Development of Foot-and-Mouth Disease Virus-Neutralizing Monoclonal Antibodies Derived From Plasmablasts of Infected Cattle and Their Germline Gene Usage

Figure 1. Workflow of cattle mAb production using singe B cell antibody techniques. Cattle were sequentially immunized with different FMDV serotype O strains, and then peripheral blood was collected to isolate PBMCs. The isolated PBMCs were stained with a panel of mouse anti-cattle CD21, anti-cattle IgM, and biotinylated antigen, followed by incubation with APC labeled anti-biotin secondary antibody. The single antigen-specific plasmablast was sorted into 96 well-plate with lysis buffer and immediately reverse-transcribed into cDNA. The paired VH and VL sequences was respectively amplified by nested PCR and then sequenced. The variable regions were codon optimized and cloned into pcDNA3.4 expression cassette containing cattle IgG constant region coding sequences. The recombinant mAbs were expressed in CHO-S and purified by affinity and further molecular sieve chromatography.

Figure 2. Cattle IgG antibody titers against FMDV in sera, and the virus-specific plasmablast-derived mAbs from 3 infected cattle. (A) A total of 3 cattle (#1217, #1234, and #0005) were sequentially infected with O/Mya/98, O/HN/CHA/93, and O/Tibet/99 strains at days 0, 35, and 132. Sera were pooled at days 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 132, 169, 223, and 225 after first infection. IgG titres were expressed as the reciprocal log10 of serum dilutions that yielded 50% of the absorbance value of the negative control wells in LPB-ELISA. IgG titres less than the sensitivity of the assay (0.9) were adjusted to 0.6 in the figure. (B) IgG plasmablasts from the 3 cattle were collected to produce mAbs, which were specific for FMDV in 23 out of 26 cells from #2334, 27 out of 37 cells from #1217, and 5 out of 6 cells from #0005.

Figure 3. Confirmation of the phenotype and proportion of FMDV-specific plasmablasts in the peripheral blood by flow cytometry. Cattle PBMCs (A) were analyzed by three-color flow cytometry and gate 1 (B) was selected to exclude cells debris with lower values of SSC-A and FSC-A, and further analyzed to gate singlets (C) based on diagonal streak of the FSC-A and FSC-H plot. The IgM− B cells were gated to check the distribution of CD21 and FMDV-specific cells, in the presence (D) or absence (F) of FMDV antigen. Similarly, IgM+ B cells were gated to check the distribution of CD21 and FMDV-specific cells, in the presence (E) or absence (G) of FMDV antigen. One million PBMCs were collected per sample. The distribution and phenotype of FMDV-specific plasmablasts in the IgM+ B cells (H) and the IgM− B cell (I) population were analyzed. The differences between two groups were marked with asterisks in t-test; wherein the alpha values were labeled as NS (P > 0.05), *P < 0.05 and **P < 0.01.

Figure 4. Characterization of FMDV-specific mAbs. The reactivity of cattle mAbs were checked by IFA, indirect ELISA and WB. The IFA (A) was performed using BHK cells infected with the O/Mya/98 strain, and the working concentration of the tested cattle mAbs was 5 μg/ml, followed by incubation with goat anti-cattle FITC (diluted 1:5,000 in PBS). The indirect ELISA (B) were carried out by incubation with different concentrations of 0–20 μg/ml of the tested mAbs with FMDV 146S antigen, followed by probing with anti-His HRP (diluted 1:6,000 in PBS). The lysis of 146S antigen (C) and GST-fused antigens (D) of FMDV were subjected to 12% SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane. The membrane was incubated with tested mAbs and then probed with a 1:5,000 dilution of HRP-conjugated goat anti-cattle IgG (Sigma-Aldrich, USA). (E) B66 stained with GST-C antigen and showed the binding with VP1 C-termial of FMDV.

Figure 5. Evaluation of the cattle-derived intra-type bnAbs-based immunodominant antigen sites of FMDV serotype O using a blocking ELISA. The 13 intra-type bnAbs were respectively coated on a 96-well plate overnight, and 20 serum samples consisting of 10 from vaccinated cattle and 10 from experimentally infected cattle were incubated with variable diluted titers of FMDV 146S antigen. The result corresponding to each tested mAb was expressed as the serum dilution that yielded 50% of the absorbance OD450 value of the PBS control wells.

Figure 6. The CDRs and germline gene usage of FMDV-specific mAbs. Of the 55 FMDV-specific mAbs, the length of the VH and VL sequences (A) and the six CDRs (B), as well as the frequency of the different length of HCDR3 (C) were analyzed. The HCDR3 length between the neutralizing and non-neutralizing FMDV-specific antibody clones (D) was examined by the Mann-Whitney test (one-tailed, P = 0.0419; *P < 0.05). Data were presented as the mean ± standard error. To determine the individual gene segments employed by V-D-J rearrangements, the VH and VL sequences were separately aligned with bovine reference germline gene segments using the online IMGT/V-QUEST program. The counts of different gene segments or recombinations were displayed for VH (E), DH (F), JH (G), Vλ (I), and Jλ (J) gene segments, VH1-7*02 containing VH-DH-JH recombinations (H) and Vλ-Jλ (K) recombinations of the neutralizing and non-neutralizing FMDV-specific antibody clones.

Figure 7. The pairings involved in the Vλ1-47*01 (or 1-52*01) containing IgG antibodies as well as the VH1-7*02 containing IgG antibodies. The Vλ gene segments (A) in the IgG antibodies pairing with VH1-7*02 (n=18) were all (100%) Vλ1-47*01 (or 1-52*01). Inversely, the proportion of VH gene segments (B) in the IgG antibodies pairing with Vλ 1-47*01 (or 1-52*01) (n = 18) was 78% for VH1-7*02, 11% for VH1-10*01/2, 6% for VH1-30*01/2 and 6% for VH1-21*01 (VH1-33*01).