CONICET | Buscador de Institutos y Recursos Humanos

Brucella lumazine synthase (BLS) is a stable protein that folds into a dimer of homopentamers. BLS activates dendritic cells (DC) via TLR4 and generates a strong long-lasting humoral immune response. In order to generate a bifunctional and polarized platform, we rationally designed two different mutant pentamers, BLSDR and BLSKE. In these mutants the association between identical pentamers is impaired but they are able to generate heterodecamers, BLSDRKE, when mixed. In order to evaluate if the pentamers are able to activate DC, bone marrow derived DC (BMDC) were stimulated with each pentamer or with decameric wild type BLS and expression of costimulatory molecules and cytokine secretion were analyzed. BMDC were stimulated with 90μg of BLSDR, BLSKE or BLS. After 18h the level of expression of CD80 was analyzed by FACS. The amount of IL-12p70 in culture supernatant was analyzed by ELISA. Results show that both pentamers and BLS induce a similar upregulation of CD80 in BMDC surface. Surprisingly, the secretion of IL12p70 induced by each pentamer is significantly higher than the amount secreted by BLS-stimulated BMDC. To assess the humoral response induced by each protein, BALB/c mice were sc immunized with 10μg of BLS, BLSDR, BLSKE or BLSDRKE. Serum was obtained every 14 days and anti-BLS humoral response was assesed by ELISA. After 14 days post-immunization specific antibodies can only be detected in mice immunized with BLS. After 42 days, mice immunized with BLSKE or BLSDRKE show a significant level of anti-BLS IgG but lower of that induced by BLS; BLSDR does not generate specific antibodies.In conclusion, these mutant pentamers are capable of activating DC and induce a TH1 response in a similar way than BLS. However, the ability to induce a humoral response is diminished in the heterodecamer and BLSKE pentamer and is impaired in BLSDR pentamer. These results are promising for future biotechnological applications in which humoral response is not desired.