Systematic Analysis of Monoclonal Antibodies against Ebola Virus GP Defines Features that Contribute to Protection

Abstract

Antibodies are promising post-exposure therapies against emerging viruses, but which antibody features and in vitro assays best forecast protection are unclear. Our international consortium systematically evaluated antibodies against Ebola virus (EBOV) using multidisciplinary assays. For each antibody, we evaluated epitopes recognized on the viral surface glycoprotein (GP) and secreted glycoprotein (sGP), readouts of multiple neutralization assays, fraction of virions left un-neutralized, glycan structures, phagocytic and natural killer cell functions elicited, and in vivo protection in a mouse challenge model. Neutralization and induction of multiple immune effector functions (IEFs) correlated most strongly with protection. Neutralization predominantly occurred via epitopes maintained on endosomally cleaved GP, whereas maximal IEF mapped to epitopes farthest from the viral membrane. Unexpectedly, sGP cross-reactivity did not significantly influence in vivo protection. This comprehensive dataset provides a rubric to evaluate novel antibodies and vaccine responses and a roadmap for therapeutic development for EBOV and related viruses.

Keywords: antibody; consortium; ebola virus; epitope; glycoprotein; neutralization; protection.

Figure 1

VIC antibody epitope distribution. (A) Recombinant full-length and fragments of Ebola virus GP (Mayinga) used to assess antibody binding by ELISA. (B) ELISA binding in which +, − and, ± indicate binding, no binding, and either binding or no binding, respectively. GP Q508R abolishes GP base binding. Some epitope classes (green labels) were later assigned by alanine scanning mutagenesis and electron microscopy analysis. (C) Space filling diagram of EBOV GPΔMuc (PDB 3CSY) with epitope classes colored according to the histogram. The mucin domain structures were not resolved and are shown as blue circles. GP1/Core encompasses several regions of the GP structure and is not shown. (D) Single-particle negative-stain reconstructions of selected VIC panel antibodies fit to EBOV GPΔMuc (PDB 3CSY). GP1 (blue and cyan) and GP2 (yellow and green) domains are represented by ribbon diagrams (top left panel). Core GPs and Fabs are shown as surfaces in white and various colors, respectively. Fab densities were fit to a model Fab and a side view is shown. (E) VIC mAb epitope classes. The total number of mAbs in each epitope class is shown; lighter shading represents murine mAbs. (F) VIC panel mAb epitope classes. The color coding scheme is used in all figures.

Figure 2

Comparison of neutralization assay results. (A) Results are color-coded according to the ranges listed. mAbs are grouped by epitope class and arranged by position on the GP structure. The EBOV GP structure is shown and the black line outlines regions remaining in GP_CL. mAbs to the right of the black triangles were donated after the epidemic. (B–E) Correlation between epitope class and neutralization assay results by parametric ANOVA shown as modified box plots. Vertical lines represent distribution between first and third quartiles; horizontal lines indicate the mean value. Asterisks, squares and carats indicate significantly different mean values with p<0.04.

Figure 3

Relationship between in vivo protection and epitope class for VIC mAb panel. (A) Groups of 10 mice were infected with ma-EBOV and treated with 100 μg/ml of the indicated mAb on day 2 post-infection. The final percentage of surviving animals is indicated. (B) Parametric analysis of correlation between epitope class and protection. Black and white bars indicate mean values. The mean values for HR2, fusion, base and GP1/Head differed significantly from those for unknown, mucin, GP1/2, GP1/Core and cap (p ≤ 0.0026).

Figure 4

K-means clustering of VIC mAb neutralization readouts. Clusters C1–C5 are based on neutralization activity (normalized with 1.0 as the maximum value). In vivo protection results are shown for reference. VIC 115, 121 and 143 (arrows) protect but do not neutralize.

Figure 5

ELISA cross-reactivity of VIC mAbs with GP of other Ebolavirus species (Bundibugyo virus, BDBV; Sudan virus, SUDV; Reston virus, RESTV) and the marburgvirus Ravn virus (RAVV). Strong, moderate and weak/no neutralization was scored as 2, 1 and 0, respectively, in the four assays.

Figure 6

Immune effector functions and VIC mAb polyfunctionality. Induction of phagocytosis activity in mouse and human monocytes and neutrophils (huADCP, huADNP and mADCP, mADNP) and NK cell activation (IFNγ and MIP-1β secretion; CD107a surface exposure) was measured for each mAb. Polyfunctionality scores ranged from 0–7, with one point given for strong or moderate activity on each of seven readouts. Tier 1 epitopes lie the farthest from the membrane.

Figure 7

Network analysis of correlations between mAb features. A) Force directed network of statistically significant pairwise Spearman’s rho correlation coefficients >0.5 (positive, blue, negative, red). Colored nodes represent mAb features and line thickness corresponds to coefficient values. (B) Coefficients of selected features in the logistic regression model with elastic net regularization calculated by: $$P(\mathit{Protection}=\mathit{High})=\frac{1}{1+e^{-({\beta }_{\mathrm{o}}+{\beta }_1{x}_1+{\beta }_2{x}_2+{\beta }_3{x}_3+\cdots )}}$$ where P is probability and β is the coefficient that weights individual (χ) features. Positive and negative coefficients imply that an increase in the value of the feature will increase and decrease, respectively, the probability of the mAb conferring “High” protection. (C) Fraction of surviving mice plotted as a function of rVSV instantaneous inhibitory potential (IIP). Circles indicating individual mAbs are colored according to protection offered. (D) rVSV IIP as a function of polyfunctionality. mAbs having high polyfunctionality but low (VIC 151) or no (VIC 143, VIC 145) rVSV neutralization are labeled.