<i>Listeria</i>-based cancer vaccines that segregate immunogenicity from toxicity

<jats:p>The facultative intracellular bacterium<jats:italic>Listeria monocytogenes</jats:italic>is being developed as a cancer vaccine platform because of its ability to induce potent innate and adaptive immunity. For successful clinical application, it is essential to develop a<jats:italic>Listeria</jats:italic>platform strain that is safe yet retains the potency of vaccines based on wild-type bacteria. Here, we report the development of a recombinant live-attenuated vaccine platform strain that retains the potency of the fully virulent pathogen, combined with a >1,000-fold reduction in toxicity, as compared with wild-type<jats:italic>Listeria</jats:italic>. By selectively deleting two virulence factors, ActA (Δ<jats:italic>actA</jats:italic>) and Internalin B (Δ<jats:italic>inlB</jats:italic>), the immunopotency of<jats:italic>Listeria</jats:italic>was maintained and its toxicity was diminished<jats:italic>in vivo</jats:italic>, largely by blocking the direct internalin B-mediated infection of nonphagocytic cells, such as hepatocytes, and the indirect ActA-mediated infection by cell-to-cell spread from adjacent phagocytic cells. In contrast, infection of phagocytic cells was not affected, leaving intact the ability of<jats:italic>Listeria</jats:italic>to stimulate innate immunity and to induce antigenspecific cellular responses.<jats:italic>Listeria</jats:italic>Δ<jats:italic>actA</jats:italic>/Δ<jats:italic>inlB</jats:italic>-based vaccines were rapidly cleared from mice after immunization and induced potent and durable effector and memory T-cell responses with no measurable liver toxicity. Therapeutic vaccination of BALB/c mice bearing murine CT26 colon tumor lung metastases or palpable s.c. tumors (>100 mm<jats:sup>3</jats:sup>) with recombinant<jats:italic>Listeria</jats:italic>Δ<jats:italic>actA</jats:italic>/Δ<jats:italic>inlB</jats:italic>expressing an endogenous tumor antigen resulted in breaking of self-tolerance and long-term survival. We propose that recombinant<jats:italic>Listeria</jats:italic>Δ<jats:italic>actA</jats:italic>/Δ<jats:italic>inlB</jats:italic>expressing human tumor-associated antigens represents an attractive therapeutic strategy for further development and testing in human clinical trials.</jats:p>