Conditional Lethality Yields a New Vaccine Strain ofListeria monocytogenesfor the Induction of Cell-Mediated Immunity

Abstract

Vaccinology is the most cost effective and efficacious of medical interventions for the eradication or management of many infectious diseases. The generation of a cellular immune response is key to survival against a variety of viral and intracellular bacterial pathogens and cancer. Therefore, the development of safe vaccines capable of inducing strong cellular immunity continues to be a pressing challenge for medicine. We have explored the use of an attenuated strain of Listeria monocytogenes as a novel live vaccine vector for this purpose. L. monocytogenes is a gram-positive intracellular bacterial pathogen whose genome has been fully sequenced (22), greatly facilitating detailed studies of its biochemistry, genetics, and physiology and contributing to the existing wealth of knowledge of its adjuvant properties and immunogenicity.Since the early work of Mackaness in the 1960s (30), L. monocytogenes has been studied extensively as a paradigm for understanding innate and cell-mediated immunity. The organism can enter phagocytic cells through Fc receptors or type I macrophage scavenger receptors (12, 48) and can invade nonphagocytic cells by the interaction of the bacterial surface proteins InlA (16) and InlB (18) with their cognate host cell receptors. Following internalization by phagocytic cells, the majority of engulfed organisms are degraded in the phagosome (11). However, listeriolysin O (LLO), a pore-forming cytolysin, and PI-PLC, a phosphatidylinositol-specific phospholipase C, mediate permeabilization of the phagosomal membrane, enabling a fraction of the organisms to enter the host cell cytosol (9,31,38,45). Subsequently, proteins secreted by the organism can be delivered directly into the major histocompatibility complex (MHC) class I pathway of antigen processing and presentation (2, 5). Mutants of L. monocytogenes unable to enter the cytosol are avirulent. These bacteria fail to stimulate protective immunity in mice, and cells infected by such mutants are not recognized by L. monocytogenes-immune CD8 ϩ T cells (7, 34). Conversely, mice infected with sublethal doses of wildtype bacteria develop long-lasting immunity, mediated predominantly by CD8 ϩ T cells (13,29). This unique property of L. monocytogenes makes it attractive as a potential live vaccine vector, and recombinant strains expressing foreign antigens have successfully protected mice against lymphocytic choriomeningitis (23, 44), papillomavirus (26) and influenza virus (25) infections, and tumor challenge (24,36,37).Because of the potential use of this organism as a vaccine vector for protection against infectious diseases and cancer, the safety of L. monocytogenes is a critical issue. While infections by L. monocytogenes are fairly rare and can readily be controlled by a number of antibiotics, the organism can cause meningitis and death, particularly in immunocompromised or pregnant patients. A vaccine strain of L. monocytogenes should therefore be avirulent but immunogenic. We previously sought to generate an attenuated strain that could e...

KBMA Listeria monocytogenes is an effective vector for DC-mediated induction of antitumor immunity

“…DCs infected with empty vector served as negative controls. Forty hours after infection, the DCs were washed and cocultured with MP 58-66 -or MART1 [26][27][28][29][30][31][32][33][34][35] specific CD8 + T cell clones for 24 hours. The number of cells synthesizing IFN-γ was enumerated by enzyme-linked immunospot (ELISPOT) assay.…”

“…Only Mart-1 [26][27][28][29][30][31][32][33][34][35] tetramer-positive cells responded to stimulation with Mart-1 [26][27][28][29][30][31][32][33][34][35] peptide-pulsed T2 cells but not to HLA-A*0201-restricted HIV Gag 77-85 peptide-pulsed T2 cells that we used as a negative control ( Figure 5C). In 2 different donors, most of the responding Mart-1 [26][27][28][29][30][31][32][33][34][35] tetramer-positive cells produced several cytokines, including IFN-γ, TNF-α, and IL-2 (24% and 17% of Mart-1 [26][27][28][29][30][31][32][33][34][35] tetramer-positive cells produced all 3 cytokines in both donors) (Figure 5D). Cytotoxicity of the Mart-1 26-35 -specific T cells primed with KBMA LmAg was tested by using a chromium release assay.…”

“…platforms that has proved to elicit protective responses in a number of experimental models, including infectious and malignant diseases (29)(30)(31). L. monocytogenes localize to T cell zones of spleen white pulp and efficiently targets DCs in vivo (26).…”

“…As shown in Figure 5A, DCs infected with KBMA LmAg expanded MP 58-66 -specific naive CD8 + T cells. Before the onset of priming, no staining of naive T We next evaluated whether live LmAg was more efficient at priming naive T cells to Mart-1 [26][27][28][29][30][31][32][33][34][35] than KBMA LmAg in our in vitro system. DCs infected with live LmAg (MOI 10) or KBMA LmAg (MOI 50) were used to stimulate T cells as described above, and priming efficacy was tested with Mart-1 26-35 tetramers.…”

“…Both strains were able to prime Mart-1 26-35 -specific T cells at similar frequencies ( Figure 5B, 4 left panels). The Mart-1 [26][27][28][29][30][31][32][33][34][35] population from the culture stimulated by KBMA LmAg-infected DCs was enriched by using a limiting dilution assay of pooled wells in which Mart-1 [26][27][28][29][30][31][32][33][34][35] tetramer-positive cells were detected ( Figure 5B, 2 right panels). Specific antigen responses were measured by intracellular cytokine secretion.…”