<b>Surface Proteome of Plasma Extracellular Vesicles differentiates between SARS-CoV-2 and Influenza Infection</b>

<p dir="ltr">Plasma samples from patients with acute COVID-19 (n=100) and Influenza (n=17) were probed for vesicle surface marker decoration on a EV antibody microarray.</p><p dir="ltr">The protein microarrays were produced in Multitarget plates (MTP, Microfluor 2, 96 wells, polystyrene, Thermo Fisher Scientific, MA, USA) and the printing was performed using a sciFLEXARRAYER S12 microarray printer installed with a Piezo Dispense Capillary (PDC) size 60 with coating type 3 (Scienion AG, DE). The printing procedure was performed under strict humidity and temperature control, as described by Jørgensen et al.(Jørgensen et al. 2021).</p><p dir="ltr">As positive controls 50, 10 and 5 µg/mL of biotinylated goat anti-mouse IgG (Novus Biologicals, CO, USA) was printed and PBS with 5% trehalose was used as negative control. After the print procedure, the MTP were left to dry at room temperature overnight prior to further analysis. Anti-human antibodies used for capturing: CD146 (P1H12), Hsp90 (IGF1), Flotillin-1, CD147, Neuropilin 1, ACE2 (EPR4435(2)), TMPRSS2 (EPR3861) (Abcam, UK); CD9 (SN4/C3-3A2), CD81 (1.3.3.22) (Ancell corporation, MN, USA); Alix (3A9), CD41 (HIP8), HLA ABC (W6/32; Biolegend, CA USA); CD63 (Bio-Rad, CA, USA); ICAM-1 (R6.5, eBioscience, CA, USA); CTLA4 (ANC152.2/8H5; LSbio, WA, USA); tissue factor / CD142 (323,514), VCAM-1 / CD106 (HAE-2Z), thrombomodulin / CD141 (501733), CD31 (AF806, R&D Systems), Annexin V, CD4 (34930), CD151 (210127), CD45 (2D1), tPA, Thrombospondin-1, CD14 (50040), CD166, CD162 (R&D Systems); EpCAM (0.N.277; Santa Cruz Biotechnology, TX, US); CD3 (Hit3a), CD16 (3G8; BD Biosciences, US); HLA DR/DP/DQ (HB-145/IVA12; Caprico Biotechnologies, GA, US); CD62E (Thermo Fisher Scientific, MA, US). All antibodies were diluted in PBS with 5% trehalose and printed in triplicates at 200 µg / mL. The EV Array was prepared as described by Jørgensen et al.(Jørgensen et al. 2013)and Jørgensen et al.(Jørgensen et al. 2021)with modifications.</p><p dir="ltr">In short, the MTP was initially blocked and the blocking buffer (50 mM ethanolamine, 100 mM Tris, 0.1% SDS, pH 9.0) was applied using a hand-held spray gun in a closed box for gentle application of the buffer. After 30 minutes of incubation the wells were emptied and additional 100 µL Liquid Plate Sealer® (Candor Bioscience GmbH, DE) was gently added each well followed by another 1-hour incubation. Subsequently, the wells were emptied and left to dry for 5 hours prior to sealing and storage until use.</p><p dir="ltr">The EV Array analysis was initiated by washing the MTPs in Buffer A (0.2% Tween20® in PBS) using a HydroFlex™ microplate washer (TecanTrading AG, CH). Then, 75 µL sample and 25 µL Buffer B (½x Casein Blocking Buffer (10x concentrate, Sigma-Aldrich, MO, USA, catalog B6429) was applied to each well and incubated for 2 hours RT at an orbital shaker (450 rpm) followed by an overnight incubation at 4°C. After a wash procedure in Buffer A, each well of the MTPs were incubated with 100 µL detection antibody cocktail (biotinylated anti-human-CD9, -CD63, and -CD81 (Ancell, MN, USA) diluted 1:1,500 in Buffer B for 2 hours RT with shaking. Following a wash in Buffer A, 100 µL streptavidin-Cy3 ((Life Technologies, MA, USA) diluted 1:3,000 in Buffer B) was added to each well and incubated for 30 min RT on the shaker. The analysis was finalized by washing with Buffer A and subsequently with MilliQ water.</p><p dir="ltr">The MTPs were dried and scanned using a sciREADER FL2 microarray scanner (Scienion AG, DE) at 535 nm and an exposure time at 2,000 millisec.</p><p><br></p><p dir="ltr">For validation of the array results, MACSPlex Analysis was carried out with additional samples. We have used the FACS-based MACSPlex EV kit and patient samples from the Bioinflame cohort (DOI: 10.1038/s41598-019-57108-0): </p><p dir="ltr">EDTA-Plasma from selected COVID-19 and influenza patients was analyzed for EV surface marker expression using the MACSPlex EV kit (MACSPlex EV Kit IO, human, 130-108-813, Miltenyi Biotec, Germany). 750 µl plasma were diluted with PBS and ultracentrifuged (Sorval Discovery 90SE and Fiberlite F50L rotor, Thermo Fisher Scientific) at 100,000 xg at 4°C for 1 h. The supernatant was carefully removed, and the pellet was resuspended in MACSPlex EV kit assay buffer to reach a final volume of 130 µl/sample. 120 µl/sample was used for EV surface marker staining according to the manufacturer’s protocol for tube format. Flow cytometric measurements and subsequent calculation of the relative EV marker expression were performed using the BD FACSCantoTM II or FACSymphony A1 flow cytometer and FACS Diva v9.0.2 software and FlowJoTM v10.10 (BD Biosciences) according to the manufacturer’s protocol.</p>