Gene Expression Profile and Immunological Evaluation of Unique Hypothetical Unknown Proteins of Mycobacterium leprae by Using Quantitative Real-Time PCR

Abstract

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. Twentysix 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.T he diagnosis of leprosy is usually based solely on clinical symptoms, requiring the presence of a neurological deficit and skin lesions (1; http://www.who.int/lep/diagnosis/en/index.html), but due to low sensitivity, physical diagnosis is applicable only to patients with actual disease. More than 70% of infected patients are negative for acid-fast bacilli (AFB) and do not present analgesic skin lesions, especially paucibacillary/tuberculoid (PB/TT) leprosy patients (1). Since the presence of skin lesions in these patients is variable, their clinical symptoms are not sufficient to specifically diagnose leprosy (1). These problems are accentuated in the case of diagnosis of individuals with subclinical Mycobacterium leprae infection, including household contacts (HHCs) of leprosy patients, regarded as the primary source of ongoing leprosy prevalence (1-5).Several M. leprae antigens have been identified and evaluated for their diagnostic potential by serological or cell-mediated immunity (CMI)-based tests (2, 4-24). Serological assay with a single M. leprae-specific antigen, phenolic glycolipid I (PGL-I), successfully detects circulating antibodies in sera of multibacillary/lepromatous leprosy (MB/LL) patients. However, this test fails to detect the majority of PB/TT patients and HHCs, though these individuals present strong CMI responses to mycobacterial antigens (3, 13). Both in vitro gamma interferon release assays (IGRAs) and a simple delayed-type hypersensitivity skin test have been developed to detect...

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