Integrated Computational and In Vivo Assessment of a Novel Multi‐Epitope Vaccine Against Dengue Virus Serotype 4 Capsid Protein

Dengue Virus Serotype 4 (DENV4) continues to contribute substantially to global dengue morbidity, yet current tetravalent vaccines provide inconsistent protection and may pose risks of antibody-dependent enhancement (ADE). This study aimed to design and evaluate a serotype-specific multi-epitope vaccine (MEV-DV4) targeting the conserved capsid protein of DENV4 using integrated reverse vaccinology and immunoinformatics approaches. Conserved, antigenic, non-allergenic, and non-toxic B-cell, cytotoxic T lymphocyte (CTL), and helper T lymphocyte (HTL) epitopes were identified and assembled with a β-defensin adjuvant and PADRE sequence using optimized linkers. The final construct demonstrated high antigenicity (0.8559), structural stability, favorable physicochemical properties, and excellent solubility. Structural validation confirmed 97.6% of residues in favored Ramachandran regions. Molecular docking revealed strong interactions with TLR4 and TLR8, particularly MEV-TLR8, and molecular dynamics simulations supported the overall stability of the complex. Immune simulations predicted robust humoral and cellular responses with strong memory formation. Experimental validation in an albino mouse model using an alum-adjuvanted MEV-DV4 formulation showed early and potent antibody responses, with peak HI titers at day 21. Notably, antibody titers induced by MEV-DV4 were statistically comparable to those produced by a commercial inactivated dengue vaccine at all tested time points (p > 0.05), while no adverse reactions were observed. These computational and experimental findings demonstrate that MEV-DV4 is a safe, immunogenic, and promising serotype-specific vaccine candidate against DENV4. Further neutralization and challenge studies are warranted to advance its preclinical development.