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.

We developed an innovative strategy to induce a cytotoxic T cell (CTL) immune response against protein antigens of choice. It relies on the production of exosomes, i.e., nanovesicles spontaneously released by all cell types. We engineered the upload of huge amounts of protein antigens upon fusion with an anchoring protein (i.e., HIV-1 Nef^mut), which is an inactive protein incorporating in exosomes at high levels also when fused with foreign proteins. We compared the immunogenicity of engineered exosomes uploading human papillomavirus (HPV)-E7 with that of lentiviral virus-like particles (VLPs) incorporating equivalent amounts of the same antigen. These exosomes, whose limiting membrane was decorated with VSV-G, i.e., an envelope protein inducing pH-dependent endosomal fusion, proved to be as immunogenic as the cognate VLPs. It is noteworthy that the immunogenicity of the engineered exosomes remained unaltered in the absence of VSV-G. Most important, we provide evidence that the inoculation in mouse of exosomes uploading HPV-E7 induces production of anti-HPV E7 CTLs, blocks the growth of syngeneic tumor cells inoculated after immunization, and controls the development of tumor cells inoculated before the exosome challenge. These results represent the proof-of-concept about both feasibility and efficacy of the Nef^mut-based exosome platform for the induction of CD8⁺ T cell immunity.

Approximately 12% of human cancers worldwide are associated with infectious agents, which are classified by the International Agency for Research on Cancer (IARC) as Group 1 within the agents that are carcinogenic to humans. Most of these agents are viruses. Group 1 oncogenic viruses include hepatitis C virus, hepatitis B virus (HBV), human T-cell lymphotropic virus type 1, Epstein-Barr virus, Kaposi sarcoma-associated herpesvirus, human immunodeficiency virus-1 and high-risk human papillomaviruses (HPVs). In addition, some human polyomaviruses are suspected of inducing cancer prevalently in hosts with impaired immune responses. Merkel cell polyomavirus has been associated with Merkel cell carcinoma and included by the IARC in Group 2A (i.e., probably carcinogenic to humans). Linking viruses to human cancers has allowed for the development of diagnostic, prophylactic and therapeutic measures. Vaccination significantly reduced tumours induced by two oncogenic viruses as follows: HBV and HPV. Herein, we focus on mucosal alpha HPVs, which are responsible for the highest number of cancer cases due to tumour viruses and against which effective prevention strategies have been developed to reduce the global burden of HPV-related cancers.

Some human papillomavirus (HPV) genotypes are universally recognized as major etiological agents not only of ano-genital tumors but also of head and neck cancers, which show increasing incidence. The evaluation of current and future therapeutic approaches against HPV-induced tumors is a global health priority, despite an effective prophylactic vaccine against 7 of the 12 genotypes involved in the etiology of tumors being currently available. In this review, we present the main anti-HPV therapeutic approaches in clinical experimentation, with a focus on a novel tumor antigen delivery method using engineered exosomes, that we recently developed. Our system allows the induction of an efficient unrestricted cytotoxic T lymphocyte (CTL) immune response against the HPV16-E7 tumor-associated antigen, with the formation of endogenously engineered exosomes, i.e., nanovesicles spontaneously released by all cell types. Immunogenic exosomes are uploaded with HPV16-E7 due to the fusion with a unique exosome-anchoring protein referred to as Nef^mut. Intramuscular injection of a DNA vector expressing the fusion protein generates exosomes sufficiently immunogenic to elicit a potent anti-16E7 CTL immune response. The approach is described here and the advantages over other existing methodologies are reported.