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. 2022 Jan 17;12:785941. doi: 10.3389/fimmu.2021.785941

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Copyright © 2022 Pesce, Manfrini, Cordiglieri, Santi, Bandera, Gobbini, Gruarin, Favalli, Bombaci, Cuomo, Collino, Cricrì, Ungaro, Lombardi, Mangioni, Muscatello, Aliberti, Blasi, Gori, Abrignani, De Francesco, Biffo and Grifantini

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Circulating exosomes of MILD COVID-19 patients carry more SARS-CoV-2-S-derived peptides than those of SEVERE COVID-19 patients and are enriched in CD9 and CD41a exosomal markers. (A, B) Tetraspanin ELISA assay performed on 20 COVID-19 patients (10 with MILD symptoms and 10 with SEVERE symptoms) and 20 COVID-19-negative HDs. Patient-recovered samples were first analyzed as a whole (A) and subsequently separated based on class (B). Positivity for S protein was calculated as the presence of the signal compared to a control well (S/Co) (Two-tailed t-test ****p < 0.0001). (C) Flow cytometry analysis. Latex beads coated with the anti-CD63 antibodies were incubated with plasma-recovered exosomes. Bead-bound exosomes were subjected to flow cytometry. Anti-CD9/AF488 antibodies were used to define and gate the specific exosome population. The percentage of exosomes positive for anti-SARS-CoV-2-S-RBD/APC is reported in RFI (value vs. isotype control) (One-way ANOVA with Tukey test ****p < 0.0001). (D) Western blot showing anti-SARS-CoV-2-S-RBD (left) and anti-SARS-CoV-2-S1 immunoblotting (right) in exosomes of COVID-19 patients or HDs. Exosomes were immunoprecipitated with anti-tetraspanin (CD63, CD9, CD81) antibodies and immunoblotted for the indicated proteins. Exosomal marker HSP70 was used as a loading control. Blots are representative of three independent experiments. (E) Western blots showing the presence of exosomal markers CD63, CD9, TSG101, and syntenin and absence of contaminant plasma-protein ApoA. (F) Scheme of the ExoView™ tetraspanin chip. EVs from plasma of COVID-19 patients or HDs were immobilized on ExoView™ chips by affinity capture against CD81, CD63, CD9, and CD41a exosomal transmembrane proteins. Once affinity-captured, the samples were incubated with fluorescent anti-SARS-CoV-2-S-RBD antibodies and analyzed using ExoView™ R100. (G) Difference in the average particle count from each antibody referred to in panel (A). (H) Colocalization of SARS-CoV-2-S on the surface of the vesicles captured on the chip with the indicated antibodies. Histograms represent the count number (expressed as protein fold change) of SARS-CoV-2-S⁺ exosomes (t-test *p < 0.05, **p < 0.01, ***p < 0.001). HD, Healthy donor; S/Co, Signal/Control; RFI, Relative Fluorescence Intensity; S, Spike; rec, recombinant; Hsp70, Heat shock protein 70; TSG101, tumour susceptibility gene 101; ApoA, Apolipoprotein A; AF488, Alexa Fluor 488; APC, Allophycocyanin; Ctrl, Control.