In silico design and validation of a novel CSP-based multi-epitope vaccine with modified melittin adjuvant against Plasmodium falciparum

Malaria caused by Plasmodium falciparum remains a major global health burden, with current vaccines offering only partial protection. We designed CSP-MelMut, a multi-epitope subunit vaccine incorporating selected high-affinity B- and T-cell epitopes from the circumsporozoite protein (CSP) fused to a detoxified melittin L16A mutant as a built-in adjuvant using rigid EAAAK linkers. Epitopes were predicted using NetMHCpan 4.1, NetMHCIIpan 4.2, ABCpred, and IFNepitope, followed by IEDB population coverage analysis. The final 262-amino-acid construct was modelled with ColabFold, refined with GalaxyRefine2, and validated through molecular docking (ClusPro and HADDOCK), dynamic simulations (iMODS and CABS-flex),a 100 ns all-atom molecular dynamics simulation, and immune simulation (C-ImmSim). Additional analyses included BLASTp, ElliPro, IL4pred, IL10pred, and sequence conservancy. Codon optimization and in silico cloning into pET-28a( +) were performed. The construct showed high structural quality (MolProbity 0.793, ERRAT 99.578, ProSA Z-score –7.54) and favourable binding to TLR4/MD-2 (ClusPro score –1013.0; best HADDOCK score –99.6). A subsequent 100 ns all-atom MD simulation confirmed complex stability, with progressive strengthening of the binding interface (hydrogen bonds + 81%, buried surface area + 39%). Immune simulation predicted a robust booster effect with increased IgG and a Th1-biased response. BLASTp confirmed specificity to P. falciparum. ElliPro identified strong conformational B-cell epitopes (highest score 0.954). Six of seven HTL epitopes were IL-4 inducers with moderate IL-10 induction. The sequence achieved CAI = 1.0 and was successfully cloned in silico. This study presents a comprehensively validated CSP-MelMut candidate integrating a novel melittin-based adjuvant strategy. Experimental validation is warranted.