Two Synthetic Antibodies that Recognize and Neutralize Distinct Proteolytic Forms of the Ebola Virus Envelope Glycoprotein

Ebola virus (EBOV) is a highly pathogenic member of the Filoviridae family of viruses that causes severe hemorrhagic fever. Infection proceeds through fusion of the host cell and viral membranes, a process that is mediated by the viral envelope glycoprotein (GP). Following endosomal uptake, a key step in viral entry is the proteolytic cleavage of GP by host endosomal cysteine proteases. Cleavage exposes a binding site for the host cell receptor Niemann‐Pick C1 (NPC1) and may induce conformational changes in GP leading to membrane fusion. However, the precise details of the structural changes in GP associated with proteolysis and the role of these changes in viral entry have not been established. Here, we have employed synthetic antibody technology to identify antibodies targeting EBOV GP prior to and following proteolysis (i.e. in the “uncleaved” [GP_UNCL] and “cleaved” [GP_CL] forms). We identified antibodies with distinct recognition profiles: Fab_CL bound preferentially to GP_CL (EC₅₀=1.7 nM ), whereas Fab_UNCL bound specifically to GP_UNCL (EC₅₀=75 nM ). Neutralization assays with GP‐containing pseudotyped viruses indicated that these antibodies inhibited GP_CL‐ or GP_UNCL‐mediated viral entry with specificity matching their recognition profiles (IC₅₀: 87 nM for IgG_CL; 1 μM for Fab_UNCL). Competition ELISAs indicate that Fab_CL binds an epitope distinct from that of KZ52, a well‐characterized EBOV GP antibody, and from that of the luminal domain of NPC1. The binding epitope of Fab_UNCL was also distinct from that of KZ52, suggesting that Fab_UNCL binds a novel neutralization epitope on GP_UNCL. Furthermore, the neutralizing ability of Fab_CL suggests that there are targets on GP_CL available for neutralization. This work showcases the applicability of synthetic antibody technology to the study of viral membrane fusion, and provides new tools for dissecting intermediates of EBOV entry.