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 GPCL) 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.
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Data availability
Coordinates and structure factors have been deposited in the Protein Data Bank under accession number 6MAM. Other data are available from corresponding author upon reasonable request.
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Acknowledgements
X-ray diffraction data were collected at the Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory. SSRL is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). Opinions, conclusions, interpretations, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army. The mention of trade names or commercial products does not constitute endorsement or recommendation for use by the Department of the Army or the Department of Defense. We acknowledge National Institutes of Health grants no. U19 AI109762 (E.O.S., K.C., J.M.D.), no. R01 AI132256 (K.C.), no. U19 AI109711 (A.B.), no. R01 AI132204 (E.O.S., M.J.A.) and no. R01 AI126587 (M.J.A.), the Defense Threat Reduction Agency HDTRA1-13-1-0034 (A.B.) and the Viral Hemorrhagic Fever Immunotherapeutic Consortium for support. This is manuscript number 29630 from The Scripps Research Institute.
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B.R.W., A.Z.W., C.L.M., M.L.F., P.A.I., K.H., A.S.W., R.M.J., A.S.H., S.H., E.G., K.A.H. and S.K. carried out the research. B.R.W., A.Z.W., K.C. and E.O.S. designed the study. M.J.A. contributed materials. L.M.W., J.M.D., A.B., K.C. and E.O.S. supervised the research. B.R.W., A.Z.W., K.C. and E.O.S. drafted the manuscript. B.R.W., A.Z.W., K.C. and E.O.S edited the manuscript. All authors analyzed data and commented on the drafts.
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M.J.A. has stock in Integrated Biotherapeutics, a company developing antibody therapeutics for ebolavirus disease. A.Z.W., E.G. and L.M.W. are employees and equity holders of Adimab. K.C. and J.M.D. are members of the Scientific Advisory Board of Integrum Scientific, LLC.
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Supplementary Figure 1 Structural modeling of potential interactions between ADI-15946 and the glycan cap.
(a) The structure of uncleaved GP (PDB 5JQ3) showing the position of the β17-β18 loop bound to the 310 pocket. The connecting residues between the bound peptide and the β-sheets are unresolved in this structure and are indicated here as dotted lines. GP1 is shown in teal, GP2 in light cyan, and the glycan cap is shown in light green. (b) Structural alignment of uncleaved GP to the EBOV GPCL–ADI-15946 complex shows that the mAb might make favorable interactions with the glycan cap even though binding displaces the glycan cap β17-β18 loop from the 310 pocket (arrow showing displacement of loop). The heavy and light chains of ADI-15946 are colored dark orange and light orange respectively. (c) The heavy and light chain of ADI-15946 may form favorable interactions with the glycan cap. For example, HC R100G and LC Y92 are oriented such that they could potentially form hydrogen bonds with the 100% conserved GP1 residue, Q251. (d) Cathepsin access to the β13-β14 loop, shown as a green cartoon with the unresolved continuation of the loop as a dotted line, is likely inhibited by the steric bulk of ADI-15946 upon binding to GP. The structural alignment shows that the β13-β14 loop passes within close proximity to ADI-15946 and highlights a possible interaction between GP1 residue S211 and ADI-15946 LC residues F67 and D70.
Supplementary Figure 2 Conformational changes influence binding of ADI-15946.
(a) Cartoon illustration of GP with mucin-like domains (MLD) and glycan cap domains (CAP) illustrated as green ovals. The β17-β18 loop of the glycan cap descends to cover the ADI-15946 epitope (orange). This loop may exist in a dynamic equilibrium between ‘tethered’ and ‘loose’ positions that mask and expose the ADI-15946 epitope. (b) The W291R point mutation in the β17-β18 loop results in enhanced exposure of the ADI-15946 epitope. (c) Antibody FVM09 (gray) which binds to the β17-β18 loop appears to lift it up and away30, also better exposing the ADI-15946 epitope. (d) Enzymatic cleavage of GP deletes the glycan cap and the β17-β18 loop, better exposing the ADI-15946 epitope. Deletion of the glycan cap and β17-β18 loop enhances neutralization by ~100-fold. Mutation of the loop and binding of the loop by FVM09 enhance ADI-15946 neutralization by ~10- and 15-fold, respectively.
Supplementary Figure 3 Binding kinetics ADI-15946 and inferred germline progenitors against EBOV GPFL/GPCL.
Somatic hypermutation of ADI-15946 LC improves binding to GPFL and GPCL.
Supplementary Figure 4 FVM09 competition biolayer interferometry assays.
EBOV GP was loaded onto Nickel-NTA biosensors followed by two association steps of test antibodies. Pre-binding of FVM09 does not interfere with subsequent association of ADI-15946 (a) or KZ52 (c). In a converse experiment, pre-binding of ADI-15946 (b) or KZ52 (d) to GP does not interfere with subsequent binding of FVM09. In the second association steps, the first binder is maintained at the same concentration as in the first association step with addition of the competing antibody. All measurements were performed with antibodies at 330 nM concentration.
Supplementary Figure 5 Clashes between the β17-β18 loop and ADI-15946, c2G4, c4G7, or KZ52.
The cartoon representation on the left shows the various antibodies’ Fragment variable (Fv) aligned to unbound, uncleaved EBOV GP (PDB 5JQ3). The zoomed-in panel shows a stereoview of each alignment. On the right is a molecular surface of GPCL with the footprints of each antibody highlighted by selecting all atoms within 4 Å of the Fv. (a) The position of the β17-β18 loop (green) in unbound, uncleaved GP sterically overlaps the position of ADI-15946 in the EBOV GPCL–ADI-15946 crystal structure. (b-c) The position of the β17-β18 loop also interferes with the binding of c2G4 (PDB 5KEL), and c4G7 (PDB 5KEN). (d) The CDRs of KZ52 (PDB 3CSY), however, do not clash with the β17-β18 loop, but rather may form favorable interactions with the loop in its tethered position.
Supplementary Figure 6 Germline neutralization data.
(a-c) Neutralization of rVSVs bearing EBOV GP, BDBV GP, and SUDV GP, respectively, by wild-type ADI-15946 (black circles), heavy chain germline-revertant (HCIGL, gray squares), light chain germline-revertant (LCIGL; light blue triangles) and the HCIGL-LCIGL combination (purple diamonds). (d-f) Neutralization of rVSV bearing GPCL of EBOV, BDBV and SUDV, respectively, by the same ADI-15946 variants. (g) Neutralization of rVSV-EBOV GP bearing a W291R point mutation by wild-type and germline-revertant ADI-15946. For comparison, neutralization of rVSV-EBOV bearing uncleaved GP by wild-type ADI-15946 (orange open circles) is also shown. Virions bearing GPCL are better neutralized by wild-type ADI-15946 and its heavy chain germline-revertant (HCIGL; gray squares). (a-g) Mean±SD; n=6 biologically independent samples in each figure panel.
Supplementary Figure 7 Neutralization and binding of rVSVs bearing ebolavirus GPs or expressed ectodomains by engineered variants of mAb ADI-15946.
(a-e) Neutralization of rVSVs bearing GP from EBOV, BDBV, SUDV, TAFV, and RESTV respectively. Wild-type ADI-15946 is in black, structure-guided mutants 46M1, 46M2 and 46M3 in light, medium and dark blue, respectively. (f-h) Neutralization of rVSV bearing GPCL from EBOV, BDBV and SUDV, respectively. Neutralization of GPCL-bearing virions by wild-type ADI-15946 is in orange. Mutants 46M1-46M3 are in light to dark blue. Neutralization of virions bearing full-length GP by wild-type ADI-15946 is shown in black for comparison. (i) Antibody variants were tested for binding to soluble EBOV, BDBV (j), or SUDV (k) GP ectodomain immobilized on ELISA plates. Mutation of the neighboring ADI-15946 residue Y100A to alanine decreases binding to SUDV GPecto. (a-h) Mean±SD; n=6 biologically independent samples in each figure panel. (i-k) mean±SD; n=3 biologically independent samples in each figure panel.
Supplementary Figure 8 Neutralization of rVSV-EBOV GP and rVSV-EBOV GPΔβ17-β18 by ADI-15946 and affinity variants.
(a) ADI-15946 shows increased neutralization of an rVSV-EBOV GP variant bearing the deletion of residues 187-198 (GPΔβ17-β18) compared to rVSV bearing wild-type GP (WT)—same as figure 3C. (b) Deletion of the β17-β18 loop does not have a major impact on neutralization by KZ52. (c-d) ADI-15946 affinity variants 46M1, 46M2, and 46M3 also show enhanced neutralization of rVSV-EBOV GPΔβ17-β18. (f) The IC50 values are tabulated from each of the experiments graphed in a-e. (a-e) Mean ± SD; n = 6 biologically independent samples in each figure panel.
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West, B.R., Wec, A.Z., Moyer, C.L. et al. Structural basis of broad ebolavirus neutralization by a human survivor antibody. Nat Struct Mol Biol 26, 204–212 (2019). https://doi.org/10.1038/s41594-019-0191-4
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DOI: https://doi.org/10.1038/s41594-019-0191-4
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