key: cord-0279433-2jg8sg1y
authors: Ferrantelli, Flavia; Manfredi, Francesco; Chiozzini, Chiara; Olivetta, Eleonora; Giovannelli, Andrea; Leone, Patrizia; Federico, Maurizio
title: Long-term antitumor CD8+ T cell immunity induced by endogenously engineered extracellular vesicles
date: 2021-02-06
journal: bioRxiv
DOI: 10.1101/2021.02.05.429897
sha: 2416cd77b62b0ba9336005b31307d60c046d7983
doc_id: 279433
cord_uid: 2jg8sg1y

We developed a novel approach to induce antigen-specific CD8+ T cytotoxic lymphocyte (CTL) immunity based on in vivo engineering of extracellular vesicles (EVs). This is an innovative vaccination approach employing a DNA vector expressing a mutated HIV-1 Nef protein (Nefmut) that has lost 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 cells expressing the Nefmut_based DNA vector. We previously provided evidence that the inoculation in mice of a DNA vector expressing the Nefmut/HPV16-E7 fusion protein induced an E7-specific CTL immune response as detected 2 weeks after the second immunization. In an effort to optimize the anti-HPV16 CD8+ T cell immune response, 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 we observed in mice injected with the single vectors. When TC-1 cells, i.e., a tumor cell line co-expressing E6 and E7, were implanted before immunization, all mice survived until day 44, whereas no mice injected with either void or Nefmut_expressing vectors survived until day 32 after tumor implantation. A substantial part of mice (7 out of 12) cleared the tumor. When cured mice were re-challenged with a second sub cute implantation of TC-1 cells, and followed for additional 135 days, whereas none of them developed tumors. Both E6- and E7-specific CD8+ T immunity was still detectable at the end of the observation time. Hence, the immunity elicited by engineered EVs, besides curing already developed tumors, is strong enough to guarantee the resistance to additional tumor attack. This results is of relevance for therapy against both metastatic and relapsing tumors.

Eukaryotic cells spontaneously release vesicles of different sizes. Extracellular vesicles (EVs) are classified as apoptotic bodies (1-5 m) , microvesicles (50-1,000 nm), and exosomes (50-200 nm) (1) .

Microvesicles (also referred to as ectosomes) shed by plasma membrane, whereas exosomes are released after inward invagination of endosome membranes and formation of intraluminal vesicles. Healthy cells constitutively release both exosomes and microvesicles, together referred to as EVs hereinafter. EVs are an important mean of intercellular communication by transporting their cargo, such as DNAs, RNAs, proteins, and lipids, from the producer cell to the recipient one (2, 3) .

EVs are abundant, stable, and highly bioavailable to tissues in vivo. They find potential applications as diagnostic biomarkers, therapeutics, drug delivery vehicles, and functional cosmetics. Several EV-based anticancer immunotherapies have been under clinical trials for different indications (4, 5) . Despite high expectations, clinical trials have not yet confirmed therapeutic application of in vitro engineered EVs, mostly because a number of drawbacks associated with functional reproducibility and loading of specific cargoes (6) .

Two types of cancer immunotherapy have emerged so far as being the most promising: i) T cellbased cancer immunotherapy, including active vaccination and adoptive cell transfer, and ii) immune modulation through monoclonal antibodies referred to as immune checkpoint blockers (ICBs) (7) .

Cancer vaccines rely on induction of antitumor immunity using whole or part of tumor antigens. In this manner, a de novo antitumor immunity can be established, and both potency and breadth of pre-existing immunity can be widened. Different approaches include the use of synthetic peptides from tumor antigens (allogenic and autologous), dendritic cell (DC)-based vaccines, and genetic vaccines (DNA/RNA/virus/bacterial) (8) .

Cancer cells express a burden of new antigens as a result of their intrinsic genetic instability typical of malignant transformation and/or of the expression of the etiologic cancer agents, as in the case of virusinduced malignancies. In non-virus-induced cancers, transformed cells can produce antigens to which the host is basically tolerant (tumor-associated self-antigens. In addition, cancer cells can express antigens to which the host does not develop tolerance, being however the immune response not effective enough to counteract the cancer cell growth. They include the so-called "tumor specific neo-antigens" as well as antigens normally produced in immune-privileged tissues, e.g., cancer-testis antigens. Hence, establishing a method to induce an adaptive immune response against both tolerogenic and non-tolerogenic TAAs would be of great relevance for the design of novel antitumor therapeutic approaches.

An explosive growth of interest in cancer immunotherapy occurred in the last decade mainly due to approval of new clinical protocols based on the use of monoclonal antibodies, referred to as ICBs (9, 10), fostering the pre-existing immune response against both tumor-associated antigens (TAAs) and neo-antigens.

The success of immunotherapies based on ICBs definitely proved that cancer can be treated and cured by manipulating the immune system. However, this strategy still suffers from some limitations, including intrinsic and/or acquired resistance, and development of hyper-immune activation, which can associate with immune-related adverse events affecting several organs including skin, gut, heart, lungs, and bone (11) . DNA vaccination has many potential advantages (12) . DNA molecules (usually plasmids) express the antigen of interest under the control of a strong promoter are transferred to cells of a vaccine recipient.

Intracellularly produced antigens are presented to the recipient's immune system, resulting in both humoral and cellular immune responses that may protect against disease in preclinical models of cancer, infectious diseases, and autoimmunity. Nevertheless, to date the efficacy of DNA vaccines in clinical trials has been disappointing, and it is uncertain whether the high expectations associated with DNA vaccines will be fulfilled. Anyway, preclinical and clinical studies have yielded many safety data (13) .

We developed a vaccine platform based on the high levels of uploading into extracellular vesicles (EVs) of a Human Immunodeficiency virus-1 Nef mutant, referred to as Nef mut (14) . In the Nef mut -based biotechnology platform, the antigen of interest is expressed by a DNA vector as product of fusion to Nef mut .

Upon intramuscular injection, the antigen is incorporated into the EVs which are spontaneously released by muscle cells. These EVs freely circulate into the body reaching also compartments distal from the injection site. When they enter professional APCs, the Nef mut -antigen product of fusion is cross-presented, thereby inducing antigen-specific CTLs (15) (16) (17) . On the contrary, no humoral response is produced most likely as a consequence of the incorporation of Nef mut -based fusion products into EVs, where they remain hidden and protected from external environment.

Here, data regarding both duration and efficacy against tumor challenge and re-challenge of an anti-HPV16 vaccine based on Nef mut in vivo-engineered EVs are reported.

The pTargeT (Invitrogen, Thermo Fisher Scientific) vectors expressing Nef mut , Nef mut /HPV16-E6 and Nef mut /HPV16-E7 were already described (15, 18) . Both E6 and E7 ORFs were synthesized by Eurofins Genomics Germany GmbH. Kozak sequences were inserted at 5' end, and ORFs were inserted in the Not I and Apa I sites of the pTargeT vector polylinker. A 6×His tag sequence (i.e., 5' CACCATCACCATCACCAT 3') was included at the 3' end just before the stop codon.

Human embryonic kidney (HEK)293T cells (ATCC, CRL-11268) were grown in DMEM (Gibco) plus 10% heat-inactivated fetal calf serum (FCS, Gibco). Transfection assays were performed using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific).

Cells transfected with vectors expressing the Nef mut -based fusion proteins were washed 24 h later, and reseeded in medium supplemented with EV-deprived FCS. The supernatants were harvested from 48 to 72 h after transfection. EVs were recovered through differential centrifugations (19) by centrifuging supernatants at 500×g for 10 min, and then at 10,000×g for 30 min. Supernatants were harvested, filtered with 0.22 m pore size filters, and ultracentrifuged at 70,000×g for l h. Pelleted vesicles were resuspended in 1×PBS, and ultracentrifuged again at 70,000×g for 1 h. Finally, pellets containing EVs were resuspended in 1:100 of the initial volume.

Western blot analyses of both cell lysates and EVs were carried out after resolving samples in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In brief, the analysis on cell lysates was performed by washing cells twice with 1×PBS (pH 7.4) and lysing them with 1x SDS-PAGE sample buffer. Samples were resolved by SDS-PAGE and transferred by electroblotting on a 0.45 μM pore size nitrocellulose membrane (Amersham) overnight using a Bio-Rad Trans-Blot. For western blot analysis of EVs, they were lysed and analyzed as described for cell lysates. For immunoassays, membranes were blocked with 5% non-fat dry milk in PBS containing 0.1% Triton X-100 for 1 h at room temperature, then incubated overnight at 4 °C with specific antibodies diluted in PBS containing 0.1% Triton X-100. Filters were revealed using 1:1,000-diluted sheep anti-Nef antiserum ARP 444 (MHRC, London, UK), 1:500diluted anti--actin AC-74 mAb from Sigma, and 1:500 diluted anti-Alix H-270 polyclonal Abs from Santa Cruz.

Six-weeks old C57 Bl/6 female mice were obtained from Charles River and placed in the Central Animal Facility of the ISS, as approved by the Italian Ministry of Health, authorization n. 950/2018. The DNA vector preparations were diluted in sterile 0.9% saline solution. Both quality and quantity of the DNA preparations were checked by 260/280 nm absorbance and electrophoresis assays. Mice were anesthetized with isoflurane as prescribed in the Ministry authorization. Each inoculum volume was injected into both quadriceps. Immediately after inoculation, electroporation was applied at the site of injection through the Agilepulse BTX device using a 4-needle array 4 mm gap, 5 mm needle length, BD, with the following parameters: 1 pulse of 450 V for 50 µsec; 0.2 msec interval; 1 pulse of 450 V for 50 µsec; 50 msec interval; 8 pulses of 110 V for 10 msec with 20 msec intervals. Immunizations were repeated identically 14 days later.

For immunogenicity studies, fourteen days after the last immunization mice were sacrificed by cervical dislocation as recommended by the Ministry authorization protocol. Spleens were then explanted and placed into a 2 mL Eppendorf tubes filled with 1 mL of RPMI 1640 (Gibco), 50 µM 2-mercaptoethanol (Sigma).

PBMCs were recovered from blood samples obtained by retro orbital bleeding. Red blood cells were eliminated through incubation with ACK lysing buffer (Gibco).

For the IFN-γ EliSpot assay, 2.5×10 5 live cells were seeded in each microwell. Cultures were run in triplicate in EliSpot multiwell plates (Millipore, cat n. MSPS4510) pre-coated with the AN18 mAb against mouse IFN-γ (Mabtech) in RPMI 1640 (Gibco) plus 10% FBS (Gibco) for 16 h in the presence or not of 5 µg/mL of the following peptides (Table 1) 

Splenocytes were seeded at 2×10 6 . The CD8 + cell population was gated against APC-Cy7, PE, and BV421 to observe changes in IFN-, IL-2, and TNF- production, respectively. Boolean gates were created in order to determine any cytokine co-expression pattern.

Six-weeks old C57 Bl/6 female mice were obtained from Charles River and placed in the Central Animal Facility of the ISS. TC-1 cells (a kind gift of Prof. Wu, Johns Hopkins University, Boston, MA) were prepared at 2×10 6 cells/mL in 1×PBS, and s.c. inoculated into mice with a volume of 100 µL. At the time of immunization, mice were anesthetized with isoflurane as prescribed in the Ministry authorization.

Immunizations were carried out as here above described and repeated after 10 days. To collect peripheral blood mononuclear cells (PBMCs) for evaluating the immune responses, seven days after the second immunization 200 L of blood were collected from each mouse through retro orbital bleeding. Tumor Nineteen weeks after the last immunization, six mice that had recovered from tumor growth were rechallenged with the same cells under the same experimental modalities used for the first implantation.

Briefly, TC-1 cells were prepared at 2×10 6 cells/mL in 1×PBS, and s.c. inoculated into mice (100 µL).

Hence, a total of 2×10 5 TC-1 cells were implanted s.c. in the opposite side respect to the previous cell implantation. As a control, two age-matched (i.e., seven months old) naïve mice were injected with TC-1 cells with identical modalities.

Tumor growth was monitored as above described. Mice were sacrificed by cervical dislocation as soon as the tumor reached the size of 1 cm 3 , or at the end of experiment, i.e., 19 weeks after tumor cell reimplantations.

When appropriate, data are presented as mean + standard deviation (SD).

Very recently, we provided evidence that the co-injection of up to three distinct DNA vectors expressing SARS-CoV-2 antigens fused with Nef mut generated an additive CD8 + T cell immune response, in the absence of evident negative interferences among the different immunogens (23) . We aimed at assessing whether a similar additive effect would take place with HPV16 antigens. To this aim, two DNA vectors expressing Nef mut /E6 and Nef mut /E7 were considered. In these DNA vectors the HPV16-related ORFs were optimized for the expression in eukaryotic cells, and the domains involved in the pathogenic interaction with cell protein targets were inactivated (24, 25) .

Both vectors were proven to express fusion proteins which are efficiently uploaded into EVs (Fig.   1A ). DNA vectors were i.m. injected in mice either singularly or in combination. Fourteen days after the second immunization, the CD8 + T cell immune response was evaluated in terms of antigen-specific activation through both IFN- EliSpot assay and ICS analysis.

We observed general stronger CD8 + T cell responses against E7 compared to E6. When the two vectors were co-injected, additive immune responses were generated (Fig. 1B) , thus excluding possible functional interference. This conclusion was also supported by the evidence that in co-injected animals not only was the E7-specific CD8 + T cell response not reduced, but resulted slightly increased compared to that of mice injected with the Nef mut /E7-expressing vector alone (Fig. 1B) .

The induction of antigen-specific polyfunctional lymphocytes is considered hallmark of efficacy for CD8 + T cell immune response. We evaluated the levels of E6-and/or E7-specific CD8 + T cells expressing IFN-, IL-2, and TNF-by ICS after overnight cultivation of splenocytes with specific nonamers.

Consistently with data obtained from IFN- EliSpot assays, the immunization with the Nef mut /E7-expressing vector resulted in higher percentages of IFN- producing CD8 + T cells compared to those from Nef mut /E6 immunized mice (Fig. 2A) . The highest percentages of IFN- producing CD8 + T cells was observed with splenocytes from mice injected with the two DNA vectors (Fig. 2A) . Similar results were obtained with the analysis of IL-2 and TNF. Most important, the percentages of triple-positive (i.e., polyfunctional) antigenspecific CD8 + T cells increased in co-injected mice compared to mice injected with single vectors (Fig. 2B) .

We concluded that an optimal CD8 + T cell immune response can be achieved using the combined immunization with Nef mut /E6-and Nef mut /E7-expressing DNA vectors.

Next, the CD8 + T cell immune response against both E6 and E7 was tested in mice immunized after the s.c. implantation of TC-1 tumor cells. The actual expression of both HPV16 E6 and -E7 genes in TC-1 cells was confirmed by qRT-PCR assay (not shown). C57 Bl/6 mice were implanted with 2×10 5 TC-1 cells and, 10 days thereafter, the first immunization was carried out in mice bearing palpable tumors. Mice were injected with both Nef mut /E6-and Nef mut /E7-expressing vectors or, as control,: i) void DNA vector; ii) a vector expressing Nef mut alone, and iii) a combination of vectors expressing E6 and E7. . As expected, neither E6 nor E7 associated with EVs, as shown by the western blot comparative analysis including a His-tagged single-chain antibody (scFv) fused with Nef mut (26) (Fig. 3A) . The immunizations were repeated after a week, and after additional seven days, 200 L of blood were recovered to evaluate the immune responses.

The analysis of E6-and E7-specific CD8 + T cell immune responses carried out by IFN- Elispot assay on PBMCs shown an immune response about 5-fold more potent in mice injected with DNA vectors expressing the HPV16 proteins fused with Nef mut than detected in mice injected with E6 and E7-expressing vectors without Nef mut (Fig. 3B) . In this experimental setting, the sole implantation of the E6-and E7expressing TC-1 cells did not result in a CD8 + T cell immune response, as indicated by the analysis carried out with PBMCs from mice injected with either void or Nef mut -expressing DNA vectors (not shown).

Overall, immune responses to Nef mut -based products were in line with previous observations in tumor-free mice.

The tumor size of injected mice was evaluated over the time. Tumors implanted in mice injected with void or Nef mutexpressing vectors grew in a very quick and uncontrolled way, rapidly leading the mice to death (Fig. 4A) . Tumor growth was less rapid in mice injected with vectors expressing E6 and E7. In this group, the mouse showing the strongest CD8 + T cell response remained tumor-free over the time, while in the remainders the tumor led the mice to reach the maximum tumor size allowed before euthanasia (i.e., 1 cm 3 ) within 60 days (Fig. 4A) . Conversely, seven of the twelve mice immunized with Nef mut /E6 plus Nef mut /E7-expressing vectors were cured and remained tumor-free over the time. In particular, at day 35 after tumor implantation (i.e., when all mice of control groups had been euthanized) no or very limited tumor growth was observed in mice co-injected with Nef mut /E6 plus Nef mut /E7 DNA vectors. At day 65 after tumor implantation, when only 1 mouse of 12 mice injected with DNA vectors expressing E6 and E7 was alive, all mice immunized with Nef mut /E6 plus Nef mut /E7 still survived. As shown by the survival curve (Fig. 4B) , seven mice of the group immunized with DNA vectors expressing Nef mut /E7 and Nef mut /E6 remained tumorfree throughout.

A good association between the levels of antigen-specific CD8 + T cell immune responses and antitumor effects was found in the group of mice injected with Nef mut /E7-and Nef mut /E6-expressing vectors.

In fact, the mean of SFUs/10 5 PBMCs measured in mice developing tumor was 115±61, whereas in cured mice increased to 187±92.

Taken together, these data demonstrated that the Nef mut /E6 plus Nef mut /E7 combined vaccine led to a tumor growth control resulting much more potent than that induced by the combined injection of DNA vectors expressing HPV16-E6 and -E7.

Next, we were interested in evaluating the persistence of the antitumor state in mice cured by the immunization with DNA vectors expressing Nef mut /E6 and Nef mut /E7. Six mice which were cured by the immunization were re-challenged by implanting 2×10 5 TC-1 cells in the opposite flank respect to the previous cell implantation. As control, age-matched, non-immunized mice were used. We observed that cured mice remained tumor-free over the 135 days of monitoring, whereas the age-matched naïve control mice developed a palpable tumor after 12 days from TC-1 cell implantation, and had to be sacrificed at day by IFN- Elispot assay to assess the presence of E6-and E7-specific CD8 + T cells. Detectable levels of both E6-and E7-specific CD8 + T cell immune responses were observed in all mice, in the presence of higher E7specific immune responses (Fig. 5B) . All re-challenged mice were confirmed tumor free and with normallooking organs (i.e., lungs, heart, liver, spleen, kidneys, stomach, intestine) by observation at necroscopy.

These results strongly suggested that the antitumor state induced by engineered EVs was strong and durable enough to counteract the proliferation of re-challenging tumor cells.

Duration and robustness of the immune response are key features for any vaccine strategy. The Nef mut -based CTL vaccine platform has been proven to induce strong immunity towards a wide range of both tumor and viral antigens (15, 16) . However, log-term efficacy against tumor cell challenge and re-challenge not evaluated yet. To fill this gap, we first tried to identify the most efficient immunogenic strategy pertaining the Nef mut technology. We found that co-injection of DNA vectors expressing different antigens fused to Nef mut resulted in an additive anti-HPV16 CD8 + T cell immune response with no interference in terms of the downstream immunogenicity. Consistently, increased percentages of antigen-specific polyfunctional CD8 + T lymphocytes compared to those induced by single vectors have been observed. These results can be considered of great relevance since they open the way towards the application of the Nef mutbased platform on multiple targets in new vaccine combination strategies.

In tumor-implanted mice, the expression of fusion to Nef mut of HPV16-E6 and -E7 antigens led to a strong increase of the antigen-specific CD8 + T cell immune response compared to that elicited by each HPV16 product expressed alone. Consistently, the antitumor effect was far more striking in mice injected with both DNA vectors expressing the products of fusion. Results from the tumor re-challenge experiment demonstrated that the Nef mut /E6 plus Nef mut /E7 vaccine conferred a potent, long lasting (at least up to 38 weeks after the last immunization), and efficacious CD8 + T cell immunity against the tumors re-implanted 19 weeks after the last immunization. At this time, immune responses against both HPV-E6 and E7 proteins were still detectable, and all mice remained tumor-free.

Taken together, these data prove that the vaccine strategy based on endogenously engineered EVs is a promising approach against HPV-antigen expressing tumors, and that it may be worth further testing against this and additional pathologies.

We previously described an antitumor therapeutic effect in mice injected with a Nef mut /E7 DNA vector (15) . However, the tumor monitoring was limited to 30 days after tumor implantation, and the tumorigenicity of TC-1 cells appeared significantly reduced compared to that observed in the here presented experiments. In fact, at day 30 after tumor implantation all mice injected with control vectors survived and the tumor mass did not exceed 0.6 cm 3 , whereas at this time point in the here presented experiment all mice had to be euthanized.

In the classic mechanism of action underlying i.m. DNA vaccination, antigens expressed by DNA recipient muscle cells can be secreted, thereby essentially generating a humoral adaptive immune response (27) . Muscle cells are not professional antigen presenting cells (APCs), and do not express co-stimulatory molecules. Hence, the CD8 + T immune response relies on the capture and expression of the DNA molecules by professional and semi-professional APCs (e.g., DCs, endothelial cells) embedded in the muscle tissue.

However, naked DNA does not spread from cell to cell in vivo, and APCs do not efficiently take up exogenous DNA to activate satisfactory immune responses. For these reasons, the delivery of DNA vaccines in DCs is most effectively achieved through subcutaneous and intradermal injections, possibly associated with gold particles and gene gun. Differently from all currently developed DNA vaccines, in the Nef mutbased platform the expressed antigen is incorporated into the EVs, which are spontaneously released by the muscle cells, and are expected to freely circulate into the body. The Nef mut -based vaccine platform combines the remarkable benefits of efficient cross-presentation of EV-associated antigens and the consequent specific induction of a potent CD8 + T immune response, with several advantages typical of DNA vaccines, including: i) simple and flexible design, so that a wide range of antigens and immunomodulatory molecules can be expressed; ii) unrestricted MHC Class I immune response; iii) no unsafe infectious agents involved in the preparation of immunogens and, consequently, no adverse clinical effects or toxicity are expected to occur; iv) great heat stability and ease of storage and transport without need for a cold chain, and v) cost effectiveness. Immunogens can be developed quickly and easily once the antigen has been identified. The production can be very rapid, reproducible and perfectly suitable for large-scale production and administration.

Patient-derived EVs were employed as a novel cancer immunotherapy in several clinical trials, but this strategy lacked sufficient efficacy (28) (29) (30) . Other lines of research have focused on modifying the content and function of EVs in various ways, toward the end-goal of specialized therapeutic EVs. Exosomes engineered to upload cargoes represent the last frontier in terms of nanoparticle-based technology (31) , and a number of companies have been established on the basis of patented technologies to engineer EVs in vitro.

Ideally, EVs could be designed for a desired immunostimulatory function and loaded with antigens or celltargeting proteins in vitro to produce a potent, antigen-specific immune response in vivo, e.g. addressed to a functional tumor therapy. Despite high expectations, however, clinical trials have not yet confirmed therapeutic applicability of in vitro engineered EVs (32) (33) (34) . Considering the underlying mechanism of action, the Nef mut vaccine approach can overcome limitations pertaining DNA-based vaccines, as well as ex vivo/in vitro engineered EVs. Fig. 1. HPV16-E6 and -E7-specific CD8 + cell immunity induced in mice co-injected with Nef mut /E6 and Nef mut /E7 DNA vectors. A. Detection of Nef mut -related fusion proteins in transfected cells and EVs. Western blot analysis from 30 g of cell lysates from 293T cells transfected with DNA vectors expressing Nef mut /E6 and Nef mut /E7, and equal volumes of buffer where purified EVs were resuspended after differential centrifugations of the respective supernatants. As control, conditions from mock-transfected cells as well as cells transfected either with Nef mut or Nef mut /E7 were included. Polyclonal anti-Nef Abs served to detect Nef mut -based products, while -actin and Alix were markers for cell lysates and exosomes, respectively. Nef protein products are indicated by arrows. Molecular markers are given in kDa. B. CD8 + T cell immune response induced in C57 Bl/6 mice inoculated with the DNA vectors expressing Nef mut /E6 and Nef mut /E7 either singularly or in combination. As control, mice were inoculated with the void vector. Values detected with splenocytes from mice injected with control vector were below the sensitivity threshold of the assay.

A. Analysis of both HPV16 E6 and E7 products in transfected cells and respective EVs. Western blot analysis from 30 g of cell lysates from 293T cells transfected with DNA vectors expressing the indicated HPV16 ORFs (left panels), and equal volumes of buffer where purified EVs were resuspended after differential centrifugations of the respective supernatants (right panels). As control, conditions from mocktransfected cells as well as cells transfected with Nef mut fused with a scFv (Nef mut GO) including an His-tag at its C-terminus were included. Polyclonal anti-His-tag Abs served to detect both HPV16-related and Nef mut-GO products, while -actin and Alix were revealed as markers for cell lysates and EVs, respectively.

Relevant protein products are highlighted. Molecular markers are given in kDa. B. CD8 + T cell immune response in C57 Bl/6 mice injected s.c. with 2×10 5 TC-1 cells, and then inoculated+EP with 10 g of both DNA vectors expressing E6 and E7 either alone or fused with Nef mut . PBMCs were isolated after retro orbital bleeding, and then incubated o.n. with or without 5 g/ml of either unrelated (not shown), E6 and E7-specific nonamers in triplicate IFN- EliSpot microwells. Shown are the number of IFN- spot-forming units (SFU)/10 5 PBMCs as mean values of triplicates. On the right, the SFU values are associated with each mouse. Intragroup mean values ± SD are also reported. Fig. 4 . Antitumor therapeutic effect induced by i.m. injection of both Nef mut /E6 and Nef mut /E7 DNA vectors. A. Tumor growth curves. C57 Bl/6 mice (12 per group) were challenged with 2×10 5 TC-1 cells and, the day after the tumor appearance, i.e., when tumor masses became detectable by palpation, co-inoculated with DNA vectors expressing Nef mut /E6, Nef mut /E7, E6, and E7, or, as control, with either Nef mut or empty vector. The DNA inoculations were repeated at day 17 after tumor cell implantation, and the growth of tumor mass was followed over the time. Shown are the data referred to each injected mouse identified by the immunized with both Nef mut /E6-and Nef mut /E7-expressing vectors were re-challenged with TC-1 tumor cells. Nineteen weeks after cell re-implantation, mice were sacrificed, and splenocytes tested for both E6and E7-specific CD8 + T cell immunity by IFN-γ EliSpot assay. Either E6, E7, or a mix of E6 and E7 peptides were used in triplicate microwells. 

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