OBJECTIVES/GOALS: An ex-vivo tissue model has been developed to predict target concentrations of tenofovir diphosphate (TFVdp; active metabolite of tenofovir) but has not been utilized to see how vaginal dysbiosis affects TFVdp/dATP exposure in female genital tract (FGT). My central hypothesis is that presence of specific anaerobic bacteria will increase dATP in FGT. METHODS/STUDY POPULATION: De-identified HIV-negative cervical tissues from women undergoing gynecological surgeries will be procured and a punch biopsy will be used to create explants. TFVdp/dATP concentrations were both tested in both aerobic and anaerobic conditions after a 24-hour incubation in tenofovir (TFV) to determine any changes between conditions. TFVdp/dATP in cervical tissue was be measured using LC-MS. Next, media and explants were collected at baseline to characterize donor microbiome for 6 donors. 16S microbiome sequencing was performed on extracted DNA to obtain the relative abundances of each bacteria species present. To test changes in dATP/TFVdp due to the microbiome, explants will be incubated in TFV for 24 hours with Prevotella and Dialister to specifically see how microbiomes dominated by these taxa affect dATP. RESULTS/ANTICIPATED RESULTS: There was no significant difference in TFVdp formation between aerobic and anaerobic conditions after a 24-hour tenofovir incubation (p = 0.2) for 8 donors. dATP was not quantifiable at 24 hours in explants, so explants are being collected before 24hrs during a TFV incubation to determine how quickly dATP depletes after collection. We were able to characterize the donor microbiome in media and tissue at baseline and 24hrs which had inter variability. We did not see any presence of Prevotella or Dialister in any donors. We are working on characterizing bacteria growth over time to see how the donor microbiome would change during a 24 hour experiment in anaerobic conditions. Finally, we anticipate seeing increases in dATP with a Prevotella or Dialister supplemented donor microbiome compared to baseline donor microbiome. DISCUSSION/SIGNIFICANCE: The addition of vaginal dysbiosis to tissue model will increase accuracy of prediction of 100% protective TFVdp concentrations and is likely to provide a translational model that can be used to improve TFV-based PrEP in women and streamline development of future PrEP candidates, bringing more prevention options to women and ending the HIV epidemic.