Isolation of potent neutralizing antibodies from a survivor of the 2014 Ebola virus outbreak

Abstract

Antibodies targeting the Ebola virus surface glycoprotein (EBOV GP) are implicated in protection against lethal disease, but the characteristics of the human antibody response to EBOV GP remain poorly understood. We isolated and characterized 349 GP-specific monoclonal antibodies (mAbs) from the peripheral B cells of a convalescent donor who survived the 2014 EBOV Zaire outbreak. Remarkably, 77% of the mAbs neutralize live EBOV, and several mAbs exhibit unprecedented potency. Structures of selected mAbs in complex with GP reveal a site of vulnerability located in the GP stalk region proximal to the viral membrane. Neutralizing antibodies targeting this site show potent therapeutic efficacy against lethal EBOV challenge in mice. The results provide a framework for the design of new EBOV vaccine candidates and immunotherapies.

Fig. 1. Antigen binding properties of anti-GP mAbs

(A) Apparent binding affinities of GP-specific IgGs to Zaire GPΔTM and Zaire GPΔmuc constructs as determined by BLI measurements. Newly discovered anti-GP mAbs are shown as red circles. KZ52 IgG (yellow diamond), 13C6 IgG (green triangle), 1H3 IgG (orange square), and 2G4 IgG (purple hexagon) are included for comparison. (B) Apparent binding affinities of GP-specific IgGs to Zaire sGP and Zaire GPΔmuc as determined by BLI measurements. (C) Pie chart summarizing antibody binding profiles. Cross-reactive mAbs refer to those that bind to both GP and sGP. N.B., non-binder; W.B., weak binder. IgG K_Ds were calculated for mAbs with BLI responses >0.1 nm. MAbs with BLI responses <0.05 nm were designated as N.B.; MAbs with BLI responses between 0.05–0.1 nm were designated as W.B. All data are representative of two or more independent experiments.

Fig. 2. Epitope mapping

(A) Percentage of sGP-reactive and sGP non-reactive mAbs directed against each antigenic site on EBOV GP. Epitope binning was performed using a previously described yeast-based competition assay (20). (B) Percentage of selected KZ52 competitors that cross-react with SUDV GP and BDBV GP. Binding cross-reactivity was assessed by ELISA. (C) ELISA binding of selected KZ52 competitors to a minimal GP core that contains deletions in the mucin-like domain and glycan cap (GP_CL). ELISA binding is expressed as the OD₄₀₅ reading at a concentration of 0.2 µg/ml. (D) Percentage of selected KZ52 non-competitors that cross-react with SUDV GP and BDBV GP. Binding cross-reactivity was assessed by ELISA. (E) Summary of the antigenic sites targeted by the anti-GP mAbs. All data are representative of two or more independent experiments.

Fig. 3. Neutralizing activity of anti-GP mAbs

(A) Percentage of mAbs in each competition group that reached PRNT₅₀ or PRNT₈₀ at concentrations ≤50 µg/ml. The total number of mAbs tested from each competition group are shown at the top of the corresponding bar. (B) PRNT₅₀ and PRNT₈₀ values of selected mAbs from each competition group. KZ52 IgG is included for comparison (green inverted triangle). Red bars indicate median PRNT₅₀ and PRNT₈₀ values. Neutralization assays were performed using a live virus plaque reduction assay. PRNT₅₀ and PRNT₈₀ values represent the concentration of IgG required to reduce viral infectivity by 50% and 80%, respectively. All data are representative of two independent experiments.

Fig. 4

Negative stain electron microscopy of Fab:EBOV GPΔTM complexes (A) A structure-based (PDB 3CSY and 3S88) (5) surface representation of the ebolavirus GP trimer. The mucin domain (grey), glycan cap domain of GP1 (aqua green), GP1 core (blue), GP2 (light blue), fusion loop region of GP2 (pink), and the stalk/HR2 region (orange) have been mapped onto the structure. The residues comprising the trimeric body and the stalk region of the ebolavirus GP are displayed on the right. The mucin domains are modeled only as spheres as they are largely unstructured and poorly defined (27). Residues 613–637 corresponding to the stalk/HR2 region were modeled in silico using 3-fold symmetry and peptide structure prediction for the HR2 region (28). (B) Corresponding three-dimensional reconstructions of four Fab:EBOV GPΔTM complexes are shown in transparent surface representation (gray) with the model from panel (A) fitted in the density. Additionally, structural models for each Fab variable region were generated using the ROSIE server (29, 30) and then fitted into the density maps as surface representations. Each structure is shown as side (left) and top (right) views with the exception of ADI-15758, which is shown from the bottom up, respective to the viral membrane.

Fig. 5. Therapeutic efficacy of NAbs against MA-EBOV

Kaplan–Meier survival curves for ADI-15974 competitor NAbs (A) KZ52 competitor NAbs (B) 13C6 competitor NAbs (C) and NAbs targeting undefined epitopes (D). Mice were infected with 100 p.f.u. of MA-EBOV and treated intraperitoneally with a single dose of the indicated mAbs at two dpi (dotted black line). Negative control mice were treated with PBS. MAb 2G4 is included for comparison. Data are representative of one experiment with 10 mice per group.