PROJECT SUMMARY Pandemic outbreaks of severe acute respiratory diseases driven by viral infections such as influenza are happening with increasing frequency. According to CDC estimates, during the 2019-2020 flu season there were > 36 million cases in the US alone, over 370,000 patients were hospitalized with pulmonary complications such as pneumonia and > 22,000 died. An emerging body of evidence suggests that the patient's own innate immune system is responsible for much of the pathology due to the rapid pulmonary influx of large numbers of activated monocytes, macrophages, and neutrophils that produce and sustain a “storm” of injurious cytokines that damage pulmonary epithelia and prevent effective resolution of ongoing inflammation. The development of host directed therapies that attenuate the “storm” without impacting the host's ability to clear the virus and infected/dead cells would have utility not just in influenza, but potentially for any respiratory infection exhibiting similar cytokine and immune cell infiltration pathology signatures. The gene for human serpin B1 is a critical survival factor during pulmonary influenza infection in mice. It regulates protease activity, cytokine production, programmed cell death pathways and inflammatory caspase activation – key inflammatory processes associated with an increased risk of fatality in severe pulmonary influenza infections in patients. Serpin B1 is predominantly an intracellular protein, highly expressed in innate immune cells but also found at low levels in the circulation. Thus it is unclear what, if any, effect an exogenously administered recombinant serpin B1 protein would have on the outcome of pulmonary influenza infection. We have made an oxidation resistant recombinant form of murine sB1 (rmsb1aOxR) and demonstrated it to have anti-inflammatory properties in an animal model of viral asthma exacerbations and to reduce weight loss, protect against morbidity and significantly decrease mortality in a mouse model of H3N2 influenza infection. We predict that rmsb1aOxR will function as a host-directed therapy to significantly improve mortality and morbidity outcomes with other type A and B influenza viral strains. We anticipate it will protect or boost early innate immune responses through specific effects on myeloid and epithelial cells and cytokines thus ameliorating the intensity of the more harmful later responses e.g. NETosis. The proposed research in phase I will focus on purification and in vitro cell-based activity of oxidation resistant mouse sb1a (Aim 1) and providing proof-of-principle in animal model(s) of severe pulmonary influenza and further insight into the mechanism of action (Aim 2). The outcome of these studies will guide the design of future confirmatory efficacy studies in other animal models of disease and clinical studies in humans.