In silico design and immunogenicity evaluation of a multi-epitope vaccine against EV-A71.

Enterovirus A71 (EV-A71), the primary causative agent of hand, foot, and mouth disease (HFMD), can cause severe neurological complications and even death, particularly in young children. Despite the availability of inactivated vaccines, their protective efficacy has been compromised due to frequent intra- and intertypic recombination events and ongoing mutations among circulating EV-A71 strains. To address this, we employed immunoinformatic approaches and identified conserved epitopes and constructed a multi-epitope vaccine (MEV) candidate against EV-A71. A total of 1,627 structural protein sequences from EV-A71 strains encompassing all major circulating subtypes were retrieved and aligned to generate a consensus sequence. With this consensus sequence, 11 conserved, antigenic, and non-allergenic epitopes capable of eliciting B-cell, T-cell, and interferon-gamma (IFN-γ) responses were identified. The constructed MEV demonstrated superior immunological potential with a high antigenicity score of 0.94 and was predicted to be non-allergenic and non-toxic. Structural characterization via AlphaFold 3 and 300 ns molecular dynamics (MD) simulations confirmed the formation of a stable β-strand framework. Molecular docking followed by trajectory-stabilized interaction analysis revealed that the MEV maintains a high-affinity and stable binding profile with Toll-like receptor 3 (TLR-3). To ensure optimal translational efficiency, the vaccine gene was codon-optimized with a GC content of 52.8%, and the protein was successfully expressed in a bacterial system. Collectively, this study provides a high-performance MEV candidate with robust structural stability and potent immunogenicity, offering a promising and cost-effective strategy for broad-spectrum protection against EV-A71.