VacBB-548, a monoclonal antibody that broadly neutralizes SARS-CoV-2, and its applications

Abstract

The present invention discloses a monoclonal antibody VacBB-548 that broadly neutralizes SARS-CoV-2 and its applications. The monoclonal antibody consists of a heavy chain and a paired light chain; the amino acid sequences of the CDR1, CDR2, and CDR3 of the heavy chain are "EFTVSSNY," "IYPGGST," and "ARDFGDLYFDY," respectively; and the amino acid sequences of the CDR1, CDR2, and CDR3 of the light chain are "QSVSSY," "GAS," and "QQYGSSPRT," respectively. VacBB-548, a novel coronavirus monoclonal antibody of the present invention, has cross-neutralizing and binding activity against eight novel coronaviruses, including multiple subtypes of Omicron variants, and its neutralizing effect is superior to that of neutralizing antibodies isolated from existing inactivated vaccines, providing a new option for the prevention and treatment of novel coronaviruses.

Description

Technical Field

[0001] The present invention belongs to the technical field of novel coronavirus treatment, and specifically relates to a monoclonal antibody VacBB-548 that broadly neutralizes SARS-CoV-2 and its application. Background Art

[0002] The Spike protein is a trimeric transmembrane glycoprotein and the most important surface membrane protein of the novel coronavirus (SARS-CoV-2). It consists of two subunits, S1 and S2. S1 primarily comprises the N-terminal domain (NTD) and receptor binding domain (RBD), responsible for recognizing and binding to host cell receptors; S2 contains essential elements required for membrane fusion. During viral infection, the S1 subunit binds to the host cell's angiotensin-converting enzyme 2 (ACE2), allowing the virus to attach to the host cell surface. The serine protease TMPRSS2 activates the S protein. The host cell's Furin enzyme then cleaves the S protein at the S1 / S2 site. Virus-receptor binding destabilizes the pre-fusion S protein trimer, causing the S1 subunit to detach and the S2 subunit to transition to a stable post-fusion conformation, driving the fusion of the viral and cellular membranes and allowing the virus to enter the host cell. Neutralizing antibodies (nAbs) inhibit viral entry into host cells by blocking the binding of the spike protein to the cellular receptor ACE2. The RBD on the spike protein directly binds to ACE2 and is the most important target for neutralizing antibodies. Mutations in the RBD often affect viral infection and transmission, and can even allow the virus to escape neutralizing antibodies induced by natural infection or vaccination.

[0003] Like natural viral infection, vaccination can induce an effective humoral immune response in the human body, and therefore plays a key role in preventing viral infection. Furthermore, multiple vaccinations can continuously stimulate the human immune system and induce increasingly higher levels of neutralizing antibodies, especially some broad-spectrum neutralizing antibodies. However, the continuous emergence of new coronavirus variants, especially the Omicron variant, has greatly undermined the immune barrier established by the vaccine and significantly escaped the neutralizing antibodies produced in the vaccine recipients. Currently, there are many literature reports that vaccine booster immunity can prevent viral escape to a certain extent, but the characteristics of the antibodies that play a key role in this are still unclear, especially in the bodies of inactivated vaccine recipients. At the same time, there are currently few reports of broad-spectrum neutralizing antibodies isolated from inactivated vaccine recipients, especially those that can neutralize multiple Omicron subtype variants.

[0004] Therefore, in order to cope with the continuous evolution and recombination of SARS-CoV-2 around the world and the potential risk of neutralizing antibody escape, the development of new novel coronavirus antibodies remains a research focus and difficulty in this field. Summary of the Invention

[0005] The purpose of the present invention is to provide a monoclonal antibody VacBB-548 that broadly neutralizes SARS-CoV-2 and its application.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention discloses a monoclonal antibody that broadly neutralizes SARS-CoV-2. The monoclonal antibody that broadly neutralizes SARS-CoV-2 is VacBB-548, which consists of a heavy chain and a paired light chain; the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain are "EFTVSSNY," "IYPGGST," and "ARDFGDLYFDY," in sequence; and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are "QSVSSY," "GAS," and "QQYGSSPRT," in sequence.

[0008] It should be noted that the key to the present invention is the cloning and identification of a monoclonal neutralizing antibody, VacBB-548, from specific single memory B cells in the peripheral blood of volunteers who received three doses of an inactivated vaccine. VacBB-548 neutralizes a variety of SARS-CoV-2 pseudovirus variants, including the novel coronavirus Omicron variant, specifically WT (wild type), Beta, Delta, BA.1, BA.1.1, BA.2, BA.2.12.1, and BA.4 / 5. Furthermore, compared to previously reported neutralizing antibodies isolated from inactivated vaccines, the VacBB-548 monoclonal antibody of the present invention exhibits a stronger neutralizing effect.

[0009] In one implementation of the present invention, the heavy chain variable region sequence of the novel coronavirus monoclonal antibody VacBB-548 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.

[0010] It should be noted that the heavy chain sequence variable region and light chain sequence variable region of the above specific sequences are only the monoclonal antibody sequences specifically adopted in one implementation of the present invention; it is understood that for monoclonal antibodies, the region that affects its precise complementarity with the antigenic determinant is the complementarity determining region (abbreviated as CDR), specifically, the CDR1, CDR2 and CDR3 of the heavy chain sequence, and the CDR1, CDR2 and CDR3 of the light chain sequence; therefore, as long as the CDR1, CDR2 and CDR3 sequences of the heavy chain sequence and light chain sequence of the present invention remain unchanged, the functions and effects of the novel coronavirus monoclonal antibody of the present invention can be basically achieved. In other words, the specific sequences of the novel coronavirus monoclonal antibody of the present invention are not limited to the heavy chain sequence variable region and light chain sequence variable region of the above specific sequences.

[0011] The second aspect of the present invention discloses a nucleic acid fragment encoding the monoclonal antibody that broadly neutralizes SARS-CoV-2 of the present invention, wherein the nucleic acid fragment encodes the heavy chain and light chain of VacBB-548.

[0012] In one implementation of the present invention, the nucleic acid sequence encoding the variable region of the heavy chain sequence shown in Seq ID No.1 is shown in Seq ID No.3, and the nucleic acid sequence encoding the variable region of the light chain sequence shown in Seq ID No.2 is shown in Seq ID No.4.

[0013] It should be noted that the above specific nucleic acid sequence is only a nucleic acid sequence specifically used in one implementation of the present invention. It can be understood that there can be multiple codons for one amino acid; therefore, based on the degeneracy of the codons, while ensuring that the coding sequence remains unchanged, in addition to the nucleic acid sequences defined above, there can also be several nucleic acid sequences encoding the same heavy chain or light chain, all of which are within the scope of protection of the present invention.

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

[0015] It should be noted that the recombinant plasmid of the present invention is designed to effectively express the nucleic acid fragment of the present invention, thereby obtaining the corresponding heavy chain, light chain or novel coronavirus monoclonal antibody; therefore, in principle, any vector that can transfect the nucleic acid fragment into a host cell for nucleic acid expression can be used in the present invention.

[0016] 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.

[0017] It should be noted that the recombinant cells of the present invention refer to host cells transfected with the nucleic acid fragments or recombinant plasmids of the present invention; the heavy chains, light chains or novel coronavirus monoclonal antibodies of the present invention can generally be obtained by directly culturing such host cells.

[0018] The fifth aspect of the present invention discloses a method for preparing a monoclonal antibody that broadly neutralizes SARS-CoV-2 according to the present invention, comprising using the recombinant cell of the present invention to express the recombinant plasmid of the present invention, extracting and purifying the expressed protein, thereby obtaining the monoclonal antibody that broadly neutralizes SARS-CoV-2 according to the present invention.

[0019] The sixth aspect of the present invention discloses the use of the monoclonal antibody of the present invention that broadly neutralizes SARS-CoV-2, 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 new coronavirus prevention and treatment drugs and new coronavirus detection reagents.

[0020] It should be noted that the novel coronavirus monoclonal antibodies of the present invention have good cross-neutralizing activity against different variants, especially against multiple subtypes of Omicron; therefore, they can be used to prepare corresponding novel coronavirus antibody drugs, or other drugs with similar functions for preventing and treating novel coronavirus. Similarly, the cross-neutralizing activity of the monoclonal antibodies of the present invention against different variants can also be used to detect corresponding novel coronavirus variants. As for nucleic acid fragments, recombinant plasmids and recombinant cells, these can be used as raw materials for preparing novel coronavirus monoclonal antibodies, and thus for preparing novel coronavirus prevention and treatment drugs and novel coronavirus detection reagents.

[0021] The seventh aspect of the present invention discloses a novel coronavirus detection reagent, which contains the monoclonal antibody of the present invention that broadly neutralizes SARS-CoV-2, or contains an antigen that can specifically bind to the monoclonal antibody of the present invention that broadly neutralizes SARS-CoV-2.

[0022] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0023] The novel coronavirus monoclonal antibody VacBB-548 of the present invention has cross-neutralizing activity against eight novel coronaviruses, including multiple sublineages of Omicron variants, and its neutralizing effect is superior to the neutralizing antibodies isolated from existing inactivated vaccines, providing a new option for the prevention and treatment of novel coronaviruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are the results of neutralization tests of VacBB-548 against different pseudoviruses of SARS-CoV-2 in the examples of the present invention.

[0025] Figure 2 This is the epitope identification result of VacBB-548 in the examples of the present invention. DETAILED DESCRIPTION

[0026] The present invention uses the novel coronavirus RBD protein as bait, separates single B cells from volunteers who have received three doses of inactivated vaccine (Sinopharm Group) by flow cytometry, and clones and identifies a monoclonal neutralizing antibody, VacBB-548, which is effective against the novel coronavirus WT strain (wild type) pseudovirus IC 50 The range is 0.007 μg / mL. The heavy chain of VacBB-548 belongs to the 3-53 family, which is often enriched in convalescent individuals and is known as a common antibody.

[0027] The present invention further used competitive ELISA to predict the binding epitope of the neutralizing antibody VacBB-548 by comparing it with ACE2 and four representative neutralizing antibodies (Class 1: P2C-1F11, Class 2: BD-368-2, Class 3: S309, and Class 4: EY6A). The results showed that VacBB-548 exhibited strong competition with both ACE2 and the Class 1 antibody P2C-1F11, indicating that it belongs to Class 1 antibodies.

[0028] Finally, the present invention tested the cross-neutralizing activity of VacBB-548 against WT, Beta, Delta, and Omicron subtype variants BA.1, BA.1.1, BA.2, BA.2.12.1, and BA.4 / 5 of the novel coronavirus. The results showed that VacBB-548 maintained high neutralizing activity against all tested pseudovirus variants, with neutralizing capacity superior to that of most reported monoclonal neutralizing antibodies.

[0029] The present invention isolated and characterized a monoclonal neutralizing antibody from individuals who received a wild-type inactivated vaccine. Its germline gene usage, epitope recognition, neutralizing potency, and cross-neutralization against SARS-CoV-2 variants were similar to those of neutralizing antibodies induced by natural viral infection. Furthermore, the present invention demonstrated the ability of the inactivated vaccine to induce broadly neutralizing monoclonal antibodies and obtained a highly potent and broadly neutralizing antibody, VacBB-548, which can be used as a candidate therapeutic antibody and also for passive prevention of various SARS-CoV-2 variants.

[0030] The present invention will be further described in detail below by means of specific embodiments in conjunction with the accompanying drawings. The following examples are only provided to further illustrate the present invention and should not be construed as limiting the present invention. Unless otherwise specified, the instruments and materials used in the following examples are all conventional laboratory equipment, and the technical solutions are all conventional techniques in the art.

[0031] Example

[0032] 1. Materials and Methods

[0033] 1. Study Approval and Biological Samples

[0034] This study was approved by the Ethics Committee of Shenzhen Third People's Hospital (approval number: 2021-030). All participants provided written informed consent for sample collection and subsequent analysis. Plasma and peripheral blood mononuclear cell (PBMC) samples were collected approximately 2 weeks after the third vaccination with the inactivated SARS-CoV-2 vaccine (Beijing Institute of Biological Products Co., Ltd. BBIBP-CorV) from Shenzhen Third People's Hospital. All plasma samples were stored at −80°C and heat-inactivated at 56°C for 1 hour before use. PBMCs were stored in liquid nitrogen.

[0035] In this example, one sample was collected. The sample collection method is as follows:

[0036] Isolation of peripheral blood mononuclear cells: Collect 10 mL of venous blood using an anticoagulant tube, transfer to a 50 mL centrifuge tube, dilute with 10 mL of PBS, and gently mix. Add 5 mL of Ficoll separation buffer to each of two 15 mL centrifuge tubes. Add 10 mL of diluted blood to the top layer of the Ficoll separation buffer. Centrifuge at 2000 rpm for 20 minutes. Aspirate the white blood cell layer into a clean 15 mL centrifuge tube. Add PBS to 10 mL, centrifuge at 1500 rpm for 10 minutes, remove the supernatant, resuspend in 3 mL of cell freezing buffer, and transfer 1 mL of cells to each of three cryovials. Place in a cryovial box and freeze at -80°C overnight. The next day, transfer the cells to liquid nitrogen for long-term storage.

[0037] 2. Isolation of Monoclonal Antibodies from B Cells of Inactivated Vaccine Recipients

[0038] Frozen peripheral blood mononuclear cells were thawed and washed twice with 10 mL of PBS. A mixture of CD3-Pacific Blue, CD8-Pacific Blue, CD14-Pacific Blue, CD19-PE-Cy7, CD27-APC-H7, IgG-FITC (all from BD Biosciences), and a His-tagged 2019-nCoV RBD (Sino Biological) probe was added to 100 μL of staining buffer (PBS + 2% fetal bovine serum). The peripheral blood mononuclear cells were resuspended and stained at 4°C for 30 minutes. After washing twice with PBS, APC- and PE-labeled anti-His tag secondary antibodies (Abcam) were added to 100 μL of staining buffer (PBS + 2% fetal bovine serum) and the cells were stained at 4°C for 30 minutes. After washing twice with PBS, 2019-nCoV RBD-specific IgG+ B cells were sorted using a BD FACS Aria II sorting flow cytometer.

[0039] Single B cells were sorted into 96-well PCR plates containing lysis buffer. RT-PCR and nested PCR were then performed according to the method described in "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" to amplify the heavy and light chain variable regions, respectively. The PCR amplification products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the resulting antibody was named VacBB-548.

[0040] The antibody variable region sequences obtained by sequencing were sent to GenScript Biotech Co., Ltd. for synthesis. The company cloned the variable regions of the antibody heavy and light chains into the full-length IgG1 heavy and light chain expression vectors pcDNA3.4 (GenScript Biotech Co., Ltd.), respectively, and prepared a large number of antibody heavy and light chain plasmids. After obtaining the plasmids prepared by GenScript, the paired heavy and light chain expression plasmids were co-transfected into 293F cells (500 mL as an example) using PEI transfection reagent for expression, and the monoclonal antibodies were purified from the culture supernatant using a protein A adsorption column. The specific steps are as follows:

[0041] 293F cells were cultured in an 8% CO2, 37°C incubator and the 293F cell concentration was adjusted to 1.2×106 / mL, continue to culture for 2 hours; prepare solution A: add 250μg antibody heavy chain plasmid and 250μg antibody light chain plasmid to 12.5mL opti-MEM (31985070, Gibco); prepare solution B: add 2.5mL 1mg / mL PEI transfection reagent (24885-2, Polyscieces) to 12.5mL opti-MEM and let it stand for 5 minutes; mix solution A and solution B, let it stand for 20 minutes to obtain AB mixed solution; add 25mL AB mixed solution dropwise to 500mL 293F cells, shaking while adding dropwise; continue to culture the cells for 5 days; then centrifuge the 293F cells at 3000g for 20 minutes, collect the supernatant, and filter with a 0.45μm filter membrane; open the lid of the ProteinA gravity column, use gravity to let the 20% ethanol solution in the column completely flow out, and use 5 times the column volume of 10mM PBS solution Balance the Protein A gravity column; 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 times the column volume of PBS solution, and then elute with 5 times the volume of 0.1M glycine-hydrochloric acid solution (pH = 3.0); place the eluate in a 30KD ultrafiltration concentration tube, fill it up 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, aspirate the concentrated and replaced antibody solution, and measure the antibody protein concentration.

[0042] 3. Competitive ELISA to detect antibody epitopes

[0043] ACE2 protein and P2C-1F11, BD-368-2, S309, and EY6A antibodies were labeled using an HRP labeling kit (ab102890, Abcam). The specific steps are as follows:

[0044] Dilute 100 μg of the protein or antibody to be labeled to 100 μL with PBS, add 10 μL Modifier reagent, and mix gently by pipetting. Open the bottle cap of the HRP coupling mixture, use a pipette tip to draw up the antibody sample (with Modifier reagent added), add it directly to the lyophilized powder material, and gently resuspend. Cover the bottle cap and place it in the dark at room temperature (20-25°C) for 3 hours. After incubation for 3 hours (or longer), add 1 μL Quencher reagent to each 10 μL of antibody in the reaction and mix gently. After 30 minutes, the coupled antibody can be used. Add 100 μL glycerol, mix well, and store at -20°C (approximately 0.5 μg / mL).

[0045] SARS-CoV-2 RBD protein (40592-V08B, Sino Biological) was added to a 96-well microtiter plate at 2 μg / mL, 100 μL per well, and coated overnight at 4°C. The plates were washed five times with PBST (PBS solution containing 0.5% Tween-20); the plates were blocked with blocking solution at room temperature for 1 hour (blocking solution formula: 5% skim milk + 2% BSA (prepared in PBS)), 200 μL per well, and then washed five times with PBST. All subsequent antibody dilutions were made with the same blocking solution formula; the antibody to be tested was prepared at 20 μg / mL and then mixed with HRP-labeled ACE2 and four representative neutralizing antibodies, namely P2C-1F11, BD-368-2, S309, and EY6A, in equal volumes. The plates were then added to a 96-well plate, 100 μL per well, and incubated at 37°C for 1 hour. The cells were then washed five times with PBST. A chromogenic solution (A) and a solution (B) (Sanggong) were mixed at a ratio of 1:1, with 100 μL added to each well and incubated at room temperature in the dark for 20 minutes. The reaction was then terminated with 50 μL of 2 M H₂SO₄. Optical density was measured at 450 nm (OD) using a Varioskan LUX multimode microplate reader (Thermo Scientific).

[0046] 4. Neutralization assay of SARS-CoV-2 pseudovirus

[0047] HEK-293T cells and HEK-293T-hACE2 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 1% HEPES buffer, and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. 20 μg of the 2019-nCoV spike protein expression plasmid and 20 μg of the env-deficient HIV-1 backbone vector plasmid (pNL4-3.Luc.RE-) were co-transfected into HEK-293T cells. The 2019-nCoV spike protein expression plasmid was synthesized by GenScript Biotech Co., Ltd.

[0048] The specific experimental steps for pseudovirus preparation are as follows: When the confluence of HEK-293T cells in the T75 cell culture flask reaches about 80%, the culture medium is aspirated and the cells are digested with trypsin. The cells are then resuspended in culture medium and centrifuged at 1000 rpm for 5 min. The supernatant is discarded and the cells are resuspended in 10 mL of culture medium. After counting, 6 × 10 6The cells were cultured overnight in a new T75 cell culture flask; 2 1.5 mL centrifuge tubes were taken, 400 μL of serum-free medium was added to each, 120 μL of PEI transfection reagent and 20 μg pNL4-3.Luc.RE- plus 10 μg of SARS-CoV-2 spike protein expression plasmid were added respectively, mixed and allowed to stand at room temperature for 5 minutes, then the two were mixed and allowed to stand at room temperature for 20 minutes; the transfection reagent was added to the HEK-293TT75 cell flask, mixed and cultured in an incubator for about 7 hours, the medium was discarded, fresh medium was added, and the culture was continued for 48 hours; the cell culture medium containing pseudovirus was aspirated and transferred to a 50 mL centrifuge tube, centrifuged at 3000 rpm for 10 minutes, the supernatant was filtered with a 0.45 μm filter, and aliquoted and frozen in a -80°C low-temperature refrigerator for later use.

[0049] To determine the neutralizing activity of the antibody, the monoclonal antibody was diluted 8 times in a 5-fold serial dilution starting at 100 μg / mL in a 96-well plate, and an equal volume of diluted pseudovirus was added and incubated at 37°C for 1 hour. Subsequently, 100 μL of HEK-293T-hACE2 cells was added to each well, containing 30,000 cells. 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 incubation at room temperature for 2 minutes, the chemiluminescent signal was detected using a multifunctional microplate reader. GraphPad Prism8.0 software was used to calculate the 50% inhibitory concentration (IC) by the logarithm (inhibitor) and standardized response-variable slope (four-parameter) model. 50 ).

[0050] 2. Results and Analysis

[0051] 1. Isolation of Monoclonal Antibodies

[0052] In this case, a monoclonal neutralizing antibody, VacBB-548, was isolated and obtained. The sequences of its heavy chain variable region and light chain variable region, as well as the sequencing results, are shown in Tables 1 and 2, respectively.

[0053] Table 1 Monoclonal antibody sequences

[0054]

[0055] Table 2 Monoclonal antibody nucleic acid sequencing results

[0056]

[0057]

[0058] The analysis results showed that the CDR1, CDR2 and CDR3 of the heavy chain sequence of VacBB-548 were "EFTVSSNY", "IYPGGST" and "ARDFGDLYFDY" in sequence, and the CDR1, CDR2 and CDR3 of the light chain sequence were "QSVSSY", "GAS" and "QQYGSSPRT" in sequence.

[0059] 2. SARS-CoV-2 pseudovirus neutralization test results

[0060] The neutralization activity of the novel coronavirus RBD protein-specific monoclonal antibody VacBB-548 against novel coronavirus variants was analyzed by pseudovirus neutralization experiments. In order to detect the effect of novel coronavirus variants (especially Omicron variants) on the neutralization activity of VacBB-548, pseudoviruses of Beta, Delta and Omicron subtype variants BA.1, BA.1.1, BA.2, BA.2.12.1, and BA.4 / 5 were prepared in this example. By comparing the neutralization activity of wild-type viruses, the neutralization effect of monoclonal antibodies on mutant strains was analyzed. The results are shown in Figure 2. Figure 1 As shown, VacBB-548 can block the infection of the 2019-nCoV pseudovirus in target cells, and its neutralizing activity is clearly concentration-dependent, with strong neutralizing effects against variant pseudoviruses. This result indicates that VacBB-548 is a broad-spectrum neutralizing antibody against the 2019-nCoV with potential application.

[0061] 3. Epitope Identification of Monoclonal Antibodies

[0062] Previous studies have shown that RBD-specific neutralizing antibodies are divided into four categories based on competition with ACE2 and the recognition of up and down conformational epitopes on RBD; direct competition with ACE2 for binding to RBD is one of the important mechanisms by which neutralizing antibodies effectively block the binding of viruses to receptors. In this case, competitive ELISA was used to predict the binding epitopes of monoclonal antibodies based on their competition with ACE2 and representative antibodies of four types of epitopes (category 1: P2C-1F11, category 2: BD-368-2, category 3: S309, category 4: EY6A). The results are as follows Figure 2 As shown, VacBB-548 can compete with ACE2 and the class 1 antibody P2C-1F11, indicating that this antibody belongs to the class 1 antibody.

[0063] In summary, VacBB-548 is a broad-spectrum neutralizing antibody with good application potential, which can effectively deal with multiple SARS-CoV-2 variants.

[0064] The above content is a further detailed description of the present application in conjunction with specific implementation methods, and the specific implementation of the present application cannot be considered to be limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A monoclonal antibody that broadly neutralizes SARS-CoV-2, characterized in that: The monoclonal antibody that broadly neutralizes SARS-CoV-2 is VacBB-548, which consists of a heavy chain and a paired light chain; the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain are "EFTVSSNY," "IYPGGST," and "ARDFGDLYFDY," respectively; and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are "QSVSSY," "GAS," and "QQYGSSPRT," respectively.

2. The monoclonal antibody that broadly neutralizes SARS-CoV-2 according to claim 1, characterized in that The heavy chain variable region sequence of VacBB-548 is shown in Seq ID No. 1, and the light chain variable region sequence is shown in Seq ID No.

2.

3. A nucleic acid fragment encoding the monoclonal antibody that broadly neutralizes SARS-CoV-2 according to claim 1 or 2, characterized in that: The nucleic acid fragment encodes the heavy chain and light chain of VacBB-548.

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

4.

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

7. The method for preparing a monoclonal antibody that broadly neutralizes SARS-CoV-2 according to claim 1 or 2, characterized in that: The method comprises using the recombinant cell according to claim 6 to express the recombinant plasmid according to claim 5, extracting and purifying the expressed protein, thereby obtaining the monoclonal antibody that broadly neutralizes SARS-CoV-2.

8. Use of the broadly SARS-CoV-2 neutralizing monoclonal 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 novel coronavirus therapeutic drugs and novel coronavirus detection reagents.

9. A novel coronavirus detection reagent, characterized in that: Containing the monoclonal antibody that broadly neutralizes SARS-CoV-2 according to claim 1 or 2.

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