WO2022144317A1 - Antigènes et dosages pour la détection d'anticorps contre le sars-cov-2 - Google Patents

Antigènes et dosages pour la détection d'anticorps contre le sars-cov-2 Download PDF

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WO2022144317A1
WO2022144317A1 PCT/EP2021/087597 EP2021087597W WO2022144317A1 WO 2022144317 A1 WO2022144317 A1 WO 2022144317A1 EP 2021087597 W EP2021087597 W EP 2021087597W WO 2022144317 A1 WO2022144317 A1 WO 2022144317A1
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protein
amino acid
seq
cov
acid sequence
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PCT/EP2021/087597
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Kerstin Dehne
Martin Ungerer
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Isar Bioscience Gmbh
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Priority to EP21847953.3A priority Critical patent/EP4271409A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
    • 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
    • C12N2770/20051—Methods of production or purification of viral material
    • 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 provides a protein (antigen), and a process of producing the protein, usable in an assay for antibodies against SARS-CoV-2, as well as a nucleic acid molecule encoding the protein.
  • the invention further provides an assay for detecting antibodies against SARS-CoV-2 after an infection of a patient with this virus, during ongoing COVID- 19 of a patient, and/or after cured COVID-19 of a patient.
  • the invention also provides an assay for estimating the time having passed since SARS-CoV-2 infection of a patient with ongoing or cured COVID-19.
  • the invention provides a solid support, notably a microarray, having attached the protein and an apparatus for performing the assays.
  • the SARS-CoV-2 pandemic has led to an enormous increase, during the year 2020, of the need for reliable assays for testing human or animal subjects on a present and/or past SARS-CoV-2 infection.
  • Tests on the presence of antibodies (Abs) that develop in mammals after infection by SARS-CoV-2 allow, in principle, determining a present infection as well as past infection, but require a blood sample to be taken from the subject to be tested.
  • Commercial SARS-CoV-2 antibody tests are available.
  • the Roche® SARS-CoV-2 Rapid Antibody Test for example, is a chromatographic immunoassay in devices for individual tests. Thus, it is not adapted for large-scale testing of many samples in parallel.
  • Another example is the Mikrogen® recomWell SARS-CoV-2 test that is in an ELISA screening format and is performed on plates for up to 96 determinations in parallel.
  • a general problem with immunoassays is insufficient specificity which may lead to false positive results and insufficient sensitivity which may lead to false negative results.
  • the Mikrogen® assay suffers from the problem of limited specificity.
  • a process of producing a protein comprising cultivating a mammalian cell containing a nucleic acid molecule comprising a polynucleotide encoding said protein and expressing said protein in said cell, wherein said protein comprises a polypeptide whose amino acid sequence is or comprises:
  • nucleotide sequence of said polynucleotide is or comprises the nucleotide sequence of SEQ ID NO: 2 (encoding RBD without His-tag) or SEQ ID NO: 4 (encoding RBD with His-tag).
  • a process of producing a protein comprising cultivating a mammalian cell containing a nucleic acid molecule comprising polynucleotide encoding said protein and expressing said protein in said cell, wherein said protein comprises a polypeptide whose amino acid sequence is or comprises:
  • nucleotide sequence of said polynucleotide is or comprises the nucleotide sequence of SEQ ID NO: 6 (encoding nucleocapsid protein without Strep-tag) or 8 (encoding nucleocapsid protein with Strep-tag).
  • a process of producing a protein comprising cultivating a mammalian cell containing a nucleic acid molecule comprising a polynucleotide encoding said protein and expressing said protein in said cell, wherein said protein comprises a polypeptide whose amino acid sequence is or comprises:
  • nucleotide sequence of said polynucleotide is or comprises the nucleotide sequence of SEQ ID NO: 22 (encoding omicron RBD with His- tag) or SEQ ID NO: 24 (encoding omicron RBD without His-tag).
  • the protein according to 7 or 8 having a protein purity of at least 98 %, preferably of at least 99 %.
  • Nucleic acid molecule comprising a polynucleotide whose nucleotide sequence is or comprises that of SEQ ID NO: 2 (encoding RBD without His-tag), SEQ ID NO: 4 (encoding RBD with His-tag), SEQ ID NO: 6 (encoding nucleocapsid protein without Strep-tag), SEQ ID NO: 8 (encoding nucleocapsid protein with Strep-tag), SEQ ID NO: 22 (encoding omikron RBD with His-tag, or SEQ ID NO: 24 (encoding omikron RBD without His-tag).
  • step (iii) detecting the presence or absence of said binding of step (ii) using a detection antibody specific for a human immunoglobulin; wherein said protein is as defined in any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • step (iii) detecting the presence or absence of said binding of step (ii) using a detection antibody specific for a human immunoglobulin M or a human immunoglobulin A; wherein said protein is as defined in any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • step (iii) detecting the presence or absence of said binding of step (ii) using a first detection antibody specific for a human immunoglobulin M and/or A and using a second detection antibody specific for a human immunoglobulin G; wherein said protein is as defined in any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the assay according to 14, comprising the following steps:
  • step (iii) detecting the presence or absence of said binding of step (ii) on said first surface area using a first detection antibody specific for a human immunoglobulin M or A, and detecting the presence or absence of said binding of step (ii) on said second surface area using a second detection antibody specific for a human immunoglobulin G.
  • said detection antibody or said detection antibodies is/are linked to a peroxidase and step (iii) comprises detecting chemiluminescence produced in the presence of hydrogen peroxide, e.g. using a camera.
  • Solid support having attached one or more spots of the protein defined in any one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, each spot preferably comprising 2 ng or less of said protein, preferably preferably comprising 1 ng or less of said protein, more preferably comprising 0.5 ng or less of said protein.
  • Apparatus for performing an assay according to any one of 12 to 22, comprising a solid support according to 23 or 24 and a microfluidic capillary and pumping system for pumping sample solution, washing solution(s), and detection solutions over said solid support that is preferably a microarray.
  • the inventors have surprisingly found that the reliability of an assay for antibodies against SARS-CoV-2 can be improved by using a protein bound to a solid support that is/was expressed in mammalian cells.
  • the inventors assume that the high quality of the protein in terms of purity and/or mammalian-type glycosylation is/are responsible for the improved reliability of the assay results, such as improved specificity, whereby false positive results are less likely to occur.
  • an assay for antibodies against SARS-CoV-2 can be improved by using a microarray as a solid support, preferable a microarray-immunoassay (MIA), more preferably when combined with a flow-through immunochip as said solid support and chemiluminescence detection.
  • MIA microarray-immunoassay
  • Using a microarray as solid support allows reducing unspecific binding of blood or serum components to the support, leading to less unspecific binding of immunoglobulins (Igs) in a sample and, thus, to a better signal-to- noise ratio for a sample that is positive for SARS-CoV-2 antibodies.
  • Igs immunoglobulins
  • the inventors have conceived this improvement from Fig.
  • Figure 1 SDS-PAGE results of purification of His-tagged RBD protein of a SARS- CoV-2 spike protein. Eluates after HisTrapFF column. Sample numbering: #1 : sample before HisTrapFF column; #2: 1st flow-through from His TrapFF column; #3: eluate 1 (1ml); #4: eluate 2 (1ml); #5: eluate 3 (1ml); #6: eluate 4 (1ml); #7: eluate 5 (1ml); #8: eluate 6 (1ml); #9: eluate 7 (1ml); #10: eluate 8 (1ml); #11: eluate 9 (1ml); #12: eluate 10 (1ml); #13: eluate 11 (1ml); #14: eluate 12 (1ml); #15: eluate 13 (1ml); #16: eluate 14 (1ml); #17: eluate 15 (1ml); #18: eluate 16 (1ml).
  • Figure 2 Results of protein concentration of His-tagged RBD protein measured with the Micro BCA Protein Kit. Measured concentration: 0.5 pg/ml.
  • Figure 3 Purification of strep-tagged nucleoprotein (N). Visualized by Coomassie Blue (Gelcode) staining.
  • Figure 4 Detection of strep-tagged nucleoprotein (N) by immunoblotting against strep- tag.
  • Figure 5 Protein concentration of strep-tagged nucleoprotein (N) measured with Mico BCA Protein Assay Kit. Measured concentration: 51 ng/pl.
  • FIG. 6 Comparison by SDS-PAGE of the RBD-His protein produced in CHO-cells with RBD-His produced in a baculoviral expression system.
  • the RBD-His protein from CHO-cells runs with higher molecular weight than the RDB-His protein from a baculoviral expression system, indicating higher glycosylation of the CHO cell-expressed RBD-His.
  • Figure 7 Comparison of the present ELISA assay of the invention of Example 4 using purified RBD-His produced in CHO-cells with a commercially available SARS-CoV-2 antibody test from Mikrogen®. Both assays were used to analyze serum from patients that had been tested for SARS-CoV-2 by RT-PCR of a throat swab sample. Patients that were positive for SARS-CoV-2 based on the RT-PCR test are indicated with plus (+). Patients that are negative for SARS-CoV-2 in the RT-PCR test are indicated with a minus (-) sign. Given is the optical density (OD) readout of the ELISA test in a plate reader at 450 nm wavelength.
  • OD optical density
  • Figure 8 Log-scale plotting of the data shown in the diagram of Figure 7.
  • Figure 9 Amino acid sequence (SEQ ID NO: 9) of the spike protein S1 of human coronavirus OC43 (OC43S1) and the coding sequence (SEQ ID NO: 10). The coding sequence is optimized for Cricetulus griseus. 1-2049 [ATG ...TGA],
  • FIG. 10 Amino acid sequence (SEQ ID NO: 11) of the spike protein S1 of human coronavirus HKLI1 (HKLI1S1) and the coding sequence (SEQ ID NO: 12).
  • the coding sequence is optimized for Cricetulus griseus. 1-2271 [ATG ...TAA],
  • FIG 11 Amino acid sequence (SEQ ID NO: 13) of the spike protein S1 of human coronavirus NL63 (NL63S1) and the coding sequence (SEQ ID NO: 14).
  • the coding sequence is optimized for Cricetulus griseus. 1-2172 [ATG ...TGA],
  • Figure 12 Amino acid sequence (SEQ ID NO: 15) of the spike protein S1 of human coronavirus 229E (229ES1) and the coding sequence (SEQ ID NO: 16).
  • the coding sequence is optimized for Cricetulus griseus. 1-1632 [ATG ...TAA],
  • FIG. 13 Amino acid sequence (SEQ ID NO: 17) of SARS-CoV-2 spike S2 protein and its coding sequence (SEQ ID NO: 18).
  • the coding sequence is optimized for Cricetulus griseus. 1-1785 [ATG ...TGA],
  • Figure 14 Amino acid sequence (SEQ ID NO: 21) of the RBD of spike protein S1 of human coronavirus omicron variant with N-terminal signal peptide and with C-terminal His- tag, and the coding sequence thereof (SEQ ID NO: 22).
  • the coding sequence is optimized for Cricetulus griseus. 1-750 [ATG ...TGA],
  • the SARS-CoV-2 genome, structure, and proteins contained in the virus particle are known e.g. from Abu Turab Naqvi et al. (BBA - Molecular Basis of Disease, 2020 Oct 1 ;1866(10):165878; doi: 10.1016/j.bbadis.2020.165878) or from Mittal et al. (PLoS Pathog. 2020 Aug; 16(8): e1008762; doi: 10.1371/journal.ppat.1008762).
  • any of the proteins contained in a SARS-CoV-2 viral particle may induce an antibody response in a mammal such as a human, whereby in principle any of these proteins, or fragments thereof, may be used in an immunoassay for antibodies against SARS-CoV-2.
  • S spike protein
  • N nucleoprotein
  • M membrane protein
  • E envelope protein
  • SARS-CoV-2 proteins or fragments thereof for the invention are the proteins S and N, and S is most preferred.
  • S protein contains the major protein subunits S1 and S2.
  • S1 comprises the receptor binding domain (RBD) that contains the receptor binding motif for host cell receptors.
  • RBD receptor binding domain
  • the RBD of protein S forms the outermost part of the spikes of SARS- CoV-2 and is thus generally believed to be highly immunogenic. Therefore, the RBD is a preferred protein to be produced in the process of producing a protein of the invention and a preferred protein to be attached to the solid support used in the assays of the invention.
  • Another preferred protein for these purposes is protein N.
  • a SARS-CoV-2 protein of the invention may, if desired or required, be produced as a fusion protein comprising the polypeptide of the protein itself and an N-terminal or C- terminal tag that may be used for purification by affinity chromatography of the protein after expression and/or for supporting binding of the protein to a solid support for the assays of the invention.
  • tags are a His-tag and the Strep-tag.
  • the protein may comprise an N-terminal signal sequence for secretion of the expressed protein from the mammalian cells in which it is expressed.
  • the protein for use in the invention comprises a polypeptide whose amino acid sequence is or comprises:
  • the protein defined in (b), (c), and (d) is capable of binding to an antibody produced in a mammal after infection with SARS-CoV-2 (Wuhan).
  • the protein for use in the invention comprises a polypeptide whose amino acid sequence is or comprises:
  • the protein defined in (b), (c), and (d) is capable of binding to an antibody produced in a mammal after infection with SARS-CoV-2 (Wuhan).
  • the protein for use in the invention comprises a polypeptide whose amino acid sequence is or comprises:
  • the protein defined in (b), (c), and (d) is capable of binding to an antibody produced in a mammal after infection with SARS-CoV-2 B1.1.529 (ZA 11/2021) (omicron variant).
  • amino acid substitutions, additions, insertions or deletions may be combined, but the given number or number range refers to the sum of all amino acid substitutions, additions, insertions and deletions.
  • amino acid substitutions, additions, insertions and deletions amino acid substitutions, additions, and deletions are preferred.
  • insertion relates to insertions within the amino acid sequence of a reference sequence, i.e. excluding additions at the C- or N-terminal end.
  • additions means additions at the C- or N-terminal end of the amino acid sequence of a reference sequence.
  • a deletion may be a deletion of a terminal or an internal amino acid residue of a reference sequence.
  • Reference sequences are amino acid sequences identified herein by a SEQ ID NO.
  • the processes of producing a protein of the invention comprise cultivating a mammalian cell containing a nucleic acid molecule comprising a polynucleotide encoding said protein and expressing said protein in said cell.
  • the mammalian cell by be a human cell or an animal cell. Widely used mammalian cells for protein expression are CHO cells that are also used in the examples. Suitable culture media for culturing mammalian cells are known in the art. Methods of expressing a protein in mammalian cells are also known in the art.
  • the polynucleotide encoding said protein may be codon-optimized for expression in the cells used for producing the protein.
  • the nucleic acid molecule may further contain genetic elements for expressing the protein from the polynucleotide, such as a promoter and/or a terminator.
  • the nucleic acid molecule may be a vector or plasmid and may further contain a selectable marker.
  • the protein of the invention as defined above preferably comprises, in addition to the polypeptide of the protein and an optional tag and/or signal peptide, glycosylation, more preferably mammalian-type glycosylation.
  • the invention provides the protein of the invention producible by or produced according to the process of the invention, i.e. expressed in a mammalian cell.
  • the protein of the invention has a higher molecular weight as judged by SDS-PAGE than a protein having the identical polypeptide expressed in a baculoviral expression system (see Figure 6).
  • the protein should have a high purity.
  • the purity is preferably at least 98 % as determined by SDS- PAGE analysis with silver staining and read-out of bands using an imaging system.
  • the purity of the protein can be analyzed by capillary gel electrophoresis (CGE).
  • Capillary gel electrophoresis (CGE)-on-a-chip analysis can be performed on an Agilent 2100 bioanalyzer (Agilent Technologies GmbH; Waldbronn, Germany) in combination with an Agilent Protein 80 Kit (sizing range: 5-80 kDa) and 2100 Expert Software (Kuschel et al. 2002). All reagents and chips were prepared according to the manufacturer's instructions. Lyophilized, buffer containing protein samples are reconstituted with water to a concentration of 1 mg protein per ml. 4 pl of each protein sample and 2 pl of reducing sample buffer are mixed and incubated at 95°C for 5 min.
  • the assays of the invention are immunochemical assays that make use of a solid support having attached one or more protein(s) of the invention.
  • the solid support may be made of plastic or glass.
  • the solid support may be a multi-well plate such as a 96-well plate or a 384-well plate. Such plates are generally known in the art and are commercially available.
  • the solid support having attached the protein is a microarray having attached multiple spots of the protein in a predetermined order, whereby the multiple protein spots are spatially separated.
  • Such solid support may be made of glass or plastic.
  • the microarray may have at least 5, preferably at least 10, and more preferably at least 20 protein spots attached to it.
  • the protein used in the invention (as well as optional additional proteins, if used) should be attached to the microarray in a small amount.
  • each protein spot on the microarray comprises 2 ng or less, preferably 1 ng or less, more preferably 0.5 ng or less protein. More preferably, the protein forming the protein spot should have a minimum purity as defined above and/or mammalian-type glycosylation for achieving particularly high specificity for avoiding false positive results.
  • the microarray may have attached multiple spots of the same protein, e.g. for making the same test multiple times in parallel to increase the reliability of the assay result.
  • the microarray may have attached two or more spots of a given protein that binds specifically to antibodies that are produced in humans upon infection with SARS- CoV-2.
  • the protein may be as defined above with reference to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:21, or SEQ ID NO:23, preferably SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7.
  • Examples of the protein are those having the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:21, and SEQ ID NO:23 that have been expressed in mammalian cells, preferably the protein whose polypeptide is that of SEQ ID NO: 1, 5, or 23, preferably 1 or 5.
  • the microarray may have attached spots of different proteins, e.g. for analyzing a given sample in parallel for two or more antibodies as analytes present in the sample.
  • the microarray may have attached one or more spots of a first protein that binds specifically to antibodies that are produced in humans upon infection with SARS-CoV-2 and one or more spots of a second protein, i.e. different protein, that binds specifically to antibodies that develop in humans upon infection with SARS-CoV- 2.
  • the first protein may be the RBD of protein S or a fragment or variant of protein S thereof (e.g. as defined above) and the second protein may be protein N of SARS-CoV-2 or a fragment or variant thereof (e.g. as defined above).
  • the first protein may be the RBD of protein S of the Wuhan SARS-CoV-2 variant (e.g. one of those defined with reference to SEQ ID NO: 1 or 3) or a fragment or variant of protein S thereof (e.g. as defined above)
  • the second protein may be the RBD of protein S of the omikron SARS-CoV-2 variant (e.g. one of those defined with reference to SEQ ID NO: 21 or 23) or a fragment or variant of protein S thereof (e.g. as defined above).
  • the assay of the invention may be used for assaying, in addition to antibodies against SARS-CoV-2, antibodies against other viral pathogens present in a patient sample.
  • examples of such other viruses are SARS-CoV, MERS-CoV, common cold-CoV, and other common respiratory viruses such as RSV, adenovirus, and influenza virus.
  • the microarray may have additionally attached spots of antigenic proteins of any one or more of these or other viruses.
  • An example of a multiple antibody microarray is described by Khan et al., doi: https://doi.org/10.1101/2020.03.24.006544.
  • the microarray may, for example, have attached protein spots from the spike protein S1 of human coronavirus OC43 (OC43S1) given in SEQ ID NO: 9 or a fragment thereof; protein spots from the spike protein S1 of human coronavirus H Kill (HKU1S1) given SEQ ID NO: 11 or a fragment or variant thereof, protein spots of the spike protein S1 of human coronavirus NL63 (NL63S1) given in SEQ ID NO: 13 or a fragment or variant thereof, protein spots of the spike protein S1 of human coronavirus 229E (229ES1) given in SEQ ID NO: 15 or a fragment or variant thereof, and/or protein spots of SARS-CoV-2 spike protein SI I given in SEQ ID NO: 17 or a fragment or variant thereof.
  • a variant of any of these proteins may be a protein comprising a polypeptide whose amino acid sequence has at least 90%, preferably at least 95% sequence identity to the (entire) amino acid sequence of SEQ ID NO: 9, 11, 13, 15, or 17, respectively.
  • a variant of these proteins may be a protein comprising a polypeptide whose amino acid sequence has: from 1 to 70, preferably from 1 to 35 amino acid substitutions, additions, deletions and/or insertions compared to the amino acid sequence of SEQ ID NO: 9; from 1 to 70, preferably from 1 to 35 amino acid substitutions, additions, deletions and/or insertions compared to the amino acid sequence of SEQ ID NO: 11 ; from 1 to 70, preferably from 1 to 35 amino acid substitutions, additions, deletions and/or insertions compared to the amino acid sequence of SEQ ID NO: 13; from 1 to 50, preferably from 1 to 25 amino acid substitutions, additions, deletions and/or insertions compared to the amino acid sequence of SEQ ID NO:
  • the microarray comprises one or more protein spots of the RBD of SARS-CoV-2 (e.g. that of SEQ ID NO: 1 or a variant as defined above, and/or of SEQ ID NO: 23 or a variant as defined above) and one or more protein spots of the S1 protein of human coronavirus HKLI1 (HKLI1S1) or a fragment thereof, and one or more protein spots of the spike protein S1 of human coronavirus NL63 (NL63S1) or a fragment thereof, and one or more protein spots of the spike protein S1 of human coronavirus 229E (229ES1) or a fragment thereof, and protein spots of SARS-CoV-2 spike protein SI I.
  • SARS-CoV-2 e.g. that of SEQ ID NO: 1 or a variant as defined above, and/or of SEQ ID NO: 23 or a variant as defined above
  • the microarray comprises one or more protein spots of the RBD of SARS-CoV-2 (e.g. that of SEQ ID NO: 1 or
  • the surface chemistry for coating a protein on a solid support to form a microarray is known in the art. These aspects are described in detail e.g. in the dissertation of Klaus Wutz at the Technical University Kunststoff, 2014. Parts of the thesis were published by Wutz et al., Analytical Chemistry 2013, 85, 5279-5285. Particularly preferred is the surface chemistry used and described in the latter publication (see also supporting information therein).
  • the assays of the invention are in vitro assays in that they are done using a sample from a mammalian subject, preferably a human subject or patient, but are done outside the human or animal body.
  • the sample is a blood sample or serum sample.
  • a blood sample may be used in the assay as it is. However, it is generally preferred to produce a serum sample from the blood sample, e.g. by centrifugation of the blood sample, to avoid interference of blood cells with the assay.
  • the invention provides an in-vitro assay for detecting antibodies against SARS-CoV-2 after an infection of a patient with this virus, during ongoing COVID-19 disease of a patient, and/or after cured COVID-19 of a patient, comprising the following steps:
  • the solid support was described above.
  • the solid support is a microarray comprising multiple spots of protein (one or more different proteins), as also described above.
  • the general assay steps are known in the art.
  • the assay may be performed as an ELISA in a multi-well plate.
  • the assay may be performed in an apparatus that facilitates addition and removal of sample, addition and removal of washing solutions, addition and removal of solutions containing the detection antibody or antibodies, and/or, as required, addition and removal of solutions containing components for detecting the detection antibody or antibodies.
  • the apparatus should further have a device for reading out the microarray in step (iii), such as a CCD camera, and a computer system and software for analyzing the images taken by the camera.
  • a device for reading out the microarray in step (iii) such as a CCD camera
  • a computer system and software for analyzing the images taken by the camera is the MCR 3 described in the dissertation of Klaus Wutz mentioned above and in Wutz et al., Analytical Chemistry 2013, 85, 5279-5285.
  • Detection methods for step (iii) of the assay are generally known in the art. For example, the methods disclosed in the above-cited documents of Wutz may be used.
  • binding of antibodies in the sample to the antigen (protein) on the solid support can be detected by using a secondary antibody (also referred to herein as detection antibody).
  • the detection antibody is capable of binding to antibodies belonging to a class of antibodies (in the sample) to be detected.
  • the detection antibody may be an anti-IgG antibody, if an IgG antibody response to a SARS-CoV-2 infection or to COVID-19 is to be detected.
  • the detection antibody used in step (iii) may be an anti-IgG antibody, an anti-lgA antibody, or an anti-IgM antibody, preferably an anti-human IgG antibody, an anti-human IgA antibody, or an anti-human IgM antibody where the subject from which the sample is derived is a human patient.
  • the detection antibody is an antibody specific for a human IgM or a human IgA.
  • IgM, IgA, and IgG antibodies occur in an infected subject of patient at different time ranges.
  • IgM antibodies and IgA antibodies occur early after infection, generally within a few days after symptom development duo to a SARS-CoV-2 infection. IgM antibodies occur about 2 days earlier than IgA antibodies. However, the concentration of IgM antibodies in blood decrease within about 2 weeks after its maximum concentration which is about 1 week after symptom development. IgA remain much longer and remain for 3 to 5 weeks after symptom development. IgG antibodies start occurring in blood of a patient about 1-2 weeks after symptom development and continue increasing up to about 4 weeks after symptom development. IgG response likely lasts for several months. Symptom development is generally assumed to occur within 5-10 days post infection.
  • assaying at least for IgA or IgM and for IgG in parallel in the assay allows estimating the time of SARS-CoV-2 infection of a patient with ongoing or cured COVID-19, or estimating the time that has passed since infection with SARS-CoV-2.
  • step (i) comprises providing a solid support having attached, on at least a first and a second surface area of said support, a protein that binds specifically to antibodies that are produced in humans upon infection with SARS- CoV-2, and step (iii) comprises detecting the presence or absence of the binding of step (ii) on said first surface area using a first detection antibody specific for a human IgM or IgA, and detecting the presence or absence of said binding of step (ii) on said second surface area using a second detection antibody specific for a human IgG.
  • the solid support is preferably a microarray.
  • said solid support of step (i) comprises providing a solid support having attached, on at least a first, a second, and a third surface area of said support, a protein that binds specifically to antibodies that are produced in humans upon infection with SARS-CoV-2, and step (iii) comprises detecting the presence or absence of the binding of step (ii) on said first surface area using a first detection antibody specific for a human immunoglobulin M, detecting the presence or absence of said binding of step (ii) on said second surface area using a second detection antibody specific for a human immunoglobulin A, and detecting the presence or absence of said binding of step (ii) on said third surface area using a third detection antibody specific for a human immunoglobulin G.
  • the solid support is preferably a microarray.
  • Binding of the detection antibody or antibodies to antibody from the sample having bound to the protein attached to the solid support can be detected using generally known means, for example by fluorescence emitted by a fluorescent dye labeled to the detection antibodies.
  • a widely used method is a color reaction or luminescence generated by an enzyme such as peroxidase (e.g. horseradish peroxidase) labeled to the detection antibodies and a substrate that can be converted by the enzyme or a reaction product thereof (e.g. by hydrogen peroxide generated by a peroxidase).
  • Developing color or luminescence may be measured by a suitable camera, and color intensity or luminescence intensity may be calculated using known methods to estimate the concentration of the Ig from the sample that was detected by the respective detection antibody.
  • the 6 x His-tagged receptor binding domain of the Sars-CoV-2 spike protein (hereinafter RBD-His) consists of the amino acid residues corresponding to the receptor binding (RBD) domain, which was derived from the S protein nucleotide sequence (positions 22517- 23183, amino acid 319 to 541, RVQP....CVNF) of the SARS-CoV-2 Wuhan Hu-1 genome (Genbank accession number MN908947) followed by six histidines.
  • NP-Strep The strep-tagged nucleocapsid protein (hereinafter NP-Strep) consists of the amino acids corresponding to the N protein nucleotide sequence (positions 28290 to 29549) of the SARS-CoV-2 Wuhan Hu-1 genome (Genbank accession number MN908947) followed by a streptavidin tag (NP-Strep).
  • the complementary DNA sequences adapted for hamster codon usage were produced synthetically by GeneArt (Life Technologies) by adding a sequence encoding a signal peptide METPAQLLFLLLLWLPDTTG (SEQ ID NO: 19) and cloned into the plasmid vector pcDNA5/FRT via BamHI and Xhol.
  • the resulting vectors were called pcDNA5/CoV-RBD-His and pcDNA5/CoV-NP-Strep, respectively, and allow for expression and secretion of RBD-His or NP-Strep into the culture medium of mammalian cells under the control of the human cytomegalovirus (CMV) immediate-early enhancer/promoter and selection for stable clones with Hygromycin B after co-transfection with plasmid pOG44.
  • CMV human cytomegalovirus
  • the vectors were transfected by using Lipofectamine 2000 Reagent (Invitrogen, #11668-019) into Flip-lnTM-Chinese hamster ovary (CHO) cells (Life Technologies), together with the plasmid pOG44, providing site-directed recombination.
  • CHO Flip-lnTM-Chinese hamster ovary
  • pOG44 Flip-lnTM-Chinese hamster ovary
  • CHO-spike-RBD-His cells and CHO-spike-NP-Strep cells were grown in suspension in ProCHO5, 4 mM L-glutamine and 600 pg/ml hygromycin B in flasks to submaximal density at 37°C and then centrifuged. The cells were continuously grown at 37°C, with splitting every 3-4 days. The supernatants were cleared by centrifugation at 400 g for 5 min and subsequent filtration with a 0.22 pm sterile filter (TPP, #99722). The resulting RBD-His or NP-Strep protein-containing medium was immediately frozen and stored at -20°C until protein purification.
  • CHO-RBD-His supernatants 0.5 L were diluted 1 :2 in 20 mM sodium phosphate, 0.3 M NaCI, pH 8.0, and loaded on an equilibrated 1 ml HisTrapTM excel column (GE Healthcare 17-3712-05). After washing the column with 20 mM sodium phosphate, 0.3 M NaCI, pH 8.0, RBD-His was eluted with 4 x 1 ml 20 mM sodium phosphate, 0.3 M NaCI, 0.25 M imidazole, pH 8.0.
  • Protein content was determined by OD 280 measurement and the relevant fractions were dialysed (Slyde-A-Lyzer Dialysis Cassette, 10000 MWCO, Thermo Scientific # 66380) against phosphate-buffered saline (PBS from Roth: 137 mM NaCI, 2.7 mM KCI, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4, 0.2 pm filtered and steam sterilized) at 4°C for 16 h.
  • PBS phosphate-buffered saline
  • CHO-NP-Strep supernatants were diluted 1 :2 in 50 mM sodium phosphate, 0.3 M NaCI, pH 8.0, and loaded on an equilibrated 1 ml StrepTrapTM HP column (GE Healthcare 28-9075-46). After washing the column with 50 mM sodium phosphate, 0.3 M NaCI, pH 8.0, NP-Strep was eluted with 4 x 1 ml 20 mM sodium phosphate, 0.3 M NaCI, 2.5 mM desthiobiotin (Sigma, # D 1411) pH 8.0.
  • Protein content was determined by OD 280 measurement and the relevant fractions were dialysed (Slyde-A-Lyzer Dialysis Cassette, 10000 MWCO, Thermo Scientific # 66380) against PBS at 4°C for 16 h.
  • washing /binding buffer 20mM sodium phosphate, 0.3 M NaCI, 20 mM imidazole (pH 8.0)
  • Washing of column wash the column with 50ml wash buffer (10x column volume) and let the buffer flow through Elution of proteins apply 20ml Elution buffer and collect eluates per 1ml volume ; store at 4°C until further analysis
  • This format is a multi-layer based format which contains the image as displayed in the software, the raw data image, the image settings and the GLP data.
  • washing /binding buffer 1x Buffer W ( fresh prepared working buffer from StrepTactin Buffer Set)
  • Blocking Buffer for Immuno Blot 3% BSA in 1X PBS-0.1% Tween
  • Washing of column wash the column 5 times with 5ml 1x Buffer W (1x column volume) and let the buffer flow through
  • the RBD of the SARS-CoV-2 omikron variant with N-terminal signal peptide and C- terminal His-tag (SEQ ID NO: 21) can be expressed and purified analogously as described in Production Example 1.
  • the coding sequence is given in (SEQ ID NO: 22).
  • the RBD of SARS-CoV-2 beta variant with N-terminal signal peptide and C-terminal His-tag can also be expressed and purified analogously as described in Production Example 1.
  • Example 1 Functionality of the RBD-His protein in an ELISA assay
  • Ni-NTA Hissorb ELISA plates (Qiagen, # 35061) were coated with 100 pl/ well serially diluted standard protein (5-5000 ng/ml RBD-His, Sino Biological, # 40592-V08B) or test samples (purified RBD-His protein produced in CHO-cells according to the present invention or CHO supernatant) in PBS for 1 h.
  • the coated plates were washed three times with PBST (PBS, 0.1 % Tween-20), blocked with 100 pl/ well of blocking solution (PBST, 3% milk powder) for 1 h and washed again.
  • PBST PBS, 0.1 % Tween-20
  • Elisa plates were incubated with anti-SARS-CoV-2 Spike Glycoprotein S1 antibody CR3022 (abeam, # ab273073) diluted 1 :2000 in PBST + 1 %BSA for 1 h. After washing with PBST, the ELISA plates were incubated for 1 h with 100 pl/well of the anti-human IgG detection antibody labelled with POD (Dianova, # 109-035-088, 1 :10000 dilution in PBST + 1 % BSA). After washing, bound POD was detected by incubation with 100 pl/ well of TMB substrate (Thermo Scientific, #34029) until a maximal optical density (OD) of about 1 to 2 was reached. Finally, the colorimetric reaction was stopped with 100 pl/well stopping solution (1M H2SO4) and the OD determined at a wavelength of 450 nm with a reference wavelength of 595 nm in a plate reader.
  • 1M H2SO4 100 pl/well stopping solution
  • Example 2 Functionality of the NP-Strep protein in an ELISA assay
  • NP-Strep protein produced in CHO-cells according to the present invention or respective CHO supernatant was analysed by SARS-CoV-2 Nucleoprotein I NP Elisa Kit (Sino Biological, # KIT40588). The results demonstrated that the NP-strep protein produced in CHO-cells according to the present invention is functional in ELISA assays.
  • the detection method is a two-step incubation antigen “sandwich” ELISA using the Receptor Binding Domain (RBD) of the Spike Protein (S1) of the SARS-CoV-2 virus. All procedures were performed at room temperature (RT) and incubations were done on a microtiter plate shaker. ELISA plates were coated with 60 pl/ well RBD-His protein (final concentration 0,45 pg/ml) in coating solution (Candor, #121125) for 1 h. The coated plates were washed three times with PBST (PBS, 0.1 % Tween-20), blocked with 100 pl/well of blocking solution (PBST, 3 % milk powder) for 1 h, and washed again.
  • PBST PBS, 0.1 % Tween-20
  • test serum samples were diluted with 30pl PBS and transferred to the blocked ELISA plates and incubated for 1h. After washing three times with PBST, the ELISA plates were incubated for 1 h with 60 pl/well of biotinylated RBD-His (Bio-RBD-His, final concentration 0.02 pg/ml, diluted in PBST). The plates were washed three times with PBST and incubated with Strep- POD (Jackson Immunoresearch, #016-030-084) diluted in PBST 1 :50000 for 1h.
  • biotinylated RBD-His Bio-RBD-His, final concentration 0.02 pg/ml, diluted in PBST.
  • Strep- POD Jackson Immunoresearch, #016-030-084
  • Example 4 Measurement of serum samples by ELISA using a secondary anti-IgG antibody All procedures were performed at room temperature (RT) and incubations were on a microtiter plate shaker. Ni-NTA Hissorb ELISA plates (Qiagen, # 35061) were coated with 100 pl/ well serially diluted standard protein (5-5000 ng/ml RBD-His, Sino Biological, # 40592-V08B) as an internal control or with purified RBD-His protein from CHO-cells according to the present invention in PBS for 1 h.
  • the coated plates were washed three times with PBST (PBS, 0.1 % Tween-20), blocked with 100 pl/ well of blocking solution (PBST, 3% milk powder) for 1 h, and washed again.
  • Elisa plates were incubated with anti- SARS-CoV-2 Spike Glycoprotein S1 antibody CR3022 (abeam, # ab273073) diluted 1:2000 in PBST + 1%BSA as internal control, or with 30pl test serum samples (diluted with 30pl PBS) for 1h.
  • the test serum samples originate from patients that have been tested for SARS-CoV-2 by a RT-PCR test from a throat swab.
  • the ELISA plates were incubated for 1 h with 100 pl/well of the anti-human IgG detection antibody labelled with POD (Dianova, # 109-035-088, 1:10000 dilution in PBST + 1% BSA). After washing, bound POD was detected by incubation with 100 pl/ well of TMB substrate (Thermo Scientific, #34029) until a maximal optical density (OD) of about 1 to 2 was reached. Finally, the colorimetric reaction was stopped with 100 pl/well stopping solution (1M H2SO4) and the OD determined at a wavelength of 450 nm with a reference wavelength of 595 nm within a plate reader.
  • POD maximal optical density
  • test serum samples from the patients tested were also analyzed by a commercially available test kit from Mikrogen® and compared with the assay applying the purified RBD-His protein from CHO-cells according to the present invention.
  • the results are shown in Figure 7 and Figure 8.
  • the assay using the purified RBD-His protein from CHO-cells according to the present invention is less likely to produce false positive results for patient samples that have been tested negative for SARS-CoV-2 by a RT-PCR test. This can be seen for example from patient samples 1 , 3, 12, 13 and I - 24.
  • SEQ ID NO: 1 RBD with N-terminal signal peptide, without C-terminal His-taq METPAQLLFLLLLWLPDTTGRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISN CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADY NYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG VEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGPKKSTNLVKNKCVNF
  • SEQ ID NO: 2 Nucleotide sequence encoding SEQ ID NO: 1 without stop codon ATGGAAACACCAGCTCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGATACCACC GGAAGAGTGCAGCCTACCGAGTCCATCGTGCGGTTCCCCAACATCACCAACCTGTGT CCTTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGG AAGCGGATCTCTAACTGCGTGGCCGACTACTCCGTGCTGTACAACTCCGCCTCCTTCA GCACCTTCAAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACCAA CGTGTACGCCGACTCCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCTCCTGG ACAGACCGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTG TGATCGCTTGGAACTCCAACAACCTGGACTCCAAAGTCGGCGGCAACTACAATTAC CTGTACCGGCTGTTCCT
  • SEQ ID NO: 3 RBD with N-terminal signal peptide and with C-terminal His-taq METPAQLLFLLLLWLPDTTGRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISN CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADY NYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFH HHHHH
  • SEQ ID NO: 4 Nucleotide sequence encoding encoding SEQ ID NO: 3 with stop codon ATGGAAACACCAGCTCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGATACCACC GGAAGAGTGCAGCCTACCGAGTCCATCGTGCGGTTCCCCAACATCACCAACCTGTGT CCTTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGG AAGCGGATCTCTAACTGCGTGGCCGACTACTCCGTGCTGTACAACTCCGCCTCCTTCA GCACCTTCAAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACCAA CGTGTACGCCGACTCCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCTCCTGG ACAGACCGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTG TGATCGCTTGGAACTCCAACAACCTGGACTCCAAAGTCGGCGGCAACTACAATTAC
  • SEQ ID NO: 5 Nucleocapsid (NP) with N-terminal signal peptide, without Strep-tag
  • SEQ ID NO: 6 Nucleotide seguence encoding SEQ ID NO: 5
  • SEQ ID NO: 7 Nucleocapsid (NP) with N-terminal signal peptide and C-terminal Strep-tag (NP-Strep) METPAQLLFLLLLWLPDTTGMSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQ
  • SEQ ID NO: 8 Nucleotide sequence encoding SEQ ID NO: 7
  • SEQ ID NO: 9 amino acid sequence of the spike protein S1 of human coronavirus 0043
  • SEQ ID NO: 10 coding sequence of SEQ ID NO: 9, shown in Fig. 9.
  • SEQ ID NO: 11 amino acid sequence of the spike protein S1 of human coronavirus HKLI1
  • SEQ ID NO: 12 coding sequence of SEQ ID NO: 11 , shown in Fig. 10
  • SEQ ID NO: 13 amino acid sequence of the spike protein S1 of human coronavirus NL63
  • SEQ ID NO: 14 coding sequence of SEQ ID NO: 13, shown in Fig. 11.
  • SEQ ID NO: 15 amino acid sequence of the spike protein S1 of human coronavirus 229E (229ES1) as shown in Fig. 12
  • SEQ ID NO: 17 amino acid sequence of SARS-CoV-2 spike Sil as shown in Fig.13.
  • SEQ ID NO: 18 coding sequence of SEQ ID NO: 17, shown in Fig.13.
  • SEQ ID NO: 19 signal peptide METPAQLLFLLLLWLPDTTG
  • SEQ ID NO: 20 RBD of Sars-CoV-2 beta variant with N-terminal signal peptide and with C- terminal His-tag (differences over Wuhan variant are shown in bold):
  • SEQ ID NO: 21 RBD of S1 protein of Sars-CoV-2 omikron variant (B1.1.529 (ZA 11/2021)) with N-terminal signal peptide and with C-terminal His-tag (differences over Wuhan variant are shown in bold):
  • SEQ ID NO: 22 coding sequence of SEQ ID NO: 21, shown in Fig.14.
  • SEQ ID NO: 24 coding sequence of SEQ ID NO: 23

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Abstract

L'invention concerne un procédé de production d'un antigène (protéine) utilisable dans un dosage d'anticorps contre le SARS-CoV-2. L'invention concerne en outre un dosage pour détecter des anticorps contre le SARS-CoV-2 après une infection d'un patient avec ce virus, pendant un COVID-19 en cours d'un patient, et/ou après un COVID-19 guéri d'un patient. L'invention concerne également un dosage pour estimer le temps ayant passé après une infection par le SARS-CoV-2 d'un patient avec COVID-19 en cours ou guéri.
PCT/EP2021/087597 2020-12-30 2021-12-23 Antigènes et dosages pour la détection d'anticorps contre le sars-cov-2 WO2022144317A1 (fr)

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WO2024067861A1 (fr) * 2022-09-29 2024-04-04 Suzhou Abogen Biosciences Co., Ltd. Vaccins d'acide nucléique multivalents pour coronavirus basés sur des séquences dérivées de souches de sars-cov-2 delta et omicron

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