Fig 1: RBCEVs prevent the adsorption and entry of viruses into cells by competing for binding to PS receptors. (a) Effect of the order of adding RBCEVs on SARS-CoV-2 spike (w/t) pseudotyped virus neutralization efficiency. RBCEVs were either added to the cells and then washed off before the virus was added (RBCEV + Cell → Virus); RBCEVs were preincubated with the virus prior to adding to cells (RBCEV + Virus → Cell), or they were added to the cells after viral infection (postinfection). The virus/virus-EV mixtures were incubated with cells for 5 h to allow time for infection. (b) RBCEV binding to Vero E6 or A549-ACE2 cells was detected using flow cytometric detection of GPA, a RBCEV specific marker. (c) Immunofluorescence imaging to show the binding and uptake of EVs by Vero E6 cells. CFSE-labeled EVs are shown in green, and cellular membranes (stained with CellMask Deep Red membrane stain) are shown in red. Scale bar: 20 μm. (d) Effect of PS or PC liposomes on the binding of RBCEVs to Vero E6 and A549-ACE2 cells. Cells were preincubated with 7.5 × 1011 liposomes prior to incubation with RBCEVs, followed by GPA staining and flow cytometric analysis. (e) Effect of equal numbers of PS liposomes, PC liposomes, or RBCEVs on the neutralization efficiency of SARS-CoV-2 spike (w/t) pseudotyped virus in Vero E6 and A549-ACE2 cells. Relative infection was determined by quantifying mCherry mRNA using RT-qPCR. (f) Relative neutralization efficiency of human RBCEVs, mouse RBCEVs, human plasma-derived EVs, and 4TO7-EVs on SARS-CoV-2 spike (w/t) pseudotyped virus infection in Vero E6 cells. (g) Relative PS exposure in each of the EVs used in (f) was determined using Annexin V staining of EVs via bead-assisted flow cytometric analysis. For b, d, e, f, and g, n = 3 biological repeats were performed using EVs from separate donors or cells from separate passages. The graphs represent the mean ± standard deviation, and data were compared using Student’s one-tailed t test such that **P < 0.01 and ***P < 0.001.