Humoral Immunity and CD4+ Th1 Cells Are Both Necessary for a Fully Protective Immune Response upon Secondary Infection with Brucella melitensis

Abstract

Brucella spp are intracellular bacteria that cause brucellosis, one of the most common zoonoses in the world. Given the serious medical consequences of this disease, a safe and effective human vaccine is urgently needed. Efforts to develop this vaccine have been hampered by our lack of understanding of what constitutes a protective memory response against Brucella. In this study, we characterize the cells and signaling pathways implicated in the generation of a protective immune memory response following priming by the injection of heat-killed or live Brucella melitensis 16M. Using a panel of gene-deficient mice, we demonstrated that during a secondary recall response, both the Brucella-specific humoral response and CD4+ Th1 cells must act together to confer protective immunity in the spleen to B. melitensis infection. Humoral protective immunity is induced by the inoculation of both heat-killed and live bacteria, and its development does not require T cells, MyD88/IL-12p35 signaling pathways, or an activation-induced deaminase–mediated isotype switch. In striking contrast, the presence of memory IFN-γ–producing CD4+ Th1 cells requires the administration of live bacteria and functional MyD88/IL-12p35 pathways. In summary, our work identifies several immune markers closely associated with protective immune memory and could help to define a rational strategy to obtain an effective human vaccine against brucellosis.

Identification of Immune Effectors Essential to the Control of Primary and Secondary Intranasal Infection with Brucella melitensis in Mice

“…At 28 d p.i., both groups were treated daily with a combination of antibiotics (see Materials and Methods) for 2 wk to eliminate the Brucella. As shown previously (17), this treatment led to the complete elimination of Brucella in all tested organs of the infected mice. After a rest period of 2 wk, control mice and previously infected mice were challenged with an mCherry-Br strain of B. melitensis.…”

“…Previous studies by our group using an i.p. B. melitensis infectious model with the same B. melitensis strain (17) showed that genetic deficiencies leading to a lack of CD4 + T cells (MHCII infected with wt B. melitensis 28 d previously and treated with antibiotics as described previously were challenged with mCherry-B. melitensis.…”

“…The oral treatment was a combination of rifampicin (12 mg/kg) and streptomycin (450 mg/kg) (adapted from Ref. 17) prepared fresh daily and given in the drinking water. To ensure that the antibiotic treatment was effective, some mice from each group were sacrificed 1 wk prior to the challenge, and the CFU counts were evaluated in the spleen.…”

“…As described previously (17), spleens were harvested, cut into small pieces, and incubated for 30 min at 37˚C with a mix of DNase I fraction IX (Sigma-Aldrich) (100 mg/ml) and 1.6 mg/ml collagenase (400 M and l U/ml). Spleen cells were washed, filtered, and incubated with saturating doses of purified 2.4G2 (anti-mouse FcR; American Type Culture Collection) in 200 ml PBS, 0.2% BSA, and 0.02% NaN3 (FACS buffer) for 20 min at 4˚C to prevent Ab binding to the FcR.…”

“…+ T cells (15)(16)(17), and specific Abs (17) in the protective immune response against Brucella has been well established. However, those results were obtained primarily through analysis of the splenic immune response following i.p.…”