Alphavirus replicons encoding IFN-γ enhance cancer virotherapy by overcoming macrophage-mediated suppression

Interference by tumor-associated macrophages may significantly reduce the efficacy of therapeutic viruses designed to infect cancer cells and activate anti-tumor T cells. Using a computational model, we hypothesized that viruses encoding a T cell-stimulating signal, like interferon-gamma (IFN-γ), could overcome this barrier. We engineered an alphavirus-based replicon expressing IFN-γ and evaluated its effect in various human-derived tumor-immune coculture systems and an in vivo murine model. While alphavirus replicons do not replicate in macrophages, macrophages acted as a barrier, limiting tumor infection in a frequency-dependent but phenotype-independent manner. Nonetheless, T cell activation occurred even when only a fraction of infected tumor cells expressed IFN-γ, regardless of macrophage presence, frequency, or phenotype. Additionally, viral stimulation drove macrophage repolarization toward a pro-inflammatory phenotype favoring T cell activation. These findings highlight a strategy for optimizing virotherapy in macrophage-rich tumors by designing viruses that stimulate T cell activation, ensuring therapeutic efficacy.