Design of Multi-Epitope Cancer Vaccine Targeting p53 Tumor Antigen using Myotis lucifugus Sequencers: An Immunoinformatics Approach.

The p53 tumor suppressor protein is dysregulated in more than 50% of human cancers, representing an attractive target for immunotherapy. However, immune tolerance to the self-antigen limits the effectiveness of traditional p53-based vaccines. The presented study is based on a comprehensive in silico approach to design a multi-epitope cancer vaccine using the p53 sequence from Myotis lucifugus (little brown bat) that would break the self-tolerance without losing cross-reactive immunogenicity. Human and bat p53 amino acid sequences were aligned using claustral omega, with a highly conserved core domain at position 120-300 with 78.3% sequence identity as the vaccine region. Cytotoxic T-lymphocyte, helper T-lymphocyte, and B-cell epitopes were predicted using METMHCpan 4.1, NETMHCIIPan 4.0, and BepiPred 2.0, respectively, followed by immunological and safety screening, including antigenicity, allergenicity, toxicity, and human proteome homology analysis. Several epitopes meeting all criteria were assembled into a 134-amino acid construct with β-defensin-derived adjuvant and function-specific linkers. The vaccine demonstrated optimal physicochemical properties and strong structural integrity, with high Ramachandran favorable residues and Verify3D scores. Immune simulation showed sustained IgG1 response up to day 350, significant TH and CTL expansion, and pronounced TH1 polarization marked by IFN- γ levels. Population analysis reached 80.84% globally, with the highest representation in Europe and North America. Docking analysis showed strong binding to TLR4-MD2 and negligible interaction with wild-type and mutant p53, supporting immune-specific activity without off-target effects. Codon optimization yielded a CAI for E. coli expression. The designed multi-epitope vaccine demonstrated excellent immunogenic potential, favorable safety profiles, and broad population applicability.