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Flavia Ferrantelli,1,* Claudia Arenaccio,1,* Francesco Manfredi,1 Eleonora Olivetta,1 Chiara Chiozzini,1 Patrizia Leone,1 Zulema Percario,2 Alessandro Ascione,1 Michela Flego,1 Paola Di Bonito,3 Luisa Accardi,3 Maurizio Federico1 1National Center for Global Health, Istituto Superiore Di Sanità (ISS), Rome, Italy; 2Department of Science, Roma Tre University, Rome, Italy; 3Department of Infectious Diseases, Istituto Superiore Di Sanità (ISS), Rome, Italy*These authors contributed equally to this workCorrespondence: Maurizio FedericoNational Center for Global Health, Istituto Superiore Di Sanità, Viale Regina Elena, 299, Rome 00161, ItalyTel +39-06-4990-3605Fax +39-06-49903210Email maurizio.federico@iss.itPurpose: 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.Keywords: single-chain variable fragments, Human Papilloma Virus E7 protein, exosomes, human immunodeficiency virus Nef protein, scFv intracellular delivery

Additional file 3: Figure S7AIII. The treatment with the divalent scFvF7-Fc Ab increases the activation of peptide-stimulated Mart1-specific CD8+ T lymphocytes in terms of IFN-Îł secretion (Exp. II and Exp. III). For details see Legend of Fig. 7a. (PPTX 41 kb)

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.

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.

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.

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.

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.

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.

We developed an innovative strategy to induce a cytotoxic T cell (CTL) immune response against protein antigens of choice. It relies on the production of exosomes, i.e., nanovesicles spontaneously released by all cell types. We engineered the upload of huge amounts of protein antigens upon fusion with an anchoring protein (i.e., HIV-1 Nefmut), which is an inactive protein incorporating in exosomes at high levels also when fused with foreign proteins. We compared the immunogenicity of engineered exosomes uploading human papillomavirus (HPV)-E7 with that of lentiviral virus-like particles (VLPs) incorporating equivalent amounts of the same antigen. These exosomes, whose limiting membrane was decorated with VSV-G, i.e., an envelope protein inducing pH-dependent endosomal fusion, proved to be as immunogenic as the cognate VLPs. It is noteworthy that the immunogenicity of the engineered exosomes remained unaltered in the absence of VSV-G. Most important, we provide evidence that the inoculation in mouse of exosomes uploading HPV-E7 induces production of anti-HPV E7 CTLs, blocks the growth of syngeneic tumor cells inoculated after immunization, and controls the development of tumor cells inoculated before the exosome challenge. These results represent the proof-of-concept about both feasibility and efficacy of the Nefmut-based exosome platform for the induction of CD8+ T cell immunity.