Screening of a cosmid library of Mycobacterium bovis BCG in Mycobacterium smegmatis for novel T-cell stimulatory antigens

We have developed a novel method for screening a Mycobacterium bovis (BCG) cosmid library in Mycobacterium smegmatis for the detection of immunostimulatory T-cell antigens (Ag). Distinctive protein banding patterns were demonstrated in culture filtrates of three of 30 recombinant M. smegmatis clones: pBCCS13 (41 and 73 kDa); pBCCS221 (30, 50 and 68 kDa); pBCCS223 (100 kDa). Western immunoblots indicated that monoclonal antibodies (mAb) directed to the previously characterized 19-, 30-, 38-, 65- and 71-kDa mycobacterial Ag were not reactive with the distinctive recombinant proteins. Furthermore, T-cell Western blots demonstrated that fractions containing the distinctive proteins were immunostimulatory. A given tuberculin-positive donor expressed unique patterns of blastogenic reactivity to protein fractions isolated from each of the three recombinant clones. Restriction enzyme digests of the three recombinant BCG inserts revealed distinctive DNA-banding patterns. The immunostimulatory Ag, therefore, are most likely encoded within different regions of the BCG genome, as contained within three distinct inserts. T-cell Western blots further indicated a heterogeneity in the repertoire of BCG-responsive T cells since tuberculin-positive donors varied in the pattern of reactivity to protein fractions isolated from the same recombinant filtrate. Most likely, immunity to M. tuberculosis results from activation of a heterogeneous array of T cells targeted to multiple immunostimulatory Ag. The method we describe should greatly enhance our ability to define the full spectrum of T-cell Ag encoded by mycobacteria, particularly those which are secreted proteins.