CD8⁺ T cells are thought to play an important role in protective immunity to tuberculosis. Although several nonprotein ligands have been identified for CD1-restricted CD8⁺ CTLs, epitopes for classical MHC class I-restricted CD8⁺ T cells, which most likely represent a majority among CD8⁺ T cells, have remained ill defined. HLA-A*0201 is one of the most prevalent class I alleles, with a frequency of over 30% in most populations. HLA-A2/K^b transgenic mice were shown to provide a powerful model for studying induction of HLA-A*0201-restricted immune responses in vivo. The Ag85 complex, a major component of secreted Mycobacterium tuberculosis proteins, induces strong CD4⁺ T cell responses in M. tuberculosis-infected individuals, and protection against tuberculosis in Ag85-DNA-immunized animals. In this study, we demonstrate the presence of HLA class I-restricted, CD8⁺ T cells against Ag85B of M. tuberculosis in HLA-A2/K^b transgenic mice and HLA-A*0201⁺ humans. Moreover, two immunodominant Ag85 peptide epitopes for HLA-A*0201-restricted, M. tuberculosis-reactive CD8⁺ CTLs were identified. These CD8⁺ T cells produced IFN-γ and TNF-α and recognized Ag-pulsed or bacillus Calmette-Guérin-infected, HLA-A*0201-positive, but not HLA-A*0201-negative or uninfected human macrophages. This CTL-mediated killing was blocked by anti-CD8 or anti-HLA class I mAb. Using fluorescent peptide/HLA-A*0201 tetramers, Ag85-specific CD8⁺ T cells could be visualized in bacillus Calmette-Guérin-responsive, HLA-A*0201⁺ individuals. Collectively, our results demonstrate the presence of HLA class I-restricted CD8⁺ CTL against a major Ag of M. tuberculosis and identify Ag85B epitopes that are strongly recognized by HLA-A*0201-restricted CD8⁺ T cells in humans and mice. These epitopes thus represent potential subunit components for the design of vaccines against tuberculosis.

During stationary growth or in vitro conditions mimicking relevant aspects of latency, the HspX protein (Rv2031c) is specifically upregulated byMycobacterium tuberculosis. In this study we compared T-cell responses against HspX and the secretedM. tuberculosisprotein Ag85B (Rv1886c) in tuberculosis (TB) patients, tuberculin skin test-positive individuals,M. bovisBCG-vaccinated individuals, and healthy negative controls. Gamma interferon responses to HspX were significantly higher inM. tuberculosis-exposed individuals than inM. tuberculosis-unexposed BCG vaccinees. In contrast, no such differences were found with respect to T-cell responses against Ag85B. Therefore, BCG-based vaccines containing relevant fragments of HspX may induce improved responses against this TB latency antigen. To identify relevant major histocompatibility complex class I- and class II-restricted HspX-specific T-cell epitopes, we immunized HLA-A2/K^band HLA-DR3.Ab⁰transgenic (tg) mice with HspX. Two new T-cell epitopes were identified, p91-105 and p31-50, restricted via HLA-A*0201 and HLA-DRB1*0301, respectively. These epitopes were recognized by human T cells as well, underlining the relevance of HspX T-cell recognition both in vivo and in vitro. In line with the data in humans, BCG immunization of both tg strains did not lead to T-cell responses against HspX-derived epitopes, whereas nonlatency antigens were efficiently recognized. These data support the notion that BCG vaccination per se does not induce T-cell responses against the latency antigen, HspX. Thus, we suggest that subunit vaccines incorporating HspX and/or other latency antigens, as well as recombinant BCG strains expressing latency antigens need to be considered as new vaccines against TB.

In this paper we describe identification and characterization ofMycobacterium lepraeESAT-6 (L-ESAT-6), the homologue ofM. tuberculosisESAT-6 (T-ESAT-6). T-ESAT-6 is expressed by all pathogenic strains belonging to theM. tuberculosiscomplex but is absent from virtually all other mycobacterial species, and it is a promising antigen for immunodiagnosis of tuberculosis (TB). Therefore, we analyzed whether L-ESAT-6 is a similarly powerful tool for the study of leprosy by examining T-cell responses against L-ESAT-6 in leprosy patients, TB patients, and exposed or nonexposed healthy controls from areas where leprosy and TB are endemic and areas where they are not endemic. L-ESAT-6 was recognized by T cells from leprosy patients, TB patients, individuals who had contact with TB patients, and healthy individuals from an area where TB and leprosy are endemic but not by T cells from individuals who were not exposed toM. tuberculosisandM. leprae. Moreover, leprosy patients who were not responsive toM. lepraefailed to respond to L-ESAT-6. A very similar pattern was obtained with T-ESAT-6. These results show that L-ESAT-6 is a potentM. lepraeantigen that stimulates T-cell-dependent gamma interferon production in a large proportion of individuals exposed toM. leprae. Moreover, our results suggest that there is significant cross-reactivity between T-ESAT-6 and L-ESAT-6, which has implications for the use of ESAT-6 as tool for diagnosis of leprosy and TB in areas where both diseases are endemic.

Leprosy is not eradicable with currently available diagnostics or interventions, as evidenced by its stable incidence. Early diagnosis of Mycobacterium leprae infection should therefore be emphasized in leprosy research. It remains challenging to develop tests based on immunological biomarkers that distinguish individuals controlling bacterial replication from those developing disease. To identify biomarkers for field-applicable diagnostics, we determined cytokines/chemokines induced by M. leprae proteins in blood of leprosy patients and endemic controls (EC) from high leprosy-prevalence areas (Bangladesh, Brazil, Ethiopia) and from South Korea, where leprosy is not endemic anymore. M. leprae-sonicate–induced IFN-γ was similar for all groups, excluding M. leprae/IFN-γ as a diagnostic readout. By contrast, ML2478 and ML0840 induced high IFN-γ concentrations in Bangladeshi EC, which were completely absent for South Korean controls. Importantly, ML2478/IFN-γ could indicate distinct degrees of M. leprae exposure, and thereby the risk of infection and transmission, in different parts of Brazilian and Ethiopian cities. Notwithstanding these discriminatory responses, M. leprae proteins did not distinguish patients from EC in one leprosy-endemic area based on IFN-γ. Analyses of additional cytokines/chemokines showed that M. leprae and ML2478 induced significantly higher concentrations of MCP-1, MIP-1β, and IL-1β in patients compared with EC, whereas IFN-inducible protein-10, like IFN-γ, differed between EC from areas with dissimilar leprosy prevalence. This study identifies M. leprae-unique Ags, particularly ML2478, as biomarker tools to measure M. leprae exposure using IFN-γ or IFN-inducible protein-10, and also shows that MCP-1, MIP-1β, and IL-1β can potentially distinguish pathogenic immune responses from those induced during asymptomatic exposure to M. leprae.

Early detection of leprosy is key to reduce the ongoing transmission. Antibodies directed against M. leprae PGL-I represent a useful biomarker for detecting multibacillary (MB) patients. Since efficient leprosy diagnosis requires field-friendly test conditions, we evaluated two rapid lateral flow assays (LFA) for detection of Mycobacterium leprae-specific antibodies: the visual immunogold OnSite Leprosy Ab Rapid test [Gold-LFA] and the quantitative, luminescent up-converting phosphor anti-PGL-I test [UCP-LFA]. Test performance was assessed in independent cohorts originating from three endemic areas. In the Philippine cohort comprising patients with high bacillary indices (BI; average:4,9), 94%(n = 161) of MB patients were identified by UCP-LFA and 78%(n = 133) by Gold-LFA. In the Bangladeshi cohort, including mainly MB patients with low BI (average:1), 41%(n = 14) and 44%(n = 15) were detected by UCP-LFA and Gold-LFA, respectively. In the third cohort of schoolchildren from a leprosy hyperendemic region in Brazil, both tests detected 28%(n = 17) seropositivity. Both rapid tests corresponded well with BI(p < 0.0001), with a fairly higher sensitivity obtained with the UCP-LFA assay. However, due to the spectral character of leprosy, additional, cellular biomarkers are required to detect patients with low BIs. Therefore, the UCP-LFA platform, which allows multiplexing with differential biomarkers, offers more cutting-edge potential for diagnosis across the whole leprosy spectrum.