Targeting TNF-a regulating genes to enhance tuberculosis vaccination

Abstract

Tumor necrosis factor-a (TNF) plays a critical role in controlling the host immune response to tuberculosis. After exposure to Mycobacterium tuberculosis (MTB), TNF is essential for the formation and maintenance of the TB granuloma. TNF blockers, prescribed for the treatment of inflammatory diseases, can cause reactivation of latent TB, indicating the importance of the cytokine for the continual control of disease latency. Recent evidence has shown that certain bacterial components can modulate TNF production, leading to the supposition that MTB has evolved the ability to manipulate host TNF.;Using a genetic screening system, we identified four genes, which when inactivated, induced infected mouse macrophages to increase TNF production. TNF is a critical factor for phagosome-lysosome maturation, an event that is pertinent to antigen presentation and activation of CD4+ T cells. Using fluorescent microscopy analysis, we showed that TNF enhancing MTB knockouts improved phago-lysosomal processing compared to macrophages infected with wildtype bacteria. Another cellular process highly dependent upon TNF is apoptosis, as TNF is a known initiator of caspase pathways. Strains engineered to enhance apoptosis have been shown to boost immunogenicity via cross-priming of CD8+ T cells. Using a fluorescent caspase inhibitor and laser scanning cytometry, we demonstrated that TNF enhancing knockouts increased apoptosis of infected macrophages. Furthermore, we have demonstrated that mice infected with TNF enhancing mutants produce CD4+ and CD8+ T cells that secrete more IFN-gamma compared to mice infected with wildtype bacteria. These results lay credence to the hypothesis that by engineering strains to specifically boost TNF production, we can potentially augment host immunity.;The ultimate goal of this work is for the rational design of a safe and effective anti-TB vaccine. Using auxotroph mutants that have been derived from virulent MTB, we have begun incorporating TNF enhancing mutations into parental auxotroph strains. Although all recently tested mutants induce protection comparable to BCG, we hope to identify a new strain that protects better, thus leading to the potential development of a new vaccine candidate.