Internalization and persistence of the vaccine carrier Brucella lumazine synthase

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

Buenos Aires

Congreso; International Joint Meeting between the Latin American Society for Immunodeficiencies (LASID-IV Meeting), the Argentinean Society for Immunology (SAI-LVIII Meeting), and the French Society for Immunology (SFI); 2015

SAI

BrucellaLumazine Synthase (BLS) is a highly immunogenic and stable decameric protein,successfully used as a carrier for several proteins. BLS immunization activatesdendritic cells (DC) and induces the cross-presentation of associated peptidesand TLR4-dependent specific cytotoxicity in mice. To measure the duration ofantigen exposure to CD8+ T cells, mice were immunized with BLS-OVA257-264 andadoptive transfer of OT-I cells was performed at different times aftervaccination. Proliferation of OT-I cells is observed even at 22 days afterimmunization, indicating that antigen presentation still takes place andsuggesting a prolonged antigen availability. After immunization with BLS-5IAF,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. In TLR4-deficient mice, fluorescence is onlydetected until 4h. In this work, the presence of BLS was demonstrated byWestern blot using a specific monoclonal antibody and a single-chain camelid antibody(VHH) that only recognizes BLS in its native conformation. Results show thatBLS is still found in native conformation at the DLNs 14 days p.i. To study themechanism of BLS internalization in bone marrow DCs, SAI 2015 35 cells werepulsed with BLS-Alexa488 in presence of different inhibitors and then analyzedby FACS. The addition of a specific inhibitor of macropinocytosis (DMA)significantly diminished BLS internalization, thus being the main mechanism ofinternalization. These results show that BLS is mainly internalized bymacropinocytosis and support the idea that BLS is a persisting antigen.