Structural and molecular basis for Ebola virus neutralization by protective human antibodies

Abstract

Ebola virus causes hemorrhagic fever with a high case fatality rate for which there is no approved therapy. Two human monoclonal antibodies, mAb100 and mAb114, in combination, protect nonhuman primates against all signs of Ebola virus disease, including viremia. Here, we demonstrate that mAb100 recognizes the base of the Ebola virus glycoprotein (GP) trimer, occludes access to the cathepsin-cleavage loop, and prevents the proteolytic cleavage of GP that is required for virus entry. We show that mAb114 interacts with the glycan cap and inner chalice of GP, remains associated after proteolytic removal of the glycan cap, and inhibits binding of cleaved GP to its receptor. These results define the basis of neutralization for two protective antibodies and may facilitate development of therapies and vaccines.

Figure 1. Binding requirements and structure of antibodies in complex with GP

(A) Schematic representation of GP monomer, colored by domain. GP1 core region (33–190) is colored blue, GP1 glycan cap is colored yellow (201–308), and the mucin-like domain is uncolored (309–501). The GP2 internal fusion loop (IFL) is colored red and the remainder of GP2 is colored orange. Glycans are shown as branched lines and proteolytic cleavage sites are labeled with arrows. Disulfide bonds within and between GP1 and GP2 are omitted for clarity. (B) Immunoprecipitation (IP) of soluble GP ectodomain containing or lacking the mucin-like domain (GP_ΔMuc) by mAb100, mAb114 or isotype control. Binding and input were analyzed using immunoblotting for GP1. * Represents a GP1 degradation product present only in mucin-containing GP. (n=3, representative image shown) (C) Crystal structure of GP_ΔMuc in ternary complex with Fab100 and Fab114. Fab100 is shown in purple (heavy chain) and white (light chain). Fab114 is shown in pink (heavy chain) and white (light chain). Molecular surfaces of two GP_ΔMuc protomers are colored in green and beige, whereas the third is shown as a ribbon representation and colored according to the schematic in panel A.

Figure 2. Cryo-EM of GP_ΔMuc–Fab100–Fab114 complex

Cryo-EM was performed on the ternary complex of GP_ΔMuc with Fab100 and Fab114 at (A) pH 7.4 and (B) pH 5. Shown are the superimpositions of the crystal structure (ribbon) into the cryo-EM density maps at their respective pH.

Figure 3. Inhibition of cathepsin cleavage of GP by mAb100

(A) Fab100 binding occludes access to the β13–β14 loop of GP1. Protomers and Fab100 heavy and light chains are colored and oriented as in Fig. 1C. The variable domain of one Fab100 is shown in a ribbon representation and all other Fabs are removed for clarity. Inset shows a zoomed view of the Fab100–GP β13–β14 loop interface with the difference map generated by masking out the cryo-EM densities of the fitted crystal structures shown as a grey transparent surface. The β13–β14 loop is shown as a dashed yellow line connecting the GP1 core (blue) and the glycan cap (yellow). (B) GP_ΔMuc was incubated with the indicated antibodies followed by cleavage at pH 5.5 by Cat L at 37 °C. Samples were removed at 5 minute intervals and analyzed by immunoblot for GP1. (n=3, representative image shown) (C) Binding kinetics of GP_ΔMuc or GP_THL (THL) with Fab100 or KZ52 at the indicated pH as determined by biolayer interferometry. Equilibrium dissociation constants (K_D) are plotted on a negative log scale. (n=2, representative experiment shown)

Figure 4. Competition of NPC1 binding to GP by mAb114

(A) Fab114 binds to regions in the glycan cap and core of GP1. Protomers are colored as in Fig. 1C and viewed with a 100° rotation about the trimeric axis with respect to the orientation in Fig. 1C. The variable domain of a single Fab114 is shown in ribbons and all other Fabs have been removed for clarity. GP residues predicted to make contact with Fab114 are shown as transparent surfaces. (B) Immunoprecipitation of GP_ΔMuc and GP_THL by the indicated antibodies. Samples were analyzed by immunoblot for GP1. (n=3, representative image shown) (C) Class averages of single particles from negative-stain electron micrographs of Fab114 in complex with GP_ΔMuc and GP_THL. (D) Binding kinetics of GP_ΔMuc or GP_THL with Fab114, 13C6 or monomeric domain C of NPC1 (NPC1-dC) at the indicated pH as measured by biolayer interferometry. Equilibrium dissociation constants (K_D) are plotted on a negative log scale. * indicates no binding. (n=2, representative experiment shown) (E) Inhibition of NPC1-dC binding to GP_THL by competitor proteins (NPC1-dC) or antibodies (mAb100, mAb114, 13C6, KZ52, isotype control) was determined by biolayer interferometry. Dashed line represents 60% inhibition of binding. (n=3, representative experiment shown)