Fig 1.
(A) Overall summary of mAb generation, characterization, in vitro neutralization of MARV GP-pseudotyped VSV, and in vivo protection in mice. Antibodies were generated from a single immunogen with the exception of 54G1, 54G2 and 54G3 which were primed with Ravn GPΔmuc and boosted with GPΔmuc 30G4 Fab complex. (B) ELISA EC50 values against different forms of purified MARV Ravn GP or against VLPs in ng/ml. Relative potency is indicated by orange highlight (high, EC50 <20ng/ml), dark yellow (medium, EC50 20-200ng/ml), or light yellow (low, EC50 >200ng/ml). (C) Relative ELISA binding to purified mucin-containing GPs from other MARV strains. No binding at 10μg/ml maximum mAb concentration is represented by >.
Fig 2.
GP schematic and GP2-wing epitope analysis.
(A) Schematic of purified GP ectodomains used in this study. Dashed lines represent deleted regions. SS, signal sequence; MLD, mucin-like domain; IFL, internal fusion loop; TM, transmembrane domain. A red triangle indicates the furin cleavage site, numbered in red. The GP2-wing region, which is unique to MARV, is colored orange. B) MARV sequence alignment of pepscan defined epitopes for anti-GP2 wing mAbs. This region has four residues unique to strain Ravn; notably, 465E is 465K in other strains. (C) ELISA binding of GP2-wing mAbs to wild type (wt) and E465K Ravn GPΔmuc at 2 and 0.2μg/ml.
Fig 3.
In vitro neutralization activity.
Neutralization potency of mAbs against VSV pseudotyped with MARV Ravn GP (VSVΔG Ravn GP) in Vero cells. VSVΔG MARV Ravn GP was incubated with 50μg/ml of the indicated mAb for 1 hour before infection, and entry efficiency was calculated based on GFP expression. Positive control is human survivor mAb MR78.
Fig 4.
Groups of BALB/c mice at 10 animals per group were injected with individual mAbs one hour after challenge with mouse-adapted MARV Ravn virus. Two separate studies are represented; treatment groups are broken up into 3 or 4 mAbs to simplify survival and health score graphs. Studies continued for 28 days total, however no additional changes were observed beyond day 14. Asterisks represent P value summaries with non-significant curves labeled ns. (A) Study #1. PBS control survival is 10%. (B) Study #2. PBS control survival is 0%.
Fig 5.
Filovirus GP cross-reactivity of 40G1 and 2D8.
(A) Reactivity of 40G1 and 2D8 mAbs to GP antigens determined by ELISA at 5μg/ml. SUDV, Sudan virus; BDBV, Bundibugyo virus; RESTV, Reston virus. (B) Binding curves determined by ELISA with mAb serial dilutions starting at 20μg/ml. Note that MARV GPΔmuc, MARV GPcl, and EBOV GPcl curves overlay in both graphs.
Fig 6.
Negative stain EM and modeling of Fabs bound to MARV GP.
Representative 2D class averages of MARV Ravn GP:Fab complexes (A) GPΔmuc + 9A11 (B) GPΔmuc + 30G5 (C) GP + 30G5. (D) The crystal structure of MARV GPcl is shown with the unresolved GP2-wing region (436–510) depicted by a dashed orange line. The footprints of 9A11 and 30G5 Fabs are unknown; possible binding areas are highlighted by gray dotted ovals. For simplification, Fab binding regions are highlighted on only one monomer of the trimer in each view.