Antigenic characterization and mapping of a conserved neutralizing epitope on VP1 of coxsackievirus A5

Coxsackievirus A5 (CV-A5) has re-emerged as an important pathogen associated with hand, foot, and mouth disease (HFMD) in young children. However, the antigenic structure of CV-A5 and the key determinants responsible for neutralizing antibody responses remain largely undefined, hindering rational vaccine design and antibody-based intervention strategies.

To investigate the relationship between viral particle composition and humoral immune responses, three forms of CV-A5 particles, including empty particles (EPs), full particles (FPs), A-particles (APs) were purified from the candidate vaccine strain CV-A5-3487, and artificial B-particles (BPs) were generated by heat treatment of FPs. These four particles were evaluated in mice. A mouse-adapted strain (CV-A5-611) was used to mimic natural infection for monoclonal antibody generation, and neutralizing hybridomas were rapidly screened using a fluorescent reporter virus (CV-A5-r611-iLOV). Linear epitope mapping was performed using truncated peptides and alanine scanning peptides, followed by sequence conservation analysis. Protective efficacy of the neutralizing antibody was evaluated in a lethal mouse challenge model.

Among the four particle types, FPs elicited the most potent neutralizing antibody responses, indicating that intact virions are critical for inducing effective humoral immunity. Hybridoma screening generated 617 ELISA-positive clones, of which 84 (13.61%) exhibited neutralizing activity against CV-A5. Ten neutralizing monoclonal antibodies (mAb) were obtained after subcloning, and only one antibody, 4D6, recognized a linear epitope. Epitope mapping identified the binding site within VP1 residues 155-169 (VPPGAPVPTGRDTFQ), with the minimal neutralizing core epitope defined as 158GAPVPTGR165. This epitope showed high sequence conservation (90.5%) among CV-A5 strains, suggesting a structurally constrained neutralization site. Passive transfer experiments demonstrated that mAb 4D6 conferred complete protection against lethal CV-A5 challenge at a dose of 20 μg per mouse, confirming its strong in vivo protective activity.

Our study provides a systematic comparison of the immunogenicity of distinct CV-A5 particle forms and identifies a highly conserved linear neutralizing epitope on VP1 for the first time. The potent protective efficacy of mAb 4D6 highlights the importance of this epitope as a target for therapeutic antibody development and rational vaccine design, and provides new insights into the mechanisms of protective humoral immunity against CV-A5 infection.