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. 2024 Nov;13(11):e70001.
doi: 10.1002/jev2.70001.

Extracellular vesicles containing SARS-CoV-2 proteins are associated with multi-organ dysfunction and worse outcomes in patients with severe COVID-19

Diego de Miguel-Perez  1   2 , Marisol Arroyo-Hernandez  3 , Sabrina La Salvia  4 , Muthukumar Gunasekaran  5   6 , Edward M Pickering  7   8 , Stephanie Avila  9 , Etse Gebru  9 , Eduardo Becerril-Vargas  10 , Sergio Monraz-Perez  10 , Kapil Saharia  11 , Alison Grazioli  12 , Michael T McCurdy  7 , Matthew Frieman  13 , Lisa Miorin  14   15 , Alessandro Russo  5 , Andrés F Cardona  16 , Adolfo García-Sastre  14   15   17   18   19   20 , Sunjay Kaushal  5   6 , Fred R Hirsch  1 , Djordje Atanackovic  9 , Susmita Sahoo  4 , Oscar Arrieta  3 , Christian Rolfo  1   2
Affiliations

Extracellular vesicles containing SARS-CoV-2 proteins are associated with multi-organ dysfunction and worse outcomes in patients with severe COVID-19

Diego de Miguel-Perez et al. J Extracell Vesicles. 2024 Nov.

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) and has been related to more than 7 million deaths globally since 2019. The association of high levels of IL-6 with severe cases led to the early evaluation of the anti-IL6 inhibitor tocilizumab as a potential treatment, which unfortunately failed to improve survival in many trials. Moreover, little is known about the development of COVID-19 sequelae, and biomarkers are needed to understand and anticipate these processes. Because extracellular vesicles (EVs) play an important role in viral infection and immune response, they could potentially serve as predictive and prognostic biomarkers. We isolated EVs from 39 patients with severe COVID-19, from which 29 received tocilizumab and 10 were considered controls. Blood samples, which were collected at hospitalisation before treatment, at Day 7, and Day 15 during follow-up, were assessed by immunoblot for longitudinal expression of spike (S) and nucleocapsid (N) proteins. Dynamic expression was calculated and compared with clinicopathological and experimental variables. Expression of EV S was validated by immunogold and imaging flow-cytometry, revealing an enrichment in CD9+ EVs. As a result, decreasing expression of EV viral proteins was observed in patients treated with tocilizumab. Moreover, higher increase in EV S was observed in patients with lower antibody response, hyperfibrinogenemia, lower respiratory function, higher blood pressure and shorter outcomes. These findings lay the foundation for future studies characterizing the role of EVs in multiorgan assessment and identifying biomarkers in patients with severe COVID-19 and possible long COVID.

Keywords: COVID‐19; biomarkers; extracellular vesicles; long‐COVID; nucleocapsid; spike protein; tocilizumab.

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Conflict of interest statement

M. Arroyo‐Hernandez reports receiving personal fees from Astra Zeneca, Bristol, and Roche outside the submitted work A. Russo reports advisory board role/consultancy from AstraZeneca, MSD, Novartis, Pfizer, BMS, Takeda, and Amgen; compensated activity for editorial projects from AstraZeneca, MSD, Novartis, and Roche unrelated to the current work. A. F. Cardona discloses financial research support from Merck Sharp & Dohme, Boehringer Ingelheim, Roche, Bristol‐Myers Squibb, Foundation Medicine, Roche Diagnostics, Thermo Fisher, Broad Institute, BioNTech, Amgen, Flatiron Health, Teva Pharma, Rochem Biocare, Bayer, INQBox and The Foundation for Clinical and Applied Cancer Research—FICMAC. Advisor role to EISAI, Merck Serono, Jannsen Pharmaceutical, Merck Sharp & Dohme, Boehringer Ingelheim, Roche, Bristol‐Myers Squibb, Pfizer, Novartis, Celldex Therapeutics, Foundation Medicine, Eli Lilly, Guardant Health, Illumina, and Foundation for Clinical and Applied Cancer Research—FICMAC. The A. Garcia‐Sastre laboratory has received research support from GSK, Pfizer, Senhwa Biosciences, Kenall Manufacturing, Blade Therapeutics, Avimex, Johnson & Johnson, Dynavax, 7Hills Pharma, Pharmamar, ImmunityBio, Accurius, Nanocomposix, Hexamer, N‐fold LLC, Model Medicines, Atea Pharma, Applied Biological Laboratories and Merck, outside of the reported work. He has consulting agreements for the following companies involving cash and/or stock: Castlevax, Amovir, Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Pagoda, Accurius, Esperovax, Applied Biological Laboratories, Pharmamar, CureLab Oncology, CureLab Veterinary, Synairgen, Paratus, Pfizer and Prosetta, outside of the reported work. He has been an invited speaker in meeting events organized by Seqirus, Janssen, Abbott, Astrazeneca and Novavax. He is inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections and cancer, owned by the Icahn School of Medicine at Mount Sinai, New York, outside of the reported work. F. R. Hirsch reports advisory boards consultancy for Bristol‐Myers Squibb, AstraZeneca/Daiichi, Sanofi/Regeneron, Novartis, Amgen, OncoCyte, Genentech, and Nectin Therapeutics, Novocure, NextCure, Merus, G1 Therapeutics, Oncohost, Agilent/DAKO. Patent (Through University of Colorado); EGFR protein‐ and EGFR gene copy number expression as predictive biomarkers for EGFR‐directed therapy. O. Arrieta reports receiving personal fees from Pfizer, Lilly, Merck, and Bristol‐Myers Squibb and grants and personal fees from AstraZeneca, Boehringer Ingelheim, and Roche outside the submitted work. Other authors declare that they have no competing interests. C. Rolfo has received speaker honoraria from AstraZeneca, Roche and MSD, advisory board honoraria from Inivata, Archer, Boston Pharmaceuticals, MD Serono and Novartis, Bayer, Invitae, Regeneron, Janssen, Bostongene, Novocure, Scientific Advisory Board member of Imagene, and institutional research funding from LCRF‐ Pfizer and NCRF, non‐renumerated research support from GuardantHealth and Foundation Medicine. He has non‐renumerated leadership roles at the International Society of Liquid Biopsy (ISLB), the International Association for Study of Lung Cancer (IASLC), the European School of Oncology (ESO), and Oncology Latin American Association (OLA). The rest of the authors declares no conflict of interests.

Figures

FIGURE 1
FIGURE 1
Study design and biomarkers analysed: patient accrual, distribution in treatment and control group, and longitudinal blood analysis of viral proteins in EVs as well as antibody titers and soluble cytokines (Credit: Created with Biorender).
FIGURE 2
FIGURE 2
EV characterization in patients with severe COVID‐19: (a) Particle size analysis by DLS showed a medium diameter of 81.7 and 158.6 nm in samples from healthy and infected patients, respectively (p = 0.080) (b) Nanoparticle tracking analysis of EVs from a patient with COVID‐19 with mode of 108 nm. (c) Representative images from immunogold TEM revealing EVs from BAL of similar diameter with presence of CD9 and S by binding to nanogold particles of 5 and 10 nm, respectively, in a patient with COVID‐19 while only presence of CD9 in EVs from an uninfected donor. (d) Immunoblot characterization of EVs from healthy donors and COVID‐19 patients depicted presence of EV markers Hsp70, Flotillin‐1, and CD9 in CD9‐enriched EVs from patients with COVID‐19 and healthy donors and absence of other non‐EV markers such as GM130 and Calnexin. (e) Immunomagnetic selection with CD9 beads of EVs from COVID‐19 patients showed enrichment of S (ab1 & ab2) and N, while lower levels of these proteins were seen in the WL or the unbound fraction resulting after selection. COVID‐19 (+) or infected VERO E6 were used as positive controls while uninfected (−) were used as negative controls. Enriched EVs exhibited bands corresponding to the 25 kDa IgG light chain and 55 kDa IgG heavy chain of the CD9 antibody‐beads. BAL, bronchoalveolar lavage; DLS, dynamic light scattering; EVs, extracellular vesicles; N, nucleocapsid; S, spike; TEM, transmission electron microscopy; WL, whole lysate.
FIGURE 3
FIGURE 3
EV S characterization by imaging flow‐cytometry: Percentages of positive and negative EVs for S (ab2) (y‐axis) and CD9 (x‐axis) in baseline samples from patients with COVID‐19 (a), (b) (c) and samples at 1 week during the follow‐up (d) (e) (f). Top‐right orange squares highlight double‐positive S & CD9 EVs. (g) Representative images from the S+ (red) EVs and S‐ EVs from a COVID‐19 patient. The EV marker CD9 is depicted in green. EVs, extracellular vesicles; S, spike.
FIGURE 4
FIGURE 4
Expression of viral proteins in plasma CD9+ EVs in patients treated with tocilizumab and controls. Patients treated with tocilizumab showed reduced expression of ΔEV S (ab1) (T1–T2) and ΔEV S (ab2) (T1–T2 & T1–T3) in comparison to controls. Moreover, more profound reduction was seen of ΔEV S (ab1) (T1–T3) and ΔEV S (ab2) (T1–T2 & T1–T3) after the second dose. No statistically significant reduction of EV N protein was observed (Mann–Whitney U‐test). EV viral protein expression was measured by western‐blot. EVs, extracellular vesicles; N, nucleocapsid; S, spike.
FIGURE 5
FIGURE 5
Correlation between viral proteins in CD9+ EVs and clinical variables. (a) and (b) Changes in plasma ΔEV S (ab2) (T1–T3) observed by immunoblot were associated to those of anti‐S2 and anti‐N antibody titers evaluated by ELISA. (c) Increase in ΔEV S (ab2) correlated positively with dynamic levels of fibrinogen in plasma (T1–T3). (d) Elevation in ΔEV S (ab1) and ΔEV S (ab2) levels were linked with increasing blood chloride (T1–T3). (e) ΔEV S (ab1) and ΔEV S (ab2) were negatively correlated with changes in P/F ratio (T1–T3). (f) An increase in ΔEV S (ab2) was associated with higher blood systolic pressure during follow‐up (Spearman's rank correlation test). Number of patients included in each analysis based on availability of data (n). EVs, extracellular vesicles; P/F, PaO2/FiO2; S, spike.
FIGURE 6
FIGURE 6
ΔEV S is a prognostic biomarker for survival. (a) Patients with increasing plasma ΔEV S (ab2) (orange) showed shorter OS than those with decreasing or undetectable levels (blue) (log‐rank test). (b) The multivariate analysis demonstrated that quantitative increases in ΔEV S (ab2) (T1–T2) and diagnosis of diabetes mellitus were independent factors associated with worse survival (Cox regression). EVs, extracellular vesicles; OS, overall survival; S, spike.
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