For biological testing 0.5 mg/ml mycolactone stock solutions were made by adding cell culture BDP5290 grade DMSO (Sigma). Extracts of mycolactone were prepared using mycobacterial pellets from cultures of strains isolated from lesions of Cameroonian BU patients (S1013, S1019, S1047). to the lipid-like nature of mycolactones, the production of antibodies against these molecules has so far been unsuccessful. By using the classical approach of fusing immortal cells with spleen cells of mice immunized with a protein conjugate of a truncated nontoxic synthetic mycolactone, we were BDP5290 able to generate hybridoma cells producing mycolactone-specific monoclonal antibodies. Mammalian cell-based cytotoxicity assays demonstrated that these antibodies have neutralizing activity and can fully block the cytotoxic activity of mycolactone, resulting in survival of the target cells. These findings support the concept to target mycolactone by a vaccine. Furthermore, the anti-mycolactone antibodies may represent useful tools for BU diagnostics development. == Introduction == Mycobacterium ulcerans, the causative agent of the neglected tropical skin disease Buruli ulcer (BU), produces the exotoxin mycolactone, which is responsible for the formation of chronic necrotizing skin lesions [1,2]. Early diagnosis followed by rapid initiation of the BDP5290 currently recommended treatment with rifampicin and streptomycin [3] is crucial to avoid massive tissue destruction and long-term disabilities. Mycolactones consist of a 12-membered macrolide core structure, a short C-linked upper side chain (comprising C12C20) and a longer C5-O-linked lower polyunsaturated acyl side chain. Mycolactones produced by differentM.ulceranslineages differ in the structure of the lower side chain, but are otherwise conserved [4]. For the lower side chain variations in length, the number of double bonds and the number and localization of hydroxyl groups have been described. WhileM.ulceransstrains may produce mixtures of several mycolactone species, the composition of these pools seems to be highly conserved for a particularM.ulceranslineage [5]. Strains belonging to the genomically monomorphic classical African lineage produce the most toxic variant, mycolactone A/B [6]. This lineage is responsible for > 95% of the BU cases reported worldwide [7]. Mycolactone-deficientM.ulceransmutants are less virulent and intradermal injection of the toxin in animal models is sufficient to induce the formation of BU-like lesions [4,8]. Since mycolactones play such a central role in the pathogenesis of BU [1,2,9], they may represent a suitable target for both vaccine development and passive immunotherapy. However, due BDP5290 to their lipid-like nature, as well as their cytotoxic and immunosuppressive [1012] activities, attempts to raise antibody responses against mycolactones have failed so far. While extracts fromM.ulceranscultures have been the only source of mycolactones for a long time, highly defined synthetic mycolactones have become available more recently [6,13,14], greatly facilitating experimental work with these compounds. Here, we generated mycolactone specific immune-sera and monoclonal antibodies (mAbs) by immunizing mice with a protein conjugate of a nontoxic synthetic truncated mycolactone derivative. == Materials and Methods == == Ethics statement == Animal experiments performed were approved by the animal welfare committee of the Canton of Basel (authorization number 2375) and were conducted in compliance with the Swiss Animal Welfare Act (TSchG), Animal Welfare Ordinance (TSchV), and the Animal Experimentation Ordinance (TVV). == Preparation of mycolactone stock solutions == Natural mycolactones A/B, C and F as well as variants PG-157, PG-165, PG-182 and core PG-119 were produced as described previously [6,14]. BDP5290 The synthesis of biotin-conjugate PG-204 and the immunogen PG-203 will be described elsewhere. All compounds were HPLC-purified. For biological testing 0.5 mg/ml mycolactone stock solutions were made by adding cell culture grade DMSO (Sigma). Extracts of mycolactone were prepared using mycobacterial pellets from cultures of strains isolated from lesions of Cameroonian BU patients (S1013, S1019, S1047). Briefly, 1 ml of a chloroform-methanol solution (2:1, v/v) was added Rabbit Polyclonal to Cyclin F to the individual pellets. Bacteria were resuspended by vortexing and lipids were extracted by incubating the samples shaking at RT for 2 h. Then, 200 L ddH2O were added to induce a phase separation. After vigorous vortexing, the samples were centrifuged for.