From Genome-Based In Silico Predictions to Ex Vivo Verification of Leprosy Diagnosis

Abstract

The detection of hundreds of thousands of new cases of leprosy every year suggests that transmission of Mycobacterium leprae infection still continues. Unfortunately, tools for identification of asymptomatic disease and/or early-stage M. leprae infection (likely sources of transmission) are lacking. The recent identification of M. leprae-unique genes has allowed the analysis of human T-cell responses to novel M. leprae antigens. Antigens with the most-promising diagnostic potential were tested for their ability to induce cytokine secretion by using peripheral blood mononuclear cells from leprosy patients and controls in five different areas where leprosy is endemic; 246 individuals from Brazil, Nepal, Bangladesh, Pakistan, and Ethiopia were analyzed for gamma interferon responses to five recombinant proteins (ML1989, ML1990, ML2283, ML2346, and ML2567) and 22 synthetic peptides. Of these, the M. leprae-unique protein ML1989 was the most frequently recognized and ML2283 the most specific for M. leprae infection/exposure, as only a limited number of tuberculosis patients responded to this antigen. However, all proteins were recognized by a significant number of controls in areas of endemicity. T-cell responses correlated with in vitro response to M. leprae, suggesting that healthy controls in areas where leprosy is endemic are exposed to M. leprae. Importantly, 50% of the healthy household contacts and 59% of the controls in areas of endemicity had no detectable immunoglobulin M antibodies to M. leprae-specific PGL-I but responded in T-cell assays to >1 M. leprae protein. T-cell responses specific for leprosy patients and healthy household contacts were observed for ML2283-and ML0126-derived peptides, indicating that M. leprae peptides hold potential as diagnostic tools. Future work should concentrate on the development of a sensitive and field-friendly assay and identification of additional peptides and proteins that can induce M. leprae-specific T-cell responses.

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

“…Only two of the selected antigens, ML0573 and ML0574, had already been studied as T cell antigens and reported in the literature, but these failed to induce M. leprae-specific T cell immunity in a Brazilian population (14,15). Recently, an IGRA validated that five of the most promising recombinant hypothetical unknowns (ML0126, ML1420, ML1989, ML2283, and ML2346) and 22 of their peptides induced IFN-␥ secretion from PB patients or HHCs in areas where leprosy is endemic in Brazil, Bangladesh, Nepal, Pakistan, and Ethiopia (16). In agreement with previous studies (14,15,17,22), the ML2283 recombinant protein and ML2283-derived peptides induced the highest M. leprae-specific T cell responses (16).…”

“…Eventually, the selected antigens in this study will allow detection of individuals exposed to and/or infected by M. leprae in regions where leprosy is endemic. Previously, 31 hypothetical unknowns, including some of those addressed in the present study, as well as their corresponding peptides, were expressed and their antigenic potentials for early diagnosis of leprosy evaluated using the IGRA format in various regions of endemicity (6,7,14,16,22). Some of these M. leprae-specific antigens were shown to be valuable diagnostic reagents for specific diagnosis of those infected with M. leprae, particularly occupational contacts or HHCs of MB or PB patients (14,15,17,22).…”

“…Therefore, it is possible that their ORFs are not genuine or are barely expressed and available for T or B cell recognition. Since the identification of these antigens was highly dependent on in silico analysis, low availability to the human immune system and HLA variability might be responsible for the variable reactivity and low sensitivity of IGRAs for different ethnic populations (14)(15)(16)22). Therefore, a major remaining challenge in the development of leprosy diagnostic assays was to select the hypothetical unknown antigens that are definitely expressed and evoke a more definite immune response in individuals with M. leprae infection/exposure, regardless of genetic or geographic situation, and can maintain the specificity of some current IGRAs.…”

“…However, most of the hypothetical unknown proteins and peptides were recognized by a considerable number of endemic controls and TB patients and showed apparent variation in reactivity to racially and geographically different study populations (14)(15)(16)(17)22). Although some of the antigens induced low levels of IFN-␥ production in some individuals in the endemic control group who had strongly responded to M. leprae lysate, the level (100 to 200 pg) of released IFN-␥ in these individuals was just above the cutoff value (100 pg), making it difficult to determine T cell positivity in individuals in the endemic control group with M. leprae exposure (16,17). The question therefore arose as to which, if any, of these proteins are expressed in M. leprae.…”

“…Recently, either recombinant proteins or synthetic peptides originating from these ORFs and containing T cell epitopes restricted via major HLA-DR alleles have been studied as M. leprae-specific antigens (6,7,(14)(15)(16)(17)22), and some have been shown by IGRAs to differentiate individuals infected with M. leprae from healthy controls in areas of endemicity (16,17). However, the levels of gamma interferon (IFN-␥) secretion in response to these antigens, particularly their peptides, were often too low to distinguish all individuals exposed to M. leprae from healthy volunteers in regions of endemicity (16,22). The low sensitivity of current IGRAs raises the question of whether any of the hypothetical unknowns are expressed or are immunologically relevant, especially considering that about 50% of M. leprae genes encoding functional proteins in other mycobacteria are deleted or are pseudogenes (26; http://genolist.pasteur.fr/Leproma/; http: //genolist.pasteur.fr/TubercuList/).…”