Mycobacterium leprae and Mycobacterium tuberculosis are successful intracellular pathogens which downregulate host immune responses. T-cell interferon-g (IFNg) and macrophage tumour necrosis factor-a (TNFa) activate chemokines such as, C-C chemokine ligand-2 (CCL2) and CCL5, which play a role in granuloma formation. Lepromatous leprosy is characterized by defective granulomas with lowered T-cell-and macrophage-mediated responses. Tuberculosis (TB) can be localized to the lung, whereby discreet granulomas are formed. The role of chemokines in leprosy infections is as yet unclear. We compared chemokine responses in lepromatous leprosy and pulmonary tuberculosis patients. Circulating serum CCL2 was raised while CCL5 was lowered in leprosy, as compared with TB patients and healthy controls. However, both Mycobacterium bovis BCG-(P ¼ 0.08) and M. leprae-induced (P ¼ 0.05) CCL2 secretion was reduced in leprosy. In leprosy, BCG induced greater CCL2 (P ¼ 0.01), TNFa (P ¼ 0.02) and somewhat greater CCL5 (P ¼ 0.08) than M. leprae, while CXCL8 induction was comparable. Overall levels of Mycobacterium-induced CCL2, TNFa and CXCL8 were two to threefold lower, and CCL5 was 10-fold lower in leprosy as compared with TB. Reduced inducible CCL2 combined with a lowered TNFa response in lepromatous leprosy may contribute to the unrestricted growth and dissemination of mycobacteria found in the disease.

Tumor necrosis factor (TNF-alpha) in conjunction with interferon-gamma (IFN-gamma) plays an important role in lymphocyte recruitment and granuloma formation in mycobacterial diseases. Lepromatous leprosy infections are typically associated with low to absent T cell responses and the absence of INF-gamma secretion. Chemokines such as IL-8, MCP-1, and MIP-1beta, have also been shown to recruit neutrophils and lymphocytes to the site of mycobacterial infections. We have studied IL-8 expression in relation to TNF-alpha and TGF-beta in monocytes from lepromatous patients (LL) as compared with healthy endemic controls. In endemic controls, no spontaneous expression of IL-8, TNF-alpha, and TGF-beta was observed, but BCG and M. leprae induced activation of all three cytokines. Lepromatous leprosy monocytes spontaneously expressed high levels of IL-8 and TGF-beta but negligible levels of TNF-alpha. A further increase in IL-8 secretion or gene expression by BCG or M. leprae was not significant. BCG, but not M. leprae, was able to stimulate TNF-alpha activation in lepromatous leprosy subjects. TGF-beta responses in LL were parallel to those of IL-8. This suggests a vigorous and active ongoing IL-8 response in lepromatous disease that is independent of TNF-alpha activation. Therefore, in the absence of IFN-gamma and TNF-alpha activation, IL-8 may assume a pivotal role in cell recruitment in leprosy patients with disseminated mycobacterial infections.

SUMMARYMycobacterium leprae (ML) GroES has been shown to induce strong T cell responses in tuberculoid as well as in exposed healthy contacts of leprosy patients, and therefore this antigen has been the focus of study as a potential vaccine candidate. Paradoxically, we have shown that ML GroES also induces extremely high titres of IgG1 antibody in leprosy patients across the disease spectrum, a response associated with disease progression. IgG1 antibodies in leprosy also show a negative association with interferon-g, a critical T cell cytokine responsible for macrophage activation and intracellular killing of mycobacteria. We therefore queried if antibody and T cell responses were being evoked by different epitopes in ML GroES proteins. To address the issue of epitope recognition in mycobacterial diseases, we have analysed 16 peptides (15-mer peptides) spanning the entire ML and M. tuberculosis GroES protein in leprosy (n 19) and tuberculosis (n 9) patients and healthy endemic controls (n 8). Our analysis demonstrates clearly that the dominant peptides evokingT cell and IgG subclass antibodies were different. The target of both T and B cell responses were cross-reactive epitopes in all groups. Differences in disease and healthy states related to the strength (mean intensity) of the T cell and antibody response. IgG1 and IgG3 antibodies were associated with disseminated disease and IgG 2 and IgG4 with disease limitation. Such comprehensive immune pro®ling of antigen-speci®c responses is critical to understanding the disease pathogenesis and also if these reagents are to be exploited for either diagnostic or vaccine purposes.

SUMMARYIn order to identify T cell epitopes within the Mycobacterium leprae 45-kD serine-rich antigen, we analysed responses to overlapping 17-mer peptides encompassing the whole antigen in non-exposed UK controls, Pakistani leprosy patients and tuberculosis patients in both the United Kingdom and Pakistan. This antigen has been described as M. leprae-specific, although it has a hypothetical homologue in M. tuberculosis. Human peripheral blood mononuclear cells were stimulated with peptide for 5 days and IFN-g measured in supernatants by ELISA. Some peptides were recognized more frequently by T cells from tuberculoid leprosy patients than those from UK controls, suggesting that such T cell epitopes might have diagnostic potential, while other peptides induced greater responses among UK control subjects. Short-term cell lines confirmed that these assays detected specific T cell recognition of these peptides. However, many tuberculosis patients also recognized these potentially specific peptides suggesting that there could be a true homologue present in M. tuberculosis.

SUMMARYT cell responses play a critical role in determining protective responses to leprosy. Patients with selflimiting tuberculoid leprosy show high T cell reactivity, while patients with disseminated lepromatous form of the disease show absent to low levels of T cell reactivity. Since the T cell reactivity of lepromatous patients to purified protein derivative (PPD), a highly cross-reactive antigen, is similar to that of tuberculoid patients, we queried if lepromatous patients could recognize cross-reactive epitopes in Mycobacterium leprae antigens as well. T cell responses were analysed to a recombinant antigen 10-kD (a heat shock cognate protein) which is available from both M. tuberculosis (MT) and M. leprae (ML) and displays 90% identity in its amino acid sequence. Lymphoproliferative responses were assessed to ML and MT 10 kD in newly diagnosed leprosy patients (lepromatous, n ¼ 23; tuberculoid, n ¼ 65). Lepromatous patients showed similar, but low, lymphoproliferative responses to ML and MT 10 kD, while tuberculoid patients showed much higher responses to ML 10 kD. This suggests that the tuberculoid patients may be recognizing both species-specific and cross-reactive epitopes in ML 10 kD, while lepromatous patients may be recognizing only cross-reactive epitopes. This was further supported by linear regression analysis. Lepromatous patients showed a high concordance in T cell responses between ML and MT 10 kD (r ¼ 0·658; P < 0·0006) not observed in tuberculoid patients (r ¼ 0·203; P > 0·1). Identification of cross-reactive T cell epitopes in M. leprae which could induce protective responses should prove valuable in designing second generation peptide-based vaccines.