Broadly neutralizing antibodies from human survivors target a conserved site in the Ebola virus glycoprotein HR2-MPER region

Abstract

Ebola virus (EBOV) in humans causes a severe illness with high mortality rates. Several strategies have been developed in the past to treat EBOV infection, including the antibody cocktail ZMapp, which has been shown to be effective in nonhuman primate models of infection ¹ and has been used under compassionate-treatment protocols in humans ² . ZMapp is a mixture of three chimerized murine monoclonal antibodies (mAbs)^3-6 that target EBOV-specific epitopes on the surface glycoprotein^7,8. However, ZMapp mAbs do not neutralize other species from the genus Ebolavirus, such as Bundibugyo virus (BDBV), Reston virus (RESTV) or Sudan virus (SUDV). Here, we describe three naturally occurring human cross-neutralizing mAbs, from BDBV survivors, that target an antigenic site in the canonical heptad repeat 2 (HR2) region near the membrane-proximal external region (MPER) of the glycoprotein. The identification of a conserved neutralizing antigenic site in the glycoprotein suggests that these mAbs could be used to design universal antibody therapeutics against diverse ebolavirus species. Furthermore, we found that immunization with a peptide comprising the HR2-MPER antigenic site elicits neutralizing antibodies in rabbits. Structural features determined by conserved residues in the antigenic site described here could inform an epitope-based vaccine design against infection caused by diverse ebolavirus species.

Figure 1. Cross-reactive neutralizing antibodies from BDBV survivors bind near the membrane proximal region of GP

a) Data from competition-binding assays using BDBV223, BDBV317 or BDBV340; antibodies from ZMapp^TM cocktail (c2G4 and c13C6). Numbers indicate the percent binding of the second mAb in the presence of the first mAb, compared to binding of second mAb alone. MAbs were judged to compete for the same site if maximum binding of the second mAb was reduced to <30% of its un-competed binding (black boxes with white numbers). MAbs were considered non-competing if maximum binding of the second mAb was >70% of its un-competed binding (white boxes with red numbers). Grey boxes with black numbers indicate an intermediate phenotype (between 30 and 70% of un-competed binding). Multiple readings were used to determine the maximum signal for each mAb alone and a single reading of a mAb in combination with each competing antibody was recorded. b) Representative negative stain class averages of antibodies that bind GP2 exclusively in the HR2/MPER region. Complexes are of BDBV Fabs bound to BDBV GPΔmuc. c) (Top) A class average of BDBV GPΔmuc bound to BDBV223 demonstrates the location of each component, with the core GP colored blue and the Fabs in green. (Middle) A class average of c13C6 Fab:c4G7 Fab bound to EBOV GPΔTM (with c13C6 in dark blue, c4G7 in yellow and GP core in light blue). (Bottom) Overlaying a class average of c13C6 Fab:c4G7 Fab bound to EBOV GPΔTM (with c13C6 in dark blue, c4G7 in yellow and GP core in light blue) over a class average of BDBV223 Fab bound to BDBV GPΔmuc (with BDBV223 in green and GP core in light blue), demonstrates that BDBV223 binds significantly lower down on GP, well below the epitope of the c4G7 site of vulnerability at the GP1/GP2 interface. d) A model of the c13C6 Fab:c4G7 Fab bound to EBOV GPΔTM (EMDB ID-6152, Fab variable regions from PDB 5KEN, EBOV GPΔMuc from PDB 5JQ3) is shown with the relative location of BDBV223/317/340 Fabs (segmented c4G7 Fab from the above map placed in the relative location on GP as indicated by class averages, representative Fab from PDB 3CSY).

Figure 2. Neutralization and protective efficacy of HR2/MPER-specific mAbs

a) Neutralization activity of BDBV223, BDBV317 or BDBV340 against BDBV, EBOV, RESTV or SUDV. Means ± SD of triplicates or quadruplicates are shown. b) Post-exposure protection of EBOV-inoculated mice treated with BDBV223, BDBV317, BDBV340 or a control mAb 2D22 one day after virus challenge (n = 5 animals/group). Kaplan-Meier survival curves are shown. c) Post-exposure protection of EBOV-inoculated guinea pigs treated with BDBV223, BDBV317, or a control mAb 2D22 one day after EBOV challenge (n = 5 animals/group). Kaplan-Meier survival curves are shown. d) Post-exposure protection of BDBV-inoculated ferrets treated with BDBV223 or a control 2D22 mAb at days 3 and 6 after BDBV challenge (n = 4 animals/group). Kaplan-Meier survival curves are shown. Separate two-sided log-rank (Mantel-Cox) tests were used for pairwise comparisons of survival curves between each BDBV mAb-treated group and control 2D22-treated mAb group (b-d). P-values were not adjusted for multiple comparisons. P ≤0.05 considered significant; ns – not significant.

Figure 3. Structural and functional analysis of GP residues important for mAb cross-reactivity and neutralization

a) Sequence alignment of GP2 from BDBV, EBOV, RESTV and SUDV. The numbers above the sequence correspond to the amino acid position in GP. Amino acids identical to BDBV are indicated by dots. Color-coded circles indicate the position of residues at which alanine substitutions disrupt mAb binding, as determined by alanine-scanning mutagenesis (ASM). Color-coded triangles indicate the residues at which mutations disrupt the binding to peptides in ELISA (PE) and color-coded squires indicate escape mutations for BDBV223, BDBV317 or BDBV340. BDBV1p, BDBV2p, EBOV2p or SUDV2p peptide sequences analyzed are indicated by grey, black, blue or purple lines, respectively. Vertical lines indicate C-terminal ends of the GP constructs used for ELISA binding assay in Figure 4. b) Neutralization activity of BDBV223, BDBV317 or BDBV340 against wild-type BDBV (black), BDBV223 (green), BDBV317 (blue) or BDBV340 (purple) escape mutants. Means ± SD of triplicates are shown. c) Binding of BDBV223, BDBV317 or BDBV340 to BDBV1p (grey), BDBV2p (black), EBOV2p (brown) or SUDV2p (orange) peptides. Means ± SD of quadruplicates are shown. d) Binding of BDBV223, BDBV317 or BDBV340 to HR2/MPER peptides. The EC₅₀ value for each peptide-mAb combination is shown. EC₅₀ values greater than 20,000 are indicated (>).

Figure 4. Immunization with HR2/MPER peptide elicits peptide and protein antigen-reactive and neutralizing antibody response

a) Pre-immune serum (black) or immune serum (grey) samples from animals were tested in ELISA for binding to BDBV2p (animals 1 and 2) or SUDV2p (animals 3 and 4) peptides. Means ± SD of quadruplicates are shown. b) Binding of rabbit polyclonal Abs to BDBV2p (black), EBOV3p (brown), SUDV2p (orange), or BDBV1p (grey) peptides. Means ± SD of quadruplicates are shown. c) Binding of rabbit polyclonal Abs to BDBV (black), EBOV (brown), SUDV (orange), or MARV (grey) glycoproteins. Means ± SD of quadruplicates are shown. d) Neutralization activity of polyclonal Abs against BDBV (black), EBOV (brown), or SUDV (orange). Means ± SD of triplicates are shown.