+ICR小鼠体内免疫原性测试的免疫方案
Figure PCTCN2021141198-appb-000010
在本公开提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本公开的上述讲授内容之后，本领域技术人员可以对本公开作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
附录：序列表信息
Figure PCTCN2021141198-appb-000011

Claims (10)

  1. 一种免疫原性肽，其包含如下部分：
    (a)免疫原茎部：其包含流感病毒血凝素HA的HA2区域；
    (b)免疫原冠部：其包含病毒膜蛋白或其免疫原性片段，其中所述病毒膜蛋白的来源病毒不同于(a)中HA2区域的来源病毒；
    (c)可任选地，与前述部分连接的其他部分。
  2. 如权利要求1所述的免疫原性肽，其中，
    所述HA2区域的来源选自下组：H1～H18中任一种，尤其是来自于广泛流行的人流感H1、H2、H3以及多次出现的人感染禽流感H5与H7，例如源自2009年流行的H1(H1N1)、2013年流行的H7(H7N9)；
    例如，所述HA2区域的氨基酸序列如SEQ ID NO：1所示，或由具有SEQ ID NO：2所示序列的核苷酸分子编码。
  3. 如权利要求1所述的免疫原性肽，其中，所述免疫原冠部包含：来源不同于(a)中HA2区域的流感病毒；非流感病毒或其他流感毒株的膜蛋白或其免疫原性片段；
    所述免疫原冠部的来源选自下组：冠状病毒、艾滋病毒、流感病毒(例如流感病毒血凝素HA的HA1区域)、狂犬病毒、猪瘟病毒、蓝耳病病毒、麻疹病毒、埃博拉病毒、疱疹病毒、虫媒病毒(例如，寨卡病毒、流行性乙型脑炎病毒、森林脑炎病毒、登革病毒、汉坦病毒、出血热病毒)；
    例如，所述免疫原冠部的来源选自：冠状病毒SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV，例如包含冠状病毒的受体结合域的S1蛋白、或受体结合域(RBD)、或经改造的受体结合域(例如RBD区经末段半胱氨酸修饰形成sRBD区)或其免疫原性片段；
    例如，所述免疫原冠部选自冠状病毒SARS-CoV-2的S1，尤其是来自于S1的RBD区(例如其序列如SEQ ID NO：3所示或由SEQ ID NO：4的核苷酸分子编码)、或其修饰的RBD区(例如经末端Cys修饰改造的sRBD区，如SEQ ID NO：5所示肽段或由SEQ ID NO：6所示核苷酸分子编码)、或者RBD区的免疫原性片段(例如如SEQ ID NO：21所示肽段或由SEQ ID NO：22所示核苷酸分子编码)。
  4. 如权利要求1所述的免疫原性肽，其中，所述其他部分选自：
    免疫调节序列，例如IL-2、IL-7、IL-12、IL-18、IL-21、GM-CSF、CD40L、CD40刺激抗体、PD-1与PD-L1抗体、CTLA4抗体、趋化因子CXCL9、CXCL10、CXCL11、CXCL12、CXCL3、XCL1、CCL4、CCL20、霍乱毒素及其亚单位、细菌鞭毛蛋白、FimH、HIV p24、HIV gp41；
    使免疫原性肽能够形成纳米颗粒的部分，例如转铁蛋白(Fn，例如SEQ ID NO：7所示的肽分子或由SEQ ID NO：8所示核苷酸分子编码)；
    信号肽，例如CD33、CD8、CD16、小鼠IgG1抗体；
    使免疫原性肽能够表达在病毒载体表面的跨膜区，例如CD8跨膜区(CD8TM，例如SEQ ID NO：17所示的肽分子或由SEQ ID NO：18所示核苷酸分子编码)、HA2跨膜区、CD4跨膜区、gp41跨膜区；
    连接肽，例如(G4S) 3、(G4S) n、GSAGSAAGSGEF、(Gly) 6、EFPKPSTPPGSSGGAP、KESGSVSSEQLAQFRSLD、(Gly) 8、EGKSSGSGSESKST；
    标签，例如His-tag、AviTag、Calmodulin tag、polyglutamate tag、E-tag、FLAG tag、HA-tag、Myc-tag、S-tag、SBP-tag、Sof-tag 1、Sof-tag3、Strep-tag、TC tag、V5 tag、T7 tag、VSV tag、Xpress tag、3X FLAG tag、Isopep tag、Spytag、Snoop tag和PNE tag。
  5. 如权利要求1-4中任一项所述的免疫原性肽，其中，所述免疫原性肽包含：与HA2区域连接的RBD区或sRBD区或其免疫原性片段，以及可任选的与前述部分连接的其他部分；
    例如，所述免疫原性肽包含：与HA2区域连接的RBD区或sRBD区(例如SEQ ID NO：5所示的肽分子或由SEQ ID NO：6所示核苷酸分子编码)以及Fn区(例如SEQ ID NO：7所示的Fn肽分子或由SEQ ID NO：8所示核苷酸分子编码)；与HA2区域连接的RBD区或sRBD区以及CD8跨膜区(例如SEQ ID NO：17所示的CD8跨膜区肽分子或由SEQ ID NO：18所示核苷酸分子编码)；与HA2区域连接的RBD区免疫原性片段(例如SEQ ID NO：21所示的肽分子或由SEQ ID NO：22所示核苷酸分子编码)以及Fn区；
    例如所述免疫原性肽的氨基酸序列如SEQ ID NO：9或15或25所示，或者所述免疫原性肽的编码序列如SEQ ID NO：10或16或26所示。
  6. 一种核苷酸分子，其编码如权利要求1-5中任一项所述的免疫原性肽；
    例如，所述HA2区域的编码序列包含SEQ ID NO：2所示的核苷酸序列；所述免疫原冠部的编码序列包含SEQ ID NO：4所示的核苷酸序列或SEQ ID NO：6所示的核苷酸序列或SEQ ID NO：22所述的核苷酸序列；和/或所述其他部分的编码序列包含SEQ ID NO：8所示或SEQ ID NO：18所示的核苷酸序列；
    例如，所述核苷酸分子的序列如SEQ ID NO：10或16或26所示。
  7. 一种载体，其包含如权利要求6所述的核苷酸分子。
  8. 一种宿主细胞，其包含如权利要求6所述的核苷酸分子或如权利要求7所述的载体或能表达如权利要求1-5中任一项所述的免疫原性肽，
    例如所述宿主细胞为哺乳动物细胞或昆虫细胞，如HEK293、HeLa、K562、CHO、NS0、SP2/0、PER.C6、Vero、RD、BHK、HT 1080、A549、Cos-7、ARPE-19和MRC-5细胞；High Five、Sf9、Se301、SeIZD2109、SeUCR1、Sf9、Sf900+、Sf21、BTI-TN-5B1-4、MG-1、Tn368、HzAm1、BM-N、Ha2302、Hz2E5以及Ao38。
  9. 一种能同时诱导针对流感病毒和另一非流感病毒的免疫应答的疫苗，其包含如权利要求1-5中任一项所述的免疫原性肽、如权利要求6所述的核苷酸分子、如权利要求7所述的载体和/或如权利要求8所述的宿主细胞；
    例如，所述疫苗为核酸疫苗(DNA或RNA疫苗)、重组蛋白亚单位疫苗、重组病毒载体疫苗、重组细菌载体疫苗、病毒样颗粒疫苗、纳米颗粒疫苗、细胞载体疫苗；
    例如，所述非流感病毒选自：冠状病毒、艾滋病毒、狂犬病毒、猪瘟病毒、蓝耳病病毒、麻疹病毒、埃博拉病毒、疱疹病毒、虫媒病毒(例如，寨卡病毒、流行性乙型脑炎病毒、森林脑炎病毒、登革病毒、汉坦病毒、出血热病毒)；
    例如，所述疫苗为病毒载体疫苗，所述病毒载体选自：痘病毒(例如天坛株、北美疫苗株、惠氏衍生株、李斯特株、安卡拉衍生株、哥本哈根株和纽约株)、腺病毒(Ad5、Ad11、Ad26、Ad35、AdC68)、腺相关病毒、单纯疱疹病毒、麻疹病毒、呼肠弧病毒、弹状病毒、森林脑炎病毒、流感病毒、呼吸道合胞病毒、 脊髓灰质炎病毒；
    例如，所述疫苗包含佐剂或与佐剂联合使用，例如所述佐剂选自：铝佐剂、霍乱毒素及其亚单位、寡脱氧核苷酸、锰离子佐剂、胶体锰佐剂、弗氏佐剂、MF59佐剂、QS-21佐剂、Poly I：C及其他TLR配体、GM-CSF、IL-2、IL-3、IL-7、IL-11、IL-12、IL-18、IL-21；
    例如，所述疫苗的形式适于肌肉接种、皮内接种、皮下接种、滴鼻、雾化吸入、生殖道、直肠、口服或上述不同接种方式的组合(例如肌肉注射+滴鼻)；
    例如，所述疫苗的形式适于进行2种或以上的组合接种(例如联合接种或序贯接种)，如与冠状病毒(例如SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV)的S或S1疫苗进行前后序贯接种，或与流感病毒的HA或HA2(例如源自H1～H18中任一种的HA或HA2)疫苗进行前后序贯接种。
  10. 如权利要求1-5中任一项所述的免疫原性肽、如权利要求6所述的核苷酸分子、如权利要求7所述的载体和/或如权利要求8所述的宿主细胞在制备用于同时预防或治疗流感病毒和另一非流感病毒的药物中的应用，
    例如，所述非流感病毒选自：冠状病毒、艾滋病毒、狂犬病毒、猪瘟病毒、蓝耳病病毒、麻疹病毒、埃博拉病毒、疱疹病毒、虫媒病毒(例如，寨卡病毒、流行性乙型脑炎病毒、森林脑炎病毒、登革病毒、汉坦病毒、出血热病毒)。
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