Immunization with VirB7 from Brucella abortus, a modulator of macrophage functions, protects mice from virulent challenge

CORA POLLAK; M. VICTORIA DELPINO; CARLOS A. FOSSATI; PABLO C. BALDI

Buenos Aires, Argentina

Congreso; 1º Congreso Franco-Argentino de Inmunología (FAIC) y LVIII Reunión Anual de la Sociedad Argentina de Inmunología.; 2010

Brucella survival inside macrophages depends on the type IV secretion system (T4SS) encoded by virB genes. We evaluated in vitro the modulation of macrophage functions by T4SS structural proteins located in Brucella external membrane (VirB7 and VirB9). Monocytic human THP-1 cells were preincubated with recombinant VirB7 or VirB9 proteins and stimulated for 24 h with agonists of TLR4 (LPS), TLR2 (Pam3Cys) or TLR5 (flagellin), and TNF-a and IL-1B levels were measured in culture supernatants. Only VirB7-pretreated cells showed a significant decrease of LPS- or flagellin-stimulated IL-1B secretion (>77% and >81%, respectively), and of LPS-, Pam3Cysor flagellin-stimulated TNF-a secretion (62%, 63% and 90% respectively). In view of these results we decided to evaluate if immunization with VirB proteins awards protection to Brucella challenge. BALB/c mice were immunized twice with either VirB7, VirB9 or PBS mixed with Freund´s adjuvant. As positive vaccination control a group received a dose of heat-killed B. melitensis H38. One month after the last dose half of the animals were challenged with B. abortus 544 and the other half was used for immune response studies. One month after challenge animals were sacrificed and spleens were extracted for bacterial counts. Specific anti-VirB7 IgG1 titers were higher than IgG2, and the opposite was true for anti-VirB9. In cellular response tests both proteins specifically stimulated IFN-g secretion (VirB7, 3223.03 ± 873.39 pg/ml vs. 83.89 ± 80.56 pg/ml in PBS control; VirB9, 477.81 ± 235.42 pg/ml vs. 30.98 ± 34.31 pg/ml). VirB proteins did not stimulate IL-4 secretion. Splenic bacterial loads after B. abortus challenge were significantly lower in mice immunized with VirB proteins than in the PBS group. These results suggest that VirB7 modulates human macrophages functions, contributing to Brucella survival inside these cells. In addition, VirB7 and VirB9 are potentially useful for developing acellular vaccines against Brucella.