Brucella spp. is a Gram negative facultative intracellular bacterium that induces chronic infectious disease by direct contact or by consumption of animal products. Brucella is considered a potential pathogen for bioterrorism. Our long-term goal is to develop a Brucella vaccine. Little is known regarding Brucella genes encoding proteins that contribute to intracellular survival and virulence. Recently, a Brucella promoter trap system has been engineered using the promoterless green fluorescence protein (GFP) gene to identify Brucella promoters and associated genes that are activated following intracellular infection. A library of Brucella genes activated within 4 hours of macrophage infection has been identified and additional genes are being determined. Also, our previous evidence indicates that CD4+, CD8+ T cells and IFN-gamma are prominent during clearance of acute infection; but little data exist to indicate the immunologic features critical to disease resolution. However, a Brucella memory response is a likely foundation for successful vaccination, and immunologic memory is hypothesized to play a key role in protection. Now, strategies will be created to protect interferon regulatory factor-1 gene knockout (IRF-1-r) mice that die within 7-10 days from virulent Brucella using attenuated Brucella mutants as vaccine candidates, and evaluate the cytokines and cell phenotypes of immune cells to understand the mechanism of protection. In addition, IRF-1-r mice can mount a protective immune response if vaccinated with certain attenuated mutants. The following specific aims are proposed.
Specific Aim 1 will identify Brucella abortus genes that affect intracellular survival and then engineer those gene deletion mutants of Brucella. Novel promoter-gene combinations activated during in vitro macrophage infection will be identified. In preliminary studies, we have isolated a library of important genes, and results obtained from these studies will be used in the design of attenuated mutant Brucella. Gene deletion mutants of Brucella will be engineered. Attenuated mutant Brucella will be used as vaccine candidates in highly susceptible IRF-1-r mice.
Specific Aim 2 will evaluate the efficacy of attenuated mutant Brucella as potential vaccines and determine the mechanisms responsible for a protective memory response in mice. Protective immunity of selected attenuated mutant Brucella in IRF-1-/- and C57BL/6 mice will be evaluated. Brucella mutants will be tested for their ability to confer protection to IRF-1-r mice as a rapid screen. Candidate mutants will then be tested in C57BL/6 mice followed by virulent B. abortus challenge. The immunologic components that induce protection will be identified using IRF-1-r and C57BL/6 mice. The change in cell phenotypes and cytokines will be monitored throughout the course of vaccination and challenge. Our goal is to use these findings to help develop a vaccine for Brucella. Importantly, no Brucella vaccine is available for humans, and there is an immediate need to protect the public against Brucella that might be used by bioterrorists.
Showing the most recent 10 out of 19 publications
