key: cord-0707129-9bi73sev
authors: Dubey, Akhilesh; Lobo, Cynthia Lizzie; GS, Ravi; Shetty, Amitha; Hebbar, Srinivas; El-Zahaby, Sally A.
title: Exosomes: Emerging Implementation of Nanotechnology for Detecting and Managing Novel Corona Virus- SARS-CoV-2
date: 2021-10-03
journal: Asian J Pharm Sci
DOI: 10.1016/j.ajps.2021.08.004
sha: 592fc250f2604cbd167c72dc626f57cf6368cfe8
doc_id: 707129
cord_uid: 9bi73sev

The spread of SARS-CoV-2 as an emerging novel coronavirus disease (COVID-19) had progressed as a worldwide pandemic since the end of 2019. COVID-19 affects firstly lungs tissues which are known for their very slow regeneration. Afterwards, enormous cytokine stimulation occurs in the infected cells immediately after a lung infection which necessitates good management to save patients. Exosomes are extracellular vesicles of nanometric size released by reticulocytes on maturation and are known to mediate intercellular communications. The exosomal cargo serves as biomarkers in diagnosing various diseases; moreover, exosomes could be employed as nanocarriers in drug delivery systems. Exosomes look promising to combat the current pandemic since they contribute to the immune response against several viral pathogens. Many studies have proved the potential of using exosomes either as viral elements or host systems that acquire immune-stimulatory effects and could be used as a vaccine or drug delivery tool. It is essential to stop viral replication, prevent and reverse the massive storm of cytokine that worsens the infected patients’ situations for the management of COVID-19. The main benefits of exosomes could be; no cells will be introduced, no chance of mutation, lack of immunogenicity and the damaged genetic material that could negatively affect the recipient is avoided. Additionally, it was found that exosomes are static with no ability for in vivo reproduction. The current review article discusses the possibilities of using exosomes for detecting novel coronavirus and summarizes state of the art concerning the clinical trials initiated for examining the use of COVID-19 specific T cells derived exosomes and mesenchymal stem cells derived exosomes in managing COVID-19.

In multicellular organisms, eukaryotic cells execute intercellular communications in order to maintain homeostasis and cell development. The local cell communications are achieved by direct cytoplasmic connection through the cell junctions, and the distant communications are established through the chemical messengers such as hormones, growth factors, cytokines, and other secreted molecules [8] . In recent times, researchers discovered extracellular vesicles (EVs) as mediators of intercellular communications with proved pathways [9] . In most eukaryotic cells, the multivesicular body (MVB) or endosomes are produced in the endosomal compartment fused with the cell plasma membrane, and released as lipid bilayer entities called EVs [10] . These multi-functional EVs are ubiquitously found in all types of biofluids which initially reported removing cellular waste. However, now we know these EVs cargo lipids, proteins, genetic materials such as messenger RNA (mRNA), small non-coding RNAs, and genomic DNA (gDNA) from mother cells to distant tissue cells and thereby transfer the cellular information [11] . Classification EVs can be based upon their cell morphology, size, biological function, origin, and the content they carry. They are more specifically categorised into two distinct classes based on their cellular origin or biogenesis, i.e. exosomes and microvesicles [12] .

As aforementioned, EVs are formed either by the development of the cell membrane or within the lumen of the MVBs, and in this case, they are called intraluminal vesicles (ILVs). The other type is known as microvesicles or ectosomes and, in the latter, a fusion of MVBs along with cell membrane (exocytosis) to deliver ILVs, which is then stated as exosomes [11, 12] . The size of microvesicles ranges from 50 to 1000 nm extensively studied due to their role in blood coagulation [13] .

However, they carry specific proteins as well as lipids to the designated receiver cell and are a crucial part of intercellular communication [14] . The diameter of exosomes, nano-sized membrane vesicles released by reticulocytes on maturation, ranges from 30 to 100 nm [15] . In other words, during the formation of MVBs, the endosomal membrane was budding to the inside forming exosomes. These ILVs are exuded upon exocytosis (merging of MVBs with the plasma membrane). Johnstone with his team first described exosomes in the 1970s [16] , and interest in these vesicles grown in recent years owing to their vast array of intriguing beneficial potential such as exosomal cargo as biomarkers in the diagnosis of various diseases and exosomes as nanocarriers in drugs delivery systems [17] .

SARS-CoV-2, a novel human-infecting Beta coronavirus, belongs to the family Coronaviridae inherently different from SARS-CoV (79% similarity) and MERS-CoV [18] . Amino acid sequence in SARS-CoV-2 varies from other coronaviruses exclusively in the areas of 1ab polyprotein as well as S-protein or surface glycoprotein. The host receptor is directly attached to one of the subunits of the Sprotein, facilitating the entry of viruses into the cells [19] . In SARS-CoV-2, the RNA binding domain of the S-protein has a stronger resemblance to SARS-CoV. It has been reported that the COVID-19 human receptor is an angiotensin-converting enzyme (ACE2). Coronaviruses, which includes SARS-CoV-2, gain access (ACE2) into human cells [20] . Structurally (Fig. 2) , SARS-CoV-2 is an enclosed, non-segmented, pleomorphic or spherical shaped, single-stranded RNA virus, ranging from 150 to 160 nm in size.

SARS-CoV-2 genome encodes approximately 25 proteins, notably, glycoproteins for instance, spike (S), envelop (E), nucleocapsid (N), and membrane (M) protein.

Further, COVID-19 encodes a supplementary glycoprotein that has acetyl esterase and hemagglutination properties [21] . N protein, the basic structural unit of SARS-CoV-2, encapsulates the RNA genome. It has been demonstrated that nucleocapsid is involved in the viral genome-related processes, viral replication, and host cell response to the viral infection. It has highly phosphorylated and prone to structural modifications, improving the affinity for viral RNA [22, 23] . There are possibilities of cross-reactivity of antibodies produced against N protein of SARS-CoV with SARS-CoV-2, but these heterophile antibodies of the SARS-CoV might not deliver crossprotection to SARS-CoV-2. However, they may be used for diagnostic purposes. N protein of SARS-CoV can counteract the immune response of the host by acting as a viral suppressor protein of RNAi (VSR). Infection occurs as VSRs repress RNAi at a pre-dicer and post-dicer level to counteract the host's defence mechanism.

SARS-CoV and SARS-CoV-2 had around 90% same sequence identity based on analysis of clustal W of N-protein of by NCBI amino acid blast. Therefore, the Nprotein of SARS-CoV-2 might similarly act as VSR to SARS-CoV to counteract the host defense mechanism [24, 25] .

The M protein determines the virus envelop, shape and affinity to bind all other structural proteins. M protein binding to N Protein provides the stable N protein-RNA complex and completes the viral assembly in the internal virion. E protein is the minor structural protein of SARS-CoV-2 which plays an important role in viral production as well as maturation [26] . Additionally, a transmembrane protein called S glycoprotein is present in the external portion of the virus, with a molecular weight of approximately 15 kDa. These S proteins help the virus bind the specific receptors, especially ACE2 of lower respiratory tract cells, by forming homotrimers protruding in the viral surface and thus entering the human cells/ host cell, which is necessary for the survival of the enveloped viruses such as SARS-CoV-2 [27] . The S protein further gets split into S1 and S2 subunits by the host cell protease, out of which S1 will involve in host cell target, receptor binding, and cellular tropism, and the S2 will mediate the virus fusion in transmitting host cells [28] .

Exosomal biogenesis occurs via the cell trafficking system in two pathways named the endosomal sorting complex needed for the transport (ESCRT) dependent and transport-independent pathways. This ESCRT, formed of four protein subunits (ESCRT 0 to IV), is considered central molecular machinery involved in forming exosomes from endosomes. ESCRT is active in local membrane remodelling in autophagy, cytokinesis, and viral budding and thus facilitates the formation of ILVs inside MVBs by stepwise action with Alg-2-interacting protein-X (Alix) and arrestin domain comprising 1 (ARRDC1), which are well known as associated proteins [29, 30] . During the formation of exosome, accumulation of proteins, lipids, and nucleic acids occurs at the cytosolic face of endocytic membrane microdomains with the aid of these associated proteins. Following these accumulations, an inward curvature occurs, then-budding of the microdomains occurs with cleaving and release of individual ILVs within the lumen of the MVB [31] . Further, ESCRT also facilitates the deubiquitination of sorted proteins within ILVs by interacting with e protein tyrosine phosphatase HD-PTP essential for the exosome functioning. Additionally, the inner curvature and the development of endocytic membrane microdomains were assisted by sphingomyelin-derivative ceramide; then the small GTPase Ral confer in the process of release of exosome through merging of MVBs along with plasma membrane [11, 32] .

In the ESCRT dependent pathway, biogenesis of exosomes occurs successively, and it is a multistep event. It involves identifying cargo by ESCRT-0, and then the cargo is sorted into the nascent ILVs. Further, invagination of MVB's membrane takes place with the aid of ESCRT-0, -I and -II. Vesicle maturation was then followed by this step and ends up due to mediation by ESCRT-III, which causes neck contraction, and finally, membrane scission is facilitated by vacuolar ATPase Vps4 and ILVs formation. Within exosomes, exosomal markers are released, namely, Alix, which is an additional protein, and Tsg101 that is a component of ESCRT-I [30, [33] [34] [35] .

ESCRT-independent pathways also produce exosomes. Ceramide, a cone-shaped lipid, was found to mediate exosome biogenesis. Independent pathway, ESCRT is activated by lipids for example cholesterol and ceramides. Ceramide was found to have a significant role in the development of MVBs. Nanovesicles of 40-150 nm size are released by docking these MVBs with the aid of SNARE (soluble Nethylmaleimide-sensitive factor attachment protein receptors) complexes along with the dominant plasma membrane. Ceramide is produced from the sphingomyelin present on the endosomal's membrane surface in the presence of neutral sphingomyelinase (nSMase). ILVs are produced after an impulsive inward curvature on MVB's membrane mediated by Ceramaides [36] . Further, tetraspanins such as CD81, CD9, CD63, and other molecules like syndecan-syntenin-ALIX complex, Tsg101, VCAM-1, phosphatidic acid, and α4 integrin had been found to have a critical part in the biogenesis and loading of exosome [30, 37, 38] . T cells slightly differ from this pathway as they produce EVs out of the cell's surface, which possesses attributes of exosomes, likely by utilizing the cell components including mechanisms at the cell membrane commonly allied with the endosomal biogenesis of ILVs [39] . Fig. 3 represents the biogenesis and structure of exosome.

Exosomes are composed of specially sorted proteins, nucleic acids, lipids and other contents majorly dependent on their site of origin. The exosome membrane lipid component is characterised by cholesterol and sphingomyelin in vital concentration and ceramide that has a vital role in the biogenesis of ILVs [40] . Furthermore, in contrast with the normal cell membrane, exosome membrane lipids contain lysobisphosphatidic acid that facilitates cholesterol accumulation [41] . Additionally, exosome membrane proteins include tetraspanins clusters and other transmembrane proteins. These tetraspanins have a role in luminal cargo loading via interaction with cytosolic proteins [42] . Additional tetraspanins act independently and are involved in the surface and intracellular trapping of signalling proteins such as b-catenin, Ecadherin, and Wnt [43] . Exosome membrane proteins also include the adhesion proteins as L1CAM (L1 cell adhesion molecule) and LAMP2 (lysosomal associated membrane protein 2); PGRL (CD81 regulatory-like protein), flotillin, and stomatin which bind lipids; the enzyme alanyl aminopeptidase N [43] ; insoluble fibronectin, a surface glycoprotein [44] and integrins (Fig. 3) . 

To survive, viruses usually evolve some mechanisms for facing the host immune system. Nevertheless, pathogens have a counteracting mechanism for each process performed by the immune system [50, 51] . Exosomes are usually injected by viral components where these viral antigens can enhance their survival via decoying the immune system, cloaking viral genomes [52, 53] . Moreover, exosomes can act as biomarkers since they carry the viral antigen, which can be used for targeted therapy and biomarkers [54] . The immune response is regulated in viral infections by the viral and host components released, including exosomes and some EVs. Exosomes are implicated in the pathogenesis of various viruses. For instance, in the case of HIV, EVs and exosomes were imprisoned by cells then viral, and host proteins and RNA contribute to the replication and infection of viral components in the recipient [55] .

CD4 + as well as CD8 + T-cell activation, are inhibited by the released exosomes. Table   1 Table 2 .

As aforementioned, EVs from almost all cell kinds lead to intercellular transmission by conveying biological entities such as lipids, proteins and nucleic acids to beneficiary cells. The cells infected with the virus release exosomes and these exosomes enable infection by transferring viral-derived miRNAs and proteins that are nothing but viral components. In addition, exosomes possess receptors for viruses which makes the recipient cell more prone to virus invasion. Earnest et al., [81] reported that tetraspanin CD9 and TMPRSS2 were found to ease the entry of MERScoronavirus and active lung infection of mouse in vivo. Exosomes and microvesicles contain CD9 molecules within them, and these molecules play a vital role in the exosome biogenesis and the loading of cargo into exosomes. Exosomes derived from infected cells transfer CD9 molecules which might contribute to enhance virus entry.

On uptake of exosomes, the delivery of exosomal cargo to recipient cells occurs and thus promotes the susceptibility of the recipient cell for virus infection. Besides, it has been confirmed that CD9 molecules participate in the entry of the protein-protein network in the MVBs membrane within exosomes [82] and thus favourably affect the loading of COVID-19 viral proteins. It was found that there is an increase in circulating exosomes that contain lung-associated self-antigens, viral antigens as well as 20S proteasome in coronavirus infections. This evidence supports the idea that the SARS-Cov-2 virus-infected cells yield exosomes containing viral proteins [83] . The greater affinity of SARS-Cov-2 virus to human ACE2 than SARS-CoV was reported by Wan et al. [84] , which augment the spread of COVID-19. Recently it has been confirmed that cleavage of spike protein by TMPRSS2 is required for the SARS-Cov-2 virus to move in and cause infection through interaction with ACE2 receptor. It has been evident that exosomes transfer ACE2 to recipient cells, providing a supporting function for the internalization and infection by the SARS-Cov-2. Following the sorting of ACE2 into exosomes, the SARS-Cov-2 virus enters the cells through internalization passage and its components including miRNAs as well as proteins may get similarly incorporated into exosomes [85] . Coronavirus internalization occurs via caveolin-1 dependent endocytosis followed by shedding of viral components from the cell membrane by the vesicles via a mechanism depending on dynamin supporting the idea of exosomes' role in spreading viral infections. Nevertheless, humoral as well as cellular response of the host can be triggered by exosomes derived from infected cells via the transfer of viral and self-antigens [86] . A549 lung epithelial cells on transduction with lentivirus overexpressing genes found in SARS-CoV-2 had led to further isolation of exosomes from A549 cells in the supernatant layer. These exosomes were found to possess the viral genome. Both Exosomes and viral genes were held by human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), this was confirmed by detecting them both in cardiac cells. Besides, the increase of inflammation-related genes in hiPSC-CMs was caused by the uptake of exosomes which contained viral genes. These outcomes suggest that cardiomyocytes can hold SARS-CoV-2 RNA-containing exosomes and produce severe cardiac problems without direct viral infection [87, 88] .

Exosomes can be utilized as biomarkers since they are found to be present in almost all of our body fluids such as, urine, saliva, semen and lung lavage fluid. Their different composition in both normal and disease state like cancer will eventually aid in detecting abnormal body conditions [89] .

In the diagnosis of infectious diseases, not much research was done on exosomes, and this area of research shall be of interest in the future. Literature suggested that the bronchoalveolar lavage fluid obtained from M bovis BCG-infected mice had exosomes containing mycobacterial proteins [90] . Additionally, the serum of patient suffering from tuberculosis (TB) had several mycobacterial proteins involved in the exosomes highlighting their importance as a biomarker for TB. Moreover, mycobacterial RNA was found to be released from infected macrophages with TB, which ensure the possibility of using this RNA as a biomarker for active TB detection [91] . Welker et al. [92] found that CD81 (exosomal protein) levels augmented in the serum of hepatitis C chronic patients. Both the level of this exosomal protein and other markers of liver inflammations like ALT were found to be positively correlated.

Therefore, detecting the level of CD81 in exosomes of these patients might help in both the diagnosis and follow up of their diseased state [92] .

Various viruses, including SARS-CoV-2, exploit or need the ESCRT pathway for entering the recipient cells. The extracted exosomes out of virus-infected cells are challenging to characterise and investigate as these vesicles possess a similar density and size range as that of the virus, which renders their separation very challenging. As an example, exosomes isolated from HCV or HIV-infected cells possess the same densities and sedimentation velocities as these viruses and cannot be readily isolated away from them. The sequential sucrose-gradient ultracentrifugation technique is considered the conventional method for exosomes' isolation from both body fluids and culture media [93] . Other approaches include; technologies based on microfiltration, microfluidic techniques, using precipitation reagents like Total Exosome Isolation reagent (Life Technologies Grand Island, USA) and ExoQuick™ (System Biosciences, Mountain View, USA), also SA, in addition to, using antibodycoated magnetic bead-based immunopurification. Different viruses, either enveloped or non-enveloped, can be purified similarly by alternating centrifugation and ultracentrifugation methods [93] [94] [95] . For the characterization and detection of exosomes when present with the virus, many methods have been utilized, which include size analysis using NanoSight tracking analysis system for nanoparticles, detection by electron microscopy examination, and by using immunoblot analysis for detecting the presence of exosome protein markers like CD81, CD63, Annexin5, ICAM1, TSG101, Alix and FLOT1 [94, 96] . Unfortunately, certain viruses contain some exosomal proteins, so the marker is crucial for characterization by immunoblot analysis. Other analysis can present data about the presence of these exosomal proteins in viruses; for instance, proteomic studies by the use of liquid chromatography and tandem mass spectrometry (LC-MS/MS) reported that influenza virus contains exosomal markers such as Annexin A3, ICAM1, CD81 and CD9, however, Alix and CD63 were not present [97] . Likewise, exosomes as well as retroviruses contain common molecules such as MHC-II, co-stimulatory molecules (CD28, CD54), integrins (CD11a, CD18) and complement neutralizing molecules (CD55, CD59) [98] . Therefore, the exosomes isolated by precipitation reagent or ultracentrifugation shall be essentially subjected to process of immunopurification such as CD63 immunomagnetic bead isolation or other efficient virus purification methods to acquire contamination-free exosomes. Further, exosomes were isolated from the serum of lung transplant beneficiaries diagnosed with respiratory viral infections by ultracentrifugation and the purity was estimated by means of a sucrose cushion [99] . The occurrence of lung self-antigens, 20S proteasome and a viral antigen for coronavirus were determined using immunoblot [99, 100] .

The classical production methodology for exosomes involves performing differential centrifugation to the supernatants of the cell-culture medium. The successive centrifugation aim at removing the unwanted debris and dead cells. Firstly, the centrifugation process yields the final form of exosomes, this step usually lasts for about 1 h, and the centrifuge apparatus operated at 100 000 rpm. This step is followed by a purification step using ultracentrifugation to remove the nucleosomal fragments that might be released from apoptotic cells and any protein aggregates. The final obtained exosomes after all of the differential centrifugation steps were found to be homogenous. Furthermore, their homogeneity was tested in some researches by examination using transmission electron microscopy. However, this method is timeconsuming because it is a multi-step procedure. Moreover, the percentage of exosome recovery ranged from 5%-25% [101] .

Another method for producing exosomes is also adopted. The technique used in this method is based on the use of antibody-coated magnetic beads and flow cytometry.

First, a coat of monoclonal antibodies for the molecules rich in exosomes is applied to the magnetic beads. This step is followed by the incubation step of beads with culture supernatants [102] .

A third method was also used for exosomes production, which is more rapid than the two previous methods. The purification in this method comprises two steps; the first one involves using a 500-kDa membrane for ultrafiltration of the supernatant, and the second step using 30% sucrose/deuterium oxide (98%) cushion. This method is characterized by higher efficiency in removing unwanted proteins from exosomes and much higher yields of 40%-50% compared to the previously discussed classical methods [103, 104] .

The use of exosomes as vaccines has been originated from the cancer field. Numerous advantages have been detected by employing exosomes as vaccines against pathogens; (i) enhanced stability in terms of conformational environments for proteins; (ii) amplified molecular distribution owing to exosomal circulatation into body fluids enabling them to reach distal organs; (iii) extremely coherent correlation with antigen-presenting cells, owing to the expression of adhesion molecules on exosomal surface; (iv) more protection of nucleic acids and proteins against DNase, RNase and proteinases thereby providing a stable environment; and (v) exosomes can act as cross-priming since they are considered as one amongst the body's natural mechanism for antigen transport amongst cells [64] . It was found that CD4 T-cell clones can be triggered by the EVs released by B-cell lines bearing MHC class-II, adhesion and co-stimulatory molecules [105, 106] . Further, tumor peptide-pulsed dendritic cells (DCs) exosomes were used as a vaccine for mice, they carried tumorspecific cytotoxic T lymphocytes (CTLs), and they successfully inhibited the growth of tumor in a T-cell dependent manner [106, 107] proteins derived from exosome were tested and established that the immune sera of pigs earlier exposed to PRRSV explicitly reacted with the exosomes from NV animals. It was found that there was a close similarity between the exosomal mediated antigenic activity and the antigenic activity enclosed in the vaccine available

commercially. An important point to be considered for developing novel vaccines is that exosomes carrying viral antigens circulated in host serum without pathogen load detected in the peripheral circulation (NV) [110, 111] .

Exosomes permit intercellular communication, and presenting antigens could induce a robust immune response. The antigen-presenting exosomes could be used as a novel strategy by modifying the exosomes to present viral antigens that would trigger high and specific CD8+ T cells also known as killer T cells as well as triggering B cell reactions. EV-based vaccines such as expression of the spike protein of SARS-CoV-2 on an exosomal surface or delivering viral protein mRNAs via EVs are being developed against COVID-19 by some biotech companies [112] .

Capricor Therapeutics is acting on two distinct EV-based vaccines which could induce a long-term protecting immune response against SARS-CoV-2 [113] . Firstly, an EV display vaccine, made up of the human HEK293 cells transfected with the vectors expressing the Spike, Nucleocapsid, Membrane and Envelope proteins, are the four structural proteins of SARS-CoV-2. It was previously established that developing a vaccine consisting of multiple forms of protein permits for the magnitude modulation in addition to the nature of the immune response involved in cytokine production and Th1 or Th2 stimulation [114] . The second type of EV contains five different mRNAs that encode for Spike, Nucleocapsid, Membrane, and Envelope proteins (LSNME) of modified SARS-CoV-2 and the full-length spike of Wuhan-1 isolate (Sw1). The vaccines were injected intramuscularly into mice and showed an immunity that lasted up to 2 months after the second booster dose. Finally, there were no vaccine-induced adverse reactions in mice, such as inflammation at the injection site, altered organ morphology, blood cell profiles, or body growth [115] . The Ciloa Company has come up with CoVEVax, a vaccine against COVID-19 based on HEK293T-derived CD81+/CD63+/CD9+ EVs. Due to merging with the patented EVsorting peptide CilPP, the EVs are modified in such a way to display the entire S protein on their surface. Mice were subcutaneously given without adjuvants both the two components and vaccine holding DNA vector for the engineered EVs, and HEK293T-derived engineered EVs. Both humoral and cellular response was evoked only by this combination, which specific IgG measured to S1 or S2 peptide levels and antigen-specific IFN-production [116] . The biotech firm, Codiak BioSciences, is developing a vaccine for COVID-19 called exoVACCTM. It is an advanced system of vaccine that takes advantage of the characteristic properties of EV, where it is possible to simultaneously deliver specific antigens besides immunostimulatory adjuvants to the antigen-presenting cells (APCs) to stimulate both innate as well as humoral immune response. On the other hand, exoVACCTM requires further research as there are possibilities of formation of various combinations of SARS-CoV-2 antigens and adjuvants, and in addition, their efficacy as well as specificity in vitro and animal models ought to be evaluated [117, 118] .

Exosomes possess immunogenic activity in managing SARS coronavirus infection, and this activity has been reported in some studies. Kuate virus-infected cells can prompt immune cell response and deliver therapeutic agents as they comprise specific targeting molecules, ACE2. Moreover, in the same context, drugs can be loaded into exosomes to limit the virus spreadability and replication in host cells. Exosomes are considered to be superior to other nano-delivery systems. For example, exosomes originate from cells, and hence they are safer and possess constant property unlike the other delivery systems such as liposomes [120] . It was found that exosomes derived from the virus-infected cells contributed to promoting virus infection as well as suppressed immune cell responses (Table 1 and 2). Therefore inhibiting the exosome uptake by the neighbouring cells may be used as a strategy to overcome virus spreading.

Stem cells showed recently a crucial therapeutic role in regenerative medicine, where, mesenchymal stem cells (MSCs) therapy has moved from preclinical trials to clinical trials for managing various diseases [121] [122] [123] [124] . For example, MSCs could be used in managing COVID-19 since they can fight inflammation; they were reported to diminish the pathological deteriorations in the lungs significantly and were found to inhibit the cell-mediated immune-inflammatory response in an animal model that was induced by the influenza virus [124] . When COVID-19 virus replicates in the infected patient's body, it induces a series of inflammatory responses that worsen the disease condition. This includes progressive damage of alveolar epithelial and capillary endothelial cells, which causes diffuse interstitial and alveolar oedema and ultimately leads to acute hypoxic respiratory insufficiency. So fighting inflammation will aid in managing severely affected patients with COVID-19, help re-establishment lung cells function, and establish lung tissue regeneration by using MSCs. Additionally, MSCs can act as an immunomodulator and having pro-angiogenic, anti-fibrotic and regenerative abilities [125] .

Based on this fact, recently, a study was recorded with an identified number that focused to investigate the aerosol inhalation of exosomes derived from allogeneic MSCs in the management of COVID-19 patients with severe pneumonia [126] . It is known that MSCs derived exosomes (MSCs-Exo) are useful in the treatment of various diseases and are well known to suppress inflammation of the lungs and the pathological damage caused due to several kinds of lung injury. Almost certainly, exosome treatment could be a valuable tool for managing or at least inhibiting the spread of COVID-19 in the host, yet, further study is necessary. In this regard, some in-progress clinical trials on the MSCs or MSCs-Exo for the COVID-19 treatment are listed in Table 3 .

MSCs were used in two recent studies in China to treat patients with COVID-19 pneumonia. A case report of a critically ill COVID-19 patient on a ventilator was one among the studies and showed evidence of liver injury despite receiving intensive therapy. Treatment was done with allogeneic human umbilical cord MSC as an intravenous infusion every three days (each time 5×10 7 cells) three times. The patient was off the ventilator and able to walk after the second administration. The T cell counts and other measured parameters returned to normal levels, and the injured tissue was repaired with no noticeable side effects [127] . Another study was conducted to examine whether MSC transplantation could improve the consequence in patients with COVID-19 pneumonia. In this study, a single dose of 1×10 6 Additionally, a dramatic rise in a group of CD14+CD11c+CD11bmid regulatory DC cell population was detected. Comparing the MSC treatment group with the placebo control group revealed a significant decrease in TNF-α level and an augmentation in IL-10 levels. Therefore the safety and efficacy of IV administration of MSCs for managing COVID-19 pneumonia were approved [128] . Furthermore, it was found that the MSCs were ACE2 negative, which proves they were free from the virus (Fig.   4) , and this makes them beneficial in COVID-19 patients through immunoregulatory function [129] .

MSCs could hold and release biologically active moieties named secretome. They can do their function via paracrine. These MSC-secretome are composed of proteins like cytokines, growth factors, chemokines, and EVs that are of micro and nano-size.

Moreover, it can manage both acute and chronic lung problems since they resemble the parental MSCs. It was found that secretome could activate endogenous stem cells besides progenitor cells, control inflammatory response, suppress apoptosis, trigger angiogenesis, stimulate remodelling of the extracellular matrix, facilitate chemoattraction and reduce fibrosis [130] . MSC-secretome surpasses the effect of monoclonal antibodies since the former can act on several cytokines. MSC-secretome showed good effectiveness in both levels in the preclinical stage, in vivo and ex vivo [131] . Following IV administration, secretome showed high stability in the bloodstream and was found distributed with the blood flow until reaching the lungs.

Following that, the secretome can penetrate inside tissues offering immune modulation, reducing inflammation, and it can restore the capillary barrier function, and finally enhance bacterial clearance. Comparing both MSCs and secretome, one can reveal that secretome cannot self-replicate, and therefore, it is safer with no probability of tumour induction. Upon IV injection of secretome, a chance for emboli formation is lesser than MSCs because secretome possesses low immunogenicity [132] . MSC-secretome possesses certain technological advantages since it could be easily stored at a low cost, and it is available as ready to use product suitable for emergency cases.

Both injection and inhalation routes are available options for MSC-secretome administration. However, the inhalation route is more preferable, and it will give rise to fast action and enable using a small amount of active moiety. A clinical trial with the title 'A Pilot clinical study on inhalation of MSCs-Exo treating severe novel coronavirus pneumonia' (NCT04276987) started and is in Phase I stage [133] . This clinical trial aimed at exploring both the safety as well as efficacy of inhaling allogenic adipose mesenchymal stem cells (MSCs-Exo) in managing hospitalized patients severely infected by a novel coronavirus and suffering from pneumonia.

Another clinical trial 'A clinical tolerance study on aerosol inhalation of MSCs exosomes in healthy volunteers' (NCT04313647) is in Phase I stage [137] . After completion, this study will evaluate both the safety as well as tolerance of inhaling exosomes that are derived from allogeneic adipose MSCs in healthy volunteers. detection, which put more responsibility on the shoulder of researchers to make available standardized techniques for ensuring the wide usage of exosomes clinically. Development in stable inner reference genes is still under study; this will ensure more accurate exosomal miRNAs quantification. The isolation and production of a large amount of exosomes are also a major burden. The available methods for exosomes isolation are costly and time-consuming. Moreover, the isolation is not easy enough, and contamination by particles of the same size as exosomes could occur.

Furthermore, developing the right dosage form for exosomes is very challenging to ensure their effectiveness in managing different types of cancer. Entrapping miRNAs in exosomes must be high enough in order to produce effective targeted cancer therapy. Cell-specific receptors for each tumor must be well identified to prepare the right targeted miRNAs exosomal preparation. This will help to diminish the off-target effects [140, 141] . After choosing the suitable dosage form and preparing it, largescale studies are needed to approve exosome usage to introduce the risk for both immunosuppression and tumorigenesis [142, 143] . 

The urgent need arises about treating COVID-19 infected patients developing pneumonia that necessitates exploring new strategies and novel delivery systems. A therapeutic strategy "without cells" is an emerging field having several advantages.

Hence, exosomes derived from MSCs and those derived from COVID-19 specific T cells could be one of the best therapeutic approaches. Clinical trials have been started, and the results are still awaited. It was found that delivering MSCs-Exo rather than live MSCs is safer; however, both significantly reduced lung inflammation and diminished other pathological conditions of various types of lung injury. The continuous and detailed study on the mechanism of viral proteins loading onto exosomes and their roles in managing viral diseases will provide more specific insights on developing exosomes as novel vaccine and drug delivery systems. HIV-1-infected cells release exosomes that inactivate CD4 + T-cells to achieve replication of HIV-1 via both a Nef-and ADAM17-dependent mechanism.

HIV [56] Exosomes can uptake transactivating response (TAR) RNA, this will aid downregulation and apoptosis and therefore contribute to HIV infection.

HIV [57] The apoptosis of T-cell was found to increase due to exosomal Nef leading to depletion of CD4 + T-cell in case of AIDS.

HIV [58] Nef supports HIV-1 infection by decreasing the HIV expression of CD4 in exosomes derived from infected cells.

HIV [59] Tropical spastic paraparesis is caused by HTLV-1 infection. HTLV-1 infected cell lines released exosomes comprising Tax, a pleiotropic transactivating protein intricated in immune dysregulation linked through infection.

HSV-1 infection releases a variety of microvesicles from cells; L particles are most prominent. L particles are made up of virus envelope as well as tegument and are devoid of viral genome as well as capsid proteins. As such they are noninfectious but have been shown to increase the susceptibility for infection in uninfected cells.

Latent membrane protein 1 which is a signal transduction protein was found in exosomes isolated from EBV + cancer cells. The uptake of LMP + exosomes inhibited the activity of natural killer cell as well as T-cell activation and proliferation.

EBV [62] Exosomes released from EBV + cells contained Galectin-9 which induces EBVspecific T-cell apoptosis and hence circumvents detection via the immune system.

EBV [63] EBV was found on packaging viral miRNAs into exosomes and the miRNAs can decrease CXCL11, a targeted immunoregulatory gene essential for antiviral activity.

EBV [64] CMV infection increases DC-SIGN release on exosomes, which belongs to the C-type lectin family and is crucial for uptake of virus. This mediates myeloid DCs infection by CMV and augmented total CMV infectivity.

CMV [65] HHV-6 infection increases MHCI transfer to released exosomes, and downregulation of MHCI is a renowned pathway for immunoevasion.

The presence of viral RNA and proteins in the extracted exosomes out of infected cells with RVFV, led to apoptosis of immune cells exposed to these exosomes.

A phelebovirus incorporated virions into CD63 + exosomes that lead to receptorindependent uptake by neighbouring cells.

SFTSV [68] Human immunodeficiency virus (HIV);Human T-cell lymphotropic virus type 1 (HTLV-1);Rift Valley fever virus (RVFV);Herpes simplex virus type 1 (HSV-1);Epstein-Barr virus (EBV);Cytomegalovirus (CMV);Human herpesvirus type 6 (HHV-6);Severe fever with thrombocytopenia syndrome virus (SFTSV); Dendritic cell-specific ICAM3-grabbing-nonintegrin (DC-SIGN)； Major histocompatibility complex (MHCI) HSV-1 incorporates STING protein into exosomes, and delivers it to uninfected cells. The viral miRNAs such as miR-H3, miR-H5 and miR-H6 were also packaged into exosomes, and these exosomes may negatively affect both the host-host infection and viral spread thereby increasing host survival.

HSV-1 [69] APOBEC3G, cGAMP, miRNA-99, and miRNA-88 incorporated into the exosomes exhibited antiviral effect.

HIV-1 [70] [71] [72] The dUTPase was found to be incorporated into exosomes which produced an antiviral effect.

EBV [73] Viral miRNAs and mitochondrial DNA loaded into exosomes exhibited an antiviral effect.

KSHV [74, 75] IFI16 and Glycoprotein B were present in the exosomes, which produced an antiviral effect.

CMV [76, 77] The extracellular IFITM3 protein present in exosomes was found to contribute to inhibitory effect of DENV entry in cell models of DENV-2 infection via interferon-induced inhibition.

DENV [78] Exosomes increase the functioning of macrophages and NK cells and deliver antiviral molecules between cells.

HBV [79] During influenza virus infection the released exosomes in the airways elicit inflammatory responses in lungs and convey viral antigen that could be exploited by antigen-presenting cells in order to induce a cellular immune response. In addition, the attachment factors α2,3 and α2,6-linked sialic acids that are present on the airway exosomal surface, and can neutralize influenza virus. Thus the virus is unable to bind and enter the target cells.

Influenza virus [80] Herpes simplex virus type 1 (HSV-1);Stimulator of INF genes (STING);Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (APOBEC3G); Interferon-inducible transmembrane 3 (IFITM3);Human immunodeficiency virus type 1 (HIV-1)Epstein-Barr virus (EBV);Kaposi's sarcoma-associated herpesvirus (KSHV);Cytomegalovirus (CMV);Dengue virus (DENV);Hepatitis B virus (HBV) Ongoing Clinical trial NCT04276987 (China) [126] A critically ill COVID-19 patient on treatment with hUCMSCs developed clinical remission.

The laboratory indices and CT images indicated decrease in inflammation symptom. Patients were shifted out of ICU, and the throat swabs tested negative 4 d later. These results showed the clinical outcome and good tolerance of allogenic hUCMSCs transfer. [127] Patients with COVID-19 pneumonia benefit from ACE2-MSC transplantation.

ACE2-MSC (IV)

After 2 d of MSC transplantation, these 7 patients had considerably improved pulmonary function and symptoms. It was found to be safe and efficacious in patients with COVID- 19 pneumonia, who had been in a critical condition. [128] A Phase I/II randomised, double-blind, placebo-controlled trial to assess the safety and potential efficacy of an IV infusion of Zofin (Organicell Flow) to treat moderate COVID-19 infection caused by SARS.

Growth factors, and other EVs/nanoparticles derived from HAF (IV)

Ongoing Clinical trial NCT04384445 (USA) [129] A single-arm, open label, combined interventional (phase I/II trials) clinical trial is being conducted to determine the safety and efficacy of inhaled CSTCexosomes in the treatment of early stage novel coronavirus pneumonia.

Ongoing Clinical trial NCT04389385 (Turkey) [130] Exosomes (ExoFlo TM ) derived from allogeneic bone marrow mesenchymal stem cells were used for treating severe COVID-19 in a non-randomized, openlabel, cohort study.

Bone marrow MSCs-Exo (IV)

All safety endpoints were met with no adverse events detected. The survival rate was 83%. ExoFlo TM is a promising therapeutic candidate for severe COVID-19 due to its safety profile, capacity to restore oxygenation, downregulate cytokine storm and reconstruct immunity. [131] ExoFlo TM , bone marrow-derived EVs on IV administration, is being evaluated as a treatment for moderate to severe ARDS in patients with severe COVID-19 in a multi-center, randomized, double-blinded, placebo-controlled clinical trial.

Ongoing Clinical trial NCT04493242 (USA) [132] Exosome inhalation was evaluated for safety and efficacy in SARS-CoV-2 associated pneumonia.

Ongoing Clinical trial NCT04491240 (Russia) [133] To determine the efficacy of MSCs infusion as a supplementary therapy to standard supportive treatment for patients with moderate/severe COVID-19.

Ongoing Clinical trial NCT04444271 (Pakistan) [134] hUMSCs and Exosomes for lung injury in patients with COVID-19. hUMSCs (IV)

Ongoing Clinical trial ChiCTR2000030484 (China) [135] CAP-1002 Allogeneic Cardiosphere-Derived Cells EVs from CDCs (IV) Ongoing Clinical trial NCT04338347 (USA) [136] human umbilical cord mesenchymal stem cells (hUCMSCs);COVID-19 Specific T Cell Derived Exosomes (CSTC-Exo);Acute Respiratory Distress Syndrome (ARDS);Cardiosphere-Derived Cells (CDCs)

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Proteomics of extracellular vesicles: Exosomes and ectosomes

Extracellular vesicles: exosomes, microvesicles, and friends

Exosomes and ectosomes in intercellular communication

Shedding light on the cell biology of extracellular vesicles

Investigation of procoagulant activity in extracellular vesicles isolated by differential ultracentrifugation

Exosomes and microvesicles in normal physiology, pathophysiology, and renal diseases

Reticulocyte maturation and exosome release: transferrin receptor containing exosomes shows multiple plasma membrane functions

Revisiting the road to the discovery of exosomes

Exosomes in diagnostic and therapeutic applications: biomarker, vaccine and RNA interference delivery vehicle

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): an overview of viral structure and host response

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

A close look at the biology of SARS-CoV-2, and the potential influence of weather conditions and seasons on COVID-19 case spread

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Coronaviruses: an overview of their replication and pathogenesis

The nucleocapsid protein of coronaviruses acts as a viral suppressor of RNA silencing in mammalian cells

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Coronavirus envelope protein: current knowledge

Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein

Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine

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Exosomes: biogenesis, biologic function and clinical potential

Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles

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The ESCRT pathway

ESCRTs are everywhere

Sorting it out: regulation of exosome loading

Ceramide: A simple sphingolipid with unique biophysical properties

Exosomes: Current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials

Syndecan-syntenin-ALIX regulates the biogenesis of exosomes

Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane

Ectosomes and exosomes: shedding the confusion between extracellular vesicles

Lysobisphosphatidic acid controls endosomal cholesterol levels

The Intracellular interactome of tetraspanin-enriched microdomains reveals their function as sorting machineries toward exosomes

Signaling pathways in exosomes biogenesis, secretion and fate

Fibronectin on the surface of myeloma cell-derived exosomes mediates exosome-cell interactions

Multivesicular bodies associate with components of miRNA effector complexes and modulate miRNA activity

KRAS-MEK signaling controls Ago2 sorting into exosomes

KRAS-MEK signaling controls Ago2 sorting into exosomes

Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes

Exosomes in viral disease

The role of extracellular vesicles in viral infection and transmission

Extracellular vesicles and viruses: Are they close relatives?

Exosome biogenesis, regulation, and function in viral Infection

Role of extracellular vesicles in viral and bacterial infections: pathogenesis, diagnostics, and therapeutics

Microvesicles and viral infection

Exosomes from human immunodeficiency virus type 1 (HIV-1)-infected cells license quiescent CD4+ T lymphocytes to replicate HIV-1 through a Nef-and ADAM17-dependent mechanism

Exosomes from HIV-1-infected cells stimulate production of pro-inflammatory cytokines through trans-activating response (TAR) RNA

HIV Nef is secreted in exosomes and triggers apoptosis in bystander CD4+ T cells

Nef neutralizes the ability of exosomes from CD4+ T cells to act as decoys during HIV-1 infection

Highlights on distinctive structural and functional properties of HTLV Tax proteins

L Particles transmit viral proteins from herpes simplex virus 1-infected mature dendritic cells to uninfected bystander cells, inducing CD83 downmodulation

Exosomes Derived from

virus-infected cells are internalized via Caveola-dependent endocytosis and promote phenotypic modulation in target cells

Blood diffusion and Th1-suppressive effects of galectin-9-containing exosomes released by Epstein-Barr virus-infected nasopharyngeal carcinoma cells

Exosomes and other extracellular vesicles in host-pathogen interactions

Pivotal advance: the promotion of soluble DC-SIGN release by inflammatory signals and its enhancement of cytomegalovirus-mediated cis -infection of myeloid dendritic cells

Expression of MHC class I molecule in HHV-6B-infected cells

Presence of viral RNA and proteins in exosomes from cellular clones resistant to rift valley fever virus infection

Extracellular vesicles mediate receptor-independent transmission of novel tick-borne bunyavirus

Extracellular vesicles during herpes simplex virus type 1 infection: an inquire

Exosomes packaging APOBEC3G confer human immunodeficiency virus resistance to recipient cells

Viruses transfer the antiviral second messenger cGAMP between cells

Novel HIV-1 miRNAs stimulate TNFα release in human macrophages via TLR8 signaling pathway

Systemically Circulating viral and tumor-derived microRNAs in KSHV-associated malignancies

Extracellular vesicles from KSHV-infected cells stimulate antiviral immune response through mitochondrial DNA

The nuclear DNA sensor IFI16 acts as a restriction factor for human papillomavirus replication through epigenetic modifications of the viral promoters

Cytomegalovirus-infected human endothelial cells can stimulate allogeneic CD4+ memory T cells by releasing antigenic exosomes

IFITM3-containing exosome as a novel mediator for anti-viral response in dengue virus infection

Exosomes Modulate the viral replication and host immune responses in HBV infection

Airway exosomes released during influenza virus infection serve as a key component of the antiviral innate immune response

The tetraspanin CD9 facilitates MERS-coronavirus entry by scaffolding host cell receptors and proteases

Exosomal cargos modulate autophagy in recipient cells via different signaling pathways

The role of extracellular vesicles in COVID-19 virus infection

Receptor recognition by the novel coronavirus from Wuhan: an analysis based on decade-long structural studies of SARS coronavirus

COVID-19 outbreak: history, mechanism, transmission, structural studies and therapeutics

Exosomes in pathogen infections: a bridge to deliver molecules and link functions

SARS-CoV-2 and the cardiovascular system

Biological function of exosomes as diagnostic markers and therapeutic deliver vehicles in carcinogenesis and infectious diseases

Exosomes released from macrophages infected with intracellular pathogens stimulate a proinflammatory response in vitro and in vivo

Exosomal RNA from Mycobacterium tuberculosisinfected cells is functional in recipient macrophages

Soluble serum CD81 is elevated in patients with chronic hepatitis C and correlates with alanine aminotransferase serum activity

Progress in exosome isolation techniques

A protocol for exosome isolation and characterization: evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods

Isolation of extracellular vesicles: general methodologies and latest trends

Exosomes: isolation methods and specific markers

Conserved and host-specific features of influenza virion architecture

Exosomes and Their Role in the Life Cycle and Pathogenesis of RNA Viruses

Respiratory viral infection in lung transplantation induces exosomes that trigger chronic rejection

Proteasome activator PA28γ-dependent degradation of coronavirus disease (COVID-19) nucleocapsid protein

Exosomes: a bubble ride for prions?

Proteomic analysis of melanoma-derived exosomes by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry

Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry

Production and characterization of clinical grade exosomes derived from dendritic cells

B lymphocytes secrete antigen-presenting vesicles

Tumorderived exosomes are a source of shared tumor rejection antigens for CTL crosspriming

Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell derived exosomes

Direct exosome stimulation of peripheral humanT cells detected by ELISPOT

Regulation of immune responses by extracellular vesicles

Serumderived exosomes from non-viremic animals previously exposed to the porcine respiratory and reproductive virus contain antigenic viral proteins

A novel role of exosomes in the vaccination approach

Promising extracellular vesiclebased vaccines against viruses, including SARS-CoV-2

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Exosomal vaccines containing the S protein of the SARS coronavirus induce high levels of neutralizing antibodies

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Map of COVID-19 affected countries reported to WHO as of 2

The ranges from deep blue to light shades as follows: > 1