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 62 EEEE 65 acidic cluster domain. In addition, we observed that ADAM17, i.e., a disintegrin and metalloprotease converting pro-tumor necrosis factor alpha (TNF- ␣
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, Nef , 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 Nef , and are uploaded in exosomes at levels comparable to Nef . 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.
We recently proved that exosomes engineered in vitro to deliver high amounts of HPV E7 upon fusion with the Nef mut 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 Nef mut fused with HPV E7. In this way, we predicted that the expression of the Nef mut /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 Nef mut /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 Nef mut /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 Nef mut /E7 vector. Finally, we provide evidence that the injection of Nef mut /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 Nef mut and its derivatives.
BackgroundCompletion 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.ResultsHIV-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.ConclusionsOur 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.
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