Immunoinformatics-based design of a multi-epitope mRNA vaccine candidate targeting the major human-infecting hepatitis E virus genotypes 1-4.

Background: Hepatitis E virus (HEV) is one of the leading causes of acute and chronic hepatitis among humans across the globe and is linked to severe diseases in immunocompromised individuals and pregnant women. Despite its substantial global burden, only one vaccine is licensed (in China and Pakistan), limiting its global accessibility. Furthermore, no globally approved mRNA vaccine against HEV is currently available, highlighting the need to develop strategies for alternative vaccines. Methods: In this study, the amino acid sequences of ORF2 capsid proteins representative of HEV genotypes 1-4 were curated, retrieved, and analyzed to generate a consensus sequence. The conserved cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B-cell epitopes were computationally screened based on their antigenicity, allergenicity, toxicity, IFN-γ induction, and human non-homology. Then, the selected epitopes, seven in total, were assembled into a computational multi-epitope mRNA construct incorporated with β-defensin adjuvant, correct linkers, and regulatory elements [Kozak, tPA, MITD, and globin untranslated regions (UTRs)]. The construct was subsequently subjected to structural modeling, refinement and validation, docked with the TLR1/TLR2 heterodimer, followed by normal mode analysis and a 100-ns molecular dynamics simulation with MM-GBSA binding free-energy estimation, immune simulations, codon optimization, and Results: The computational analyses predicted that the proposed epitopes possessed a favorable antigenicity (VaxiJen score ≥0.4-1), with non-allergenicity and epitope conservancy (80%-100%) across the analyzed HEV genotypes (1-4), and that the assembled vaccine construct was structurally stable. Protein-protein docking of the vaccine construct candidate with the TLR1/TLR2 heterodimer and normal mode analysis of the docked complex indicated a stable structural interface rather than a confirmation of biological receptor engagement; a subsequent 100-ns molecular dynamics simulation supported a persistent, increasingly hydrogen-bonded interface with a favorable MM-GBSA binding free-energy (-91.96 kcal/mol). Computational immune simulations predicted cellular and humoral immune responses with B- and T-cell interaction activity and cytokine production levels. Conclusion: This study describes a computationally designed multi-epitope mRNA vaccine candidate with predicted immunogenicity and structural stability against the major human-infecting HEV genotypes 1-4. These computational results support the proposed vaccine construct as a promising candidate for further research to assess its immunogenicity; safety and protective efficacy will require experimental validation before its translational potential is verified.