Diagnosis of leprosy is a major obstacle to disease control and has been compromised in the past due to the lack of specific reagents. We have used comparative genome analysis to identify genes that are specific to Mycobacterium leprae and tested both recombinant proteins and synthetic peptides from a subset of these for immunological reactivity. Four unique recombinant proteins (ML0008, ML0126, ML1057, and ML2567) and a panel of 58 peptides (15 and 9 mer) were tested for IFN-γ responses in PBMC from leprosy patients and contacts, tuberculosis patients, and endemic and nonendemic controls. The responses to the four recombinant proteins gave higher levels of IFN-γ production, but less specificity, than the peptides. Thirty-five peptides showed IFN-γ responses only in the paucibacillary leprosy and household contact groups, with no responses in the tuberculosis or endemic control groups. High frequencies of IFN-γ-producing CD4+ and CD8+ T cells specific for the 15- and 9-mer peptides were observed in the blood of a paucibacillary leprosy patient. 9-mer peptides preferentially activated CD8+ T cells, while the 15-mer peptides were efficient in inducing responses in both the CD4+ and CD8+ T cell subsets. Four of the six 9-mer peptides tested showed promising specificity, indicating that CD8+ T cell epitopes may also have diagnostic potential. Those peptides that provide specific responses in leprosy patients from an endemic setting could potentially be developed into a rapid diagnostic test for the early detection of M. leprae infection and epidemiological surveys of the incidence of leprosy, of which little is known.

Culture filtrate protein 10 (CFP-10) from Mycobacterium tuberculosis is a well-characterized immunodominant 10-kDa protein antigen known to elicit a very potent early gamma interferon response in T cells from M. tuberculosis-infected mice and humans. The sequence of the Mycobacterium leprae homologue of CFP-10 shows only 40% identity (60% homology) at the protein level with M. tuberculosis CFP-10 and thus has the potential for development as a T-or B-cell reactive antigen for specific diagnosis of leprosy. Antisera raised in mice or rabbits against recombinant M. leprae and M. tuberculosis CFP-10 proteins reacted only with homologous peptides from arrays of overlapping synthetic peptides, indicating that there was no detectable cross-reactivity at the antibody level. Sera from leprosy and tuberculosis patients were also specific for the homologous protein or peptides and showed distinct patterns of recognition for either M. leprae or M. tuberculosis CFP-10 peptides. At the cellular level, only 2 of 45 mouse T-cell hybridomas raised against either M. leprae or M. tuberculosis CFP-10 displayed a cross-reactive response against the N-terminal heterologous CFP-10 peptide, the region that exhibits the highest level of identity in the two proteins; however, the majority of peptide epitopes recognized by mouse T-cell hybridomas specific for each protein did not cross-react with heterologous peptides. Coupled with the human serology data, these results raise the possibility that peptides that could be used to differentiate infections caused by these two related microorganisms could be developed. Immunohistochemical staining of sections of M. leprae-infected nude mouse footpads resulted in strongly positive staining in macrophages and dendritic cells, as well as weaker staining in extracellular areas, suggesting that M. leprae CFP-10, like its homologue in M. tuberculosis, is a secreted protein.The number of cases of leprosy worldwide has been reduced dramatically through aggressive World Health Organizationsponsored multidrug therapy, from approximately 10 million cases in 1985 to a little fewer than 700,000 cases today (46-48). However, with the unabated emergence of more than 600,000 new cases per year, there is the possibility that the source of infection is not being addressed (16). The single greatest need in leprosy research is the development of definitive diagnostic tools that identify sources of leprosy and routes of transmission. The recent completion of the genome sequences of both Mycobacterium tuberculosis (8) and Mycobacterium leprae (9) provides an opportunity to identify leprosy-specific antigens that might serve this purpose. A similar comparative approach has allowed identification of genes in the RD1 region that are deleted from Mycobacterium bovis BCG but present in M. tuberculosis and can therefore distinguish between infection with M. tuberculosis and vaccination with BCG (22). Among the deleted antigens are two low-molecular-weight proteins, culture filtrate protein 10 (CFP-10) (Rv3874) and ESAT-6...

The sequence of the Mycobacterium leprae homologue of ESAT-6 shows only 36% amino acid correspondence to that from Mycobacterium tuberculosis. Anti-M. leprae ESAT-6 polyclonal and monoclonal antibodies and T-cell hybridomas reacted only with the homologous protein and allowed identification of the B-and T-cell epitopes. The protein is expressed in M. leprae and appears in the cell wall fraction. Thus, M. leprae ESAT-6 shows promise as a specific diagnostic agent for leprosy.The global implementation of effective chemotherapy for leprosy has resulted in the diminution of cases from approximately 10 million in 1985 to about 800,000 today (27). However, there is no evidence as yet of any reduction in the number of new cases (28), and we know little about the transmission of leprosy or the time elapsed between infection and disease. The single greatest need from leprosy research is definitive diagnostic tools to help understand transmission and allow early detection of disease. The recent completion of the sequencing of the genomes of Mycobacterium tuberculosis (4) and Mycobacterium leprae (5) provides the opportunity to identify leprosy-specific antigens. An analogous approach applied to Mycobacterium bovis BCG allowed the identification of deleted genes and the development of antigens that can distinguish between M. tuberculosis infection and vaccination with BCG (17). Among those antigens were two low-molecular-weight M. tuberculosis culture filtrate proteins, ESAT-6 and CFP10 (2, 10), both encoded by genes in the RD1 region, a genetic segment that has been deleted from all strains of BCG. When tested together in a gamma interferon assay of peripheral blood mononuclear cells from M. tuberculosis-infected and BCG-vaccinated individuals, the sensitivity and specificity of the response were 84 and 100%, respectively, with no responses in purified protein derivative-negative individuals (1).Although previous studies have identified a number of M. leprae proteins (7,11,19) and peptides (6, 26) capable of inducing gamma interferon responses in leprosy patients, a comparative analysis of the M. tuberculosis and M. leprae genomes should reveal new specific antigens, potential diagnostic and epidemiological tools for leprosy. In this report, comparative analysis of the M. leprae and M. tuberculosis ESAT-6 homologues suggests that the M. leprae product holds promise in this respect.Comparison of the sequences of ESAT-6 from M. leprae and M. tuberculosis. Whereas M. tuberculosis contains 14 members of the ESAT-6 family (23), the M. leprae genome shows evidence of only 4 (5, 8). A comparison of the alignment of the sequences of the 95-amino-acid (aa)-length ESAT-6 protein from M. tuberculosis (22) with its counterpart from M. leprae showed 36% homology overall (Fig. 1). Although there was identity between 9 out of 13 amino acids (69% homology) in the region bounded by aa 34 and 46, this is the only instance with more than 4 consecutive, identical amino acids. The rest of the sequence shows only one or two identical amino aci...