Circulating Extracellular Vesicle Levels in Patients with Coronavirus Disease 2019 Coagulopathy: A Prospective Cohort Study.

Coronavirus disease 2019 (COVID-19) is associated with coagulopathy. However, the underlying mechanisms are not completely understood. We evaluated the association between COVID-19 coagulopathy and extracellular vesicle (EV) levels. We hypothesized that several EV levels would be higher in COVID-19 coagulopathy patients than in non-coagulopathy patients. This prospective observational study was conducted in four tertiary care faculties in Japan. We enrolled 99 COVID-19 patients (48 with coagulopathy and 51 without coagulopathy) aged ≥20 years who required hospitalization, and 10 healthy volunteers; we divided the patients into coagulopathy and non-coagulopathy groups according to the D-dimer levels (≥1 µg/mL and <1 µg/mL, respectively). We used flow cytometry to measure the tissue-factor-bearing, endothelium-derived, platelet-derived, monocyte-derived, and neutrophil-derived EV levels in platelet-free plasma. The EV levels were compared between the two COVID-19 groups as well as among the coagulopathy patients, non-coagulopathy patients, and healthy volunteers. No significant difference was found in EV levels between the two groups. Meanwhile, the cluster of differentiation (CD) 41 + EV levels were significantly higher in COVID-19 coagulopathy patients than in healthy volunteers (549.90 [255.05-984.65] vs. 184.3 [150.1-254.1] counts/µL, p = 0.011). Therefore, CD41+ EVs might play an essential role in COVID-19 coagulopathy development.

GlycoEVLR: Glycosylated extracellular vesicle-like receptors for targeting and sensing viral antigen

Biosensors are rapid and portable detection devices with great potential for the instant screening of infectious diseases. Receptors are the critical element of biosensors. They determine the specificity, sensitivity and stability. However, current receptors are mainly limited to antibodies and aptamers. Herein, we developed a glycosylated extracellular vesicle-like receptor (GlycoEVLR) for the rapid detection of virus antigens, specifically using SARS-CoV-2 as a model. The human angiotensin-converting enzyme 2 (ACE2)-overexpressed and heparin-functionalized HEK-293T cell membrane-cloaked Fe3O4 nanoparticles (NPs) were prepared as functionalizing GlycoEVLR. They were characterized as spherical core–shell structures with a diameter of around 100 nm, which were perfectly comparable to natural extracellular vesicles. Binding affinities between GlycoEVLR and spike1 (S1) antigen were demonstrated using surface plasmon resonance (SPR). The GlycoEVLR was fixed on magnetic electrodes to construct electrochemical biosensors. Using electrochemical impedance spectroscopy (EIS) as a measurement technique, the S1 antigen was detected down to 1 pg/mL within 20 min and showed a good linearity range from 1 pg/mL to 1 ng/mL. Also, the GlycoEVLR-based electrochemical biosensors showed excellent antifouling performance and stability. Overall, our work provides a useful methodology for developing extracellular vesicle-like receptors for biosensors. Combining the inherit natural receptor proteins and antifouling lipids from the host cells with engineered glycan motifs to target and sense viral antigens will open a newavenue for biosensors.

Investigational Use of Mesenchymal Stem/Stromal Cells and Their Secretome as Add-On Therapy in Severe Respiratory Virus Infections: Challenges and Perspectives.

Serious manifestations of respiratory virus infections such as influenza and coronavirus disease 2019 (COVID-19) are associated with a dysregulated immune response and systemic inflammation. Treating the immunological/inflammatory dysfunction with glucocorticoids, Janus kinase inhibitors, and monoclonal antibodies against the interleukin-6 receptor has significantly reduced the risk of respiratory failure and death in hospitalized patients with severe COVID-19, but the proportion of those requiring invasive mechanical ventilation (IMV) and dying because of respiratory failure remains elevated. Treatment of severe influenza-associated pneumonia and acute respiratory distress syndrome (ARDS) with available immunomodulators and anti-inflammatory compounds is still not recommended. New therapies are therefore needed to reduce the use of IMV and the risk of death in hospitalized patients with rapidly increasing oxygen demand and systemic inflammation who do not respond to the current standard of care. This paper provides a critical assessment of the published clinical trials that have tested the investigational use of intravenously administered allogeneic mesenchymal stem/stromal cells (MSCs) and MSC-derived secretome with putative immunomodulatory/antiinflammatory/regenerative properties as add-on therapy to improve the outcome of these patients. Increased survival rates are reported in 5 of 12 placebo-controlled or open-label comparative trials involving patients with severe and critical COVID-19 and in the only study concerning patients with influenza-associated ARDS. Results are encouraging but inconclusive for the following reasons: small number of patients tested in each trial; differences in concomitant treatments and respiratory support; imbalances between study arms; differences in MSC source, MSC-derived product, dosing and starting time of the investigational therapy; insufficient/inappropriate reporting of clinical data. Solutions are proposed for improving the clinical development plan, with the aim of facilitating regulatory approval of the MSC-based investigational therapy for life-threatening respiratory virus infections in the future. Major issues are the absence of a biomarker predicting responsiveness to MSCs and MSC-derived secretome and the lack of pharmacoeconomic evaluations.

Antigen Delivery Systems: Past, Present, and Future.

The COVID-19 pandemic has increased demand for safe and effective vaccines. Research to develop vaccines against diseases including Middle East respiratory syndrome, Ebolavirus, human immunodeficiency virus, and various cancers would also contribute to global well-being. For successful vaccine development, the advancement of technologies such as antigen (Ag) screening, Ag delivery systems and adjuvants, and manufacturing processes is essential. Ag delivery systems are required not only to deliver a sufficient amount of Ag for vaccination, but also to enhance immune response. In addition, Ag types and their delivery systems determine the manufacturing processes of the vaccine product. Here, we analyze the characteristics of various Ag delivery systems: plasmids, viral vectors, bacterial vectors, nanoparticles, self-assembled particles, natural and artificial cells, and extracellular vesicles. This review provides insight into the current vaccine landscape and highlights promising avenues of research for the development and improvement of Ag delivery systems.

Extracellular Vesicles and Their Membranes: Exosomes vs. Virus-Related Particles.

Cells produce nanosized lipid membrane-enclosed vesicles which play important roles in intercellular communication. Interestingly, a certain type of extracellular vesicle, termed exosomes, share physical, chemical, and biological properties with enveloped virus particles. To date, most similarities have been discovered with lentiviral particles, however, other virus species also frequently interact with exosomes. In this review, we will take a closer look at the similarities and differences between exosomes and enveloped viral particles, with a focus on events taking place at the vesicle or virus membrane. Since these structures present an area with an opportunity for interaction with target cells, this is relevant for basic biology as well as any potential research or medical applications.

Extracellular vesicles in vaccine development and therapeutic approaches for viral diseases

Extracellular vesicles (EVs) are lipid bilayer nanovesicles generated by almost all living cells which possess various size ranges depending on producer cells and biogenesis mechanisms. Several EV markers were determined including tetraspanins (e.g., CD9, CD63 and CD81), heat shock proteins (HSP70 and HSP90), some 14–3–3 proteins (a family of conserved regulatory molecules), major histocompatibility complex molecules (MHC-I/-II), and enzymes (Glyceraldehyde 3-phosphate dehydrogenase and enolase-1). EVs are known as an abundant source of antigens and immune molecules that can be used for vaccine development in human and animals. EV-based immunization could significantly activate immune responses in different infections such as Porcine reproductive and respiratory syndrome virus (PRRSV), Lymphocytic choriomeningitis virus (LCMV), Marek's disease virus (MDV), and SARS-CoV-2 infections. The engineered and modified EVs showed a promising potential in development of anti-tumor vaccines and therapeutics, protection against parasitic diseases (e.g., Eimeria, and Plasmodium yoelii) and viral diseases (e.g., COVID-19), and improvement of biomarkers. Also, EVs possess a crucial role in antigen presentation in vivo. In this review, we describe the roles of EVs in vaccine development and therapeutic approaches for viral diseases. © 2023 Elsevier Ltd

Erythrocyte-Derived microRNAs: Emerging Players in Cardiovascular and Metabolic Disease.

Recent studies have demonstrated a novel function of red blood cells (RBCs) beyond their classical role as gas transporters, that is, RBCs undergo functional alterations in cardiovascular and metabolic disease, and RBC dysfunction is associated with hypertension and the development of cardiovascular injury in type 2 diabetes, heart failure, preeclampsia, familial hypercholesterolemia/dyslipidemia, and COVID-19. The underlying mechanisms include decreased nitric oxide bioavailability, increased arginase activity, and reactive oxygen species formation. Of interest, RBCs contain diverse and abundant micro (mi)RNAs. miRNA expression pattern in RBCs reflects the expression in the whole blood, serum, and plasma. miRNA levels in RBCs have been found to be altered in various cardiovascular and metabolic diseases, which contributes to the development of cardiovascular complications. Evidence has shown that RBC-derived miRNAs interact with the cardiovascular system via extracellular vesicles and argonaute RISC catalytic component 2 as carriers. Alteration of RBC-to-vascular communication via miRNAs may serve as potential disease mechanism for vascular complications. The present review summarizes RBCs and their released miRNAs as potential mediators of cardiovascular injury. We further focus on the possible mechanisms by which RBC-derived miRNAs regulate cardiovascular function. A better understanding of the function of RBC-derived miRNAs will increase insights into the disease mechanism and potential targets for the treatment of cardiovascular complications.

Plant-Derived Extracellular Vesicles as a Delivery Platform for RNA-Based Vaccine: Feasibility Study of an Oral and Intranasal SARS-CoV-2 Vaccine.

Plant-derived extracellular vesicles (EVs) may represent a platform for the delivery of RNA-based vaccines, exploiting their natural membrane envelope to protect and deliver nucleic acids. Here, EVs extracted from orange (Citrus sinensis) juice (oEVs) were investigated as carriers for oral and intranasal SARS-CoV-2 mRNA vaccine. oEVs were efficiently loaded with different mRNA molecules (coding N, subunit 1 and full S proteins) and the mRNA was protected from degrading stress (including RNase and simulated gastric fluid), delivered to target cells and translated into protein. APC cells stimulated with oEVs loaded with mRNAs induced T lymphocyte activation in vitro. The immunization of mice with oEVs loaded with S1 mRNA via different routes of administration including intramuscular, oral and intranasal stimulated a humoral immune response with production of specific IgM and IgG blocking antibodies and a T cell immune response, as suggested by IFN-γ production by spleen lymphocytes stimulated with S peptide. Oral and intranasal administration also triggered the production of specific IgA, the mucosal barrier in the adaptive immune response. In conclusion, plant-derived EVs represent a useful platform for mRNA-based vaccines administered not only parentally but also orally and intranasally.

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.

Protective role of engineered extracellular vesicles loaded quercetin nanoparticles as anti-viral therapy against SARS-CoV-2 infection: A prospective review.

Quercetin (QCT) is a naturally occurring phenolic flavonoid compound with inbuilt characteristics of antioxidant, anti-inflammatory, and immune protection. Several recent studies have shown that QCT and QCTits nanoparticles have therapeutic potential against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Novel therapeutics also include the implication of extracellular vesicles (EVs) to protect from SARS-CoV-2 viral infection. This article highlighted the therapeutic/prophylactic potential of engineered EVs loaded with QCT against SARS-CoV-2 infection. Several biotechnological engineering approaches are available to deliver EVs loaded with QCT nanoparticles. Among these biotechnological advances, a specific approach with significantly higher efficiency and yield has to be opted to fabricate such drug delivery of nano molecules, especially to combat SARS-CoV-2 infection. The current treatment regime protects the human body from virus infection but has some limitations including drugs and long-term steroid side effects. However, the vaccine strategy is somehow effective in inhibiting the spread of coronavirus disease-19 (COVID-19) infection. Moreover, the proposed exosomal therapy met the current need to repair the damaged tissue along with inhibition of COVID-19-associated complications at the tissue level. These scientific findings expand the possibilities and predictability of developing a novel and cost-effective therapeutic approach that combines the dual molecule, EVs and QCT nanoparticles, to treat SARS-CoV-2 infection. Therefore, the most suitable engineering method to fabricate such a drug delivery system should be better understood before developing novel therapeutics for clinical purposes.

SARS-CoV-2 Spike Protein-Activated Dendritic Cell-Derived Extracellular Vesicles Induce Antiviral Immunity in Mice.

The onset and spread of the SARS-CoV-2 virus have created an unprecedented universal crisis. Although vaccines have been developed against the parental SARS-CoV-2, outbreaks of the disease still occur through the appearance of different variants, suggesting a continuous need for improved and effective therapeutic strategies. Therefore, we developed a novel nanovesicle presenting Spike protein on the surface of the dendritic cell-derived extracellular vesicles (DEVs) for use as a potential vaccine platform against SARS-CoV-2. DEVs express peptide/MHC-I (pMHC-I) complexes, CCR-7, on their surface. The immunogenicity and efficacy of the Spike-activated DEVs were tested in mice and compared with free Spike protein. A 1/10 Spike equivalent dose of DEVs showed a superior potency in inducing anti-Spike IgG titers in blood of mice when compared to dendritic cells or free Spike protein treatment. Moreover, DEV-induced sera effectively reduced viral infection by 55-60% within 15 days of booster dose administration. Furthermore, a 1/10 Spike equivalent dose of DEV-treated mice was found to be equally effective in inducing CD19+CD38+ T-cells in the spleen and lymph node; CD8 cells in the bone marrow, spleen, and lymph node; and CD4+CD25+ T-cells in the spleen and lymph node after 90 days of treatment. Thus, our results support the immunogenic nature of DEVs, demonstrating that a low dose of DEVs induces antibodies to inhibit SARS-CoV-2 infection in vitro, therefore warranting further investigations.

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.

Type 2 alveolar epithelial cell-derived circulating extracellular vesicle-encapsulated surfactant protein C as a mediator of cardiac inflammation in COVID-19.

Among the countless endeavours made at elucidating the pathogenesis of COVID-19, those aimed at the histopathological alterations of type 2 alveolar epithelial cells (AT2) are of outstanding relevance to the field of lung physiology, as they are the building blocks of the pulmonary alveoli. A merit of high regenerative and proliferative capacity, exocytotic activity resulting in the release of extracellular vesicles (EVs) is particularly high in AT2 cells, especially in those infected with SARS-CoV-2. These AT2 cell-derived EVs, containing the genetic material of the virus, might enter the bloodstream and make their way into the cardiovascular system, where they may infect cardiomyocytes and bring about a series of events leading to heart failure. As surfactant protein C, a marker of AT2 cell activity and a constituent of the lung surfactant complex, occurs abundantly inside the AT2-derived EVs released during the inflammatory stage of COVID-19, it could potentially be used as a biomarker for predicting impending heart failure in those patients with a history of cardiovascular disease.

Peritoneal M2 macrophage-derived extracellular vesicles as natural multitarget nanotherapeutics to attenuate cytokine storms after severe infections.

Cytokine storms are a primary cause of multiple organ damage and death after severe infections, such as SARS-CoV-2. However, current single cytokine-targeted strategies display limited therapeutic efficacy. Here, we report that peritoneal M2 macrophage-derived extracellular vesicles (M2-EVs) are multitarget nanotherapeutics that can be used to resolve cytokine storms. In detail, primary peritoneal M2 macrophages exhibited superior anti-inflammatory potential than immobilized cell lines. Systemically administered M2-EVs entered major organs and were taken up by phagocytes (e.g., macrophages). M2-EV treatment effectively reduced excessive cytokine (e.g., TNF-α and IL-6) release in vitro and in vivo, thereby attenuating oxidative stress and multiple organ (lung, liver, spleen and kidney) damage in endotoxin-induced cytokine storms. Moreover, M2-EVs simultaneously inhibited multiple key proinflammatory pathways (e.g., NF-κB, JAK-STAT and p38 MAPK) by regulating complex miRNA-gene and gene-gene networks, and this effect was collectively mediated by many functional cargos (miRNAs and proteins) in EVs. In addition to the direct anti-inflammatory role, human peritoneal M2-EVs expressed angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2 spike protein, and thus could serve as nanodecoys to prevent SARS-CoV-2 pseudovirus infection in vitro. As cell-derived nanomaterials, the therapeutic index of M2-EVs can be further improved by genetic/chemical modification or loading with specific drugs. This study highlights that peritoneal M2-EVs are promising multifunctional nanotherapeutics to attenuate infectious disease-related cytokine storms.

Pivoting Novel Exosome-Based Technologies for the Detection of SARS-CoV-2.

The National Institutes of Health (NIH) launched the Rapid Acceleration of Diagnostics (RADx) initiative to meet the needs for COVID-19 diagnostic and surveillance testing, and to speed its innovation in the development, commercialization, and implementation of new technologies and approaches. The RADx Radical (RADx-Rad) initiative is one component of the NIH RADx program which focuses on the development of new or non-traditional applications of existing approaches, to enhance their usability, accessibility, and/or accuracy for the detection of SARS-CoV-2. Exosomes are a subpopulation of extracellular vesicles (EVs) 30-140 nm in size, that are critical in cell-to-cell communication. The SARS-CoV-2 virus has similar physical and molecular properties as exosomes. Therefore, the novel tools and technologies that are currently in development for the isolation and detection of exosomes, may prove to be invaluable in screening for SARS-CoV-2 viral infection. Here, we describe how novel exosome-based technologies are being pivoted for the detection of SARS-CoV-2 and/or the diagnosis of COVID-19. Considerations for these technologies as they move toward clinical validation and commercially viable diagnostics is discussed along with their future potential. Ultimately, the technologies in development under the NIH RADx-Rad exosome-based non-traditional technologies toward multi-parametric and integrated approaches for SARS-CoV-2 program represent a significant advancement in diagnostic technology, and, due to a broad focus on the biophysical and biochemical properties of nanoparticles, the technologies have the potential to be further pivoted as tools for future infectious agents.

Evaluation of a new bead-based assay to measure levels of human tissue factor antigen in extracellular vesicles in plasma.

Background: Circulating tissue factor (TF)-expressing extracellular vesicles (EVs) are associated with thrombosis in several diseases, such as coronavirus disease 2019 (COVID-19). Activity assays have higher sensitivity and specificity compared to antigen assays for measuring TF+ EVs in plasma. The MACSPlex Exosome Kit is designed to detect 37 exosomal surface epitopes, including TF, on EVs in plasma using various fluorescently labeled beads. The different EV-bead complexes are detected by flow cytometry. A recent study used the MACSPlex Exosome Kit to measure levels of TF+ EVs in serum from patients with COVID-19. Objectives: To evaluate the ability of the MACSPlex Exosome Kit to detect TF on EVs in plasma. Methods: We measured levels of TF+ EVs isolated from plasma with or without TF detected using our in-house EVTF activity assay and the MACSPlex Exosome Kit. Results: The MACSPlex Exosome Kit gave a very low TF antigen signal (TF bead signal) compared to platelet-derived CD41b+ EVs, which was used as a control. Lipopolysaccharide (LPS) increased levels of EVTF activity but not TF bead signal in four donors. Inhibition of TF reduced levels of EVTF activity but did not affect the TF bead signal in EVs isolated from plasma from LPS-treated blood. Finally, we found no correlation between levels of EVTF activity and TF bead signal in EVs isolated from plasma from ovarian cancer patients (r = .16, P = .62). Conclusion: Our data suggest that the MACSPlex Exosome Kit gives a nonspecific signal for TF and does not have the sensitivity to detect TF+ EVs in plasma.

A Defective Viral Particle Approach to COVID-19.

The novel coronavirus SARS-CoV-2 has caused a pandemic resulting in millions of deaths worldwide. While multiple vaccines have been developed, insufficient vaccination combined with adaptive mutations create uncertainty for the future. Here, we discuss novel strategies to control COVID-19 relying on Defective Interfering Particles (DIPs) and related particles that arise naturally during an infection. Our intention is to encourage and to provide the basis for the implementation of such strategies by multi-disciplinary teams. We therefore provide an overview of SARS-CoV-2 for a multi-disciplinary readership that is specifically tailored to these strategies, we identify potential targets based on the current knowledge of the properties and functions of coronaviruses, and we propose specific strategies to engineer DIPs and other interfering or therapeutic nanoparticles.

Mesenchymal Stem Cell-Derived Extracellular Vesicles to the Rescue of Renal Injury.

Acute kidney injury (AKI) and chronic kidney disease (CKD) are rising in global prevalence and cause significant morbidity for patients. Current treatments are limited to slowing instead of stabilising or reversing disease progression. In this review, we describe mesenchymal stem cells (MSCs) and their constituents, extracellular vesicles (EVs) as being a novel therapeutic for CKD. MSC-derived EVs (MSC-EVs) are membrane-enclosed particles, including exosomes, which carry genetic information that mimics the phenotype of their cell of origin. MSC-EVs deliver their cargo of mRNA, miRNA, cytokines, and growth factors to target cells as a form of paracrine communication. This genetically reprograms pathophysiological pathways, which are upregulated in renal failure. Since the method of exosome preparation significantly affects the quality and function of MSC-exosomes, this review compares the methodologies for isolating exosomes from MSCs and their role in tissue regeneration. More specifically, it summarises the therapeutic efficacy of MSC-EVs in 60 preclinical animal models of AKI and CKD and the cargo of biomolecules they deliver. MSC-EVs promote tubular proliferation and angiogenesis, and inhibit apoptosis, oxidative stress, inflammation, the epithelial-to-mesenchymal transition, and fibrosis, to alleviate AKI and CKD. By reprogramming these pathophysiological pathways, MSC-EVs can slow or even reverse the progression of AKI to CKD, and therefore offer potential to transform clinical practice.

Potential Therapeutic Effect of Micrornas in Extracellular Vesicles from Mesenchymal Stem Cells against SARS-CoV-2.

Extracellular vesicles (EVs) are cell-released, nanometer-scaled, membrane-bound materials and contain diverse contents including proteins, small peptides, and nucleic acids. Once released, EVs can alter the microenvironment and regulate a myriad of cellular physiology components, including cell-cell communication, proliferation, differentiation, and immune responses against viral infection. Among the cargoes in the vesicles, small non-coding micro-RNAs (miRNAs) have received attention in that they can regulate the expression of a variety of human genes as well as external viral genes via binding to the complementary mRNAs. In this study, we tested the potential of EVs as therapeutic agents for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. First, we found that the mesenchymal stem-cell-derived EVs (MSC-EVs) enabled the rescue of the cytopathic effect of SARS-CoV-2 virus and the suppression of proinflammatory responses in the infected cells by inhibiting the viral replication. We found that these anti-viral responses were mediated by 17 miRNAs matching the rarely mutated, conserved 3'-untranslated regions (UTR) of the viral genome. The top five miRNAs highly expressed in the MSC-EVs, miR-92a-3p, miR-26a-5p, miR-23a-3p, miR-103a-3p, and miR-181a-5p, were tested. They were bound to the complemented sequence which led to the recovery of the cytopathic effects. These findings suggest that the MSC-EVs are a potential candidate for multiple variants of anti-SARS-CoV-2.