Neutralizing antibody W322-3E1 for neutralizing coronavirus in broad spectrum and application of neutralizing antibody W322-3E1

Abstract

The invention discloses a neutralizing antibody W322-3E1 for neutralizing coronavirus in a broad spectrum and application of the neutralizing antibody W322-3E1. The invention provides an IgG (immunoglobulin G) antibody, which is named as a W322-3E1 antibody and consists of a light chain and a heavy chain, cDR1, CDR2 and CDR3 in a heavy chain variable region in the heavy chain are sequentially shown as the 45 to 52 sites, the 70 to 77 sites and the 116 to 136 sites in SEQ ID NO: 1; cDR1, CDR2 and CDR3 in a light chain variable region in the light chain are sequentially shown as the 45 to 53 sites, the 71 to 73 sites and the 110 to 121 sites in SEQ ID NO: 3. The W322-3E1 antibody not only has the effect of neutralizing the atypical pneumovirus, but also has broad-spectrum neutralizing activity on other coronaviruses. The invention has great application value for prevention and control of beta coronavirus, and can generate profound social significance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a neutralizing antibody W322-3E1 for broadly neutralizing coronaviruses and an application thereof. Background Art

[0002] Severe acute respiratory syndrome virus (SARS-CoV-1) is a type of beta coronavirus that is transmitted through the respiratory tract. SARS virus infection has 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. The technical reserve for the prevention and treatment of SARS virus strains is of great value and significance in responding to 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 neutralizing antibody W322-3E1 for neutralizing a broad spectrum of coronaviruses and an application thereof.

[0006] The present invention provides an IgG antibody, named W322-3E1 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 116-136, 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 110-121, respectively.

[0007] Specifically, the heavy chain variable region is shown in positions 20-147 of SEQ ID NO:1.

[0008] Specifically, the light chain variable region is shown at positions 20-131 in SEQ ID NO:3.

[0009] Specifically, the heavy chain is as follows (a) or (b): (a) the protein shown at positions 20-477 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 20-237 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 1434 of 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 to 714 of 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 present invention also protects a product, the active ingredient of which is any one of the above-mentioned IgG antibodies.

[0020] The product is (e) or (f) or (g) or (h) as follows:

[0021] (e) vaccines against coronavirus;

[0022] (f) medicines for the prevention and / or treatment of coronavirus infection;

[0023] (g) Viral inhibitors targeting coronaviruses;

[0024] (h) Preparations for neutralizing coronaviruses.

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

[0026] Specifically, the coronavirus is a β coronavirus.

[0027] Specifically, the coronavirus is atypical pneumonia virus, novel coronavirus, bat coronavirus or pangolin coronavirus.

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

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

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

[0031] 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 W322-3E1 antibody. The W322-3E1 antibody provided by the present invention not only has the effect of neutralizing atypical pneumonia virus, but also has broad-spectrum neutralizing activity against other coronaviruses. The present invention has great application value for the prevention and control of β-coronaviruses and will have far-reaching social significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The neutralizing activity of W322-3E1 antibody against SARS virus pseudovirus.

[0033] Figure 2 The W322-3E1 antibody has a broad-spectrum neutralizing activity against multiple coronavirus pseudoviruses. DETAILED DESCRIPTION

[0034] 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.

[0035] 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.

[0036] hACE2-hela cells (i.e. “HeLa cell lines stably expressing the ACE2molecules"), recorded in the following literature: Wang, R., Zhang, Q., Ge, J., Ren, W., Zhang, R., Lan, J., Ju, B., Su, B., Yu, F., Chen ,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 reveals neutralization escape mechanisms and the ability to use ACE2 receptors from additional species. Immunity 54, 1611-1621.e1615, doi:10.1016 / j.immuni.2021.06.003(2021).

[0037] Example 1. Discovery and preparation of W322-3E1 antibody

[0038] Memory B cells were isolated from peripheral blood mononuclear cells of patients in the recovery stage of SARS-CoV-1 infection and disease, 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 SARS-CoV-1 IgG antibody with excellent activity, which was named W322-3E1 antibody.

[0039] The amino acid sequence of the full-length heavy chain of the W322-3E1 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 20-147 constitute a heavy chain variable region, and amino acid residues 148-477 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 (GDTFSSNG), 70-77 (ITPILRVA), and 116-136 (AREEGGYSGYDTHFFDNWFDT), respectively.

[0040] The amino acid sequence of the full-length light chain of the W322-3E1 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 20-131 constitute a light chain variable region, and amino acid residues 132-237 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 (SSNIGAGYN), 71-73 (GSN), and 110-121 (QSFDSSLSGSNV).

[0041] Example 2. Preparation of W322-3E1 antibody

[0042] 1. Construction of recombinant plasmid

[0043] 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.

[0044] 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.

[0045] Both the heavy chain DNA molecule and the light chain DNA molecule are double-stranded DNA molecules.

[0046] 2. Construction of recombinant cells

[0047] The heavy chain expression vector and the light chain expression vector were co-transfected into 293F cells to obtain recombinant cells.

[0048] 3. Antibody Preparation

[0049] 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.

[0050] 2. Affinity chromatography

[0051] Affinity chromatography filler: Protein A Resin (GenScript Biotech, product catalog number L00210);

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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 W322-3E1 antibody solution.

[0056] Example 3. Neutralizing activity of W322-3E1 antibody against SARS-CoV-1

[0057] 1. Preparation of SARS-CoV-1 pseudovirus

[0058] 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.

[0059] 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).

[0060] 2. Neutralizing activity detection of antibodies

[0061] Test virus solution: SARS-CoV-1 virus solution prepared in step 1.

[0062] 1. Take the W322-3E1 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.

[0063] 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 4TCID50 / 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.

[0064] 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.

[0065] 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.

[0066] 5. After completing step 4, take the cell culture plate and detect the luciferase activity.

[0067] Multiple replicate wells were set up for each treatment.

[0068] 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%.

[0069] 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.

[0070] 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.

[0071] The IC50 value of W322-3E1 antibody against SARS-CoV-1 pseudovirus is 74.76 ng / ml.

[0072] Example 4: Broad-spectrum neutralizing activity of W322-3E1 antibody against coronaviruses

[0073] 1. Preparation of coronavirus pseudovirus

[0074] The coding gene of coronavirus membrane protein is inserted between the BamHII and EcoRI restriction sites of pcDNA3.1(+) vector to obtain a plasmid expressing coronavirus membrane protein. The plasmid expressing coronavirus membrane protein and the backbone plasmid pNL4-3R-E-luciferase are co-transfected into 293T cells, incubated at 37°C (using DMEM medium containing 10% fetal bovine serum), and the cell culture supernatant is collected 60 hours after transfection to obtain the virus solution containing coronavirus pseudovirus.

[0075] Virus solutions of pseudoviruses of 18 coronavirus strains were prepared respectively, namely, virus solutions of pseudoviruses of 14 novel coronavirus strains, virus solutions of pseudoviruses of 2 bat coronavirus strains, and virus solutions of pseudoviruses of 2 pangolin coronavirus strains. The 14 novel coronavirus strains refer to the novel coronavirus wild strain, novel coronavirus Alpha strain, novel coronavirus Beta strain, novel coronavirus Gamma strain, novel coronavirus Delta strain, novel coronavirus Omicron BA.1 strain, novel coronavirus Omicron BA.2 strain, novel coronavirus Omicron BA.4 / 5 strain, novel coronavirus Omicron BF.7 strain, novel coronavirus Omicron BQ.1 strain, novel coronavirus Omicron BQ.1.1 strain, novel coronavirus Omicron XBB strain, novel coronavirus Omicron XBB.1 strain, and novel coronavirus Omicron XBB.1.5 strain. The 2 bat coronavirus strains refer to bat coronavirus WIV16 strain and bat coronavirus RaTG13 strain. 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 the genes encoding the membrane proteins are shown in Table 1.

[0076] Table 1

[0077]

[0078]

[0079] 2. Neutralizing activity detection of antibodies

[0080] Test virus solutions: virus solutions of pseudoviruses of 18 coronavirus strains prepared in step 1.

[0081] The method is the same as step 2 of Example 3.

[0082] Neutralization activity results are shown in Figure 2 . Figure 2Middle: 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.

[0083] The IC50 values ​​of W322-3E1 antibody against various strains are shown in Table 2.

[0084] Table 2

[0085] IC50 value (μg / ml) Novel coronavirus wild strain (WT) 0.708296 Novel coronavirus Alpha strain 0.717837 Novel coronavirus Beta strain 0.14827 Novel coronavirus Gamma strain 0.170688 Novel coronavirus Delta strain 0.154248 Novel coronavirus Omicron BA.1 strain 0.982062 Novel coronavirus Omicron BA.2 strain >100 Novel coronavirus Omicron BA.4 / 5 strain >100 Novel coronavirus Omicron BF.7 strain >100 Novel coronavirus Omicron BQ.1 strain >100 Novel coronavirus Omicron BQ.1.1 strain >100 Novel coronavirus Omicron XBB strain >100 Novel coronavirus Omicron XBB.1 strain >100 Novel coronavirus Omicron XBB.1.5 strain >100 Bat coronavirus WIV16 strain 0.007235 Bat coronavirus RaTG13 strain 0.013586 Pangolin coronavirus GD strain 0.040913 Pangolin coronavirus P4L strain >10

[0086] 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 116-136, 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 110-121, respectively.

2. The IgG antibody according to claim 1, characterized in that: The heavy chain variable region is shown in positions 20-147 of SEQ ID NO: 1; The light chain variable region is shown in positions 20-131 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 20-477 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 237 in SEQ ID NO: 3; (d) the protein shown in 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) or (g) or (h) as follows: (e) vaccines against coronavirus; (f) medicines for the prevention and / or treatment of coronavirus infection; (g) Viral inhibitors targeting coronaviruses; (h) Preparations for neutralizing coronaviruses.

6. The use according to claim 5, characterized in that: The coronavirus is a β coronavirus.

7. The use according to claim 5, characterized in that: The coronavirus is atypical pneumonia virus, new coronavirus, bat coronavirus or pangolin coronavirus.

8. A product, the active ingredient of which is the IgG antibody according to claim 1, 2 or 3; The product is (e) or (f) or (g) or (h) as follows: (e) vaccines against coronavirus; (f) medicines for the prevention and / or treatment of coronavirus infection; (g) Viral inhibitors targeting coronaviruses; (h) Preparations for neutralizing coronaviruses.

9. The product according to claim 8, characterized in that: The coronavirus is a β coronavirus.

10. The product according to claim 8, characterized in that: The coronavirus is atypical pneumonia virus, new coronavirus, bat coronavirus or pangolin coronavirus.

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