WO2023104154A1 - 抗原性多肽及其用途 - Google Patents

抗原性多肽及其用途 Download PDF

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WO2023104154A1
WO2023104154A1 PCT/CN2022/137554 CN2022137554W WO2023104154A1 WO 2023104154 A1 WO2023104154 A1 WO 2023104154A1 CN 2022137554 W CN2022137554 W CN 2022137554W WO 2023104154 A1 WO2023104154 A1 WO 2023104154A1
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seq
polypeptide
amino acid
acid sequence
antigenic fragment
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PCT/CN2022/137554
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English (en)
French (fr)
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胡兰靛
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安达生物药物开发(深圳)有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • the present disclosure relates to isolated polypeptides and antigenic fragments thereof, and their use for inducing an immune response, diagnosing and/or treating disease.
  • Novel coronavirus pneumonia has become a global pandemic, and its pathogen is SARS-CoV-2 (Zhou, P., et al., A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature, 2020.579(7798):p.270-273). As of February 24, 2021, a total of 111,762,993 confirmed cases have been reported worldwide, and 101,778 confirmed cases in China. To control this pandemic, one of the fundamental tasks is to perform rapid, reliable and affordable diagnostics. Until now, detection of viral infection has relied on extensive nucleic acid testing screening of isolated individuals.
  • the sensitivity of nucleic acid testing largely depends on the course and type of clinical COVID-19 syndrome, the site of collection, and the transportation and storage of the specimen. According to reports, in COVID-19 patients, the false negative rate of nucleic acid testing is about 30% (Ai,T.,et al.,Correlation of Chest CT and RT-PCR Testing for Coronavirus Disease 2019(COVID-19) in China : A Report of 1014 Cases. Radiology, 2020.296(2): p.E32-E40). Recent nucleic acid test positivity surveys have shown that COVID-19 cases in different countries and regions, especially asymptomatic cases, have been greatly underestimated. Therefore, due to the limited sensitivity of nucleic acid testing, some asymptomatic infection cases may have been overlooked.
  • Serum antibody detection which is easier to detect and interpret, can effectively improve the reliability of the results and can be used as a double insurance for diagnosis.
  • serum samples from COVID-19 patients were collected within 19 days after the onset of symptoms, the sensitivity of the IgG antibody response to the new coronavirus could be close to 100% (Long, Q.X., et al., Antibody responses to SARS-CoV-2 in patients with COVID -19. Nat Med, 2020.26(6): p.845-848).
  • antibodies with strong neutralizing responses against viral proteins can be effective against viruses.
  • SARS-CoV-2 is similar to bat SARS-CoV and SARS-CoV, belongs to the genus Betacoronavirus, and is most closely related to the Bat-CoV RaTG13 virus (Zhou, P., et al., supra).
  • the genome sequence of the new coronavirus is about 80% similar to that of the SARS coronavirus.
  • the new coronavirus can encode 29 proteins: 4 structural proteins, 9 auxiliary proteins and 16 non-structural proteins (Nsp) (Wu, A., et al., Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China.
  • Structural proteins include membrane protein (M protein), nucleocapsid protein (N protein), spike protein (S protein), and envelope protein (E protein), which are exposed on the surface of the virus, while non-structural proteins wrap the inside of the virus .
  • M protein membrane protein
  • N protein nucleocapsid protein
  • S protein spike protein
  • E protein envelope protein
  • Nsp1 inhibits the antiviral response
  • Nsp15 and Orf3a activate interferon respectively
  • NLRP3 and Orf7a are viral proteins responsible for inducing cell death.
  • the current serum antibody detection generally uses the S protein, especially the RBD region, as the target of the antibody response (Long, Q.X., et al., Antibody responses to SARS-CoV-2 in patients with COVID- 19. Nat Med, 2020.26(6): p.845-848).
  • Therapeutic antibodies and vaccines used to treat COVID-19 also mainly target the S protein, especially the RBD region (Cao, Y., et al., Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent Patients'B Cells. Cell, 2020.182(1):p.73-84.e16).
  • S protein-based serological detection The key reagent of S protein-based serological detection is recombinant S protein.
  • S protein The key reagent of S protein-based serological detection is recombinant S protein.
  • the production of S protein is difficult and expensive (Petherick, A., Developing antibody tests for SARS-CoV-2. Lancet, 2020.395(10230): p.1101-1102).
  • inconsistencies between different manufacturers or even between batches may lead to differences in test results (Lisboa, B.M., et al., Diagnostic accuracy of serological tests for covid-19: systematic review and meta-analysis.BMJ,2020.370:p.m2516).
  • the present disclosure provides an isolated polypeptide or an antigenic fragment thereof, the polypeptide has an amino acid sequence selected from the following amino acid sequence, or one or several amino acid residues have been substituted or deleted in the following amino acid sequence , amino acid sequence obtained after insertion and/or addition:
  • LGLAAIMQLFFSYFA SEQ ID NO: 2
  • VLSTFISAARQGFVD (SEQ ID NO: 3);
  • EKYCALAPNMMVTNN (SEQ ID NO: 12);
  • ATYKPNTWCIRCLWS (SEQ ID NO: 13);
  • KVDTANPKTPK (SEQ ID NO: 17);
  • LGSALLEDEFTPFDV (SEQ ID NO: 18);
  • VPLNIIPLTTAAKLM SEQ ID NO: 21
  • FCAFAVDAAKAYKDY (SEQ ID NO: 23);
  • NLDKSAGFPFNKWGK (SEQ ID NO: 25);
  • VVYRGTTTYKLNVGD (SEQ ID NO: 26);
  • PAEIVDTVSALVYDN SEQ ID NO: 28
  • TAFVTNVNASSSEAF (SEQ ID NO: 32);
  • AALALLLLDRLNQLE (SEQ ID NO: 37);
  • the present disclosure provides an isolated polypeptide or an antigenic fragment thereof, the polypeptide has an amino acid sequence selected from the following amino acid sequence, or one or several amino acid residues have been substituted or deleted in the following amino acid sequence , amino acid sequence obtained after insertion and/or addition:
  • LGLAAIMQLFFSYFA SEQ ID NO: 2
  • VLSTFISAARQGFVD (SEQ ID NO: 3);
  • EKYCALAPNMMVTNN (SEQ ID NO: 12);
  • ATYKPNTWCIRCLWS (SEQ ID NO: 13);
  • KVDTANPKTPK (SEQ ID NO: 17);
  • LGSALLEDEFTPFDV (SEQ ID NO: 18);
  • VPLNIIPLTTAAKLM SEQ ID NO: 21
  • FCAFAVDAAKAYKDY (SEQ ID NO: 23);
  • NLDKSAGFPFNKWGK (SEQ ID NO: 25);
  • VVYRGTTTYKLNVGD (SEQ ID NO: 26);
  • AKHYVYIGDPAQLPA SEQ ID NO: 27
  • PAEIVDTVSALVYDN SEQ ID NO: 28
  • TAFVTNVNASSSEAF (SEQ ID NO: 32);
  • the antigenic fragment is at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, or at least 10 amino acids in length. amino acid.
  • the present disclosure provides an isolated polypeptide or an antigenic fragment thereof, the polypeptide has an amino acid sequence selected from the following amino acid sequence, or one or several amino acid residues have been substituted or deleted in the following amino acid sequence , amino acid sequence obtained after insertion and/or addition:
  • GWEIVKFISTCACEI SEQ ID NO: 40
  • WADNNCYLATALLTL (SEQ ID NO: 41);
  • ASDTYACWHHSIGFD (SEQ ID NO: 48).
  • the antigenic fragment is at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids or at least 10 amino acids in length.
  • the present disclosure provides an isolated antibody that binds a polypeptide or an antigenic fragment thereof according to the first, second or third aspect of the present disclosure.
  • the antibody is selected from an IgG antibody, an IgM antibody, and an IgA antibody. In some embodiments, the antibody is selected from IgG1, IgG2, IgG3 and IgG4 antibodies. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibodies are human antibodies.
  • the present disclosure provides a composition comprising one or more polypeptides or antigenic fragments thereof according to the first, second or third aspect of the present disclosure.
  • the composition comprises a polypeptide having the amino acid sequence shown in SEQ ID NO: 10 or an antigenic fragment thereof and a polypeptide having the amino acid sequence selected from SEQ ID NO: 1-9 and 11-48 One or more of the polypeptides of the indicated amino acid sequences or antigenic fragments thereof.
  • the composition comprises a polypeptide having an amino acid sequence shown in SEQ ID NO: 10 or an antigenic fragment thereof and a polypeptide having an amino acid sequence selected from SEQ ID NO: 1-9 and 11-48.
  • the composition comprises a polypeptide having an amino acid sequence shown in SEQ ID NO: 10 or an antigenic fragment thereof and a polypeptide having an amino acid sequence selected from SEQ ID NO: 1-9 and 11-39.
  • the composition comprises a polypeptide having an amino acid sequence shown in SEQ ID NO: 10 or an antigenic fragment thereof and a polypeptide having an amino acid sequence selected from SEQ ID NO: 1-9 and 11-39. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38.
  • the composition comprises a polypeptide having an amino acid sequence shown in SEQ ID NO: 10 or an antigenic fragment thereof and a polypeptide having an amino acid sequence selected from SEQ ID NO: 1-9 and 11-35.
  • the composition comprises a polypeptide having an amino acid sequence shown in SEQ ID NO: 10 or an antigenic fragment thereof and a polypeptide having an amino acid sequence selected from SEQ ID NO: 1-9 and 11-35. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 types.
  • the composition comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10, 7, 35, 29, 23, 30, 2, 21, and 12, or an antigenic fragment thereof.
  • the composition comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10, 12, 35, 14, 30, 2, 3, 13, and 19, or an antigenic fragment thereof.
  • the composition comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10, 35, 12, and 19, or an antigenic fragment thereof.
  • the composition comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10 and 33, or an antigenic fragment thereof.
  • the composition comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10, 32, and 15, or an antigenic fragment thereof.
  • the composition comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10, 21, 30, 15, 12, 35, 23, 19, and 20, or an antigenic fragment thereof.
  • the antigenic fragment is at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, or at least 10 amino acids in length.
  • the composition is a vaccine composition, and optionally further comprises one or more adjuvants.
  • the present disclosure provides a composition comprising one or more antibodies according to the fourth aspect of the present disclosure, and optionally a carrier and/or excipient.
  • the present disclosure provides a nucleotide sequence encoding a polypeptide or an antigenic fragment thereof according to the first, second or third aspect of the present disclosure, or according to the present disclosure The antibody of the fourth aspect.
  • the present disclosure also provides a vector comprising the nucleotide sequence.
  • the present disclosure provides an array, the array comprising a solid substrate, and a polypeptide immobilized on the solid substrate, the polypeptide comprising at least one polypeptide according to the first or second aspect of the present disclosure or an antigenic fragment thereof.
  • the polypeptides in the array comprise at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, or at least 35 polypeptides according to the present disclosure.
  • the polypeptide or antigenic fragment thereof described in the first or second aspect are examples of the polypeptides in the array.
  • the polypeptides in the array comprise a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10 or an antigenic fragment thereof and a polypeptide having an amino acid sequence set forth in SEQ ID NO: 1-9 and 11-39.
  • the polypeptides in the array comprise a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10 or an antigenic fragment thereof and a polypeptide having an amino acid sequence set forth in SEQ ID NO: 1-9 and 11-39. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38.
  • the polypeptides in the array comprise a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10 or an antigenic fragment thereof and a polypeptide having an amino acid sequence set forth in SEQ ID NO: 1-9 and 11-35.
  • the polypeptides in the array comprise a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10 or an antigenic fragment thereof and a polypeptide having an amino acid sequence set forth in SEQ ID NO: 1-9 and 11-35. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 types.
  • the polypeptides in the array comprise a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10, 7, 35, 29, 23, 30, 2, 21, and 12, or an antigenic fragment thereof.
  • the polypeptides in the array comprise a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10, 12, 35, 14, 30, 2, 3, 13, and 19, or an antigenic fragment thereof.
  • polypeptides in the array comprise polypeptides having the amino acid sequences set forth in SEQ ID NOs: 10, 35, 12, and 19, or antigenic fragments thereof.
  • polypeptides in the array comprise polypeptides having the amino acid sequences set forth in SEQ ID NOs: 10 and 33, or antigenic fragments thereof.
  • polypeptides in the array comprise polypeptides having the amino acid sequences set forth in SEQ ID NOs: 10, 32, and 15, or antigenic fragments thereof.
  • the polypeptides in the array comprise a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10, 21, 30, 15, 12, 35, 23, 19, and 20, or an antigenic fragment thereof.
  • the antigenic fragment is at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, or at least 10 amino acids in length.
  • the polypeptides in the array comprise a plurality of polypeptides or antigenic fragments thereof according to the first or second aspect of the present disclosure, wherein the plurality of polypeptides or antigenic fragments thereof comprise at least one derived from SARS- A polypeptide or an antigenic fragment thereof of the CoV-2 ORF1ab polyprotein, and at least one polypeptide derived from the SARS-CoV-2 S protein or an antigenic fragment thereof.
  • the plurality of polypeptides further comprises at least one polypeptide derived from a SARS-CoV-2 N protein or an antigenic fragment thereof.
  • the present disclosure provides an array, the array comprising a solid substrate, and a polypeptide immobilized on the solid substrate, the polypeptide comprising at least one polypeptide or its antigen according to the third aspect of the present disclosure sex clips.
  • the polypeptides in the array comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or all 9 polypeptides according to the third aspect of the present disclosure.
  • the polypeptides in the array comprise a plurality of polypeptides or antigenic fragments thereof according to the third aspect of the present disclosure, wherein the plurality of polypeptides or antigenic fragments thereof comprise at least one polypeptide derived from SARS-CoV-2 A polypeptide of ORF1ab polyprotein or its antigenic fragment, at least one polypeptide derived from SARS-CoV-2 S protein or its antigenic fragment, at least one polypeptide derived from SARS-CoV-2 N protein or its antigenicity fragment.
  • the polypeptide is immobilized on a solid substrate by coupling with BSA.
  • the array is a polypeptide chip.
  • the present disclosure provides a method of detecting the presence of antibodies to SARS-CoV-2 in a subject comprising:
  • the method is used to diagnose whether said subject is infected with SARS-CoV-2.
  • the present disclosure provides a method for prognosing a subject infected with SARS-CoV-2 comprising:
  • the antibodies are IgG antibodies and/or IgM antibodies.
  • the sample is a blood sample, such as serum or plasma.
  • the present disclosure provides a method of inducing an immune response against SARS-CoV-2 in a subject, comprising administering to the subject a method according to the first, second or third aspect of the present disclosure.
  • the polypeptide or antigenic fragment thereof, or the composition according to the fifth aspect of the present disclosure are particularly useful as immunosuppression kits.
  • the present disclosure provides a method for preventing and/or treating a disease caused by SARS-CoV-2 infection in a subject, which includes administering to the subject the first, second or The polypeptide or antigenic fragment thereof in the third aspect, or the composition according to the fifth aspect of the present disclosure.
  • the method comprises administering to the subject at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, or at least 30 of the first, A combination of polypeptides or antigenic fragments thereof as described in the second or third aspect.
  • the method comprises administering to a subject a polypeptide comprising an amino acid sequence shown in SEQ ID NO: 10 or an antigenic fragment thereof and a polypeptide having an amino acid sequence selected from SEQ ID NO: 1-9 and 11-48 A combination of one or more of amino acid sequence polypeptides or antigenic fragments thereof.
  • the method comprises administering to a subject a polypeptide comprising an amino acid sequence shown in SEQ ID NO: 10 or an antigenic fragment thereof and a polypeptide having an amino acid sequence selected from SEQ ID NO: 1-9 and 11-48 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 of a polypeptide of amino acid sequence or an antigenic fragment thereof , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 , 46 or 47 combinations.
  • the method comprises administering to a subject a polypeptide comprising an amino acid sequence shown in SEQ ID NO: 10 or an antigenic fragment thereof and a polypeptide having an amino acid sequence selected from SEQ ID NO: 1-9 and 11-39 A combination of one or more of amino acid sequence polypeptides or antigenic fragments thereof.
  • the method comprises administering to a subject a polypeptide comprising an amino acid sequence shown in SEQ ID NO: 10 or an antigenic fragment thereof and a polypeptide having an amino acid sequence selected from SEQ ID NO: 1-9 and 11-35 A combination of one or more of amino acid sequence polypeptides or antigenic fragments thereof.
  • the method comprises administering to a subject a polypeptide comprising an amino acid sequence shown in SEQ ID NO: 10 or an antigenic fragment thereof and a polypeptide having an amino acid sequence selected from SEQ ID NO: 1-9 and 11-35 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 of a polypeptide of amino acid sequence or an antigenic fragment thereof , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 combinations.
  • the method comprises administering to the subject a combination comprising a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10, 7, 35, 29, 23, 30, 2, 21, and 12, or an antigenic fragment thereof .
  • the method comprises administering to the subject a combination comprising a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10, 12, 35, 14, 30, 2, 3, 13, and 19, or an antigenic fragment thereof .
  • the method comprises administering to the subject a combination comprising a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10, 35, 12, and 19, or an antigenic fragment thereof.
  • the method comprises administering to the subject a combination comprising a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10 and 33, or an antigenic fragment thereof.
  • the method comprises administering to the subject a combination comprising a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10, 32, and 15, or an antigenic fragment thereof.
  • the method comprises administering to the subject a combination comprising a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10, 21, 30, 15, 12, 35, 23, 19, and 20, or an antigenic fragment thereof .
  • the antigenic fragment is at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, or at least 10 amino acids in length.
  • the method comprises the step of administering to the subject a plurality of polypeptides or antigenic fragments thereof according to the first, second or third aspect of the present disclosure, wherein the plurality of polypeptides or antigenic fragments thereof comprise At least one polypeptide derived from SARS-CoV-2 ORF1ab polyprotein or its antigenic fragment, at least one polypeptide derived from SARS-CoV-2 S protein or its antigenic fragment, and at least one polypeptide derived from SARS-CoV-2 A polypeptide of CoV-2 N protein or an antigenic fragment thereof.
  • the present disclosure provides a method of treating a disease caused by SARS-CoV-2 infection in a subject, comprising administering to the subject an antibody according to the fourth aspect of the present disclosure .
  • FIG. 2 Schematic diagram of the detection of serum samples using the peptide chip.
  • the peptides of the peptide library were connected to BSA and printed on the chip. After adding serum samples to react, the chip was scanned with LuxScan10K-A (CapitalBio Company, Beijing, China), and the parameters were set to 95% laser power/PMT550 and 95% laser power/ PMT480. Fluorescence intensity data were obtained by GenePixPro6.0 software (Molecular Devices, CA, USA), where green fluorescence indicated the intensity of IgG, red fluorescence indicated the intensity of IgM, and yellow was the superposition of green and red.
  • ORF open reading frame
  • S spike protein
  • E envelope protein
  • N nucleocapsid protein
  • M membrane protein.
  • Figure 4 Signal intensity of dominant and persistent IgG and IgM epitopes. Each point represents pooled sera from healthy donors or serum samples from different patients collected from indicated time points after symptom onset.
  • Figure 5 Schematic representation of the location of dominant and persistent epitopes on the 3D structure of monomeric (a) and trimeric (b) S protein (PBD ID: 6VXX).
  • FIG. 6 Epitope conservation analysis of early SARS-CoV-2 (Wuhan-Hu-1 strain) and six emerging variant strains. Black dots represent the same amino acid residues as the Wuhan-Hu-1 strain.
  • Figure 7 a. IgG and IgM recognition frequency of subdominant epitopes in patient serum samples collected at various time points after disease onset. b. Signal intensity kinetics of identified subdominant epitopes over time. Each point represents a different patient's serum sample obtained from a COVID-19 individual at the indicated time point after the onset of symptoms. Dotted lines indicate positive response cutoffs for each peptide.
  • FIG. 8 Detailed structural analysis of the C-terminal dimerization domain epitope of N protein (PDB ID: 6YUN).
  • Figure 10 Signal intensities of epitopes at three sampled time points after symptom onset. Each point represents sera from an individual patient collected at the indicated time point after symptom onset, and dashed lines indicate the positive response cutoff for each peptide. "*” represents p ⁇ 0.05, and "**” represents p ⁇ 0.01.
  • the present disclosure relates to isolated polypeptides or antigenic fragments thereof, antibodies that bind the isolated polypeptides or antigenic fragments thereof, compositions comprising the isolated polypeptides or antigenic fragments thereof, and compositions comprising the isolated polypeptides or antigenic fragments thereof array of .
  • the present disclosure also provides methods for detecting the presence of antibodies against SARS-CoV-2 in a subject, methods for prognosing a subject infected with SARS-CoV-2, and inducing antibodies against SARS-CoV-2 in a subject. 2, and methods of preventing and/or treating diseases caused by SARS-CoV-2 infection in a subject.
  • polypeptide refers to a compound composed of amino acid residues covalently linked by peptide bonds, and does not limit the minimum length of the product.
  • the above terms include peptides, oligopeptides, polypeptides, dimers (heterologous and homologous), multimers (heterologous and homologous) and the like.
  • An “isolated” polypeptide refers to a polypeptide that has been removed from its natural environment or has been artificially synthesized using standard molecular biology techniques and expression systems.
  • polypeptides of the present disclosure can be isolated from wild-type or mutant SARS-CoV-2 viral proteins, or can be synthesized using commercially automated methods (e.g., total solid-phase synthesis, partial solid-phase synthesis, fragment combination, or solution synthesis) Chemically synthesized.
  • Protein and polypeptide encompass full-length proteins and fragments thereof.
  • the term also includes post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like.
  • a polypeptide also refers to a variant obtained by modifying the amino acid sequence of a native protein or polypeptide.
  • amino acid sequence modifications include substitutions, deletions, insertions and additions.
  • substitution means that at least one residue has been removed and a different residue inserted in its place.
  • Amino acid substitutions are usually single residues but can occur at many different positions simultaneously.
  • a “deletion” is characterized by the removal of one or more amino acid residues from the sequence. Typically no more than about 2-6 residues will be deleted at any one point within the protein molecule.
  • “Insertion” refers to intrasequence insertion of single or multiple amino acid residues, while “addition” refers to amino and/or carboxy terminal fusions. Insertions will generally be smaller insertions than additions, eg, about 1-4 residues.
  • variants with respect to an amino acid sequence includes any substitution, change, modification, replacement, deletion or addition from that sequence or one (or more) amino acid(s) to that sequence. Substitutions, deletions, insertions, additions, or any combination thereof may be contained in a single variant so long as the polypeptide variant is antigenic.
  • Immunogenicity also known as “antigenicity” and “immunoreactivity” refers to the ability of a protein or polypeptide to generate antibodies or induce an antibody response in a subject.
  • Antigenic fragment refers to a polypeptide fragment capable of inducing an immune response.
  • An antigenic fragment may be, for example, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids or at least 10 amino acids in length, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.
  • Antigenic fragments can be produced by methods known to those skilled in the art.
  • epitope or “antigenic epitope” are used interchangeably, are terms of the art, and refer to a localized region of an antigen to which an antibody can specifically bind.
  • An epitope can be, for example, contiguous amino acids of a polypeptide (linear or continuous epitope) or an epitope can be, for example, from two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous or non-contiguous). gauge).
  • binding or “specifically binding” generally refer to a non-covalent association between two or more entities, which is defined as the separation between an antibody and the corresponding epitope at less than about 10 -5 M , eg, binds the antigen with an affinity (K D ) of less than about 10 ⁇ 6 M, 10 ⁇ 7 M, 10 ⁇ 8 M, 10 ⁇ 9 M, or 10 ⁇ 10 M or less.
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. Determination of binding constants is known to those skilled in the art.
  • an “antibody” is an immunoglobulin molecule that is capable of specifically binding to a target through at least one antigen recognition site located in the variable region of the immunoglobulin molecule.
  • References to antibodies herein should be understood in their broadest sense.
  • Antibodies include antibodies of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), and the antibody need not be of any particular class. Any antibody referred to in this disclosure may be monoclonal or polyclonal.
  • the term “monoclonal antibody” refers to a homogeneous population of antibodies, while the term “polyclonal antibody” refers to a heterogeneous population of antibodies. These two terms should not be construed as limiting the source of the antibody or the manner in which it was prepared.
  • neutralizing refers to an antigen binding molecule, scFv, antibody or fragment thereof that binds a ligand and prevents or reduces the biological effect of the ligand.
  • the antigen binding molecule, scFv, antibody or fragment thereof directly blocks the binding site on the ligand or alters the binding ability of the ligand through indirect means (eg, structural or energetic changes in the ligand).
  • the antigen binding molecule, scFv, antibody or fragment thereof prevents the biological function of the protein to which it binds.
  • nucleic acid refers to a sequence of any length consisting essentially of nucleotides, such as deoxyribonucleotides and/or ribonucleotides. Oligomers and Polymers. Nucleic acids may contain purine and/or pyrimidine bases and/or other natural (e.g. xanthine, inosine, hypoxanthine), chemically or biochemically modified (e.g. methylation), non-natural or derived nucleotide bases .
  • natural e.g. xanthine, inosine, hypoxanthine
  • biochemically modified e.g. methylation
  • the backbone of a nucleic acid may comprise sugar and phosphate groups normally found in RNA or DNA, and/or one or more modified or substituted sugars and/or one or more modified or substituted phosphate groups. Modifications of phosphate groups or sugars can be introduced to improve stability, resistance to enzymatic degradation, or some other useful property.
  • a "nucleic acid” can be, for example, double-stranded, partially double-stranded or single-stranded. When single-stranded, the nucleic acid can be the sense or antisense strand.
  • a "nucleic acid” can be circular or linear.
  • nucleic acid encompasses DNA and RNA, including genomic, pre-mRNA, mRNA, cDNA, recombinant or synthetic nucleic acids comprising vectors.
  • RNA including genomic, pre-mRNA, mRNA, cDNA, recombinant or synthetic nucleic acids comprising vectors.
  • polynucleotides may be modified by any method available in the art.
  • an infection means invasion by proliferation and/or presence of a virus in a cell or individual.
  • an infection refers to an "active" infection, that is, an infection in which the virus is replicating in the cell or individual.
  • Such infections are characterized by the spread of the virus from the cells, tissues and/or organs it originally infected into other cells, tissues and/or organs.
  • Infection may also refer to a latent infection, ie an infection in which the virus is not replicating.
  • an infection refers to a pathological state caused by a virus present in a cell or an individual, or refers to a pathological state caused by a virus attacking a cell or an individual.
  • proteins and polypeptides described herein may refer to variants of native proteins or polypeptides having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% %, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, provided that the variant retains the original function or activity of the native protein or polypeptide.
  • the present disclosure provides an isolated polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 1-39, or an antigenic fragment thereof.
  • the present disclosure provides an isolated polypeptide, or an antigenic fragment thereof, comprising an amino acid having at least 50% identity to an amino acid sequence selected from any one of SEQ ID NOs: 1-39 Sequences, such as amino acid sequences having at least 60% identity, at least 70% identity, at least 80% identity, at least 90% identity or 100% identity, such as having 50%, 55%, 60%, 65%, Amino acid sequences that are 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.
  • the antigenic fragment of the polypeptide is at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids or at least 10 amino acids in length, e.g., 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.
  • the present disclosure also provides an isolated polypeptide or an antigenic fragment thereof, the polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 1-35.
  • the present disclosure provides an isolated polypeptide, or an antigenic fragment thereof, comprising an amino acid having at least 50% identity to an amino acid sequence selected from any one of SEQ ID NOs: 1-35 Sequences, such as amino acid sequences having at least 60% identity, at least 70% identity, at least 80% identity, at least 90% identity or 100% identity, such as having 50%, 55%, 60%, 65%, Amino acid sequences that are 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.
  • the antigenic fragment of the polypeptide is at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids or at least 10 amino acids in length, e.g., 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.
  • the present disclosure also provides an isolated polypeptide or an antigenic fragment thereof, the polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 40-48.
  • the present disclosure provides an isolated polypeptide, or an antigenic fragment thereof, comprising an amino acid having at least 50% identity to an amino acid sequence selected from any one of SEQ ID NOs: 40-48 Sequences, such as amino acid sequences having at least 60% identity, at least 70% identity, at least 80% identity, at least 90% identity or 100% identity, such as having 50%, 55%, 60%, 65%, Amino acid sequences that are 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.
  • the antigenic fragment of the polypeptide is at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids or at least 10 amino acids in length, e.g., 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.
  • sequence identity refers to the degree of identity between any given query sequence and the subject sequence.
  • identity can be calculated after the two sequences have been aligned to achieve the highest level of identity, eg gaps can be introduced.
  • Another method of computing identity can be implemented by published algorithms. Non-limiting examples of such mathematical algorithms include the algorithm of Myers and Miller (1988) CABIOS 4:11-17, the local homology algorithm of Smith et al.
  • the program can be suitably executed by a computer.
  • Examples of such programs include, but are not limited to, the CLUSTAL of the PC/Gene programs, the ALIGN program (Version 2.0), and the GAP, BESTFIT, BLAST, FASTA, and TFASTA of the Wisconsin Genetics Package. Alignments using these programs can be performed, for example, by using initial parameters.
  • isolated polypeptides of the present disclosure and antigenic fragments thereof include variants, which may comprise one or more amino acid modifications. Substitutions, deletions, insertions, additions, or any combination thereof may be contained in a single variant so long as the polypeptide variant is antigenic. Methods and techniques for making variants are well known to those skilled in the art.
  • Amino acid substitutions as used herein may be conservative or non-conservative substitutions.
  • amino acids can be divided into groups based on their physical properties. Examples of such groupings include, but are not limited to, charged amino acids, uncharged amino acids, polar uncharged amino acids, and hydrophobic amino acids.
  • Conservative amino acid substitutions may involve substituting a natural amino acid residue with a non-natural residue such that there is little or no effect on the size, polarity, charge, hydrophobicity or hydrophilicity of the amino acid residue at that position, specifically, No reduction in antigenicity.
  • substitution with a conservative amino acid refers to exchangeability with residues having similar side chains.
  • Families of amino acid residues having side chains have been defined in the art. These families include amino acids with the following side chains: basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar Side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g.
  • substitutions are not conservative.
  • substitutions may be made with amino acids that alter some property or aspect of the polypeptide.
  • non-conservative amino acid substitutions can be made, e.g., to alter the structure of the polypeptide, to alter the binding properties of the polypeptide (e.g., to increase or decrease the binding affinity of the polypeptide, and/or to increase or decrease the binding specificity of the polypeptide).
  • the present disclosure also relates to an isolated antibody that binds a polypeptide described in the present disclosure or an antigenic fragment thereof.
  • antibody is understood in its broadest sense and encompasses not only intact (i.e., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (e.g., Fab, Fab', F(ab' )2, Fv), single chain (scFv), mutants thereof, fusion proteins comprising antibody parts, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single chain antibodies, multispecific antibodies ( For example, bispecific antibodies), and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site with the desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and modified Covalently modified antibodies.
  • Antibodies include antibodies of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), and the antibody need not be of any particular class.
  • Immunoglobulins can be assigned to different classes based on the antibody amino acid sequence of the constant domain of their heavy chains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), such as IgGl, IgG2, IgG3, and IgG4.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.
  • any of the antibodies described herein may be monoclonal or polyclonal.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • Monoclonal antibodies are highly specific, directed against a single antigen. Furthermore, each antibody in a monoclonal preparation is directed against the same single determinant on the antigen, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes).
  • Monoclonal antibodies are not limited to antibodies produced by hybridoma technology.
  • polyclonal antibody refers to a heterogeneous antibody population, which is a mixture of antibodies containing multiple types of antibodies, usually produced by different types of plasma cells. These two terms should not be construed as limiting the source of the antibody or the manner in which it was prepared.
  • monoclonal antibodies include chimeric antibodies, such as humanized forms of murine monoclonal antibodies.
  • humanized antibodies can be prepared by known techniques and offer the advantage of reduced immunogenicity when the antibody is administered to humans.
  • a humanized monoclonal antibody comprises the variable regions (or only the antigen binding site thereof) of a murine antibody and the constant regions derived from a human antibody.
  • a humanized antibody fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen binding site) derived from a human antibody.
  • Procedures for generating chimeric monoclonal antibodies and further engineered monoclonal antibodies include those described in: Riechmann et al., (Nature 332:323, 1988); Liu et al., (PNAS 84 :3439, 1987); Larrick et al., (Bio/Technology 7:934, 1989); and Winter and Harris (TIPS 14:139, May, 1993). Procedures for transgenic production of antibodies can be found in GB 2,272,440, US Patent Nos. 5,569,825 and 5,545,806.
  • Antibodies produced by genetic engineering methods eg, chimeric and humanized monoclonal antibodies
  • chimeric and humanized monoclonal antibodies which contain both human and non-human portions
  • Such chimeric and humanized monoclonal antibodies can be produced by genetic engineering using standard DNA techniques known in the art.
  • Human monoclonal antibodies having human constant and variable regions can be produced by immunizing transgenic animals containing human immunoglobulin genes. See Jakobovits et al., Ann NY Acad Sci 764:525-535 (1995).
  • Anti-PLA2-GIB can also be prepared by constructing combinatorial immunoglobulin libraries (such as Fab phage-display libraries or scFv phage-display libraries) using immunoglobulin light and heavy chain cDNAs prepared from mRNA derived from subject's lymphocytes. Human monoclonal antibodies to polypeptides.
  • variable regions of human antibodies known to bind PLA2-GIB can be mutated, e.g., by using randomly altered mutagenic oligonucleotides, to generate a library of mutated variable regions that can then be screened for binding to PLA2-GIB. Gib.
  • Powerful neutralizing antibodies could provide therapeutic and prophylactic reagents to combat COVID-19.
  • only focusing on the S protein and the RBD region of the S protein may increase the evolutionary selection pressure on this region, resulting in the enrichment of mutations in this region, resulting in reduced effectiveness of therapeutic antibodies and vaccines centered on the RBD region (Wang, L., et al., Importance of Neutralizing Monoclonal Antibodies Targeting Multiple Antigenic Sites on the Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein To Avoid Neutralization Escape. J Virol, 2018.92(10)) .
  • S protein regions or epitopes of the S protein are known to elicit neutralizing antibodies in addition to the RBD.
  • Other proteins such as N protein, Orf9b, and Nsp5 protein, also elicited significant antibody neutralization.
  • the antibody response induced by E protein and Orf7b protein is also significantly different between healthy people and virus-infected people (Jiang, H.W., et al., SARS-CoV-2 proteome microarray for global profiling of COVID-19 specific IgG and IgM responses. Nat Commun, 2020.11(1): p.3581).
  • the present disclosure also relates to a nucleotide sequence encoding a polypeptide according to the present disclosure or an antigenic fragment thereof, or an antibody according to the present disclosure.
  • the present disclosure also relates to a vector according to the nucleotide sequence described in the present disclosure.
  • a “vector” as used herein refers to a nucleic acid delivery vehicle into which nucleotides can be inserted. When the vector is capable of expressing the protein encoded by the inserted nucleotide, the vector is called an expression vector. Vectors can be introduced into host cells by methods such as transformation, transduction, or transfection, and then the genetic material elements carried by them can be expressed in the host cells.
  • Vectors are recognized by those skilled in the art, including but not limited to: (1) plasmid; (2) phagemid; (3) cosmid; (4) artificial chromosome, such as yeast artificial chromosome, bacterial artificial chromosome or P1 source (5) bacteriophages such as lambda phages or M13 phages and (6) animal viruses such as retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, poxviruses, and baculoviruses.
  • a vector may contain various expression-controlling elements, including but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene; in addition, the vector may also contain a replication initiation site.
  • SARS-CoV-2 virus namely Severe Acute Respiratory Syndrome Coronavirus 2
  • Severe Acute Respiratory Syndrome Coronavirus 2 is an enveloped positive-sense single-stranded RNA virus belonging to the Coronaviridae Betacoronavirus genus Severe Acute Respiratory Syndrome-associated Coronavirus species . Its gene sequence and SARS virus and MERS virus belong to the same lineage but different clades. It is the seventh known coronavirus that can infect humans. Hosts of viruses include mammals and birds. The virus can invade the human body through the upper respiratory tract of humans, and infects through the ACE2 expressed on the surface of various cells; the main infected organs include the lungs, heart, kidneys and other major organs. At present, the mainstream detection method for SARS-CoV-2 infection is RT-PCR. "SARS-CoV-2 virus” and "coronavirus” are used interchangeably herein.
  • the genome sequence of SARS-CoV-2 virus is 5'UTR-ORF1a/b-structural protein gene-3'UTR. It is predicted that the SARS-CoV-2 virus can encode 29 proteins, including 4 structural proteins, 9 accessory proteins and 16 nonstructural proteins (Nsp). Structural proteins include membrane proteins (M proteins), nucleocapsid proteins (N proteins), spike proteins (S proteins), and envelope proteins (E proteins). Among them, the non-structural protein sequence of SARS-CoV-2 virus is relatively conservative, and the ORF1ab gene encodes a multifunctional protein involved in viral RNA transcription and replication, including proteases responsible for cutting polyproteins.
  • the S protein belongs to the trimeric class I fusion protein, which contains two subunits, S1 and S2, and a transmembrane anchor.
  • the S1 subunit contains a signal peptide, N-terminal domain (NTD) and receptor binding domain (RBD), while the S2 subunit contains fusion peptide (FP), heptad repeat (HR) 1 and 2, transmembrane structure domains and intracellular domains.
  • N protein sequence is highly conserved and plays an important role in the process of virus replication.
  • the N protein combines with the viral RNA to form a complex, and then under the joint action of the M protein and the E protein, it is wrapped and enters the viral capsid.
  • N protein contains N1 and N2 epitopes. Epitope N1 can stimulate the body to produce high-affinity antibodies, but generally has no neutralizing activity.
  • the SARS-CoV-2 virus can cause an infectious disease COVID-19 (coronavirus disease 2019, also known as "new coronavirus pneumonia”). Its common clinical symptoms include fever, cough and shortness of breath. Fatigue, phlegm and other symptoms. Pathological studies on COVID-19 have shown that SARS-CoV-2 virus infection can lead to respiratory system damage (such as acute lung lesions), cytokine release syndrome (such as cytokine storm and acute inflammatory response), circulatory system damage (such as Cardiovascular injury (such as arrhythmia, vascular embolism, pulmonary vasoconstriction, etc.), digestive system injury (such as pancreas, liver and other organs), genitourinary system injury (such as acute renal failure), nervous system injury (such as loss of sense of smell, taste) , eye injury (such as conjunctival infection) and so on. In addition, some COVID-19 patients continue to experience a series of effects after recovery (COVID-19 long-term syndrome).
  • compositions and vaccine compositions are provided.
  • compositions comprising one or more polypeptides as described in the present disclosure, or antigenic fragments thereof.
  • the composition is a vaccine composition.
  • a "vaccine composition” is a composition that, when administered to a subject (eg, a mammal), induces or enhances an immune response against antigenic material contained within the composition.
  • the response can include inducing antibody production (eg, by stimulating B cells) or a T cell-based response (eg, cytolytic response). These responses may or may not be protective or neutralizing.
  • a protective or neutralizing immune response is one that is detrimental to an infecting organism consistent with an antigen (eg, the organism from which the antigen was derived) but beneficial to the subject (eg, by reducing or preventing infection).
  • Immune response also known as immune response, refers to the cells of the immune system (such as T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils granulocytes) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, which result in the selective targeting, binding, damage, destruction, and/or exclusion of vertebrates Invading pathogens in the body, cells or tissues infected by pathogens, cancer cells or other abnormal cells, or in the case of autoimmunity or pathological inflammation, normal human cells or tissues.
  • T lymphocytes such as T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils granulocytes
  • soluble macromolecules including antibodies, cytokines, and complement
  • composition comprises an amount of a polypeptide of the present disclosure, or an antigenic fragment thereof, sufficient to elicit an immune response when administered to a subject.
  • compositions for use as vaccines comprise a polypeptide of the present disclosure, or an antigenic fragment thereof, and any other components required. "Immunologically effective amount” means that the amount administered to a subject, either in a single dose or as part of a series, is effective for treatment or prophylaxis.
  • compositions of the present disclosure further comprise one or more adjuvants.
  • adjuvant generally refers to an agent that increases, stimulates, activates, potentiates or modulates the immune response to an active ingredient of a composition at the cellular or humoral level. Adjuvants can play a role in both acquired and innate immunity and function in a variety of ways.
  • adjuvants Many substances (natural or synthetic) have been shown to function as adjuvants.
  • suitable adjuvants include aluminum salts (aluminum phosphate, aluminum hydroxide, aluminum oxyhydroxide, etc.), alum, cholera toxin, salmonella toxin, IFA (incomplete Freund's adjuvant), CFA (complete Freund's adjuvant), ISCOMatrix , GM-CSF and other immunostimulatory cytokines, oligodeoxynucleotides containing CpG motifs (CpG7909, etc.), oil-in-water emulsions, saponins or their derivatives (QS21, etc.), lipopolysaccharides such as lipid A or its derivatives substances (MPL, RC529, GLA, E6020, etc.), lipopeptide, lactoferrin, flagellin, double
  • compositions comprising the antibodies described herein further comprise carriers and/or excipients.
  • Carriers include any and all pharmaceutically acceptable solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids wait.
  • the use of such media and agents for pharmaceutically active substances is well known in the art.
  • Supplementary active ingredients can also be incorporated into the compositions.
  • compositions as disclosed herein, a pharmaceutically acceptable carrier or excipient must be compatible with the other ingredients of the composition and not deleterious to, or harmful to, the recipients thereof. The benefits outweigh any detrimental effects.
  • pharmaceutically acceptable carrier refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, medium, encapsulating material, manufacturing aid (such as lubricants, magnesium talc, calcium or zinc stearate, or stearic acid) or solvent-encapsulating materials that participate in the carrying or transport of agents from one part of the body to another (for example, from one organ to another) .
  • manufacturing aid such as lubricants, magnesium talc, calcium or zinc stearate, or stearic acid
  • solvent-encapsulating materials that participate in the carrying or transport of agents from one part of the body to another (for example, from one organ to another) .
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials that may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as Sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) excipients (8) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (9) glycols, such as propylene glycol; (10) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (11) esters such as ethyl oleate and ethyl laurate; (12) agar; (13) buffers such as magnesium hydroxide and hydrogen (14) Alginic acid; (15) Pyrogen-free
  • excipients will be compatible with the antibodies of the disclosure.
  • Excipients known in the art include, for example, fillers, binders, disintegrants, coating agents, adsorbents, anti-adhesive agents, glidants, preservatives, antioxidants, flavoring agents, coloring agents, sweeteners Agents, solvents, co-solvents, buffers, chelating agents, viscosity imparting agents, surfactants, diluents, wetting agents, carriers, preservatives, emulsifiers, stabilizers and tonicity regulators.
  • the excipients included in the compositions of the present disclosure depend on the route of administration and suitable excipients for use in the compositions of the present disclosure are known in the art.
  • compositions according to the present disclosure may be formulated in the form of ointments, gels, pastes, liquid solutions, suspensions, tablets, gelatin capsules, capsules, suppositories, powders, nasal drops Or aerosol, preferably formulated as an injection solution or suspension.
  • the composition is usually packaged in the form of a liquid suspension, which can be injected by syringe or infusion.
  • compositions are generally dissolved in saline, physiological, isotonic or buffered solutions compatible with pharmaceutical use and known to those skilled in the art.
  • the present disclosure provides an array comprising a solid substrate, and polypeptides immobilized on the solid substrate, wherein the polypeptides comprise at least one polypeptide described in the present disclosure or an antigenic fragment thereof.
  • the polypeptide comprises at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, or at least 35 polypeptides or Antigenic fragments thereof, for example comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 polypeptides of the present disclosure or antigenic fragments thereof.
  • the polypeptide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or all 9 polypeptides or antigens thereof according to the present disclosure
  • a fragment for example comprising 2, 3, 4, 5, 6, 7, 8 or 9 polypeptides or antigenic fragments thereof of the present disclosure.
  • an "array” is generally formed of linear or two-dimensional structures with spaced apart (i.e., discrete) regions ("spots"), each array having a finite area and formed on a solid on the surface of the substrate.
  • Arrays can also be bead structured, where each bead can be identified by a molecular or color code, or in a continuous fluid. Analysis can also be performed sequentially, where the sample passes through a series of points, each point adsorbing the molecule from solution.
  • the solid substrate used in the present disclosure is not particularly limited as long as it is a substrate on which a polypeptide can be immobilized.
  • the solid substrate is usually glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • Solid substrates can be tubes, beads, discs, silicon chips, microwell plates, polyvinylidene fluoride (PVDF) membranes, nitrocellulose membranes, nylon membranes, other porous membranes, non-porous membranes (e.g., plastics, polymers, perspex, silicon, etc.), a mass of polymer needles, or a mass of microtiter wells, or any other surface form suitable for immobilizing polypeptides, polynucleotides, and other suitable molecules and/or performing immunoassays.
  • PVDF polyvinylidene fluoride
  • Binding methods are well known in the art and generally include crosslinking, covalently binding or physically adsorbing polypeptides, polynucleotides, etc. to a solid substrate.
  • the position of each point can be determined (Jenkins, R.E., Pennington, S.R. (2001, Proteomics, 2, 13-29) and Lal et al. People (2002, Drug Discov Today 15;7(18 Suppl):S143-9)).
  • the array is typically a microarray.
  • "Microarray” means an array of areas having a density of discrete areas of at least about 100/ cm2 , preferably at least about 1000/ cm2 . Areas in a microarray have typical dimensions, eg, in the range of about 10-250 ⁇ m in diameter, and are separated from other areas in the array by approximately the same distance. Arrays can also be macroarrays or nanoarrays. Methods of fabricating arrays are well known to those skilled in the art.
  • the array is a polypeptide chip.
  • Polypeptide chip refers to a biochip suitable for capturing polypeptides, which can be used for high-throughput screening.
  • the surface of the polypeptide chip contains a plurality of addressable locations, each addressable location having a biologically specific component, such as a polypeptide or an antigenic fragment, bound thereto.
  • IgG and IgM antibodies play an important role in targeting viral infections such as SARS coronavirus and MERS coronavirus infection.
  • Routine techniques used to study IgG and IgM responses in patients include ELISA and immunocolloidal gold techniques. These techniques typically only test a single protein or antibody of interest in a single reaction.
  • peptide chips enable proteome-wide measurement of antibody responses in a high-throughput format, thereby providing a more systematic picture of these important antibody responses.
  • the polypeptides or antigenic fragments thereof contained in the array may be derived from a polypeptide or antigenic fragment thereof of SARS-CoV-2 ORF1ab polyprotein, a polypeptide derived from SARS-CoV-2 S protein or an antigen thereof sexual fragments and/or polypeptides or antigenic fragments derived from SARS-CoV-2 N protein.
  • the polypeptides or antigenic fragments thereof contained in the array can be derived from polypeptides or antigenic fragments thereof of SARS-CoV-2 ORF1ab polyprotein, polypeptides of SARS-CoV-2 S protein or antigenic fragments thereof and at least one of the polypeptides or antigenic fragments thereof of the SARS-CoV-2 N protein, for example, the polypeptides or antigenic fragments thereof included in the array may be derived from the polypeptides of the SARS-CoV-2 ORF1ab polyprotein or their antigenicity Fragments, polypeptides of SARS-CoV-2 S protein or antigenic fragments thereof, and polypeptides of SARS-CoV-2 N protein or antigenic fragments thereof, can be derived from polypeptides of SARS-CoV-2 ORF1ab polyprotein or antigenic fragments thereof, polypeptides of SARS-CoV-2 S protein or antigenic fragments thereof, and polypeptides of SARS-CoV-2 N protein or antigenic fragments thereof
  • the present disclosure provides methods for diagnosing whether a subject is infected by SARS-CoV-2 using the polypeptides or antigenic fragments thereof of the present disclosure, or the arrays of the present disclosure.
  • the method comprises contacting the subject's sample with the polypeptide or antigenic fragment thereof of the present disclosure, or the array described in the present disclosure, and detecting the presence or absence of an antibody specifically binding to the polypeptide in the sample, thereby conducting an anti-SARS- Diagnosis or prognosis of CoV-2 infection.
  • Diagnosing means identifying the presence or nature of a pathological condition, or a subtype of a pathological condition, ie the presence or risk of COVID-19.
  • Prognosis is used herein to refer to the prediction of the likelihood of disease or disease progression, including recurrence and response to treatment.
  • the terms "subject”, “individual”, “host” and “patient” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, prognosis or treatment is desired, in particular is human.
  • the methods described herein are suitable for both human therapy and veterinary applications.
  • the subject is a mammal, and in specific embodiments, the subject is a human.
  • sample of a subject refers to a biological sample from a subject that is available for analysis, including tissue or fluid samples taken from or derived from an individual. Suitable biological samples include, for example, whole blood, serum, plasma, blood fractions, sputum, urine, biopsy samples, crude lysates, and the like. Preferably the sample from the subject to be assayed is from the same species as the control sample.
  • the present disclosure provides methods for inducing an immune response in a subject against SARS-CoV-2 using the polypeptides described herein, or antigenic fragments thereof, or compositions described herein.
  • the method comprises administering to a subject a polypeptide of the present disclosure or an antigenic fragment thereof, or a composition of the present disclosure (eg, a vaccine composition), such that the polypeptide or antigenic fragment of the present disclosure and/or The adjuvant is exposed to the subject's immune system.
  • inducing an immune response refers to the first encounter of the immune system with at least one antigenic protein or antigenic fragment thereof and the subsequent induction of an antigen-specific immune response within a defined period of time.
  • the time period is prior to induction eg, at least 1 year, at least 2 years, at least 3 years, at least 5 years or at least 10 years.
  • an encounter of an individual's or subject's immune system with an antigenic protein or antigenic fragment thereof that does not induce an antigen-specific immune response is not considered “inducing an immune response.”
  • an encounter of an individual's immune system with an antigenic protein or antigenic fragment thereof that does not induce durable immunity is not considered “inducing an immune response" according to the present disclosure.
  • the induction of durable immunity is mediated by the generation of memory B cells and/or memory T cells.
  • specific antigens can be expressed by cancer cells without eliciting an immune response. The mere presence of the antigen is not "inducing an immune response" against said antigen as understood herein.
  • the individual or subject has not been intentionally immunized with an antigenic protein or antigenic fragment thereof, or a vector comprising a nucleic acid encoding such protein or fragment, for the treatment or prevention of a disease within the time period given above.
  • Administration can occur once or can occur multiple times. In one embodiment, one or more administrations may occur as part of a so-called "prime-boost" regimen. Other administration systems may include delayed-release or sustained-release delivery systems.
  • compositions of the present invention can be stored in solution or in lyophilized form. Solutions are preferably lyophilized in the presence of sugars such as sucrose or lactose. It is still preferred that they are lyophilized and reconstituted before use.
  • compositions may be presented as a kit comprising the composition and an adjuvant or a reconstitution solution comprising one or more pharmaceutically acceptable diluents to facilitate the combination for administration to a mammal using conventional or other equipment. reconstitution of the substance.
  • kits may optionally include a device for administering the composition in liquid form (eg, hypodermic syringe, microneedle array) and/or instructions for use.
  • compositions disclosed herein can be incorporated into various delivery systems.
  • the composition can be applied to a "microneedle array” or "microneedle patch” delivery system for administration.
  • the present disclosure provides methods of preventing and/or treating disease caused by SARS-CoV-2 infection in a subject.
  • the method comprises administering to the subject a polypeptide of the present disclosure or an antigenic fragment thereof, a composition of the present disclosure, or an antibody of the present disclosure.
  • prevention means administering to a subject a therapeutically effective amount of a polypeptide of the present disclosure, or an antigenic fragment thereof, a composition or an antibody, in order to protect the subject from the occurrence of SARS-CoV-2 infection. disease.
  • prevention in relation to a given treatment for a given condition (e.g. prevention of SARS-CoV-2 infection)
  • it is intended to mean that the treated subject does not develop clinically observable levels of the condition at all, or Progression was slower and/or to a lesser extent than subjects in the absence of treatment.
  • a treatment is considered to be a treatment if given during a patient's exposure to a stimulus expected to produce the manifestations of a given condition, and the treatment causes the subject to experience fewer and/or milder symptoms of the condition than would be expected if it were not administered.
  • the condition has been prevented.
  • Treatment is said to "prevent" infection by causing the subject to exhibit only mild overt symptoms of infection, which does not mean that there must not be any cells infiltrated by the virus.
  • treatment refers to methods of obtaining beneficial or desired results, including clinical results.
  • a beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: alleviation of one or more symptoms caused by the disease, reduction of the extent of the disease, stabilization of the disease (e.g., prevention or delay the progression of the disease), prevent or delay the spread of the disease, prevent or delay the recurrence of the disease, delay or slow the progression of the disease, improve the disease state, provide remission (partial or complete), reduce the need for treatment of the disease Dosage of various other drugs, delaying disease progression, increasing or improving quality of life, weight gain and/or prolonging survival.
  • the methods of the invention contemplate any one or more of these aspects of treatment.
  • treatment may be administered after one or more symptoms have occurred.
  • treatment can be administered in the absence of symptoms.
  • treatment can be administered to a susceptible individual prior to the onset of symptoms, or can be treated with another damaging agent (eg, based on history of symptoms, based on genetic or other predisposing factors, disease therapy, or any combination thereof).
  • Treatment can also be continued after symptoms have subsided, for example, to prevent or delay their recurrence.
  • a reduction in the risk of infection with a given treatment refers to a reduction in the risk of infection compared to a control or in the absence of treatment (e.g., administration of a polypeptide of the disclosure).
  • the subject develops the infection more slowly or to a lesser extent than the basal level of development.
  • a reduction in the risk of infection may cause the subject to exhibit only mild overt symptoms of infection or delayed symptoms of infection, which does not mean that any cells must not be infiltrated by the virus.
  • administering refers to introducing a composition or agent into a subject, and includes concurrent or sequential introduction of compositions or agents.
  • administering can refer to, for example, treatment, pharmacokinetic, diagnostic, research, placebo, and experimental procedures.
  • administering also encompasses in vitro and ex vivo treatments.
  • the composition or agent is introduced into a subject by any suitable route, examples of suitable methods for administering a polypeptide, antibody or composition of the present disclosure include oral, intradermal, subcutaneous, intramuscular, intraosseous, intraperitoneal and intravenous injection, and systemic administration or local administration to the vicinity of the target site, but not limited thereto.
  • Administration includes self-administration and administration by others.
  • Administration can be by any suitable route.
  • a suitable route of administration allows the composition or agent to perform its intended function. For example, if the appropriate route is intravenous, the composition is administered by introducing the composition or agent into the subject's vein.
  • a polypeptide, antibody or composition of the present disclosure is packaged with or stored in a device for administration.
  • Devices for injectable formulations include, but are not limited to, injection ports, auto-injectors, syringe pumps, and injection pens.
  • Devices for aerosolized or powdered formulations include, but are not limited to, inhalers, insufflators, aspirators, and the like. Accordingly, the present disclosure includes administration devices comprising a polypeptide, antibody or composition of the present disclosure for the treatment or prevention of one or more of the disorders described herein.
  • Subjects to which administration is contemplated include, but are not limited to, humans (i.e., male or female, of any age group, such as pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., youth, middle-aged, or elderly people)) and/or other non-human animals such as mammals (eg primates).
  • humans i.e., male or female, of any age group, such as pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., youth, middle-aged, or elderly people)
  • other non-human animals such as mammals (eg primates).
  • Serum samples from patients infected with SARS-CoV-2 were first tested for overall neutralizing antibodies.
  • the results showed that compared with healthy donors, COVID-19 patients all produced significantly more anti-N protein and anti-S protein IgG antibodies within 30 days after the onset of symptoms (Fig. 1A and 1B).
  • the inventors Based on the amino acid sequence of the SARS-CoV-2 Wuhan-Hu-1 strain, the inventors designed a total of 515 polypeptides covering the entire SARS-CoV-2 proteome, wherein each polypeptide is 15 amino acids in length, and each polypeptide There are 11 amino acid overlaps with adjacent polypeptides.
  • Peptides were synthesized by GL Biochemical Co., Ltd. (Shanghai, China). Polypeptides were coupled to BSA using Sulfo-SMCC (ThermoFisher, MA, USA). Briefly, BSA was activated with Sulfo-SMCC at a molar ratio of 1:30, followed by dialysis in PBS buffer.
  • a cysteine residue is added to the C-terminus of the polypeptide to couple its sulfhydryl group to BSA. Cysteine-containing peptides were added at a ratio of 1:1 (w/w), incubated for 2 hours, and then dialyzed against PBS to remove free peptides.
  • the peptide chip was heated to room temperature, and then incubated in blocking buffer (PBS buffer containing 3% BSA and PBS buffer containing 0.1% Tween20) for 3 hours respectively. Serum samples from COVID-19 patients or pooled serum samples from 20 healthy donors (control group) were diluted in PBS containing 0.1% Tween20, and then incubated with the peptide chip at 4°C for 2 hours.
  • the peptide chip was washed with 1 ⁇ PBST, and then cy3-coupled goat anti-human IgG and AlexaFluor647-coupled donkey anti-human IgM (Jackson Immunological Research Corporation, Pennsylvania, USA, Cat#109) were mixed with 1 ⁇ PBST at a dilution of 1:1000, respectively.
  • the average signal intensity of each peptide in the three sampling time point groups was calculated separately, and the peptides with high signal intensity (ie, higher than the mean plus standard deviation of signal intensity) were selected.
  • R software version 3.6.3 was used for data processing and analysis, and significant signal intensity changes between groups at different sampling time points were evaluated based on the Limma algorithm. A p value of less than 0.05 indicates a statistically significant difference.
  • the epitopes that consistently maintained strong antibody responses in more than 80% of the samples in the three sampling groups were selected, which were called dominant and persistent epitopes.
  • ORF1ab polyprotein has the most dominant epitopes, and its epitopes are widely distributed in the regions of nonstructural protein (nsp)2-5, nsp8-10, nsp12-14 and nsp16.
  • an immunodominant epitope No.2073 (SEQ ID NO.7, ORF1ab, aa 5801-5815) was identified, which can be detected by 100% of COVID-19 individuals regardless of the time point of serum sampling. IgG and IgM antibody recognition in serum ( Figure 4). This highly reactive peptide is located in the helicase (nsp13) region of the ORF1ab polyprotein and is essential for unwinding double-stranded RNA templates during SARS-CoV-2 replication (Chen, J. et al. Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex. Cell 182, 1560-1573 e1513, doi:10.1016/j.cell.2020.07.033(2020)).
  • the fold change in signal intensity for each peptide relative to healthy controls was further calculated.
  • the immunodominant peptides with the highest average signal intensity recognized by two IgG antibodies——No.1985 (SEQ ID NO.6, ORF1ab, aa 5449-5463) and No.2073 (SEQ ID NO.7, ORF1ab , aa 5801-5815) are located on nsp13.
  • Predominant and persistent epitopes as defined above could not be found on the N protein (positive rates were above 80% at all three sampling time points). Subsequently, a second round of epitope screening was performed based on the data obtained from the peptide chip to select subdominant and persistent epitopes that consistently maintained positive reactions in more than 60% of the COVID-19 samples at three sampling time points.
  • Example 6 Identification of epitopes with high signal intensity and decreased reactivity over time
  • Example 7 Construction of a new coronavirus infection diagnostic model based on dominant and persistent epitopes
  • Example 2 Collect 75 human serum samples, and use the new coronavirus polypeptide chip manufactured in Example 2 to detect the antibody response of the polypeptide in the sample.
  • the 75 samples included 24 samples from healthy donors who were not infected with the new coronavirus, 18 samples from the recovery period of 1 month after infection with the new coronavirus, 18 samples from the recovery period of 4 months after the infection with the new coronavirus and 15 samples from the recovery period of 7 months after being infected with the new coronavirus.
  • the serum samples of patients who had been infected with the new coronavirus were taken as the case group in January, April and July respectively, and were combined with uninfected samples (as the control group) in sequence for analysis.
  • the correlation of antibody response intensity between pairs of 35 polypeptides was analyzed, and the data of one of the two peptides with similar correlation was eliminated.
  • feature screening was performed to explore predictors that can accurately predict patients with new crown infection using some peptides.
  • the predictors are calculated using randomForest, lasso, elasticNet, svmLinear and svmRadial algorithms respectively.
  • the panel-based score of each sample was calculated according to the optimal polypeptide subset produced by each algorithm. Use this score to draw the ROC curve, and use the AUC score (the closer the AUC is to 1, the higher the accuracy), specificity, sensitivity and the number of polypeptides included in the subset (size) as the model for evaluating each algorithm. standard.
  • the number of subset peptides obtained by the randomForest algorithm is moderate, and the prediction accuracy of the model is high.
  • Using the randomForest algorithm based on IgM to predict the model of new crown infection samples in January, April and July is as follows:
  • the number of subset peptides obtained by the randomForest algorithm is moderate, and the prediction accuracy of the model is high.
  • Using the randomForest algorithm based on IgG to predict the model of new crown infection samples in January, April and July is as follows:

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Abstract

提供了分离的多肽或其抗原性片段，结合该分离的多肽或其抗原性片段的抗体，包含该分离的多肽或其抗原性片段的组合物，以及包含该分离的多肽或其抗原性片段的阵列。还提供了检测受试者中针对SARS-CoV-2的抗体的存在的方法，对被SARS-CoV-2感染的受试者进行预后的方法，诱导受试者中针对SARS-CoV-2的免疫反应的方法，以及预防和/或治疗受试者中由SARS-CoV-2感染引起的疾病的方法。

Description

抗原性多肽及其用途 发明领域
本公开涉及分离的多肽及其抗原性片段，以及其用于诱导免疫反应、诊断和/或治疗疾病的用途。
发明背景
新型冠状病毒肺炎(COVID-19)目前已经在全球范围大流行，其病原体是SARS-CoV-2(Zhou,P.,et al.,A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature,2020.579(7798):p.270-273)。截至到2021年2月24日，全球已经累计报告了111,762,993例确诊病例，中国累积101,778例确诊病例。为了控制这种大流行，基本任务之一是执行快速，可靠和负担得起的诊断。到目前为止，对病毒感染的检测一直依赖于对被隔离个体进行广泛的核酸检测筛查。核酸检测的敏感性在很大程度上取决于临床COVID-19综合征的病程和类型，采集部位，标本的运输和存储。据报道，在COVID-19患者中，核酸检测的假阴性率约为30％(Ai,T.,et al.,Correlation of Chest CT and RT-PCR Testing for Coronavirus Disease 2019(COVID-19)in China:A Report of 1014 Cases.Radiology,2020.296(2):p.E32-E40)。最近的核酸检测阳性率调查表明，不同国家和地区的COVID-19病例，特别是无症状的病例，被大大低估了。因此，由于核酸检测的敏感性有限，可能忽视了部分无症状的感染病例。
采用检测和判读更加简便的血清抗体检测，可以有效提高结果的可靠性，可以作为诊断的双保险。当症状发作后19天内采集COVID-19患者的血清样品时，新冠病毒的IgG抗体反应的敏感性可接近100％(Long,Q.X.,et al.,Antibody responses to SARS-CoV-2 in patients with COVID-19.Nat Med,2020.26(6):p.845-848)。此外，针对病毒蛋白质有强中和反应的抗体可以有效地抵抗病毒。恢复期患者的血清中抗体应答在其恢复中起到重要作用，并且这些抗体对于其他感染患者也具有同样的治疗效果(Jiang,L.,et al.,Potent neutralization of MERS-CoV by human neutralizing monoclonal antibodies to the viral spike glycoprotein.Sci Transl Med,2014.6(234):p.234ra59)。因此， 无论是诊断检测、筛选治疗性抗体还是用于制备疫苗，都需要充分了解新冠病毒各种蛋白质和各个区域的抗原性，以及患者对各种蛋白质的抗体反应随疾病进程的变化。
病毒的序列分析表明，SARS-CoV-2与蝙蝠SARS冠状病毒和SARS病毒相似，属于β冠状病毒属，与Bat-CoV RaTG13病毒最密切相关(Zhou,P.,et al.，同上)。其中，新冠病毒的基因组与SARS冠状病毒的基因组序列相似度约有80％。通过与其他已知冠状病毒的蛋白质序列进行比较，预测新冠病毒可以编码29种蛋白质：4种结构蛋白，9种辅助蛋白和16种非结构蛋白(Nsp)(Wu,A.,et al.,Genome Composition and Divergence of the Novel Coronavirus(2019-nCoV)Originating in China.Cell Host Microbe,2020.27(3):p.325-328)。结构蛋白包括膜蛋白(M蛋白)、核衣壳蛋白(N蛋白)、刺突蛋白(S蛋白)和包膜蛋白(E蛋白)，这些蛋白质暴露在病毒表面，而非结构蛋白包裹着病毒内部。目前功能研究已知，内部的蛋白质中Orf6、Orf9b抑制干扰素，Nsp1抑制抗病毒应答，而Nsp15、Orf3a则分别激活干扰素，并且NLRP3、Orf7a是负责诱导细胞死亡的病毒蛋白。
已有证据表明，新冠病毒与SARS冠状病毒利用相同的机制进入宿主细胞，即通过S蛋白的受体结合域(RBD)和宿主的血管紧张素转化酶2(ACE2)基因之间的高亲和力结合(Li,W.,et al.,Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus.Nature,2003.426(6965):p.450-4)。
由于S蛋白与宿主的相互作用关系，目前血清抗体检测普遍采用S蛋白特别是RBD区域作为抗体反应的靶点(Long,Q.X.,et al.,Antibody responses to SARS-CoV-2 in patients with COVID-19.Nat Med,2020.26(6):p.845-848)。用于治疗COVID-19的治疗性抗体和疫苗也主要针对S蛋白尤其是RBD区域(Cao,Y.,et al.,Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent Patients'B Cells.Cell,2020.182(1):p.73-84.e16)。基于S蛋白的血清学检测的关键试剂是重组S蛋白。然而，S蛋白的生产是困难且昂贵的(Petherick,A.,Developing antibody tests for SARS-CoV-2.Lancet,2020.395(10230):p.1101-1102)。此外，虽然都使用重组S蛋白，但不同制造商之间甚至批次之间的不一致可能会导致检测结果出现差异(Lisboa,B.M.,et al.,Diagnostic accuracy of serological tests for  covid-19:systematic review and meta-analysis.BMJ,2020.370:p.m2516)。另外，由于其他人类冠状病毒感染引起的交叉反应可能也会导致假阳性结果，特别是对于在人群中流行的四种常见的感冒冠状病毒(HCoV-OC43，HKU1，NL63和229E)(Petherick,A.，同上)。为了开发高度特异性的血清抗体测试，需要鉴定更具特异性的强抗原性的区域并且与相关冠状病毒的同源性较低的部分。
发明概述
在第一方面，本公开提供了一种分离的多肽或其抗原性片段，所述多肽具有选自以下的氨基酸序列，或在以下氨基酸序列中进行了一个或几个氨基酸残基的取代、缺失、插入和/或添加后获得的氨基酸序列：
SFKWDLTAFGLVAEW(SEQ ID NO:1)；
LGLAAIMQLFFSYFA(SEQ ID NO:2)；
VLSTFISAARQGFVD(SEQ ID NO:3)；
SFCYMHHMELPTGVH(SEQ ID NO:4)；
MQTMLFTMLRKLDND(SEQ ID NO:5)；
TCTERLKLFAAETLK(SEQ ID NO:6)；
KGVITHDVSSAINRP(SEQ ID NO:7)；
DQFKHLIPLMYKGLP(SEQ ID NO:8)；
SSVLHSTQDLFLPFF(SEQ ID NO:9)；
SKRSFIEDLLFNKVT(SEQ ID NO:10)；
YELQTPFEIKLAKKF(SEQ ID NO:11)；
EKYCALAPNMMVTNN(SEQ ID NO:12)；
ATYKPNTWCIRCLWS(SEQ ID NO:13)；
LKTLVATAEAELAKN(SEQ ID NO:14)；
MYLKLRSDVLLPLTQ(SEQ ID NO:15)；
YEDLLIRKSNHNFLV(SEQ ID NO:16)；
KVDTANPKTPK(SEQ ID NO:17)；
LGSALLEDEFTPFDV(SEQ ID NO:18)；
SEVVLKKLKKSLNVA(SEQ ID NO:19)；
LEKMADQAMTQMYKQ(SEQ ID NO:20)；
VPLNIIPLTTAAKLM(SEQ ID NO:21)；
LNRGMVLGSLAATVR(SEQ ID NO:22)；
FCAFAVDAAKAYKDY(SEQ ID NO:23)；
FFKFRIDGDMVPHIS(SEQ ID NO:24)；
NLDKSAGFPFNKWGK(SEQ ID NO:25)；
VVYRGTTTYKLNVGD(SEQ ID NO:26)；
AKHYVYIGDPAQLPA(SEQ ID NO:27)；
PAEIVDTVSALVYDN(SEQ ID NO:28)；
KDKSAQCFKMFYKGV(SEQ ID NO:29)；
GFDYVYNPFMIDVQQ(SEQ ID NO:30)；
LIGDCATVHTANKWD(SEQ ID NO:31)；
TAFVTNVNASSSEAF(SEQ ID NO:32)；
LSSYSLFDMSKFPLK(SEQ ID NO:33)；
IDGYFKIYSKHTPIN(SEQ ID NO:34)；
ALQIPFAMQMAYRFN(SEQ ID NO:35)；
GGDAALALLLLDRL(SEQ ID NO:36)；
AALALLLLDRLNQLE(SEQ ID NO:37)；
GMEVTPSGTWLTYTG(SEQ ID NO:38)；和
IDAYKTFPPTEPKKD(SEQ ID NO:39)。
在第二方面，本公开提供了一种分离的多肽或其抗原性片段，所述多肽具有选自以下的氨基酸序列，或在以下氨基酸序列中进行了一个或几个氨基酸残基的取代、缺失、插入和/或添加后获得的氨基酸序列：
SFKWDLTAFGLVAEW(SEQ ID NO:1)；
LGLAAIMQLFFSYFA(SEQ ID NO:2)；
VLSTFISAARQGFVD(SEQ ID NO:3)；
SFCYMHHMELPTGVH(SEQ ID NO:4)；
MQTMLFTMLRKLDND(SEQ ID NO:5)；
TCTERLKLFAAETLK(SEQ ID NO:6)；
KGVITHDVSSAINRP(SEQ ID NO:7)；
DQFKHLIPLMYKGLP(SEQ ID NO:8)；
SSVLHSTQDLFLPFF(SEQ ID NO:9)；
SKRSFIEDLLFNKVT(SEQ ID NO:10)；
YELQTPFEIKLAKKF(SEQ ID NO:11)；
EKYCALAPNMMVTNN(SEQ ID NO:12)；
ATYKPNTWCIRCLWS(SEQ ID NO:13)；
LKTLVATAEAELAKN(SEQ ID NO:14)；
MYLKLRSDVLLPLTQ(SEQ ID NO:15)；
YEDLLIRKSNHNFLV(SEQ ID NO:16)；
KVDTANPKTPK(SEQ ID NO:17)；
LGSALLEDEFTPFDV(SEQ ID NO:18)；
SEVVLKKLKKSLNVA(SEQ ID NO:19)；
LEKMADQAMTQMYKQ(SEQ ID NO:20)；
VPLNIIPLTTAAKLM(SEQ ID NO:21)；
LNRGMVLGSLAATVR(SEQ ID NO:22)；
FCAFAVDAAKAYKDY(SEQ ID NO:23)；
FFKFRIDGDMVPHIS(SEQ ID NO:24)；
NLDKSAGFPFNKWGK(SEQ ID NO:25)；
VVYRGTTTYKLNVGD(SEQ ID NO:26)；
AKHYVYIGDPAQLPA(SEQ ID NO:27)；
PAEIVDTVSALVYDN(SEQ ID NO:28)；
KDKSAQCFKMFYKGV(SEQ ID NO:29)；
GFDYVYNPFMIDVQQ(SEQ ID NO:30)；
LIGDCATVHTANKWD(SEQ ID NO:31)；
TAFVTNVNASSSEAF(SEQ ID NO:32)；
LSSYSLFDMSKFPLK(SEQ ID NO:33)；
IDGYFKIYSKHTPIN(SEQ ID NO:34)；和
ALQIPFAMQMAYRFN(SEQ ID NO:35)。
在本公开第一方面和第二方面的一些实施方案中，抗原性片段的长度为至少5个氨基酸、至少6个氨基酸、至少7个氨基酸、至少8个氨基酸、至少9个氨基酸或至少10个氨基酸。
在第三方面，本公开提供了一种分离的多肽或其抗原性片段，所述多肽具有选自以下的氨基酸序列，或在以下氨基酸序列中进行了一个或几个氨基酸残基的取代、缺失、插入和/或添加后获得的氨基酸序列：
GWEIVKFISTCACEI(SEQ ID NO:40)；
WADNNCYLATALLTL(SEQ ID NO:41)；
KDYLASGGQPITNCV(SEQ ID NO:42)；
DKSAFVNLKQLPFFY(SEQ ID NO:43)；
FIEDLLFNKVTLADA(SEQ ID NO:44)；
LLLLDRLNQLESKMS(SEQ ID NO:45)；
LKKLKKSLNVAKSEF(SEQ ID NO:46)；
RLKLFAAETLKATEE(SEQ ID NO:47)；或
ASDTYACWHHSIGFD(SEQ ID NO:48)。
在本公开第三方面的一些实施方案中，抗原性片段的长度为至少5个氨基酸、至少6个氨基酸、至少7个氨基酸、至少8个氨基酸、至少9个氨基酸或至少10个氨基酸。
在第四方面，本公开提供了一种分离的抗体，所述抗体结合根据本公开第一、第二或第三方面中所述的多肽或其抗原性片段。
在一些实施方案中，抗体选自IgG抗体、IgM抗体和IgA抗体。在一些实施方案中，抗体选自IgG1、IgG2、IgG3和IgG4抗体。在一些实施方案中，抗体为单克隆抗体。在一些实施方案中，抗体为人抗体。
在第五方面，本公开提供了一种组合物，所述组合物包含一种或多种根据本公开第一、第二或第三方面中所述的多肽或其抗原性片段。
在本公开第五方面的一些实施方案中，所述组合物包含具有SEQ ID NO:10所示的氨基酸序列的多肽或其抗原性片段以及具有选自SEQ ID NO:1-9和11-48所示的氨基酸序列的多肽或其抗原性片段中的一种或多种。在一些实施方案中，所述组合物包含具有SEQ ID NO:10所示的氨基酸序列的多肽或其抗原性片段以及具有选自SEQ ID NO:1-9和11-48所示的氨基酸序列的多肽或其抗原性片段中的1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46或47种。
在一些实施方案中，所述组合物包含具有SEQ ID NO:10所示的氨基酸序列的多肽或其抗原性片段以及具有选自SEQ ID NO:1-9和11-39所示的氨基酸序列的多肽或其抗原性片段中的一种或多种。在一些实施方案中，所述组合物包含具有SEQ ID NO:10所示的氨基酸序列的多肽或其抗原性片段以及具有选自SEQ ID NO:1-9和11-39所示的氨基酸序列的多肽或其抗原性片段中的1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37或38种。
在一些实施方案中，所述组合物包含具有SEQ ID NO:10所示的氨基酸序列的多肽或其抗原性片段以及具有选自SEQ ID NO:1-9和11-35所示的氨基酸序列的多肽或其抗原性片段中的一种或多种。在一些实施方案中，所述组合物包含具有SEQ ID NO:10所示的氨基酸序列的多肽或其抗原性片段以及具有选自SEQ ID NO:1-9和11-35所示的氨基酸序列的多肽或其抗原性片段中的1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33或34种。
在一些实施方案中，所述组合物包含具有SEQ ID NO:10、7、35、29、23、30、2、21和12所示的氨基酸序列的多肽或其抗原性片段。
在一些实施方案中，所述组合物包含具有SEQ ID NO:10、12、35、14、30、2、3、13和19所示的氨基酸序列的多肽或其抗原性片段。
在一些实施方案中，所述组合物包含具有SEQ ID NO:10、35、12和19所示的氨基酸序列的多肽或其抗原性片段。
在一些实施方案中，所述组合物包含具有SEQ ID NO:10和33所示的氨基酸序列的多肽或其抗原性片段。
在一些实施方案中，所述组合物包含具有SEQ ID NO:10、32和15所示的氨基酸序列的多肽或其抗原性片段。
在一些实施方案中，所述组合物包含具有SEQ ID NO:10、21、30、15、12、35、23、19和20所示的氨基酸序列的多肽或其抗原性片段。
在一些实施方案中，抗原性片段的长度为至少5个氨基酸、至少6个氨基酸、至少7个氨基酸、至少8个氨基酸、至少9个氨基酸或至少10个氨基酸。
在一些实施方案中，组合物是疫苗组合物，并且任选地还包含一种或多种佐剂。
在第六方面，本公开提供了一种组合物，所述组合物包含一种或多种根据本公开第四方面的抗体，以及任选的载体和/或赋形剂。
在第七方面，本公开提供了一种核苷酸序列，所述核苷酸序列编码根据本公开第一、第二或第三方面中所述的多肽或其抗原性片段，或根据本公开第四方面所述的抗体。本公开还提供了包含所述核苷酸序列的载体。
在第八方面，本公开提供了一种阵列，所述阵列包含固体基质，以及固定于固体基质上的多肽，所述多肽包含至少一种根据本公开第一或第二方面中所述的多肽或其抗原性片段。
在一些实施方案中，阵列中的多肽包含至少2种、至少3种、至少5种、至少10种、至少15种、至少20种、至少25种、至少30种或至少35种根据本公开第一或第二方面中所述的多肽或其抗原性片段。
在一些实施方案中，阵列中的多肽包含具有SEQ ID NO:10所示的氨基酸序列的多肽或其抗原性片段以及具有选自SEQ ID NO:1-9和11-39所示的氨基酸序列的多肽或其抗原性片段中的一种或多种。在一些实施方案中，阵列中的多肽包含具有SEQ ID NO:10所示的氨基酸序列的多肽或其抗原性片段以及具有选自SEQ ID NO:1-9和11-39所示的氨基酸序列的多肽或其抗原性片段中的1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37或38种。
在一些实施方案中，阵列中的多肽包含具有SEQ ID NO:10所示的氨基酸序列的多肽或其抗原性片段以及具有选自SEQ ID NO:1-9和11-35所示的氨基酸序列的多肽或其抗原性片段中的一种或多种。在一些实施方案中，阵列中的多肽包含具有SEQ ID NO:10所示的氨基酸序列的多肽或其抗原性片段以及具有选自SEQ ID NO:1-9和11-35所示的氨基酸序列的多肽或其抗原性片段中的1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33或34种。
在一些实施方案中，阵列中的多肽包含具有SEQ ID NO:10、7、35、29、 23、30、2、21和12所示的氨基酸序列的多肽或其抗原性片段。
在一些实施方案中，阵列中的多肽包含具有SEQ ID NO:10、12、35、14、30、2、3、13和19所示的氨基酸序列的多肽或其抗原性片段。
在一些实施方案中，阵列中的多肽包含具有SEQ ID NO:10、35、12和19所示的氨基酸序列的多肽或其抗原性片段。
在一些实施方案中，阵列中的多肽包含具有SEQ ID NO:10和33所示的氨基酸序列的多肽或其抗原性片段。
在一些实施方案中，阵列中的多肽包含具有SEQ ID NO:10、32和15所示的氨基酸序列的多肽或其抗原性片段。
在一些实施方案中，阵列中的多肽包含具有SEQ ID NO:10、21、30、15、12、35、23、19和20所示的氨基酸序列的多肽或其抗原性片段。
在一些实施方案中，抗原性片段的长度为至少5个氨基酸、至少6个氨基酸、至少7个氨基酸、至少8个氨基酸、至少9个氨基酸或至少10个氨基酸。
在一些实施方案中，阵列中的多肽包含多种根据本公开第一或第二方面中所述的多肽或其抗原性片段，其中多种多肽或其抗原性片段包含至少一种来源于SARS-CoV-2 ORF1ab多聚蛋白的多肽或其抗原性片段，以及至少一种来源于SARS-CoV-2 S蛋白的多肽或其抗原性片段。
在一些实施方案中，多种多肽还包含至少一种来源于SARS-CoV-2 N蛋白的多肽或其抗原性片段。
在第九方面，本公开提供了一种阵列，所述阵列包含固体基质，以及固定于固体基质上的多肽，所述多肽包含至少一种根据本公开第三方面中所述的多肽或其抗原性片段。
在一些实施方案中，阵列中的多肽包含至少2种、至少3种、至少4种、至少5种、至少6种、至少7种、至少8种或全部9种根据本公开第三方面中所述的多肽或其抗原性片段。
在一些实施方案中，阵列中的多肽包含多种根据本公开第三方面中所述的多肽或其抗原性片段，其中多种多肽或其抗原性片段包含至少一种来源于SARS-CoV-2 ORF1ab多聚蛋白的多肽或其抗原性片段，至少一种来源于SARS-CoV-2 S蛋白的多肽或其抗原性片段，至少一种来源于SARS-CoV-2 N蛋白的多肽或其抗原性片段。
在一些实施方案中，多肽通过与BSA偶联后固定化于固体基质上。
在一些实施方案中，阵列是多肽芯片。
在第十方面，本公开提供了一种检测受试者中针对SARS-CoV-2的抗体的存在的方法，其包括：
(a)使来自所述受试者的样品与根据本公开第一或第二方面中所述的多肽或其抗原性片段，或根据本公开第八或第九方面中所述的阵列接触；
(b)检测样品中与多肽或阵列上的多肽特异性结合的抗体的存在。
在一些实施方案中，方法用于诊断所述受试者是否被SARS-CoV-2感染。
在第十一方面，本公开提供了一种用于对被SARS-CoV-2感染的受试者进行预后的方法，其包括：
(a)使来自所述受试者的样品与根据本公开第三方面中所述的多肽或其抗原性片段，或根据本公开第九方面中所述的阵列接触；
(b)检测样品中与多肽或阵列上的多肽特异性结合的抗体的存在。
在一些实施方案中，抗体是IgG抗体和/或IgM抗体。
在一些实施方案中，样品是血液样品，例如血清或血浆。
在第十二方面，本公开提供了一种诱导受试者中针对SARS-CoV-2的免疫反应的方法，其包括对受试者施用根据本公开第一、第二或第三方面中所述的多肽或其抗原性片段，或根据本公开第五方面中所述的组合物。
在第十三方面，本公开提供了一种预防和/或治疗受试者中由SARS-CoV-2感染引起的疾病的方法，其包括对受试者施用根据本公开第一、第二或第三方面中所述的多肽或其抗原性片段，或根据本公开第五方面中所述的组合物。
在一些实施方案中，方法包括对受试者施用至少2种、至少3种、至少5种、至少10种、至少15种、至少20种、至少25种或至少30种根据本公开第一、第二或第三方面中所述的多肽或其抗原性片段的组合。
在一些实施方案中，方法包括对受试者施用包含具有SEQ ID NO:10所示的氨基酸序列的多肽或其抗原性片段以及具有选自SEQ ID NO:1-9和11-48所示的氨基酸序列的多肽或其抗原性片段中的一种或多种的组合。在一些实施方案中，方法包括对受试者施用包含具有SEQ ID NO:10所示的氨基酸序列的多肽或其抗原性片段以及具有选自SEQ ID NO:1-9和11-48所示的 氨基酸序列的多肽或其抗原性片段中的1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46或47种的组合。
在一些实施方案中，方法包括对受试者施用包含具有SEQ ID NO:10所示的氨基酸序列的多肽或其抗原性片段以及具有选自SEQ ID NO:1-9和11-39所示的氨基酸序列的多肽或其抗原性片段中的一种或多种的组合。在一些实施方案中，方法包括对受试者施用包含具有SEQ ID NO:10所示的氨基酸序列的多肽或其抗原性片段以及具有选自SEQ ID NO:1-9和11-39所示的氨基酸序列的多肽或其抗原性片段中的1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37或38种的组合。
在一些实施方案中，方法包括对受试者施用包含具有SEQ ID NO:10所示的氨基酸序列的多肽或其抗原性片段以及具有选自SEQ ID NO:1-9和11-35所示的氨基酸序列的多肽或其抗原性片段中的一种或多种的组合。在一些实施方案中，方法包括对受试者施用包含具有SEQ ID NO:10所示的氨基酸序列的多肽或其抗原性片段以及具有选自SEQ ID NO:1-9和11-35所示的氨基酸序列的多肽或其抗原性片段中的1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33或34种的组合。
在一些实施方案中，方法包括对受试者施用包含具有SEQ ID NO:10、7、35、29、23、30、2、21和12所示的氨基酸序列的多肽或其抗原性片段的组合。
在一些实施方案中，方法包括对受试者施用包含具有SEQ ID NO:10、12、35、14、30、2、3、13和19所示的氨基酸序列的多肽或其抗原性片段的组合。
在一些实施方案中，方法包括对受试者施用包含具有SEQ ID NO:10、35、12和19所示的氨基酸序列的多肽或其抗原性片段的组合。
在一些实施方案中，方法包括对受试者施用包含具有SEQ ID NO:10和33所示的氨基酸序列的多肽或其抗原性片段的组合。
在一些实施方案中，方法包括对受试者施用包含具有SEQ ID NO:10、32和15所示的氨基酸序列的多肽或其抗原性片段的组合。
在一些实施方案中，方法包括对受试者施用包含具有SEQ ID NO:10、21、30、15、12、35、23、19和20所示的氨基酸序列的多肽或其抗原性片段的组合。
在一些实施方案中，抗原性片段的长度为至少5个氨基酸、至少6个氨基酸、至少7个氨基酸、至少8个氨基酸、至少9个氨基酸或至少10个氨基酸。
在一些实施方案中，方法包括对受试者施用多种根据本公开第一、第二或第三方面中所述的多肽或其抗原性片段的步骤，其中多种多肽或其抗原性片段包含至少一种来源于SARS-CoV-2 ORF1ab多聚蛋白的多肽或其抗原性片段，至少一种来源于SARS-CoV-2 S蛋白的多肽或其抗原性片段，以及至少一种来源于SARS-CoV-2 N蛋白的多肽或其抗原性片段。
在第十四方面，本公开提供了一种治疗受试者中由SARS-CoV-2感染引起的疾病的方法，其包括对所述受试者施用根据本公开第四方面中所述的抗体。
附图说明
图1A和1B：血清样品中抗SARS-CoV-2的N蛋白(A)或S蛋白(B)的抗原特异性IgG抗体在SARS-CoV-2感染症状出现后不同时间点的滴度(第1-30天，n＝36；第31-61天，n＝18；第100-150天，n＝21；第180-220天，n＝26)，对照组为20名健康供体(HD)的血清样品。
图2：使用多肽芯片检测血清样品的示意图。将多肽库的肽连接BSA后打印到芯片上，加入血清样品反应后，用LuxScan10K-A(中国北京CapitalBio公司)对芯片进行扫描，参数分别设置为95％激光功率/PMT550和95％激光功率/PMT480。荧光强度数据由GenePixPro6.0软件(Molecular Devices,CA,USA)获得，其中绿色荧光指示IgG强度，红色荧光指示IgM强度，黄色为绿色和红色的叠加。
图3：在疾病发作后10-60天(n＝18)、100-150天(n＝18)和180-220天(n＝15)收集的至少一个样品中可检测到针对其阳性反应的肽计数和分布。数字表示来自每个蛋白鉴定出的IgG和IgM表位的总数。缩略词：ORF，开放阅读框； S，刺突蛋白；E，包膜蛋白；N，核衣壳蛋白；M，膜蛋白。
图4：优势和持久性IgG和IgM表位的信号强度。每个点表示来自健康供体的混合血清或从症状出现后指定时间点收集的不同患者的血清样品。
图5：单体(a)和三聚体(b)S蛋白(PBD ID：6VXX)的3D结构上的优势和持久性表位的位置的示意图。
图6：早期SARS-CoV-2(Wuhan-Hu-1毒株)和六个新出现的变异株的表位保守性分析。黑点代表与Wuhan-Hu-1毒株相同的氨基酸残基。
图7：a.在疾病发作后多个时间点收集的患者血清样品中，亚优势表位的IgG和IgM识别频率。b.经鉴定的亚优势表位随时间的信号强度动力学。每个点代表在症状出现后的指定时间点从COVID-19个体获得的不同患者的血清样品。虚线表示每个肽的阳性反应截止值。
图8：N蛋白(PDB ID：6YUN)C端二聚化结构域表位的详细结构分析。
图9：高结合信号强度肽的识别频率随着时间的推移阳性率降低。
图10：表位在症状发作后的三个采样时间点的信号强度。每个点代表在症状出现后指定时间点收集的个体患者的血清，虚线表示每个肽的阳性反应截止值。“*”代表p<0.05，“**”代表p<0.01。
发明详述
本公开涉及分离的多肽或其抗原性片段，结合该分离的多肽或其抗原性片段的抗体，包含该分离的多肽或其抗原性片段的组合物，以及包含该分离的多肽或其抗原性片段的阵列。本公开还提供了检测受试者中针对SARS-CoV-2的抗体的存在的方法，对被SARS-CoV-2感染的受试者进行预后的方法，诱导受试者中针对SARS-CoV-2的免疫反应的方法，以及预防和/或治疗受试者中由SARS-CoV-2感染引起的疾病的方法。
本文描述的任何方面或实施方案可以与本文公开的任何其他方面或实施方案组合。虽然已经结合本公开的详细描述描述了本公开，但是前面的描述旨在说明而不是限制本公开的范围，本公开的范围由所附权利要求的范围限定。其他方面、优点和修改在以下权利要求的范围内。本文提及的专利和科学文献建立了本领域技术人员可获得的知识。本文引用的所有美国专利和公开或未公开的美国专利申请通过引用并入本文。本文引用的所有公开的外 国专利和专利申请在此通过引用并入本文。本文引用的所有其他公开的参考文献、词典、文件、手稿和科学文献在此通过引用并入本文。从包括实施方案和权利要求的以下详细描述中，本公开的其他特征和优点将是显而易见的。
定义
为了更容易理解本发明公开，首先在下面定义某些术语。在整个说明书中阐述了对以下术语和其他术语的附加定义。
除非另有限定，否则本文所使用的全部技术和科学术语的含义与本领域普通技术人员通常所理解的含义相同。例如，本文所使用的术语如Janeway CA Jr，Travers P，Walport M等的《免疫生物学》(Immunobiology)，第五版，New York：GarlandScience(2001)和“A multilingual glossary of biotechnological terms:(IUPAC Recommendations)”,Leuenberger,H.G.W,Nagel,B.和
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H.编辑(1995),Helvetica Chimica Acta,CH-4010 Basel,Switzerland中所述的定义。
应当注意，如本文中及所附权利要求书中使用的，单数形式“一个”、“一种”和“该/所述”包括复数提及物，除非上下文另有明确规定。因此，术语“一个”、“一种”、“一个/种或多个/种”和“至少一个/种”可以互换使用。类似地，术语“包含”、“包括”和“具有”可以互换使用。
在本文中及所附权利要求书中使用术语“包含”时，其不排除其他元素。为了本发明的目的，术语“由……组成”被认为是术语“包含”的优选实施方案。
在本文中，术语“多肽”、“肽”、“蛋白”和“蛋白质”可交换地使用，是指通过肽键共价连接的由氨基酸残基组成的化合物，并且不限制产物的最小长度。因此，上述术语包含肽、寡肽、多肽、二聚体(异源和同源)、多聚体(异源和同源)等。“分离的”多肽是指从其天然环境中取出的多肽，或者是指使用标准分子生物学技术和表达系统人工合成的多肽。例如，本公开的多肽可以从野生型或突变体SARS-CoV-2病毒蛋白中分离获得，或者使用商业自动化方法(例如，全部固相合成、部分固相法、片段组合法或溶液合成)以化学方式合成。
“蛋白质”和“多肽”涵盖了全长蛋白质及其片段。该术语还包括多肽的表达后修饰，如糖基化、乙酰化、磷酸化等。此外，为了本公开的目的，多肽还指对天然蛋白质或多肽的氨基酸序列进行修饰后所得的变体。
典型地，氨基酸序列修饰包括取代、缺失、插入和添加。如本文所用，“取代”是指至少一个残基已被移除并在该位置上插入不同的残基。氨基酸取代通常为单残基但可同时在许多不同位置发生。“缺失”的特征是从序列中移除一个或多个氨基酸残基。通常在蛋白质分子内的任何一个位点上缺失不超过约2-6个残基。“插入”是指单个或多个氨基酸残基的序列内插入，而“添加”是指氨基和/或羧基端融合物。与添加相比，插入通常将为较小的插入，例如大约1-4个残基。
关于氨基酸序列的术语“变体”包括从该序列或对该序列的一个(或多个)氨基酸进行的任何取代、变化、修饰、替换、缺失或添加。在单个变体中可以包含取代、缺失、插入、添加或其任何组合，只要多肽变体具有抗原性。
“免疫原性”又称“抗原性”、“免疫反应性”，是指蛋白质或多肽产生抗体或者诱导受试者的抗体反应的能力。“抗原性片段”是指能够诱导免疫反应的多肽片段。抗原性片段的长度可以为，例如至少5个氨基酸、至少6个氨基酸、至少7个氨基酸、至少8个氨基酸、至少9个氨基酸或至少10个氨基酸，例如5、6、7、8、9、10、11、12、13、14或15个氨基酸。抗原性片段可以通过本领域技术人员已知的方法产生。
在本文中，“表位”或“抗原表位”可交换地使用，其是本领域的术语，并且是指抗体可以特异性结合的抗原的局部区域。表位可以是例如多肽的连续氨基酸(线性或连续表位)或者表位可以例如来自多肽或多个多肽的两个或更多个非连续区域(构象、非线性、不连续的或非连续的表位)。
在该背景下，术语“结合”或“特异性结合”通常是指两个或多个实体之间的非共价缔合，其定义为抗体和相应表位之间以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的亲和力(K D)结合该抗原。本文所用“K D”是指，特定抗体-抗原相互作用的解离平衡常数，用于描述抗体与抗原间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。结合常数的测定是本领域技术人员已知的。
如本文所用，“抗体”是一种免疫球蛋白分子，其能够通过位于免疫球蛋白分子的可变区中的至少一个抗原识别位点与靶标特异性结合。本文中提 及的抗体应以其最广泛的含义理解。抗体包括任何类别的抗体，如IgD、IgE、IgG、IgA或IgM(或其子类别)，并且该抗体不必是任何特定类别。本公开涉及的任何抗体可以是单克隆的或多克隆的。术语“单克隆抗体”是指同质性抗体群，而术语“多克隆抗体”是指异质性抗体群。这两个术语不应被解释为对抗体的来源或制备方式的限制。
术语“中和”是指结合配体并防止或降低该配体的生物效应的抗原结合分子、scFv、抗体或其片段。在一些实施方案中，抗原结合分子、scFv、抗体或其片段直接阻断配体上的结合位点或者通过间接方式改变配体的结合能力(例如配体的结构或能量改变)。在一些实施方案中，抗原结合分子、scFv、抗体或其片段防止与其结合的蛋白质行使生物学功能。
术语“核酸”、“核苷酸”、“核苷酸序列”可互换使用，是指基本上由核苷酸(如脱氧核糖核苷酸和/或核糖核苷酸)组成的任何长度的寡聚物和聚合物。核酸可以包含嘌呤和/或嘧啶碱基和/或其他天然(例如黄嘌呤、肌苷、次黄嘌呤)，化学或生物化学修饰(例如甲基化)，非天然或衍生的核苷酸碱基。核酸的骨架可以包含通常存在于RNA或DNA中的糖和磷酸基团，和/或一种或多种经修饰或取代的糖和/或一种或多种经修饰或取代的磷酸基团。可以引入磷酸基团或糖的修饰以改善稳定性、对酶促降解的抗性，或一些其他有用的特性。“核酸”可以是例如双链的，部分双链的或单链的。当是单链时，核酸可以是有义链或反义链。“核酸”可以是环形或线形的。如本文所用，术语“核酸”涵盖DNA和RNA，包括基因组、pre-mRNA、mRNA、cDNA、包含载体的重组或合成核酸。出于本文所述用途的目的，应理解，可以通过本领域可用的任何方法修饰多核苷酸。
本领域技术人员将理解，由于遗传密码的简并性，许多不同的多核苷酸和核酸可以编码相同的多肽。另外，应当理解，技术人员可以使用常规技术进行不影响本文所述多核苷酸编码的多肽序列的核苷酸取代，以反映要在其中表达多肽的任何具体宿主生物体的密码子使用。
如本文所用，术语“感染”意指通过病毒在细胞或个体中增殖和/或存在而侵袭。在一些实施方案中，感染是指“活性”感染，也就是说，病毒在细胞或个体中复制的感染。此类感染的特征在于病毒从其最初感染的细胞、组织和/或器官传播至其他细胞、组织和/或器官中。感染也可以指潜伏性感染， 即病毒不复制的感染。在某些实施方案中，感染是指由细胞或个体中存在的病毒引起的病理状态，或者是指通过病毒对细胞或个体的侵袭引起的病理状态。
抗原性多肽及其变体
本文所述的蛋白质和多肽可以指天然蛋白质或多肽的变体，其具有与天然蛋白质或多肽至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％的序列同一性，条件是所述变体保持天然蛋白质或多肽的初始功能或活性。
本公开提供了一种分离的多肽或其抗原性片段，所述多肽具有选自SEQ ID NO:1-39中任一项的氨基酸序列。在一些实施方案中，本公开提供了一种分离的多肽或其抗原性片段，所述多肽包含与选自SEQ ID NO:1-39中任一项的氨基酸序列具有至少50％同一性的氨基酸序列，例如具有至少60％同一性、至少70％同一性、至少80％同一性、至少90％同一性或100％同一性的氨基酸序列，例如具有50％、55％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。在一些实施方案中，所述多肽的抗原性片段的长度为至少5个氨基酸、至少6个氨基酸、至少7个氨基酸、至少8个氨基酸、至少9个氨基酸或至少10个氨基酸，例如5、6、7、8、9、10、11、12、13、14或15个氨基酸。
本公开还提供了一种分离的多肽或其抗原性片段，所述多肽具有选自SEQ ID NO:1-35中任一项的氨基酸序列。在一些实施方案中，本公开提供了一种分离的多肽或其抗原性片段，所述多肽包含与选自SEQ ID NO:1-35中任一项的氨基酸序列具有至少50％同一性的氨基酸序列，例如具有至少60％同一性、至少70％同一性、至少80％同一性、至少90％同一性或100％同一性的氨基酸序列，例如具有50％、55％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。在一些实施方案中，所述多肽的抗原性片段的长度为至少5个氨基酸、至少6个氨基酸、至少7个氨基酸、至少8个氨基酸、至少9个氨基酸或至少10个氨基酸，例如5、6、7、8、9、10、11、12、13、14或15个氨基酸。
本公开还提供了一种分离的多肽或其抗原性片段，所述多肽具有选自SEQ ID NO:40-48中任一项的氨基酸序列。在一些实施方案中，本公开提供了一种分离的多肽或其抗原性片段，所述多肽包含与选自SEQ ID NO:40-48中任一项的氨基酸序列具有至少50％同一性的氨基酸序列，例如具有至少60％同一性、至少70％同一性、至少80％同一性、至少90％同一性或100％同一性的氨基酸序列，例如具有50％、55％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。在一些实施方案中，所述多肽的抗原性片段的长度为至少5个氨基酸、至少6个氨基酸、至少7个氨基酸、至少8个氨基酸、至少9个氨基酸或至少10个氨基酸，例如5、6、7、8、9、10、11、12、13、14或15个氨基酸。
如本文所用，“序列同一性”是指任何给定的查询序列和主题序列之间的同一性程度。本领域的技术人员将容易理解如何确定两个多肽(例如未经修饰的肽和肽变体)的同一性。例如，可以在对齐两条序列使它们的同一性达到最高水平之后再计算同一性，例如可以引入空位。计算同一性的另一个方法可以通过公开的算法实施。此类数学算法的非限制性实例包括Myers和Miller(1988)CABIOS 4:11-17的算法、Smith等(1981)Adv.Appl.Math.2:482的局部同源性算法、Needleman和Wunsch(1970)J.Mol.Biol.48:443-453的同源性比对算法、Pearson和Lipman(1988)Proc.Natl.Acad.Sci.85:2444-2448的用于搜索同源性的方法、和Karlin和Altschul(1990)Proc.Natl.Acad.Sci.USA 87:2264的算法的修改形式，记载于Karlin和Altschul(1993)Proc.Natl.Acad.Sci.USA 90:5873-5877的算法。通过使用基于此类数学算法的程序，可以实施用于测定序列同一性的序列比较(即比对)。程序可以由计算机适当执行。此类程序的实例包括但不限于PC/Gene程序的CLUSTAL、ALIGN程序(Version 2.0)、和Wisconsin遗传学软件包的GAP、BESTFIT、BLAST、FASTA、和TFASTA。可以例如通过使用初始参数实施使用这些程序的比对。
在一些实施方案中，本公开的分离的多肽及其抗原性片段包括变体，变体可以包含一种或多种氨基酸修饰。在单个变体中可以包含取代、缺失、插入、添加或其任何组合，只要多肽变体具有抗原性。制备变体的方法和技术是本领域技术人员公知的。
本文使用的氨基酸取代可以为保守或非保守取代。在这一点上，本领域中理解，氨基酸可以基于其物理特性而分成组。此类分组的实例包括但不限于带电荷的氨基酸、不带电荷的氨基酸、极性不带电荷的氨基酸和疏水性氨基酸。保守氨基酸取代可以涉及将天然氨基酸残基用非天然残基取代，使得对该位置上氨基酸残基的尺寸、极性、电荷、疏水性或亲水性有很少或没有影响，具体而言，不导致抗原性降低。
在本文中，用保守氨基酸取代是指与具有相似侧链的残基的可交换性。本领域已经定义了具有侧链的氨基酸残基家族。这些家族包括具有以下侧链的氨基酸：碱性侧链(例如赖氨酸、精氨酸、组氨酸)，酸性侧链(例如天冬氨酸、谷氨酸)，不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)，非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)，beta分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。
在一些实施方案中，取代并不保守。例如，可以用改变该多肽的一些性质或方面的氨基酸进行取代。在一些实施方案中，可以实施非保守的氨基酸取代，例如，改变多肽的结构，改变多肽的结合性质(例如，增加或降低多肽的结合亲和力，和/或增加或降低多肽的结合特异性)。
抗体
本公开还涉及一种分离的抗体，其结合本公开所述的多肽或其抗原性片段。如本文所用，术语“抗体”以其最广泛的含义理解，其不仅涵盖完整(即，全长)多克隆或单克隆抗体，而且涵盖其抗原结合片段(如Fab、Fab'、F(ab')2、Fv)、单链(scFv)、其突变体、包含抗体部分的融合蛋白、人源化抗体、嵌合抗体、双抗体、纳米抗体、线性抗体、单链抗体、多特异性抗体(例如，双特异性抗体)、以及包含具有所需特异性的抗原识别位点的免疫球蛋白分子的任何其他修饰构造，其包括抗体的糖基化变体、抗体的氨基酸序列变体、以及经共价修饰的抗体。抗体包括任何类别的抗体，如IgD、IgE、IgG、IgA或IgM(或其子类别)，并且该抗体不必是任何特定类别。免疫球蛋白可以根据其重链恒定结构域的抗体氨基酸序列划分为不同的类别。免疫球蛋白有五 种主要类别：IgA、IgD、IgE、IgG和IgM，并且这些中的若干可以进一步分为子类别(同种型)，例如IgG1、IgG2、IgG3和IgG4。对应于不同类别的免疫球蛋白的重链恒定结构域分别被称为α、δ、ε、γ和μ。不同类别的免疫球蛋白的亚基结构和三维构型是众所周知的。
本文描述的任何抗体可以是单克隆的或多克隆的。如本文所用，术语“单克隆抗体”是指从基本均质的抗体群体获得的抗体，即构成群体的各抗体是相同的，除了可以少量存在的可能的天然存在的突变外。单克隆抗体是高度特异性的，针对单一抗原。此外，与通常包括针对不同决定簇(表位)的不同抗体的多克隆抗体制剂相反，单克隆制剂中的每种抗体针对抗原上相同的单一决定簇。单克隆抗体不限于通过杂交瘤技术产生的抗体。术语“多克隆抗体”是指异质性抗体群，其是含有多种类型抗体的抗体混合物，通常由不同类型的浆细胞生产而来。这两个术语不应被解释为对抗体的来源或制备方式的限制。
例如，在一些实施方案中，单克隆抗体包括嵌合抗体，如鼠源性单克隆抗体的人源化形式。此类人源化抗体可以通过已知技术制备，并且在将抗体施用于人时提供降低免疫原性的优点。在一个实施方案中，人源化单克隆抗体包含鼠源性抗体的可变区(或仅其抗原结合位点)和衍生自人抗体的恒定区。替代地，人源化抗体片段可以包含鼠源性单克隆抗体的抗原结合位点和衍生自人抗体的可变区片段(缺乏抗原结合位点)。用于产生嵌合的单克隆抗体和进一步经工程化改造的单克隆抗体的程序包括描述在以下中的那些程序：Riechmann等人，(Nature 332:323,1988)；Liu等人，(PNAS 84:3439,1987)；Larrick等人，(Bio/Technology 7:934,1989)；以及Winter and Harris(TIPS 14:139,May,1993)。用于转基因生成抗体的程序可以见于GB 2,272,440、美国专利号5,569,825和5,545,806。
可以使用通过基因工程方法产生的抗体(如嵌合和人源化单克隆抗体)，其包含人和非人部分二者，可以使用标准重组DNA技术来制造。可以使用本领域已知的标准DNA技术，通过基因工程来产生此类嵌合和人源化单克隆抗体。
可以通过对含有人免疫球蛋白基因的转基因动物进行免疫来生成具有人恒定区和可变区的人单克隆抗体。参见Jakobovits等人，Ann NY Acad Sci  764:525-535(1995)。还可以使用从衍生自受试者淋巴细胞的mRNA制备的免疫球蛋白轻链和重链cDNA，通过构建组合免疫球蛋白文库(如Fab噬菌体展示文库或scFv噬菌体展示文库)来制备针对PLA2-GIB多肽的人单克隆抗体。参见，例如，McCafferty等人，PCT公布WO 92/01047；Marks等人，(1991)J.Mol.Biol.222:581 597；以及Griffths等人，(1993)EMBO J 12:725 734。另外，可以通过对已知的人抗体进行突变来产生抗体可变区的组合文库。例如，可以通过例如使用随机改变的诱变寡核苷酸来突变已知结合PLA2-GIB的人抗体的可变区，以生成突变可变区的文库，然后可以筛选该文库以结合至PLA2-GIB。在免疫球蛋白重链和/或轻链的CDR区内诱导随机诱变的方法、将随机化重链和轻链进行杂交以形成配对的方法、以及筛选方法可以见于例如Barbas等人，PCT公布WO 96/07754；Barbas等人，(1992)Proc.Nat'l Acad.Sci.USA 89:4457 4461。
强大的中和抗体可以提供治疗和预防试剂以对抗COVID-19。然而仅关注S蛋白和S蛋白的RBD区域可能会增加对该区域的进化选择压力，从而导致该区域突变的富集，致使以RBD区域为中心的治疗性抗体和疫苗的有效性降低(Wang,L.,et al.,Importance of Neutralizing Monoclonal Antibodies Targeting Multiple Antigenic Sites on the Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein To Avoid Neutralization Escape.J Virol,2018.92(10))。
实际上，已知除了RBD之外，S蛋白的其他区域或表位也可以引发中和抗体。其他蛋白质，如N蛋白质、Orf9b和Nsp5蛋白质也有可以引发明显的抗体中和反应。此外，E蛋白质和Orf7b蛋白诱导的抗体反应在健康人群和病毒感染者之间也有显著差别(Jiang,H.W.,et al.,SARS-CoV-2 proteome microarray for global profiling of COVID-19 specific IgG and IgM responses.Nat Commun,2020.11(1):p.3581)。
核苷酸和载体
本公开还涉及一种核苷酸序列，其编码根据本公开所述的多肽或其抗原性片段，或根据本公开所述的抗体。在另一方面，本公开还涉及一种载体，其根据本公开所述的核苷酸序列。
本文所用“载体”是指可以将核苷酸插入其中的一种核酸运载工具。而 当载体能使插入的核苷酸编码的蛋白获得表达时，该载体称为表达载体。载体可以通过转化、转导或者转染等方法导入宿主细胞，继而使其携带的遗传物质元件在宿主细胞内获得表达。载体是本领域技术人员公认的、包括但不限于：(1)质粒；(2)噬菌粒；(3)柯斯质粒；(4)人工染色体，如酵母人工染色体、细菌人工染色体或P1来源的人工染色体；(5)噬菌体如λ噬菌体或M13噬菌体及(6)动物病毒，如逆转录酶病毒、腺病毒、腺相关病毒、孢疹病毒、痘病毒、杆状病毒。一种载体可以含有多种控制表达的元件，包括但不局限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因；此外，载体还可以含有复制起始位点。
SARS-CoV-2病毒和COVID-19
SARS-CoV-2病毒，即严重急性呼吸系统综合征冠状病毒2，是一种具有包膜的正链单股RNA病毒，属于冠状病毒科乙型冠状病毒属严重急性呼吸道综合征相关冠状病毒种。它的基因序列和SARS病毒及MERS病毒属于同一谱系但不同进化枝，是已知的第七种可感染人类的冠状病毒。病毒的宿主包括哺乳动物和禽类动物。该病毒可通过人类上呼吸道入侵人体，以多种细胞表面表达的ACE2为受体达到感染；主要感染器官包括肺部、心脏、肾脏等多个主要器官。目前针对感染SARS-CoV-2病例的主流检测手段为RT-PCR。“SARS-CoV-2病毒”和“新冠病毒”在本文中可交换地使用。
SARS-CoV-2病毒基因组序列为5’UTR-ORF1a/b-结构蛋白基因-3’UTR。预测SARS-CoV-2病毒可以编码29种蛋白质，其中包括4种结构蛋白，9种辅助蛋白和16种非结构蛋白(Nsp)。结构蛋白包括膜蛋白(M蛋白)、核衣壳蛋白(N蛋白)、刺突蛋白(S蛋白)和包膜蛋白(E蛋白)。其中SARS-CoV-2病毒的非结构蛋白序列相对保守，ORF1ab基因编码参与病毒RNA转录和复制的多功能蛋白，包含负责切割多聚蛋白的蛋白酶。S蛋白属于三聚体I类融合蛋白，包含S1和S2两个亚基以及一个跨膜锚。S1亚基包含1个信号肽、N末端结构域(NTD)和受体结合域(RBD)，而S2亚基包含融合肽(FP)、七肽重复序列(HR)1和2、跨膜结构域和胞内结构域。N蛋白序列保守程度高，在病毒复制过程中发挥重要作用。N蛋白与病毒RNA结合形成复合体，随后在M蛋白和E蛋白的共同作用下，包裹后进入病毒衣壳中。N蛋白含有N1和N2表位，表位 N1能刺激机体产生高亲和力的抗体，但是一般没有中和活性。
SARS-CoV-2病毒会造成一种感染性疾病COVID-19(2019冠状病毒病，又称“新型冠状病毒肺炎”)，其常见临床症状包括发烧、咳嗽以及呼吸急促，部分可见咽喉酸痛、肌肉乏力、积痰等症状。对COVID-19的病理研究表明，SARS-CoV-2病毒感染会导致呼吸系统损伤(如肺部急性病变)、细胞因子释放综合征(如细胞因子风暴和急性炎症反应)、循环系统损伤(例如心血管损伤如心律失常、血管栓塞、肺血管收缩等)、消化系统损伤(例如胰脏、肝脏等器官)、泌尿生殖系统损伤(如急性肾衰竭)、神经系统损伤(如丧失嗅觉、味觉)、眼部损伤(如结膜感染)等。此外，部分COVID-19患者在康复后仍会经历一系列影响(2019冠状病毒病长期综合症)。
组合物和疫苗组合物
本公开提供了包含一种或多种如本公开所述的多肽或其抗原性片段的组合物。在一些实施方案中，该组合物为疫苗组合物。“疫苗组合物”是当施用于受试者(例如，哺乳动物)时诱导或增强针对组合物内所包含抗原性物质的免疫反应的组合物。该反应可以包括诱导抗体的产生(例如，通过刺激B细胞)或基于T细胞的反应(例如，溶细胞反应)。这些反应可以是或可以不是保护性或中和性的。保护性或中和性免疫反应是对与抗原相一致的感染生物体(例如抗原所源自的生物体)有害但对受试者有益(例如通过降低或预防感染)的免疫反应。
免疫反应，又称免疫应答，是指免疫系统的细胞(例如T淋巴细胞、B淋巴细胞、自然杀伤(NK)细胞、巨噬细胞、嗜酸性粒细胞、肥大细胞、树突状细胞以及中性粒细胞)和由这些细胞中的任一种或肝脏产生的可溶性大分子(包括抗体、细胞因子和补体)的作用，该作用导致选择性靶向、结合、损害、破坏和/或排除脊椎动物体内的入侵病原体、被病原体感染的细胞或组织、癌细胞或其他异常细胞，或者在自身免疫或病理性炎症的情况下，正常的人细胞或组织。当施用于受试者时，组合物包含一定量的足以引起免疫反应的本公开的多肽或其抗原性片段。用作疫苗的组合物包含本公开的多肽或其抗原性片段以及所需的任何其他组分。“免疫有效量”意指以单剂量或作为一系列的部分将该量施用于受试者对治疗或预防有效。
在一些实施方案中，本公开的组合物还包含一种或多种佐剂。“佐剂”通常是指在细胞水平或体液水平增加、刺激、活化、加强或调节针对组合物的活性成分的免疫反应的试剂。佐剂可以在获得性和先天性免疫两者中起作用并以多种方式发挥功能。
许多物质(天然或合成的)已显示作为佐剂起作用。可以使用描述于文献中的已知佐剂，例如Clin Microbiol Rev 1994,7:277-89。合适佐剂的实例包括铝盐(磷酸铝、氢氧化铝、羟基氧化铝等)、明矾、霍乱毒素、沙门氏菌毒素、IFA(不完全弗氏佐剂)、CFA(完全弗氏佐剂)、ISCOMatrix、GM-CSF和其他免疫刺激细胞因子、含有CpG基序的寡脱氧核苷酸(CpG7909等)、水包油乳液、皂苷或其衍生物(QS21等)、脂多糖例如脂质A或其衍生物(MPL、RC529、GLA、E6020等)、脂肽、乳铁蛋白、鞭毛蛋白、双链RNA或其衍生物(poli IC等)、细菌DNA、咪唑并喹啉(Imiquimod、R848等)、C型凝集素配体(海藻糖-6,6’-二山嵛酸/酯(TDB)等)、CD1d配体(alpha-半乳糖神经酰胺等)、角鲨烯乳液(MF59、AS03、AF03等)、PLGA等，但不限于此。佐剂和组合物可以连续地施用于受试者，或者在即将施用给受试者之前混合在一起。
在另一方面，本公开提供包含本文所述的抗体的组合物还包含载体和/或赋形剂。
载体包括任何和所有药学上可接受的溶剂、分散介质、媒介物、包衣、稀释剂、抗细菌剂和抗真菌剂、等渗剂和延迟吸收剂、缓冲液、载体溶液、悬浮液、胶体等。此类介质和试剂对于药学活性物质的使用在本领域中是公知的。补充的活性成分也可以被引入组合物中。
术语“药学上可接受的”是指一种分子或组合物，当其施用于接受者时，对其接受者无害，或者对其接受者的益处超过任何有害作用。关于用于配制如本文公开的组合物的载体或赋形剂，药学上可接受的载体或赋形剂必须与组合物的其他成分相容并且对其接受者无害，或对其接受者的益处超过任何有害作用。术语“药学上可接受的载体”是指药学上可接受的材料、组合物或媒介物，如液体或固体填充剂、稀释剂、赋形剂、溶剂、介质、包封材料、制造助剂(例如润滑剂、滑石镁、硬脂酸钙或锌或硬脂酸)或溶剂包封材料，其参与将药剂从身体的一个部分携带或运输到另一部分(例如，从一个器官到另一个器官)。在与配制剂的其他成分相容并且不对患者有害的意义上， 每种载体必须是“可接受的”。可充当药学上可接受的载体的材料的一些实例包括：(1)糖，如乳糖、葡萄糖和蔗糖；(2)淀粉，如玉米淀粉和马铃薯淀粉；(3)纤维素及其衍生物，如羧甲基纤维素钠、甲基纤维素、乙基纤维素、微晶纤维素和乙酸纤维素；(4)粉状黄蓍胶；(5)麦芽；(6)明胶；(7)赋形剂，如可可脂和栓剂蜡；(8)油，如花生油、棉籽油、红花油、芝麻油、橄榄油、玉米油和大豆油；(9)二醇，如丙二醇；(10)多元醇，如甘油、山梨糖醇、甘露醇和聚乙二醇(PEG)；(11)酯，如油酸乙酯和月桂酸乙酯；(12)琼脂；(13)缓冲剂，如氢氧化镁和氢氧化铝；(14)海藻酸；(15)无热原水；(16)等张盐水；(17)林格氏溶液；(19)pH缓冲溶液；(20)聚酯、聚碳酸酯和/或聚酐；(21)填充剂，如多肽和氨基酸(22)血清组分，如血清白蛋白、HDL和LDL；(23)C2-C12醇，如乙醇；和(24)药物配制剂中使用的其他无毒相容物质。
合适的赋形剂将与本公开的抗体相容。本领域公知的赋形剂包括例如填充剂、粘合剂、崩解剂、包衣剂、吸附剂、抗粘附剂、助流剂、防腐剂、抗氧化剂、调味剂、着色剂、甜味剂、溶剂、共溶剂、缓冲剂、螯合剂、粘度赋予剂、表面活性剂、稀释剂、润湿剂、载体、防腐剂、乳化剂、稳定剂和张力调节剂。本公开的组合物所包含的赋形剂取决于施用途径，适合用于本公开组合物的赋形剂是本领域已知的。
在一些实施方案中，根据本公开的组合物可以配制成以下形式：软膏剂、凝胶、糊剂、液体溶液、悬浮液、片剂、明胶胶囊剂、胶囊剂、栓剂、粉剂、滴鼻剂或气雾剂，优选地配制成注射溶液或悬浮液的形式。例如，对于注射剂，组合物通常包装成液体悬浮液的形式，其可以通过注射器或灌注来注入。在这方面，通常将组合物溶解在与药物用途相容且本领域技术人员已知的盐水、生理、等渗或缓冲的溶液中。
阵列
本公开提供了一种阵列，其包含固体基质，以及固定于固体基质上的多肽，其中多肽包含至少一种本公开所述的多肽或其抗原性片段。在一些实施方案中，多肽包含至少2种、至少3种、至少5种、至少10种、至少15种、至少20种、至少25种、至少30种或至少35种本公开所述的多肽或其抗原性片段，例如包含2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、 19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34或35种本公开所述的多肽或其抗原性片段。在另一些实施方案中，多肽包含至少2种、至少3种、至少4种、至少5种、至少6种、至少7种、至少8种或全部9种根据本公开所述的多肽或其抗原性片段，例如包含2、3、4、5、6、7、8或9种本公开所述的多肽或其抗原性片段。
如本领域所熟知的，“阵列”通常是由具有分开的间距(即离散的)区域(“点”)的线性的或二维结构形成的，每个阵列均具有有限的面积并形成在固体基质的表面上。阵列还可以是微珠结构的，其中每个微珠可以通过分子密码或颜色密码识别，或在连续流体中被识别。还可以序贯实施分析，其中样品通过一系列点，每个点从溶液中吸附该类分子。本公开中使用的固体基质没有特别限定，只要是能够将多肽固定于其上的基质即可。固体基质通常为玻璃或聚合物，最常用的聚合物是纤维素、聚丙烯酰胺、尼龙、聚苯乙烯、聚氯乙烯或聚丙烯。固体基质可以为管、珠子、盘、硅芯片、微孔板、聚偏氟乙烯(PVDF)膜、硝基纤维素膜、尼龙膜、其他多孔膜、非多孔膜(例如，塑料、聚合物、有机玻璃(perspex)、硅等)、大量聚合物针、或大量微滴定孔、或适合用于固定多肽、多核苷酸和其他合适的分子和/或进行免疫分析的任何其他表面形式。结合方法是本领域所熟知的并且通常包括交联共价结合或物理吸附多肽、多核苷酸等至固体基质。通过使用熟知的技术，例如接触式或非接触式印刷、掩膜或光刻，可以确定每个点的位置(Jenkins,R.E.,Pennington,S.R.(2001,Proteomics,2,13-29)和Lal等人(2002,Drug Discov Today 15；7(18 Suppl):S143-9))。
阵列通常为微阵列。“微阵列”是指具有至少约100/cm 2，优选至少约1000/cm 2离散区域密度的区域的阵列。微阵列中的区域具有通常的维度，例如直径在约10-250μm的范围，并通过大致相同的距离与阵列中的其他区域分开。阵列还可以是宏阵列或纳米阵列。制造阵列的方法是本领域技术人员所熟知的。
在一些实施方案中，阵列是多肽芯片。“多肽芯片”是指适于多肽捕捉的生物芯片，其可以用于进行高通量筛选。多肽芯片表面包含多个可寻址位置，每个可寻址位置具有与其结合的生物特异性组分，例如多肽或抗原性片段。
众所周知，IgG和IgM抗体在针对病毒感染如SARS冠状病毒和MERS冠 状病毒感染中发挥重要的作用。用于研究患者的IgG和IgM反应的常规技术包括ELISA和免疫胶体金技术。这些技术通常只能在单个反应中测试单个目标蛋白或抗体。相比之下，多肽芯片能够以高通量格式对抗体反应进行蛋白质组范围的测定，从而提供了对这些重要抗体反应的更系统的描述。
如本文所公开的，阵列中包含的多肽或其抗原性片段可以来源于SARS-CoV-2 ORF1ab多聚蛋白的多肽或其抗原性片段、来源于SARS-CoV-2 S蛋白的多肽或其抗原性片段和/或来源于SARS-CoV-2 N蛋白的多肽或其抗原性片段。在一些实施方案中，阵列中包含的多肽或其抗原性片段可以来源于SARS-CoV-2 ORF1ab多聚蛋白的多肽或其抗原性片段、SARS-CoV-2 S蛋白的多肽或其抗原性片段和SARS-CoV-2 N蛋白的多肽或其抗原性片段中的至少一种，例如阵列中包含的多肽或其抗原性片段可以来源于SARS-CoV-2 ORF1ab多聚蛋白的多肽或其抗原性片段、SARS-CoV-2 S蛋白的多肽或其抗原性片段和SARS-CoV-2 N蛋白的多肽或其抗原性片段中的一种，可以来源于SARS-CoV-2 ORF1ab多聚蛋白的多肽或其抗原性片段、SARS-CoV-2 S蛋白的多肽或其抗原性片段和SARS-CoV-2 N蛋白的多肽或其抗原性片段中的两种，或者SARS-CoV-2 ORF1ab多聚蛋白的多肽或其抗原性片段、SARS-CoV-2 S蛋白的多肽或其抗原性片段和SARS-CoV-2 N蛋白的多肽或其抗原性片段中的全部三种。
诊断和预后
本公开提供了使用本公开所述的多肽或其抗原性片段，或本公开所述的阵列诊断受试者是否被SARS-CoV-2感染的方法。该方法包括通过使受试者的样品与本公开所述的多肽或其抗原性片段，或本公开所述的阵列接触，检测样品中与多肽特异性结合的抗体是否存在，从而进行针对SARS-CoV-2感染的诊断或预后。
“诊断”是指鉴定病理状况的存在或性质，或病理状况的亚型，即COVID-19的存在或风险。“预后”在本文中用来指疾病或疾病进展(包括复发和治疗反应)的可能性的预测。
如本文所用，术语“受试者”、“个体”、“宿主”和“患者”在本文可互换使用，并且是指期望对其进行诊断、预后或治疗的任何哺乳动物受试者， 特别是人类。本文描述的方法适用于人类疗法和兽医应用两者。在一些实施方案中，受试者是哺乳动物，并且在具体的实施方案中，受试者是人类。
“受试者的样品”是指来自受试者的可用于分析的生物样品，包括取自或来源于个体的组织或流体样品。合适的生物样品包括，例如，全血、血清、血浆、血液部分、痰液、尿液、活检样品和粗裂解物等。优选待测定受试者的样品与对照样品来源于相同的物种。
诱导免疫反应
本公开提供了使用本公开所述的多肽或其抗原性片段，或本公开所述的组合物诱导受试者产生针对SARS-CoV-2的免疫反应的方法。该方法包括对受试者施用本公开所述的多肽或其抗原性片段，或本公开所述的组合物(例如疫苗组合物)，以使本公开所述的多肽或抗原性片段和/或佐剂暴露于受试者的免疫系统。
术语“诱导免疫反应”指免疫系统第一次与至少一种抗原性蛋白或其抗原性片段相遇和随后在限定的时间段内诱导抗原特异性的免疫反应。所述时间段为诱导前例如，至少1年、至少2年、至少3年、至少5年或至少10年。在一个实施方案中，个体或受试者的免疫系统与抗原性蛋白或其抗原性片段的不诱导抗原特异性免疫反应的相遇不视为“诱导免疫反应”。例如，个体的免疫系统与抗原性蛋白或其抗原性片段的不诱导持久免疫的相遇依照本公开不视为“诱导免疫反应”。在又一个实施方案中，持久免疫的诱导由记忆B细胞和/或记忆T细胞的生成介导。在癌症的情况中，例如，特定的抗原可由癌细胞表达而不引发免疫反应。仅存在该抗原不是本申请所理解的针对所述抗原“诱导免疫反应”。在一个实施方案中，个体或受试者尚未有意用抗原性蛋白或其抗原性片段或包含编码这类蛋白质或片段的核酸的载体进行免疫以在前文给定的时间段内治疗或预防疾病。
施用可以一次发生或者可以多次发生。在一个实施方案中，一次或多次施用可以作为所谓的“初免-加强”方案的一部分发生。其他施用系统可以包括延迟释放或持续释放递送系统。
本发明的组合物可以以溶液或者冻干形式保存。优选在糖诸如蔗糖或者乳糖存在的条件下冻干溶液。仍然优选的是它们被冻干，并在使用前复溶。
组合物可以以包含组合物和佐剂或复溶溶液的药盒形式呈现，复溶溶液包含一种或多种药学上可接受的稀释剂以促进用于使用常规或其他设备施用于哺乳动物的组合物的复溶。这种药盒可任选包括用于给予液体形式的组合物的设备(例如皮下注射器、微针阵列)和/或使用说明书。
本文公开的组合物可以结合到各种递送系统中。例如，在一些实施方案中，可以将组合物应用于“微针阵列”或“微针贴片”递送系统以用于施用。
预防和治疗
本公开提供了预防和/或治疗受试者中由SARS-CoV-2感染引起的疾病的方法。该方法包括对受试者施用本公开所述的多肽或其抗原性片段、本公开所述的组合物或本公开所述的抗体。
如本文所用，“预防”意指将治疗有效量的本公开的多肽或其抗原性片段、组合物或抗体施用于受试者以便保护受试者免于发生由SARS-CoV-2感染引起的疾病。当本文使用的术语“预防”与对给定病况的给定治疗有关(例如预防SARS-CoV-2感染)时，意在表达治疗的受试者根本不发展临床上可观察的病况水平，或与不存在治疗的受试者相比发展更缓慢和/或至较轻的程度。这些术语不仅限于其中受试者不经历任何病况方面的情况。例如，如果在患者暴露于预期产生给定病况表现的刺激期间给予治疗，并且该治疗导致受试者与不给予时的预期相比经历较少和/或更温和的病况症状，那么可以认为治疗已预防病况。通过引起受试者呈现仅有的温和明显感染症状，则认为治疗可以“预防”感染，这不意味着必须没有任何细胞为病毒所渗透。
如本文所用，“治疗”是指获得有益或期望的结果(包括临床结果)的方法。出于本发明的目的，有益的或期望的临床结果包括但不限于以下的一项或多项：减轻由疾病引起的一种或多种症状、降低疾病的程度、稳定疾病(例如，防止或延迟疾病的恶化)、防止或延迟疾病的传播、防止或延迟疾病的复发、延迟或减缓疾病的进展、改善疾病状态、提供疾病的缓解(部分或完全)、减少治疗疾病所需的一种或多种其他药物的剂量、延缓疾病的进展、提高或改善生活质量、体重增加和/或延长生存期。本发明的方法设想了治疗的这些方面中的任一个或多个方面。
在某些实施方案中，治疗可以在已经出现一种或多种症状之后给予。在 其他实施方案中，治疗可以在没有症状的情况下给予。例如，治疗可以在症状发作之前给予易感个体，或者可以用另一种损伤剂进行治疗(例如，根据症状史，根据遗传或其他易感因素、疾病疗法或它们的任何组合)。还可以在症状已经消退之后继续治疗，例如，以预防或延迟它们的复发。
类似地，与具有给定治疗的感染风险有关的本文使用的降低(例如，降低SARS-CoV-2感染的风险)，指与对照或不存在治疗(例如，施用本公开的多肽)时感染所发展的基础水平相比，受试者发展感染更缓慢或至较轻的程度。感染风险的降低可引起受试者呈现仅有的温和明显感染症状或延缓感染的症状，这不意味着必须没有任何细胞为病毒所渗透。
如本文所用，“施用”是指将组合物或药剂引入到受试者中，并且包括并行或顺序引入组合物或药剂。“施用”可以指例如治疗、药代动力学、诊断、研究、安慰剂和实验方法。“施用”还涵盖体外和离体治疗。通过任何合适的途径将组合物或药剂引入到受试者中，用于施用本公开的多肽、抗体或组合物的合适方法的实例包括口服、皮内、皮下、肌肉内、骨内、腹膜内和静脉内注射，以及全身施用或局部施用至靶位点附近，但不限于此。施用包括自我施用和他人施用。可以通过任何合适的途径进行施用。合适的施用途径允许组合物或药剂进行其预期功能。例如，如果合适的途径是静脉内，则通过将组合物或药剂引入到受试者的静脉中来施用组合物。
在本公开的某些实施方案中，本公开的多肽、抗体或组合物与用于施用的装置包装在一起或储存在用于施用的装置中。用于可注射制剂的装置包括但不限于注射口，自动注射器，注射泵和注射笔。用于雾化或粉末制剂的装置包括但不限于吸入器，吹入器，抽吸器等。因此，本公开包括包含本公开的多肽、抗体或组合物的施用装置，用于治疗或预防本文所述的一种或多种病症。
预期所施用的“受试者”包括但不限于人(即任何年龄组的男性或女性，例如儿童受试者(例如婴儿、少儿、青少年)或成人受试者(例如青年、中年人、或老年人))和/或其他非人动物，例如哺乳动物(例如灵长类)。
实施例
下面结合具体实施例来进一步描述本公开，本公开的优点和特点将会随 着描述而更为清楚。但这些实施例仅是范例性的，并不对本公开的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本公开的精神和范围下可以对本公开技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本公开的保护范围内。
实施例1.SARS-CoV-2感染诱导持续的中和抗体
首先对SARS-CoV-2感染者的血清样品进行了总体中和抗体的检测。使用ELISA测定血清样品中抗SARS-CoV-2的N或S蛋白的抗原特异性IgG抗体在症状出现后不同时间点的滴度(第1-30天，n＝36；第31-61天，n＝18；第100-150天，n＝21；第180-220天，n＝26)，对照组为20名健康供体(HD)的血清样品。结果表明，与健康供体相比，COVID-19患者在症状出现后30天内均产生显著更多的抗N蛋白和抗S蛋白的IgG抗体(图1A和1B)。
除了测定结合抗体之外，还使用假型病毒进一步确定了COVID-19个体中功能性中和抗体的动态反应。在症状出现后4至30天期间收集的36个患者血清样品中，超过94％(34/36)表现出针对SARS-CoV-2假病毒的强的抗体中和活性。该结果说明，感染SARS-CoV-2后患者产生特异性抗体中和反应。
实施例2.新冠病毒多肽的合成及多肽芯片的制造
发明人根据SARS-CoV-2 Wuhan-Hu-1毒株的氨基酸序列，设计了覆盖整个SARS-CoV-2蛋白质组的共515个多肽，其中每个多肽长度为15个氨基酸，并且每个多肽与相邻多肽之间有11个氨基酸重叠。多肽由GL生物化学有限公司(中国上海)合成。使用Sulfo-SMCC(ThermoFisher，MA，美国)将多肽与BSA偶联。简言之，用Sulfo-SMCC以1：30的摩尔比激活BSA，然后在PBS缓冲液中进行透析。在多肽C端添加半胱氨酸残基，以利用其巯基与BSA偶联。将含有半胱氨酸的肽以1：1(w/w)的比例加入，孵育2小时，然后用PBS透析去除游离肽。
将合成的每个多肽以及阴性(BSA)和阳性对照(抗人IgG Cat#I2136和IgM抗体Cat#I2386)的三份重复印在PATH底片(GraceBio-Labs，美国)上，使用Super Marathon打印机(Arrayjet，英国)生成芯片。然后将多肽芯片存储在-80℃下，直到使用。
实施例3.血清抗体检测和数据分析
将多肽芯片加热至室温，然后在封闭缓冲液(含3％BSA的PBS缓冲液和0.1％Tween20的PBS缓冲液)中分别孵育3小时。将来自COVID-19患者的血清样品或来自20个健康供体(对照组)的合并血清样品在含有0.1％Tween20的PBS中稀释，然后与多肽芯片在4℃下孵育2小时。用1×PBST洗涤多肽芯片，然后与分别用1×PBST以1：1000稀释的cy3偶联的山羊抗人IgG和AlexaFluor647偶联的驴抗人IgM(美国宾夕法尼亚州杰克逊免疫研究公司，Cat#109-165-008和Cat#709-605-073)在室温下孵育1h。孵育后，用1×PBST洗涤，室温离心干燥。然后用LuxScan10K-A(中国北京CapitalBio公司)对芯片进行扫描，参数分别设置为95％激光功率/PMT550和95％激光功率/PMT480。荧光强度数据由GenePixPro6.0软件(Molecular Devices，CA，美国)获得。图2显示了获得的荧光强度数据的示意性图片，其中绿色荧光指示IgG信号强度，红色荧光指示IgM信号强度，黄色为绿色和红色的叠加。
分别分析IgG和IgM的数据。对于每个点，将信号强度定义为前景减去背景，并取每个多肽的三个点的平均值。将COVID-19个体样品阳性肽反应的临界值设为健康供体对照组信号强度的两倍。将在所有三个采样时间点(发病后10-60天、100-150天、180-220天)的样品中，阳性率超过80％的肽定义为“优势和持久性表位”，将三个时间点阳性率超过60％的肽定义为“亚优势和持久性表位”。分别计算三个采样时间点组中每个肽的平均信号强度，并选择信号强度高(即高于信号强度平均值加标准差)的肽。采用R软件(第3.6.3版)进行数据处理和分析，并基于Limma算法评估不同采样时间点组间的显著信号强度变化。p值小于0.05表示差异有统计学意义。
实施例4.优势抗原表位鉴定
使用多肽芯片共检测了19例轻度至中度(n＝17)、无症状(n＝1)和严重(n＝1)SARS-CoV-2感染患者的51份纵向血清样品。除1例无症状感染的患者外，其他患者均诱导并维持了中和抗体(数据未显示)。在从每个COVID-19患者症状出现后16天到219天期间的2或3个时间点依次收集血清样品。根据不同的采样时间点，将样品分为第10-60天(n＝18)、第100-150天(n＝18)和第180-220天 (n＝15)三组。以20名健康供体的合并血清为阴性对照，对COVID-19个体的血清IgG和IgM抗体的病毒表位谱进行了纵向评估。
利用SARS-CoV-2多肽芯片，测定肽特异性抗体反应动力学，并分析结合信号强度，以及每个肽的阳性反应样品的百分比(即阳性率)。基于阳性肽结合反应的临界值(设置为阴性对照信号强度的两倍)，并在要求至少一个患者血清样品为阳性反应的情况下，共鉴定出460个IgG阳性肽和479个IgM阳性肽。结果表明，SARS-CoV-2在不同ORF蛋白质中的反应分布随时间变化相对稳定，其中复制酶多蛋白ORF1ab具有最多的阳性反应(图3)。
在所鉴定出的阳性表位的基础上，选择了在三个采样组中超过80％的样品始终保持强抗体反应的表位，将其称为优势和持久性表位。结果显示，这些能够介导长期体液免疫反应的高度优势的表位位于SARS-CoV-2的ORF1ab多聚蛋白和S蛋白，IgM抗体识别的表位(n＝33)多于IgG抗体识别的表位(n＝10)(表1，图4)。其中ORF1ab多聚蛋白具有最多的优势表位，其表位广泛分布在非结构蛋白(nsp)2-5、nsp8-10、nsp12-14和nsp16的区域。
值得注意的是，其中鉴定了一个免疫优势表位No.2073(SEQ ID NO.7，ORF1ab，aa 5801-5815)，无论血清采样时间点如何，该表位可以被100％的COVID-19个体血清中的IgG和IgM抗体识别(图4)。这种高反应性肽位于ORF1ab多聚蛋白的解旋酶(nsp13)区域，对于SARS-CoV-2复制过程中解开双链RNA模板是必不可少的(Chen,J.et al.Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex.Cell 182,1560-1573 e1513,doi:10.1016/j.cell.2020.07.033(2020))。
进一步计算了每个肽相对于健康对照组的信号强度的倍数变化。在ORF1ab肽中，两种IgG抗体识别的平均信号强度最高的免疫优势肽——No.1985(SEQ ID NO.6，ORF1ab，aa 5449-5463)和No.2073(SEQ ID NO.7，ORF1ab，aa 5801-5815)均位于nsp13上。对于IgM反应，nsp2上的No.685(SEQ ID NO.11，ORF1ab，aa 249-263)和nsp13上的No.1985(SEQ ID NO.6，ORF1ab，aa 5449-5463)具有最强的结合强度(图4)。
表1.在所有三个采样时间点阳性率超过80％的表位
Figure PCTCN2022137554-appb-000002
从SARS-CoV-2的S蛋白中共鉴定出4个优势和持久性表位：肽编号No.318(SEQ ID NO.9，S，aa 45-59)和No.356(SEQ ID NO.34，S，aa 197-211)，位于NTD结构区域；No.510(SEQ ID NO.10，S，aa 813-827)，其覆盖了S2的裂解位点和部分S2亚基的融合肽(FP)区域；以及No.530(SEQ ID NO.35，S，aa 893-907)，其位于FP与S2亚基的第一个七步重复序列(HR1)之间的连接区域。在这些肽中，位于S蛋白NTD上的No.318肽具有最强的结合强度。
结构分析显示，在S蛋白单体中，这些表位均暴露在S蛋白单体的表面，然而，在三聚体S蛋白中，多肽No.318、No.356和No.530被隐藏在三聚体蛋白内部(图5)。这些肽被鉴定为优势和持久性表位的结果表明，S蛋白单体和三聚体结构在体内都能够被宿主免疫系统有效识别。
考虑到全球范围内出现的新的SARS-CoV-2变异，进一步对早期SARS-CoV-2(Wuhan-Hu-1毒株)和6个变异株(B.1.1.7、B.1.351、B.1.429、P.1、B.1.525和B.1.617)的S蛋白中的优势表位No.318(SEQ ID NO.9)、No.356(SEQ ID NO.34)、No.510(SEQ ID NO.10)以及No.530(SEQ ID NO.35)进行了序列比对。结果表明，除B.1.525病毒株在No.318上有一个Q52R变异发生以外，这些优势和持久性表位的序列在不同病毒株中相同(图6)。表明这些表位呈高度保守，潜在介导SARS-CoV-2变异感染后持续的长期抗体反应。
实施例5.亚优势和持久性表位的鉴定
N蛋白上未能找到如上定义的优势和持久性表位(在所有三个采样时间点的阳性率均在80％以上)。随后，根据多肽芯片获得的数据进行了第二轮表位筛选，以选择在三个采样时间点的COVID-19样品中，60％以上样品始终保持阳性反应的亚优势和持久性表位。
共鉴定出4个N蛋白的亚优势和持久性表位(表2，图7)，其中肽No.2455(SEQ ID NO.36，N，aa 213-227)对COVID-19患者的IgG和IgM抗体均有反应，信号强度较高；两个重叠的肽No.2455(SEQ ID NO.36，N，aa 213-227)和No.2456(SEQ ID NO.37，N，aa 217-231)位于N蛋白N端RNA结合域和C端二聚化结构域之间的富Ser/Arg连接区；多肽No.2482(SEQ ID NO.38，N，aa 321-335)和No.2491(SEQ ID NO.39，N，aa 357-371)完全或部分位于N蛋白的二聚化结构域内。
表2.在所有三个采样时间点阳性率超过60％的表位
Figure PCTCN2022137554-appb-000003
对位于N蛋白C端的二聚化结构域内的表位No.2482和No.2491进行了结构分析。No.2482的氨基酸序列形成两条β链，以反平行的方式排列在二聚体中，而肽No.2491的357-364氨基酸序列形成了位于二聚体相反两端的螺旋基结构(图8)。
实施例6.具有高信号强度且反应性随着时间降低的表位鉴定
除了鉴定上述负责持续体液免疫反应的高反应性表位外，进一步鉴定一组9个阳性肽(表3)，其显示出强大的结合强度(即高于所有测试样品的信号强度的平均值+SD)，但是血清样品的反应性随时间呈下降的趋势(阳性率变化在20％以上)。这种变化趋势与患者体内体液免疫反应下降的总体趋势一致。这9个表位位于三个优势反应的抗原S、N和ORF1ab蛋白内(图9)。
表3.具有高信号强度但随时间阳性率下降的表位
Figure PCTCN2022137554-appb-000004
在这9个表位中，ORF1ab中的3个肽(No.784，SEQ ID NO.40-IgG、No.1617，SEQ ID NO.46-IgM、No.1986，SEQ ID NO.47-IgM)和N蛋白中的1个肽(No.2457，SEQ ID NO.45-IgG)的信号强度在采样时间点早期(发病后10-60天)和采样时间点后期(症状出现后100-150天或180-220天)的样品之间显著降低(p<0.05)(图10)。基于这种特性，对这些肽的体液免疫反应可以作为监控SARS-CoV-2感染后的疾病进程，和/或对患者进行预后的指标。
实施例7.基于优势和持久性表位构建新冠病毒感染诊断模型
收集75份人的血清样品，利用实施例2中制造的新冠病毒多肽芯片检测样品中多肽的抗体反应。75份样品包含24份来自未感染新冠病毒的健康供体的样品，18份来自感染过新冠病毒后恢复期1个月的样品，18份来自感染过新冠病毒后恢复期4个月的样品和15份来自感染过新冠病毒后恢复期7个月的样品。
从多肽芯片检测结果中提取出实施例4中所鉴定的35种优势和持久性表位肽(表1)在每份样品中的抗体反应强度，以探索在新冠病毒感染者中有高抗体反应的35条多肽中是否存在子集(panel)，可以准确地区分或预测感染和未感染新冠病毒的样品，并以此子集构建新冠诊断模型。
具体而言，将感染过新冠病毒的患者的血清样品按照1月、4月和7月分别作为病例Case组，与未感染的样品(作为对照control组)依次合并分析。首先分析35条多肽两两之间的抗体反应强度的相关性，并剔除相关性相近的两条肽中的一条肽的数据。随后，进行特征筛选，以探索用部分多肽可以准确预测新冠感染患者的预测器。预测器分别采用randomForest、lasso、elasticNet、svmLinear和svmRadial算法进行运算。根据每种算法产出的最优多肽子集的情况再计算出每个样品的分数(panel-based score)。利用该分数绘制ROC曲线，以AUC得分(AUC越接近1准确度越高)，特异性(specificity)、敏感性(sensitivity)和子集包含多肽的数量(size)作为评价每种算法得到的模型的标准。
结果：
1.依据IgM反应强度得到的结果：
汇总不同算法筛选到的多肽子集和基于这些多肽的IgM反应强度构建的预测模型的表现如下表：
Figure PCTCN2022137554-appb-000005
其中randomForest算法得到的子集肽数量适中，并且模型预测准确度高。采用randomForest算法基于IgM预测1月、4月和7月新冠感染样品的模型如下表：
Figure PCTCN2022137554-appb-000006
2.依据IgG反应强度得到的结果：
汇总不同算法筛选到的多肽子集和基于这些多肽的IgG反应强度构建的预测模型的表现如下表：
Figure PCTCN2022137554-appb-000007
其中randomForest算法得到的子集肽数量适中，并且模型预测准确度高。采用randomForest算法基于IgG预测1月、4月和7月新冠感染样品的模型如下表：
Figure PCTCN2022137554-appb-000008

Claims (50)

  1. 一种分离的多肽或其抗原性片段，所述多肽具有选自以下的氨基酸序列，或在以下氨基酸序列中进行了一个或几个氨基酸残基的取代、缺失、插入和/或添加后获得的氨基酸序列：
    SFKWDLTAFGLVAEW(SEQ ID NO:1)；
    LGLAAIMQLFFSYFA(SEQ ID NO:2)；
    VLSTFISAARQGFVD(SEQ ID NO:3)；
    SFCYMHHMELPTGVH(SEQ ID NO:4)；
    MQTMLFTMLRKLDND(SEQ ID NO:5)；
    TCTERLKLFAAETLK(SEQ ID NO:6)；
    KGVITHDVSSAINRP(SEQ ID NO:7)；
    DQFKHLIPLMYKGLP(SEQ ID NO:8)；
    SSVLHSTQDLFLPFF(SEQ ID NO:9)；
    SKRSFIEDLLFNKVT(SEQ ID NO:10)；
    YELQTPFEIKLAKKF(SEQ ID NO:11)；
    EKYCALAPNMMVTNN(SEQ ID NO:12)；
    ATYKPNTWCIRCLWS(SEQ ID NO:13)；
    LKTLVATAEAELAKN(SEQ ID NO:14)；
    MYLKLRSDVLLPLTQ(SEQ ID NO:15)；
    YEDLLIRKSNHNFLV(SEQ ID NO:16)；
    KVDTANPKTPK(SEQ ID NO:17)；
    LGSALLEDEFTPFDV(SEQ ID NO:18)；
    SEVVLKKLKKSLNVA(SEQ ID NO:19)；
    LEKMADQAMTQMYKQ(SEQ ID NO:20)；
    VPLNIIPLTTAAKLM(SEQ ID NO:21)；
    LNRGMVLGSLAATVR(SEQ ID NO:22)；
    FCAFAVDAAKAYKDY(SEQ ID NO:23)；
    FFKFRIDGDMVPHIS(SEQ ID NO:24)；
    NLDKSAGFPFNKWGK(SEQ ID NO:25)；
    VVYRGTTTYKLNVGD(SEQ ID NO:26)；
    AKHYVYIGDPAQLPA(SEQ ID NO:27)；
    PAEIVDTVSALVYDN(SEQ ID NO:28)；
    KDKSAQCFKMFYKGV(SEQ ID NO:29)；
    GFDYVYNPFMIDVQQ(SEQ ID NO:30)；
    LIGDCATVHTANKWD(SEQ ID NO:31)；
    TAFVTNVNASSSEAF(SEQ ID NO:32)；
    LSSYSLFDMSKFPLK(SEQ ID NO:33)；
    IDGYFKIYSKHTPIN(SEQ ID NO:34)；
    ALQIPFAMQMAYRFN(SEQ ID NO:35)；
    GGDAALALLLLDRL(SEQ ID NO:36)；
    AALALLLLDRLNQLE(SEQ ID NO:37)；
    GMEVTPSGTWLTYTG(SEQ ID NO:38)；和
    IDAYKTFPPTEPKKD(SEQ ID NO:39)。
  2. 一种分离的多肽或其抗原性片段，所述多肽具有选自以下的氨基酸序列，或在以下氨基酸序列中进行了一个或几个氨基酸残基的取代、缺失、插入和/或添加后获得的氨基酸序列：
    SFKWDLTAFGLVAEW(SEQ ID NO:1)；
    LGLAAIMQLFFSYFA(SEQ ID NO:2)；
    VLSTFISAARQGFVD(SEQ ID NO:3)；
    SFCYMHHMELPTGVH(SEQ ID NO:4)；
    MQTMLFTMLRKLDND(SEQ ID NO:5)；
    TCTERLKLFAAETLK(SEQ ID NO:6)；
    KGVITHDVSSAINRP(SEQ ID NO:7)；
    DQFKHLIPLMYKGLP(SEQ ID NO:8)；
    SSVLHSTQDLFLPFF(SEQ ID NO:9)；
    SKRSFIEDLLFNKVT(SEQ ID NO:10)；
    YELQTPFEIKLAKKF(SEQ ID NO:11)；
    EKYCALAPNMMVTNN(SEQ ID NO:12)；
    ATYKPNTWCIRCLWS(SEQ ID NO:13)；
    LKTLVATAEAELAKN(SEQ ID NO:14)；
    MYLKLRSDVLLPLTQ(SEQ ID NO:15)；
    YEDLLIRKSNHNFLV(SEQ ID NO:16)；
    KVDTANPKTPK(SEQ ID NO:17)；
    LGSALLEDEFTPFDV(SEQ ID NO:18)；
    SEVVLKKLKKSLNVA(SEQ ID NO:19)；
    LEKMADQAMTQMYKQ(SEQ ID NO:20)；
    VPLNIIPLTTAAKLM(SEQ ID NO:21)；
    LNRGMVLGSLAATVR(SEQ ID NO:22)；
    FCAFAVDAAKAYKDY(SEQ ID NO:23)；
    FFKFRIDGDMVPHIS(SEQ ID NO:24)；
    NLDKSAGFPFNKWGK(SEQ ID NO:25)；
    VVYRGTTTYKLNVGD(SEQ ID NO:26)；
    AKHYVYIGDPAQLPA(SEQ ID NO:27)；
    PAEIVDTVSALVYDN(SEQ ID NO:28)；
    KDKSAQCFKMFYKGV(SEQ ID NO:29)；
    GFDYVYNPFMIDVQQ(SEQ ID NO:30)；
    LIGDCATVHTANKWD(SEQ ID NO:31)；
    TAFVTNVNASSSEAF(SEQ ID NO:32)；
    LSSYSLFDMSKFPLK(SEQ ID NO:33)；
    IDGYFKIYSKHTPIN(SEQ ID NO:34)；和
    ALQIPFAMQMAYRFN(SEQ ID NO:35)。
  3. 根据权利要求1或2所述的多肽或其抗原性片段，其中所述抗原性片段的长度为至少5个氨基酸、至少6个氨基酸、至少7个氨基酸、至少8个氨基酸、至少9个氨基酸或至少10个氨基酸。
  4. 一种分离的多肽或其抗原性片段，所述多肽具有选自以下的氨基酸序列，或在以下氨基酸序列中进行了一个或几个氨基酸残基的取代、缺失、插 入和/或添加后获得的氨基酸序列：
    GWEIVKFISTCACEI(SEQ ID NO:40)；
    WADNNCYLATALLTL(SEQ ID NO:41)；
    KDYLASGGQPITNCV(SEQ ID NO:42)；
    DKSAFVNLKQLPFFY(SEQ ID NO:43)；
    FIEDLLFNKVTLADA(SEQ ID NO:44)；
    LLLLDRLNQLESKMS(SEQ ID NO:45)；
    LKKLKKSLNVAKSEF(SEQ ID NO:46)；
    RLKLFAAETLKATEE(SEQ ID NO:47)；和
    ASDTYACWHHSIGFD(SEQ ID NO:48)。
  5. 根据权利要求4所述的多肽或其抗原性片段，其中所述抗原性片段的长度为至少5个氨基酸、至少6个氨基酸、至少7个氨基酸、至少8个氨基酸、至少9个氨基酸或至少10个氨基酸。
  6. 一种分离的抗体，所述抗体结合根据权利要求1-5中任一项所述的多肽或其抗原性片段。
  7. 根据权利要求6的抗体，其中所述抗体选自IgG抗体、IgM抗体和IgA抗体。
  8. 根据权利要求7的抗体，其中所述抗体选自IgG1、IgG2、IgG3和IgG4抗体。
  9. 根据权利要求6-8中任一项所述的抗体，其中所述抗体为单克隆抗体。
  10. 根据权利要求6-9中任一项所述的抗体，其中所述抗体为人抗体。
  11. 一种组合物，所述组合物包含一种或多种根据权利要求1-5中任一项所述的多肽或其抗原性片段。
  12. 根据权利要求11的组合物，其中所述组合物包含具有SEQ ID NO:10所示的氨基酸序列的多肽或其抗原性片段以及具有选自SEQ ID NO:1-9和11-48所示的氨基酸序列的多肽或其抗原性片段中的一种或多种。
  13. 根据权利要求11或12的组合物，其中所述组合物包含具有SEQ ID NO:10、7、35、29、23、30、2、21和12所示的氨基酸序列的多肽或其抗原性片段。
  14. 根据权利要求11或12的组合物，其中所述组合物包含具有SEQ ID NO:10、12、35、14、30、2、3、13和19所示的氨基酸序列的多肽或其抗原性片段。
  15. 根据权利要求11或12的组合物，其中所述组合物包含具有SEQ ID NO:10、35、12和19所示的氨基酸序列的多肽或其抗原性片段。
  16. 根据权利要求11或12的组合物，其中所述组合物包含具有SEQ ID NO:10和33所示的氨基酸序列的多肽或其抗原性片段。
  17. 根据权利要求11或12的组合物，其中所述组合物包含具有SEQ ID NO:10、32和15所示的氨基酸序列的多肽或其抗原性片段。
  18. 根据权利要求11或12的组合物，其中所述组合物包含具有SEQ ID NO:10、21、30、15、12、35、23、19和20所示的氨基酸序列的多肽或其抗原性片段。
  19. 根据权利要求11-18中任一项的组合物，其中所述抗原性片段的长度为至少5个氨基酸、至少6个氨基酸、至少7个氨基酸、至少8个氨基酸、至少9个氨基酸或至少10个氨基酸。
  20. 根据权利要求11-19中任一项的组合物，其中所述组合物是疫苗组合物，并且任选地还包含一种或多种佐剂。
  21. 一种组合物，所述组合物包含一种或多种根据权利要求6-10中任一项的抗体，以及任选的载体和/或赋形剂。
  22. 一种核苷酸序列，所述核苷酸序列编码根据权利要求1-5中任一项所述的多肽或其抗原性片段，或根据权利要求6-10中任一项所述的抗体。
  23. 一种载体，所述载体包含根据权利要求22所述的核苷酸序列。
  24. 一种阵列，所述阵列包含固体基质，以及固定于所述固体基质上的多肽，所述多肽包含至少一种根据权利要求1-3中任一项所述的多肽或其抗原性片段。
  25. 根据权利要求24所述的阵列，其中所述多肽包含至少2种、至少3种、至少5种、至少10种、至少15种、至少20种、至少25种、至少30种或至少35种根据权利要求1-3中任一项所述的多肽或其抗原性片段。
  26. 根据权利要求24或25的阵列，其中所述多肽包含具有SEQ ID NO:10所示的氨基酸序列的多肽或其抗原性片段以及具有选自SEQ ID NO:1-9和11-48所示的氨基酸序列的多肽或其抗原性片段中的一种或多种。
  27. 根据权利要求24-26中任一项的阵列，其中所述多肽包含具有SEQ ID NO:10、7、35、29、23、30、2、21和12所示的氨基酸序列的多肽或其抗原性片段。
  28. 根据权利要求24-26中任一项的阵列，其中所述多肽包含具有SEQ ID NO:10、12、35、14、30、2、3、13和19所示的氨基酸序列的多肽或其抗原性片段。
  29. 根据权利要求24-26中任一项的阵列，其中所述多肽包含具有SEQ ID NO:10、35、12和19所示的氨基酸序列的多肽或其抗原性片段。
  30. 根据权利要求24-26中任一项的阵列，其中所述多肽包含具有SEQ ID NO:10和33所示的氨基酸序列的多肽或其抗原性片段。
  31. 根据权利要求24-26中任一项的阵列，其中所述多肽包含具有SEQ ID NO:10、32和15所示的氨基酸序列的多肽或其抗原性片段。
  32. 根据权利要求24-26中任一项的阵列，其中所述多肽包含具有SEQ ID NO:10、21、30、15、12、35、23、19和20所示的氨基酸序列的多肽或其抗原性片段。
  33. 根据权利要求24-32中任一项的阵列，其中所述抗原性片段的长度为至少5个氨基酸、至少6个氨基酸、至少7个氨基酸、至少8个氨基酸、至少9个氨基酸或至少10个氨基酸。
  34. 根据权利要求24所述的阵列，其中所述多肽包含多种根据权利要求1-3中任一项的多肽或其抗原性片段，其中所述多种多肽或其抗原性片段包含至少一种来源于SARS-CoV-2 ORF1ab多聚蛋白的多肽或其抗原性片段，以及至少一种来源于SARS-CoV-2 S蛋白的多肽或其抗原性片段。
  35. 根据权利要求34所述的阵列，其中所述多种多肽还包含至少一种来源于SARS-CoV-2 N蛋白的多肽或其抗原性片段。
  36. 一种阵列，所述阵列包含固体基质，以及固定于所述固体基质上的多肽，所述多肽包含至少一种根据权利要求4或5所述的多肽或其抗原性片段。
  37. 根据权利要求36所述的阵列，其中所述多肽包含至少2种、至少3种、 至少4种、至少5种、至少6种、至少7种、至少8种或全部9种根据权利要求4或5所述的多肽或其抗原性片段。
  38. 根据权利要求36所述的阵列，其中所述多肽包含多种根据权利要求4或5的多肽或其抗原性片段，其中所述多种多肽或其抗原性片段包含至少一种来源于SARS-CoV-2 ORF1ab多聚蛋白的多肽或其抗原性片段，至少一种来源于SARS-CoV-2 S蛋白的多肽或其抗原性片段，至少一种来源于SARS-CoV-2 N蛋白的多肽或其抗原性片段。
  39. 根据权利要求24-38中任一项所述的阵列，其中所述多肽通过与BSA偶联后固定化于所述固体基质上。
  40. 根据权利要求24-39中任一项所述的阵列，其中所述阵列是多肽芯片。
  41. 一种检测受试者中针对SARS-CoV-2的抗体的存在的方法，所述方法包括：
    (a)使来自所述受试者的样品与根据权利要求1-3中任一项的多肽或其抗原性片段，或根据权利要求24-40中任一项的阵列接触；
    (b)检测所述样品中与所述多肽或所述阵列上的多肽特异性结合的抗体的存在。
  42. 根据权利要求41所述的方法，其中所述方法用于诊断所述受试者是否被SARS-CoV-2感染。
  43. 一种用于对被SARS-CoV-2感染的受试者进行预后的方法，所述方法包括：
    (a)使来自所述受试者的样品与根据权利要求4或5的多肽或其抗原性片段，或根据权利要求36-40中任一项的阵列接触；
    (b)检测所述样品中与所述多肽或所述阵列上的多肽特异性结合的抗体的存在。
  44. 根据权利要求41-43中任一项的方法，其中所述抗体是IgG抗体和/或IgM抗体。
  45. 根据权利要求41-44中任一项的方法，其中所述样品是血液样品，例如血清或血浆。
  46. 一种诱导受试者中针对SARS-CoV-2的免疫反应的方法，所述方法包括对所述受试者施用根据权利要求1-5中任一项的多肽或其抗原性片段，或根据权利要求11-20中任一项的组合物。
  47. 一种预防和/或治疗受试者中由SARS-CoV-2感染引起的疾病的方法，所述方法包括对所述受试者施用根据权利要求1-5中任一项所述的多肽或其抗原性片段，或根据权利要求11-20中任一项所述的组合物。
  48. 根据权利要求46或47的方法，所述方法包括对所述受试者施用至少2种、至少3种、至少5种、至少10种、至少15种、至少20种、至少25种或至少30种根据权利要求1-5中任一项所述的多肽或其抗原性片段的组合。
  49. 根据权利要求46或47的方法，所述方法包括对所述受试者施用多种根据权利要求1-5中任一项的多肽或其抗原性片段的步骤，其中所述多种多肽或其抗原性片段包含至少一种来源于SARS-CoV-2 ORF1ab多聚蛋白的多肽或其抗原性片段，至少一种来源于SARS-CoV-2 S蛋白的多肽或其抗原性片段，以及至少一种来源于SARS-CoV-2 N蛋白的多肽或其抗原性片段。
  50. 一种治疗受试者中由SARS-CoV-2感染引起的疾病的方法，所述方法包括对所述受试者施用根据权利要求6-10中任一项所述的抗体。
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112034174A (zh) * 2020-03-20 2020-12-04 中国人民解放军军事科学院军事医学研究院 一种多肽芯片及其在病毒检测上的应用
WO2021163371A1 (en) * 2020-02-12 2021-08-19 La Jolla Institute For Immunology Coronavirus t cell epitopes and uses thereof
WO2021163427A1 (en) * 2020-02-14 2021-08-19 Epivax, Inc. Regulatory t cell epitopes and detolerized sars-cov-2 antigens
WO2021188969A2 (en) * 2020-03-20 2021-09-23 Biontech Us Inc. Coronavirus vaccines and methods of use
WO2021198999A1 (en) * 2020-04-03 2021-10-07 Axon Neuroscience Se Epitope-based vaccines for treatment of coronavirus associated diseases
WO2021239666A1 (en) * 2020-05-26 2021-12-02 Diaccurate Therapeutic methods

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021163371A1 (en) * 2020-02-12 2021-08-19 La Jolla Institute For Immunology Coronavirus t cell epitopes and uses thereof
WO2021163427A1 (en) * 2020-02-14 2021-08-19 Epivax, Inc. Regulatory t cell epitopes and detolerized sars-cov-2 antigens
CN112034174A (zh) * 2020-03-20 2020-12-04 中国人民解放军军事科学院军事医学研究院 一种多肽芯片及其在病毒检测上的应用
WO2021188969A2 (en) * 2020-03-20 2021-09-23 Biontech Us Inc. Coronavirus vaccines and methods of use
WO2021198999A1 (en) * 2020-04-03 2021-10-07 Axon Neuroscience Se Epitope-based vaccines for treatment of coronavirus associated diseases
WO2021239666A1 (en) * 2020-05-26 2021-12-02 Diaccurate Therapeutic methods

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CROOKE STEPHEN N., OVSYANNIKOVA INNA G., KENNEDY RICHARD B., POLAND GREGORY A.: "Immunoinformatic identification of B cell and T cell epitopes in the SARS-CoV-2 proteome", SCIENTIFIC REPORTS, vol. 10, no. 1, 1 December 2020 (2020-12-01), XP055949643, DOI: 10.1038/s41598-020-70864-8 *
LI MIN; LIU JIAOJIAO; LU RENFEI; ZHANG YUCHAO; DU MENG; XING MAN; WU ZHENCHUAN; KONG XIANGYIN; ZHU YUFEI; ZHOU XIANCHAO; HU LANDIA: "Longitudinal immune profiling reveals dominant epitopes mediating long-term humoral immunity in COVID-19–convalescent individuals", JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 149, no. 4, 21 January 2022 (2022-01-21), AMSTERDAM, NL , pages 1225 - 1241, XP087013519, ISSN: 0091-6749, DOI: 10.1016/j.jaci.2022.01.005 *
NELSON, C.W.: "Revision d7b99170d99133396c63f706a21383dc542a0075 : MHCII_epitope_summary.tsv", SOFTWARE HERITAGE, XP009546775, Retrieved from the Internet <URL:https://archive.softwareheritage.org/browse/revision/d7b99170d99133396c63f706a21383dc542a0075/?path=data/MHCII_epitope_summary.tsv> *

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