L Monocytogenes As Delivery Vehicle For Eukaryotic Expression Plasmids

The ability of L. monocytogenes to transfer eukaryotic expression plasmids into host cells has been mainly explored in vitro so far. However, vast experience exists for the use of such bacteria as a carrier for heterologous antigens, especially in murine tumor systems.

A. L. monocytogenes-mediated DNA Transfer In Vitro

L. monocytogenes infects many cell types in a wide range of different species, including mice and humans. Its intracellular infection cycle, as well as innate and adaptive immune responses against this pathogen in mice, have been extensively studied since the 1970s (35,36). Due to its capacity to induce phagocytosis, L. monocytogenes can efficiently infect a variety of cells, including nonphagocytic cells. After its uptake, the bacterium quickly escapes from the phagosome into the cytosol of the host cell, where it replicates. Therefore heterolo-gous plasmid DNA carried by Listeria can be delivered directly into the cytosol of the mammalian cell and only has to cross the membrane barrier of the nucleus for expression. Highly efficient plasmid transfer to epithelial cell lines from different species, including fish, mouse, rat, hamster, monkey, and human has been described (16,17,24,25). In addition,

Figure 2 T cell responses to tumors in the presence of bacterial pathogens. Tumors release antigens that are picked up by dendritic cells of the immature phenotype. Presentation of antigens by immature DCs leads to the induction of T cell tolerance. In contrast, bacterial infection is accompanied by the release of inflammatory cytokines, which induce maturation of DCs. In addition, molecules representing conserved pathogen-associated patterns (e.g., unmethylated CpG motifs of bacterial DNA) can be released from infected cells. Alternatively, DCs themselves can be infected and activated. Tumor antigens that are taken up and presented by immature DCs that are activated to mature by the infection will lead to the induction of tumor-specific T cell responses.

Figure 2 T cell responses to tumors in the presence of bacterial pathogens. Tumors release antigens that are picked up by dendritic cells of the immature phenotype. Presentation of antigens by immature DCs leads to the induction of T cell tolerance. In contrast, bacterial infection is accompanied by the release of inflammatory cytokines, which induce maturation of DCs. In addition, molecules representing conserved pathogen-associated patterns (e.g., unmethylated CpG motifs of bacterial DNA) can be released from infected cells. Alternatively, DCs themselves can be infected and activated. Tumor antigens that are taken up and presented by immature DCs that are activated to mature by the infection will lead to the induction of tumor-specific T cell responses.

professional APC-like murine macrophage-like cell lines, primary macrophages, and human monocyte-derived DCs can be recipients of DNA delivered by Listeria, although the frequencies observed vary among studies (17,21-25). This might be due to differences in the experimental settings of infection experiments, but might also be attributed to the mechanism applied to induce bacterial cell death. So far, two strategies have been followed to induce cytosolic plasmid liberation by recombinant L. monocytogenes: bacteria were engineered to lyse due to the activity of a bacteriophage autolysin. The expression of this lytic system was under the control of the listerial actA promotor and hence was activated preferentially in the cytosol of the host cell (21,23). Alternatively, bacteria were killed by antibiotic treatment of the infected cells (24,25). Interestingly, the way by which an antibiotic interferes with the bacterial metabolism greatly influences the out come of gene transfer efficiency. Penicillin, an inhibitor of cell wall synthesis, was superior to substances blocking protein synthesis or DNA replication (25). However, gene delivery also occurs using wild-type bacteria without further manipulation, probably as a result of death of the microorganism caused by natural host cell defense activities (17,25).

The efficiency of gene transfer by L. monocytogenes is dramatically dependent on the capability of the bacteria to escape from the phagosome into the cytosol of the target cell. Three virulence factors have been demonstrated to be involved in this phagosomal escape—the pore-forming listeriolysin (LLO; encoded by the hly gene) and 2 phospholipases (PlcA/ PlcB). For some host cells of epithelial origin and for human DC phospholipase activity seems to be sufficient to mediate bacteria entry into the cytosol. In contrast, in the majority of target cells, LLO activity is absolutely required (25,37,38).

However, LLO exhibits severe toxic effects on some cells. Therefore, the bacteria-to-target cell ratios (MOI, multiplicity of infection) normally need to be kept low, which limits the DNA transfer. To overcome this barrier, Listeria mutants were constructed that expressed a listeriolysin of reduced cytolytic activity. Indeed, by increasing of the MOI, such strains were able to mediate greatly enhanced plasmid transfer without inducing adverse effects (25).

B. In Vivo Application of L. monocytogenes as Vector System

An attenuated strain of L. monocytogenes, impaired in intra-and intercellular spreading, has been demonstrated to be capable of plasmid delivery in vivo. Green fluorescent protein (GFP) expressing macrophages could be isolated from cotton rats after intraperitoneal infection with inducible autolyic bacteria that carried a GFP reporter gene plasmid.

Studies exploiting recombinant Listeria in gene therapy of cancer have not been published so far, but the therapeutic potential of Listeria in tumor therapy was already impressively demonstrated. Wild-type bacteria and attenuated strains of L. monocytogenes have been used as an oral vaccine carrier. Listeria modified to express p-galactosidase of E. coli as surrogate tumor antigen conferred protective immunity in BALB/ c mice against a challenge with p-galactosidase (p-gal) expressing fibrosarcoma cells and reduced the growth of established tumors (39).

Even more impressive were the results obtained by the group of Y. Paterson (40,41). In these studies, bacteria were genetically modified with a prokaryotic expression plasmid that integrated into the bacterial chromosome. The plasmid encoded the influenza nucleoprotein (NP) as a model tumor antigen. The NP gene was fused to a deletion variant of the hly gene in order to achieve secretion of the LLO-NP gene product. Intraperitoneal (i.p.) immunization of BALB/c mice with LLO-NP expressing Listeria conferred complete protection against an otherwise lethal challenge with tumor cells. Colon and renal carcinoma cells that were retrovirally transduced with the NP encoding gene had been used in these studies. In immunized mice, NP-specific CD8 + T cells were identified as main effectors, but CD4 + T cells also contributed to antitumor immunity. The antitumor reactivity was of sufficient potency to even induce regression of macroscopically established tumors (40). In such model systems, tumor protection and regression could also be achieved when LLO-NP expressing bacteria were administered orally (41). Intraperitoneal immunization of C57BL/6 mice with the same bacteria mediated regression of primary tumors and of established lung metastasis by the B16F10 melanoma, when these tumor cells were modified to express the NP gene (42).

Recombinant Listeria have also been successfully used to induce protective immunity against viral antigens associated with cancer development. Bacteria, expressing and secreting the E7 protein from the human papilloma virus-16 in the form of an LLO-E7 fusion protein, induced regression of E7-ex-pressing tumors in C57BL/6 mice (43). Similarly, protective and therapeutic immunity against cottontail rabbit papillo-mavirus induced papillomas, which progress with high frequency to carcinoma, was established when rabbits were immunized with a cocktail of recombinant L. monocytogenes expressing and secreting deletion variants of the viral E1 protein (44). Again the efficacy of this carrier system, even in a therapeutical setting, was demonstrated.

Furthermore, recombinant Listeria could induce protective immunity against gliomas expressing the nucleoprotein of the lymphocytic choriomeningitis virus (LCMV-NP) as surrogate tumor antigen (45). Subcutaneous administration of bacteria genetically modified to secrete the LCMV-NP conferred antigen-specific CD8 + T cell-dependent protection against a subcutaneous challenge with NP-expressing glioma cells in rats. After tumor rejection, enhanced tumor immunity was observed due to epitope spreading. Such mice were protected against a lethal intracerebral challenge with NP-expressing and parental glioma cells. Parental cells obviously did not express the recombinant surrogate tumor antigen. This indicates that recombinant Listeria can initiate antitumor immunity that is even protective within the central nervous system. This anatomical site is considered immunoprivileged (i.e., it is usually not accessible to immunocytes).

Based on these encouraging data an initial clinical safety study of an attenuated L. monocytogenes strain in humans has been performed recently (46). Twenty healthy volunteers received orally escalating doses of bacteria that were attenuated by deletions of the actA and plcB genes. No long-term health sequelae were observed in these studies. This proves the potential applicability of this bacterial carrier system for extended human trials.

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