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Mutants ofListeria monocytogeneswith deletions in genes of the common branch of the biosynthesis pathway leading to aromatic compounds were constructed as possible virulence-attenuated carrier strains for protein antigens or vaccine DNA.aroA,aroB, and in particulararoEmutants showed strongly reduced growth rates in epithelial cells and even in rich culture media. The metabolism of thearomutants under these conditions was predominantly anaerobic. Aerobic metabolism and a wild-type growth rate were, however, regained upon the addition of vitamin K2, suggesting that thearomutants are deficient in oxidative respiration due to the lack of menaquinone. Replication of thearomutants in the host cell's cytosol and cell-to-cell spread were drastically slowed down, and allaromutants showed high virulence attenuation in mice, i.e., the 50% lethal dose in BALB/c mice was increased at least 104-fold for thearoA,aroB, andaroA/Bmutants and >105-fold for thearoEmutant compared to the parent strain. Nevertheless, mice preimmunized witharomutant bacteria elicited good T-cell response and full protection against a subsequent challenge with the virulent wild-type strain. A total of 5 × 106aroA,aroB, andaroA/Bmutant bacteria were sufficient to obtain a protective T-cell response, while 5 × 108aroEoraroA/Emutants were necessary to achieve comparable numbers of antigen-specific T cells. These numbers were well tolerated without causing any signs of disease, indicating thatListeriastrains with deletions in genes of the basic branch of the aromatic amino acid pathway could be useful vaccine carriers for inducing T-cell immunity.

Bacteria-mediated transfer of plasmid DNA into mammalian cells (bactofection) is a potent approach to express plasmid-encoded heterologous proteins (protein antigens, toxins or enzymes) in a large set of different cell types including phagocytic and nonphagocytic mammalian cells. Previously, we have described a Listeria monocytogenes-mediated DNA delivery system, which releases plasmid DNA directly into the cytosol of mammalian cells by partial self-destruction of the carrier bacteria. Here we report on a second generation of this phage lysin supported bactofection system, which is greatly improved with respect to plasmid stability, transfer efficacy and biosafety. In this case, DNA release is initiated by spontaneous bacterial lysis in the infected cells cytosol which is subsequently enhanced by the simultaneously released phage lysin produced by the intracellular carrier bacteria. Bacteria that are capable of cell-to-cell spread are found to be much more efficient in bactofection than their non spreading counterparts.

Protein p60 encoded by theiapgene is regarded as an essential gene product ofListeria monocytogenes. Here we report, however, the successful construction of a viableiapdeletion mutant ofL. monocytogenesEGD. The mutant, which produces no p60, shows abnormal septum formation and tends to form short filaments and hooked forms during logarithmic growth. These abnormal bacterial cells break into almost normal sized single bacteria in the late-stationary-growth phase. Theiapmutant is strongly attenuated in a mouse model after intravenous injection, demonstrating the importance of p60 during infection, and the invasiveness of the Δiapmutant for 3T6 fibroblasts and Caco-2 epithelial cells is slightly reduced. Upon uptake by epithelial cells and macrophages, theiapmutant escapes from the phagosome into the cytosol with the same efficiency as the wild-type strain, and the mutant bacteria also grow intracellularly at a rate similar to that of the wild-type strain. Intracellular movement and cell-to-cell spread are drastically reduced in various cell lines, since theiap-negative bacteria fail to induce the formation of actin tails. However, the bacteria are covered with actin filaments. Most intracellular bacteria show a nonpolar and uneven distribution of ActA around the cell, in contrast to that for the wild-type strain, where ActA is concentrated at the old pole. In aniap+revertant strain that produces wild-type levels of p60, intracellular movement, cell-to-cell spread, and polar distribution of ActA are fully restored. In vitro analysis of ActA distribution on the filaments of the Δiapstrain shows that the loss of bacterial septum formation leads to ActA accumulation at the presumed division sites. In the light of data presented here and elswhere, we propose to renameiap(invasion-associated protein)cwhA(cell wall hydrolase A).

Human-monocyte-derived dendritic cells (MoDC) are very efficient in the uptake ofListeria monocytogenes, a gram-positive bacterium which is an important pathogen in humans and animals causing systemic infections with symptoms such as septicemia and meningitis. In this work, we analyzed the influence of blood plasma on the internalization ofL. monocytogenesinto human MoDC and compared the uptake ofL. monocytogeneswith that ofSalmonella entericaserovar Typhimurium andYersinia enterocolitica. While human plasma did not significantly influence the uptake of serovar Typhimurium andY. enterocoliticaby human MoDC, the efficiency of the uptake ofL. monocytogenesby these phagocytes was strongly enhanced by human plasma. In plasma-free medium the internalization ofL. monocytogeneswas very low, whereas the addition of pooled human immunoglobulins resulted in the internalization of these bacteria to a degree comparable to the highly efficient uptake observed with human plasma. All human plasma tested contained antibodies against the 60-kDa extracellular protein ofL. monocytogenes(p60), and anti-p60 antibodies were also found in the commercially available pooled immunoglobulins. Strikingly, in contrast toL. monocytogeneswild type, aniapdeletion mutant (totally deficient in p60) showed only a minor difference in the uptake by human MoDC in the presence or the absence of human plasma. These results support the assumption that antibodies against the listerial p60 protein may play an important role in Fc-receptor-mediated uptake ofL. monocytogenesby human MoDC via opsonization of the bacteria. This process may have a major impact in preventing systemic infection inL. monocytogenesin immunocompetent humans.

Listeria monocytogenes is a facultative intracellular bacterium that enters a variety of non-professional mammalian cells by triggered phagocytosis ("zipper mechanism") and replicates in the cytosol of the infected host cells. Therefore, it is a promising vaccine vector for the presentation of passenger antigens to the MHC class II and especially class I pathways. Here, we review recent progress made in our laboratory on the development of novel attenuated L. monocytogenes carrier strains for the delivery of heterologous antigens or antigen-encoding DNA and RNA to eukaryotic host cells. Based on the deletion of the chromosomal copy of the tryptophanyl-tRNA synthetase gene (trpS) and plasmid-based in trans complementation of the same, we were able to establish a balanced-lethal plasmid system in L. monocytogenes. Safety concerns in the antigen delivery in vivo were addressed by chromosomal deletion of genes in the basic branch of the aromatic amino acid pathway, resulting in safe, attenuated L. monocytogenes carrier strains. Furthermore, plasmid-based expression of a cytosolically expressed phage lysin resulted in a self-destructing carrier strain that has been successfully used for the delivery of antigens as well as antigen-encoding plasmid DNA and particularly mRNA, therefore overcoming bottlenecks that have been shown to exist for bacteria-mediated DNA delivery.

Listeria monocytogenescan be used to deliver protein antigens or DNA and mRNA encoding such antigens directly into the cytosol of host cells because of its intracellular lifestyle. In this study, we compare the in vivo efficiencies of activation of antigen-specific CD8 and CD4 T cells when the antigen is secreted byL. monocytogenesor when antigen-encoding plasmid DNA or mRNA is released by self-destructing strains ofL. monocytogenes. Infection of mice with self-destructingL. monocytogenescarriers delivering mRNA that encodes a nonsecreted form of ovalbumin (OVA) resulted in a significant OVA-specific CD8 T-cell response. In contrast, infection withL. monocytogenesdelivering OVA-encoding DNA failed to generate specific T cells. Secretion of OVA by the carrier bacteria yielded the strongest immune response involving OVA-specific CD8 and CD4 T cells. In addition, we investigated the antigen delivery capacity of a self-destructing, virulence-attenuatedL. monocytogenes aroA/Bmutant. In contrast to the wild-type strain, this mutant exhibited only marginal liver toxicity when high doses (5 × 107CFU per animal administered intravenously) were used, and it was also able to deliver sufficient amounts of secreted OVA into mice. Therefore, the results presented here could lay the groundwork for a rational combination ofL. monocytogenesas an attenuated carrier for the delivery of protein and nucleic acid vaccines in novel vaccination strategies.

The limited access to the nuclear compartment may constitute one of the major barriers after bacteria-mediated expression plasmid DNA delivery to eukaryotic cells. Alternatively, a self-destructing Listeria monocytogenes strain was used to release translation-competent mRNA directly into the cytosol of epithelial cells, macrophages and human dendritic cells. Enhanced green fluorescent protein (EGFP)-encoding mRNA, adapted for translation in mammalian cells by linking an IRES element to the 5'-end of the egfp coding sequence, was produced by T7 RNA polymerase in the carrier bacteria upon entry into the cytosol where the mRNA is efficiently released from the lysed bacteria and immediately translated in eukaryotic host cells. Besides the much earlier expression of EGFP being detectable already 4 h after infection, the number of EGFP expressing mammalian cells obtained with this novel RNA delivery technique is comparable to or - especially in phagocytic cells - even higher than that obtained with the expression plasmid DNA delivery strategy. Accordingly, bacteria-mediated delivery of ovalbumin-encoding mRNA to macrophages resulted in efficient antigen processing and presentation in vitro indicating that this approach may also be adapted for the in vivo delivery of antigen-encoding mRNA leading to a more efficient immune response when applied to vaccine development.

Genetic immunization with plasmid DNA vaccines has proven to be a promising tool in conferring protective immunity in various experimental animal models of infectious diseases or tumors. Recent research focuses on the use of bacteria, in particular enteroinvasive species, as effective carriers for DNA vaccines. Attenuated strains of Shigella flexneri, Salmonella spp., Yersinia enterocolitica or Listeria monocytogenes have shown to be attractive candidates to target DNA vaccines to immunological inductive sites at mucosal surfaces. This review summarizes recent progress in bacteria-mediated delivery of plasmid DNA vaccines in the field of infectious diseases and cancer.

On the basis of attenuated intracellular bacteria, we have developed two delivery systems for either heterologous proteins or DNA vaccine vectors. The first system utilizes attenuated strains of Gram-negative bacteria which are engineered to secrete heterologous antigens via the α-hemolysin secretion system (type I) of Escherichia coli. The second system is based on attenuated suicide strains of Listeria monocytogenes, which are used for the direct delivery of eukaryotic antigen expression vectors into professional antigen-presenting cells (APC) like macrophages and dendritic cells in vitro and can be also used in animal models.