Hand, foot and mouth disease (HFMD) is mainly caused by EV-A71 and CV-A16. An increasing number of cases have been found to be caused by CV-A10, CV-A6, CV-B3 and the outbreaks are becoming increasingly more complex, often accompanied by the prevalence of a variety of enteroviruses. Based on the principle of synthetic peptide vaccines, we applied immune-informatics to design a highly efficient and safe multivalent epitope-based vaccine against EV-A71, CV-A16, CV-A10, CV-A6 and CV-B3. By screening B-cells, HTL and CTL cell antigen epitopes with high conservativity and immunogenicity that have no toxic effect on the host, further analysis confirmed that the vaccine built was IFN-γ inductive and IL-4 non-inductive HTL cell epitopes and had population coverage corresponding to MHC molecular alleles associated with T-cell phenotype. The multivalent enterovirus vaccine was constructed to connect the 50 s ribosomal protein L7/L12 adjuvant and candidate epitopes sequentially through appropriate linkers. Then, the antigenic, allergen and physical properties of the vaccine were evaluated, followed by a secondary structure analysis and tertiary structure modeling, disulfide engineering, refinement and validation. Moreover, the conformational B cell epitope of the vaccine was analyzed. The stability of the TLR4/MD2/Vaccine complex and details at atomic level were investigated by docking and molecular dynamics simulation. Finally, in silico immune simulation and in vivo immune experiments were done. This study provides a high cost-effective method of designing a multivalent enterovirus vaccine protect against a wide range of enterovirus pathogens.

手足口病（HFMD）主要由EV-A71和CV-A16引起。已经发现越来越多的病例是由CV-A10，CV-A6，CV-B3引起的，并且爆发变得越来越复杂，通常伴随着多种肠病毒的流行。基于合成肽疫苗的原理，我们应用免疫信息学设计了针对EV-A71，CV-A16，CV-A10，CV-A6和CV-B3的高效，安全的多价表位疫苗。通过筛选对宿主无毒作用的高保守性和免疫原性的B细胞，HTL和CTL细胞抗原表位，进一步分析证实所构建的疫苗是IFN-γ诱导型和IL-4非诱导性HTL细胞表位，并具有人群覆盖率对应于与T细胞表型相关的MHC分子等位基因。构建了多价肠病毒疫苗，以通过适当的接头依次连接50 s核糖体蛋白L7 / L12佐剂和候选表位。然后，评估疫苗的抗原，过敏原和物理特性，然后进行二级结构分析和三级结构建模，二硫键工程，精制和验证。此外，分析了疫苗的构象B细胞表位。通过对接和分子动力学模拟研究了TLR4 / MD2 /疫苗复合物的稳定性和原子水平的细节。最后，进行了计算机免疫模拟和体内免疫实验。这项研究为设计针对多种肠道病毒病原体的多价肠病毒疫苗提供了一种经济高效的方法。