A Postgenomic Approach to Identification ofMycobacterium leprae-Specific Peptides as T-Cell Reagents

Abstract

To identify Mycobacterium leprae-specific human T-cell epitopes, which could be used to distinguish exposure to M. leprae from exposure to Mycobacterium tuberculosis or to environmental mycobacteria or from immune responses following Mycobacterium bovis BCG vaccination, 15-mer synthetic peptides were synthesized based on data from the M. leprae genome, each peptide containing three or more predicted HLA-DR binding motifs. Eighty-one peptides from 33 genes were tested for their ability to induce T-cell responses, using peripheral blood mononuclear cells (PBMC) from tuberculoid leprosy patients (n ‫؍‬ 59) and healthy leprosy contacts (n ‫؍‬ 53) from Brazil, Ethiopia, Nepal, and Pakistan and 20 United Kingdom blood bank donors. Gamma interferon (IFN-␥) secretion proved more sensitive for detection of PBMC responses to peptides than did lymphocyte proliferation. Many of the peptides giving the strongest responses in leprosy donors compared to subjects from the United Kingdom, where leprosy is not endemic, have identical, or almost identical, sequences in M. leprae and M. tuberculosis and would not be suitable as diagnostic tools. Most of the peptides recognized by United Kingdom donors showed promiscuous recognition by subjects expressing differing HLA-DR types. The majority of the novel T-cell epitopes identified came from proteins not previously recognized as immune targets, many of which are cytosolic enzymes. Fifteen of the tested peptides had >5 of 15 amino acid mismatches between the equivalent M. leprae and M. tuberculosis sequences; of these, eight gave specificities of >90% (percentage of United Kingdom donors who were nonresponders for IFN-␥ secretion), with sensitivities (percentage of responders) ranging from 19 to 47% for tuberculoid leprosy patients and 21 to 64% for healthy leprosy contacts. A pool of such peptides, formulated as a skin test reagent, could be used to monitor exposure to leprosy or as an aid to early diagnosis.The completion of the sequencing of the genome of Mycobacterium tuberculosis (7) and the availability of almost 98% of the genome sequence of Mycobacterium leprae (http://www .sanger.ac.uk) provide a unique opportunity to identify specific antigens within these pathogens, which could be used as diagnostic tools. One approach, which has been used previously to develop M. tuberculosis-specific diagnostic antigens, is to identify genes present in M. tuberculosis which have been deleted from Mycobacterium bovis BCG (20) Previous studies of the human T-cell response in leprosy patients have identified a number of antigens that induce T-cell responses, measured by lymphocyte proliferation or gamma interferon (IFN-␥) secretion, in patients with tuberculoid leprosy. Such antigens include the M. leprae 70-kDa, 65-kDa, 45-kDa, 35-kDa, 18-kDa, and 10-kDa antigens (1,2,6,12,16,29,31). The members of the heat shock family of proteins are highly conserved, and homologues of the M. leprae 70-kDa, 65-kDa, and 10-kDa antigens show over 90% homology between M. leprae and M. tuberculosis. It is the...

Specific T-Cell Epitopes for Immunoassay-Based Diagnosis ofMycobacterium tuberculosisInfection

“…In the present study, instead of abandoning the nonspecific molecules as diagnostic reagents, we dissected the proteins by testing overlapping peptides spanning their sequence and divided them into specific and nonspecific moieties. A peptide-based diagnostic approach has also been attempted for leprosy, but although high specificity was demonstrated, the selected specific peptides gave a relatively low sensitivity (14). We made the same observation in the present study; selecting only the regions of the protein without cross-reactive epitopes decreased the sensitivity.…”

Postgenomic Approach To Identify Novel Mycobacterium leprae Antigens with Potential To Improve Immunodiagnosis of Infection

“…Scrutiny of the M. leprae genome revealed the presence of two candidate genes, ML0049 and ML0050, that encode the M. leprae homologs of ESAT-6 and CFP-10, respectively (16,17,21,22). We recently reported that recombinant M. leprae ESAT-6 and CFP-10 proteins were efficiently recognized by T cells from the majority of M. leprae responsive leprosy patients (12,16). Despite limited sequence identity with their M. tuberculosis homologues Rv3875 and Rv3874 (36 and 40%, respectively), however, significant immunologic cross-reactivity with M. tuberculosis proteins was found for human T cells.…”

“…Prior to the completion of the M. leprae genome sequence, the search for M. leprae-specific antigens that stimulate cellular rather than humoral immunity led to the identification of several antigens and peptides that were able to induce T-cell responses in vitro (12,16,17). Unfortunately, for most of these antigens, homologues were later found in other mycobacteria, including M. tuberculosis, which limits the diagnostic potential of these antigens for leprosy (6,21,22).…”

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

“…A previous study using M. leprae-derived peptides showed considerable variation in peptide reactivity at different sites (8). Thus, in order to estimate the potential of the peptides for detecting M. lepraespecific T-cell immunity in the context of genetically different backgrounds, we selected the most promising peptides (n ϭ 22) and proteins (n ϭ 5) from the previous studies in Brazil, four other countries of leprosy endemicity in Asia (Nepal, Bangladesh, and Pakistan) and Africa (Ethiopia), and an additional site in west central Brazil (Goiás State).…”