An engineered probiotic co-expressing dual melanoma epitopes for therapeutic cancer vaccination.

Therapeutic cancer vaccines face challenges in tumor targeting, adjuvant dependency, and inefficient antigen cross-presentation. We engineered a probiotic vaccine using Escherichia coli Nissle 1917 co-expressing MHC-I epitopes from melanoma antigens Pmel17 and TYRP2. The fusion protein, designed via AlphaFold3, incorporating an IgG Fc domain for stability and Listeriolysin O (LLO) for cytosolic delivery, with expression controlled by an L-arabinose-inducible promoter. The strain retained probiotic properties, was efficiently phagocytosed by antigen-presenting cells, and enhanced their maturation and antigen presentation. It selectively accumulated in B16 melanoma tumors and elicited potent antigen-specific CD8+ T cell responses, including tumor-specific cytotoxicity, IFN-γ production, and memory T cell expansion. In a subcutaneous melanoma model, the vaccine suppressed tumor growth, prolonged survival, and remodeled the tumor microenvironment by increasing effector cell infiltration and reducing immunosuppressive regulatory T cells. This work presents a rationally designed bacterial vaccine platform for targeted cancer immunotherapy.