Comparison of viral inactivation methods on the characteristics of extracellular vesicles from SARS-CoV-2 infected human lung epithelial cells.

The interaction of SARS-CoV-2 infection with extracellular vesicles (EVs) is of particular interest at the moment. Studying SARS-CoV-2 contaminated-EV isolates in instruments located outside of the biosafety level-3 (BSL-3) environment requires knowing how viral inactivation methods affect the structure and function of extracellular vesicles (EVs). Therefore, three common viral inactivation methods, ultraviolet-C (UVC; 1350 mJ/cm2 ), ß-propiolactone (BPL; 0.005%), heat (56°C, 45 min) were performed on defined EV particles and their proteins, RNAs, and function. Small EVs were isolated from the supernatant of SARS-CoV-2-infected human lung epithelial Calu-3 cells by stepwise centrifugation, ultrafiltration and qEV size-exclusion chromatography. The EV isolates contained SARS-CoV-2. UVC, BPL and heat completely abolished SARS-CoV-2 infectivity of the contaminated EVs. Particle detection by electron microscopy and nanoparticle tracking was less affected by UVC and BPL than heat treatment. Western blot analysis of EV markers was not affected by any of these three methods. UVC reduced SARS-CoV-2 spike detectability by quantitative RT-PCR and slightly altered EV-derived ß-actin detection. Fibroblast migration-wound healing activity of the SARS-CoV-2 contaminated-EV isolate was only retained after UVC treatment. In conclusion, specific viral inactivation methods are compatible with specific measures in SARS-CoV-2 contaminated-EV isolates. UVC treatment seems preferable for studying functions of EVs released from SARS-CoV-2 infected cells.

An intra-cytoplasmic route for SARS-CoV-2 transmission unveiled by Helium-ion microscopy.

SARS-CoV-2 virions enter the host cells by docking their spike glycoproteins to the membrane-bound Angiotensin Converting Enzyme 2. After intracellular assembly, the newly formed virions are released from the infected cells to propagate the infection, using the extra-cytoplasmic ACE2 docking mechanism. However, the molecular events underpinning SARS-CoV-2 transmission between host cells are not fully understood. Here, we report the findings of a scanning Helium-ion microscopy study performed on Vero E6 cells infected with mNeonGreen-expressing SARS-CoV-2. Our data reveal, with unprecedented resolution, the presence of: (1) long tunneling nanotubes that connect two or more host cells over submillimeter distances; (2) large scale multiple cell fusion events (syncytia); and (3) abundant extracellular vesicles of various sizes. Taken together, these ultrastructural features describe a novel intra-cytoplasmic connection among SARS-CoV-2 infected cells that may act as an alternative route of viral transmission, disengaged from the well-known extra-cytoplasmic ACE2 docking mechanism. Such route may explain the elusiveness of SARS-CoV-2 to survive from the immune surveillance of the infected host.

A new strategy to count and sort neutrophil-derived extracellular vesicles: Validation in infectious disorders.

Newly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcellular mediators such as neutrophil-derived extracellular vesicles (NDEVs) involved in immune and thrombo-inflammatory responses. Elevation of their plasmatic level has been reported in a variety of infectious and cardiovascular disorders, but the clinical use of this potential biomarker is hampered by methodological issues. Although flow cytometry (FCM) is currently used to detect NDEVs in the plasma of patients, an extensive characterization of NDEVs has never been done. Moreover, their detection remains challenging because of their small size and low antigen density. Therefore, the objective of the present study was first to establish a surface antigenic signature of NDEVs detectable by FCM and therefore to improve their detection in biological fluids by developing a strategy allowing to overcome their low fluorescent signal and reduce the background noise. By testing a large panel of 54 antibody specificities already reported to be positive on PMNs, we identified a profile of 15 membrane protein markers, including 4 (CD157, CD24, CD65 and CD66c) never described on NDEVs. Among them, CD15, CD66b and CD66c were identified as the most sensitive and specific markers to detect NDEVs by FCM. Using this antigenic signature, we developed a new strategy combining the three best antibodies in a cocktail and reducing the background noise by size exclusion chromatography (SEC). This strategy allowed a significant improvement in NDEVs enumeration in plasma from sepsis patients and made it feasible to efficiently sort NDEVs from COVID-19 patients. Altogether, this work opens the door to a more valuable measurement of NDEVs as a potential biomarker in clinical practice. A similar strategy could also be applied to improve detection by FCM of other rare subpopulations of EVs generated by tissues with limited access, such as vascular endothelium, cancer cells or placenta.

Repurposing Antiviral Protease Inhibitors Using Extracellular Vesicles for Potential Therapy of COVID-19.

In January 2020, Chinese health agencies reported an outbreak of a novel coronavirus-2 (CoV-2) which can lead to severe acute respiratory syndrome (SARS). The virus, which belongs to the coronavirus family (SARS-CoV-2), was named coronavirus disease 2019 (COVID-19) and declared a pandemic by the World Health Organization (WHO). Full-length genome sequences of SARS-CoV-2 showed 79.6% sequence identity to SARS-CoV, with 96% identity to a bat coronavirus at the whole-genome level. COVID-19 has caused over 133,000 deaths and there are over 2 million total confirmed cases as of April 15th, 2020. Current treatment plans are still under investigation due to a lack of understanding of COVID-19. One potential mechanism to slow disease progression is the use of antiviral drugs to either block the entry of the virus or interfere with viral replication and maturation. Currently, antiviral drugs, including chloroquine/hydroxychloroquine, remdesivir, and lopinavir/ritonavir, have shown effective inhibition of SARS-CoV-2 in vitro. Due to the high dose needed and narrow therapeutic window, many patients are experiencing severe side effects with the above drugs. Hence, repurposing these drugs with a proper formulation is needed to improve the safety and efficacy for COVID-19 treatment. Extracellular vesicles (EVs) are a family of natural carriers in the human body. They play a critical role in cell-to-cell communications. EVs can be used as unique drug carriers to deliver protease inhibitors to treat COVID-19. EVs may provide targeted delivery of protease inhibitors, with fewer systemic side effects. More importantly, EVs are eligible for major aseptic processing and can be upscaled for mass production. Currently, the FDA is facilitating applications to treat COVID-19, which provides a very good chance to use EVs to contribute in this combat.

Extracellular vesicle-mediated endothelial apoptosis and EV-associated proteins correlate with COVID-19 disease severity.

Coronavirus disease-2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has lead to a global pandemic with a rising toll in infections and deaths. Better understanding of its pathogenesis will greatly improve the outcomes and treatment of affected patients. Here we compared the inflammatory and cardiovascular disease-related protein cargo of circulating large and small extracellular vesicles (EVs) from 84 hospitalized patients infected with SARS-CoV-2 with different stages of disease severity. Our findings reveal significant enrichment of proinflammatory, procoagulation, immunoregulatory and tissue-remodelling protein signatures in EVs, which remarkably distinguished symptomatic COVID-19 patients from uninfected controls with matched comorbidities and delineated those with moderate disease from those who were critically ill. Specifically, EN-RAGE, followed by TF and IL-18R1, showed the strongest correlation with disease severity and length of hospitalization. Importantly, EVs from COVID-19 patients induced apoptosis of pulmonary microvascular endothelial cells in the order of disease severity. In conclusion, our findings support a role for EVs in the pathogenesis of COVID-19 disease and underpin the development of EV-based approaches to predicting disease severity, determining need for patient hospitalization and identifying new therapeutic targets.

Extracellular vesicles carry SARS-CoV-2 spike protein and serve as decoys for neutralizing antibodies.

In late 2019, a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China. SARS-CoV-2 and the disease it causes, coronavirus disease 2019 (COVID-19), spread rapidly and became a global pandemic in early 2020. SARS-CoV-2 spike protein is responsible for viral entry and binds to angiotensin converting enzyme 2 (ACE2) on host cells, making it a major target of the immune system - particularly neutralizing antibodies (nAbs) that are induced by infection or vaccines. Extracellular vesicles (EVs) are small membraned particles constitutively released by cells, including virally-infected cells. EVs and viruses enclosed within lipid membranes share some characteristics: they are small, sub-micron particles and they overlap in cellular biogenesis and egress routes. Given their shared characteristics, we hypothesized that EVs released from spike-expressing cells could carry spike and serve as decoys for anti-spike nAbs, promoting viral infection. Here, using mass spectrometry and nanoscale flow cytometry (NFC) approaches, we demonstrate that SARS-CoV-2 spike protein can be incorporated into EVs. Furthermore, we show that spike-carrying EVs act as decoy targets for convalescent patient serum-derived nAbs, reducing their effectiveness in blocking viral entry. These findings have important implications for the pathogenesis of SARS-CoV-2 infection in vivo and highlight the complex interplay between viruses, extracellular vesicles, and the immune system that occurs during viral infections.

Early prediction of COVID-19 severity using extracellular vesicle COPB2.

The clinical manifestations of COVID-19 vary broadly, ranging from asymptomatic infection to acute respiratory failure and death. But the predictive biomarkers for characterizing the variability are still lacking. Since emerging evidence indicates that extracellular vesicles (EVs) and extracellular RNAs (exRNAs) are functionally involved in a number of pathological processes, we hypothesize that these extracellular components may be key determinants and/or predictors of COVID-19 severity. To test our hypothesis, we collected serum samples from 31 patients with mild COVID-19 symptoms at the time of their admission for discovery cohort. After symptomatic treatment without corticosteroids, 9 of the 31 patients developed severe/critical COVID-19 symptoms. We analyzed EV protein and exRNA profiles to look for correlations between these profiles and COVID-19 severity. Strikingly, we identified three distinct groups of markers (antiviral response-related EV proteins, coagulation-related markers, and liver damage-related exRNAs) with the potential to serve as early predictive biomarkers for COVID-19 severity. As the best predictive marker, EV COPB2 protein, a subunit of the Golgi coatomer complex, exhibited significantly higher abundance in patients remained mild than developed severe/critical COVID-19 and healthy controls in discovery cohort (AUC 1.00 (95% CI: 1.00-1.00)). The validation set included 40 COVID-19 patients and 39 healthy controls, and showed exactly the same trend between the three groups with excellent predictive value (AUC 0.85 (95% CI: 0.73-0.97)). These findings highlight the potential of EV COPB2 expression for patient stratification and for making early clinical decisions about strategies for COVID-19 therapy.

Extracellular Vesicle Capture by AnTibody of CHoice and Enzymatic Release (EV-CATCHER): A customizable purification assay designed for small-RNA biomarker identification and evaluation of circulating small-EVs.

Circulating nucleic acids, encapsulated within small extracellular vesicles (EVs), provide a remote cellular snapshot of biomarkers derived from diseased tissues, however selective isolation is critical. Current laboratory-based purification techniques rely on the physical properties of small-EVs rather than their inherited cellular fingerprints. We established a highly-selective purification assay, termed EV-CATCHER, initially designed for high-throughput analysis of low-abundance small-RNA cargos by next-generation sequencing. We demonstrated its selectivity by specifically isolating and sequencing small-RNAs from mouse small-EVs spiked into human plasma. Western blotting, nanoparticle tracking, and transmission electron microscopy were used to validate and quantify the capture and release of intact small-EVs. As proof-of-principle for sensitive detection of circulating miRNAs, we compared small-RNA sequencing data from a subset of small-EVs serum-purified with EV-CATCHER to data from whole serum, using samples from a small cohort of recently hospitalized Covid-19 patients. We identified and validated, only in small-EVs, hsa-miR-146a and hsa-miR-126-3p to be significantly downregulated with disease severity. Separately, using convalescent sera from recovered Covid-19 patients with high anti-spike IgG titers, we confirmed the neutralizing properties, against SARS-CoV-2 in vitro, of a subset of small-EVs serum-purified by EV-CATCHER, as initially observed with ultracentrifuged small-EVs. Altogether our data highlight the sensitivity and versatility of EV-CATCHER.

Capturing Cytokines with Advanced Materials: A Potential Strategy to Tackle COVID-19 Cytokine Storm.

The COVID-19 pandemic, induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused great impact on the global economy and people's daily life. In the clinic, most patients with COVID-19 show none or mild symptoms, while approximately 20% of them develop severe pneumonia, multiple organ failure, or septic shock due to infection-induced cytokine release syndrome (the so-called "cytokine storm"). Neutralizing antibodies targeting inflammatory cytokines may potentially curb immunopathology caused by COVID-19; however, the complexity of cytokine interactions and the multiplicity of cytokine targets make attenuating the cytokine storm challenging. Nonspecific in vivo biodistribution and dose-limiting side effects further limit the broad application of those free antibodies. Recent advances in biomaterials and nanotechnology have offered many promising opportunities for infectious and inflammatory diseases. Here, potential mechanisms of COVID-19 cytokine storm are first discussed, and relevant therapeutic strategies and ongoing clinical trials are then reviewed. Furthermore, recent research involving emerging biomaterials for improving antibody-based and broad-spectrum cytokine neutralization is summarized. It is anticipated that this work will provide insights on the development of novel therapeutics toward efficacious management of COVID-19 cytokine storm and other inflammatory diseases.

Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis.

Since the outbreak of the COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2-positive individuals demanded the use of inactivation protocols to ensure laboratory operators' safety. While not standardized, these practices can be roughly divided into two categories, namely heat inactivation and solvent-detergent treatments. These routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus-inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the analytical community, yet deep investigations in this direction have not yet been reported. We here provide insights into SARS-CoV-2 inactivation practices to be adopted prior to serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entails the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of a set of complementary analytical techniques: Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat treatment; however, it is accompanied by a marked enrichment in EVs-associated contaminants. On the other hand, solvent/detergent treatment is promising for small EVs (<150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.

Challenges in Biomaterial-Based Drug Delivery Approach for the Treatment of Neurodegenerative Diseases: Opportunities for Extracellular Vesicles.

Biomaterials have been the subject of numerous studies to pursue potential therapeutic interventions for a wide variety of disorders and diseases. The physical and chemical properties of various materials have been explored to develop natural, synthetic, or semi-synthetic materials with distinct advantages for use as drug delivery systems for the central nervous system (CNS) and non-CNS diseases. In this review, an overview of popular biomaterials as drug delivery systems for neurogenerative diseases is provided, balancing the potential and challenges associated with the CNS drug delivery. As an effective drug delivery system, desired properties of biomaterials are discussed, addressing the persistent challenges such as targeted drug delivery, stimuli responsiveness, and controlled drug release in vivo. Finally, we discuss the prospects and limitations of incorporating extracellular vesicles (EVs) as a drug delivery system and their use for biocompatible, stable, and targeted delivery with limited immunogenicity, as well as their ability to be delivered via a non-invasive approach for the treatment of neurodegenerative diseases.