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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., Competing Interests: HP, MMo, and LF are shareholders of Innovex Therapeutics. SM-T was a former employee of Innovex Therapeutics. JM-P has received institutional grants and educational/consultancy fees from AbiVax; AstraZeneca; Gilead Sciences; Grifols; Janssen; Merck Sharp & Dohme; and ViiV Healthcare; all outside the submitted work. JC and JB are shareholders of Albajuna Therapeutics SL, NI-U reports institutional grants from Grifols, Dentaid, Hipra and Amassence. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Gualdrón-López, Ayllon-Hermida, Cortes-Serra, Resa-Infante, Bech-Serra, Aparici-Herraiz, Nicolau-Fernandez, Erkizia, Gutierrez-Chamorro, Marfil, Pradenas, Ávila Nieto, Cucurull, Montaner-Tarbés, Muelas, Sotil, Ballana, Urrea, Fraile, Montoya, Vergara, Segales, Carrillo, Izquierdo-Useros, Blanco, Fernandez-Becerra, de La Torre, Pinazo, Martinez-Picado and del Portillo.)

Recent studies indicate that human spleen contains over 95% of the total parasite biomass during chronic asymptomatic infections caused by Plasmodium vivax . Previous studies have demonstrated that extracellular vesicles (EVs) secreted from infected reticulocytes facilitate binding to human spleen fibroblasts (hSFs) and identified parasite genes whose expression was dependent on an intact spleen. Here, we characterize the P. vivax spleen-dependent hypothetical gene (PVX_114580). Using CRISPR/Cas9, PVX_114580 was integrated into P. falciparum 3D7 genome and expressed during asexual stages. Immunofluorescence analysis demonstrated that the protein, which we named P. vivax Spleen-Dependent Protein 1 (PvSDP1), was located at the surface of infected red blood cells in the transgenic line and this localization was later confirmed in natural infections. Plasma-derived EVs from P. vivax -infected individuals (PvEVs) significantly increased cytoadherence of 3D7_PvSDP1 transgenic line to hSFs and this binding was inhibited by anti-PvSDP1 antibodies. Single-cell RNAseq of PvEVs-treated hSFs revealed increased expression of adhesion-related genes. These findings demonstrate the importance of parasite spleen-dependent genes and EVs from natural infections in the formation of intrasplenic niches in P. vivax , a major challenge for malaria elimination., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ayllon-Hermida, Nicolau-Fernandez, Larrinaga, Aparici-Herraiz, Tintó-Font, Llorà-Batlle, Orban, Yasnot, Graupera, Esteller, Popovici, Cortés, del Portillo and Fernandez-Becerra.)

Parasitic diseases constitute a major global health problem, affecting millions of people worldwide. Recent advances in the study of extracellular vesicles (EVs) have opened up new strategies for biomarker discovery in protozoan and helminth infections. Analyses of EVs in cultures and biological fluids have identified numerous potential biomarkers that could be useful for early and differential diagnosis, monitoring therapeutic responses, and the overall management and control of these diseases. Despite the potential of these biomarkers, several challenges must be addressed, including limited research, the need for standardized protocols, and the reproducibility of results across studies. In many parasitic infections, EVs have been obtained from various sample types, including plasma from human patients and mouse models, as well as cultures of the parasites at different stages. EVs were isolated by various methods and predominantly characterized through proteomic analysis or RNA sequencing to assess their cargo and identify potential biomarkers. These biomarker candidates were investigated and validated using different assays such as ELISA, Western Blot, and ROC curves. Overall, the use of EVs is considered a promising new diagnostic strategy for parasite infections, but further research with larger cohorts, standardized methods, and additional validation tests are essential for effective diagnosis and management of these diseases., (Copyright © 2024. Published by Elsevier Inc.)