Enterotoxigenic E. coli (ETEC) is a major cause of bacterial infectious diarrhea in children, travelers and deployed military personnel in risk areas. As such, development of a vaccine would be advantageous for public health. One strategy is to use subunits of colonization factors combined with toxoids of heat-labile toxin (LT). Recently, a first-in-humans safety and immunogenicity Phase 1 vaccine trial (NCT03404674) was conducted. with dose- escalating intramuscular delivery of CS6-subunit antigen CssBA combined with LT-R192G/L211A (dmLT). No serious adverse events were reported and we observed strong humoral immunogenicity in several cohorts, notably related to dmLT dose. Yet a complete analysis of clinical trial samples, including humoral and cellular memory is lacking. As this vaccine trial indicates, dmLT is not only an LT toxoid but also a potent adjuvant that stimulates immunity to co-delivered antigens; however, there is a gap in our understanding of molecular mechanisms responsible for initiating vaccination outcomes with antigens co-delivered with dmLT. The objective of this proposal is to expand analysis on serum and PBMC samples from an ETEC Phase 1 clinical trial and to define the key biomarkers and molecular mechanisms directing vaccine outcomes. In the proposed studies we aim to explore (1) how vaccination doses modulated development of memory, longevity and diversity of the humoral response; (2) how vaccination altered development of durable cellular immunity; (3) whether early signaling events can serve as biomarkers of immunity; and (4) what molecular mechanisms during immunization shape vaccination outcomes. To do so we will analyze our stored clinical trial samples and perform a number of sophisticated analyses, including transcriptional and metabolomics assays using samples from a related ETEC Phase 2b vaccine- efficacy trail to define signatures of vaccine induced protection which we will then validate using the Phase 1 trial samples. In addition, we will validate findings with cellular analyses and mouse models. These findings will help with the development of an ETEC vaccine for human use by parenteral route and also provide mechanistic insight into key events directing vaccination outcomes.