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

Background: 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. Methods: 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. Results: 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 Conclusion: 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.