(224) Immunological and Molecular Characterization of Circulating Exosomes Induced by Lung Transplant Recipients with Covid-19

PURPOSE: Exosomes are nano-vesicles of <200 nm released by cells. We have demonstrated the presence of circulating exosomes containing lung self-antigens (SAgs) (Collagen-V, Kα1-Tubulin) and respiratory viral (RVI) antigens in lung transplant recipients (LTxRs) with RVI and rejection. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in COVID-19 disease which can result in high morbidity and mortality in LTxRs. The goal of this study is to determine role of exosomes induced after SARS-CoV-2 infection in LTxRs, their origin and immune as well as molecular characteristics. METHODS: We analyzed exosomes from 20 LTxRs with SARS-CoV-2 infection. Exosomes were isolated from plasma by Exosome Precipitation Kit and characterized by western blot for SARS-CoV-2 Spike (CSP), nucleocapsid protein (CNP), lung SAgs, cardiac SAgs (Myosin, Vimentin), transcription factors CIITA and NFkB, 20S proteasome, endothelial marker Von Willebrand Factor (VWF, VEGFR1 and V-CADHERIN), epithelial marker (beta catenin, MUC1 and FOX A1), angiotensin II type-I receptor (AT1R), macrophage stimulating factor 1 (MST1) and GRANZYME B (GRA-B). Isolated exosomes were also analyzed by transmission electron microscopy (TEM) for the presence of CSP, VWF, lung SAgs and cardiac SAgs. Mice were immunized with isolated exosomes containing CSP and CNP. Immune responses and histopathology of the mice lung tissues were analyzed. RESULTS: Exosomes from SARS-CoV-2 infected LTxRs contained CSP, nucleocapsid, VWF, VEGFR1, V-CADHERIN, beta catenin, MUC1, FOX A1 lung SAgs, cardiac SAgs, transcription factors, MST1, GRA-B and AT1R. TEM of exosomes also revealed the surface expression of spike protein, VWF, Kα1-Tubulin and Vimentin. C57BL/6 mice immunized with exosomes developed antibodies to CSP and CNP. Histopathology of the lungs demonstrated inflammation and lung fibrosis. CONCLUSION: We demonstrated that SARS-CoV-2 infected LTxRs induced circulating exosomes with CSP, CNP, lung SAgs, epithelial and endothelial markers suggesting that SARS-CoV-2 infection occurs in both endothelial and epithelium of the host. Therefore, we propose that the induced exosomes can activate both endothelium and epithelium leading to cytokine release. Immunized mice developed antibodies specific to CSP and CNP resulting in inflammation in the lung and fibrosis.