Endocytosis blocks the vesicular secretion of exosome marker proteins.

Exosomes are secreted vesicles of ~30 to 150 nm diameter that play important roles in human health and disease. To better understand how cells release these vesicles, we examined the biogenesis of the most highly enriched human exosome marker proteins, the exosomal tetraspanins CD81, CD9, and CD63. We show here that endocytosis inhibits their vesicular secretion and, in the case of CD9 and CD81, triggers their destruction. Furthermore, we show that syntenin, a previously described exosome biogenesis factor, drives the vesicular secretion of CD63 by blocking CD63 endocytosis and that other endocytosis inhibitors also induce the plasma membrane accumulation and vesicular secretion of CD63. Finally, we show that CD63 is an expression-dependent inhibitor of endocytosis that triggers the vesicular secretion of lysosomal proteins and the clathrin adaptor AP-2 mu2. These results suggest that the vesicular secretion of exosome marker proteins in exosome-sized vesicles occurs primarily by an endocytosis-independent pathway.

Antigen-display exosomes provide adjuvant-free protection against SARS-CoV-2 disease at nanogram levels of spike protein.

As the only bionormal nanovesicle, exosomes have high potential as a nanovesicle for delivering vaccines and therapeutics. We show here that the loading of type-1 membrane proteins into the exosome membrane is induced by exosome membrane anchor domains, EMADs, that maximize protein delivery to the plasma membrane, minimize protein sorting to other compartments, and direct proteins into exosome membranes. Using SARS-CoV-2 spike as an example and EMAD13 as our most effective exosome membrane anchor, we show that cells expressing a spike-EMAD13 fusion protein produced exosomes that carry dense arrays of spike trimers on 50% of all exosomes. Moreover, we find that immunization with spike-EMAD13 exosomes induced strong neutralizing antibody responses and protected hamsters against SARS-CoV-2 disease at doses of just 0.5-5 ng of spike protein, without adjuvant, demonstrating that antigen-display exosomes are particularly immunogenic, with important implications for both structural and expression-dependent vaccines.

Exomap1 mouse: a transgenic model for in vivo studies of exosome biology.

Exosomes are small extracellular vesicles (sEVs) of ~30-150 nm in diameter that have the same topology as the cell, are enriched in selected exosome cargo proteins, and play important roles in health and disease. To address large unanswered questions regarding exosome biology in vivo, we created the exomap1 transgenic mouse model. In response to Cre recombinase, exomap1 mice express HsCD81mNG, a fusion protein between human CD81, the most highly enriched exosome protein yet described, and the bright green fluorescent protein mNeonGreen. As expected, cell type-specific expression of Cre induced the cell type-specific expression of HsCD81mNG in diverse cell types, correctly localized HsCD81mNG to the plasma membrane, and selectively loaded HsCD81mNG into secreted vesicles that have the size (~80 nm), topology (outside out), and content (presence of mouse exosome markers) of exosomes. Furthermore, mouse cells expressing HsCD81mNG released HsCD81mNG-marked exosomes into blood and other biofluids. Using high-resolution, single-exosome analysis by quantitative single molecule localization microscopy, we show here that that hepatocytes contribute ~15% of the blood exosome population whereas neurons contribute <1% of blood exosomes. These estimates of cell type-specific contributions to blood EV population are consistent with the porosity of liver sinusoidal endothelial cells to particles of ~50-300 nm in diameter, as well as with the impermeability of blood-brain and blood-neuron barriers to particles >5 nm in size. Taken together, these results establish the exomap1 mouse as a useful tool for in vivo studies of exosome biology, and for mapping cell type-specific contributions to biofluid exosome populations. In addition, our data confirm that CD81 is a highly-specific marker for exosomes and is not enriched in the larger microvesicle class of EVs.

The D614G mutation redirects SARS-CoV-2 spike to lysosomes and suppresses deleterious traits of the furin cleavage site insertion mutation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) egress occurs by lysosomal exocytosis. We show that the Spike D614G mutation enhances Spike trafficking to lysosomes, drives Spike-mediated reprogramming of lysosomes, and reduces cell surface Spike expression by ~3-fold. D614G is not a human-specific adaptation. Rather, it is an adaptation to the earlier furin cleavage site insertion (FCSI) mutation that occurred at the genesis of SARS-CoV-2. While advantageous to the virus, furin cleavage of spike has deleterious effects on spike structure and function, inhibiting its trafficking to lysosomes and impairing its infectivity by the transmembrane serine protease 2(TMPRSS2)-independent, endolysosomal pathway. D614G restores spike trafficking to lysosomes and enhances the earliest events in SARS-CoV-2 infectivity, while spike mutations that restore SARS-CoV-2's TMPRSS2-independent infectivity restore spike's trafficking to lysosomes. Together, these and other results show that D614G is an intragenic suppressor of deleterious traits linked to the FCSI and lend additional support to the endolysosomal model of SARS-CoV-2 egress and entry.

A shared, stochastic pathway mediates exosome protein budding along plasma and endosome membranes.

Exosomes are small extracellular vesicles of ∼30 to 150 nm that are secreted by all cells, abundant in all biofluids, and play important roles in health and disease. However, details about the mechanism of exosome biogenesis are unclear. Here, we carried out a cargo-based analysis of exosome cargo protein biogenesis in which we identified the most highly enriched exosomal cargo proteins and then followed their biogenesis, trafficking, and exosomal secretion to test different hypotheses for how cells make exosomes. We show that exosome cargo proteins bud from cells (i) in exosome-sized vesicles regardless of whether they are localized to plasma or endosome membranes, (ii) ∼5-fold more efficiently when localized to the plasma membrane, (iii) ∼5-fold less efficiently when targeted to the endosome membrane, (iv) by a stochastic process that leads to ∼100-fold differences in their abundance from one exosome to another, and (v) independently of small GTPase Rab27a, the ESCRT complex-associated protein Alix, or the cargo protein CD63. Taken together, our results demonstrate that cells use a shared, stochastic mechanism to bud exosome cargoes along the spectrum of plasma and endosome membranes and far more efficiently from the plasma membrane than the endosome. Our observations also indicate that the pronounced variation in content between different exosome-sized vesicles is an inevitable consequence of a stochastic mechanism of small vesicle biogenesis, that the origin membrane of exosome-sized extracellular vesicles simply cannot be determined, and that most of what we currently know about exosomes has likely come from studies of plasma membrane-derived vesicles.

Continued Virus-Specific Antibody-Secreting Cell Production, Avidity Maturation and B Cell Evolution in Patients Hospitalized with COVID-19.

Understanding the development and sustainability of the virus-specific protective immune response to infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains incomplete with respect to the appearance and disappearance of virus-specific antibody-secreting cells (ASCs) in circulation. Therefore, we performed cross-sectional and longitudinal analyses of peripheral blood mononuclear cells and plasma collected from 55 hospitalized patients up to 4 months after onset of COVID-19 symptoms. Spike (S)- and nucleocapsid (N)-specific IgM and IgG ASCs appeared within 2 weeks accompanied by flow cytometry increases in double negative plasmablasts consistent with a rapid extrafollicular B cell response. Total and virus-specific IgM and IgG ASCs peaked at 3-4 weeks and were still being produced at 3-4 months accompanied by increasing antibody avidity consistent with a slower germinal center B cell response. N-specific ASCs were produced for longer than S-specific ASCs and avidity maturation was greater for antibody to N than S. Patients with more severe disease produced more S-specific IgM and IgG ASCs than those with mild disease and had higher levels of N- and S-specific antibody. Women had more B cells in circulation than men and produced more S-specific IgA and IgG and N-specific IgG ASCs. Flow cytometry analysis of B cell phenotypes showed an increase in circulating B cells at 4-6 weeks with decreased percentages of switched and unswitched memory B cells. These data indicate ongoing antigen-specific stimulation, maturation, and production of ASCs for several months after onset of symptoms in patients hospitalized with COVID-19.

Degron tagging of BleoR and other antibiotic-resistance genes selects for higher expression of linked transgenes and improved exosome engineering.

Five antibiotic resistance (AR) genes have been used to select for transgenic eukaryotic cell lines, with the BleoR, PuroR, HygR, NeoR, and BsdR cassettes conferring resistance to zeocin, puromycin, hygromycin, geneticin/G418, and blasticidin, respectively. We recently demonstrated that each AR gene establishes a distinct threshold of transgene expression below which no cell can survive, with BleoR selecting for the highest level of transgene expression, nearly ∼10-fold higher than in cells selected using the NeoR or BsdR markers. Here, we tested the hypothesis that there may be an inverse proportionality between AR protein function and the expression of linked, transgene-encoded, recombinant proteins. Specifically, we fused each AR protein to proteasome-targeting degron tags, used these to select for antibiotic-resistant cell lines, and then measured the expression of the linked, recombinant protein, mCherry, as a proxy marker of transgene expression. In each case, degron-tagged AR proteins selected for higher mCherry expression than their cognate WT AR proteins. ER50BleoR selected for the highest level of mCherry expression, greater than twofold higher than BleoR or any other AR gene. Interestingly, use of ER50BleoR as the selectable marker translated to an even higher, 3.5-fold increase in the exosomal loading of the exosomal cargo protein, CD63/Y235A. Although a putative CD63-binding peptide, CP05, has been used to decorate exosome membranes in a technology known as "exosome painting," we show here that CP05 binds equally well to CD63-/- cells, WT 293F cells, and CD63-overexpressing cells, indicating that CP05 may bind membranes nonspecifically. These results are of high significance for cell engineering and especially for exosome engineering.

Exosome-mediated mRNA delivery in vivo is safe and can be used to induce SARS-CoV-2 immunity.

Functional delivery of mRNA has high clinical potential. Previous studies established that mRNAs can be delivered to cells in vitro and in vivo via RNA-loaded lipid nanoparticles (LNPs). Here we describe an alternative approach using exosomes, the only biologically normal nanovesicle. In contrast to LNPs, which elicited pronounced cellular toxicity, exosomes had no adverse effects in vitro or in vivo at any dose tested. Moreover, mRNA-loaded exosomes were characterized by efficient mRNA encapsulation (∼90%), high mRNA content, consistent size, and a polydispersity index under 0.2. Using an mRNA encoding the red light-emitting luciferase Antares2, we observed that mRNA-loaded exosomes were superior to mRNA-loaded LNPs at delivering functional mRNA into human cells in vitro. Injection of Antares2 mRNA-loaded exosomes also led to strong light emission following injection into the vitreous fluid of the eye or into the tissue of skeletal muscle in mice. Furthermore, we show that repeated injection of Antares2 mRNA-loaded exosomes drove sustained luciferase expression across six injections spanning at least 10 weeks, without evidence of signal attenuation or adverse injection site responses. Consistent with these findings, we observed that exosomes loaded with mRNAs encoding immunogenic forms of the SARS-CoV-2 Spike and Nucleocapsid proteins induced long-lasting cellular and humoral responses to both. Taken together, these results demonstrate that exosomes can be used to deliver functional mRNA to and into cells in vivo.

Extracellular vesicles derived from cardiosphere-derived cells as a potential antishock therapeutic.

BACKGROUND: Extracellular vesicles (EVs) isolated from cardiosphere-derived cells (CDC-EVs) are coming to light as a unique cell-free therapeutic. Because of their novelty, however, there still exist prominent gaps in knowledge regarding their therapeutic potential. Herein the therapeutic potential of CDC-EVs in a rat model of acute traumatic coagulopathy induced by multiple injuries and hemorrhagic shock is outlined. METHODS: Extracellular vesicle surface expression of procoagulant molecules (tissue factor and phosphatidylserine) was evaluated by flow cytometry. Extracellular vesicle thrombogenicity was tested using calibrated thrombogram, and clotting parameters were assessed using a flow-based adhesion model simulating blood flow over a collagen-expressing surface. The therapeutic efficacy of EVs was then determined in a rat model of acute traumatic coagulopathy induced by multiple injuries and hemorrhagic shock. RESULTS: Extracellular vesicles isolated from cardiosphere-derived cells are not functionally procoagulant and do not interfere with platelet function. In a rat model of multiple injuries and hemorrhagic shock, early administration of EVs significantly reduced the elevation of lactate and creatinine and did not significantly enhance coagulopathy in rats with acute traumatic coagulopathy. CONCLUSION: The results of this study are of great relevance to the development of EV products for use in combat casualty care, as our studies show that CDC-EVs have the potential to be an antishock therapeutic if administered early. These results demonstrate that research using CDC-EVs in trauma care needs to be considered and expanded beyond their reported cardioprotective benefits.

Choice of selectable marker affects recombinant protein expression in cells and exosomes.

Transgenic mammalian cells are used for numerous research, pharmaceutical, industrial, and clinical purposes, and dominant selectable markers are often used to enable the selection of transgenic cell lines. Using HEK293 cells, we show here that the choice of selectable marker gene has a significant impact on both the level of recombinant protein expression and the cell-to-cell variability in recombinant protein expression. Specifically, we observed that cell lines generated with the NeoR or BsdR selectable markers and selected in the antibiotics G418 or blasticidin, respectively, displayed the lowest level of recombinant protein expression as well as the greatest cell-to-cell variability in transgene expression. In contrast, cell lines generated with the BleoR marker and selected in zeocin yielded cell lines that expressed the highest levels of linked recombinant protein, approximately 10-fold higher than those selected using the NeoR or BsdR markers, as well as the lowest cell-to-cell variability in recombinant protein expression. Intermediate yet still-high levels of expression were observed in cells generated with the PuroR- or HygR-based vectors and that were selected in puromycin or hygromycin, respectively. Similar results were observed in the African green monkey cell line COS7. These data indicate that each combination of selectable marker and antibiotic establishes a threshold below which no cell can survive and that these thresholds vary significantly between different selectable markers. Moreover, we show that choice of selectable marker also affects recombinant protein expression in cell-derived exosomes, consistent with the hypothesis that exosome protein budding is a stochastic rather than determinative process.

The D614G Mutation Enhances the Lysosomal Trafficking of SARS-CoV-2 Spike.

The spike D614G mutation increases SARS-CoV-2 infectivity, viral load, and transmission but the molecular mechanism underlying these effects remains unclear. We report here that spike is trafficked to lysosomes and that the D614G mutation enhances the lysosomal sorting of spike and the lysosomal accumulation of spike-positive punctae in SARS-CoV-2-infected cells. Spike trafficking to lysosomes is an endocytosis-independent, V-ATPase-dependent process, and spike-containing lysosomes drive lysosome clustering but display poor lysotracker labeling and reduced uptake of endocytosed materials. These results are consistent with a lysosomal pathway of coronavirus biogenesis and raise the possibility that a common mechanism may underly the D614G mutation's effects on spike protein trafficking in infected cells and the accelerated entry of SARS-CoV-2 into uninfected cells.

Exosomes.

Exosomes are small, single-membrane, secreted organelles of ∼30 to ∼200 nm in diameter that have the same topology as the cell and are enriched in selected proteins, lipids, nucleic acids, and glycoconjugates. Exosomes contain an array of membrane-associated, high-order oligomeric protein complexes, display pronounced molecular heterogeneity, and are created by budding at both plasma and endosome membranes. Exosome biogenesis is a mechanism of protein quality control, and once released, exosomes have activities as diverse as remodeling the extracellular matrix and transmitting signals and molecules to other cells. This pathway of intercellular vesicle traffic plays important roles in many aspects of human health and disease, including development, immunity, tissue homeostasis, cancer, and neurodegenerative diseases. In addition, viruses co-opt exosome biogenesis pathways both for assembling infectious particles and for establishing host permissiveness. On the basis of these and other properties, exosomes are being developed as therapeutic agents in multiple disease models.

Advances, challenges, and opportunities in extracellular RNA biology: insights from the NIH exRNA Strategic Workshop.

Extracellular RNA (exRNA) has emerged as an important transducer of intercellular communication. Advancing exRNA research promises to revolutionize biology and transform clinical practice. Recent efforts have led to cutting-edge research and expanded knowledge of this new paradigm in cell-to-cell crosstalk; however, gaps in our understanding of EV heterogeneity and exRNA diversity pose significant challenges for continued development of exRNA diagnostics and therapeutics. To unravel this complexity, the NIH convened expert teams to discuss the current state of the science, define the significant bottlenecks, and brainstorm potential solutions across the entire exRNA research field. The NIH Strategic Workshop on Extracellular RNA Transport helped identify mechanistic and clinical research opportunities for exRNA biology and provided recommendations on high priority areas of research that will advance the exRNA field.

Extracellular vesicles carry microRNA-195 to intrahepatic cholangiocarcinoma and improve survival in a rat model.

The cancer microenvironment plays a central role in cancer development, growth, and homeostasis. This paradigm suggests that cancer fibroblasts support cancers, probably in response to stimuli received from the cancer cells. We aimed at investigating whether extracellular vesicles (EVs) can shuttle microRNA (miR) species between cancer-associated fibroblasts (CAFs) and cancer cells. To this end, we extracted EVs according to published protocols. EVs were studied for their miR content by quantitative reverse-transcription polymerase chain reaction. EVs were transfected with select miR species and utilized in vitro as well as in vivo in a rat model of cholangiocarcinoma (CCA). We found that miR-195 is down-regulated in CCA cells, as well as in adjoining fibroblasts. Furthermore, we report that EVs shuttle miR-195 from fibroblasts to cancer cells. Last, we show that fibroblast-derived EVs, loaded with miR-195, can be administered in a rat model of CCA, concentrate within the tumor, decrease the size of cancers, and improve survival of treated rats. CONCLUSION: EVs play a salient role in trafficking miR species between cancer cells and CAFs in human CCA. Understanding of these mechanisms may allow devising of novel therapeutics. (Hepatology 2017;65:501-514).

The cis-acting signals that target proteins to exosomes and microvesicles.

Proteins bud from cells in small single-membraned vesicles (~50-250 nm) that have the same topology as the cell. Known variously as exosomes and microvesicles (EMVs), these extracellular organelles are enriched for specific proteins, lipids, carbohydrates and nucleic acids. EMV biogenesis plays critical roles in protein quality control and cell polarity, and, once released, EMVs can transmit signals and molecules to neighbouring cells via a non-viral pathway of intercellular vesicle traffic. In the present paper, we discuss the cis-acting targeting signals that target proteins to EMVs and mediate protein budding from the cell.

HIV Pol inhibits HIV budding and mediates the severe budding defect of Gag-Pol.

The prevailing hypothesis of HIV budding posits that the viral Gag protein drives budding, and that the Gag p6 peptide plays an essential role by recruiting host-cell budding factors to sites of HIV assembly. HIV also expresses a second Gag protein, p160 Gag-Pol, which lacks p6 and fails to bud from cells, consistent with the prevailing hypothesis of HIV budding. However, we show here that the severe budding defect of Gag-Pol is not caused by the absence of p6, but rather, by the presence of Pol. Specifically, we show that (i) the budding defect of Gag-Pol is unaffected by loss of HIV protease activity and is therefore an intrinsic property of the Gag-Pol polyprotein, (ii) the N-terminal 433 amino acids of Gag and Gag-Pol are sufficient to drive virus budding even though they lack p6, (iii) the severe budding defect of Gag-Pol is caused by a dominant, cis-acting inhibitor of budding in the HIV Pol domain, and (iv) Gag-Pol inhibits Gag and virus budding in trans, even at normal levels of Gag and Gag-Pol expression. These and other data support an alternative hypothesis of HIV budding as a process that is mediated by the normal, non-viral pathway of exosome/microvesicle biogenesis.

Biogenesis of the posterior pole is mediated by the exosome/microvesicle protein-sorting pathway.

Biogenesis of the posterior pole is critical to directed cell migration and other polarity-dependent processes. We show here that proteins are targeted to the posterior pole on the basis of higher order oligomerization and plasma membrane binding, the same elements that target proteins to exosomes/microvesicles (EMVs), HIV, and other retrovirus particles. We also demonstrate that the polarization of the EMV protein-sorting pathway can occur in morphologically non-polarized cells, defines the site of uropod formation, is induced by increased expression of EMV cargo proteins, and is evolutionarily conserved between humans and the protozoan Dictyostelium discoideum. Based on these results, we propose a mechanism of posterior pole biogenesis in which elevated levels of EMV cargoes (i) polarize the EMV protein-sorting pathway, (ii) generate a nascent posterior pole, and (iii) prime cells for signal-induced biogenesis of a uropod. This model also offers a mechanistic explanation for the polarized budding of EMVs and retroviruses, including HIV.

Protein targeting to exosomes/microvesicles by plasma membrane anchors.

Animal cells secrete small vesicles, otherwise known as exosomes and microvesicles (EMVs). A short, N-terminal acylation tag can target a highly oligomeric cytoplasmic protein, TyA, into secreted vesicles (Fang, Y., Wu, N., Gan, X., Yan, W., Morell, J. C., and Gould, S. J. (2007) PLoS Biol. 5, 1267-1283). However, it is not clear whether this is true for other membrane anchors or other highly oligomeric, cytoplasmic proteins. We show here that a variety of plasma membrane anchors can target TyA-GFP to sites of vesicle budding and into EMVs, including: (i) a myristoylation tag; (ii) a phosphatidylinositol-(4,5)-bisphosphate (PIP(2))-binding domain; (iii), a phosphatidylinositol-(3,4,5)-trisphosphate-binding domain; (iv) a prenylation/palmitoylation tag, and (v) a type-1 plasma membrane protein, CD43. However, the relative budding efficiency induced by these plasma membrane anchors varied over a 10-fold range, from 100% of control (AcylTyA-GFP) for the myristoylation tag and PIP(2)-binding domain, to one-third or less for the others, respectively. Targeting TyA-GFP to endosome membranes by fusion to a phosphatidylinositol 3-phosphate-binding domain induced only a slight budding of TyA-GFP, ∼2% of control, and no budding was observed when TyA-GFP was targeted to Golgi membranes via a phosphatidylinositol 4-phosphate-binding domain. We also found that a plasma membrane anchor can target two other highly oligomeric, cytoplasmic proteins to EMVs. These observations support the hypothesis that plasma membrane anchors can target highly oligomeric, cytoplasmic proteins to EMVs. Our data also provide additional parallels between EMV biogenesis and retrovirus budding, as the anchors that induced the greatest budding of TyA-GFP are the same as those that mediate retrovirus budding.