Computational Design and Biophysical Evaluation of VC032: A Multi-Epitope Vaccine Candidate Targeting Four Human-Pathogenic Orthoebolaviruses

Ebola Virus Disease (EVD), caused by four human-pathogenic Orthoebolavirus species (Bundibugyo, Sudan, Taï Forest, and Zaire ebolaviruses), presents severe mortality (40%–90%). Licensed vaccines confer protection exclusively against Zaire ebolavirus, leaving persistent vulnerabilities against heterologous species due to 30%–40% envelope glycoprotein divergence. To address this, we executed proteome-wide immunoinformatics screening across 20,723 viral sequences encompassing seven structural and non-structural proteins. Multi-tier filtering for MHC restriction, conservancy, non-allergenicity, and human proteome non-homology identified 24 conserved epitopes across five viral targets (GP, VP35, VP24, L-polymerase, NP). From a combinatorial screen of 48 unique vaccine candidates, construct VC032 was prioritized, integrating a human β-defensin-3 adjuvant via specialized linkers. VC032 achieved ≥99.8% HLA coverage across African outbreak epicenters and globally. Tertiary structure prediction and refinement confirmed favorable stereochemical quality (92.5% Ramachandran favored). Molecular docking and 100-ns all-atom molecular dynamics with the human TLR1/TLR2 receptor complex demonstrated stable interfacial engagement (mean 977 contacts, 29 hydrogen bonds) and robust binding free energies (MM-GBSA: −216.84 ± 15.27 kcal/mol). Immune simulations in V032 predicted coordinated B-cell and Th1-skewed memory responses. In silico codon optimization and virtual cloning into pET-28a(+) established translational feasibility for bacterial expression, delivering a broad-spectrum pan-orthoebolavirus vaccine blueprint. Further experimental validation is required.