Abstract

Introduction:Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor and is highly resistant to conventional therapies. Designing multi-epitope peptide vaccines represents a promising strategy to elicit tumor antigen-specific immune responses.

Methods:Using immunoinformatics tools, cytotoxic T lymphocyte (CTL) and B-cell epitopes were identified from the IL13Rα2 and EPHA2 tumor-associated antigens and filtered based on antigenicity, non-allergenicity, and non-toxicity. The final vaccine construct comprised 16 CTL epitopes and 6 B-cell epitopes. Physicochemical properties, structural features, and interactions of the vaccine with Toll-like receptors (TLRs) were evaluated.

Results: The final vaccine consisted of 281 amino acids, with a molecular weight of 31,579.57 Da, pI = 8.98, and an instability index of 26.10, indicating high stability. A GRAVY value of −0.463 and a QuerySol score of 0.539 suggested favorable hydrophilicity and solubility. The refined 3D structure showed 93.6% of residues in favored regions of the Ramachandran plot, and a Z-score of −1.63 confirmed good structural quality. Molecular docking analyses revealed the strongest binding affinity toward TLR3 (ΔG = −11.1 kcal/mol; Kd ≈ 1.6×10⁻⁸ M). Immune simulation demonstrated robust induction of both humoral and cellular immune responses: rapid IgM production after the first immunization followed by isotype switching to IgG1/IgG2 in subsequent doses. In silico immune simulation with C-ImmSim suggested a potential for robust induction of both humoral and cellular immune responses.
Conclusion: The designed multi-epitope vaccine exhibits favorable immunogenicity, stability, and strong interaction with TLR3, positioning it as a promising candidate for further preclinical development as a therapeutic vaccine against glioblastoma.