Chlamydia trachomatis (CT) is the most common bacterial sexually transmitted infection. In the U.S., women aged 15-24 have the highest CT burden: 782,697 cases in 2019. Up to 80% of urogenital CT infections are asymptomatic, and untreated persistent infection can cause pelvic inflammatory disease, infertility, and ectopic pregnancy. Risk of reproductive sequelae rises with duration of CT persistence, likely due to chronic inflammation in the fallopian tubes. In contrast, untreated CT can spontaneously clear without antibiotic use. Clearance rate estimates vary: it appears 7-44% of women clear CT between testing and initiating treatment (~1-2 weeks), but mechanisms of this natural resolution are poorly understood. Studies show spontaneous clearance may reduce reinfection, and antibiotics may impede development of lasting immunity. We have strong preliminary data that bacterial vaginosis, a condition characterized by a low-Lactobacillus vaginal microbiota, is associated with CT persistence. This project will further identify specific features of the vaginal metagenome and immune milieu that contribute to spontaneous CT clearance and assess if these features differ by infection duration. Antibiotic-sparing approaches to promote CT clearance will offer the most protection from reproductive sequelae when clearance is rapid and occurs early in infection. The proposed work will leverage a unique set of cervicovaginal lavage samples from a large longitudinal study (n=1,053 samples, n=431 women). Quarterly samples were collected for 1 year. Due to historical reasons, a majority of the samples were screened for CT at study end. We identified 310 spontaneous clearance events: 247 were rapid and 63 delayed. Clearance events were matched to control persistence events on infection duration. This proposal focuses on epidemiologically robust analyses of the vaginal metagenome and immune milieu at CT+ visits (before clearance) to identify features that may functionally contribute to clearance. Specific aims will evaluate whether the following are associated with spontaneous CT clearance and if associations differ by infection duration: (1) species- and strain- specific vaginal bacteria growth rates, (2) interplay of Th1 and Th17 immune responses, and (3) interactions between the vaginal metagenome and immune milieu. Bacterial growth rates may reflect metabolic activity and allow identification of bacteria that functionally contribute to CT clearance. Spontaneous clearance is likely mediated by IFNγ and Th1 immune responses; Th17 responses may promote persistence and pathology. However, data suggest IFNγ’s role in clearance may depend on timing and duration of its production, and Th17 and Th1 responses may regulate each other. The microbiome and host constantly interact, and multi-omic analyses of microbiome and host immune data can yield insights that are missed in single-omic analyses. This work will highlight potential targets to promote rapid CT clearance, whi...