The current global outbreak of monkeypox (MPX) disease, caused by Monkeypox virus (MPXV), has resulted in 16 thousand infection cases, five deaths, and has been declared a global health emergency of international concern by the World Health Organization. Given current challenges in the safety of existing vaccines, a vaccine to prevent MPX infection and/or onset of symptoms would significantly advance disease management. In this context, a multi-epitope-based vaccine could be a well-suited approach. Herein, we searched a publicly accessible database (Virus Pathogen Database and Analysis Resource) for MPXV immune epitopes from various antigens. We prioritized a group of epitopes (10 CD8+ T cells and four B-cell epitopes) using a computer-aided technique based on desirable immunological and physicochemical properties, sequence conservation criteria, and non-human homology. Three multi-epitope vaccines were constructed (MPXV-1–3) by fusing finalized epitopes with the aid of appropriate linkers and adjuvant (beta-defensin 3, 50S ribosomal protein L7/L12, and Heparin-binding hemagglutinin). Codon optimization and in silico cloning in the pET28a (+) expression vector ensure the optimal expression of each construct in the Escherichia Coli system. Two and three-dimensional structures of the constructed vaccines were predicted and refined. The optimal binding mode of the construct with immune receptors [Toll-like receptors (TLR2, TLR3, and TLR4)] was explored by molecular docking, which revealed high docking energies of MPXV-1–TLR3 (–99.09 kcal/mol), MPXV-2–TLR3 (–98.68 kcal/mol), and MPXV-3–TLR2 (–85.22 kcal/mol). Conformational stability and energetically favourable binding of the vaccine-TLR2/3 complexes were assessed by performing molecular dynamics simulations and free energy calculations (Molecular Mechanics/Generalized Born Surface Area method). In silico immune simulation suggested that innate, adaptive, and humoral responses will be elicited upon administration of such potent multi-epitope vaccine constructs. The vaccine constructs are antigenic, non-allergen, non-toxic, soluble, topographically exposed, and possess favourable physicochemical characteristics. These results may help experimental vaccinologists design a potent MPX vaccine.
