The Attenuated Brucella abortus Strain 19 Invades, Persists in, and Activates Human Dendritic Cells, and Induces the Secretion of IL-12p70 but Not IL-23.

BACKGROUND: Bacterial vectors have been proposed as novel vaccine strategies to induce strong cellular immunity. Attenuated strains of Brucella abortus comprise promising vector candidates since they have the potential to induce strong CD4(+) and CD8(+) T-cell mediated immune responses in the absence of excessive inflammation as observed with other Gram-negative bacteria. However, some Brucella strains interfere with the maturation of dendritic cells (DCs), which is essential for antigen-specific T-cell priming. In the present study, we investigated the interaction of human monocyte-derived DCs with the smooth attenuated B. abortus strain (S) 19, which has previously been employed successfully to vaccinate cattle. METHODOLOGY/PRINCIPAL FINDINGS: We first looked into the potential of S19 to hamper the cytokine-induced maturation of DCs; however, infected cells expressed CD25, CD40, CD80, and CD86 to a comparable extent as uninfected, cytokine-matured DCs. Furthermore, S19 activated DCs in the absence of exogeneous stimuli, enhanced the expression of HLA-ABC and HLA-DR, and was able to persist intracellularly without causing cytotoxicity. Thus, DCs provide a cellular niche for persisting brucellae in vivo as a permanent source of antigen. S19-infected DCs produced IL-12/23p40, IL-12p70, and IL-10, but not IL-23. While heat-killed bacteria also activated DCs, soluble mediators were not involved in S19-induced activation of human DCs. HEK 293 transfectants revealed cellular activation by S19 primarily through engagement of Toll-like receptor (TLR)2. CONCLUSIONS/SIGNIFICANCE: Thus, as an immunological prerequisite for vaccine efficacy, B. abortus S19 potently infects and potently activates (most likely via TLR2) human DCs to produce Th1-promoting cytokines.

Cytotoxic activity of murine resident peritoneal cells against Listeria monocytogenes-infected hepatocytes in vitro.

Sterile immunity to the Gram-positive facultative intracellular bacterium Listeria monocytogenes critically depends on cytotoxic CD8+ T lymphocytes. However, the cytotoxic cell population able to kill infected cells before specific T cells are generated is not well characterised. Based on histological observations and the use of monoclonal antibodies abrogating the CD11b/CD18-dependent cellular influx into infected organs as well as granulocyte-depleting antibodies, some authors favour PMNs as being most important in the preimmune lysis of L. monocytogenes-infected hepatocytes. However, these experiments do not conclusively demonstrate that this cell population is indeed directly cytotoxic to infected cells. The aim of this study was to establish an experimental approach for a more detailed analysis of cell-cell interactions in the preimmune phase of listeriosis. We here present an ex vivo-in vitro plaque-forming assay, which enables the quantitative analysis and experimental modulation of the cytotoxic activity of murine (BALB/c) peritoneal cells (PC) against L. monocytogenes-infected hepatocytes (ATCC TIB 73). Using this system, we show that naive resident peritoneal macrophages are able to kill L. monocytogenes-infected hepatocytes. The plaque-forming capacity increased in the presence of interferon gamma (IFN-gamma) and was dissociable from nitric oxide (NO) production. These findings suggest a new, i.e. cytotoxic role for macrophages in the early phase of host defence against a facultative intracellular bacterium.

Tailoring host immune responses to Listeria by manipulation of virulence genes -- the interface between innate and acquired immunity.

Although attenuated strains of microbial pathogens have triggered vaccine development from its origin, the role of virulence factors in determining host immunity has remained largely unexplored. Using the murine listeriosis model, we investigated whether the induction and expansion of protective and inflammatory T cell responses may be modified by selective manipulation of virulence genes. We intentionally deleted specific genes of Listeria monocytogenes, including those encoding the positive regulatory factor (prfA), hemolysin (hly), the actin nucleator (actA), and phospholipase B (plcB). The resulting strains showed decisive differences in their immunogenic properties. In particular, we identified a double-deletion mutant that retained Listeria's profound ability to induce protective CD8(+) T cells, but that is strongly attenuated and exhibits a significantly reduced ability to induce CD4(+) T cell-mediated inflammation. We conclude that this mutant, L. monocytogenes DeltaactADeltaplcB, is at present the most promising mutant for a bacterial vaccine vector and is able to safely induce potent CD8(+) T cell-mediated immunity.