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Inducing immunogenic cell death (ICD) is a critical strategy for enhancing cancer immunotherapy. However, inefficient and risky ICD inducers along with a tumor hypoxia microenvironment seriously limit the immunotherapy efficacy. Non-specific delivery is also responsible for this inefficiency. In this work, we report a drug-free bacteria-derived outer membrane vesicle (OMV)-functionalized Fe 3 O 4 -MnO 2 (FMO) nanoplatform that realized neutrophil-mediated targeted delivery and photothermally enhanced cancer immunotherapy. In this system, modification of OMVs derived from Escherichia coli enhanced the accumulation of FMO NPs at the tumor tissue through neutrophil-mediated targeted delivery. The FMO NPs underwent reactive decomposition in the tumor site, generating manganese and iron ions that induced ICD and O 2 that regulated the tumor hypoxia environment. Moreover, OMVs are rich in pathogen-associated pattern molecules that can overcome the tumor immunosuppressive microenvironment and effectively activate immune cells, thereby enhancing specific immune responses. Photothermal therapy (PTT) caused by MnO 2 and Fe 3 O 4 can not only indirectly stimulate systemic immunity by directly destroying tumor cells but also promote the enrichment of neutrophil-equipped nanoparticles by enhancing the inflammatory response at the tumor site. Finally, the proposed multi-modal treatment system with targeted delivery capability realized effective tumor immunotherapy to prevent tumor growth and recurrence.

Sphingolipids (SLs) are essential structural components of mammalian cell membranes. Our group recently determined that the oral anaerobe Porphyromonas gingivalis delivers its SLs to host cells and that the ability of P. gingivalis to synthesize SLs limits the elicited host inflammatory response during cellular infection. As P. gingivalis robustly produces outer membrane vesicles (OMVs), we hypothesized that OMVs serve as a delivery vehicle for SLs, that the SL status of the OMVs may impact cargo loading to OMVs, and that SL-containing OMVs limit elicited host inflammation similar to that observed by direct bacterial challenge. Transwell cell culture experiments determined that in comparison to the parent strain W83, the SL-null mutant elicited a hyperinflammatory immune response from THP-1 macrophage-like cells with elevated tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), and IL-6. Targeted assessment of Toll-like receptors (TLRs) identified elevated expression of TLR2, unchanged TLR4, and elevated expression of the adaptor molecules MyD88 and TRIF (Toll/IL-1 receptor domain-containing adaptor-inducing beta interferon) by SL-null P. gingivalis No significant differences in gingipain activity were observed in our infection models, and both strains produced OMVs of similar sizes. Using comparative two-dimensional gel electrophoresis, we identified differences in the protein cargo of the OMVs between parent and SL-null strain. Importantly, use of purified OMVs recapitulated the cellular inflammatory response observed in the transwell system with whole bacteria. These findings provide new insights into the role of SLs in P. gingivalis OMV cargo assembly and expand our understanding of SL-OMVs as bacterial structures that modulate the host inflammatory response., (Copyright © 2021 Rocha et al.)

Plague caused by the Gram-negative bacterium, Yersinia pestis, is still endemic in parts of the world today. Protection against pneumonic plague is essential to prevent the development and spread of epidemics. Despite this, there are currently no licensed plague vaccines in the western world. Here we describe the means of delivering biologically active plague vaccine antigens directly to mucosal sites of plague infection using highly stable microvesicles (outer membrane vesicles; OMVs) that are naturally produced by the abundant and harmless human commensal gut bacterium Bacteroides thetaiotaomicron (Bt). Bt was engineered to express major plague protective antigens in its OMVs, specifically Fraction 1 (F1) in the outer membrane and LcrV (V antigen) in the lumen, for targeted delivery to the gastrointestinal (GI) and respiratory tracts in a non-human primate (NHP) host. Our key findings were that Bt OMVs stably expresses F1 and V plague antigens, particularly the V antigen, in the correct, immunogenic form. When delivered intranasally V-OMVs elicited substantive and specific immune and antibody responses, both in the serum [immunoglobulin (Ig)G] and in the upper and lower respiratory tract (IgA); this included the generation of serum antibodies able to kill plague bacteria. Our results also showed that Bt OMV-based vaccines had many desirable characteristics, including: biosafety and an absence of any adverse effects, pathology or gross alteration of resident microbial communities (microbiotas); high stability and thermo-tolerance; needle-free delivery; intrinsic adjuvanticity; the ability to stimulate both humoral and cell-mediated immune responses; and targeting of primary sites of plague infection., (© 2019 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)

Many microbial pathogens produce a β-(1→6)-linked poly- N -acetyl-d-glucosamine (PNAG) surface capsule, including bacterial, fungal, and protozoan cells. Broadly protective immune responses to this single conserved polysaccharide antigen in animals are possible but only when a deacetylated poly- N -acetyl-d-glucosamine (dPNAG; <30% acetate) glycoform is administered as a conjugate to a carrier protein. Unfortunately, conventional methods for natural extraction or chemical synthesis of dPNAG and its subsequent conjugation to protein carriers can be technically demanding and expensive. Here, we describe an alternative strategy for creating broadly protective vaccine candidates that involved coordinating recombinant poly- N -acetyl-d-glucosamine (rPNAG) biosynthesis with outer membrane vesicle (OMV) formation in laboratory strains of Escherichia coli The glycosylated outer membrane vesicles (glycOMVs) released by these engineered bacteria were decorated with the PNAG glycopolymer and induced high titers of PNAG-specific IgG antibodies after immunization in mice. When a Staphylococcus aureus enzyme responsible for PNAG deacetylation was additionally expressed in these cells, glycOMVs were generated that elicited antibodies to both highly acetylated PNAG (∼95-100% acetate) and a chemically deacetylated dPNAG derivative (∼15% acetate). These antibodies mediated efficient in vitro killing of two distinct PNAG-positive bacterial species, namely S. aureus and Francisella tularensis subsp. holarctica , and mice immunized with PNAG-containing glycOMVs developed protective immunity against these unrelated pathogens. Collectively, our results reveal the potential of glycOMVs for targeting this conserved polysaccharide antigen and engendering protective immunity against the broad range of pathogens that produce surface PNAG., Competing Interests: Conflict of interest statement: C.C.-B. is an inventor of intellectual properties (use of human mAb to PNAG and use of PNAG vaccines) that are licensed by Brigham and Women’s Hospital to Alopexx Vaccine, LLC, and Alopexx Pharmaceuticals, LLC. As an inventor of intellectual properties, C.C.-B. also has the right to receive a share of licensing-related income (royalties, fees) through Brigham and Women’s Hospital from Alopexx Pharmaceuticals, LLC, and Alopexx Vaccine, LLC. D.P. and M.P.D. have a financial interest in Versatope, Inc., and M.P.D. also has a financial interest in Glycobia, Inc. The interests of D.P. and M.P.D. are reviewed and managed by Cornell University in accordance with their conflict of interest policies. G.B.P. is an inventor of intellectual properties (human mAb to PNAG and PNAG vaccines) that are licensed by Brigham and Women’s Hospital to Alopexx Vaccine, LLC, and Alopexx Pharmaceuticals, LLC, entities, in which G.B.P. also holds equity. As an inventor of intellectual properties, G.B.P. also has the right to receive a share of licensing-related income (royalties, fees) through Brigham and Women’s Hospital from Alopexx Pharmaceuticals, LLC, and Alopexx Vaccine, LLC. The interests of G.B.P. are reviewed and managed by Brigham and Women’s Hospital and Partners Healthcare in accordance with their conflict of interest policies.

During infection, the release of nano-sized membrane vesicle is a process which is common both for bacteria and host cells. Host cell-derived membrane vesicles can be involved in innate and adaptive immunity whereas bacterial membrane vesicles can contribute to bacterial pathogenicity. To study the contribution of both membrane vesicle populations during infection is highly complicated as most vesicles fall within a similar size range of 30-300nm. Specialized techniques for purification are required and often no single technique complies on its own. Moreover, techniques for vesicle quantification are either complicated to use or do not distinguish between host cell-derived and bacterial membrane vesicle subpopulations. Here we demonstrate a bead-based platform that allows a semi-quantitatively analysis by flow-cytometry of bacterial and host-cell derived membrane vesicles. We show this method can be used to study heterogeneous and complex vesicle populations composed of bacterial and host-cell membrane vesicles. The easy accessible design of the protocol makes it also highly suitable for screening procedures to assess how intrinsic and environmental factors affect vesicle release., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Outer membrane vesicles (OMVs) have proven to be highly immunogenic and induced an immune response against bacterial infection in human clinics and animal models. We sought to investigate whether engineered OMVs can be a feasible antigen-delivery platform for efficiently inducing specific antibody responses. In this study, Omp22 (an outer membrane protein of A. baumannii) was displayed on E. coli DH5α-derived OMVs (Omp22-OMVs) using recombinant gene technology. The morphological features of Omp22-OMVs were similar to those of wild-type OMVs (wtOMVs). Immunization with Omp22-OMVs induced high titers of Omp22-specific antibodies. In a murine sepsis model, Omp22-OMV immunization significantly protected mice from lethal challenge with a clinically isolated A. baumannii strain, which was evidenced by the increased survival rate of the mice, the reduced bacterial burdens in the lung, spleen, liver, kidney, and blood, and the suppressed serum levels of inflammatory cytokines. In vitro opsonophagocytosis assays showed that antiserum collected from Omp22-OMV-immunized mice had bactericidal activity against clinical isolates, which was partly specific antibody-dependent. These results strongly indicated that engineered OMVs could display a whole heterologous protein (~22 kDa) on the surface and effectively induce specific antibody responses, and thus OMVs have the potential to be a feasible vaccine platform.

Trivalent native outer membrane vesicles (nOMVs) derived from three genetically modified Neisseria meningitidis serogroup B strains have been previously evaluated immunologically in mice and rabbits. This nOMV vaccine elicited serum bactericidal activity (SBA) against multiple N. meningitidis serogroup B strains as well as strains from serogroups C, Y, W, and X. In this study, we used trivalent nOMVs isolated from the same vaccine strains and evaluated their immunogenicity in an infant Rhesus macaque (IRM) model whose immune responses to the vaccine are likely to be more predictive of the responses in human infants. IRMs were immunized with trivalent nOMV vaccines and sera were evaluated for exogenous human serum complement-dependent SBA (hSBA). Antibody responses to selected hSBA generating antigens contained within the trivalent nOMVs were also measured and we found that antibody titers against factor H binding protein variant 2 (fHbpv2) were very low in the sera from animals immunized with these original nOMV vaccines. To increase the fHbp content in the nOMVs, the vaccine strains were further genetically altered by addition of another fHbp gene copy into the porB locus. Trivalent nOMVs from the three new vaccine strains had higher fHbp antigen levels and generated higher anti-fHbp antibody responses in immunized mice and IRMs. As expected, fHbp insertion into the porB locus resulted in no PorB expression. Interestingly, higher expression of PorA, an hSBA generating antigen, was observed for all three modified vaccine strains. Compared to the trivalent nOMVs from the original strains, higher PorA levels in the improved nOMVs resulted in higher anti-PorA antibody responses in mice and IRMs. In addition, hSBA titers against other strains with PorA as the only hSBA antigen in common with the vaccine strains also increased., (Copyright © 2016 Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ. Published by Elsevier Ltd.. All rights reserved.)

Outer membrane vesicles (OMVs) are vesicle-like structures, widely present in gram-negative bacteria and even in some gram-positive bacteria. OMVs contain biological active substances and their sizes are normally between 20 to 250 nm. Components of OMVs include lipopolysaccharide, outer membrane protein, phospholipids, DNA, as well as the periplasmic components produced during their formation. OMVs are non-viable vesicles that contain multiple antigenic proteins from the bacterial outer membrane, and are capable of activating the immune system, therefore they are considered to be potential vaccine candidates. Although outer membrane vesicles were discoverd more than 50 years ago, hardly any reports were published in China. In this review, we summarized the progress of outer membrane vesicles as a novel strategy for disease prevention and control in two aspects: the mechanism of the outer membrane vesicle-induced immune response and the advances in OMVs vaccine. This review provides some information on outer membrane vesicles as vaccine development.

Survival of Mycobacterium tuberculosis (Mtb) within the host macrophage is mediated through pathogen-dependent inhibition of phagosome-lysosome fusion, which enables bacteria to persist within the immature phagosomal compartment. By employing ultrastructural examination of different field isolates supported by biochemical analysis, we found that some of the Mtb strains were in fact poorly adapted for subsistence within endocytic vesicles of infected macrophages. Instead, through a mechanism involving activation of host cytosolic phospholipase A2, these bacteria rapidly escaped from phagosomes, and established residence in the cytoplasm of the host cell. Interestingly, by facilitating an enhanced suppression of host cellular autophagy, this translocation served as an alternate virulence acquisition mechanism. Thus, our studies reveal plasticity in the adaptation strategies employed by Mtb, for survival in the host macrophage.

Infection of fish with the facultative intracellular bacterium Francisella noatunensis remains an unresolved problem for aquaculture industry worldwide as it is difficult to vaccinate against without using live attenuated vaccines. Outer membrane vesicles (OMVs) are biological structures shed by Gram-negative bacteria in response to various environmental stimuli. OMVs have successfully been used to vaccinate against both intracellular and extracellular pathogens, due to an ability to stimulate innate, cell-mediated and humoral immune responses. We show by using atomic force and electron microscopy that the fish pathogenic bacterium F. noatunensis subspecies noatunensis (F.n.n.) shed OMVs both in vitro into culture medium and in vivo in a zebrafish infection model. The main protein constituents of the OMV are IglC, PdpD and PdpA, all known Francisella virulence factors, in addition to the outer membrane protein FopA and the chaperonin GroEL, as analyzed by mass spectrometry. The vesicles, when used as a vaccine, reduced proliferation of the bacterium and protected zebrafish when subsequently challenged with a high dose of F.n.n. without causing adverse effects for the host. Also granulomatous responses were reduced in F.n.n.-challenged zebrafish after OMV vaccination. Taken together, the data support the possible use of OMVs as vaccines against francisellosis in fish., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The traditional view of how intracellular effector proteins are recruited to the B cell antigen receptor (BCR) complex at the plasma membrane is based on the occurrence of direct protein-protein interactions, as exemplified by the recruitment of the tyrosine kinase Syk (spleen tyrosine kinase) to phosphorylated motifs in BCR signaling subunits. By contrast, the subcellular targeting of the cytosolic adaptor protein SLP-65 (Src homology 2 domain-containing leukocyte adaptor protein of 65 kD), which serves as a proximal Syk substrate, is unclear. We showed that SLP-65 activation required its association at vesicular compartments in resting B cells. A module of ~50 amino acid residues located at the amino terminus of SLP-65 anchored SLP-65 to the vesicles. Nuclear magnetic resonance spectroscopy showed that the SLP-65 amino terminus was structurally disordered in solution but could bind in a structured manner to noncharged lipid components of cellular membranes. Our finding that preformed vesicular signaling scaffolds are required for B cell activation indicates that vesicles may deliver preassembled signaling cargo to sites of BCR activation., (Copyright © 2014, American Association for the Advancement of Science.)

The discovery that submicron-sized extracellular vesicles (EVs) are generated by both prokaryotic and eukaryotic cells might have a profound effect on experimental and clinical sciences, and could pave the way for new strategies to combat various diseases. EVs are carriers of pathogen-associated and damage-associated molecular patterns, cytokines, autoantigens and tissue-degrading enzymes. In addition to a possible role in the pathogenesis of a number of inflammatory conditions, such as infections and autoimmune diseases, EVs, including microvesicles (also known as microparticles), exosomes and apoptotic vesicles, have therapeutic potential and might be used as biomarkers for inflammatory diseases. Therefore, molecular diagnostics and targeted therapy could benefit from expanding knowledge in the field. In this Review, we summarize important developments and propose that extracellular vesicles could be used as therapeutic vehicles and as targets for the treatment and prevention of inflammatory diseases.

Recognition of phosphatidylserine (PS) lipids exposed on the extracellular leaflet of plasma membranes is implicated in both apoptotic cell removal and immune regulation. The PS receptor T cell immunoglobulin and mucin-domain-containing molecule 4 (Tim4) regulates T-cell immunity via phagocytosis of both apoptotic (high PS exposure) and nonapoptotic (intermediate PS exposure) activated T cells. The latter population must be removed at lower efficiency to sensitively control immune tolerance and memory cell population size, but the molecular basis for how Tim4 achieves this sensitivity is unknown. Using a combination of interfacial X-ray scattering, molecular dynamics simulations, and membrane binding assays, we demonstrate how Tim4 recognizes PS in the context of a lipid bilayer. Our data reveal that in addition to the known Ca(2+)-coordinated, single-PS binding pocket, Tim4 has four weaker sites of potential ionic interactions with PS lipids. This organization makes Tim4 sensitive to PS surface concentration in a manner capable of supporting differential recognition on the basis of PS exposure level. The structurally homologous, but functionally distinct, Tim1 and Tim3 are significantly less sensitive to PS surface density, likely reflecting the differences in immunological function between the Tim proteins. These results establish the potential for lipid membrane parameters, such as PS surface density, to play a critical role in facilitating selective recognition of PS-exposing cells. Furthermore, our multidisciplinary approach overcomes the difficulties associated with characterizing dynamic protein/membrane systems to reveal the molecular mechanisms underlying Tim4's recognition properties, and thereby provides an approach capable of providing atomic-level detail to uncover the nuances of protein/membrane interactions.

Oral delivery of Gram positive bacteria, often derived from the genera Lactobacillus or Bifidobacterium, can modulate immune function. Although the exact mechanisms remain unclear, immunomodulatory effects may be elicited through the direct interaction of these bacteria with the intestinal epithelium or resident dendritic cell (DC) populations. We analyzed the immune activation properties of Lactobacilli and Bifidobacterium species and made the surprising observation that cellular responses in vitro were differentially influenced by the presence of serum, specifically the extracellular vesicle (EV) fraction. In contrast to the tested Lactobacilli species, tested Bifidobacterium species induce TLR2/6 activity which is inhibited by the presence of EVs. Using specific TLR ligands, EVs were found to enhance cellular TLR2/1 and TLR4 responses while TLR2/6 responses were suppressed. No effect could be observed on cellular TLR5 responses. We determined that EVs play a role in bacterial aggregation, suggesting that EVs interact with bacterial surfaces. EVs were found to slightly enhance DC phagocytosis of Bifidobacterium breve whereas phagocytosis of Lactobacillus rhamnosus was virtually absent upon serum EV depletion. DC uptake of a non-microbial substance (dextran) was not affected by the different serum fractions suggesting that EVs do not interfere with DC phagocytic capacity but rather modify the DC-microbe interaction. Depending on the microbe, combined effects of EVs on TLR activity and phagocytosis result in a differential proinflammatory DC cytokine release. Overall, these data suggest that EVs play a yet unrecognized role in host-microbe responses, not by interfering in recipient cellular responses but via attachment to, or scavenging of, microbe-associated molecular patterns. EVs can be found in any tissue or bodily fluid, therefore insights into EV-microbe interactions are important in understanding the mechanism of action of potential probiotics and gut immune homeostasis.

Francisella spp. are highly infectious and virulent bacteria that cause the zoonotic disease tularemia. Knowledge is lacking for the virulence factors expressed by Francisella and how these factors are secreted and delivered to host cells. Gram-negative bacteria constitutively release outer membrane vesicles (OMV), which may function in the delivery of virulence factors to host cells. We identified growth conditions under which Francisella novicida produces abundant OMV. Purification of the vesicles revealed the presence of tube-shaped vesicles in addition to typical spherical OMV, and examination of whole bacteria revealed the presence of tubes extending out from the bacterial surface. Recently, both prokaryotic and eukaryotic cells have been shown to produce membrane-enclosed projections, termed nanotubes, which appear to function in cell-cell communication and the exchange of molecules. In contrast to these previously characterized structures, the F. novicida tubes are produced in liquid as well as on solid medium and are derived from the OM rather than the cytoplasmic membrane. The production of the OMV and tubes (OMV/T) by F. novicida was coordinately regulated and responsive to both growth medium and growth phase. Proteomic analysis of purified OMV/T identified known Francisella virulence factors among the constituent proteins, suggesting roles for the vesicles in pathogenesis. In support of this, production of OM tubes by F. novicida was stimulated during infection of macrophages and addition of purified OMV/T to macrophages elicited increased release of proinflammatory cytokines. Finally, vaccination with purified OMV/T protected mice from subsequent challenge with highly lethal doses of F. novicida.

Accumulating evidence underscores the immune synapse (IS) of naive T cells as a site of intense vesicular trafficking. At variance with helper and cytolytic effectors, which use the IS as a secretory platform to deliver cytokines and/or lytic granules to their cellular targets, this process is exploited by naive T cells as a means to regulate the assembly and maintenance of the IS, on which productive signaling and cell activation crucially depend. We have recently identified a role of the intraflagellar transport (IFT) system, which is responsible for the assembly of the primary cilium, in the non-ciliated T-cell, where it controls IS assembly by promoting polarized T-cell receptor recycling. This unexpected finding not only provides new insight into the mechanisms of IS assembly but also strongly supports the notion that the IS and the primary cilium, which are both characterized by a specialized membrane domain highly enriched in receptors and signaling mediators, share architectural similarities and are homologous structures. Here, we review our current understanding of vesicular trafficking in the regulation of the assembly and maintenance of the naive T-cell IS and the primary cilium, with a focus on the IFT system., (© 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.)

Campylobacter jejuni is the most prevalent cause of food-borne gastroenteritis in the developed world; however, the molecular basis of pathogenesis is unclear. Secretion of virulence factors is a key mechanism by which enteric bacterial pathogens interact with host cells to enhance survival and/or damage the host. However, C. jejuni lacks the virulence-associated secretion systems possessed by other enteric pathogens. Many bacterial pathogens utilize outer membrane vesicles (OMVs) for delivery of virulence factors into host cells. In the absence of prototypical virulence-associated secretion systems, OMVs could be an important alternative for the coordinated delivery of C. jejuni proteins into host cells. Proteomic analysis of C. jejuni 11168H OMVs identified 151 proteins, including periplasmic and outer membrane-associated proteins, but also many determinants known to be important in survival and pathogenesis, including the cytolethal distending toxin (CDT). C. jejuni OMVs contained 16 N-linked glycoproteins, indicating a delivery mechanism by which these periplasm-located yet immunogenic glycoproteins can interact with host cells. C. jejuni OMVs possess cytotoxic activity and induce a host immune response from T84 intestinal epithelial cells (IECs), which was not reduced by OMV pretreatment with proteinase K or polymyxin B prior to coincubation with IECs. Pretreatment of IECs with methyl-beta-cyclodextrin partially blocks OMV-induced host immune responses, indicating a role for lipid rafts in host cell plasma membranes during interactions with C. jejuni OMVs. OMVs isolated from a C. jejuni 11168H cdtA mutant induced interleukin-8 (IL-8) to the same extent as did wild-type OMVs, suggesting OMV induction of IL-8 is independent of CDT.

Immunological control of infections or tumors depends on the release of effector cytokines and polarized secretion of cytotoxic granules from T cells and natural killer cells. Here we show that the sorting receptor Sortilin controlled both processes. In murine Sortilin-deficient cytotoxic T lymphocytes, regulated secretion of granzyme A and cytotoxic killing was enhanced and correlated with increased vesicle-associated membrane protein 7 availability. In contrast, loss of Sortilin reduced the release of interferon-γ upon infections and in autoimmune colitis. Exit of interferon-γ from the Golgi apparatus required the presence of Sortilin. Furthermore, we tracked the transport route of interferon-γ beyond this Sortilin-dependent Golgi to early endosome step. In wild-type T cells, trafficking of interferon-γ from the endosomal sorting platform to the plasma membrane proceeded independently of recycling endosomes, and interferon-γ remained excluded from late endosomes. Our results suggest that Sortilin modulates systemic immune responses through exocytic sorting of immunological effector molecules., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Microtubule cytoskeleton is a dynamic structure involved in the maintenance of eukaryote cell shape, motion of cilia and flagellum, and intracellular movement of vesicles and organelles. Many antibodies against tubulins have been described, most of them against the C-terminal portion, which is exposed at the outside of the microtubules. By generating a novel set of monoclonal antibodies against the cytoskeleton of Trypanosoma cruzi, a flagellate protozoan that causes Chagas' disease, we selected a clone (mAb 3G4) that recognizes β-tubulin. The epitope for mAb 3G4 was mapped by pepscan to a highly conserved sequence motif found between α-helices 11 and 12 of the C-terminus of β-tubulin in eukaryotes. It labels vesicular structures in both T. cruzi and mammalian cells, colocalizing respectively with a major cysteine protease (Cruzipain) and lysosome associated protein (LAMP2) respectively, but it does not label regular microtubules on these cellular models. We propose that the epitope recognized by mAb 3G4 is exposed only in a form of tubulin associated with endosomes.

Outer membrane vesicles (OMVs) released by some gram-negative bacteria have been shown to exert immunomodulatory effects that favor the establishment of the infection. The aim of the present study was to assess the interaction of OMVs from Brucella abortus with human epithelial cells (HeLa) and monocytes (THP-1), and the potential immunomodulatory effects they may exert. Using confocal microscopy and flow cytometry, FITC-labeled OMVs were shown to be internalized by both cell types. Internalization was shown to be partially mediated by clathrin-mediated endocytosis. Pretreatment of THP-1 cells with Brucella OMVs inhibited some cytokine responses (TNF-α and IL-8) to E. coli LPS, Pam3Cys or flagellin (TLR4, TLR2 and TLR5 agonists, respectively). Similarly, pretreatment with Brucella OMVs inhibited the cytokine response of THP-1 cells to B. abortus infection. Treatment of THP-1 cells with OMVs during IFN-γ stimulation reduced significantly the inducing effect of this cytokine on MHC-II expression. OMVs induced a dose-dependent increase of ICAM-1 expression on THP-1 cells and an increased adhesion of these cells to human endothelial cells. The addition of OMVs to THP-1 cultures before the incubation with live B. abortus resulted in increased numbers of adhered and internalized bacteria as compared to cells not treated with OMVs. Overall, these results suggest that OMVs from B. abortus exert cellular effects that promote the internalization of these bacteria by human monocytes, but also downregulate the innate immune response of these cells to Brucella infection. These effects may favor the persistence of Brucella within host cells.

Background and Objectives: Damage to intestinal mucosa in celiac disease (CD) is mediated both by inflammation due to adaptive and innate immune responses, with IL-15 as a major mediator of the innate immune response, and by proliferation of crypt enterocytes as an early alteration of CD mucosa causing crypts hyperplasia. We have previously shown that gliadin peptide P31-43 induces proliferation of cell lines and celiac enterocytes by delaying degradation of the active epidermal growth factor receptor (EGFR) due to delayed maturation of endocytic vesicles. IL-15 is increased in the intestine of patients affected by CD and has pleiotropic activity that ultimately results in immunoregulatory cross-talk between cells belonging to the innate and adaptive branches of the immune response. Aims of this study were to investigate the role of P31-43 in the induction of cellular proliferation and innate immune activation., Methods/principal Findings: Cell proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation both in CaCo-2 cells and in biopsies from active CD cases and controls. We used real-time PCR to evaluate IL-15 mRNA levels and FACS as well as ELISA and Western Blot (WB) analysis to measure protein levels and distribution in CaCo-2 cells. Gliadin and P31-43 induce a proliferation of both CaCo-2 cells and CD crypt enterocytes that is dependent on both EGFR and IL-15 activity. In CaCo-2 cells, P31-43 increased IL-15 levels on the cell surface by altering intracellular trafficking. The increased IL-15 protein was bound to IL15 receptor (IL-15R) alpha, did not require new protein synthesis and functioned as a growth factor., Conclusion: In this study, we have shown that P31-43 induces both increase of the trans-presented IL-15/IL5R alpha complex on cell surfaces by altering the trafficking of the vesicular compartments as well as proliferation of crypt enterocytes with consequent remodelling of CD mucosa due to a cooperation of IL-15 and EGFR.

As new disease threats arise and existing pathogens grow resistant to conventional interventions, attention increasingly focuses on the development of vaccines to induce protective immune responses. Given their admirable safety records, protein subunit vaccines are attractive for widespread immunization, but their disadvantages include poor immunogenicity and expensive manufacture. We show here that engineered Escherichia coli outer membrane vesicles (OMVs) are an easily purified vaccine-delivery system capable of greatly enhancing the immunogenicity of a low-immunogenicity protein antigen without added adjuvants. Using green-fluorescent protein (GFP) as the model subunit antigen, genetic fusion of GFP with the bacterial hemolysin ClyA resulted in a chimeric protein that elicited strong anti-GFP antibody titers in immunized mice, whereas immunization with GFP alone did not elicit such titers. Harnessing the specific secretion of ClyA to OMVs, the ClyA-GFP fusion was found localized in OMVs, resulting in engineered recombinant OMVs. The anti-GFP humoral response in mice immunized with the engineered OMV formulations was indistinguishable from the response to the purified ClyA-GFP fusion protein alone and equal to purified proteins absorbed to aluminum hydroxide, a standard adjuvant. In a major improvement over current practice, engineered OMVs containing ClyA-GFP were easily isolated by ultracentrifugation, effectively eliminating the need for laborious antigen purification from cell-culture expression systems. With the diverse collection of heterologous proteins that can be functionally localized with OMVs when fused with ClyA, this work signals the possibility of OMVs as a robust and tunable technology platform for a new generation of prophylactic and therapeutic vaccines.

Gangliosides are glycolipids mainly present at the plasma membrane (PM). Antibodies to gangliosides have been associated with a wide range of neuropathy syndromes. Particularly, antibodies to GM1 ganglioside are present in patients with Guillain-Barré syndrome (GBS). We investigated the binding and intracellular fate of antibody to GM1 obtained from rabbits with experimental GBS in comparison with the transport of cholera toxin (CTx), which binds with high affinity to GM1. We demonstrated that antibody to GM1 is rapidly and specifically endocytosed in CHO-K1 cells. After internalization, the antibody transited sorting endosomes to accumulate at the recycling endosome. Endocytosed antibody to GM1 is recycled back to the PM and released into the culture medium. In CHO-K1 cells, antibody to GM1 colocalized with co-endocytosed CTx at early and recycling endosomes, but not in Golgi complex and endoplasmic reticulum, where CTx was also located. Antibody to GM1, in contraposition to CTx, showed a reduced internalization to recycling endosomes in COS-7 cells and neural cell lines SH-SY5Y and Neuro2A. Results from photobleaching studies revealed differences in the lateral mobility of antibody to GM1 in the PM of analyzed cell lines, suggesting a relationship between the efficiency of endocytosis and lateral mobility of GM1 at the PM. Taken together, results indicate that two different ligands of GM1 ganglioside (antibody and CTx) are differentially endocytosed and trafficked, providing the basis to gain further insight into the mechanisms that operate in the intracellular trafficking of glycosphingolipid-binding toxins and pathological effects of neuropathy-associated antibodies.

Release of outer membrane vesicles is a common feature of Gram negative bacteria. There is growing interest in the use of these vesicles in the development of affordable vaccines. However, to exploit their full potential a convenient system to generate recombinant vesicles would be highly desirable. Here, we report the design of a versatile system for preparation of recombinant outer membrane vesicles based on an engineered autotransporter. Two model vaccine antigens of Leishmania were expressed as fusion proteins with the transporter domain of AIDA, the Escherichia coli adhesin involved in diffuse adhesion. Single subcutaneous injections of recombinant vesicles boosted vaccine antigen-specific antibody responses in mice primed with live recombinant salmonella vaccines by 6-40-fold. The results further show an expansion of only the vaccine antigen-specific antibody response indicating a great potential of this approach for prime boost vaccination strategies.

To develop low endotoxic and multi-immunogenic outer membrane vesicles (OMVs), a deletion mutant of the msbB gene in Salmonella enterica serovar Typhimurium (S. Typhimurium) was used as a source of low endotoxic OMV, and an expression vector of the canine parvovirus (CPV) VP2 epitope fused to the bacterial OmpA protein was constructed and transformed into the Salmonella deltaAmsbB mutant. In a lethality test, BALB/c mice injected intraperitoneally with the Salmonella deltaAmsbB mutant survived for 7 days, whereas mice injected intraperitoneally with the wild type survived for 3 days. Moreover, all mice inoculated orally with the deltaAmsbB mutant survived for 30 days, but 80% of mice inoculated orally with the wild type survived. The OmpA::CPV VP2 epitope fusion protein was expressed successfully and associated with the outer membrane and OMV fractions from the mutant S. Typhimurium transformed with the fusion protein-expressing vector. In immunogenicity tests, sera obtained from the mice immunized with either the Salmonella msbB mutant or its OMVs containing the OmpA::CPV VP2 epitope showed bactericidal activities against wild-type S. Typhimurium and contained specific antibodies to the CPV VP2 epitope. In the hemagglutination inhibition (HI) assay as a measurement of CPV-neutralizing activity in the immune sera, there was an 8-fold increase of HI titer in the OMV-immunized group compared with the control. These results suggested that the CPV-neutralizing antibody response was raised by immunization with OMV containing the OmpA::CPV VP2 epitope, as well as the protective immune response against S. Typhimurium in BALB/c mice.

Cytotoxic lymphocytes kill target cells by releasing the content of secretory lysosomes at the immune synapse. To understand the dynamics and control of cytotoxic immune synapses, we imaged human primary, live natural killer cells on lipid bilayers carrying ligands of activation receptors. Formation of an organized synapse was dependent on the presence of the beta2 integrin ligand ICAM-1. Ligands of coactivation receptors 2B4 and NKG2D segregated into central and peripheral regions, respectively. Lysosomal protein LAMP-1 that was exocytosed during degranulation accumulated in a large and spatially stable cluster, which overlapped with a site of membrane internalization. Lysosomal compartments reached the plasma membrane at focal points adjacent to centrally accumulated LAMP-1. Imaging of fixed cells revealed that perforin-containing granules were juxtaposed to an intracellular compartment where exocytosed LAMP-1 was retrieved. Thus, cytotoxic immune synapses include a central region of bidirectional vesicular traffic, which is controlled by integrin signaling.

Dendritic cells (DCs) are able to orchestrate innate and acquired immunity and can activate and sustain a long-lasting anti-tumor immune response in vivo when used as anti-tumor cell therapy. The selection of the antigen and the choice of its formulation are key points in designing anti-cancer DC-based vaccines. Cell released vesicles/exosomes have been shown to transfer antigens, HLAI/peptide complexes and co-stimulatory molecules to recipient cells. In this study we describe the generation of an allogenic microvesicle cell factory in which the expression of a specific tumor antigen was combined to the expression of co-stimulatory and allogeneic molecules. The DG75 lymphoblastoid cell line was selected as microvesicle producer and transfected with ErbB2, as tumor antigen prototype. The shed microvesicles transferred antigenic components to recipient DCs, increasing their immunogenicity. DC pulsing resulted in cross-presentation of ErbB2 both in HLAI and HLAII compartments, and ErbB2-specific CD8+ T cells from cancer patients were activated by DCs pulsed with vesicle-bound ErbB2. The microvesicle cell factory proposed may represent a source of cell free immunogen to be used for DC-based cancer therapy.

By presenting antigenic peptides on the cell surface, human leukocyte antigen (HLA) class I molecules are critical for immune defense. Their surface density determines, to a large extent, the level of CD8(+) T cell-dependent immune reactions; their loss is a major mechanism of immune escape. Therefore, powerful processes should regulate their surface expression. Here we document the mechanisms used by CD99 to mediate HLA class I modulation. Up-regulation of HLA class I by IFN-gamma requires CD99. In the trans Golgi network (TGN), and up to the cell surface, CD99 and HLA class I are physically associated via their transmembrane domain. CD99 also binds p230/golgin-245, a coiled-coil protein that recycles between the cytosol and buds/vesicles of the TGN and which plays a fundamental role in trafficking transport vesicles. p230/golgin-245 is anchored within TGN membranes via its Golgin-97, RanBP1, IMh1p, P230 (GRIP) domain and the overexpression of which leads to surface and intracellular down-modulation of HLA class I molecules.

In eukaryotes, proteins of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family are believed to have a general role for the fusion of intracellular transport vesicles with acceptor membranes. Arabidopsis thaliana PEN1 syntaxin resides in the plasma membrane and was previously shown to act together with its partner SNAREs, the adaptor protein SNAP33, and endomembrane-anchored VAMP721/722 in the execution of secretory immune responses against powdery mildew fungi. We conducted a structure-function analysis of PEN1 and show that N-terminal phospho-mimicking and non-phosphorylatable variants neither affected binary nor ternary SNARE complex formation with cognate partners in vitro. However, expression of these syntaxin variants at native protein levels in a pen1 mutant background suggests that phosphorylation is required for full resistance activity in planta. All tested site-directed substitutions of SNARE domain or "linker region" residues reduced PEN1 defense activity. Two of the variants failed to form ternary complexes with the partner SNAREs in vitro, possibly explaining their diminished in planta activity. However, impaired pathogen defense in plants expressing a linker region variant is likely because of PEN1 destabilization. Although Arabidopsis PEN1 and SYP122 syntaxins share overlapping functions in plant growth and development, PEN1 activity in disease resistance is apparently the result of a complete functional specialization. Our findings are consistent with the hypothesis that PEN1 acts in plant defense through the formation of ternary SNARE complexes and point to the existence of unknown regulatory factors. Our data indirectly support structural inferences that the four-helical coiled coil bundle in ternary SNARE complexes is formed in a sequential order from the N- to C-terminal direction.

Nepmucin/CLM-9 is an Ig domain-containing sialomucin expressed in vascular endothelial cells. Here we show that, like CD31, nepmucin was localized to interendothelial contacts and to vesicle-like structures along the cell border and underwent intracellular recycling. Functional analyses showed that nepmucin mediated homotypic and heterotypic cell adhesion via its Ig domain. Nepmucin-expressing endothelial cells showed enhanced lymphocyte transendothelial migration (TEM), which was abrogated by anti-nepmucin mAbs that block either homophilic or heterophilic binding. Notably, the mAbs that inhibited homophilic binding blocked TEM without affecting lymphocyte adhesion. These results suggest that endothelial nepmucin promotes lymphocyte TEM using multiple adhesion pathways.

Staphylococcus aureus colonizes the lungs of cystic fibrosis patients and treatment with antibiotics usually results in recurrent and relapsing infections. We have shown that S. aureus can invade and replicate within a cystic fibrosis epithelial cell line (CFT-1), and that these internalized bacteria subsequently escape from the endocytic vesicle. The accessory gene regulator, agr, in S. aureus has been shown to control the expression of a large number of secreted toxins involved in virulence. Here we show that an agr mutant of S. aureus strain RN6390 was unable to escape from the endocytic vesicle after invasion of the CFT-1 cells using markers of vesicular trafficking (LAMP-1 and 2, LysoTracker and Vacuolar-ATPase). Trafficking analysis of live S. aureus which did not express alpha-haemolysin, a specific agr regulated toxin, revealed a defect in vesicular escape that was undistinguishable from the trafficking defect exhibited by the agr mutant. Furthermore, overexpression of alpha-haemolysin under an inducible promoter in an agr mutant of S. aureus partially restored the phagosome-escaping phenotype of an agr mutant. These results demonstrate that the expression of agr is required for vesicular escape, and that biologically active alpha-haemolysin is required for S. aureus escape from the endocytic vesicle into the cytosol of CFT-1 cells.

Microvesicles (MVs) can derive from several cell types and their membranes contain cell surface elements. Their role is increasingly recognized in cell-to-cell communication, as they act as both paracrine and remote messengers, occurring in circulating form as well as in plasma. Successful pregnancy requires a series of interactions between the maternal immune system and the implanted fetus, such that the semi-allograft will not be rejected. These interactions occur at the materno-placental interface and/or at a systemic level. In the present study we identified for the first time the in vivo plasma pattern of the MVs of third-trimester, healthy pregnant women, their cellular origin, and their target cells using flow cytometry and confocal laser microscopy. We searched for the cellular target molecules of thrombocyte-derived MVs with the help of neutralizing antibodies. We examined the in vitro effects of MVs on STAT3 phosphorylation of primary lymphocytes and Jurkat cells. We found that both placental trophoblast-derived and maternal thrombocyte-derived MVs bind to circulating peripheral T lymphocytes, but not to B lymphocytes or NK cells. We were able to show that the P-selectin (CD62P)-PSGL-1 (CD162) interaction is one mechanism binding platelet-derived MVs to T cells. We were also able to demonstrate that MV-lymphocyte interactions induce STAT3 phosphorylation in T cells. Our findings indicate that both thrombocyte- and trophoblast-derived MVs may play an important role in the immunomodulation of pregnancy. We suggest that the transfer of different signals via MVs represents a novel form of communication between the placenta and the maternal immune system, and that MVs contribute to the establishment of stable immune tolerance to the semi-allograft fetus.

Exosomes are nanovesicles originating from multivesicular bodies that are secreted by a variety of cell types. The dual capability of exosomes to promote immunity or to induce tolerance has prompted their clinical use as vehicles for vaccination against different human diseases. In the present study, the effect of allergen-specific exosomes from tolerized mice on the development of allergen-induced allergic response was determined using a mouse model. Mice were tolerized by respiratory exposure to the olive pollen allergen Ole e 1. Exosome-like vesicles were isolated from bronchoalveolar lavage fluid of the animals by the well-established filtration and ultracentrifugation procedure, characterized by electron microscopy, Western blot, and FACS analyses, and assessed in a prophylactic protocol. To this end, BALB/c mice were intranasally treated with tolerogenic exosomes or naive exosomes as control, 1 wk before sensitization/challenge to Ole e 1. Blood, lungs, and spleen were collected and analyzed for immune responses. Intranasal administration of tolerogenic exosomes inhibited the development of IgE response, Th2 cytokine production, and airway inflammation--cardinal features of allergy--and maintained specific long-term protection in vivo. This protective effect was associated with a concomitant increase in the expression of the regulatory cytokine TGF-beta. These observations demonstrate that exosomes can induce tolerance and protection against allergic sensitization in mice. Thus, exosome-based vaccines could represent an alternative to conventional therapy for allergic diseases in humans.

The CXC chemokine CXCL12 and its cognate receptor CXCR4 play an important role in inflammation, human immunodeficiency virus (HIV) infection and cancer metastasis. The signal transduction and intracellular trafficking of CXCR4 are involved in these functions, but the underlying mechanisms remain incompletely understood. In the present study, we demonstrated that the CXCR4 formed a complex with the cytolinker protein plectin in a ligand-dependent manner in HEK293 cells stably expressing CXCR4. The glutathione-S-transferase (GST)-CXCR4 C-terminal fusion proteins co-precipitated with the full-length and the N-terminal fragments of plectin isoform 1 but not with the N-terminal deletion mutants of plectin isoform 1, thereby suggesting an interaction between the N-terminus of plectin and the C-terminus of CXCR4. This interaction was confirmed by confocal microscopic reconstructions showing co-distribution of these two proteins in the internal vesicles after ligand-induced internalization of CXCR4 in HEK293 cells stably expressing CXCR4. Knockdown of plectin with RNA interference (RNAi) significantly inhibited ligand-dependent CXCR4 internalization and attenuated CXCR4-mediated intracellular calcium mobilization and activation of extracellular signal regulated kinase 1/2 (ERK1/2). CXCL12-induced chemotaxis of HEK293 cells stably expressing CXCR4 and of Jurkat T cells was inhibited by the plectin RNAi. Moreover, CXCR4 tropic HIV-1 infection in MAGI (HeLa-CD4-LTR-Gal) cells was inhibited by the RNAi of plectin. Thus, plectin appears to interact with CXCR4 and plays an important role in CXCR4 signaling and trafficking and HIV-1 infection.

Background: Exosomes are vesicles of 30 to 100 nm produced by inward budding of endosomal compartments and are released by a range of different cell types. Exosomes from antigen-presenting cells carry immunorelevant molecules like MHC class I and II and costimulatory molecules and thus are suggested to have a role in immune modulation., Objective: To investigate the role of antigen-presenting cell derived exosomes in allergen presentation and T-cell stimulation., Methods: Exosomes were isolated from supernatants of B-cell lines derived from patients with birch pollen allergy. The exosomes were characterized with regard to the expression of surface molecules by flow cytometry. Moreover, exosomes were loaded with T-cell-activating peptides from the major birch allergen Bet v 1, and binding was tested with ELISA. Loaded exosomes were used for stimulation of Bet v 1-specific T-cell lines. Cell proliferation and cytokine production were assessed., Results: The exosomes had a phenotype typical of B cell-derived exosomes with expression of MHC, costimulatory molecules like CD86, tetraspanin proteins such as CD81, and CD19. Furthermore, B cell-derived exosomes bound Bet v 1-derived peptides and subsequently induced a dose-dependent T-cell proliferation. In addition to proliferation, T cells synthesized the cytokines IL-5 and IL-13 in response to peptide-loaded exosomes., Conclusion: These results demonstrate for the first time that exosomes isolated from B cells can present allergen-derived peptides and thereby induce T-cell proliferation and T(H)2-like cytokine production., Clinical Implications: Our data suggest that exosomes from B lymphocytes are an immunostimulatory factor in allergic immune responses.

Exosomes are 50- to 100-nm vesicles that are formed within the late endocytic compartment and released from a variety of cell types. Previously, we demonstrated that exosomes derived from dendritic cells transduced with adenoviral vectors expressing IL-10, IL-4, or Fas ligand (FasL) produce anti-inflammatory exosomes able to reduce inflammation in a murine paw delayed-type hypersensitivity model, suppress the onset on murine collagen-induced arthritis, and reduce the severity of established collagen-induce arthritis. In this study, we examined the ability of endogenous, blood-borne exosomes to regulate the immune response. Exosomes isolated from plasma of mice immunized to keyhole limpet hemocyanin, but not from naive or OVA-immunized mice, were able to suppress the keyhole limpet hemocyanin-specific delayed-type hypersensitivity inflammatory response. The anti-inflammatory effect was mediated by MHC class II(+) plasma exosomes that were also FasL(+) and CD11b(+), but CD11c(-). Moreover, the anti-inflammatory effect of the MHC class II(+) plasma-derived exosomes was, in part, dependent upon the presence of FasL in the exosomes and Fas in the recipient mouse. These results suggest that exosomes in the plasma, produced by MHC class II(+) and CD11b(+) cells, have the ability to suppress the immune response in an Ag-specific manner in part through a Fas/FasL-dependent manner.

Picornaviruses carry a small number of proteins with diverse functions that subvert and exploit the host cell. We have previously shown that three coxsackievirus B3 (CVB3) proteins (2B, 2BC, and 3A) target the Golgi complex and inhibit protein transit. Here we investigate these effects in more detail and evaluate the distribution of major histocompatibility complex (MHC) class I molecules, which are critical mediators of the CD8(+) T-cell response. We report that concomitant with viral protein synthesis, MHC class I surface expression is rapidly downregulated during infection. However, this phenomenon may not result solely from inhibition of anterograde trafficking; we propose a new mechanism whereby the CVB3 2B and 2BC proteins upregulate the internalization of MHC class I (and possibly other surface proteins), perhaps by focusing of endocytic vesicles at the Golgi complex. Thus, our findings indicate that CVB3 carries at least three nonstructural proteins that directionally complement one another; 3A disrupts the Golgi complex to inhibit anterograde transport, while 2B and/or 2BC upregulates endocytosis, rapidly removing proteins from the cell surface. Taken together, these effects may render CVB3-infected cells invisible to CD8(+) T cells and untouchable by many antiviral effector molecules. This has important implications for immune evasion by CVB3.

Purpose: Tumor-derived exosomes (TEX) have been proposed as a new kind of cancer vaccine; however, their in vivo antitumor effects are not satisfactory. In order to further improve the efficacy of vaccination with TEX, we investigated whether interleukin-2 (IL-2) genetic modification of tumor cells can make IL-2 presence in the exosomes, thus increasing antitumor effects of the TEX., Methods: E.G7-OVA tumor cells expressing Ovalbumin (OVA) as a tumor model antigen were used to prepare TEX by serial centrifugation and sucrose gradients ultracentrifugation. To demonstrate their antitumor effects, IL-2-containing exosomes (Exo/IL-2) were injected subcutaneously into C57BL/C mice: either bearing tumor or followed by tumor inoculation., Results: We found IL-2 within those exosomes as detected by both ELISA and Western blot. Vaccination with these Exo/IL-2 could induce antigen-specific Th1-polarized immune response and Cytotoxic T lymphocytes (CTL) more efficiently, resulting in more significant inhibition of tumor growth. CD8(+) T cells are the main effector cells, however, CD4(+) T cells, and NK cells are also involved in the induction of antitumor response of this approach., Conclusions: Our results demonstrate that IL-2 genetic modification of tumor cells can make the TEX contain IL-2 with the increased antitumor effects, representing a promising way of exosome-based tumor vaccine.

Infection with the SARS-associated coronavirus (SARS-CoV) induces an atypical pulmonary disease with a high lethality rate. Although the initial SARS epidemic was contained, sporadic outbreaks of the disease still occur, suggesting a continuous need for a vaccine against this virus. We therefore explored exosome-based vaccines containing the spike S proteins of SARS-CoV. S-containing exosomes were obtained by replacing the transmembrane and cytoplasmic domains of the S protein by those of VSV-G. The immunogenicity and efficacy of the S-containing exosomes were tested in mice and compared to an adenoviral vector vaccine expressing the S protein. Both, S-containing exosomes and the adenoviral vector vaccine induced neutralizing antibody titers. After priming with the SARS-S exosomal vaccine and boosting with the adenoviral vector the neutralizing antibody titers exceeded those observed in the convalescent serum of a SARS patient. Both approaches were effective in a SARS-S-expressing tumor challenge model and thus warrant further investigation.

Tumor-derived exosomes have been regarded as a new kind of cancer vaccine; however, their therapeutic efficacy needs to be further improved. Superantigen staphylococcal enterotoxin A (SEA)-coated tumor cells have been shown to potently induce tumor-specific T cell response. To increase efficacy of tumor-derived exosomes to induce antitumor immune response, we modified the exosomes by protein transfer of SEA tailed with a highly hydrophobic transmembrane domain (SEA-TM) and designated those SEA-TM-anchored exosomes as Exo/SEA-TM. We found the exosomes secreted from murine lymphoma E.G7-OVA cell line were round vesicles with the sizes of 40-100 nm limited by a bilayer membrane. Interestingly, the inner structure of the exosomes were visible under the transmission electron microscope; those "honeycomb-like" inner structure has not been described by other labs. Immunization with Exo/SEA-TM inhibited tumor growth and prolonged survival of the mice challenged with parental tumor cells more significantly than with exosomes (Exo) and even more than with the mixture of exosomes and SEA-TM. The results of mixed lymphocyte-tumor reaction (MLTR) showed that the increased IL-2, IFN-gamma secretion, and specific cytotoxic T lymphocyte (CTL) could be effectively induced from the splenic lymphocytes of the mice immunized with Exo/SEA-TM. In vivo depletion experiments showed that CD8(+) T cells are the main effector cells, and both CD4(+) T cells and NK cells are also involved in the antitumor effect of Exo/SEA-TM immunization. Therefore, tumor-derived exosomes surface anchored with SEA-TM can efficiently induce tumor-specific CTL thereby resulting in more potent inhibition of tumor growth. Our data provide an efficient and novel approach to tumor immunotherapy by protein modification of tumor-derived exosomes.

Tumor-released microvesicles, or exosomes, which are abundant in the body fluids of patients with cancer, are likely to be involved in tumor progression. We recently showed that microvesicles released by human melanoma and colorectal carcinoma cells can promote the differentiation of monocytes to myeloid-derived suppressor cells which support tumoral growth and immune escape. These findings underscore an important role for these extracellular organelles in remodeling tumor-stromal interactions to promote malignancy.

Antigen (Ag) capture and presentation onto major histocompatibility complex (MHC) class II molecules by B lymphocytes is mediated by their surface Ag receptor (B cell receptor [BCR]). Therefore, the transport of vesicles that carry MHC class II and BCR-Ag complexes must be coordinated for them to converge for processing. In this study, we identify the actin-associated motor protein myosin II as being essential for this process. Myosin II is activated upon BCR engagement and associates with MHC class II-invariant chain complexes. Myosin II inhibition or depletion compromises the convergence and concentration of MHC class II and BCR-Ag complexes into lysosomes devoted to Ag processing. Accordingly, the formation of MHC class II-peptides and subsequent CD4 T cell activation are impaired in cells lacking myosin II activity. Therefore, myosin II emerges as a key motor protein in BCR-driven Ag processing and presentation.

Exosomes (EXO) derived from dendritic cells (DC), which express major histocompatibility complex (MHC) and costimulatory molecules, have been used for antitumour vaccines. However, they are still less effective by showing only prophylatic immunity in animal models or very limited immune responses in clinical trials. In this study, we showed that ovalbumin (OVA) protein-pulsed DC (DC(OVA))-derived EXO (EXO(OVA)) displayed MHC class I-OVA I peptide (pMHC I) complexes, CD11c, CD40, CD80, CCR7, DEC205, Toll-like receptor 4 (TLR4), TLR9, MyD88 and DC-SIGN molecules, but at a lower level than DC(OVA). EXO(OVA) can be taken up by DC through LFA-1/CD54 and C-type lectin/mannose (glucosamine)-rich C-type lectin receptor (CLR) interactions. Mature DC pulsed with EXO(OVA), which were referred to as mDC(EXO), expressed a higher level of pMHC I, MHC II, and costimulatory CD40, CD54 and CD80 than DC(OVA). The mDC(EXO) could more strongly stimulate OVA-specific CD8(+) T-cell proliferation in vitro and in vivo, and more efficiently induce OVA-specific cytotoxic T-lymphocyte responses, antitumour immunity and CD8(+) T-cell memory in vivo than EXO(OVA) and DC(OVA). In addition, mDC(EXO) could also more efficiently eradicate established tumours. Therefore, mature DC pulsed with EXO may represent a new, highly effective DC-based vaccine for the induction of antitumour immunity.

Objective: In addition to releasing proteins and mediators, cells also release membrane vesicles (exosomes and apoptotic blebs) into the extracellular environment. Apoptotic blebs contain multiple autoantigens, but few data are available concerning the protein content of exosomes. Exosomes are formed during an immune response and can directly stimulate T cells or bind to dendritic cells. The aim of this study was to identify the nature of synovial exosomes from patients with different rheumatic diseases and to examine their potential autoantigenic content, which may be involved in the induction of an autoimmune response., Methods: Synovial exosomes from patients with rheumatoid arthritis (RA), patients with reactive arthritis, and patients with osteoarthritis were purified, analyzed by electron microscopy, and labeled with immunogold to detect IgG and IgM molecules. Autoantigen content was identified by 2-dimensional electrophoresis-immunoblotting and subsequent mass spectrometry. In order to investigate the presence of citrullinated proteins, immunoblotting with anticitrulline antibodies was performed., Results: Citrullinated proteins were observed in all exosome preparations, in contrast to other autoantigenic proteins (e.g., BiP and heterogeneous nuclear RNP A2) that were previously observed in RA and other autoimmune diseases. These citrullinated proteins included the fibrin alpha-chain fragment, fibrin beta-chain, fibrinogen beta-chain precursor, fibrinogen D fragment, and the Sp alpha (CD5 antigen-like protein) receptor. Purification of synovial exosomes led to the detection of citrullinated fibrinogen and citrullinated Sp alpha associated with IgM and IgG., Conclusion: Synovial exosomes contain citrullinated proteins, which are known to be autoantigens in RA. Although immune mechanisms in which exosomes carry citrullinated peptides could play an important role in the induction and distribution of citrullinated proteins, there must be a specific recognition of these proteins that is unique to the RA immune system.

Exosomes derived from dendritic cells (DC) activate T cells in vivo, but whether exosomes are able to induce and/or modulate humoral immune responses is still unknown. We show that murine bone marrow DC pulsed in vitro with an intact protein (diphtheria toxoid (DT)) produce exosomes that induce, in the absence of free protein, in vivo Ig responses specific for DT in naive recipients. Furthermore, these exosomes stimulate secondary IgG anti-DT responses in mice primed with intact DT. Exosomes from mature, relative to immature, DC were more effective at inducing primary, although not secondary, IgG anti-DT responses. Whereas intact DT preferentially induced a type 2 (IgG1) anti-DT response, exosomes from DT-pulsed bone marrow DC favored induction of type 1 (IgG2b and IgG2a) DT-specific IgG. These results are the first to demonstrate the ability of exosomes derived from Ag-pulsed DC to induce and modulate Ag-specific humoral immunity in vivo.

Background Information: Exosomes are small membrane vesicles secreted by several cell types during exocytic fusion of multivesicular bodies with the plasma membrane. Exosomes from tumour cells can transfer antigens from cell to cell, a property favouring antigen-specific immune responses in vitro and in vivo, and are thus an interesting putative therapeutic tool in human cancers. Exosomes have been well studied in EBV (Epstein-Barr virus)-transformed human B-cell lines; however, biological stimuli regulating exosome secretion quantitatively and/or qualitatively still remain poorly defined., Results: We analysed the effect of the BCR stimulation on exosome release in the human follicular lymphoma B-cell line DOHH2. We found that BCR (B-cell receptor) triggering of DOHH2 cells induced the polarization of CD63(+) MHC class II compartments. Moreover, BCR stimulation increased the release of exosome-associated proteins in the extracellular space. Finally, we found that the BCR was expressed at the surface of exosomes, and could target a bound anti-human IgG to these vesicles., Conclusions: BCR can modulate the protein content of exosomes upon stimulation, and can target its bound antigen to these vesicles.

Transcutaneous immunization (TCI) is a novel vaccination strategy based on the application of antigen together with an adjuvant onto hydrated bare skin. This simple and non-invasive immunization procedure elicits systemic and cell mediated immune responses and therefore, it provides a viable and cost-effective strategy for disease prevention. In the present study, novel fusogenic vesicular carrier constructs, i.e. vesosomes were developed and evaluated for topical delivery of vaccines using tetanus toxoid (TTx) as a model antigen. Prepared vesosomes were characterized for size, shape, entrapment efficiency and zeta potential. The prepared novel systems were examined for in process antigen stability and long-term storage stability studies. In vitro skin permeation and fluorescence microscopy study were also preformed for prepared novel vesicular systems for the evaluation of skin penetration efficiency. The immune stimulating activity of these vesicles was studied by measuring the serum anti-tetanus toxoid IgG titer and isotype ratio IgG2a/IgG1 following topical immunization in three different protocols and results were compared with the alum adsorbed tetanus toxoid given intramuscularly and topically administered plain tetanus toxoid solution, plain liposomes and cationic fusogenic liposomes. Serum IgG titers after three consecutive topical administrations were significantly better (*P < 0.05) than single administration of TTx antigen with vesosomal systems, suggesting an effective stimulation of serum immune response. Furthermore, notable serum anti-TTx antibody titers also occurred in animals primed with alum adsorbed TTx and subsequently boosted with topical administration of novel vesosomal systems. In each immunization studies, the vesosomal systems could elicit combined Th1 and Th2 immune responses following topical administration. These results suggest that the investigated vesosomal systems can be effective as topical delivery of vaccines.

Exosomes (EXO) derived from dendritic cells (DC) and tumor cells have been used to stimulate antitumor immune responses in animal models and in clinical trials. However, there has been no side-by-side comparison of the stimulatory efficiency of the antitumor immune responses induced by these two commonly used EXO vaccines. In this study, we selected to study the phenotype characteristics of EXO derived from a transfected EG7 tumor cells expressing ovalbumin (OVA) and OVA-pulsed DC by flow cytometry. We compared the stimulatory effect in induction of OVA-specific immune responses between these two types of EXO. We found that OVA protein-pulsed DCOVA-derived EXO (EXODC) can more efficiently stimulate naïve OVA-specific CD8+ T cell proliferation and differentiation into cytotoxic T lymphocytes in vivo, and induce more efficient antitumor immunity than EG7 tumor cell-derived EXO (EXOEG7). In addition, we elucidated the important role of the host DC in EXO vaccines that the stimulatory effect of EXO is delivered to T cell responses by the host DC. Therefore, DC-derived EXO may represent a more effective EXO-based vaccine in induction of antitumor immunity.

Because dendritic cell (DC)-derived exosomes (EXO) harbor many important DC molecules involved in inducing immune responses, EXO-based vaccines have been extensively used to induce antitumor immunity in different animal tumor models. However, it is not clear which route of EXO administration can induce more efficient antitumor immune responses. In this study, we compared the antitumor immunity derived from EXO vaccine by way of the two common administration routes, the subcutaneous (s.c.) and the intradermal (i.d.) administrations. Our data showed that the i.d. EXO administration resulted in more EXO-absorbed DC migrating into the T-cell areas of draining lymph nodes than the s.c. administration. Interestingly, the i.d. EXO administration also resulted in an enhanced ovalbumin (OVA)-specific CD8(+) T-cell proliferation and CD8(+) CTL effector responses in vivo, compared to the s.c. administration. Similarly, compared to the s.c. vaccination, the i.d. vaccination induced stronger antitumor immunity in the animal tumor model. Therefore, the i.d. EXO vaccination is superior to the s.c. one and should be considered when EXO-based vaccine is designed.