Extracellular Vesicles (EVs) Innate Immune Cargo in Respiratory Viral Infections - PROJECT SUMMARY/ABSTRACT Developmental delay in the maturation of the innate immune system and in particular reduced functionality of interferon (IFN) pathways are recognized mechanisms of disease severity following viral respiratory infections in early life. Respiratory syncytial virus (RSV) in particular, represents the leading cause of severe lower respiratory tract illness (LRTI) in young infants and children, with no therapies or vaccines currently available. As part of our investigations focused on the isolation and structural characterization of epithelial-derived subcellular fractions called extracellular vesicles (EVs) we discovered that EVs released from viral-infected human airway epithelial cells contain biologically active type I and III IFNs, while EVs isolated from the upper airways of patients with respiratory viral infections contain only type III IFN. EVs, a type of secretory vehicle released from cells, are characterized by size and specific markers which has allowed their identification in various human bio-fluids. EVs contain nucleic acids, lipids, and proteins, and have been shown to transfer this biologically active cargo between neighboring cells and to distant sites, therefore participating in processes of cell-to-cell communication, inflammation, and disease pathogenesis. Overall, little is known regarding the characteristics of EVs generated in vivo in human airways, how viral respiratory infections such as those caused by RSV shape the EVs structural and cargo-related characteristics, and whether they contribute to the antiviral immune response. Based on our work and this gap in the knowledge, Specific Aim 1 will characterize the innate immune cargo of EVs isolated from the nasopharyngeal secretions (NPS) of children with acute RSV infections and from cultures of human nose organoids (HNOs)-derived epithelial cells. The biological activity of NPS and HNO EVs and EV-expressed IFNs will be tested by state-of-the art antiviral assays in RSV- infected recipient lung epithelial cells. Mechanistic studies in Specific Aim 2 will determine the contribution of RSV F and G proteins carried by EVs and EV cargo of nucleic acids and their respective cytosolic receptor(s), in the process of EV internalization, antiviral gene expression and overall induction of an antiviral status in target/recipient cells. Our published work and preliminary data support the concept that the packaging of innate immune mediators in EVs secreted in the upper airway mucosa represents an important and currently unknown antiviral mechanism directed not only to adjacent areas but also to sites far from of initial viral entry or infection, such as the lower respiratory tract and lung. The results of studies proposed in this exploratory R21 will inform a future R01, which will include studies in a large cohort of viral-infected infants to determine correlation between EV-associated IFNs/innate mediators and viral load in the nasal mucosa, disease severity and progression (from upper to lower respiratory tract), and testing the hypothesis that upper airway EVs reach the lung where they modulate antiviral responses using experimental animal models.
