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Keywords:

  • human papillomavirus;
  • cervical cancer;
  • genital tumor model;
  • therapeutic vaccination;
  • mucosal vaccination

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Cervical cancer is a public health concern as it represents the second cause of cancer death in women worldwide. High-risk human papillomaviruses (HPV) are the etiologic agents, and HPV E6 and/or E7 oncogene-specific therapeutic vaccines are under development to treat HPV-related lesions in women. Whether the use of mucosal routes of immunization may be preferable for inducing cell-mediated immune responses able to eradicate genital tumors is still debated because of the uniqueness of the female genital mucosa (GM) and the limited experimentation. Here, we compared the protective activity resulting from immunization of mice via intranasal (i.n.), intravaginal (IVAG) or subcutaneous (s.c.) routes with an adjuvanted HPV type 16 E7 polypeptide vaccine. Our data show that s.c. and i.n. immunizations elicited similar frequencies and avidity of TetE7+CD8+ and E7-specific Interferon-gamma-secreting cells in the GM, whereas slightly lower immune responses were induced by IVAG immunization. In a novel orthotopic murine model, both s.c. and i.n. immunizations allowed for complete long-term protection against genital E7-expressing tumor challenge. However, only s.c. immunization induced complete regression of already established genital tumors. This suggests that the higher E7-specific systemic response observed after s.c. immunization may contribute to the regression of growing genital tumors, whereas local immune responses may be sufficient to impede genital challenges. Thus, our data show that for an efficiently adjuvanted protein-based vaccine, parenteral vaccination route is superior to mucosal vaccination route for inducing regression of established genital tumors in a murine model of HPV-associated genital cancer.

Vaccination strategies that elicit cell-mediated immunity in the genital mucosa (GM) may be critical for protection against pathogens that use this mucosa as entry and replication site. This may be especially relevant in the case of genital human papillomavirus (HPV), as persistent infection with the high-risk types, most often HPV type 16 (HPV16), may lead to malignant diseases, including cervical cancer,1 the second leading cause of cancer death in women worldwide.2 The available prophylactic vaccines prevent HPV infections through the induction of neutralizing antibodies, but they possess no therapeutic effects on already established HPV infections or associated lesions.3 Control of these lesions by cell-mediated immune responses is demonstrated by their increased prevalence when cell-mediated immunity is impaired4–6 and by the finding that early infiltration of highly cytotoxic effector T cells in low-grade genital lesions appears to provide protection against tumor progression.7 Thus, induction of cell-mediated immune responses against the HPV oncogenes (E6 and/or E7), which are implicated in initiation and maintenance of high-grade anogenital lesions, became an attractive approach, though with limited clinical success to date.8 The most promising results [i.e., 47% regression of vulvar intraepithelial neoplasia (VIN)] were recently achieved after subcutaneous (s.c.) vaccination with synthetic E7 long peptides in incomplete Freund's adjuvant.9 However, it is still unclear whether a mucosal or systemic route of vaccine delivery is superior for generating high-frequency, functional, avid, and long-term cellular immune responses in the GM; as most of the therapeutic trials against anogenital HPV-associated lesions have used systemic routes of vaccination and determined the immune responses in the blood and not in the GM.8 We have recently shown that a single parenteral immunization with an adjuvanted HPV16 E7 synthetic polypeptide vaccine was able to induce high-avidity E7-specific IFN-γ-secreting CD8 T cells in the GM of vaccinated mice.10 However, two mucosal immunization routes, intranasal (i.n.) and intravaginal (IVAG), were previously shown to induce cell-mediated protective immunity in genital sites in challenge studies with mucosal pathogens.11–15 Here, we have thus examined whether these mucosal immunization routes may be more effective to induce vaccine-specific CD8 responses in the GM. More importantly, we have taken advantage of a novel orthotopic murine model, in which genital tumors develop in the vaginal epithelium after an IVAG instillation of E7- and luciferase-expressing tumor cells,16 to compare parenteral and mucosal vaccination for their ability to prevent or induce regression of these genital tumors.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Immunization of mice and preparation of murine cells

Female C57Bl/6 mice (Charles River Laboratories, L'arbresle, France) were used according to the Swiss veterinary authorities. Either E71-98 or E734-98 peptides (hereafter referred as E7) were used for immunizations as they induced similar E7-specific responses in all organs examined (data not shown). Fifty micrograms of E7 was injected s.c. at the base of the tail or administered by the i.n. and IVAG routes under deep anesthesia.17–19 The following adjuvants were coadministered in combinations as mentioned in the Results section: Resiquimod (R-848, 75 μg per dose; Huang Lisheng Pharmatec, China), heat-labile enterotoxin (HLT,20 0.4 μg per dose; kindly provided by C. Gremion, Pevion Biotech, Bern, Switzerland) and CpG oligodeoxynucleotides 1826 (10 μg per dose; Coley Pharmaceutical Group, Wellesley, MA). Murine cells from blood, spleen, lymph nodes and genital tract were prepared as previously described.21 Bone marrow-derived dendritic cells (BMDC) were generated using bone marrow cells collected from tibias and femurs and were cultured in the presence of 150 U/ml of recombinant mouse granulocyte–macrophage colony-stimulating factor (R&D Systems, Basel, Switzerland).22

IFN-γ ELISPOT assay and fluorescent tetramer labeling

IFN-γ ELISPOT assays were performed as described in detail in Ref. 21 using anti-IFN-γ monoclonal antibody (R4-6A2, Beckton Dickinson Pharmingen, Allschwill, Switzerland) and biotinylated anti-IFN-γ monoclonal antibody [AN 18.03.C12 (Ref. 23) or XMG1.2; Beckton Dickinson Pharmingen]. In ELISPOT assays revealed with XMG1.2 antibody, 30,000 BMDC per well were loaded with 1 μg/ml of E749-57 peptide for 1 hr before adding 100,000 CV cells per well. Similar E7-specific responses were measured in CV tissues with the two protocols. The phycoerythrin tetramers used in this study were formed from H-2Db HPV16 E749-57 or L1165-173 peptide monomeric complexes (from the Ludwig Institute for Cancer Research, Tetramer Production Facility, Epalinges, Switzerland). Tetramer E7 (TetE7), tetramer L1 (TetL1, used here as negative control) and Allophycocyanin-conjugated-conjugated anti-CD8 antibodies (eBioscience, Vienna, Austria) labeling were performed as previously described.10 Cells were analyzed using FACS Calibur and CellQuest Pro software (BD Biosciences). Double-positive TetE7+CD8+ cells were expressed as percentage among recovered cells and/or as percentage among total CD8+ cells.

Tumor protection assays

The HPV16 E6/E7-expressing TC-1 and luciferase-expressing TC-1-luc cell lines are described in Refs. 16 and 24. To induce s.c. tumors, 2 × 104 TC-1 cells were s.c. injected into the flank of mice. In the prophylactic setting, mice were vaccinated once with the adjuvanted E7 vaccines 6–8 days before s.c. challenge with TC-1 cells, whereas in the therapeutic setting, vaccination was performed 8 days after TC-1 s.c. implantation.

To induce genital tumors, anesthetized diestrus synchronized mice pretreated with 4% nonoxynol-9 (N9, Igepal, Sigma, Buchs, Switzerland) were IVAG challenged with 12,500–20,000 TC-1-luc cells.16 Genital tumor growth was monitored by bioluminescence in a Xenogen imaging system (Xenogen/Caliper Life Science, kindly provided by CIF, UNIL, Lausanne, Switzerland) as described in Ref. 16. In the therapeutic setting, vaccination was performed 9–10 days after genital tumor implantation, whereas in the long-term prophylactic setting, IVAG TC-1 challenge was performed at Day 111 after mice had received two vaccine doses at Days 1 and 31.

Statistical analysis

Statistical analyses were performed using Prism 5.00 for Windows (GraphPad Software, San Diego, CA) as indicated in the text or in the figure legends.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

E7-specific IFN-γ-secreting CD8 T-cell responses after s.c. or i.n. immunization

We first characterized the amplitude and kinetics of E7-specific CD8 T-cell responses after s.c. and i.n. vaccinations to investigate the potential differences between these immunization routes. Groups of mice received E7 adjuvanted with CpG and HLT for s.c. immunization or with HLT and R-848 for i.n. immunization, as these combinations proved to be more effective in pilot experiments (data not shown). Mice were sacrificed at Days 7, 9 or 11. Peripheral blood mononuclear cells (PBMC) or cells recovered from spleen, cervix-vagina (CV) and genital lymph nodes (GLN, draining the CV) were analyzed by ex vivo IFN-γ ELISPOT assays. After s.c. immunization (Fig. 1, black circles), the highest E7-specific CD8 T cell responses were measured at Day 9 in PBMC, spleen and CV. The responses were similar at Day 7, except in CV which exhibited a significantly lower response (p < 0.01). At Day 11, the responses were significantly lower than at Day 9 in all these organs (p < 0.05 for PBMC, spleen and CV or p < 0.01 for GLN). After i.n. immunization (Fig. 1, white circles), although the highest E7-specific CD8 T-cell responses were measured at Day 7 in PBMC, spleen and GLN, the responses were similar at Day 9 in CV and nonsignificantly decreased in PBMC and spleen. At Day 11, the responses were lower in these organs though not significantly different from Day 7 or 9. This suggests different kinetics of the CD8 T cell response after s.c. or i.n. immunizations, the latter appearing slower, though comparisons performed between Day 9 for s.c. immunization and Day 7 or 9 for i.n. immunization seem appropriate.

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Figure 1. E7-specific IFN-γ-secreting CD8 T cells induced in different organs after s.c. or i.n. E7 immunization. Groups of five to nine mice were immunized with E7+HLT+CpG s.c. (left column, black circles) or E7+HLT+R-848 i.n. (right column, white circles). After 6, 8 or 10 days of vaccination, the indicated organs from individual mice were analyzed by ex vivo IFN-γ ELISPOT. The numbers of E749-57-specific IFN-γ-secreting cells per 105 cells are indicated for each mouse/organ. The horizontal bars represent the mean responses. Significant differences among organs analyzed at different time points for each vaccination or between vaccinations at peak of responses (Day 9 for s.c. and Day 7 or 9 for i.n. immunization) are indicated by “*” for p < 0.05 or “**” for p < 0.01 following Student's t-test.

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Comparison between the two routes of immunization showed significantly higher responses after s.c. immunization in PBMC and GLN (mean E7-specific IFN-γ-secreting cells ± SEM of 346.9 ± 84.1 and 31.9 ± 4.1 at Day 9 as compared to 77.9 ± 17.7 and 6.7 ± 2.8 at Day 7 of i.n. immunization for PBMC and GLN, respectively, p < 0.05; see Fig. 1). The responses were similar after i.n. immunization in spleen (78.9 ± 11.5 at Day 7 as compared to 88.3 ± 14.6 at Day 9 of s.c. immunization) and nonsignificantly lower in CV (6.4 ± 3.0 at Day 9 as compared to 12.8 ± 3.3 of s.c. immunization). Because the site of s.c. immunization in the tail area also drains to the GLN, like CV, we further s.c. immunized mice in the neck area and showed that the site of s.c. injection did not account for the immune response measured in GM after s.c. immunization (Supporting Information Fig. S1).

In an attempt to optimize the nonsignificantly lower response induced in CV by i.n. immunization, different protocols were tested (Table 1). Administration of a third adjuvant, CpG, in addition to HLT and R-848, as well as a 150 μg E7 dose (administered as one dose or three 50 μg doses at Days 1, 2 and 3) with the three adjuvants augmented the genital immune response, though not significantly. Interestingly, a booster i.n. immunization (administered at Week 4) did not significantly improve the immune response, similarly to what was previously observed using the s.c. route.10 Because of limitations in the peptide availability and insolubility of the vaccine inoculum, we used 50 μg E7 formulated with the three adjuvants for further i.n. immunizations, as a single dose yielded about 12 E7-specific IFN-γ-secreting cells per 105 cells in CV, similarly to s.c. immunization. Using the different individual mice analyzed, our data show that even when using a mucosal immunization route, only poor correlation could be found between the response in CV with those measured in PBMC or spleen of the same mice (Spearman r = 0.34 and 0.26, respectively; Supporting Information Fig. S2), confirming that the vaccine response in the GM cannot be predicted by the immune responses measured in the periphery.

Table 1. E7-specific CD8 T-cell responses after different protocols of i.n. vaccination
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Functional avidity of the E7-specific CD8 T cells in CV induced by i.n. immunization were determined and compared to those observed in s.c. vaccinated mice10 (see Supporting Information Table S1). The ratio of high-avidity effector T cells varied nonsignificantly among the organs examined, though higher ratios of high-avidity effector T cells were measured after s.c. immunization in GLN and spleen (p < 0.05 compared to spleen of i.n. immunized mice). However, almost identical ratio of high-avidity CTL were generated in CV by both routes (mean of 0.49 ± 0.12 for i.n. as compared to 0.42 ± 0.07 for s.c. immunization), suggesting a similar effector activity in the GM.

Flow cytometry analysis of E7-specific CD8 T-cell responses to s.c. and i.n. vaccinations

Groups of mice were either s.c. or i.n. immunized with the adjuvanted E7 vaccine, and 8 days later, cells recovered from individual or pools of two organs were analyzed by flow cytometry (Fig. 2). DoubleTetE7+CD8+ cells were significantly higher after s.c. immunization in spleen (mean percentage of TetE7+CD8+ and TetE7+CD8+/total CD8+ ± SEM of 0.63 ± 0.14 and 4.67 ± 0.88, respectively, as compared to 0.07 ± 0.01 and 0.65 ± 0.12 after i.n. immunization, p < 0.01 and p < 0.001, respectively) and in GLN (mean percentage of TetE7+CD8+ and TetE7+CD8+/total CD8+ ± SEM of 0.19 ± 0.06 and 0.96 ± 0.27, respectively, as compared to 0.05 ± 0.01 and 0.23 ± 0.05 after i.n. immunization, p < 0.05 for both comparisons). In contrast, the percentage of TetE7+CD8+ cells was similar in CV regardless of the immunization routes (0.13 ± 0.03 after s.c. as compared to 0.09 ± 0.03 after i.n. immunization) confirming the ELISPOT data, though the numbers of tetramer positive cells appeared about 10-fold higher than the numbers of E7-specific IFN-γ-secreting cells on average. Interestingly, the percentage of TetE7+CD8+/total CD8+ appeared higher after i.n. immunization (mean percentage ± SEM of 26.50 ± 2.29 as compared to 16.83 ± 1.93 after s.c. immunization, p < 0.01), possibly in line with the preferential trafficking of mucosally induced vaccine-specific CD8 T cells to the GM.25

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Figure 2. E7-specific CD8 T cells induced by s.c. or i.n. immunization visualized using fluorescent tetramer labeling. Groups of 10–20 mice were either s.c. or i.n. immunized with E7+HLT+CpG or E7+HLT+R-848+CpG, respectively. Eight days later, cells recovered from individual or pool of two spleens, GLN and CV, were stained with anti-CD8 and TetE749-57 or TetL1165-173 (as control) and analyzed by flow cytometry. The percentage of TetE7+CD8+ cells among recovered cells (left panels) are calculated after subtraction of the background measured with control TetL1165-173. The percentage of TetE7+CD8+/total CD8+ cells are also represented (right panels). The horizontal bars represent the mean responses.

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Both s.c. and i.n. vaccination are able to prevent and regress s.c. TC-1 tumors while only the s.c. immunization can efficiently regress genital TC-1 tumors

Antitumor activity of the s.c. and i.n. E7 vaccines was first assayed using the common s.c. TC-1 tumor protection assay in a prophylactic setting.24 All s.c. and i.n. vaccinated mice remained tumor free for at least 150 days, whereas control unvaccinated mice rapidly developed a tumor (p < 0.0001 with a χ2 test; Fig. 3a). A tumor protection assay was further performed in a therapeutic setting (Fig. 3b). At Day 20, all unvaccinated mice harbored a tumor that never regressed, whereas both s.c. and i.n. vaccinated mice underwent regression of growing tumors and/or remained tumor free, at least until Day 70. Two new tumors then appeared in s.c. immunized and one in i.n. immunized mice resulting in 4/6 and 5/6 mice, respectively, that remained tumor free until Day 280 (both significantly different from unvaccinated mice p < 0.05 with a χ2 test). Thus, our data show that a single i.n. or s.c. vaccination with the adjuvanted E7 vaccine was able to fully prevent and cure the majority of s.c. E7-tumors.

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Figure 3. Tumor protection assays in the s.c. TC-1 or genital TC-1-luc mouse models. (a) Naive mice (black diamond, n = 8), mice s.c. immunized with E7+HLT+CpG (black cross, n = 8) or mice i.n. immunized with E7+HLT+CpG+R-848 (white circle, n = 8) were s.c. challenged into their flank with TC-1 cells 6 days later. Percentage of tumor-free mice in each group upon time is represented. (b) Mice s.c. challenged with TC-1 cells received 8 days later the s.c. E7 vaccine (black cross, n = 6), the i.n. E7 vaccine (white circle, n = 6) or left untreated (black diamond, n = 6). (c) Mice IVAG challenged with TC-1-luc cells received the s.c. E7 vaccine 9 days later (black cross, n = 10) or left unvaccinated (black square, n = 10). Tumor growth/regression was followed twice a week by bioluminescence measurement (p/sec/cm2/sr), and representations of all s.c. E7-vaccinated mice at days 13 and 30 are shown (right). (d) Mice IVAG challenged with TC-1-luc cells received the i.n. vaccine 8 days later (white circle, n = 8) or left unvaccinated (black square, n = 8). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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We recently reported an orthotopic murine model where tumors develop in the genital epithelium after an IVAG instillation of luciferase-expressing TC-1-luc cells.16 Herein, we used for the first time this novel model to test the ability of therapeutic vaccines to induce regression of preimplanted genital tumors. At Days 9 and 10, when the first tumors could be detected by bioluminescence, mice were either s.c or i.n. immunized with the adjuvanted E7 vaccines. Between Days 13 and 16, most of the unvaccinated or vaccinated mice harbored a bioluminescent genital tumor (for instance, 90% of s.c. immunized mice at Day 13, as shown in Fig. 3c, right panel). A single s.c. immunization with the adjuvanted E7 vaccine induced regression of these established genital tumors in 90% of mice (see bioluminescence imaging of s.c. vaccinated mice at Day 30 in Fig. 3c, right panel) that remained tumor free for at least 150 days, whereas only 1/10 of the unvaccinated mice never developed a tumor (p = 0.0003 with a χ2 test; Fig. 3c, left panel). Although i.n. immunization resulted in significant but transient regression of genital tumors in 50% of the mice (see between Days 26 and 55 in Fig. 3d, p = 0.02 with a χ2 test at Day 55), in two mice, tumors started to grow again, resulting in only 25% of the i.n. vaccinated mice being tumor free from Day 80. A second attempt to induce regression of genital tumors (n = 9) with i.n. immunization was similarly ineffective in the long term (data not shown). Unexpectedly, our data thus show that s.c. immunization with the adjuvanted E7 vaccine could more efficiently provoke the regression of established genital tumors than did i.n. immunization.

Induction of E7-specific IFN-γ-secreting cells and genital tumor regression on IVAG immunization

Given the relatively poor efficacy of i.n. immunization, we wondered whether a local immunization might be a more efficient mucosal route of immunization to induce genital tumor regression. We IVAG pretreated groups of mice with N9 before IVAG administration of 50 μg E7 adjuvanted with CpG and HLT, similarly to a protocol used to induce humoral responses,19 and found that cell-mediated immune responses could also be induced as E7-specific T cells that were detected both in the periphery and locally (mean ± SEM of E7-specific IFN-γ secreting cells per 105 cells of 71.9 ± 19.8 in PBMC, 17.4 ± 6.5 in spleen, 8.4 ± 2.7 in GLN and 7.3 ± 1.4 in CV; see Fig. 4a, black circles). These responses were then compared to those obtained after a single s.c. (Day 9; Fig. 1) or i.n. immunization (Table 1, see i.n. immunization 50 μg E7 with the three adjuvants) using one-way ANOVA, followed by a Bonferroni post-test. The E7-specific effector responses after IVAG immunization were similar or not significantly lower than those measured after i.n. immunization, whereas they were significantly lower than the responses induced by a single s.c. immunization in spleen (p < 0.001), PBMC (p < 0.05) and in GLN (p < 0.001), the two latter also being higher than after i.n. immunization (p < 0.01 and p < 0.001, respectively). In contrast, the responses obtained by the three immunization routes did not significantly differ in CV (ranging from a mean of ca. 12 E7-specific IFN-γ-secreting cells per 105 cells after s.c. and i.n. immunizations to a mean of ca. 7 E7-specific IFN-γ-secreting cells per 105 cells after IVAG immunization). Comparative analysis (data not shown) performed on the E7-responses measured with tetramers (Figs. 2 and 4b) yielded similar results for PBMC, spleen and GLN. The percentage of TetE7+CD8+ in CV also did not significantly differ after the three immunization routes (ranging from mean percentage of 0.13 for s.c., 0.09 for i.n. to 0.03 for IVAG immunizations). A group of 10 other mice received two IVAG immunizations (at Weeks 0 and 4; Fig. 4a white circles), but the IVAG booster immunization only slightly increased the E7-specific CD8 responses in GLN (p < 0.05), but not in PBMC, spleen or CV, which is similar to the data obtained after s.c. or i.n. booster immunizations with this adjuvanted E7 vaccine.

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Figure 4. E7-specific CD8 T cells and genital tumor regression induced on IVAG vaccination. Groups of mice were IVAG immunized with E7+HLT+CpG once (black circle, n = 14) or boosted 4 weeks later (white circle, n = 10). Eight days after immunization, the indicated organs from individual or pool of two mice were analyzed by ex vivo IFN-γ ELISPOT (a) and by flow cytometry (b). The percentage of TetE7+CD8+ cells (left panel) are calculated after subtraction of the background measured with control TetL1165-173. The percentage of TetE7+CD8+/total CD8+ cells are also represented (right panel). The horizontal bars represent the mean responses or percentages. Mice IVAG challenged with TC-1-luc cells received the IVAG vaccine 8 days later (white triangle, n = 9), only pretreated with N9 (black triangle, n = 8) or left unvaccinated (black square, n = 8). (c) Percentage of tumor-free mice upon time is represented.

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We examined whether IVAG immunization with the E7 vaccine could induce regression of genital tumor (Fig. 4c). However, this mucosal route, similarly to i.n. immunization, was only partially efficient as genital tumors regressed in only 5/9 mice (42%), which remained tumor free at least until Day 158. Similar partial regression (2/8 mice) was obtained in a second experiment (data not shown). As expected, no effect on tumor growth or regression was observed in mice only IVAG pretreated with N9, as all rapidly developed a tumor similarly to unvaccinated mice, thus confirming that regression of tumors after IVAG immunization is mediated by vaccine-specific immunity (Fig. 4c).

E7-specific long-term immunity afforded by the three immunization routes

To get more insight in the immunity induced by these three immunization routes, we examined their ability to prevent at long-term genital tumor implantation (Fig. 5). Our data show that all s.c. and i.n. immunized mice remained tumor free at least until Day 230 (p = 0.0001 as compared to unvaccinated mice with a χ2 test), whereas only 33% (2/6) of IVAG immunized mice were protected from tumor growth (statistically not different from unvaccinated mice at Day 230). This suggests that both s.c. and i.n. immunizations were able to induce an effective long-term immunity, but not the IVAG route.

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Figure 5. Genital tumor protection assay in long-term prophylactic setting. Naive mice (black square, n = 7), mice s.c. immunized with E7+HLT+CpG (black cross, n = 8), i.n. immunized with E7+HLT+CpG+R-848 (white circle, n = 8) or IVAG immunized with E7+HLT+CpG (white triangle, n = 6) and boosted at Day 31 were IVAG challenged with TC-1-luc cells at Day 111. Tumor growth was followed by bioluminescence. Percentage of tumor-free mice in each group on time is represented.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We demonstrated here that both s.c. and i.n. administrations of an adjuvanted E7 polypeptide vaccine induced similar frequencies of TetE7+CD8+ and high-avidity E7-specific IFN-γ-secreting cells in GM of mice. However, only s.c. immunization was able to induce regression of already implanted genital tumors in mice. Trafficking of antigen-specific CD8 T cells to the GM have been reported after either parenteral or mucosal infections/immunization routes with variable results. Thus, the vaccination route that may be better suited to induce effective protective immunity in the genital tract remains controversial (reviewed in Refs. 26 and 27). To address this issue, we combined a well-defined protein antigen (50 μg of HPV16 E7 synthetic polypeptide) with adjuvants able to exert their immunopotentiating activity by either mucosal or parenteral immunization routes. CpG, a toll-like receptor (TLR) 9 agonist28, HLT (a heat-labile enterotoxin)20, 29 and R848 (a TLR7/8 agonist29, only necessary for i.n. immunization) turned out to be effective in this regard, inducing similar peak effector E7-specific CD8 T-cell responses in the GM of mice vaccinated by either s.c., i.n. or IVAG routes, though systemic responses were higher after s.c. immunization. Direct comparison of parenteral and mucosal routes for their ability to induce CTL in the GM was recently reported. Intramuscular delivery of recombinant adenovirus vector vaccines30 induced higher CTL responses in both systemic and vaginal compartments than the i.n. immunization route. Using naked DNA vaccines, Bivas-Benita et al.31 reported similar systemic and genital vaccine-specific CD8 responses with either i.m. or pulmonary vaccination routes. Higher in vivo-specific lysis in GM and spleen were shown after i.n. immunization with ovalbumine adjuvanted with CT than after IVAG or sublingual immunizations.32, 33 Similarly to our findings, Manuri et al.34 reported relatively higher E7-specific CD8 T-cell responses in the vagina than in the spleen of mice i.n. immunized with two doses of another adjuvanted E7 peptide. However, most of the mice vaccination studies did address the induction of specific CD8 T cells in the GM after mixed prime-boost immunizations35, 36 or subsequent IVAG infection with recombinant vaccinia.12, 13 As a result, a side-by-side comparison of our results is not possible. In our case, the finding that similar E7-specific CD8 T cell responses were induced in the GM with i.n., IVAG and s.c. immunization routes and that systemic responses were higher after s.c. immunization may suggest a preferential homing of CD8 T cells to the GM after mucosal immunizations. These findings are similar to data obtained for humoral (reviewed in Refs. 25 and 37) and some CTL responses (reviewed in Ref. 27) and in line with the concept of a common mucosal immune system, where antigen presentation occurring in a mucosal site lead to priming of lymphocytes with a tendency to selectively home to other mucosal sites. However, the difficulty to keep solubility while augmenting antigen and/or adjuvant doses in the small volume that can be administered to mice by i.n. and IVAG routes, as well as the low efficacy of homologous booster immunizations, limited our potential to further increase the vaccine-specific CD8 T cells in the GM after mucosal immunization using this animal model.

Nevertheless, almost identical number of E7-specific IFN-γ CD8 T cells in the GM could be generated by a single s.c. and i.n. immunization. In contrast to our hypothesis that this would reflect protective potential against tumor located in this mucosa, our data show that only s.c. immunization induced effective regression of genital tumors. However, E7-specific CD8 T cells in the GM were not only similar in magnitude, but also in quality, at least as judged by the equal proportion of high-avidity IFN-γ-secreting CD8 T cells present in GM after either s.c. or i.n. immunization. Although good correlations between IFN-γ secretion and cytotoxicity have been reported,38 this may not be always the case30 and we cannot exclude that the killing potential of E7-specific CD8 T cells in the GM was greater after s.c. immunization, thus explaining the better tumor regression achieved. Vaccination has also been often reported to increase T-regulatory cells (Treg) able to counteract vaccination benefits.39–42 However, our preliminary analysis showed no differences in the percentage of FoxP3+CD4+/total CD4+ cells in the spleen (ca. 9%) of naive as compared to s.c. or i.n. vaccinated mice, though it is possible that this may differ in the GM, where higher percentage of FoxP3+CD4+/total CD4+ (ca. 22%) are already found in naive mice.16 An additional complication may be the influence of the growing genital tumor on vaccine-induced immune responses. In the case of VIN, it was recently shown that local treatment with imiquimod to modify the tumor microenvironment was beneficial to a subsequent vaccination leading to higher infiltration of lymphocytes and vaccine-specific responses in imiquimod responders.43 CTL expansion may also be inhibited in the presence of an established tumor44; however, the numbers of E7-specific CD8 T cells that we measured in PBMC 8 days after i.n. vaccination of genital tumor-bearing mice were similar to those found in only-vaccinated mice (ca. 40/105 E7-specific IFN-γ-secreting cells), and lower responses did not correlate with larger tumors as shown in the case of s.c. ovalbumin-expressing EG7 tumors.45 Further investigations are however needed to exclude differences in both Treg and E7-specific CD8 T cells that may have arisen locally in the GM after either s.c. or i.n. immunizations. Finally, it is noteworthy that s.c. immunization with the adjuvanted E7 vaccine induced higher numbers of E7-specific CD8 T cells in PBMC, spleen and GLN than the i.n. and IVAG routes, thus possibly leading to a higher tumor infiltration that may account for the effective regression of the genital tumors. However, it is unclear as why i.n. immunization was able to induce regression of TC-1 tumor when located s.c. but not when in the GM. This may highlight not only differences in requirements for homing to dermis or GM epithelium and/or differences in local tumor microenvironments but also emphasizes the desirability to use a biologically relevant tumor model for preclinical investigation of HPV therapeutic vaccines.

To date, efficacy of vaccination strategies to protect the GM through cell-mediated immune responses were only examined in mice in the setting of the prevention from genital challenge with herpes simplex virus14, 46, 47 or vaccinia recombinant virus.36, 47, 48 This may clearly differ from attempting regression of aggressively growing tumors established in the GM that probably require acute effector immune responses in contrast to the memory responses involved in prevention from a genital infection. Indeed, in a prophylactic setting, our data showed that both s.c. and i.n. immunizations were able to fully prevent implantation of genital tumors, suggesting that similar E7-specific long-term memory responses were induced by both immunization routes in the GM. This is in agreement with the notion that the size of the memory CD8+ T-cell pool is directly proportional to the size of the effector T-cell pool generated after primary vaccination49 and may suggest that the poor effectiveness of IVAG immunization to prevent genital tumor implantation is linked to the twofold lower E7-specific effector immune response generated in CV by this immunization route. Interestingly, for another mucosal tumor location, in a spontaneous colorectal cancer murine model, both systemic (intravenous) and rectal immunization were able to decrease adenoma formation and prevent progression to invasive colorectal cancer.50

In conclusion, the availability of a genital HPV-tumor model provided a unique opportunity to test efficacy of different immunization routes to induce regression of tumors located in the genital tract. Our data show that, for an efficiently adjuvanted protein-based vaccine, parenteral is superior to mucosal vaccination route for inducing regression of established genital tumor in this animal model. Future vaccination trials in patients suffering from HPV-induced lesions may confirm both the predictability of vaccine efficacy using this novel murine model and the potency of parenterally administered HPV16 E7 therapeutic vaccines.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank Dr. Laurent Derré for his critical reading of the manuscript, and Ana-Rita Gonçalves and Christelle Pythoud for their technical help.

References

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  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
IJC_25973_sm_SuppFig1.tif395KSupporting Information Figure 1.
IJC_25973_sm_SuppFig2.tif285KSupporting Information Figure 2.
IJC_25973_sm_SuppTable1.doc36KSupporting Information Table 1.
IJC_25973_sm_SuppMaterials.doc26KSupporting Information Materials.

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