Vaccines and drugs against SARS-CoV-1 and neutralizing antibody W322-3D10 as active ingredient thereof

Abstract

The application discloses a vaccine and medicine for SARS-CoV-1 and a neutralizing antibody W322-3D10 as an active component thereof. The application provides an IgG antibody, named W322-3D10 antibody, which is composed of a light chain and a heavy chain; CDR1, CDR2 and CDR3 in the heavy chain variable region are sequentially shown in the 45th-52nd, 70th-77th and 116th-130th positions of SEQ ID NO:1; CDR1, CDR2 and CDR3 in the light chain variable region are sequentially shown in the 45th-53rd, 71st-73rd and 110th-117th positions of SEQ ID NO:3. The application also protects the application of the IgG antibody in the preparation of products; the products are: a vaccine for atypical pneumonia virus; a medicine for inhibiting atypical pneumonia virus. The application has great application value for the prevention and control of beta genus coronavirus and will produce far-reaching social significance.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to vaccines and drugs against SARS-CoV-1, as well as the neutralizing antibody W322-3D10 as its active ingredient. Background Technology

[0002] Severe Acute Respiratory Syndrome (SARS-CoV-1) is a 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 prepare for 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 vaccines and drugs against SARS-CoV-1, as well as the neutralizing antibody W322-3D10 as its active ingredient.

[0006] This invention provides an IgG antibody, named W322-3D10 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 116-130 of SEQ ID NO: 1, respectively; the CDR1, CDR2, and CDR3 in the variable region of the light chain are as shown in positions 45-53, 71-73, and 110-117 of SEQ ID NO: 3, respectively.

[0007] Specifically, the heavy chain variable region is shown in positions 20-141 of SEQ ID NO: 1.

[0008] Specifically, the variable region of the light chain is shown in positions 20-127 of SEQ ID NO: 3.

[0009] Specifically, the heavy chain is either (a) or (b) as follows: (a) the protein represented by positions 20-471 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 58-1416 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 above-described IgG antibodies in the preparation of products;

[0019] The product is either (e) or (f) as follows:

[0020] (e) Vaccines against SARS virus;

[0021] (f) Drugs used to suppress SARS virus.

[0022] This invention also protects a product whose active ingredient is any of the IgG antibodies described above;

[0023] The product is either (e) or (f) as follows:

[0024] (e) Vaccines against SARS virus;

[0025] (f) Drugs used to suppress SARS virus.

[0026] This invention also protects the use of any of the above-described IgG antibodies in the preparation of formulations for use against SARS and ASARS viruses. The formulations are vaccine formulations or pharmaceutical formulations.

[0027] This invention also protects a formulation for use against SARS and atypical pneumonia viruses, wherein the active ingredient is any of the IgG antibodies described above. The formulation is a vaccine formulation or a pharmaceutical formulation.

[0028] The present invention also protects the use of any of the above-described IgG antibodies in the preparation of medicaments for the prevention and / or treatment of SARS virus infection.

[0029] The present invention also protects a medicament for the prevention and / or treatment of SARS virus infection, wherein the active ingredient is any of the IgG antibodies described above.

[0030] This invention screens memory B cells generated from peripheral blood mononuclear cells of patients infected with SARS-CoV-2, obtaining a monoclonal antibody that specifically binds to the spike protein, named W322-3D10 antibody. The W322-3D10 antibody provided by this invention is effective against SARS-CoV-2. This invention has significant application value for the prevention and control of β-coronaviruses and will have profound social implications. Attached Figure Description

[0031] Figure 1 The neutralizing activity of the W322-3D10 antibody against SARS-CoV-1 pseudovirus was measured. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. 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 invention in any way.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Unless otherwise specified, all quantitative experiments in the following examples were performed in triplicate, and the results were averaged. 293F cells and 293T cells were commercially available human embryonic kidney epithelial cells. The pMD18-T vector was a commercially available plasmid vector. The pcDNA3.1(+) vector was from Invitrogen, catalog number V790-20.

[0034] 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).

[0035] Example 1: Discovery of the W322-3D10 antibody

[0036] 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 IgG antibody with excellent activity against SARS-CoV-1, and named this IgG antibody W322-3D10 antibody.

[0037] The full-length heavy chain amino acid sequence of the W322-3D10 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 20-141 constitute the variable region of the heavy chain, and amino acid residues 142-471 constitute the constant region of the heavy chain. CDR1, CDR2, and CDR3 in the variable region of the heavy chain are shown as follows: positions 45-52 (GFTFDAYA), positions 70-77 (INEDGGST), and positions 116-130 (GKDFGGAYSGTLVDS) in SEQ ID NO: 1, respectively.

[0038] The full-length light chain amino acid sequence of the W322-3D10 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 be secreted extracellularly), amino acid residues 20-127 constitute the variable region of the light chain, and amino acid residues 128-233 constitute the constant region of the light chain. CDR1, CDR2, and CDR3 in the variable region of the light chain are shown as positions 45-53 (SSNIGAGYG), 71-73 (GDT), and 110-117 (LSYDGSLV) in SEQ ID NO: 3, respectively.

[0039] Example 2: Preparation of W322-3D10 antibody

[0040] I. Construction of Recombinant Plasmids

[0041] 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.

[0042] 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.

[0043] Both heavy chain DNA molecules and light chain DNA molecules are double-stranded DNA molecules.

[0044] II. Construction of Recombinant Cells

[0045] The heavy chain expression vector and the light chain expression vector were co-transfected into 293F cells to obtain recombinant cells.

[0046] III. Antibody Preparation

[0047] 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.

[0048] 2. Affinity chromatography

[0049] Affinity chromatography packing material: Protein A Resin (Genscript Biotechnology, product catalog number L00210);

[0050] 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.

[0051] 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.

[0052] Elution buffer: Dissolve 7.5g of glycine in water and bring the volume to 500ml. Adjust the pH to 3.0 with hydrochloric acid.

[0053] 3. Take the column pass 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 W322-3D10 antibody solution.

[0054] Example 3, Neutralization Test

[0055] I. Preparation of SARS-CoV-1 pseudovirus

[0056] Co-transfection of 293T cells with a plasmid expressing the SARS-CoV-1 membrane protein and the backbone plasmid pNL4-3R-E-luciferase yielded an infectious but non-replicating SARS-CoV-1 pseudovirus, with infectivity similar to live SARS-CoV-1 virus. The backbone plasmid pNL4-3R-E-luciferase, i.e., the backbone plasmid pNL4-3R-E containing luciferase (i.e., vector with the luciferase gene containing backbone pNL4-3R-E in the literature), 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.

[0057] 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).

[0058] II. Detection of antibody neutralizing activity

[0059] Test virus solution: SARS-CoV-1 virus solution prepared in step one.

[0060] 1. Take the W322-3D10 antibody solution prepared in Example 2 and perform serial dilution with PBS buffer (pH 7.2, 10mM) to obtain antibody dilutions of various concentrations.

[0061] 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 using TCID50 / ml. Use an equal volume of PBS buffer (pH 7.2, 10mM) instead of the antibody dilution solution 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.

[0062] 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.

[0063] 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.

[0064] 5. After completing step 4, take the cell culture plate and detect the luciferase activity.

[0065] Each process is configured with multiple duplicate holes.

[0066] Neutralization activity (%) = [1 - (fluorescence intensity of experimental group - fluorescence intensity of cell control) / (fluorescence intensity of virus control - fluorescence intensity of cell control)] × 100%.

[0067] 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.

[0068] The antibody concentration at which the neutralizing activity is 50% was calculated using Prism 8 software, i.e., the IC50 value of the antibody.

[0069] The IC50 value of the W322-3D10 antibody against SARS-CoV-1 pseudovirus was 7.146 ng / ml.

[0070] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended 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 116-130 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 45-53, 71-73, and 110-117 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 positions 20-141 of SEQ ID NO: 1; The variable region of the light chain is shown in positions 20-127 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 20-471 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. Use of the IgG antibody according to claim 1, 2 or 3 in the preparation of a medicament for inhibiting SARS-CoV-1.

6. A drug for inhibiting SARS-CoV-1, wherein the active ingredient is the IgG antibody as described in claim 1, 2 or 3.

7. Use of the IgG antibody of claim 1, 2 or 3 in the preparation of a formulation for neutralizing SARS-CoV-1.

8. A formulation for neutralizing SARS-CoV-1, wherein the active ingredient is the IgG antibody as described in claim 1, 2 or 3.

9. The use of the IgG antibody according to claim 1, 2 or 3 in the preparation of a medicament for the prevention and / or treatment of SARS-CoV-1 infection.

10. A medicament for the prevention and / or treatment of SARS-CoV-1 infection, wherein the active ingredient is the IgG antibody as described in claim 1, 2 or 3.

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