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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.

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.