Structure-function characterization of three human antibodies targeting the vaccinia virus adhesion molecule D8

Abstract

Vaccinia virus (VACV) envelope protein D8 is one of three glycosaminoglycan adhesion molecules and binds to the linear polysaccharide chondroitin sulfate (CS). D8 is also a target for neutralizing antibody responses that are elicited by the smallpox vaccine, which has enabled the first eradication of a human viral pathogen and is a useful model for studying antibody responses. However, to date, VACV epitopes targeted by human antibodies have not been characterized at atomic resolution. Here, we characterized the binding properties of several human anti-D8 antibodies and determined the crystal structures of three VACV-mAb variants, VACV-66, VACV-138, and VACV-304, separately bound to D8. Although all these antibodies bound D8 with high affinity and were moderately neutralizing in the presence of complement, VACV-138 and VACV-304 also fully blocked D8 binding to CS-A, the low affinity ligand for D8. VACV-138 also abrogated D8 binding to the high-affinity ligand CS-E, but we observed residual CS-E binding was observed in the presence of VACV-304. Analysis of the VACV-138- and VACV-304-binding sites along the CS-binding crevice of D8, combined with different efficiencies of blocking D8 adhesion to CS-A and CS-E allowed us to propose that D8 has a high- and low-affinity CS-binding region within its central crevice. The crevice is amenable to protein engineering to further enhance both specificity and affinity of binding to CS-E. Finally, a wild-type D8 tetramer specifically bound to structures within the developing glomeruli of the kidney, which express CS-E. We propose that through structure-based protein engineering, an improved D8 tetramer could be used as a potential diagnostic tool to detect expression of CS-E, which is a possible biomarker for ovarian cancer.

Keywords: X-ray crystallography; antibody; glycosaminoglycan; protein crystallization; protein structure; vaccine; viral protein.

Figure 1.

Cross-blocking of human and mouse D8 Abs reveal a novel epitope. A, human antibody cross-blocking reveals two specificity groups (columns labeled 1 and 2). B, overlap between mouse and human mAbs identify a separate epitope for VACV-66. Black cells correspond to Abs that fully block D8 binding of each other and are thus assumed to share overlapping epitopes. Empty cells correspond to Abs that did not affect the D8 binding of each other. Relative strength of cross-blocking is indicated in shades of gray, because epitopes may overlap partially and allow simultaneous binding of both antibodies. Columns 1–4 indicate the four different specificity groups.

Figure 2.

Human anti-D8 mAbs are moderately neutralizing. Shown is VACV IMV neutralization activity of purified human (VACV-249, VACV-304, and VACV-66) and mouse (JE11, JF11, and LA5) anti-D8 mAbs in the absence (black bars) or presence (red bars) of complement. Murine anti-L1 mAb (M12B9) was used as positive control and anti-L1 mAb 39D4 as control for a weakly neutralizing mAb.

Figure 3.

Real-time binding kinetics using biolayer interferometry. mAbs were immobilized on anti-human Fc capture antibody sensors and the 1:1 binding of each Fab to monomeric D8 was measured by dipping into increasing concentrations (colored curves) of D8. Kinetic measurements were unaffected by antibody avidity. All antibodies bind with high affinity.

Figure 4.

Interference of D8 binding to CS-A or CS-E by human mAbs. Hexameric D8 was incubated with mAbs VACV-66, VACV-138, VACV-304, or alone prior to binding to GAG microarrays. Antibody blocking was assessed by comparing the amount of D8 and D8–mAb complex bound to the microarray. Blocking experiments were performed in triplicate (± S.E., error bars). Although VACV-66 does not block D8 binding to CS-A or CS-E, VACV-138 blocks binding to both. VACV-304 fully blocks binding to CS-A, whereas slight binding to CS-E was observed (∼25% of maximum binding).

Figure 5.

Structural overview of human mAb–D8 complexes. A, binding of VACV-66, VACV-138, or VACV-304 to D8. B, mAb footprint on D8 is colored by chain on the D8 protein otherwise shown in gray. Heavy-chain footprint is shown in orange and light-chain in green. The CS-binding site is indicated with a black dot (A) and a yellow line (B). Both VACV-138 and VACV-304 bind above the CS-binding crevice, whereas VACV-66 binds on the side of D8 away from the crevice.

Figure 6.

Interactions between mAbs and D8. A–C, binding VACV-66 (A), VACV-138 (B), or VACV-304 (C) to D8. D8 is shown in gray and the mAb CDRs are colored as follows: L1 (green), L2 (light green), L3 (dark green), H1 (yellow), H2 (cyan), and H3 (orange). Potential H-bonds are indicated with blue dotted lines.

Figure 7.

Footprint of CS-blocking mAbs on D8. Shown is a schematic representation of mAb binding sites (circles) and illustration of a high- (H) and low- (L) affinity binding region for CS within the central binding crevice (blue rectangle) of D8.

Figure 8.

D8 tetramers specifically detect chondroitin sulfate E in tissues. Kidney tissue samples were flash-frozen, sectioned, fixed in 4% paraformaldehyde and reacted with 156 ng/ml of either D8 or D8 control tetramers coupled to Alexa Fluor 488. Samples were counterstained with phalloidin to reveal dense F-actin in the glomerulus and with the DNA stain Hoechst. The D8 tetramer immunoreactivity was localized within the glomeruli. Negative control with the mutated D8 tetramer revealed the absence of binding in the glomerulus but low-grade immunoreactivity in adjacent regions. Bar = 20 μm.