Long-Term Antitumor CD8+ T Cell Immunity Induced by Endogenously Engineered Extracellular Vesicles.

We developed an innovative method to induce antigen-specific CD8+ T cytotoxic lymphocyte (CTL) immunity based on in vivo engineering of extracellular vesicles (EVs). This approach employs a DNA vector expressing a mutated HIV-1 Nef protein (Nefmut) deprived of the anti-cellular effects typical of the wild-type isoform, meanwhile showing an unusual efficiency of incorporation into EVs. This function persists even when foreign antigens are fused to its C-terminus. In this way, Nefmut traffics large amounts of antigens fused to it into EVs spontaneously released by the recipient cells. We previously provided evidence that mice injected with a DNA vector expressing the Nefmut/HPV16-E7 fusion protein developed an E7-specific CTL immune response as detected 2 weeks after the second immunization. Here, we extended and optimized the anti-HPV16 CD8+ T cell immune response induced by the endogenously engineered EVs, and evaluated the therapeutic antitumor efficacy over time. We found that the co-injection of DNA vectors expressing Nefmut fused with E6 and E7 generated a stronger anti-HPV16 immune response compared to that observed in mice injected with the single vectors. When HPV16-E6 and -E7 co-expressing tumor cells were implanted before immunization, all mice survived at day 44, whereas no mice injected with either void or Nefmut-expressing vectors survived until day 32 after tumor implantation. A substantial part of immunized mice (7 out of 12) cleared the tumor. When the cured mice were re-challenged with a second tumor cell implantation, none of them developed tumors. Both E6- and E7-specific CD8+ T immunities were still detectable at the end of the observation time. We concluded that the immunity elicited by engineered EVs, besides counteracting and curing already developed tumors, was strong enough to guarantee the resistance to additional tumor attacks. These results can be of relevance for the therapy of both metastatic and relapsing tumors.

Simultaneous CD8+ T-Cell Immune Response against SARS-Cov-2 S, M, and N Induced by Endogenously Engineered Extracellular Vesicles in Both Spleen and Lungs.

Most advanced vaccines against severe acute respiratory syndrome coronavirus (SARS-CoV)-2 are designed to induce antibodies against spike (S) protein. Differently, we developed an original strategy to induce CD8+ T cytotoxic lymphocyte (CTL) immunity based on in vivo engineering of extracellular vesicles (EVs). This is a new vaccination approach based on intramuscular injection of DNA expression vectors coding for a biologically inactive HIV-1 Nef protein (Nefmut) with an unusually high efficiency of incorporation into EVs, even when foreign polypeptides are fused to its C-terminus. Nanovesicles containing Nefmut-fused antigens released by muscle cells can freely circulate into the body and are internalized by antigen-presenting cells. Therefore, EV-associated antigens can be cross-presented to prime antigen-specific CD8+ T-cells. To apply this technology to a strategy of anti-SARS-CoV-2 vaccine, we designed DNA vectors expressing the products of fusion between Nefmut and different viral antigens, namely N- and C-terminal moieties of S (referred to as S1 and S2), M, and N. We provided evidence that all fusion products are efficiently uploaded in EVs. When the respective DNA vectors were injected in mice, a strong antigen-specific CD8+ T cell immunity became detectable in spleens and, most important, in lung airways. Co-injection of DNA vectors expressing the diverse SARS-CoV-2 antigens resulted in additive immune responses in both spleen and lungs. Hence, DNA vectors expressing Nefmut-based fusion proteins can be proposed for new anti-SARS-CoV-2 vaccine strategies.

The C-Terminal Domain of Nefmut Is Dispensable for the CD8+ T Cell Immunogenicity of In Vivo Engineered Extracellular Vesicles.

Intramuscular injection of DNA vectors expressing the extracellular vesicle (EV)-anchoring protein Nefmut fused at its C-terminus to viral and tumor antigens elicit a potent, effective, and anti-tolerogenic CD8+ T cell immunity against the heterologous antigen. The immune response is induced through the production of EVs incorporating Nefmut-derivatives released by muscle cells. In the perspective of a possible translation into the clinic of the Nefmut-based vaccine platform, we aimed at increasing its safety profile by identifying the minimal part of Nefmut retaining the EV-anchoring protein property. We found that a C-terminal deletion of 29-amino acids did not affect the ability of Nefmut to associate with EVs. The EV-anchoring function was also preserved when antigens from both HPV16 (i.e., E6 and E7) and SARS-CoV-2 (i.e., S1 and S2) were fused to its C-terminus. Most important, the Nefmut C-terminal deletion did not affect levels, quality, and diffusion at distal sites of the antigen-specific CD8+ T immunity. We concluded that the C-terminal Nefmut truncation does not influence stability, EV-anchoring, and CD8+ T cell immunogenicity of the fused antigen. Hence, the C-terminal deleted Nefmut may represent a safer alternative to the full-length isoform for vaccines in humans.

KSHV G-protein coupled receptor vGPCR oncogenic signaling upregulation of Cyclooxygenase-2 expression mediates angiogenesis and tumorigenesis in Kaposi's sarcoma.

Kaposi's sarcoma-associated herpesvirus (KSHV) vGPCR is a constitutively active G protein-coupled receptor that subverts proliferative and inflammatory signaling pathways to induce cell transformation in Kaposi's sarcoma. Cyclooxygenase-2 (COX-2) is an inflammatory mediator that plays a key regulatory role in the activation of tumor angiogenesis. Using two different transformed mouse models and tumorigenic full KSHV genome-bearing cells, including KSHV-Bac16 based mutant system with a vGPCR deletion, we demostrate that vGPCR upregulates COX-2 expression and activity, signaling through selective MAPK cascades. We show that vGPCR expression triggers signaling pathways that upregulate COX-2 levels due to a dual effect upon both its gene promoter region and, in mature mRNA, the 3'UTR region that control mRNA stability. Both events are mediated by signaling through ERK1/2 MAPK pathway. Inhibition of COX-2 in vGPCR-transformed cells impairs vGPCR-driven angiogenesis and treatment with the COX-2-selective inhibitory drug Celecoxib produces a significant decrease in tumor growth, pointing to COX-2 activity as critical for vGPCR oncogenicity in vivo and indicating that COX-2-mediated angiogenesis could play a role in KS tumorigenesis. These results, along with the overexpression of COX-2 in KS lesions, define COX-2 as a potential target for the prevention and treatment of KSHV-oncogenesis.

N-Terminal Fatty Acids of NEFMUT Are Required for the CD8+ T-Cell Immunogenicity of In Vivo Engineered Extracellular Vesicles.

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.

The Intracellular Delivery Of Anti-HPV16 E7 scFvs Through Engineered Extracellular Vesicles Inhibits The Proliferation Of HPV-Infected Cells.

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.

Tumor cells endowed with professional antigen-presenting cell functions prime PBLs to generate antitumor CTLs.

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.

Exosomes in Therapy: Engineering, Pharmacokinetics and Future Applications.

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.

DNA Vectors Generating Engineered Exosomes Potential CTL Vaccine Candidates Against AIDS, Hepatitis B, and Tumors.

Eukaryotic cells constitutively produce nanovesicles of 50-150 nm 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.

An Exosome-Based Vaccine Platform Imparts Cytotoxic T Lymphocyte Immunity Against Viral Antigens.

Exosomes are 50-150 nm sized nanovesicles released by all eukaryotic cells. The authors very recently described a method to engineer exosomes in vivo with the E7 protein of Human Papilloma Virus (HPV). This technique consists in the intramuscular injection of a DNA vector expressing HPV-E7 fused at the C-terminus of an exosome-anchoring protein, that is, Nefmut , the authors previously characterized for its high levels of incorporation in exosomes. In this configuration, the ≈11 kDa E7 protein elicited a both strong and effective antigen-specific cytotoxic T lymphocyte (CTL) immunity. Attempting to establish whether this method could have general applicability, the authors expanded the immunogenicity studies toward an array of viral products of various origin and size including Ebola Virus VP24, VP40 and NP, Influenza Virus NP, Crimean-Congo Hemorrhagic Fever NP, West Nile Virus NS3, and Hepatitis C Virus NS3. All antigens appeared stable upon fusion with Nefmut , and are uploaded in exosomes at levels comparable to Nefmut . When injected in mice, DNA vectors expressing the diverse fusion products elicited a well detectable antigen-specific CD8+ T cell response associating with a cytotoxic activity potent enough to kill peptide-loaded and/or antigen-expressing syngeneic cells. These data definitely proven both effectiveness and flexibility of this innovative CTL vaccine platform.

Engineered exosomes emerging from muscle cells break immune tolerance to HER2 in transgenic mice and induce antigen-specific CTLs upon challenge by human dendritic cells.

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. KEY MESSAGES: We established a novel, exosome-based method to produce unrestricted CTL vaccines. This strategy is effective in breaking the tolerance towards tumor self-antigens. Our method is also useful to elicit antigen-specific CTL immunity in humans. These findings open the way towards the use of this antitumor strategy in clinic.

Antitumor HPV E7-specific CTL activity elicited by in vivo engineered exosomes produced through DNA inoculation.

We recently proved that exosomes engineered in vitro to deliver high amounts of HPV E7 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 HPV E7. 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.

The CD8⁺ T Cell-Mediated Immunity Induced by HPV-E6 Uploaded in Engineered Exosomes Is Improved by ISCOMATRIXTM Adjuvant.

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 ISCOMATRIXTM adjuvant, i.e., an adjuvant composed of purified ISCOPREPTM 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 ISCOMATRIXTM 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.

Latent HIV-1 is activated by exosomes from cells infected with either replication-competent or defective HIV-1.

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-TNFα-processing ADAM17 enzyme, or anti-TNFα Abs abolished HIV-1 activation. Hence, similar to what previously demonstrated for the exosome-mediated activation of uninfected CD4(+) T lymphocytes, the Nef-ADAM17-TNFα 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 TNFα 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.

HPV-E7 delivered by engineered exosomes elicits a protective CD8⁺ T cell-mediated immune response.

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.

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

UNLABELLED: 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 62EEEE65 acidic cluster domain. In addition, we observed that ADAM17, i.e., a disintegrin and metalloprotease converting pro-tumor necrosis factor alpha (TNF-α) 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-α is the downstream effector of ADAM17 since the treatment of resting lymphocytes with anti-TNF-α 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. IMPORTANCE: Overall, our findings support the idea that HIV evolved to usurp the exosome-based intercellular communication network to favor its spread in infected hosts.

Cell activation and HIV-1 replication in unstimulated CD4+ T lymphocytes ingesting exosomes from cells expressing defective HIV-1.

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-α 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-α appeared to be the downstream effector of ADAM17 since the treatment of unstimulated lymphocytes with antibodies against TNF-α or its receptors blocked the HIV-1 replication. Finally, we found that the expression of NefF12 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.

HIV-1 Nef impairs key functional activities in human macrophages through CD36 downregulation.

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-α release in MDMs. Although these data suggest a possible involvement of TNF-α in mediating Nef activity, our results exclude a possible relationship between Nef-induced TNF-α 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.

The HIV protease inhibitor indinavir down-regulates the expression of the pro-angiogenic MT1-MMP by human endothelial cells.

In addition to contrast human immunodeficiency virus (HIV) replication, the HIV protease inhibitors (HIV-PI) have reduced tumour incidence or clinical progression in infected patients. In this regard, we have previously shown that, independently of its anti-viral activity, the HIV-PI indinavir (IDV) directly blocks matrix metalloproteinase (MMP)-2 proteolytic activation, thus efficiently inhibiting tumour angiogenesis in vitro, in animal models, and in humans. Herein we investigated the molecular mechanism for IDV anti-angiogenic effect. We found that treatment of human primary endothelial cells with therapeutic IDV concentrations decreases the expression of membrane type (MT)1-MMP, which is the major activator of MMP-2. This occurs for both the constitutive expression of MT1-MMP and that up-regulated by angiogenic factors. In either cases, reduction of MT1-MMP levels by IDV is preceded by the inhibition of the binding of the specificity protein (Sp)1 transcription factor to the promoter region of the MT1-MMP gene in endothelial cell nuclei. As MT1-MMP is key for tumour angiogenesis, these results support the use of IDV or its derivatives in anti-cancer therapy. This is recommended by the low toxicity of the drug, and the large body of data on its pharmacokinetic.

A role for virally induced reactive oxygen species in Kaposi's sarcoma herpesvirus tumorigenesis.

AIMS: Kaposi's sarcoma (KS), caused by the Kaposi's sarcoma herpesvirus (KSHV), is an AIDS-associated cancer characterized by angiogenesis and proliferation of spindle cells. Rac1-activated reactive oxygen species (ROS) production has been implicated in KS tumorigenesis. We used an animal model of KSHV-induced Kaposi's sarcomagenesis (mECK36) to study the role of ROS in KS and the efficacy of N-acetyl l-cysteine (NAC) in inhibiting or preventing KS. RESULTS: Signaling by the KSHV early lytic gene viral G protein-coupled receptor (vGPCR) activated ROS production in mECK36 cells via a Rac1-NADPH oxidase pathway. Induction of the lytic cycle in KSHV-infected KS spindle cells upregulated ROS along with upregulation of vGPCR expression. We also found that expression of the major latent transcript in 293 cells increased ROS levels. ROS scavenging with NAC halted mECK36 tumor growth in a KSHV-specific manner. NAC inhibited KSHV latent gene expression as well as tumor angiogenesis and lymphangiogenesis. These effects correlated with the reduction of vascular endothelial growth factor (VEGF), c-myc, and cyclin D1, and could be explained on the basis of inhibition of STAT3 tyrosine phosphorylation. NAC prevented mECK36 de novo tumor formation. Molecular analysis of NAC-resistant tumors revealed a strong upregulation of Rac1 and p40(PHOX). INNOVATION AND CONCLUSION: Our results demonstrate that ROS-induction by KSHV plays a causal role in KS oncogenesis by promoting proliferation and angiogenesis. Our results show that both ROS and their molecular sources can be targeted therapeutically using NAC or other Food and Drug Administration (FDA)-approved inhibitors for prevention and treatment of AIDS-KS.