Epitope of an antibody against a structural protein of SARS-CoV-2, an antibody reactive with said epitope, a method for detecting SARS-CoV-2 using said antibody, a SARS-CoV-2 detection kit containing said antibody, a method for detecting an anti-SARS-CoV-2 antibody containing a polypeptide of said epitope, an anti-SARS-CoV-2 antibody detection kit containing a polypeptide of said epitope, a SARS-CoV-2 vaccine containing a polypeptide of said epitope, and a therapeutic agent for SARS-CoV-2 infection containing said antibody

Abstract

To provide an antibody for detecting SARS-CoV-2, a method for detecting SARS-CoV-2 using the antibody, and a detection kit comprising the antibody, and also to provide an antibody for simultaneously detecting a human infectious coronavirus such as MERS-CoV or SARS-CoV in addition to SARS-CoV-2 that cause severe respiratory symptoms, a method for detecting SARS-CoV-2 and a human infectious coronavirus simultaneously using the antibody, and a detection kit comprising the antibody.SOLUTION: The present invention relates to a monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or the antigen-binding fragment thereof specifically reacts with a structural protein of SARS-CoV-2, and the structural protein of SARS-CoV-2 is N-protein, and a hapten that specifically reacts with an antibody reactive with a protein of SARS-CoV-2, wherein the protein of SARS-CoV-2 is N-protein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an epitope recognized by an antibody against a structural protein of SARS-CoV-2, an antibody that reacts with the epitope, a method for detecting SARS-CoV-2 using the antibody, a SARS-CoV-2 detection kit containing the antibody, a method for detecting an anti-SARS-CoV-2 antibody containing a polypeptide of the epitope, an anti-SARS-CoV-2 antibody detection kit containing a polypeptide of the epitope, a SARS-CoV-2 vaccine containing the polypeptide of the epitope, and a therapeutic drug for SARS-CoV-2 infection containing the antibody. [Background technology]

[0002] Coronaviruses are enveloped viruses with a single-stranded RNA genome and a nucleocapsid with helical symmetry. Coronaviruses that infect humans include betacoronaviruses, among which the MERS coronavirus (MERS-CoV) and SARS coronavirus (SARS-CoV) are known to cause severe respiratory symptoms.

[0003] The novel coronavirus (SARS-CoV-2) emerged in China in 2019, and as of February 2020, the infection continues to spread primarily in East Asia, Southeast Asia, the Middle East, and Europe. Therefore, in addition to developing treatments and vaccines for SARS-CoV-2, there is an urgent need to develop a rapid and simple detection method for SARS-CoV-2 in order to detect it early and prevent its spread.

[0004] Since the emergence of SARS-CoV-2, reports have been published on the antigenicity and candidate epitopes of SARS-CoV-2 (e.g., Non-Patent Document 1), but these candidate epitopes are aimed at the development of a vaccine against SARS-CoV-2, and epitopes suitable for detecting SARS-CoV-2 and antibodies that recognize these epitopes have not yet been reported. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Suresh Kumar, Preprints, doi:https: / / www.preprints.org / manuscript / 202002.0071 / v1 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide an antibody that detects SARS-CoV-2, a method for detecting SARS-CoV-2 using the antibody, and a detection kit containing the antibody.The present invention also aims to provide an antibody that simultaneously detects SARS-CoV-2 as well as human infectious coronaviruses that cause severe respiratory symptoms, such as MERS-CoV and SARS-CoV, a method for simultaneously detecting SARS-CoV-2 and human infectious coronaviruses using the antibody, and a detection kit containing the antibody. [Means for solving the problem]

[0007] Several algorithms for predicting antigenicity have been reported (e.g., Chou PY, Fasman GD. 1978. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol 47:45-148). However, because these algorithms contain contradictory indicators, the accuracy of antigenicity prediction by each algorithm is poor. The inventors have integrated these algorithms to improve the accuracy of antigenicity prediction. Based on the antigenicity strength scores (Figures 1-4) obtained using this improved algorithm for the entire structural protein molecule constituting SARS-CoV-2, they discovered that a region containing a specific amino acid sequence in the structural protein constituting SARS-CoV-2 has strong viral antigenicity, and that this specific amino acid sequence is useful as an epitope (polypeptide) recognized by antibodies that detect SARS-CoV-2, thereby completing the present invention.

[0008] That is, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof that specifically reacts with a structural protein of SARS-CoV-2, and a SARS-CoV-2 detection kit comprising the antibody, wherein the structural protein of SARS-CoV-2 is at least one protein selected from the group consisting of S-protein, N-protein, M-protein, and E-protein.

[0009] The present invention also provides a method for detecting SARS-CoV-2 in a sample, which comprises contacting the sample with a monoclonal antibody or an antigen-binding fragment thereof that specifically reacts with a structural protein of SARS-CoV-2 and detecting SARS-CoV-2 by immunoassay. The method for detecting SARS-CoV-2 in a sample is preferably a sandwich method using at least two monoclonal antibodies or antigen-binding fragments thereof for each structural protein.

[0010] The present invention also provides a monoclonal antibody or an antigen-binding fragment thereof that specifically reacts with a structural protein of SARS-CoV-2 and a structural protein of a human infectious coronavirus other than SARS-CoV-2 (preferably, MERS-CoV and / or SARS-CoV), and a detection kit for SARS-CoV-2 and a human infectious coronavirus other than SARS-CoV-2 (preferably, MERS-CoV and / or SARS-CoV), comprising the antibody. The SARS-CoV-2 structural protein is at least one protein selected from the group consisting of S-protein, N-protein, M-protein, and E-protein, and the structural protein of the human infectious coronavirus other than SARS-CoV-2 is at least one protein selected from the group consisting of S-protein, N-protein, M-protein, and E-protein.

[0011] The present invention also provides an epitope (polypeptide) recognized by an antibody that detects SARS-CoV-2. Furthermore, the present invention also provides a method for detecting an anti-SARS-CoV-2 antibody comprising the polypeptide of the epitope, an anti-SARS-CoV-2 antibody detection kit comprising the polypeptide of the epitope, a SARS-CoV-2 vaccine comprising the polypeptide of the epitope, and a therapeutic agent for SARS-CoV-2 infection comprising the antibody. [Effects of the Invention]

[0012] The present invention provides an antibody that detects SARS-CoV-2, a method for detecting SARS-CoV-2 using the antibody, and a detection kit containing the antibody. The present invention also provides an antibody that simultaneously detects SARS-CoV-2 as well as coronaviruses that cause severe respiratory symptoms, such as MERS-CoV and SARS-CoV, and a detection kit containing the antibody. Furthermore, the present invention provides an epitope (polypeptide) recognized by the antibody that detects SARS-CoV-2. [Brief explanation of the drawings]

[0013] [Figure 1] This is a graph scoring the antigenic strength of the entire S-protein molecule of SARS-CoV-2. [Figure 2] This is a graph scoring the antigenic strength of the entire N-protein molecule of SARS-CoV-2. [Figure 3] This is a graph scoring the antigenic strength of the entire SARS-CoV-2 M-protein molecule. [Figure 4] This is a graph scoring the antigenic strength of the entire SARS-CoV-2 E protein molecule. [Figure 5a] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO:5 when immunized with the polypeptide. [Figure 5b] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO:6 when immunized with the polypeptide. [Figure 5c] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO: 7 when immunized with the polypeptide. [Figure 5d] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO:8 when immunized with the polypeptide. [Figure 5e] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO:9 when immunized with the polypeptide. [Figure 5f] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO: 10 when immunized with the polypeptide. [Figure 5g] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO: 11 when immunized with the polypeptide. [Figure 5h] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO: 13 when immunized with the polypeptide. [Figure 5i]1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO: 15 when immunized with the polypeptide. [Figure 5j] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO: 16 when immunized with the polypeptide. [Figure 5k] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO: 18 when immunized with the polypeptide. [Figure 5l] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO: 19 when immunized with the polypeptide. [Figure 5m] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO: 20 when immunized with the polypeptide. [Figure 5n] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO: 21 when immunized with the polypeptide. [Figure 5o] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO: 23 when immunized with the polypeptide. [Figure 5p] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO: 24 when immunized with the polypeptide. [Figure 5q] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO: 25 when immunized with the polypeptide. [Figure 5r] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO: 26 when immunized with the polypeptide. [Figure 5s] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO: 27 when immunized with the polypeptide. [Figure 5t] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO: 30 when immunized with the polypeptide. [Figure 5u]1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO: 31 when immunized with the polypeptide. [Figure 5v] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO: 32 when immunized with the polypeptide. [Figure 5w] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO: 33 when immunized with the polypeptide. [Figure 5x] 1 is a graph showing the specific reactivity to a polypeptide having the sequence of SEQ ID NO: 34 when immunized with the polypeptide. [Figure 6] 1 is a graph showing the reactivity of a total of 24 types of antibodies obtained in Example 1 to inactivated SARS-CoV-2 antigens. [Figure 7] This is a graph showing the results of analyzing the epitopes of antibodies present in antisera obtained by immunization with SARS-CoV-2 inactivated antigen. [Figure 8] 1 is a graph showing the results of analyzing the epitopes of antibodies present in the antiserum obtained by immunization with the recombinant N protein (full-length) shown in SEQ ID NO:2. [Figure 9] 1 is a graph showing the reactivity of antibodies in the antisera obtained in Examples 3 and 4 to the N protein epitope sequences of SEQ ID NOs: 17 to 25. DETAILED DESCRIPTION OF THE INVENTION

[0014] [Antibody against SARS-CoV-2, method for detecting SARS-CoV-2 using said antibody, and detection kit containing said antibody] SARS-CoV-2 is a virus of the betacoronavirus genus, composed mainly of the S protein (surface protein), N protein (nucleocapsid protein), M protein (membrane protein), and E protein (envelope protein). The monoclonal antibody of this embodiment specifically reacts with at least one protein selected from the group consisting of the major structural proteins that make up SARS-CoV-2, i.e., the S protein, N protein, M protein, and E protein.

[0015] In this embodiment, "specifically" means that in a liquid system in which a protein and the monoclonal antibody of this embodiment are mixed, the antibody does not undergo a detectable antigen-antibody reaction with protein components other than SARS-CoV-2 proteins, or even if it does undergo some kind of binding or association reaction, the reaction is clearly weaker than the antigen-antibody reaction of the antibody with SARS-CoV-2 proteins.

[0016] The S-protein (surface protein; hereinafter also referred to simply as "S-protein" in this specification) of SARS-CoV-2 typically consists of a 1273 amino acid sequence represented by SEQ ID NO: 1. The S-protein may be a protein consisting of the amino acid sequence of SEQ ID NO: 1, or may be a protein having 90% or more (preferably 95% or more) sequence identity with SEQ ID NO: 1.

[0017] In this embodiment, the monoclonal antibody that specifically reacts with the S-protein of SARS-CoV-2 binds to the amino acid region of the S-protein at positions 66 to 85 (SEQ ID NO: 5), 87 to 107 (SEQ ID NO: 6), 105 to 131 (SEQ ID NO: 7), 140 to 157 (SEQ ID NO: 8), 155 to 170 (SEQ ID NO: 9), 522 to 541 (SEQ ID NO: 10), 538 to 562 (SEQ ID NO: 11), 595 to 611 (SEQ ID NO: 12), 670 to 693 (SEQ ID NO: 13), 761 to 797 (SEQ ID NO: 14), 802 to 818 (SEQ ID NO: 15), or 1235 to 1273 (SEQ ID NO: 16). These amino acid regions are polypeptides consisting of at least 16 amino acid sequences whose antigenicity scores are higher than the threshold score in Figure 1, which scores the antigenicity strength of the entire S-protein molecule.

[0018] According to this embodiment, the monoclonal antibody that specifically reacts with the S-protein of SARS-CoV-2 preferably binds to the amino acid regions of the S-protein at positions 71-83, 89-103, 107-127, 143-154, 158-167, 524-538, 546-560, 598-608, 672-691, 772-796, 806-816, or 1238-1269. These amino acid regions are polypeptides consisting of a sequence of at least 10 amino acids, and have antigenicity scores higher than a threshold score in Figure 1, which scores the antigenicity of the entire S-protein molecule.

[0019] The monoclonal antibody according to this embodiment that specifically reacts with S-protein more preferably binds to the amino acid regions of S-protein at positions 73 to 79, 93 to 99, 109 to 115, 145 to 152, 160 to 165, 526 to 531, 553 to 558, 599 to 607, 674 to 684, 773 to 795, 808 to 814, or 1257 to 1261. These amino acid regions are polypeptides consisting of a sequence of at least five amino acids that have the highest antigenicity score compared to a threshold score in Figure 1, which scores the antigenicity of the entire S-protein molecule.

[0020] The N protein (nucleocapsid protein; hereinafter also referred to simply as "N protein" in this specification) of SARS-CoV-2 consists of a 419 amino acid sequence represented by SEQ ID NO:2.

[0021] According to this embodiment, the monoclonal antibody that specifically reacts with the N-protein binds to the amino acid regions of positions 1 to 18 (SEQ ID NO: 17), 19 to 49 (SEQ ID NO: 18), 174 to 207 (SEQ ID NO: 19), 230 to 252 (SEQ ID NO: 20), 247 to 267 (SEQ ID NO: 21), 336 to 350 (SEQ ID NO: 22), 362 to 379 (SEQ ID NO: 23), 377 to 395 (SEQ ID NO: 24), or 401 to 419 (SEQ ID NO: 25) of the N-protein. These amino acid regions are polypeptides consisting of an amino acid sequence of at least 15 sequences, and have an antigenicity score higher than a threshold score in Figure 2, which scores the antigenicity of the entire N-protein molecule.

[0022] According to this embodiment, the monoclonal antibody that specifically reacts with the N-protein preferably binds to the amino acid region of the N-protein at positions 1 to 12, 20 to 48, 175 to 206, 234 to 250, 254 to 263, 338 to 348, 366 to 377, 381 to 391, or 403 to 417. These amino acid regions are polypeptides consisting of a sequence of at least 10 amino acids, and have an antigenicity score higher than a threshold score in Figure 2, which scores the antigenicity of the entire N-protein molecule.

[0023] The monoclonal antibody according to this embodiment that specifically reacts with the N-protein more preferably binds to the amino acid regions of the N-protein at positions 4 to 11, 27 to 41, 185 to 196, 237 to 247, 256 to 261, 340 to 347, 367 to 375, 384 to 390, or 406 to 415. These amino acid regions are polypeptides consisting of a sequence of at least six amino acids that have the highest antigenicity score compared to a threshold score in Figure 2, which scores the antigenicity of the entire N-protein molecule.

[0024] The M-protein (membrane protein; hereinafter also referred to simply as "M-protein" in this specification) of SARS-CoV-2 consists of a 222 amino acid sequence represented by SEQ ID NO:3.

[0025] According to this embodiment, the monoclonal antibody that specifically reacts with M-protein binds to the amino acid regions of M-protein at positions 1 to 24 (SEQ ID NO: 26), 33 to 50 (SEQ ID NO: 27), 101 to 120 (SEQ ID NO: 28), 121 to 139 (SEQ ID NO: 29), 146 to 171 (SEQ ID NO: 30), 169 to 195 (SEQ ID NO: 31), or 193 to 222 (SEQ ID NO: 32). These amino acid regions are polypeptides consisting of a sequence of at least 18 amino acids, and have an antigenicity score higher than a threshold score in Figure 3, which scores the antigenicity of the entire M-protein molecule.

[0026] According to this embodiment, the monoclonal antibody that specifically reacts with M-protein preferably binds to the amino acid region of M-protein at positions 1 to 19, 38 to 45, 103 to 118, 122 to 138, 155 to 168, 172 to 195, or 196 to 216. These amino acid regions are polypeptides consisting of a sequence of at least eight amino acids, and have antigenicity scores higher than a threshold score in Figure 3, which scores the antigenicity of the entire M-protein molecule.

[0027] The monoclonal antibody according to this embodiment that specifically reacts with M-protein more preferably binds to the amino acid regions of M-protein at positions 1 to 16, 40 to 44, 105 to 117, 123 to 136, 160 to 167, 173 to 179, or 205 to 215. These amino acid regions are polypeptides consisting of an amino acid sequence of at least 5 sequences, and have the highest antigenicity score compared to a threshold score in Figure 3, which scores the antigenicity of the entire M-protein molecule.

[0028] The E protein (envelope protein; hereinafter also referred to simply as "E protein" in this specification) of SARS-CoV-2 consists of a 75 amino acid sequence represented by SEQ ID NO:4.

[0029] According to this embodiment, the monoclonal antibody that specifically reacts with E-protein binds to the amino acid region of E-protein from positions 1 to 19 (SEQ ID NO: 33) or from positions 50 to 75 (SEQ ID NO: 34). These amino acid regions are polypeptides consisting of a sequence of at least 19 amino acids, and have an antigenicity score higher than the threshold score in Figure 4, which scores the antigenicity of the entire E-protein molecule.

[0030] The monoclonal antibody according to this embodiment that specifically reacts with E-protein preferably binds to the region of amino acids 4 to 13 or 51 to 72 of E-protein. These amino acid regions are polypeptides consisting of a sequence of at least 10 amino acids, and have an antigenicity score higher than a threshold score in Figure 4, which scores the antigenicity of the entire E-protein molecule.

[0031] The monoclonal antibody according to this embodiment that specifically reacts with E-protein more preferably binds to the region of amino acids 5 to 10 or 60 to 71 of E-protein. These amino acid regions are polypeptides consisting of a sequence of at least six amino acids that have the highest antigenicity score compared to a threshold score in Figure 4, which scores the antigenicity of the entire E-protein molecule.

[0032] The antigen-binding site can also be isolated and used based on the monoclonal antibody of this embodiment. In other words, fragments (antigen-binding fragments) having specific antigen-binding ability, such as Fab, Fab', F(ab')2, single-chain antibody (scFv), and VHH, produced by known methods, are included within the scope of the present invention. Furthermore, the class of the monoclonal antibody of this embodiment is not limited to IgG, but may also be IgY, IgM, camel Ig, or Ig NAR. In this specification, the term "antibody" refers to an "antibody or an antigen-binding fragment thereof," unless otherwise clear from the context.

[0033] The monoclonal antibody of this embodiment can be obtained by using known immunological techniques to immunize an animal with a SARS-CoV-2 protein or a partial peptide thereof (for example, a polypeptide consisting of a specific amino acid region in the above-mentioned S-protein, N-protein, M-protein, or E-protein), and then producing a hybridoma using cells from the immunized animal, or by producing a recombinant antibody using genetic engineering techniques. The length of the peptide used for immunization is not particularly limited, but a peptide of preferably 5 or more amino acids, more preferably 10 or more amino acids, and even more preferably 13 or more amino acids can be used as an immunogen.

[0034] The SARS-CoV-2 protein used as an immunogen can be obtained from cultured virus fluid, but it can also be obtained by incorporating DNA encoding the SARS-CoV-2 protein into a plasmid vector and introducing it into host cells for expression.

[0035] The SARS-CoV-2 protein or its partial peptide to be used as an immunogen can be expressed as a fusion protein with the following proteins and used as an immunogen either after purification or unpurified. Fusion proteins can be produced using tags commonly used by those skilled in the art as "protein expression and purification tags," such as glutathione S-transferase (GST), maltose-binding protein (MBP), thioredoxin (TRX), Nus tag, S tag, HSV tag, FLAG tag, polyhistidine tag, Strep tag, Strep-II tag, Myc tag, HA tag, V5 tag, E tag, T7 tag, VSV-G tag, Glu-Glu tag, and Avi tag. Fusion proteins with these tags are preferably cleaved using digestive enzymes to separate the SARS-CoV-2 protein or its partial peptide from the remaining tag, followed by separation and purification before use as an immunogen.

[0036] Monoclonal antibodies can be easily prepared from immunized animals using the well-known method of Kohler et al. (Kohler et al., Nature, vol. 256, pp. 495-497 (1975)). Specifically, antibody-producing cells such as splenocytes and lymphocytes are collected from the immunized animal and fused with mouse myeloma cells by standard methods to produce hybridomas. The resulting hybridomas are then cloned by limiting dilution or other methods, and monoclonal antibodies that undergo an antigen-antibody reaction with SARS-CoV-2 proteins are selected from the monoclonal antibodies produced by each cloned hybridoma.

[0037] Monoclonal antibodies can be purified from ascites or culture supernatant using known immunoglobulin purification methods. Examples include fractionation by salting out using ammonium sulfate or sodium sulfate, PEG fractionation, ethanol fractionation, DEAE ion exchange chromatography, and gel filtration. Purification can also be performed by affinity chromatography using a carrier bound to Protein A, Protein G, or Protein L, depending on the immunized animal species and the class of the monoclonal antibody.

[0038] The method for detecting SARS-CoV-2 according to this embodiment includes contacting a sample with a monoclonal antibody or an antigen-binding fragment thereof that specifically reacts with at least one protein selected from the group consisting of SARS-CoV-2 proteins, i.e., S-protein, N-protein, M-protein, and E-protein, and detecting SARS-CoV-2 by immunoassay.

[0039] Examples of specimens used in the detection method of this embodiment include human or animal body fluids such as blood, serum, plasma, urine, semen, cerebrospinal fluid, saliva, sweat, tears, ascites, or amniotic fluid; mucus; feces; organs such as blood vessels or the liver; tissues; cells; or extracts thereof, all of which may contain SARS-CoV-2 proteins. Preferred specimens are easily collected, such as cells and secretions from the oral cavity, tonsils, nasal cavity, pharynx, larynx, trachea, bronchi, or lungs, nasal swabs, pharyngeal swabs, gargle, sputum, tracheal aspirates, and bronchoalveolar lavage fluid. The method for collecting these specimens is not particularly limited, and known methods can be used. Specifically, a method using a cotton swab is one example.

[0040] The SARS-CoV-2 protein according to this embodiment can be measured by any immunoassay method known to those skilled in the art, including competitive assays, agglutination assays, Western blotting, immunostaining, and sandwich assays.

[0041] The method for measuring SARS-CoV-2 proteins according to this embodiment is preferably a sandwich method using at least two types of monoclonal antibodies. The sandwich method itself is well known in the field of immunoassays and can be performed, for example, by immunochromatography, ELISA, etc. The measurement method according to this embodiment can be performed using the well-known sandwich method, except for using the monoclonal antibodies that specifically react with the SARS-CoV-2 proteins described above.

[0042] In the sandwich method, when the SARS-CoV-2 protein to be measured is S-protein, it is preferable to use at least two types of monoclonal antibodies that can simultaneously bind to amino acid regions selected from the group consisting of: 66 to 85 (SEQ ID NO: 5), 87 to 107 (SEQ ID NO: 6), 105 to 131 (SEQ ID NO: 7), 140 to 157 (SEQ ID NO: 8), 155 to 170 (SEQ ID NO: 9), 522 to 541 (SEQ ID NO: 10), 538 to 562 (SEQ ID NO: 11), 595 to 611 (SEQ ID NO: 12), 670 to 693 (SEQ ID NO: 13), 761 to 797 (SEQ ID NO: 14), 802 to 818 (SEQ ID NO: 15), and 1235 to 1273 (SEQ ID NO: 16).

[0043] In the sandwich method, when the SARS-CoV-2 protein to be measured is the N-protein, it is preferable to use at least two types of monoclonal antibodies that can simultaneously bind to amino acid regions selected from the group consisting of 1 to 18 (SEQ ID NO: 17), 19 to 49 (SEQ ID NO: 18), 174 to 207 (SEQ ID NO: 19), 230 to 252 (SEQ ID NO: 20), 247 to 267 (SEQ ID NO: 21), 336 to 350 (SEQ ID NO: 22), 362 to 379 (SEQ ID NO: 23), 377 to 395 (SEQ ID NO: 24), and 401 to 419 (SEQ ID NO: 25).

[0044] In the sandwich method, when the SARS-CoV-2 protein to be measured is M-protein, it is preferable to use at least two types of monoclonal antibodies that can simultaneously bind to amino acid regions selected from the group consisting of 1 to 24 (SEQ ID NO: 26), 33 to 50 (SEQ ID NO: 27), 101 to 120 (SEQ ID NO: 28), 121 to 139 (SEQ ID NO: 29), 146 to 171 (SEQ ID NO: 30), 169 to 195 (SEQ ID NO: 31), and 193 to 222 (SEQ ID NO: 32).

[0045] In the sandwich method, when the SARS-CoV-2 protein to be measured is E-protein, it is preferable to use two types of monoclonal antibodies that can simultaneously bind to the amino acid regions 1 to 19 (SEQ ID NO: 33) and 50 to 75 (SEQ ID NO: 34).

[0046] The SARS-CoV-2 detection kit of this embodiment comprises a monoclonal antibody or an antigen-binding fragment thereof that specifically reacts with at least one protein selected from the group consisting of SARS-CoV-2 proteins, i.e., S-protein, N-protein, M-protein, and E-protein.

[0047] The SARS-CoV-2 detection kit of this embodiment can be in any form that is used in the above-mentioned immunological assay method, but preferably is in the form of an immunochromatographic strip that can accurately, quickly, and easily measure SARS-CoV-2 proteins.

[0048] A preferred embodiment of the SARS-CoV-2 detection kit according to this embodiment comprises at least two types of monoclonal antibodies or antigen-binding fragments thereof that can simultaneously bind to amino acid regions selected from the group consisting of amino acid regions 66 to 85 (SEQ ID NO: 5), 87 to 107 (SEQ ID NO: 6), 105 to 131 (SEQ ID NO: 7), 140 to 157 (SEQ ID NO: 8), 155 to 170 (SEQ ID NO: 9), 522 to 541 (SEQ ID NO: 10), 538 to 562 (SEQ ID NO: 11), 595 to 611 (SEQ ID NO: 12), 670 to 693 (SEQ ID NO: 13), 761 to 797 (SEQ ID NO: 14), 802 to 818 (SEQ ID NO: 15), and 1235 to 1273 (SEQ ID NO: 16) of the S-protein.

[0049] A preferred embodiment of the SARS-CoV-2 detection kit according to this embodiment comprises at least two types of monoclonal antibodies or antigen-binding fragments thereof that can simultaneously bind to amino acid regions selected from the group consisting of amino acid regions 1 to 18 (SEQ ID NO: 17), 19 to 49 (SEQ ID NO: 18), 174 to 207 (SEQ ID NO: 19), 230 to 252 (SEQ ID NO: 20), 247 to 267 (SEQ ID NO: 21), 336 to 350 (SEQ ID NO: 22), 362 to 379 (SEQ ID NO: 23), 377 to 395 (SEQ ID NO: 24), and 401 to 419 (SEQ ID NO: 25) of the N protein.

[0050] A preferred embodiment of the SARS-CoV-2 detection kit according to this embodiment comprises at least two types of monoclonal antibodies or antigen-binding fragments thereof that can simultaneously bind to amino acid regions selected from the group consisting of amino acid regions 1 to 24 (SEQ ID NO: 26), 33 to 50 (SEQ ID NO: 27), 101 to 120 (SEQ ID NO: 28), 121 to 139 (SEQ ID NO: 29), 146 to 171 (SEQ ID NO: 30), 169 to 195 (SEQ ID NO: 31), and 193 to 222 (SEQ ID NO: 32) of the M-protein.

[0051] A preferred embodiment of the SARS-CoV-2 detection kit of this embodiment comprises two types of monoclonal antibodies or antigen-binding fragments thereof that can simultaneously bind to the amino acid regions 1 to 19 (SEQ ID NO: 33) and 50 to 75 (SEQ ID NO: 34) of the E protein.

[0052] [Antibodies against SARS-CoV-2 and human infectious coronaviruses other than SARS-CoV-2; a method for detecting SARS-CoV-2 and human infectious coronaviruses other than SARS-CoV-2 using the antibodies; and a detection kit containing the antibodies] The monoclonal antibody of this embodiment specifically reacts with at least one protein selected from the group consisting of structural proteins of SARS-CoV-2, i.e., S-protein (surface protein), N-protein (nucleocapsid protein), M-protein (membrane protein), and E-protein (envelope protein) of SARS-CoV-2, and structural proteins of human infectious coronaviruses other than SARS-CoV-2, i.e., S-protein (surface protein), N-protein (nucleocapsid protein), M-protein (membrane protein), and E-protein (envelope protein) of human infectious coronaviruses other than SARS-CoV-2.

[0053] In this embodiment, "specifically" means that in a liquid system in which a protein and the monoclonal antibody of this embodiment are mixed, the antibody does not undergo a detectable antigen-antibody reaction with protein components other than SARS-CoV-2 proteins and proteins of human infectious coronaviruses other than SARS-CoV-2, or even if some binding reaction or association reaction occurs, the reaction is clearly weaker than the antigen-antibody reaction of the antibody with proteins other than SARS-CoV-2 proteins and proteins of human infectious coronaviruses other than SARS-CoV-2.

[0054] The human-infectious coronavirus other than SARS-CoV-2 is not particularly limited as long as it is a coronavirus that causes severe respiratory symptoms in humans, but is preferably MERS-CoV and / or SARS-CoV, and more preferably SARS-CoV.

[0055] The monoclonal antibody of this embodiment can be prepared by a method similar to that described above in the section "Antibody against SARS-CoV-2, method for detecting SARS-CoV-2 using the antibody, and detection kit containing the antibody."

[0056] The method for detecting SARS-CoV-2 and human infectious coronaviruses other than SARS-CoV-2 according to this embodiment includes contacting a sample with a monoclonal antibody or an antigen-binding fragment thereof that specifically reacts with a structural protein of SARS-CoV-2, i.e., at least one protein selected from the group consisting of the S-protein, N-protein, M-protein, and E-protein of SARS-CoV-2, and a structural protein of a human infectious coronavirus other than SARS-CoV-2, i.e., at least one protein selected from the group consisting of the S-protein, N-protein, M-protein, and E-protein of a human infectious coronavirus other than SARS-CoV-2, and detecting SARS-CoV-2 and human infectious coronaviruses other than SARS-CoV-2 by immunoassay.

[0057] The samples used in the method of this embodiment, as well as the measurement of SARS-CoV-2 proteins and proteins of human infectious coronaviruses other than SARS-CoV-2, are the same as those described above in the sections "Antibodies against SARS-CoV-2, methods for detecting SARS-CoV-2 using the antibodies, and detection kits containing the antibodies."

[0058] The detection kit for SARS-CoV-2 and human infectious coronaviruses other than SARS-CoV-2 according to this embodiment comprises a monoclonal antibody or an antigen-binding fragment thereof that specifically reacts with a structural protein of SARS-CoV-2, i.e., at least one protein selected from the group consisting of the S-protein, N-protein, M-protein, and E-protein of SARS-CoV-2, and a structural protein of a human infectious coronavirus other than SARS-CoV-2, i.e., at least one protein selected from the group consisting of the S-protein, N-protein, M-protein, and E-protein of a human infectious coronavirus other than SARS-CoV-2.

[0059] [Epitopes (polypeptides) recognized by antibodies that detect SARS-CoV-2] A polypeptide according to this embodiment, which consists of any one of the amino acid sequences of SEQ ID NOs: 5 to 34, can be used as an epitope recognized by an antibody that detects SARS-CoV-2 present in a sample. Furthermore, the polypeptide according to this embodiment can be used as an epitope recognized by an antibody that detects human infectious coronaviruses other than SARS-CoV-2 in addition to SARS-CoV-2 present in a sample.

[0060] The polypeptides of this embodiment can be used as haptens to detect antibodies against SARS-CoV-2 (i.e., anti-SARS-CoV-2 antibodies produced by the body's defense mechanism against SARS-CoV-2) present in a sample (preferably blood). Therefore, one embodiment of the present invention provides a method for detecting anti-SARS-CoV-2 antibodies in a sample, comprising contacting one or more polypeptides having the amino acid sequences of SEQ ID NOs: 5 to 34 with the sample (preferably blood) and detecting the anti-SARS-CoV-2 antibodies by immunoassay. Another embodiment of the present invention provides a kit for detecting anti-SARS-CoV-2 antibodies, comprising one or more polypeptides having the amino acid sequences of SEQ ID NOs: 5 to 34.

[0061] The polypeptides of this embodiment can be used as haptens to detect antibodies against SARS-CoV-2 and antibodies against human infectious coronaviruses other than SARS-CoV-2 (i.e., anti-human infectious coronavirus antibodies produced by the body's defense mechanism against human infectious coronaviruses other than SARS-CoV-2) present in a sample (preferably blood). Therefore, one embodiment of the present invention provides a method for detecting anti-SARS-CoV-2 antibodies and anti-human infectious coronavirus antibodies other than anti-SARS-CoV-2 antibodies in a sample, comprising contacting the sample (preferably blood) with one or more polypeptides having the amino acid sequences of SEQ ID NOs: 5 to 34 and detecting the anti-SARS-CoV-2 antibodies and anti-human infectious coronavirus antibodies other than anti-SARS-CoV-2 antibodies by immunoassay. Another embodiment of the present invention provides a kit for detecting anti-SARS-CoV-2 antibodies and anti-human infectious coronavirus antibodies other than anti-SARS-CoV-2 antibodies, comprising one or more polypeptides having the amino acid sequences of SEQ ID NOs: 5 to 34.

[0062] The polypeptide according to this embodiment can be used as a basic framework for vaccines (e.g., peptide vaccines, cocktail peptide vaccines, fusion peptides, or fragment domains containing the peptides) intended to prevent infection with SARS-CoV-2. Furthermore, the polypeptide according to this embodiment can be used as a basic framework for vaccines intended to prevent infection with human-infectious coronaviruses other than SARS-CoV-2, in addition to SARS-CoV-2.

[0063] The polypeptide according to this embodiment can be used as the basic framework of a vaccine (e.g., a peptide vaccine, a cocktail peptide vaccine, a fusion peptide, or a fragment domain containing the peptide) intended to prevent the aggravation of SARS-CoV-2 infection. Furthermore, the polypeptide according to this embodiment can be used as the basic framework of a vaccine intended to prevent the aggravation of infection with human-infectious coronaviruses other than SARS-CoV-2, in addition to SARS-CoV-2 infection.

[0064] A monoclonal antibody or antigen-binding fragment thereof that specifically reacts with the polypeptide of this embodiment can be used as an antibody drug (neutralizing antibody) for the treatment of SARS-CoV-2 infection. Furthermore, a monoclonal antibody or antigen-binding fragment thereof that specifically reacts with the polypeptide of this embodiment can be used as an antibody drug (neutralizing antibody) for the treatment of SARS-CoV-2 infection as well as infections with human coronaviruses other than SARS-CoV-2.

[0065] In each of the above-mentioned embodiments, the human-infectious coronavirus other than SARS-CoV-2 is not particularly limited as long as it is a coronavirus that causes severe respiratory symptoms in humans, but is preferably MERS-CoV and / or SARS-CoV, and more preferably SARS-CoV. [Example]

[0066] Example 1: Measurement of antibody titer Twenty-four polypeptides, each having the sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 13, 15, 16, 18, 19, 20, 21, 23, 24, 25, 26, 27, 30, 31, 32, 33, or 34, were individually covalently linked to Blue Carrier Protein (mollusk-derived hemocyanin) and BSA (bovine serum albumin) using the MBS method (Syed Salman Lateef, et al. J Biomol Tech. 2007 Jul;18(3):173-176.). Each Blue Carrier Protein-based peptide conjugate was administered intraperitoneally to three BALB / c mice at a dose of 100 μg / 200 μL / head. Sigma adjuvant system was used as the adjuvant. Blood samples were collected via the tail vein over time during the immunization period. The antibody titer was confirmed by peptide conjugate solid-phase ELISA (Enzyme-Linked Immunosorbent Assay) using BSA according to the following procedure. 1) A total of 24 types of BSA peptide conjugate solutions diluted with PBS to 2 μg / mL were added to a 96-well immunoplate at 100 μL / well and immobilized overnight at 4°C. 2) The BSA peptide conjugate solution was discarded, and the plate was washed twice with wash buffer (0.05% Tween 20 / PBS). Blocking was then performed by adding 250 μL / well of Blocking One (Nacalai Tesque) diluted 5-fold with MilliQ water and incubating at 25°C for approximately 1 hour. 3) The blocking solution was discarded, and the plate was washed twice with wash buffer. Mouse plasma diluted 100 to 100,000 times with wash buffer was added at 100 μL / well, and the plate was incubated at 25°C for 2 hours. 4) The plasma solution was discarded, and the plate was washed three times with wash buffer. Peroxidase-Conjugated Goat Anti-Mouse Immunoglobulins (DAKO) diluted 5,000-fold with wash buffer was added at 100 μL / well, and the plate was incubated at room temperature for 1 hour. 5) The antibody solution was discarded, and after washing three times with wash buffer, 100 μL / well of TMB solution (TMB One-Step Substrate System: manufactured by DAKO) was added. 6) After incubation at 25°C for 20 minutes, 100 μL / well of 2N H2SO4 was added to stop the reaction. 7) The absorbance at 450 nm / 630 nm was measured using a plate reader. The results are shown in Figures 5a to 5x. The error bar (SD) in the figures represents the inter-individual difference between immunized mice (n=3).

[0067] As shown in Figures 5a to 5x, it was confirmed that the antibody titers against the epitope peptides of the S-protein, N-protein, M-protein, and E-protein were increased.

[0068] Example 2: Antibody reactivity to inactivated SARS-CoV-2 antigen Using a microtiter plate immobilized with inactivated SARS-CoV-2 antigen (EPI_ISL_406034), the reactivity of a total of 24 antibodies obtained in Example 1 to the inactivated SARS-CoV-2 antigen was confirmed by ELISA (Enzyme-Linked Immunosorbent Assay) using the following procedure. 1) A total of 24 antibodies obtained in Example 1 were added at 100 μL / well to a 96-well immunoplate on which SARS-CoV-2 inactivated antigen had been immobilized, and the plate was incubated at 37°C for 1 hour. 2) The plasma solution was discarded, and the plate was washed twice with wash buffer (0.05% Tween 20 / PBS). Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (subclasses 1+2a+2b+3) and Fcγ Fragment Specific (Jackson ImmunoResearch) diluted 25,000-fold with dilution buffer (0.05% Tween 20, 0.5% BSA / PBS) were added at 100 μL / well, and the plate was incubated at 37°C for 1 hour. 3) The antibody solution was discarded, and after washing twice with wash buffer, 100 μL / well of TMB solution (TMB One-Step Substrate System: manufactured by DAKO) was added. 4) After incubation at 25°C for 20 minutes, 100 μL / well of 2N H2SO4 was added to stop the reaction. 5) The absorbance at 450 nm / 630 nm was measured using a plate reader. The results are shown in Figure 6. The error bar (1.96 × SD) in the figure represents the difference between measurements (n = 3).

[0069] As shown in Figure 6, all 24 types of antibodies obtained in Example 1 were confirmed to react with the inactivated SARS-CoV-2 antigen.

[0070] Example 3: Analysis of epitopes of antibodies present in antisera when SARS-CoV-2 inactivated antigen is used as an immunogen BALB / c mice were immunized using the inactivated SARS-CoV-2 antigen used in Example 2 as an immunogen in the same manner as in Example 1. The epitopes of the antibodies present in the obtained antisera were analyzed by the following procedure. 1) SARS-CoV-2 N-protein peptide set (BioTides) is a polypeptide consisting of 15 consecutive amino acids, in which the sequence of the SARS-CoV-2 N-protein (SEQ ID NO: 2) is shifted from the N-terminus to the C-terminus one amino acid at a time, and each polypeptide chain is biotin-labeled at the N-terminus and one glycine residue is added at the C-terminus of each polypeptide chain. TM Biotinylated Peptides (JPT Peptide Technologies GmbH) were used. 2) BioTides described in 1) above TM Biotinylated peptides were dissolved in DMF (N,N-dimethylformamide) and transferred to LumAvidin Microspheres (Liminex) via biotin-avidin reaction. TM Biotinylated peptide-immobilized LumAvidin microspheres were prepared according to the details of the Luminex xMAP (registered trademark) Cookbook (Chapter 4.2.1). 3) BALB / c mice were immunized with SARS-CoV-2 inactivated antigen, and the antiserum was diluted 100-fold with PBS-TBN (0.02% Tween 20, 0.1% BSA / PBS) to obtain 50 μL of the solution. TM Biotinylated peptide-immobilized LumAvidin microspheres were mixed with 500 beads of LumAvidin microspheres for each type of SARS-CoV-2 N protein peptide, and 50 μL of the beads mix was mixed and incubated at 37°C for 1 hour. 4) After centrifugation at 3000 rpm for 5 minutes, the supernatant of the reaction solution was discarded and the plate was washed twice with wash buffer (0.02% Tween 20 / PBS). 5) R-Phycoerythrin AffiniPure Goat Anti-Mouse IgG (subclasses 1+2a+2b+3) and Fcγ Fragment Specific (Jackson ImmunoResearch) were diluted 200-fold with PBS-TBN, and 100 μL of this solution was added to the beads mixed after the reaction in 4) above, followed by incubation at 37°C for 1 hour. 6) After centrifugation at 3000 rpm for 5 minutes, the supernatant of the reaction solution was discarded and the mixture was washed twice with wash buffer. 7) 50 μL of PBS-BN (1% BSA / PBS) was added to the bead mix after the reaction in 6) above to prepare a bead suspension. 8) The bead suspension prepared in 7) above was measured for MFI (mean fluorescence intensity) using Bio-Plex 200 (Bio-Rad). As a blank (reference control), measurements were also performed under the same conditions as in 3) above, except that the diluent PBS-TBN was used instead of the antiserum. The value obtained by subtracting the measurement value for the blank from the measurement value for the antiserum was expressed as ΔMFI(-Blank). The results are shown in Figure 7.

[0071] As shown in FIG. 7, it was confirmed that the antisera obtained in Example 3 had significant antibody titers against N-protein epitope sequences other than those of SEQ ID NOs: 21 and 22.

[0072] Example 4: Analysis of epitopes of antibodies present in antisera when recombinant N-protein is used as an immunogen BALB / c mice were immunized using the recombinant N-protein (full-length; SEQ ID NO: 2) as an immunogen using the same procedure as in Example 1. The epitopes of the antibodies present in the obtained antisera were analyzed using the same procedure as in Example 3. The results are shown in Figure 8.

[0073] As shown in FIG. 8, it was confirmed that the antisera obtained in Example 4 had significant antibody titers against all epitope sequences of the N-protein represented by SEQ ID NOs: 17 to 25.

[0074] Example 5: Reactivity of antibodies in antisera to epitope sequences of N-protein Using the antisera obtained in Examples 3 and 4, the reactivity of antibodies in the antisera to the epitope sequences of the N protein of SEQ ID NOs: 17 to 25 was confirmed by the following procedure. 1) The N-terminus of each polypeptide of SEQ ID NOs: 17 to 25 was biotinylated, dissolved in DMF (N,N-dimethylformamide), and then added to LumAvidin Microspheres (manufactured by Liminex) via a biotin-avidin reaction to prepare biotinylated epitope-peptides-immobilized LumAvidin Microspheres. Details of this preparation were in accordance with Luminex's xMAP (registered trademark) Cookbook (Chapter 4.2.1). 2) 50 μL of a solution prepared by diluting the antisera obtained in Examples 3 and 4 100-fold with PBS-TBN (0.02% Tween 20, 0.1% BSA / PBS) was mixed with 50 μL of a beads mix prepared above using the biotinylated epitope-peptides-immobilized LumAvidin microspheres (500 beads of LumAvidin microspheres for each polypeptide of SEQ ID NOs: 17 to 25), and the mixture was incubated at 37°C for 1 hour. 3) After centrifugation at 3000 rpm for 5 minutes, the supernatant of the reaction solution was discarded and the plate was washed twice with wash buffer (0.02% Tween 20 / PBS). 4) R-Phycoerythrin AffiniPure Goat Anti-Mouse IgG (subclasses 1+2a+2b+3) and Fcγ Fragment Specific (Jackson ImmunoResearch) were diluted 200-fold with PBS-TBN, and 100 μL of this solution was added to the beads mixed after the reaction in 3) above, followed by incubation at 37°C for 1 hour. 5) After centrifugation at 3000 rpm for 5 minutes, the supernatant of the reaction solution was discarded and the mixture was washed twice with wash buffer. 6) 50 μL of PBS-BN (1% BSA / PBS) was added to the bead mix after the reaction in 5) above to prepare a bead suspension. 7) The bead suspension prepared in 6) above was measured for MFI (mean fluorescence intensity) using a Bio-Plex 200 (Bio-Rad). As a blank (reference control), measurements were also performed under the same conditions as in 2) above, except that the diluent PBS-TBN was used instead of the antiserum. The results are shown in Figure 9. The error bar (SD) in the figure indicates the difference between measurements (n=3).

[0075] As shown in FIG. 9, it was confirmed that the antisera obtained in Examples 3 and 4 had significant antibody titers against all epitope sequences of the N-protein represented by SEQ ID NOs: 17 to 25.

Claims

1. A polypeptide having the amino acid sequence of any one of SEQ ID NOs: 18, 25, 17, 23, 24, 21, 19, 20 and 22.

2. A method for detecting an anti-SARS-CoV-2 antibody in a sample, comprising contacting the sample with the polypeptide of claim 1 and detecting the anti-SARS-CoV-2 antibody by immunoassay.

3. A kit for detecting an anti-SARS-CoV-2 antibody, comprising the polypeptide of claim 1.

4. A vaccine for SARS-CoV-2 comprising a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 18, 25, 23, 24, 21, 19, and 20.

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