Background/Objectives: African Swine Fever (ASF) represents one of the most serious threats to animal health and global food security. The causative agent of ASF is the African swine fever virus (ASFV), a DNA virus belonging to the Asfarviridae family. Here, we describe ex vivo results for an original anti-ASFV vaccine approach based on the cellular immune response induced by extracellular vesicles (EVs) engineered to express four ASFV proteins. EV engineering was achieved by expressing a DNA vector encoding a biologically inactive HIV-1 Nef protein (Nefmut), which exhibits unusually high efficiency of incorporation into EVs, even when fused to foreign proteins. Previous studies have demonstrated that intramuscular injection of Nefmut-based vectors leads to the engineering of Evs, spontaneously released by muscle cells, and induction of antigen-specific CD8+ T cell immunity. Methods: We designed DNA vectors expressing the fusion products between Nefmut and each of the four ASFV structural proteins p30, p54, pp62, and p72. Engineered EVs were molecularly characterized by Western blot and nanotrack analysis, and their potential immunogenicity was assessed by priming and cross-presentation assays. Results: We assessed that the four fusion proteins were successfully expressed in transfected mammalian cells, with the release of valuable amounts of engineered EVs. When immature swine dendritic cells were challenged with the engineered EVs and then co-cultivated with autologous peripheral blood lymphocytes in priming assays, lymphocyte subpopulations specifically reacting against each ASFV antigen were elicited, as detected by an IFN-γ ELISpot assay. In addition, we provide evidence that the Nefmut-based fusion products incorporated into the engineered EVs can be cross-presented by professional antigen-presenting cells, leading to cross-priming of autologous lymphocytes. Conclusions: These results represent the best premise to go forward with experiments examining immunogenicity and antiviral efficiency in pigs.

We previously developed an innovative strategy to induce CD8+ T lymphocyte-immunity through in vivo engineering of extracellular vesicles (EVs). This approach relies on intramuscular injection of DNA expressing antigens of interest fused at a biologically-inactive HIV-1 Nef protein mutant (Nefmut). Nefmut is very efficiently incorporated into EVs, thus conveying large amounts of fusion proteins into EVs released by transfected cells. This platform proved successful against highly immunogenic tumor-specific antigens. Here, we tested whether antigen-specific CD8+ T cell immune responses induced by engineered EVs can counteract the growth of tumors expressing two “self” tumor-associated antigens (TAAs): HOXB7 and Her2/neu. FVB/N mice were injected with DNA vectors expressing Nefmut fused to HOXB7 or Her2/neu, singly and in combination, before subcutaneous implantation of breast carcinoma cells co-expressing HOXB7 and Her2/neu. All mice immunized with the combination vaccine remained tumor-free, whereas groups vaccinated with single Nefmut-fused antigens were only partly protected, with stronger antitumor effects in Her2/neu-immunized mice. Double-vaccinated mice also controlled tumor growth upon a later tumor cell re-challenge. Importantly, co-vaccination also contained tumors in a therapeutic immunization setting. These results showed the efficacy of EV-based vaccination against two TAAs, and represent the first demonstration that HOXB7 may be targeted in multi-antigen immunotherapy strategies.

Induction of effective immunity in the lungs should be a requisite for any vaccine designed to control the severe pathogenic effects generated by respiratory infectious agents. We recently provided evidence that the generation of endogenous extracellular vesicles (EVs) engineered for the incorporation of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 Nucleocapsid (N) protein induced immunity in the lungs of K18-hACE2 transgenic mice, which then can survive the lethal virus infection. However, nothing is known about the ability of the N-specific CD8+ T cell immunity in controlling viral replication in the lungs, a major pathogenic signature of severe disease in humans. To fill the gap, we investigated the immunity generated in the lungs by N-engineered EVs in terms of induction of N-specific effectors and resident memory CD8+ T lymphocytes before and after virus challenge carried out three weeks and three months after boosting. At the same time points, viral replication extents in the lungs were evaluated. Three weeks after the second immunization, virus replication was reduced in mice best responding to vaccination by more than 3-logs compared to the control group. The impaired viral replication matched with a reduced induction of Spike-specific CD8+ T lymphocytes. The antiviral effect appeared similarly strong when the viral challenge was carried out 3 months after boosting, and associated with the persistence of N-specific CD8+ T-resident memory lymphocytes. In view of the quite low mutation rate of the N protein, the present vaccine strategy has the potential to control the replication of all emerging variants.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 enters the host by infecting nasal ciliated cells. Then, the virus can spread towards the oropharyngeal cavity and the pulmonary tissues. The antiviral adaptive immunity is promptly induced in response to the virus’s detection, with virus-specific T-lymphocytes appearing before antiviral antibodies. Both the breadth and potency of antiviral CD8+ T-cell immunity have a key role in containing viral spread and disease severity. Current anti-SARS-CoV-2 vaccines do not impede the virus’s replication in the upper respiratory tract, and there is consensus on the fact that the best potency of the antiviral immune response in both blood and the upper respiratory tract can be reached upon infection in vaccinees (i.e., breakthrough infection). However, whether the antiviral CD8+ T-cells developing in response to the breakthrough infection in the upper respiratory tract diffuse to the lungs is also still largely unknown. To fill the gap, we checked the CD8+ T-cell immunity elicited after infection of K18-hACE2 transgenic mice both at 3 weeks and 3 months after anti-spike vaccination. Virus-specific CD8+ T-cell immunity was monitored in both blood and the lungs before and after infection. By investigating the de novo generation of the CD8+ T-cells specific for SARS-CoV-2 viral proteins, we found that both membrane (M) and/or nucleocapsid (N)-specific CD8+ T-cells were induced at comparable levels in the blood of both unvaccinated and vaccinated mice. Conversely, N-specific CD8+ T-cells were readily found in the lungs of the control mice but were either rare or absent in those of vaccinated mice. These results support the idea that the hybrid cell immunity developing after asymptomatic/mild breakthrough infection strengthens the antiviral cell immunity in the lungs only marginally, implying that the direct exposition of viral antigens is required for the induction of an efficient antiviral cell immunity in the lungs.

Induction of effective immunity in lungs should be a pre-requisite for any vaccine designed to control the severe pathogenic effects generated by respiratory infectious agents. In the case of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 infection, vaccination is expected to associate with significant inhibition of viral replication in lungs. We recently provided evidence that the generation of endogenous extracellular vesicles (EVs) engineered for the incorporation of SARS-CoV-2 Nucleocapsid (N) protein can protect K18-hACE2 transgenic mice from the lethal intranasal infection with the ancestral Wuhan isolate. Actually, it was widely demonstrated that these transgenic mice succumb to SARS-CoV-2 intranasal infection mainly as a consequence of the viral invasiveness of central nervous system, a pathogenetic mechanism almost absent in humans. On the other hand, K18-hACE2 transgenic mice support viral replication in lungs, an event strictly mirroring the major pathogenic signature linked to the severe disease in humans. However, nothing is known about the ability of N-specific CD8 + T cell immunity induced by engineered EVs in controlling viral replication in lungs. To fill the gap, we investigated the immunity generated in lungs by N-engineered EVs in terms of induction of N-specific effectors and resident memory CD8 + T lymphocytes before and after virus challenge carried out three weeks and three months after boosting. At the same time points, viral replication extents in lungs were evaluated. We found that three weeks after second immunization, virus replication was reduced in mice best responding to vaccination by more than 3-logs compared to control group. The impaired viral replication matched with a reduced induction of Spike-specific CD8 + T lymphocytes. The antiviral effect appeared similarly strong when the viral challenge was carried out 3 months after boosting. This inhibitory effect associated with the persistence of a N-specific CD8 + T-resident memory lymphocytes in lungs of N-immunized mice. In view of the quite conserved sequence of the N protein among SARS-CoV-2 variants, these results support the idea that a vaccine strategy focused on the induction of anti-N CD8 + T cell immunity in lungs has the potential to control the replication of emerging variants.

Abstract Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 enters the host by infecting nasal ciliated cells. The virus then spreads toward the oropharyngeal cavity, and, in most severe cases, the pulmonary tissues. The antiviral adaptive response is promptly induced in response to virus detection, with virus-specific T lymphocytes appearing before antiviral antibodies. Both breadth and potency of the antiviral CD8+ T cell immunity have a key role in containing viral spread and disease severity. However, how much the spread of infecting SARS-CoV-2 into the respiratory tracts influences the potency of the antiviral CD8+ T-cell immunity is still largely unknown. To fill the gap, we checked the CD8+ T cell immunity induced after infection of K18-hACE2 transgenic mice in conditions where the virus spread is impeded. In detail, mice were infected with SARS-CoV-2 both 3 weeks and 3 months after anti-Spike vaccinations, and virus-specific CD8+ T cell immunity was monitored both before and after infection. We noticed a strong increase of the Spike-specific CD8+ T cell immunity in vaccinated mice six days after infection despite a nearly-full inhibition of the viral replication. Most important, both kinetics and efficiency of the induction of SARS-CoV-2 N-and M-specific CD8+ T cell immunity in vaccinated mice appeared not inferior to those induced in control mice. These results support the idea that the SARS-CoV-2 replication in the lungs does not relevantly influence the generation of virus-specific CD8+ T cell immunity.

Extracellular vesicles (EVs) are membranous particles released by all cells in the external milieu. Depending on their origin, they are given different names: exosomes are nanovesicles that originate from the endosomal compartment, whereas microvesicles bud from plasma membrane. Both contain molecules that are crucial for the onset and spreading of different pathologies, from neurodegenerative diseases to cancer, and are considered promising disease markers. On the other hand, EVs are often used as therapeutic tools, and can be engineered to carry drugs and chemicals. This chapter describes a method to produce EVs, mainly exosomes, containing the green fluorescent protein (GFP) linked to an exosome anchoring protein (Nefmut). This enables counting and tracing of fluorescent EVs by different methods, including conventional flow cytometry.

We propose an innovative anti-SARS-CoV-2 immune strategy based on extracellular vesicles (EVs) inducing an anti-SARS-CoV-2 N CD8+ T cytotoxic lymphocyte (CTL) immune response. We previously reported that the SARS-CoV-2 N protein can be uploaded at high levels in EVs upon fusion with Nefmut, i.e., a biologically inactive HIV-1 Nef mutant incorporating into EVs at quite high levels. Here, we analyze the immunogenic properties in human cells of EVs engineered with SARS-CoV-2 N fused at the C-terminus of either Nefmut or a deletion mutant of Nefmut referred to as NefmutPL. The analysis of in vitro-produced EVs has supported the uploading of N protein when fused with truncated Nefmut. Mice injected with DNA vectors expressed each fusion protein developed robust SARS-CoV-2 N-specific CD8+ T cell immune responses. When ex vivo human dendritic cells were challenged with EVs engineered with either fusion products, the induction of a robust N-specific CTL activity, as evaluated by both CD107a and trogocytosis assays, was observed. Through these data we achieved the proof-of-principle that engineered EVs can be instrumental to elicit anti-SARS-CoV-2 CTL immune response in human cells. This achievement represents a mandatory step towards the upcoming experimentations in pre-clinical models focused on intranasal administration of N-engineered EVs.

SARS-CoV-2-specific CD8+ T cell immunity is expected to counteract viral variants in both efficient and durable ways. We recently described a way to induce a potent SARS-CoV-2 CD8+ T immune response through the generation of engineered extracellular vesicles (EVs) emerging from muscle cells. This method relies on intramuscular injection of DNA vectors expressing different SARS-CoV-2 antigens fused at their N-terminus with the Nefmut protein, i.e., a very efficient EV-anchoring protein. However, quality, tissue distribution, and efficacy of these SARS-CoV-2-specific CD8+ T cells remained uninvestigated. To fill the gaps, antigen-specific CD8+ T lymphocytes induced by the immunization through the Nefmut-based method were characterized in terms of their polyfunctionality and localization at lung airways, i.e., the primary targets of SARS-CoV-2 infection. We found that injection of vectors expressing Nefmut/S1 and Nefmut/N generated polyfunctional CD8+ T lymphocytes in both spleens and bronchoalveolar lavage fluids (BALFs). When immunized mice were infected with 4.4 lethal doses of 50% of SARS-CoV-2, all S1-immunized mice succumbed, whereas those developing the highest percentages of N-specific CD8+ T lymphocytes resisted the lethal challenge. We also provide evidence that the N-specific immunization coupled with the development of antigen-specific CD8+ T-resident memory cells in lungs, supporting the idea that the Nefmut-based immunization can confer a long-lasting, lung-specific immune memory. In view of the limitations of current anti-SARS-CoV-2 vaccines in terms of antibody waning and efficiency against variants, our CD8+ T cell-based platform could be considered for a new combination prophylactic strategy.

Intramuscular injection of DNA vectors expressing the extracellular vesicle (EV)-anchoring protein Nefmut fused at its C-terminus to viral and tumor antigens elicit a potent, effective, and anti-tolerogenic CD8+ T cell immunity against the heterologous antigen. The immune response is induced through the production of EVs incorporating Nefmut-derivatives released by muscle cells. In the perspective of a possible translation into the clinic of the Nefmut-based vaccine platform, we aimed at increasing its safety profile by identifying the minimal part of Nefmut retaining the EV-anchoring protein property. We found that a C-terminal deletion of 29-amino acids did not affect the ability of Nefmut to associate with EVs. The EV-anchoring function was also preserved when antigens from both HPV16 (i.e., E6 and E7) and SARS-CoV-2 (i.e., S1 and S2) were fused to its C-terminus. Most important, the Nefmut C-terminal deletion did not affect levels, quality, and diffusion at distal sites of the antigen-specific CD8+ T immunity. We concluded that the C-terminal Nefmut truncation does not influence stability, EV-anchoring, and CD8+ T cell immunogenicity of the fused antigen. Hence, the C-terminal deleted Nefmut may represent a safer alternative to the full-length isoform for vaccines in humans.

Most advanced vaccines against severe acute respiratory syndrome coronavirus (SARS-CoV)-2 are designed to induce antibodies against spike (S) protein. Differently, we developed an original strategy to induce CD8+ T cytotoxic lymphocyte (CTL) immunity based on in vivo engineering of extracellular vesicles (EVs). This is a new vaccination approach based on intramuscular injection of DNA expression vectors coding for a biologically inactive HIV-1 Nef protein (Nefmut) with an unusually high efficiency of incorporation into EVs, even when foreign polypeptides are fused to its C-terminus. Nanovesicles containing Nefmut-fused antigens released by muscle cells can freely circulate into the body and are internalized by antigen-presenting cells. Therefore, EV-associated antigens can be cross-presented to prime antigen-specific CD8+ T-cells. To apply this technology to a strategy of anti-SARS-CoV-2 vaccine, we designed DNA vectors expressing the products of fusion between Nefmut and different viral antigens, namely N- and C-terminal moieties of S (referred to as S1 and S2), M, and N. We provided evidence that all fusion products are efficiently uploaded in EVs. When the respective DNA vectors were injected in mice, a strong antigen-specific CD8+ T cell immunity became detectable in spleens and, most important, in lung airways. Co-injection of DNA vectors expressing the diverse SARS-CoV-2 antigens resulted in additive immune responses in both spleen and lungs. Hence, DNA vectors expressing Nefmut-based fusion proteins can be proposed for new anti-SARS-CoV-2 vaccine strategies.

We developed an innovative method to induce antigen-specific CD8+ T cytotoxic lymphocyte (CTL) immunity based on in vivo engineering of extracellular vesicles (EVs). This approach employs a DNA vector expressing a mutated HIV-1 Nef protein (Nefmut) deprived of the anti-cellular effects typical of the wild-type isoform, meanwhile showing an unusual efficiency of incorporation into EVs. This function persists even when foreign antigens are fused to its C-terminus. In this way, Nefmut traffics large amounts of antigens fused to it into EVs spontaneously released by the recipient cells. We previously provided evidence that mice injected with a DNA vector expressing the Nefmut/HPV16-E7 fusion protein developed an E7-specific CTL immune response as detected 2 weeks after the second immunization. Here, we extended and optimized the anti-HPV16 CD8+ T cell immune response induced by the endogenously engineered EVs, and evaluated the therapeutic antitumor efficacy over time. We found that the co-injection of DNA vectors expressing Nefmut fused with E6 and E7 generated a stronger anti-HPV16 immune response compared to that observed in mice injected with the single vectors. When HPV16-E6 and -E7 co-expressing tumor cells were implanted before immunization, all mice survived at day 44, whereas no mice injected with either void or Nefmut-expressing vectors survived until day 32 after tumor implantation. A substantial part of immunized mice (7 out of 12) cleared the tumor. When the cured mice were re-challenged with a second tumor cell implantation, none of them developed tumors. Both E6- and E7-specific CD8+ T immunities were still detectable at the end of the observation time. We concluded that the immunity elicited by engineered EVs, besides counteracting and curing already developed tumors, was strong enough to guarantee the resistance to additional tumor attacks. These results can be of relevance for the therapy of both metastatic and relapsing tumors.

Intramuscular injection of DNA vectors expressing the extracellular vesicle (EV)-anchoring protein Nef mut fused at its C-terminus to viral and tumor antigens elicits a potent, effective, and anti-tolerogenic CD8 + T cell immunity against the heterologous antigen. The immune response is induced through the production of EVs incorporating Nef mut -derivatives released by muscle cells. In the perspective to a possible translation into the clinic of the Nef mut -based vaccine platform, we aimed at increasing its safety profile by identifying the minimal part of Nef mut retaining the EV-anchoring protein property. We found that a C-terminal deletion of 29-amino acids did not affect the ability of Nef mut to associate with EVs. Furthermore, the EV-anchoring function was preserved when antigens from both HPV16 (i.e., E6 and E7) and SARS-CoV-2 (i.e., S1 and S2) were fused to its C-terminus. By analyzing the immune responses induced after intramuscular injection of DNA vectors expressing fusion products based on the four viral antigens, we found that the Nef mut C-terminal deletion did not impact on the levels of antigen –specific CD8 + T lymphocytes as evaluated by IFN-γ EliSpot analysis and intracellular cytokine staining. In addition, immune responses at distal sites remained unaffected, as indicated by the similar percentages of SARS-CoV-2 S1- and S2-specific CD8 + T cells detected in spleens and lung airways of mice injected with DNA vectors expressing the viral antigens fused with either Nef mut or Nef mut PL. We concluded that the C-terminal Nef mut truncation does not affect stability, EV-anchoring, and CD8 + T cell immunogenicity of the fused antigen. Hence, Nef mut PL represents a safer alternative to full-length Nef mut for the design of CD8 + T cell vaccines for humans.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is spreading rapidly in the absence of validated tools to control the growing epidemic besides social distancing and masks. Many efforts are ongoing for the development of vaccines against SARS-CoV-2 since there is an imminent need to develop effective interventions for controlling and preventing SARS-CoV-2 spread. Essentially all vaccines in most advanced phases are based on the induction of antibody response against either whole or part of spike (S) protein. Differently, we developed an original strategy to induce CD8+ T cytotoxic lymphocyte (CTL) immunity based on in vivo engineering of extracellular vesicles (EVs). We exploited this technology with the aim to identify a clinical candidate defined as DNA vectors expressing SARS-CoV-2 antigens inducing a robust CD8+ T-cell response. This is a new vaccination approach employing a DNA expression vector encoding a biologically inactive HIV-1 Nef protein (Nefmut) showing an unusually high efficiency of incorporation into EVs even when foreign polypeptides are fused to its C-terminus. Nanovesicles containing Nefmut-fused antigens released by muscle cells are internalized by antigen-presenting cells leading to cross-presentation of the associated antigens thereby priming of antigen-specific CD8+ T-cells. To apply this technology to a design of anti-SARS-CoV-2 vaccine, we recovered DNA vectors expressing the products of fusion between Nefmut and four viral antigens, namely N- and C-terminal moieties of S (referred to as S1 and S2), M, and N. All fusion products are efficiently uploaded in EVs. When the respective DNA vectors were injected in mice, a strong antigen-specific CD8+ T cell immunity was generated. Most important, high levels of virus-specific CD8+ T cells were found in bronchoalveolar lavages of immunized mice. Co-injection of DNA vectors expressing the diverse SARS-CoV-2 antigens resulted in additive immune responses in both spleen and lung. EVs engineered with SARS-CoV-2 antigens proved immunogenic also in the human system through cross-priming assays carried out with ex vivo human cells. Hence, DNA vectors expressing Nefmut-based fusion proteins can be proposed as anti-SARS-CoV-2 vaccine candidates. ### Competing Interest Statement The authors have declared no competing interest.

Neurodegenerative diseases are commonly generated by intracellular accumulation of misfolded/aggregated mutated proteins. These abnormal protein aggregates impair the functions of mitochondria and induce oxidative stress, thereby resulting in neuronal cell death. In turn, neuronal damage induces chronic inflammation and neurodegeneration. Thus, reducing/eliminating these abnormal protein aggregates is a priority for any anti-neurodegenerative therapeutic approach. Although several antibodies against mutated neuronal proteins have been already developed, how to efficiently deliver them inside the target cells remains an unmet issue. Extracellular vesicles/exosomes incorporating intrabodies against the pathogenic products would be a tool for innovative therapeutic approaches. In this review/perspective article, we identify and describe the major molecular targets associated with neurodegenerative diseases, as well as the antibodies already developed against them. Finally, we propose a novel targeting strategy based on the endogenous engineering of extracellular vesicles/exosomes constitutively released by cells of the central nervous system.

HIV-1 infection is efficiently controlled by combination anti-retroviral therapy (cART). However, despite preventing disease progression, cART does not eradicate virus infection which persists in a latent form for an individual's lifetime. The latent reservoir comprises memory CD4(+) T lymphocytes, macrophages, and dendritic cells; however, for the most part, the reservoir is generated by virus entry into activated CD4(+) T lymphocytes committed to return to a resting state, even though resting CD4(+) T lymphocytes can be latently infected as well. The HIV-1 reservoir is not recognized by the immune system, is quite stable, and has the potential to re-seed systemic viremia upon cART interruption. Viral rebound can occur even after a long period of cART interruption. This event is most likely a consequence of the extended half-life of the HIV-1 reservoir, the maintenance of which is not clearly understood. Several recent studies have identified extracellular vesicles (EVs) as a driving force contributing to HIV-1 reservoir preservation. In this review, we discuss recent findings in the field of EV/HIV-1 interplay, and then propose a mechanism through which EVs may contribute to HIV-1 persistence despite cART. Understanding the basis of the HIV-1 reservoir maintenance continues to be a matter of great relevance in view of the limitations of current strategies aimed at HIV-1 eradication.

Immunosuppression at tumor microenvironment (TME) is one of the major obstacles to be overcome for an effective therapeutic intervention against solid tumors. Tumor-associated macrophages (TAMs) comprise a sub-population that plays multiple pro-tumoral roles in tumor development including general immunosuppression, which can be identified in terms of high expression of mannose receptor (MR or CD206). Immunosuppressive TAMs, like other macrophage sub-populations, display functional plasticity that allows them to be re-programmed to inflammatory macrophages. In order to mitigate immunosuppression at the TME, several efforts are ongoing to effectively re-educate pro-tumoral TAMs. Extracellular vesicles (EVs), released by both normal and tumor cells types, are emerging as key mediators of the cell to cell communication and have been shown to have a role in the modulation of immune responses in the TME. Recent studies demonstrated the enrichment of high mannose glycans on the surface of small EVs (sEVs), a subtype of EVs of endosomal origin of 30–150 nm in diameter. This characteristic renders sEVs an ideal tool for the delivery of therapeutic molecules into MR/CD206-expressing TAMs. In this review, we report the most recent literature data highlighting the critical role of TAMs in tumor development, as well as the experimental evidences that has emerged from the biochemical characterization of sEV membranes. In addition, we propose an original way to target immunosuppressive TAMs at the TME by endogenously engineered sEVs for a new therapeutic approach against solid tumors.

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.

Purpose: Single-chain variable fragments (scFvs) are one of the smallest antigen-binding units having the invaluable advantage to be expressed by a unique short open reading frame (ORF). Despite their reduced size, spontaneous cell entry of scFvs remains inefficient, hence precluding the possibility to target intracellular antigens. Here, we describe an original strategy to deliver scFvs inside target cells through engineered extracellular vesicles (EVs). This approach relies on the properties of a Human Immunodeficiency Virus (HIV)-1 Nef mutant protein referred to as Nef(mut). It is a previously characterized Nef allele lacking basically all functions of wt Nef, yet strongly accumulating in the EV lumen also when fused at its C-terminus with a foreign protein. To gain the proof-of-principle for the efficacy of the proposed strategy, the tumor-promoting Human Papilloma Virus (HPV)16-E7 protein was considered as a scFv-specific intracellular target. The oncogenic effect of HPV16-E7 relies on its binding to the tumor suppressor pRb protein leading to a dysregulated cell duplication. Interfering with this interaction means impairing the HPV16-E7-induced cell proliferation. Methods: The Nef(mut) gene was fused in frame at its 3'-terminus with the ORF coding for a previously characterized anti-HPV16-E7 scFv. Interaction between the Nef(mut)-fused anti-HPV16-E7 scFv and the HPV16-E7 protein was tested by both confocal microscope and co-immunoprecipitation analyses on co-transfected cells. The in cis anti-proliferative effect of the Nef(mut)/anti-HPV16-E7 scFv was assayed by transfecting HPV16-infected cells. The anti-proliferative effect of EVs engineered with Nef(mut)/anti-HPV16-E7 scFv on HPV16-E7-expressing cells was evaluated in two ways: i) through challenge with purified EVs by a Real-Time Cell Analysis system and ii) in transwell co-cultures by an MTS-based assay. Results: The Nef(mut)/anti-HPV16-E7 scFv chimeric product is efficiently uploaded in EVs, binds HPV16-E7, and inhibits the proliferation of HPV16-E7-expressing cells. Most important, challenge with cell-free EVs incorporating the Nef(mut)/anti-HPV16-E7 scFv led to the inhibition of proliferation of HPV16-E7-expressing cells. The proliferation of these cells was hindered also when they were co-cultured in transwells with cells producing EVs uploading Nef(mut)/anti-HPV16-E7 scFv. Conclusion: Our data represent the proof-of-concept for the possibility to target intracellular antigens through EV-mediated delivery of scFvs. This finding could be relevant to design novel methods of intracellular therapeutic interventions.

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. KEY MESSAGES: We established a novel method to induce antitumor CTLs without a need to identify TAAs and/or tumor neoantigens. This strategy relies on transducing tumor cells with a retroviral vector expressing both CD80 and CD86. In this way, tumor cells prime naïve CD8+ T lymphocytes in a way that CTLs killing the same tumor cells are generated. These findings open the way towards preclinical assays in the perspective to introduce this antitumor immunotherapy strategy in clinic.