Mapping of foot-and-mouth disease virus antigenic sites recognized by single-domain antibodies reveals different 146S particle specific sites and particle flexibility

Abstract

Vaccination with intact (146S) foot-and-mouth disease virus (FMDV) particles is used to control FMD. However, 146S particles easily dissociate into stable pentameric 12S particles which are less immunogenic. We earlier isolated several single-domain antibody fragments (VHHs) that specifically bind either 146S or 12S particles. These particle-specific VHHs are excellent tools for vaccine quality control. In this study we mapped the antigenic sites recognized by these VHHs by competition ELISAs, virus neutralization, and trypsin sensitivity of epitopes. We included two previously described monoclonal antibodies (mAbs) that are either 12S specific (mAb 13A6) or 146S specific (mAb 9). Although both are 12S specific, the VHH M3F and mAb 13A6 were found to bind independent antigenic sites. M3F recognized a non-neutralizing and trypsin insensitive site whereas mAb 13A6 recognized the trypsin sensitive VP2 N-terminus. The Asia1 146S-specific site was trypsin sensitive, neutralizing and also recognized by the VHH M8F, suggesting it involves the VP1 GH-loop. The type A 146S-specific VHHs recognized two independent antigenic sites that are both also neutralizing but trypsin insensitive. The major site was further mapped by cross-linking mass spectrometry (XL-MS) of two broadly strain reactive 146S-specific VHHs complexed to FMDV. The epitopes were located close to the 2-fold and 3-fold symmetry axes of the icosahedral virus 3D structure, mainly on VP2 and VP3, overlapping the earlier identified mAb 9 site. Since the epitopes were located on a single 12S pentamer, the 146S specificity cannot be explained by the epitope being split due to 12S pentamer dissociation. In an earlier study the cryo-EM structure of the 146S-specific VHH M170 complexed to type O FMDV was resolved. The 146S specificity was reported to be caused by an altered conformation of this epitope in 12S and 146S particles. This mechanism probably also explains the 146S-specific binding by the two type A VHHs mapped by XL-MS since their epitopes overlapped with the epitope recognized by M170. Surprisingly, residues internal in the 146S quaternary structure were also cross-linked to VHH. This probably reflects particle flexibility in solution. Molecular studies of virus-antibody interactions help to further optimize vaccines and improve their quality control.

Keywords: ELISA; VHH; XL-MS; epitope; foot-and-mouth disease virus (FMDV); nanobody; neutralizing antibody.

Figure 1

Epitope mapping of FMDV binding VHHs and mAbs by competition ELISAs using FMDV strains A/TUR (A), A24Cru (B), A10/HOL/1/42 (C), and Asia1 Shamir (D). A red-blue coloring is used to visualize differences in percentage inhibition of biotinylated VHH binding. The 14 VHHs of the 7 CDR3 groups comprising the 12 VHHs that bind specifically to 146S of serotype A or Asia1 strains are color-coded by their CDR3 group. VNT titres and binding to trypsin sensitive epitopes of VHHs is also indicated (A, B, D). The number of serotype A strains recognized in ELISA by serotype A binding VHHs (A, B) was derived from ELISA data obtained earlier (8) using 15 serotype A strains assuming an absorbance > 0.5 indicative of binding. VHH binding is either sensitive to trypsin treatment of FMDV (Y) or not (N). Competition of mAb 9 and mAb 32 by unlabeled mAbs (C) could not be determined (ND) due to using RaM-HRP for mAb detection. However, mAb 13A6 was used in biotinylated form, enabling detection of competition with unlabeled mAb. Roman numerals indicate VHH epitope bins.

Figure 2

Affinity binding curves and deduced affinity constants of VHHs. Using Biolayer Interferometry on an Octet Red96 system, association and dissociation rates were determined by tight multivalent capturing of FMDV A/TUR on optical streptavidin sensors that were loaded with biotinylated M678F VHH. The FMDV-bound sensors were incubated with specific concentrations of M691F (A) or M702F (B) to allow association at time = 0 s. After 300 s the sensors were then moved to VHH-free solution and allowed to dissociate over a time interval. Curve fitting using a 1:1 interaction model (red lines) allows for the affinity constant (K_D) to be measured for each VHH (C).

Figure 3

Location of VP2 residues cross-linked to M702F relative to VP3 cross-linked residues. Cross-linked residues are mapped onto a cartoon presentation of the A22IRQ structure (PDB: 4GH4). Six protomers that originate from three different 12S pentamers surrounding the 3-fold symmetry axis are shown. M702F cross-linked residues are shown for VP3 of the protomer on top and the 4 VP2 molecules closest to this VP3 molecule. The VP2 residues closest to the cross-linked VP3 residues (bold) most likely form the same antigenic site (dashed oval). VP1, blue; VP2, green; VP3, red; VP4, yellow. Cross-linked residues are shown with side chains as yellow (VP2) or cyan/pink (VP3) spheres. Symmetry axes: 5-fold, pentagon; 3-fold, triangle; 2-fold, oval.

Figure 4

Surface accessibility of M702F cross-linked residues mapped on a 12S pentamer of the A22IRQ structure (PDB: 4GH4). The M702F cross-linked residues forming a single antigenic site on VP3 of the protomer at the bottom and the relevant VP2 residues adjacent to this VP3 molecule are shown as cartoon graph (A). Cross-linked residues are shown with side chains as yellow (VP2) or cyan/pink (VP3) spheres. The surface accessible area of a pentamer is shown from a top view (B) or side view (C, D). The nine residues that are surface accessible even when 12S pentamers associate are indicated by arrows (B). Cross-linked residues surface exposed at the 2-fold symmetry axis are shown by a side view of a single pentamer (C) or this same pentamer with a protomer of an adjacent 12S pentamer that associates at the 2-fold symmetry axis in a 146S particle (D). Among the 5 cross-linked residues at this 2-fold symmetry axis (arrows), R120, T191 and T115 are only surface accessible in a 12S pentamer (C) but not a 146S particles (D). VP1, blue; VP2, green; VP3, red; VP4, yellow. Fivefold symmetry axis, pentagon; 3-fold symmetry axis, triangle; 2-fold symmetry axis, oval.

Figure 5

Surface accessibility of M691F cross-linked residues mapped on a 12S pentamer of the A22IRQ structure (PDB: 4GH4) with superimposed GH loops of reduced O1K (PDB: 1FOD). The M691F cross-linked residues forming a single antigenic site on the protomer at the bottom is shown as a cartoon graph (A). Side chains of cross-linked residues are shown as light blue (VP1) yellow (VP2) or cyan/pink (VP3) spheres. The surface accessible area of a pentamer (B, C) is shown for the inner surface (B), outer surface (C) or from a side view looking at the 2-fold symmetry axis (D). The 8 residues that are surface accessible are indicated by arrows (B–D). VP1, blue; VP1 GH-loop, purple; VP2, green; VP3, red; VP4, yellow. The side chains of VP3-K139 and VP3-E70 that are mutated in mAb 9 escape mutants are indicated in magenta spheres. Fivefold symmetry axis, pentagon; 3-fold symmetry axis, triangle; 2-fold symmetry axis, oval.

Figure 6

Comparison of antigenic site of VHH M170 determined by cryo-EM and M702F and M691F determined by XL-MS. Protomers of O/BY/CHA/2010 [PDB: 7DST; (A, B)] or A22IRQ [PDB: 4GH4; (C–F)] are shown as cartoon with footprints of M170 (A, B), M702F (C, D) or M691F (E, F) shown by indicating the side chains of residues within 4 Å of the VHH (A, B) or cross-linked to the VHH (C–F) as light blue (VP1), yellow (VP2), or cyan/pink (VP3) spheres. VPs and VHHs are shown as cartoon: VP1, blue; VP1 of O1K GH-loop (E, F), purple; VP2, green; VP3, red; VP4, yellow; M170, magenta. The C-terminal 20 amino acids of VP1 from an adjacent protomer are shown as light blue cartoon to visualize the M170 footprint (A, B). Part of VP2 from an adjacent protomer is shown to visualize the M702F cross-linked residues (C, D). An alignment of the structures of A22IRQ (VP coloring as done throughout this paper) with M702F or M691F cross-linked residues and O/BY/CHA/2010 (gray) complexed to M170 is shown (D, F). Fivefold symmetry axis, pentagon; 3-fold symmetry axis, triangle; 2-fold symmetry axis, oval.

Figure 7

Antigenic sites 1-5 as earlier identified by sequence analysis of mAb escape mutants mapped onto the 3D structure of an O1K (PDB: 1FOD) pentamer. VPs are shown as cartoon: VP1, blue; VP2, green; VP3, red; VP4, yellow. Side chains of residues defining the antigenic sites are shown as spheres in different colors for sites 1-5 and in case of the novel site (16, 38) also VP2 and VP3 residues. Fivefold symmetry axis, pentagon; 3-fold symmetry axis, triangle; 2-fold symmetry axis, oval.