Melisa Gualdrón-López ^1,2 Alberto Ayllon-Hermida ^1,2 Nuria Cortes-Serra ^1,2 Patricia Resa-Infante ^2,3,4,5 Joan Josep Bech-Serra ⁶ Iris Aparici Herraiz ^1,2 Marc Nicolau-Fernández ^1,2 Itziar Erkizia ³ Lucia Gutierrez-Chamorro ³ Silvia Marfil ³ Edwards Pradenas ³ Carlos Avila Nieto ³ Bernat Cucurull ⁶ Sergio R. Montaner Tarbes ⁷ Magdalena Muelas ⁸ Ruth Sotil ⁸ Ester Ballana ^2,3 Victor Urrea ³ Lorenzo Fraile ⁹ Maria Montoya ¹⁰ Júlia Vergara-Alert ^11,12 Joaquim Segalés ^11,12 Jorge Carrillo ^3,4 Nuria Izquierdo-Useros ^3,4 Julià Blanco ^13,2,3,4 Carmen Fernandez-Becerra ^1,2,4 Carolina De La Torre Gómez ⁶ Maria-Jesus Pinazo ¹⁴ Javier Martinez-Picado ^13,2,3,4,5* Hernando A. Del Portillo ^15*

• ¹ ISGlobal, Hospital Clínic - Universitat de Barcelona, Barcelona, Spain, Barcelona, Catalonia, Spain
• ² Germans Trias i Pujol Health Science Research Institute (IGTP), Barcelona, Catalonia, Spain
• ³ IrsiCaixa AIDS Research Institute, Badalona, Spain
• ⁴ Networked Biomedical Research Center for Infectious Diseases (CIBERINFEC), Madrid, Asturias, Spain
• ⁵ University of Vic - Central University of Catalonia, Barcelona, Catalonia, Spain
• ⁶ Josep Carreras Leukaemia Research Institute (IJC), Badalona, Catalonia, Spain
• ⁷ Innovex Therapeutics SL, Badalona, Spain
• ⁸ International Health Service, Hospital Clínic, Barcelona, Spain
• ⁹ Agrotecnio Center, Lleida, Catalonia, Spain
• ¹⁰ Margarita Salas Center for Biological Research, Spanish National Research Council (CSIC), Madrid, Catalonia, Spain
• ¹¹ Other, Bellaterra, Spain
• ¹² Independent researcher, Bellaterra, Spain
• ¹³ Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Catalonia, Spain
• ¹⁴ ISGlobal, Hospital Clínic - Universitat de Barcelona, Barcelona, Spain
• ¹⁵ Instituto Salud Global Barcelona (ISGlobal), Barcelona, Spain

Extracellular vesicles (EVs) released by virus-infected cells have the potential to encapsulate viral peptides, a characteristic that could facilitate vaccine development. Furthermore, plasma-derived EVs may elucidate pathological changes occurring in distal tissues during viral infections. We hypothesized that molecular characterization of EVs isolated from COVID-19 patients would reveal peptides suitable for vaccine development. Blood samples were collected from three cohorts: severe COVID-19 patients (G1), mild/asymptomatic cases (G2), and SARS-CoV-2-negative healthcare workers (G3). Samples were obtained at two time points: during the initial phase of the pandemic in early 2020 (m0) and eight months later (m8). Clinical data analysis revealed elevated inflammatory markers in G1. Notably, non-vaccinated individuals in G1 exhibited increased levels of neutralizing antibodies at m8, suggesting prolonged exposure to viral antigens. Proteomic profiling of EVs was performed using three distinct methods: immunocapture (targeting CD9), ganglioside-capture (utilizing Siglec-1) and size-exclusion chromatography (SEC). Contrary to our hypothesis, this analysis failed to identify viral peptides. These findings were subsequently validated through Western blot analysis targeting the RBD of the SARS-CoV-2 Spike protein’s and comparative studies using samples from experimentally infected Syrian hamsters. Furthermore, analysis of the EV cargo revealed a diverse molecular profile, including components involved in the regulation of viral replication, systemic inflammation, antigen presentation, and stress responses. These findings underscore the potential significance of EVs in the pathogenesis and progression of COVID-19.