Tuberculosis results in 3 million deaths annually world-wide and is likely to cause increasing morbidity and mortality in the United States, given the magnitude of the HIV epidemic. This proposal addresses issues critical to eventual development of a recombinant anti-tuberculosis vaccine encoding antigens recognized by human T lymphocytes. Our goals are to understand the human immune response to Mycobacterium tuberculosis at the site of disease activity and gain insight into protective immunologic mechanisms. We will characterize potentially protective mycobacterial antigens and genes with emphasis on cell-wall associated proteins which we hypothesize to be of major immunologic significance. We will achieve our goals through the following specific aims: (1) Identification of phenotypes and functional markers of lymphocytes that are important in the cellular immune response in vivo. We will characterize immunoregulatory CD4+ and CD8+ subpopulations by immunostaining of pleural fluid and tissue of tuberculous pleuritis patients, as well as pulmonary tissue of individuals with fulminant tuberculosis. The importance of various cytokines will be assessed by direct measurement of cytokine levels in pleural fluid and study of cellular mRNA by Northern blot analysis and in situ hybridization. (2) Determination of the in vitro antigen specificity and function of T-lymphocyte clones important in the cellular immune response to tuberculosis. We plan to establish T-cell clones bearing the phenotype and functional markers found to be important by immunohistologic studies and then define the range of antigens and epitopes recognized by these clones. (3) Study the immunologic significance of cell-wall associated proteins in tuberculosis. We will identify cell wall antigens that stimulate potentially protective immune responses to M. tuberculosis utilizing T-cell clones and limited dilution analysis. Identification of the genes encoding protective antigens and determination of the DNA sequences controlling protein incorporation into cell walls of mycobacteria will greatly enhance our ability to develop an anti-tuberculosis vaccine.
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