Structural insight into rabies virus neutralization revealed by an engineered antibody scaffold
Host-cell entry of the highly pathogenic rabies virus (RABV) is mediated by trimeric glycoprotein (G) spikes, which also represent the primary target for the humoral immune response. RABV-G displays several antigenic sites targeted by neutralizing antibodies, including monoclonal antibodies (mAbs) which have been proposed as quality-controlled alternatives to traditional polyclonal rabies immunoglobulin treatment. In this study, we determine the epitope of a potently neutralizing human anti-rabies mAb, CR57, which we engineered into a diabody to facilitate crystallization. We report the crystal structure of the CR57 diabody alone at 2.38 [A] resolution, and in complex with RABV-G domain III at 3.15 [A] resolution. CR57 is demonstrated to bind RABV through a predominantly hydrophobic interface, with essential interactions targeting a conserved six-residue peptide sequence KLCGVL on the RABV-G. Further, our structural analysis suggests that CR57 sterically hinders receptor recognition and the fusogenic transitions of the spike protein that are required for host-cell entry. Altogether, this investigation provides a structural perspective on rabies inhibition by a potent antibody and delineates a functionally significant region in the spike. This understanding could pave the way for the development of prophylactic antibodies and other therapeutic strategies. Author summaryRabies virus (RABV) and many other lyssaviruses possess the ability to invade the central nervous system, leading to fatal encephalitis in mammals. Initiation of the infectious cycle depends on host cell recognition and entry, which is mediated by viral surface glycoprotein (G) spikes and can be inhibited by spike-targeting neutralizing antibodies. In our study, we elucidated the crystal structure of an antigenic domain from RABV-G in complex with a diabody derived from the potently neutralizing antibody CR57. This investigation revealed the molecular interactions by which CR57 binds to RABV-G and outlined a site of vulnerability comprising a conserved peptide in RABV-G domain III, where antibody binding is likely to inhibit RABV by obstructing host cell entry. Insights into the binding modalities of antibodies like CR57 deepen our understanding of how RABV and other lyssaviruses are neutralized, aiding the development of potential therapeutics. Furthermore, our study showcases the utility of engineering antibodies into diabodies to obtain crystal structures of antibody-antigen complexes.