SARS-CoV-2 Spike Protein and Viral RNA Persist in the Lung of Patients with Post-COVID Lung Disease.

Abstract

SARS-CoV-2 has resulted in over 100 million cases with ∼2% of cases severe enough to merit hospitalization. In severe cases, up to 70% have persistent respiratory symptoms or imaging findings consistent with ongoing inflammation. We and others have described the presence of usual interstitial pneumonia and cryptogenic organizing pneumonia in patients with post-viral lung disease, including COVID-19 infection. In this work, we hypothesize that persistent SARS-CoV-2 antigens well after acute infection may be driving this response. METHODS: Bronchoalveolar lavage (BAL) samples sent to the Clinical BAL Laboratory at Indiana University for evaluation of possible post-COVID lung disease were studied. BAL was filtered through nylon gauze and then centrifuged at 400 g for 10 minutes to pellet cells and obtain acellular supernatants.Acellular BAL fluid was analyzed for spike protein using a commercially available ELISA kit (Kerafast) and for spike protein RNA using a laboratory-developed SARS-CoV-2 qualitative realtime PCR to determine the presence of viral RNA. In preliminary work, extracellular vesicles (EVs) were isolated from three BAL samples by ultracentrifugation and also analyzed for spike protein by ELISA. RESULTS: Thirty-seven subjects a median of 178 (range 28-730) days post documentation of acute SARS-CoV-2 infection were included. Patients with suspected post-COVID lung disease had spike protein concentrations in acellular BAL higher than non-COVID patient controls by ELISA (p=0.009), with over half having values above the highest negative control. Preliminary data in three subjects suggested EVs were a rich source of spike protein. 83.7% (31/37) of patients with suspected post-COVID lung disease had detectable RNA using PCR against spike protein. CONCLUSION: Spike protein and RNA persists in BAL from patients with post-COVID lung disease up to two years after acute infection. Continued analysis of EVs from these samples is ongoing to determine if other viral proteins are present (i.e. nucleocapsid), what the cells of origin are (epithelial cells, immune cells), and to link persistence of viral proteins and RNA to immune responses and clinical outcomes.