Monoclonal antibody VacBB-551 neutralizing a broad spectrum of SARS-CoV-2 and uses thereof

Abstract

The application discloses a monoclonal antibody VacBB-551 for neutralizing SARS-CoV-2 in a broad spectrum and an application thereof, which is composed of a heavy chain and a light chain matched with the heavy chain; amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain are 'EIIVSRNY', 'IYAGGST' and 'ARSLGDRFDF' in sequence, and amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are 'QGIPSY', 'AAS' and 'QHEDT' in sequence. The novel coronavirus monoclonal antibody VacBB-551 of the application has cross-neutralization and binding activity to nine kinds of novel coronaviruses including various subgroups of the Omicron variant, and the neutralization effect is better than that of the neutralizing antibody separated from an existing inactivated vaccine, thereby providing a new choice for prevention and treatment of the novel coronavirus.

Description

Technical Field

[0001] This invention belongs to the field of novel coronavirus treatment technology, specifically relating to a broad-spectrum neutralizing monoclonal antibody VacBB-551 for SARS-CoV-2 and its applications. Background Technology

[0002] Spike protein is a trimeric transmembrane glycoprotein and the most important surface membrane protein of the novel coronavirus (SARS-CoV-2). It contains two subunits, S1 and S2. S1 mainly contains an N-terminal domain (NTD) and a receptor-binding domain (RBD), responsible for recognizing and binding to host cell receptors. S2 contains the 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. Then, the host cell's furin enzyme cleaves the S protein at the S1 / S2 site. Virus-receptor binding destabilizes the pre-fused S protein trimer, causing the S1 subunit to detach and the S2 subunit to transform into a stable fused conformation. This drives the fusion of the viral membrane with the cell membrane, 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 recognition target of neutralizing antibodies. Mutations in the RBD often affect viral infection and transmission, and may even lead to viral escape and natural infection or antibody neutralization induced by vaccination.

[0003] Similar to natural viral infection, vaccination induces an effective humoral immune response in the human body, thus playing a crucial role in preventing viral infection. Multiple vaccinations continuously stimulate the immune system and induce increasingly higher levels of neutralizing antibodies, especially broad-spectrum neutralizing antibodies. However, the emergence of new SARS-CoV-2 variants, particularly the Omicron variant, significantly undermines the immune barrier established by vaccines, allowing neutralizing antibodies produced in vaccine recipients to escape. While numerous studies report that booster immunization can combat viral escape to some extent, the key antibody characteristics remain unclear, especially in recipients of inactivated vaccines. Furthermore, there are few reports on broad-spectrum neutralizing antibodies isolated from inactivated vaccine recipients, particularly those capable of neutralizing multiple Omicron subtype variants.

[0004] Therefore, in order to address the ongoing evolution and recombination of SARS-CoV-2 around the world and the potential risk of neutralizing antibody escape, developing new novel coronavirus antibodies remains a key research focus and challenge in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a broad-spectrum neutralizing monoclonal antibody, VacBB-551, for SARS-CoV-2 and its applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention discloses a broad-spectrum neutralizing monoclonal antibody against SARS-CoV-2, wherein the broad-spectrum neutralizing monoclonal antibody against SARS-CoV-2 is VacBB-551, and the monoclonal antibody is composed of a heavy chain and a light chain paired therewith; the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain are “EIIVSRNY”, “IYAGGST” and “ARSLGDRFDF” in sequence, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are “QGIPSY”, “AAS” and “QHEDT” in sequence.

[0008] It should be noted that the key to this invention lies in the cloning and identification of a monoclonal neutralizing antibody, VacBB-551, from peripheral blood-specific single memory B cells of volunteers who received three doses of inactivated vaccine. This antibody is capable of neutralizing various SARS-CoV-2 variant pseudoviruses, including the Omicron variant of the novel coronavirus, specifically WT (wild-type), Beta, Delta, BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4 / 5, and BA.2.75. Furthermore, compared to previously reported neutralizing antibodies isolated from inactivated vaccines, the VacBB-551 monoclonal antibody of this invention exhibits stronger neutralizing activity.

[0009] In one implementation of the present invention, the heavy chain variable region sequence of the novel coronavirus monoclonal antibody VacBB-551 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 variable regions of the heavy chain and light chain sequences in the above-mentioned specific sequences are merely monoclonal antibody sequences specifically used in one implementation of the present invention. It can be understood that for monoclonal antibodies, the regions affecting their precise complementarity with antigenic determinants are complementarity-determining regions (CDRs), specifically CDR1, CDR2, and CDR3 of the heavy chain sequence, and CDR1, CDR2, and CDR3 of the light chain sequence. Therefore, as long as the CDR1, CDR2, and CDR3 sequences of the heavy chain and light chain sequences of the present invention remain unchanged, the function and role of the novel coronavirus monoclonal antibody of the present invention can be basically achieved. In other words, the specific sequence of the novel coronavirus monoclonal antibody of the present invention is not limited to the variable regions of the heavy chain and light chain sequences in the above-mentioned specific sequences.

[0011] A second aspect of the invention discloses a nucleic acid fragment encoding a broad-spectrum neutralizing monoclonal antibody against SARS-CoV-2 of the invention, said nucleic acid fragment encoding the heavy and light chains of VacBB-551.

[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 specific nucleic acid sequences mentioned above are only the nucleic acid sequences 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 codons, in addition to the nucleic acid sequences defined above, there can be several other nucleic acid sequences that encode the same heavy or light chain, all of which are within the scope of protection of the present invention, while ensuring that the coding sequence remains unchanged.

[0014] A 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 plasmids of the present invention are designed to effectively express the nucleic acid fragments of the present invention, thereby obtaining the corresponding heavy chain, light chain, or novel coronavirus monoclonal antibody; therefore, in principle, any vector capable of transfecting the nucleic acid fragment into host cells for nucleic acid expression can be used in the present invention.

[0016] A 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; generally, the heavy chain, light chain or novel coronavirus monoclonal antibody of the present invention can be obtained by directly culturing such host cells.

[0018] The fifth aspect of the present invention discloses a method for preparing a broad-spectrum neutralizing monoclonal antibody against SARS-CoV-2, comprising expressing a protein in a recombinant plasmid of the present invention using recombinant cells of the present invention, extracting and purifying the expressed protein, thereby obtaining a broad-spectrum neutralizing monoclonal antibody against SARS-CoV-2 of the present invention.

[0019] The sixth aspect of the present invention discloses the use of the broad-spectrum neutralizing SARS-CoV-2 monoclonal antibody 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 the prevention and treatment of novel coronavirus and reagents for the detection of novel coronavirus.

[0020] It should be noted that the novel coronavirus monoclonal antibody of this invention exhibits good cross-neutralizing activity against different variants, especially against multiple Omicron subspecies; therefore, it can be used to prepare corresponding novel coronavirus antibody drugs, or other drugs with similar functions for the prevention and treatment of novel coronavirus. Similarly, the cross-neutralizing activity of the monoclonal antibody of this 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 the preparation of novel coronavirus monoclonal antibodies, thereby enabling the preparation of 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 containing a broad-spectrum neutralizing monoclonal antibody of the present invention for SARS-CoV-2, or containing an antigen capable of specifically binding to the broad-spectrum neutralizing monoclonal antibody of the present invention for SARS-CoV-2.

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

[0023] The novel coronavirus monoclonal antibody VacBB-551 of this invention has cross-neutralizing activity against nine novel coronaviruses, including multiple subspecies of the Omicron variant, 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 coronavirus. Attached Figure Description

[0024] Figure 1 The results of the neutralization test of VacBB-551 against different pseudoviruses of SARS-CoV-2 in this embodiment of the invention are shown.

[0025] Figure 2 The epitope identification results of VacBB-551 in this embodiment of the invention. Detailed Implementation

[0026] This invention uses the novel coronavirus RBD protein as bait to isolate single B cells from volunteers who have received three doses of an inactivated vaccine (Sinopharm) using flow cytometry. From these cells, a monoclonal neutralizing antibody, VacBB-551, was cloned and identified. This antibody targets the novel coronavirus WT strain (wild-type) pseudovirus IC. 50 The range is 0.004 μg / mL. The heavy chain of VacBB-551 belongs to family 3-53, whose antibodies are typically enriched in individuals recovering from COVID-19 and are referred to as common antibodies.

[0027] This invention further employs a competitive ELISA to predict the binding epitopes of the monoclonal antibody VacBB-551 by comparing it with ACE2 and four representative neutralizing antibodies (Category 1: P2C-1F11, Category 2: BD-368-2, Category 3: S309, and Category 4: EY6A). The results show that VacBB-551 exhibits high-intensity competition with both ACE2 and the Category 1 antibody P2C-1F11, indicating that it belongs to the Category 1 antibody group.

[0028] Finally, this invention tested the cross-neutralizing activity of VacBB-551 against the novel coronavirus WT, Beta, Delta, and Omicron subtype variants BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4 / 5, and BA.2.75. The results showed that VacBB-551 maintained high neutralizing activity against all tested variant pseudoviruses, exhibiting superior neutralizing capacity compared to most reported monoclonal neutralizing antibodies.

[0029] This invention isolated and identified a monoclonal neutralizing antibody from individuals receiving a wild-type inactivated vaccine. Its germline gene usage, epitope recognition, neutralizing potency, and cross-neutralization against SARS-CoV-2 variants are similar to those of neutralizing antibodies induced by natural viral infection. Furthermore, this invention demonstrates the ability of inactivated vaccines to induce a wide range of monoclonal neutralizing antibodies and yielded a highly effective, broad-spectrum neutralizing antibody, VacBB-551, which can serve as a candidate therapeutic antibody and also for passive protection against various SARS-CoV-2 variants.

[0030] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. The following embodiments are only for further illustration of 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 all commonly used laboratory equipment, and the technical solutions described are all conventional techniques in the field.

[0031] Example

[0032] I. Materials and Methods

[0033] 1. Research approval and biological samples

[0034] This study was approved by the Ethics Committee of Shenzhen Third People's Hospital (Approval No.: 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 two weeks after the third dose of the SARS-CoV-2 inactivated vaccine (BBIBP-CorV, Beijing Institute of Biological Products Co., Ltd.) administered at 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] This example specifically collected one sample, and the sample collection method is as follows:

[0036] Peripheral blood mononuclear cell isolation: Collect 10 mL of venous blood using an anticoagulant tube, transfer it to a 50 mL centrifuge tube, dilute with 10 mL of PBS solution, and mix gently. Take two 15 mL centrifuge tubes and add 5 mL of Ficoll separation solution to each. Then add 10 mL of diluted blood to the supernatant of the Ficoll separation solution. Centrifuge at 2000 rpm for 20 minutes. Transfer the leukocyte layer to a clean 15 mL centrifuge tube. Add 10 mL of PBS, centrifuge at 1500 rpm for 10 minutes, remove the supernatant, resuspend the cells in 3 mL of cell cryopreservation solution, and transfer 1 mL of cells from each tube to three cryovials. Place the tubes in a cryovial box and incubate 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 resuscitated and washed twice with 10 mL of PBS. Peripheral blood mononuclear cells were resuspended in 100 μL of staining buffer (PBS + 2% fetal bovine serum) with 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 novel coronavirus WT strain RBD (Sino Biological) probe. The cells were stained at 4°C for 30 minutes. After washing twice with PBS, cells were stained at 4°C for 30 minutes with APC and PE-labeled anti-His-tagged secondary antibody (Abcam) in 100 μL of staining buffer (PBS + 2% fetal bovine serum). After washing twice with PBS, novel coronavirus WT strain 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. Then, RT-PCR and nested PCR were performed according to the method described 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." to amplify the variable regions of the heavy and light chains, respectively. The PCR amplification products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the resulting antibody was named VacBB-551.

[0040] The antibody variable region sequence obtained from sequencing was sent to GenScript Biotech for synthesis. GenScript then 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), respectively, and prepared large quantities 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. The monoclonal antibody was then 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 cell concentration was adjusted to 1.2 × 10⁻⁶ cells / year.6 Cells were cultured at 1 / mL for 2 hours. Solution A was prepared by adding 250 μg of antibody heavy chain plasmid and 250 μg of antibody light chain plasmid to 12.5 mL of Opti-MEM (31985070, Gibco). Solution B was prepared by adding 2.5 mL of 1 mg / mL PEI transfection reagent (24885-2, Polysciences) to 12.5 mL of Opti-MEM and incubating for 5 minutes. Solution A and Solution B were mixed and incubated for 20 minutes to obtain the AB mixture. 25 mL of the AB mixture was added dropwise to 500 mL of 293F cells, shaking constantly. Cells were cultured for 5 days. The 293F cells were then centrifuged at 3000g for 20 minutes. The supernatant was collected and filtered through a 0.45 μm filter. The cap of the Protein A gravity column was opened, and gravity was used to allow the 20% ethanol solution in the column to completely drain. The solution was then diluted with 5 column volumes of 10 mM PBS. Equilibrate the Protein A gravity column; add filtered cell supernatant to the Protein A gravity column, allowing it to flow out under gravity; wash the Protein A gravity column with 3 column volumes of PBS solution, then elute with 5 volume volumes of 0.1M glycine-hydrochloric acid solution (pH=3.0); place the eluent in a 30KD ultrafiltration concentration tube, fill with PBS, centrifuge at 3500rpm for 40 minutes at 4°C, discard the waste liquid in the collection tube, add 20mL of PBS solution, centrifuge at 3500rpm for 40 minutes at 4°C, aspirate the concentrated and replaced antibody solution, and determine the antibody protein concentration.

[0042] 3. Competitive ELISA method for detecting antibody epitopes

[0043] ACE2 protein and antibodies against P2C-1F11, BD-368-2, S309, and EY6A 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 of modifier reagent, and gently pipette to mix. Open the cap of the HRP conjugation mixture bottle, and use a pipette tip to draw up the antibody sample (with modifier reagent added), adding it directly to the lyophilized powder material and gently resuspending. Cap the bottle and incubate at room temperature (20–25°C) in the dark for 3 hours. After incubation for 3 hours (or longer), add 1 μL of Quencher reagent to every 10 μL of antibody in the reaction and gently mix. After 30 minutes, the conjugated antibody is ready for use. Add 100 μL of 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 96-well microplates at a concentration of 2 μg / mL, 100 μL per well, and incubated overnight at 4°C. The plates were washed 5 times with PBST (PBS solution containing 0.5% Tween-20); then blocked at room temperature for 1 hour with blocking buffer (blocking buffer formulation: 5% skim milk + 2% BSA (prepared in PBS)), 200 μL per well, followed by 5 washes with PBST. All subsequent antibody dilution buffer formulations were the same as the blocking buffer. The antibody to be tested was prepared to 20 μg / mL, and then mixed with equal volumes of HRP-labeled ACE2 and four representative neutralizing antibodies, namely P2C-1F11, BD-368-2, S309, and EY6A. This mixture was then added to 96-well plates at 100 μL per well and incubated at 37°C for 1 hour. Wash five times with PBST; mix 100 μL of chromogenic solution A and solution B (Sangon Biotech) at a 1:1 ratio and incubate 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).

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

[0047] 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 novel coronavirus pseudovirus was generated by co-transfecting HEK-293T cells with 10 μg of the novel coronavirus spike protein expression plasmid and 20 μg of the env-deficient HIV-1 backbone vector plasmid (pNL4-3.Luc.RE-). The novel coronavirus spike protein expression plasmid was synthesized and prepared 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 approximately 80%, the culture medium is discarded, the cells are digested with trypsin, and then resuspended in culture medium. The cells are centrifuged at 1000 rpm for 5 minutes, the supernatant is discarded, and the cells are resuspended in 10 mL of culture medium. After counting, 6 × 10⁶ cells are collected. 6Cells were cultured overnight in new T75 cell culture flasks. Two 1.5 mL centrifuge tubes were used, each containing 400 μL of serum-free medium, 120 μL of PEI transfection reagent, 20 μg of pNL4-3.Luc.RE, and 10 μg of SARS-CoV-2 spike protein expression plasmid. After mixing, the mixture was incubated at room temperature for 5 minutes, then combined and incubated at room temperature for 20 minutes. The transfection reagent was added to HEK-293TT75 cell culture flasks, mixed, and incubated for about 7 hours. The medium was discarded, fresh medium was added, and the cells were cultured for another 48 hours. The cell culture medium containing pseudovirus was transferred to 50 mL centrifuge tubes, centrifuged at 3000 rpm for 10 minutes, and the supernatant was filtered through a 0.45 μm filter, aliquoted, and stored at -80°C for later use.

[0049] To determine antibody neutralizing activity, monoclonal antibodies were serially diluted 8 times (5-fold) from 100 μg / mL in 96-well plates, and an equal volume of diluted pseudovirus was added. The plates were incubated at 37°C for 1 hour. Subsequently, 100 μL of HEK-293T-hACE2 cells (containing 30,000 cells) were added to each well. After incubation at 37°C with 5% CO2 for 48 hours, the cell culture medium was removed, and 100 μL of Bright Lite luciferase reagent (Vazyme Biotech) was added to each well. After incubation at room temperature for 2 minutes, the chemiluminescence signal was detected using a multi-mode microplate reader. The 50% inhibitory concentration (IC50) was calculated using GraphPad Prism 8.0 software using a logarithmic (inhibitor) and normalized reaction-variable slope (four-parameter) model. 50 ).

[0050] II. Results and Analysis

[0051] 1. Isolation of monoclonal antibodies

[0052] In this case, a monoclonal neutralizing antibody, VacBB-551, was isolated. The sequences of its heavy chain variable region and light chain variable region, as well as the sequencing results, are shown in Table 1 and Table 2, respectively.

[0053] Table 1 Monoclonal antibody sequences

[0054]

[0055] Table 2. Results of monoclonal antibody nucleic acid sequencing

[0056]

[0057]

[0058] The analysis results show that the CDR1, CDR2 and CDR3 of the heavy chain sequence of VacBB-551 are “EIIVSRNY”, “IYAGGST” and “ARSLGDRFDF”, respectively, while the CDR1, CDR2 and CDR3 of the light chain sequence are “QGIPSY”, “AAS” and “QHEDT”, respectively.

[0059] 2. Results of SARS-CoV-2 pseudovirus neutralization test

[0060] The neutralizing activity of the novel coronavirus RBD protein-specific monoclonal antibody VacBB-551 against novel coronavirus variants was analyzed using a pseudovirus neutralization assay. To examine the effect of novel coronavirus variants (especially the Omicron variant) on the neutralizing activity of VacBB-551, pseudoviruses of Beta, Delta, and Omicron subtype variants BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4 / 5, and BA.2.75 were prepared. The neutralizing activity of the monoclonal antibody against the mutant strains was analyzed by comparing it with that of the wild-type (Wuhan strain, WT) virus. The results are as follows: Figure 1 As shown, VacBB-551 can block the infection of target cells by novel coronavirus pseudoviruses, and its neutralizing activity is significantly concentration-dependent. It can neutralize all detected variant pseudoviruses, exhibiting good broad-spectrum neutralization. These results indicate that VacBB-551 is a promising broad-spectrum neutralizing antibody against the novel coronavirus.

[0061] 3. Epitope identification of monoclonal antibodies

[0062] Previous studies have shown that RBD-specific neutralizing antibodies are classified into four classes based on their competition with ACE2 and the conformational epitopes on the RBD that recognize up and down. Direct competitive binding to the RBD with ACE2 is one of the important mechanisms by which neutralizing antibodies effectively block viral-receptor binding. In this study, a competitive ELISA was used to predict the binding epitopes of monoclonal antibodies based on their competition with ACE2 and representative antibodies of the four epitopes (Class 1: P2C-1F11, Class 2: BD-368-2, Class 3: S309, Class 4: EY6A). The results are as follows: Figure 2 As shown, VacBB-551 can compete with ACE2 and the class 1 antibody P2C-1F11, indicating that this antibody belongs to class 1 antibodies.

[0063] In conclusion, VacBB-551 is a broad-spectrum neutralizing antibody with good application potential and can effectively combat multiple SARS-CoV-2 variants.

[0064] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.

Claims

1. A monoclonal antibody that broadly neutralizes SARS-CoV-2, characterized in that, The broad-spectrum neutralizing SARS-CoV-2 monoclonal antibody is VacBB-551, 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 "EIIVSRNY", "IYAGGST" and "ARSLGDRFDF" in sequence, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are "QGIPSY", "AAS" and "QHEDT" in sequence.

2. The broad-spectrum neutralizing monoclonal antibody against SARS-CoV-2 according to claim 1, characterized in that, The heavy chain variable region sequence of VacBB-551 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 a broad-spectrum neutralizing monoclonal antibody against SARS-CoV-2 as described in claim 1 or 2, characterized in that, The nucleic acid fragment encodes the heavy and light chains of VacBB-551.

4. The nucleic acid fragment according to claim 3, characterized in that, The nucleic acid sequence encoding the heavy chain variable region shown in Seq ID No. 1 is shown in Seq ID No. 3; the nucleic acid sequence encoding the light chain variable region shown in Seq ID No. 2 is shown in Seq ID No.

4.

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

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

7. The method for preparing a broad-spectrum neutralizing monoclonal antibody against SARS-CoV-2 according to claim 1 or 2, characterized in that, This includes using the recombinant cells described in claim 6 to express proteins from the recombinant plasmid described in claim 5, extracting and purifying the expressed protein, thereby obtaining the broad-spectrum neutralizing monoclonal antibody against SARS-CoV-2.

8. The use of the broad-spectrum neutralizing monoclonal antibody against SARS-CoV-2 as described in claim 1 or 2, or the nucleic acid fragment as described in claim 3 or 4, or the recombinant plasmid as described in claim 5, or the recombinant cell as described in claim 6, in the preparation of drugs for the prevention and treatment of novel coronavirus and reagents for the detection of novel coronavirus.

9. A novel coronavirus detection reagent, characterized in that, Contains a broad-spectrum neutralizing monoclonal antibody against SARS-CoV-2 as described in claim 1 or 2.

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