Vaccine and medicament against SARS-CoV-1 and neutralizing antibody W322-3D10 as active ingredient thereof

Abstract

The invention discloses a vaccine and a medicine aiming at SARS-CoV-1 and a neutralizing antibody W322-3D10 serving as an active ingredient of the vaccine and the medicine. The invention provides an IgG antibody which is named as a W322-3D10 antibody and consists of a light chain and a heavy chain, cDR1, CDR2 and CDR3 in the heavy chain variable region are sequentially shown as the 45 to 52 , the 70 to 77 and the 116 to 130 in SEQ ID NO: 1; cDR1, CDR2 and CDR3 in the light chain variable region are sequentially shown as the 45th site to the 53rd site, the 71st site to the 73rd site and the 110th site to the 117th site in SEQ ID NO: 3. The invention also discloses application of the IgG antibody in preparation of products. The product is: a vaccine against atypical pneumovirus; the invention relates to a medicament for inhibiting atypical pneumovirus. The invention has great application value for prevention and control of beta coronavirus, and can generate profound social significance.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to vaccines and drugs targeting SARS-CoV-1 and neutralizing antibody W322-3D10 as active ingredients thereof. Background Art

[0002] Severe acute respiratory syndrome (SARS-CoV-1) is a beta coronavirus that is transmitted through the respiratory tract. SARS infection causes an acute onset, rapid spread, and high mortality rate. The main symptoms are hypoxia, cyanosis, and high fever. It often develops into a severe infection with accelerated breathing or respiratory distress syndrome, accompanied by septic shock, metabolic acidosis, coagulation dysfunction, and multiple organ failure.

[0003] In the 20 years since the outbreak of the SARS virus, two waves of coronavirus outbreaks have occurred, namely the Middle East Respiratory Syndrome (MERS-CoV) and the new coronavirus (SARS-CoV-2). β-coronaviruses have a wide range of similar conservative regions in sequence and structure. It is of great value and significance to prepare for the prevention and treatment of SARS virus strains to deal with new coronavirus infectious diseases that may emerge in the future.

[0004] Monoclonal antibodies can be industrially mass-produced. Their high affinity and specificity for binding to antigens greatly reduce adverse reactions during clinical application. At the same time, antibody molecules can be modified to increase their antiviral efficacy. Antibodies have become a very promising means of treating infectious diseases due to their specificity and flexibility of use. The storage of antibody sequences against atypical pneumonia viruses can provide effective prevention and treatment methods in the first place in possible new coronavirus outbreaks in the future. Summary of the invention

[0005] The purpose of the present invention is to provide a vaccine and a medicine against SARS-CoV-1 and a neutralizing antibody W322-3D10 as an active ingredient thereof.

[0006] The present invention provides an IgG antibody, named W322-3D10 antibody, which consists of a light chain and a heavy chain; CDR1, CDR2 and CDR3 in the heavy chain variable region of the heavy chain are shown in SEQ ID NO: 1 at positions 45-52, 70-77 and 116-130, respectively; CDR1, CDR2 and CDR3 in the light chain variable region of the light chain are shown in SEQ ID NO: 3 at positions 45-53, 71-73 and 110-117, respectively.

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

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

[0009] Specifically, the heavy chain is as follows (a) or (b): (a) the protein shown at positions 20-471 in SEQ ID NO: 1; (b) the protein shown in SEQ ID NO: 1;

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

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

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

[0013] (1) the DNA molecule represented by nucleotides 58 to 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] The present invention also protects the use of any of the above IgG antibodies in preparing products;

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

[0020] (e) Vaccines against SARS virus;

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

[0022] The present invention also protects a product, the active ingredient of which is any of the above-mentioned IgG antibodies;

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

[0024] (e) Vaccines against SARS virus;

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

[0026] The present invention also protects the use of any of the above IgG antibodies in the preparation of a preparation for neutralizing SARS virus. The preparation is a vaccine preparation or a pharmaceutical preparation.

[0027] The present invention also protects a preparation for neutralizing SARS virus, wherein the active ingredient is any one of the above IgG antibodies. The preparation is a vaccine preparation or a pharmaceutical preparation.

[0028] The present invention also protects the use of any of the above IgG antibodies in the preparation of a medicament for preventing and / or treating atypical pneumonia virus infection.

[0029] The present invention also protects a drug for preventing and / or treating atypical pneumonia virus infection, wherein the active ingredient is any one of the above-mentioned IgG antibodies.

[0030] The present invention screens antibody-producing memory B cells from peripheral blood mononuclear cells of patients infected with atypical pneumonia virus, and obtains a monoclonal antibody that can specifically bind to the spike protein, which is named W322-3D10 antibody. The W322-3D10 antibody provided by the present invention has the effect of neutralizing atypical pneumonia virus. The present invention has great application value for the prevention and control of beta coronaviruses and will have far-reaching social significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The neutralizing activity of W322-3D10 antibody against SARS-CoV-1 pseudovirus. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0033] The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. Unless otherwise specified, the quantitative tests in the following examples are set up for three repeated experiments, and the results are averaged. 293F cells and 293T cells are both 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.

[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 W322-3D10 antibody

[0036] Memory B cells were isolated from peripheral blood mononuclear cells of patients in the recovery period after infection with SARS-CoV-1, and the antibody genes of memory B cells were amplified to obtain antibody sequences. Through a large number of comparisons, analyses, preparations and effect verifications, the inventors of the present invention discovered a new IgG antibody with excellent activity against SARS-CoV-1, and named the IgG antibody W322-3D10.

[0037] The amino acid sequence of the full-length heavy chain of the W322-3D10 antibody is shown in SEQ ID NO: 1. In SEQ ID NO: 1, amino acid residues 1-19 constitute a signal peptide (to guide protein secretion outside the cell), amino acid residues 20-141 constitute a heavy chain variable region, and amino acid residues 142-471 constitute a heavy chain constant region. CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ ID NO: 1 at positions 45-52 (GFTFDAYA), 70-77 (INEDGGST), and 116-130 (GKDFGGAYSGTLVDS), respectively.

[0038] The amino acid sequence of the full-length light chain of the W322-3D10 antibody is shown in SEQ ID NO: 3; in SEQ ID NO: 3, amino acid residues 1-19 constitute a signal peptide (to guide protein secretion outside the cell), amino acid residues 20-127 constitute a light chain variable region, and amino acid residues 128-233 constitute a light chain constant region. CDR1, CDR2, and CDR3 in the light chain variable region are shown in SEQ ID NO: 3, respectively, at positions 45-53 (SSNIGAGYG), 71-73 (GDT), and 110-117 (LSYDGSLV).

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

[0040] 1. Construction of recombinant plasmid

[0041] The heavy chain DNA molecule was inserted into the pMD18-T vector to obtain a 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 encoding gene shown in SEQ ID NO: 2, and the terminator shown in SEQ ID NO: 6.

[0042] Insert the light chain DNA molecule into the pMD18-T vector to obtain a 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 encoding gene shown in SEQ ID NO: 4, and the terminator shown in SEQ ID NO: 6.

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

[0044] 2. 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] 3. Antibody Preparation

[0047] 1. Take the recombinant cells obtained in step 2, culture them in DMEM medium containing 2% fetal bovine serum for 72 hours, then centrifuge them at 4°C and 4000 rpm for 30 minutes, collect the supernatant, and filter it with a 0.45 μm filter membrane.

[0048] 2. Affinity chromatography

[0049] Affinity chromatography filler: Protein A Resin (GenScript Biotech, product catalog number L00210);

[0050] Operation steps: ① Mix 300 mL of the filtrate obtained in step 1 with 3 mL of affinity chromatography filler and incubate at 4°C for 16 h; ② Add the mixed solution to a 25 mL gravity flow column to retain the filler; ③ Wash the column with 60 mL of binding buffer; elute the target protein with 30 mL of elution buffer, collect the solution after passing through the column, and adjust the pH to neutral.

[0051] Binding buffer: Take 112.6 g of glycine and 175.2 g of sodium chloride, dissolve them in water and make up to 1 L with water, and adjust the pH to 8.0 with sodium hydroxide.

[0052] Elution buffer: Take 7.5 g of glycine, dissolve it in water and make up to 500 ml with water, and adjust the pH to 3.0 with hydrochloric acid.

[0053] 3. Take the column solution obtained in step 2, concentrate it with an ultrafiltration concentration tube and replace the system with PBS buffer (pH 7.2, 10 mM) to obtain a W322-3D10 antibody solution.

[0054] Embodiment 3, neutralization test

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

[0056] The plasmid expressing the SARS-CoV-1 membrane protein and the backbone plasmid pNL4-3R-E-luciferase were co-transfected into 293T cells, and after incubation, a SARS-CoV-1 pseudovirus with infectivity but no replication ability was obtained, and its infectivity was similar to that of the live SARS-CoV-1 virus. The backbone plasmid pNL4-3R-E-luciferase, i.e., the backbone plasmid pNL4-3R-E containing Luciferase (i.e., the vector with the luciferase gene containing backbone pNL4-3R-E in the literature) is recorded 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 coding gene of SARS-CoV-1 membrane protein was inserted between the BamHII and EcoRI restriction sites of pcDNA3.1(+) vector to obtain a plasmid expressing SARS-CoV-1 membrane protein. The plasmid expressing SARS-CoV-1 membrane protein and the backbone plasmid pNL4-3R-E-luciferase were co-transfected into 293T cells, incubated at 37°C (using DMEM medium containing 10% fetal bovine serum), and the cell culture supernatant was collected 60 hours after transfection, which was the virus liquid containing SARS-CoV-1 pseudovirus, referred to as SARS-CoV-1 virus liquid. SARS-CoV-1 membrane protein is shown in SEQ ID NO: 7. The coding gene of SARS-CoV-1 membrane protein is shown in SEQ ID NO: 8 (codon-optimized sequence).

[0058] 2. Neutralizing activity detection of antibodies

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

[0060] 1. Take the W322-3D10 antibody solution prepared in Example 2 and perform gradient dilution with PBS buffer (pH 7.2, 10 mM) to obtain antibody dilution solutions 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 4 TCID50 / ml), incubate at 37°C for 1 hour. Replace the antibody diluent with an equal volume of PBS buffer (pH 7.2, 10 mM) as a virus control. Replace the test virus solution with an equal volume of DMEM medium containing 10% fetal bovine serum as a cell control.

[0062] 3. After completing step 2, take the cell culture plate and inoculate 100 μl of hACE2-hela cell suspension in each well (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 5 cells / ml) and incubated at 37°C for 64 hours.

[0063] 4. After completing step 3, take the cell culture plate, discard the supernatant, add 150 μl lysis solution (Micrograss Biotechnology, product 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] Multiple replicate wells were set up for each treatment.

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

[0067] Neutralization activity results are shown in Figure 1 . Figure 1 Middle: The ordinate is the neutralization activity (%); the abscissa is the logarithm of the antibody concentration (μg / ml) to the base 10, and the antibody concentration refers to the antibody concentration in the mixed system consisting of 100 μl of antibody diluent and 50 μl of test virus solution in step 2.

[0068] Prism 8 software was used to calculate the antibody concentration at which the neutralization activity was 50%, i.e., the IC50 value of the antibody.

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

[0070] 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 implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. An IgG antibody, consisting of a light chain and a heavy chain; the CDR1, CDR2 and CDR3 in the heavy chain variable region of the heavy chain are shown in SEQ ID NO: 1 at positions 45-52, 70-77 and 116-130, respectively; the CDR1, CDR2 and CDR3 in the light chain variable region of the light chain are shown in SEQ ID NO: 3 at positions 45-53, 71-73 and 110-117, respectively.

2. The IgG antibody according to claim 1, characterized in that: The heavy chain variable region is shown in positions 20-141 of SEQ ID NO: 1; The light chain variable region is shown in positions 20-127 of SEQ ID NO:

3.

3. The IgG antibody according to claim 2, characterized in that: The heavy chain is (a) or (b) as follows: (a) the protein shown at positions 20-471 in SEQ ID NO: 1; (b) the protein shown in SEQ ID NO: 1; The light chain is (c) or (d) as follows: (c) the protein shown at positions 20 to 233 of SEQ ID NO: 3; (d) the protein shown by SEQ ID NO:

3.

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

5. Use of the IgG antibody according to claim 1, 2 or 3 in preparing a product; The product is (e) or (f) as follows: (e) Vaccines against SARS virus; (f) Drugs used to inhibit SARS virus.

6. A product, the active ingredient of which is the IgG antibody according to claim 1, 2 or 3; The product is (e) or (f) as follows: (e) Vaccines against SARS virus; (f) Drugs used to inhibit SARS virus.

7. Use of the IgG antibody according to claim 1, 2 or 3 in the preparation of a preparation for neutralizing SARS virus.

8. A preparation for neutralizing SARS virus, wherein the active ingredient is the IgG antibody according to claim 1, 2 or 3.

9. Use of the IgG antibody according to claim 1, 2 or 3 in the preparation of a medicament for preventing and / or treating atypical pneumonia virus infection.

10. A drug for preventing and / or treating atypical pneumonia virus infection, wherein the active ingredient is the IgG antibody according to claim 1, 2 or 3.

Images