Abstract: To evaluate the heterologous cross-neutralizing capacity of antisera induced by an inactivated EV-A71 vaccine (FY-23K-B strain) against common non-EV-A71 enteroviruses associated with hand-foot-mouth disease (HFMD), Wistar rats were immunized with the vaccine, and microneutralization assays were performed against 18 clinical enterovirus isolates, comprising one EV-A71 isolate and 17 non-EV-A71 isolates. P1-region phylogeny and sequence identity were analyzed, together with the local sequence conservation and structural localization of the EV-A71 VP1-derived linear epitope K1, which has previously been reported to be associated with enterovirus cross-neutralization. The antiserum showed a homologous neutralizing titer of 1 024 against EV-A71. Low-titer neutralizing activity was detected against eight heterologous enteroviruses, including CV-A6 of the EV-A species and CV-A9, CV-B4, ECHO6, ECHO11, ECHO14, ECHO30, and ECHO33 of the EV-B species. Among these, CV-A9 and ECHO14 showed relatively higher titers of 16 and 12, respectively, whereas no detectable neutralizing activity was observed against the remaining heterologous serotypes. Although most isolates with detectable cross-neutralization belonged to the EV-B species, neutralizing titers showed no clear correspondence with overall P1 sequence identity. The K1 epitope showed partial conservation in isolates with and without detectable cross-neutralization, suggesting that a single linear epitope is insufficient to determine the cross-neutralization phenotype. Overall, the inactivated EV-A71 vaccine elicited only limited, low-titer in vitro neutralizing activity against heterologous HFMD-associated pathogens, which may involve the coordinated conservation of multiple local antigenic epitopes and their spatial conformational accessibility. This study provides experimental evidence for understanding the heterologous cross-neutralizing characteristics and potential molecular basis of the inactivated EV-A71 vaccine, and may serve as a reference for future screening of broadly neutralizing epitopes and the design of multivalent HFMD vaccines.
Keywords: hand-foot-mouth disease; EV-A71; inactivated vaccine; cross-neutralization; enterovirus
（Acta Laser Biology Sinica, 2026, 35(4): 326-336）