Computational design and immunoinformatics validation of a T cell multi-epitope vaccine targeting glioblastoma stem cells

Glioblastoma multiforme is a highly aggressive and lethal primary brain tumor in adults, with a poor prognosis and high recurrence rate driven by glioblastoma stem cells (GSCs). The primary objective of this study is to design and characterize a novel multi-epitope vaccine, Gliostem-MultiVax, targeting GSC-specific antigens to overcome tumor recurrence and treatment resistance. These biomarkers are essential for tumor initiation, progression, resistance to standard treatments, and the recurrence of cancer. Six well-established GSC-specific markers (CD133, CD44, Sox2, Nestin, CD15, and Musashi-1) were selected as antigen sources. Using comprehensive immunoinformatic tools, CD8 + and CD4 + T-cell epitopes were predicted. The epitopes were linked with appropriate linkers and combined with the adjuvant Rv3628 to construct the multi-epitope vaccine. Physicochemical characteristics, population coverage, and 2D and 3D structures of the vaccine were analyzed. Molecular docking and molecular dynamics (MD) simulations were performed to evaluate the interaction model of the vaccine–TLR2 complex. The immune simulation to evaluate the immunogenicity and in silico cloning of the designed vaccine were conducted. Nine CD8+ T-cell and seven CD4+ T-cell epitopes demonstrating significant HLA binding, immunogenicity, antigenicity, and the ability to elicit IL-4 and IFN-γ were identified. These epitopes exhibited high affinity for HLA alleles, achieving 94.25% global population coverage. The designed Gliostem-MultiVax was antigenic, non-allergenic, hydrophilic (GRAVY: -0.426), soluble, and stable. The refined 3D model showed 96.4% residues in Ramachandran-favored regions with a Z-score of -7.53. Molecular docking and molecular dynamics simulations revealed that the Gliostem-MultiVax-TLR2 complex is compact and stable (ΔG = − 20.4 kcal/mol, RMSD = 0.65 nm, Rg = 3.36 nm, 31 hydrogen bonds), with an optimal interaction network and flexibility. Immune simulation demonstrated strong humoral and cellular responses, including elevated IgG and IgM levels, proliferation of CD4⁺ and CD8⁺ T cells, and increased production of IFN-γ and IL-12. These comprehensive findings suggest that Gliostem-MultiVax is a promising candidate for further preclinical and clinical investigation as a therapeutic vaccine against GBM.