In this new grant proposal, investigators with expertise in cellular immunology, molecular biology, purification of mycobacterial antigens and diagnostic techniques propose to use human T-cell lines and clones to screen a lambda gtll genomic DNA expression library for M. tuberculosis The goal of this screening will be to identify recombinant antigens of potential use in protection against tuberculosis and in the diagnosis of infection and disease due to the tubercle bacillus. Since tuberculosis is a leading cause of morbidity and mortality worldwide, this is a health related project. Direct T-cell screening of recombinant mycobacterial antigens is proposed, a novel approach consonant with the importance of cell-mediated immunity in protection against and delayed-type hypersensitivity skin testing in diagnosis of tuberculous infection. Different bacteriophage will be amplified in E. coli Y1090 and used to prepare lysogens in E. coli Y1089. phage multiplication and production of fusion proteins will be induced. Then, fusion proteins will be purified from bacterial lysates by immunoabsorbent and molecular sieve chromatography. Initial screening will be for recombinant mycobacterial antigens capable of inducing 3H-thymidine incorporation in antigen-specific T-cell lines from healthy tuberculin reactors. Bacteriophage encoding positive fusion proteins will be screened in smaller batches by diluting the E. coli lysogens and rescreening until relevant clones of recombinant bacteriophage are identified. Screening for possible protective antigens will involve testing individual recombinant antigens for induction of interleukin-2 and interferon-gamma production in antigen-specific T-helper cell clones. Screening for antigens useful in skin testing will consist of demonstrating species restriction. Such antigens will induce DNA synthesis in antigen specific T-cell lines and may be capable of activating T-cell clones specific for M. tuberculosis or reacting with antibody to species-specific epitopes. They must not, however, activate T-cells or react with antibodies which reflect hypersensitivity to nontuberculous mycobacteria. Candidate diagnostic antigens will be evaluated for specificity in elicitation of delayed-type hypersensitivity skin test responses in sensitized guinea pigs.
