Recombinant proteins encoded by genes present in Mycobacterium tuberculosis-specific regions of difference induce delayed-type hypersensitivity skin responses (43.31)

Abstract

Abstract The world-wide control of tuberculosis (TB) requires affordable and easy to apply test(s), which could differentiate TB from BCG vaccination and exposure to environmental mycobacteria. In this study, the proteins encoded by Mycobacterium tuberculosis-specific regions of difference (RD) genes, which are absent in BCG and most environmental mycobacteria, were studied for delayed-type hypersensitivity (DTH) skin responses in guinea pigs. The genes of RD1 (CFP10 and ESAT-6), RD7 (Rv2346 and Rv2347) and RD9 (Rv3619 and Rv3620) proteins were cloned in plasmid vectors and expressed in Escherichia coli. The expressed recombinant proteins were purified to homogeneity using affinity columns. Guinea pigs were injected with 1x1010 heat-killed M. tuberculosis, and phosphate buffered saline (PBS), as a placebo control. After four weeks of injection, all the animals were tested for DTH skin responses by intradermal injection with PBS (negative control), purified protein derivative (PPD) and sonicates of M. tuberculosis (positive control), and purified recombinant proteins. The results showed that PBS failed to induced DTH responses in any group, whereas PPD, M. tuberculosis sonicates and the recombinant proteins induced positive DTH responses in animals injected with heat-killed M. tuberculosis, but not in PBS-injected animals. These results suggest that purified RD proteins are active in inducing DTH responses in vivo, and may be useful in specific diagnosis of TB.