Broad-spectrum neutralizing antibody W328-6E10 and its application in the preparation of vaccines against coronaviruses

Abstract

This invention discloses a broad-spectrum neutralizing antibody, W328-6E10, and its application in the preparation of vaccines against coronaviruses. This invention provides an IgG antibody, named W328-6E10 antibody, composed of a light chain and a heavy chain; the CDR1, CDR2, and CDR3 in the variable region of the heavy chain are as shown in positions 45-52, 70-77, and 116-129 of SEQ ID NO: 1, respectively; the CDR1, CDR2, and CDR3 in the variable region of the light chain are as shown in positions 46-51, 69-71, and 108-116 of SEQ ID NO: 3, respectively. The W328-6E10 antibody is effective against not only SARS and atypical pneumonia viruses, but also exhibits broad-spectrum neutralizing activity against other coronaviruses. This invention has significant application value for the prevention and control of β-coronaviruses and will have profound social implications.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the broad-spectrum neutralizing antibody W328-6E10 and its application in the preparation of vaccines against coronaviruses. Background Technology

[0002] Severe Acute Respiratory Syndrome (SARS-CoV-1) is a type of beta coronavirus that is transmitted through the respiratory tract. SARS-CoV infection is characterized by rapid onset, rapid spread, and high mortality. The main symptoms include hypoxia, cyanosis, and high fever. It often progresses to severe infection, leading to rapid breathing or acute respiratory distress syndrome, accompanied by septic shock, metabolic acidosis, coagulation disorders, and multiple organ failure.

[0003] In the 20 years following the outbreak of SARS, two more coronavirus outbreaks occurred: Middle East Respiratory Syndrome (MERS-CoV) and the novel coronavirus (SARS-CoV-2). Beta coronaviruses share extensive conserved regions in sequence and structure, making it crucial to have a robust reserve of technologies for the prevention and treatment of SARS virus strains to address potential future emerging coronavirus infectious diseases.

[0004] Monoclonal antibodies can be mass-produced industrially. Their high affinity and specificity for binding to antigens significantly reduce adverse reactions in clinical applications. Furthermore, antibody molecules can be modified to increase their antiviral efficacy. Due to their specificity and flexibility of use, antibodies are a very promising tool in the treatment of infectious diseases. Stockpiling antibody sequences against SARS virus can provide effective prevention and treatment measures in the immediate event of a potential new coronavirus outbreak. Summary of the Invention

[0005] The purpose of this invention is to provide a broad-spectrum neutralizing antibody, W328-6E10, and its application in the preparation of vaccines against coronaviruses.

[0006] This invention provides an IgG antibody, named W328-6E10 antibody, composed of a light chain and a heavy chain; the CDR1, CDR2, and CDR3 in the variable region of the heavy chain are shown as positions 45-52, 70-77, and 116-129 in SEQ ID NO: 1, respectively; the CDR1, CDR2, and CDR3 in the variable region of the light chain are shown as positions 46-51, 69-71, and 108-116 in SEQ ID NO: 3, respectively.

[0007] Specifically, the heavy chain variable region is shown in positions 20-140 of SEQ ID NO: 1.

[0008] Specifically, the variable region of the light chain is shown in positions 20-126 of SEQ ID NO: 3.

[0009] Specifically, the heavy chain is either (a) or (b) as follows: (a) the protein represented by positions 20-470 of SEQ ID NO: 1; (b) the protein represented by SEQ ID NO: 1;

[0010] Specifically, the light chain is either (c) or (d) as follows: (c) the protein shown at positions 20-233 of SEQ ID NO: 3; (d) the protein shown in SEQ ID NO: 3.

[0011] The gene encoding the IgG antibody is also within the scope of protection of this invention.

[0012] Specifically, the gene encoding the heavy chain is as follows (1) or (2):

[0013] (1) The DNA molecule represented by nucleotides 58-1413 in SEQ ID NO: 2;

[0014] (2) The DNA molecule shown in SEQ ID NO: 2.

[0015] Specifically, the gene encoding the light chain is as follows (3) or (4):

[0016] (3) The DNA molecule represented by nucleotides 58-702 in SEQ ID NO: 4;

[0017] (4) The DNA molecule shown in SEQ ID NO: 4.

[0018] This invention also protects the use of any of the IgG antibodies described above in the preparation of products.

[0019] This invention also protects a product whose active ingredient is any of the IgG antibodies described above.

[0020] The product is one of the following (e), (f), (g), or (h):

[0021] (e) Vaccines against the coronavirus;

[0022] (f) Medicines used to prevent and / or treat coronavirus infection;

[0023] (g) Virus inhibitors targeting coronaviruses;

[0024] (h) Preparations used to neutralize coronaviruses.

[0025] The preparation is a vaccine preparation or a pharmaceutical preparation.

[0026] Specifically, the coronavirus in question is a beta coronavirus.

[0027] Specifically, the coronaviruses mentioned are SARS virus, novel coronavirus, bat coronavirus, or pangolin coronavirus.

[0028] Specifically, the novel coronavirus is any of the following strains: wild-type novel coronavirus, novel coronavirus Alpha strain, novel coronavirus Beta strain, novel coronavirus Gamma strain, novel coronavirus Delta strain, novel coronavirus Omeprone BA.1 strain, novel coronavirus Omeprone BA.2 strain, novel coronavirus Omeprone BA.4 / 5 strain, novel coronavirus Omeprone BF.7 strain, novel coronavirus Omeprone BQ.1 strain, novel coronavirus Omeprone BQ.1.1 strain, novel coronavirus Omeprone XBB strain, novel coronavirus Omeprone XBB.1 strain, or novel coronavirus Omeprone XBB.1.5 strain.

[0029] Specifically, the bat coronavirus is either the bat coronavirus WIV16 strain or the bat coronavirus RaTG13 strain.

[0030] Specifically, the pangolin coronavirus is either the pangolin coronavirus GD strain or the pangolin coronavirus P4L strain.

[0031] This invention screens memory B cells generated from antibody-producing peripheral blood mononuclear cells of patients infected with SARS-CoV-2, obtaining a monoclonal antibody that specifically binds to the spike protein, named W328-6E10 antibody. The W328-6E10 antibody provided by this invention not only neutralizes SARS-CoV-2 but also exhibits broad-spectrum neutralizing activity against other coronaviruses. This invention has significant application value for the prevention and control of β-coronaviruses and will have profound social implications. Attached Figure Description

[0032] Figure 1 The neutralizing activity of the W328-6E10 antibody against atypical pneumonia virus pseudoviruses was measured.

[0033] Figure 2 The W328-6E10 antibody exhibits broad-spectrum neutralizing activity against various coronavirus pseudoviruses. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the present invention in any way. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments are all commercially available. Unless otherwise specified, the quantitative experiments in the following embodiments are all set up with three replicate experiments, and the results are averaged. 293F cells and 293T cells are commercially available human embryonic kidney epithelial cells. pMD18-T vector is a commercially available plasmid vector. pcDNA3.1(+) vector: Invitrogen, product catalog number V790-20.

[0035] hACE2-hela cells (ie "HeLa cell lines stably expressing theACE2molecules"), recorded in the following literature: Wang, R., Zhang, Q., Ge, J., Ren, W., Zhang, R., Lan, J., Ju, B., Su, B., Yu, F., Ch. en,P.,Liao,H.,Feng,Y.,Li,X.,Shi,X.,Zhang,Z.,Zhang,F.,Ding,Q.,Zhang,T.,Wang,X.&Zhang,L.Analysis of SARS-CoV-2 variant mutations reveal neutralization escape mechanisms and the ability to use ACE2receptors from additional species. Immunity 54, 1611-1621.e1615, doi:10.1016 / j.immuni.2021.06.003(2021).

[0036] Example 1: Discovery and preparation of W328-6E10 antibody

[0037] Memory B cells were isolated from peripheral blood mononuclear cells of patients recovering from SARS-CoV-1 infection and disease, and the antibody gene of the memory B cells was amplified to obtain the antibody sequence. Through extensive comparison, analysis, preparation, and efficacy verification, the inventors of this invention discovered a new SARS-CoV-1 IgG antibody with excellent activity, which was named W328-6E10 antibody.

[0038] The full-length heavy chain amino acid sequence of the W328-6E10 antibody is shown in SEQ ID NO: 1. In SEQ ID NO: 1, amino acid residues 1-19 constitute the signal peptide (guiding the protein to be secreted extracellularly), amino acid residues 20-140 constitute the variable region of the heavy chain, and amino acid residues 141-470 constitute the constant region of the heavy chain. CDR1, CDR2, and CDR3 in the variable region of the heavy chain are shown as positions 45-52 (GYTFTSYD), 70-77 (MNPNSGDT), and 116-129 (ARESHRHYYGSGSF) in SEQ ID NO: 1, respectively.

[0039] The full-length light chain amino acid sequence of the W328-6E10 antibody is shown in SEQ ID NO: 3. In SEQ ID NO: 3, amino acid residues 1-19 constitute the signal peptide (guiding the protein to the extracellular space), amino acid residues 20-126 constitute the variable region of the light chain, and amino acid residues 127-233 constitute the constant region of the light chain. CDR1, CDR2, and CDR3 in the variable region of the light chain are shown in SEQ ID NO: 3. ID The numbers in NO:3 are shown in bits 46-51 (QSISYY), 69-71 (AAS), and 108-116 (QQSYSTPYN).

[0040] Example 2: Preparation of W328-6E10 antibody

[0041] I. Construction of Recombinant Plasmids

[0042] The heavy chain DNA molecule was inserted into the pMD18-T vector to obtain the heavy chain expression vector. The heavy chain DNA molecule consists of the following three elements from upstream to downstream: the promoter shown in SEQ ID NO: 5, the full-length heavy chain coding gene shown in SEQ ID NO: 2, and the terminator shown in SEQ ID NO: 6.

[0043] The light chain DNA molecule was inserted into the pMD18-T vector to obtain the light chain expression vector. The light chain DNA molecule consists of the following three elements from upstream to downstream: the promoter shown in SEQ ID NO: 5, the full-length light chain coding gene shown in SEQ ID NO: 4, and the terminator shown in SEQ ID NO: 6.

[0044] Both heavy chain DNA molecules and light chain DNA molecules are double-stranded DNA molecules.

[0045] II. Construction of Recombinant Cells

[0046] The heavy chain expression vector and the light chain expression vector were co-transfected into 293F cells to obtain recombinant cells.

[0047] III. Antibody Preparation

[0048] 1. Take the recombinant cells obtained in step 2 and culture them in DMEM medium containing 2% fetal bovine serum for 72 h. Then centrifuge at 4℃ and 4000 rpm for 30 min, collect the supernatant and filter it through a 0.45 μm filter membrane.

[0049] 2. Affinity chromatography

[0050] Affinity chromatography packing material: Protein A Resin (Genscript Biotechnology, product catalog number L00210);

[0051] Operating steps: ① Mix 300 mL of the filtrate obtained in step 1 with 3 mL of affinity chromatography packing material and incubate at 4 °C for 16 h; ② Add the mixture to a 25 mL gravity flow empty column to trap the packing material; ③ Wash the column with 60 mL of binding buffer; elute the target protein with 30 mL of elution buffer, collect the post-column solution, and adjust the pH to neutral.

[0052] Binding buffer: Dissolve 112.6g of glycine and 175.2g of sodium chloride in water and bring the volume to 1L. Adjust the pH to 8.0 with sodium hydroxide.

[0053] Elution buffer: Dissolve 7.5g of glycine in water and bring the volume to 500ml. Adjust the pH to 3.0 with hydrochloric acid.

[0054] 3. Take the post-column solution obtained in step 2, concentrate it with an ultrafiltration concentrator and replace the system with PBS buffer (pH 7.2, 10mM) to obtain the W328-6E10 antibody solution.

[0055] Example 3: Neutralizing activity of W328-6E10 antibody against SARS-CoV-1

[0056] I. Preparation of SARS-CoV-1 pseudovirus

[0057] Co-transfection of 293T cells with a plasmid expressing SARS-CoV-1 membrane proteins and a backbone plasmid pNL4-3R-E-luciferase yielded infectious but non-replicating SARS-CoV-1 pseudoviruses after incubation, with infectivity similar to live SARS-CoV-1 virus. The backbone plasmid pNL4-3R-E-luciferase is a backbone plasmid containing Luciferase, pNL4-3R-E (i.e., the vector in the literature). with the luciferase gene containing The backbone pNL4-3R-E is described in the following literature: Wang Q, Liu L, Ren W, Gettie A, Wang H, Liang Q, Shi X, Montefiori DC, Zhou T, Zhang L. Cell Rep. 2019.

[0058] The gene encoding the SARS-CoV-1 membrane protein was inserted between the BamHII and EcoRI restriction sites of the pcDNA3.1(+) vector to obtain a plasmid expressing the SARS-CoV-1 membrane protein. The plasmid expressing the SARS-CoV-1 membrane protein and the backbone plasmid pNL4-3R-E-luciferase were co-transfected into 293T cells and incubated at 37°C in DMEM medium containing 10% fetal bovine serum. The cell culture supernatant was collected 60 hours after transfection; this was the viral fluid containing SARS-CoV-1 pseudovirus, referred to as SARS-CoV-1 viral fluid. The SARS-CoV-1 membrane protein is shown in SEQ ID NO: 7. The gene encoding the SARS-CoV-1 membrane protein is shown in SEQ ID NO: 8 (the codon-optimized sequence).

[0059] II. Detection of antibody neutralizing activity

[0060] Test virus solution: SARS-CoV-1 virus solution prepared in step one.

[0061] 1. Take the W328-6E10 antibody solution prepared in Example 2 and perform serial dilution with PBS buffer (pH 7.2, 10mM) to obtain antibody dilutions of various concentrations.

[0062] 2. Take a 96-well cell culture plate and add 100 μl of antibody diluent and 50 μl of test virus solution to each well (the virus concentration in 50 μL of test virus solution is 1×10⁻⁶). 4 Incubate at 37°C for 1 hour with TCID50 / ml. Use an equal volume of PBS buffer (pH 7.2, 10mM) instead of antibody dilution as a virus control. Use an equal volume of DMEM medium containing 10% fetal bovine serum instead of the test virus solution as a cell control.

[0063] 3. After completing step 2, take the cell culture plate and seed each well with 100 μl of hACE2-hela cell suspension (the solvent used to prepare the cell suspension is DMEM medium containing 10% fetal bovine serum, and the concentration of hACE2-hela cells in the cell suspension is 2 × 10⁻⁶). 5 (cells / ml), incubated at 37°C for 64 hours.

[0064] 4. After completing step 3, take the cell culture plate, discard the supernatant, add 150 μl of lysis buffer (Microglass Biotechnology, catalog number T003, follow the instructions) to each well, and incubate at 37°C for 5 minutes.

[0065] 5. After completing step 4, take the cell culture plate and detect the luciferase activity.

[0066] Each process is configured with multiple duplicate holes.

[0067] Neutralization activity (%) = [1 - (fluorescence intensity of experimental group - fluorescence intensity of cell control) / (fluorescence intensity of virus control - fluorescence intensity of cell control)] × 100%.

[0068] Neutralization activity results are shown in Figure 1 . Figure 1 The vertical axis represents neutralizing activity (%), and the horizontal axis represents the antibody concentration (μg / ml) logarithm to base 10. The antibody concentration refers to the antibody concentration in the mixed system consisting of 100 μl of antibody dilution and 50 μl of test virus solution in step 2.

[0069] The antibody concentration at which the neutralizing activity is 50% was calculated using Prism 8 software, i.e., the IC50 value of the antibody.

[0070] The IC50 value of the W328-6E10 antibody against SARS-CoV-1 pseudovirus was 91.56 ng / ml.

[0071] Example 4: Broad-spectrum neutralizing activity of W328-6E10 antibody against coronaviruses

[0072] I. Preparation of Coronavirus Pseudoviruses

[0073] The gene encoding the coronavirus membrane protein was inserted between the BamHII and EcoRI restriction sites of the pcDNA3.1(+) vector to obtain a plasmid expressing the coronavirus membrane protein. The plasmid expressing the coronavirus membrane protein and the backbone plasmid pNL4-3R-E-luciferase were co-transfected into 293T cells and incubated at 37°C (using DMEM medium containing 10% fetal bovine serum). The cell culture supernatant was collected 60 hours after transfection; this was the viral solution containing coronavirus pseudoviruses.

[0074] Viral solutions of pseudoviruses from 18 coronavirus strains were prepared, specifically pseudoviruses of 14 novel coronavirus strains, 2 bat coronavirus strains, and 2 pangolin coronavirus strains. The 14 novel coronavirus strains refer to the following strains: wild-type novel coronavirus, novel coronavirus Alpha strain, novel coronavirus Beta strain, novel coronavirus Gamma strain, novel coronavirus Delta strain, novel coronavirus Omeprone BA.1 strain, novel coronavirus Omeprone BA.2 strain, novel coronavirus Omeprone BA.4 / 5 strain, novel coronavirus Omeprone BF.7 strain, novel coronavirus Omeprone BQ.1 strain, novel coronavirus Omeprone BQ.1.1 strain, novel coronavirus Omeprone XBB strain, novel coronavirus Omeprone XBB.1 strain, and novel coronavirus Omeprone XBB.1.5 strain. The 2 bat coronavirus strains refer to bat coronavirus WIV16 and bat coronavirus RaTG13. The two pangolin coronavirus strains refer to the pangolin coronavirus GD strain and the pangolin coronavirus P4L strain. The membrane proteins of each coronavirus and their coding genes are shown in Table 1.

[0075] Table 1

[0076]

[0077]

[0078] II. Detection of antibody neutralizing activity

[0079] Test virus solutions: virus solutions of the 18 coronavirus strains prepared in step one.

[0080] The method is the same as step two of Example 3.

[0081] Neutralization activity results are shown in Figure 2 . Figure 2 The vertical axis represents neutralizing activity (%); the horizontal axis represents the antibody concentration (μg / ml) logarithm to base 10. The antibody concentration refers to the antibody concentration in the mixed system consisting of 100 μL of antibody dilution and 50 μL of test virus solution in step 2.

[0082] The IC50 values ​​of the W328-6E10 antibody against each strain are shown in Table 2.

[0083] Table 2

[0084] IC50 value (μg / ml) Wild-type novel coronavirus (WT) 2.764759 Novel Coronavirus Alpha Strain 3.129607 Novel Coronavirus Beta Strain 10.45797 Novel Coronavirus Gamma Strain 2.440765 novel coronavirus Delta strain 5.468884 Novel coronavirus Omeprone BA.1 strain 4.330305 Novel coronavirus Omeprone BA.2 strain 0.396063 Novel coronavirus Omeprone BA.4 / 5 strain 3.393191 Novel coronavirus Omeprone BF.7 strain 1.670593 Novel coronavirus Omeprone BQ.1 strain 5.001401 Novel coronavirus Omeprone BQ.1.1 strain 6.829785 Novel coronavirus Omeprone XBB strain 5.582714 Novel coronavirus Omeprone XBB.1 strain 5.043248 Novel coronavirus Omeprone XBB.1.5 strain 0.947161 bat coronavirus WIV16 strain 0.018367 bat coronavirus RaTG13 strain 0.052255 Pangolin coronavirus GD strain 0.647881 Pangolin coronavirus P4L strain 0.066291

[0085] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. An IgG antibody that binds to the SARS-CoV-1 spike protein, comprising a light chain and a heavy chain; wherein CDR1, CDR2, and CDR3 in the variable region of the heavy chain are as shown in positions 45-52, 70-77, and 116-129 of SEQ ID NO: 1, respectively; and wherein CDR1, CDR2, and CDR3 in the variable region of the light chain are as shown in positions 46-51, 69-71, and 108-116 of SEQ ID NO: 3, respectively.

2. The IgG antibody as described in claim 1, characterized in that: The heavy chain variable region is shown in bits 20-140 of SEQ ID NO: 1; The variable region of the light chain is shown in positions 20-126 of SEQ ID NO:

3.

3. The IgG antibody as described in claim 2, characterized in that: The heavy chain is either (a) or (b) as follows: (a) the protein represented by positions 20-470 of SEQ ID NO: 1; (b) the protein represented by SEQ ID NO: 1; The light chain is either (c) or (d) as follows: (c) the protein shown at positions 20-233 of SEQ ID NO: 3; (d) the protein shown in SEQ ID NO:

3.

4. A gene encoding the IgG antibody of any one of claims 1 to 3.

5. The use of the IgG antibody according to claim 1, 2, or 3 in the preparation of the product; The product is one of the following (f), (g), or (h): (f) Medicines used to prevent and / or treat coronavirus infection; (g) Virus inhibitors targeting coronaviruses; (h) Preparations used to neutralize coronaviruses; The coronaviruses mentioned are SARS-CoV-1, wild-type novel coronavirus, novel coronavirus Alpha strain, novel coronavirus Beta strain, novel coronavirus Gamma strain, novel coronavirus Delta strain, novel coronavirus Omeprone BA.1 strain, novel coronavirus Omeprone BA.2 strain, novel coronavirus Omeprone BA.4 / 5 strain, novel coronavirus Omeprone BF.7 strain, novel coronavirus Omeprone BQ.1 strain, novel coronavirus Omeprone BQ.1.1 strain, novel coronavirus Omeprone XBB strain, novel coronavirus Omeprone XBB.1 strain, novel coronavirus Omeprone XBB.1.5 strain, bat coronavirus WIV16 strain, bat coronavirus RaTG13 strain, pangolin coronavirus GD strain, or pangolin coronavirus P4L strain.

6. A product wherein the active ingredient is the IgG antibody as described in claim 1, 2, or 3; The product is one of the following (f), (g), or (h): (f) Medicines used to prevent and / or treat coronavirus infection; (g) Virus inhibitors targeting coronaviruses; (h) Preparations used to neutralize coronaviruses; The coronaviruses mentioned are SARS-CoV-1, wild-type novel coronavirus, novel coronavirus Alpha strain, novel coronavirus Beta strain, novel coronavirus Gamma strain, novel coronavirus Delta strain, novel coronavirus Omeprone BA.1 strain, novel coronavirus Omeprone BA.2 strain, novel coronavirus Omeprone BA.4 / 5 strain, novel coronavirus Omeprone BF.7 strain, novel coronavirus Omeprone BQ.1 strain, novel coronavirus Omeprone BQ.1.1 strain, novel coronavirus Omeprone XBB strain, novel coronavirus Omeprone XBB.1 strain, novel coronavirus Omeprone XBB.1.5 strain, bat coronavirus WIV16 strain, bat coronavirus RaTG13 strain, pangolin coronavirus GD strain, or pangolin coronavirus P4L strain.

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