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.

Cryo-EM Studies of Virus-Antibody Immune Complexes

“…Compared with 1D5, Fab MA28-7 is closer to the symmetry axis, which renders steric hindrance between possible Fabs, thereby limiting the number of bound Fabs. As another example, the bivalent binding pattern of D5 was characterized in which the two Fab IgG fragments could bind to the GH loops of neighboring VP1 molecules related by twofold symmetry, a finding consistent with the observation that D5 IgG was able to neutralize EV-A71 much more potently than D5 Fab (Ye et al 2016). Contrastingly, the 22A12 binding sites near twofold axes on EV-A71 are further apart and bivalent binding of an antibody cannot occur (Shingler et al 2015).…”

“…1A, 1B), a situation that could result in highly heterogeneous antigen-antibody complexes if one antigen particle becomes crosslinked with another. Except for SPA studies on IgG bivalency (Ye et al 2016), IgG is usually used in cryo-ET studies at medium resolution for HIV-1 (Tran et al 2012), influenza (Tran et al 2016b) and Ebola viruses (Tran et al 2016a). Compared with the intact IgG molecule, the monovalent Fab fragments generated by papain digestion of whole IgG molecules are more commonly used for structural studies on virus-antibody complexes (Tables 1, 2, 3).…”

“…Site 1 is located near the icosahedral fivefold axis of EV-A71 (MA28-7), CV-A6 (1D5)) and EV-D68 (11G1)(Zheng et al 2019). While site 2 maps to the VP1 GH-loop across the twofold axis of EV-A71 (22A12 and D5)(Shingler et al 2015;Ye et al 2016), site 3 is situated near the threefold axis of EV-A71 (E18, E19 and A9)(Plevka et al 2014;Zhu L et al 2018b) and EV-D68 (15C5)(Zheng et al 2019). Site 4 is adjacent to the quasi threefold axis of CV-A10 (2G8)(Zhu R et al 2018).Some clinically relevant viruses in the Flaviviridae family, such as dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV) and Zika virus (ZIKV)…”

Rational Engineering and Characterization of an mAb that Neutralizes Zika Virus by Targeting a Mutationally Constrained Quaternary Epitope

“…The spacing between the bound Fab fragments suggests the possibility of a bivalent binding pattern of ZAb_FLEP antibody, wherein the two arms (Fab) of a single IgG contact the epitopes near the 2-fold and 3-fold icosahedral axes, respectively ( Figure 6B ). The binding mode of the Fabs and the distances between the heavy-chain C terminus of adjacent Fab fragments have been used by others to evaluate such a bivalent mode of engagement (Ye et al, 2016). The epitope specificity of ZAb_FLEP mapping to the fusion loop peptide combined with its ability to lock the intra-dimer and inter-dimer interfaces suggests that the monoclonal antibody (mAb) neutralizes by preventing the necessary conformational changes required for the fusion event.…”

Enterovirus 71 infection impairs the reproductive capacity of female mice

“…Enterovirus (EV) 71 is a member of the picornaviridae family of viruses that contain single-stranded positive-sense RNA (1), which primarily infects children <5 years old (2). Infants with EV71 infection develop sores on their hands, feet, buttocks and mouth; therefore, the infection is referred to as hand, foot and mouth disease (HFMD) (3).…”