Prospects for the development of a subunit vaccine against Mycobacterium ulcerans disease (Buruli ulcer)

Abstract

Buruli ulcer (BU) is a slow progressing disease of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. It presents in different clinical forms ranging from small non-ulcerative nodules to large ulcers and sometimes multiple ulcerations. The highest prevalence of the focally occurring disease is found in rural areas of West African countries. Both the mode of transmission as well as the potential environmental reservoir of M. ulcerans remain unidentified to date. In Cameroon, the remote Mape dam region has recently been identified as a new BU endemic area. To assess the age-adjusted prevalence and local geographic distribution of BU, a house-by-house survey in the Bankim health district was conducted. Results showed that children between the age of five to 15 and elderly people were over proportionally affected by BU. To confirm the clinical diagnosis of BU during and after the health survey in Bankim, fine needle aspirates and swabs from undermined ulcer edges were transported to the Swiss Tropical and Public Health Institute for laboratory confirmation by quantitative real time PCR. In parallel we developed a protocol for primary culture initiation of M. ulcerans from patient lesions after a long time span between sampling and processing in a BSL3 culture laboratory. The established two sets of Cameroonian patient isolates from the Mape and the Nyong river valleys were used for a comparative genome sequencing study revealing the presence of two distinct phylogenetic clonal complexes in Cameroon. Despite the fact that BU can be treated with antibiotics, the socioeconomic impact of the disease on affected populations remains devastating. As long as it is not clearly known how the disease is contracted, interruption of transmission is not an option. A vaccine against M. ulcerans on the other hand could be used both as preventive measure and therapeutically. While sero-epidemiological studies imply the presence of protective immunity in some individuals, no vaccine is available to date. Within the framework of the EU funded collaborative project BuruliVac we investigated the potential for developing a protein subunit vaccine against M. ulcerans by delivering vaccine candidate antigens with three different systems: i. as recombinant proteins with an adjuvant, ii. as vesicular stomatitis virus replicon and iii. incorporated into a genetically modified mouse malaria parasite (Plasmodium berghei) in an infection - treatment - approach. All three formulations were assessed for their immunogenicity and their protective potential in a mouse model of BU. Although all three vaccination approaches elicited strong humoral immune responses, no full protection was observed for any of the formulations. However a slight partial protection was seen for a replicon - prime / recombinant protein boost regimen with a vesicular stomatitis virus replicon incorporating an expression cassette for the M. ulcerans protein MUL2232. Additionally, a transient delay of foot pad swelling was observed in mice receiving infection - treatment - vaccination with P. berghei expressing MUL4987. Despite the mainly extracellular nature of M. ulcerans in infected tissue, antibody production against the protein vaccine candidates thus does not seem to be sufficient for protection. Considering marked differences between the mouse footpad model of BU and the disease in humans, we aimed at developing an animal model that better reflects local pathogenesis and host-pathogen interactions in the human BU lesions. Hence, we developed the pig as novel animal model for BU and showed that the observed histopathological changes in the infected pig skin closely represent those of human BU. Therefore the pig model has great potential for the validation of new therapeutic and prophylactic interventions.