Immunodominant IgM and IgG Epitopes Recognized by Antibodies Induced in Enterovirus A71-Associated Hand, Foot and Mouth Disease Patients

Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD). Unlike other enteroviruses that cause HFMD, EV-A71 is more frequently associated with severe neurological complications and fatality. To date, no effective licensed antivirals are available to combat EV-A71 infection. Little is known about the immunogenicity of viral non-structural proteins in humans. Previous studies have mainly focused on characterization of epitopes of EV-A71 structural proteins by using immunized animal antisera. In this study, we have characterized human antibody responses against the structural and non-structural proteins of EV-A71. Each viral protein was cloned and expressed in either bacterial or mammalian systems, and tested with antisera by western blot. Results revealed that all structural proteins (VP1-4), and non-structural proteins 2A, 3C and 3D were targets of EV-A71 IgM, whereas EV-A71 IgG recognized all the structural and non-structural proteins. Sixty three synthetic peptides predicted to be immunogenic in silico were synthesized and used for the characterization of EV-A71 linear B-cell epitopes. In total, we identified 22 IgM and four IgG dominant epitopes. Synthetic peptide PEP27, corresponding to residues 142–156 of VP1, was identified as the EV-A71 IgM-specific immunodominant epitope. PEP23, mapped to VP1 41–55, was recognized as the EV-A71 IgG cross-reactive immunodominant epitope. The structural protein VP1 is the major immunodominant site targeted by anti-EV-A71 IgM and IgG antibodies, but epitopes against non-structural proteins were also detected. These data provide new understanding of the immune response to EV-A71 infection, which benefits the development of diagnostic tools, potential therapeutics and subunit vaccine candidates.

肠病毒A71（EV-A71）是手足口病（HFMD）的主要致病因子之一。与其他导致HFMD的肠道病毒不同，EV-A71更常与严重的神经系统并发症和死亡率相关联。迄今为止，尚无有效的授权抗病毒药物可用于对抗EV-A71感染。关于病毒非结构蛋白在人类中的免疫原性知之甚少。先前的研究主要集中于使用免疫动物抗血清对EV-A71结构蛋白表位进行表征。在本研究中，我们对EV-A71的结构蛋白和非结构蛋白的人类抗体反应进行了表征。每种病毒蛋白均被克隆并在细菌或哺乳动物系统中表达，并通过Western blot与抗血清进行测试。结果显示，所有结构蛋白（VP1-4）和非结构蛋白2A、3C和3D均为EV-A71 IgM的靶点，而EV-A71 IgG则识别所有结构蛋白和非结构蛋白。合成了63个预测在计算机模拟中具有免疫原性的合成肽，用于EV-A71线性B细胞表位的表征。总共，我们鉴定出22个IgM和4个IgG优势表位。与VP1的142-156位氨基酸残基相对应的合成肽PEP27被鉴定为EV-A71 IgM特异性的免疫优势表位。PEP23，定位在VP1的41-55位，被识别为EV-A71 IgG交叉反应的免疫优势表位。结构蛋白VP1是抗-EV-A71 IgM和IgG抗体靶向的主要免疫优势位点，但针对非结构蛋白的表位也已被检测到。这些数据为EV-A71感染免疫反应的新理解提供了依据，这对诊断工具、潜在治疗药物和亚单位疫苗候选物的开发具有重要意义。