Identification of specific and shared epitopes at the extreme N-terminal VP1 of Coxsackievirus A4, A2 and A5 by monoclonal antibodies

Hand, foot and mouth disease (HFMD) is caused by a variety of serotypes in species A of the Enterovirus genus, including recently re-emerged Coxsackievirus A2 (CV-A2), CV-A4 and CV-A5. For development of diagnostic reagents, for surveillance, and the development of multivalent vaccines against HFMD, the antigenicity of HFMDassociated enteroviruses warrants investigation. The purified virions of CV-A4 were inoculated into Balb/c mice and hybridomas were obtained secreting monoclonal antibodies (mAbs) directed against CV-A4 and crossreacting with other closely related species A enteroviruses. The mAbs were characterized by ELISA, Western blotting and in vitro neutralizing assays. The majority of mAbs was non-neutralizing, with only 2% of the mAbs neutralizing CV-A4 specifically. Most of mAbs bound to linear VP1 epitopes of CV-A4. Interestingly, four types of mAbs were obtained which bound specifically to CV-A4 or were broadly to CV-A4/-A2, CV-A4/-A5 and CV-A4/A2/-A5, respectively. Mapping with overlapping or single-amino-acid mutant peptides revealed that the four types of mAbs all bound to the first 15 amino acids at the N-terminus of the VP1. This region of picornaviruses is functionally important as it is involved in uncoating and releasing of viral RNA into the cytosol. The binding footprints of four type mAbs are composed of conserved and variable residues and are different from each other. The newly discovered broadly cross-reactive mAbs reflect the high homology of CV-A4/ CV-A2/CV-A5. The results also demonstrate that it is possible and beneficial to develop the diagnostic reagents to detect rapidly the main pathogens of enteroviruses associated with HFMD cause by CV-A4/CV-A2/CV-A5.