Listeria-based cancer vaccines that segregate immunogenicity from toxicity

Abstract

The facultative intracellular bacterium Listeria monocytogenes 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 Listeria 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 Listeria. By selectively deleting two virulence factors, ActA (DeltaactA) and Internalin B (DeltainlB), the immunopotency of Listeria was maintained and its toxicity was diminished in vivo, 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 Listeria to stimulate innate immunity and to induce antigenspecific cellular responses. Listeria DeltaactA/DeltainlB-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(3)) with recombinant Listeria DeltaactA/DeltainlB expressing an endogenous tumor antigen resulted in breaking of self-tolerance and long-term survival. We propose that recombinant Listeria DeltaactA/DeltainlB expressing human tumor-associated antigens represents an attractive therapeutic strategy for further development and testing in human clinical trials.

Figures

Fig. 1.

Recombinant Listeria strains induce a potent OVA-specific effector T cell response that is associated with transient liver toxicity. (A) Female C57BL/6 mice were vaccinated i.v. (IV) with 0.1 LD50 of indicated attenuated Listeria-OVA strain. OVA-specific immunity was assessed by intracellular cytokine staining on day 7 after a single vaccination. Representative dot blots of SL8-stimulated spleen cells gated on CD8+ T cells are shown. The percentage of IFN-γ+CD8+ T cells in the absence of SL8 stimulation was <0.03% (data not shown). (B) Vaccination of mice with wild type (wt) or the attenuated Listeria ΔactA mutant induces transient liver toxicity. Three C57BL/6 mice per group were vaccinated i.v. with 5 × 104 colony-forming units (CFU) of wild type or 1 × 107 colony-forming units of ΔactA. Serum levels of the liver enzyme AST (in units per liter) were determined at different times after infection.

Fig. 2.

The introduction of the inlB deletion on the ΔactA mutant does not abrogate the ability of strains to induce potent antigen-specific T cell responses. C57BL/6 mice were vaccinated i.v. with 1 × 107 colony-forming units (CFU) of the indicated strain. OVA-specific CD8+ T cell immunity was assessed on day 7 after vaccination by using intracellular cytokine staining. (A) Representative dot blots of SL8-stimulated spleen cells are shown. (Left) ΔactA/ΔinlB. (Center) ΔactA-OVA. (Right) ΔactA/ΔinlB-OVA. PerCP, peridinin chlorophyll protein. (B) The average percentage of OVA-specific CD8+ T cells of eight mice is shown.

Fig. 3.

The inability of Listeria ΔactA/ΔinlB to infect nonphagocytic cells in vitro results in accelerated clearance in vivo. (A) In vitro infectivity of the human hepatocyte cell line (HepG2), fresh human hepatocytes (two donors), a human monocyte cell line (THP-1), and primary human monocytes (three donors) is shown. For all strains, the rate of infection is normalized to the rate of infection by wild-type (wt) Listeria. The averages of three (or two for primary human hepatocytes) independent experiments are shown. (B) The in vivo growth kinetic in liver was assessed for wild type (Upper Left), ΔactA (Lower Left), ΔinlB (Upper Right), and ΔactA/ΔinlB (Lower Right). C57BL/6 mice were injected i.v. with 0.1 LD50 of the indicated strain and bacteria per organ were determined from three mice at each time point. A representative of two experiments is shown.

Fig. 4.

Recombinant attenuated Listeria ΔactA/ΔinlB AH1-A5 breaks tolerance to self that results in potent therapeutic antitumor activity and prolongation of life. (A) Female BALB/c mice were vaccinated once with 0.1 LD50 of the indicated strain. Cytotoxic activity was determined by in vivo cytotoxicity assay. CFSEhi AH1 (Upper) or AH1-A5 (Lower) peptide-pulsed BALB/c splenocytes (3 × 106) and CFSElow nonpulsed splenocytes (3 × 106) were coinjected i.v. into BALB/c mice that were naïve or 7 days prior to vaccination with the indicated Listeria strain. In vivo killing of CFSE-labeled target cells was assessed 18 h later. Histograms are gated on CFSE+ cells in host mice. The number represents the average percentage of specific killing of three individual mice normalized to HBSS-injected mice. (B) Female BALB/c mice were implanted i.v. with 2 × 105 CT26 cells on day 0. Four days later, mice received a single vaccination with a dose equal to 0.1 LD50 of the indicated strain. Lungs were harvested on day 19, fixed in Bouin's solution, and the number of surface lung metastases were counted. Representative lungs are shown. (C) Female BALB/c mice were implanted i.v. with 2 × 105 CT26 cells on day 0. Four days later, mice were vaccinated with 0.1 LD50 of the indicated strain or vehicle control (n = 10). Survival was monitored over the course of the experiment. Therapeutic vaccination of mice with ΔactA/ΔinlB AH1-A5 both resulted in a significant survival benefit (one-way ANOVA, P < 0.0001) compared with HBSS or ΔactA controls.