Brucella spp. lumazine synthase (BLS) vaccine carrier as a model to study antigen processing and persistance.

ROSSI ANDRÉS; ALZOGARAY VANINA; GOLDBAUM FERNANDO; BERGUER PAULA

Milano

Congreso; 15th International Congress of Immunology; 2013

BLS is a highly immunogenic stable decamer, successfully used as a carrier for several proteins. BLS immunization activates murine dendritic cells (DC) and recruits DC, B cells, CD8+ and CD4+ cells at the draining lymph nodes (LN) via TLR4. BLS induces the cross-presentation of associated peptides and a TLR4-dependent specific cytotoxicity. Here, we show which cells BLS is associated with after immunization and its intracellular localization in vitro. Mice were immunized in the hind footpad with BLS-Fluorophore. At 4hs BLS was found in 30±4% of DC and in 10±2% of B lymphocytes at popliteal LN, and in 14±3% of the DC at the inguinal LN. At 120h BLS was still found within DC. In TLR4-deficient mice similar results were observed until 4hs; at longer times BLS was not observed. These results show that BLS resides for longer periods in DC and suggest that DC carry BLS to the inguinal LN. Subcellular localization of BLS was studied by confocal microscopy. Peritoneal macrophages (Mo) and BMDC were incubated with BLS-Fluorophore. Only minor colocalization was observed with classical antigen presentation markers. BLS colocalized with TLR4 at the cell membrane at 5min and with cytoplasmic TLR4 at 6h. BLS was observed at least until 6hs in BALB/c BMDC or Mo; in TLR4-deficient mice it was found only for 1h. These results suggest that BLS binds to TLR4 at the plasma membrane and subsequently enters to the cytoplasm. BLS would escape the endocytic pathway of antigen processing. BLS persistence is dependent on TLR4 signaling.