Clostridium difficile disease is mediated primarily by toxins A and B (TcdA and TcdB). The receptor binding domains (RBD) of TcdA and TcdB are immunogenic and anti-RBD antibodies are protective. Since these toxins act locally, an optimal C. difficile vaccine would generate both systemic and mucosal responses. We have repurposed an attenuated Salmonella enterica serovar Typhimurium strain (YS1646) to produce such a vaccine. Plasmid-based candidates expressing either the TcdA or TcdB RBD were screened. Different vaccine routes and schedules were tested to achieve detectable serum and mucosal antibody titers in C57BL/6J mice. When given in a multimodality schedule over 1 week (day 0 IM+PO, days 2 and 4 PO), several candidates provided 100% protection against lethal challenge. Substantial protection (82%) was achieved with combined PO TcdA/TcdB vaccination alone (d0, 2 and 4). These data demonstrate the potential of the YS1646-based vaccines for C. difficile and strongly support their further development. Copyright 2019 Winter et al.