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Purpose Single-chain variable fragments (scFvs) are one of the smallest antigen-binding units having the invaluable advantage to be expressed by a unique short open reading frame (ORF). Despite their reduced size, spontaneous cell entry of scFvs remains inefficient, hence precluding the possibility to target intracellular antigens. Here, we describe an original strategy to deliver scFvs inside target cells through engineered extracellular vesicles (EVs). This approach relies on the properties of a Human Immunodeficiency Virus (HIV)-1 Nef mutant protein referred to as Nefmut. It is a previously characterized Nef allele lacking basically all functions of wt Nef, yet strongly accumulating in the EV lumen also when fused at its C-terminus with a foreign protein. To gain the proof-of-principle for the efficacy of the proposed strategy, the tumor-promoting Human Papilloma Virus (HPV)16-E7 protein was considered as a scFv-specific intracellular target. The oncogenic effect of HPV16-E7 relies on its binding to the tumor suppressor pRb protein leading to a dysregulated cell duplication. Interfering with this interaction means impairing the HPV16-E7-induced cell proliferation. Methods The Nefmut gene was fused in frame at its 3'-terminus with the ORF coding for a previously characterized anti-HPV16-E7 scFv. Interaction between the Nefmut-fused anti-HPV16-E7 scFv and the HPV16-E7 protein was tested by both confocal microscope and co-immunoprecipitation analyses on co-transfected cells. The in cis anti-proliferative effect of the Nefmut/anti-HPV16-E7 scFv was assayed by transfecting HPV16-infected cells. The anti-proliferative effect of EVs engineered with Nefmut/anti-HPV16-E7 scFv on HPV16-E7-expressing cells was evaluated in two ways: i) through challenge with purified EVs by a Real-Time Cell Analysis system and ii) in transwell co-cultures by an MTS-based assay. Results The Nefmut/anti-HPV16-E7 scFv chimeric product is efficiently uploaded in EVs, binds HPV16-E7, and inhibits the proliferation of HPV16-E7-expressing cells. Most important, challenge with cell-free EVs incorporating the Nefmut/anti-HPV16-E7 scFv led to the inhibition of proliferation of HPV16-E7-expressing cells. The proliferation of these cells was hindered also when they were co-cultured in transwells with cells producing EVs uploading Nefmut/anti-HPV16-E7 scFv. Conclusion Our data represent the proof-of-concept for the possibility to target intracellular antigens through EV-mediated delivery of scFvs. This finding could be relevant to design novel methods of intracellular therapeutic interventions.

HIV-1 infection is efficiently controlled by combination anti-retroviral therapy (cART). However, despite preventing disease progression, cART does not eradicate virus infection which persists in a latent form for an individual’s lifetime. The latent reservoir comprises memory CD4+ T lymphocytes, macrophages, and dendritic cells; however, for the most part, the reservoir is generated by virus entry into activated CD4+ T lymphocytes committed to return to a resting state, even though resting CD4+ T lymphocytes can be latently infected as well. The HIV-1 reservoir is not recognized by the immune system, is quite stable, and has the potential to re-seed systemic viremia upon cART interruption. Viral rebound can occur even after a long period of cART interruption. This event is most likely a consequence of the extended half-life of the HIV-1 reservoir, the maintenance of which is not clearly understood. Several recent studies have identified extracellular vesicles (EVs) as a driving force contributing to HIV-1 reservoir preservation. In this review, we discuss recent findings in the field of EV/HIV-1 interplay, and then propose a mechanism through which EVs may contribute to HIV-1 persistence despite cART. Understanding the basis of the HIV-1 reservoir maintenance continues to be a matter of great relevance in view of the limitations of current strategies aimed at HIV-1 eradication.

Eukaryotic cells constitutively produce nanovesicles of 50-150nm of diameter, referred to as exosomes, upon release of the contents of multivesicular bodies (MVBs). We recently characterized a novel, exosome-based way to induce cytotoxic T lymphocyte (CTL) immunization against full-length antigens. It is based on DNA vectors expressing products of fusion between the exosome-anchoring protein Nef mutant (Nefmut) with the antigen of interest. The strong efficiency of Nefmut to accumulate in MVBs results in the production of exosomes incorporating huge amounts of the desired antigen. When translated in animals, the injection of Nefmut-based DNA vectors generates engineered exosomes whose internalization in antigen-presenting cells induces cross-priming and antigen-specific CTL immunity. Here, we describe the molecular strategies we followed to produce DNA vectors aimed at generating immunogenic exosomes potentially useful to elicit a CTL immune response against antigens expressed by the etiologic agents of major chronic viral infections, i.e., HIV-1, HBV, and the novel tumor-associated antigen HOXB7. Unique methods intended to counteract intrinsic RNA instability and nuclear localization of the antigens have been developed. The success we met with the production of these engineered exosomes opens the way towards pre-clinic experimentations devoted to the optimization of new vaccine candidates against major infectious and tumor pathologies.

Paola Di Bonito,1 Chiara Chiozzini,2 Claudia Arenaccio,2 Simona Anticoli,2 Francesco Manfredi,2 Eleonora Olivetta,2 Flavia Ferrantelli,2 Emiliana Falcone,3 Anna Ruggieri,3 Maurizio Federico2 1Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Rome, Italy; 2National AIDS Center, Istituto Superiore di Sanità, Rome, Italy; 3Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome, Italy Abstract: We recently proved that exosomes engineered in vitro to deliver high amounts of HPVE7 upon fusion with the Nefmut exosome-anchoring protein elicit an efficient anti-E7 cytotoxic T lymphocyte immune response. However, in view of a potential clinic application of this finding, our exosome-based immunization strategy was faced with possible technical difficulties including industrial manufacturing, cost of production, and storage. To overcome these hurdles, we designed an as yet unproven exosome-based immunization strategy relying on delivery by intramuscular inoculation of a DNA vector expressing Nefmut fused with HPVE7. In this way, we predicted that the expression of the Nefmut/E7 vector in muscle cells would result in a continuous source of endogenous (ie, produced by the inoculated host) engineered exosomes able to induce an E7-specific immune response. To assess this hypothesis, we first demonstrated that the injection of a Nefmut/green fluorescent protein-expressing vector led to the release of fluorescent exosomes, as detected in plasma of inoculated mice. Then, we observed that mice inoculated intramuscularly with a vector expressing Nefmut/E7 developed a CD8+ T-cell immune response against both Nef and E7. Conversely, no CD8+ T-cell responses were detected upon injection of vectors expressing either the wild-type Nef isoform of E7 alone, most likely a consequence of their inefficient exosome incorporation. The production of immunogenic exosomes in the DNA-injected mice was formally demonstrated by the E7-specific CD8+ T-cell immune response we detected in mice inoculated with exosomes isolated from plasma of mice inoculated with the Nefmut/E7 vector. Finally, we provide evidence that the injection of Nefmut/E7 DNA led to the generation of effective antigen-specific cytotoxic T lymphocytes whose activity was likely part of the potent, therapeutic antitumor effect we observed in mice implanted with TC-1 tumor cells. In summary, we established a novel method to generate immunogenic exosomes in vivo by the intramuscular inoculation of DNA vectors expressing the exosome-anchoring protein Nefmut and its derivatives. Keywords: nanovesicles, cytotoxic T lymphocytes, HIV-1 Nef, DNA vectors

Purpose Single-chain variable fragments (scFvs) are one of the smallest antigen-binding units having the invaluable advantage to be expressed by a unique short open reading frame (ORF). Despite their reduced size, spontaneous cell entry of scFvs remains inefficient, hence precluding the possibility to target intracellular antigens. Here, we describe an original strategy to deliver scFvs inside target cells through engineered extracellular vesicles (EVs). This approach relies on the properties of a Human Immunodeficiency Virus (HIV)-1 Nef mutant protein referred to as Nefmut. It is a previously characterized Nef allele lacking basically all functions of wt Nef, yet strongly accumulating in the EV lumen also when fused at its C-terminus with a foreign protein. To gain the proof-of-principle for the efficacy of the proposed strategy, the tumor-promoting Human Papilloma Virus (HPV)16-E7 protein was considered as a scFv-specific intracellular target. The oncogenic effect of HPV16-E7 relies on its binding to the tumor suppressor pRb protein leading to a dysregulated cell duplication. Interfering with this interaction means impairing the HPV16-E7-induced cell proliferation. Methods The Nefmut gene was fused in frame at its 3ʹ-terminus with the ORF coding for a previously characterized anti-HPV16-E7 scFv. Interaction between the Nefmut-fused anti-HPV16-E7 scFv and the HPV16-E7 protein was tested by both confocal microscope and co-immunoprecipitation analyses on co-transfected cells. The in cis anti-proliferative effect of the Nefmut/anti-HPV16-E7 scFv was assayed by transfecting HPV16-infected cells. The anti-proliferative effect of EVs engineered with Nefmut/anti-HPV16-E7 scFv on HPV16-E7-expressing cells was evaluated in two ways: i) through challenge with purified EVs by a Real-Time Cell Analysis system and ii) in transwell co-cultures by an MTS-based assay. Results The Nefmut/anti-HPV16-E7 scFv chimeric product is efficiently uploaded in EVs, binds HPV16-E7, and inhibits the proliferation of HPV16-E7-expressing cells. Most important, challenge with cell-free EVs incorporating the Nefmut/anti-HPV16-E7 scFv led to the inhibition of proliferation of HPV16-E7-expressing cells. The proliferation of these cells was hindered also when they were co-cultured in transwells with cells producing EVs uploading Nefmut/anti-HPV16-E7 scFv. Conclusion Our data represent the proof-of-concept for the possibility to target intracellular antigens through EV-mediated delivery of scFvs. This finding could be relevant to design novel methods of intracellular therapeutic interventions.

Background Lymphocyte-activation gene (LAG)3 is a 498 aa transmembrane type I protein acting as an immune inhibitory receptor. It is expressed on activated lymphocytes, natural killer cells and plasmacytoid dendritic cells. In activated lymphocytes, LAG3 expression is involved in negative control of cell activation/proliferation to ensure modulation and control of immune responses. In view of its deregulated expression in tumor-infiltrating lymphocytes, LAG3, together with the additional immune checkpoint inhibitors CTLA4 and PD1, is considered a major target in order to reverse the immunosuppression typically mounting in oncologic diseases. Since many patients still fail to respond to current immune checkpoints-based therapies, the identification of new effective immune inhibitors is a priority in the ongoing fight against cancer. Results We identified a novel human single-chain variable fragment (scFv) Ab against a conformational epitope of LAG3 by in vitro phage display technology using the recombinant antigen as a bait. This scFv (referred to as F7) was characterized in terms of binding specificity to both recombinant antigen and human LAG3-expressing cells. It was then rebuilt into an IgG format pre-optimized for clinical usage, and the resulting bivalent construct was shown to preserve its ability to bind LAG3 on human cells. Next, we analyzed the activity of the anti-LAG3 scFvF7 using two different antigen-specific CD8+ T lymphocyte clones as target cells. We proved that the reconstituted anti-LAG3 F7 Ab efficiently binds the cell membrane of both cell clones after peptide-activation. Still more significantly, we observed a striking increase in the peptide-dependent cell activation upon Ab treatment as measured in terms of IFN-γ release by both ELISA and ELISPOT assays. Conclusions Overall, the biotechnological strategy described herein represents a guiding development model for the search of novel useful immune checkpoint inhibitors. In addition, our functional data propose a novel candidate reagent for consideration as a cancer treatment.

Background:Eukaryotic cells release vesicles of different sizes under both physiological and pathological conditions. On the basis of the respective biogenesis, extracellular vesicles are classified as apoptotic bodies, microvesicles, and exosomes. Among these, exosomes are considered tools for innovative therapeutic interventions, especially when engineered with effector molecules. The delivery functions of exosomes are favored by a number of typical features. These include their small size (i.e., 50-200 nm), the membrane composition tightly similar to that of producer cells, lack of toxicity, stability in serum as well as other biological fluids, and accession to virtually any organ and tissue including central nervous system. However, a number of unresolved questions still affects the possible use of exosomes in therapy. Among these are the exact identification of both in vitro and ex vivo produced vesicles, their large-scale production and purification, the uploading efficiency of therapeutic macromolecules, and the characterization of their pharmacokinetics. Objective: Here, we discuss two key aspects to be analyzed before considering exosomes as a tool of delivery for the desired therapeutic molecule, i.e., techniques of engineering, and their in vivo biodistribution/ pharmacokinetics. In addition, an innovative approach aimed at overcoming at least part of the obstacles towards a safe and efficient use of exosomes in therapy will be discussed.Conclusion:Several biologic features render exosomes an attractive tool for the delivery of therapeutic molecules. They will surely be a part of innovative therapeutic interventions as soon as few still unmet technical hindrances will be overcome.

Intrinsic genetic instability of tumor cells leads to continuous production of mutated proteins referred to as tumor-specific neoantigens. Generally, they are recognized as nonself products by the host immune system. However, an effective adaptive response clearing neoantigen-expressing cells is lost in tumor diseases. Most advanced therapeutic strategies aim at inducing neoantigen-specific immune activation through personalized approaches. They include tumor cell exome sequencing, human leukocyte antigen (HLA) typing, synthesis, and injection of peptides/RNA with adjuvants. Here, we propose an innovative method to induce a CD8+ T cytotoxic lymphocyte (CTL) immune response against tumor neoantigens bypassing the steps needed in current therapeutic strategies of personalized vaccination. We assumed that tumor cells can be the most efficient and precise factory of major histocompatibility complex (MHC) class I-associated, tumor neoantigen-derived peptides. Hence, endowing tumor cells with professional antigen-presenting functions would prime CD8+ T lymphocytes towards a response against nonself tumor antigens. To explore this possibility, both adenocarcinoma and melanoma human cells were engineered to express both CD80 and CD86 costimulatory molecules. HLA-matched lymphocytes were then primed through cocultivation with the engineered tumor cells. The generation of tumor-specific CD8+ T lymphocytes was tested through the combined analysis of cell activation markers, formation of immunologic synapses, generation of tumor antigen-specific CD8+ T lymphocytes, and cytotoxic activity. Our data consistently indicate that tumor cells endowed with professional antigen-presenting functions can generate an effective tumor-specific CTL immune response. This finding may open avenues towards the development of innovative antitumor immunotherapies. Key messages: We established a novel method to induce antitumor CTLs without a need to identify TAAs and/or tumor neoantigens.This strategy relies on transducing tumor cells with a retroviral vector expressing both CD80 and CD86.In this way, tumor cells prime naïve CD8+ T lymphocytes in a way that CTLs killing the same tumor cells are generated.These findings open the way towards preclinical assays in the perspective to introduce this antitumor immunotherapy strategy in clinic.

We recently described a cytotoxic CD8+ T lymphocyte (CTL) vaccine platform based on the intramuscular (i.m.) injection of DNA eukaryotic vectors expressing antigens of interest fused at the C-terminus of HIV-1 Nefmut, i.e., a functionally defective mutant that is incorporated at quite high levels into exosomes/extracellular vesicles (EVs). This system has been proven to elicit strong CTL immunity against a plethora of both viral and tumor antigens, as well as inhibit both transplantable and orthotopic tumors in mice. However, a number of open issues remain regarding the underlying mechanism. Here we provide evidence that hindering the uploading into EVs of Nefmut-derived products by removing the Nefmut N-terminal fatty acids leads to a dramatic reduction of the downstream antigen-specific CD8+ T-cell activation after i.m. injection of DNA vectors in mice. This result formally demonstrates that the generation of engineered EVs is part of the mechanism underlying the in vivo induced CD8+ T-cell immunogenicity. Gaining new insights on the EV-based vaccine platform can be relevant in view of its possible translation into the clinic to counteract both chronic and acute infections as well as tumors.

We recently described a novel biotechnological platform for the production of unrestricted cytotoxic T lymphocyte (CTL) vaccines. It relies on in vivo engineering of exosomes, i.e., nanovesicles constitutively released by all cells, with full-length antigens of choice upon fusion with an exosome-anchoring protein referred to as Nefmut. They are produced upon intramuscular injection of a DNA vector and, when uploaded with a viral tumor antigen, were found to elicit an immune response inhibiting the tumor growth in a model of transplantable tumors. However, for a possible application in cancer immunotherapy, a number of key issues remained unmet. Among these, we investigated: (i) whether the immunogenic stimulus induced by the engineered exosomes can break immune tolerance, and (ii) their effectiveness when applied in human system. As a model of immune tolerance, we considered mice transgenic for the expression of activated rat HER2/neu which spontaneously develop adenocarcinomas in all mammary glands. When these mice were injected with a DNA vector expressing the product of fusion between Nefmut and the extracellular domain of HER2/neu, antigen-specific CD8+ T lymphocytes became readily detectable. This immune response associated with a HER2-directed CTL activity and a significant delay in tumor development. On the other hand, through cross-priming experiments, we demonstrated the effectiveness of the engineered exosomes emerging from transfected human primary muscle cells in inducing antigen-specific CTLs. We propose our CTL vaccine platform as part of new immunotherapy strategies against tumors expressing self-antigens, i.e., products highly expressed in oncologic lesions but tolerated by the immune system.

Nef is an accessory protein expressed exclusively in primate lentiviruses. It is devoid of enzymatic activities while interacting with several cell proteins as an adaptor/scaffold protein. Intracellular functions of Nef largely account for many pathogenic effects observed in AIDS disease. Nef, despite lacking known secretory pathways, can be detected in plasma of HIV-1-infected patients at the concentration varing from 5 to 10 ng/ml. Remarkably, the levels of Nef in plasma of HIV patients do not correlate with viral load or number of CD4(+) T lymphocytes, and persist during antiretroviral therapy. Here, we review literature data describing how Nef can be transmitted from HIV-1- infected cells to bystander ones, and the effects of extracellular Nef in different cell types. Overall, large part of experimental evidences supports the idea that extracellular Nef plays a relevant role in AIDS pathogenesis. Hence, efforts focused on the identification of Nef-inhibiting drugs would be of relevance to establish new therapeutic approaches supporting current antiretroviral therapies.

Adenosine deaminase acting on RNA1 (ADAR1) was previously reported to affect HIV-1 replication. We report data showing that ADAR1 interacts with the HIV-1 p55 Gag protein, the major structural protein of the immature virus capsid. Furthermore, we found that the endogenous ADAR1 is incorporated into virions purified from the supernatant of primary HIV-1-infected CD4+ T lymphocytes. Additional experiments demonstrated that the expression of the p55 Gag protein is sufficient for ADAR1 incorporation into virus-like particles (VLPs).Overall, our data originally support the evidence that ADAR1 can be part of the cell protein array uploaded in HIV-1 particles.

ABSTRACT Defective HIV-1 genomes populate blood cells of HIV-1 infected patients, especially during HAART treatment. They can express viral proteins which, if released, may induce bystander effects favoring viral spread. Here, we review recent literature regarding the effects of extracellular HIV-1 proteins which can act as effectors of transcriptionally active, defective HIV-1, including Gag p17, Env gp120, Vpr, Tat and Nef. It has been very recently described that, different to the other HIV products, the bystander effects of Nef can be mediated by exosomes, that is, nanovesicles constitutively released by all cell types. Exosomes from Nef-expressing cells induce cell activation and HIV-1 susceptibility in resting CD4+ T lymphocytes in a TNF-α-dependent way. This mechanism likely contributes to virus persistence in HAART-treated patients.

Exosomes are extracellular vesicles of 50-150 nm in diameter secreted by basically all cell types. They mediate micro-communication among cells, tissues, and organs under both healthy and disease conditions by virtue of their ability to deliver macromolecules to target cells. Research on exosomes is a rapidly growing field, however many aspects of their biogenesis and functions still await a complete clarification. In our review we summarize most recent findings regarding biogenesis, structure, and functions of exosomes. In addition, an overview regarding the role of exosomes in both infectious and non-infectious diseases is provided. Finally, the use of exosomes as biomarkers and delivery tools for therapeutic molecules is addressed. Considering the body of literature data, exosomes have to be considered key components of the intercellular communication in both health and disease.

Intact HIV-1 and exosomes can be internalized by dendritic cells (DCs) through a common pathway leading to their transmission to CD4

Exosomes are extracellular vesicles of 50–150 nm in diameter secreted by basically all cell types. They mediate micro-communication among cells, tissues, and organs under both healthy and disease conditions by virtue of their ability to deliver macromolecules to target cells. Research on exosomes is a rapidly growing field, however many aspects of their biogenesis and functions still await a complete clarification. In our review we summarize most recent findings regarding biogenesis, structure, and functions of exosomes. In addition, an overview regarding the role of exosomes in both infectious and non-infectious diseases is provided. Finally, the use of exosomes as biomarkers and delivery tools for therapeutic molecules is addressed. Considering the body of literature data, exosomes have to be considered key components of the intercellular communication in both health and disease.

Intact HIV-1 and exosomes can be internalized by dendritic cells (DCs) through a common pathway leading to their transmission to CD4+ T lymphocytes by means of mechanisms defined as trans-infection and trans-dissemination, respectively. We previously reported that exosomes from HIV-1-infected cells activate both uninfected quiescent CD4+ T lymphocytes, which become permissive to HIV-1, and latently infected cells, with release of HIV-1 particles. However, nothing is known about the effects of trans-dissemination of exosomes produced by HIV-1-infected cells on uninfected or latently HIV-1-infected CD4+ T lymphocytes. Here, we report that trans-dissemination of exosomes from HIV-1-infected cells induces cell activation in resting CD4+ T lymphocytes, which appears stronger with mature than immature DCs. Using purified preparations of both HIV-1 and exosomes, we observed that mDC-mediated trans-dissemination of exosomes from HIV-1-infected cells to resting CD4+ T lymphocytes induces efficient trans-infection and HIV-1 expression in target cells. Most relevant, when both mDCs and CD4+ T lymphocytes were isolated from combination anti-retroviral therapy (ART)-treated HIV-1-infected patients, trans-dissemination of exosomes from HIV-1-infected cells led to HIV-1 reactivation from the viral reservoir. In sum, our data suggest a role of exosome trans-dissemination in both HIV-1 spread in the infected host and reactivation of the HIV-1 reservoir.

Resting CD4(+) T lymphocytes resist human immunodeficiency virus (HIV) infection. Here, we provide evidence that exosomes from HIV-1-infected cells render resting human primary CD4(+) T lymphocytes permissive to HIV-1 replication. These results were obtained with transwell cocultures of HIV-1-infected cells with quiescent CD4(+) T lymphocytes in the presence of inhibitors of exosome release and were confirmed using exosomes purified from supernatants of HIV-1-infected primary CD4(+) T lymphocytes. We found that the expression of HIV-1 Nef in exosome-producing cells is both necessary and sufficient for cell activation as well as HIV-1 replication in target CD4(+) T lymphocytes. We also identified a Nef domain important for the effects we observed, i.e., the (EEEE65)-E-62 acidic cluster domain. In addition, we observed that ADAM17, i.e., a disintegrin and metalloprotease converting pro-tumor necrosis factor alpha (TNF-alpha) in its mature form, associates with exosomes from HIV-1-infected cells, and plays a key role in the HIV-1 replication in quiescent CD4(+) T lymphocytes. Treatment with an inhibitor of ADAM17 abolished both activation and HIV-1 replication in resting CD4(+) T lymphocytes. TNF-alpha is the downstream effector of ADAM17 since the treatment of resting lymphocytes with anti-TNF-alpha antibodies blocked the HIV-1 replication. The data presented here are consistent with a model where Nef induces intercellular communication through exosomes to activate bystander quiescent CD4(+) T lymphocytes, thus stimulating viral spread. Resting CD4(+) T lymphocytes resist human immunodeficiency virus (HIV) infection. Here, we provide evidence that exosomes from HIV-1-infected cells render resting human primary CD4(+) T lymphocytes permissive to HIV-1 replication. These results were obtained with transwell cocultures of HIV-1-infected cells with quiescent CD4(+) T lymphocytes in the presence of inhibitors of exosome release and were confirmed using exosomes purified from supernatants of HIV-1-infected primary CD4(+) T lymphocytes. We found that the expression of HIV-1 Nef in exosome-producing cells is both necessary and sufficient for cell activation as well as HIV-1 replication in target CD4(+) T lymphocytes. We also identified a Nef domain important for the effects we observed, i.e., the (EEEE65)-E-62 acidic cluster domain. In addition, we observed that ADAM17, i.e., a disintegrin and metalloprotease converting pro-tumor necrosis factor alpha (TNF-alpha) in its mature form, associates with exosomes from HIV-1-infected cells, and plays a key role in the HIV-1 replication in quiescent CD4(+) T lymphocytes. Treatment with an inhibitor of ADAM17 abolished both activation and HIV-1 replication in resting CD4(+) T lymphocytes. TNF-alpha is the downstream effector of ADAM17 since the treatment of resting lymphocytes with anti-TNF-alpha antibodies blocked the HIV-1 replication. The data presented here are consistent with a model where Nef induces intercellular communication through exosomes to activate bystander quiescent CD4(+) T lymphocytes, thus stimulating viral spread.

ABSTRACT: Aims: Exosomes are lipid bilayer vesicles of 50–100 nm released by basically all cell types. We recently reported that full-length HIV-1 RNA and lentiviral vector (LV) genome associate with exosomes through similar mechanisms. Here, we investigated the fate of lentiviral RNA shuttled by exosomes in target cells. Material & methods: Exosomes from cells transduced by a LV expressing green fluorescent protein under the control of an heterologous promoter were purified by iodixanol gradients and used to evaluate the LV expression in target cells. Results: The genome of LV incorporated in exosomes can be expressed in HIV-1-infected cells, but not in those that are uninfected, despite apparently similar levels of exosome internalization. The expression disappeared 2–3 days after challenge, and was blocked by pre-treatment with azidothymidine. Conclusion: Lentiviral genome incorporated in exosomes can be expressed in target cells having reverse transcriptase activity.

We recently described the induction of an efficient CD8⁺ T cell-mediated immune response against a tumor-associated antigen (TAA) uploaded in engineered exosomes used as an immunogen delivery tool. This immune response cleared tumor cells inoculated after immunization, and controlled the growth of tumors implanted before immunization. We looked for new protocols aimed at increasing the CD8⁺ T cell specific response to the antigen uploaded in engineered exosomes, assuming that an optimized CD8⁺ T cell immune response would correlate with a more effective depletion of tumor cells in the therapeutic setting. By considering HPV-E6 as a model of TAA, we found that the in vitro co-administration of engineered exosomes and ISCOMATRIX(TM) adjuvant, i.e., an adjuvant composed of purified ISCOPREP(TM) saponin, cholesterol, and phospholipids, led to a stronger antigen cross-presentation in both B- lymphoblastoid cell lines ( and monocyte-derived immature dendritic cells compared with that induced by the exosomes alone. Consistently, the co-inoculation in mice of ISCOMATRIX(TM) adjuvant and engineered exosomes induced a significant increase of TAA-specific CD8⁺ T cells compared to mice immunized with the exosomes alone. This result holds promise for effective usage of exosomes as well as alternative nanovesicles in anti-tumor therapeutic approaches.

Engineering exosomes to upload heterologous proteins represents the last frontier in terms of nanoparticle-based technology. A limited number of methods suitable to associate proteins to exosome membrane has been described so far, and very little is known regarding the possibility to upload proteins inside exosomes. We optimized a method of protein incorporation in exosomes by exploiting the unique properties of a nonfunctional mutant of the HIV-1 Nef protein referred to as Nef(mut). It incorporates at high extents in exosomes meanwhile acting as carrier of protein antigens fused at its C-terminus. Manipulating Nef(mut) allows the incorporation into exosomes of high amounts of heterologous proteins which thus remain protected from external neutralization/degradation factors. These features, together with flexibility in terms of incorporation of foreign antigens and ease of production, make Nef(mut)-based exosomes a convenient vehicle for different applications (e.g., protein transduction, immunization) whose performances are comparable with those of alternative, more complex nanoparticle-based delivery systems.

Engineering exosomes to upload heterologous proteins represents the last frontier in terms of nanoparticle-based technology. A limited number of methods suitable to associate proteins to exosome membrane has been described so far, and very little is known regarding the possibility to upload proteins inside exosomes. We optimized a method of protein incorporation in exosomes by exploiting the unique properties of a nonfunctional mutant of the HIV-1 Nef protein referred to as Nef(mut). It incorporates at high extents in exosomes meanwhile acting as carrier of protein antigens fused at its C-terminus. Manipulating Nef(mut) allows the incorporation into exosomes of high amounts of heterologous proteins which thus remain protected from external neutralization/degradation factors. These features, together with flexibility in terms of incorporation of foreign antigens and ease of production, make Nef(mut)-based exosomes a convenient vehicle for different applications (e.g., protein transduction, immunization) whose performances are comparable with those of alternative, more complex nanoparticle-based delivery systems.

Objective:The identification of still unrevealed mechanisms affecting the anti-HIV CD8(+) T-cell response in HIV-1 infection.Design:Starting from the observation that anti-Tat immunization is associated with improved CD8(+) T-cell immunity, we developed both in-vitro and ex-vivo assays to characterize the effects of extra-cellular Tat on the adaptive CD8(+) T-cell response.Methods:The effects of Tat on CD8(+) T-cell activation were assayed using CD8(+) T-cell clones specific for either cellular (MART-1) or viral (HIV-1 Nef) antigens, and HIV-1 Gag-specific CD8(+) T cells from HIV-1 patients.Results:The interaction between CD8(+) T lymphocytes and immobilized Tat, but not its soluble form, inhibits peptide-specific CD8(+) T-lymphocyte activation. The inhibition does not depend on Tat trans-activation activity, but on the interaction of the Tat RGD domain with 51 and v3 integrins. Impaired CD8(+) T-cell activation was also observed in cocultures of CD8(+) T cells with HIV-1-infected cells. Anti-Tat Abs abrogate the inhibitory effect, consistently with the evidence that extracellular Tat accumulates on the cell membrane of virus-producing cells. The Tat-induced inhibition of cell activation associates with increased apoptosis of CD8(+) T cells. Finally, the inhibition of cell activation also takes place in Gag-specific CD8(+) T lymphocytes from HIV-1-infected patients.Conclusion:Our results support the idea that CD8(+) T-cell apoptosis induced by surface-bound extracellular Tat can contribute to the dysregulation of the CD8(+) T-cell adaptive response against HIV as well as other pathogens present in AIDS patients.

Background: A relevant burden of defective HIV-1 genomes populates PBMCs from HIV-1 infected patients, especially during HAART treatment. These viral genomes, although unable to codify for infectious viral particles, can express viral proteins which may affect functions of host cells as well as bystander ones. Cells expressing defective HIV-1 have a lifespan longer than that of cells producing infectious particles. Hence, their interaction with other cell types, including resting lymphocytes, is expected to occur frequently in tissues where HIV actively replicates. We investigated the effects of the expression of a prototype of functionally defective HIV-1 on bystander, unstimulated CD4(+) T lymphocytes. Results: We observed that unstimulated human primary CD4(+) T lymphocytes were activated and became permissive for HIV-1 replication when co-cultivated with cells expressing a functionally defective HIV-1 (F12/Hut-78 cells). This effect depended on the presence in F12/Hut-78 supernatants of nanovesicles we identified as exosomes. By inspecting the underlying mechanism, we found that ADAM17, i.e., a disintegrin and metalloprotease converting pro-TNF-alpha in its mature form, associated with exosomes from F12/Hut-78 cells, and played a key role in the HIV-1 replication in unstimulated CD4(+) T lymphocytes. In fact, the treatment with an inhibitor of ADAM17 abolished both activation and HIV-1 replication in unstimulated CD4(+) T lymphocytes. TNF-alpha appeared to be the downstream effector of ADAM17 since the treatment of unstimulated lymphocytes with antibodies against TNF-alpha or its receptors blocked the HIV-1 replication. Finally, we found that the expression of Nef(F12) in exosome-producing cells was sufficient to induce the susceptibility to HIV-1 infection in unstimulated CD4(+) T lymphocytes. Conclusions: Exosomes from cells expressing a functionally defective mutant can induce cell activation and HIV-1 susceptibility in unstimulated CD4(+) T lymphocytes. This evidence highlights the relevance for AIDS pathogenesis of the expression of viral products from defective HIV-1 genomes.

Monocytes and macrophages utilize the class A and B scavenger receptors to recognize and perform phagocytosis of invading microbes before a pathogen-specific immune response is generated. HIV-1 Nef protein affects the innate immune system impairing oxidative burst response and phagocytic capacity of macrophages. Our data show that exogenous recombinant myristoylated Nef protein induces a marked CD36 downregulation in monocytes from Peripheral Blood Mononuclear Cells, in Monocyte-Derived Macrophages (MDMs) differentiated by cytokines and in MDMs contained in a mixed culture obtained expanding PBMCs under Human Erythroid Massive Amplification condition. Under the latter culture condition we identify three main populations after 6 days of expansion: lymphocytes (37.8+/-14.7%), erythroblasts (46.7+/-6.1%) and MDMs (15.7+/-7.5%). The Nef addition to the cell culture significantly downregulates CD36 expression in MDMs, but not in erythroid cells. Furthermore, CD36 inhibition is highly specific since it does not modify the expression levels of other MDM markers such as CD14, CD11c, CD86, CD68, CD206, Toll-like Receptor 2 and Toll-like Receptor 4. Similar results were obtained in MDMs infected with VSV-G pseudotyped HIV-1-expressing Nef. The reduced CD36 membrane expression is associated with decrease of correspondent CD36 mRNA transcript. Furthermore, Nef-induced CD36 downregulation is linked to both impaired scavenger activity with reduced capability to take up oxidized lipoproteins and to significant decreased phagocytosis of fluorescent beads and GFP-expressing Salmonella tiphymurium. In addition we observed that Nef induces TNF-alpha release in MDMs. Although these data suggest a possible involvement of TNF-alpha in mediating Nef activity, our results exclude a possible relationship between Nef-induced TNF-alpha release and Nef-mediated CD36 downregulation. The present work shows that HIV-1 Nef protein may have a role in the strategies elaborated by HIV-1 to alter pathogen disease outcomes, by modulating CD36 expression in macrophages, favoring the onset of opportunistic infections in HIV-1 infected people.

MicroRNA miR-146a and PLZF are reported as major players in the control of hematopoiesis, immune function and cancer. PLZF is described as a miR-146a repressor, whereas CXCR4 and TRAF6 were identified as miR-146a direct targets in different cell types. CXCR4 is a co-receptor of CD4 molecule that facilitates HIV-1 entry into T lymphocytes and myeloid cells, whereas TRAF6 is involved in immune response. Thus, the role of miR-146a in HIV-1 infection is currently being thoroughly investigated.In this study, we found that PLZF mediates suppression of miR-146a to control increases of CXCR4 and TRAF6 protein levels in human primary CD4+ T lymphocytes. We show that miR-146a upregulation by AMD3100 treatment or PLZF silencing, decreases CXCR4 protein expression and prevents HIV-1 infection of leukemic monocytic cell line and CD4+ T lymphocytes.Our findings improve the prospects of developing new therapeutic strategies to prevent HIV-1 entry via CXCR4 by using the PLZF/miR-146a axis.

Background: Completion of HIV life cycle in CD4+ T lymphocytes needs cell activation. We recently reported that treatment of resting CD4+ T lymphocytes with exosomes produced by HIV-1 infected cells induces cell activation and susceptibility to HIV replication. Here, we present data regarding the effects of these exosomes on cells latently infected with HIV-1. Results: HIV-1 latently infecting U937-derived U1 cells was activated upon challenge with exosomes purified from the supernatant of U937 cells chronically infected with HIV-1. This effect was no more detectable when exosomes from cells infected with HIV-1 strains either nef-deleted or expressing a functionally defective Nef were used, indicating that Nef is the viral determinant of exosome-induced HIV-1 activation. Treatment with either TAPI-2, i.e., a specific inhibitor of the pro-TNFaα-processing ADAM17 enzyme, or anti-TNFaα Abs abolished HIV-1 activation. Hence, similar to what previously demonstrated for the exosome-mediated activation of uninfected CD4+ T lymphocytes, the Nef-ADAM17-TNFaα axis is part of the mechanism of latent HIV-1 activation. It is noteworthy that these observations have been reproduced using: (1) primary CD4+ T lymphocytes latently infected with HIV-1; (2) exosomes from both primary CD4+ T lymphocytes and macrophages acutely infected with HIV-1; (3) co-cultures of HIV-1 acutely infected CD4+ T lymphocytes and autologous lymphocytes latently infected with HIV-1, and (4) exosomes from cells expressing a defective HIV-1. Conclusions: Our results strongly suggest that latent HIV-1 can be activated by TNFaα released by cells upon ingestion of exosomes released by infected cells, and that this effect depends on the activity of exosome-associated ADAM17. These pieces of evidence shed new light on the mechanism of HIV reactivation in latent reservoirs, and might also be relevant to design new therapeutic interventions focused on HIV eradication.