CN112010963B - SARS-COV-2 antibody and use thereof - Google Patents

Abstract

The present invention relates to SARS-COV-2 antibody and its use. The present invention provides an isolated or non-naturally occurring SARS-CoV-2 monoclonal antibody comprising a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOS: 1-17 and a light chain variable region comprising an amino acid sequence selected from SEQ ID NOS: 18-26. The invention also relates to an isolated or non-naturally occurring monoclonal antibody comprising a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOS: 18-76. The antibodies of the invention may be used as therapeutic agents for treating COVID-19 or as diagnostic tools for assessing COVID-19 infection in a subject.

Description

SARS-COV-2 antibody and use thereof

Technical Field

The present invention relates to virus antibody and its use, and is especially SARS-CoV-2 monoclonal antibody and its medical use.

Background

Coronavirus diseases (Covid-19 ) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were reported in 2019. This virus was highly related to SARS-CoY in 2003. The virus mainly affects the lung, but also affects multiple organs such as kidney, liver, brain, gastrointestinal tract, etc. The virus is transmitted through respiratory droplets, urine and feces. Clinical symptoms of SARS-CoV-2 include fever, cough, shortness of breath, absence of other pathogens, and chest CT imaging examination of pneumonia characterized by bilateral vitreous opacities. Most patients with COVID-19 present with mild or moderate symptoms that recover after appropriate clinical care, while some patients with COVID-19 develop severe pneumonia and multiple organ failure rapidly.

At present, the immune response mechanism to host SARS-CoV-2 infection is not well understood. Lymphopenia is common in SARS-CoV-2 infected patients as well as in SARS-CoV patients and Middle East Respiratory Syndrome (MERS) patients. It was observed that in severe cases of COVID-19, the number of total T cells, CD4+ and CD8+ subtype T cells decreased dramatically, while programmed death ligand 1(PD-1) and T cell immunoglobulin mucin 3(Tim-3) increased, indicating that T cells had depleted. Some researchers have suggested monitoring lymphocyte proportion dynamics as an index for predicting the severity of COVID-19. Elderly patients with comid-19 are at higher risk of developing severe pneumonia and death. Defects in T cell and B cell function in elderly patients may lead to insufficient capacity to clear the virus and a long-term pro-inflammatory response.

At present, no vaccine is available for preventing COVID-19. Therefore, there is a need to identify and produce specific binding and neutralizing antibodies effective against SARS-CoV-2, as well as elucidating the targets and antigenic determinants to which the antibodies bind.

Disclosure of Invention

Dynamic adaptive immune responses in patients with COVID-19 have been revealed herein by analysis of the peripheral blood T cell receptor and B cell receptor repertoire. In some embodiments, to understand T cell and B cell immune responses at a systemic level, the inventors used next generation sequencing to analyze T Cell Receptor (TCR) and B Cell Receptor (BCR) repertoires of patients at different stages of COVID-19 disease. The adaptive immune response in early onset and in recovery of the patient is presented in a panoramic visualization. The TCR repertoire is dramatically reduced at early onset of severe disease, but is restored at the recovery stage. In some embodiments, monitoring a TCR repertoire can serve as an indicator biomarker for predicting disease progression. The BCR repertoire has isotype switching from IgM to IgG and transient significant IgA amplification. Clonal expansion of dominant B cells occurs after infection with a concomitant reduction in diversity. The studies herein indicate that immunohistological analysis is a promising approach to evaluate host immunity to novel viral infections. In some embodiments, the use of NGS of BCRs and TCRs for immune repertoire analysis allows for the overall observation of immune responses following SARS-CoV-2 infection. In some embodiments, the TCR repertoire has been found to decline dramatically in severe cases, but to recover during the recovery phase. In some embodiments, it has been found that the BCR repertoire shows dominant clonal expansion with IgM/IgG isotype switching and transient IgA surge after disease onset.

In some embodiments, the invention relates to isolated or non-naturally occurring monoclonal antibodies capable of binding to one or more epitopes of SARS-CoV-2. In some embodiments, the invention relates to an isolated or non-naturally occurring monoclonal antibody comprising: a light chain variable region comprising a sequence selected from SEQ ID NOS: 1-17; and a heavy chain variable region comprising a sequence selected from SEQ ID NOS: 18-26.

In some embodiments, the invention relates to an isolated or non-naturally occurring monoclonal antibody comprising: a light chain variable region comprising a sequence selected from SEQ ID NOS: 1-4; and a heavy chain variable region comprising SEQ ID NO: 18.

In some embodiments, the invention relates to an isolated or non-naturally occurring monoclonal antibody comprising: comprises the amino acid sequence shown in SEQ ID NO: 5 in the light chain variable region; and a polypeptide comprising SEQ ID NO: 19 in a heavy chain variable region of the amino acid sequence of seq id no.

In some embodiments, the invention relates to an isolated or non-naturally occurring monoclonal antibody comprising: comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 6-8; and a polypeptide comprising SEQ ID NO: 20, or a heavy chain variable region of the amino acid sequence of seq id no.

In some embodiments, the invention relates to an isolated or non-naturally occurring monoclonal antibody comprising: comprises the amino acid sequence shown in SEQ ID NO: 9, a light chain variable region of the amino acid sequence of seq id no; and a polypeptide comprising SEQ ID NO: 21, or a heavy chain variable region of the amino acid sequence of seq id No. 21.

In some embodiments, the invention relates to an isolated or non-naturally occurring monoclonal antibody comprising: comprises the amino acid sequence shown in SEQ ID NO: 10, a light chain variable region of the amino acid sequence of seq id no; and a polypeptide comprising SEQ ID NO: 22.

In some embodiments, the invention relates to an isolated or non-naturally occurring monoclonal antibody comprising: comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 11-12; and a polypeptide comprising SEQ ID NO: 23, or a heavy chain variable region of the amino acid sequence of seq id no.

In some embodiments, the invention relates to an isolated or non-naturally occurring monoclonal antibody comprising: comprises the amino acid sequence shown in SEQ ID NO: 13, a light chain variable region of the amino acid sequence of seq id no; and a polypeptide comprising SEQ ID NO: 24.

In some embodiments, the invention relates to an isolated or non-naturally occurring monoclonal antibody comprising: comprises the amino acid sequence shown in SEQ ID NO: 14, a light chain variable region of the amino acid sequence of seq id no; and a polypeptide comprising SEQ ID NO: 25.

In some embodiments, the invention relates to an isolated or non-naturally occurring monoclonal antibody comprising: comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 15-17, and a light chain variable region of an amino acid sequence; and a polypeptide comprising SEQ ID NO: 26.

In some embodiments, the invention relates to an isolated or non-naturally occurring monoclonal antibody comprising a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOS: 18-76.

In some embodiments, the invention relates to an isolated or non-naturally occurring monoclonal antibody comprising:

(a) kabat numbering and SEQ ID NOS: 78, heavy chain CDR1, CDR2, and CDR3 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the heavy chain CDR1, CDR2, and CDR3 set forth in SEQ ID NOS: 80, a light chain CDR1, CDR2, CDR3 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the light chain CDR1, CDR2, CDR 3;

(a) and SEQ ID NOS: 78, and a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a heavy chain variable region set forth in SEQ ID NOS: 80, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; or

(a) And SEQ ID NOS: 78, and a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the heavy chain set forth in SEQ ID NOS: 80, or a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

In some embodiments, the invention includes amino acid sequences having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid mutations (preferably conservative mutations, preferably substitutions, insertions, or deletions) compared to the amino acid sequence set forth in the above-described SEQ ID NOs.

In some embodiments, the antibodies of the invention are present at 10^-9M or less KD binds to SARS-CoV-2. In some embodiments, the antibodies of the invention are present at 10^-10M or less KD binds to SARS-CoV-2.

In some embodiments, the antibodies of the invention can be human antibodies, humanized antibodies, chimeric antibodies, or antibody fragments (e.g., fragments having antibody activity, e.g., binding or inhibitory activity). In some embodiments, antigen-binding fragments of the antibodies of the invention may include Fab, Fab ', F (ab')₂Fd, Fv, Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), diabodies, or domain antibodies.

In some embodiments, the antibodies or fragments thereof of the invention can specifically bind to SARS-CoV-2, e.g., an NP protein of SARS-CoV-2, e.g., a Spike protein of SARS-CoV-2, e.g., an RBD domain of a Spike protein of SARS-CoV-2, and/or inhibit the activity of SARS-CoV-2, e.g., inhibit the replication of SARS-CoV-2, e.g., inhibit the binding of SARS-CoV-2 to a receptor, e.g., reduce the burden of SARS-CoV-2.

In some embodiments, the invention relates to a nucleic acid encoding an antibody described herein.

In some embodiments, the invention relates to compositions comprising any of the above antibodies. In some embodiments, the invention relates to immortalized B cell clones expressing any of the above antibodies. In some embodiments, the invention relates to a pharmaceutical composition comprising any of the above antibodies and a pharmaceutically acceptable carrier. In some embodiments, the invention relates to a method of detecting COVID-19 in a biological sample, the method comprising contacting the sample with an antibody, wherein the antibody is a monoclonal antibody or antibody fragment of the invention, and determining the presence or absence of SARS-CoV-2 virus. In some embodiments, the invention relates to the use of an antibody or fragment thereof described herein in the preparation of a detection agent for detecting the presence of SARS-CoV-2 virus in a biological sample or a diagnostic agent for diagnosing COVID-19 or a therapeutic agent for treating COVID-19.

The present invention relates to isolated or non-naturally occurring SARS-CoV-2 monoclonal antibodies. In particular, the invention relates to an isolated or non-naturally occurring monoclonal antibody comprising a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOS: 1-17 (see table 1) and a light chain variable region comprising an amino acid sequence selected from SEQ ID NOS: 18-26 (see table 2). The invention also relates to an isolated or naturally occurring monoclonal antibody comprising a heavy chain variable region comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 18-76 (see table 2).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, the individual antibodies comprising the population being identical except for possible small amounts of naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, unlike polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized uncontaminated by other antibodies. The modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present invention can be prepared by the hybridoma method first described by Kohler et al, Nature, 256: 495(1975), or by using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). "monoclonal antibodies" can also be isolated from phage antibody libraries.

Typically, the antibodies of the invention herein are recombinantly produced using vectors and methods available in the art, as described further below. The antibodies disclosed herein can also be produced by B cells activated in vitro (see, e.g., U.S. Pat. No. 5,567,610). The antibodies of the invention herein can also be produced in transgenic animals, such as mice, which are capable of producing a full antibody repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that homologous deletion of the antibody heavy chain joining region (JH) gene in chimeras and germ line mutant mice results in complete inhibition of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays into such germline mutant mice results in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, proc.natl.acad.sci.usa, 90: 2551 (1993); U.S. Pat. nos. 5,545,806, 5,545,807, 5,569,825 and 5,591,669; and WO 97/17852. Such animals can be genetically engineered to produce human antibodies comprising the polypeptides of the invention.

The "CDRs" ("complementarity determining regions", also referred to as "hypervariable regions" or "HVRs") of the antibodies described herein refer to each region of the variable region of the antibody which is hypervariable in sequence and/or forms structurally defined loops ("hypervariable loops") and/or contains residues for contact with an antigen ("antigen-contact points"). Typically, an antibody comprises six CDRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). As known to those skilled in the art, CDR residues and other residues (e.g., FR residues) can be numbered according to different numbering systems. For example, the protein may be encoded according to the IMGT numbering system, the Kabat numbering system (Kabat et al, Sequences of Proteins of Immunological Interest, Public Health Service, National Institutes of Health, Bethesda, MD (1991)), Chothia and Lesk, J.mol.biol.196: 901-917(1987)), etc. As known to those skilled in the art, the "framework" or "FR" of an antibody refers to the residues of the variable region, excluding the residues of the hypervariable region (HVR). The FRs of the variable region typically comprise four FR domains: FR1, FR2, FR3 and FR 4. Thus, HVR and FR sequences typically occur in VH (or VL) in the following sequences: FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

"human antibodies" as described herein can include amino acid sequences corresponding to those of antibodies produced by humans or human cells or antibodies derived from non-human sources using repertoires of human antibodies or other human antibody coding sequences. "humanized" antibodies described herein may include chimeric antibodies of amino acid residues derived from non-human CDRs and amino acid residues derived from human FRs. A "chimeric" antibody described herein means an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

As is known to those skilled in the art, an alignment of percent (%) amino acid sequence identity can be achieved in a number of ways known to those skilled in the art, e.g., an alignment of amino acid sequences can be performed using the computer program ALIGN-2 or the like. In some embodiments, sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but retain the activity of the original sequence, e.g., binding or inhibitory activity. Antibody binding activity or inhibitory activity can be determined by any method known in the art.

Amino acid substitutions may be made to antibodies of the invention based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine, and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Variants may also or alternatively comprise non-conservative changes. Preferably, the variant polypeptide differs from the original sequence by substitution, deletion or addition of 5 or fewer amino acids. Variants may also or alternatively be modified by deletion or addition of amino acids that have minimal impact on the immunogenicity, secondary structure and water solubility of the polypeptide.

Alternative EBV immortalization methods described in WO2004/076677 may be used to generate immortalized B cell clones. In this way, B cells producing the antibodies of the invention can be transformed with EBV in the presence of a polyclonal B cell activator. Transformation with EBV can also be adapted to include a polyclonal B cell activator. Additional stimulators of cell growth and differentiation, such as IL-2 and IL-15, may be added during the transformation step to increase efficiency.

The invention also provides antibody fragments comprising a polypeptide of the invention. In some cases, it may be advantageous to use antibody fragments rather than whole antibodies. For example, the smaller size of the segments allows for rapid clearance and may result in improved access to some tissues. For all the examples describing the preparation of antibody fragments of monoclonal antibodies herein, the antibody fragments were screened to ensure that antigen binding was not disrupted. This can be accomplished by any of a variety of means known in the art, such as methods using phage display libraries.

As understood by those of skill in the art, an "antibody fragment" includes a portion of an intact antibody, preferably the antigen binding or variable region of an intact antibody, and retains an acceptable percentage of binding activity to the antigen of interest. As understood by those skilled in the art, the acceptable percentage depends on the particular intended use. Examples of antibody fragments include, for example, single chain antibody molecules (e.g., scFv), Fab ', F (ab') 2, and Fv fragments; a single chain antibody molecule; multispecific antibodies formed from antibody fragments; and other fragments known to those skilled in the art. By "antibody fragment" is meant a molecule other than an intact antibody, which molecule comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds.

Several techniques are known for the production of antibody fragments. Although antibody fragments have traditionally been obtained by proteolytic digestion of intact antibodies, they can now be produced directly by recombinant host cells. For example, Fab, Fv and ScFv antibody fragments can be expressed in E.coli and secreted from E.coli, and therefore these fragments can be easily produced in large quantities. Other techniques for producing antibody fragments are known to those skilled in the art.

Any of the above antibodies or antibody fragments thereof may be formulated as a pharmacotherapeutic agent for providing passive immunity to a subject suspected of having or at risk of having COVID-19, comprising a therapeutically effective amount of the antibody. The pharmaceutical formulation may include a suitable excipient or carrier. As will be appreciated by those skilled in the art, the total dose may vary depending on the weight, health and condition of the subject and the therapeutic effect of the antibody. Formulations for in vivo administration are preferably sterile, which may be achieved, for example, by filtering the formulation through a sterile filtration membrane.

As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers which are non-toxic to the cells or mammal to which they are exposed at the dosages and concentrations employed. Typically the physiologically acceptable carrier is a pH buffered aqueous solution. Examples of physiologically acceptable carriers include buffers such as phosphoric acid, citric acid and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone. Amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, e.g. TWEEN^TMPolyethylene glycol (PEG) and PLURONICS^TM。

Passive immunization has proven to be an effective and safe strategy for the prevention and treatment of viral diseases. Thus, passive immunization using the antibodies and antibody fragments thereof disclosed herein may provide a therapeutic strategy for COVID-19.

In various embodiments, the antibodies disclosed herein have intrinsic therapeutic activity. Alternatively, or in addition, the antibodies of the invention herein may be conjugated to a cytotoxic or growth inhibitory agent, such as a radioisotope or toxin, for use in treating infected cells to which the antibody is conjugated or contacted.

Subjects at high risk for COVID-19 include those who have been exposed to infections, particularly those with potential health problems, such as respiratory diseases like diabetes, heart disease or asthma or COPD. The prophylactic agent may be administered prior to manifestation of symptoms characteristic of COVID-19, thereby preventing the onset or delaying the progression of COVID-19.

For in vivo treatment of human and non-human subjects, a pharmaceutical formulation comprising at least one antibody disclosed herein is typically administered or provided to the subject. When used for in vivo therapy, the antibodies of the invention are administered to a subject in a therapeutically effective amount. As used herein, "therapeutically effective amount" refers to an amount that eliminates or reduces the viral load in a subject. The antibodies can be administered to a human subject according to known methods, for example, intravenously (as a suppository or by continuous infusion) or by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intramuscular, intrathecal, oral, topical or inhalation routes. The antibody may be administered parenterally, as much as possible at the site of the target cell, or by intravenous injection. In some cases, intravenous or subcutaneous administration of the antibody is preferred. The therapeutic compositions of the present invention may be administered to a subject systemically, parenterally or topically.

For parenteral administration, the antibodies of the invention herein are formulated in unit dose injectable forms (solutions, suspensions, emulsions) in combination with a pharmaceutically acceptable parenteral carrier. Examples of such carriers are water, physiological saline, ringer's solution, dextrose solution and 5% human serum albumin. Non-aqueous carriers such as oils and ethyl oleate may also be used. Liposomes can be used as carriers. The carrier may contain minor amounts of additives such as substances which enhance isotonicity and chemical stability, e.g. buffers and preservatives. The antibody may be formulated in such a vehicle at a concentration of about 1mg/ml to 10mg/ml, although other concentrations may be used.

The dosage and dosage regimen may be determined in accordance with a variety of factors which are readily ascertainable by a physician, such as the nature of the infection and the identity of the particular cytotoxic agent or growth inhibitory agent optionally used in conjunction with the antibody, such as the therapeutic index and the subject's medical history. Generally, a therapeutically effective amount of the antibody is administered to the subject. In particular instances, the amount of antibody administered is in the range of about 0.1mg/kg to about 50mg/kg of the subject's body weight. Depending on the type and severity of the infection, about 0.1mg/kg to about 50mg/kg body weight (about 0.1-15 mg/kg/dose) of antibody is an initial candidate dose for administration to a subject, whether, for example, by one or more separate administrations, or by continuous infusion. The progress of such treatment is readily monitored by routine methods and based on criteria known to the physician or to those skilled in the art.

In a particular embodiment, an immunoconjugate comprising an antibody conjugated to a cytotoxic agent disclosed herein is administered to a subject. Preferably, the immunoconjugate is internalized by the cell, resulting in an increased therapeutic effect of the immunoconjugate in killing the cell to which it binds. In one embodiment, the cytotoxic agent is directed against or interferes with nucleic acid in the infected cell. Examples of such cytotoxic agents are described above, and include, but are not limited to maytansinoids (maytansinoids), calicheamicins (calicheamicins), ribonucleases, and DNA endonucleases.

In some embodiments, other treatment regimens may be combined with the administration of one or more SARS-CoV-2 antibodies of the invention. Co-administration includes co-administration using separate formulations or a single pharmaceutical formulation and sequential administration in either order, preferably over a period of time, with two or all of the active agents exerting their biological activities simultaneously. Preferably such combination therapy results in a synergistic therapeutic effect.

In some embodiments, it is preferred to administer one or more of the antibodies disclosed herein in combination. In some embodiments, it is desirable to administer one or more antibodies configured to bind to an antigen associated with SARS-CoV-2 in combination with one or more other antibodies directed against a different antigen associated with SARS-CoV-2.

In addition to administering the antibody to a subject, the invention also provides methods of administering the antibody by gene therapy. The invention of the "administering a therapeutically effective amount of an antibody" includes administering a nucleic acid encoding an antibody. See, for example, PCT patent application publication WO96/07321, which relates to the production of intracellular antibodies using gene therapy.

In another embodiment, the SARS-CoV-2 antibodies disclosed herein are used to determine the structure of the bound antigen, e.g., conformational epitopes, which structure is then used to develop vaccines with or mimicking such structure, e.g., by chemical modeling and SAR methods. Such a vaccine may be used to prevent COVID-19.

In some embodiments, the present invention provides methods for detecting a polypeptide having a sequence selected from the group consisting of SEQ ID NOS: 1-17 or a polypeptide having an amino acid sequence selected from SEQ ID NOS: 18-26 in the preparation of a detection agent for detecting the presence of SARS-CoV-2 virus in a biological sample or a diagnostic agent (composition and kit) for diagnosing COVID-19. As shown herein, the most influential sequences in the largest cluster have been obtained from the COVID-19 patient cohort and are listed in tables 1 and 2. Thus, in some embodiments, specific detection of the presence of these sequences in a patient may be indicative of SARS-CoV-2 viral infection or of COVID-19 in the patient. Specific detection has the sequence selected from SEQ ID NOS: 1-17 or a polypeptide having an amino acid sequence selected from SEQ ID NOS: 18-26 can be prepared by methods known in the art, for example, by immunizing an animal with a polypeptide having the sequence as an antigen to obtain a corresponding antibody, e.g., a monoclonal antibody.

In some embodiments, the invention relates to the use of an agent (e.g., an antibody such as a monoclonal antibody that specifically binds IgA) for detecting IgA from a subject (e.g., a subject suspected of being infected with SARS-CoV-2) in the preparation of a composition or kit for detecting SARS-CoV-2 infection. In some embodiments, the compositions or kits can be used to detect early infection with SARS-CoV-2, e.g., infection at days 4-7 (e.g., days 4, 5, 6, 7) post-infection. In some embodiments, the composition or kit can further include reagents (e.g., SARS-CoV-2 specific primers, IgM, and/or IgG specific antibodies) for detecting nucleic acids, IgM, and/or IgG from the subject. Such reagents can be prepared by methods known in the art based on the published SARS-CoV-2 sequence.

In some embodiments, the invention relates to the use of an agent for determining the T Cell Receptor (TCR) and B Cell Receptor (BCR) repertoire from a subject (e.g., a subject suspected of being infected with SARS-CoV-2) in the manufacture of a composition or kit for monitoring the progress of COVID-19, for predicting the progression of COVID-19, or a prognostic assay. In some embodiments, reagents for assaying a T Cell Receptor (TCR) and B Cell Receptor (BCR) repertoire from a subject (e.g., a subject suspected of being infected with SARS-CoV-2) can include reagents for performing NGS. In some embodiments, the reagents may be used to analyze a TCR repertoire that has been found to be drastically reduced at early onset of severe disease, but restored at the recovery stage. Thus, monitoring the TCR repertoire can serve as an indicative biomarker for predicting disease progression. In some embodiments, the reagents may be used to analyze BCR repertoires that have been found to have IgM to IgG isotype switching and transient IgA surges in viral infections, and also to undergo dominant B cell clonal expansion following infection with reduced diversity. In some embodiments, the immune repertoire assay reagents can be used to evaluate the immunity of a host to infection by a new virus.

In another embodiment, the SARS-CoV-2 antibodies disclosed herein are capable of distinguishing between subjects having COVID-19 and subjects without COVID-19 and determining whether the patient has an infection. According to one method, a biological sample is obtained from a subject suspected of being infected or known to be infected with SARS-CoV-2. In a preferred embodiment, the biological sample comprises cells from the subject. The sample is contacted with the SARS-CoV-2 antibodies disclosed herein for a time and under conditions sufficient for the SARS-CoV-2 antibodies to bind to infected cells present in the sample. For example, the sample may be contacted with the SARS-CoV-2 antibody for 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 3 days, or any time in between. The amount of bound SARS-CoV-2 antibody is determined and compared to a control value, which may be a predetermined value or a value determined from a normal tissue sample. An increased amount of antibody bound to the subject sample as compared to the control sample indicates the presence of infected cells in the subject sample. The bound antibody is detected using procedures described herein and known in the art. In some embodiments, the diagnostic methods of the invention are practiced using the SARS-CoV-2 antibodies disclosed herein conjugated to a detectable label, such as a fluorophore, to facilitate detection of the conjugated antibody. However, they are also practiced using secondary detection methods for SARS-CoV-2 antibodies. These methods include, for example, RIA, ELISA, precipitation, agglutination, complement fixation, and immunofluorescence.

In another embodiment, the invention relates to a kit suitable for diagnostic and prognostic testing using the antibodies of the invention. The kits of the invention include a suitable container comprising the SARS-CoV-2 antibody of the invention in labeled or unlabeled form. In addition, when the antibody is provided in a labeled form suitable for an indirect binding assay, the kit also includes reagents for performing an appropriate indirect assay. For example, the kit includes one or more suitable containers, including enzyme substrates or derivatizing reagents. In some embodiments, a control sample and/or instructions may also be included.

Drawings

The disclosure may be better understood with reference to the following drawings.

FIG. 1 shows TCR-alpha (TRA), -beta (TRB), -delta (TRD), -gamma (TRG), BCR IgH, IgK, IgL in PBMCs of patients with COVID-19 at different disease stages. The proportion of each immune chain is indicated as mean + SD in the block diagram, and the average for each chain is shown in the pie chart. Samples from patients with COVID-19 were divided into three groups: (A) samples 4-7 days after onset of symptoms (n-8); (B) samples 8-15 days after onset of symptoms (n ═ 10); (C) samples 17-22 days after symptom onset (n-5); (D) sample asian healthy control (n ═ 15).

FIG. 2 immunoglobulin isotypes from the BCR repertoire in PBMCs from different disease stage COVID-19 patients. The proportions of IgA, IgG, IgD, IgE and IgM are indicated in the bar graph as mean + SD, while the bar graph shows the mean for each isotype. Samples from patients with COVID-19 were divided into three groups: (A) samples 4-7 days after onset of symptoms (n-8); (B) samples 8-15 days after onset of symptoms (n ═ 10); (C) samples 17-22 days after symptom onset (n-5); (D) asian healthy control sample (n ═ 15).

Figure 3 figure 1 is a graph showing longitudinal analysis of the TCR and BCR repertoire (repotore) of 4 subjects. Samples were collected from 3 patients in the early-to-mid period ( days 3, 5, 7, 12, 14) to the recovery period (days 17, 19) after the onset of symptoms. Samples before and after vaccination ( days 0, 4, 14, 28) of healthy seasonal tetravalent influenza vaccinees were used as controls. (A) TCR-TRA, -TRB, -TRD, -TRG and BRC-IgH, -IgK, -IgL in the adaptive immune repertoire. (B) At a designated time point after the onset of symptoms, a dendrogram (treemap) representing a unique CDR3 clonotype in each of the 7 strands. 7 dendrograms represent TCR-beta, -alpha, -delta, -gamma, and BCR-IgH, -IgK, -IgL, respectively. A rectangle in the dendrogram represents a unique clonotype. The size of the rectangle indicates the relative frequency of the individual CDR3 sequences, while the different square sizes reflect the clonally amplified regions in the sampled immune repertoire. From the top left to the bottom clockwise. IgH, IgK, IgL, TRB, TRA, TRD and TRG.

FIG. 4 shows a dendrogram of the TCR and BCR repertoire in ICU patient PBMCs. (A) Dendrograms containing 7 adaptive immune chains from patient 4 and patient 5 on day 4 after symptoms occurred. The control dendrogram was from the same healthy person prior to vaccination with seasonal tetravalent influenza vaccine. B) A dendrogram containing clones of total TCR-beta unique CDR3, each representing patient 4, patient 5, and healthy controls. A rectangle in the dendrogram represents a unique clonotype. The size of the rectangle indicates the relative frequency of the individual CDR3 sequences. The colors of the various CDR3 sequences in each tree plot are randomly selected, and thus, the colors between the plots do not match.

FIG. 5: summary of 10 diagnosed Covid-19 patients.

FIG. 6: comparison of immune repertoires in COVID-19 patients with healthy control PBMCs. In connection with fig. 1. The ratios of IgH, IgK and IgL of BCR and TCR-alpha (TRA), TCR-beta (TRB), TCR-delta (TRD) and TCR-gamma (TRG) are indicated as the median values in the above graph, and the pie chart shows the median values for each chain. Samples from COVID-19 patients are shown as median (a) sample (n-23), (B) sample of healthy control (n-15).

FIG. 7: comparison of Ig isotypes in PBMCs between COVID-19 patients and healthy controls. In connection with fig. 2. The proportions of IgA, IgG, IgD, IgE and IgM are indicated as medians in the above figures, and the medians for each isotype are shown in the pie charts. (A) Samples from patients with COVID-19 (n-23), (B) healthy controls.

Detailed Description

The following examples, applications, illustrations and descriptions are exemplary and explanatory and should not be construed as limiting the scope of the invention.

The TCR and BCR repertoire showed dynamic changes with the progress of COVID-19 disease

In this study, we investigated PBMC samples from patients with different stages of the COVID-19 disease. By immunohistochemical library analysis, we determined patterns of TCR and BCR dynamic changes during disease. Results the TCR and BCR libraries showed dynamic changes over the course of COVID-19. We collected 23 samples from patients with COVID-19 at different disease stages (fig. 5). The cohort consisted of 4 males and 6 females with a median age of 57 years (33-81 years). Peripheral Blood Mononuclear Cells (PBMCs) were collected at least one time point. From 3 patients 4 samples were collected, ranging from early to convalescent stages (fig. 5). A control group of 15 asian healthy donors of peripheral blood samples, 9 males and 6 females, aged between 22-67 years, was collected normally. Inclusion criteria for healthy donors were: 1) chronic disease has not been previously diagnosed; and 2) no diagnosis of any acute disease in the last three months. In addition, we used 4 PBMC samples from a 57-year-old healthy seasonal influenza vaccine as longitudinal healthy controls. We amplified an immune panel including all TCR chains (TCR-alpha, TRA, TCR-beta, TRB; TCR-delta, TRD; TCR-gamma, TRG) and BCR chains (IgH, including various IgH isotypes IgK; and IgL) unbiased in one PCR reaction. Approximately 500 million total sequencing reads were performed per sample. The data analysis pipeline of iResertire further analyzes the data (Wang et al, 2010; Yang et al, 2015).

Previously, lymphopenia was considered a common manifestation in patients with COVID-19, especially T lymphocytes (Guan W et al, NEJM, 2020; Huang et al, Lancet, 2020). Consistent with these reports, the absolute lymphocyte counts of the selected samples were also reduced, but the detailed TCR and BCR expression was not clear. We found that the mRNA expression of the TCR (n ═ 23) was significantly lower than that of the healthy control (n ═ 15) (fig. 1D, fig. 6). Compared with the control group, the average proportion of TCR-alpha expression in the immune group library is reduced from 28.3% to 4.9%, and the average proportion of TCR-beta expression is reduced from 18.7% to 5.2%, which are respectively reduced by 5.8 times and 3.6 times. The ratios of IgH, IgK and IgL in the COVID-19 samples (FIG. 6) were all higher than those in the healthy control group (FIG. 1D), especially the average ratio of IgL increased from 8.5% to 23.5%.

Furthermore, we divided these samples into several groups according to the course of the disease: group 1, samples 4-7 days after symptom onset (n-8); group 2, sample episodes 8-15 days after symptom onset (n ═ 10); group 3, 17-22 days after onset of symptoms (n-5). Analysis showed that on samples corresponding to groups 1 and 2, mRNA expression frequencies of TCR-alpha and TCR-beta chains were lower, but these expressed TCR chains increased on days 17-22 of group 3 (convalescent period). Surprisingly, TCR-beta/alpha expression in group 3 was similar to that of the normal control group. This indicates that TCR-beta/alpha expression can be used as a reference index for recovery from SARS-COV-2 virus infection. At all time points, the percentage of IgH/IgK/IgL expression relative to the overall adaptive group (adaptome) was higher than in the healthy control group, but the IgK expression at days 17-22 was lower than in the control group. In conclusion, TCR expression is significantly reduced during the early disease stage and increased IgH/IgK/IgL expression is likely to be the result of the humoral immune system responding to viral infection.

After onset, the BCR repertoire has isotype switching from IgM to IgG and a transient IgA surge

For SARS-COV-2 infection, IgM was first mobilized and its class switched to IgG isotype. In samples from day 4 to 7 of COVID-19 patients (n ═ 8), the median percentage of IgM was 19.5%, while the control group was 43.6% (fig. 2). IgG expression increased by about 10% in the early stages of infection compared to healthy controls, increased to 40% of total IgH expression at the apex of infection, and slightly decreased to 33.5% during convalescence (fig. 2). Another notable change was a significant increase in IgA proportion during early infection (days 4-7) compared to 42.6% in the healthy control group to 60.1% compared to 43.3% to 46.4% in later stage COVID-19 patients (FIG. 2, FIG. 7). Control levels to which IgA levels rapidly declined in samples from day 8-15. These results indicate that IgM/IgG/IgA was mobilized in response to coronavirus infection.

The dynamic changes in the immune repertoire revealed the progression of the COVID-19 disease.

To observe the immune repertoire dynamics of COVID-19 patients, we longitudinally followed the immune repertoire of 3 patients, each with 4 time points covering the period from onset to recovery. We found that TCR-beta is very low in early stages of disease. As the overall disease improves, TCR-beta increases, especially during the convalescent phase (fourth time point). The percentage of TCR-beta increased to the same level as the healthy controls (figure 1, figure 3). This result indicates that normal level of TCR-beta recovery is a reference indicator of recovery. We also found that IgH in the repertoire gradually decreased as the disease subsided. It was at its highest level on days 4-7 after symptom onset and returned to about 20% level on day 19 or day 20 (fig. 3). The proportion of IgL is highest on days 4-7 after the onset of symptoms and shows a decreasing trend throughout the course of the disease (Pt2 and Pt 3). The ratio of TCR and BCR chains was relatively stable before vaccination (day 0) and 28 days after vaccination (figure 3) compared to the group pool of healthy seasonal influenza vaccines vaccinated annually with influenza vaccine.

Longitudinal analysis of these three patients using a dendrogram showed that TCR-beta expression increased to normal levels during the convalescent period. In addition to the presence of some dominant clones in the TCR-beta expression profile, the diversity of clonotypes was significantly improved during the recovery phase (time point 4). A significant feature of IgH/IgK/IgL expression was significant clonal expansion around two weeks post infection (third time point, Pt1 at day 12, Pt2 and Pt3 at day 14), indicating that coronavirus-specific B cell clones predominated in the repertoire. This is a very important finding as it shows that the immune system will mount a humoral response to this new virus. It also offers the possibility of using some or several of the clonally amplified IgH sequences as a source of neutralizing antibodies.

Approximately 15% of patients with COVID-19 develop severe disease (Guan et al, 2020). Thus, we analyzed two samples obtained by 2 patients on days 4 and 6 after the onset of symptoms, which were sent to the Intensive Care Unit (ICU) due to the severity of the disease. Similar to what we found in other early infection samples, IgH/IgK/IgL predominates in the dendrogram, whereas TCR-beta expression is significantly reduced compared to healthy humans (FIG. 4). Many dominant clones were found on IgK expression and the diversity of this chain was lower than healthy controls in the repertoire.

Discussion of TCR and BCR dynamics in COVID-19 patients research provides valuable insight into clinical treatment. Here we show at the molecular level how the immune system should deal with SARS-COV-2 infection. Dynamic panoramagram visualizing TCR and BCR demonstrates how the adaptive immune system and disease progress until recovery. The profile of TCRs and BCRs in early stages of disease that are aberrantly expressed, particularly TCR-alpha and TCR-beta expression, and the restoration of such expression during convalescent stages may be a reference indicator of potential disease recovery. The initial absence of TCR alpha and TCR-beta expression is consistent with clinical data showing lymphopenia in Covid-19 patients. This means that these T-cells may leave the circulation and enter tissues such as lungs or lymph organs, or die due to viral infection. T cells play a role in viral clearance and subsequent establishment of antibody-mediated protection against viral infection (Schmidt and Varga, 2018; Zhao et al, 2014). T-cell deficient mice with MERS-CoV infection, but not B-cell deficient mice, resulted in persistence of the virus in the lungs, suggesting a direct effect of T-cell clearance of the virus (ZHao et al, 2014). Depletion of CD8+ T cells in patients with COVID-19 results in depletion, an indicator of the need for ICU therapy (Diao et al, 2020). Consistent with evidence of the lowest lymphocyte count is that on day 4 after the onset of symptoms (Zhou et al, 2020a), we found that expression of TCRs is markedly reduced in patients with moderate or severe levels at early stages of infection (days 4-7). One explanation is that virus-induced pneumonia increases vascular permeability and T cells recruited by chemokines leak indiscriminately into the lungs (Russell et al, 2017). Further studies are needed to elucidate the presence of T cells in lung bronchoalveolar lavage fluid in persons with severe and mild initial infection.

In terms of B cell response, IgM/IgG and IgA were mobilized against viral infection. Clonal expansion within the IgH chains was evident after two weeks. Importantly, clonal expansion within the B cell subpopulation provides evidence that the human adaptive immune system is initiating a response to this novel pathogenic virus. IgM is considered the first antibody isotype to initiate antigen exposure, and measurement of SARS-CoV-2 specific IgM antibodies can help in the rapid diagnosis of viral infections (sherdan, 2020). Abnormalities in IgM and IgG transcript levels in this study were found as early as day 4 of SARS-CoV-2 infection, while IgM/IgG serum titers appeared at 7-14 days post-pathogenesis (Long et al, 2020; Mo et al, 2006; Zhao et al, 2020). Thus, abnormalities in IgM/IgG/IgA can be rapidly examined to diagnose unknown pathogens. Evidence from other sources shows that relative IgG expression may be higher even after viral clearance for more than six months, suggesting that virus-specific antibodies persist in an affinity matured state at germinal centers (Davis et al, 2019; Niu et al, 2020; Wec et al, 2020). Another surprising finding is the IgA advantage early in infection. To our knowledge, this is the first report that IgA can be activated in peripheral blood following infection with the novel virus. Understanding IgA activation and identifying IgA antibodies in peripheral blood is very useful for the isolation of IgA-specific antibodies and diagnostics. IgA plays an important role in mucosal immunity (Gleeson et al, 1995). An increase in the percentage of IgA expression indicates that IgA can synthesize and migrate, and exert immune functions on the respiratory or gastrointestinal tract to clear the virus (Macpherson et al, 2008).

Previous studies have primarily been directed to the TCR-beta or IgH chain repertoire to show adaptive immune responses to cancer immunotherapy, autoimmune diseases or viral infections (Davis et al, 2019; Hopkins et al, 2018; Niu et al, 2020; Wendel et al, 2018; Wu et al, 2018). The multiplex PCR method used here-multiplex PCR (dam-PCR) avoiding dimers-allows amplification of TCR-alpha, -beta, -gamma, -delta chain and BCR-IgH, -IgK, -IgL (Han and Lotze, 2020) while having an inclusive and quantitative mode in one PCR reaction. To our knowledge, this is the first report that systematically elucidates the BCR and TCR repertoire of COVID-19 patients.

Longitudinal analysis of all IgH/IgK/IgL chains can show an overall view of the repertoire of antibodies over time. SARS-CoV-2 specific B cell single cell PCR combined with BCR repertoire analysis will speed the identification of lineage development and neutralizing antibodies by NGS and classical antibody cloning (Niu et al, 2020; Setliff et al, 2019). In summary, the results of this study provide a thorough understanding of the immune kinetics of patients with COVID-19. Overall and longitudinal analysis of adaptive immunity in COVID-19 patients can provide insight into the mechanisms of viral infection, provide guidance for clinical treatment, and assist in the development of antiviral therapies and vaccines.

Principal materials and reagents

Sample collection

Blood samples were taken from the eighth national hospital of Guangzhou City, affiliated Guangzhou medical university. All patients signed informed consent to donate blood samples. The study was approved by the ethical review committee of Guangzhou medical university. All healthy controls signed written informed consent before peripheral blood was collected. The work of designing and recruiting 15 Asian healthy donors in this study resulted in the New England independent review Committee

(NEIRB) approval (IRB Nos. 14-378). All experiments were performed according to relevant guidelines and regulations.

PBMC isolation and RNA extraction.

PBMCs were isolated by density gradient separation on Ficoll-Hypaque gradients as described previously by Niu et al, emery Microbes Infect 9, 111-. (GE Healthcare, Chicago, IL, USA). Invitrogen company TRIzol was used according to the manufacturer's protocol^TMLS reagent extracts total RNA.

Unbiased amplification of TCRs and BCRs.

iR-RepSeq-plus 7-Chain Cassette (iRepertore) was used to generate NGS libraries covering all TCR and BCR chains, including TCR-beta, -alpha, -delta, -gamma, and BCR-IgH, -IgK, -IgL. All 7 strands were amplified in one assay, using a strategy that allowed the addition of a unique molecular identifier during the Reverse Transcription (RT) step. A disposable cartridge for library preparation of a sample; all reagents required for amplification and purification are preloaded into the cassette. 1000ng of the extracted RNA was added to the cassette with an appropriate amount of RT mixture and nuclease-free water and processed by an iR-Processor. The instrument was automatically set up to perform all amplification and purification. Briefly, RT was performed using Qiagen OneStep RT-PCR mix (Qiagen). First strand cDNA was selected, unused primers removed by SPRISELECT bead selection (Beckman Coulter), and then a second round of binding and extension was performed with V-gene primer mix. After binding and extension, the first and second strand synthesis products were purified using SPRIselect beads. Library amplification is performed with a pair of primers, which are specific common sites, engineered to the 5' ends of the C-and V-primers used in the first and second strand synthesis. The final constructed library included an Illumina double-indexed sequencing linker, a 10-nucleotide unique molecular identifier region, and an 8-nucleotide internal barcode associated with the C-gene primers. The amplified libraries were multiplexed and pool sequenced by a commercial sequencing service laboratory (Personal Biotechnology co., ltd., Shanghai, China) on the Illumina NovaSeq platform using a 500-cycle kit (250 paired end reads). The exported immune receptor sequences cover the first framework region to the beginning of the constant region, including CDR1, CDR2, and CDR 3.

Data collection and bioinformatics analysis

The raw data were analyzed using the iRmap program described by Wang et al, Proc Natl Acad Sci USA 107, 1518-. Briefly, sequence reads are de-multiplexed (de-multiplexed) according to the barcode sequence at the 5' end of the reads from the constant region. Reads were then trimmed with a 2 base sliding window according to their base mass. If any of the quality values in the window is below 20, the sequences extending from the window to the 3' end are clipped from the original reads. The trimmed paired end reads were joined together using an overlap alignment by a modified Needleman-Wunsch algorithm. If the paired forward and reverse readings in the overlap region do not match exactly, both the forward and reverse readings are discarded without further consideration. The merged reads were mapped to germline V, D, J and C reference sequences downloaded from the IMGT website using the Smith-Waterman algorithm (Lefranc, 2003). To define the CDR3 regions, the CDR3 boundary positions of the reference sequences in the IMGT database were migrated onto the reads by mapping results, and the resulting CDR3 regions were extracted and translated into amino acids. The data set was condensed by a combination of Unique Molecular Identifiers (UMIs) and CDR3 regions to remove incorrect CDR3 introduced by sequencing and amplification. The reading with the same combination of CDR3 and UMI was concentrated to one UMI count.

The most influential CDR3 sequence (IMGT) in the largest cluster was obtained from the COVID-19 patient cohort and is listed in tables 1 and 2. The inventors also performed gene synthesis of 10 light chain and 10 heavy chain genes (some of which residues may be appropriately modified, e.g., inserted, deleted and/or substituted) selected from tables 1 and 2, respectively. The genes are cloned into an expression vector, and after cloning is successful, the coding region and the reading frame are sequenced and confirmed. And purifying the obtained product by using an in vitro transcription and translation kit and a modified vector to obtain the antibody. Exemplary antibodies of the invention were detected by ELISA detection experiments (results are shown in the following Table), which showed high specificity and high affinity binding (e.g., at 10) to the antigenic protein of SARS-CoV-2^-10M or less KD binds to SARS-CoV-2 antigen). The SARS-CoV-2 pseudovirus invaded cell in vitro experiment shows that the inventionThe antibody has excellent activity of inhibiting the invasion of pseudoviruses into cells. Table 3 lists exemplary antibody sequences of the invention (which bind the NP protein of SARS-CoV-2 with high specificity and high affinity).

TABLE 1

TABLE 2

Table 3: an exemplary antibody (B280108) sequence of the invention.

The heavy chain gene sequence, the heavy chain amino acid sequence, the light chain gene sequence and the light chain amino acid sequence of the antibody are respectively numbered as SEQ ID NOS: 77, 78, 79, and 80, the light and heavy chain CDRs, respectively, according to Kabat numbering are shown in table 4 below.

CDRs	Sequence of	SEQ ID Nos：
			CDR-H1	ELSIY	81
CDR-H2	NIDAVYGETTYAQKFEG	82
			CDR-H3	DSPRPIVVYAFAM	83
CDR-L1	RASQSVSSNLA	84
			CDR-L2	GASARAT	85
CDR-L3	QQYNDWPRT	86

ELISA detection experiment

Antigen: 2019-nCoV-NP-His, Bioimtron Cat. # B233501)

SARS-CoV-2 S protein，Acor，Cat.#SPN-C52H4

S1-RBD-His Bioimtron Cat.#B232004

Secondary antibody: goat Anti-Human IgG Fc HRP, Jackson Immuno Research, 109-

1 coating:

1.1 according to the concentration of the antigen protein, diluting the antigen protein to 2ug/ml by using a coating buffer solution, preparing 20ml, uniformly mixing, adding 100ul of diluted antigen protein (2ug/ml) into each hole of the ELISA plate, and slightly oscillating the ELISA plate to enable the antibody to cover the bottom of the hole of the ELISA plate.

1.2 plates closed, incubated overnight at 4 ℃.

2, sealing:

after washing the ELISA plate for 3 times with 2.11XPBS buffer, add 200ul of blocking solution to block.

2.2 Add blocking solution and incubate 1h at 37 ℃ in incubator.

The microplate was washed 5 times with 2.31 XPBS/Tween buffer.

3 add sample to be tested (expression antibody):

the initial concentration of the first well antibody was: 10ug/ml, diluted with PBS and added, 1/5 gradient down to 7 wells, set up duplicate wells.

3.2 sealing plates and then acting at 37 ℃ for 1 hour.

3.3 spin-drying the liquid in the ELISA plate, and washing the ELISA plate for 5 times by 200ul of 1 XPBS/Tween buffer solution.

4 secondary antibody (coat Anti-Human IgG HRP):

4.1 preparing a secondary antibody solution according to the required amount of the experiment, adding 1ul of secondary antibody into every 5ml of 1XPBS, uniformly mixing, and adding 100ul of diluted secondary antibody into each hole of the ELISA plate.

4.2 sealing the plate and then acting at 37 ℃ for 30 min.

4.3 spin-drying the liquid in the ELISA plate, and washing the ELISA plate for 5 times by 200ul of 1 XPBS/Tween buffer solution.

5, adding a substrate:

5.1 Add 100ul OPD chromogenic solution (OPD preparation: 10ml substrate buffer +40ul OPD +5ul H2O2) per well, incubate 10min at 37 deg.C, add 50ul stop solution (0.25M HCl) to stop chromogenic reaction

The optical density values were read at 492nm with a microplate reader 5.2.

Antibody neutralization assay (detection of neutralizing effect of antibody on virus)

Preparation of pseudovirus: the day before transfection, 293T cells were seeded in 75cm dish flasks. Cells were transfected at 80% confluence. Preparing a plasmid mixture: 3ug PVRC8304, 9ug PCMV8.2, 12ug PHR-CMV-Luciferase and 62ul 2M CaCl2, adding water to 500ul, mixing, adding dropwise and slowly into 500ul2XHEBS, mixing with vortex device, and standing on ice for 20 min. During which the 293T cell culture medium was discarded and replaced with 10ml of fresh medium. 5min before transfection, 25ul of 50mM chloroquine was added to the cell flask, and then the precipitate was added dropwise to 293T cells, and the culture was continued at 37 ℃ after 8h of 5% CO2 by replacing fresh DMEM medium. Respectively harvesting the supernatant at 48h and 72h, centrifuging at 2500r/min for 10min at 4 ℃, filtering the supernatant by using a 0.45um filter, and subpackaging to obtain the pseudovirus containing SARS-COV-2 gene, and freezing and storing at minus 80 ℃ for later use. Measuring with Vironostika HIV-1 antigen micro ELISA kit of BIOMERIIEUX, calculating P24 content, and determining the required dosage of pseudovirus for micro neutralization test according to P24 content.

Pseudovirus microneutralization assay: the day before the experiment, 293T-ACE2 cells (293T cells over-expressing ACE2 protein) were seeded in 96-well flat-bottom plates, and the seeded cells were observed the next day, and the experiment was performed until the cells spread over more than 80% of the well area. The antibodies were diluted 1: 100, 1: 300, 1: 900 and 1: 2700 in cell culture medium, 50ul of the diluted antibodies at different concentrations were mixed with an equal volume of pseudovirus and incubated at 37 ℃ for 1 h. The above mixture was then seeded at 100 ul/well into 96-well plates of 293T-ACE2 cells plated, 3 wells for each dilution, while setting 3-well negative controls and 3-well pseudovirus positive controls. Placing a 96-hole culture plate in a 37-degree and 5% CO2 incubator for incubation for 48h, removing culture solution in the culture plate, gently patting the 96-hole plate on facial tissue, adding cell lysate (30 ul/hole), and allowing the action time to be longer than 10 min; then 70ul Luciferase reagent is added into each hole, the solution is evenly blown and beaten by a discharging gun, 70ul mixed solution is absorbed into a new 96-hole culture plate, and the new 96-hole culture plate is placed on a multifunctional photometer of a Modulus 96-hole micropore plate type to measure the fluorescence value. The final calculation was performed to calculate the neutralization efficiency of the experimental wells and the negative serum control wells, respectively, with reference to the fluorescence of the cells in the positive control wells.

Although the present invention and its advantages have been described in detail, it should be understood that various changes and substitutions may be made herein without departing from the spirit of the invention. The methods described herein and the various embodiments thereof are exemplary and other embodiments may be included.

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Claims (8)

1. An isolated or non-naturally occurring antibody comprising:

(a) heavy chain CDR1, CDR2, and CDR3 shown in SEQ ID NOS:81-83, as indicated by Kabat numbering, and (b) light chain CDR1, CDR2, and CDR3 shown in SEQ ID NOS:84-86, as indicated by Kabat numbering, wherein the antibody specifically binds to the NP protein of SARS-CoV-2.

2. The antibody of claim 1, comprising:

(a) the heavy chain variable region in the heavy chain shown in SEQ ID NO:78, and (b) the light chain variable region in the light chain shown in SEQ ID NO: 80.

3. The antibody of claim 1, comprising:

(a) the heavy chain shown in SEQ ID NO:78, and (b) the light chain shown in SEQ ID NO: 80.

4. The antibody of any one of claims 1-3, which is a human, humanized, chimeric, or antigen-binding fragment, wherein the antigen-binding fragment is Fab, Fab ', F (ab')₂Fd, or an Fv fragment.

5. A nucleic acid encoding the antibody of any one of claims 1-4.

6. An immortalized B-cell clone expressing the antibody of any one of claims 1 to 4.

7. A diagnostic composition comprising the antibody of any one of claims 1-4.

8. The antibody of any one of claims 1-4 orUse of an antigen binding fragment thereof, wherein the antigen binding fragment is Fab, Fab ', F (ab')₂Fd, or an Fv fragment.

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