PROJECT SUMMARY Adenovirus (Ad) normally induces mild, self-limited infections in immunocompetent human hosts. A more virulent strain of Ad14, Ad14p1, first emerged in the U.S. military and has since spread globally to civilian populations resulting in severe infections, sometimes leading to acute respiratory distress syndrome (ARDS). The incidence of ARDS in the U.S. is ~200,000 cases annually, with a hospital mortality rate of ~40%. Most ARDS treatment measures target the inflammatory response; however, all have failed to show a mortality benefit. We have shown that the Ad E1B 20K (20K) gene product controls modulation of the macrophage inflammatory response to cells dying from Ad infection (Ad CPE corpses). Absent or low level 20K gene expression generates Ad CPE cells that are pro-inflammatory, whereas normal (wild type virus) 20K gene expression generates anti-inflammatory Ad CPE cells. Ad14p1 expresses only 20% of the 20K expressed by wild type Ad14. This reduced 20K expression induces pro-inflammatory Ad14p1 CPE corpses, whereas wild type Ad14 CPE corpses are anti- inflammatory. The central hypothesis of this application is that this viral genetic change in the immunomodulatory effect of infection with emergent Ad14p1 is the key biological difference through which this emergent virus increases the incidence and severity of acute lung injury (ALI) that can result in ARDS and death. The Syrian hamster, Mesocricetus auratus, is permissive for infection with human adenoviruses. Syrian hamsters are also susceptible to many other human viruses such as influenza, hantavirus, SARS-CoV, SARS-CoV-2, Marburg and Ebola. We have shown that infection of hamsters with Ad14p1 replicates many of the key features of human ALI/ARDS including patchy bronchopneumonia, increased pro-inflammatory cytokine expression, edema and neutrophil infiltration into the lung and airways. A problem with the Syrian hamster model system has been that there are no transgenic hamsters and that there is a lack of immunological reagents available, such as those for the mouse and human. Development of the CRISPR/Cas9 Syrian hamster has removed one of those obstacles. This project addresses the other problem by creating antibodies that are specific for hamster cell surface markers expressed on macrophages. These antibodies will allow identification and isolation of macrophage sub- populations and comparative characterization of macrophage activation in response to Ad14 and Ad14p1 infections. The antibodies will complement the small number of existing hamster-specific antibodies available for flow cytometry studies. These antibodies will allow us to begin to understand the effects of Ad14 and Ad14p1 infection on the innate immune response and to develop mechanistic studies of how these two viruses generate such different immune responses. Understanding those key factors will allow development of targeted therapeutics to treat not only Ad-induced ALI/ARDS but potentially ARDS triggered by other causes of ALI. In addition, these antibodies can be used to study the effects of other human pathogens on macrophages and neutrophils in the Syrian hamster.

PROJECT NARRATIVE Human adenovirus (Ad) infection can cause acute lung injury and acute respiratory distress syndrome (ARDS), with few treatment options for infection-induced ARDS that has a fatality rate approaching 40%. The Syrian hamster, Mesocricetus auratus, is permissive for human Ad infection and can be used as a model of Ad-induced ARDS. This proposal will develop immunological reagents specific for hamster cell surface markers expressed on macrophages that will be essential for the comparative study of the effects of wild type Ad14 versus emergent Ad14p1 infection on the inflammatory state to help understand how the Ad14p1 variant induces ARDS and to strengthen the Syrian hamster as a small animal model for other human infectious diseases including SARS- CoV-2.