Immunoreactivity of peptides generated by limited proteolysis of 71-kDa cell wall protein of Mycobacterium tuberculosis H37Ra using PLG-microparticles

Peptide mapping by limited proteolysis of a highly protective 71-kDa cell w all-associated protein of Mycobacterium tuberculosis H37Ra was carried out in order to identify key protective determinants within the native protein. The 71-kDa protein, which had an isoelectric point of 4.25, was digested i nto eight major bands at 48 h using trypsin and pepsin at equal enzyme to p rotein ratios (pH 5.5). The in vitro lymphocyte reactivity of individual pe ptides suggested P-1, P-2 and P-5 to be significantly immunoreactive in mic e immunized with native 71-kDa-polylactide-coglyeolide (PLG); however, the reactivity was significantly lower than that of the native 71-kDa protein. Immunization of mice with a pooled fraction (upper fraction-71 kDa) of more immunoreactive peptides (consisting of P-1 and P-2) did not further boost their immunoreactivity. However, P-1 and P-2 exhibited comparable or even h igher lymphocyte proliferation in human tuberculous and control subjects. T hese data suggest distinct antigenic specificities in humans and mice and f urther substantiate the use of the 71-kDa protein or its peptides P-1 and P -2 as potential vaccine candidates for tuberculosis.