Neutralizing antibodies against sars-cov-2 and variants gr75 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 GR75 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 GR75 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] The present application relates to a novel coronavirus neutralizing antibody, in particular, a neutralizing antibody capable of specifically binding to the RBD domain of SARS-CoV-2 and the S-Trimer domain of the Omicron variant, thereby preventing the infection of cells by the novel coronavirus and its variants in a broad spectrum, and use thereof. BACKGROUND

[0002] The novel coronavirus (SARS-CoV-2) belongs to the beta coronavirus genus, and its genome is a single-stranded RNA that encodes various structural proteins including the 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), mediates viral invasion, and is the core target for neutralizing antibody and vaccine design. The N protein is highly conserved and is the main target for nucleic acid detection and antigen detection. Since the outbreak of the epidemic, the continuous evolution of the virus has led to the emergence of various variants (such as Delta, Omicron and its sub-lineages), which has put higher requirements on detection and treatment technologies.

[0003] Neutralizing antibodies are specific antibodies produced by the human immune system or through in vitro screening techniques, which can block viral invasion of host cells by binding to viral surface proteins (such as the RBD or NTD region of the S protein). The development path mainly includes: (1) Antibody source: Early studies isolated high-titer neutralizing antibodies from the sera of recovered patients, and then screened candidate antibodies through monoclonal antibody techniques (such as phage display, hybridoma technology). (2) Target selection: The RBD region of the S protein is the main strategy, as it is directly involved in ACE2 receptor binding; some antibodies target the conserved epitopes of the NTD or S2 subunit to deal with viral mutations. (3) Function verification: The antibody activity is preliminarily evaluated through pseudovirus neutralization test (based on VSV or lentivirus vector), and then confirmed through live virus neutralization test (which needs to be performed in a biosafety level three laboratory BSL-3). Neutralizing antibodies have important 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, which can reduce the risk of hospitalization and severe illness. However, the phenomenon of antibody escape caused by viral mutation (such as the Omicron strain mutation) has seriously weakened the effectiveness of some antibodies, and it is urgent to develop broad-spectrum neutralizing antibodies.

[0004] The technical problems of the existing virus neutralizing antibodies are: (1) limitation: antibody escape phenomenon caused by virus variation seriously weakens the effectiveness of some antibodies, for example, frequent mutation of virus S protein (such as L452Q of Omicron BA.2 and F486V of BA.4 / 5) may change the antibody binding epitope, resulting in the failure of the existing antibodies. (2) Antibody-dependent enhancement (ADE) risk: 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 Fc segment engineering. (3) Pharmacokinetic defects: the half-life of natural antibodies is short (about 21 days), and the stability is poor. (4) Sensitivity and specificity bottleneck of detection technology: false negative problem of nucleic acid detection: low viral load samples (Ct value > 35), sampling errors (such as insufficient virus RNA in nasal swabs) or primer probe mismatch with variant strain sequence (such as D3L mutation of N gene of Alpha strain) may lead to missed detection. (5) Insufficient sensitivity of antigen detection: the sensitivity of existing reagents is only 60%-80%, which cannot replace nucleic acid detection; in addition, N protein mutation (such as P13L mutation of Omicron BA.1) may affect the detection performance. (6) Decline of neutralizing antibody efficacy: most approved antibodies are ineffective against Omicron strain because they target RBD epitopes; only a few broad-spectrum antibodies remain active, but their potency still needs to be improved.

[0005] In summary, the research and development of new coronavirus neutralizing antibodies and detection kits are the core technical means to cope with severe illness caused by viral infection, and are the focus of research for those skilled in the art. SUMMARY

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

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

[0008] Still another technical problem to be solved by the present application is to provide an antibody expression vector obtained from the above-mentioned neutralizing antibody GR75 that binds to the new coronavirus.

[0009] Still another technical problem to be solved by the present application is to provide the use of the above-mentioned neutralizing antibody GR75 that binds to the new coronavirus.

[0010] To achieve the above technical purposes, the application adopts the following technical solutions:

[0011] A neutralizing antibody GR75 binding 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 shown as SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown as 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 Omicron variant.

[0013] The nucleotide sequence encoding the neutralizing antibody GR75 binding to the novel coronavirus described above, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 3, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region is shown as 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 neutralizing antibody GR75 binding to the novel coronavirus described above, the method comprising PBMC collection, antigen labeling, magnetic bead enrichment, and B cell sorting by flow cytometry, PCR and vector construction, HEK293 transient cell culture, culture supernatant ELISA binding detection, culture supernatant ELISA neutralization detection, and pseudovirus neutralization detection; preferably, the method further comprises a bioinformatics analysis step, after the B cells are sorted by flow cytometry, based on high-throughput sequencing of individual B cell gene expression, analyzing its heterogeneity, clonal evolution and immune response mechanism, the steps are:

[0017] S1. IGHG subtype VDJ distribution screening: by high-throughput sequencing analysis of the combination frequency of IGHG heavy chain VDJ gene fragments in B cells, screening out IGHG antibody sequences with similarity of 95% or more to the overall B cell population VDJ distribution.

[0018] S2. CDR3 length distribution optimization: screening antibody sequences with heavy chain CDR3 amino acid length of 16 to 17, requiring that the sequence accounts for not less than 60% in the sample. At the same time, exclude abnormal sequences in light chain CDR3 sequence with length deviating from the mean value ± 2 times standard deviation (SD);

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

[0020] (1) Screening antibody sequences with somatic hypermutation frequency in heavy chain reaching or exceeding 15%, and somatic hypermutation frequency in light chain reaching or exceeding 10%;

[0021] (2) Using sequencing data and original V gene alignment, marking and confirming mutation hot spot region.

[0022] S4. Isotype-specific CDR3 length difference utilization:

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

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

[0025] The application of the above-mentioned neutralizing antibody GR75 binding to the novel coronavirus, or the above-mentioned nucleotide sequence, or the above-mentioned expression vector, or the above-mentioned host cell in preparing a reagent for blocking novel coronavirus infection or preparing a medicine for preventing and / or treating novel coronavirus infection.

[0026] The application of the above-mentioned neutralizing antibody GR75 binding to the novel coronavirus, or the above-mentioned nucleotide sequence, or the above-mentioned expression vector, or the above-mentioned host cell in preparing a kit for detecting neutralizing antibodies against the novel coronavirus and / or evaluating the immune effect of the 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 capturing novel coronavirus antigens, a biotin-labeled neutralizing antibody and an enzyme-labeled avidin.

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

[0030] a) Coating SARS-CoV-2 S-trimer protein and RBD protein on an enzyme-labeled plate;

[0031] b) After diluting the antibody culture supernatant, add it to the enzyme-labeled plate, incubate and then wash;

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

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

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

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

[0036] (1) The present application optimizes the antibody screening method, which screens the affinity matured antibody through bioinformatics analysis of single B cells, combines single cell RNA sequencing, VDJ rearrangement analysis and somatic hypermutation research, optimizes the antibody screening process, avoids the blindness of traditional methods, and improves the accuracy and effectiveness of antibody screening.

[0037] (2) Based on the optimized screening method of the present application, a novel neutralizing antibody GR75 is screened, the heavy chain and light chain variable region of which can specifically bind to the RBD domain of SARS-CoV-2 and the S-Trimer domain of Omicron mutant strain, realizing the broad-spectrum neutralization effect on SARS-CoV-2 virus and its mutant strains.

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

[0039] (4) The neutralizing antibody GR75 provided by the present application can simultaneously target SARS-CoV-2 RBD and Omicron S-Trimer, effectively inhibit a variety of mutant strains (such as JN.1, WT pseudovirus inhibition rate > 90%), and overcome the failure problem of existing antibodies caused by virus mutation. Antibody GR75 shows strong blocking ACE2 receptor binding and pseudovirus neutralization ability in in vitro experiments, providing a core tool for developing therapeutic drugs (such as reducing the rate of severe cases) and evaluating the immune effect of vaccines. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 Gate setting diagram for flow cytometry sorting B cells;

[0041] Figure 2 Expression plasmid map;

[0042] Figure 3 Heavy chain and light chain expression level of IGHG;

[0043] Figure 4 Heavy chain and light chain expression level of whole 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] The application screens memory B cells of people infected with new coronavirus, selects people recovered from new coronavirus infection by using single B cell antibody preparation technology, collects peripheral blood in blood of the people, separates PBMCs from whole blood by using density gradient centrifugation method, enriches B cells by using magnetic beads and flow sorting, analyzes heterogeneity, clonal evolution and immune response mechanism of the B cells based on high-throughput sequencing single B cell gene expression, and accurately screens neutralizing antibodies with excellent efficiency, and the specific method is described as follows.

[0059] Example 1 Screening and efficacy verification method of neutralizing antibody provided by the present application

[0060] 1. PBMC collection

[0061] Sample collection and pretreatment

[0062] Subject preparation: 10 eligible new coronavirus recovered patients (nucleic acid test negative and symptoms disappeared for more than 14 days) were selected. The informed consent was signed and the ethical review was passed.

[0063] Blood collection requirements: EDTA anticoagulant vacuum blood collection tube was used, and 10 mL of peripheral venous blood was extracted from each case. The blood was treated within 2 hours after blood collection, and cold storage or freezing was avoided.

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

[0065] Blood dilution: the whole blood was mixed with sterile PBS at a ratio of 1:1 (such as 10 mL blood + 10 mL PBS).

[0066] Ficoll layering: 3 mL of Ficoll separation solution (density 1.077 g / mL, pre-equilibrated to 20-25℃) was added to a 15 mL centrifuge tube. Diluted blood was slowly added along the tube wall to the upper layer of the separation solution, and the liquid level was layered clearly (avoid mixing).

[0067] Centrifugation parameters: horizontal centrifuge setting: 500xg (or 1500rpm), 25 minutes, the rise / fall speed was set to the lowest gear (avoid disturbing the layering) 14.

[0068] Collecting the PBMC layer: after centrifugation, the layers from top to bottom are: plasma layer, PBMC white membrane layer, Ficoll layer, red blood cell layer. The white membrane layer was gently sucked into a new centrifuge tube with a pipette (avoid sucking the upper plasma or lower separation solution).

[0069] Washing and purifying: 10 mL of PBS was added, and 250xg centrifugation was performed for 10 minutes, and the supernatant was discarded; the washing was repeated once

[0070] 2. Antigen labeling

[0071] 2.1 PE labeling

[0072] 1) PE is concentrated to 3mg / mL, UV is measured for quantification, then a certain ratio of SMCC is added, the NHS group on SMCC will react with the primary amine group on PE, and the MAL group on the other end will react with the -SH on the protein.

[0073] 2) Recombinant Omicron EG.5.1 Spike S1+S2 Trimer protein 40589-V08H55 is concentrated to 2mg / ml, a certain volume of 2-IT solution is added, mixed well, and reacted at room temperature for 2h.

[0074] 3) The activated protein is desalted into PBS, concentrated, and UV is measured.

[0075] 4) The activated protein with -SH is added to the activated APC with MAL at a molar ratio of 1:1, mixed well, and reacted overnight at 4°C.

[0076] 5) The reaction sample is purified with an S300 purification column, the coupled protein is collected, UV is measured, and the amount of protein is calculated.

[0077] 2.2 APC labeling

[0078] 1) APC is concentrated to 3mg / mL, UV is measured for quantification, then a certain ratio of SMCC is added, the NHS group on SMCC will react with the primary amine group on APC, and the MAL group on the other end will react with the -SH on the polypeptide.

[0079] 2) Recombinant Omicron BA.2.86 Spike RBD protein (40592-V08H152) is concentrated to 2mg / ml, a certain volume of 2-IT solution is added, mixed well, and reacted at room temperature for 2h.

[0080] 3) The activated protein is desalted into PBS, concentrated, and UV is measured.

[0081] 4) The activated protein with -SH is added to the activated APC with MAL at a molar ratio of 1:1, mixed well, and reacted overnight at 4°C.

[0082] 5) The reaction sample is purified with an S300 purification column, the coupled protein is collected, UV is measured, and the amount of protein is calculated.

[0083] 3. Magnetic bead enrichment and flow sorting of B cells

[0084] 3.1 Magnetic bead enrichment

[0085] (1) PBMC isolation: Human EDTA anti-coagulation whole blood was added to a centrifuge tube containing Ficoll, centrifuged, and the intermediate white membrane layer containing lymphocytes and mononuclear cells was taken to a new centrifuge tube, and PMBC cells were obtained by centrifugation (see Example 1 for specific steps).

[0086] (2) B Cell Biotin-Antibody Cocktail was added to the cells, incubated at 4°C in the dark for 10 min, Anti-Biotin MicroBeads were added, incubated at 4°C in the dark for 15 min, and sorting Buffer was added. The cells were washed by centrifugation, resuspended in sorting buffer, and then added to a sorting column. The flow-through was collected.

[0087] (3) CD27 MicroBeads were added to the flow-through cells, incubated at 4°C in the dark for 15 min, and the cells were washed by centrifugation. After resuspension in sorting buffer, the cells were added to a sorting column, and the cells in the sorting column were collected.

[0088] 3.2 Flow sorting

[0089] (1) Flow staining

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

[0091] (2) Flow sorting

[0092] The flow sorting circuit, delay, and sorting angle were adjusted, and the fluorescence compensation was adjusted. Through flow cytometry, we successfully sorted IgG-positive, CD 19-positive, and SARS-CoV-2 antigen-positive memory B cells. Specifically, we first identified the B cell population using CD19 labeling, then screened for IgG-expressing B cells using IgG labeling, and finally detected the specific reactivity of these cells using SARS-CoV-2 antigen. This strategy ensured that the sorted memory B cells had specificity for SARS-CoV-2, laying the foundation for subsequent research. The sorting results are shown in Figure 1 Figure 1 A gate map for flow sorting levels was set up, and IgG-positive, CD 19-positive, and SARS-CoV-2 antigen-positive memory B cells were sorted.

[0093] 4. PCR and vector construction

[0094] After lysing the sorted B cells, cDNA was obtained by reverse transcription using a reverse transcription kit, and 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 sequencing-corrected heavy and light chain expression plasmids, which were then transferred to HEK 293 for transient expression.

[0095] ​(1) Cell lysis

[0096] Lysis Solution was aliquoted into 96-well plates, and the cells were fully lysed.

[0097] (2) Reverse transcription

[0098] Two-step reverse transcription was used. The reverse transcription system was aliquoted into the PCR plate, mixed well by blowing, and then the reverse transcription reaction was performed.

[0099] (3) Heavy and light chain variable region preparation

[0100] Using cDNA as a template, the antibody heavy and light chain variable region genes were gradually amplified by nested PCR and multiple rounds of progressive amplification.

[0101] (4) Expression vector construction

[0102] The heavy and light chain variable region fragments were added to the membrane binding solution, mixed well, and then added to the purification column. Nuclease-Free Water was used for elution, and the purified heavy and light chain variable region gene fragments were obtained. The heavy and light chain variable region fragments were constructed into CMV expression vectors, respectively. The ligation products were transformed into competent cells and cultured at 37°C overnight. The obtained monoclonal was amplified and sequenced to obtain the antibody heavy and light chain sequences. The correct vector was extracted and the plasmid was passed to a small test expression. The expression plasmid map is shown in Figure 2 .

[0103] 5. HEK293 transient cell culture

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

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

[0106] 6. Culture supernatant ELISA binding detection

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

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

[0109] (3) Washing: 300 μL / well washing solution, washing 2 times, the last time pat dry;

[0110] (4) Sample dilution: dilute the culture supernatant 2 times and 300 times with sample diluent, mix well, and reserve;

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

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

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

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

[0115] (9) Color development: mix A and B liquids at 1:1, add 200 μL per well,

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

[0117] (10) Termination: add 50 μL of termination solution per well, and immediately measure the OD value at 450 nm.

[0118] 7. Culture supernatant ELISA neutralization detection

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

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

[0121] (3) Washing: 300 μL / well washing solution, washing 2 times, the last time pat dry;

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

[0123] (5) Sample addition: add the supernatant to the enzyme-labeled plate at 100 μL / well, and the blank control is sample diluent; then add the diluted RBD protein to the enzyme-labeled plate at 100 μL / well, and the final sample is 200 μL / well, mix well, and incubate at room temperature for 1 h;

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

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

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

[0127] (9) Color development: mix A and B liquids at a ratio of 1:1, add 200 pL per well, 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 pL per well of termination solution, and immediately measure the OD value at 450 nm wavelength.

[0129] 8. Pseudovirus neutralization detection

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

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

[0132] (2) Sample-pseudovirus neutralization: each sample is divided into 2 parts, 50 pL each; mix 50 pL of the sample stock solution with 50 pL of the WT pseudovirus diluent, and mix 50 pL of the sample stock solution with 50 pL 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 culture medium to a cell suspension with a density of 30000 cells / 100mL.

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

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

[0136] Example 2 Bioinformatics analysis and screening experimental results

[0137] As Figure 16As shown, the inventors initially did not use bioinformatics analysis screening method after sorting out IgG positive, CD19 positive and positive for coronavirus antigen memory B cells, and the screened antibodies were all negative. Subsequently, after successfully sorting out IgG positive, CD19 positive and positive for coronavirus antigen memory B cells in Example 1 step 3, the inventors further improved the screening method, and first carried out bioinformatics analysis on individual B cells, and used the analysis results to accurately screen neutralizing antibodies with good effect.

[0138] The bioinformatics analysis method is as follows:

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

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

[0141] Objective: To retain the diversity generation mode consistent with other subtypes of IGHG subtype, and ensure that the constructed antibody library has broad spectrum function and high efficiency of antigen recognition ability.

[0142] First, the distribution of B cell antigen receptor (BCR) is shown in Table 1: it can be seen that the BCR distribution is mainly IGHG, among which IGHG1 accounts for the highest proportion in IGHG; followed by IGHM, IGH A, and IGH D. IGHG accounts for the highest proportion, among which IGHG1 is the main subtype of IGHG, suggesting its core role in humoral immunity such as neutralizing pathogens in antibodies. The high proportion of IGHG1 may be related to its long half-life, complement activation ability and placental penetration (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％ Cell number 3401 63 1814 1554 229 137 2704

[0145] Based on the above analysis results, we further analyzed the expression level of the heavy chain and light chain of IGHG, and the results showed that the expression level of the heavy chain and light chain of IGHG BCR was consistent, as shown in Figure 3 .

[0146] We further analyzed the expression level of the heavy chain and light chain of all cell BCR, and the results showed that the expression level of the heavy chain and light chain of all cell BCR was consistent, as shown in 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 somatic hypermutation frequency in heavy chain reaching or exceeding 15%, and in light chain reaching or exceeding 10%;

[0155] (2) Mark and confirm mutation hotspots using sequencing data and original V gene alignment (see Figure 9 circled area).

[0156] Objective: Simulate the process of affinity maturation 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, and the results showed that different levels of hypermutation occurred in both heavy and light chains, and the hypermutation is circled in the figure. Somatic hypermutation is a key process by which B cells enhance antibody affinity under antigen stimulation. Hypermutation occurs in both heavy and light chains, indicating that B cells undergo affinity maturation during immune response, thereby optimizing antibody function.

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

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

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

[0161] Objective: Utilize the structural advantage of longer heavy chain CDR3 (see Figure 10A and 10B ) to enhance the complexity of the antigen binding interface, further improving 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 heavy chain CDR3 sequences is longer than that of light chain ( Figure 10A mean 50 or so vs. Figure 10B mean 30 or so). Heavy chain CDR3 is usually longer than light chain CDR3, which is consistent with the structural and functional requirements of antibodies. Heavy chain CDR3 plays a dominant role in antigen binding, and its longer length helps to form a more complex binding site, thereby enhancing the specificity and affinity of the antibody.

[0163] Single-cell RNA-seq results:

[0164] As Figure 11To illustrate, we performed single-cell RNA seq sequencing, mainly focusing on CD74, CD79a, and CXCR4 (dark part of the graph), and pseudotime analysis showed the developmental trajectory of memory B cells. CD74 is mainly involved in antigen presentation and as a receptor for MIF, affecting immune response and cell survival. CD79a is an important part of the B cell antigen receptor complex, which is crucial for B cell development and signaling. In B cells, CXCR4 signals regulate their migration to lymphoid organs and bone marrow, which is an important part of the immune response. These findings not only deepen our understanding of the function and regulatory mechanisms of B cells in the immune system, but also provide an important basis for further studying the role of B cells in immune response and their potential application in SARS-COV-2. Example 3 Obtain memory B cell heavy and light chain variable regions and construct new coronavirus neutralizing antibody heavy and light chain vector plasmid

[0165] After sorting and deriving human B cells, cDNA was obtained after reverse transcription after lysis, PCR amplification of variable region fragments, identification of correct fragments to construct full-length expression vectors and sequencing.

[0166] The main goal of this study is to screen specific neutralizing antibodies from post-COVID-19 infected individuals. The specific steps are as follows: single B cell sorting and RNA extraction Specific memory B cells were sorted from individuals who recovered from COVID-19 infection by flow cytometry (FACS). The sorted B cells were CD19+, CD27+, IgG+ memory B cell population, and 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 reverse transcription, specific primers were used for PCR amplification of the immunoglobulin heavy and light chain variable regions. Successful amplification of antibody variable region fragments of the expected size was confirmed.

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

[0170] After confirming the correctness of the PCR product, fragments containing the correct antibody variable region sequence were selected, subjected to restriction enzyme digestion, and ligated into expression vectors. The constructed vector contains the full-length antibody gene. The cloned antibody gene was subjected to Sanger sequencing to verify the correctness of its sequence.

[0171] 3. HEK293 cell transient transfection 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 after 24-48 hours, and the antibody yield and function were preliminarily verified by ELISA and neutralization experiments.

[0173] The resulting sequence is shown below:

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

[0175] MGWSLILLFLVAVATRVLSQVQLVESGGGLVKPGGSLRLSCEASGFTFSSHDMHWVRQTTGK

[0176] SLEWLSLIGTAGDTFYPDSVKGRFTISRDNAKNSLFLQMNSLRVGDTAVYYCVRAHYDDSGF

[0177] FSYFDSWGQGAQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA

[0178] LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH

[0179] TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN

[0180] AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

[0181] YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

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

[0183] ATGGGCTGGTCCCTGATTCTGCTGTTCCTGGTGGCTGTGGCTACCAGGGTGCTGAGTCAGGT

[0184] GCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCACTGAGACTCTCCTGTG

[0185] AGGCCTCTGGATTCACCTTCAGTAGCCACGACATGCACTGGGTCCGCCAAACGACAGGAAAG

[0186] AGTCTGGAGTGGCTCTCACTGATTGGTACGGCTGGTGACACATTTTATCCGGACTCCGTGAA

[0187] GGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCCTTGTTTCTTCAAATGAACAGCC

[0188] TGAGAGTCGGGGACACGGCTGTGTATTACTGTGTTAGAGCTCACTATGATGATAGTGGTTTT

[0189] TTCTCCTACTTTGACTCCTGGGGCCAGGGAGCCCAGGTCACCGTCTCTTCAGCAAGCACCAA

[0190] GGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCC

[0191] TGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCC

[0192] CTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAG

[0193] CAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC

[0194] ACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCAC

[0195] ACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCC

[0196] GAGAAGAGCCGGCTGTGGTTTCTGGCTGTGGCTCTGCCTGTGGAGCCGGAGACGGCCGTG

[0197] TGAGCCACGAAGACCCCGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAAT

[0198] GCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCAC

[0199] CGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCC

[0200] TCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG

[0201] TACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGT

[0202] CAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACA

[0203] ACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTC

[0204] ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGC

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

[0206] MGWSCIILFLVATATGVHSDIVMTQSPFTLSASVGDRVTITCRASQSIGTYLNWYQQKPGKA

[0207] PKVLIYATSNLQTGVPSRFSGSGSGTDFTLTISSLQREDFATYYCQQSYTTPGLTFGGGTKL

[0208] EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ

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

[0210] ATGGGCTGGTCCTGTATCATCCTGTTCCTGGTGGCTACAGCCACAGGAGTGCATAGTGATAT

[0211] TGTGATGACCCAGTCTCCATTCACCCTGTCTGCATCTGTAGGAGACAGAGTTACCATCACTT

[0212] GCCGGGCAAGTCAGAGCATTGGCACCTATTTAAATTGGTATCAGCAGAAGCCAGGGAAAGCC

[0213] CCAAAGGTCCTGATCTATGCTACATCCAATTTGCAAACTGGGGTCCCATCAAGATTCAGTGG

[0214] CAGTGGTTCTGGGACAGATTTCACTCTCACCATTAGCAGTCTGCAACGTGAAGACTTTGCAA

[0215] CTTACTACTGTCAACAGAGTTACACTACCCCTGGACTCACTTTCGGCGGAGGGACCAAGCTG

[0216] GAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTT

[0217] GAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG

[0218] TACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG

[0219] GACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGA

[0220] GAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGA

[0221] GCTTCAACAGGGGAGAGTGTTAG

[0222] Example 4 Affinity detection and neutralization activity detection of neutralizing antibody screened by the present application 1. Culture supernatant neutralizing antibody ELISA binding detection

[0223] To further study the antigen epitope characteristics and neutralizing antibody screening strategy of the Omicron variant of the new coronavirus, the 18 neutralizing antibodies screened were detected by ELISA method.

[0224] The specific experimental results are shown in the following table: Figure 12 Under the condition of 2-fold dilution, the binding activity of antibody supernatant with Omicron S-Trimer and RBD was detected, and it was found that the neutralizing activity of GR75 provided by the application was much stronger than that of other neutralizing antibodies with Omicron S-Trimer and RBD. As shown in the following table: Figure 13 Further dilution of the antibody supernatant by 300 times, the neutralizing antibody GR75 provided by the application still maintains strong neutralizing activity compared with other neutralizing antibodies with Omicron S-Trimer and RBD.

[0225] The above results show that although most of the antibodies have strong binding activity under low dilution conditions, with the increase of dilution times, only part of the antibodies can maintain high affinity, especially the antibodies against RBD. This suggests that there is a significant difference in the binding strength of the antibodies, and they may have specificity for different antigen epitopes. These high-affinity antibodies provide high-quality candidates for further neutralization experiments and structural analysis, and lay a foundation for the optimization design of new coronavirus vaccines and the development of antibody drugs. The above results also demonstrate the effectiveness of the method of screening antibodies by bioinformatics analysis, and provide new data support for understanding the antigen characteristics of the Omicron variant, and provide important inspiration for basic research and clinical application in related fields.

[0226] 2. Culture supernatant ELISA neutralization detection

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

[0228] The experimental results are shown in Figure 14 The neutralizing antibody GR75 supernatant provided by the present application can effectively block the binding of SARS-CoV-2 S protein or RBD to ACE2 receptor at the same time, and has broad spectrum. Thus significantly inhibiting the key step of virus invasion of host cells. These antibodies further showed good neutralizing activity in in vitro verification, and could protect host cells from SARS-CoV-2 infection.

[0229] The above results show that the GR75 antibody supernatant has high neutralizing activity and clinical application potential, laying a foundation for further in-depth study. They can not only be used 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.

[0230] 3. Pseudovirus neutralization detection

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

[0232] The detection results are shown in Figure 15 The neutralizing antibody GR75 provided by the present application has an inhibition rate of more than 90% on JN.1 and WT pseudovirus. This provides important candidate resources for subsequent antibody function verification and its application in therapeutic drug development, and also provides strong data support for studying the characteristics of SARS-CoV-2 mutant strains and their immune escape mechanism.

Claims

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

2.

2. The neutralizing antibody GR75 against 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 mutant Omicron.

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

4.

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

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

6. The use of the neutralizing antibody GR75 binding to the novel coronavirus as described in claim 1 or 2, or the nucleotide as described in claim 3, or the expression vector as described in claim 4, or the host cell as described in claim 5 in the preparation of reagents for blocking novel coronavirus infection or in the preparation of drugs for the prevention and / or treatment of novel coronavirus infection.

7. The use of the neutralizing antibody GR75 against the novel coronavirus as described in claim 1 or 2, or the nucleotide as described in claim 3, or the expression vector as described in claim 4, or the host cell as described in claim 5 in the preparation of a kit for detecting neutralizing antibodies against the novel coronavirus and / or evaluating the immunization effect of a vaccine.

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

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