Use of a whole blood assay to evaluatein vitroT cell responses to new leprosy skin test antigens in leprosy patients and healthy subjects

Abstract

Development of an immunological tool to detect infection with Mycobacterium leprae would greatly benefit leprosy control programmes, as demonstrated by the contribution of the tuberculin test to tuberculosis control. In a new approach to develop a 'tuberculin-like' reagent for use in leprosy, two new fractions of M. leprae depleted of cross-reactive and immunomodulatory lipids- MLSA-LAM (cytosol-derived) and MLCwA (cell wall-derived)-have been produced in a form suitable for use as skin test reagents. T cell responses (interferon-gamma (IFN-gamma) and lymphoproliferation) to these two new fractions were evaluated in a leprosy-endemic area of Nepal using a simple in vitro whole blood test. The two fractions were shown to be highly potent T cell antigens in subjects exposed to M. leprae-paucibacillary leprosy patients and household contacts. Responses to the fractions decreased towards the lepromatous pole of leprosy. Endemic control subjects also showed high responses to the fractions, indicating high exposure to M. leprae, or cross-reactive mycobacterial antigens, in this Nepali population. The new fractions, depleted of lipids and lipoarabinomannan (LAM) gave enhanced responses compared with a standard M. leprae sonicate. The cell wall fraction appeared a more potent antigen than the cytosol fraction, which may be due to the predominance of the 65-kD GroEL antigen in the cell wall. The whole blood assay proved a robust field tool and a useful way of evaluating such reagents prior to clinical trials.

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

“…A number of current initiatives therefore aim to develop new skin test reagents for leprosy. One approach is to fractionate the antigens of the leprosy bacillus to provide a skin test reagent (4); such fractions are highly antigenic but may not have the requisite specificity (30). Another approach is to identify synthetic peptide epitopes specific for M. leprae, which could be combined to give a reagent capable of inducing a positive skin test response in the majority of individuals from varying ethnic backgrounds and HLA types.…”

“…Previous studies using M. leprae antigens fractionated on nitrocellulose indicated that many additional proteins might be recognized as antigens (4). It is also likely that within fractionated M. leprae preparations such as the M. leprae cell wall antigenic fraction and the M. leprae cytosolic antigen fraction, which induce strong T-cell responses in peripheral blood cells from tuberculoid leprosy patients (21,30), there are many as yet unidentified antigens. The objective of the current study was to utilize available genomic information from M. leprae to identify some of these additional, as yet unidentified antigens and, in particular, those which might be specific for M. leprae.…”

Antigen Discovery: a Postgenomic Approach to Leprosy Diagnosis

“…IFN-␥ measurements in whole blood restimulation assays were performed as described previously (17,28,29). Briefly, 5 ml of blood was taken by venipuncture from each patient and immediately mixed with heparin at a concentration of 20 U/ml.…”

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

“…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 individuals in the early stages of leprosy, using highly antigenic M. leprae fractions and the major individual immunogenic proteins (4,8,14,22,25). However, an obstacle in the application of IGRAs to the major M. leprae protein antigens is that most of these antigens share appreciable homology with orthologues in Mycobacterium spp.…”

“…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)(2)(3)(4)(5).…”