SEARCH

SEARCH BY CITATION

Keywords:

  • human papilloma virus;
  • integrase;
  • lentiviral vector;
  • vaccine;
  • tumor

Abstract

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

Persistent infection with high risk genotypes of human papillomavirus (HPV) is the cause of cervical cancer, one of most common cancer among woman worldwide, and represents an important risk factor associated with other anogenital and oropharyngeal cancers in men and women. Here, we designed a therapeutic vaccine based on integrase defective lentiviral vector (IDLV) to deliver a mutated nononcogenic form of HPV16 E7 protein, considered as a tumor specific antigen for immunotherapy of HPV-associated cervical cancer, fused to calreticulin (CRT), a protein able to enhance major histocompatibility complex class I antigen presentation (IDLV-CRT/E7). Vaccination with IDLV-CRT/E7 induced a potent and persistent E7-specific T cell response up to 1 year after a single immunization. Importantly, a single immunization with IDLV-CRT/E7 was able to prevent growth of E7-expressing TC-1 tumor cells and to eradicate established tumors in mice. The strong therapeutic effect induced by the IDLV-based vaccine in this preclinical model suggests that this strategy may be further exploited as a safe and attractive anticancer immunotherapeutic vaccine in humans.

High-risk human papillomaviruses (HR-HPV) have been recognized as causal agents of cervical cancer (CC), the second most common cancer in women worldwide, and represent an important risk factor associated with anogenital and oropharyngeal cancers in both men and women.1, 2 Importantly, a recent report showed that in the United States overall prevalence of oral HPV infection among men and women aged 14–69 years was 6.9%, and that prevalence was higher among men than women.3

Two prophylactic vaccines inducing neutralizing antibodies against the HR-HPV are now available, GARDASIL, against HPV genotypes 6, 11, 16 and 18, and CEVARIX, against HPV genotypes 16 and 18. These HPV VLP-based vaccines have been shown to be 100% effective in preventing high-grade cervical lesions associated with HPV16 and HPV18, and thus, they are used in several countries to vaccinate young women before of onset of sexual activity. Unfortunately, they are not effective on established infections4 and it has been predicted that it will take decades to detect their quantifiable effect on CC rate in the population. Thus, the development of therapeutic strategies to treat HPV-associated cancer lesions remains an area of great importance and interest.

The success of any targeted cancer immunotherapy approach is dependent on the induction of an immune response against a tumor specific antigen which, directly or indirectly, would induce cell death in tumours.5 In the case of HPV-induced diseases, the viral E6 and E7 oncoproteins represent ideal targets for immunotherapeutic approaches. These early viral antigens are constitutively expressed in HPV-associated cancers and contribute to the progression of HPV-associated malignancies by interacting with cellular partners involved in cell cycle control such as pRb and p53.6–10 Different immunotherapeutic vaccine platforms targeting E7 and/or E6 have been developed over the last decade including peptide/protein, dendritic cell (DC), plasmid DNA and viral vector-based therapies,11–13 but current strategies have met with only limited success in preclinical and clinical research. Concerning genetic immunization, a promising approach involves the use of a nononcogenic E7-mutant unable to bind the Rb protein due to three amino acid substitutions within the Rb protein binding site (D21G, C24G and E26G),14 fused to intracellular targeting sequences that are able to route HPV16 E7 to desired subcellular compartments, in order to enhance antigen processing and presentation to T cells.15 These targeting sequences include calreticulin (CRT), essential for loading of major histocompatibility complex class I molecule (MHC-I) with the appropriate peptide.16, 17

Several studies suggest that immunization with lentiviral vectors (LV) results in the induction of a potent and durable antigen-specific T-cell immunity against the delivered antigen and low interfering vector-specific responses.18 Important advantages of LV are their ability to infect a variety of cell types including DC and their efficiency in transducing and expressing heterologous genes. However, the use of LV in humans poses serious safety concerns; among these, there is the potential for oncogenesis following insertional mutagenesis.19 To minimize the risk of insertional mutagenesis, integrase defective lentiviral vectors (IDLV) have been engineered to present viral antigens in a similar but safer manner than the integrase competent counterpart.20 In the absence of integration, reverse transcribed vector DNA is circularized in two different forms containing either a single LTR (1-LTR), or two tandem LTRs (2-LTR). Importantly, the level of gene expression driven by the circular extra-chromosomal forms of vector DNA (E-DNA) remains long-lasting in antigen presenting cells (APC), such as DC and macrophages.21, 22 This allows having the same advantages in terms of antigen presentation and expression but major safety advantage compared to the integrase competent vector. Moreover, we and others have shown that a single immunization with a relatively low dose of IDLV provided prolonged immunization against viral and tumoral antigens23–27 and was effective in protecting mice against challenge with wild-type virus and tumor cell lines.24–26

Here, we report the efficient in vitro expression of HPV16 E7 fused to CRT by IDLV (IDLV-CRT/E7) and the use of this recombinant vector as a therapeutic vaccine against HPV16 E7 expressing TC-1 tumor in an animal model. A single intramuscular immunization with IDLV-CRT/E7 induced a persistent E7-specific CD8 T cell response which lasted up to 1 year from the vaccination. Importantly, we provide evidence that a single immunization with IDLV-CRT/E7 generated a potent therapeutic effect able to eradicate established tumors in TC-1 injected mice.

Material and Methods

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

Animals and cell lines

Female C57BL/6 mice (6–8 weeks old) were purchased from Charles River (Calco, Como, Italy) and kept in pathogen-free condition in the animal facility of the Istituto Superiore di Sanità (ISS). All animal procedures have been performed according to the institutional guidelines for animal care and according to approved protocols. Lenti-X human embryonic kidney 293T cell line was obtained from Clontech (Mountain View, CA) and was chosen due to its high titer lentiviral production. Cells were maintained in DMEM (Euroclone, LifeSciences Division, Italy) supplemented with 10% fetal bovine serum (Lonza, Treviglio, Milan, Italy), 2 mmol/l L-glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin (Lonza). TC-1 cell line, derived from primary C57BL/6 mouse lung epithelial cells and expressing HPV16 E6 and E7 proteins, was kindly provided by Dr T.C. Wu (Johns Hopkins University, Baltimore, MD).28 Expression of MHC-I molecules in all TC-1 cells was confirmed by FACS using a biotinilated anti-H2-Db antibody followed by Streptavidin-PE (BD Pharmingen; data not shown). TC-1 tumor cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum (Lonza), 2 mmol/l L-glutamine (Lonza), 50 U/ml penicillin (Lonza), 50 μg/ml streptomycin (Lonza) and 0.4 mg/ml Geneticine (Invitrogen, Carlsbad, CA).

Vectors construction and production

Transfer vector plasmid pTY2CMV-GFPW expressing GFP (pLenti-GFP) has been already described.22 For construction of transfer vector expressing the CRT/E7 fusion protein, the coding sequence of the CRT/E7 fusion protein was excised from plasmid pUMC4a (pNGVL4a-CRT/E7(detox) gently provided by Dr. T.C. Wu (Johns Hopkins, Medical Institutions, Baltimore, MD) using SnaBI/BamHI and cloned into pTY2CMV-GFPW by replacing the GFP coding sequence, thus obtaining the transfer vector plasmid pLenti-CRT/E7. The expressed E7GGG protein is a nononcogenic mutant (D21G, C24G and E26G) preventing binding to the Rb protein.14 The accuracy of DNA construction was confirmed by DNA sequencing. The HIV-based packaging plasmid IN defective (pcHelp/IN-) and the VSV.G envelope-expressing pMD.G plasmids have been already described.23, 29

For production of recombinant IDLV expressing CRT/E7 (IDLV-CRT/E7) and GFP (IDLV-GFP), 293T cells were transiently transfected on 10-cm Petri dishes using the Calcium Phosphate-based Profection Mammalian Transfection System (Promega Corporation, Madison, WI) as previously described.23, 27 A total of 12 μg of plasmid DNA were used for each plate in a ratio 6:4:2 (transfer vector:packaging plasmid:VSV.G-envelope plasmid). After 48 hr, cell culture supernatants were recovered, cleared from cellular debris and passed through a 0.45-μM pore size filter (Millipore Corporation, Billerica, MA). For concentration, vector containing supernatants were ultracentrifuged (Beckman Coulter, Fullerton, CA) on a 20% sucrose gradient (Sigma Chemical Co., St. Louis, MO) and viral pellets were resuspended in 1× PBS. Viral titers for the GFP-coding vectors were normalized by exogenous reverse transcriptase (RT) activity assay30 and titration on 293T cells.23, 31 For IDLV-CRT/E7, titration was performed by the reverse transcriptase (RT) activity assay over standards of known infectivity and the vector-associated RT activity was compared to the one of IDLV-GFP coding virions of known infectious titers, thus allowing for the determination of their infectious titer units.

Analysis of E7 expression by western blotting and immunofluorescence

293T cells were seeded in six well plates and transduced with normalized amounts of IDLV-CRT/E7 or IDLV-GFP at 37°C in atmosphere containing 5% CO2. Twenty-four hour postinfection cells were lysed and the proteins separated on 12% SDS polyacrylamide gel along with a recombinant purified HPV16 E7 protein, used as a positive control and transferred to a PVDF membrane (Millipore). For evaluating the presence of HPV16 E7 and HIV-1 Gag in IDLV-CRT/E7 preparations, 25 μl of viral pellets were separated on 16% SDS polyacrylamide gel along with recombinant purified HPV16 E7 and HIV-1 Gag proteins, used as positive controls, and transferred to a PVDF membrane (Millipore). The filters were saturated for 1 hr with 5% nonfat dry milk (NFDM) in PBST (PBS with 0.1% Tween 20) and then incubated with an anti-E7 monoclonal antibody (Zymed, USA) and an anti-Gag polyclonal antibody (NIH Repository Reagents, Cat. #4250), as primary antibodies, for 1 hr at room temperature, followed by incubation for 1 hr at room temperature with an anti-mouse HRP-conjugated IgG, for the anti-E7 monoclonal antibody, or an anti-rabbit HRP-conjugated IgG, for the anti-Gag polyclonal antibody (Sigma Aldrich, USA). The immunocomplexes were visualized using chemiluminescence ECL detection system (Super Signal West Dura Extended Duration Substrate-PIERCE/Thermo Scientific, USA).32 Immunofluorescence analysis was carried out on infected 293T cells at 48 hr postinfection. Transduced cells were fixed with 3% paraformaldehyde for 30 min at room temperature, permeabilized for 10 min with 0.1% Triton X-100, saturated with 3% BSA-PBS and incubated with anti-E7 monoclonal antibody (Zymed, USA) in 1% BSA-PBST for 1 hr at room temperature, followed by an anti-mouse FITC-conjugated IgG (Sigma Aldrich, USA) in 1% BSA for 1 hr at room temperature. Finally, cells were washed with PBS and mounted with a drop of Vectashield mounting medium with DAPI (Vector Laboratories, USA). Fluorescent cells were evaluated on a Leitz fluorescence microscope.

Therapeutic immunization

Therapeutic potential of lentiviral vaccination was tested in two experimental settings (Fig. 1). In both cases, C57BL/6 mice were injected into the right flank with 2 × 105 TC-1 cells in 200 μl of PBS. In the experimental therapeutic setting no. 1 (early-stage tumor), groups of mice (5 mice per group) received a single intramuscular injection of 1 × 107 RT Units of IDLV-CRT/E7 or IDLV-GFP in 200 μl of PBS at 2 weeks after TC-1 cell injection. In the experimental therapeutic setting no. 2 (established tumor), TC-1 cells injected mice (8 mice per group) were vaccinated intramuscularly once with 1 × 107 RT Units of IDLV-CRT/E7 or IDLV-GFP in 200 μl of PBS only when a palpable tumor of at least 3–4 mm in diameter was present. Tumor growth was monitored by palpation at the side of TC-1 injection every 5–7 days. Tumor size was measured with a caliper spanning the shortest and longest tumor diameters at different time points. Mice were sacrificed when the tumor size reached 16–18 mm or an ulceration of tumor was observed or for evaluating the immune response.

thumbnail image

Figure 1. Schematic diagram of vaccination protocols. Groups of C57BL/6 were subcutaneously inoculated with 2 × 105/mouse of TC-1 tumor cells. Experimental setting no. 1 (early-stage tumor): mice were injected with TC-1 at day-14 and vaccinated at day 0 with either IDLV-CRT/E7 or IDLV-GFP. Experimental setting no. 2 (established tumor): mice with 3–4 mm tumor size were vaccinated with either IDLV-CRT/E7 or IDLV-GFP. At different time points, E7-specific immune responses were evaluated.

Download figure to PowerPoint

Evaluation of antibody responses against E7 by ELISA

The presence of HPV16 E7 antibodies in the plasma of immunized mice was detected by in-house made ELISA as described previously.32 Briefly, recombinant HPV16 E7 protein (0.25 μg/well) or HIV-Gag protein (NIH Repository Reagents, Bethesda, MD) diluted in carbonate buffer (pH 9.4) was adsorbed at 4°C overnight into 96 well Maxisorp plates (Nunc Rochester, NY). The next day, wells were washed with PBST (PBS with 0.05% Tween 20) and blocked with 3% NFDM in PBS for 2 hr at 37°C. After washing with PBST, the plate was incubated for 2 hr at 37°C with serially diluted mouse sera in 1% NFDM-PBS. Serum from mice immunized with recombinant HPV16 E7 protein was used as positive control. After washing with PBST, the plate was incubated for 2 hr at 37°C with 1:20,000 dilution of a peroxidase conjugated rabbit anti-mouse Ab (Sigma Aldrich, USA). Specific antigen–antibody complexes were detected by the addition of tetramethyl benzidine substrate (DBA Italia S.R.L., Milan, Italy). After 30 min at room temperature, the enzymatic reaction was stopped by adding 50 μl of 1 M sulfuric acid/well and the plate was read with a standard ELISA reader at 450 nm. Cut-off values were established for each plate and in each run and were calculated as the average value of serum samples from 4 naive mice (Δ value) plus 2 standard deviations (SD). Serum samples with Δ values higher than the cut-off value were considered positive.

Evaluation of E7-specific CD8+ T cell immune response by IFNγ ELISPOT

The presence of E7-specific CD8+ T cells was evaluated at different time points after vaccination in blood or in spleen of sacrificed animals. Mice were orbitally bled collecting 200 μl of whole blood in the presence of K-EDTA anticoagulant. Plasma were separated from cell fractions by low speed centrifugation and kept at −80°C for further analysis. Leukocytes, obtained after ammonium chloride potassium (ACK) treatment, were counted and resuspended in complete medium (RPMI 1640 supplemented with 10% FBS, 2 mmol/l L-glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin). Single cell suspension from each spleen was prepared by mechanical disruption and passage through cell strainers (BD Biosciences, San Diego, CA). A cytokine enzyme-linked immunospot (ELISPOT) assay designed for measuring the number of interferon (IFN)-γ-secreting cells in PBMCs and splenocytes in response to specific antigen peptides was performed using reagents from BD Biosciences. Briefly, blood leukocytes or splenocytes were incubated with or without E749-57 peptide (UFPeptides s.r.l., Italy), the known immunodominant H2-Db-restricted epitope.33 Positive control included incubation of cells with Concanavalin A (Sigma Aldrich, USA). After 24 hr, the plate was developed according to manufacturer's protocol and Spot Forming Cells (SFC) were counted with an ELISPOT reader (A.EL.VIS, Hannover, Germany) and expressed as SFC/1 × 106 cells.

Analysis of cytokine production by intracellular staining

Splenocytes from single mice were cultured with or without the specific E7-9mer (5 μg/mL) in the presence of anti-mouse CD28 mAb (BDclone 37.51) at 2 μg/mL; PMA (50 ng/mL) in combination with Ionomicin (2 μg/mL) was used as positive control. After 1 hr from the stimulation, 10 μg/ml of Brefeldin A was added to the cultures as a protein transport inhibitor. After 6 hr of incubation, cells were stained with PercP anti-mouse CD8a, or the isotype matched mAb. Then, cells were permeabilized and stained with PE-labeled anti-mouse IL2, APC-labeled IFNγ and PECy7-labeled TNFα mAbs or their isotype-matched controls in PBS-0.5% saponin and analyzed by flow cytometry. All monoclonal antibodies were from BD Pharmingen (San Diego, CA), and all chemicals were from Sigma Aldrich Co. (St. Louis, MO). Acquisition and analysis of data were performed using FACSCanto and FACS Diva software (BD Biosciences, USA).

Statistical analysis

Kaplan-Meier plots were used to analyze survival. SPSS for Windows (SPSS, Chicago, IL) was used to determine the significance of differences in survival curves with a log-rank test.

Results

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

CRT/E7 fusion protein is efficiently expressed by IDLV

To construct the IDLV expressing HPV16 E7, we used a nononcogenic form of HPV16 E7 gene expressing a protein unable to bind the cellular pRb protein. Moreover, to optimize protein immunogenicity, E7 gene was fused to the CRT, a Ca2+ binding protein, shown to target the fused protein to the endoplasmic reticulum avoiding its degradation and favoring its presentation by the MHC system.16, 17 A schematic representation of the transfer vector pLenti-CRT/E7 is depicted in Figure 2a. Production of IDLV-CRT/E7 and control IDLV-GFP was performed as described in “Material and Method” section and according to established protocols.23 To confirm the in vitro expression of CRT/E7 fusion protein, 293T cells were transduced with IDLV-CRT/E7 or as a control IDLV-GFP and cell lysates were analyzed by Western blotting assay. As shown in the Figure 2b, a band of the expected molecular size for the CRT/E7 fusion protein was detected in cells infected with IDLV-CRT/E7 but not in those transduced with IDLV-GFP, lacking the CRT/E7 coding sequence. Expression and cytoplasmic localization of CRT/E7 fusion protein was also shown by immunofluorescence microscopy after transduction of 293T cells with IDLV-CRT/E7 (Fig. 2c). These results demonstrated that CRT/E7 fusion protein was efficiently expressed in vitro by the IDLV, validating IDLV-CRT/E7 as a suitable candidate for in vivo vaccination studies.

thumbnail image

Figure 2. Layout of pLenti-CRT/E7 transfer vector and expression of the CRT/E7 fusion protein. (a) Depiction of the plasmid used for the production of IDLV-CRT/E7. The CRT/E7 fusion gene is under the control of cytomegalovirus (CMV) promoter. The packaging signal (Ψ), primer binding site (PBS), major splice donor (SD) and acceptor (SA) sites, bovine growth hormone polyadenylation signal (bHGpA), central polypurine tract (cPPT) and Woodchuck post-transcriptional regulatory element (WPRE) are indicated. (b) Western blot analysis of lysates of 293T cell transduced with IDLV-CRT/E7 or IDLV-GFP as control using an anti-E7 monoclonal antibody. A band of 18 kDa of molecular mass is visible in the line where was run a recombinant E7 protein (rE7) and a band of about 65 kDa of molecular mass, corresponding to the molecular mass of the CRT/E7 fusion protein, is visible in lysate of 293T cells transduced with IDLV-CRT/E7. (c) Fluorescence microscopy analysis on 293T cells transduced with IDLV-CRT/E7 using anti-E7 monoclonal antibody. The photograph shows the nuclei colored in blue by DAPI staining and the fusion protein (green fluorescence) exclusively localized in the cytoplasm of the cells transduced with IDLV-CRT/E7.

Download figure to PowerPoint

Vaccination with IDLV-CRT/E7 protects TC-1 injected mice

To assess the efficacy of the IDLV-based vaccine in a therapeutic setting, an early-stage tumor model was initially evaluated (Fig. 1). Groups of mice were inoculated with 2 × 105 TC-1 cells and immunized 2 weeks later with one injection of equal amounts of either purified IDLV-CRT/E7 or IDLV-GFP as a control. For evaluating the therapeutic effect of IDLV-CRT/E7 vaccination and the persistence of immune response, growth of tumors was followed in these TC-1 inoculated mice for up to 11 months. As shown in Figure 3a, 5 out of 5 mice injected with the control vector IDLV-GFP developed tumors showing an increase in tumor size up to 15 mm in average size at 49 days from tumor injection. All IDLV-GFP injected mice were sacrificed by day 49 from injection. On the contrary, in the IDLV-CRT/E7 vaccinated group (Fig. 3b), only 1 out of 5 mice developed a large tumor, starting at 3 weeks from TC-1 cells injection, whereas 1 mouse transiently developed a small tumor (1.5 mm size) which rapidly regressed. The survival curve showed that all control mice died within 49 days after the tumor injection, whereas 80% of IDLV-CRT/E7 immunized mice remained tumor-free up to 11 months (Fig. 3c).

thumbnail image

Figure 3. Inhibition of tumor growth by IDLV-CRT/E7 vaccination in early-stage tumors. Two weeks after injection with 2 × 105 TC-1 cells, groups of 5 mice each were vaccinated (arrow) with IDLV-GFP (a) or IDLV-CRT/E7 (b). Tumor growth was followed over time and mice were euthanized when tumor size, calculated as mean of the two perpendicular diameter, approached 12–14 mm. (c) Kaplan-Meier plot of mice survival for each treatment is depicted. Survival was monitored up to 330 days (*p < 0.001 when comparing mice treated with IDLV-CRT/E7 [circles] and mice treated with IDLV-GFP [squares]).

Download figure to PowerPoint

IDLV-CRT/E7 vaccine induces persistent E7-specific humoral and cellular immune response: kinetic study of antigen-specific CD8+ T cell responses in vivo

Immunogenicity of IDLV-CRT/E7 vaccine was evaluated at different time points after challenge with TC-1 cells on blood in terms of both cellular and humoral E7-specific responses. Low levels anti-E7 specific antibodies were present in mice injected with IDLV-CRT/E7 for up to 4 months from the immunization, whereas absent in the control group mice injected with IDLV-GFP (Fig. 4a). It is possible that protein contamination in the vaccine rather than E7 expressed by the vaccine is responsible for this effect. However, we did not find evidence of E7 protein contamination in the injected vector preparations (Fig. 4d). To verify that the mice in both groups had been injected with the same amount of IDLV vectors, anti-HIV Gag antibody response was evaluated. As shown in Figure 4b, anti-Gag antibodies were present in all IDLV-injected mice and persisted up to 11 months. As expected, the titers of anti-Gag antibodies were higher than those directed against HPV-E7. Indeed, the amount of Gag protein present in the vector particles is much higher than that of E7 protein transcribed by the vector and eventually released from the transduced cells.

thumbnail image

Figure 4. Kinetic of E7 specific immune response in mice with early-stage tumors immunized with IDLV-CRT/E7 or IDLV-GFP. Anti-E7 (black bars) and anti-Gag (gray bars) IgG titers measured by ELISA in plasma samples of (a) mice immunized with IDLV-CRT/E7 at 22, 64, 106 and 316 days from the immunizations or in (b) IDLV-GFP injected mice at 22 days after immunization. Plasma samples from immunized mice were analyzed separately. Bars represent the geometric mean values in each group with indicated the standard error (SE). (c) IFN-γ responses measured by ELISPOT on whole blood cells at 22, 64, 106 and 316 days from the immunization with IDLV-CRT/E7. Analysis was performed on cells stimulated overnight with E7-specific peptide (black bar) or medium alone (white bar). The number of IFN-γ producing cells is expressed as SFC/106 cells. Error bars represent the SE of the average. (d) Western blot analysis of lysates of IDLV-CRT/E7 vector particles used in the immunizations. Left, a band of 18 kDa of molecular mass is visible in the lines where was run a recombinant E7 protein (rE7), whereas the corresponding CRT/E7 fusion protein is absent in virions. Right, a band corresponding to the recombinant p24 Gag protein is present in virions. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Download figure to PowerPoint

Because tumor-specific CD8+ T cells have a recognized role as effectors in anticancer responses, the induction of E7-specific CD8+ T cells was investigated in the vaccinated animals of each group by ELISPOT for IFNγ-producing T cells. Kinetic study of antigen-specific CD8+ T cell responses was performed by analyzing E7-specific T cells in blood samples recovered at different time points. As shown in Figure 4c, a high number of E7-specific IFNγ-producing T cells was present in all animals vaccinated with IDLV-CRT/E7 at 22 days after immunization (equivalent to 36 days after TC-1 injection), showing an average ± standard error (SE) of 1294 ± 288 SFC/106 cells. Although the number of E7-specific T cells decreased with time, the immune response was still present at 64, 106 and 316 days after vaccination (639 ± 74, 653 ± 121 and 218 ± 84 SFC/106 cells, respectively). No E7-specific IFNγ-producing T cells were detected in IDLV-GFP vaccinated mice (data not shown). These results indicate that a single immunization with IDLV-CRT/E7 was able to induce both anti-E7 specific antibodies and E7-specific CD8+ T cells detectable up to 11 months.

Vaccination with IDLV-CRT/E7 is able to cure tumor-bearing mice

To further test the antitumor therapeutic potential of IDLV-CRT/E7 as a vaccine in a more stringent setting, mice were first injected with 2 × 105 TC-1 cells per mouse and then vaccinated when established tumors reached a size of 3–4 mm (Fig. 1). As expected, all control animals developed a large tumor and were sacrificed starting from day 20 to day 30 after the immunization (Fig. 5). In vaccinated animals, the tumor continued to grow following immunization with IDLV-CRT/E7, reaching a 6 mm average-size ∼7 days after the therapeutic vaccination (Fig. 5a). Then, the tumors decreased in size and at day 22 after immunization the tumors were not measurable (Fig. 5a). In 1 out 8 vaccinated mice, this therapeutic effect was transient, and after a first regression, tumor recurrence was observed starting from day 30 and, at day 40, this animal was sacrificed (Fig. 5b). These results indicate that a single immunization with IDLV expressing E7 was able to cure 87.5% of mice (7/8) with established tumor. Four IDLV-CRT/E7 treated mice showing tumor eradication were sacrificed at 40 days from the immunization, to evaluate the frequency and quality of E7 specific T cells by ELISPOT and intracellular staining (ICS) for cytokines. The three remaining vaccinated mice remained tumor free for up to 90 days from immunization (Fig. 5b).

thumbnail image

Figure 5. Therapeutic effect of IDLV-CRT/E7 vaccination. (a) C57BL/6 mice were inoculated subcutaneously with 2 × 105 TC-1 cells. Mice with 3–4 mm size tumors were vaccinated with IDLV-CRT/E7 (circles) or with IDLV-GFP (squares). Tumor size, as mean of two perpendicular diameter, was examined over a 1-month period. (b) Kaplan-Meier plot of mice survival for each treatment is depicted. Survival was monitored up to 90 days (*p < 0.05 when compared mice treated with IDLV-CRT/E7 and mice treated with IDLV-GFP).

Download figure to PowerPoint

To evaluate the E7-specific immune response after the vaccination and during the reduction of tumor growth, ELISPOT for IFNγ was performed on blood samples. After 17 days from IDLV injection, a high number of E7-specific IFNγ producing T cells was detected in all IDLV-CRT/E7 vaccinated mice (953 ± 105 SFC/106 cells), whereas no specific response was detected in IDLV-GFP treated mice or in naïve animals (Fig. 6a).

thumbnail image

Figure 6. E7-specific CD8 T cell immune response after IDLV-CRT/E7 vaccination in mice with established tumors. (a) IFN-γ responses measured by ELISPOT on whole blood cells at 17 days from the immunization with IDLV-CRT/E7 or IDLV-GFP and from Naïve mice. (b) IFN-γ responses measured by ELISPOT on splenocytes from mice sacrificed at 40 days from the immunization with IDLV-CRT/E7 and from Naïve mice. Analysis was performed on cells stimulated overnight with E7-specific peptide (black bar) or medium alone (white bar). The number of IFN-γ producing cells is expressed as SFC/106 cells. Error bars represent the SE of the average. (c) Analysis of cytokine production by intracellular staining (ICS) on splenocytes from mice sacrificed at 40 days from the immunization with IDLV-CRT/E7 and from Naïve mice. Splenocytes were cultured in vitro with E7-specific 9mer peptide and then stained for IFNγ, TNFα and IL2. Error bars indicate the average and SE among mice of the same group. Data are expressed as percentage of single cytokine-producing T cells by gating the CD8+ T cell population. (d) IDLV-CRT/E7 vaccination induced polyfunctional antigen-specific CD8+ T cells producing IFNγ and TNFα but not IL-2. A representative experiment is shown. The analysis was performed on CD8+ T cells from Naïve mice (upper panels) or from mice immunized with IDLV-CRT/E7 (lower panels). The percentages of double cytokine producing cells were calculated on CD8+ T cells and are indicated within the dot plots.

Download figure to PowerPoint

To assess the persistence and quality of the E7-specific immune response, at 40 days from the immunization, an ICS for IFNγ, TNFα and IL2 was performed in splenocytes of sacrificed mice immunized with IDLV-CRT/E7 and naive mice, as negative controls. As shown in Figure 6b, the production of IFNγ seen in blood cells at earlier time point was confirmed in splenocytes by ELISPOT. In addition, ICS analysis showed that CD8+ T splenocytes from IDLV-CRT/E7 immunized animals produced IFNγ and TNFα, but not IL2, when cultured in vitro in the presence of E7 specific peptide (Fig. 6c). Of note, 50% of the IFNγ-producing CD8+ T cells were able to produce both IFNγ and TNFα (Fig. 6d, lower left panel).

Discussion

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

The development of an immunotherapeutic vaccine able to induce a tumor specific cytotoxicity with long benefits and without side effects remains a very attractive prospective in the field of tumor therapy. In this context, we have developed an HPV therapeutic vaccine based on IDLV delivering a nononcogenic mutated HPV16 E7, a recognized tumor-specific antigen in HPV-associated lesions. This vector has been used in preventive vaccine strategies in different preclinical models.34–36 We and other groups previously demonstrated its efficacy in inducing a strong and long lasting immune response directed versus viral and tumor antigens. In the present report, we demonstrated that IDLV is able to eradicate established tumors after a single immunization in about 90% of tumor-bearing mice.

Our results indicate that a single administration of IDLV-CRT/E7 at early stage of tumor elicited a potent therapeutic effect able to prevent or control the tumor growth. It has been shown that E7-specific CD8+ T cell response is correlated to the control of tumor growth in this preclinical model.37 To determine the ability of IDLV in inducing a tumor-specific immune response in a therapeutic setting, we first set up an early-stage tumor model, by vaccinating mice injected with TC-1 tumor cell line without measurable tumor masses. We investigated the presence of E7-specific T cells in peripheral blood at different time points from therapeutic immunization. All mice immunized with IDLV-CRT/E7 developed high E7-specific CD8+ T cell responses reaching the peak soon after the immunization. This response decreased with time but was still detectable 11 months after vaccination.

A significant contribution of antibody-dependent cell-mediated cytotoxicity (ADCC) in tumor cell death is very unlikely to occur in vivo, because HPV-E7 is not present on the cell surface. Nevertheless, the presence of E7-specific antibodies in plasma samples was assessed to validate the immunogenicity of the vector. As expected, low but detectable levels of E7-specific IgG antibodies in IDLV-CRT/E7 immunized mice were detected. These levels are consistent with those observed in other experiments using other nonsecreted proteins as antigens delivered by IDLV (data not shown).25 On the contrary, we found higher and homogeneous levels of anti-Gag antibodies, which give evidence of a correct inoculum of the vector.

To determine the ability of IDLV in controlling the tumor growth, the vaccination was performed in mice bearing a tumor mass averaging 3–4 mm of diameter. Our results indicate that a single immunization with IDLV-CRT/E7 was able to eradicate the tumor mass in the majority of treated mice. The strong specific immune response measured in immunized mice is certainly related to the potent anti-tumor effect seen in our experiments. The IDLV-based vaccination induced a strong polyfunctional E7-specific effector T cells able to produce both IFNγ and TNFα. These data are consistent with results already published using IDLV as a delivery system in vaccine studies.27, 38 Considering the immunotherapy approaches used in HPV preclinical model in mice by other groups, the strong effectiveness of this immunotherapeutic vaccine is evident. Indeed, it has been shown that to eradicate the tumor mass more than one therapeutic vaccination with DNA expressing CRT/E7 by electroporation was needed in addition to chemotherapeutic treatment.39 In this context, we previously demonstrated that a single immunization with IDLV is more efficient in inducing antigen-specific immune responses compared to two immunizations with DNA plasmid expressing the same antigen.38 Although it has been shown that multiple intratumoral administrations of adenoviral vector expressing E7 were able to cure TC-1 tumor bearing mice,40 a single intramuscular immunization with IDLV might be more feasible and applicable than repeated intratumoral administration. To our knowledge, this is the first report showing that in this tumor model a single systemic immunization without adjuvants and/or drug treatments is able to eradicate an established tumor mass in vaccinated mice.

In summary, this study shows the potential of IDLV as a vaccine for cancer immunotherapy. IDLV was able to efficiently express CRT/E7 and generate both cellular and humoral tumor specific immune responses in vivo. One round of immunization, without adjuvants, chemotherapy or any boost was able to elicit long-lasting high levels of E7-specific polyfunctional CD8+ T cell responses sufficient to control tumor growth in an early-stage tumor model and completely eradicate the tumor in tumor-bearing mice. This efficiency, combined with its inherent safety feature, makes IDLV a promising and intriguing vector system for immunotherapy. However, more studies are needed to fully determine the utility of this vector system for therapeutic vaccine applications. For example, the use of IDLV in a more aggressive tumor model should validate this approach and allow testing the combination with a chemotherapy regimen able to increase the efficiency of the immunotherapy. Another useful approach in cancer immunotherapy is represented by injection of DC transduced with IDLV expressing tumor antigens. We previously demonstrated that IDLV-transduced human DC was able to induce a strong expansion of autologous primed T cells using Flu-M1 as a model antigen.22 The ability to induce or recall ex vivo a HPV-specific response in patients with HPV-induced lesions will be useful for the further development of a therapeutic vaccination in humans using IDLV-based vaccination.

Acknowledgements

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

This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) to A.C. and the Italian AIDS National Program to D.R.M.N. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 Gag protein and HIV-1SF2 p24 Antiserum.

References

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