Gene expression profile and immunological evaluation of unique hypothetical unknown proteins of Mycobacterium leprae by using quantitative real time-PCR.

Cell mediated immunity (CMI) based in vitro interferon-γ released assay (IGRA) to Mycobacterium leprae specific antigens has potential as a promising diagnostic means to detect those individuals in the early stages of M. leprae infection. Diagnosis of leprosy is a major obstacle toward ultimate disease control, and has been compromised in the past by the lack of specific markers. Comparative bioinformatic analysis among mycobacterial genomes identified potential M. leprae unique proteins called "hypothetical unknowns". Due to massive gene decay and the prevalence of pseudogenes, it is unclear whether any of these proteins are expressed or are immunologically relevant. Here, we performed cDNA based quantitative real time PCR to investigate the expression status of 131 putative open reading frames (ORFs) encoding hypothetical unknowns. Twenty six of the M. leprae specific antigen candidates showed significant levels of gene expression compared to that of ESAT-6 (ML0049) which is an important T cell antigen of low abundance in M. leprae. Fifteen out of 26 selected antigen candidates were expressed and purified in Escherichia coli. The seroreactivity to these proteins using the pooled sera of lepromatous leprosy patients and cavitary tuberculosis patients revealed that 9 out of 15 recombinant hypothetical unknowns elicited M. leprae specific immune responses. These nine proteins may be good diagnostic reagents to improve both sensitivity and specificity in detection of individuals with asymptomatic leprosy.