Project Summary: Diarrhea is the second leading cause of death in children <5 years, and remains a major public health problem. Enterotoxigenic Escherichia coli (ETEC), the most common bacterial cause of diarrhea, causes 280 to 400 million diarrheal cases in children <5 years and 100 million more cases in children >5 years annually, resulting in 150,000 to 300,000 deaths annually. ETEC also commonly cause travellers? diarrhea. While improved sanitation is the eventual solution, vaccination will be the best preventive measure for many decades. Key virulence factors of ETEC are bacterial adhesins and enterotoxins. Adhesins mediate bacterial attachment to host receptors and colonization in small intestines. Enterotoxins, heat-labile toxin (LT) and heat- stable toxin (STa), disrupt intestinal fluid homeostasis to cause fluid hyper-secretion and diarrhea. A vaccine preventing bacteria colonization and neutralizing toxin enterotoxicity could effectively prevent ETEC diarrhea, but until now, a safe and effective ETEC vaccine has not been developed. Developing ETEC vaccines is challenging since the heterogeneous ETEC strains express at least 23 adhesins and 2 distinct toxins. As ETEC strains producing any one or two adhesins and either toxin (LT or STa) cause diarrhea, ideally, an effective ETEC vaccine should protect against all adhesins and both toxins. But protecting against 23 adhesins is not feasible and protecting against 2 toxins is very difficult. Additionally, suitable animal models to unambiguously assess efficacy of ETEC vaccine candidates have been lacking. In this study, we will develop a safe and broadly protective ETEC vaccine. We will apply a multiepitope fusion antigen (MEFA) method to create an adhesin MEFA to induce antibodies against the 7 key adhesins associates with 80% of ETEC diarrhea cases (caused by ETEC strains with known adhesins), and fuse an LT toxoid and a STa toxoid for a safe toxoid fusion to induce antibodies neutralizing both toxins. The adhesin MEFA and the toxoid fusion will be combined (co-administration) or further fused together (as a CFA-toxoid MEFA) to induce broadly protective anti-adhesin and antitoxin antibodies. We will then use a novel piglet and rabbit dual-challenge model to assess efficacy of a subunit vaccine candidate against ETEC diarrhea. Our preliminary studies show LT-STa toxoid fusions, adhesin MEFA and adhesin-toxoid fusion induce antibodies neutralizing both toxins and 7 adhesins and protecting pigs against ETEC diarrhea, and rabbits and pigs are ideal animal models for ETEC vaccine efficacy assessment. A broadly protective subunit ETEC vaccine will benefit millions of children and travelers. The innovative approaches developed in this research can be generally applied to multivalent vaccine development against other diseases.