Expression-Guided Immunoinformatics Design of a Recombinant Multi-Epitope Vaccine Candidate for Triple-Negative Breast Cancer Incorporating a MyD88-Derived Immunomodulatory Domain

Abstract

Triple-negative breast cancer (TNBC) remains a major therapeutic challenge because of its aggressive behavior, molecular heterogeneity, and limited subtype-specific targets. This study used an integrated immunoinformatics workflow to design a recombinant tumor-associated antigen-derived multi-epitope vaccine based on MMP1, CXorf61/CT83, and COL11A1, prioritized according to their tumor-to-normal transcript-expression ratios. Candidate cytotoxic T-lymphocyte, helper T-lymphocyte, and linear B-cell epitopes were screened for predicted HLA binding, antigenicity, allergenicity, toxicity, and IFN-γ-induction potential. The selected epitopes were assembled with PADRE, a MyD88-derived exploratory immunomodulatory domain, class-specific linkers, and a C-terminal histidine tag to generate a 621-amino-acid construct. Combined HLA class I and II analysis predicted 99.36% worldwide population coverage, with 3.14 epitope–HLA hits per individual. The construct was predicted to be antigenic, non-allergenic, soluble, and physicochemically compatible with recombinant production. Structural modeling yielded a ProSA Z-score of −7.43 and 97.45% of residues in favored Ramachandran regions, while disulfide engineering identified ten candidate intramolecular bridges. Docking produced an HDOCK score of −298.65 for the modeled vaccine–TLR4 complex, and flexibility analysis showed an average RMSF of 1.93 Å. Immune simulation predicted Th1-associated cytokine production, T-cell expansion, antibody responses, and memory-cell formation. Codon optimization generated a CAI of 0.95 and a GC content of 53.2%, followed by virtual cloning into pET-28a(+). These findings support the computational feasibility of the proposed vaccine candidate, which requires experimental validation of expression, antigen processing, HLA presentation, immunogenicity, safety, and antitumor activity.