Background: In 2022, the World Health Organisation (WHO) announced new cases of the developing Monkeypox Virus (MPXV), a zoonotic orthopoxvirus viral infection that mimics smallpox signs. Despite the ongoing infection, no proper medication is available to completely overcome this infection.

Aim: The study aims to construct a multi-epitope vaccine targeting Monkeypox Virus (MPXV) membrane glycoprotein to provoke robust immune responses.

Objective: To construct a potential immuno-dominant epitope vaccine to combat MPXV.

Methods: The target sequence, sourced from the UAE-to-India travel case, was analyzed to identify potential B-cell and T-cell epitopes (MHC-I and MHC-II). Immunodominant epitopes were selected and fused with β-defensin-I and PADRE to increase immunogenicity. The vaccine was modeled, docked with TLR3, and subjected to a 500 ns molecular dynamics simulation for stability analysis. Immune responses and bacterial expression were also evaluated.

Results: The vaccine, comprising 230 amino acids, demonstrated antigenicity (0.6620), non-allergenicity, and broad population coverage. Selected epitopes included 3 B-cells, 4 MHC-I, and 2 MHC-II, ensuring a potent immunodominant profile. Docking with TLR3 revealed a binding affinity of -17.2 kcal/mol, while simulations confirmed their stability. Cloning (pET28a (+)) and immune response analyses showed a strong immunogenic profile, including elevated IgG1, IgM, and antigen levels, supported by a Codon Adaptation Index (CAI) of 1.0.

Conclusion: The proposed multi-epitope vaccine shows promise against MPXV. However, further in vivo and in vitro investigations are essential to confirm its immune efficacy.