Novel coronavirus monoclonal neutralizing antibody ConBA-998 and application thereof

Abstract

The invention discloses a novel coronavirus monoclonal neutralizing antibody ConBA-998 and application thereof. The ConBA-998 is composed of a heavy chain and a light chain paired with the heavy chain; the sequences of the CDR1, the CDR2 and the CDR3 of the ConBA-998 heavy chain are 'GFTVSSNY', 'IYSGGDT' and 'ARSPTPPDGFDI' in sequence, and the sequences of the CDR1, the CDR2 and the CDR3 of the light chain are 'NIGSYS', 'YDS' and 'QVWDSSSDWV' in sequence. The monoclonal neutralizing antibody encoded by the public antibody family IGHV3-66 is obtained by sorting from volunteers which are not inoculated with new crown vaccines and are initially infected with new crown virus Omicro BA.1 mutant strains, the monoclonal neutralizing antibody has efficient neutralizing activity on the Omicro BA.1 mutant strains, and a new choice is provided for prevention and treatment of novel coronavirus.

Description

Technical Field

[0001] The present invention belongs to the technical field of novel coronavirus treatment, and particularly relates to a novel coronavirus monoclonal neutralizing antibody ConBA-998 and its application. Background Art

[0002] The novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) has spread rapidly worldwide, and the Corona Virus Disease 2019 (COVID-19) caused by it seriously threatens human health and social and economic activities. The new coronavirus binds to the receptor angiotensin-converting enzyme 2 (ACE2) on the surface of human host cells by means of its surface spike glycoprotein. Through the cleavage of the Spike protein by host proteases, a series of conformational changes occur, releasing the S2 membrane fusion peptide, thereby fusing with the cell and entering the host cell, and injecting the viral genetic material into the host cell to complete the infection process. Neutralizing antibodies target the Spike protein, can effectively block the virus from invading host cells, and have good specificity, high efficacy and safety, so they have become a hot spot in the development of COVID-19 drugs. At present, a large number of literatures have reported the selection of COVID-19 neutralizing antibodies from recovered natural infected patients. Among them, some monoclonal antibodies have obtained emergency use authorization (EUA) for clinical treatment and have achieved good efficacy in the early stage.

[0003] However, as an RNA virus, the novel coronavirus has a high mutation frequency during infection and transmission, resulting in many mutant strains of the novel coronavirus. Currently, the Omicron strain is the main strain prevalent globally, and it has derived multiple subclasses, such as BA.1, BA.2, BA.2.12.1, BA.3, BA.4, BA.5, BA.2.75, BQ.1.1, XBB.1, CH.1.1, BA.2.86, JN.1, etc. The emergence of these mutant strains has significantly escaped most of the neutralizing antibodies in the bodies of recovered individuals from natural infection and vaccinated individuals. Many therapeutic antibodies used or approved for marketing clinically are also inactivated against the mutant strains. During the COVID-19 pandemic, the population widely received COVID-19 vaccines based on the original strain. When stimulated by Omicron again, most of the activated memory B cells are vaccine-induced, and the antibodies produced mainly target the conserved epitopes between WT and Omicron, rather than Omicron-specific neutralizing antibodies. Given that most monoclonal neutralizing antibodies discovered early were induced by the original strain WT and are insufficient to combat the later-prevailing Omicron mutant strain. Therefore, it is urgent to conduct research on Omicron-specific antibodies, hoping to screen out highly effective neutralizing antibodies against the Omicron mutant strain as a strategic reserve for future responses to the COVID-19 pandemic. At the same time, by studying the characteristics and action mechanisms of specific neutralizing antibodies, key immune in-situ points can be found to provide reference guidance for the design of a new generation of vaccines. Summary of the Invention

[0004] The object of the present invention is to provide a new monoclonal neutralizing antibody ConBA-998 against the novel coronavirus and its applications.

[0005] To achieve the above object, the present invention adopts the following technical solutions: In the first aspect of the present invention, a monoclonal neutralizing antibody against the novel coronavirus is disclosed. The monoclonal neutralizing antibody against the novel coronavirus is ConBA-998; the monoclonal neutralizing antibody is composed of a heavy chain and a paired light chain; the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain of ConBA-998 are "GFTVSSNY", "IYSGGDT", and "ARSPTPPDGFDI" in sequence, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are "NIGSYS", "YDS", and "QVWDSSSDHWV" in sequence.

[0006] It should be noted that the key of the present invention lies in cloning and identifying a monoclonal neutralizing antibody, namely ConBA-998, from the peripheral blood specific memory B cells of volunteers who have never been vaccinated against COVID-19 and have only been initially infected with the Omicron BA.1 mutant strain of the novel coronavirus. This monoclonal antibody is encoded by the public antibody family IGHV3-66 and can efficiently neutralize the Omicron BA.1 mutant strain of the novel coronavirus.

[0007] In one implementation of the present invention, the heavy chain variable region sequence of the novel coronavirus monoclonal neutralizing antibody ConBA-998 is the sequence shown in Seq ID No.1, and the light chain variable region sequence is the sequence shown in Seq ID No.2.

[0008] It should be noted that the heavy chain variable region and light chain variable region of the above specific sequences are only the monoclonal neutralizing antibody sequences specifically adopted in one implementation of the present invention; it can be understood that for a monoclonal neutralizing antibody, the regions that affect its precise complementarity with the antigen determinant are the complementarity-determining regions (abbreviated as CDR), specifically, CDR1, CDR2, and CDR3 of the heavy chain, and CDR1, CDR2, and CDR3 of the light chain; therefore, as long as the sequences of CDR1, CDR2, and CDR3 of the heavy chain and light chain of the present invention remain unchanged, the functions and effects of the novel coronavirus monoclonal neutralizing antibody of the present invention can be basically achieved. That is to say, for the novel coronavirus monoclonal neutralizing antibody of the present invention, its specific sequence is not limited to the heavy chain variable region and light chain variable region of the above specific sequences.

[0009] The second aspect of the present invention discloses a nucleic acid fragment encoding the novel coronavirus monoclonal neutralizing antibody of the present invention, and this nucleic acid fragment encodes the heavy chain and light chain of ConBA-998.

[0010] In one implementation of the present invention, the nucleic acid fragment encoding the heavy chain variable region shown in Seq ID No.1 has the sequence shown in Seq ID No.3; the nucleic acid fragment encoding the light chain variable region shown in Seq ID No.2 has the sequence shown in SeqID No.4.

[0011] It should be noted that the above specific nucleic acid sequences are only the nucleic acid sequences specifically adopted in one implementation of the present invention. It can be understood that there can be multiple codons for one amino acid; therefore, according to the degeneracy of codons, in addition to the above-defined nucleic acid sequences, there can be several nucleic acid sequences encoding the same heavy chain or light chain under the condition of ensuring the encoding sequence remains unchanged, and they are all within the protection scope of the present invention.

[0012] The third aspect of the present invention discloses a recombinant plasmid containing the nucleic acid fragment of the present invention.

[0013] It should be noted that the recombinant plasmid of the present invention is for the effective expression of the nucleic acid fragment of the present invention, so as to obtain the corresponding heavy chain, light chain or monoclonal neutralizing antibody against SARS-CoV-2; therefore, in principle, any vector that can transfect the nucleic acid fragment into host cells for nucleic acid expression can be used in the present invention.

[0014] The fourth aspect of the present invention discloses a recombinant cell containing the nucleic acid fragment of the present invention or the recombinant plasmid of the present invention.

[0015] It should be noted that the recombinant cell of the present invention refers to a host cell transfected with the nucleic acid fragment or recombinant plasmid of the present invention; generally, the heavy chain, light chain or monoclonal neutralizing antibody against SARS-CoV-2 of the present invention can be obtained by directly culturing such host cells.

[0016] The fifth aspect of the present invention discloses a method for preparing a monoclonal neutralizing antibody against SARS-CoV-2 of the present invention, which includes protein expression of the recombinant plasmid of the present invention using the recombinant cell of the present invention, extracting and purifying the expressed protein, and thus obtaining the monoclonal neutralizing antibody against SARS-CoV-2 of the present invention.

[0017] The sixth aspect of the present invention discloses the application of the monoclonal neutralizing antibody against SARS-CoV-2 of the present invention, or the nucleic acid fragment of the present invention, or the recombinant plasmid of the present invention, or the recombinant cell of the present invention, in the preparation of drugs for preventing and treating SARS-CoV-2 or SARS-CoV-2 detection reagents.

[0018] It should be noted that the monoclonal neutralizing antibody against SARS-CoV-2 of the present invention can have a highly efficient neutralizing effect on Omicron BA.1; therefore, it can be used to prepare corresponding drugs for preventing and treating SARS-CoV-2. Similarly, the monoclonal antibody of the present invention has a highly efficient binding activity to Omicron BA.1 and can also be used to detect the corresponding SARS-CoV-2. As for the nucleic acid fragment, recombinant plasmid and recombinant cell, these can be used as raw materials for preparing monoclonal antibodies against SARS-CoV-2, and thus be used to prepare drugs for preventing and treating SARS-CoV-2 or SARS-CoV-2 detection reagents.

[0019] The seventh aspect of the present invention discloses a drug for preventing and treating SARS-CoV-2, which contains the monoclonal neutralizing antibody against SARS-CoV-2 of the present invention, or a substance that can induce the production or in vivo expression of the monoclonal neutralizing antibody against SARS-CoV-2 of the present invention.

[0020] The eighth aspect of the present invention discloses a SARS-CoV-2 detection reagent, which contains the monoclonal neutralizing antibody against SARS-CoV-2 of the present invention, or an antigen that can specifically bind to the monoclonal neutralizing antibody against SARS-CoV-2 of the present invention.

[0021] Advantages of the present invention are as follows due to the above technical solution: The novel coronavirus monoclonal neutralizing antibody ConBA-998 of the present invention has high neutralizing and binding activities against the Omicron BA.1 mutant strain, providing a new option for the prevention and treatment of the novel coronavirus.

[0022] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 It is the neutralization experiment results of the monoclonal neutralizing antibody ConBA-998 against the SARS-CoV-2 wild-type (WT) original strain and Omicron BA.1 pseudovirus in the embodiments of the present invention.

[0025] Figure 2 It is the binding activity detection results of the monoclonal neutralizing antibody ConBA-998 against the SARS-CoV-2 BA.1-Spike, BA.1-S1, BA.1-NTD, BA.1-RBD, and WT-Spike proteins in the embodiments of the present invention.

[0026] Figure 3 It is the SPR experiment results of the monoclonal neutralizing antibody ConBA-998 against the SARS-CoV-2 BA.1-Spike and WT-Spike proteins in the embodiments of the present invention.

[0027] Figure 4 It is the neutralization experiment results of the monoclonal neutralizing antibody ConBA-998 against 23 single-site revertant mutants of BA.1-Spike at different sites in the embodiments of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The continuously emerging mutant strains of SARS-CoV-2, especially Omicron-related variants, have significantly escaped neutralizing antibodies in both naturally infected individuals and vaccinated individuals. In the context of widespread vaccination with vaccines based on the original strain of the novel coronavirus, when Omicron breakthrough infections occur in the population, the immune memory of the original antigen produced after vaccination is mainly evoked in the body, while the neutralizing activity against other Omicron subtype variants is relatively low. At present, it is very difficult to use Omicron vaccines as booster shots to induce broad-spectrum neutralizing antibodies and improve the population's resistance to continuously emerging variants of the novel coronavirus; in contrast, designing more unique immunogens to induce highly efficient antibodies specifically against Omicron is a new strategy. Therefore, we urgently need to explore Omicron-specific antibodies and also use reverse vaccinology to find key immune in situ sites to provide reference guidance for the design of new-generation vaccines.

[0029] In the present invention, the Omicron BA.1-S1 protein of the novel coronavirus is used as a bait, and single B cells are isolated from volunteers who have never been vaccinated against the novel coronavirus and have only been initially infected with the Omicron BA.1 strain by flow cytometry, and a monoclonal neutralizing antibody, namely ConBA-998, is cloned and identified therefrom. Its IC 50 against the Omicron BA.1 pseudovirus of the novel coronavirus is 0.003 μg / mL. The heavy chain of ConBA-998 belongs to the IGHV3-66 family, and antibodies of this family are usually enriched in different individuals during the convalescent period of novel coronavirus infection and are called public antibody families.

[0030] In the present invention, the binding activities of ConBA-998 with BA.1-S1, BA.1-NTD, BA.1-RBD, BA.1-Spike and WT-Spike are determined by ELISA. The results show that ConBA-998 can strongly bind to BA.1-S1, BA.1-RBD and BA.1-Spike, indicating that this antibody is a monoclonal antibody targeting BA.1-RBD.

[0031] In the present invention, the affinity of this antibody with WT-Spike and BA.1-Spike is further determined by SPR. The results show that ConBA-998 has a relatively high affinity with BA.1-Spike, and the KD value is 1.41 nM; while the affinity of ConBA-998 with WT-Spike cannot be detected.

[0032] Finally, we performed single-point revertant mutations on BA.1-Spike to construct 23 single-point mutant pseudoviruses and evaluated the neutralizing activity of ConBA-998. The results showed that the S496G and R498Q mutations significantly decreased the neutralizing activity of ConBA-998, while the R493Q, Y501N, and H505Y mutations completely inactivated ConBA-998. This result revealed the key sites on BA.1 that affect the activity of ConBA-998, demonstrating that R493, S496, R498, Y501, and H505 have certain immunogenicity and can induce effective neutralizing antibodies.

[0033] Based on the above understanding and research, a novel coronavirus monoclonal neutralizing antibody ConBA-998 of the present invention has specific neutralizing and binding activities against Omicron BA.1, providing a new candidate therapeutic antibody for the prevention and treatment of novel coronavirus.

[0034] The present invention will be further described in detail below through specific embodiments in conjunction with the accompanying drawings. The following embodiments are only for further illustrating the present invention and should not be construed as limiting the present invention. Unless otherwise specified, the instruments and materials used in the following embodiments are conventional laboratory equipment, and the technical solutions are all conventional techniques in the art. Examples

[0035] I. Materials and Methods 1. Research Approval and Biological Samples This study was approved by the Ethics Committee of the Third People's Hospital of Shenzhen (Approval No.: 2021-030). The participants provided written informed consent for sample collection and subsequent analysis. Peripheral blood mononuclear cell (PBMC) samples were collected from patients who had not been vaccinated against COVID-19 and had only been initially infected with Omicron BA.1 about 2 weeks later and stored in the biobank of the Third People's Hospital of Shenzhen. The PBMC samples were stored in liquid nitrogen before use.

[0036] Specifically, 1 sample was collected in this example, and the sample collection method is as follows: Isolation of peripheral blood mononuclear cells: 10 mL of venous blood was collected in an anticoagulant tube and transferred to a 50 mL centrifuge tube, and diluted with 10 mL of PBS solution and gently mixed; Two 15 mL centrifuge tubes were taken, and 5 mL of Ficoll separation solution was added to each. Then 10 mL of the diluted blood was added to the upper layer of the Ficoll separation solution; Centrifuge at 2000 rpm for 20 minutes; Aspirate the leukocyte layer into a clean 15 mL centrifuge tube; Add PBS to 10 mL, centrifuge at 1500 rpm for 10 minutes, then discard the supernatant, resuspend with 3 mL of cell cryopreservation solution, take 1 mL of cells into 3 cryotubes each, place them in a cryobox and then put them in a -80 °C refrigerator overnight, and transfer the cells to liquid nitrogen for long-term storage the next day.

[0037] 2. Isolation of monoclonal antibodies from B cells of peripheral blood mononuclear cell samples Resuscitate cryopreserved peripheral blood mononuclear cells and wash them twice with 10 mL of PBS. Add a probe mixture of CD3-Pacific Blue, CD8-Pacific Blue, CD14-Pacific Blue, CD19-PE-Cy7, CD27-APC-H7, IgG-FITC (all from BD Biosciences), IgA-FITC (Miltenyi Biotec), and SARS-CoV-2 Omicron BA.1-S1 with His tag (Sino Biological) to 50 μL of staining buffer PBS, resuspend the peripheral blood mononuclear cells, and stain them at 4°C for 30 minutes. After washing twice with PBS, add APC- and PE-labeled anti-His tag secondary antibody (Abcam) to 50 μL of staining buffer PBS and stain the cells at 4°C for 30 minutes. After washing twice with PBS, sort SARS-CoV-2 BA.1-S1-specific IgG+ or IgA+ memory B cells using a BD FACS Aria II cell sorter flow cytometer.

[0038] Sort single B cells into a 96-well PCR plate containing lysis buffer, and then perform RT-PCR and nested PCR to amplify the variable regions of the heavy and light chains respectively according to the method in the literature (Liao HX, Levesque MC, Nagel A, Dixon A, Zhang R, Walter E, et al. High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies. Journal of virological methods. 2009;158:171-9.). Send the PCR amplification products to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the obtained antibody is named ConBA-998.

[0039] The antibody variable region sequences obtained by sequencing were sent to GenScript Biotech Corporation for synthesis. The company cloned the variable regions of the heavy and light chains of the antibody into the full-length IgG1 heavy and light chain expression vectors pCDNA3.4 (GenScript Biotech Corporation) respectively, and prepared a large amount of plasmids of the heavy and light chains of the antibody. After obtaining the plasmids prepared by GenScript, the paired heavy and light chain expression plasmids were co-transfected into 293F cells (taking 500 mL as an example) using PEI transfection reagent for expression, and the monoclonal antibody was purified from the culture supernatant using a protein A adsorption column. The specific steps are as follows: The 293F cells were cultured in an 8% CO2, 37 °C incubator, and the concentration of 293F cells was adjusted to 1.2×10 6 cells / mL and cultured for another 2 hours; Prepare Solution A: Add 250 μg of the heavy chain plasmid of the antibody and 250 μg of the light chain plasmid of the antibody to 12.5 mL of opti-MEM (31985070, Gibco). Prepare Solution B: Add 2.5 mL of 1 mg / mL PEI transfection reagent (24885-2, Polyscieces) to 12.5 mL of opti-MEM and let it stand for 5 minutes; Mix Solution A and Solution B and let it stand for 20 minutes to obtain the AB mixture; Slowly add 25 mL of the AB mixture drop by drop to 500 mL of 293F cells, shaking well while adding; Continue to culture the cells for 5 days; Then centrifuge the 293F cells at 3000 g for 20 minutes, collect the supernatant, and filter it using a 0.45 μm filter membrane; Open the lid of the Protein A gravity column and let the 20% ethanol solution in the column flow out completely by gravity, and equilibrate the Protein A gravity column with 5 column volumes of 10 mM PBS solution; Add the filtered cell supernatant to the Protein A gravity column and let it flow out by gravity; Wash the Protein A gravity column with 3 column volumes of PBS solution, and then elute with 5 volumes of 0.1 M glycine-hydrochloric acid solution (pH = 3.0); Place the eluate in a 30KD ultrafiltration concentration tube, fill it with PBS, centrifuge at 3500 rpm at 4 °C for 40 minutes, discard the waste liquid in the collection tube, add 20 mL of PBS solution, centrifuge at 3500 rpm at 4 °C for 40 minutes, and aspirate the concentrated and replaced antibody solution to measure the antibody protein concentration.

[0040] 3. Neutralization experiment of SARS-CoV-2 pseudovirus HEK-293T cells and HEK-293T-hACE2 cells were cultured in DMEM medium containing 10% fetal bovine serum, 1% HEPES buffer, and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator. The SARS-CoV-2 pseudovirus was generated by co-transfecting HEK-293T cells with 10 μg of the SARS-CoV-2 spike protein expression plasmid and 20 μg of the env-deficient HIV-1 backbone vector plasmid (pNL4-3.Luc.R-E-). The SARS-CoV-2 spike protein expression plasmid was synthesized and prepared by GenScript Biotech Corporation; the point mutation plasmid was constructed using a mutation kit (Vazyme).

[0041] The specific experimental steps for pseudovirus preparation were as follows: When the confluence of HEK-293T cells in a T75 cell culture flask reached about 80%, the culture medium was aspirated, the cells were digested with trypsin, resuspended in the culture medium, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the cells were resuspended in 10 mL of the culture medium, and after counting, 6×10 6 cells were transferred to a new T75 cell culture flask and cultured overnight; Two 1.5 mL centrifuge tubes were taken, each containing 400 μL of serum-free medium, 120 μL of PEI transfection reagent and 20 μg of pNL4-3.Luc.R-E- plus 10 μg of the SARS-CoV-2 spike protein expression plasmid were added respectively, after mixing, they were left standing at room temperature for 5 minutes and then the two were mixed, and left standing at room temperature for 20 minutes; The transfection reagent was added to the HEK-293T T75 cell flask, after mixing, it was cultured in the incubator for about 7 hours, the culture medium was aspirated, fresh culture medium was added, and the culture was continued for 48 hours; The cell culture medium containing the pseudovirus was aspirated, transferred to a 50 mL centrifuge tube, centrifuged at 3000 rpm for 10 minutes, the supernatant was taken and filtered through a 0.45 μm filter, and then aliquoted and stored frozen at -80°C in a low-temperature refrigerator for later use.

[0042] To determine the antibody neutralization activity, in a 96-well plate, the monoclonal antibody was serially diluted 8 times at a 5-fold dilution starting from 100 μg / mL, an equal volume of diluted pseudovirus was added, and incubated at 37°C for 1 hour. Subsequently, 100 μL of HEK-293T-hACE2 cells, i.e., containing 30,000 cells, were added to each well. After culturing in a 37°C, 5% CO2 incubator for 48 hours, the cell culture medium was removed and 100 μL of Bright Lite luciferase reagent (Vazyme Biotech) was added to the cell wells. After incubating at room temperature for 2 minutes, the chemiluminescence signal was detected using a multifunctional microplate reader. Using GraphPad Prism8.0 software, the 50% inhibitory concentration (IC 50 ) was calculated by the logarithm (inhibitor) vs. normalized response - variable slope (four-parameter) model.

[0043] 4. Detection of antibody binding activity by ELISA Add SARS-CoV-2 BA.1-Spike, BA.1-S1, BA.1-NTD, BA.1-RBD (Sino Biological) and WT-Spike proteins into a 96-well ELISA plate at 2 μg / mL and 100 μL per well respectively, and coat overnight at 4°C. Wash 5 times with PBST (PBS solution containing 0.5% Tween-20); Block at room temperature for 1 hour with the blocking solution (blocking solution formula: 5% non-fat milk + 2% BSA (prepared with PBS)), 200 μL per well, then wash 3 times with PBST. The formula of all subsequent antibody diluents is the same as that of the blocking solution; Prepare the antibody to be detected at 10 μg / mL, add it to the 96-well plate, 100 μL per well, and incubate at 37°C for 1 hour. Wash 5 times with PBST; Add HRP-labeled goat anti-human IgG antibody (diluted 1:5000), 100 μL per well, and incubate at 37°C for 1 hour. Then wash 5 times with PBST; Add 100 μL of TMB chromogenic solution (Sangon Biotech) to each well and react at room temperature in the dark for 20 minutes; Then terminate the reaction with 50 μL of 2M H2SO4. Measure the optical density at 450 nm (OD) using a Varioskan LUX multimode microplate reader (Thermo Scientific).

[0044] 5. Surface Plasmon Resonance (SPR) Binding Analysis Use a Biacore 8K system (GE Healthcare) to perform the binding experiment of SARS-CoV-2 BA.1-Spike (SinoBiological) and WT-Spike proteins with ConBA-998 antibody. The specific steps are as follows: Dissolve SARS-CoV-2 BA.1-Spike (Sino Biological) and WT-Spike proteins with 10 mM sodium acetate buffer (pH = 5.0), and covalently coat one flow cell of a CM5 sensor chip to obtain a final response unit (RU) of about 500, while the other flow cell remains uncoated and is used as a control after blocking. All analyses are performed at a flow rate of 30 μL / min in HBS-EP buffer (10 mM HEPES pH = 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% Tween-20). Inject serial dilutions of ConBA-998 antibody for 60 seconds respectively, and the obtained data are fitted in a 1:1 binding model using Biacore evaluation software (GE Healthcare). Each measurement is performed twice, and a single value is used to generate the average affinity constant.

[0045] II. Results and Analysis 1. Isolation of Monoclonal Antibodies SARS-CoV-2-specific IgG+ or IgA+ single memory B cells were isolated from the peripheral blood of a volunteer who had not been vaccinated against COVID-19 and had been infected with the SARS-CoV-2 Omicron BA.1 strain once by flow cytometry. A fully human monoclonal antibody, ConBA-998, was finally obtained using RT-PCR and nested PCR techniques.

[0046] The sequences of the heavy-chain variable region and the light-chain variable region of the monoclonal neutralizing antibody ConBA-998 isolated in this case, as well as the sequencing results, are shown in Tables 1 and 2. Among them, Table 1 shows the amino acid sequences of the heavy-chain variable region and the light-chain variable region of the monoclonal antibody, and Table 2 shows the nucleotide sequencing results after PCR amplification of the heavy-chain variable region and the light-chain variable region of the monoclonal antibody.

[0047] Table 1 Amino acid sequences of monoclonal antibodies

[0048] Table 2 Nucleotide sequencing results of monoclonal antibodies

[0049] The analysis results showed that the sequences of CDR1, CDR2, and CDR3 of the heavy chain of ConBA-998 were "GFTVSSNY", "IYSGGDT", and "ARSPTPPDGFDI" in sequence, and the sequences of CDR1, CDR2, and CDR3 of the light chain were "NIGSYS", "YDS", and "QVWDSSSDHWV" in sequence.

[0050] 2. Results of SARS-CoV-2 pseudovirus neutralization experiment The neutralizing activity of the monoclonal antibody ConBA-998 against the original strain WT and Omicron BA.1 of SARS-CoV-2 was analyzed by pseudovirus neutralization experiment. The results are as Figure 1 shown. The monoclonal antibody ConBA-998 could block the infection of the target cells by the SARS-CoV-2 pseudovirus BA.1, and the neutralizing activity had an obvious concentration dependence. This antibody had strong neutralizing activity against BA.1, with an IC 50 of 0.003 μg / mL, but did not neutralize WT. The results indicated that the monoclonal antibody ConBA-998 was a specific neutralizing antibody against the SARS-CoV-2 Omicron BA.1.

[0051] 3. Detection of the binding activity of monoclonal antibodies In this case, ELISA was used to detect the binding activity of the monoclonal antibody ConBA-998 to BA.1-Spike, BA.1-S1, BA.1-NTD, BA.1-RBD, and WT-Spike. The results are as Figure 2As shown, ConBA-998 can effectively bind to BA.1-Spike, BA.1-S1, and BA.1-RBD, but not to BA.1-NTD and WT-Spike, indicating that this antibody targets the RBD region of BA.1.

[0052] 4. Surface Plasmon Resonance (SPR) Results The affinity of ConBA-998 for SARS-CoV-2 BA.1-Spike and WT-Spike proteins was detected by surface plasmon resonance technology, and the results are as Figure 3 shown (K on : association rate constant; K off : dissociation rate constant; K D : affinity constant). ConBA-998 has a high affinity for BA.1-Spike, with a KD of 1.41 nM, which is consistent with the neutralization results, indicating a certain correlation between affinity and neutralization.

[0053] 5. Neutralization Experiment Results of Monoclonal Neutralizing Antibody ConBA-998 against 23 SARS-CoV-2 BA.1 Point Mutation Pseudoviruses Carrying Single-Point Reverse Mutations To identify the key sites affecting the neutralization of ConBA-998 antibody, in this example, 23 pseudoviruses with single-point amino acid reverse mutations were constructed based on SARS-CoV-2 BA.1-Spike, and the neutralization of monoclonal neutralizing antibody ConBA-998 against these 23 point mutation pseudoviruses was evaluated. The results are as Figure 4 shown. Most point mutations do not affect the neutralization activity of ConBA-998. S496G and R498Q significantly decrease the neutralization activity of ConBA-998, while R493Q, Y501N, and H505Y completely inactivate ConBA-998. This study shows that the neutralization of ConBA-998 depends on the sites R493, S496, R498, Y501, and H505, which to a certain extent proves the immunogenicity of these sites and can induce effective neutralizing antibodies.

[0054] In summary, the monoclonal neutralizing antibody ConBA-998 induced by the initial infection of SARS-CoV-2 BA.1 is a highly efficient BA.1 neutralizing antibody, and its recognition mainly depends on the sites R493, S496, R498, Y501, and H505, providing a new candidate and reference for the prevention and treatment of the novel coronavirus.

[0055] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A novel coronavirus monoclonal neutralizing antibody, characterized in that, The novel coronavirus monoclonal neutralizing antibody is ConBA-998, and this monoclonal neutralizing antibody consists of a heavy chain and a paired light chain; The amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain of ConBA-998 are "GFTVSSNY", "IYSGGDT", and "ARSPTPPDGFDI" in sequence, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are "NIGSYS", "YDS", and "QVWDSSSDHWV" in sequence.

2. The novel coronavirus monoclonal neutralizing antibody according to claim 1, characterized in that, The variable region sequence of the heavy chain of ConBA-998 is the sequence shown in Seq ID No.1, and the variable region sequence of the light chain is the sequence shown in Seq ID No.

2.

3. A nucleic acid fragment encoding the novel coronavirus monoclonal neutralizing antibody according to claim 1 or 2, characterized in that, The nucleic acid fragment encodes the heavy chain and light chain of ConBA-998.

4. The nucleic acid fragment according to claim 3, wherein It contains a nucleic acid fragment encoding the variable region of the heavy chain shown in Seq ID No.1, and its sequence is the sequence shown in Seq ID No.3; a nucleic acid fragment encoding the variable region of the light chain shown in Seq ID No.2, and its sequence is the sequence shown in Seq ID No.

4.

5. A recombinant plasmid containing the nucleic acid fragment according to claim 3 or 4.

6. A recombinant cell containing the nucleic acid fragment according to claim 3 or 4 or the recombinant plasmid according to claim 5.

7. The preparation method of the monoclonal neutralizing antibody against the novel coronavirus according to claim 1 or 2, characterized in that, It includes performing protein expression on the recombinant plasmid according to claim 5 using the recombinant cell according to claim 6, extracting and purifying the expressed protein, and thus obtaining the novel coronavirus monoclonal neutralizing antibody.

8. The use of the novel coronavirus monoclonal neutralizing antibody according to claim 1 or 2, or the nucleic acid fragment according to claim 3 or 4, or the recombinant plasmid according to claim 5, or the recombinant cell according to claim 6 in the preparation of a drug for preventing and treating novel coronavirus or a novel coronavirus detection reagent.

9. A drug for preventing and treating novel coronavirus, characterized in that, The drug for preventing and treating novel coronavirus contains the novel coronavirus monoclonal neutralizing antibody according to claim 1 or 2.

10. A novel coronavirus detection reagent, characterized in that, The novel coronavirus detection reagent contains the novel coronavirus monoclonal neutralizing antibody according to claim 1 or 2.

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