Functional and in silico Characterization of Neutralizing Interactions Between Antibodies and the Foot-and-Mouth Disease Virus Immunodominant Antigenic Site

Figures

• 

  Figure 1. Multiple-sequence alignments of anti-foot-and-mouth disease virus (FMDV) mAb Fv sequences. Amino acid Fv sequences of 4A2, 1E12, and reference 4C4 mAbs. Invariant and conserved residues are highlighted in red and yellow, respectively. Residues are numbered according to the Kabat numbering system and secondary structure elements for variable sequences are indicated above the sequence [spirals, α, and 310(η)-helices; arrows, β-strands; T, turns]. Antigen-binding regions (ABR) are underlined for H (A) and L (B) chains.

• 

  Figure 2. Peptide ELISA mapping of functional epitopes for two anti-FMDV Ab. ELISA results between 15 peptides and mAbs 4A2 or 1E12 are presented in horizontal panels (A,B), respectively. Each panel: Left, the panel displays the specific aa sequences of each peptide; the point mutation is indicated with a box. Right, the panel shows the influence of the point mutations according to the ELISA data; the colors (in the columns) represent IIR50 relative to the wt peptide as follows: in green, the wt peptide and peptides with mutations of IIR50 (relative interaction index 50) equal to wt; in blue, peptides with IIR50 mutations lower than wt (improve interaction) and in red, peptides with IIR50 mutations greater than wt (worsen interaction). The horizontal bar on each peptide matrix summarizes the positions most affected by the mutations in the functional epitope for each mAb, in red and yellow; positions of low and medium tolerance to mutations, respectively, in blue; non-mutated positions and near-native mutations, in white.

• 

  Figure 3. Ag–Ab focused-docking recapitulation for an FMDV interaction complex of reference. The figure displays data for 16 Ag–Ab candidate interaction complexes. (A) Regression plot between interface energy score (dG) and RMSD for each Ag–Ab candidate solutions. (B,C) exemplifies the structural alignment (of the main chains) between mAbs, revealing the structural distance of FMDV peptide in the wt complex (orange), compared with a top (pale blue), and to a poor (gray) dG scored solution.

• 

  Figure 4. Focused-docking experiments for candidate Ag–Ab complexes. Plots of five different docking experiments using mAb 1E12 or 4A2; dots composed by dG values (Docking score) vs. structural homology values (RMSD, on the horizontal axis). A characteristic pattern called energy funnel (A–D), except for (E), which was a negative control) emerges at near-native docking solutions. Green circles at (B,E) denotes the presence or absence of the energy funnel pattern.

• 

  Figure 5. Top docking solutions emerging from the integral modeling protocol. Two perspectives of the interaction topologies between the FMDV AgSA peptide (pink) and mAbs 1E12 (red) and 4A2 (blue) are shown in vertical panels (A,B), respectively. Note that the peptide has a flatter interaction with 1E12 mAb, unlike the interaction with 4A2, where the peptide would be more embedded in the paratope.

• 

  Figure 6. AgSA mutational profile for three FMDV Ag–Ab complexes. Computational mining and comparison of the influence of single point mutations at the FMDV Ag partner for two Ag–Ab complexes and one Ag–Ab complex of reference. Horizontally, each panel contains the native FMDV AgSA sequence (see the conserved RGD motif in yellow) and, vertically, the energetic effect (dG) of single point aa mutations. (A,B) correspond to mAbs 4A2 and 1E12 interaction complexes, respectively, and (C) displays reference 4C4 mAb (pdb 1EJO) interaction data. Color variation from blue to red implies a worsening of the interaction energy and a less tolerated mutation at a given Ag–Ab. See the similarities between (A,C) involving IgG Abs and a different mutational profile for the IgM Ab (B).