Exosome-Based COVID-19 Vaccine.

Extracellular vesicles (EVs) enable cell-to-cell communication and, by delivering antigens, can stimulate the immune response strongly. Approved in use SARS-CoV-2 vaccine, candidates immunize with the viral spike protein delivered via viral vectors, translated by injected mRNAs, or as a pure protein. Here, we outline a novel methodological approach for generating SARS-CoV-2 vaccine using exosome that delivers antigens from the SARS-CoV-2 structural proteins. Engineered EVs can be loaded with viral antigens, thus acting as antigens presenting EVs, eliciting strong and targeted CD8(+) T cell and B cell, offering a unique approach to vaccine development. Engineered EVs thus portray a safe, adaptable, and effective approach for a virus-free vaccine development.

Extracellular Vesicles of COVID-19 Patients Reflect Inflammation, Thrombogenicity, and Disease Severity.

Severe COVID-19 infections present with cytokine storms, hypercoagulation, and acute respiratory distress syndrome, with extracellular vesicles (EVs) being involved in coagulation and inflammation. This study aimed to determine whether coagulation profiles and EVs reflect COVID-19 disease severity. Thirty-six patients with symptomatic COVID-19 infection with mild/moderate/severe disease (12 in each group) were analyzed. Sixteen healthy individuals served as controls. Coagulation profiles and EV characteristics were tested by nanoparticle tracking analysis (NTA), flow cytometry, and Western blot. While coagulation factors VII, V, VIII, and vWF were comparable, significant differences were found in patients' D-Dimer/fibrinogen/free protein S levels compared to controls. Severe patients' EVs displayed higher percentages of small EVs (<150 nm) with increased expression of exosome marker CD63. Severe patients' EVs displayed high levels of platelet markers (CD41) and coagulation factors (tissue factor activity, endothelial protein C receptor). EVs of patients with moderate/severe disease expressed significantly higher levels of immune cell markers (CD4/CD8/CD14) and contained higher levels of IL-6. We demonstrated that EVs, but not the coagulation profile, may serve as biomarkers for COVID-19 severity. EVs demonstrated elevated levels of immune- and vascular-related markers in patients with moderate/severe disease, and may play a role in disease pathogenesis.

Stem cell- derived extracellular vesicles as new tools in regenerative medicine - Immunomodulatory role and future perspectives.

In the last few decades, the practical use of stem cells (SCs) in the clinic has attracted significant attention in the regenerative medicine due to the ability of these cells to proliferate and differentiate into other cell types. However, recent findings have demonstrated that the therapeutic capacity of SCs may also be mediated by their ability to secrete biologically active factors, including extracellular vesicles (EVs). Such submicron circular membrane-enveloped vesicles may be released from the cell surface and harbour bioactive cargo in the form of proteins, lipids, mRNA, miRNA, and other regulatory factors. Notably, growing evidence has indicated that EVs may transfer their bioactive content into recipient cells and greatly modulate their functional fate. Thus, they have been recently envisioned as a new class of paracrine factors in cell-to-cell communication. Importantly, EVs may modulate the activity of immune system, playing an important role in the regulation of inflammation, exhibiting broad spectrum of the immunomodulatory activity that promotes the transition from pro-inflammatory to pro-regenerative environment in the site of tissue injury. Consequently, growing interest is placed on attempts to utilize EVs in clinical applications of inflammatory-related dysfunctions as potential next-generation therapeutic factors, alternative to cell-based approaches. In this review we will discuss the current knowledge on the biological properties of SC-derived EVs, with special focus on their role in the regulation of inflammatory response. We will also address recent findings on the immunomodulatory and pro-regenerative activity of EVs in several disease models, including in vitro and in vivo preclinical, as well as clinical studies. Finally, we will highlight the current perspectives and future challenges of emerging EV-based therapeutic strategies of inflammation-related diseases treatment.

Peripheral blood mononuclear cell tissue factor (F3 gene) transcript levels and circulating extracellular vesicles are elevated in severe coronavirus 2019 (COVID-19) disease.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with excessive coagulation, thrombosis, and mortality. OBJECTIVE: To provide insight into mechanisms that contribute to excessive coagulation in coronavirus 2019 (COVID-19) disease. PATIENTS/METHODS: Blood from COVID-19 patients was investigated for coagulation-related gene expression and functional activities. RESULTS: Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells from severe COVID-19 patients revealed a 5.2-fold increase in tissue factor (TF [F3 gene]) transcript expression levels (P < .05), the trigger of extrinsic coagulation; a 7.7-fold increase in C1-inhibitor (SERPING1 gene; P < .01) transcript expression levels, an inhibitor of intrinsic coagulation; and a 4.4-fold increase in anticoagulant thrombomodulin (TM [THBD gene]) transcript expression levels (P < .001). Bulk RNA-seq analysis of sorted CD14+ monocytes on an independent cohort of COVID-19 patients confirmed these findings (P < .05). Indicative of excessive coagulation, 41% of COVID-19 patients' plasma samples contained high D-dimer levels (P < .0001); of these, 19% demonstrated extracellular vesicle TF activity (P = .109). COVID-19 patients' ex vivo plasma-based thrombin generation correlated positively with D-dimer levels (P < .01). Plasma procoagulant extracellular vesicles were elevated ∼9-fold in COVID-19 patients (P < .01). Public scRNA-seq data sets from bronchoalveolar lung fluid and our peripheral blood mononuclear cell scRNA-seq data show CD14+ monocytes/macrophages TF transcript expression levels are elevated in severe but not mild or moderate COVID-19 patients. CONCLUSIONS: Beyond local lung injury, SARS-CoV-2 infection increases systemic TF (F3) transcript levels and elevates circulating extracellular vesicles that likely contribute to disease-associated coagulation, thrombosis, and related mortality.

Lipid Nanoparticles Deliver the Therapeutic VEGFA mRNA In Vitro and In Vivo and Transform Extracellular Vesicles for Their Functional Extensions.

Lipid nanoparticles (LNPs) are currently used to transport functional mRNAs, such as COVID-19 mRNA vaccines. The delivery of angiogenic molecules, such as therapeutic VEGF-A mRNA, to ischemic tissues for producing new blood vessels is an emerging strategy for the treatment of cardiovascular diseases. Here, the authors deliver VEGF-A mRNA via LNPs and study stoichiometric quantification of their uptake kinetics and how the transport of exogenous LNP-mRNAs between cells is functionally extended by cells' own vehicles called extracellular vesicles (EVs). The results show that cellular uptake of LNPs and their mRNA molecules occurs quickly, and that the translation of exogenously delivered mRNA begins immediately. Following the VEGF-A mRNA delivery to cells via LNPs, a fraction of internalized VEGF-A mRNA is secreted via EVs. The overexpressed VEGF-A mRNA is detected in EVs secreted from three different cell types. Additionally, RNA-Seq analysis reveals that as cells' response to LNP-VEGF-A mRNA treatment, several overexpressed proangiogenic transcripts are packaged into EVs. EVs are further deployed to deliver VEGF-A mRNA in vitro and in vivo. Upon equal amount of VEGF-A mRNA delivery via three EV types or LNPs in vitro, EVs from cardiac progenitor cells are the most efficient in promoting angiogenesis per amount of VEGF-A protein produced. Intravenous administration of luciferase mRNA shows that EVs could distribute translatable mRNA to different organs with the highest amounts of luciferase detected in the liver. Direct injections of VEGF-A mRNA (via EVs or LNPs) into mice heart result in locally produced VEGF-A protein without spillover to liver and circulation. In addition, EVs from cardiac progenitor cells cause minimal production of inflammatory cytokines in cardiac tissue compared with all other treatment types. Collectively, the data demonstrate that LNPs transform EVs as functional extensions to distribute therapeutic mRNA between cells, where EVs deliver this mRNA differently than LNPs.

Saliva and Saliva Extracellular Vesicles for Biomarker Candidate Identification-Assay Development and Pilot Study in Amyotrophic Lateral Sclerosis.

Saliva is gaining increasing attention as a source of biomarkers due to non-invasive and undemanding collection access. Extracellular vesicles (EVs) are nano-sized, cell-released particles that contain molecular information about their parent cells. In this study, we developed methods for saliva biomarker candidate identification using EV-isolation and proteomic evaluation. We used pooled saliva samples for assay development. EVs were isolated using membrane affinity-based methods followed by their characterization using nanoparticle tracking analysis and transmission electron microscopy. Subsequently, both saliva and saliva-EVs were successfully analyzed using proximity extension assay and label-free quantitative proteomics. Saliva-EVs had a higher purity than plasma-EVs, based on the expression of EV-proteins and albumin. The developed methods could be used for the analysis of individual saliva samples from amyotrophic lateral sclerosis (ALS) patients and controls (n = 10 each). The starting volume ranged from 2.1 to 4.9 mL and the amount of total isolated EV-proteins ranged from 5.1 to 42.6 µg. Although no proteins were significantly differentially expressed between the two groups, there was a trend for a downregulation of ZNF428 in ALS-saliva-EVs and an upregulation of IGLL1 in ALS saliva. In conclusion, we have developed a robust workflow for saliva and saliva-EV analysis and demonstrated its technical feasibility for biomarker discovery.

COVID-19 Plasma Extracellular Vesicles Increase the Density of Lipid Rafts in Human Small Airway Epithelial Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is the causative agent of the COVID-19 disease. COVID-19 viral infection can affect many cell types, including epithelial cells of the lungs and airways. Extracellular vesicles (EVs) are released by virtually all cell types, and their packaged cargo allows for intercellular communication, cell differentiation, and signal transduction. Cargo from virus-infected cells may include virally derived metabolites, miRNAs, nucleic acids, and proteins. We hypothesized that COVID-19 plasma EVs can induce the formation of signaling platforms known as lipid rafts after uptake by normal human small airway epithelial cells (SAECs). Circulating EVs from patients with or without COVID-19 were characterized by nanoparticle tracking analysis, Western blotting using specific antibodies, and transmission electron microscopy. Primary cultures of normal human small airway epithelial cells were challenged with EVs from the two patient groups, and lipid raft formation was measured by fluorescence microscopy and assessed by sucrose density gradient analysis. Collectively, our data suggest that circulating EVs from COVID-19-infected patients can induce the formation of lipid rafts in normal human small airway epithelial cells. These results suggest the need for future studies aimed at investigating whether the increased density of lipid rafts in these cells promotes viral entry and alteration of specific signaling pathways in the recipient cells.

Extracellular vesicle proteomics and phosphoproteomics identify pathways for increased risk in patients hospitalized with COVID-19 and type 2 diabetes mellitus.

Recent studies suggest that extracellular vesicles (EVs) play a role in the pathogenesis of SARS-CoV-2 infection and the severity of COVID-19. However, their role in the interaction between COVID-19 and type 2 diabetes (T2D) has not been addressed. Here, we characterized the circulating EV proteomic and phosphoproteomic landscape in patients with and without T2D hospitalized with COVID-19 or non-COVID-19 acute respiratory illness (RSP). We detected differentially expressed protein and phosphoprotein signatures that effectively characterized the study groups. The trio of immunomodulatory and coagulation proteins C1QA, C1QB, and C1QC appeared to be a central cluster in both the COVID-19 and T2D functional networks. PKCß appeared to be retained in cells by being diverted from EV pathways and contribute to the COVID-19 and T2D interaction via a PKC/BTK/TEC axis. EV-shuttled CASP3 and ROCK1 appeared to be coregulated and likely contribute to disease interactions in patients with COVID-19 and T2D. Predicted activation of AMPK, MAPK, and SYK appeared to also play important roles driving disease interaction. These results suggest that activated cellular kinases (i.e., PKC, AMPK, MAPK, and SYK) and multiple EV-shuttled kinases (i.e., PKCß, BTK, TEC, MAP2K2, and ROCK1) may play key roles in severe COVID-19, particularly in patients with comorbid diabetes.

Mesenchymal Stem Cell-Derived Extracellular Vesicles for Therapeutic Use and in Bioengineering Applications.

Extracellular vesicles (EVs) are small lipid bilayer-delimited particles that are naturally released from cells into body fluids, and therefore can travel and convey regulatory functions in the distal parts of the body. EVs can transmit paracrine signaling by carrying over cytokines, chemokines, growth factors, interleukins (ILs), transcription factors, and nucleic acids such as DNA, mRNAs, microRNAs, piRNAs, lncRNAs, sn/snoRNAs, mtRNAs and circRNAs; these EVs travel to predecided destinations to perform their functions. While mesenchymal stem cells (MSCs) have been shown to improve healing and facilitate treatments of various diseases, the allogenic use of these cells is often accompanied by serious adverse effects after transplantation. MSC-produced EVs are less immunogenic and can serve as an alternative to cellular therapies by transmitting signaling or delivering biomaterials to diseased areas of the body. This review article is focused on understanding the properties of EVs derived from different types of MSCs and MSC-EV-based therapeutic options. The potential of modern technologies such as 3D bioprinting to advance EV-based therapies is also discussed.

Anticarcinogenic impact of extracellular vesicles (exosomes) from cord blood stem cells in malignant melanoma: A potential biological treatment.

Incidence of Malignant Melanoma has become the 5th in the UK. To date, the major anticancer therapeutics include cell therapy, immunotherapy, gene therapy and nanotechnology-based strategies. Recently, extracellular vesicles, especially exosomes, have been highlighted for their therapeutic benefits in numerous chronic diseases. Exosomes display multifunctional properties, including inhibition of cancer cell proliferation and initiation of apoptosis. In the present in vitro study, the antitumour effect of cord blood stem cell (CBSC)-derived exosomes was confirmed by the CCK-8 assay (p < 0.05) on CHL-1 melanoma cells and improve the repair mechanism on lymphocytes from melanoma patients. Importantly, no significant effect was observed in healthy lymphocytes when treated with the exosome concentrations at 24, 48 and 72 h. Comet assay results (OTM and %Tail DNA) demonstrated that the optimal exosome concentration showed a significant impact (p < 0.05) in lymphocytes from melanoma patients whilst causing no significant DNA damage in lymphocytes of healthy volunteers was 300 µg/ml. Similarly, the Comet assay results depicted significant DNA damage in a melanoma cell line (CHL-1 cells) treated with CBSC-derived exosomes, both the cytotoxicity of CHL-1 cells treated with CBSC-derived exosomes exhibited a significant time-dependent decrease in cell survival. Sequencing analysis of CBSC exosomes showed the presence of the let-7 family of miRNAs, including let-7a-5p, let-7b-5p, let-7c-5p, let-7d-3p, let-7d-5p and two novel miRNAs. The potency of CBSC exosomes in inhibiting cancer progression in lymphocytes from melanoma patients and CHL-1 cells whilst causing no harm to the healthy lymphocytes makes it a potential candidate as an anticancer therapy.

Coagulation potential and the integrated omics of extracellular vesicles from COVID-19 positive patient plasma.

Extracellular vesicles (EVs) participate in cell-to-cell communication and contribute toward homeostasis under physiological conditions. But EVs can also contribute toward a wide array of pathophysiology like cancer, sepsis, sickle cell disease, and thrombotic disorders. COVID-19 infected patients are at an increased risk of aberrant coagulation, consistent with elevated circulating levels of ultra-high molecular weight VWF multimers, D-dimer and procoagulant EVs. The role of EVs in COVID-19 related hemostasis may depend on cells of origin, vesicular cargo and size, however this is not well defined. We hypothesized that the procoagulant potential of EV isolates from COVID-19 (+) patient plasmas could be defined by thrombin generation assays. Here we isolated small EVs (SEVs) and large EVs (LEVs) from hospitalized COVID-19 (+) patient (n = 21) and healthy donor (n = 20) plasmas. EVs were characterized by flow cytometry, Transmission electron microscopy, nanoparticle tracking analysis, plasma thrombin generation and a multi-omics approach to define coagulation potential. These data were consistent with differences in EV metabolite, lipid, and protein content when compared to healthy donor plasma isolated SEVs and LEVs. Taken together, the effect of EVs on plasma procoagulant potential as defined by thrombin generation and supported by multi-omics is enhanced in COVID-19. Further, we observe that this effect is driven both by EV size and phosphatidyl serine.

Circulating tissue factor-positive extracellular vesicles and their association with thrombosis in different diseases.

Tissue factor (TF) is a procoagulant protein released from activated host cells, such as monocytes, and tumor cells on extracellular vesicles (EVs). TF + EVs are observed in the circulation of patients with various types of diseases. In this review, we will summarize the association between TF + EVs and activation of coagulation and survival in different types of diseases, including cancer, sepsis, and infections with different viruses, such as human immunodeficiency virus (HIV), influenza A virus (IAV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We will also discuss the source of TF + EVs in various diseases. EVTF activity is associated with thrombosis in pancreatic cancer patients and coronavirus disease 2019 patients (COVID-19) and with disseminated intravascular coagulation in cancer patients. EVTF activity is also associated with worse survival in patients with cancer and COVID-19. Monocytes are the major sources of TF + EVs in sepsis, and viral infections, such as HIV, Ebola virus, and SARS-CoV-2. In contrast, alveolar epithelial cells are the major source of TF + EVs in bronchoalveolar lavage fluid in COVID-19 and influenza A patients. These studies indicate that EVTF activity could be used as a biomarker to identify patients that have an increased risk of coagulopathy and mortality.

Proviral role of human respiratory epithelial cell-derived small extracellular vesicles in SARS-CoV-2 infection.

Small Extracellular Vesicles (sEVs) are 50-200 nm in diameter vesicles delimited by a lipid bilayer, formed within the endosomal network or derived from the plasma membrane. They are secreted in various biological fluids, including airway nasal mucus. The goal of this work was to understand the role of sEVs present in the mucus (mu-sEVs) produced by human nasal epithelial cells (HNECs) in SARS-CoV-2 infection. We show that uninfected HNECs produce mu-sEVs containing SARS-CoV-2 receptor ACE2 and activated protease TMPRSS2. mu-sEVs cleave prefusion viral Spike proteins at the S1/S2 boundary, resulting in higher proportions of prefusion S proteins exposing their receptor binding domain in an 'open' conformation, thereby facilitating receptor binding at the cell surface. We show that the role of nasal mu-sEVs is to complete prefusion Spike priming performed by intracellular furin during viral egress from infected cells. This effect is mediated by vesicular TMPRSS2 activity, rendering SARS-CoV-2 virions prone to entry into target cells using the 'early', TMPRSS2-dependent pathway instead of the 'late', cathepsin-dependent route. These results indicate that prefusion Spike priming by mu-sEVs in the nasal cavity plays a role in viral tropism. They also show that nasal mucus does not protect from SARS-CoV-2 infection, but instead facilitates it.

Role of Mesenchymal Stem Cells and Extracellular Vesicles in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial fibrotic disease that leads to disability and death within 5 years of diagnosis. Pulmonary fibrosis is a disease with a multifactorial etiology. The concept of aberrant regeneration of the pulmonary epithelium reveals the pathogenesis of IPF, according to which repeated damage and death of alveolar epithelial cells is the main mechanism leading to the development of progressive IPF. Cell death provokes the migration, proliferation and activation of fibroblasts, which overproduce extracellular matrix, resulting in fibrotic deformity of the lung tissue. Mesenchymal stem cells (MSCs) and extracellular vesicles (EVs) are promising therapies for pulmonary fibrosis. MSCs, and EVs derived from MSCs, modulate the activity of immune cells, inhibit the expression of profibrotic genes, reduce collagen deposition and promote the repair of damaged lung tissue. This review considers the molecular mechanisms of the development of IPF and the multifaceted role of MSCs in the therapy of IPF. Currently, EVs-MSCs are regarded as a promising cell-free therapy tool, so in this review we discuss the results available to date of the use of EVs-MSCs for lung tissue repair.

Hepatic, Extrahepatic and Extracellular Vesicle Cytochrome P450 2E1 in Alcohol and Acetaminophen-Mediated Adverse Interactions and Potential Treatment Options.

Alcohol and several therapeutic drugs, including acetaminophen, are metabolized by cytochrome P450 2E1 (CYP2E1) into toxic compounds. At low levels, these compounds are not detrimental, but higher sustained levels of these compounds can lead to life-long problems such as cytotoxicity, organ damage, and cancer. Furthermore, CYP2E1 can facilitate or enhance the effects of alcohol-drug and drug-drug interactions. In this review, we discuss the role of CYP2E1 in the metabolism of alcohol and drugs (with emphasis on acetaminophen), mediating injury/toxicities, and drug-drug/alcohol-drug interactions. Next, we discuss various compounds and various nutraceuticals that can reduce or prevent alcohol/drug-induced toxicity. Additionally, we highlight experimental outcomes of alcohol/drug-induced toxicity and potential treatment strategies. Finally, we cover the role and implications of extracellular vesicles (EVs) containing CYP2E1 in hepatic and extrahepatic cells and provide perspectives on the clinical relevance of EVs containing CYP2E1 in intracellular and intercellular communications leading to drug-drug and alcohol-drug interactions. Furthermore, we provide our perspectives on CYP2E1 as a druggable target using nutraceuticals and the use of EVs for targeted drug delivery in extrahepatic and hepatic cells, especially to treat cellular toxicity.

Interrelation between extracellular vesicles miRNAs with chronic lung diseases.

Extracellular vehicles (EVs) are nanoscale lipid bilayer vesicles that carry biologically active biomolecule cargos like proteins, lipids, and nucleic acids (DNA, RNA) outside of the cell. Blood (serum/plasma), urine, and bronchoalveolar lavage fluid are all examples of biofluids from which they may be collected. EVs play a vital role in intracellular communication. The molecular signature of EVs largely depends on the parental cell's status. EVs are classified into two groups, (1) exosomes (originated by endogenous route) and (2) microvesicles (originated from the plasma membrane, also known as ectosomes). The quantity and types of EV cargo vary during normal conditions compared to pathological conditions (chronic inflammatory lung diseases or lung cancer). Consequently, EVs contain novel biomarkers that differ based on the cell type of origin and during lung diseases. Small RNAs (e.g., microRNAs) are transported by EVs, which is one of the most rapidly evolving research areas in the field of EVs biology. EV-mediated cargos transport small RNAs that can result in reprograming the target/recipient cells. Multiple chronic inflammatory lung illnesses, such as chronic obstructive pulmonary disease, asthma, pulmonary hypertension, pulmonary fibrosis, cystic fibrosis, acute lung injury, and lung cancer, have been demonstrated to be regulated by EV. In this review, we will consolidate the current knowledge and literature on the novel role of EVs and their small RNAs concerning chronic lung diseases (CLDs). Additionally, we will also provide better insight into the clinical and translational impact of mesenchymal stem cells-derived EVs as novel therapeutic agents in treating CLDs.

Association of extracellular vesicle inflammatory proteins and mortality.

Even before the COVID-19 pandemic declines in life expectancy in the United States were attributed to increased mortality rates in midlife adults across racial and ethnic groups, indicating a need for markers to identify individuals at risk for early mortality. Extracellular vesicles (EVs) are small, lipid-bound vesicles capable of shuttling functional proteins, nucleic acids, and lipids. Given their role as intercellular communicators and potential biomarkers of disease, we explored whether circulating EVs may be markers of mortality in a prospective, racially, and socioeconomically diverse middle-aged cohort. We isolated plasma EVs from 76 individuals (mean age = 59.6 years) who died within a 5 year period and 76 surviving individuals matched by age, race, and poverty status. There were no significant differences in EV concentration, size, or EV-associated mitochondrial DNA levels associated with mortality. We found that several EV-associated inflammatory proteins including CCL23, CSF-1, CXCL9, GDNF, MCP-1, STAMBP, and 4E-BP1 were significantly associated with mortality. IL-10RB and CDCP1 were more likely to be present in plasma EVs from deceased individuals than in their alive counterparts. We also report differences in EV-associated inflammatory proteins with poverty status, race, and sex. Our results suggest that plasma EV-associated inflammatory proteins are promising potential clinical biomarkers of mortality.

The Role of Extracellular Vesicles in COVID-19 Pathology.

Extracellular vesicles (EVs) have become a trending topic in recent years; they constitute a new intercellular communication paradigm. Extracellular vesicles are 30-4000 nanometers in diameter particles that are limited by a phospholipid bilayer and contain functional biomolecules, such as proteins, lipids, and nucleic acids. They are released by virtually all types of eukaryotic cells; through their cargoes, EVs are capable of triggering signaling in recipient cells. In addition to their functions in the homeostatic state, EVs have gained attention because of their roles in pathological contexts, eventually contributing to disease progression. In the Coronavirus disease 2019 (COVID-19) pandemic, aside from the scientific race for the development of preventive and therapeutic interventions, it is critical to understand the pathological mechanisms involved in SARS-CoV-2 infection. In this sense, EVs are key players in the main processes of COVID-19. Thus, in this review, we highlight the role of EVs in the establishment of the viral infection and in the procoagulant state, cytokine storm, and immunoregulation of innate and adaptive immune responses.

Proteomic Profile of Procoagulant Extracellular Vesicles Reflects Complement System Activation and Platelet Hyperreactivity of Patients with Severe COVID-19.

Background: Extracellular vesicles (EVs) are a valuable source of biomarkers and display the pathophysiological status of various diseases. In COVID-19, EVs have been explored in several studies for their ability to reflect molecular changes caused by SARS-CoV-2. Here we provide insights into the roles of EVs in pathological processes associated with the progression and severity of COVID-19. Methods: In this study, we used a label-free shotgun proteomic approach to identify and quantify alterations in EV protein abundance in severe COVID-19 patients. We isolated plasma extracellular vesicles from healthy donors and patients with severe COVID-19 by size exclusion chromatography (SEC). Then, flow cytometry was performed to assess the origin of EVs and to investigate the presence of circulating procoagulant EVs in COVID-19 patients. A total protein extraction was performed, and samples were analyzed by nLC-MS/MS in a Q-Exactive HF-X. Finally, computational analysis was applied to signify biological processes related to disease pathogenesis. Results: We report significant changes in the proteome of EVs from patients with severe COVID-19. Flow cytometry experiments indicated an increase in total circulating EVs and with tissue factor (TF) dependent procoagulant activity. Differentially expressed proteins in the disease groups were associated with complement and coagulation cascades, platelet degranulation, and acute inflammatory response. Conclusions: The proteomic data reinforce the changes in the proteome of extracellular vesicles from patients infected with SARS-CoV-2 and suggest a role for EVs in severe COVID-19.

Genetically Engineered MRI-Trackable Extracellular Vesicles as SARS-CoV-2 Mimetics for Mapping ACE2 Binding In Vivo.

The elucidation of viral-receptor interactions and an understanding of virus-spreading mechanisms are of great importance, particularly in the era of a pandemic. Indeed, advances in computational chemistry, synthetic biology, and protein engineering have allowed precise prediction and characterization of such interactions. Nevertheless, the hazards of the infectiousness of viruses, their rapid mutagenesis, and the need to study viral-receptor interactions in a complex in vivo setup call for further developments. Here, we show the development of biocompatible genetically engineered extracellular vesicles (EVs) that display the receptor binding domain (RBD) of SARS-CoV-2 on their surface as coronavirus mimetics (EVsRBD). Loading EVsRBD with iron oxide nanoparticles makes them MRI-visible and, thus, allows mapping of the binding of RBD to ACE2 receptors noninvasively in live subjects. Moreover, we show that EVsRBD can be modified to display mutants of the RBD of SARS-CoV-2, allowing rapid screening of currently raised or predicted variants of the virus. The proposed platform thus shows relevance and cruciality in the examination of quickly evolving pathogenic viruses in an adjustable, fast, and safe manner. Relying on MRI for visualization, the presented approach could be considered in the future to map ligand-receptor binding events in deep tissues, which are not accessible to luminescence-based imaging.