Conserved antigen structures and antibody-driven variations on foot-and-mouth disease virus serotype A revealed by bovine neutralizing monoclonal antibodies

Fig 1. Sorting and proportion of FMDV serotype A-specific B cells via different bait antigens by flow cytometry. Bovine PBMCs (A) were analyzed by 3-color flow cytometry and the gate P1 (B) was selected to exclude cells debris with lower values of SSC-A and FSC-A, and further analyzed singlets in gate P2 (C) based on diagonal streak of the FSC-A and FSC-H plot. The class-switched (IgM-) B cells in gate P3 were used to check the distribution of CD21 and FMDV-specific cells, in the presence of FMDV bait antigen A/GDMM/2013 (D) and A/AF72 (E), respectively. The demarcation line between FMDV-specific binding and nonbinding B cells was set according to the FMO control (F) in absence of bait antigen. The CD21+/- Antigen+ B cells in gate P4 were separately sorted for preparation of bovine mAbs. Appropriately one million bovine PBMCs were collected to analyze the distribution proportion of A/GDMM/2013-binding (G) and A/AF72-binding (H) B cells in circulating peripheral blood.

Fig 2. Cryo-EM structures of the FMDV-AWH-W125 complex and FMDV-AWH-W2 complex. The central cross-sections through cryo-EM maps of the FMDV-AWH-W125 complex (A) and FMDV-AWH-W2 complex (D) are shown with icosahedral 2-, 3- and 5-fold axes. Each image in the 480-pixel boxes corresponds to 446 Å in each dimension. Scale bars, 100 Å. Rendered images of the FMDV-AWH-W125 complex (B) and FMDV-AWH-W2 complex (E). Depth cueing with color is used to indicate the radius (< 120 Å, blue; 130–150 Å, from cyan to yellow; > 160 Å, red). The icosahedral five- and threefold axes are represented by pentagons and triangles, respectively. Footprints of W125 (C) and W2 (F) on the FMDV surface. Each figure shows a 2D projection of the FMDV surface produced using RIVEM [43]. The 5- and 3-fold icosahedral symmetry axes are marked as pentagons and triangles, respectively, on one icosahedral asymmetrical unit. The spherical polar angles (θ, ϕ) define the location on the icosahedral surface. The depictions are radially depth cued from blue (radius = 130 Å) to red (radius = 155 Å). The W125 and W2 footprints are pink-colored.

Fig 3. Structure of the FMDV-AWH-W125 complex and key determinations on VP2 as well as VP3 of FMDV serotype A. (A) Cartoon representation of one protomer showing the interaction interface between W125 scFv and the capsid. The heavy chain and light chain of W125 are colored purple and orange, respectively. The capsid proteins VP1 to VP4 are colored blue, green, red and yellow, respectively. (B to D) Expanded views of the interaction interface highlighting the Ββ, E-F loop (B), B-C loop and H-I loop (C) of VP2 as well as B-B knob and H-I loop (D) of VP3 within one protomer. Presumable hydrogen bonds and salt bridges in the interaction interface are marked by black dashed lines. (E) Identification of rescued single-substitution mutants by immunofluorescence analysis. BHK-21 cells were infected with rescue mutants at an MOI of 10 for 4 h. FMDV protein 3A was detected using mouse mAb 3A24 and an Alexa Fluor 561-conjugated secondary antibody. (F) Sequence alignment of VP2/VP3 of A/AF72, A/WH/CHA/09 and A/GDMM/2013 strains. The critical residues in interactive interfaces are indicated with black triangles. (G) The neutralization efficacy of W125 against wildtype (A/WH/CHA/09) and mutants…

Fig 4. Structure of the FMDV-AWH-W2 complex and key determinations on VP3 of FMDV serotype A. (A) Cartoon representation of one protomer showing the interaction interface between W2 scFv and the capsid. The heavy chain and light chain of W2 are colored sky blue and rose madder, respectively. The capsid proteins VP1 to VP4 are colored blue, green, red and yellow, respectively. (B to D) Expanded views of the interaction interface highlighting the B-B knob, CD loop (C), βC, EF/GH loops (B), BC loop (D) on VP3. Presumable hydrogen bonds and salt bridges in the interaction interface are marked by black dashed lines. (E) Identification of rescued single-substitution mutants by immunofluorescence analysis. BHK-21 cells were infected with rescue mutants at an MOI of 10 for 4 h. FMDV protein 3A was detected using mouse mAb 3A24 and an Alexa Fluor 561-conjugated secondary antibody. (F) Amino acid sequence alignment of VP3 of A/AF72, A/WH/CHA/09 and A/GDMM/2013 strains. The critical residues in interactive interfaces are indicated with black triangles. (G) The neutralization efficacy of W2 against wildtype (A/WH/CHA/09) and mutants corresponding to interactive residues (VP3 D59A, VP3 Q71A, VP3…

Fig 5. Sequence alignment of VP1 of representative FMDV serotype A strains in ASIA, EURO-SA and AFRICA topotypes. The VP1 amino acids sequences of A/AF72, A/WH/CHA/09 and A/GDMM/2013 strains (marked with diamond) were aligned with that of 48 serotype A strains retrieved from NCBI, including sevens lineages from ASIA topotype, four lineages from EURO-SA topotype and four lineages from AFRICA topotype. The identified key determinations at positions 58 and 147 were circled with blue box and the strains with varied residues involving determinations were indicated with red circle.

Fig 6. The strain-specific epitopes identified on FMDV serotype A A/AF72 strain and A/GDMM/2013 strain. The antibody-driven variations on A/AF72 strain (A) and A/GDMM/2013 strain (B) were separately determined by selection of neutralization-escape mutants using 20 A/AF72-specific and A/GDMM/2013-specific neutralizing mAbs individually. The proportion of pin chart indicated each mAb-driven variation accounted for the five known antigen sites encompassing VP1 GH loop and C-terminus (site 1/5), VP1 B-C loop (site 3), VP2 B-C loop (site 2) and VP3 B-B knob (site 4), as well as the other unidentified site. (C) Amino acid sequence alignment of VP3 of A/AF72, A/WH/CHA/09 and A/GDMM/2013 strains. The VP3 68 and 175 positions that formed strain-specific epitopes were framed with black oval circles. (D) Immunofluorescence analysis of rescued VP3 68 (T→A) mutant that was constructed basis on entire P1 gene of A/GDMM/2013 strain. BHK-21 cells were infected with the rescue mutant or wildtype virus (A/GDMM/2013) at an MOI of 10 for 4 h. FMDV protein 3A was detected using mouse mAb 3A24 and an Alexa Fluor 561-conjugated secondary antibody. (E) The neutralization efficacy of the A/AF72-specific mAbs W3…

Fig 7. The distribution characterization of antibody-driven variations on viral capsid surface of FMDV serotype A between A/AF72 strain and A/GDMM/2013 strain. A total of 135 A/AF72-neutralizaiton escape mutants were obtained by antibody pressure selection using 20 A/AF72-specific neutralization antibodies. The antibody-driven variations on A/AF72 strain were identified by sequence alignment of the 135 mutants and the frequency of varied amino acid residues at positions on VP3 (A), VP1 (B) and VP2 (C) were separately displayed. A total of 117 A/GDMM/2013-neutralizaiton escape mutants were obtained by antibody pressure selection using 19 A/GDMM/2013-specific neutralization antibodies. The antibody-driven variations on A/GDMM/2013 strain were identified by sequence alignment of the 117 mutants and the frequency of varied amino acid residues at positions on VP1 (D), VP2 (E) and VP3 (F) were separately displayed. (G) Footprint of key determinations on VP2 recognized by broad neutralization antibody (W125) and strain-specific mAbs (W121, R55, R95 and R127). The conserved determinations (VP2T70, VP2H77, VP2Q196 and VP3K61) recognized by W125 were circled with black dotted line and highlighted…

Fig 8. The conservative and variable antigen sites of FMDV serotype A recognized by bovine neutralization antibodies. Four conservative antigen sites were indicated with pink and respectively determined by bnAb W125 (site 2), W153 (site 3), W2 (site 4) and W145 (site 5). The variable antigen sites involving the position 68 and 175 on VP3 that were recognized by A/AF72-specific and A/GDMM/2013-specific antibodies were marked with oranges.