Identification of key amino acids defining conformational neutralizing epitopes of coxsackievirus A5 using monoclonal antibody escape mutants.

BACKGROUND

Coxsackievirus A5 (CVA5) is an emerging pathogen associated with severe hand, foot, and mouth disease. Protective antibodies are critical for protection from CVA5 infection, yet the antigenic determinants of CVA5 remain poorly defined. In this study, we aimed to map the conformational neutralizing epitopes of CVA5 using monoclonal antibodies (mAbs) and escape mutant analysis.

METHODS

Two IgG, one IgA, and five IgM neutralizing mAbs were purified and characterized for binding affinity and neutralization potency. Antibody pressure selection was applied to generate immune escape mutants, and comparative sequence analysis with wild-type virus identified potential critical residues. Reverse genetics was used to confirm key sites pivotal for mAb recognition. Structural mapping was performed to localize these sites on the viral capsid.

RESULTS

Ten key sites (K1103, V1215, N1282, F1288, T1291, K2076, E2160, T3059, D3060, E3139) were confirmed to be pivotal for mAb recognition. Structural mapping revealed distinct localization of these sites on the viral capsid: V1215, N1282, F1288, T1291, K2076, E2160, T3059, D3060 at the southern rim; K1103 at the canyon's northern rim; and E3139 near the two-fold axis.

CONCLUSION

This study provides the first evidence of multiple conformational neutralizing epitopes on CVA5. These findings define the antigenic basis of CVA5 neutralization and offer a structural framework for vaccine and antibody-based therapeutic development.