Structure-Guided Design of a Next-Generation Multi-Epitope Subunit Vaccine Targeting the Monkeypox Virus H3L Glycoprotein with Predicted Innate and Adaptive Immune Engagement

<title>Abstract</title> <p>Purpose The monkeypox virus (MPXV) represents an escalating public health concern globally, necessitating the development of tailored vaccination strategies that function independently of prior smallpox immunization history. This investigation introduces a mechanistically-grounded, multi-epitope-based subunit vaccine formulation directed against the H3L surface-exposed glycoprotein, which fulfills critical biological functions, including viral-host cell interactions, nuclear chromatin remodeling, and IL1A-dependent inflammatory cascade triggering that results in multi-organ pathology. Methods The computational design pipeline integrated multiple complementary immunoinformatics tools namely IEDB, BepiPred-2.0, NetMHCpan EL 4.1, VaxiJen v2.0, AllerTOP and ToxinPred—to generate a curated collection of immunologically functional, non-allergenic, non-toxic CTL, HTL and B-cell determinants with anticipated coverage across approximately &gt; 90% of global HLA allele distributions. An N-terminal β-defensin immunological adjuvant connected via an EAAAK linker was incorporated to potentiate innate immunity activation. Computational 3D structure prediction utilizing RoseTTAFold confirmed high-quality stereochemical properties. Results Molecular docking via HDOCK demonstrated energetically favorable, stable interactions between the vaccine construct and critical immune recognition molecules (TLR2, TLR4-MD2, MHC-I, MHC-II). Subsequent molecular dynamics simulations spanning 100 nanoseconds using GROMACS revealed sustained structural integrity and binding persistence across all vaccine-receptor pairings. Conclusion This construct is positioned as a highly promising immunogenic candidate for preclinical MPXV vaccine evaluation.</p>