Cytokine (IFN- γ, IL-5) response to infection with Mycobacterium Ulcerans

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Buruli ulcer is a disease of the skin and soft tissue caused by the pathogen Mycobacterium ulcerans. Mycolactone, a lipid toxin has been identified as the main bacterial virulence factor for the disease. This toxin has been shown to be responsible for the immunosuppression and tissue necrosis which is characteristic of the disease. There is currently no vaccine for Buruli ulcer disease. In this study, the systemic immune responses of M. ulcerans infected patients to polyketide synthase domains were investigated. The effect of paradoxical reactions on the Th1 and Th2 responses of M. ulcerans infected patients were also evaluated. This was a prospective observational study. All clinically suspected cases of Buruli ulcer were confirmed by standard PCR. Interferon gamma (interferon-γ) secretion following whole blood stimulation with polyketide synthase domains(PKS) using heparinised blood samples from patients with PCR confirmed Buruli ulcer, endemic controls and non-endemic controls were investigated using the ELISA. The effectiveness of 12 recombinant antigens and Ag85Aulc as potential vaccines were tested using the ELISA method. Also, concentrations of cytokines (interferon-γ, IL-5) in overnight supernates of whole blood assays in Buruli ulcer patients who developed paradoxical reaction, after stimulation with M. ulcerans sonicate antigens were measured using ELISA. The results show that responses to antigens were generally high (above 80% responders) with the exception of ACP3 where active Buruli ulcer cases had 47% and endemic controls 71% responders. The highest percentage responders in both participant groups were observed in reaction to Ksalt (100% responders) and ER (100% responders) antigens. A higher proportion of endemic controls responded with higher interferon-γ responses than the Buruli ulcer cases to all the PKS domain antigens and to DNA encoding mycolyltransferase Ag85A of M. ulcerans (Ag85Aulc). There was no response to any of the antigens in all but one of the control participants from non-endemic areas. There was a trend to increasing interferon-γ responses with treatment; this was not statistically significant except for ACP2 and ATac2. Patients with non-ulcerative lesions mounted generally higher interferon-γ responses than those with ulcerative disease. There was a positive but not statistically significant association between interferon-γ responses and time to healing of the patients to the PKS antigens. Although patients who developed paradoxical reactions mounted a lower interferon-γ response [median (range) 754.32 (50.93-4190.4) pg/ml] at baseline compared to patients who had no paradoxical reactions [1246.93 (81.11-6969) pg/ml], there was no statistically significant difference between the two groups. By contrast, these two groups of patients elicited comparable median interleukin-5 (IL-5) response levels 35.61 (24.79-67.83) pg/ml vs 35.52 (11.82-993.90) pg/ml). Patients who developed paradoxical reaction mounted a consistently low Th1 and Th2 responses when compared with patients who did not develop paradoxical reactions. Th1 and Th2 responses of patients who developed paradoxical reaction improved with treatment. These results suggests that the immune response of patients to PKS domains was lower compared to that of the contacts, which could be suggestive to the immunosuppressive effect of mycolactone on immune response. ER and Ksalt were the most immunogenic antigens. A vaccine made up of the most immunogenic plasmid DNA encoding mycolactone polyketide synthase domains and Ag85Aulc is an interesting possibility that require further study. We have confirmed that paradoxical reaction has an effect on the immune response of patients.
A thesis submitted in fulfillment of the requirements for the degree of Master of Philosophy, Immunology in the Department of Molecular Medicine