Immunoinformatic Approaches to Explore Monkeypox Virus to Design B and T Cell Multi-epitope Subunit Vaccine

Background: Monkeypox virus (MPXV), a member of the Orthopoxvirus genus, is closely related to the variola and vaccinia viruses and has emerged as a significant global public health concern. The virus exists in two infectious forms, intracellular mature virion (IMV) and extracellular enveloped virion (EEV), which play critical roles in inter-host and intra-host transmission, respectively. Given the urgent need for effective therapeutic and preventive strategies, this study aimed to design a multi-epitope therapeutic vaccine against MPXV using a reverse vaccinology and immunoinformatics approach targeting key antigenic proteins involved in viral infectivity and spread. <div> <br> </div> <div> Methods: Four antigenic proteins of MPXV, including A27, A33, B5, and L1, were selected for epitope prediction. Cytotoxic T lymphocyte (CTL) and helper T lymphocyte (HTL) epitopes were screened and selected based on antigenicity, allergenicity, and toxicity profiles. Selected epitopes were assembled using suitable linkers and adjuvants to construct a multi-epitope vaccine candidate. The final construct was evaluated for physicochemical characteristics, antigenicity, allergenicity, and immunogenic potential. Tertiary structure prediction, molecular docking analyses, immune simulation, and in silico cloning were additionally performed to assess structural stability, receptor binding affinity, and expression feasibility. </div> <div> <br> </div> <div> Findings: The designed multi-epitope vaccine construct demonstrated favorable physicochemical stability, strong antigenicity, non-allergenic properties, and the potential to induce both cellular and humoral immune responses. Structural modeling predicted a stable tertiary conformation of the vaccine candidate. Molecular docking analyses indicated strong interactions between the vaccine construct and immune receptors, supporting its potential immunostimulatory activity. In silico cloning further suggested efficient expression feasibility in a suitable expression vector system. Overall, computational analyses supported the potential effectiveness of the proposed vaccine candidate against MPXV infection. </div> <div> <br> </div> <div> Interpretation: This study presents a promising immunoinformatics-driven multi-epitope vaccine candidate targeting major MPXV antigens associated with viral transmission and pathogenicity. The findings highlight the utility of reverse vaccinology approaches for accelerating vaccine development against emerging infectious diseases such as monkeypox. Nevertheless, further experimental validation through in vitro and in vivo studies is required to confirm the immunogenicity, safety, and protective efficacy of the proposed vaccine construct. </div>