“Reverse Vaccinology and Immunoinformatics Based Design of an mRNA Multi-Epitope Vaccine against MPXV”

• Reverse vaccinology and Immunoinformatics based design an mRNA multi-epitope vaccine. • LBL, CTL, and HTL epitopes were identified and refined for constructed vaccine. • A final set of 8 LBL, 17 CTL, 4HTL, and 7 discontinuous B-cell epitopes (DSE) was identified. • CTL, HTL, and constructed vaccine showed strong binding affinity with MHC-I, MHC-II, TLR2, TLR3, and TLR4 in molecular docking analyses. • mRNA multi-epitope vaccine exhibits stable, antigenic, hydrophilic, and thermally tolerant properties In silico. In 2024, the World Health Organization classified MPXV infection as a public health emergency of international concern, underscoring the need for effective vaccine strategies. This study employed reverse vaccinology and immunoinformatics to design an mRNA-based multi-epitope vaccine candidate targeting extracellular enveloped virus (EEV), intracellular mature virion (IMV), and membrane-associated MPXV proteins. Cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and linear B-cell (LBL) epitopes were identified and screened using predefined immunological criteria. Selected epitopes were assembled into a multi-epitope construct using immunostimulatory adjuvants (β-defensin and LL-37) and suitable linkers, yielding a final set of 8 LBL, 17 CTL, and 4 HTL epitopes. The construct was computationally evaluated through sequence-based profiling and structural modeling, refinement, and validation. Receptor-interaction feasibility was assessed via docking with innate immune receptors (TLR2, TLR3, and TLR4) and supported by molecular dynamics analysis of complex stability. Immune simulation was performed to generate trend-level predictions of humoral and cellular response dynamics under a prime–boost schedule. Compared with prior MPXV in-silico vaccine studies, this work integrates a multi-protein target set with an mRNA-ready construct design and a consolidated computational evaluation pipeline. Overall, these findings present a hypothesis-generating MPXV mRNA-based multi-epitope vaccine candidate which can be strengthen by experimental validation to confirm safety and efficacy.