In a subset of Chlamydia trachomatis-infected women, the infection escapes immune control and ascends to the upper reproductive tract. There is a fundamental gap in understanding how pathways activated by chlamydial infection lead to immune pathology and reproductive morbidity in women. Its continued existence is a significant barrier to identification of biomarkers to diagnose or predict risk for reproductive sequelae after exposure. The long-term goal of this program is to develop vaccines that reduce transmission and prevent reproductive tract sequelae after exposure to C. trachomatis. The overall objective of this proposal is to identify biomarkers of asymptomatic ascending infection and endometritis in women and genetic biomarkers of susceptibility/risk for disease. The central hypothesis is that protective and immunopathologic responses elicited in response to STI associate with characteristic transcriptional signatures and genetic variance. The rationale for this project is that identifying biomarkers of susceptibility to ascending infection and risk for subsequent immune pathology will accelerate rational vaccine design and testing by (i) identifying individuals most likely to benefit from immunization, to facilitate appropriately powered studies, (ii) enabling balanced group selection for vaccine trials, and (iii) serving as clinical measures of vaccine efficacy. Blood-borne transcriptional profiling of women with a spectrum of chlamydial genital tract infection revealed specific inflammatory pathways associated with disease and enabled definition of a biomarker panel that diagnoses endometritis in asymptomatically-infected women with high chlamydial burden. Guided by strong preliminary data, the following three specific aims will test the hypothesis: 1) Determine candidate biomarkers positively- or negatively associated with ascending chlamydial infection in asymptomatic women; 2) Identify genetic biomarkers of susceptibility to ascending chlamydial infection and risk for subsequent reproductive sequelae; and 3) Identify disease-causing pathways and their key regulators engaged during ascending chlamydial infection, using causal network analysis. The first aim will profile cervical inflammatory responses from women with local infection or upper tract involvement in a new cohort of highly-exposed women (TRAC2, Core B). Pathogen load, co-infection, cervicovaginal microbiome and hormonal contraceptives will be assessed as confounders or additional biomarkers. The remaining aims will focus on integrating genotype, gene expression and disease phenotypes from TRAC2 and previously-profiled cohorts via expression quantitative trait locus (eQTL) mapping and causal mediation test, and on identification of disease- causal genes, using graphical models. The research proposed is innovative, in our opinion, because it will implement a comprehensive, non-biased approach to the identification of molecular biomarkers in a highly disease-relevant clinical population. The proposed research is significant because it is expected to translate directly to anti-chlamydial vaccine evaluation in humans and to have broad importance for diagnosis and for evaluation of novel therapeutics.