Control of HPV-associated tumors by innovative therapeutic HPV DNA vaccine in the absence of CD4+ T cells
© Peng et al.; licensee BioMed Central Ltd. 2014
Received: 3 December 2013
Accepted: 24 December 2013
Published: 4 March 2014
Human papillomavirus (HPV) infections are particularly problematic for HIV + and solid organ transplant patients with compromised CD4+ T cell-dependent immunity as they produce more severe and progressive disease compared to healthy individuals. There are no specific treatments for chronic HPV infection, resulting in an urgent unmet need for a modality that is safe and effective for both immunocompromised and otherwise normal patients with recalcitrant disease. DNA vaccination is attractive because it avoids the risks of administration of live vectors to immunocompromised patients, and can induce potent HPV-specific cytotoxic T cell responses. We have developed a DNA vaccine (pNGVL4a-hCRTE6E7L2) encoding calreticulin (CRT) fused to E6, E7 and L2 proteins of HPV-16, the genotype associated with approximately 90% vaginal, vulvar, anal, penile and oropharyngeal HPV-associated cancers and the majority of cervical cancers. Administration of the DNA vaccine by intramuscular (IM) injection followed by electroporation induced significantly greater HPV-specific immune responses compared to IM injection alone or mixed with alum. Furthermore, pNGVL4a-hCRTE6E7L2 DNA vaccination via electroporation of mice carrying an intravaginal HPV-16 E6/E7-expressing syngeneic tumor demonstrated more potent therapeutic effects than IM vaccination alone. Of note, administration of the DNA vaccine by IM injection followed by electroporation elicited potent E6 and E7-specific CD8+ T cell responses and antitumor effects despite CD4+ T cell-depletion, although no antibody response was detected. While CD4+ T cell-depletion did reduce the E6 and E7-specific CD8+ T cell response, it remained sufficient to prevent subcutaneous tumor growth and to eliminate circulating tumor cells in a model of metastatic HPV-16+ cancer. Thus, the antibody response was CD4-dependent, whereas CD4+ T cell help enhanced the E6/E7-specific CD8+ T cell immunity, but was not required. Taken together, our data suggest that pNGVL4a-hCRTE6E7L2 DNA vaccination via electroporation warrants testing in otherwise healthy patients and those with compromised CD4+ T cell immunity to treat HPV-16-associated anogenital disease and cancer.
KeywordsDNA vaccine Human papillomavirus Immunosuppression CD4 depletion Calreticulin Immunotherapy
Of the over one hundred known Human papillomaviruses (HPVs), a dozen high risk types are associated with cancer. These oncogenic types infect the genital mucosa and are sexually transmitted. The oncogenic HPVs are the primary etiologic agents of cervical cancer and are also known to cause a subset of head and neck, vaginal, vulvar, anal and penile cancers [1–3]. HPV-16 is the most problematic type as it causes at least half of all cervical cancers and the great majority (approximately 90%) of the HPV-associated cancers at the other anogenital sites and oral cavity. HPV-associated diseases are a significant problem for HIV+ and otherwise immunocompromised patients who are less able to clear their HPV infections than healthy individuals. HPV-associated cancer incidence is significantly elevated at multiple sites in HIV + patients, notably cervical cancer, which has been designated an HIV-associated malignancy , and anal cancer that is predominantly driven by HPV-16 . In addition, HIV + patients more frequently acquire multi-type infections; many of which are infrequently seen in healthy individuals, and consequently are not targeted by the current HPV vaccines . Furthermore, benign HPV disease is also more severe and intractable in HIV + and solid organ transplant patients . There are no specific treatments for chronic HPV infection, and warts are typically treated with limited success by surgery, ablation via cryotherapy or non-specific immune modulators. Therefore, there remains an urgent need for a therapy to effectively treat chronic oncogenic HPV infection, particularly HPV-16, and associated diseases using an approach that is safe and effective even for immunocompromised patients .
One potential strategy to treat HPV infection and disease in immunocompromised patients is DNA vaccination. DNA vaccines elicit cell-mediated and/or humoral immune responses and are considered safe even for immunocompromised individuals because they do not contain live pathogen . Although intramuscular (IM) DNA injection has a low efficiency of host cell transduction and encoded antigen expression on its own, delivery is greatly enhanced with immediate electroporation at the injection site . We have previously generated various therapeutic HPV DNA vaccines encoding HPV antigens and including various enhancement strategies such as targeting the antigen to the endosomal/lysosomal compartment , as well as linkage to HSP70 , to the extracellular domain of Fms-like tyrosine kinase 3-ligand  and to the translocation domain of a bacterial toxin . In particular, we have found that linkage to the heat shock protein calreticulin (CRT) in DNA vaccines potently enhances the immune response to heterologous antigens [14–16]. Indeed, one of these DNA vaccines encoding CRT linked to HPV-16 E7 antigen (CRT/E7(detox)) has advanced to clinical trials. The CRT/E7(detox) DNA vaccine is currently being tested in Phase I clinical trials in patients with HPV-16+ head and neck cancer (NCT01493154) and in a trial in HPV-16+ grade 2/3 cervical intraepithelial neoplasia (NCT00988559).
Although the clinical grade CRT/E7 (detox) DNA vaccine has been used in the clinic, it could be potentially improved by the fusion of additional HPV antigens. It has been shown that T cell-mediated immune responses against HPV E6 antigen are associated with a favorable clinical outcome, and may be immunodominant in some patients [17, 18]. Furthermore, humoral immune responses against the L2 capsid protein have been shown to provide broad protection against infection by a wide spectrum of HPV subtypes in animal models [19, 20]. Therefore, we developed a preventive and therapeutic HPV DNA vaccine encoding CRT linked to HPV-16 E6, E7 and L2 proteins (pNGVL4a-hCRTE6E7L2) . The cross-protective effects of L2-specific neutralizing antibody have the potential to prevent the unusual HPV types and multi-type HPV infections seen in the HIV + population. Indeed, we previously found that vaccination with pNGVL4a-hCRTE6E7L2 DNA vaccine was capable of generating HPV neutralizing antibodies as well as control of HPV-16 E6/E7-expressing tumors in a preclinical model . In contrast to patients, E6 is poorly immunogenic in the C57/BL6 mouse, but immunization with a DNA vaccine expressing E6 fused to CRT elicits therapeutic responses . Taken together, the pNGVL4a-hCRTE6E7L2 DNA vaccine has promise for the treatment of HPV16-associated lesions as well as to generate a pan-HPV protective effect against new or re-infection. However, it is unclear whether the vaccine will be active in patients with compromised CD4+ T cell-dependent immunity who so urgently need such a treatment. In the current study, we test the immunogenicity and therapeutic effects of the pNGVL4a-hCRTE6E7L2 DNA vaccine against an E6/E7-expressing murine model of anogenital HPV-16+ cancer in both immunocompetent and immunosuppressed settings.
In vivo electroporation enhances cell-mediated and humoral HPV antigen-specific immune responses to intramuscular vaccination with CRTE6E7L2 DNA
CRTE6E7L2 DNA vaccine administered intramuscularly followed by electroporation generates potent antitumor effects
CRTE6E7L2 DNA vaccine administered intramuscularly followed by electroporation elicits antigen-specific CD8+ T cells systemically and locally
CRTE6E7L2 DNA vaccine generates potent protective antigen-specific immune responses and antitumor effects against E6/E7-expressing tumors in CD4-depleted mice
CRTE6E7L2 DNA vaccine generates potent therapeutic antigen-specific immune responses and antitumor effects in CD4-depleted tumor-bearing mice
In the current study, we examined the protective and therapeutic effects of CRTE6E7L2 DNA vaccine against E6/E7-expressing tumors in immunocompetent and immunosuppressed mice. We first demonstrated that administration of the CRTE6E7L2 DNA vaccine by IM injection followed by electroporation generated greater HPV neutralizing antibody titers and cell-mediated immune responses compared to IM injection alone. Although the use of alum enhanced the HPV neutralizing antibody titers, it was no more effective than electroporation alone, and the combination did not yield a clear advantage. In addition, the use of alum failed to enhance the HPV-specific cellular immune responses elicited by IM vaccination. IM injection of CRTE6E7L2 DNA vaccine followed by electroporation elicited more potent E7-specific CD8+ T cell responses and therapeutic antitumor effects in tumor-bearing mice compared to IM injection without electroporation, and these responses effectively targeted tumors in the vaginal tract. Notably, we also observed these potent cell-mediated immune responses and antitumor effects in CD4-depleted tumor-bearing mice. Taken together, our data suggest that CRTE6E7L2 DNA vaccine may be appropriate for use in immunocompromised patients, including HIV + patients, to control of HPV-16-associated cancer.
Here we observed that mice vaccinated with CRTE6E7L2 DNA vaccine were capable of generating potent HPV antigen-specific CD8+ T cell-mediated immune responses in the absence of CD4+ T cells. We have previously shown that DNA vaccines encoding CRT linked to the target antigen are capable of generating high levels of antigen-specific CD8+ T cell responses as well as significant antitumor immunity [14, 15]. CRT is a heat shock protein that associates with peptides delivered into the ER  as well as with MHC class I-²2 microglobulin molecules to aid in antigen presentation . Furthermore, CRT has been shown to bind with CD91 (also known as ±2-macroglobulin receptor or low density lipoprotein-related protein), a cell surface receptor on antigen presenting cells . Thus, CRT may be able to deliver the linked antigens to dendritic cells through their specific binding with CD91 to facilitate cross priming activities. Furthermore, CRT has been shown to activate dendritic cells (DCs) .
A critical safety feature of the CRTE6E7L2 DNA vaccine is that it contains mutated (detox) versions of the E6 and E7 oncogenes. This is essential for the clinical translation of the CRTE6E7L2 DNA vaccine. Previously, it has been shown that mutation of E7 at position 24 and/or 26 disrupts the Rb binding site of E7 thus alleviating the concerns for the oncogenicity of E7  and that mutation at position 91 destroys the single zinc finger in E7 . This construct is being used in ongoing clinical studies. Additionally, mutation of E6 in positions that have been shown to destroy several key oncogenic functions, including the ability to bind p53, which prevent the E6 (detox) protein from immortalizing human epithelial cells [27, 28], were included. We further deleted the PDZ protein-binding domain at the C-terminus of E6 (aa146-151)  as this is also critical for transformation. The DNA construct used in the current study includes HPV-16 E6 and E7 genes that contain the mutations described above, thus minimizing safety concerns for clinical translation.
In the clinical setting, DNA vaccines can be delivered by a variety of methods, which include particle-mediated intradermal delivery by gene gun, intralesional injection, intramuscular injection, and intramuscular injection followed by electroporation [30–33]. The gene gun enables delivered DNA to directly transfect keratinocytes and epidermal Langerhans cells (immature DCs). This stimulates DC maturation and migration to the local lymphoid tissue, where DCs prime T cells for HPV antigenâ€“specific immune responses. DNA vaccine administration with electroporation increases the number of HPV DNAâ€“transfected cells and enhances the magnitude of gene expression, while requiring less time to reach a maximal immune response compared to conventional intramuscular vaccine injection. The use of an electroporation device may be even more important in non-human primates and humans than mice . Recent reports from clinical trials indicated that delivery of naked DNA vaccines with electroporation was capable of generating potent cellular as well as humoral immune responses against encoded HPV antigens . Additionally, the CRT/E7 (detox) DNA vaccine, administered by IM injection and electroporation, is being tested in HPV-16-associated head and neck cancer patients (NCT01493154), as well as via intracervical injection or a gene gun like device (PMED) (NCT00988559).
The current study suggests the potential of CRTE6E7L2 DNA vaccination to treat HPV-16 infections or HPV-16-associated diseases in both HIV- and in HIV + patients. HIV + patients in particular need a preventive and therapeutic HPV vaccine because they are more susceptible to HPV infection and often have frequent multi-type infections including uncommon HPV subtypes. Furthermore, HIV + patients exhibit more severe and progressive HPV infections compared to healthy individuals. The CRTE6E7L2 DNA vaccine has the potential to fulfill this urgent need because it safe, effective in the absence of CD4+ T cells and elicits potent cell-mediated immune responses and therapeutic antitumor effects in murine studies.
5 ~ 8 week old female C57BL/6 mice were purchased from the National Cancer Institute (Frederick, MD). All mice were housed at Johns Hopkins University School of Medicine cancer center animal facility under specific-pathogen free conditions, and all procedures were performed according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals.
Peptides, antibodies and regents
The H-2Db-restricted HPV16 E7aa49-57 peptide, RAHYNIVTF and H-2Kb-restricted HPV16 E6aa50-57 peptide, YDFAFRDL, were synthesized by Macromolecular Resources (Denver, CO) at a purity of ≥ 80%. FITC-conjugated anti-mouse CD3 (clone 145-2C11), FITC, PE and APC-conjugated anti-mouse CD8a (clone 53.6.7), and FITC-conjugated anti-mouse IFN-³ (clone XMG1.2) antibodies were purchased from BD Pharmingen (BD Pharmingen, San Diego, CA). PE-conjugated anti-mouse CD4 (clone RM4-4) antibody was purchased from Biolegend (San Diego, CA). PE-conjugated, HPV16 E7aa49-57 peptide and RAHYNIVTF loaded H2-Db tetramers were obtained from the National Institute of Allergy and Infectious Diseases Tetramer Facility (Atlanta, GA). Medroxyprogesterone acetate was purchased from Greenstone LLC (Peapack, NJ), and 4% Nonoxynol-9 (N-9) was purchased from Revive personal products company (Madison, NJ).
HPV-16 E6 and E7-expressing TC-1 cells were generated as previously described . TC-1-Luc cells were established by transducing TC-1 cells with luciferase. The cells were maintained in RPMI medium supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 100IUml−1 penicillin, 100μgml−1 streptomycin and 10% fetal bovine serum (FBS). The creation of 293TTF and Lovo-T has been described  and cultured in DMEM medium containing 2 mM glutamine, 1 mM sodium pyruvate, 100IUml−1 penicillin, 100μgml−1 streptomycin and 10% FBS.
The DNA vaccine, pNGVL4a-CRTE6E7L2 used in this study was constructed as follows. Briefly, CRTdE6E7L2 contains human calreticulin (CRT), E7 with three mutations , E6 with two mutations  and deletion of aa146-151 , and 11-200aa of HPV16 L2. To clone pNGVL4a-CRTdE6dE7L2, human CRT was isolated from pNGVL4a-hCRTE6E7L2  by Sal I/EcoRI. DE6E7L2 codon, modified and synthesized by Genescript (Piscataway, NJ), was cut EcoRI/BamHI. The digested human CRT and dE6E7L2 were cloned into pNGVL4a vector digested with Sal/BamHI. The mutation and deletion sequences of E6 and E7 are detailed in Additional file 2: Figure S2.
Electroporation-mediated DNA vaccination
pNGVL4a-CRT/E6E7L2 DNA vaccine was administered to C57BL/6 mice via intramuscular (IM) injection in the flank with or without subsequent electroporation with Ichor TriGridâ„¢ Electroporation Delivery System (TDS) (Ichor Medical Systems Inc., San Diego, CA) as described previously . When aluminum phosphate was used, DNA was mixed with 22.5 μg of aluminum phosphate with a final volume of 50 μl. The DNA/aluminum phosphate mixture was used within half hour after preparation. When vaccination schedules required a booster vaccination, the contralateral leg was used for vaccination, and subsequent vaccinations used alternating hind legs.
In vivo CD4+ T cell depletion
To deplete CD4+ T cells, mice were injected with a depleting anti-mouse CD4 antibody (Clone GK1.5) for 3 days via intraperitoneal injection. The depletion was maintained through the experiment by injecting the antibody once per week. The success of depletion was confirmed by staining peripheral blood cells with an anti-mouse CD4 antibody that recognizes a different epitope than the depleting antibody.
Production of furin-cleaved HPV16 pseudovirus
Production of furin-cleaved HPV16 pseudoviruses (fcPsV) is described elsewhere . Briefly, fcPsV were produced by following the standard PsV production protocol [39, 40] with the following modifications: (1) instead of 293TT cells, 293TTF cells were used; (2) ammonium sulfate was excluded from the maturation buffer; (3) the concentration of CaCl2 in the maturation buffer was increased to 5 mM; and (4) maturation time was increased from 24 hours to 48 hours.
In vitro HPV pseudovirus neutralization assay
The details of the in vitro HPV pseudovirus neutralization assay are described elsewhere . Briefly, 1.5 × 104 LoVoT cells were plated into 96-well tissue culture plate. The next day, mouse serum was serially diluted and mixed with fcPsV containing luciferase, incubated at 37°C for 2 hours before adding to the cells. These plates were then incubated at 37°C for 72 hours. Luciferase activity was analyzed and a 50% reduction in luciferase activity was considered as positive for neutralization.
HPV16 E7-specific antibody response was determined by an enzyme-linked immunosorbent assay (ELISA) as described previously  and the optical density (OD) value was read with xMark microplate spectrophotometer (BioRad, Hercules, CA) ELISA reader at 450 nm.
Preparation of single-cell suspensions from spleens and TC-1/luciferase tumors
Single splenocyte suspensions were prepared by meshing spleens and lysing red blood cells. To prepare single cells from vaginal TC-1-Luc tumors, tumors were surgically resected under sterile conditions and placed in RPMI 1640 medium containing 100Uml−1 penicillin, 100μgml−1 streptomycin on ice, and washed with phosphate-buffered saline (PBS). The solid tumors were then minced into 1- to 2-mm pieces and incubated with serum-free RPMI 1640 medium containing 1mgml−1 collagenase D, and 0.25mgml−1 DNase I (both from Roche, Indianapolis, IN), 100Uml−1 penicillin, 100μgml−1 streptomycin and incubated at 37°C with periodic agitation. The cells were then filtered through a 70-μm nylon filter mesh to remove undigested tissue fragments. The resultant single tumor cell suspensions were washed twice in Hank’s buffered salt solution (HBSS) (400 g for 10 min), and viable cells were determined using Trypan blue dye exclusion.
For tetramer staining, 1 × 106 splenocytes or tumor infiltrating lymphocytes were stained with purified anti-mouse CD16/32 (Fc block, BD Pharmingen, San Diego, CA) first, and then stained with anti-mouse CD8-FITC, PE-conjugated, HPV16 E7aa49-57 peptide, RAHYNIVTF loaded H2-Db tetramer at 4°C. After the wash, cells were stained with 7-AAD prior to flow cytometry analysis to exclude dead cells. The cells were acquired with FACSCalibur and analyzed with CellQuest (BD Bioscience, Mountain View, CA) or FlowJo software (Tree Star, Ashland, OR).
Intracellular cytokine staining and flow cytometry analysis
To detect HPV16 E6 or E7-specific CD8+ T cell responses by IFN-³ intracellular staining, splenocytes were stimulated with either HPV16 E6aa50-57 or E7aa49-57 peptide (1 μg/ml) at the presence of GolgiPlug (BD Pharmingen, San Diego, CA) at 37°C overnight. The stimulated splenocytes were then washed once with FACScan buffer and stained with PE-conjugated monoclonal rat anti-mouse CD8a. Cells were subjected to intracellular cytokine staining using the Cytofix/Cytoperm kit according to the manufacturer’s instruction (BD Pharmingen, San Diego, CA). Intracellular IFN-³ was stained with FITC-conjugated rat anti-mouse IFN-³. Flow cytometry analysis was performed using FACSCalibur with CELLQuest software.
In vivo tumor protection experiment
Mice were depleted of CD4+ T cells, mice were injected with a depleting anti-mouse CD4 antibody (Clone GK1.5) for 3 days via intraperitoneal injection as described above. Mice were then vaccinated with 20 μg pNGVL4a-CRT/E6E7L2 DNA by IM injection followed by electroporation three times at two-week intervals. 8 days after last vaccination, mice were injected with 5x104 TC-1 cells subcutaneously. The tumor growth was monitored by palpation twice a week.
In vivo tumor treatment experiment in a vaginal tumor model
To test whether pNGVL4a-CRT/E6E7L2 DNA vaccine-induced T cell responses could cure existing HPV16 E6E7-expressing tumors, an orthotopic vaginal tumor model was used. CD4 depletion was performed as described above. Briefly, female C57BL/6 mice were treated with 3 mg/mouse of medroxyprogesterone acetate via subcutaneous injection. 4 days later, the mice were treated intravaginally with 4% N-9 overnight. After wash, 2x104 TC-1/luciferase cells were instilled into intravaginal cavity. Seven days after tumor challenge, mice were vaccinated with 10 μg pNGVL4a-CRT/E6E7L2 DNA by IM injection followed by electroporation three times at three-day intervals. Tumor growth was monitored by luminescence imaging at indicated time points.
In vivo tumor treatment experiment in a hematologic spread model
After CD4+ T cell depletion, mice were injected with 1x105 TC-1 cells intravenously via the tail vein. 3 days later, mice were vaccinated with pNGVL4a-CRT/E6E7L2 DNA vaccine via IM administration followed by electroporation and boosted twice with the same regimen at one-week intervals. 30 days after TC-1 cell challenge, mice were sacrificed, splenocytes were prepared to detect HPV-16 E6- and E7-specific CD8+ T cell responses and lungs were harvested to examine the tumor growth.
All data were expressed as means ± standard deviations (SD). Comparisons between individual data point was analyzed by 2-tailed Student’s t test. Data for tumor treatment experiments were evaluated by analysis of variance (ANOVA). Survival distributions for mice in different groups were compared by the Kaplanâ€“Meier curves and by use of the long-rank tests. A P value of less than 0.05 was considered significant.
Human immunodeficiency virus
This work was supported by the National Institutes of Health/National Cancer Institute Cervical Cancer SPORE P50CA098252, R43CA174436 and 2R01CA114425-06 grants.
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