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

• Published in: International journal of nanomedicine Vol. 12; pp. 4579 - 4591
• Main Authors: Di Bonito, Paola, Chiozzini, Chiara, Arenaccio, Claudia, Anticoli, Simona, Manfredi, Francesco, Olivetta, Eleonora, Ferrantelli, Flavia, Falcone, Emiliana, Ruggieri, Anna, Federico, Maurizio
• Format: Journal Article
• Language: English
• Published: New Zealand Dove Medical Press Limited 01.01.2017; Taylor & Francis Ltd; Dove Medical Press
• Subjects: Animals; Antigens; Antineoplastic Agents - immunology; Antineoplastic Agents - pharmacology; Biosynthesis; CD8-Positive T-Lymphocytes - immunology; Cell culture; Complications and side effects; Cytotoxic T-lymphocytes; Deoxyribonucleic acid; DNA; DNA - administration & dosage; DNA vectors; Drug therapy; Exosomes - genetics; Exosomes - immunology; Exosomes - metabolism; Female; Genes, nef; Genetic Engineering - methods; Genetic Vectors - immunology; Health aspects; HIV; HIV-1 Nef; Human immunodeficiency virus; Human papillomavirus; Laboratory animals; Mice, Inbred C57BL; Nanoparticles; Nanovesicles; Original Research; Papillomavirus E7 Proteins - genetics; Papillomavirus E7 Proteins - pharmacology; Papillomavirus infections; Physiological aspects; Properties; Proteins; T cells; T-Lymphocytes, Cytotoxic - immunology; Vaccines; Italy; United Kingdom > UK; United States > US; HIV-1 Nef; cytotoxic T lymphocytes; nanovesicles; DNA vectors
• ISSN: 1178-2013; 1176-9114; 1178-2013
• DOI: 10.2147/IJN.S131309

We recently proved that exosomes engineered in vitro to deliver high amounts of HPV E7 upon fusion with the Nef 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 fused with HPV E7. In this way, we predicted that the expression of the Nef /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 /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 /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 /E7 vector. Finally, we provide evidence that the injection of Nef /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 and its derivatives.