Structural basis for broad neutralization of ebolaviruses by an antibody targeting the glycoprotein fusion loop

Abstract

The severity of the 2014-2016 ebolavirus outbreak in West Africa expedited clinical development of therapeutics and vaccines though the countermeasures on hand were largely monospecific and lacked efficacy against other ebolavirus species that previously emerged. Recent studies indicate that ebolavirus glycoprotein (GP) fusion loops are targets for cross-protective antibodies. Here we report the 3.72 Å resolution crystal structure of one such cross-protective antibody, CA45, bound to the ectodomain of Ebola virus (EBOV) GP. The CA45 epitope spans multiple faces of the fusion loop stem, across both GP1 and GP2 subunits, with ~68% of residues identical across > 99.5% of known ebolavirus isolates. Extensive antibody interactions within a pan-ebolavirus small-molecule inhibitor binding cavity on GP define this cavity as a novel site of immune vulnerability. The structure elucidates broad ebolavirus neutralization through a highly conserved epitope on GP and further enables rational design and development of broadly protective vaccines and therapeutics.

Fig. 1

Crystal structure of EBOV GPΔMuc bound to CA45 Fab. a Schematic of the EBOV GPΔMuc construct used in crystallizations, with the mucin-like domain (MLD) and transmembrane (TM) domain deleted (gray hatch). b Side view of crystal structure of trimeric EBOV GPΔMuc (GP1, green, with glycan cap yellow; GP2, orange, with fusion loop (FL) blue) bound to CA45 Fab (heavy chain purple; light chain gray). Protomer III in the back is shown in light blue surface representation for clarity. c Top and bottom views of structure in b, shown in combined surface and cartoon representations with all protomers colored by subunit and domain

Fig. 2

CA45-EBOV GP interactions. a Single protomer of EBOV GPΔMuc bound to CA45 Fab, colored as in Fig. 1. b Sequence alignment of representative sequences of the five ebolavirus species at selected regions of GP. Buried surface areas on GP residues at the CA45-binding interface are plotted for each residue of the epitope. Residues of EBOV GP that ablate CA45 recognition upon alanine mutation or those associated with viral escape are highlighted in red and yellow, respectively. Sequence logo reflects sequence variation across representative ebolavirus sequences from previous outbreaks, with residues colored by amino acid type. GP subdomains, secondary structure elements, and N-linked glycosylation sequon sites are annotated below the sequence alignment. c Close-up view of CA45 interactions with fusion loop stem. Residues sensitive to alanine mutation and viral escape are shown in stick representation on GP along with select interacting CA45 residues. 2fofc composite omit electron density contoured at 1σ is shown for the HDCR3 in gray. d Close-up view of light chain interactions with fusion loop base

Fig. 3

Structural basis for broad ebolavirus neutralization by antibody CA45. a Surface representation of CA45-bound EBOV GPΔMuc trimer (left) colored by Shannon sequence entropy on a scale of increasing entropy from white to green (GP1) and white to orange (GP2), with border of CA45 footprint colored raspberry. Close-up view rotated by 60° (right), with bound CA45 heavy and light chains shown in purple and gray, respectively. b Comparison of mean entropies of CA45 epitope with those of other neutralizing antibodies and inhibitors that target the GP1–GP2 interface, by mean entropy per GP-binding site residue (left) or normalized by individual residue buried surface area (right). c Surface representation of CA45-bound EBOV GPΔMuc trimer (left), colored by subunit and subdomain, with CA45 epitope residues conserved in > 99.5% of ebolavirus isolates colored white and variable residues colored dark gray. Close-up and rotated view of CA45 epitope with bound antibody shown as colored in a. d Sequence alignment of CA45 epitope residues across representative ebolavirus sequences. Residues are listed in order of increasing sequence entropy, with entropy values listed and graphed above. Sequence logo reflects sequence variation across representative ebolavirus sequences from previous outbreaks, with residues colored by amino acid type. CA45-specific alanine sensitive mutations and escape mutation are boxed in red and yellow, respectively. Residues unique in RESTV and TAFV colored teal and violet, respectively. e “Open-book” view of right panel in c with surface representation of antibody shown rotated by 180° relative to view in c. Alanine sensitive mutations and escape mutation mapped onto surface of GP in red and yellow respectively (left), with corresponding interface residues on antibody colored similarly (right). f “Open-book” view of right panel in c, with residue positions unique to RESTV and TAFV mapped onto surface of GP in teal and violet, respectively, with corresponding interface residues on antibody colored similarly (right)

Fig. 4

Comparison with other neutralizing antibodies and inhibitors that target the GP1–GP2 interface. a Epitope footprints of antibodies and small-molecule inhibitors that target the GP1–GP2 interface mapped onto the surface of the CA45-bound EBOV GPΔMuc trimer. Footprints of each respective ligand colored raspberry. Lower panels rotated by 30° relative to CA45 panel. b Comparison of number of shared epitope residues with CA45, total (left) and by GP subunit (right). c Pairwise linear regressions of log transformed buried surface areas (BSA) of shared GP interface residues of CA45 with toremifene (TOR; left) and ibuprofen (IBP; right). Statistical correlations were determined under the null hypothesis of zero correlation against the alternative hypothesis of non-zero correlation. R², coefficient of determination; p, p-value; n, sample size. d ELISA competition analysis of toremifene against neutralizing antibodies CA45 and FVM04 for binding to recombinant EBOV GPΔMuc, at pHs 7.4 and 5.2. Data were normalized to percent binding relative to no competitor control and error bars represent SD of sample duplicates from plotted mean

Fig. 5

CA45 defines an inhibitor-binding cavity on GP as a site of immune vulnerability. a The CA45 HCDR3 loop (purple) inserts into the DFF inhibitor-binding cavity located beneath strand β19–β20 of the fusion loop stem (blue). GP1 is colored green, GP2 orange, and fusion loop (FL) blue. Cartoon representation of front of cavity with apical residues of HCDR3 shown (left) and rotated with a more extensive view of HCDR3 (right). b Cross-sectional view of right panel in a, with GP shown in surface representation to reveal DFF cavity. c Superposition of unliganded GP (gray; PDB ID 5JQ3) onto CA45-bound GP (representations in left and right panels are as described in a). Residues of the “DFF lid” of unliganded GP1 are shown in yellow, with residues F193 and F194 shown in stick representation. d Cross-sectional view of right panel in c, with GP shown in surface representation. e Superposition of toremifene- and ibuprofen-bound GP structures (gray; PDB IDs 5JQ7 and 5JQB, respectively) onto CA45-bound GP structure, with toremifene (TOR) shown in pale cyan and ibuprofen (IBP) shown in salmon (representations in left and right panels are as described in a). f Cross-sectional view of right panel in e, with GP shown in surface representation

Fig. 6

Somatically matured CA45 bridges the termini of the cathepsin cleavage loop. a A close-up view of CA45 bound to EBOV GPΔMuc, with Cα atoms of residues that flank germline deletion G31_GLΔ shown in red spheres. CA45 mediates interactions with both termini (green and yellow spheres) of the disordered cathepsin loop (dashed line). b Sequence alignment of mature CA45 heavy and light chains against cynomolgus macaque germline precursors. Residues that undergo somatic mutation in CA45 are shaded in cyan and those that are buried at the interface with GP1 or GP2 are labeled with green and orange asterisks, respectively. Asterisks shaded yellow mediate interface with glycan cap. c CA45 paratope with full GP interface mapped onto CA45 Fab shown in surface representation (heavy chain, purple; light chain, gray), rotated by 90° from view in a. Footprints of GP1 are shown in green, of glycan cap yellow, and of GP2 orange. d CA45 residues that are somatically hypermutated (SHM) are mapped onto CA45 surface in cyan with unmutated heavy and light chain residues colored in purple and gray, respectively. Shown in same orientation as in c. Residues that flank the G31_GLΔ germline deletion are shown in red