Cellular immune response to mycobacterium ulcerans infection

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Background: M. ulcerans infection (Buruli ulcer) is a disease of the skin and soft tissue endemic in sub-Sahara Africa, with the major burden in West Africa. It belongs to a large group of environmental mycobacteria. Generally, protection against mycobacterial infection is thought to be based on cell-mediated immunity, specifically Th-1 type cellular immune responses are essential for control of mycobacterial infections, while humoral response have little benefit. The reasons why only some individuals in endemic areas who are exposed to M. ulcerans develop lesions are not known but are likely to reflect individual differences in the immune response to infections with this mycobacterium. Aim of study: These study aims at describing the cellular immune response to Mycobacterium ulcerans infection (Buruli ulcer) associated with protection and determine the efficacy of two subunit proteins (MUL 4978 and MUL 3720) as potential candidates for vaccine against Buruli ulcer. And finally investigate the effect of co-infection with M. perstans on BU susceptibility. Methods: All clinically suspected cases of Buruli ulcer were confirmed by standard PCR and ZN microscopy. Interferon gamma (IFN-γ) secretion following stimulation with mycobacterial antigens of peripheral blood mononuclear cells (PBMC) and whole blood from Buruli ulcer confirmed patients and household contacts were investigated using IFN-γ ELISA. The effectiveness of two subunit protein vaccines (MUL 3720 and MUL 4978) as potential vaccine candidates was tested using the method mention above. Also CD4+, CD8+ T-cell profiles and CD19 + cell populations in patients with Buruli ulcer were determined by flow cytometry analysis. A case control of 66 M. ulcerans (Mu) disease patients and 30 household contacts were investigated for Mansonella perstans (Mp) co-infection. Patients confirmed to have Mu disease vi by PCR were given the WHO recommended combination of rifampicin 10mg/kg and streptomycin 15mg/kg for 8 weeks. Ivermectin 150ug/kg and doxycycline 200mg were administered for 6 weeks as treatment of Mp infection when present. Results: The results showed that following stimulation with M. ulcerans antigens, PBMC from Buruli ulcer patients and their household contacts mounted high IFN-γ response. Also the Buruli ulcer patients with ulcerative lesions produced more IFN-γ than those with pre-ulcerative lesions (p = 0.026). IFN-γ secretion increased with treatment, with significant difference (p = 0.0078) at 6 weeks compared to baseline (pre-treatment), corresponding to a decrease in patients’ lesion sizes. Patients with severe forms of Buruli lesions (Category II and III) had a significantly decreased CD4+ T-cell population compared with healthy contacts (p = 0.0395). There were no statistically significant differences in the populations of CD8+ T-cell and B cell (CD3-CD19+) populations. There were high IFN-γ responses to subunit protein vaccines and IFN-γ levels in both candidates vaccine antigens (MUL 3720 and MUL 4978) were significantly high after 6 weeks of treatment compared to before treatment (p = 0.03 and 0.005) respectively. Fifteen out of 66 (23%) patients with Mu disease were co-infected with Mp while 4 out of 30 (13%) of the household contacts had infection with Mp. While filarial infection was more common among Buruli ulcer patients than household contacts it did not influence healing time of Buruli lesions (p = 0.93) or predispose patients to more severe forms of the disease. Conclusions: These findings suggest that T helper cell-1 (Th-1) immune response to M. ulcerans may play a protective role in the control of the disease. Also patients with severe forms of Buruli ulcer had depleted CD4+ T-lymphocyte populations. Furthermore, the results indicate that subunit protein vaccines MUL 3720 and MUL 4978 are immunogenic in human ex-vivo assays. This study also provided clear evidence of M. perstans co-infection in Buruli ulcer patients.
A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy, Immunology in the Department of Molecular Medicine.