Conservation guided reverse vaccinology for the design of a broadly immunogenic multi-epitope vaccine against monkeypox virus

Mpox, caused by the Monkeypox virus (MPXV), has emerged as a global public health concern following outbreaks beyond its traditional endemic regions. Although existing smallpox-derived vaccines provide partial protection, their limitations and declining population immunity highlight the need for safer and more targeted vaccine strategies. This study aimed to identify conserved antigenic targets and design a multi-epitope vaccine candidate against MPXV using a conservation-guided immunoinformatics approach. A dataset of 27 complete MPXV genomes (2001–2025) from diverse geographic regions was analyzed to identify highly conserved viral genes. Conserved surface-exposed proteins were prioritized for epitope prediction, including cytotoxic T-lymphocyte, helper T-lymphocyte, and B-cell epitopes. High-confidence epitopes were selected based on binding affinity, antigenicity, immunogenicity, toxicity, allergenicity, and absence of human sequence homology. Promiscuous epitopes with broad human leukocyte antigen (HLA) binding were curated to maximize population coverage. Three multi-epitope vaccine constructs were designed using appropriate linkers and adjuvants. Structural modeling, molecular docking with innate immune receptors, immune response simulation, and codon optimization were performed to evaluate the constructs. All constructs were predicted to be stable, antigenic, and non-allergenic. Molecular docking indicated strong interactions of vaccine construct 2 (VC2) with TLR2 and the TLR4–MD-2 complex, achieving the highest binding affinities (ΔG = − 19.6 and − 27.8 kcal/mol, respectively). Immune simulations predicted robust humoral and cellular responses, including strong antibody production and interferon-γ induction for VC2. Population coverage analysis estimated 99.27% global coverage, and codon optimization suggested efficient expression in Escherichia coli . Overall, VC2 emerges as a promising multi-epitope vaccine candidate with strong predicted immunogenic and structural properties. While these computational findings lay a solid foundation, VC2 require further experimental validation and preclinical development as a targeted vaccine strategy against MPXV.