A BRUCELLA MEMBRANE PROTEIN IMPROVES THE INMUNOGENICITY OF THE CO-ADMINISTERED ANTIGEN BY INCREASING ITS HALF LIFE IN ENDOSOMAL COMPARTMENTS.

CORIA LM; DELPINO MV; IBAÑEZ AE; BRUNO L; RISSO, GS; BARRIONUEVO P.; GIAMBARTALOMEI G. H.; CASSATARO J

Congreso; XXXVII Congreso de la Sociedad Brasilera de Inmunología; 2012

Introduction: In previous studies we have shown that a protein from Brucella spp. (Bp) co-administered as adjuvant increases antigen (Ag) cross-presentation by dendritic cells (DCs) and induces IFN-g producing CD8+ and cytotoxic T cells. We also found that this protein has significant sequence homology with protease inhibitors from other bacteria, therefore we hypothesized that it would inhibit lysosomal proteases activities in endosomal compartments increasing the half life of the co-administered Ag thus increasing its immunogenicity.Methods and results: We analyzed the biological activity of Bp against cysteine and aspartyl proteases (cathepsins) that constitute a subset of endocytic proteases using a fluorometric assay kit (Enzcheck) which contains casein as substrate. We found that Bp inhibited partially the activity of cathepsin D, L, C, and B (p<0.05). Next, we examined whether Bp would limit the different lysosomal proteolytic capacities of APCs. Bp partially inhibited (30%) the proteolytic activity of crude lysosomal extracts derived from macrophages and DCs. To examine if Bp affects Ag proteolysis in the context of whole cell, purified DCs and macrophages from spleen of mice were incubated in vitro with the fluorescent Ag (OVADQ or CASEINBODIPY) and then its intracellular degradation was analyzed by flow cytometry. We have found that Bp limited Ag proteolysis inside DCs or macrophages in vitro (p<0.05). Moreover, Bp was able to inhibit Ag degradation by APCs from spleen and lymph nodes after subcutaneous immunization with the Ag (p<0.05, n=5). Finally, we analyzed if DCs incubated with OVA in presence of Bp were able to induce a sustained cross-presentation of OVA in the context of MHCI using an antibody that recognizes OVA257-264 (SIINFEKL) peptide bound to H-2Kb molecules. DCs pulsed with OVA plus Bp facilitates a greater and maintained cross-presentation of OVA along the time compared with DCs incubated with OVA alone (p<0.05).Conclusion: These results suggest that Bp increases the half life of the Ag inside the endosomal compartments of APCs by limiting its proteolysis. A controlled degradation of the Ag would promote a sustained cross presentation of the Ag, thus increasing its immunogenicity.