Viral anti-inflammatory serpin reduces immuno-coagulopathic pathology in SARS-CoV-2 mouse models of infection.

SARS-CoV-2 acute respiratory distress syndrome (ARDS) induces uncontrolled lung inflammation and coagulopathy with high mortality. Anti-viral drugs and monoclonal antibodies reduce early COVID-19 severity, but treatments for late-stage immuno-thrombotic syndromes and long COVID are limited. Serine protease inhibitors (SERPINS) regulate activated proteases. The myxoma virus-derived Serp-1 protein is a secreted immunomodulatory serpin that targets activated thrombotic, thrombolytic, and complement proteases as a self-defense strategy to combat clearance. Serp-1 is effective in multiple animal models of inflammatory lung disease and vasculitis. Here, we describe systemic treatment with purified PEGylated Serp-1 as a therapy for immuno-coagulopathic complications during ARDS. Treatment with PEGSerp-1 in two mouse-adapted SARS-CoV-2 models in C57Bl/6 and BALB/c mice reduced lung and heart inflammation, with improved outcomes. PEGSerp-1 significantly reduced M1 macrophages in the lung and heart by modifying urokinase-type plasminogen activator receptor (uPAR), thrombotic proteases, and complement membrane attack complex (MAC). Sequential changes in gene expression for uPAR and serpins (complement and plasminogen inhibitors) were observed. PEGSerp-1 is a highly effective immune-modulator with therapeutic potential for severe viral ARDS, immuno-coagulopathic responses, and Long COVID.

PEGylated Serp-1 Markedly Reduces Pristane-Induced Experimental Diffuse Alveolar Hemorrhage, Altering uPAR Distribution, and Macrophage Invasion.

Diffuse alveolar hemorrhage (DAH) is one of the most serious clinical complications of systemic lupus erythematosus (SLE). The prevalence of DAH is reported to range from 1 to 5%, but while DAH is considered a rare complication there is a reported 50-80% mortality. There is at present no proven effective treatment for DAH and the therapeutics that have been tested have significant side effects. There is a clear necessity to discover new drugs to improve outcomes in DAH. Serine protease inhibitors, serpins, regulate thrombotic and thrombolytic protease cascades. We are investigating a Myxomavirus derived immune modulating serpin, Serp-1, as a new class of immune modulating therapeutics for vasculopathy and lung hemorrhage. Serp-1 has proven efficacy in models of herpes virus-induced arterial inflammation (vasculitis) and lung hemorrhage and has also proved safe in a clinical trial in patients with unstable coronary syndromes and stent implant. Here, we examine Serp-1, both as a native secreted protein expressed by CHO cells and as a polyethylene glycol modified (PEGylated) variant (Serp-1m5), for potential therapy in DAH. DAH was induced by intraperitoneal (IP) injection of pristane in C57BL/6J (B6) mice. Mice were treated with 100 ng/g bodyweight of either Serp-1 as native 55 kDa secreted glycoprotein, or as Serp-1m5, or saline controls after inducing DAH. Treatments were repeated daily for 14 days (6 mice/group). Serp-1 partially and Serp-1m5 significantly reduced pristane-induced DAH when compared with saline as assessed by gross pathology and H&E staining (Serp-1, p = 0.2172; Serp-1m5, p = 0.0252). Both Serp-1m5 and Serp-1 treatment reduced perivascular inflammation and reduced M1 macrophage (Serp-1, p = 0.0350; Serp-1m5, p = 0.0053), hemosiderin-laden macrophage (Serp-1, p = 0.0370; Serp-1m5, p = 0.0424) invasion, and complement C5b/9 staining. Extracellular urokinase-type plasminogen activator receptor positive (uPAR+) clusters were significantly reduced (Serp-1, p = 0.0172; Serp-1m5, p = 0.0025). Serp-1m5 also increased intact uPAR+ alveoli in the lung (p = 0.0091). In conclusion, Serp-1m5 significantly reduces lung damage and hemorrhage in a pristane model of SLE DAH, providing a new potential therapeutic approach.