Immune Responses and Therapeutic Antitumor Effects of an Experimental DNA Vaccine Encoding Human Papillomavirus Type 16 Oncoproteins Genetically Fused to Herpesvirus Glycoprotein D

Mice.Female C57BL/6 mice at 6 to 8 weeks of age were used for these studies. Animals were supplied by the Animal Breeding Center of the Biomedical Sciences Institute of the University of São Paulo and Charles River Laboratories and housed at the Microbiology Department of the University of São Paulo or at the Animal Facility of The Wistar Institute, respectively. All procedures involving the handling and sacrifice of animals were performed according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals at the Biomedical Sciences Institute of the University of São Paulo or at The Wistar Institute.

Cell lines.The TC-1 cell line was most kindly provided by T. C. Wu, Johns Hopkins University, Baltimore, MD. The cells had been transfected with a triple promoter retroviral vector carrying v-HA-ras together with E6 and E7 carrying genes of HPV-16 (26). TC-1 cells were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM l-glutamine, 1 mM sodium pyruvate, 2 mM nonessential amino acids, 10 mM HEPES buffer, 50 units/ml penicillin-streptomycin, and 10% fetal bovine serum (FBS) and kept at 37°C in 5% CO₂. Before inoculation, TC-1 cells were harvested by trypsinization, washed twice, and suspended in serum-free media at 5 × 10⁶ cells/ml.

DNA vaccines.The generation of the DNA vaccines encoding the in tandem-fused HPV-16 E7, E6, and E5 oncoproteins (pE7E6E5) or the three oncoproteins genetically fused after amino acid 244 of the HSV-1 gD protein (pgD-E7E6E5) has been described previously (25). The correct in-frame cloning of E7, E6, and E5-encoding genes was confirmed by nucleotide sequencing. The DNA vaccine (pgD) encoding the complete nonfused HSV-1 gD has been described previously (24).

Adenovirus vectors.Vectors expressing either pE7E6E5 or pgD-E7E6E5 were constructed as described. pE7E6E5 and pgD-E7E6E5 chimeric genes were subcloned into an E1-deleted molecular clones of chimpanzee-derived adenovirus 68 (AdC68) as described previously (13). Recombinant Ad vectors were rescued and propagated on HEK 293 cells and purified by CsCl gradient centrifugation.

Immunization and tumor cell challenge.Groups of 5 to 10 mice were vaccinated intramuscularly (i.m.) with the DNA vaccines. Each dose contained 100 μg of DNA divided in two 50-μl aliquots and applied into the tibialis anterior muscle of each hind limb. Mice were i.m. immunized with 5 × 10¹⁰ virus particles (VP) of AdC68 vectors diluted in 100 μl of phosphate-buffered saline (PBS) into the tibialis anterior muscle of each hind limb. Mice were challenged subcutaneously (s.c.) with 5 × 10⁵ TC-1 cells suspended in 100 μl of serum-free media and injected into the left rear flank of the animals 2 weeks after the last vaccination. To perform the long-term protection assay, mice were immunized with one dose of the vaccine vectors and challenged 100 days later. To determine the effect of postchallenge vaccination, mice were vaccinated on the same day 8 h after challenge with 5 × 10⁵ TC-1 cells. One or two additional vaccine doses were given to the animals in weekly intervals thereafter. The postchallenge protocol was also performed with the three doses of immunization protocol starting at the following different time points: 0 (8 h later), 3, 5, 7 or 10 days after challenge. For the postchallenge experiments with coadministration of plasmids expressing cytokines, mice were immunized with 100 μg of DNA of the vaccine vectors admixed with 100 μg of DNA of the plasmid-expressing cytokine divided in two 50-μl aliquots. Tumor growth was monitored by visual inspection and palpation three times a week after challenge. Animals were scored as tumor bearing when tumors reached a size of approximately 1 to 2 mm in diameter. Mice were euthanized once tumors exceeded a diameter of 1 cm or became necrotic or burdensome to the animals. Tumor growth was otherwise followed for a period of 60 days after the challenge.

Intracellular cytokine staining.Intracellular gamma interferon (IFN-γ) staining was performed using blood samples or spleen cells treated for 5 min on ice with Ack lysing buffer (BioSource International, Camarillo, CA) to rupture red blood cells and then centrifuged at 1,000 × g for 5 min. Peripheral blood mononuclear cells (PBMC) or splenocytes were treated again with the lysis buffer, centrifuged, and suspended in DMEM. Cells were cultured at a concentration of 10⁶ cells/well for 5 h at 37°C in a 96-well round-bottom microtiter plate in 200 μl of DMEM supplemented with 2% FBS and 10⁻⁶ M 2-mercaptoethanol. Brefeldin A (GolgiPlug; BD Bioscience, San Jose, CA) was added at 1 μl/ml (24, 25). The E7-specific RAHYNIVTF peptide, E6-specific VYDFAFRDL peptide, E5-specific VCLLIRPLL peptide, or the V3 control peptide, delineated from the sequence of the envelope protein of HIV-1 clade B (VVEDEGCTNLSGF) (4, 12, 30, 24), were used as stimuli at concentrations of 3 μg/ml. After being washed, cells were incubated for 30 min at 4°C with 100 μl of a 1:100 dilution of a fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody to mouse CD8a (BD Bioscience, San Jose, CA). Cells were washed once with PBS, followed by permeabilization with Cytofix/Cytoperm (BD Bioscience, San Jose, CA) for 20 min at 4°C, washed twice with Perm/Wash buffer (BD Bioscience, San Jose, CA), and incubated in the same buffer for 30 min at 4°C with 50 μl of a 1:100 dilution of a phycoerythrin (PE)-labeled monoclonal antibody to mouse IFN-γ (BD Bioscience, San Jose, CA). After being washed, cells were suspended in PBS and examined by two-color flow cytometry using Epics Elite XL (Beckman Coulter, Fullerton, CA) or FACSCalibur (BD Bioscience, San Jose, CA). Data were analyzed by FlowJo software. The percentages of CD8⁺ cells that stained positive for IFN-γ over all CD8⁺ T cells were determined. Expression of the CD107a degranulation marker was evaluated as previously described (8). Spleen cells of vaccinated mice were stimulated with the E5-, E6-, or E7-specific peptide in the presence of brefeldin A (GolgiPlug; BD Bioscience, San Jose, CA), monensin (GolgiStop; BD Bioscience, San Jose, CA), and PE-conjugated monoclonal antibody to mouse CD107a (BD Bioscience, San Jose, CA). Five hours later, cells were washed and stained with PE Cy5-conjugated monoclonal antibody to mouse CD8a (BD Bioscience, San Jose, CA) and, after following fixation and permeabilization procedures, labeled with FITC-conjugated monoclonal antibody to mouse IFN-γ (BD Bioscience, San Jose, CA).

T-cell-mediated in vivo cell lysis.The in vivo cell lysis assay was carried out with splenocytes from naive mice as target cells, as previously described (3, 8). The cells were stained with 0.5 μM or 5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen, Carlsbad, CA) and then, the population stained with 5 μM CFSE was pulsed for 40 min at 37°C with 1 μM peptides carrying epitopes of the HPV oncoproteins that had previously been defined as the immunodominant major histocompatibility complex class I (MHC-I) epitopes for the H-2D^b mouse haplotype, as follows: RAHYNIVTF (E7), VYDFAFRDL (E6), or VCLLIRPLL (E5) (4, 12, 30). Control cells were labeled with 0.5 μM CFSE but not pulsed with the peptides. Both peptide-pulsed and nonpulsed cells were injected intravenously (i.v.) at 2 × 10⁷ cells each into mice that had previously been challenged with 5 × 10⁵ TC-1 cells and then immunized with three doses of pgD, pE7E6E5, or pgD-E7E6E5. One day after transfer of CFSE-labeled cells, splenocytes were harvested and analyzed for CFSE expression by flow cytometry in a multivariant FACScan analyzer (FACSCalibur; BD Biosciences, San Jose, CA). The percentages of target-specific cell killing were calculated as previously described (8).

Statistical analyses.All data expressed as means ± standard deviations (SD) are representative of results from at least two independent experiments. Student's t tests or analyses of variance (ANOVA) were employed whenever individual data points were compared.

Activation of E7-specific CD8⁺ T-cell responses and protection from TC-1 cell challenge in mice immunized with pgD-E7E6E5.In the present study, we investigated the activation of E7-specific CD8⁺ T-cell responses and induction of antitumor preventive effects in mice vaccinated with one or two doses of the pgD-E7E6E5 vector. Approximately 50% of the mice immunized with one dose and all animals vaccinated with two doses of pgD-E7E6E5 developed significant amounts of E7-specific IFN-γ-producing CD8⁺ T cells (Fig. 1 A and B). Mice immunized with pgD or pE7E6E5 did not develop any detectable IFN-γ⁺ CD8⁺ T-cell responses. Despite the different magnitude of the E7-specific CD8⁺ T-cell responses, all animals immunized with either one or two pgD-E7E6E5 doses were protected from a subsequent challenge with TC-1 cells (25; data not shown). In vivo depletion of CD8⁺ T cells, but not CD4⁺ T cells, abrogated protection conferred by pgD-E7E6E5, suggesting that CD4⁺ T helper cells do not play a relevant role in the induction of protective effects in vaccinated mice (data not shown). To determine the longevity of the pgD-E7E6E5-induced protective response, mice vaccinated with one dose of pgD-E7E6E5 were analyzed for E7-specific CD8⁺ T-cell activation and preventive antitumor responses for 6 months. Mice were immunized with either pgD or pE7E6E5 as a control. Frequencies of E7-specific CD8⁺ T cells in mice immunized with pgD-E7E6E5 reached a peak 14 days after vaccination and then gradually declined (Fig. 2 A). By day 100, when the E7-specific CD8⁺ T cells were barely detectable, mice were challenged with TC-1 cells. The frequencies of E7-specific CD8⁺ T cells increased sharply to levels similar to those seen at the initial response 21 days after challenge (Fig. 2B). After peaking on day 121, the numbers of E7-specific CD8⁺ T cells contracted rapidly and remained at low levels until the end of the observation period (Fig. 2A). Mice challenged 100 days after pgD-E7E6E5 administration showed only a partial protection (50%) from tumor development (Fig. 2C). No protection was recorded in mice immunized with pgD or pE7E6E5.

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FIG. 1.

Activation of E7-specific CD8⁺ T-cell precursors and preventive anticancer responses in mice immunized with pgD-E7E6E5 administered via the i.m. route. Individual (A) and pooled (B) CD8⁺ T-cell responses in mice immunized with one or two doses of pgD, pE7E6E5, or pgD-E7E6E5 delivered via the i.m. route (100 μg/dose). The level of E7-specific CD8⁺ T cells was determined 2 weeks after the last vaccine dose. Detection of IFN-γ-producing E7-specific CD8⁺ T cells was carried out with PBMC stimulated with the MHC-I-restricted E7 peptide (⁴⁹RAHYNIVTF⁵⁷) and stained for CD8 (with FITC) and intracellular IFN-γ (with PE).

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FIG. 2.

E7-specific CD8⁺ T-cell response and preventive antitumor effects in pgD-E7E6E5-vaccinated mice. Mice were immunized with one dose of pgD-E7E6E5, pE7E6E5, or pgD and challenged with 5 × 10⁵ TC-1 tumor cells 100 days after vaccine administration. Long-term (A) and time point-specific (day 121) (B) detection of IFN-γ-producing E7-specific CD8⁺ T cells in pooled PBMC. E7-specific CD8⁺ T cells were stimulated with the E7 peptide (⁴⁹RAHYNIVTF⁵⁷) and stained for CD8 (with FITC) and intracellular IFN-γ (with PE). (C) Long-term preventive antitumor responses in vaccinated mice. Tumor growth was monitored up to 60 days after challenge. The arrow indicates when the TC-1 cell challenge occurred.

Priming-boosting effects of pgD-E7E6E5 on the CD8⁺ T-cell responses.The capability of pgD-E7E6E5 to prime and boost E7-specific CD8⁺ T-cell responses in heterologous immunization regimens in mice submitted to immunization regimens employing a recombinant E1-deleted chimpanzee-derived serotype 68 adenovirus (AdC68) vector encoding the same target antigen, i.e., the three HPV-16 antigens genetically fused into gD, was measured. Mice primed with one dose of the recombinant adenovirus vector and subsequently boosted 3 months later with a single dose of pgD-E7E6E5 showed, 14 days later, a slight enhancement of E7-specifc IFN-γ-producing CD8⁺ T cells that gradually declined (Fig. 3). On the other hand, mice primed with the pgD-E7E6E5 vector showed a dramatic increase (47.4% of the total CD8⁺ T-cell population) in the number of IFN-γ-producing CD8⁺ T cells following a boost with a single dose of the recombinant adenovirus vector at 3 months after the priming with the DNA vaccine (Fig. 3). Both the priming effects of the DNA vaccine and the boosting effects of the adenovirus vector required fusion of the target antigens to the gD protein (Table 1). Collectively, these results indicate that the pgD-E7E6E5 vector has a remarkable priming effect on induced CD8⁺ T-cell responses when used in a heterologous priming-boosting immunization regimen and further emphasize the strong adjuvant effects of the gD protein when encoded by a DNA vaccine vector as a chimeric protein genetically fused to the target antigen.

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FIG. 3.

Priming/boosting effects on E7-specific CD8⁺ T-cell responses in mice submitted to a heterologous prime-boost immunization regimen using pgD-E7E6E5 and a recombinant adenovirus vector encoding the same antigen. Groups of mice (n = 5) were primed with pgD-E7E6E5 and subsequently boosted with the recombinant adenovirus vector (AdC68gD-E7E6E5) 3 months later (▴). The same procedure was repeated in mice immunized with AdC68gD-E7E6E5 and subsequently boosted with pgD-E7E6E5 (▪). Detection of IFN-γ-producing E7-specific CD8⁺ T cells was carried out with pooled PBMC stimulated with the MHC-I-restricted E7 peptide (⁴⁹RAHYNIVTF⁵⁷) and stained for CD8 (with FITC) and intracellular IFN-γ (with PE).

Therapeutic antitumor effects of the pgD-E7E6E5 vaccine.We also investigated the CD8⁺ T-cell responses to the three HPV-16 oncoproteins under a scenario compatible with the therapeutic use of the pgD-E7E6E5 vector. Mice were challenged with TC-1 tumor cells and then immunized with three doses of the DNA vaccine, or control vectors, administered 1 week apart. Splenocytes were harvested 10 days after the last vaccine dose, stimulated with peptides from E5, E6, or E7, and stained for detection of intracellular IFN-γ and CD107a, a degranulation marker of activated antigen-specific CD8⁺ T cells (1). Mice immunized with the pgD-E7E6E5 DNA vaccine showed increased frequencies of E6- and E7-specific IFN-γ-producing CD8⁺ T cells with regard to those of mice immunized with pE7E6E5 (Fig. 4 A). E5-specific CD8⁺ T-cell responses were also detected in mice immunized with pgD-E7E6E5 but did not reach a statistically significant difference compared to those of mice immunized with pE7E6E5. Detection of the CD107a marker further demonstrated that immunization with pgD-E7E6E5 resulted in positive responses to all three HPV-16 oncoproteins in vaccinated mice, with regard to animals immunized with pE7E6E5 (Fig. 4B). These results indicated that mice therapeutically treated with pgD-E7E6E5 manage to circumvent the immune-suppressive conditions induced by the tumor growth and develop significant CD8⁺ T-cell responses against all three HPV-16 oncoproteins encoded by the DNA vaccine.

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FIG. 4.

Activation of antigen-specific cytotoxic CD8⁺ T cells in mice immunized with pgD-E7E6E5 after challenge with TC-1 cells. (A) Frequencies of IFN-γ-producing CD8⁺ T cells following in vitro stimulation of splenocytes harvested from mice therapeutically treated with three doses of pgD, pE7E6E5, or pgD-E7E6E5. Fourteen days after the last vaccine dose, splenocytes were stimulated with the E5, E6, or E7 peptide and stained for CD8 (with FITC) and intracellular IFN-γ (with PE). Mice were challenged with 5 × 10⁵ TC-1 tumor cells. (B) Frequencies of CD107a-producing CD8⁺ T cells following in vitro stimulation of splenocytes harvested from mice therapeutically treated with three doses of pgD, pE7E6E5, or pgD-E7E6E5. Stimulation with the specific E5, E6, or E7 peptide was performed in the presence of anti-CD107a (with PE), and after the incubation period, spleen cells were stained for CD8 (with PE Cy5) and intracellular IFN-γ (with FITC). Mice were challenged with 5 × 10⁵ TC-1 tumor cells. (C) In vivo cytotoxic activities of antigen-specific CD8⁺ T cells induced in mice immunized with different DNA vectors. Ten days after the last vaccine dose, mice received target spleen cells pulsed with E5, E6, or E7 CD8⁺ T-cell-specific peptide and stained with 5 mM CSFE (right-side curves) or 0.5 mM CSFE (left-side curves) but not pulsed with the target peptides. One day later, total spleen cells were harvested and monitored by flow cytometry. Results from representative mice immunized with one of the tested DNA vectors. Reduction of the peak area corresponding to the cell population labeled with antigen-specific derived peptides (right-side curve), with regard to CFSE-labeled cells not exposed to the peptides (left-side curve), reflects the in vivo cytotoxic activity of antigen-specific CD8⁺ T cells. (D) Individual in vivo cytotoxic activities detected in mice immunized with different DNA vaccine vectors. CFSE-labeled spleen cells were pulsed with MCH-I-restricted peptides derived from E5 (•), E6 (▴), and E7 (⧫) oncoproteins. Values detected only in mice immunized with pgD-E7E6E5 after stimulation with E7- or E6-specific peptides showed statistically significant differences (P < 0.0001) with regard to mice immunized with pgD or pE7E6E5.

To further demonstrate that the CD8⁺ T-cell responses developed in mice therapeutically treated with the DNA vaccine show in vivo cytolytic activity to cells expressing the HPV-16 oncoproteins, we conducted experiments in which CFSE-stained cells were pulsed with E5-, E6-, or E7-derived peptides representing the immunodominant H-2D^b-restricted epitopes of each oncoprotein. As in the previously described experiments, mice were challenged with TC-1 cells, vaccinated at weekly intervals with 3 doses of pgD, pE7E6E5, or pgD-E7E6E5 vector, and injected with CFSE-labeled target cells 14 days after the last vaccine dose. Peptide-pulsed cells were brightly labeled with the dye, while control cells were labeled with a 10-fold-lower dose of CFSE. Splenocytes were harvested 24 h later from individual mice and tested for loss of the brightly stained cells as a measure for the specific CD8⁺ T-cell-mediated lysis (Fig. 4C and D). In vivo cell lysis was not observed in mice immunized with the pgD or pE7E6E5 vector. In contrast, mice immunized with pgD-E7E6E5 clearly showed a reduction in the number of peptide-pulsed cells, particularly those labeled with peptides derived from the E6 or E7 peptides. All pgD-E7E6E5-immunized animals had reduced numbers of E7-pulsed target cells, 3 out of 4 animals showed a reduction in the number of target cells pulsed with the E6-specific peptide, and only 1 animal developed a specific lysis response to E5-pulsed cells (Fig. 4D). Collectively, these results demonstrate that postchallenge-vaccinated mice developed functionally active CD8⁺ T-cell responses to the HPV-16 oncoproteins.

We next measured the therapeutic antitumor effects of pgD-E7E6E5 in vaccinated mice previously inoculated with TC-1 cells. Mice were challenged with TC-1 cells and treated on the same day with one dose of pgD-E7E6E5 or the control vectors. Other groups received a second dose or a third dose of each DNA vaccine given at weekly intervals. As shown in Fig. 5, one dose of the pgD-E7E6E5 vector failed to prevent tumor progression. However, administration of a second dose and a third dose conferred only 50% and 70% protection, respectively, to mice immunized with pgD-E7E6E5 (Fig. 5A). Monitoring of the E7-specific CD8⁺ T-cell responses in mice immunized with pgD-E7E6E5 showed that a single dose of the DNA vaccine failed to induce a statistically significant CD8⁺ T-cell response. Nonetheless, mice treated with two or three doses of pgD-E7E6E5 showed a significant increase in the number of E7-specific CD8⁺ T cells, which reached maximum values 20 days after the tumor challenge (Fig. 5B). The CD8⁺ T-cell responses quickly declined and reached baseline values 1 week later. Animals receiving one or two doses of the pgD or pE7E6E5 vectors did not develop measurable E7-specific CD8⁺ T-cell responses (data not shown).

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FIG. 5.

Therapeutic antitumor effects and induced E7-specific CD8⁺ T-cell responses in mice immunized with pgD-E7E6E5. (A) Dose-dependent antitumor protection in mice immunized with one, two, or three i.m. doses of pgD-E7E6E5. (B) Frequencies of E7-specific IFN-γ-producing CD8⁺ T cells in mice challenged with TC-1 cells and i.m immunized 8 h later with one, two, or three doses of pgD-E7E6E5 delivered at weekly intervals. (C) Therapeutic antitumor effects of pgD-E7E6E5 in mice challenged with TC-1 cells and subsequently immunized with three doses of the DNA vaccine on the same day (day 0) of the TC-1 challenge or 3, 5, 7, or 10 days after challenge.

We also measured the therapeutic antitumor effects of the DNA vaccine in animals challenged up to 10 days before the first vaccine dose. As shown in Fig. 5C, mice treated with three pgD-E7E6E5 doses up to 3 days after the TC-1 cell challenge showed 70% protection to tumor development. When the interval between the tumor challenge and vaccine treatment was increased to 5 or 7 days, the antitumor protection effect was observed in only 50% and 25% of the vaccinated mice, respectively. No antitumor effect in mice vaccinated 10 days after the TC-1 challenge was observed.

In an attempt to improve the therapeutic effects generated by the DNA vaccine, we combined the pgD-E7E6E5 vector with plasmids encoding interleukin-12 (IL-12) or granulocyte-macrophage colony-stimulating factor (GM-CSF), known to exert adjuvant effects on immune responses induced by DNA vaccines or synthetic peptides (10, 23, 35). Mice were challenged with TC-1 cells and immunized with 3 doses of pgD-E7E6E5 or pE7E6E5 mixed with 100 μg of pcDNA3.1 (empty vector), pIL-12 encoding murine IL-12, or pGM-CSF encoding murine GM-CSF. As shown in Fig. 6, mice that received pgD-E7E6E5 combined with pIL-12 or with pGM-CSF increased the antitumor response to 100%. Control groups immunized with pgD-E7E6E5 and pcDNA3.1 did not showed further enhancement of the therapeutic protection observed in mice immunized only with pgD-E7E6E5. Mice immunized with pcDNA3.1, pIL-12, or pGM-CSF, in combination or not with pE7E6E5, did not show any antitumor protection (data not shown).

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FIG. 6.

Coadministration of pgD-E7E6E5 and IL-12- or GM-CSF-encoding plasmid confers full therapeutic protection to the TC-1 cell challenge. Mice were i.m. immunized with three doses (100 μg) of pE7E6E5 or pgD-E7E6E5 admixed with 100 μg of pcDNA3.1 (empty vector), pGM-CSF, or pIL-12 vector. The vaccines were administered 8 h after challenge with 5 × 10⁵ TC-1 cells. Tumor growth was monitored up to 60 days after challenge. Statistically significant differences were noted (P = 0.0023) using the log rank test with regard to mice immunized with pgD-E7E6E5 plus pcDNA3.1 and pgD-E7E6E5 plus pIL-12 or pgD-E7E6E5 plus pGM-CSF vectors.