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-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.

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. 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. 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.

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.