Project Summary Diarrhea is the second leading cause of death in children aged <5 years in developing countries, more than AIDS, malaria, and measles combined. Our long-term goal is to develop effective vaccines against diarrheal bacteria. The objective of this industrial partnership project is to combine expertise and leadership from academic vaccine research and industrial vaccine development and manufacturing to accelerate the development of MecVax, a multivalent cross-protective subunit vaccine for enterotoxigenic Escherichia coli (ETEC). ETEC bacteria are one of the top five causes of children's diarrhea and the most common cause of travelers' diarrhea. ETEC is listed as a category B priority pathogen (NIH) and a serious threat of antibiotic resistance (CDC), and causes >220 million diarrhea clinical cases annually, resulting in stunting and poor cognitive development in diarrheal children, 1,065,000 years lost due to disability (YLD), 6,894,000 years to disability-adjusted life-years (DALY), and about 100,000 deaths (many are children < 5 years). ETEC is also a primary cause of diarrhea in young animals and causes significant economic losses worldwide. Currently, there are no licensed vaccines against ETEC diarrhea. The development of effective vaccines for ETEC is a top priority for WHO, UNICEF, and many other public health institutions. By applying an innovative vaccinology platform, we have constructed two polyvalent proteins and developed a protein-based multivalent ETEC vaccine candidate, MecVax. MecVax is the only ETEC vaccine candidate that induces protective antibodies against both ETEC toxins (LT and STa) and the seven most important ETEC adhesins (CFA/I, CS1 - CS6). Since ETEC bacteria producing LT and/or STa toxin are associated with all ETEC diarrhea cases and strains expressing adhesin CFA/I or CS1 - CS6 cause > 66% of ETEC clinical cases, the synergy of antitoxin and anti-adhesin immunity from MecVax provides truly broad protection against ETEC children's diarrhea and travelers' diarrhea. MecVax is demonstrated to broadly protect against ETEC clinical diarrhea and colonization of small intestines preclinically. The central goal of this milestone-oriented vaccine development project is to optimize MecVax vaccine formulation, analytical development, and production processing. The rationale is that the completion of this application will identify MecVax optimal formulation, optimize upstream and downstream processing and analytical development, and manufacture vaccine cGLP products, - essential to accelerate MecVax development. To achieve these goals, we will 1) evaluate MecVax protection against ETEC adherence and enterotoxicity at different antigen doses, adjuvants, buffers, and shelving conditions, 2) optimize analytical assays and upstream and downstream processing, 3) manufacture and test MecVax master cell banks, and 4) perform product production engineering runs and submit MecVax pre-IND application. The positive impact is...