WO2021209925A1 - Test sérologique de coronavirus - Google Patents

Test sérologique de coronavirus Download PDF

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Publication number
WO2021209925A1
WO2021209925A1 PCT/IB2021/053080 IB2021053080W WO2021209925A1 WO 2021209925 A1 WO2021209925 A1 WO 2021209925A1 IB 2021053080 W IB2021053080 W IB 2021053080W WO 2021209925 A1 WO2021209925 A1 WO 2021209925A1
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WIPO (PCT)
Prior art keywords
sample
peptide
sars
cov
polypeptide
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PCT/IB2021/053080
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English (en)
Inventor
Philip Ralph Dormitzer
Bridget Yih Jin HUANG
Alicia SOLORZANO QUIJANO
Kena Anne SWANSON
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Pfizer Inc.
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Publication of WO2021209925A1 publication Critical patent/WO2021209925A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the invention relates to compositions, such as nucleic acids, polypeptides, and fragments thereof, and methods of use thereof relating to Severe Acute Respiratory Syndrome (SARS) coronavirus, preferably SARS-CoV-2.
  • SARS Severe Acute Respiratory Syndrome
  • the compositions may be useful as diagnostic reagents, in kits comprising such reagents, and for methods to identify a history of a SARS or SARS-like coronavirus infection in a person or animal.
  • Coronaviruses are a large family of viruses, some of which cause respiratory illnesses in humans ranging from common colds to more severe conditions such as Severe Acute Respiratory Syndrome (SARS) and Middle Eastern Respiratory Syndrome (MERS).
  • SARS Severe Acute Respiratory Syndrome
  • MERS Middle Eastern Respiratory Syndrome
  • SARS-CoV-2 The novel coronavirus involved in the outbreak starting in 2019-2020 has been named SARS-CoV-2 by the World Health Organization (WHO).
  • WHO World Health Organization
  • coronavirus disease 2019 or “coronavirus disease 2019” (or “COVID-19”).
  • COVID-19 There are currently no specific treatments, drugs, or vaccines available to treat or prevent COVID-19.
  • SARS-CoV-2 protein other than spike (S) protein that is highly immunogenic and well enough behaved biochemically to be an easily produced target antigen for immunoassays is sought.
  • the invention relates to a peptide having the sequence SEQ ID NO: 1.
  • the invention relates to a peptide having the sequence SEQ ID NO: 5 (also shown in FIG. 3B).
  • the invention relates to a peptide having the sequence SEQ ID NO: 2.
  • the invention relates to a peptide having the sequence SEQ ID NO: 6, also shown in FIG. 6.
  • the invention relates to a nucleic acid molecule that encodes any one of the peptides described herein.
  • the peptide further includes a linker sequence.
  • the linker sequence is GRS.
  • the peptide further includes a 3C protease cleavage sequence.
  • the peptide further includes a peptide sequence tag.
  • the invention in another aspect, relates to a method of detecting a previous SARS-CoV- 2 infection in a biological sample.
  • the method includes a) contacting the sample with an N- terminal domain-modified nucleocapsid polypeptide or variant thereof under conditions conducive to binding; and b) measuring binding between the sample and the polypeptide or variant thereof; wherein detection of binding between the sample and the polypeptide or variant thereof indicates a previous SARS-CoV-2 infection in the person or animal from which the sample was obtained.
  • the invention in another aspect, relates to a method of detecting a SARS-CoV-2 specific antibody in a biological sample.
  • the method includes a) contacting the sample with an N- terminal domain-modified nucleocapsid polypeptide or variant thereof under conditions conducive to binding; and b) measuring binding between the sample and the polypeptide or variant thereof; wherein detection of binding between the sample and the polypeptide or variant thereof indicates the presence of a SARS-CoV-2 specific antibody in the sample.
  • the invention in yet another aspect, relates to a method of detecting a previous SARS- CoV-2 infection in a human or animal from which a biological sample was obtained.
  • the method includes a) contacting the sample with a C-terminal domain dimer nucleocapsid peptide or variant thereof under conditions conducive to binding; and b) measuring binding between the sample and the polypeptide or variant thereof; wherein detection of binding between the sample and the polypeptide or variant thereof indicates a previous SARS-CoV-2 infection in the person or animal from which the sample was obtained.
  • the invention in yet another aspect, relates to a method of detecting a SARS-CoV-2 antibody in a biological sample.
  • the method includes a) contacting the sample with a C- terminal domain dimer nucleocapsid peptide or variant thereof under conditions conducive to binding; and b) measuring binding between the sample and the polypeptide or variant thereof; wherein detection of binding between the sample and the polypeptide or variant thereof indicates the presence of a SARS-CoV-2 specific antibody in the sample.
  • the N-terminal domain-modified nucleocapsid polypeptide includes the sequence SEQ ID NO: 1 . In some embodiments, the N-terminal domain-modified nucleocapsid polypeptide includes the sequence SEQ ID NO: 5 (also shown in FIG. 3B).
  • the C-terminal domain dimer nucleocapsid peptide includes the sequence SEQ ID NO: 2. In some embodiments, the C-terminal domain dimer nucleocapsid peptide includes the sequence SEQ ID NO: 6, also shown in FIG. 6.
  • the sample is from a human or animal to which an immunogenic composition eliciting an immune response against a SARS-CoV spike polypeptide has been administered.
  • the sample is from a human or animal prior to administration of an immunogenic composition eliciting an immune response against a SARS-CoV spike polypeptide.
  • the sample is from a human or animal to which an immunogenic composition eliciting an immune response against a SARS-CoV spike polypeptide had not been administered.
  • the polypeptide or variant thereof is attached to a solid support prior to contact with the sample.
  • the sample is selected from any one of blood, serum, plasma, saliva, urine, mucus, fecal matter, and tissue extract.
  • FIG. 1 depicts sequence features of SARS-CoV-2 nucleocapsid protein.
  • FIG. 1 is taken from Figure 1A-C of Kang et al., "Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites," version posted March 7, 2020, on bioRxiv preprint server, Figure 1A-C of which is incorporated by reference.
  • FIG. 2 depicts conserveed sequence motif of N protein. Only the N-terminal region is shown. SEQ ID Nos of the motifs from the respective viruses listed in FIG. 2 are as follows:
  • SARS-CoV2 Wuhan-Hu-1 (SEQ ID NO: 10); Bat-SL-CoV-ZC45 (SEQ ID NO: 10); Bat-SL-CoV-ZX21(SEQ ID NO: 10); SARS-CoV Tor2 (SEQ ID NO: 11); SARS-CoV BJ01 (SEQ ID NO: 11); SARS-CoV INP_828858.1 (SEQ ID NO: 11); HCoV-OC43 Human Coronavirus OC43 (SEQ ID NO: 12); Bat Coronavirus HKU9-1 (SEQ ID NO: 13); HCoV-NL63 Human Coronavirus NL63 (SEQ ID NO: 14); HCoV229E Human Coronavirus 229E (SEQ ID NO: 15); HCoV-HKU-1 Human Coronavirus (SEQ ID NO: 16); SARS-CoV GZ02 (SEQ ID NO: 17); SARS-CoV Rs_672/2006 (SEQ ID NO: 18); Bat-SL-CoV RsSHC014 (SEQ ID NO:
  • FIG. 3A depicts an exemplary full length N protein with a linker, 3C protease cleavage sequence, twin-strep tag, and His-8 tag (SEQ ID NO: 8).
  • FIG. 3B depicts an exemplary NTD-modified Full length N protein with a linker, 3C protease cleavage sequence, twin-strep tag, and His-8 tag (SEQ ID NO: 5).
  • FIG. 4 depicts structural coordinates for the SARS-CoV-2 N-terminal domain.
  • FIG. 5 depicts a structure of the C-terminal domain of SARS-CoV N.
  • FIG. 6 depicts an exemplary CTD-dimer N protein construct, including a dimerization interface, 3C protease cleavage sequence, twin-strep tag, and His-8 tag (SEQ ID NO: 6).
  • SEQ ID NO: 1 sets forth the amino acid sequence for an N-terminal domain (NTD)-modified full length Nucleocapsid (N) protein.
  • SEQ ID NO: 2 sets forth the amino acid sequence for a C-terminal domain (CTD)-dimer N protein.
  • SEQ ID NO: 3 sets forth the amino acid sequence for a full length N protein, wild-type.
  • SEQ ID NO: 4 sets forth the amino acid sequence corresponding to residues 110-117 of the sequence shown in SEQ ID NO: 3.
  • SEQ ID NO: 5 sets forth the amino acid sequence for an exemplary N-terminal domain (NTD)- modified full length Nucleocapsid (N) protein, shown in FIG. 3B.
  • SEQ ID NO: 6 sets forth the amino acid sequence for an exemplary C-terminal domain (CTD)- dimer N protein, shown in FIG. 6.
  • SEQ ID NO: 7 sets forth the amino acid sequence for an exemplary CTD-dimer N protein.
  • SEQ ID NO: 8 sets forth the amino acid sequence for an exemplary full length N protein, shown in FIG. 3A.
  • SEQ ID NO: 9 and SEQ ID NO: 26 set forth the respective 9 amino acid sequence in the N protein.
  • SEQ ID Nos: 10-25 set forth the respective amino acid sequences shown in FIG. 2.
  • the major structural proteins common to coronaviruses include the spike (S), membrane (M), and nucleocapsid (N).
  • S spike glycoprotein
  • M membrane
  • N nucleocapsid
  • S spike glycoprotein
  • S membrane glycoprotein
  • N nucleocapsid
  • the spike glycoprotein (S) has been characterized as a surface antigen of Coronaviridae viruses and it binds to the host cell receptor, angiotensin converting enzyme-2 (ACE-2).
  • ACE-2 angiotensin converting enzyme-2
  • the S protein is a known target for neutralizing immune responses.
  • the membrane (M) glycoproteins of coronaviruses are also surface exposed and may be useful for immunoassays.
  • the nucleocapsid (N) protein is the most abundant protein of coronaviruses. It packages the viral genome into a helical ribonucleocapsid (RNP) and plays a fundamental role during viral self-assembly, among other functions. Its N-terminal region includes mostly positively charged amino acids, and is responsible for RNA binding. The C-terminus is capable of self-association. Between these two structural domains, there lies a highly disordered region, which serves as a linker. Accordingly, the N-protein can be classified into three distinct regions, referred to herein as the NTD (N terminal domain), a SR rich linker, and the CTD (C-terminal domain).
  • a variant of the N protein includes a modification by amino acid additions to the N-terminus, C-terminus, and/or middle of the peptide.
  • additions are to the N-terminus or C- terminus of the peptide. Additions can be of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16,
  • additions may constitute amino acid sequences that are present in SARS-CoV-2 in their entirety or in part.
  • additions of amino acid sequences that are present in SARS-CoV-2 N protein are of 15 amino acids or less.
  • Such additions may also constitute amino acid sequences which are not present in SARS-CoV- 2 N protein.
  • Addition of sequences which are not present in SARS-CoV-2 N protein include, but are not limited to, small charged sequences (e.g., lysine-lysine-lysine) and sequences that enable the formation of branched structures (e.g., lysine or methionine).
  • additions of amino acid sequences that are not present in SARS-CoV-2 N protein are of 5 amino acids or less. Residue additions can be either classical or non-classical amino acids or a mixture thereof.
  • a variant of the N protein includes a modification by amino acid deletions to the N-terminus, C-terminus, and/or middle of the peptide.
  • deletions are to the N-terminus or C-terminus of the peptide.
  • Deletions can be of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid resides.
  • deletions of amino acid sequences are of 10 amino acids or less.
  • the peptide includes a deletion of the sequence FYYLGTGP (SEQ ID NO: 4) (corresponding to residues 110-117 of the sequence shown in SEQ ID NO: 3) in an N protein.
  • the invention relates to a peptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to SEQ ID NO: 1.
  • the invention relates to a peptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to the sequence shown in SEQ ID NO: 5 (also shown in FIG. 3B).
  • the peptide includes a deletion of the sequence FYYLGTGP (SEQ ID NO: 4) (corresponding to residues 110-117 of the sequence shown in SEQ ID NO: 3) in an N protein.
  • Another exemplary peptide sequence includes:
  • variant peptide includes a peptide having at least 80%, 81 %,
  • the invention relates to a fragment of any one of the peptides described herein, including a fragment of a peptide having the sequence SEQ ID NO: 1 .
  • the invention relates to a nucleic acid molecule that encodes any one of the peptides described herein.
  • the peptide further includes a linker sequence.
  • the linker sequence is GRS.
  • the peptide further includes a 3C protease cleavage sequence.
  • the peptide further includes a peptide sequence tag.
  • the invention relates to a peptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to SEQ ID NO: 2.
  • SEQ ID NO: 2 are modified, as compared to SEQ ID NO: 2.
  • the invention relates to a peptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to the sequence SEQ ID NO: 6, also shown in FIG. 6.
  • the invention relates to a fragment of any one of the peptides described herein, including a fragment of a peptide having the sequence SEQ ID NO: 2.
  • the invention relates to a nucleic acid molecule that encodes any one of the peptides described herein.
  • the peptide further includes a linker sequence.
  • the linker sequence is GRS.
  • the peptide further includes a 3C protease cleavage sequence.
  • the peptide further includes a peptide sequence tag.
  • a peptide or variant thereof described herein may be further modified by covalent attachment to another moiety.
  • the covalent attachment between the peptide and moiety may be direct or indirect (e.g. through a linker molecule) by methods known in the art.
  • the moiety is a bead.
  • the moiety to which the peptide is attached includes a carrier molecule (e.g. bovine serum albumin or human serum albumin).
  • the moiety is a red blood cell.
  • the moiety is a latex particle.
  • the peptide further includes a linker sequence.
  • Suitable linkers may include a peptide sequence including alternating sets of glycine and serine residues, and may include an glutamic acid or lysine residue.
  • the linker sequence is Gly-Arg-Ser.
  • the methods of detecting described herein includes contacting a known quantity of SARS protease in solution to a peptide or variant thereof described herein further including a detectable marker and cleavage site for SARS protease, wherein SARS protease activity is monitored by measuring the intensity of the marker on the cleaved product.
  • the peptide further includes a 3C protease cleavage sequence.
  • the peptide further includes a peptide sequence tag.
  • the peptides described herein may be produced by recombinant expression or chemical synthesis. Host cells suitable for recombinant expression include expression in bacterial, mammalian, insect, yeast, etc. Recombinant expression may be used to produce any peptide or variant thereof described herein.
  • peptide tags include a polyarginine tag (Arg-tag), polyhistidine tag (His-tag), FLAG-tag, Strep-tag, c-myc-tag, S-tag, calmodulin-binding peptide, cellulose-binding domain, SBP-tag increment chitin- binding domain, glutathione S-transferase-tag (GST), maltose-binding protein, transcription termination antitermination factor (NusA), E.
  • TrxA coli thioredoxin
  • DsbA protein disulfide isomerase I
  • a preferred peptide tag includes His-tag and GST. After purification, the peptide tag may be removed from the expressed fusion protein by methods known in the art.
  • any one peptide or variant thereof described herein may be used in assays to detect whether a human has had a previous SARS-CoV-2 infection and in assays to detect the presence of a SARS-CoV-2 specific antibody in a biological sample.
  • the peptides described herein may be used in assays to identify individuals exposed to SARS-CoV-2 and to identify biological samples containing SARS-CoV-2 antigens or antibodies to SARS-CoV-2.
  • the methods described herein detects all immunoglobulin classes, including IgM and IgG. Positive sera could be present in individuals with a current or previous infection.
  • the methods described herein includes a multiplexed assay with an N protein from SARS-CoV-2 and from seasonal coronaviruses to allow differentiation of seroconversion against the pandemic of seasonal strains.
  • the methods described herein may include use of solid supports, or immune- precipitation.
  • the method includes use of a labeled antibody or polypeptide.
  • the label may be, for example, enzymatic, fluorescent, chemiluminescent, radioactive, or a dye molecule.
  • the methods described herein may include an element to amplify a signal from a resultant immune complex.
  • Such elements are known in the art, such as, for example, an enzyme, radioisotope, fluorophore, bioluminescent molecule, chemiluminescent molecule, biotin, avidin, streptavidin or the like.
  • the peptide or variant thereof described herein is bound to a solid matrix or support to facilitate separation of the sample from the peptide or variant thereof described herein after incubation.
  • solid supports that may be used include nitrocellulose (e.g., in membrane or microtiter well form), polyvinyl chloride (e.g., in sheets or microtiter wells), polystyrene latex (e.g., in beads or microtiter plates, polyvinylidine fluoride, diazotized paper, nylon membranes, microchips, high or low density biochips, recombinant immunoassays, microfluidity devices, micromagnetic beads, activated beads, and Protein A beads.
  • the solid support containing the peptide or variant thereof described herein is typically washed after separating it from the test sample, and prior to detection of a bound antibody.
  • the biological sample is contacted with a combination of proteins in addition to a peptide or variant thereof described herein. a) Detecting a previous SARS-CoV-2 infection by contacting a sample with an N-terminal domain-modified nucleocapsid polypeptide or variant thereof
  • the invention relates to a method of detecting a previous SARS-CoV-2 infection in a biological sample.
  • the method includes a) contacting the sample with an N- terminal domain-modified nucleocapsid polypeptide or variant thereof under conditions conducive to binding; and b) measuring binding between the sample and the polypeptide or variant thereof; wherein detection of binding between the sample and the polypeptide or variant thereof indicates a previous SARS-CoV-2 infection in the sample.
  • the N-terminal domain-modified nucleocapsid polypeptide includes the sequence SEQ ID NO: 1 . In some embodiments, the N-terminal domain-modified nucleocapsid polypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to SEQ ID NO: 1.
  • the N-terminal domain-modified nucleocapsid polypeptide has a sequence, wherein the first 175, 174, 173, 172, 171 , 170, 169, 168, 167, 166, 165, 164, 163, 162, 161 , 160, 159, 158, 157, 156, 155, 154, 153, 152, 151 , 150, 149, 148, 147, 146, 145, 144, 143, 142, 141 , 140, 139, 138, 137, 136, 135, 134, 133, 132, 131 , 130, 129, 128, 127, 126, 125, 124, 123, 122, 121 , 120, 119, 118, preferably the first 117, 116, 115, 114, 113, 112, 111 , 110, 109, 108, 107, 106, 105, 104, 103, 102, 101
  • the N-terminal domain-modified nucleocapsid polypeptide has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to the sequence SEQ ID NO: 5 (also shown in FIG. 3B).
  • the invention in another aspect, relates to a method of detecting a previous SARS-CoV- 2 infection in a biological sample.
  • the method includes a) contacting the sample with a C- terminal domain dimer nucleocapsid peptide or variant thereof under conditions conducive to binding; and b) measuring binding between the sample and the polypeptide or variant thereof; wherein detection of binding between the sample and the polypeptide or variant thereof indicates a previous SARS-CoV-2 infection in the sample.
  • the C-terminal domain dimer nucleocapsid peptide includes the sequence SEQ ID NO: 2.
  • the N-terminal domain-modified nucleocapsid polypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to SEQ ID NO: 2.
  • the C-terminal domain dimer nucleocapsid peptide has a sequence, wherein the first 150, 149, 148, 147, 146, 145, 144, 143, 142, 141 , 140, 139, 138, 137, 136, 135, 134, 133, 132, 131 , 130, 129, 128, 127, 126, 125, 124, 123, 122, 121 , 120, 119, 118, preferably the first 117, 116, 115, 114, 113, 112, 111 , 110, 109, 108, 107, 106, 105, 104, 103, 102, 101 , or 100 amino acid residues of SEQ ID NO: 2 are modified, as compared to SEQ ID NO: 2.
  • the C-terminal domain dimer nucleocapsid peptide has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to the sequence SEQ ID NO: 6, also shown in FIG. 6. c) Detecting a SARS-CoV-2 antibody by contacting a sample with an N- terminal domain-modified nucleocapsid polypeptide or variant thereof
  • the invention in another aspect, relates to a method of detecting a SARS-CoV-2 antibody in a biological sample.
  • the method includes a) contacting the sample with an N- terminal domain-modified nucleocapsid polypeptide or variant thereof under conditions conducive to binding; and b) measuring binding between the sample and the polypeptide or variant thereof; wherein detection of binding between the sample and the polypeptide or variant thereof indicates the presence of a SARS-CoV-2 antibody in the sample.
  • the N-terminal domain-modified nucleocapsid polypeptide includes the sequence SEQ ID NO: 1 . In some embodiments, the N-terminal domain-modified nucleocapsid polypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to SEQ ID NO: 1.
  • the N-terminal domain-modified nucleocapsid polypeptide has a sequence, wherein the first 175, 174, 173, 172, 171 , 170, 169, 168, 167, 166, 165, 164, 163, 162, 161 , 160, 159, 158, 157, 156, 155, 154, 153, 152, 151 , 150, 149, 148, 147,
  • ID NO: 1 are modified, as compared to SEQ ID NO: 1.
  • the N-terminal domain-modified nucleocapsid polypeptide has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to the sequence SEQ ID NO: 5 (also shown in FIG. 3B).
  • the invention in yet another aspect, relates to a method of detecting a SARS-CoV-2 antibody in a biological sample.
  • the method includes a) contacting the sample with a C- terminal domain dimer nucleocapsid peptide or variant thereof under conditions conducive to binding; and b) measuring binding between the sample and the polypeptide or variant thereof; wherein detection of binding between the sample and the polypeptide or variant thereof indicates the presence of a SARS-CoV-2 antibody in the sample.
  • the C-terminal domain dimer nucleocapsid peptide includes the sequence SEQ ID NO: 2.
  • the N-terminal domain-modified nucleocapsid polypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to SEQ ID NO: 2.
  • the C-terminal domain dimer nucleocapsid peptide has a sequence, wherein the first 150, 149, 148, 147, 146, 145, 144, 143, 142, 141 , 140, 139, 138, 137, 136,
  • the C-terminal domain dimer nucleocapsid peptide has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to the sequence SEQ ID NO: 6, also shown in FIG. 6.
  • the biological sample may be from any mammal, including humans, non-primates (e.g., a bat, pangolin, cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, etc.) and non-human primates (e.g., mammals including cynomolgus monkeys, chimpanzees, etc.).
  • non-primates e.g., a bat, pangolin, cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, etc.
  • non-human primates e.g., mammals including cynomolgus monkeys, chimpanzees, etc.
  • the sample is from a human or animal to which an immunogenic composition eliciting an immune response against a SARS-CoV-2 spike polypeptide has been administered.
  • the sample is from a human or animal prior to administration of an immunogenic composition eliciting an immune response against a SARS-CoV-2 spike polypeptide.
  • the sample is from a human or animal to which an immunogenic composition eliciting an immune response against a SARS-CoV-2 spike polypeptide had not been administered.
  • the sample is from a human or animal who has not been exposed to SARS-CoV-2 virus.
  • the sample is from a human or animal who has been exposed to SARS-CoV-2 virus and is or has recovered from a SARS-CoV-2 infection, such as, for example, convalescent serum.
  • the sample is from a human or animal that has been administered with at least one dose of an immunogenic composition, e.g., a vaccine, against SARS-CoV-2 virus.
  • an immunogenic composition e.g., a vaccine, against SARS-CoV-2 virus.
  • the sample is from a human or animal that has been administered with at least two doses of an immunogenic composition, e.g., a vaccine, against SARS-CoV-2 virus.
  • an immunogenic composition e.g., a vaccine, against SARS-CoV-2 virus.
  • the polypeptide or variant thereof is attached to a solid support prior to contact with the sample.
  • the sample is selected from any one of blood, serum, plasma, saliva, urine, mucus, fecal matter, and tissue extract.
  • EXAMPLE 1 Strategy for a non-vaccine antigen-specific antibody binding assay (NVA) to distinguish a history of SARS-CoV-2 infection from a history of other coronavirus infections in recipients of a COVID-19 vaccine candidates
  • NAA non-vaccine antigen-specific antibody binding assay
  • SARS-CoV-2 spike (S) glycoprotein To detect a history of SARS-CoV-2 infection serologically in a subject who has been immunized with a vaccine that includes or expresses an antigen that is derived from the SARS-CoV-2 spike (S) glycoprotein, we seek a SARS-CoV-2 protein other than S that is highly immunogenic and well enough behaved biochemically to be an easily produced target antigen for immunoassays. The assay should distinguish a history of infection with SARS-CoV-2 from a history of infection with common seasonal coronaviruses.
  • N The nucleocapsid (N) of SARS-CoV-2 is the most abundant protein of coronaviruses. N coats the viral RNA and forms a helical nucleocapsid. As might be expected from an abundant protein that is in complex with RNA and forms a highly multivalent, regular array, N is highly immunogenic and, therefore, is often chosen as a target antigen for diagnostic assays.
  • Table 2 Percentage amino acid identity of coronavirus spike and nudeocapsid proteins to SARS-CoV-2 proteins.
  • N has 3 domains, the NTD (N terminal domain), a SR rich linker, and the CTD (C-terminal domain; Figure 1).
  • the domain structure is conserved among the different coronavirus N’s, but the homology is low (Table 2 above).
  • the C-terminal domain is relatively variable between coronavirus strains and is responsible for dimerization.
  • the N-terminal domain interacts with RNA and is more conserved, and there is a stretch of amino acids in the NTD that is very conserved ( Figure 2). Due to these conserved regions, it is possible that infections by any human coronavirus could elicit antibodies that cross- react with SARS-CoV-2 N as has been shown in the past for SARS.
  • NTD-modified full length N protein This protein will be similar to the previous one but the sequence FYYLGTGP (SEQ ID NO: 4) will be substituted by FAYAGAG (SEQ ID NO: 29), to disrupt the conserved epitope without altering tertiary structure. Other substitutions for FYYLGTGP (SEQ ID NO: 4) may be used. This design is guided by the known structure of the NTD ( Figure 4). a. Exemplary NTD-modified full length N protein sequence includes:
  • CTD domain This construct includes only the CTD of N since this domain is less conserved. This construct will include the same tags as the previous versions. Its design is guided by the known structure of the CTD ( Figure 5). a. Exemplary CTD-dimer N protein sequence includes:
  • Figure 4 Structure guided design of a modified N-terminal domain of SARS-CoV-2 N to reduce cross-reactivity with other coronaviruses. (Structural coordinates for the SARS-CoV-2 N- terminal domain from PDB ID: 6M3M; S. Kang et al. , Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites. 2020).
  • the assay is being developed on an ELISA or a Luminex platform and will be tested initially with human non-immune and convalescent sera for SARS-CoV-2.
  • SARS-CoV-2 N alone shows insufficient specificity
  • N proteins of seasonal coronavirus strains are being synthesized.
  • a multiplexed assay with N’s of the pandemic and of seasonal coronavirus strains should allow the differentiation of seroconversion against the pandemic distinct from common seasonal strains.
  • the SARS-CoV N polypeptides may be detected using enzyme-linked immunosorbent assay (ELISA), enzyme-based histochemical assays, using fluorescent, radioactive, and/or luminescent systems.
  • ELISA enzyme-linked immunosorbent assay
  • enzyme-based histochemical assays using fluorescent, radioactive, and/or luminescent systems.
  • FIG. 1 Structure of the C-terminal domain of SARS-CoV N. PDB ID: 2GIB. I. M. Yu, M. L. Oldham, J. Zhang, J. Chen, Crystal structure of the severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein dimerization domain reveals evolutionary linkage between corona- and arteriviridae. J Biol Chem 281 , 17134-17139 (2006).
  • SARS severe acute respiratory syndrome
  • EXAMPLE 2 A single-plex SARS CoV-2 full-length (FL) Nucleoprotein Ig Direct Luminex Immunoassay was developed. Magnetic beads coated with SARS-CoV-2 Nucleocapsid protein are contacted with respective serum dilutions, and binding of antibodies, if any from the respective serum dilutions, to the SARS-CoV-2 Nucleocapsid protein are detected using PE goat anti-human specific antibodies, and analyzed in a BioPlex reader.
  • the serum samples may include any one of a standard, convalescent serum, and test serum.
  • the incubation magnetic beads coated with SARS-CoV-2 Nucleocapsid protein and the serum dilutions occurs at room temperature for about 120 minutes, whereby an antibody, if any, in the respective serum samples bind to the SARS-CoV-2 Nucleocapsid protein.
  • PE-goat anti-human Ig secondary antibodies are added for detection of anti-N antibodies in the sera samples and incubated with the sera samples at room temperature for about 90 minutes.
  • Results (positive or negative) from the single-plex SARS CoV-2 assay described herein were identical to at least 91- 100% of the results observed from the ELECSYS (Roche) immunoassay intended for qualitative detection of antibodies to SARS-CoV-2 in human serum and plasma.

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Abstract

Selon un aspect, l'invention concerne un peptide présentant la séquence SEQ ID NO : 1 et des procédés d'utilisation associés. Selon un autre aspect, l'invention concerne un peptide présentant la séquence SEQ ID NO : 5 et des procédés d'utilisation associés. Selon un autre aspect, l'invention concerne un peptide présentant la séquence SEQ ID NO : 2 et des procédés d'utilisation associés. Selon un autre aspect, l'invention concerne un peptide présentant la séquence SEQ ID NO : 6 et des procédés d'utilisation associés. Selon un autre aspect, l'invention concerne une molécule d'acide nucléique qui code l'un quelconque des peptides décrits et des procédés d'utilisation associés.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112500494A (zh) * 2020-11-09 2021-03-16 昆明市妇幼保健院 用于新型冠状病毒检测的抗原及其制备方法
CN112500494B (zh) * 2020-11-09 2023-01-24 昆明市妇幼保健院 用于新型冠状病毒检测的抗原及其制备方法

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