INTERNALIZATION AND PERSISTENCE OF THE VACCINE CARRIER BRUCELLA LUMAZINE SYNTHASE (BLS).

ANDRÉS H. ROSSI; VANINA A. ALZOGARAY; ANA FARIAS; FERNANDO A. GOLDBAUM; PAULA M. BERGUER

Ciudad Autónoma de Buenos Aires

Congreso; Latin American Society Immunodeficiencies; 2015

BLS is a highly immunogenic and stable decameric protein, successfully used as a carrier for several proteins. BLS immunization activates dendritic cells (DC) and induces the cross-presentation of associated peptides and TLR4-dependent specific cytotoxicity in mice. To measure the duration of antigen exposure to CD8+ T cells, mice were immunized with BLS-OVA257-264 and adoptive transfer of OT-I cells was performed at different times after vaccination. Proliferation of OT-I cells is observed even at 22 days after immunization, indicating that antigen presentation still takes place and suggesting a prolonged antigen availability. We have shown that fluorescence can be detected by FACS at the DLNs in B lymphocytes at 4h p.i. and in DC from 4h to 6 days p.i after immunization with fluorescent BLS. In TLR4-deficient mice, fluorescence is only detected until 4h. In this work, the presence of BLS was demonstrated by Western blot using a specific monoclonal antibody and a single-chain camelid antibody (VHH) that only recognizes BLS in its native conformation. Results show that BLS is still found in native conformation at the DLNs 14 days p.i. To study the BLS internalization mechanism in bone marrow DCs, cells were pulsed with BLS-Alexa488 in presence of different inhibitors and then analyzed by FACS. The addition of a specific inhibitor of macropinocytosis (DMA) significantly diminished BLS internalization, thus being the main mechanism of internalization. These results show that BLS is mainly internalized by macropinocytosis and support the idea that this antigen persists for long periods.These results show that BLS is internalized by at least two different mechanisms and support the idea that BLS is a persisting antigen.