Project Summary In early 2020, a new virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), generated headlines due to its unprecedented rate of transmission. SARS-CoV-2 caused the first reported cases of coronavirus disease 2019 (COVID-19) in December 2019 and continues to spread worldwide. As a family of RNA viruses, SARS-CoV-2 is prone to mutate at a rate up to a million times faster than its hosts1,2. These rapid genomic alterations have already generated highly transmissible variants, and have raised concerns that the virus will evade vaccine-induced immunity. In addition, a large percentage of the global population remains unvaccinated, due to the challenges of production and mass distribution, vaccine hesitancy, and pending approval status for patients under age 12. Therefore, an effective antiviral has the potential to relieve suffering for millions—not only helping individual patients recover and reducing the number of deaths, but also limiting the number of positive carriers and thereby curbing the spread of the pandemic. This proposal aims to develop an efficient antiviral to impede the virus’ entry into cells, specifically into lung alveolar type II (AT2) cells, the stem cells of the distal lung. Thanks to recent studies, we know which “door” (a receptor called ACE2) and “key” (a protease called TMPRSS2) the virus uses to enter cells. Our goal is to remove the key so the virus cannot open the door and enter host cells. We will use a conventional air-liquid interface (ALI) culture that is representative of the in vivo airway and a recently developed 3-dimensional (3D) in vitro lung organoid model that recapitulates many aspects of lung structure and the cellular environment and that has been used to study respiratory viruses, including SARS-CoV-2. These systems represent tissues better than cell lines, but offers the benefit of being less complex than tissue explants or animal models. In addition, we have generated a panel of highly sensitive and specific mouse monoclonal antibodies (mAbs) directed against TMPRSS2. In preliminary studies, the lead TMPRSS2 mAb, AL20, shows no signs of cytotoxicity with a trend towards inhibition of SARS-CoV-2 pseudovirus entry in cell lines and in lung organoids. Furthermore, we have identified at least two serine protease inhibitors (serpins) that form complexes with TMPRSS2, and the presence of these complexes is inversely correlated with the SARS-CoV-2 infection rate. These findings lead to our hypothesis that targeting TMPRSS2 can inhibit SARS-CoV-2 viral entry and spread. To test our hypothesis, we will first test the efficacy of AL20 for blocking the entry of SARS-CoV-2 into AT2 cells in lung organoids and in airway epithelial cells in ALI cultures, and elucidate the underlying mechanisms. We will then evaluate the effects of serpins on TMPRSS2 activity and SARS-CoV-2 viral entry and spread. Finally, to explore the feasibility of advancing AL20 to human trials, we will test humani...