Computational design and validation of a multi-epitope subunit vaccine against the neglected parasite blastocystis sp

Blastocystis sp. is a neglected, globally prevalent intestinal parasite infecting over one billion people with no available vaccine. This study describes an immunoinformatics framework for developing a multi-epitope subunit vaccine against α-L-fucosidase from Blastocystis sp. Immunogenic B-cell and T-cell epitopes were identified through systematic prediction and rigorous allergenicity, toxicity, and human homology screening. Epitopes were assembled into a vaccine with human β-defensin-3 as an N-terminal adjuvant. The vaccine displayed favorable physicochemical properties including optimal molecular weight (~ 38 kDa), stability index (< 40) and negative GRAVY score. Population coverage analysis achieved 97.13% global coverage with 24.75 epitope-HLA hits per individual and a PC90 of 14.42. Structural validation of predicted 3D structure using Ramachandran analysis (99.7% residues in favored regions) and ProSA-web evaluation (Z-score: - 5.07) confirmed high-quality tertiary structure. Molecular docking revealed extensive binding with TLR-4, MHC Class I, and MHC Class II receptors, with interface areas of 1379-2198 Ų and 13-19 hydrogen bonds per complex. VD-MM/GBSA analysis reported favorable binding energies (- 295 to - 370 kcal/mol), while NMA confirmed appropriate dynamic flexibility for biological activity. Immune simulations predicted robust humoral and Th1-biased cellular responses with sustained IgG/IgM antibody production and maintained immune homeostasis. In silico cloning into pET-28a( +) showed optimal codon adaptation (CAI: 0.97) and GC content (53.31%), providing a pathway toward experimental validation of the first Blastocystis vaccine.