Claudia Arenaccio

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 Nef^mut, 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 Nef^mut-derived products by removing the Nef^mut 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.

Intrinsic genetic instability of tumor cells leads to continuous production of mutated proteins referred to as tumor-specific neoantigens. Generally, they are recognized as nonself products by the host immune system. However, an effective adaptive response clearing neoantigen-expressing cells is lost in tumor diseases. Most advanced therapeutic strategies aim at inducing neoantigen-specific immune activation through personalized approaches. They include tumor cell exome sequencing, human leukocyte antigen (HLA) typing, synthesis, and injection of peptides/RNA with adjuvants. Here, we propose an innovative method to induce a CD8⁺ T cytotoxic lymphocyte (CTL) immune response against tumor neoantigens bypassing the steps needed in current therapeutic strategies of personalized vaccination. We assumed that tumor cells can be the most efficient and precise factory of major histocompatibility complex (MHC) class I-associated, tumor neoantigen-derived peptides. Hence, endowing tumor cells with professional antigen-presenting functions would prime CD8⁺ T lymphocytes towards a response against nonself tumor antigens. To explore this possibility, both adenocarcinoma and melanoma human cells were engineered to express both CD80 and CD86 costimulatory molecules. HLA-matched lymphocytes were then primed through cocultivation with the engineered tumor cells. The generation of tumor-specific CD8⁺ T lymphocytes was tested through the combined analysis of cell activation markers, formation of immunologic synapses, generation of tumor antigen-specific CD8⁺ T lymphocytes, and cytotoxic activity. Our data consistently indicate that tumor cells endowed with professional antigen-presenting functions can generate an effective tumor-specific CTL immune response. This finding may open avenues towards the development of innovative antitumor immunotherapies.

Background: 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. Objective: Here, we discuss two key aspects to be analyzed before considering exosomes as a tool of delivery for the desired therapeutic molecule, i.e., techniques of engineering, and their in vivo biodistribution/ pharmacokinetics. In addition, an innovative approach aimed at overcoming at least part of the obstacles towards a safe and efficient use of exosomes in therapy will be discussed.Conclusion: Several biologic features render exosomes an attractive tool for the delivery of therapeutic molecules. They will surely be a part of innovative therapeutic interventions as soon as few still unmet technical hindrances will be overcome.

Eukaryotic cells constitutively produce nanovesicles of 50–150 nm of diameter, referred to as exosomes, upon release of the contents of multivesicular bodies (MVBs). We recently characterized a novel, exosome-based way to induce cytotoxic T lymphocyte (CTL) immunization against full-length antigens. It is based on DNA vectors expressing products of fusion between the exosome-anchoring protein Nef mutant (Nef^mut) with the antigen of interest. The strong efficiency of Nef^mut to accumulate in MVBs results in the production of exosomes incorporating huge amounts of the desired antigen. When translated in animals, the injection of Nef^mut-based DNA vectors generates engineered exosomes whose internalization in antigen-presenting cells induces cross-priming and antigen-specific CTL immunity. Here, we describe the molecular strategies we followed to produce DNA vectors aimed at generating immunogenic exosomes potentially useful to elicit a CTL immune response against antigens expressed by the etiologic agents of major chronic viral infections, i.e., HIV-1, HBV, and the novel tumor-associated antigen HOXB7. Unique methods intended to counteract intrinsic RNA instability and nuclear localization of the antigens have been developed. The success we met with the production of these engineered exosomes opens the way towards pre-clinic experimentations devoted to the optimization of new vaccine candidates against major infectious and tumor pathologies.

Exosomes are 50–150 nm sized nanovesicles released by all eukaryotic cells. The authors very recently described a method to engineer exosomes in vivo with the E7 protein of Human Papilloma Virus (HPV). This technique consists in the intramuscular injection of a DNA vector expressing HPV‐E7 fused at the C‐terminus of an exosome‐anchoring protein, that is, Nef^mut, the authors previously characterized for its high levels of incorporation in exosomes. In this configuration, the ≈11 kDa E7 protein elicited a both strong and effective antigen‐specific cytotoxic T lymphocyte (CTL) immunity. Attempting to establish whether this method could have general applicability, the authors expanded the immunogenicity studies toward an array of viral products of various origin and size including Ebola Virus VP24, VP40 and NP, Influenza Virus NP, Crimean–Congo Hemorrhagic Fever NP, West Nile Virus NS3, and Hepatitis C Virus NS3. All antigens appeared stable upon fusion with Nef^mut, and are uploaded in exosomes at levels comparable to Nef^mut. When injected in mice, DNA vectors expressing the diverse fusion products elicited a well detectable antigen‐specific CD8⁺ T cell response associating with a cytotoxic activity potent enough to kill peptide‐loaded and/or antigen‐expressing syngeneic cells. These data definitely proven both effectiveness and flexibility of this innovative CTL vaccine platform.

We recently described a novel biotechnological platform for the production of unrestricted cytotoxic T lymphocyte (CTL) vaccines. It relies on in vivo engineering of exosomes, i.e., nanovesicles constitutively released by all cells, with full-length antigens of choice upon fusion with an exosome-anchoring protein referred to as Nef^mut. They are produced upon intramuscular injection of a DNA vector and, when uploaded with a viral tumor antigen, were found to elicit an immune response inhibiting the tumor growth in a model of transplantable tumors. However, for a possible application in cancer immunotherapy, a number of key issues remained unmet. Among these, we investigated: (i) whether the immunogenic stimulus induced by the engineered exosomes can break immune tolerance, and (ii) their effectiveness when applied in human system. As a model of immune tolerance, we considered mice transgenic for the expression of activated rat HER2/neu which spontaneously develop adenocarcinomas in all mammary glands. When these mice were injected with a DNA vector expressing the product of fusion between Nef^mut and the extracellular domain of HER2/neu, antigen-specific CD8⁺ T lymphocytes became readily detectable. This immune response associated with a HER2-directed CTL activity and a significant delay in tumor development. On the other hand, through cross-priming experiments, we demonstrated the effectiveness of the engineered exosomes emerging from transfected human primary muscle cells in inducing antigen-specific CTLs. We propose our CTL vaccine platform as part of new immunotherapy strategies against tumors expressing self-antigens, i.e., products highly expressed in oncologic lesions but tolerated by the immune system.

Exosomes are extracellular vesicles of 50–150 nm in diameter secreted by basically all cell types. They mediate micro-communication among cells, tissues, and organs under both healthy and disease conditions by virtue of their ability to deliver macromolecules to target cells. Research on exosomes is a rapidly growing field, however many aspects of their biogenesis and functions still await a complete clarification. In our review we summarize most recent findings regarding biogenesis, structure, and functions of exosomes. In addition, an overview regarding the role of exosomes in both infectious and non-infectious diseases is provided. Finally, the use of exosomes as biomarkers and delivery tools for therapeutic molecules is addressed. Considering the body of literature data, exosomes have to be considered key components of the intercellular communication in both health and disease.

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.

Intact HIV-1 and exosomes can be internalized by dendritic cells (DCs) through a common pathway leading to their transmission to CD4⁺ T lymphocytes by means of mechanisms defined as trans-infection and trans-dissemination, respectively. We previously reported that exosomes from HIV-1-infected cells activate both uninfected quiescent CD4⁺ T lymphocytes, which become permissive to HIV-1, and latently infected cells, with release of HIV-1 particles. However, nothing is known about the effects of trans-dissemination of exosomes produced by HIV-1-infected cells on uninfected or latently HIV-1-infected CD4⁺ T lymphocytes. Here, we report that trans-dissemination of exosomes from HIV-1-infected cells induces cell activation in resting CD4⁺ T lymphocytes, which appears stronger with mature than immature DCs. Using purified preparations of both HIV-1 and exosomes, we observed that mDC-mediated trans-dissemination of exosomes from HIV-1-infected cells to resting CD4⁺ T lymphocytes induces efficient trans-infection and HIV-1 expression in target cells. Most relevant, when both mDCs and CD4⁺ T lymphocytes were isolated from combination anti-retroviral therapy (ART)-treated HIV-1-infected patients, trans-dissemination of exosomes from HIV-1-infected cells led to HIV-1 reactivation from the viral reservoir. In sum, our data suggest a role of exosome trans-dissemination in both HIV-1 spread in the infected host and reactivation of the HIV-1 reservoir.