This proposal is in response to RFA-AI-09-016 requesting applications to develop vaccines for selected pathogens including Clostridium difficile, the cause of pseudomembranous colitis, which accounts for a quarter of all cases of antibiotic-associated diarrhea. With the recent emergence of hypervirulent strains, the incidence of C. difficile infection (CDI) has increased significantly in both North America and Europe, causing lengthy hospitalization, substantial morbidity and mortality. CDI is thought to be mainly mediated by exotoxins TcdA and TcdB, which glucosylate low molecular mass GTPases of the Rho family, leading to massive fluid secretion, acute inflammation, and necrosis of the colonic mucosa. Protection against CDI was shown to be mediated through systemic and mucosal antibodies against the 2 key toxins, although other virulence attributes are known to exist which may also contribute to the manifestation of CDI. The goal of this proposal is to design a vaccine that targets both TcdA and TcdB, with a view to elicit strong systemic and mucosal immunity to prevent CDI, reduce the severity, or eliminate an ongoing chronic disease. We propose to exploit the recently expressed atoxic C. difficile holotoxin proteins in an endotoxin-free Bacillus megaterium system. Two immunogens will be evaluated: a mixture of atoxic full-length C. difficile toxin A and B generated by point mutations (designated as aTxAB), and a chimera protein containing full-length TcdB but with its receptor binding domain replaced with that of TcdA (designated as cTxAB). cTxAB has a small deletion (97 amino acids) in transmembrane domain rendering it non-toxic. Preliminary studies showed that atoxic TcdB vaccination induced antibody responses against a wide-spectrum of epitopes and potent protective immunity superior to toxoid;cTxAB immunization induced antibody and protective responses against both TcdA and TcdB. In this project, we will first evaluate the ability of these atoxic recombinant proteins to induce protective antibody responses following parenteral immunization followed by challenge with wild type toxins (Aim 1). This will be followed by evaluating several regimens of mucosal immunizations (oral, intranasal and sublingual) designed to induce protection against systemic and mucosal challenges with wild type toxins (Aim 2). We will test the protective efficacy of the various immunization regimens developed in Aims 1 and 2 in the recently described mouse acute infection model (Aim 3a), and the most efficient immunization method resulting from the mouse infection studies will undergo preclinical evaluation in the chronic piglet model of CDI developed in this laboratory (Aim 3b). Because at this early stage the nature of the candidate vaccine is unknown, nor is the adjuvant required, we are not in a position to form a suitable partnership. Abstract Narrative: Clostridium difficile-associated diarrhea and enteric inflammatory diseases are caused primarily by two secretory toxins. This project will use recombinant atoxic holotoxins produced in this lab as a basis for a vaccine to elicit strong systemic and mucosal immunity to prevent C. difficile infection, reduce the severity, or eliminate an ongoing chronic disease.