Identification of four neutralizing antigenic sites on the enterovirus D68 capsid

Abstract

Enterovirus D68 (EV-D68) is an emerging human pathogen associated with respiratory diseases and/or acute flaccid myelitis. Neutralizing antigenic sites of EV-D68 have not yet been comprehensively studied. In this study, we generated multiple neutralizing monoclonal antibodies (MAbs) directed against EV-D68 prototype or clinical strains. All these antibodies can inhibit EV-D68 attachment. The antibody epitopes were identified by selection and sequence analysis of prototype or clinical strain-derived neutralization-resistant mutants. The epitopes were then grouped into four distinct neutralizing antigenic sites (I to IV) by cross-neutralization analysis of the mutants with the MAbs and by spatial considerations. Site I, including residues 81, 85, and 87 of VP1 protein, is located in the VP1 BC loop, near the fivefold axis, and at the north rim of the canyon (the receptor binding site). Site II, involving residues 137, 139, and 142 of VP2, is situated in the VP2 EF loop and at the south rim of the canyon. Site III is composed of VP1 C-terminal residues 285 and 293 and resides on the south side of the canyon of neighboring asymmetric unit. Site IV contains residue 70 (βB strand) of VP2 from an asymmetric unit and residues 74 and 79 (BC loop) of VP3 from an adjacent unit and is located around the threefold axis. The four antigenic sites show various degrees of sequence variation. The identification of the four neutralizing antigenic sites on EV-D68 capsid provides a better understanding of the recognition of EV-D68 by neutralizing antibodies and viral evolution and immune escape. IMPORTANCE Enterovirus D68 (EV-D68) is an emerging respiratory pathogen associated with acute flaccid myelitis. Currently, no approved vaccines or antiviral drugs are available. Here, we report four functionally independent neutralizing antigenic sites (I to IV) by analyses of neutralizing monoclonal antibody (MAb)-resistant mutants. Site I is located in the VP1 BC loop near the fivefold axis. Site II resides in the VP2 EF loop, and site III is situated in VP1 C-terminus; both sites are located at the south rim of the canyon. Site IV is composed of residue in VP2 βB strand and residues in the VP3 BC loop and resides around the threefold axis. The developed MAbs targeting the antigenic sites can inhibit viral binding to cells. These findings advance the understanding of the recognition of EV-D68 by neutralizing antibodies and viral evolution and immune escape and also have important implications for the development of novel EV-D68 vaccines.

Structure and dynamics of enterovirus genotype networks

“…It is notable that the shared sites of variability in EV-A71 and EV-D68, which were observed here in conditions of relaxed immune selection, overlap with those identified under antibody selection ( 17 ). It might suggest that these sites, which are polymorphic in both species, have evolved to be mutationally robust in response to past immune selection at this immunodominant site, in a sense, positioning drift and immune escape as parallel mutational forces as described for murine norovirus, a relative in the order Picornavirales ( 32 ) Moreover, most of the non-synonymous mutations that we observed in our genotypes were identified in previous studies of EV-A71 and EV-D68 adaptation in cultured cells or in vivo models ( 33 , 34 ).…”

“…However, mapping these substitutions on the structure of the EV-D68 capsid ( 16 ) revealed that they either directly overlap with (T208A, VP2.T139) or are located nearby to escape variants identified in a study of neutralizing antigenic sites (K834R and Q835E, near the antigenic site at VP1.285). The T208 site in VP2 represents a shared antigenic site between the EV-D68 capsid and those of EV-A71 (at T210) and CV-A10 ( 17 ).…”