Neutralizing antibody gr12 against novel coronavirus sars-cov-2 and variants and uses thereof

Abstract

The application discloses a novel coronavirus neutralizing antibody, a detection kit and application thereof, an amino acid sequence of a heavy chain variable region of the neutralizing antibody is shown as SEQ ID No. 1, and an amino acid sequence of a light chain variable region is shown as SEQ ID No. 2. The application screens the affinity-matured antibody through bioinformatics analysis of a single B cell, combines single-cell RNA sequencing, VDJ rearrangement analysis and somatic hypermutation research, optimizes the antibody screening process, avoids blindness of a traditional method, and improves the accuracy and effectiveness of antibody screening. The neutralizing antibody GR12 provided by the application can specifically combine with a RBD domain of SARS-CoV-2 and a S-Trimer domain of an Omicron mutant strain through the heavy chain and light chain variable regions, realizes broad-spectrum neutralization of SARS-CoV-2 and mutant strains thereof, and the antibody GR12 is significantly superior to other antibodies in the binding activity of S-Trimer and RBD under 2-fold and 300-fold dilution conditions, indicating that the antibody has high affinity and dilution stability, and is suitable for clinical large-dose administration requirements.

Description

Technical Field

[0001] This invention relates to a neutralizing antibody against the novel coronavirus, specifically a neutralizing antibody capable of specifically binding to the RBD domain of SARS-CoV-2 and the S-Trimer domain of the variant Omicron, thereby broadly blocking the infection of cells by the novel coronavirus and its variants, and its uses. Background Technology

[0002] The novel coronavirus (SARS-CoV-2) belongs to the genus β-coronavirus. Its genome is a single-stranded RNA that encodes various structural proteins, including the spike protein (S protein) and the nucleocapsid protein (N protein). The S protein binds to the host cell's ACE2 receptor through its 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 a major target for nucleic acid and antigen detection.

[0003] Neutralizing antibodies are specific antibodies produced by the human immune system or through in vitro screening techniques. They can block viral invasion of host cells by binding to viral surface proteins (such as the RBD or NTD regions of the S protein). Their development pathway mainly includes: (1) Antibody source: Early studies isolated high-titer neutralizing antibodies from the serum of recovered patients, and then screened candidate antibodies using monoclonal antibody techniques (such as phage display and hybridoma techniques). (2) Target screening: Targeting the RBD region of the S protein is the main strategy, as it directly participates in ACE2 receptor binding; some antibodies target conserved epitopes of the NTD or S2 subunit to address viral mutations. (3) Functional verification: Antibody activity is initially assessed through a pseudovirus neutralization test (based on VSV or lentiviral vectors), and then its effectiveness is confirmed through a live virus neutralization test (which must be performed in a biosafety level 3 laboratory, BSL-3). Neutralizing antibodies have significant value in clinical treatment. For example, antibody drugs such as REGEN-COV (Casirivimab / Imdevimab) and Sotrovimab have been granted Emergency Use Authorization (EUA) for high-risk patients, 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 broad-spectrum neutralizing antibodies.

[0004] The existing technical problems with virus neutralizing antibodies are: (1) Limitations: Antibody escape caused by viral mutations severely weakens the effectiveness of some antibodies. For example, frequent mutations of the viral S protein (such as L452Q of Omicron BA.2 and F486V of BA.4 / 5) may change the antibody binding epitope, causing existing antibodies to become ineffective. (2) Risk of antibody-dependent enhancement (ADE): Some non-neutralizing antibodies may enter immune cells through the Fc receptor, enhancing infection. Epitope screening (such as prioritizing non-ACE2 competitive epitopes of RBD) or Fc segment engineering are needed to avoid this risk. (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 negatives in nucleic acid detection: Low viral load samples (Ct value > 35), sampling errors (such as insufficient viral RNA obtained from nasal swabs) or mismatch between primer probes and mutant strain sequences (such as the D3L mutation of the N gene in Alpha strains) 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 of Omicron BA.1) may affect detection performance. (6) Decreased efficacy of neutralizing antibodies: Most approved antibodies are ineffective against Omicron strains because they target RBD epitopes; only a few broad-spectrum antibodies maintain activity, but their titers still need to be improved.

[0005] In summary, the development of neutralizing antibodies and detection kits for the novel coronavirus is a core technological means to combat severe illness caused by viral infection and is a hot research topic for those skilled in the art. Summary of the Invention

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

[0007] Another technical problem to be solved by the present invention is to propose a neutralizing antibody GR12 that binds to the novel coronavirus obtained by screening using the above method. This neutralizing antibody can specifically bind to the RBD domain of SARS-CoV-2 and the S-Trimer domain of the mutant Omicron, thereby broadly and effectively preventing the infection of cells by the novel coronavirus and its mutants, 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 by the above-mentioned neutralizing antibody GR12 that binds to the novel coronavirus.

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

[0010] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution:

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

[0014] An antibody expression vector comprising the above-mentioned nucleotide sequence, wherein the vector is a mammalian expression vector.

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

[0016] A method for screening the aforementioned neutralizing antibody GR12 against the novel coronavirus, the method comprising: PBMC collection, antigen labeling, magnetic bead enrichment and flow cytometry sorting of B cells, PCR and vector construction, HEK293 transient transfection cell culture, ELISA binding detection of culture supernatant, ELISA neutralization detection of culture supernatant, and pseudovirus neutralization detection; preferably, the method further comprises a bioinformatics analysis step, wherein after sorting B cells by flow cytometry, high-throughput sequencing of single B cell gene expression is used to analyze their heterogeneity, clonal evolution, and immune response mechanisms, the steps of which are as follows:

[0017] S1. Screening of VDJ distribution of IGHG subtypes: The combination frequency of IGHG heavy chain VDJ gene fragments in B cells was analyzed by high-throughput sequencing to screen for IGHG antibody sequences that have a similarity of 95% or more to the VDJ distribution of the overall B cell population.

[0018] S2. CDR3 Length Distribution Optimization: Antibody sequences with a heavy chain CDR3 amino acid length of 16 to 17 were screened, requiring that such sequences account for no less than 60% of the samples. At the same time, abnormal sequences in the light chain CDR3 sequence that deviated from the mean ± 2 standard deviations (SD) were excluded.

[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 a hypermutation frequency of 10% or more in the light chain.

[0021] (2) By comparing the sequencing data with the original V gene, mutation hotspot regions were marked and confirmed.

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

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

[0024] (2) For the light chain CDR3 sequence, the screening range was set 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, are used in the preparation of reagents to block novel coronavirus infection or in the preparation of drugs for the prevention and / or treatment of novel coronavirus infection.

[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, are used in the preparation of kits for detecting neutralizing antibodies against the novel coronavirus and / or for evaluating the immunization effect of vaccines.

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

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

[0029] An ELISA method for detecting the binding activity of neutralizing antibodies against COVID-19 includes the following steps:

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

[0031] b) Dilute the antibody culture supernatant and add it to the ELISA plate, incubate, and then wash.

[0032] c) Inject HRP-labeled secondary antibody, develop colorimetric results, and measure the OD450 value;

[0033] The antibody can be detected to bind 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 aforementioned novel coronavirus-binding neutralizing antibody GR 12, or a nucleotide sequence encoding the aforementioned novel coronavirus-binding neutralizing antibody GR 12, or the aforementioned expression vector.

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

[0036] (1) This invention optimizes the antibody screening method by screening antibodies with mature affinity through bioinformatics analysis of single B cells, combined with single-cell RNA sequencing, VDJ rearrangement analysis and somatic hypermutation studies, to optimize the antibody screening process, avoid the blindness of traditional methods, and improve 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 was obtained. Its 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, thereby achieving broad-spectrum neutralization of SARS-CoV-2 virus and its variants.

[0038] (3) The neutralizing antibody GR 12 provided by the present invention exhibits significantly better binding activity against S-Trimer and RBD than other antibodies under 2-fold and 300-fold dilution conditions, indicating its high affinity and dilution stability, making it suitable for clinical high-dose administration needs.

[0039] (4) The neutralizing antibody GR12 provided by this invention can simultaneously target SARS-CoV-2 RBD and Omicron S-Trimer, effectively inhibiting multiple variants (such as JN.1 and WT pseudoviruses with an inhibition rate >90%), overcoming the problem of existing antibodies becoming ineffective due to viral mutations. In in vitro experiments, antibody GR12 demonstrated a strong ability to block ACE2 receptor binding and neutralize pseudoviruses, providing a core tool for developing therapeutic drugs (such as reducing the rate of severe illness) and evaluating the immunization effect of vaccines. Attached Figure Description

[0040] Figure 1 A gate diagram for flow cytometry sorting of B-cell hierarchies;

[0041] Figure 2 To express plasmid maps;

[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 throughout the entire cell;

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

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

[0046] Figure 7A This represents the overall cellular VDJ distribution level.

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

[0048] Figure 8 The length distribution is CDR3;

[0049] Figure 9 For somatic cell hypermutation analysis;

[0050] Figure 10A and 10B The results of the CDR3 distribution are shown below;

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

[0052] Figure 12 This assay was performed to determine the affinity of the neutralizing antibody under a 2-fold dilution.

[0053] Figure 13 The affinity of neutralizing antibodies was determined under a 300-fold dilution condition.

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

[0055] Figure 15 To assess the neutralizing capacity of neutralizing antibodies against pseudoviruses;

[0056] Figure 16 This shows the antibody results obtained without using bioinformatics analysis. Detailed Implementation

[0057] The present invention is further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0058] This invention screens memory B cells from individuals infected with the novel coronavirus. Using single B cell antibody preparation technology, peripheral blood is collected from recovered COVID-19 patients. Peripheral blood cells (PBMCs) are separated from whole blood using density gradient centrifugation. B cells are enriched using magnetic beads and sorted by flow cytometry. Based on high-throughput sequencing of single B cell gene expression, heterogeneity, clonal evolution, and immune response mechanisms are analyzed to accurately screen for highly effective neutralizing antibodies. The specific method is described below.

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

[0060] 1. PBMC Collection

[0061] Sample collection and preprocessing

[0062] Subject preparation: Ten eligible recovered COVID-19 patients (negative nucleic acid test and symptom disappearance for ≥14 days) were selected. Informed consent was obtained and ethical review was conducted.

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

[0064] 1.1 PBMC Separation Procedure (Ficoll Density Gradient Centrifugation)

[0065] Blood dilution: 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 buffer (density 1.077 g / mL, equilibrated to 20-25°C beforehand) to a 15 mL centrifuge tube. Slowly add diluted blood along the tube wall to the top layer of the separation buffer, maintaining clear separation (avoiding mixing).

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

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

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

[0070] 2. Antigen labeling

[0071] 2.1 PE Marking

[0072] 1) Concentrate PE to 3 mg / mL, measure UV quantification, 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 at the other end will react with the -SH group on the protein.

[0073] 2) Concentrate the recombinant Omicron EG.5.1Spike 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 h.

[0074] 3) Desalt the activated protein into PBS, concentrate it, and measure the 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 overnight at 4°C.

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

[0077] 2.2 APC Marking

[0078] 1) Concentrate APC to 3 mg / mL, measure UV quantification, 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 at the other end will react with the -SH group on the peptide.

[0079] 2) Concentrate the recombinant Omicron BA.2.86Spike 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 h.

[0080] 3) Desalt the activated protein into PBS, concentrate it, and measure the 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 overnight at 4°C.

[0082] 5) Purify the reaction sample using an S300 purification column, collect the coupled protein, measure the 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: Add human EDTA anticoagulated whole blood to a centrifuge tube containing Ficoll, centrifuge, take the white membrane layer containing lymphocytes and monocytes in the middle to a new centrifuge tube, centrifuge to obtain PMBC 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, centrifuge and wash the cells. After resuspending in sorting buffer, add to the sorting column and collect flowthrough.

[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 them in the sorting buffer, add them to the sorting column, and collect the cells in the sorting column.

[0088] 3.2 Flow sorting

[0089] (1) Flow cytometry staining

[0090] Cells were incubated with IgG, IgM, CD19 antibodies and fluorescently labeled proteins, centrifuged, washed, and resuspended in PBS.

[0091] (2) Flow sorting

[0092] Adjusting the flow cytometry sorting path, delay, and sorting angle, and modifying fluorescence compensation, we successfully sorted IgG-positive, CD19-positive memory B cells that were also positive for the SARS-CoV-2 antigen using flow cytometry. Specifically, we first identified the B cell population using CD19 labeling, then screened for B cells expressing immunoglobulin G using IgG labeling, and finally tested the specific reactivity of these cells using the SARS-CoV-2 antigen. This strategy ensured that the sorted memory B cells were specific for SARS-CoV-2, laying the foundation for subsequent research. The sorting results are shown below. Figure 1 As shown, Figure 1 A gate diagram was set up for the flow cytometry sorting hierarchy to sort out memory B cells that were positive for IgG, CD19, and SARS-CoV-2 antigen.

[0093] 4. PCR and vector construction

[0094] After lysing the sorted B cells, 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 to obtain correctly sequenced heavy and light chain expression plasmids, which were then delivered for transient HEK 293 expression.

[0095] (1) Cell lysis

[0096] Add equal volumes of Lysis Solution to 96-well plates to allow for complete cell lysis.

[0097] (2) Reverse transcription

[0098] A two-step reverse transcription method was used. The reverse transcription system was sequentially aliquoted into a PCR plate, mixed by pipetting, and then loaded onto the PCR machine for reverse transcription.

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

[0100] Using cDNA as a template, nested PCR was employed to gradually amplify the variable region gene of the antibody heavy and light chains using a multi-round progressive amplification method.

[0101] (4) Construction of expression vector

[0102] The heavy and light chain variable region fragments were added to membrane binding buffer, mixed thoroughly, and then added to a purification column. Elution with Nuclease-Free Water yielded purified heavy and light chain variable region gene fragments. The heavy and light chain variable region fragments were separately constructed into CMV expression vectors, and the ligation products were transformed into competent cells and cultured overnight at 37°C. The obtained single clones were amplified and sequenced to obtain the antibody heavy and light chain sequences. Plasmids were extracted from vectors with correct sequencing results and expressed in a small-scale trial. A schematic diagram of the expression plasmid map is shown below. Figure 2 As shown.

[0103] 5. HEK293 transient transduction cell culture

[0104] HEK293 cells were passaged in 293 serum-free CD medium. The plasmid DNA to be expressed was mixed with 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, temperature 37℃, shaker speed 175 rpm. Culture for 3-7 days, then collect the culture supernatant for ELISA detection. Collect 1-2 mL of the supernatant from ELISA-positive binding clones for testing.

[0106] 6. ELISA combined with culture supernatant for detection.

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

[0108] (2) Sealing: Shake off the 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: 300 μL / well washing solution, wash the plate twice, and pat dry after the last wash;

[0110] (4) Sample dilution: Dilute the culture supernatant by 2 times and 300 times with sample dilution solution and mix thoroughly for later use;

[0111] (5) Sample addition: Add 100 μL of the diluted supernatant to the microplate at room temperature for 2 hours after mixing.

[0112] (6) Washing the plate: 300 μL / well washing solution, wash the plate 3 times, and pat dry after the last wash;

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

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

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

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

[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 detection using culture supernatant

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

[0120] (2) Sealing: Shake off the 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: 300 μL / well washing solution, wash the plate twice, and pat dry after the last wash;

[0122] (4) Sample dilution: Dilute the supernatant to its original volume and set aside; dilute the RBD protein to 0.05 μg / mL with sample dilution buffer and set aside;

[0123] (5) Sample addition: Add the supernatant to the microplate at 100 μL / well, and use the blank control as the sample diluent; then add the diluted RBD protein to the microplate at 100 μL / well, and finally add 200 μL / well of sample. Mix well and incubate at room temperature for 1 h.

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

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

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

[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 termination solution to each well and immediately measure the OD value at a wavelength of 450 nm.

[0129] 8. Detection of neutralized pseudoviruses

[0130] (1) Dilution of pseudoviruses: The pseudoviruses SARS-CoV-2 (2019-nCoV) Spike Ps eudovirus (hereinafter referred to as WT pseudovirus) and SARS-CoV-2 JN.1 were diluted.

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

[0132] (2) Sample-pseudovirus neutralization: Each sample was divided into two portions, each 50 μL; one portion of the original sample solution (50 μL) was mixed with WT pseudovirus diluent (50 μL), and the other portion of the original sample solution (50 μL) was mixed with JN.1 pseudovirus diluent (50 μL); 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 with a density of 30,000 cells / 100 mL.

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

[0135] (5) Lysis detection: After incubation for 48-72 hours, 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, the inventors initially did not use bioinformatics analysis to screen memory B cells that were IgG positive, CD19 positive, and positive for the SARS-CoV-2 antigen, resulting in all antibodies being negative. Subsequently, after successfully screening IgG positive, CD19 positive, and SARS-CoV-2 positive memory B cells in step 3 of Example 1, the inventors further improved the screening method by performing bioinformatics analysis on individual B cells and using the analysis results to accurately screen and obtain effective neutralizing antibodies.

[0138] Bioinformatics analysis methods are as follows:

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

[0140] S1. Screening of VDJ distribution of IGHG subtypes: The combination frequency of IGHG heavy chain VDJ gene fragments in B cells was analyzed by high-throughput sequencing to screen for IGHG antibody sequences that have a similarity of 95% or more to the VDJ distribution of the overall B cell population.

[0141] Objective: To preserve the consistent diversity generation pattern of IGHG subtypes with other subtypes, and to ensure that the constructed antibody library has broad-spectrum functionality and efficient antigen recognition capability.

[0142] First, the distribution of B-cell antigen receptors (BCRs) was obtained, as shown in Table 1. It can be seen that the BCR distribution is mainly dominated by IGHG, with IGHG1 having the highest proportion; followed by IGHM, IGHA, and IGHD. IGHGs have the highest proportion, with IGHG1 being the major subtype, suggesting its core role in humoral immunity, such as antibody neutralization of pathogens. The high proportion of IGHG1 may be related to its long half-life, complement activation capacity, and placental penetration (e.g., immune regulation 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 count 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 in IGHG. The results showed that the expression levels of the heavy and light chains in IGHG BCR were relatively consistent, such as... Figure 3 As shown.

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

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

[0148] like Figure 6 As shown, the expression level of UMI in IGHG cells indicates that most cells express BCR UMI at around 25 UMI, while a few cells express high levels of BCR UMI. This suggests 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 cells are not significantly different. VDJ rearrangement is a core mechanism for B cells to generate diverse antibodies. IGHG, as a major antibody subtype, exhibits a VDJ distribution consistent with that in whole cells. This conclusion indicates that in the studied B cell population, the antibody diversity generation pattern of the IGHG subtype is not significantly different from other subtypes or whole cells.

[0150] S2. CDR3 length distribution optimization: Screen antibody sequences with a heavy chain CDR3 amino acid length of 16 to 17, requiring that such sequences account for no less than 60% of the samples. At the same time, abnormal sequences in the light chain CDR3 sequence that deviate from the mean ± 2 standard deviations (SD) are excluded.

[0151] Objective: To confirm, through structural biology validation, that the 16-17 amino acid heavy chain CDR3 can form a more stable antigen-binding epitope, thereby enhancing antibody binding affinity, such as... Figure 8 As shown.

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

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

[0154] (1) Screen 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) By comparing sequencing data with the original V gene, mutation hotspot regions were identified and confirmed (see [link to original text]). Figure 9 (The area circled in the middle).

[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] like Figure 9 As shown, we also performed somatic hypermutation analysis, and the results showed that both the heavy and light chains underwent hypermutation at different levels. The circled areas in the figure represent hypermutations. Somatic hypermutation is a key process by which B cells enhance antibody affinity in response to antigen stimulation. The presence of hypermutations in both the heavy and light chains indicates 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 the heavy chain CDR3 sequence, the screening range was set to 48 to 52 amino acids (based on mean ± SD);

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

[0161] Objective: To utilize the structural advantage of the longer heavy chain CDR3 (see...) Figure 10A and 10B This increases the complexity of the antigen-binding interface, further improving antibody binding efficiency and functional stability.

[0162] like Figure 10A and 10B As shown, the Isotype CDR3 distribution results indicate that the heavy chain CDR3 sequence length is longer than that of the light chain. Figure 10A The average is around 50 compared to Figure 10B (The average length is around 30). Heavy chain CDR3s are typically longer than light chain CDR3s, a characteristic consistent with the structural and functional requirements of antibodies. Heavy chain CDR3s play a dominant role in antigen binding, and their longer length facilitates the formation of more complex binding sites, thereby enhancing antibody specificity and affinity.

[0163] Single-cell RNA-seq results:

[0164] like Figure 11As shown, we performed single-cell RNA seq sequencing, primarily detecting CD74, CD79a, and CXCR4 (the darker areas in the figure). Pseudo-temporal analysis revealed the developmental trajectory of memory B cells. CD74 is mainly involved in antigen presentation and acts as a receptor for MIFs, influencing immune responses and cell survival. CD79a is a crucial component of the B cell antigen receptor complex, playing a key role in B cell development and signal transduction. Within B cells, CXCR4 signaling regulates their migration to lymphoid organs and bone marrow, a vital 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 into the role of B cells in the immune response and their potential applications in SARS-CoV-2.

[0165] Example 3: Obtaining the variable regions of heavy and light chains from memory B cells and constructing a heavy and light chain vector plasmid for SARS-CoV-2 neutralizing antibodies.

[0166] Human single B cells were sorted, lysed, and reverse transcribed to obtain cDNA. The variable region fragment was amplified by PCR, and the correct fragment was identified to construct a full-length expression vector and sequenced.

[0167] The main objective of this study was to screen for specific neutralizing antibodies from individuals who had recovered from COVID-19 infection. The specific steps were as follows: Individual B cell sorting and RNA extraction: Specific memory B cells were sorted from recovered individuals using flow cytometry (FACS). The sorted B cells were a population of CD19+, CD27+, and IgG+ memory B cells, from which total RNA was further extracted.

[0168] 1. cDNA synthesis and PCR amplification

[0169] 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 variable regions of the immunoglobulin heavy and light chains. Successful amplification of the expected size of the antibody variable region fragment was confirmed.

[0170] 2. Cloning of variable region fragments and construction of expression vectors

[0171] After confirming the correctness of the PCR product, the fragment containing the correct antibody variable region sequence was selected, digested with restriction endonucleases, and ligated into the expression vector. The constructed vector contained the full-length antibody gene. The cloned antibody gene was sequenced using Sanger sequencing, which verified the sequence's correctness.

[0172] 3. Transient transfection of HEK293 cells and antibody expression

[0173] 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 antibody production and function were preliminarily verified using ELISA and neutralization assays.

[0174] The resulting sequence is shown below:

[0175] H chain amino acid sequence SEQ ID No. 1:

[0176] MGWSLILLFLVAVATRVLSQVQLVESGGILVQPGGSLRLSCAASGFSFSNYDMHWVRQTTGR

[0177] GLDWVSTIGTSGDTYYADSVKGRFTISREDAKKSLYLQMNSLTVGDTAVYYCARGSDDWTGW

[0178] YFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT

[0179] SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC

[0180] PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK

[0181] TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT

[0182] LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0183] H-chain nucleotide sequence SEQ ID No. 3:

[0184] ATGGGCTGGTCCCTGATTCTGCTGTTCCTGGTGGCTGTGGCTACCAGGGTGCTGAGTCAGGT

[0185] GCAGCTGGTGGAGTCTGGGGGAATCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG

[0186] CAGCCTCTGGATTCTCCTTCAGTAACTACGACATGCACTGGGTCCGCCAAACTACAGGAAGA

[0187] GGTCTGGATTGGGTCTCAACTATTGGCACTTCTGGTGACACATACTATGCGGACTCCGTGAA

[0188] GGGCCGATTCACCATCTCCAGAGAAGATGCCAAGAAGTCCCTGTATCTTCAAATGAACAGTC

[0189] TGACAGTCGGGGACACGGCTGTATATTACTGTGCAAGAGGGAGCGACGACTGGACCGGGTGG

[0190] TACTTTGATTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCAAGCACCAAGGGCCC

[0191] ATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT

[0192] GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACC

[0193] AGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGT

[0194] GGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGC

[0195] CCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGC

[0196] CCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACC

[0197] CAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCC

[0198] ACGAAGACCCCGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG

[0199] ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT

[0200] GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAG <00oooo469>CCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC

[0202] CTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG

[0203] CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA

[0204] AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG

[0205] GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCA

[0206] CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

[0207] Kappa chain amino acid sequence SEQ ID No.2:

[0208] MGWSCIILFLVATATGVHSDIQLTQSPVTLSASVGDRVTITCRASQGIRGSVAWYQQKPGEG

[0209] PKLLLFAANKLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYYLNPPPAFGQGTKV

[0210] EVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ

[0211] DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0212] Kappa chain nucleotide sequence SEQ ID No. 4:

[0213] ATGGGCTGGTCCTGTATCATCCTGTTCCTGGTGGCTACAGCCACAGGAGTGCATAGTGACAT

[0214] CCAGTTGACCCAGTCTCCAGTCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTT

[0215] GCCGGGCGAGTCAGGGCATTAGGGGTTCTGTTGCCTGGTATCAACAAAAACCAGGAGAAGGC

[0216] CCTAAGCTCCTGCTCTTTGCTGCAAACAAATTGCAAAGTGGGGTCCCGTCCAGGTTCAGTGG

[0217] CAGTGGATCTGGGACGGATTACACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAA

[0218] CTTATTACTGTCAACAGTATTATCTTAATCCCCCTCCGGCGTTCGGCCAAGGGACCAAGGTG

[0219] GAAGTCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTT

[0220] GAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG

[0221] TACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG

[0222] GACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGA

[0223] GAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGA

[0224] GCTTCAACAGGGGAGAGTGTTAG

[0225] Example 4: Affinity and neutralizing activity detection of the neutralizing antibodies obtained by screening in this invention.

[0226] 1. Detection of neutralizing antibody binding in culture supernatant using ELISA

[0227] To further investigate the antigenic epitope characteristics and neutralizing antibody screening strategies of the Omicron variant of SARS-CoV-2, we used ELISA to detect 18 neutralizing antibodies obtained through screening.

[0228] Specific experimental results: such as Figure 12 As shown, the binding activity of the antibody supernatant with Omicron S-Trimer and RBD was detected under 2-fold dilution conditions. The results indicate that the GR12 antibody of this invention exhibits significantly stronger neutralizing activity with Omicron S-Trimer and RBD compared to other neutralizing antibodies. Figure 13 As shown, after further 300-fold dilution of the antibody supernatant, the neutralizing antibody GR12 provided by this invention still maintains strong neutralizing activity compared to other neutralizing antibodies, Omicron S-Trimer, and RBD.

[0229] The results indicate that while most antibodies exhibit strong binding activity at low dilutions, only a portion maintain high affinity with increasing dilution, particularly antibodies targeting the RBD. This suggests significant differences in antibody binding strength and potential specificity for different antigenic epitopes. These high-affinity antibodies provide excellent candidates for further neutralization experiments and structural analysis, laying the foundation for optimized design of COVID-19 vaccines and antibody drug development. These results also demonstrate the effectiveness of the bioinformatics analysis method for antibody screening and provide new data support for understanding the antigenic characteristics of Omicron variants, offering important insights for basic research and clinical applications in related fields.

[0230] 2. ELISA neutralization detection using culture supernatant

[0231] The ability of antibodies in the culture supernatant to block S / RBD-ACE2 interaction was evaluated by ELISA to screen for antibody candidates with neutralizing activity.

[0232] Experimental results are as follows Figure 14 As shown, the neutralizing antibody GR12 antibody supernatant provided by this invention can simultaneously and effectively block the binding of SARS-CoV-2 S protein or RBD to the ACE2 receptor, exhibiting broad-spectrum activity. This significantly inhibits key steps in viral invasion of host cells. These antibodies further demonstrated good neutralizing activity in in vitro validation, protecting host cells from SARS-CoV-2 infection.

[0233] The above results demonstrate that the GR12 antibody supernatant possesses high neutralizing activity and clinical application potential, laying the foundation for further in-depth research. They not only serve as important candidates for developing novel antibody drugs but also provide crucial clues for understanding the infection mechanism and antiviral strategies of SARS-CoV-2.

[0234] 3. Detection of neutralized pseudoviruses

[0235] The neutralizing capacity of antibodies in culture supernatant was evaluated using a pseudovirus neutralization assay in order to further screen candidate strains with broad-spectrum neutralizing activity.

[0236] Test results as follows Figure 15 The results show that the neutralizing antibody GR12 provided by this invention has an inhibition rate of over 90% against JN.1 and WT pseudoviruses. This provides an important candidate resource for subsequent functional verification of antibodies and their 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 GR12 that binds to the novel coronavirus, characterized in that... It includes a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID No. 1 and the amino acid sequence of the light chain is shown in SEQ ID No.

2.

2. The nucleic acid encoding the neutralizing antibody GR12 against the novel coronavirus as described in claim 1, characterized in that: The nucleotide sequence encoding the heavy chain amino acid sequence is shown in SEQ ID No. 3, and the nucleotide sequence encoding the light chain amino acid sequence is shown in SEQ ID No.

4.

3. An antibody expression vector, characterized in that... It comprises the nucleic acid of claim 2, and the vector is a mammalian expression vector.

4. A host cell, characterized in that... A host cell comprising the nucleic acid of claim 2 or the expression vector of claim 3.

5. The use of the neutralizing antibody GR12 against the novel coronavirus as described in claim 1, or the nucleic acid as described in claim 2, or the expression vector as described in claim 3, or the host cell as described in claim 4 in the preparation of reagents for blocking novel coronavirus infection or in the preparation of drugs for preventing and / or treating novel coronavirus infection.

6. The use of the neutralizing antibody GR12 against the novel coronavirus as described in claim 1, or the nucleic acid as described in claim 2, or the expression vector as described in claim 3, or the host cell as described in claim 4 in the preparation of a kit for detecting the novel coronavirus.

7. A novel coronavirus neutralizing antibody ELISA detection reagent, detection kit, or drug, characterized in that... Includes the neutralizing antibody GR12 that binds to the novel coronavirus as described in claim 1.

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