These are expected to be internalized by antigen-presenting cells. 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. strong class=”kwd-title” Keywords: nanovesicles, cytotoxic T lymphocytes, HIV-1 Nef, DNA vectors Introduction Exosomes are vesicles ranging 50C150 nm and are released constitutively by all cell types.1 They are generated by inward invagination of endosome membranes. These intraluminal vesicles form the multivesicular bodies (MVBs) which can traffic to the plasma membrane to which they fuse, thereby releasing their vesicular contents in the extracellular milieu. Exosomes are part of the intercellular communication network. They incorporate messenger RNAs, microRNAs, DNA, and proteins which can be functional in target cells. Nanovesicles showing both physical and biochemical Candesartan cilexetil (Atacand) features resembling exosomes, but those generated through direct extrusion of plasma membrane have also been described in muscle cells. 2C4 The exosome immunogenicity basically relies on Candesartan cilexetil (Atacand) the amounts and quality of antigens they incorporate. Exosomes were tested as antitumor immunostimulatory agents, and clinical trials demonstrated that they are well tolerated as cell-free vaccines.5,6 However, their therapeutic Candesartan cilexetil (Atacand) efficacy appeared quite limited, posing the need of new methods to increase their immunogenicity. This issue has been addressed through in vitro methods devoted to engineer foreign antigens to increase their display on the exosome membrane.7,8 Exosome biogenesis and HIV budding share the functions of a number of cell proteins including Alix, Tsg101, and several components of the endosomal sorting complex required for transport.9 The convergence of exosome and HIV biogenesis implies the possibility that viral products incorporate in exosomes. Such is the case for HIV-1 Nef, which associates with exosomes through anchoring its N-terminal myristoylation to lipid raft microdomains at the endosome membranes.10 Nef is a 27 kilodalton (kDa) protein lacking enzymatic activities. However, it can act as a scaffold/adaptor element to trigger activation of signal transducing molecules.11 We previously identified a G3C V153L E177G Nef mutant incorporating in exosomes at quite high levels.12 This Nef mutant (referred to as Nefmut) is defective for basically all Nef functions. Its efficiency of incorporation in nanovesicles does not change significantly when fused at its C-terminus with foreign proteins. Manipulating Nefmut allows the incorporation of high amounts of antigens of choice into exosomes, which thus remain protected from external neutralization/degradation factors. We recently reported that the inoculation in mice of exosomes carrying HPV E7 fused with Nefmut induces an effective E7-specific CD8+ T cytotoxic lymphocyte (CTL) response.13 This result demonstrates that, through the Nefmut-based engineering strategy, Mouse monoclonal to SHH the already proven poor CTL immunogenicity of exosomes can be overcome. However, this strategy would face possible difficulties in view of potential clinical applications, including in areas of standardization of industrial manufacturing, high cost-effectiveness, and storage of the immunogen. For these reasons, we conceived a still unproven, exosome-based vaccine strategy relying on delivery of DNA vectors expressing Nefmut-based fusion proteins into host animal by intramuscular (IM) inoculation. This strategy relies on the very recent observation that muscle mass cells also, both proliferating and differentiated, constitutively release exosome-like vesicles. Considering that Nefmut and derivatives thereof associate with exosomes with high effectiveness, we predicted the manifestation of Nefmut-based vectors in muscle mass cells would be sufficient to create a continuous source of manufactured, immunogenic exosomes. Here, we demonstrate that IM inoculation of mice having a DNA vector expressing Nefmut/E7 elicits a potent CTL immune response, therefore obstructing the growth of already implanted tumor cells. We provide evidence the production of endogenously manufactured exosomes was the basis of the observed antitumor effect. Materials and methods Molecular constructs and cell ethnicities All molecular constructs were based on IE-CMV-promoted vectors. Vectors expressing Nefmut, Nefmut/green fluorescent protein (GFP),12 NefG2A/GFP,14 wild-type (wt) Nef,15 and HPV E716.
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