A broad-spectrum non-neutralizing antibody to foot-and-mouth disease virus and its use in the preparation of reagents for enhancing the induction of neutralizing antibodies

Abstract

This invention provides a broad-spectrum non-neutralizing antibody against foot-and-mouth disease virus (FMD) and its application in the preparation of reagents that enhance the production of neutralizing antibodies, belonging to the field of antibody technology. The FMD virus broad-spectrum non-neutralizing antibody has the amino acid sequence of its heavy chain variable region as shown in SEQ ID NO:1 and the amino acid sequence of its light chain variable region as shown in SEQ ID NO:2. This invention utilizes single B-cell antibody technology to screen and obtain a porcine broad-spectrum non-neutralizing monoclonal antibody SP1C6, which can recognize a conformational epitope on VP2. The broad-spectrum non-neutralizing antibody SP1C6 increases the level of neutralizing antibodies induced by FMD virus in pigs by blocking the non-neutralizing epitopes of the FMD virus antigen.

Description

Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to a broad-spectrum non-neutralizing antibody against foot-and-mouth disease virus and its application in the preparation of reagents that enhance the induction of neutralizing antibodies. Background Technology

[0002] Foot-and-mouth disease (FMD) primarily affects cloven-hoofed animals such as cattle, pigs, and sheep, significantly impacting international trade in livestock and their products. The pathogen, foot-and-mouth disease virus (FMDV), comprises seven serotypes: O, A, C, Asia 1, SAT1, SAT2, and SAT3. Within the same serotype, there are further subdivisions based on geographical topology, lineage, and genetic lineage. Different FMDV serotypes do not provide cross-immunity. Under natural infection conditions, it is difficult to produce broadly neutralizing antibodies (bnAbs) that can neutralize different serotypes of the virus; however, it is possible to produce broadly reactive non-neutralizing antibodies (brAbs).

[0003] Antibody-mediated humoral immunity is an important antiviral immune mechanism. The antiviral effect of antibodies has two aspects: firstly, neutralizing antibodies bind to viruses, preventing them from binding to cell surface receptors and invading cells; secondly, non-neutralizing antibodies, through their Fc-terminus-mediated opsonization and antibody-dependent cytotoxicity, promote the clearance of viruses or virus-infected cells by macrophages, natural killer cells, and other cells. However, non-neutralizing antibodies may also enhance the ability of viruses to infect cells through antibody-dependent invasion mechanisms, i.e., antibody-dependent enhancement (ADE).

[0004] Neutralizing antibodies are a major protective immune component against FMDV infection. Under natural infection conditions, viruses preferentially produce non-neutralizing antibodies and strain-specific antibodies, which is a mechanism for viral evolution and evasion of the host's immune system. This mechanism has been extensively studied in enveloped viruses, such as HIV, influenza virus, and porcine reproductive and respiratory syndrome virus (PRRSV). Many glycosylated antigenic sites in viral envelope proteins have immunogenicity and influence the neutralizing antibody response. However, whether the non-neutralizing antibodies produced by infection with non-enveloped viruses affect the level, affinity, and broad spectrum of neutralizing antibody responses remains poorly studied. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a broad-spectrum non-neutralizing antibody against foot-and-mouth disease virus, which increases the level of neutralizing antibody production induced by foot-and-mouth disease virus by blocking non-neutralizing epitopes on the foot-and-mouth disease virus.

[0006] This invention provides a broad-spectrum non-neutralizing antibody against foot-and-mouth disease virus, wherein 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.

[0007] This invention provides a single-chain antibody for a broad-spectrum non-neutralizing antibody against foot-and-mouth disease virus, which is prepared based on the heavy chain variable region and the light chain variable region of the aforementioned broad-spectrum non-neutralizing antibody against foot-and-mouth disease virus.

[0008] This invention provides the application of the broad-spectrum non-neutralizing antibody against foot-and-mouth disease virus or the single-chain antibody in the preparation of reagents or vaccines that enhance the production of neutralizing antibodies induced by foot-and-mouth disease virus.

[0009] Preferably, the foot-and-mouth disease virus that induces the production of neutralizing antibodies includes type O foot-and-mouth disease virus and / or type A foot-and-mouth disease virus.

[0010] Preferably, the type A foot-and-mouth disease virus includes the SEA strain and / or the A22 strain;

[0011] The O-type foot-and-mouth disease virus includes at least one of the following lineages: Cathay topology, SEA topology, and ME-SA topology IND2001 lineage.

[0012] The present invention provides a vaccine that enhances the production of neutralizing antibodies, comprising foot-and-mouth disease virus, an adjuvant, and a broad-spectrum non-neutralizing antibody against the foot-and-mouth disease virus or the single-chain antibody.

[0013] Preferably, the vaccine comprises an inactivated foot-and-mouth disease virus vaccine;

[0014] The foot-and-mouth disease virus includes type O foot-and-mouth disease virus and / or type A foot-and-mouth disease virus.

[0015] Preferably, the type A foot-and-mouth disease virus includes the SEA lineage and / or the A22 lineage.

[0016] Preferably, the type O foot-and-mouth disease virus includes at least one of the following lineages: Cathay topology, SEA topology, and ME-SA topology IND2001 lineage.

[0017] This invention provides a broad-spectrum non-neutralizing antibody against foot-and-mouth disease virus (FMDV). 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. This invention utilizes single B-cell sorting technology to obtain the monoclonal antibody SP1C6. Western blotting results show that the monoclonal antibody SP1C6 recognizes epitopes on VP2 of FMDV type A and O antigens, and reacts only with intact VP2, not with truncated VP2 protein. This indicates that the epitopes on VP2 recognized by SP1C6 are discontinuous and may be conformational epitopes. Alanine scanning results show that K63, H65, L66, F67, and D68 residues on the β-sheet and L81 residue on the C-sheet are clearly exposed on the FMDV capsid, which are key amino acids for the conformational epitopes recognized by SP1C6. Virus neutralization assay results show that the monoclonal antibody SP1C6 does not produce FMDV neutralizing activity, indicating it is a non-neutralizing monoclonal antibody. This invention also employs both NAC-ELISA and VNT methods to detect the induction results of neutralizing antibodies. NAC-ELISA results showed that the antibody levels in both the SP1C6 monoclonal antibody and its corresponding single-chain antibody groups were higher than those in the antigen-only immunization group. VNT results showed that the antibody levels in both the SP1C6 monoclonal antibody and its corresponding single-chain antibody groups were higher than those in the antigen-only immunization group, and the results were completely consistent. This indicates that blocking the non-neutralizing epitopes of foot-and-mouth disease virus strains with the broad-spectrum non-neutralizing antibody SP1C6 can improve the level of neutralizing antibody response. Attached Figure Description

[0018] Figure 1 The results of NAC-ELISA detection of antibodies in immunized pig serum;

[0019] Figure 2 The results are from the detection of VNT antibodies in immunized pig serum. Detailed Implementation

[0020] This invention provides a broad-spectrum non-neutralizing antibody against foot-and-mouth disease virus, with the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:1 (EEKLVESGGGLVQPGGSLRLSCVGSGWTLSFGGFPIG).

[0021] The amino acid sequence of the light chain variable region is shown in SEQ ID NO:2 (QTVIQEPAMTVSLGGTVTLTCAYRSGS).

[0022] As shown in VTTSNYPDWYQQTPGQPPRLLIYSTNNRPTGVPSRFSGAISGNKAALTITGAQAEDEADYFCGLHKISGNDRPFGGGTHLTVLDYKDDDDKGG).

[0023] In this invention, the foot-and-mouth disease virus broad-spectrum non-neutralizing antibody is preferably the monoclonal antibody SP1C6 obtained based on single B cell sorting technology. The monoclonal antibody SP1C6 has no neutralizing effect (IC50) against the representative A strains of FMDV A (A / GDMM / CHA / 2013, A / WH / CHA / 09, A / AF72) and the representative O strains of FMDV O (O / HN / CHA / 93, O / Mya / 98, O / XJ / CHA / 2017) circulating in China. 50 The concentration (>50 μg / mL) indicates that the monoclonal antibody SP1C6 is a non-neutralizing antibody. Furthermore, Western blotting experiments showed that SP1C6 recognizes epitopes on VP2 of FMDV type A and type O antigens, reacting only with intact VP2 and not with truncated VP2 protein. This suggests that the epitopes recognized by SP1C6 on VP2 are discontinuous and may be conformational epitopes. Simultaneously, alanine substitution assays revealed that K63, H65, L66, F67, and D68 residues on the β-sheet and L81 residue on the C-sheet are clearly exposed on the FMDV capsid, representing key amino acids for the conformational epitopes recognized by SP1C6.

[0024] This invention provides a single-chain antibody for a broad-spectrum non-neutralizing antibody against foot-and-mouth disease virus, which is prepared based on the heavy chain variable region and the light chain variable region of the aforementioned broad-spectrum non-neutralizing antibody against foot-and-mouth disease virus.

[0025] In this invention, the method for preparing the single-chain antibody preferably involves using a linker peptide (GGGGSGGGG) to connect the coding sequences of the heavy chain variable region and the light chain variable region of the foot-and-mouth disease virus broad-spectrum non-neutralizing antibody.

[0026] The fusion protein was ligated into the eukaryotic expression vector pcDNA3.4 and expressed in CHO-S cells. The resulting fusion protein was then isolated and purified. The coding sequence of the heavy chain variable region of the broad-spectrum non-neutralizing antibody against foot-and-mouth disease virus is shown in SEQ ID NO:3 (GAGGAGAAGCTGGTGGAGTC).

[0027] The coding sequence of the light chain variable region of the foot-and-mouth disease virus broad-spectrum non-neutralizing antibody is shown in SEQ ID NO:4 (CAGACTGTGATCCAGGAGCCGGCGATGACAGTGTCCTACTGCCGGAGGTGCGTTCACCGTGGACCTTGTCCTTCGGTGGTTTCCCCATAGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGCTGGCAGTTATTACTGCCGGAGGTGCGTTCACCGACTACTCAGGTTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACTCCCAGAACGCGGCCTATCTGCAAATGAACAAACTTAGAGCTGAGGACACGGCCCGCTATTACTGTGTAAGAGAAATGGTCGTTGCCGATGCCCTGGATCTCTGGGGCCCAGGCGTTGAAGTCGTCGTGTCCTCA);

[0028] TTGGAGGGACCGTCACACTCACCTGTGCCTATAGGTCTGGGTCAGTCACCACTAGTAACTACCCTGACTGGTACCAACAGACGCCAGGCCAGCCTCCCCGACTGCTGATCTATAGCACAAACAACCGCCCGACTGGGGTCCCCAGTCGCTTCTCTGGAGCCAT CTCTGGGAACAAAGCCGCCCTCACCATCACGGGGGCCCAGGCTGAGGATGAGGCCGACTACTTCTGTGGTCTGCATAAAATTAGTGGTAATGATCGTCCCTTCGGCGGTGGGACCCATCTGACCGTCCTCGATTATAAGGACGATGACGATAAAGGCGGC).

[0029] In this invention, the monoclonal antibody SP1C6 or its single-chain antibody is used in combination with the antigen to prepare a vaccine, resulting in antibody levels higher than those in the antigen-induced group alone. At the same time, the results of the virus micro-neutralization assay are consistent with the results of the neutralizing antibody competitive ELISA detection. This indicates that blocking the non-neutralizing epitopes of the FMDV A / WH / CHA / 09 strain with the broad-spectrum non-neutralizing antibody SP1C6 can improve the neutralizing antibody response level.

[0030] Based on the aforementioned performance of the monoclonal antibody SP1C6 or its single-chain antibody in promoting the expression of neutralizing antigens and inducing the production of neutralizing antibodies by blocking non-neutralizing epitopes, this invention provides the application of the foot-and-mouth disease virus broad-spectrum non-neutralizing antibody or the single-chain antibody in the preparation of reagents or vaccines that enhance the production of neutralizing antibodies induced by foot-and-mouth disease virus.

[0031] In this invention, the foot-and-mouth disease virus that induces the production of neutralizing antibodies preferably includes type O foot-and-mouth disease virus and / or type A foot-and-mouth disease virus. The type A foot-and-mouth disease virus preferably includes the SEA lineage and / or the A22 lineage. The type O foot-and-mouth disease virus preferably includes at least one of the following lineages: Cathay topology, SEA topology, and ME-SA topology IND2001 lineage. In this embodiment of the invention, representative strains from all topologies / lineages of the prevalent FMDV A and O types in my country were used to demonstrate the broad-spectrum binding of monoclonal antibody SP1C6. These strains were A / GDMM / CHA / 2013 (A / SEA / 97 / G2 genotype, SEA lineage), A / WH / CHA / 09 A / SEA / 97 / G1 genotype, AF72 (A22 lineage), O / HN / CHA / 93 (Cathay topology), O / Mya / 98 (SEA topology), and O / XJ / CHA / 2017 (ME-SA IND2001 lineage). Indirect immunofluorescence assay results showed that monoclonal antibody SP1C6 could bind to all of the above-mentioned strains.

[0032] The present invention provides a vaccine that enhances the production of neutralizing antibodies, comprising foot-and-mouth disease virus, an adjuvant, and a broad-spectrum non-neutralizing antibody against the foot-and-mouth disease virus or the single-chain antibody.

[0033] In this invention, the final concentration of the foot-and-mouth disease virus 146S antigen is preferably 5 μg / ml. The final concentration of the broad-spectrum non-neutralizing antibody against foot-and-mouth disease virus is preferably 2 μg / ml, or the final concentration of the single-chain antibody is preferably 1 μg / ml. The vaccine preferably comprises an inactivated foot-and-mouth disease virus vaccine. The foot-and-mouth disease virus preferably comprises type O foot-and-mouth disease virus and / or type A foot-and-mouth disease virus. The type A foot-and-mouth disease virus preferably comprises the SEA lineage and / or the A22 lineage. The type O foot-and-mouth disease virus preferably comprises at least one of the following lineages: Cathay topology, SEA topology, and ME-SA topology IND2001 lineage. This invention does not impose any special restrictions on the type of adjuvant; any adjuvant well known in the art can be used, such as adjuvant 201.

[0034] In this embodiment of the invention, FMDV A / WH / CHA / 09 was used as the representative immunogen to prepare an inactivated vaccine. Experiments showed that adding the aforementioned broad-spectrum non-neutralizing antibody against foot-and-mouth disease virus or the aforementioned single-chain antibody to the inactivated vaccine, compared to the inactivated vaccine, not only increased antibody content but also improved the neutralizing antibody response level. Therefore, the vaccine is beneficial for improving the prevention and control of foot-and-mouth disease.

[0035] The following detailed description, in conjunction with embodiments, illustrates a broad-spectrum non-neutralizing antibody against foot-and-mouth disease virus provided by the present invention and its application in the preparation of reagents for enhancing the induction of neutralizing antibodies. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1

[0037] Preparation and identification of broad-spectrum reactive antibody SP1C6

[0038] 1) Animal Immunization: Three healthy 6-month-old Large White pigs were immunized with 2 mL of bivalent inactivated FMDV O and A strains (O / Mya98 / BY / 2010 and A / WH / 09 strains), with immunizations every 2 months for a total of 3 immunizations. Peripheral blood mononuclear cells were isolated 3-5 days after the third immunization, and O / MYA / 98 FMDV 146S antigen-specific single B cells were sorted using flow cytometry (BD Aria II, USA) for screening and preparation of broad-spectrum FMDV reactive antibodies. The primers used for nested RT-PCR amplification of the heavy chain in porcine single B cells were: outer-F: GTTCGGCTGAACTGGGTGGTC (SEQ ID NO:7), outer-R: GGTCACTGRCTCGGGGAAGTAGC (SEQ ID NO:8) and inner-F: GGTGGAGTSTGGRGGAGGCCTG (SEQ ID NO:9), inter-R: CAGGGGG CCAGAGGGTAGACC (SEQ ID NO:10); the primers used for amplification of the light chain were: outer-F: ATGGCCTGGAYCCCTCTCCTGCTC (SEQ ID NO:11), outer-R: CCTCCAGG TCACSGTCACG (SEQ ID NO:12) and inter-F: TCCTGTGAGCTGACTCAGCC (SEQ ID NO:13), inter-R: GTCACTTATTAGACACACCAGGGTG (SEQ ID NO:14). In the primer sequences above, S represents C or G, Y represents C or T, and R represents A or G. For specific antibody preparation methods, please refer to the description in application number CN201810929067.1, patent titled "Preparation Method of a Broad-Spectrum Neutralizing Antibody for Bovine Foot-and-Mouth Disease Virus of Type O," which yielded the SP1C6 monoclonal antibody.

[0039] The heavy chain variable region HV1 and light chain variable region LV1 sequences of the SP1C6 monoclonal antibody were obtained by sequencing analysis. Specifically, the amino acid sequence of the heavy chain variable region HV1 is shown in SEQ ID NO: 1; and the amino acid sequence of the light chain variable region LV1 is shown in SEQ ID NO: 2.

[0040] The coding sequences of the heavy chain variable region (SEQ ID NO:3) and light chain variable region (SEQ ID NO:4) of the SP1C6 monoclonal antibody were respectively coupled with the porcine IgG2 heavy chain (SEQ ID NO:5: GCCCCAAAGACTGCCCCTTCCGTGTACCCACTGGCTCCTT GCGGAAGG).

[0041] GATACTAGCGGCCCAAATGTCGCTCTGGGCTGTCTGGCCAGCAGCTACTTCCCAGAGCCAGTGACAATGACATGGAACAGCGGAGCTCTGACATCCGGCGTGCACACTTTTCCAAGCGTGCTGCAACCAAGCGGACTGTACTCTCTGAGCTCCATGGTGACAGTCCCAGCCAGCTCCCTCTCCAGCAAGAGCTACACATGCAACGTGAATCACCCAGCCACTACAACTAAGGTCGACAAGAGGGTGGGCACTAAGACTAAACCTCCATGTCCTATTTGCCCCGGCTGCGAAGTGGCCGGACCAAGCGTGTTTATTTTCCCACCTAAGCCAAAGGACACTCTCATGATCAGCCAGACACCAGAGGTCACTTGCGTCGTCGTCGACGTGTCCAAGGAACATGCCGAGGTCCAGTTTAGCTGGTATGTGGACGGCGTCGAAGTGCATACTGCTGAGACAAGGCCTAAGGAGGAGCAGTTTAACTCCACATATAGGGTCGTCTCCGTCCTCCCAATCCAGCATCAAGATTGGCTGAAGGGCAAAGAGTTCAAGTGCAAGGTGAACAACGTGGATCTGCCAGCCCCTATCACAAGGACAATCTCCAAGGCCATTGGCCAGTCTAGGGAGCCACAAGTGTACACTCTGCCACCACCAGCCGAGGAGCTGTCTAGGAGCAAGGTCACTGTGACATGTCTCGTCATCGGCTTCTACCCACCAGATATTCACGTCGAGTGGAAGTCCAACGGACAACCAGAACCAGAGGGCAATTATAGGACTACACCTCCACAGCAAGATGTGGACGGAACATTCTTTCTGTACAGCAAGCTCGCCGTCGACAAAGCTAGGTGGGACCATGGCGAAACATTCGAGTGTGCCGTGATGCACGAGGCTCTGCACAACCACTACACACAGAAGAGCATCTCCAAAACTCAAGGCAAG)(insert His tag at the C-terminus) and the constant region sequence of the light chain (SEQ ID NO:6: CAGCCAAAGGCTGCCCCTACAGTGAA

[0042] The plasmids (TCTCTTCCCTCCTAGCTCCGAAGAACTGGGCACTAACAAGGCCACCCTGGTGTGTCTCATCAGCGACTTCTACCCCGGCGCCGTGACTGTGACTTGGAAGGCTGGCGGAACTACTGTGACACAAGGCGTGGAGACTACTAAGCCTAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTATCTGGCTCTGTCCGCCAGCGATTGGAAGTCCAGCTCCGGATTCACTTGTCAAGTGACACACGAAGGAACTATCGTGGAGAAGACTGTGACTCCTTCCGAGTGTGCC) (with a His tag inserted at the C-terminus) were ligated and then inserted into the pcDNA3.4 eukaryotic expression vector. The heavy and light chain expression plasmids were mixed at a mass ratio of 2:3 and transfected into CHO suspension culture cells to obtain a complete IgG2 subtype antibody. The antibody was purified by affinity chromatography. For specific methods of antibody preparation, please refer to patent application number CN201810929067.1.

[0043] Preparation of single-chain antibody (SP1C6-SCFV): The coding sequences of the heavy chain variable region (SEQ ID NO:3) and the light chain variable region (SEQ ID NO:4) of the SP1C6 monoclonal antibody were linked by a linker peptide (GGGGSGGGGSGGGGS). A His tag sequence was inserted at the end of the sequence. The resulting fusion gene fragment was inserted into the eukaryotic expression vector pcDNA3.4 and expressed in CHO-S cells. The expressed fusion protein was purified by affinity chromatography.

[0044] 2) Indirect immunofluorescence (IFA) detection of antibody reactivity

[0045] BHK21 cells were inoculated into 24-well plates. When the cells reached 80% confluence, different strains of FMDV were seeded into the 24-well plates, with a normal cell control included. After incubation at 37°C for 4 hours, the supernatant was collected and inactivated. Cells were then fixed with a mixture of methanol and acetone pre-chilled at -20°C (methanol:acetone = 1:1, v / v) and incubated at room temperature for 20 minutes. Cells were washed three times with PBS, and purified SP1C6 monoclonal antibody was added at a concentration of 5 μg / ml. Cells were incubated at 37°C for 1 hour. After washing three times with PBS, fluorescein-labeled rabbit anti-porcine IgG at a working concentration (dilution 1:5000) was added, and cells were incubated at 37°C for 30 minutes. Cells were washed five times with PBS, and fluorescence signals were observed under a fluorescence microscope.

[0046] The results showed that SP1C6 monoclonal antibody reacted with representative strains of all topotypes / lineages of FMDV A and O types prevalent in my country, namely A / GDMM / CHA / 2013 (A / SEA / 97 / G2 genotype, SEA lineage), A / WH / CHA / 09 (A / SEA / 97 / G1 genotype), AF72 (A22 lineage), O / HN / CHA / 93 (Cathay topotype), O / MYA98 / BY / 2010 (SEA topotype), and O / XJ / CHA / 2017 (ME-SA IND2001 lineage), indicating that SP1C6 is a broad-spectrum reactive antibody against FMDV.

[0047] 3) Virus neutralization test (VNT) to detect antibody neutralizing activity

[0048] Serially dilute the antibody with cell maintenance medium. Add the diluted antibody to a 96-well cell culture plate, repeating each dilution twice, with 50 μl per well. Add 100 TCID to each well. 50 / 50 μl of FMDV strain, with TCID values ​​of 100, 10, 1 and 0.1 TCID. 50 50 μl of virus control was added, with each dilution performed in triplicate. After mixing the SP1C6 monoclonal antibody with the virus solution, the mixture was incubated at 37°C for 1 hour. Then, cells at a concentration of 1–2 × 10⁶ cells per well were added. 6 50 μl of BHK21 cells per ml were cultured in a 37°C incubator for 2–3 days. After fixation and staining, the antibody concentration (IC50) of 50% of cells without disease progression was observed. 50 ). Use 50 μg / mL IC. 50 The value is used as the critical value for neutralization, and >50 μg / mL is defined as non-neutralizing activity.

[0049] The results showed that the SP1C6 monoclonal antibody had no neutralizing effect on the prevalent FMDV A-type strains A / GDMM / CHA / 2013, A / WH / CHA / 09, A / AF72 and O-type strains O / HN / CHA / 93, O / MYA98 / BY / 2010, O / XJ / CHA / 2017 in China (IC50, 100% CERT ... 50 (>50μg / mL), indicating that SP1C6 is a non-neutralizing antibody.

[0050] 4) Western blotting and alanine scanning to identify antibody-recognized antigen epitopes.

[0051] Using E. coli, GST fusion proteins of VP1, VP2, or VP3 of the FMDV A / GDMM / CHA / 2013 strain were expressed (VP1+GST, VP2+GST, VP3+GST) or a truncated VP2 fusion protein was expressed (VP2+GST).1-15aa +GST, VP2 1-100aa +GST, VP2 1-165aa +GST, VP2 1-168aa +GST, VP2 1-171aa +GST, VP2 1-174aa +GST, VP2 1-177aa +GST, VP2 1-180aa +GST, VP2 1-183aa +GST, VP2 1-186aa +GST, VP2 1-192aa +GST, VP2 1-195aa +GST, VP2 1-198aa +GST, VP2 1-200aa +GST, VP2 25-100aa +GST, VP2 69-200aa +GST, VP2 165-200aa +GST, VP2 185-200aa +GST, and performed SDS-PAGE electrophoresis on the O-type (O / Mya / 98) and A-type (A / SEA / 97) FMDV 146S antigens. The separated protein bands were then transferred to a nitrocellulose (NC) membrane and blocked with TBST buffer containing 5% skim milk powder for 2 h. After washing, the SP1C6 monoclonal antibody was diluted to the working concentration (2 μg / ml) with TBST buffer containing 5% skim milk powder and incubated at room temperature for 2 h. After washing the membrane with TBST, HRP-labeled anti-His tag antibody (1:4000) was added and incubated at room temperature for 1 h. After washing the membrane with TBST, ECL chemiluminescent substrate was added and the membrane was exposed and imaged on a medium-pressure X-ray film in a dark room.

[0052] Western blotting results showed that SP1C6 monoclonal antibody recognizes epitopes on VP2 of FMDV type A and type O antigens, and does not react with truncated VP2 protein, but only with intact VP2. This indicates that the epitopes on VP2 recognized by SP1C6 are discontinuous and may be conformational epitopes.

[0053] To investigate the conformational epitopes of FMDV VP2, this embodiment also employed the alanine substitution method to scan the entire VP2 region. Specifically, the gene containing VP2 (A / GDMM / CHA / 2013 strain) (with a FLAG tag at the C-terminus) and its three alanine or monoalanine substitution sequences spanning the entire VP2 region were synthesized by GenScript (www.gscript.com) and ligated into the pcDNA3.4 vector. Each recombinant plasmid was transfected into a 24-well plate containing 2.5 ml of ExpiCHO-S cells per well using the ExpiFectamine CHO transfection kit (Invitrogen), following the kit instructions. Forty-eight hours post-transfection, cells were fixed and infiltrated with fixation and permeation buffer (BD Biosciences) for 20 min. After washing with PBS buffer, 1 × 10⁻⁶ cells were transferred to each sample. 6 Infected cells were first detected using the porcine monoclonal antibody SP1C6, and then incubated for 30 min at room temperature (20-25°C) with Alexa647 anti-Flag antibody (Sigma-Aldrich) and FITC rabbit anti-porcine IgG. After two washes, the stained cells were loaded into a flow cytometer (FC) (BD LSR Fortessa), and 10,000 Alexa647-positive cells were recorded. If there was no FITC-positive count in the Alexa647-positive cell sample transfected with the mutant plasmid, it indicated that the substituted amino acid on VP2 disrupted the binding to the test monoclonal antibody. The corresponding amino acid substitution on VP2 is considered a key amino acid of the conformational epitope.

[0054] Alanine scanning results showed that K63, H65, L66, F67 and D68 residues on the β-sheet and L81 residue on the C-sheet were clearly exposed on the FMDV capsid, which are key amino acids of the conformational epitopes recognized by SP1C6.

[0055] Example 2

[0056] The effect of broad-spectrum non-neutralizing antibody SP1C6 on the induction of neutralizing antibodies in pigs by FMDV inactivated vaccine.

[0057] 1) Vaccine preparation and animal immunization

[0058] FMDV A / WH / CHA / 09 was inactivated to prepare antigen (Ag), which was mixed with monoclonal antibody SP1C6 or a single-chain antibody of monoclonal antibody SP1C6 (SP1C6-SCFV) at a molar ratio of 1:60 and incubated at 37°C for 1 hour. The mixture was then emulsified with 201 adjuvant at a volume ratio of 1:1 to prepare an oil-adjuvanted vaccine. Simultaneously, the antigen and PBS were emulsified with 201 adjuvant at a 1:1 ratio and used as the vaccine control group and negative control group. Eighteen healthy 2-month-old FMDV-unvaccinated large white pigs were divided into four groups. The immunization groupings and immunization doses are shown in Table 1. A booster immunization with the same dose was given 28 days after the primary immunization. Blood samples were collected on day 0 (0 dpv), day 7 (7 dpv), day 14 (14 dpv), day 21 (21 dpv), day 28 (28 dpv), day 35 (35 dpv), day 42 (42 dpv), day 49 (49 dpv), and day 56 (56 dpv) post-immunization to separate serum for neutralizing antibody detection.

[0059] Table 1. Animal grouping and immunization dosage

[0060]

[0061] Note: Ag is inactivated FMDV A / WH / CHA / 09, SP1C6-SCFV is a single-chain antibody, and SP1C6 is an intact antibody.

[0062] 2) FMDV Type A Neutralizing Antibody Competitive ELISA Detection Kit for Neutralizing Antibody Titer

[0063] The immunized swine serum samples were tested using the FMDV type A neutralizing antibody competitive ELISA kit (NAC-ELISA) (ZL202011285447.X). The procedure was performed according to the kit instructions; the specific experimental steps are as follows:

[0064] A) Diluting serum

[0065] Add serum diluent (containing 2% goat serum and 1% bovine serum albumin PBST) to a U-shaped 96-well serum dilution plate, 90 μl / well in the first row and 60 μl / well in the remaining rows. Then add the serum sample to be tested and the negative and positive control serum to the first row, 30 μl / well. Serial dilution (1:4~1:512) is performed until the final volume is 60 μl / well. The negative control is diluted in only 4 wells, and 4 wells are set up as 146S antigen control, i.e. no serum is added.

[0066] B) Add biotin-labeled monoclonal antibody

[0067] Dilute 100× biotinylated monoclonal antibody SP1C6 to the working concentration at a ratio of 1:100 using serum diluent, and add 60 μl / well to the U-shaped 96-well serum dilution plate described in step A). ​​After adding an equal volume of biotinylated monoclonal antibody, the serum dilution ratio is doubled. Gently vortex or pipette mix when transferring to the reaction plate.

[0068] C) Transfer to reaction plate

[0069] Transfer the mixture from the U-shaped 96-well serum dilution plate described in step B to the 146S antigen-captured reaction plate, 100 μl / well. Seal the plate and incubate at 37°C for 1 h. Carefully remove the sealing film, wash the plate 5 times with washing buffer, 300 μl / well, and pat dry for the last time.

[0070] D) Add enzyme-labeled avidin

[0071] Dilute 100× enzyme-labeled avidin to the working concentration at a ratio of 1:100 using serum diluent, 100 μl / well, seal the plate, and incubate at 37°C for 15 min. Carefully remove the sealing film, wash the plate 5 times with washing buffer, 300 μl / well, and pat dry after the last wash.

[0072] E) Add substrate solution

[0073] Add 100 μl of TMB substrate solution per well, seal the plate, and incubate at 37°C in the dark for 10–15 min.

[0074] F) Termination of reaction

[0075] After color development, add stop solution, 100 μl / well, and read the OD on a microplate reader. 450nm value.

[0076] G) Calculation of antibody titer in serum samples

[0077] 146S antigen control (4 wells), discard the highest and lowest OD values. 450nm Value, calculate the average OD of the remaining 2 wells. 450nm The value, divided by 2, is the critical value, representing the control OD that inhibits the response by 50%. 450nm Value. Detection well OD 450nm A value greater than the threshold indicates a negative well, and a value less than or equal to the threshold indicates a positive well. The highest dilution factor of the positive wells in the tested sample is used as the antibody titer of that serum sample.

[0078] NAC-ELISA test results ( Figure 1 The results showed that, starting 28 days after the first immunization, the antibody levels in the Ag+SP1C6-SCFV and Ag+SP1C6 immunization groups were higher than those in the Ag immunization group.

[0079] 3) Virus micro-neutralization test (VNT)

[0080] Virus neutralization experiments were performed on immunized serum samples using the FMDV A / WH / CHA / 09 strain. The specific experimental steps are as follows:

[0081] A) Add 50 μL of the serum sample to be tested to each well and serially dilute it with the inoculation solution in a 96-well plate. Then, add 50 μL of a solution containing 100 TCID to each well. 50 The FMDV was applied at 37°C for 1 hour. Simultaneously, TCID values ​​of 0.1, 1, 10, and 100 were set. 50 Control wells (without serum sample).

[0082] B) Add 50 μL of 1×10 to each well. 6 Complete culture medium for BHK21 cells was placed in an incubator at 37°C with 5% CO2 for 48 hours.

[0083] C) Discard the supernatant cell solution, add pre-cooled fixative (methanol:acetone = 1:1), and fix at -20℃ for 20 min.

[0084] D) Discard the fixative and add 100 μL of crystal violet solution to each well for staining. After 30 min, wash the 96-well plate and observe the maximum dilution of serum with 50% non-pathogenic cells. The virus neutralizing antibody titer is represented by log10, the reciprocal of the maximum serum dilution.

[0085] VNT test results ( Figure 2 The results showed that, starting from day 28 after the first immunization, the antibody levels in the Ag+SP1C6-SCFV and Ag+SP1C6 immunization groups were higher than those in the Ag immunization group. The results of NAC-ELISA and VNT detection were completely consistent, indicating that blocking the non-neutralizing epitopes of the FMDV A / WH / CHA / 09 strain with the broad-spectrum non-neutralizing antibody SP1C6 can improve the level of neutralizing antibody response.

[0086] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A broad spectrum non-neutralizing antibody to foot-and-mouth disease virus, characterized in that, 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. A single chain antibody to a broad spectrum non-neutralizing antibody to foot-and-mouth disease virus, characterized in that, It is prepared from the heavy chain variable region and light chain variable region of the foot-and-mouth disease virus broad-spectrum non-neutralizing antibody as described in claim 1; The method for preparing the single-chain antibody involves linking the coding sequences of the heavy chain variable region and the light chain variable region of the foot-and-mouth disease virus broad-spectrum non-neutralizing antibody with the sequence encoding a linker peptide, inserting the linker peptide into the eukaryotic expression vector pcDNA3.4, expressing the linker peptide in CHO-S cells, and then separating and purifying the resulting fusion protein.

3. The use of the broad-spectrum non-neutralizing antibody against foot-and-mouth disease virus according to claim 1 or the single-chain antibody according to claim 2 in the preparation of a vaccine that enhances the production of neutralizing antibodies induced by foot-and-mouth disease virus, wherein the foot-and-mouth disease virus that induces the production of neutralizing antibodies is type O foot-and-mouth disease virus and / or type A foot-and-mouth disease virus.

4. Use according to claim 3, characterized in that, The type A foot-and-mouth disease virus includes the SEA lineage and / or the A22 lineage.

5. Use according to claim 3, characterized in that, The O-type foot-and-mouth disease virus includes at least one of the following lineages: Cathay topology, SEA topology, and ME-SA topology IND2001 lineage.

6. A vaccine that enhances the induction of neutralizing antibodies, characterized in that, Includes foot-and-mouth disease virus, adjuvant, and the broad-spectrum non-neutralizing antibody against foot-and-mouth disease virus as described in claim 1 or the single-chain antibody as described in claim 2.

7. The vaccine according to claim 6, characterized in that, The vaccine in question is an inactivated foot-and-mouth disease virus vaccine. The foot-and-mouth disease virus includes type O foot-and-mouth disease virus and / or type A foot-and-mouth disease virus.

8. The vaccine of claim 7, wherein the vaccine is characterized by, The type A foot-and-mouth disease virus includes the SEA lineage and / or the A22 lineage.

9. The vaccine according to claim 7, characterized in that, The O-type foot-and-mouth disease virus includes at least one of the following lineages: Cathay topology, SEA topology, and ME-SA topology IND2001 lineage.

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