摘 要：为评估肠道病毒A种71型（EV-A71）灭活疫苗对手足口病（HFMD）常见非EV-A71病原的异源交叉中和能力，本研究以FY-23K-B株灭活疫苗免疫Wistar大鼠，采用微量中和试验检测疫苗免疫血清对18株肠道病毒临床分离株的中和活性，其中包括1株EV-A71及17株非EV-A71肠道病毒，结合各病毒型别P1区系统进化分析结果，同时对既往报道中与EV交叉中和相关的EV-A71 VP1来源线性表位K1开展局部序列保守性和结构定位分析。结果显示：抗血清对同源EV-A71的中和效价为1 024；仅对EV-A种的CV-A6以及EV-B种的CV-A9、CV-B4、ECHO6、ECHO11、ECHO14、ECHO30和ECHO33有弱中和能力，其中对CV-A9和ECHO14的中和效价相对较高，分别为16和12；其余9株异源肠道病毒未检出中和活性。尽管交叉中和阳性毒株多属于EV-B种，但其与P1区整体序列的一致性无明确对应关系。K1对应区域在部分EV-B种交叉中和阳性和阴性毒株中均呈现出一定的序列保守性，提示单一线性表位不足以决定交叉中和表型。综上，EV-A71灭活疫苗对异源HFMD病原仅显示有限、低效价的体外中和作用，这一现象可能与多个局部抗原表位的协同保守性及空间构象可及性相关。本研究为阐明EV-A71灭活疫苗的异源交叉中和特征及其潜在分子基础提供了一定的试验依据，为后续广谱中和表位筛选和多价HFMD疫苗设计提供了参考。

关键词：手足口病；肠道病毒A种71型；灭活疫苗；交叉中和；肠道病毒
中图分类号：R373.2；R512.5                    文献标志码：A                DOI：10.3969/j.issn.1007-7146.2026.04.005

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）