In Silico Design and Computational Characterization of Novel Chimeric Multiepitope Antigens for Mpox Serosurveillance: An Immunoinformatics Approach

Abstract

Background and Aims: The Monkeypox (Mpox) virus is a zoonotic Orthopoxvirus with a global outbreak that began in 2022, with more than 132,000 confirmed cases and 1500 deaths across over 128 countries. The pandemic preparedness pipeline emphasizes the importance of diagnostic surveillance of pathogens in at-risk populations to monitor transmission and mitigate the impact on public health. Current PCR-based diagnostics are limited in field-applicability, creating an urgent need for deployable serosurveillance tools. This is the first study aimed at designing and computationally characterizing novel multiepitope antigens (MP-MEDA-1 and MP-MEDA-2) for Mpox serodiagnostics using immunoinformatics approaches and combining these with molecular docking to the Fab regions of IgA, IgG, and IgM. Methods: Linear B-epitopes from previously validated Mpox diagnostic proteins were predicted using the BepiPred-3.0 and LBtope servers. Antigenic epitopes were assembled into two multiepitope constructs using GSGSG linkers. The three-dimensional structures of both antigens were predicted by AlphaFold2, refined using FG-MD and GalaxyRefine servers, and validated via ProSA-web, ERRAT, and Ramachandran analysis. Protein-protein docking with the Fab regions of human IgA, IgG, and IgM was performed using the ClusPro 2.0 server, and binding affinities were calculated using the PRODIGY server. Molecular dynamics was assessed via Normal Mode Analysis using the iMODS server. Results: Both antigens were predicted to be antigenic (VaxiJen scores: 0.61-0.75; ANTIGENpro scores: 0.95) with favorable physico-chemical properties (hydrophilic and thermostable). with desirable physicochemical properties. In addition, both antigens also demonstrated stable interactions with the Fab regions of selected immunoglobulins, with several residues interacting at the interfaces of all the docked complexes. Conclusions: These preliminary findings demonstrate that MP-MEDA-1 and MP-MEDA-2 are promising diagnostic antigens for Mpox serosurveillance. Future work will focus on the expression and serological characterization of both antigens to determine their diagnostic parameters (sensitivity, specificity, and others).