TY - JOUR
AU1 - Milligan, Jacob, C
AU2 - Parekh, Diptiben, V
AU3 - Fuller, Katherine, M
AU4 - Igarashi,, Manabu
AU5 - Takada,, Ayato
AU6 - Saphire, Erica, Ollmann
AB - Abstract Ebola virus infection causes severe disease in humans and represents a global health threat. Candidates for immunotherapeutics and vaccines have shown promise in clinical trials, although they are ineffective against other members of the Ebolavirus genus that also cause periodic, lethal outbreaks. In this study, we present a crystal structure of a pan-ebolavirus antibody, 6D6, as well as single-particle electron microscopy reconstructions of 6D6 in complex with Ebola and Bundibugyo virus glycoproteins. 6D6 binds to the conserved glycoprotein fusion peptide, implicating it as a site of immune vulnerability that could be exploited to reliably elicit a pan-ebolavirus neutralizing antibody response. antibody, broadly neutralizing, cross-reactive, Ebola, immunotherapeutic The 2014–2016 outbreak of Ebola virus disease (EVD) in West Africa resulted in over 28000 cases and 11000 deaths [1] and accelerated the development and provision of therapeutics and vaccines, some of which were deployed in 2 separate outbreaks in the Democratic Republic of Congo in 2018. The etiological agent of EVD, Ebola virus (EBOV), is a member of the genus Ebolavirus, which also includes the human pathogens Sudan virus (SUDV), Bundibugyo virus (BDBV), and Taï Forest virus (TAFV), as well as Reston virus (RESTV), which does not appear to cause disease in humans [2]. There are no US Food and Drug Administration-approved treatments or vaccines for EVD, although several candidates are currently in clinical development [3–5]. However, almost all of the candidates currently in clinical trials are specific for EBOV and are ineffective against SUDV and BDBV, which were responsible for over 40% of all EVD cases before 2013 [2]. Thus, a need remains for broadly reactive, pan-ebolavirus treatments to rapidly contain future outbreaks. In the time since the West African EVD outbreak, a few monoclonal antibodies (mAbs) with broadly neutralizing activity have been described against the surface glycoprotein (GP), the primary antigen on the surface of ebolavirus virions [6–8]. One of the first mAbs with pan-ebolavirus neutralization activity to be described was 6D6 [9], which was elicited by sequentially immunizing a mouse with EBOV and SUDV virus-like particles (VLPs). Remarkably, 6D6 effectively neutralizes vesicular stomatitis virus pseudotyped with GPs from all known ebolaviruses with half-maximal inhibitory concentrations (IC50) for EBOV, SUDV, TAFV, BDBV, and RESTV of 0.12, 0.19, 0.33, 0.24, and 0.62 μg/mL, respectively; in addition, 6D6 provides high levels of protection in mouse models of both EBOV and SUDV infection [9]. Structural analysis of 6D6 was undertaken to illuminate its mode of broad recognition and whether GP targeting differed across the antigenically distinct ebolaviruses. In this study, we present the crystal structure of the antigen-binding fragment (Fab) of 6D6 as well as single-particle electron microscopy (EM) reconstructions of 6D6 Fab in complex with GPs of EBOV and BDBV. We find that 6D6 recognizes a quaternary assembly, bridging 2 monomers in the GP trimer, and that the angle of recognition is identical between EBOV and BDBV GPs. The striking similarity between the footprint of 6D6 (elicited by immunization of a mouse) and 2 human-derived pan-ebolavirus neutralizing mAbs (elicited in natural infection) strongly implicates the hydrophobic fusion peptide as a site of immune vulnerability capable of eliciting a repeatable, pan-ebolavirus antibody response. MATERIALS AND METHODS Recombinant, chimeric 6D6 Fab was expressed in Drosophila S2 cells using 2 coexpressed pMT-puro plasmids, one encoding a C-terminally Strep-tagged heavy chain variable region and the other encoding the light chain variable region. Cysteine 109 in complementarity-determining region 3 of the heavy chain (CDR H3) was mutated to serine to aid in expression and purification. The Fab was purified using affinity chromatography followed by cleavage of the Strep tag at an Enterokinase cleavage site using EKMax (Thermo Fisher Scientific). The tagless protein was further purified using a Superdex 75 10/300 GL size-exclusion chromatography (SEC) column (GE Healthcare Lifesciences). 6D6 Fab was screened for crystallization using a Douglas Instruments Oryx8, and the protein was crystallized in a solution of 0.1 M Tris pH 7.6 with 25% w/v polyethylene glycol 6000. Diffraction data to 1.96 Å resolution were collected at beamline 23-ID-D at the Advanced Photon Source, and the structure was solved by molecular replacement using chains H and L of the Protein Data Bank entry 1I9R as a search model. Two Fab molecules are contained in the asymmetric unit of the P21 crystals. Residues 1–220 are visible in heavy chain 1, residues 2–213 are visible in light chain 1, residues 1–133 and 142–219 are visible in heavy chain 2, and residues 2–212 are visible in light chain 2. Molecular replacement, model building, and structure refinement were carried out using the PHENIX suite of programs [10]. Mucin-deleted GPs (GPΔMLD) of EBOV and BDBV were separately expressed in Drosophila S2 cells using a single plasmid encoding a C-terminally Strep-tagged construct lacking the transmembrane domain. GPΔMLDs were purified using affinity chromatography followed by cleavage of the Strep tag at an Enterokinase cleavage site using EKMax. The tagless proteins were further purified using a Superose 6 10/300 GL SEC column. All purification steps for 6D6 Fab and GPΔMLDs were facilitated via an Äkta Pure FPLC system. Glycoprotein-6D6 complexes were obtained by incubating each GPΔMLD with a 3-fold molar excess of 6D6 Fab overnight followed by purification using a Superdex 200 Increase 10/300 GL SEC column. The complexes were diluted to a concentration of 0.01 mg/mL, and 4 μL of the complex solutions were each applied to freshly plasma-cleaned carbon-coated 400 mesh copper grids (Electron Microscopy Sciences) for 1 minute. The solutions were blotted from the grids, followed by staining with 1% uranyl formate for 1 minute. The stain was then blotted from the grids, and the grids were allowed to air dry before imaging. TEM images were collected automatically using EPU on a FEI Titan Halo 300 kV electron microscope at a magnification of ×57000 with a Falcon II camera. CTF correction, particle picking, 2D class averaging, and 3D reconstruction and refinement were all carried out using cisTEM [11]. Data Availability Coordinates and structure factors for 6D6 Fab have been deposited into the Protein Databank under accession code 6DG2. Single-particle electron microscopy reconstructions of Ebolavirus GPs in complex with 6D6 Fab have been deposited into the Electron Microscopy Databank website under accession codes EMD-9048 and EMD-9049. RESULTS Crystal Structure of the Antigen Binding Fragment of 6D6 The crystal structure of unbound 6D6 Fab provides some insights into the nature of its binding site on the surface of the GP (Supplementary Figures S1 and S2 and Supplementary Table S1). It is notable that a majority of the CDRs contain hydrophobic, aromatic side chains. The CDR H3 contains 3 tyrosine residues, whereas CDR L3 contains a tyrosine as well as 2 prolines; in addition, CDR H1 contains 1 tyrosine and 1 phenylalanine. Also of note, CDR H2 contains 2 positively charged arginine side chains that can also stack with aromatic side chains through cation-π interactions. These structural features suggest a hydrophobic epitope on the GP surface, consistent with previously characterized escape mutants within the hydrophobic internal fusion loop (IFL) [9]; a negatively charged region within the epitope is also likely given the positive patch in CDR H2. Single-Particle Electron Microscopy Reconstructions of 6D6 Bound to Ebolavirus Glycoproteins To determine the epitope of 6D6, single-particle EM was used to generate reconstructions of 6D6 Fab in complex with GPΔMLD from EBOV and BDBV (Figure 1 and Supplementary Figure S3 ). These structures allow for the first side-by-side comparison of a pan-ebolavirus antibody bound to multiple Ebolavirus GPs. By docking the crystal structures of EBOV GPΔMLD [12] and the variable domain of 6D6 Fab into the reconstruction, the fusion peptide and the area surrounding were confirmed as the targeted footprint of 6D6. The antibody buries surface area on both the GP1 and GP2 subunits of the GP monomer, and the footprint is quaternary in nature, involving molecular surface belonging to GP2 of 2 adjacent monomers in the GP trimer. The interface predicted by the model displays a high degree of electrostatic complementarity (Supplementary Figure S4): the positively charged patch on CDR H2 binds near a negatively charged surface of GP1, whereas the hydrophobic CDRs pack against the hydrophobic fusion peptide. 6D6 binding to EBOV and BDBV GPΔMLD appears identical with similar electrostatic and hydrophobic complementarity. Fifty-eight percent of the proposed footprint residues are identical across the 5 known ebolaviruses, and 80% are identical or similar (Figure 2). Figure 1. View largeDownload slide Reconstruction by single-particle negative-stain electron microscopy of the antigen-binding fragment (Fab) of 6D6 bound to Ebola virus (EBOV) glycoprotein (GP). Glycoprotein and Fab densities are rendered as surfaces: GP densities are shown in gray, and the Fab densities are shown in light blue. The EBOV GP density is fit with the EBOV mucin-deleted GP (GPΔMLD) crystal structure shown in gray ribbon, and the Fab densities are fit with the crystal structure of 6D6 Fab shown in blue ribbon. (A) 6D6 Fab in complex with EBOV GPΔMLD shown from the side. (B) 6D6 Fab in complex with EBOV GPΔMLD shown from the top. The approximate location of where the mucin-like domain would be located for one of the monomers is indicated within the dashed circle. Figure 1. View largeDownload slide Reconstruction by single-particle negative-stain electron microscopy of the antigen-binding fragment (Fab) of 6D6 bound to Ebola virus (EBOV) glycoprotein (GP). Glycoprotein and Fab densities are rendered as surfaces: GP densities are shown in gray, and the Fab densities are shown in light blue. The EBOV GP density is fit with the EBOV mucin-deleted GP (GPΔMLD) crystal structure shown in gray ribbon, and the Fab densities are fit with the crystal structure of 6D6 Fab shown in blue ribbon. (A) 6D6 Fab in complex with EBOV GPΔMLD shown from the side. (B) 6D6 Fab in complex with EBOV GPΔMLD shown from the top. The approximate location of where the mucin-like domain would be located for one of the monomers is indicated within the dashed circle. Figure 2. View largeDownload slide Comparison of cross-reactive internal fusion loop antibody epitopes. The mucin-deleted Ebola virus (EBOV) glycoprotein (GPΔMLD) crystal structure is rendered as a surface shown in gray with antibody footprints colored as indicated. The path of the IFL base and stem is traced in light pink arrows, and the path of the fusion loop is traced in magenta arrows. A “*” indicates identical residues, a “:” indicates very similar residues, and a “.” indicates somewhat similar residues. (A) Sequence alignment of Ebolavirus GPs with antibody footprints of 6D6, ADI-15946, and CA45 colored in blue, red, and yellow, respectively. The shared footprint of ADI-15946 and CA45 is shown in orange. (B) Sequence alignment of Ebolavirus GPs with antibody footprints of 6D6 and ADI-15878 colored in blue and green, respectively. The shared footprint of 6D6 and ADI-15878 is shown in cyan. Figure 2. View largeDownload slide Comparison of cross-reactive internal fusion loop antibody epitopes. The mucin-deleted Ebola virus (EBOV) glycoprotein (GPΔMLD) crystal structure is rendered as a surface shown in gray with antibody footprints colored as indicated. The path of the IFL base and stem is traced in light pink arrows, and the path of the fusion loop is traced in magenta arrows. A “*” indicates identical residues, a “:” indicates very similar residues, and a “.” indicates somewhat similar residues. (A) Sequence alignment of Ebolavirus GPs with antibody footprints of 6D6, ADI-15946, and CA45 colored in blue, red, and yellow, respectively. The shared footprint of ADI-15946 and CA45 is shown in orange. (B) Sequence alignment of Ebolavirus GPs with antibody footprints of 6D6 and ADI-15878 colored in blue and green, respectively. The shared footprint of 6D6 and ADI-15878 is shown in cyan. Comparison of 6D6 With Other Antibodies Against the Internal Fusion Loop The IFL of GP is composed of a base and stem that together form an antiparallel β-sheet anchoring a central loop of ~25 residues that serves as the fusion peptide [12]. The base and stem affix the IFL assembly to the protein core through several hydrophobic interactions and hydrogen bonds, and the central fusion loop packs along the surface in the GP1/GP2 interface and extends to contact the neighboring GP monomer. The 6D6 footprint directly includes the hydrophobic fusion loop itself, in contrast to antibodies CA45 and ADI-15946, which bind to the IFL base and stem (Figure 2A) [6, 7]. CA45, a macaque-derived antibody, is broadly cross-reactive [7]. ADI-15946, a human antibody from natural infection, neutralizes EBOV and BDBV, but it only weakly neutralizes SUDV [6]. Ebola virus-specific antibodies KZ52, c4G7, and c2G4 also bind in this base and stem region [12, 13]. Their narrow breadth of recognition likely results from their slightly lower position on GP and resulting recognition of EBOV-specific residues in the GP2 N-terminal region. The footprint of 6D6 over the hydrophobic fusion loop instead more closely resembles that of the pan-ebolavirus antibody ADI-15878 and clonally related antibody ADI-15742, which also include the fusion loop in their footprints (Figure 2B) [6]. Indeed, the binding sites of 6D6, 15878, and 15742 overlap extensively, their angle of approach is highly similar, and the single particle EM reconstructions are nearly indistinguishable. The binding site and approach angle of mouse-derived REGN 3479 also appears highly similar to 6D6 and ADI-15878/ADI-15742 [5]. The EBOV-specific antibody mAb-100 also binds in this general area [14], although its binding site is lower on the GP and does not completely bury the fusion peptide. In addition, the approach angle of mAb-100 is rotated by ~90° relative to 6D6 and ADI-15878/ADI-15742. Hence, the higher position and recognition of the hydrophobic loop itself appear key for broad neutralization. DISCUSSION The IFL region of ebolavirus GPs has emerged as a potent site of immune vulnerability that is capable of eliciting a broadly neutralizing antibody response. However, the epitope typically referred to as the IFL is actually a collection of related yet distinct antigenic sites. The base/stem portion of the IFL, for example, has shown mixed results in eliciting a pan-ebolavirus neutralizing antibody response. One pan-ebolavirus antibody (CA45 [7]), an antibody of limited breadth (ADI-15946 [6]), and several EBOV-specific antibodies (KZ52 [12], c4G7, c2G4 [13]) bind near the fusion loop base and stem, with the EBOV-specific mAbs binding lower and the more broadly reactive antibodies binding higher on GP. In contrast, the fully cross-reactive 6D6 and ADI-15878/ADI-15742 recognize a distinct footprint that involves the central hydrophobic fusion loop itself and is almost completely conserved across the ebolaviruses. Strikingly similar antibodies of nearly identical footprint and orientation have been elicited against this site in both naturally infected humans (ie, ADI-15878/ADI-15742 [6]) as well as mice immunized by VLPs (ie, 6D6 [9]) or purified GP (ie, REGN 3479 [5]). As evidenced by 6D6 and ADI-15878/ADI-15742, the GP fusion peptide represents a site on the GP surface that should be a focal point in structure-based vaccine design. 6D6, however, has no binding activity to a synthetic peptide with the sequence of the EBOV GP fusion peptide [9], suggesting that the surface area surrounding the fusion peptide itself is also required for eliciting a 6D6-like pan-ebolavirus antibody response. Surrounding residues may maintain the fusion loop in the proper orientation and structure for recognition, or, as this structural data suggests, recognition likely extends beyond the fusion loop to additional conserved surfaces surrounding it. 6D6 and similar antibodies have additional potential for use in point-of-care (POC) diagnostics because their broad reactivity would allow identification of any known or possibly even novel emerging ebolavirus. Traditionally, most POC diagnostic assays for EVD utilize antibodies targeting internal viral proteins that are generally more conserved than GP [15]. The existence of antibodies such as 6D6, however, allows complementary GP-based identification. Furthermore, assays incorporating a combination of pan-ebolavirus and species-specific antibodies may be useful in rapid identification of a particular ebolavirus species. CONCLUSIONS In the future, high-resolution structural studies should aim to uncover the atomic-level differences between recognition by antibodies such as 6D6 and mAb-100 with overlapping epitopes but different levels of cross-reactivity. This structural information will aid in the design of optimized immunogens capable of reliably eliciting 6D6-like pan-ebolavirus antibodies. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Acknowledgments. We acknowledge Marnie Fusco and Kathleen Pommert for technical assistance, as well as the support staff of Beamline 23-ID-D at APS for assistance with diffraction data collection. This is manuscript no. 29724 from The Scripps Research Institute. Financial support. This work was funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health (Grants R01AI132204 and U19AI109762 [to E. O. S.] and 5T32AI007354-27 to [J. C. M.]), the Japan Agency for Medical Research and Development (AMED; Grant JP17fk0108101 [to A. T.]), and the Japan Society for the Promotion of Science , Ministry of Education, Culture, Sports, Science and Technology (KAKENHI; Grant 16H02627 [to A. T.]). 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TI - Structural Characterization of Pan-Ebolavirus Antibody 6D6 Targeting the Fusion Peptide of the Surface Glycoprotein
JF - The Journal of Infectious Diseases
DO - 10.1093/infdis/jiy532
DA - 2019-01-09
UR - https://www.deepdyve.com/lp/oxford-university-press/structural-characterization-of-pan-ebolavirus-antibody-6d6-targeting-7IFNMux58P
SP - 415
VL - 219
IS - 3
DP - DeepDyve
ER -