Dr. Navalka proposes, characterize recombinants that express immunoreactive Mycobacterium tuberculosis proteins in E. coli to characterize the role(s) of these proteins from recombinants in the immune response to mycobacterial infection, to characterize the distribution, expression and immunoreactivity of the groE operon, and to evaluate antibodies and antigens from these studies for use in the immunodiagnosis of mycobacterial infections.Specifically, this will involve the generation of recombinants expressing mycobacterial proteins fused to an oligohistidine tail that will be purified by immobilized metal ion affinity chromatography (IMAC), screening these purified proteins for ability to elicit proliferation or cytokine production. The ability of these cloned proteins to react with serum antibodies from 10 tuberculosis patients will be examined by immunoblot in both native and denaturing polyacrylamide gels, mice will be immunized and antibodies and plaque-forming cells will be reacted with wholesale lysates from other mycobacteria (leprae, avium, kansasii, intracellulare) in ELISA, immunoblot and Jerne plaque-forming assays; and DTH responses to these antigens in guinea pigs will, also, be examined in animals immunized with various other mycobacteria. In addition, mononuclear cells from tuberculosis patients, BCG-vaccines, healthy PPD+ and PPD- individuals will be studied for proliferative responses as well as lymphokine and monokine production and cytotoxic activity, and mini-libraries and chemically synthesized peptides will be used to epitope mapping. They will also characterize the groE operon with respect to heat shock stimulation as well as the distribution of GroEL homologs from related strains. Finally, they will examine the use of antibodies and antigens generated in these studies as immunodiagnostic reagents for the deduction of cellular and humoral responses and of antigens in clinical samples.
