Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody

Abstract

Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection.

Author Summary

Hand, foot and mouth disease (HFMD) caused by enterovirus infection is an infectious disease affecting millions of young children annually. Enterovirus 71 (EV71) is the major causative agent of severe HFMD with central nervous system complications. However, no prophylactic vaccine or therapeutic drug is available against EV71 so far. We previously identified a murine monoclonal antibody D5 with potent neutralization effect on EV71, yet its working mechanism remains elusive. In the current study we aim to unravel the structural basis of D5-mediated neutralization of EV71. Relative high-resolution cryo-EM analysis of EV71 particles in complex with IgG or Fab of D5 revealed a bivalent binding mode of D5 across the 2-fold axis of EV71 virion. We also found that the CDR3 of D5 heavy chain bound the VP1 GH-loop of EV71, which represents a broadly neutralizing epitope and is thought to mediate EV71 binding with its uncoating receptor SCARB2. Based on these observations, we propose that D5 neutralizes EV71 infection through competing with SCARB2 for a binding site at the VP1 GH-loop and/or inhibiting conformational change of the virus. We further showed that D5 treatment efficiently protected mice from lethal EV71 infection. Our work provides information that may facilitate the development of D5 antibody-derived anti-EV71 drugs.