Broadly neutralizing swine monoclonal antibody against foot-and-mouth disease virus type a and application thereof

Abstract

The application discloses a broad-spectrum neutralizing pig-derived monoclonal antibody of type A foot-and-mouth disease virus (FMDV) and application thereof. The antibody PAF56 has an amino acid sequence of a heavy chain variable region as shown in SEQ ID No. 12, a heavy chain CDR1 sequence of GFGFSNTY, a heavy chain CDR2 sequence of VFTGGGAT, and a heavy chain CDR3 sequence of VSNRDGFGGVGNV. The antibody PAF56 has an amino acid sequence of a light chain variable region as shown in SEQ ID No. 13, a light chain CDR1 sequence of SGSVTSLNY, a light chain CDR2 sequence of RTN, and a light chain CDR3 sequence of ALYKGSGTDTNI. The antibody PAF56 can simultaneously neutralize an AF72 strain of an A22 lineage and A / WH / CHA / 09 and A / GDMM / 2013 epidemic strains of a domestic SEA97 lineage, and has broad-spectrum neutralization activity of type A FMDV. The antibody PAF56 recognizes an antigen site 2 of a VP2 B-C loop, and a key amino acid is VP2 D72; the recognized site is highly conserved in type A FMDV. The application first obtains a broad-spectrum neutralizing antibody of type A specific FMDV from a natural host and identifies a key antigen site recognized by the antibody, thereby providing a powerful tool for antigen structure analysis of FMDV and establishment of a serological detection method.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and relates to a porcine-derived monoclonal antibody with broad-spectrum neutralization against type A foot-and-mouth disease virus and application thereof. BACKGROUND

[0002] Foot-and-mouth disease (FMD) is a highly contagious disease that affects cattle, pigs and sheep, and limits the development of international trade in animals and animal products, and seriously affects the breeding industry of various countries. The pathogenic agent, foot-and-mouth disease virus (FMDV), has seven serotypes, A, O, C, Asia1, SAT1, 2 and 3, among which the antigenic structure of type A FMDV varies the most, and it is therefore more difficult to obtain broad-spectrum neutralizing antibodies. This brings difficulties to the diagnosis of FMDV by using immune detection technology, and it is impossible to achieve the purpose of preventing and controlling foot-and-mouth disease by using broad-spectrum neutralizing antibodies. In the present application, a high-throughput method for establishing a single B cell antibody library is used to screen a porcine-derived single B cell antibody with broad-spectrum neutralization activity against type A FMDV. SUMMARY

[0003] The application aims to provide a porcine-derived monoclonal antibody with broad-spectrum neutralization against type A foot-and-mouth disease virus and application thereof.

[0004] The application provides a porcine-derived monoclonal antibody with broad-spectrum neutralization against type A foot-and-mouth disease virus, which comprises a heavy chain variable region CDR1, CDR2 and CDR3 and a light chain variable region CDR1, CDR2 and CDR3.

[0005] The amino acid sequence of the heavy chain variable region CDR1 is GFGFSNTY (SEQ ID No. 1).

[0006] The amino acid sequence of the heavy chain variable region CDR2 is VFTGGGAT (SEQ ID No. 2).

[0007] The amino acid sequence of the heavy chain variable region CDR3 is VSNRDGFGGVGNV (SEQ ID No. 3).

[0008] The amino acid sequence of the light chain variable region CDR1 is SGSVTSLNY (SEQ ID No. 4).

[0009] The amino acid sequence of the light chain variable region CDR2 is RTN.

[0010] The amino acid sequence of the light chain variable region CDR3 is ALYKGSGTDTNI (SEQ ID No. 5).

[0011] The antibody PAF56 also comprises a heavy chain variable region FR1, FR2, FR3 framework region and a light chain variable region FR1, FR2, FR3 framework region;

[0012] Amino acid sequence of the heavy chain variable region FR1: EEKLVESGGGLVQPGGSLSLSCVGS (SEQ ID No. 6)

[0013] Amino acid sequence of the heavy chain variable region FR2: MTWVRQAPGKGLEWLAS (SEQ ID No. 7)

[0014] Amino acid sequence of the heavy chain variable region FR3:

[0015] YASSVKGRFTISRDNSQNTAYLQMNSLRTEDTARYYCA (SEQ ID No. 8)

[0016] Amino acid sequence of the light chain variable region FR1: QTVIQEPAMSVSLGGTVTLSCAFS (SEQ ID No. 9)

[0017] Amino acid sequence of the light chain variable region FR2: PSWFQQTPGQPPRTMVY (SEQ ID No. 10)

[0018] Amino acid sequence of the light chain variable region FR3:

[0019] SRPAGVPSRFSGAISGNKATLTITGAQAEDEADYFC (SEQ ID No. 11).

[0020] Amino acid sequence of the heavy chain variable region of the antibody PAF56: EEKLVESGGGLVQPGGSLRLSCVGSGFGFSNTYINWVRQAPGKGLEWLAAVFTGGGATGYAGSVKGRFTISSDNSQNTAYLQMNSLITEDTARYYCVSNRDGFGGVGNVWGPGVEVVVSS (SEQ ID No. 12)

[0021] Amino acid sequence of the light chain variable region of the antibody PAF56:

[0022] QTVIQEPAMSVSLGGTVTLSCAFSSGSVTSLNYPSWFQQTPGQPPRTMVYRTNSRPAGVPSRFSGAISGNKATLTITGAQAEDEADYFCALYKGSGTDTNIFGRGTHLTVL (SEQ ID No. 13).

[0023] The application also provides application of the porcine-derived monoclonal antibody PAF56 with broad-spectrum neutralization to type A foot-and-mouth disease virus in preparation of a medicine for preventing or treating foot-and-mouth disease caused by type A foot-and-mouth disease virus, and in preparation of a diagnostic reagent for type A foot-and-mouth disease virus.

[0024] The antibody PAF56 provided by the application can simultaneously neutralize the AF72 strain of the A22 lineage, and the A / WH / CHA / 09 and A / GDMM / 2013 epidemic strains of the domestic SEA97 lineage, and has broad-spectrum neutralization activity to type A FMDV. PAF56 recognizes the antigen site 2 of the VP2 B-C loop, and the key amino acid is VP2 D72, and the recognized site is highly conserved in type A FMDV. PAF56 can bind to the three antigens of A / AF72, A / WH / CHA / 09 and A / GDMM / 2013, and has high affinity, and the affinities are 8.89 nM, 6.20 nM and 8.11 nM, respectively. The application first obtains a natural host-derived broad-spectrum neutralizing antibody specific to type A FMDV and identifies the key antigen site recognized thereby, and provides a powerful tool for antigen structure analysis of FMDV and establishment of a serological detection method. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Distribution of SHM of four types of isotype antibodies in the antibody library;

[0026] Figure 2 Thermogram of V-J genes of heavy chains and light chains;

[0027] Figure 3 SDS-PAGE detection of expression and purification of the antibody PAF56;

[0028] Figure 4 Indirect immunofluorescence detection of the reactivity of PAF56 with FMDV;

[0029] Figure 5 Indirect ELISA detection of the reactivity of PAF56 with FMDV antigens;

[0030] Figure 6 WB detection of the antigen epitope type recognized by the antibody PAF56 with FMDV;

[0031] Figure 7 Key amino acid pattern diagram of the recognition site of the antibody PAF56;

[0032] Figure 8 BLI detection of the affinity of the antibody PAF56 with three type A antigens. DETAILED DESCRIPTION

[0033] The application will be further described below through a specific embodiment.

[0034] BHK-21 cells, HEK293F cells, FMDV type A strains (A / AF72, A / WH / CHA / 09, A / GDMM / 2013, A / AF72) were preserved by the National Foot-and-Mouth Disease Reference Laboratory, and the pVH-pcDNA3.4 containing the porcine IgG H chain gene and the pVL-pcDNA3.4 containing the porcine IgG λ chain gene were constructed and preserved by the Host Anti-Virus Infection and Immunobiology Team of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0035] Histopaque-1077, 1.077 g / mL, FITC Rat anti mouse IgG antibody were purchased from Sigma-Aldrich. Biotin labeling kit (EZ-Link™ Sulfo-NHS-LC-Biotin), Polyethylenimine, Linear, MW 25000, Transfection Grade (PEI25K™) were purchased from Polysciences. One-step RT-PCR amplification kit (HiScipt II One Step RT-PCR Kit) was purchased from Nanjing Novi Biological Technology Co., Ltd. Mouse anti-porcine IgG-FITC antibody was purchased from Mybiosource. Horseradish peroxidase (HRP) labeled mouse anti-porcine tag antibody was purchased from Nanjing Kingsriver Biological Technology Co., Ltd. Endotoxin-free plasmid extraction kit was purchased from Tiangeng Biochemical Technology Co., Ltd.

[0036] Example 1 Preparation of porcine-derived monoclonal antibody with intra-type A foot-and-mouth disease virus broad-spectrum neutralization

[0037] 1. Animal immunization and establishment of porcine FMDV-specific antibody library

[0038] A healthy piglet (3 months old) with negative foot-and-mouth disease antibody was selected, and the neck muscle was injected with O / HN / CHA / 93 inactivated vaccine at a dose of 5 µg / head; after 28 days, the same dose of O / HN / CHA / 93 inactivated vaccine was used for immunization again; after 129 days of interval between the second immunization, O type (O / 18074 and O / xizang / CN / 99) and A type (A / AF72 and A / WH / CHA / 09) inactivated vaccine were used for immunization, 5 µg / head for each antigen.

[0039] Five days after inactivated vaccine immunization, 250 mL of animal anti-coagulation blood was collected, and PBMCs were isolated according to the instruction of lymphocyte separation medium. 3 mL of 1640 medium was used to resuspend PBMCs in a centrifuge tube, and mouse anti-pig antibodies (IgM, CD14, CD3, CD335) were added and incubated on ice for 30 min; after washing twice, Goat Anti-Mouse IgG MicroBeads were added and incubated on ice for 30 min; after negative screening by LD column, the enriched B cells were divided into two sample tubes and one control tube, 5 μL of FITC Rat antimouse IgG was added to the three tubes, the sample tubes were added with biotin-labeled A / AF72 146S antigen, and incubated on ice for 20 min; finally, Anti-Biotin APC was added, and the cells were washed after incubation on ice for 20 min; the cells were resuspended in 1640 medium, and A-FMDV-Biotin specifically bound to B cells. The enriched A / AF72-specific B cells were resuspended in PBS, and immediately used for single-cell capture and antibody library construction by Chromium Next GEM Single Cell 5' Kit v2 (10x Genomics) (PN-1000263).

[0040] Five days after the booster immunization of the inactivated vaccine type A, the animal anti-coagulation blood was collected to isolate PBMCs, and after enrichment of B cells, staining and flow sorting were performed to sort all antigen-specific B cells into flow tubes, and the sequencing of antibody variable region genes was performed. The pig-derived A-type FMDV-specific antibody library was successfully obtained by high-throughput sequencing. The antibody library capacity is shown in Table 1, and 8883 pairs of sequences of A / AF72-specific antibody library were obtained, including IgG, IgA, IgD, IgE, of which 8060 pairs of IgG accounted for 90%, as shown in Table 1. The somatic hypermutation rate SHM of IgA and IgE was 0.10, and the SHM of IgD and IgG was 0.07, as shown in Table 2. Figure 1 The usage of V and J genes of light chain and heavy chain is shown in Table 3. Figure 2

[0041]

[0042] 2 Expression plasmid construction and antibody expression and purification

[0043] ​One strain of IgG was screened from them, named PAF56. The variable region gene sequences of the heavy and light chains of the antibody were codon-optimized (using Cricetulus griseus) by GenScript Incorporation (www.genscript.com) in reference to CHO-S cells, with a signal peptide added at the front, in reference to the literature

Wei Delong. Development of PRRSV-specific monoclonal antibodies based on single B cell antibody technology[D]. Chinese Academy of Agricultural Sciences, 2021. DOI:10.27630 / d.cnki.gznky.2020.000282.

Li K, Zhu G, Zhou S, et al. Isolation and characterization of porcine monoclonal antibodies revealed two distinct serotype-independent epitopes on VP2 of foot-and-mouth disease virus[J]. J Gen Virol. 2021;102(7):10.1099 / jgv.0.001608

[0044] The antibody PAF56 plasmid was expressed by HEK293F cells, affinity chromatography purified, and the expressed antibody was verified by SDS-PAGE. The results showed that the porcine IgG molecule was successfully expressed, with a heavy chain molecular weight of about 50 KDa and a light chain molecular weight of about 25 KDa Figure 3 .

[0045]

[0046] Example 2 Indirect immunofluorescence test IFA detection of antibody reactivity

[0047] A / AF72 virus liquid was inoculated into BHK-21 cells in a 24-well cell culture plate at a dose of MOI = 1, and a blank control was set up. After 3 h ~ 4 h of incubation at 37 ℃, pre-cooled fixing solution (methanol: acetone = 1:1) was added and fixed at room temperature for 30 min, and then indirect immunofluorescence detection was performed according to the conventional method. The primary antibody was the expressed antibody (5 µg / mL), and the secondary antibody was FITC-labeled rabbit anti-swine IgG.

[0048] The IFA detection results showed that the antibody PAF56 could bind to BHK cells infected with A / AF72 virus, and specific green fluorescence appeared Figure 4 ) was the A type FMDV reactive antibody.

[0049] Example 3 Indirect ELISA detection of antibody reactivity

[0050] A / AF72 inactivated antigen was added to the enzyme-labeled plate at a dose of 100 ng / well, and coated on the enzyme-labeled plate at 4 ℃ overnight; the antibody to be tested was diluted to 20 µg / mL, and 10 gradient dilutions were added to the enzyme-labeled plate at a dilution ratio of 2 times, with PBS as a negative control, and incubated at 37 ℃ for 1 h. 100 µL of rabbit anti-swine IgG-HRP antibody (1:5000 dilution) was added to each well, and incubated at 37 ℃ for 30 min. TMB substrate was added for color development at 37 ℃ for 15 min, and after the reaction was terminated, the OD 450 nm wavelength absorbance was detected by an enzyme-labeled instrument. When the antibody concentration was diluted to 5 µg / mL, P / N ≥ 2.1 was determined as positive (P is the average value of the OD 450 nm values of 2 repeated wells at each dilution of the antibody to be tested, and N is the average value of the OD 450 nm values of all wells of the PBS control group).

[0051] The indirect ELISA results showed that when the antibody concentration was diluted to 5 µg / mL and above, the OD 450 nm value of the antibody PAF56 was higher than the Cut Off value Figure 5 ), and the Cut Off value was C.O (positive determination value), C.O = 2.1 × N (N is the OD 450 nm value of the negative control), which could specifically react with A / AF72 antigen.

[0052] Example 4 Neutralization test detection of antibody neutralization activity

[0053] The antibody to be tested was diluted in a 96-well plate at a dilution ratio of 2 times, 8 wells; 50 µL of virus diluent (titer of 200 TCID 50 / 0.1 mL, and cell control and virus back control were set up. After 1 h incubation at 37 °C, 100 μL of BHK-21 cell suspension (density 1 x 10 6 / mL) was added to each well. After 48 h ~ 72 h incubation, CPE was observed, and the neutralization titer of serum or antibody was calculated by Reed-Muench method, and the neutralization potency of antibody (IC50 = antibody concentration / reciprocal of neutralization titer) was calculated. If IC50≤50 μg / mL, it was determined as neutralizing antibody.

[0054] The neutralization potency of antibody PAF56 was determined by virus neutralization test. The results showed that antibody PAF56 could neutralize three strains of type A FMDV (A / AF72, A / WH / CHA / 09 and A / GDMM / 2013), but could not neutralize O / 18074 and O / ZK93 strains of type O FMDV (Table 2). This result indicated that PAF56 was a type A FMDV intra-type broad-spectrum neutralizing antibody.

[0055]

[0056] Example 5 Western-blot analysis of antigen epitope recognized by antibody

[0057] The purified A / AF72 146S antigen or GST-fused structural protein VP1-3 was subjected to SDS-PAGE, and then the protein was transferred to PVDF membrane for Western blot verification. The primary antibody was pig-derived monoclonal antibody PAF56 (working concentration 2 μg / mL), incubated at room temperature for 2 h; the secondary antibody was Anti-pig-IgG-HRP antibody, incubated at room temperature for 1 h, and then exposed using an exposure instrument. The bin control of GST-fused VP1-3 monomer protein was exposed after incubation with Anti-GST-HRP at room temperature for 1 h, see Figure 6 A or 6C. The results showed that PAF56 could react with whole antigen A / AF72, but could not react with GST-fused monomer structural protein, indicating that PAF56 recognized a conformation-dependent linear epitope in AF72 antigen. Figure 6 Figure 6 .

[0058] Example 6 Screening of antigen mutation sites for antibody escape

[0059] A / AF72 was diluted by 10-fold gradient to 10 -6 μL of each dilution of virus was taken in a 96-well plate, and an equal volume of neutralizing antibody with a concentration of 5 μg / mL was added, and virus control wells without antibody were set up; 100 μL of BHK-21 cell suspension (concentration 10 5 ​BHK-21 cells at 1 x 106cells / ml were mixed and incubated at 37 °C for 2-3 days. The maximum dilution virus from 8-well was taken and the antibody concentration was increased in 24-well plate for continuous passage. The virus RNA was extracted from the escape strain of the 5th passage, and the FMDV structural protein gene was amplified and sent to the company for sequencing. By comparing the amino acid differences between the escape strain and the parent strain, the key amino acids recognized by the neutralizing antibody to the virus epitope were found.

[0060] By screening and comparing the amino acid differences between the neutralizing antibody escape mutant and the parent strain of the structural protein, the key amino acids recognized by the antibody were identified. The results (Table 3) showed that the key amino acid recognized by the antibody PAF56 was VP2 72 (D), which was highly conserved in the sequence of A-type FMDV, as shown in Figure 7

[0061]

[0062] Example 7 Biofilm interference assay of antigen-antibody affinity

[0063] Biotinylated A / WH / CHA / 09, A / AF72 and A / GDMM / 2013 antigens were diluted to 5 μg / mL, and antibody PAF56 was diluted to 100 nM. The sample was added according to the pre-designed plate diagram, 200 μL per well. The program was set: equilibration probe (60 s), solidification of antigen (120 s), twice equilibration probe (60 s each), binding of antibody (300 s) and dissociation of antibody (180 s). The kinetic constant of antigen-antibody binding was calculated, and the binding kinetic curve was fitted, as shown in Figure 8 The results showed that the antibody PAF56 could bind to A / AF72, A / WH / CHA / 09 and A / GDMM / 2013 antigens, and the affinities were 8.89 nM, 6.20 nM and 8.11 nM, respectively.​

Claims

1. A broadly neutralizing porcine monoclonal antibody PAF56 against foot-and-mouth disease virus type A, characterized by: The antibody PAF56 comprises heavy chain variable regions CDR1, CDR2, CDR3 and light chain variable regions CDR1, CDR2, CDR3; Amino acid sequence of heavy chain variable region CDR1: GFGFSNTY Amino acid sequence of heavy chain variable region CDR2: VFTGGGAT The amino acid sequence of the heavy chain variable region CDR3 is: VSNRDGFGGVGNV Amino acid sequence of light chain variable region CDR1: SGSVTSLNY Amino acid sequence of light chain variable region CDR2: RTN The amino acid sequence of the light chain variable region CDR3 is: ALYKGSGTDTNI.

2. The broadly neutralizing porcine monoclonal antibody PAF56 against foot-and-mouth disease virus type A according to claim 1, characterized in that: The antibody PAF56 further comprises heavy chain variable region FR1, FR2, FR3 framework regions and light chain variable region FR1, FR2, FR3 framework regions; Amino acid sequence of the heavy chain variable region FR1: EEKLVESGGGLVQPGGSLSLSCVGS Amino acid sequence of heavy chain variable region FR2: MTWVRQAPGKGLEWLAS The amino acid sequence of the heavy chain variable region FR3 is: YASSVKGRFTISRDNSQNTAYLQMNSLRTEDTARYYCA The amino acid sequence of the light chain variable region FR1 is: QTVIQEPAMSVSLGGTVTLSCAFS The amino acid sequence of the light chain variable region FR2 is: PSWFQQTPGQPPRTMVY The amino acid sequence of the light chain variable region FR3 is: SRPAGVPSRFSGAISGNKATLTITGAQAEDEADYFC.

3. The broadly neutralizing porcine monoclonal antibody PAF56 against foot-and-mouth disease virus type A according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody PAF56 is shown in SEQ ID No. 12, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

13.

4. Use of the broadly neutralizing porcine monoclonal antibody PAF56 against foot-and-mouth disease virus type A according to any one of claims 1 to 3 in the preparation of a medicament for preventing or treating foot-and-mouth disease caused by foot-and-mouth disease virus type A.

5. Use of the broadly neutralizing porcine monoclonal antibody PAF56 against foot-and-mouth disease virus type A according to any one of claims 1 to 3 in the preparation of a detection or diagnostic reagent for foot-and-mouth disease virus type A.

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