While BCG vaccination extends the time to appreciable footpad swelling, protection ultimately wanes and animal euthanasia is required. to BCG or BCG MU-Ag85A. Finally, mice challenged with MU following a single subcutaneous vaccination with BCG MU-Ag85B-EsxH display significantly less bacterial burden at 6 and 12 weeks post-infection, reduced histopathological tissue damage, and significantly longer survival occasions compared to vaccination with either BCG or BCG MU-Ag85A. These results further support the potential of BCG as a foundation for BU vaccine design, whereby discovery and recombinant expression of novel immunogenic antigens could lead to greater anti-MU efficacy using this Macitentan (n-butyl analogue) highly safe and ubiquitous vaccine. Author Summary (MU) contamination causes a highly disfiguring, necrotic skin disease known as Buruli ulcer (BU). Antibiotic treatments have low efficacy if the infection is usually diagnosed after ulceration begins, leading to frequent dependence on surgical Macitentan (n-butyl analogue) removal of infected tissues. A prophylactic vaccine for BU does not exist and several attempts to create an effective vaccine have shown limited success. We recently exhibited that a recombinant strain of BCG expressing the immunodominant MU-Ag85A conferred significantly enhanced protection against experimental BU compared to the standard BCG vaccine. Here we show that BCG expression of a fusion between two option MU antigens, Ag85B and EsxH, can promote antigen-specific T cell and humoral immune response capable of significantly improving survival and protection Macitentan (n-butyl analogue) against BU pathology, compared to BCG MU-Ag85A alone. These results support the potential for using the highly safe and ubiquitous BCG vaccine as a platform for further BU vaccine development. Introduction Subcutaneous skin contamination by (MU) leads to a potentially disfiguring, necrotic condition known as Buruli ulcer (BU) [1]. What often begins as an indolent skin nodule or small lesion can ultimately progress to expanding ulcerations, body-wide scarring, loss of limbs or eyes, and osteomyelitis [2]. These infections disproportionately affect children and are largely endemic to Sub-Saharan Africa, Australia, and Japan, where the unconfirmed mode of transmission is usually thought to be dependent on exposure to contaminated wetland areas and insect vectors [3, 4]. Treatment regimens include lengthy combination anti-mycobacterial therapies, however, lack of medical access, absence of rapid and accurate diagnostics, and the often misleading symptoms of BU frequently lead to significant delays in therapeutic action [5, 6]. At the point of extensive tissue damage, surgical debridement and skin grafting is required, resulting in significant morbidity and interpersonal stigmatization [7, 8]. Antibiotics can be effective against MU if administered at an early time point prior to ulceration, and side effects of treatment can include nephrotoxicity and hearing loss [9]. While there is increasing promise for less toxic antibiotic therapies, currently no prophylactic vaccine is usually available to prevent BU in the areas with best prevalence [10]. BU vaccine research strategies have largely focused on prime-boost regimens using recombinant DNA and CRYAA MU proteins, however, the efficacy of these approaches has not surpassed the transient, cross-reactive protection observed during experimental vaccination with tuberculosis vaccine strain, bacillus Calmette- Gurin (BCG) [11C18]. BCG, the most ubiquitous World Health Organization-approved vaccine administered across the world, possesses a promising safety profile but low efficacy against pulmonary tuberculosis afflicting millions of people [19, 20]. Experimental BCG vaccination has been studied using BU animal models and has been shown to confer protection by delaying ulceration after murine footpad challenge with MU [11]. While BCG vaccination extends the time to appreciable footpad swelling, protection ultimately wanes and animal euthanasia is required. Retrospective studies in humans also Macitentan (n-butyl analogue) provide support for the potential use of BCG as a foundation for an effective BU vaccine. Patients previously vaccinated with BCG were shown to have delayed onset to ulceration after contamination with MU, as well as significant protection against developing complications of MU contamination, such as osteomyelitis [21C23]. These lines of evidence further support the potential of BCG as a foundation for BU vaccine design, whereby improvement of BCG immunogenicity could lead to greater efficacy using this highly safe and ubiquitous vaccine. BCG has previously been designed to express various recombinant immunogenic antigens and.