Reprogramming Listeria monocytogenes flavin metabolism to improve its therapeutic safety profile and broaden innate...

Listeria monocytogenes is a facultative intracellular bacterial pathogen that is a potent inducer of cell-mediated immunity, which has led to the development of attenuated, Listeria-based cancer vaccines. L. monocytogenes strains, such as live-attenuated double-deleted Listeria (LADD), lacking two key virulence factors, ΔactA and ΔinlB, have been used safely in clinical trials and showed promising anti-tumor activity. Despite early clinical success, improving potency and safety by preventing extracellular bacterial growth is paramount for the development of further clinical applications. We describe a quadruple attenuated intracellular Listeria (QUAIL) strain that, in addition to ΔactAΔinlB, lacks ribC and ribF, which encode enzymes required for generating the essential flavin cofactors flavin mononucleotide (FMN) and flavin adenine nucleotide (FAD). QUAIL imported FMN and FAD during intracellular growth but was unable to grow extracellularly in blood or on vascular catheters in mice, which reduced its lethality. Despite its lack of extracellular growth, QUAIL maintained its immunoprotective properties, which were comparable to LADD. Furthermore, we showed that QUAIL can be engineered to synthesize riboflavin, leading to expansion and activation of mucosal-associated invariant T cells. Together, our data support the use of QUAIL as a promising therapeutic platform with an improved safety profile that is amenable to further modifications to expand its immune-activating potential.IMPORTANCEListeria-based live-attenuated cancer vaccines represent a promising therapy in many different pre-clinical tumor models and in clinical trials. Enhancing its anti-cancer immunity and increasing its safety profile will advance the clinical applications of Listeria vaccines. By manipulating Listeria monocytogenes flavin metabolism, we engineered a quadruple attenuated intracellular Listeria (QUAIL) vaccine candidate strain that has limited toxicity associated with extracellular growth in major extracellular niches in vivo, including blood and implanted catheter ports. Furthermore, we showed that QUAIL can be effectively programmed to engage innate-like T cells known as mucosal-associated invariant T cells, which could be harnessed for future cancer immunotherapies. The results presented here lay the foundation for further analysis of QUAIL as a safer, yet immunopotent L. monocytogenes vaccine or therapeutic vector.