Neutralizing antibody GR75 for resisting novel coronavirus SARS-CoV-2 and variant and application of neutralizing antibody GR75

Abstract

The invention discloses a novel coronavirus neutralizing antibody, a detection kit and application of the novel coronavirus neutralizing antibody. The amino acid sequence of a heavy chain variable region of the neutralizing antibody is shown as SEQ ID No.1, and the amino acid sequence of a light chain variable region of the neutralizing antibody is shown as SEQ ID No.2. The antibody with mature affinity is screened through bioinformatics analysis of a single B cell, the antibody screening process is optimized by combining single cell RNA sequencing, VDJ rearrangement analysis and somatic cell hypermutation research, blindness of a traditional method is avoided, and the accuracy and effectiveness of antibody screening are improved. According to the neutralizing antibody GR75 provided by the invention, a heavy chain variable region and a light chain variable region of the neutralizing antibody GR75 can be specifically combined with an RBD structural domain of the SARS-CoV-2 and an S-Trimer structural domain of an Omicro variant, so that a broad-spectrum neutralizing effect on the SARS-CoV-2 virus and the variant thereof is realized. The binding activity of the antibody GR75 to S-Trimer and RBD under 2-fold and 300-fold dilution conditions is obviously superior to that of other antibodies, which indicates that the antibody GR75 has high affinity and dilution stability and is suitable for clinical large-dose administration.

Description

Technical Field

[0001] The present invention relates to a novel coronavirus neutralizing antibody, specifically a neutralizing antibody that can specifically bind to the RBD domain of SARS-CoV-2 and the S-Trimer domain of the variant Omicron, thereby broadly preventing the novel coronavirus and its variants from infecting cells, and uses thereof. Background Art

[0002] The novel coronavirus (SARS-CoV-2) belongs to the genus Betacoronavirus. Its genome is a single-stranded RNA that encodes multiple structural proteins, including the Spike Protein (S protein) and the nucleocapsid protein (N protein). Among them, the S protein binds to the host cell ACE2 receptor through the receptor binding domain (RBD), mediating viral invasion and serving as a core target for neutralizing antibody and vaccine design. The N protein is highly conserved and is the primary target for nucleic acid and antigen detection. Since the outbreak, the virus has continued to evolve, resulting in the emergence of multiple variants (such as Delta, Omicron, and its sublineages), placing higher demands on detection and treatment technologies.

[0003] Neutralizing antibodies are specific antibodies produced by the human immune system or through in vitro screening technology, which can block the virus from invading host cells by binding to viral surface proteins (such as the RBD or NTD region of the S protein). Its research and development path mainly includes: (1) Antibody source: Early studies isolated high-titer neutralizing antibodies from the serum of recovered patients, and then screened candidate antibodies through monoclonal antibody technology (such as phage display and hybridoma technology). (2) Target screening: Targeting the RBD region of the S protein is the main strategy because it is directly involved in ACE2 receptor binding; some antibodies target conserved epitopes of the NTD or S2 subunit to respond to viral mutations. (3) Functional verification: Preliminary evaluation of antibody activity through pseudovirus neutralization test (based on VSV or lentiviral vector), and then confirming effectiveness through live virus neutralization test (which must be conducted in a biosafety level 3 laboratory BSL-3). Neutralizing antibodies are of great value in clinical treatment. For example, antibody drugs such as REGEN-COV (Casirivimab / Imdevimab) and Sotrovimab have been approved for use in high-risk patients through Emergency Use Authorization (EUA), reducing hospitalization rates and the risk of severe illness. However, antibody escape caused by viral mutations (such as the Omicron strain mutation) has severely weakened the effectiveness of some antibodies, necessitating the development of broadly neutralizing antibodies.

[0004] The technical problems of existing virus neutralizing antibodies are: (1) Limitations: Antibody escape caused by viral mutations seriously weakens the effectiveness of some antibodies. For example, frequent mutations in the viral S protein (such as L452Q in Omicron BA.2 and F486V in BA.4 / 5) may change the antibody binding epitope, causing the existing antibodies to fail. (2) Antibody-dependent enhancement (ADE) risk: Some non-neutralizing antibodies may mediate the virus into immune cells through Fc receptors, enhancing infection. Risks need to be avoided through epitope screening (such as prioritizing RBD non-ACE2 competitive epitopes) or Fc segment engineering. (3) Pharmacokinetic defects: Natural antibodies have a short half-life (about 21 days) and poor stability. (4) Bottlenecks in the sensitivity and specificity of detection technology: False negative problems in nucleic acid detection: Low viral load samples (Ct value > 35), sampling errors (such as insufficient viral RNA in nasal swabs), or mismatch between primer probes and variant sequences (such as the D3L mutation in the N gene of the Alpha strain) may lead to missed detection. (5) Insufficient sensitivity of antigen detection: The sensitivity of existing reagents is only 60%-80%, which cannot replace nucleic acid detection; in addition, N protein mutations (such as the P13L mutation in Omicron BA.1) may affect detection performance. (6) Decreased efficacy of neutralizing antibodies: Most approved antibodies are ineffective against the Omicron strain because they target the RBD epitope; only a few broad-spectrum antibodies remain active, but their potency still needs to be improved.

[0005] In summary, the research and development of neutralizing antibodies and detection kits for the new coronavirus is a core technical means to deal with severe illness caused by viral infection and is a hot topic of research for technicians in this field. Summary of the Invention

[0006] The primary technical problem to be solved by the present invention is to propose a method for predicting and screening neutralizing antibodies against the new coronavirus using bioinformatics analysis.

[0007] Another technical problem to be solved by the present invention is to propose a neutralizing antibody GR75 that binds to the new coronavirus and is screened using the above method. The neutralizing antibody can specifically bind to the RBD domain of SARS-CoV-2 and the S-Trimer domain of the variant Omicron, thereby effectively preventing the infection of cells by the new coronavirus and its variants in a broad spectrum, and ultimately achieving a protective effect.

[0008] Another technical problem to be solved by the present invention is to provide an antibody expression vector obtained from the neutralizing antibody GR75 that binds to the new coronavirus.

[0009] Another technical problem to be solved by the present invention is to provide the use of the neutralizing antibody GR75 that binds to the new coronavirus.

[0010] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0011] A neutralizing antibody GR75 that binds to the new coronavirus, comprising a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region being shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region being shown in SEQ ID No. 2.

[0012] Preferably, the antibody can specifically bind to the RBD domain of SARS-CoV-2 and the S-Trimer domain of the variant Omicron.

[0013] The nucleotide sequence encoding the neutralizing antibody GR75 that binds to the new coronavirus, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 3, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region is shown in SEQ ID No. 4.

[0014] An antibody expression vector comprises the above nucleotide sequence, and the vector is a mammalian expression vector.

[0015] A host cell comprising the above nucleotide sequence or the above expression vector.

[0016] A method for screening the neutralizing antibody GR75 that binds to the novel coronavirus, the method comprising PBMC collection, antigen labeling, magnetic bead enrichment and flow cytometric sorting of B cells, PCR and vector construction, HEK293 transient cell culture, culture supernatant ELISA binding detection, culture supernatant ELISA neutralization detection, and pseudovirus neutralization detection; preferably, the method further comprises a bioinformatics analysis step, after sorting B cells by flow cytometry, analyzing their heterogeneity, clonal evolution, and immune response mechanism based on high-throughput sequencing of single B cell gene expression, the steps are:

[0017] S1. Screening of IGHG subtype VDJ distribution: High-throughput sequencing is used to analyze the combination frequency of IGHG heavy chain VDJ gene segments in B cells, and to screen out IGHG antibody sequences with a similarity of 95% or more to the VDJ distribution of the overall B cell population.

[0018] S2. CDR3 length distribution optimization: Screen antibody sequences with heavy chain CDR3 amino acid lengths of 16 to 17, requiring that such sequences account for no less than 60% of the sample. At the same time, exclude abnormal sequences in light chain CDR3 sequences whose length deviates from the mean ± 2 standard deviations (SD);

[0019] S3. Somatic Hypermutation (SHM) Enrichment:

[0020] (1) Screening for antibody sequences with a somatic hypermutation frequency of 15% or more in the heavy chain and a hypermutation frequency of 10% or more in the light chain;

[0021] (2) Use sequencing data to compare with the original V gene to mark and confirm mutation hotspot areas.

[0022] S4. Utilization of Isotype-Specific CDR3 Length Differences:

[0023] (1) For heavy chain CDR3 sequences, the screening range was set to 48 to 52 amino acids (based on mean ± SD);

[0024] (2) For light chain CDR3 sequences, the screening range was set to 28 to 32 amino acids (based on mean ± SD).

[0025] The use of the above-mentioned neutralizing antibody GR75 that binds to the new coronavirus, or the above-mentioned nucleotide sequence, or the above-mentioned expression vector, or the above-mentioned host cell in the preparation of an agent that blocks new coronavirus infection or the preparation of a drug for preventing and / or treating new coronavirus infection.

[0026] The use of the above-mentioned neutralizing antibody GR75 that binds to the new coronavirus, or the above-mentioned nucleotide sequence, or the above-mentioned expression vector, or the above-mentioned host cell in the preparation of a kit for detecting neutralizing antibodies against the new coronavirus and / or evaluating the immune effect of vaccines.

[0027] Preferably, the novel coronavirus in the above application includes the following serotypes: SARS-CoV-2 and its variant Omicron.

[0028] A novel coronavirus neutralizing antibody ELISA detection kit comprises the following components: a detection plate for capturing novel coronavirus antigens, the aforementioned neutralizing antibody labeled with biotin, and enzyme-labeled avidin.

[0029] An ELISA method for detecting the binding activity of SARS-CoV-2 neutralizing antibodies comprises the following steps:

[0030] a) Coating SARS-CoV-2 S-Trimer protein and RBD protein to the ELISA plate;

[0031] b) diluting the antibody culture supernatant and adding it to the ELISA plate, incubating, and then washing;

[0032] c) Add HRP-labeled secondary antibody and measure OD450 value after color development;

[0033] The antibody can detect binding activity with SARS-CoV-2 RDB, Omicron S-trimer or RDB.

[0034] A novel coronavirus neutralizing antibody ELISA detection reagent, detection kit or drug, comprising the above-mentioned novel coronavirus-binding neutralizing antibody GR75, or a nucleotide sequence encoding the above-mentioned novel coronavirus-binding neutralizing antibody GR75 or the above-mentioned expression vector.

[0035] Compared with the prior art, the present invention has the following technical effects:

[0036] (1) The present invention optimizes the antibody screening method by screening affinity-matured antibodies through bioinformatics analysis of single B cells. Combined with single-cell RNA sequencing, VDJ rearrangement analysis and somatic hypermutation research, the antibody screening process is optimized, avoiding the blindness of traditional methods and improving the accuracy and effectiveness of antibody screening.

[0037] (2) Based on the optimized screening method of the present invention, a new neutralizing antibody GR75 was screened, whose heavy chain and light chain variable regions can specifically bind to the RBD domain of SARS-CoV-2 and the S-Trimer domain of the Omicron variant, achieving a broad-spectrum neutralizing effect on the SARS-CoV-2 virus and its variants.

[0038] (3) The neutralizing antibody GR75 provided by the present invention has significantly better binding activity to S-Trimer and RBD than other antibodies under 2-fold and 300-fold dilution conditions, indicating its high affinity and dilution stability, which is suitable for clinical large-dose administration needs.

[0039] (4) The neutralizing antibody GR75 provided by the present invention can simultaneously target SARS-CoV-2 RBD and Omicron S-Trimer, effectively inhibiting multiple variants (e.g., JN.1 and WT pseudovirus inhibition rates >90%), overcoming the failure of existing antibodies due to viral mutations. Antibody GR75 demonstrated potent blocking of ACE2 receptor binding and pseudovirus neutralization in in vitro experiments, providing a core tool for the development of therapeutic drugs (e.g., reducing the rate of severe illness) and evaluating the immune effects of vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Set gates for flow cytometry sorted B cell levels;

[0041] Figure 2 For the expression plasmid map;

[0042] Figure 3 The expression levels of the heavy and light chains of IGHG;

[0043] Figure 4 The expression levels of heavy and light chains in whole cells;

[0044] Figure 5is the number of clones of IGHG BCR;

[0045] Figure 6 is the UMI expression level in IGHG cells;

[0046] Figure 7A is the overall cellular VDJ distribution level;

[0047] Figure 7B is the VDJ distribution level in IGHG;

[0048] Figure 8 is the CDR3 length distribution;

[0049] Figure 9 For somatic hypermutation analysis;

[0050] Figure 10A and 10B Shown are the CDR3 distribution results;

[0051] Figure 11 Single-cell RNA seq sequencing to simulate temporal analysis of the developmental trajectory of memory B cells;

[0052] Figure 12 Neutralizing antibody affinity determination under 2-fold dilution conditions;

[0053] Figure 13 Neutralizing antibody affinity determination under 300-fold dilution conditions;

[0054] Figure 14 To evaluate the ACE2 blocking ability of neutralizing antibodies;

[0055] Figure 15 To evaluate the neutralizing ability of neutralizing antibodies against pseudoviruses;

[0056] Figure 16 This is the situation of antibodies obtained by screening without using bioinformatics analysis. DETAILED DESCRIPTION

[0057] The present invention is further described below with reference to specific examples. These examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. Experimental methods in the following examples, where specific conditions are not specified, are generally performed under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred embodiments and materials described herein are for exemplary purposes only.

[0058] The present invention screens memory B cells in people infected with the new coronavirus, uses single B cell antibody preparation technology to select people who have recovered from the new coronavirus infection, collects peripheral blood from the blood, uses density gradient centrifugation to separate PBMCs from the whole blood, uses magnetic beads to enrich and flow cytometry to sort B cells, and analyzes the heterogeneity, clonal evolution and immune response mechanism of single B cell gene expression based on high-throughput sequencing to accurately screen for neutralizing antibodies with excellent efficacy. The specific method is described below.

[0059] Example 1 Neutralizing Antibody Screening and Efficacy Verification Methods Provided by the Present Invention

[0060] 1. PBMC Collection

[0061] Sample collection and pretreatment

[0062] Subject preparation: Ten eligible recovered COVID-19 patients (negative nucleic acid test and symptom-free for ≥14 days) were selected. All patients signed informed consent and passed ethical review.

[0063] Blood collection requirements: Use EDTA anticoagulant vacuum blood collection tubes and draw 10 mL of peripheral venous blood per patient. Process blood within 2 hours of collection and avoid refrigeration or freezing.

[0064] 1.1 PBMC Isolation Steps (Ficoll Density Gradient Centrifugation)

[0065] Hemodilution: Mix whole blood with sterile PBS at a 1:1 ratio (e.g., 10 mL blood + 10 mL PBS).

[0066] Ficoll separation: Add 3 mL of Ficoll separation solution (density 1.077 g / mL, pre-equilibrated to 20-25°C) to a 15 mL centrifuge tube. Slowly add diluted blood along the tube wall to the upper layer of the separation solution, maintaining clear layers (avoid mixing).

[0067] Centrifugation parameters: Horizontal centrifuge settings: 500×g (or 1500 rpm), 25 minutes, with the speed increase / deceleration set to the lowest setting (to avoid disturbing the layers) 14.

[0068] Collect the PBMC layer: After centrifugation, the layers from top to bottom are: plasma layer, PBMC buffy coat layer, Ficoll layer, and red blood cell layer. Gently pipette the buffy coat layer into a new centrifuge tube (avoid aspirating the upper plasma layer or the lower separation solution).

[0069] Wash and purify: add 10 mL PBS, centrifuge at 250 × g for 10 minutes, discard the supernatant; repeat the washing once

[0070] 2. Antigen Labeling

[0071] 2.1PE Marking

[0072] 1) Concentrate PE to 3 mg / mL, measure the quantification by UV, and then add a certain proportion of SMCC. The NHS group on SMCC will react with the primary amine group on PE, and the MAL group on the other end will react with the -SH group on the protein.

[0073] 2) Concentrate the recombinant Omicron EG.5.1 Spike S1+S2 Trimer protein 40589-V08H55 to 2 mg / ml, add a certain volume of 2-IT solution, mix thoroughly, and react at room temperature for 2 hours.

[0074] 3) Desalt the activated protein into PBS, concentrate, and measure UV.

[0075] 4) Add the activated protein with -SH and the activated PE with MAL at a molar ratio of 1:1, mix thoroughly, and react at 4°C overnight.

[0076] 5) Purify the reaction sample using an S300 purification column, collect the coupled protein, measure UV, and calculate the amount of protein.

[0077] 2.2APC Marking

[0078] 1) Concentrate APC to 3 mg / mL, measure the quantification by UV, and then add a certain proportion of SMCC. The NHS group on SMCC will react with the primary amine group on APC, and the MAL group on the other end will react with the -SH group on the peptide.

[0079] 2) Concentrate the recombinant Omicron BA.2.86 Spike RBD protein (40592-V08H152) to 2 mg / ml, add a certain volume of 2-IT solution, mix thoroughly, and react at room temperature for 2 hours.

[0080] 3) Desalt the activated protein into PBS, concentrate, and measure UV

[0081] 4) Add the activated protein with -SH and the activated APC with MAL at a molar ratio of 1:1, mix thoroughly, and react at 4°C overnight.

[0082] 5) Purify the reaction sample using an S300 purification column, collect the coupled protein, measure UV, and calculate the amount of protein.

[0083] 3. Magnetic Bead Enrichment and Flow Cytometry Sorting of B Cells

[0084] 3.1 Magnetic Bead Enrichment

[0085] (1) PBMC separation: human EDTA anticoagulated whole blood was added to a centrifuge tube containing Ficoll, centrifuged, and the middle buffy coat layer containing lymphocytes and monocytes was transferred to a new centrifuge tube and centrifuged to obtain PBMC cells (see Example 1 for specific steps).

[0086] (2) Add B Cell Biotin-Antibody Cocktail to the cells and incubate at 4°C in the dark for 10 min. Add Anti-Biotin MicroBeads and incubate at 4°C in the dark for 15 min. Add sorting buffer and wash the cells by centrifugation. Resuspend in sorting buffer and add to the sorting column to collect the flow-through.

[0087] (3) Add CD27 MicroBeads to the flow-through cells, incubate at 4°C in the dark for 15 min, wash the cells by centrifugation, resuspend in sorting buffer, add to the sorting column, and collect the cells in the sorting column.

[0088] 3.2 Flow sorting

[0089] (1) Flow cytometry

[0090] IgG, IgM, CD 19 antibodies and fluorescent-labeled proteins were added to the cells for incubation, the cells were washed by centrifugation, and resuspended in PBS.

[0091] (2) Flow sorting

[0092] Adjust the flow sorting fluid path, delay, sorting angle, and fluorescence compensation. Through flow cytometry, we successfully sorted out memory B cells that were IgG-positive, CD 19-positive, and positive for the new coronavirus antigen. Specifically, the B cell population was first identified using the CD19 marker, and then the B cells expressing immunoglobulin G were screened out using the IgG marker, and finally the new coronavirus antigen was used to detect the specific reactivity of these cells. This strategy ensures that the sorted memory B cells are specific to the new coronavirus, laying the foundation for subsequent research. The sorting results are as follows: Figure 1 As shown, Figure 1 Set gates for the flow cytometry sorting layer to isolate memory B cells that are IgG-positive, CD19-positive, and positive for SARS-CoV-2 antigens.

[0093] 4. PCR and vector construction

[0094] After the sorted B cells were lysed, cDNA was obtained by reverse transcription using a reverse transcription kit. The antibody heavy and light chain variable region sequences were obtained by single B cell amplification technology. The heavy and light chain variable region fragments were constructed into expression vectors respectively to obtain the correctly sequenced heavy and light chain expression plasmids, which were then delivered to HEK 293 for transient expression.

[0095] (1) Cell lysis

[0096] Aliquot equal volumes of Lysis Solution into 96-well plates to fully lyse the cells.

[0097] (2) Reverse transcription

[0098] A two-step reverse transcription method was used. The reverse transcription system was dispensed and added to the PCR plate in sequence. After mixing by pipetting, the reverse transcription reaction was performed on the machine.

[0099] (3) Preparation of heavy and light chain variable regions

[0100] Using cDNA as a template, nested PCR was used to gradually amplify the antibody heavy and light chain variable region genes using multiple rounds of progressive amplification.

[0101] (4) Expression vector construction

[0102] The heavy and light chain variable region fragments were added to the membrane binding solution, mixed thoroughly and added to the purification column, eluted with Nuclease-Free Water to obtain the purified heavy and light chain variable region gene fragments. The heavy and light chain variable region fragments were constructed into CMV expression vectors respectively, and the ligated products were transformed into competent cells and cultured at 37°C overnight. The obtained monoclonal was amplified and sequenced to obtain the antibody heavy and light chain sequences. The plasmids were extracted from the correctly sequenced vectors and delivered for small-scale expression. The expression plasmid map is shown in the figure. Figure 2 shown.

[0103] 5. HEK293 transient cell culture

[0104] HEK293 cells were subcultured in 293 serum-free CD medium. The plasmid DNA to be expressed was mixed with the transfection reagent TF2 and added to the cells. 293 serum-free feed solution was added on days 1, 3, and 5 after transfection.

[0105] Shake flask culture conditions: 5% CO2, 37°C, shaker speed 175 rpm. Culture for 3-7 days. Collect culture supernatant for ELISA analysis. Collect 1-2 mL of supernatant from ELISA-positive binding clones for analysis.

[0106] 6. ELISA Binding Detection of Culture Supernatant

[0107] (1) Coating: Coating protein S-Trimer and protein RBD 0.1 μg / mL and 1 μg / mL, 100 μL / well, coating overnight at 4°C;

[0108] (2) Blocking: Shake off all liquid in the plate and pat dry, add 2% BSA blocking buffer, 300 μL / well, seal and incubate at room temperature for 1 h;

[0109] (3) Washing: Wash the plate twice with 300 μL / well washing solution and pat dry for the last time;

[0110] (4) Sample dilution: Dilute the culture supernatant 2-fold and 300-fold with sample diluent, mix thoroughly, and set aside;

[0111] (5) Sample addition: Add the diluted supernatant to the ELISA plate at 100 μL / well, mix well, and incubate at room temperature for 2 h;

[0112] (6) Washing: Wash the plate three times with 300 μL / well washing solution and pat dry for the last time;

[0113] (7) Adding secondary antibody: Dilute the secondary antibody Goat Anti-Human IgG (H+L) / HRP to the working concentration, mix well, add 100 μL / well, and incubate at room temperature for 1 h;

[0114] (8) Washing: 300 μL / well washing solution, wash the plate 3 times, and pat dry the last time;

[0115] (9) Color development: Mix solution A and solution B at a ratio of 1:1 and add 200 μL to each well.

[0116] Incubate at room temperature in the dark for 20 min;

[0117] (10) Termination: Add 50 μL of stop solution to each well and immediately measure the OD value at a wavelength of 450 nm.

[0118] 7. ELISA Neutralization Detection of Culture Supernatant

[0119] (1) Coating: Coating protein ACE2-mFc 2 μg / mL, 100 μL / well, coating overnight at 4°C;

[0120] (2) Blocking: Shake off all liquid in the plate and pat dry, add 2% BSA blocking buffer, 300 μL / well, seal and incubate at room temperature for 1 h;

[0121] (3) Washing: Wash the plate twice with 300 μL / well washing solution and pat dry for the last time;

[0122] (4) Sample dilution: Discard the supernatant and set aside; dilute the RBD protein to 0.05 μg / mL with sample diluent and set aside;

[0123] (5) Sample addition: Add 100 μL / well of the supernatant to the ELISA plate, with the blank control being the sample diluent. Then, add 100 μL / well of the diluted RBD protein to the ELISA plate, for a final sample volume of 200 μL / well. Mix well and incubate at room temperature for 1 h.

[0124] (6) Washing: Wash the plate three times with 300 μL / well washing solution and pat dry for the last time;

[0125] (7) Adding secondary antibody: dilute the secondary antibody Anti-his / HRP to 0.15 μg / mL, mix well, add 100 μL / well, and incubate at room temperature for 1 h;

[0126] (8) Washing: 300 μL / well washing solution, wash the plate 3 times, and pat dry the last time;

[0127] (9) Color development: Mix solution A and solution B at a ratio of 1:1, add 200 μL to each well, and incubate at room temperature in the dark.

[0128] (10) Termination: When the OD value of the control group is between 1.00 and 1.50, add 50 μL of stop solution to each well and immediately measure the OD value at a wavelength of 450 nm.

[0129] 8. Pseudovirus Neutralization Detection

[0130] (1) Pseudovirus dilution: Pseudovirus SARS-CoV-2 (2019-nCoV) Spike Ps eudovirus (hereinafter referred to as WT pseudovirus) and SARS-CoV-2JN.1

[0131] (Omicron) Spike Pseudovirus (hereinafter referred to as JN.1 pseudovirus) was diluted to prepare pseudovirus dilution solution.

[0132] (2) Sample-pseudovirus neutralization: Each sample was divided into two portions, each 50 μL; 50 μL of the sample stock solution of one portion was mixed with 50 μL of the WT pseudovirus diluent, and 50 μL of the sample stock solution of the other portion was mixed with 50 μL of the JN.1 pseudovirus diluent; incubated at 37°C for 1 h.

[0133] (3) Cell digestion and dilution: 293T / (ACE2, TMPRSS2) cells were digested with trypsin and diluted with complete culture medium to a cell suspension density of 30,000 cells / 100 mL.

[0134] (4) Cell inoculation: After incubating the sample-pseudovirus mixture for 1 hour, add 100 μL of the above cell suspension to each well and incubate at 37°C for 48-72 hours.

[0135] (5) Lysis detection: After incubation for 48-72 h, cells were lysed using cell lysis buffer, the chemiluminescence value was detected, and the inhibition rate was calculated.

[0136] Example 2 Bioinformatics Analysis and Screening Experiment Results

[0137] like Figure 16As shown in Figure 1, the inventors initially did not use the bioinformatics analysis screening method after sorting out IgG-positive, CD19-positive, and SARS-CoV-2 antigen-positive memory B cells, and the resulting antibodies were all negative. Subsequently, after successfully sorting out IgG-positive, CD19-positive, and SARS-CoV-2 antigen-positive memory B cells in Step 3 of Example 1, the inventors further improved the screening method, first performing bioinformatics analysis on individual B cells, and using the analysis results to accurately screen and obtain effective neutralizing antibodies.

[0138] The bioinformatics analysis method is as follows:

[0139] Analysis of single B cell sequencing results: Single B cell gene expression analysis method based on high-throughput sequencing and its application in revealing heterogeneity, clonal evolution and immune response mechanisms.

[0140] S1. Screening of IGHG subtype VDJ distribution: High-throughput sequencing is used to analyze the combination frequency of IGHG heavy chain VDJ gene segments in B cells, and to screen out IGHG antibody sequences with a similarity of 95% or more to the VDJ distribution of the overall B cell population.

[0141] Objective: To maintain the diversity generation pattern of IGHG subtype consistent with other subtypes, and ensure that the constructed antibody library has a broad spectrum of functions and efficient antigen recognition ability.

[0142] First, the distribution of B-cell antigen receptors (BCRs) is shown in Table 1. As can be seen, BCRs are primarily distributed among IGHGs, with IGHG1 having the highest prevalence, followed by IGHM, IGHA, and IGHD. IGHGs have the highest prevalence, with IGHG1 being the predominant isoform within the class, suggesting their central role in humoral immunity, such as antibody neutralization of pathogens. The high prevalence of IGHG1 may be related to its long half-life, complement activation capacity, and placental penetration (e.g., immunomodulation during pregnancy).

[0143] Table 1

[0144] IGHM IGHD IGHG1 IGHG2 IGHG3 IGHG4 IGHA ％ 32.38％ 0.60％ 17.27％ 14.79％ 2.18％ 1.30％ 25.74％ Cell number 3401 63 1814 1554 229 137 2704

[0145] Based on the above analysis results, we further analyzed the expression levels of IGHG heavy and light chains. The results showed that the expression levels of IGHG BCR heavy and light chains were relatively consistent. Figure 3 shown.

[0146] We further analyzed the expression levels of heavy and light chains of BCR in all cells. The results showed that the expression levels of heavy and light chains of BCR in all cells were relatively consistent. Figure 4The high synchronization of heavy chain (HC) and light chain (LC) expression levels may be due to the allelic exclusion mechanism during B cell development, ensuring single BCR specificity.

[0147] like Figure 5 As shown in Figure 3, we further analyzed the number of IGHG BCR clones and found that BCR expression had a low correlation with the number of IGHG BCR clones, suggesting that there was no significant association between BCR expression level and IGHG BCR clonal diversity.

[0148] like Figure 6 As shown in the figure, the UMI expression levels in IGHG cells show that most cells express BCR UMI at around 25 UMI, while a few cells express high levels of BCR UMI, indicating that most cells are in an inactive state.

[0149] like Figure 7A and Figure 7B As shown, the VDJ distribution levels in IGHG and whole-cell populations are similar. VDJ rearrangement is a core mechanism for B cell production of diverse antibodies. As a major antibody subtype, IGHG has a VDJ distribution consistent with that of whole-cell populations. This conclusion suggests that within the B cell population studied, the pattern of antibody diversity generated by the IGHG subtype is not significantly different from that of other subtypes or whole-cell populations.

[0150] S2. CDR3 length distribution optimization: Screen antibody sequences with heavy chain CDR3 amino acid lengths of 16 to 17, requiring this sequence to account for at least 60% of the sample. Also exclude light chain CDR3 sequences with lengths that deviate from the mean by ±2 standard deviations (SD).

[0151] Objective: Through structural biology verification, confirm that the heavy chain CDR3 with a length of 16-17 amino acids can form a more stable antigen binding epitope, thereby improving the binding affinity of the antibody, such as Figure 8 shown.

[0152] like Figure 8 As shown in Figure 2, we further analyzed the distribution of CDR3 lengths. Antibody sequences with heavy chain CDR3 lengths of 16 and 17 amino acids were the most common. CDR3 is the key region for antibody-antigen binding, and its length and sequence diversity directly influence antibody specificity and affinity. Heavy chain CDR3 lengths of 16 and 17 amino acids predominated, suggesting that these CDR3 lengths play an important role in the B cell antibody repertoire and are closely related to the functional requirements of antibodies.

[0153] S3. Somatic Hypermutation (SHM) Enrichment:

[0154] (1) Screening for antibody sequences with a somatic hypermutation frequency of 15% or more in the heavy chain and a hypermutation frequency of 10% or more in the light chain;

[0155] (2) Use sequencing data to compare with the original V gene to mark and confirm the mutation hotspot region (see Figure 9 circled area).

[0156] Purpose: To simulate the affinity maturation process under antigen stimulation, optimize antibody function, and ensure that the obtained antibody molecules have high affinity and excellent specificity.

[0157] like Figure 9 As shown, we also performed somatic hypermutation analysis, which revealed varying levels of hypermutation in both the heavy and light chains. Hypermutations are circled in the figure. Somatic hypermutation is a key process by which B cells enhance antibody affinity in response to antigen stimulation. Hypermutation in both heavy and light chains suggests that B cells undergo affinity maturation during the immune response, thereby optimizing antibody function.

[0158] S4. Utilization of Isotype-Specific CDR3 Length Differences:

[0159] (1) For heavy chain CDR3 sequences, the screening range was set to 48 to 52 amino acids (based on mean ± SD);

[0160] (2) For light chain CDR3 sequences, the screening range was set to 28 to 32 amino acids (based on mean ± SD).

[0161] Purpose: To take advantage of the longer structure of heavy chain CDR3 (see Figure 10A and 10B ), enhance the complexity of the antigen binding interface and further improve the binding efficiency and functional stability of the antibody.

[0162] like Figure 10A and 10B As shown, the Isotype CDR3 distribution results show that the heavy chain CDR3 sequence length is longer than that of the light chain ( Figure 10A The average is around 50 Figure 10B Heavy chain CDR3s are typically longer than light chain CDR3s, a feature consistent with the structural and functional requirements of antibodies. Heavy chain CDR3s play a dominant role in antigen binding, and their longer length contributes to the formation of a more complex binding site, thereby enhancing the antibody's specificity and affinity.

[0163] Single-cell RNA-seq results:

[0164] like Figure 11As shown, we performed single-cell RNA seq sequencing, mainly focusing on CD74, CD79a, and CXCR4 (the dark part in the figure), and pseudo-time analysis showed the developmental trajectory of memory B cells. CD74 is mainly involved in antigen presentation and serves as a receptor for MIF, affecting immune response and cell survival. CD79a is an important component of the B cell antigen receptor complex and is key to B cell development and signal transduction. In B cells, CXCR4 signaling regulates their migration to lymphoid organs and bone marrow, which is an important part of the immune response. These findings not only deepen our understanding of the function and regulatory mechanisms of B cells in the immune system, but also provide an important foundation for further research on the role of B cells in immune responses and their potential application in SARS-COV-2. Example 3 Obtaining the heavy and light chain variable regions of memory B cells and constructing the heavy and light chain vector plasmids of the new crown neutralizing antibody

[0165] The sorted human single B cells were lysed and reverse transcribed to obtain cDNA, and the variable region fragments were amplified by PCR. The correct fragments were identified to construct the full-length expression vector and sequenced.

[0166] The primary goal of this study was to screen for specific neutralizing antibodies in individuals who had recovered from COVID-19 infection. The specific steps were as follows: Single B cell sorting and RNA extraction. Specific memory B cells were sorted by flow cytometry (FACS) from individuals who had recovered from COVID-19 infection. The sorted B cells were CD19+, CD27+, IgG+ memory B cell populations, from which total RNA was extracted.

[0167] 1. cDNA Synthesis and PCR Amplification

[0168] The extracted RNA was transcribed into complementary DNA (cDNA) using reverse transcriptase. Following the reverse transcription reaction, PCR amplification was performed using specific primers targeting the immunoglobulin heavy and light chain variable regions. Successful amplification of antibody variable region fragments of the expected size was confirmed.

[0169] 2. Cloning of variable region fragments and construction of expression vectors

[0170] After confirming the correctness of the PCR product, a fragment containing the correct antibody variable region sequence is selected, digested with restriction endonucleases, and ligated into an expression vector. The constructed vector contains the full-length antibody gene. The cloned antibody gene is then subjected to Sanger sequencing to verify its sequence accuracy.

[0171] 3. Transient transfection of HEK293 cells and antibody expression

[0172] The antibody gene vector with the correct sequence was transfected into HEK293 cells for transient expression. The transfected cells secreted the antibody 24-48 hours later, and the antibody production and function were preliminarily verified by ELISA and neutralization assays.

[0173] The obtained sequence is as follows:

[0174] H chain amino acid sequence SEQ ID No.1:

[0175] MGWSLILLFLVAVATRVLSQVQLVESGGGLVKPGGSLRLSCEASGFTFSSHDMHWVRQTTGK

[0176] SLEWLSLIGTAGDTFYPDSVKGRFTISRDNAKNSLFLQMNSLRVGDTAVYYCVRAHYDDSGF

[0177] FSYFDSWGQGAQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA

[0178] LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH

[0179] TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN

[0180] AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

[0181] YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0182] H chain nucleotide sequence SEQ ID No.3:

[0183] ATGGGCTGGTCCCTGATTCTGCTGTTCCTGGTGGCTGTGGCTACCAGGGTGCTGAGTCAGGT

[0184] GCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCACTGAGACTCTCCTGTG

[0185] AGGCCTCTGGATTCACCTTCAGTAGCCACGACATGCACTGGGTCCGCCAAACGACAGGAAAG

[0186] AGTCTGGAGTGGCTCTCACTGATTGGTACGGCTGGTGACACATTTTATCCGGACTCCGTGAA

[0187] GGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCCTTGTTTCTTCAAATGAACAGCC

[0188] TGAGAGTCGGGGACACGGCTGTGTATTACTGTGTTAGAGCTCACTATGATGATAGTGGTTTT

[0189] TTCTCCTACTTTGACTCCTGGGGCCAGGGAGCCCAGGTCACCGTCTCTTCAGCAAGCACCAA

[0190] GGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCC

[0191] TGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCC

[0192] CTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAG

[0193] CAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC

[0194] ACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCAC

[0195] ACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCC

[0196] AAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACG

[0197] TGAGCCACGAAGACCCCGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAAT

[0198] GCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCAC

[0199] CGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCC

[0200] TCCCAGCCCCCATCGGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG

[0201] TACACCCTGCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGGT

[0202] CAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACA

[0203] ACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTCACAGCAAGCTC

[0204] ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGC

[0205] TCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA Kappa chain amino acid sequence SEQ ID No. 2:

[0206] MGWSCIILFLVATATGVHSDIVMTQSPFTLSASVGDRVTITCRASQSIGTYLNWYQQKPGKA

[0207] PKVLIYATSNLQTGVPSRFSGSGSGTDFTLTISSLQREDFATYYCQQSYTTPGLTFGGGTKL

[0208] EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ

[0209] DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Kappa chain nucleotide sequence SEQ ID No.4:

[0210] ATGGGCTGGTCCTGTATCATCCTGTTCCTGGTGGCTACAGCCACAGGAGTGCATAGTGATAT

[0211] TGTGATGACCCAGTCTCCATTCACCCTGTCTGCATCTGTAGGAGACAGAGTTACCATCACTT

[0212] GCCGGGCAAGTCAGAGCATTGGCACCTATTTAAATTGGTATCAGCAGAAGCCAGGGAAAGCC

[0213] CCAAAGGTCCTGATCTATGCTACATCCAATTTGCAAACTGGGGTCCCATCAAGATTCAGTGG

[0214] CAGTGGTTCTGGGACAGATTTCACTCTCACCATTAGCAGTCTGCAACGTGAAGACTTTGCAA

[0215] CTTACTACTGTCAACAGAGTTACACTACCCCTGGACTCACTTTCGGCGGAGGGACCAAGCTG

[0216] GAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTT

[0217] GAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG

[0218] TACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG

[0219] GACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGA

[0220] GAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGA

[0221] GCTTCAACAGGGGAGAGTGTTAG

[0222] Example 4 Affinity detection and neutralization activity detection of neutralizing antibodies obtained by screening of the present invention 1. Neutralizing antibody ELISA binding test in culture supernatant

[0223] In order to further study the antigenic epitope characteristics and neutralizing antibody screening strategy of the 2019-nCoV Omicron variant, we used the ELISA method to detect the 18 neutralizing antibodies screened.

[0224] Specific experimental results: Figure 12 As shown in the figure, under 2-fold dilution conditions, the binding activity of the antibody supernatant with Omicron S-Trimer and RBD was detected. From the results, it can be seen that GR75 of the present invention has a stronger neutralizing activity with Omicron S-Trimer and RBD than other neutralizing antibodies. Figure 13 As shown, after further diluting the antibody supernatant 300 times, the neutralizing antibody GR75 provided by the present invention still maintained a strong neutralizing activity compared with other neutralizing antibodies, Omicron S-Trimer and RBD.

[0225] The above results show that although most antibodies have strong binding activity under low dilution conditions, only some antibodies can maintain high affinity as the dilution factor increases, especially antibodies against RBD. This suggests that there are significant differences in the binding strength of antibodies and they may have specificity for different antigenic epitopes. These high-affinity antibodies provide high-quality candidates for further neutralization experiments and structural analysis, and lay the foundation for the optimization design of new crown vaccines and the development of antibody drugs. The above results also demonstrate the effectiveness of the method of bioinformatics analysis to screen antibodies, and provide new data support for understanding the antigenic characteristics of Omicron variants, providing important inspiration for basic research and clinical applications in related fields.

[0226] 2. ELISA neutralization test of culture supernatant

[0227] The blocking ability of antibodies in the culture supernatant on the S / RBD-ACE2 interaction was evaluated by ELISA detection method to screen antibody candidates with neutralizing activity.

[0228] The experimental results are as follows Figure 14 As shown, the neutralizing antibody GR75 provided by the present invention can effectively block the binding of SARS-CoV-2 S protein or RBD to the ACE2 receptor at the same time, showing broad spectrum activity. This significantly inhibits the key step in the virus's invasion of host cells. These antibodies further demonstrated good neutralizing activity in in vitro validation, and can protect host cells from SARS-CoV-2 infection.

[0229] These results demonstrate that the GR75 antibody supernatant has high neutralizing activity and potential for clinical application, laying the foundation for further research. They not only serve as important candidates for the development of new antibody drugs, but also provide key clues for understanding the infection mechanism and antiviral strategies of SARS-CoV-2.

[0230] 3. Pseudovirus Neutralization Detection

[0231] The neutralizing ability of antibodies in the culture supernatant was evaluated by pseudovirus neutralization experiments to further screen candidate strains with broad-spectrum neutralizing activity.

[0232] Test results such as Figure 15 The results show that the neutralizing antibody GR75 provided by the present invention has an inhibition rate of over 90% against JN.1 and WT pseudoviruses. This provides an important candidate resource for subsequent functional validation of the antibody and its application in therapeutic drug development, and also provides strong data support for studying the characteristics of SARS-CoV-2 variants and their immune escape mechanisms.

Claims

1. A neutralizing antibody GR75 that binds to the novel coronavirus, characterized in that It includes a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

2.

2. The neutralizing antibody GR75 that binds to the novel coronavirus according to claim 1, characterized in that: The antibody can specifically bind to the RBD domain of SARS-CoV-2 and the S-Trimer domain of the variant strain Omicron.

3. The nucleotide sequence encoding the neutralizing antibody GR75 that binds to the novel coronavirus according to claim 1, characterized in that: The nucleotide sequence encoding the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 3, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region is shown in SEQ ID No.

4.

4. An antibody expression vector, characterized in that The vector comprises the nucleotide sequence of claim 3, and the vector is a mammalian expression vector.

5. A host cell, characterized in that A host cell comprising the nucleotide sequence of claim 3 or the expression vector of claim 4.

6. A method for screening the neutralizing antibody GR75 binding to the novel coronavirus according to claim 1 or 2, characterized in that This involves sorting B cells using flow cytometry, then sequencing the gene expression of individual B cells using high-throughput sequencing to analyze their heterogeneity, clonal evolution, and immune response mechanisms. The specific method is as follows: S1. IGHG subtype VDJ distribution screening: High-throughput sequencing was used to analyze the combination frequency of IGHG heavy chain VDJ gene segments in B cells, and to screen for IGHG antibody sequences with a similarity of 95% or more to the VDJ distribution of the overall B cell population; S2. CDR3 length distribution optimization: Screen antibody sequences with heavy chain CDR3 amino acid lengths of 16 to 17, requiring this sequence to account for no less than 60% of the sample; also exclude light chain CDR3 sequences with lengths that deviate from the mean by ±2 standard deviations; S3. Somatic hypermutation (SHM) enrichment: Screen antibody sequences with a somatic hypermutation frequency of 15% or greater in the heavy chain and 10% or greater in the light chain. Use sequencing data to align with the original V gene to identify and confirm mutation hotspots. S4. Utilization of isotype-specific CDR3 length differences: For heavy chain CDR3 sequences, the screening range is set to 48 to 52 amino acids; for light chain CDR3 sequences, the screening range is set to 28 to 32 amino acids.

7. Use of the neutralizing antibody GR75 that binds to the new coronavirus according to claim 1 or 2, or the nucleotide sequence according to claim 3, or the expression vector according to claim 4, or the host cell according to claim 5 in the preparation of an agent that blocks infection with the new coronavirus or in the preparation of a drug for preventing and / or treating infection with the new coronavirus.

8. Use of the neutralizing antibody GR75 that binds to the new coronavirus according to claim 1 or 2, or the nucleotide sequence according to claim 3, or the expression vector according to claim 4, or the host cell according to claim 5 in the preparation of a kit for detecting neutralizing antibodies against the new coronavirus and / or evaluating the immune effect of a vaccine.

9. The use according to claim 7 or 8, characterized in that The novel coronavirus includes the following serotypes: SARS-CoV-2 and its variant Omicron.

10. A novel coronavirus neutralizing antibody ELISA detection reagent, detection kit or drug, characterized in that Including the neutralizing antibody GR75 that binds to the new coronavirus as described in claim 1 or 2.

Images