Neutralizing antibody GR12 for resisting novel coronavirus SARS-CoV-2 and variant and application of neutralizing antibody GR12

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 GR12 provided by the invention, a heavy chain variable region and a light chain variable region of the neutralizing antibody GR12 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 GR12 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 GR12 has high affinity and dilution stability and is suitable for clinical large-dose administration.

Description

Technical Field

[0001] The present invention relates to a neutralizing antibody against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), specifically a neutralizing antibody that can specifically bind to the receptor-binding domain (RBD) of SARS-CoV-2 and the S-Trimer domain of the variant Omicron, thereby broadly preventing the infection of cells by SARS-CoV-2 and its variants, and its uses. Background Art

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to the genus Betacoronavirus, and its genome is a single-stranded RNA encoding various structural proteins including spike protein (S protein), nucleocapsid protein (N protein), etc. Among them, the S protein binds to the host cell ACE2 receptor through the receptor-binding domain (RBD), mediating virus invasion, and is the core target for the design of neutralizing antibodies and vaccines. The N protein is highly conserved and is the main target for nucleic acid detection and antigen detection.

[0003] Neutralizing antibodies are specific antibodies produced by the human immune system or through in vitro screening techniques, which can block virus invasion of host cells by binding to virus surface proteins (such as the RBD or NTD regions of the S protein). The main research and development paths include: (1) Antibody source: In early studies, high-titer neutralizing antibodies were isolated from the sera of recovered patients, and then candidate antibodies were screened through monoclonal antibody techniques (such as phage display, 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 in the NTD or S2 subunit to cope with virus mutations. (3) Functional verification: The antibody activity is initially evaluated through pseudovirus neutralization assays (based on VSV or lentiviral vectors), and then the effectiveness is confirmed through live virus neutralization assays (which need to be carried out in a biosafety level 3 laboratory, BSL-3). Neutralizing antibodies have important value in clinical treatment. For example, antibody drugs such as REGEN-COV (Casirivimab / Imdevimab) and Sotrovimab are used for high-risk patients through emergency use authorization (EUA), which can reduce the hospitalization rate and the risk of severe illness. However, the antibody escape phenomenon caused by virus mutations (such as mutations in the Omicron strain) has severely weakened the effectiveness of some antibodies, and there is an urgent need to develop broad-spectrum neutralizing antibodies.

[0004] The existing technical problems of virus-neutralizing antibodies are as follows: (1) Limitations: The antibody escape phenomenon caused by virus mutations severely weakens the effectiveness of some antibodies. For example, frequent mutations in the virus S protein (such as L452Q in Omicron BA.2 and F486V in BA.4 / 5) may change the antibody-binding epitope, resulting in the invalidation of existing antibodies. (2) Risk of antibody-dependent enhancement (ADE): Some non-neutralizing antibodies may mediate virus entry into immune cells through Fc receptors, enhancing infection. The risk needs to be avoided through epitope screening (such as preferentially selecting RBD non-ACE2 competitive epitopes) or engineering modification of the Fc segment. (3) Pharmacokinetic defects: The half-life of natural antibodies is relatively short (about 21 days), and their stability is poor. (4) Bottlenecks in the sensitivity and specificity of detection techniques: False-negative problems in nucleic acid detection: Low-virus-load samples (Ct value > 35), sampling errors (such as insufficient virus RNA obtained by nasal swabs), or mismatches between primers and probes and variant sequences (such as the D3L mutation in the N gene of the Alpha strain) may lead to missed detections. (5) Insufficient sensitivity of antigen detection: The sensitivity of existing reagents is only 60%-80%, which cannot replace nucleic acid detection; in addition, mutations in the N protein (such as the P13L mutation in Omicron BA.1) may affect the detection performance. (6) Decrease in the 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 titers still need to be improved.

[0005] In summary, the research and development of novel coronavirus-neutralizing antibodies and detection kits are the core technical means to address severe cases caused by virus infections and are a research hotspot for those skilled in the art. 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 novel coronavirus using bioinformatics analysis.

[0007] Another technical problem to be solved by the present invention is to propose a neutralizing antibody GR12 against novel coronavirus screened by the above method. This 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 novel coronavirus and its variants to cells in a broad-spectrum manner and finally 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 above neutralizing antibody GR12 against novel coronavirus.

[0009] Another technical problem to be solved by the present invention is to provide the use of the above neutralizing antibody GR12 against novel coronavirus.

[0010] To achieve the above technical objectives, the present invention adopts the following technical solutions:

[0011] A neutralizing antibody GR12 that binds to the novel coronavirus, comprising a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is as 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 above-mentioned neutralizing antibody GR12 that binds to the novel coronavirus. The nucleotide sequence encoding the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No.3, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region is as shown in SEQ ID No.4.

[0014] An antibody expression vector comprising 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 above-mentioned neutralizing antibody GR12 that binds to the novel coronavirus. The method includes PBMC collection, antigen labeling, magnetic bead enrichment and flow cytometry 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 includes a bioinformatics analysis step. After sorting B cells by flow cytometry, based on high-throughput sequencing of single B cell gene expression, analyze its heterogeneity, clonal evolution and immune response mechanism. The steps are as follows:

[0017] S1. IGHG subtype VDJ distribution screening: Analyze the combined frequency of IGHG heavy chain VDJ gene segments in B cells by high-throughput sequencing, and screen out IGHG antibody sequences whose VDJ distribution similarity to the overall B cell population reaches or exceeds 95%.

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

[0019] S3. Somatic hypermutation (SHM) enrichment:

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

[0021] (2) Use the sequencing data to align with the original V gene, mark and confirm the mutation hotspots.

[0022] S4. Utilization of isotype-specific CDR3 length differences:

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

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

[0025] The above-mentioned neutralizing antibody GR12 that binds to the novel coronavirus, or the above-mentioned nucleotide sequence, or the above-mentioned expression vector, or the above-mentioned host cell in the preparation of a reagent for blocking the infection of the novel coronavirus or in the preparation of a drug for preventing and / or treating the infection of the novel coronavirus.

[0026] The above-mentioned neutralizing antibody GR12 that binds to the novel 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 novel coronavirus and / or evaluating the immune effect of a vaccine.

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

[0028] A novel coronavirus neutralizing antibody ELISA detection kit, comprising the following components: a detection plate for capturing the novel coronavirus antigen, the above-mentioned biotin-labeled neutralizing antibody, and enzyme-labeled avidin.

[0029] An ELISA method for detecting the binding activity of a novel coronavirus neutralizing antibody, comprising the following steps:

[0030] a) Coating the SARS-CoV-2 S-Trimer protein and RBD protein onto an enzyme-labeled plate;

[0031] b) Diluting the antibody culture supernatant and adding it to the enzyme-labeled plate, incubating and then washing;

[0032] c) Adding an HRP-labeled secondary antibody, developing color and then measuring the OD450 value;

[0033] Among them, the antibody can detect the binding activity to 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 neutralizing antibody GR 12 that binds to the novel coronavirus, or a nucleotide sequence encoding the above-mentioned neutralizing antibody GR 12 that binds to the novel coronavirus, 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 performing bioinformatics analysis on single B cells to screen for affinity-matured antibodies, 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 novel neutralizing antibody GR 12 is obtained. The variable regions of its heavy chain and light chain can specifically bind to the RBD domain of SARS-CoV-2 and the S-Trimer domain of the Omicron variant, achieving broad-spectrum neutralization of SARS-CoV-2 virus and its variants.

[0038] (3) The neutralizing antibody GR 12 provided by the present invention has significantly better binding activity to S-Trimer and RBD under the dilution conditions of 2-fold and 300-fold, indicating its high affinity and dilution stability, and is suitable for the clinical demand of large-dose drug administration.

[0039] (4) The neutralizing antibody GR 12 provided by the present invention can simultaneously target SARS-CoV-2 RBD and Omicron S-Trimer, effectively inhibiting multiple variants (such as the inhibition rate of JN.1 and WT pseudovirus > 90%), overcoming the problem of failure of existing antibodies due to virus mutation. Antibody GR12 shows strong blocking of ACE2 receptor binding and pseudovirus neutralization ability in in vitro experiments, providing a core tool for the development of therapeutic drugs (such as reducing the severe disease rate) and evaluating the immune effect of vaccines. Description of the Drawings

[0040] Figure 1 It is a gating map for flow cytometry sorting of B cell hierarchies;

[0041] Figure 2 It is a plasmid map of the expression plasmid;

[0042] Figure 3 It is the expression level of the heavy chain and light chain of IGHG;

[0043] Figure 4 It is the expression level of the heavy chain and light chain of whole cells;

[0044] Figure 5The number of clones of IGHG BCR;

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

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

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

[0048] Figure 8 The CDR3 length distribution;

[0049] Figure 9 The somatic hypermutation analysis;

[0050] Figure 10A and 10B The CDR3 distribution results are shown as;

[0051] Figure 11 Pseudotime analysis of single-cell RNA seq sequencing for the developmental trajectory of memory B cells;

[0052] Figure 12 The neutralizing antibody affinity assay under 2-fold dilution conditions;

[0053] Figure 13 The neutralizing antibody affinity assay under 300-fold dilution conditions;

[0054] Figure 14 The evaluation of the ACE2 blocking ability of neutralizing antibodies;

[0055] Figure 15 The evaluation of the pseudovirus neutralization ability of neutralizing antibodies;

[0056] Figure 16 The situation of antibodies obtained by screening methods without using bioinformatics analysis. Detailed implementation mode

[0057] The present invention will be further described below in conjunction with specific embodiments. These embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out 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 meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are only for illustrative purposes.

[0058] The present invention screens memory B cells in populations infected with the novel coronavirus, uses single B cell antibody preparation technology, selects recovered individuals after COVID-19 infection, collects peripheral blood from the blood, isolates PBMCs from whole blood using density gradient centrifugation, enriches B cells using magnetic beads and sorts them by flow cytometry, analyzes their heterogeneity, clonal evolution and immune response mechanisms based on high-throughput sequencing of single B cell gene expression, and precisely screens neutralizing antibodies with excellent efficacy. The specific method is described as follows.

[0059] Example 1 Method for screening and efficacy verification of neutralizing antibodies provided by the present invention

[0060] 1. PBMC collection

[0061] Sample collection and pretreatment

[0062] Subject preparation: Select 10 eligible recovered COVID-19 patients (negative nucleic acid test and symptoms disappeared for ≥14 days). Sign the informed consent form and pass the ethical review.

[0063] Blood collection requirements: Use an EDTA anticoagulant vacuum blood collection tube and draw 10 mL of peripheral venous blood from each case. Process within 2 hours after blood collection to avoid refrigeration or freezing.

[0064] 1.1 PBMC separation steps (Ficoll density gradient centrifugation method)

[0065] Blood dilution: Mix whole blood and sterile PBS at a ratio of 1:1 (e.g., 10 mL of blood + 10 mL of PBS).

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

[0067] Centrifugation parameters: Horizontal centrifuge settings: 500 × g (or 1500 rpm), 25 minutes, acceleration / deceleration set to the lowest gear (avoid disturbing the layering) 14.

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

[0069] Washing and purification: Add 10 mL of PBS, centrifuge at 250 × g for 10 minutes, and discard the supernatant; repeat the washing once.

[0070] 2. Antigen labeling

[0071] 2.1 PE labeling

[0072] 1) Concentrate PE to 3 mg / mL, measure UV for quantification, and then add a certain proportion of SMCC. The NHS groups on SMCC will react with the primary amino groups on PE, and the MAL groups at the other end will react with the -SH 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 well, and react at room temperature for 2 h.

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

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

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

[0077] 2.2 APC Labeling

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

[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 well, and react at room temperature for 2 h.

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

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

[0082] 5) Purify the reaction sample with an S300 purification column, collect the conjugated 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 isolation: Add human EDTA-anticoagulated whole blood into a centrifuge tube containing Ficoll, centrifuge, and transfer the buffy coat layer containing lymphocytes and monocytes in the middle to a new centrifuge tube, and centrifuge to obtain PMBC cells (see the specific steps described in Example 1).

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

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

[0088] 3.2 Flow cytometry sorting

[0089] (1) Flow cytometry staining

[0090] Add IgG, IgM, CD19 antibody and fluorescently labeled protein to the cells for incubation, centrifuge to wash the cells, and resuspend with PBS.

[0091] (2) Flow cytometry sorting

[0092] Adjust the flow cytometry sorting liquid path, delay, sorting angle, and adjust the fluorescence compensation. By flow cytometry, we successfully sorted out memory B cells that are IgG positive, CD19 positive, and positive for the SARS-CoV-2 antigen. Specifically, first, the B cell population was identified using CD19 labeling, followed by screening for B cells expressing immunoglobulin G using IgG labeling, and finally, the specific reactivity of these cells was detected using the SARS-CoV-2 antigen. This strategy ensured that the sorted memory B cells are specific for the SARS-CoV-2, laying the foundation for subsequent research. The sorting results are shown as Figure 1 shown, Figure 1 a flow cytometry sorting hierarchical gating diagram, sorting out memory B cells that are IgG positive, CD19 positive, and positive for the SARS-CoV-2 antigen.

[0093] 4. PCR and vector construction

[0094] After lysing the sorted B cells, use a reverse transcription kit to obtain cDNA by reverse transcription. Obtain the heavy and light chain variable region sequences of the antibody through single B cell amplification technology. After separately constructing the heavy and light chain variable region fragments into expression vectors, obtain correctly sequenced heavy and light chain expression plasmids and transfer them for transient expression in HEK 293.

[0095] (1) Cell Lysis

[0096] Aliquot an equal volume of Lysis Solution into a 96-well plate to fully lyse the cells.

[0097] (2) Reverse Transcription

[0098] Adopt the two-step reverse transcription method. Aliquot the reverse transcription system into a PCR plate in sequence, mix well by pipetting, and then perform the reverse transcription reaction on the machine.

[0099] (3) Preparation of Heavy and Light Chain Variable Regions

[0100] Using cDNA as a template, through nested PCR and adopting a multi-round progressive amplification method, gradually amplify the heavy and light chain variable region genes of the antibody.

[0101] (4) Construction of Expression Vector

[0102] Add the heavy and light chain variable region fragments to the membrane-binding solution, mix well, and then add them to the purification column. Elute with Nuclease-Free Water to obtain the purified heavy and light chain variable region gene fragments. Construct the heavy and light chain variable region fragments into the CMV expression vector respectively. Transform the ligation product into competent cells and culture overnight at 37°C. Amplify the obtained monoclonal and sequence it to obtain the heavy and light chain sequences of the antibody. Extract the plasmid from the vector with correct sequencing and transfer it for small-scale expression. The schematic diagram of the expression plasmid map is as Figure 2 shown.

[0103] 5. HEK293 Transient Cell Culture

[0104] Passage and culture HEK293 cells with 293 serum-free CD medium. Mix the plasmid DNA to be expressed with the transfection reagent TF2 and then add it to the cells. Add 293 serum-free feeding solution on the 1st, 3rd, and 5th days after transfection.

[0105] Shake flask culture conditions: 5% CO2, temperature 37°C, shaker speed 175 rpm. Culture for 3 - 7 days, take the culture supernatant for ELISA detection. Take 1 - 2 mL of the positive binding clone supernatant of ELISA detection and deliver it for testing.

[0106] 6. ELISA Binding Detection of Culture Supernatant

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

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

[0109] (3) Plate washing: 300 μL / well of washing solution, wash the plate 2 times, and pat dry for the last time;

[0110] (4) Sample dilution: Dilute the culture supernatant 2-fold and 300-fold with sample dilution solution, mix well, 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) Plate washing: 300 μL / well of washing solution, wash the plate 3 times, and pat dry for the last time;

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

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

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

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

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

[0118] 7. ELISA neutralization assay of culture supernatant

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

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

[0121] (3) Plate washing: 300 μL / well of washing solution, wash the plate 2 times, and pat dry for the last time;

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

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

[0124] (6) Plate washing: 300 μL / well of washing solution, wash the plate 3 times, and pat dry for the last time;

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

[0126] (8) Wash the plate: Add 300 μL per well of washing solution, wash the plate 3 times, and pat dry for the last time;

[0127] (9) Color development: After mixing solution A and solution B at a ratio of 1:1, add 200 μL per well and incubate at room temperature in the dark;

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

[0129] 8. Pseudovirus neutralization assay

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

[0131] (Omicron) Spike Pseudovirus (hereinafter referred to as JN.1 pseudovirus) into pseudovirus diluent.

[0132] (2) Sample-pseudovirus neutralization: Divide each sample into 2 portions, 50 μL each; Mix 50 μL of the undiluted sample with 50 μL of the WT pseudovirus diluent, and mix 50 μL of the undiluted sample with 50 μL of the JN.1 pseudovirus diluent; Incubate at 37 °C for 1 h.

[0133] (3) Cell digestion and dilution: Digest 293T / (ACE2, TMPRSS2) cells using trypsin and dilute with complete medium to a cell suspension with a density of 30000 cells / 100 mL.

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

[0135] (5) Lysis detection: After incubating for 48 - 72 h, lyse the cells using cell lysis buffer, detect the chemiluminescence value, and calculate the inhibition rate.

[0136] Example 2 Results of bioinformatics analysis and screening experiments

[0137] As Figure 16As shown, the inventors initially did not use the bioinformatics analysis screening method after sorting out memory B cells that were IgG positive, CD19 positive, and positive for the new coronavirus antigen. As a result, all the antibodies screened were negative. Subsequently, after successfully sorting out memory B cells that were IgG positive, CD19 positive, and positive for the new coronavirus antigen in step 3 of Example 1, the inventors further improved the screening method. First, bioinformatics analysis was performed on individual B cells, and neutralizing antibodies with good effects were accurately screened out using the analysis conclusions.

[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. IGHG subtype VDJ distribution screening: Analyze the combined frequency of IGHG heavy chain VDJ gene segments in B cells by high-throughput sequencing, and screen out IGHG antibody sequences with a similarity to the VDJ distribution of the overall B cell population reaching or exceeding 95%.

[0141] Purpose: Retain the diversity generation pattern consistent with other subtypes of IGHG to ensure that the constructed antibody library has broad-spectrum functions and efficient antigen recognition capabilities.

[0142] First, the distribution of B cell antigen receptor (BCR) is obtained as shown in Table 1: It can be seen that the BCR distribution is mainly dominated by IGHG, among which IGHG1 has the highest proportion in IGHG; followed by IGHM, IGHA, and IGHD in turn. The IGHG class has the highest proportion, and IGHG1 is the main subtype in IGHG, suggesting its core role in humoral immunity, such as antibodies neutralizing pathogens. The high proportion of IGHG1 may be related to its long half-life, complement activation ability, and placental penetrability (such as pregnancy immune regulation).

[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％ Number of cells 3401 63 1814 1554 229 137 2704

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

[0146] We further analyzed the expression levels of the heavy and light chains of BCR in all cells. The results showed that the expression levels of the heavy and light chains of BCR in all cells were relatively consistent, as Figure 4As shown, the expression levels of heavy chain (HC) and light chain (LC) are highly synchronized, possibly due to the allelic exclusion mechanism during B cell development, ensuring the specificity of a single BCR.

[0147] As Figure 5 shown, we further analyzed the number of clones of IGHG BCR, and the results showed a low correlation between BCR expression and the number of clones of IGHG BCR. This indicates that there is no significant association between the expression level of BCR and the clonal diversity of IGHG BCR.

[0148] As Figure 6 shown, the UMI expression levels in IGHG cells showed that the BCR UMI expression levels of most cells were around 25 UMI, and a few cells expressed high levels of BCR UMI. This indicates that most cells are in an inactive state.

[0149] As Figure 7A and Figure 7B shown, the VDJ distribution levels in IGHG and overall cells are not significantly different. VDJ rearrangement is the core mechanism for B cells to generate diverse antibodies. As a major antibody subtype, the VDJ distribution of IGHG is consistent with that of overall cells. This conclusion indicates that in the B cell population studied, the antibody diversity generation pattern of the IGHG subtype is not significantly different from that of other subtypes or overall cells.

[0150] S2. Optimization of CDR3 length distribution: Screen antibody sequences with a heavy chain CDR3 amino acid length of 16 to 17, and require that the proportion of this sequence in the sample is not less than 60%. At the same time, exclude abnormal sequences in the light chain CDR3 sequences 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 enhancing the binding affinity of the antibody, as Figure 8 shown.

[0152] As Figure 8 shown, we further analyzed the CDR3 length distribution, and the antibody sequences with a heavy chain CDR3 amino acid length of 16 and 17 were the most numerous. CDR3 is the key region for antibody-antigen binding, and its length and sequence diversity directly affect the specificity and affinity of the antibody. The dominance of sequences with a heavy chain CDR3 length of 16 and 17 indicates that CDR3s of these lengths play an important role in the B cell antibody repertoire and are closely related to the functional requirements of the antibody.

[0153] S3. Somatic hypermutation (SHM) enrichment:

[0154] (1) Screen antibody sequences with somatic hypermutation frequencies in the heavy chain reaching or exceeding 15% and in the light chain reaching or exceeding 10%.

[0155] (2) Use the sequencing data to align with the original V gene, mark and confirm the hypermutation hot regions (see the circled regions in Figure 9 ).

[0156] Objective: 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] As Figure 9 shown, we also performed somatic hypermutation analysis. The results showed that different levels of hypermutation occurred in both the heavy chain and the light chain. The hypermutations are circled in the figure. Somatic hypermutation is a key process for B cells to enhance antibody affinity under antigen stimulation. The occurrence of hypermutation in both the heavy chain and the light chain indicates that B cells have experienced affinity maturation in the immune response, thus optimizing the function of antibodies.

[0158] S4. Utilization of Isotype-specific CDR3 length differences:

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

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

[0161] Objective: Utilize the structural advantage of the longer 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] As Figure 10A and 10B shown, the Isotype CDR3 distribution results show that the length of the heavy chain CDR3 sequence is longer than that of the light chain ([[]] Figure 10A mean around 50 is longer than Figure 10B mean around 30). The heavy chain CDR3 is usually longer than the light chain CDR3, and this feature is consistent with the structural and functional requirements of antibodies. The heavy chain CDR3 plays a dominant role in antigen binding, and its longer length helps to form a more complex binding site, thus enhancing the specificity and affinity of the antibody.

[0163] Single-cell RNA-seq results:

[0164] As Figure 11As shown, we performed single-cell RNA seq sequencing, mainly focusing on CD74, CD79a, and CXCR4 (the dark parts in the figure). Pseudotime analysis showed the developmental trajectory of memory B cells. CD74 is mainly involved in antigen presentation and acts as a receptor for MIF, affecting immune responses and cell survival. CD79a is an important component of the B cell antigen receptor complex, crucial for B cell development and signal transduction. In B cells, CXCR4 signaling regulates their migration to lymphoid organs and the bone marrow, which is an important part of the immune response. These findings not only deepen our understanding of the functions and regulatory mechanisms of B cells in the immune system but also provide an important basis for further studying the role of B cells in immune responses and their potential applications in SARS-COV-2. Example 3 Obtaining the variable regions of the heavy and light chains of memory B cells and constructing the plasmid vectors for the heavy and light chains of the novel coronavirus neutralizing antibody

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

[0166] The main objective of this study was to screen for specific neutralizing antibodies from the population after COVID-19 infection. The specific steps were as follows: Sorting and RNA extraction of single B cells Specific memory B cells were sorted from the recovered individuals after COVID-19 infection by flow cytometry (FACS). The sorted B cells were a population of memory B cells that were CD19+, CD27+, and IgG+. Total RNA was further extracted from them.

[0167] 1. Synthesis of cDNA and PCR amplification

[0168] The extracted RNA was transcribed into complementary DNA (cDNA) using reverse transcriptase. After the reverse transcription reaction, specific primers were used to perform PCR amplification for the variable regions of the immunoglobulin heavy and light chains. The successful amplification of the 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 products, the fragments containing the correct antibody variable region sequences were selected, digested with restriction enzymes, and ligated into the expression vectors. The constructed vectors contained the full-length antibody genes. The cloned antibody genes were sequenced by Sanger sequencing to verify the correctness of their sequences.

[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 antibodies 24 - 48 hours later, and the antibody yield and function were preliminarily verified by ELISA and neutralization experiments.

[0173] The obtained sequence is as follows:

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

[0175] MGWSLILLFLVAVATRVLSQVQLVESGGILVQPGGSLRLSCAASGFSFSNYDMHWVRQTTGR

[0176] GLDWVSTIGTSGDTYYADSVKGRFTISREDAKKSLYLQMNSLTVGDTAVYYCARGSDDWTGW

[0177] YFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT

[0178] SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC

[0179] PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK

[0180] TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT

[0181] LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

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

[0183] ATGGGCTGGTCCCTGATTCTGCTGTTCCTGGTGGCTGTGGCTACCAGGGTGCTGAGTCAGGT

[0184] GCAGCTGGTGGAGTCTGGGGGAATCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG

[0185] CAGCCTCTGGATTCTCCTTCAGTAACTACGACATGCACTGGGTCCGCCAAACTACAGGAAGA

[0186] GGTCTGGATTGGGTCTCAACTATTGGCACTTCTGGTGACACATACTATGCGGACTCCGTGAA

[0187] GGGCCGATTCACCATCTCCAGAGAAGATGCCAAGAAGTCCCTGTATCTTCAAATGAACAGTC

[0188] TGACAGTCGGGGACACGGCTGTATATTACTGTGCAAGAGGGAGCGACGACTGGACCGGGTGG

[0189] TACTTTGATTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCAAGCACCAAGGGCCC

[0190] ATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT

[0191] GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACC

[0192] AGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGT

[0193] GGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGC

[0194] CCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGC

[0195] CCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACC

[0196] CAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCC

[0197] ACGAAGACCCCGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG

[0198] ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT

[0199] GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAG

[0200] CCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC

[0201] CTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG

[0202] CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA

[0203] AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG

[0204] GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCA

[0205] CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

[0206] Kappa chain amino acid sequence SEQ ID No.2:

[0207] MGWSCIILFLVATATGVHSDIQLTQSPVTLSASVGDRVTITCRASQGIRGSVAWYQQKPGEG

[0208] PKLLLFAANKLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYYLNPPPAFGQGTKV

[0209] EVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ

[0210] DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Kappa chain nucleotide sequence SEQ ID No.4:

[0211] ATGGGCTGGTCCTGTATCATCCTGTTCCTGGTGGCTACAGCCACAGGAGTGCATAGTGACAT

[0212] CCAGTTGACCCAGTCTCCAGTCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTT

[0213] GCCGGGCGAGTCAGGGCATTAGGGGTTCTGTTGCCTGGTATCAACAAAAACCAGGAGAAGGC

[0214] CCTAAGCTCCTGCTCTTTGCTGCAAACAAATTGCAAAGTGGGGTCCCGTCCAGGTTCAGTGG

[0215] CAGTGGATCTGGGACGGATTACACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAA

[0216] CTTATTACTGTCAACAGTATTATCTTAATCCCCCTCCGGCGTTCGGCCAAGGGACCAAGGTG

[0217] GAAGTCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTT

[0218] GAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG

[0219] TACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG

[0220] GACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGA

[0221] GAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGA

[0222] GCTTCAACAGGGGAGAGTGTTAG

[0223] Example 4 Detection of the Affinity and Neutralizing Activity of the Neutralizing Antibodies Screened in the Present Invention 1. Detection of the Binding of Neutralizing Antibodies in Culture Supernatant by ELISA

[0224] To deeply study the antigenic epitope characteristics of the SARS-CoV-2 Omicron variant and the neutralizing antibody screening strategy, we detected 18 neutralizing antibodies screened by the ELISA method.

[0225] Specific experimental results: As Figure 12 shown, under the 2-fold dilution condition, the binding activities of the antibody supernatant to Omicron S-Trimer and RBD were detected. From the results, it can be seen that GR12 of the present invention has a strong neutralizing activity compared with other neutralizing antibodies against Omicron S-Trimer and RBD. As Figure 13 shown, after further diluting the antibody supernatant 300-fold, the neutralizing antibody GR12 provided by the present invention still maintains a strong neutralizing activity compared with other neutralizing antibodies against Omicron S-Trimer and RBD.

[0226] The above results indicate that although most antibodies have strong binding activity under low dilution conditions, as the dilution factor increases, only some antibodies can maintain high affinity, especially antibodies against RBD. This suggests that there are significant differences in the binding strength of antibodies and that they may have specificity for different antigenic epitopes. These high-affinity antibodies provide excellent candidates for further neutralization experiments and structural analysis, and lay the foundation for the optimized design of COVID-19 vaccines and the development of antibody drugs. The above results also demonstrate the effectiveness of the method for screening antibodies obtained by bioinformatics analysis, provide new data support for understanding the antigenic characteristics of the Omicron variant, and offer important implications for basic research and clinical applications in related fields.

[0227] 2. Neutralization Detection of Culture Supernatant by ELISA

[0228] The blocking ability of antibodies in the culture supernatant against the S / RBD-ACE2 interaction was evaluated by ELISA to screen candidate antibody strains with neutralizing activity.

[0229] The experimental results are as Figure 14 shown. The neutralizing antibody GR12 antibody supernatant provided by the present invention can effectively block the binding of SARS-CoV-2 S protein or RBD to the ACE2 receptor simultaneously, showing broad-spectrum activity. Thus, it significantly inhibits the key step of virus invasion into host cells. These antibodies further showed good neutralizing activity in in vitro verification and were able to protect host cells from SARS-CoV-2 infection.

[0230] The above results indicate that the GR12 antibody supernatant has high neutralizing activity and clinical application potential, laying the foundation for further in-depth research. They can not only serve as important candidates for the development of new antibody drugs but also provide key clues for understanding the infection mechanism of SARS-CoV-2 and antiviral strategies.

[0231] 3. Pseudovirus Neutralization Detection

[0232] 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.

[0233] The detection results are as Figure 15 shown. The neutralizing antibody GR12 antibody provided by the present invention has an inhibition rate of more than 90% against JN.1 and WT pseudoviruses. This provides an important candidate resource for the subsequent functional verification of antibodies and their application in the development of therapeutic drugs, 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 GR12 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 as shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No.

2.

2. The neutralizing antibody GR12 that binds to the novel coronavirus as described in 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 Omicron.

3. The nucleotide sequence encoding the neutralizing antibody GR12 that binds to the novel coronavirus, characterized in that: The nucleotide sequence encoding the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No.3, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region is as shown in SEQ ID No.

4.

4. An antibody expression vector, characterized in that It contains the nucleotide sequence described in claim 3, and the vector is a mammalian expression vector.

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

6. A method for screening the neutralizing antibody GR12 that binds to the novel coronavirus as described in claim 1 or 2, characterized in that It includes the steps of sorting B cells by flow cytometry and then analyzing the heterogeneity, clonal evolution and immune response mechanism of single B cell gene expression based on high-throughput sequencing. The specific method is as follows: S1. Screening of IGHG subtype VDJ distribution: Analyze the combination frequency of IGHG heavy chain VDJ gene segments in B cells by high-throughput sequencing, and screen out IGHG antibody sequences with a similarity to the VDJ distribution of the overall B cell population reaching or exceeding 95%. S2. Optimization of CDR3 length distribution: Screen antibody sequences with a heavy chain CDR3 amino acid length of 16 to 17, and require that the proportion of this sequence in the sample is not less than 60%; at the same time, exclude abnormal sequences in the light chain CDR3 sequence with a length deviating from the mean by ±2 standard deviations. S3. Enrichment of somatic hypermutation SHM: Screen antibody sequences with a somatic hypermutation frequency in the heavy chain reaching or exceeding 15% and a hypermutation frequency in the light chain reaching or exceeding 10%; use the sequencing data to align with the original V gene to mark and confirm the mutation hot spot regions. S4. Utilization of Isotype-specific CDR3 length difference: For the heavy chain CDR3 sequence, set the screening range to 48 to 52 amino acids; for the light chain CDR3 sequence, set the screening range to 28 to 32 amino acids.

7. Use of the neutralizing antibody GR12 that binds to the novel coronavirus described in claim 1 or 2, or the nucleotide sequence described in claim 3, or the expression vector described in claim 4, or the host cell described in claim 5 in the preparation of a reagent for blocking novel coronavirus infection or a drug for preventing and / or treating novel coronavirus infection.

8. Use of the neutralizing antibody GR12 that binds to the novel coronavirus described in claim 1 or 2, or the nucleotide sequence described in claim 3, or the expression vector described in claim 4, or the host cell described in claim 5 in the preparation of a kit for detecting novel coronavirus neutralizing antibody and / or evaluating the immune effect of a vaccine.

9. The application 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 It includes the neutralizing antibody GR12 that binds to the novel coronavirus described in claim 1 or 2.

Images