OBJECTIVES/GOALS: The goals of this project are to: i) investigate the cargo such as immune mediators (cytokines) and small non coding RNAs (sncRNAs) of EVs derived from nasopharyngeal secretions (NPS) of children with episodes of viral infections and exposed to SHTS; ii) test the biological activity of EVs released from upper airway mucosa on target/recipient lung cells METHODS/STUDY POPULATION: EVs were isolated from ten NPS samples of children with episodes of acute respiratory infections. EVs were characterized by particle sizing (size and concentration), EV markers, and protein arrays for interferons, cytokines, and other immune mediators content. RNA was extracted from ten samples of NPS-derived EVs by column for next generation high throughput sequencing (NGS) to identify sncRNAs in EVs. In studies currently in progress, EVs will be isolated from RSV-infected human nose organoids (HNOs) cells in air liquid interface (ALI) culture, with or without pre-exposure to tobacco smoke. EVs will be then tested for antiviral activity on recipient RSV-infected lower airway cells. Viral titers will be measured in recipient infected lung cells. RESULTS/ANTICIPATED RESULTS: We isolated EVs from NPS samples and confirmed by immunoblot EV markers CD63 and Alix. The average size of NPS-derived EVs of virus positive and negative patients was 170 nm and 145 nm, respectively. We determined the particles number of EVs, concentrations of IFN-βand IFN-λin NPS and NPS-derived EVs of these children. While IFN-βlevels were below the limit of detection in both NPS and NPS-derived EVs of all children, IFN-λwas detected in NPS and NPS-derived EVs from infected patients, except for the two patients with no viral infections. We extracted RNA from control-, virus infected- and/or SHTS- EVs and found that piR-36511, piR-40926, piR-49645, piR-32679 and piR- 53263 were all significantly enriched in EVs derived from NPS of children exposed to TS compared to those not exposed. DISCUSSION/SIGNIFICANCE: RSV leads to approximately 22,000 hospitalizations of children due to second-hand smoke. A vaccine is not currently available for RSV infection. EVs represent a novel translational approach to target undruggable. Airway mucosa EVs in viral respiratory infection function as antiviral messengers and tobacco smoke impairs the EV antiviral activity.