Leprosy and its associated disabilities will be with us well into the future, as recognized in the World Health Organization's global strategy for 2006 to 2010 (1
). Clearly, the methods and knowledge available to date have not been sufficient to eliminate leprosy. Transmission continues because tools for early, preclinical diagnosis of M. leprae
infection, which is likely to be a major source of unidentified transmission, are lacking. Thus, the design of better tools for detection of preclinical M. leprae
infection, which would allow introduction of multidrug therapy at an early stage, has been an important goal in leprosy research since the beginning of the 21st century.
The successful use of the M. tuberculosis
-specific peptides ESAT-6 (Rv3875), CFP-10 (Rv3874), and TB7.7 (Rv2654) for TB diagnostics among humans (11
) supported our belief in the possibilities for using peptides in CMI response-based diagnostic tests for leprosy. The availability of the genome sequence of M. leprae
), together with new techniques in bioinformatics, has thus enabled us to identify M. leprae
-unique candidate proteins (2
), as well as peptides (20
) or combinations of peptides (14
), that can be applied to detect M. leprae
-specific T-cell responses.
In earlier studies situated in Brazil (14
), M. leprae
proteins induced higher levels of IFN-γ, but T-cell responses to peptides were found to be more specific. Therefore, both M. leprae
proteins and peptides that had shown promising specificity in these studies were analyzed in the current multicenter study, which used samples from Brazil, Nepal, Bangladesh, Pakistan, and Ethiopia.
The results of our study show that all proteins were indeed recognized strongly by PBMC of BT/TT patients and HHCs, with ML2283 being the most specific protein, as it was not frequently recognized in TB patients (Fig. ). However, the majority of the EC group responded as well to the M. leprae
proteins and peptides (Fig. and ) as did the individuals known to have been exposed to or infected by M. leprae
, despite the fact that all the antigens had been selected on the basis of their unique sequences in M. leprae
. In addition, T-cell responses against M. leprae
antigens were observed in TB patients. Since M. leprae
-positive EC individuals and TB patients were also responding to M. leprae
whole-cell sonicate and none of the antigens induced any IFN-γ in healthy controls derived from countries of nonendemicity (see Fig. S1 in the supplemental material), the possibility remains that the response against M. leprae
-unique antigens is caused by previous exposure of EC individuals and TB patients to M. leprae
. This would then indicate that exposure to/infection by M. leprae
may be occurring in this population at much higher rates than previously thought (17
). Importantly, three M. leprae
-unique peptides, ML2283 p19 (14
), ML2283 p20 (14
), and ML0126 p81 (20
), were specific in all five areas of endemicity, as these peptides were recognized only in BL/LL patients, BT/TT patients, and HHCs but to a much lesser extent (50 to 100 pg/ml) in EC individuals or not at all in TB patients. Since HLA restriction allows a relatively low number of individuals to respond to these peptides, additional M. leprae
peptides will have to be analyzed in order for a combination of peptides that detects T-cell responses to M. leprae
in a specific fashion to be obtained. Furthermore, the variability of T-cell responses in cultures stimulated with peptides at the five different test sites (Fig. ) highlighted the necessity of testing M. leprae
peptides in different populations in order to design diagnostic tools that are applicable in various areas of endemicity.
Since a combination of PGL-I serology with assays based on CMI responses against M. leprae antigens may allow detection of most forms of leprosy (PB and MB), including preclinical leprosy, we analyzed whether these five M. leprae proteins represented potential added value in diagnosing early infection: T-cell responses against these proteins were detected in 59% of M. leprae-exposed HHCs that did not have antibodies to PGL-I (Fig. ), indicating a serologically undetected but potentially M. leprae-infected group.
The development of a sensitive, specific, and field- and user-friendly test (7
) which is also affordable can have a significant impact on leprosy control programs in countries of endemicity. In search of new diagnostic tools for leprosy, we have thus far found that T-cell responses to M. leprae
proteins and peptides can be detected in a 6-day PBMC assay. In future studies, we will aim to maintain the M. leprae
specificity of the peptides as observed here in the PBMC stimulation assay for ML2283 p19, ML2283 p20, and ML0126 p81 and, in addition, screen more M. leprae
proteins and peptides to identify sequences that together induce CMI responses in the context of multiple HLA alleles, thereby providing coverage for diagnostics in different regions of endemicity. Since T-cell responses to M. leprae
antigens are more sensitive in assays using PBMC than in whole-blood assays (data not shown), we will also assess whether the conditions of M. leprae
peptide-based whole-blood assays can be optimized.
Finally, it is crucial that follow-up studies be carried out to determine whether T-cell responses as observed here are related to M. leprae infection or exposure and whether the presence of this CMI response is indicative of protection against leprosy or disease development.