02499nas a2200337   4500000000100000008004100001260001300042653002400055653002300079653002000102653003000122653001100152653001600163653002300179653001200202653002600214653002500240653004000265653002500305653001700330100001000347700001800357700001700375700001500392700001500407245015800422300001100580490000700591520154900598022001402147       2013                            d  c2013 Feb10aAntigens, Bacterial10aBacterial Proteins10aGene Expression10aGene Expression Profiling10aHumans10aImmune Sera10aImmunity, Cellular10aleprosy10aLymphocyte Activation10aMycobacterium leprae10aReal-Time Polymerase Chain Reaction10aRecombinant Proteins10aTuberculosis1 aKim H1 aPrithiviraj K1 aGroathouse N1 aBrennan PJ1 aSpencer JS00aGene expression profile and immunological evaluation of unique hypothetical unknown proteins of Mycobacterium leprae by using quantitative real-time PCR.  a181-900 v203 a<p>The cell-mediated immunity (CMI)-based in vitro gamma interferon release assay (IGRA) of 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-specific 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. In this study, 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 of 26 selected antigen candidates were expressed and purified in Escherichia coli. The seroreactivity to these proteins of pooled sera from lepromatous leprosy patients and cavitary tuberculosis patients revealed that 9 of 15 recombinant hypothetical unknowns elicited M. leprae-specific immune responses. These nine proteins may be good diagnostic reagents to improve both the sensitivity and specificity of detection of individuals with asymptomatic leprosy.</p>  a1556-679X