Rational In Silico Design of a Multiepitope Marburg Virus Vaccine Candidate

Marburg virus (MARV) represents a critical public health threat with mortality rates of 24–88%. Here, we present an in silico vaccine design pipeline targeting MARV nucleoprotein. The integrated workflow encompassed: (1) sequence retrieval and physicochemical characterization via ExPASy ProtParam; (2) secondary structure prediction using PSIPRED; (3) antigenicity screening using VaxiJen; (4) allergenicity assessment via AllerTOP and toxicity screening via ToxDL; (5) B cell and T cell (MHC-I/II) epitope prediction using IEDB Analysis Resource; (6) population coverage analysis; (7) vaccine construct design incorporating 50S ribosomal protein L7/L12 adjuvant with epitopes linked by EAAAK, CPGPG, and AAY linkers with His6-tag; (8) construct validation for antigenicity, allergenicity, toxicity, solubility (SOLUPROT), and physicochemical properties; (9) 3D structure prediction via trRosetta, refinement with GalaxyRefine, and quality assessment via UCLA-DOE LAB PROCHECK Ramachandran plots; (10) molecular docking validation using PatchDock; (11) DNA sequence optimization via EMBOSS Backtranseq and JCat, with gene synthesis and pET vector cloning via SnapGene; and (12) immune dynamics simulation via C-IMMSIM. Results: The optimized 681 aa vaccine construct achieved 36% improvement in antigenicity (0.6416 vs. 0.4699), improved stability (instability index 44.61 vs. 51.27), predicted solubility (0.811), non-allergenic/non-toxic profiles, 95.2% Ramachandran-favored residues, favorable molecular docking (ΔG −7.2 to −8.4 kcal/mol), exceptional population coverage (70% MHC-I, 85% MHC-II globally), and robust predicted immune responses (IgM peak 500K cells/mm³, sustained IgG, Th cell expansion to 3500 cells/mm³, balanced Th1 cytokine profile). The optimized construct is suitable for recombinant E. coli expression via the engineered pET-28a vector. This multiepitope vaccine candidate represents a rationally designed, broadly applicable, and immunologically promising prototype for preclinical development.