Exploring Diverse Clinical Presentations in COVID-19 through Circulating Extracellular Vesicle Proteomics Analysis

Extracellular vesicles (EVs) released by virus-infected cells are susceptible to incorporate viral peptides. Here, we hypothesized that the molecular characterization of EVs obtained from COVID-19 patients will reveal novel immunogenic viral peptides. Blood from COVID-19 patients with severe (G1), mild/asymptomatic disease (G2) and controls (G3), were collected during active infection (m0) in early 2020 and eight months (m8) post-infection. Clinical data showed increased inflammation markers in G1. Neutralizing antibodies in non-vaccinated G1 individuals increased at m8, indicating sustained exposure to viral antigens. Proteomic profiling of EVs isolated by three different methods, immunocapture (CD9), ganglioside-capture (Siglec-1) and size-exclusion chromatography (SEC), failed in identifying viral peptides. These results were validated by Western blot against Spike-RBD and EV proteomics of hamsters with induced viral infection. Noticeably, G3 individuals showed dramatic changes in EV-associated protein abundance when comparing m0-m8, likely due to vaccination. Human EV cargo identified proteins with prognostic potential in severe disease.