Structural basis of broad ebolavirus neutralization by a human survivor antibody

Abstract

The structural features that govern broad-spectrum activity of broadly neutralizing anti-ebolavirus antibodies (Abs) outside of the internal fusion loop epitope are currently unknown. Here we describe the structure of a broadly neutralizing human monoclonal Ab (mAb), ADI-15946, which was identified in a human survivor of the 2013-2016 outbreak. The crystal structure of ADI-15946 in complex with cleaved Ebola virus glycoprotein (EBOV GP_CL) reveals that binding of the mAb structurally mimics the conserved interaction between the EBOV GP core and its glycan cap β17-β18 loop to inhibit infection. Both endosomal proteolysis of EBOV GP and binding of mAb FVM09 displace this loop, thereby increasing exposure of ADI-15946's conserved epitope and enhancing neutralization. Our work also mapped the paratope of ADI-15946, thereby explaining reduced activity against Sudan virus, which enabled rational, structure-guided engineering to enhance binding and neutralization of Sudan virus while retaining the parental activity against EBOV and Bundibugyo virus.

Figure 1.

Structure of ADI-15946 in complex with Ebola virus GP_CL. (a) Domain architecture of EBOV GP full length and the construct crystallized here, which is the ectodomain of an enzymatically cleaved GP (GP_CL) resembling the form of GP generated in endosomes during viral entry lacking the glycan cap. IFL, internal fusion loop; HR1 and HR2, heptad repeat 1 and heptad repeat 2, respectively. Glycosylation sites are represented above the domains. (b) Crystal structure of the trimeric EBOV GP_CL–ADI-15946 complex. The inset table shows the contribution of each CDR to the buried surface area (BSA) on the surface of GP_CL. (c) The interaction bridges the fusion loop and other portions of GP2 primarily via light chain and CDR H3 contacts. CDRs H1 and H2 are not involved. (d) The footprint of ADI-15946 (orange) is shifted up and to the left compared to that of the base binding mAb, KZ52 (pink).

Figure 2.

The K510E escape mutation likely clashes with ADI-15946 CDR H3. (a) Stereoview of the EBOV GP_CL–ADI-15946 complex (left; ADI-15946 in orange, and GP_CL in dark and light teal for GP1 and GP2, respectively) and electrostatic surface potential (right; color scale shown in panel c) showing that residue K510 of GP2 binds into a negatively charged pocket created by ADI-15946 CDR H3. (b) Similar views to (a), with modeling of an escape mutant of ADI-15946, GP K510E, suggesting that K510E clashes with CDR H3 and introduces conflicting negative charge into the CDR H3 pocket. (c) Open-book representation of EBOV GP_CL and ADI-15946 showing electrostatic surface potential colored according to included scale. GP_CL is shown on the left with the epitope outlined in black. ADI-15946 is shown on the right with the paratope outlined in black. (d) Binding and neutralization assays showing the capacity of ADI-15946 variants containing either the D100^CA or the L100^FA mutation to bind to rVSV-BDBV GP (WT or K510E) in an ELISA (top, mean± s.d., n=4 biologically independent samples) and neutralize infection by these viruses (bottom, mean± s.d., n=6 biologically independent samples). Electrostatic surface potentials in a and c were generated using the APBS plugin with Pymol.

Figure 3.

ADI-15946 binds a highly conserved epitope shielded by the mobile β17-β18 loop of the glycan cap. (a) Structures show that ADI-15946 (orange, left) binds the hydrophobic 3₁₀ pocket of GP (teal) that is usually occupied by the β17-β18 loop of the glycan cap (green, middle). Right, binding NPC1 loop C (magenta) to GP induces a conformational change in which the GP1 3₁₀ helix unwinds and asparagine 73 (N73) becomes solvent exposed while GP2 lysine 510 (K510) inserts into the cavity left behind after the unwinding of the 3₁₀ helix. ADI-15946 may prevent these conformational changes from occurring by locking down the 3₁₀ helix with residues that mimic those of the β17-β18 loop. (b) An enlarged view of the 3₁₀ pocket shows that CDR H3 of ADI-15946 positions similar residues in similar orientations to that of the β17-β18 loop. (c) Neutralization assay showing that ADI-15946 has enhanced neutralization of a GP construct lacking the β17-β18 loop compared to wild type (WT), likely due to increased access to the 3₁₀ pocket. Data are mean± s.d., n=6 biologically independent samples. (d) Binding assays showing that a point mutation in the β17-β18 loop (W291R) results in enhanced binding to rVSV-EBOV GP in an ELISA. Data are mean± s.d., n=4 biologically independent samples. (e) Neutralization assays showing that the W291R mutation in the β17-β18 loop or its proteolytic removal (CL) enhance the capacity of ADI-15946 to neutralize rVSV-EBOV GP. Data are mean± s.d., n=6 biologically independent samples. (f) Kinetic binding studies by biolayer interferometry reveal enhanced association rate and slower dissociation rate of ADI-15946 to GP_CL compared to uncleaved GP.

Figure 4.

mAb FVM09 potentiates ADI-15946 neutralization of EBOV GP in a dose-dependent manner. (a) Infection assays showing that addition of increasing concentrations of ADI-15946 (left), but not KZ52 (right), to fixed concentrations of FVM09 (5, 20, or 80 nM) promotes rVSV-EBOV GP neutralization. The reciprocal fold change in neutralization IC₅₀ is shown (inset). (b) Infection assays showing that addition of increasing concentrations of FVM09 to a fixed, subneutralizing concentration of ADI-15946 enhances rVSV-EBOV GP neutralization. The same experiment against KZ52 shows inhibition of neutralization as the concentration of FVM09 is increased. (c-f) Infection assays as in a-b, with authentic EBOV (c-d) showing similar trends; this trend is present but less pronounced with rVSV-SUDV GP (e-f). Data in a–f are mean± s.d., n=6 biologically independent samples. (g) A cartoon showing the proposed relationship between FVM09 binding and subsequent exposure of the ADI-15946 binding site.

Figure 5.

Genesis of ADI-15946. (a) Models showing differences between the ADI-15946 IGL sequence (left) and the mature antibody (WT, right). GP1 is shown in grey and GP2 in white. (b) Comparison of the apparent equilibrium dissociation constant (1/K_Dapp; higher value is tighter binding) for binding of ADI-15946 variants (WT, IGL, and WT:IGL chimeras) to GP_CL to their capacity to neutralize rVSV-EBOV GP infection (1/IC₅₀; higher value is more potent neutralization). (c) Heat maps for neutralization of rVSVs bearing ebolavirus GP and GP_CL proteins by the indicated ADI-15946 variants.

Figure 6.

Structure-guided affinity maturation of ADI-15946. (a) Molecular models showing the locations of mutations in ADI-15946 variants 46M1, 46M2, and 46M3 in relation to the surface of GP. CDRs are illustrated in dark orange for the heavy chain and light orange for the light chain respectively; engineered side chains that differ from wild-type are colored in green. GP1 and GP2 are shown as a grey and white surface respectively. (b) Binding assays of recombinant EBOV, BDBV, and SUDV GP ectodomains by the indicated ADI-15946 variants determined by ELISA. Data are mean± s.d., n=3 biologically independent samples. (c) Neutralization assays of authentic EBOV, BDBV and SUDV by the indicated ADI-15946 variants. Data are mean± s.d., n=6 biologically independent samples. (d-e) Heat maps for neutralization potency (IC₅₀) of each ADI-15946 variant against rVSVs (d) and authentic filoviruses (e). In panel d, neutralization of rVSVs bearing full-length GPs and cleaved GPs is shown on the top and bottom, respectively. (f) Molecular surface of EBOV GP_CL with the ADI-15946 footprint outlined in orange. Differences at five sites are listed on the left. The panel on the right shows the which CDRs are in proximity to these nonconserved sites. (g) Neutralization assays of rVSV-EBOV GP by 46M3 in the presence of increasing concentrations of FVM09 (0–100 nM). Increasing amounts of FVM09 improved 46M3 neutralization. Data are mean± s.d., n=6 biologically independent samples.