Project Summary/Abstract The long-term objective of this project is to develop a safe and broadly protective vaccine for enterotoxigenic E. coli (ETEC), a common cause of diarrhea worldwide. This highly collaborative project will rely on the combined resources, and innovation of investigators with complementary expertise in molecular microbiology and microbial pathogenesis, vaccinology, immunology, and advanced state-of-the-art structural biology to investigate novel targets for ETEC vaccine development. Specifically, the project will: (1) examine whether two key surface proteins of ETEC can provoke immune recall (memory) responses that are thought to be involved in sustained protection against infection (2) develop monoclonal antibodies from circulating immune cells obtained from blood samples following natural infections (3) map where these antibodies bind on the two surface proteins that we plan to use in vaccines (4) determine the overall structure of the proteins that we plan to use in vaccines (5) test the ability of these monoclonal antibodies to neutralize protein function and the ability of the bacteria to deliver toxins (6) vaccinate mice with these proteins to test protection against infection (7) test the activity of antibodies from mice and map where antibodies from mice bind the vaccine proteins relative to the human antibodies. The project will answer fundamental questions about the nature of the two proteins that are relevant not only to ETEC but to other important pathogens that secrete similar proteins. Most importantly the project is expected to provide critical information that can be translated into efficient design of a multi-featured vaccine for ETEC that provides broad-based long-term protection.