HIV-1-infected cells transiently express lentiviral RNA shuttled by exosomes

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

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

Exosomes in Therapy: Engineering, Pharmacokinetics and Future Applications

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

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-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