Human Papillomavirus Type 16 E7 Research Articles

Human Papillomavirus Type 16 (HPV16) E6/E7-Specific Cytotoxic T Lymphocytes (CTLs) for Immunotherapy of HPV-Associated Malignancies

Abstract 1913Vaccines effectively prevent HPV-associated cancers, but their use as therapy for established neoplasms has been disappointing. Although the target tumors express the viral E6 and E7 antigens, patients' immune responses against virally infected cells are limited, and active immunization fails to generate effective immunity to these antigens. Ineffective endogenous immunity to E6/E7 is likely due to negative environmental cues that block initial tumor cell recognition and subsequent T cell activation, expansion or persistence in vivo. We postulated that ex vivo stimulation of patient T cells in an immunologically favorable milieu would allow us to reactivate sufficient numbers of active tumor-directed CTLs for adoptive transfer to patients with HPV-associated tumors.We studied 67 patients with HPV-associated neoplasms (16 with cervical and 51 with oropharyngeal carcinoma). To investigate the presence of HPV16 E6- and E7-specific T cells in peripheral blood, we performed a γ-IFN ELISpot assay targeting those antigens, measuring both baseline immunity detectable in peripheral blood mononuclear cells (PBMCs) and the responses of T cells stimulated for 9 days by monocyte-derived dendritic cells (DCs) loaded with pepmixes (peptide libraries of 15-mers overlapping by 11 aa) spanning E6 and E7. We anticipated finding HPV-reactive T cells in a significant proportion of patients with oropharyngeal cancer, given that approximately 75% of these tumors are associated with HPV16, and a lesser rate of responsiveness in subjects with cervical neoplasia, since only half of subjects have HPV16-positive tumors. However, we found no PBMC or T cell reactivity against E6 or E7 under the tested conditions. Because HPV-specific T cells from patients with HPV-associated cancers may be anergized by their tumors, we therefore postulated that potent antigen presenting strategies might be required to reactivate them. We have previously shown that in vitro stimulation in the presence of the accessory cytokines IL-6, -7, -12 and -15 is able to reactivate T cell responses even to poorly immunogenic non-viral tumor antigens. Indeed, in combination with these cytokines, DCs successfully reactivated and expanded HPV16 E6- and/or E7-specific T cells detectable by ELISpot in 8/16 cervical and 32/51 oropharyngeal carcinoma patients.To develop a technology that would allow us to consistently produce and expand these HPV-specific T cells to the number needed for clinical use, given that sufficient DCs may be difficult to obtain when sourced from the peripheral blood of patients with cancer, we prepared substitute antigen presenting cells, B-blasts (BBs), derived from the subjects’ autologous B cells. These BBs were generated by culturing patient PBMCs in the presence of IL-4 and cyclosporine on a CD40 ligand-positive fibroblast feeder layer. Stimulation of DC-stimulated HPV-specific T cells by E6/E7 pepmix-loaded BBs allowed further expansion of HPV-specific T cells (3.8 ± 1.5 fold per round of stimulation), with successive increase in antigen specificity. Analysis of the Vβ-chain T cell receptor (TCR) repertoire established that the cell lines were polyclonal. Phenotypic analysis of the cell lines showed them to be almost exclusively composed of T cells (97.5 ± 3.4% CD3+), with a variable proportion of CD4+ and CD8+ cells (36.7 ± 28.2% and 49.4 ± 27.0%, respectively) displaying a predominantly effector memory phenotype (CD45RA–, CD45RO+, CD62L– and CCR7–); and minimal CD3– CD56+ NK cells (1.5 ± 0.9%). ELISpot assays using mini-pools of peptides from each antigen allowed identification of the epitopes recognized, which mapped to aa 49–71, 77–91 and 125–143 for E6, and aa 1–19 and 73–87 for E7. Cytotoxicity assays using these HPV-specific cell lines as effectors (E) against autologous pepmix-loaded PHA blasts as targets (T) demonstrated for several of these lines dose-dependent specific killing of antigen-loaded targets (specific lysis range 45–61% versus 0–8% in controls at 40:1 E:T ratio). Hence, these cells are true CTLs.In summary, we have developed a system that for the first time allows the robust generation of HPV-directed CTLs from the peripheral blood of patients with an HPV16-associated cancer, and shown that these lines recognize specific epitopes in tumor-associated molecules. Our cell lines have the potential to be used for adoptive cellular immunotherapy of HPV-associated carcinomas. Disclosures:No relevant conflicts of interest to declare.

Impacts of HIV infection and long‐term use of antiretroviral therapy on the prevalence of oral human papilloma virus type 16

The objectives of this study were to determine (i) the prevalence and the copy numbers of oral human papilloma virus type 16 (HPV-16) in HIV-infected patients compared with non-HIV controls, and (ii) the effects of antiretroviral therapy (ART) and its duration on the virus. A cross-sectional study was carried out in HIV-infected patients with and without ART and in non-HIV controls. Saliva samples were collected, and the DNA extracted from those samples was used as a template to detect HPV-16 E6 and E7 by quantitative polymerase chain reaction. Student's t-test and ANOVA test were performed to determine the prevalence rates among groups. Forty-nine HIV-infected patients: 37 on ART (age range, 23-54 years; mean, 37 years), 12 not on ART (age range, 20-40 years; mean, 31 years), and 20 non-HIV controls (age range, 19-53 years; mean, 31 years) were enrolled. The prevalence of oral HPV-16 infection and the copy numbers of the virus were significantly higher in HIV-infected patients than in non-HIV controls when using E6 assay (geometric mean = 10696 vs. 563 copies/10(5) cells, P < 0.001), but not E7 assay. No significant difference was observed between those who were and were not on ART. Long-term use of ART did not significantly change the prevalence of oral HPV-16 infection and the copy numbers of the virus (P = 0.567). We conclude that the prevalence of oral HPV-16 infection and the copy numbers of the virus are increased by HIV infection. Neither the use of ART nor its duration significantly affected the virus.

Abstract PL04-04: Synthetic long peptide vaccines against established disease caused by high-risk human papillomavirus.

Abstract Therapeutic vaccination of persistent virus infections and associated diseases including (pre-)cancer so far has largely evaded success, mainly due to the fact that insufficiently consistent and robust effector T cell responses were induced by the commonly used vaccine constructs and formulations such as recombinant viruses and bacteria, recombinant proteins, DNA constructs, Dendritic Cell (DC) vaccines and exact HLA class I binding peptides (short peptides). Problems have been severe antigenic competion from vector sequences by recombinant viruses and bacteria, insufficiently powerful T cell generation by DNA constructs, insufficient homing to lymph nodes injected DC and antigen presentation of short peptides by non-professional antigen presenting cells in vivo, causing tolerance instead of immunity. Much more robust and consistent T cell responses can be obtained by vaccination with long (28–35 amino acid long) synthetic peptides. Such immunogens are more efficiently processed and presented than intact proteins by DC and only DC can efficiently perform this task. Moreover only concentrated antigen of choice is offered and antigenic competition therefore plays no role. In earlier work we showed that therapeutic vaccination with a synthetic long peptide (SLP®) vaccine mediated the eradication of established human papilloma virus type 16 (HPV16)-positive tumors in mice and controlled wart growth and latent virus infection in rabbits persistently infected with cottontail rabbit papilloma virus. Subsequent phase I/II studies with an HPV16 SLP® vaccine, consisting of 13 long peptides covering the HPV16 E6 and E7 antigens, in patients with advanced HPV16-positive cervical cancer, revealed that this vaccine was safe and highly immunogenic. We then tested the clinical efficacy of this HPV16 SLP® vaccine in HPV16-induced high grade vulvar intraepithelial neoplasia (VIN3), a premalignant epithelial disorder, spontaneous regression of which occurs in less than 2% of patients and in which recurrence after standard treatment is high. In a phase 2 trial, 20 women with VIN3 were vaccinated three times sc in the limbs with a mix of the HPV16 E6 and E7 synthetic long peptides formulated in Montanide ISA-51. The endpoints were objective clinical responses, defined as reduction of at least 50% in lesion size (partial response) or complete regressions, and HPV16-specific T-cell responses. The vaccine was safe. At 3 and 12 months after the last vaccination an objective response was observed in 12/20 (60%) and 15/19 (79%) patients respectively. Nine of them showed a complete and durable regression of the lesions at 12 months and at 24 months. The strength of the vaccine-induced HPV16-specific T-cell response was significantly higher in the group of patients with a complete regression of their lesions compared to non-responders. Patients with large lesions were less likely to experience a complete clinical response than patients with small lesions and we ascribe this to a larger proportion of vaccine induced HPV-specific regulatory cells in the patients with large lesions. In conclusion, treatment with the HPV16 SLP vaccine is clearly effective in patients with established VIN disease. The SLP platform lends itself for development of therapeutic vaccines against many other chronic infections and non-viral cancers. In patients with cancer, it is attractive to combine this type of vaccination with immunogenic forms of cancer chemotherapy and with immuno-modulatory drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PL04-04.

Novel oligomannose liposome-DNA complex DNA vaccination efficiently evokes anti-HPV E6 and E7 CTL responses

The aim of this study was to establish an efficient human papilloma virus (HPV) type 16-targeting cancer immunotherapy. Persistent high-risk HPV infection causes cervical intra-epithelial neoplasia (CIN) and subsequent cervical carcinoma. HPV type16 (HPV16) is one of the common carcinogenic types and is found in about 50% of invasive cervical carcinomas. HPV16-derived viral proteins E6 and E7 are expressed in cancerous cells through the progression of the disease and have a role in carcinogenesis but are not expressed in normal cells. Thus, these proteins are regarded as ideal antigens for cervical carcinoma immunotherapy. In this study, we generated a novel HPV 16 E6 and E7 gene plasmid containing oligomannose liposomes (OML-HPV). We compared the cytotoxic T lymphocyte (CTL) induction efficiency of OML-HPV and that of standard liposome-HPV16 E6 and E7 DNA complex. HPV16 E6-specific CTLs could be generated from HPV 16-positive cervical carcinoma patient's peripheral blood mononuclear cells (PBMCs) by stimulating OML-HPV, but could not by stimulating standard liposome-HPV 16 E6, E7 DNA complex. Furthermore, we screened HLA-A24-restricted HPV16 E6- and E7-derived peptides, and found that one E6-derived peptide (E6 66–74) showed the highest immunogenicity with ELISPOT assay from 100% of HPV16-positive patients (4 out of 4). On the other hand, other E6- or E7-derived peptides, including E6 49–57, E6 82–90, E6 87–95, E6 98–106 and E7 83–93, showed less frequent reactivity. These results indicate that OML-HPV is a more effective approach than DNA vaccination using standard liposomes, and that a novel HLA-A24-restricted peptide, E6 66–74, might be a suitable target of cervical cancer immunotherapy.

Abstract C31: DMBA-induced skin tumors in HPV16 E7 transgenic mice are affected by bovine α-lactalbumin made lethal to tumor cells (BAMLET)

Abstract Background and Purpose: Human papillomavirus Type 16 (HPV16) infection is necessary but not alone a sufficient causal risk factor for cervical and other anogenital cancers and nearly 25% of head and neck malignancies (1, 2). HPV16 E7 is one of the important oncoproteins that promotes carcinogenesis. Most treatments for cervical precancers are ablative and few therapeutic drugs are available. Human α-Lactalbumin Made Lethal to Tumor Cells (HAMLET), a complex of Human α-Lactalbumin and oleic acid shows promise for treating skin papillomas by differentially inducing apoptosis in transformed cells without adverse effects on normal cells. Bovine α-Lactalbumin Made Lethal to Tumor Cells (BAMLET) has been shown to be biologically equivalent to HAMLET (3) and in vitro studies suggest BAMLET and HAMLET show similar cytotoxic effects in tumor cell lines (4). Further, in vitro studies suggest both a dose- and time-dependent response to BAMLET treatment (3–6). To determine the effect of BAMLET on tumor number, size and histological characteristics in vivo, we compared two groups of BAMLET-treated with normal saline (NS)-treated HPV16 E7 transgenic C57/BL6 mice (TG) and syngeneic mice receiving chemical carcinogen DMBA topically to induce skin tumor. Methods: For 56 TG and syngeneic mice, in six treatment groups, 100nmol/200μl DMBA was applied topically on the dorsal thoracic flank for up to 20 weeks. BAMLET, 0.7 mM, was applied daily for 7d before and either 11d (18d BAMLET) or up to 23 weeks (24w BAMLET) following DMBA initiation. As a comparison, NS was similarly applied to 10 TG and syngeneic mice for a total of 24 weeks. A series of bivariate zero-inflated repeated-measures Poisson (ZIP) models predicted the number of papillomas based on TG/syngeneic groupings, and treatment group. The fully adjusted model evaluated the effect of the TG, treatment group and duration, and time on study for predicting the number of tumors. Least-squares linear regression (LSLR) was used to estimate the effect of treatment on average tumor size at censoring or death, controlling for the effect of gender, among the transgenic mice. Results: On average, papillomas developed after 9 weeks of DMBA treatment. Analyses showed TG-mice developed more tumors than did syngeneic mice (p&amp;lt;0.0001), controlling for the effect of time on study (p&amp;lt;0.0001). In the fully adjusted analyses, the number of tumors was most affected by the TG (p&amp;lt;0.0001) and time on study (p&amp;lt;0.0001), but treatment showed no effect: 18d (p=0.9) and 24w (p=0.8) vs. normal saline (NS). Among transgenic mice, time on study was shorter than for syngeneic comparators and was a proxy indicator across treatment groups, confounding the ZIP analyses. Among transgenic mice, BAMLET treatment predicted average tumor size at censoring or death in the LSLR analysis. Specifically, among transgenic mice, the average tumor size was greatest for the 18d (μ=0.24 cm, p=0.003) and 24w BAMLET-treated (μ=0.20 cm, p=0.03) mice when compared to NS-treated controls (μ=0.12 cm). Discussion and Conclusion: These preliminary data suggest the effect of the transgene is strong on the number of tumors the result from DMBA treatment and on survival. This strong effect suggests we lack the power to detect an effect of BAMLET in transgenic mice. However, some data herein suggest BAMLET may increase average tumor size among HPV16-E7 transgenic mice. In light of positive findings in human clinical studies using the human analogue, HAMLET, understanding whether the effects of α-Lactalbumins and oleic acid complexes are modulated by HPV E6, E7, or E6/E7 oncogenes warrants further exploration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C31.

CD4+ T-cell response against human papillomavirus type 16 E6 protein is associated with a favorable clinical trend

The association between the CD8+ T-cell responses to human papillomavirus type 16 (HPV-16) E6 protein and a favorable clinical trend has been demonstrated previously. The roles of human papillomavirus (HPV)-specific CD4+ T-cell responses and of regulatory T-cells (Tregs) were examined. Subjects with a recent history of abnormal Papanicolaou smear were eligible, and colposcopy-guided biopsy was performed at enrollment. Interferon-γ enzyme-linked immunospot assay and fluorescent-activated cell sorter analysis to measure the frequencies of Tregs were performed. Subjects with histological diagnoses of cervical intraepithelial neoplasia 1, 2, or 3 were considered to have short-term persistence of cervical abnormality and were called "persistors" (n=51) while those of normal histology were designated to be "regressors" (n=33). A significantly higher percentage CD4+ T-cell response was detected in the regressors (15/33 or 45.5%) compared with the persistors (10/51 or 19.6%) (P=.015) for the E6 peptides but not for the E7 peptides. The CD4+ responses to certain E6 regions [E6(16-40), E6(91-115), E6(106-130), and E6(136-158)] were also significantly higher in the regressors. Although there was no difference in the frequencies of Tregs between the two groups, low frequencies of Tregs were significantly associated with positive CD4+ T-cell responses within certain E6 regions [E6(16-40), E6(31-55), E6(76-100), E6(91-115), and E6(106-130)]. The CD4+ and CD8+ T-cell responses to the HPV-16 E6 protein are associated with a favorable clinical trend. The HPV-16 E6 protein should be incorporated in the design of an HPV therapeutic vaccine.

E6 and E7 from Human Papillomavirus Type 16 Cooperate To Target the PDZ Protein Na/H Exchange Regulatory Factor 1

Previous studies have shown that the PDZ-binding motif of the E6 oncoprotein from the mucosal high-risk (HR) human papillomavirus (HPV) types plays a key role in HPV-mediated cellular transformation in in vitro and in vivo experimental models. HR HPV E6 oncoproteins have the ability to efficiently degrade members of the PDZ motif-containing membrane-associated guanylate kinase (MAGUK) family; however, it is possible that other PDZ proteins are also targeted by E6. Here, we describe a novel interaction of HPV type 16 (HPV16) E6 with a PDZ protein, Na(+)/H(+) exchange regulatory factor 1 (NHERF-1), which is involved in a number of cellular processes, including signaling and transformation. HPV16 E6 associates with and promotes the degradation of NHERF-1, and this property is dependent on the C-terminal PDZ-binding motif of E6. Interestingly, HPV16 E7, via the activation of the cyclin-dependent kinase complexes, promoted the accumulation of a phosphorylated form of NHERF-1, which is preferentially targeted by E6. Thus, both oncoproteins appear to cooperate in targeting NHERF-1. Notably, HPV18 E6 is not able to induce NHERF-1 degradation, indicating that this property is not shared with E6 from all HR HPV types. Downregulation of NHERF-1 protein levels was also observed in HPV16-positive cervical cancer-derived cell lines, such as SiHa and CaSki, as well as HPV16-positive cervical intraepithelial neoplasia (CIN). Finally, our data show that HPV16-mediated NHERF-1 degradation correlates with the activation of the phosphatidylinositol-3'-OH kinase (PI3K)/AKT signaling pathway, which is known to play a key role in carcinogenesis.

Establishment of a humankeratinocyte cell line (HaCaT) stably expressing human papillomavirus type 16 E7 (HPV16E7)protein

：Objective To establish ahuman keratinocyte cell line (HaCaT) stably expressing HPV16E7 protein. Methods HPV16E7gene was amplified from CaSki cells using PCR and inserted into the eukaryotic expressionplasmid pcDNA3.1. Then, the recombinant expression plasmid pcDNA3.1-HPV16E7 wastransfected into HaCaT cells followed by G418 selection and identification by RT-PCR andWestern blot. Results The recombinant eukaryotic expression plasmid pcDNA3.1-HPV16E7 wassuccessfully identified by restriction enzyme digestion pattern and sequence analysis.Agarose gel electrophoresis of RT-PCR products detected the 297-bp fragment of HPV16E7cDNA, and Western blot confirmed the stable expression of HPV16E7 protein. Conclusion Ahuman keratinocyte cell line (HaCaT) stably expressing HPV16E7 protein is successfullyestablished.

Abstract 2100: Role of pocket proteins in HPV-associated head and neck squamous cell carcinomas

Abstract High-risk human papillomaviruses(HPVs) are associated with a subset of head and neck squamous cell carcinomas (HNSCC) as well as most of cervical cancers. HPV type 16 (HPV-16), which is found in the vast majority of HPV-positive HNSCC, encodes for three oncogenes: E5, E6, and E7. Previously, we learned that HPV-16 E7 is the dominant oncoprotein in head and neck cancer in mice. More recently, we learned that tissue specific inactivation of the retinoblastoma-susceptibility gene, pRb, a well known cellular target of HPV E7 and a member of the “pocket protein’ family, was not sufficient to account fully for the oncogenic properties of E7 in HNSCC. E7 is known to inactivate not just pRb, but also other related cellular proteins, specifically p107 and p130. This led to our current study in which we have observed that the combined loss of pRb and p130, or pRb and p107 leads to increased susceptibility to head and neck cancers in comparison to mice conditionally disrupted only for pRb, yet are not sufficient to account fully for E7's oncogenic potential in HPV-associated head and neck carcinogenesis. Additionally, combinatorial loss of pRb and p130, or pRb and p107 partially mediates E7-induced acute phenotypes in head and neck epithelia. Taken together, our results indicate that the combined inactivation of two pocket proteins are not sufficient to fully recapitulate E7 oncogenic properties in HNSCC. We conclude that inactivation of all three pocket proteins is necessary to account for E7's carcinogenic properties in HNSCC or that other targets of E7 contribute to its carcinogenic potential in HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2100. doi:10.1158/1538-7445.AM2011-2100

Apoptosis induced by an antagonist peptide against HPV16 E7 in vitro and in vivo via restoration of p53

Human papilloma virus type 16 (HPV16) E7 is a viral oncoprotein that is believed to play a major role in cervical neoplasia. A novel antagonist peptide against HPV16 E7 was previously selected by phage display screening and the selected peptide was found to have anti-tumor efficacy against HPV16-positive cervical carcinoma through induction of cell cycle arrest. In the current study, to further elucidate the mechanisms of the antagonist peptide, the effects of the peptide on apoptosis are investigated by RT-PCR, Western blotting, MTT assay, TUNEL staining, Annexin V apoptosis assay, flow cytometry, and animal experiments. The antagonist peptide showed obvious anti-tumor efficacy through apoptosis induction, both in HPV16-positive cervical cancer cell lines and tumor xenografts. Our results also revealed that the peptide induced accumulation of cellular p53 and p21, and led to HPV16 E7 protein degradation. In the case of mRNA levels, it resulted in unaltered p53 and HPV16 E7 expression, but increased expression of p21. In contrast, the induction of apoptosis and p53 reactivation effects by the selected peptide were abolished after E7 knocked down with siRNA. These results demonstrate that the selected peptide can induce E7 degradation and lead to marked apoptosis in HPV16-related cancer cells by activating cellular p53 and its target genes, such as p21. Furthermore, the evident therapeutic efficacy obtained from the subcutaneous tumor model experiments in nude mice suggests a therapeutic potential for HPV16-related cancers of the selected peptide. Therefore, this specific peptide may be used to create specific biotherapies for the treatment of HPV 16-positive cervical cancers.

Exogenous and endogenous virus interactions in head and neck cancer immunity (105.34)

Abstract We report that two common tumor viruses etiologically associated with head and neck cancer, Epstein-Barr virus (EBV) with undifferentiated nasopharyngeal carcinoma, and human papillomavirus (HPV) with oropharyngeal squamous cell carcinoma, transactivate an endogenous retrovirus (HERV-K18), which encodes a superantigen that activates TCRBV13 T cells. EBV LMP-2 transactivated the HERV-K18 superantigen in nasopharyngeal epithelial cells, and EBV-associated nasopharyngeal carcinomas expressed the superantigen. HPV type-16 E6 and E7 proteins also transactivated the HERV-K18 superantigen in oropharyngeal epithelial cells, and superantigen expression was significantly increased in HPV16-associated carcinomas. Serum cytokine levels in head and neck carcinoma patients indicated that there were distinct inflammatory profiles associated with exogenous and endogenous viruses. HPV-associated patients had increased levels of serum IL-8, while HPV-negative patients had higher serum IL-6 and G-CSF levels. Interestingly, HERV-K18 superantigen levels correlated independently with serum levels of the Th2 cytokine IL-13. Among HPV+ tumors, gene expression array analyses indicated that B cell specific genes were significantly upregulated in the superantigen high group. Our results suggest that endogenous superantigen expression in HPV+ tumors was associated with a Th2 immune response, demonstrating that chronic and endogenous viruses comprising the virome affect both host and tumor immunity.

Control of Cervicovaginal HPV-16 E7-Expressing Tumors by the Combination of Therapeutic HPV Vaccination and Vascular Disrupting Agents

Abstract Antigen-specific immunotherapy and vascular disrupting agents, such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA), have emerged as attractive approaches for the treatment of cancers. In the current study, we tested the combination of DMXAA treatment with therapeutic human papillomavirus type 16 (HPV-16) E7 peptide-based vaccination for their ability to generate E7-specific CD8+ T-cell immune responses, as well as their ability to control E7-expressing tumors in a subcutaneous and a cervicovaginal tumor model. We found that the combination of DMXAA treatment with E7 long peptide (amino acids 43-62) vaccination mixed with polyriboinosinic:polyribocytidylic generated significantly stronger E7-specific CD8+ T-cell immune responses and antitumor effects compared with treatment with DMXAA alone or HPV peptide vaccination alone in the subcutaneous model. Additionally, we found that the DMXAA-mediated enhancement of E7-specific CD8+ T-cell immune responses generated by the therapeutic HPV peptide-based vaccine was dependent on the timing of administration of DMXAA. Treatment with DMXAA in tumor-bearing mice was also shown to lead to increased dendritic cell maturation and increased production of inflammatory cytokines in the tumor. Furthermore, we observed that the combination of DMXAA with HPV-16 E7 peptide vaccination generated a significant enhancement in the antitumor effects in the cervicovaginal TC-1 tumor growth model, which closely resembles the tumor microenvironment of cervical cancer. Taken together, our data demonstrated that administration of the vascular disrupting agent, DMXAA, enhances therapeutic HPV vaccine-induced cytotoxic T-lymphocyte responses and antitumor effects against E7-expressing tumors in two different locations. Our study has significant implications for future clinical translation.

Isolation and Characterization of an RNA Aptamer for the HPV-16 E7 Oncoprotein

Cervical cancer is a common neoplastic disease affecting women worldwide. Expression of human papillomavirus type 16 (HPV-16) E6/E7 genes is frequently associated with cervical cancer, representing ideal targets for diagnostic and therapeutic strategies. Aptamers are oligonucleotide ligands capable of binding with high affinity and specificity to relevant markers in therapeutics and disease detection. The aim of the study was to isolate an RNA aptamer specific for the HPV-16 E7 protein. Aptamers were selected from a randomized oligonucleotide library using a modified SELEX method and recombinant HPV-16 E7 protein. Isolated aptamers were cloned and sequenced for in silico analysis. Interaction and electromobility shift assays (EMSA) were performed to establish aptamer specificity and affinity for E7. RNase footprinting and serial deletions of the aptamer and the E7 protein were made to characterize the aptamer-protein complex. Sandwich slot-blot assays were used for K(D) determination. After several rounds of SELEX, an aptamer (G5α3N.4) exhibited specificity for E7 using cell-free and protein extracts. G5α3N.4 binding yielded a K(D) comparable to aptamers directed to other small targets. Enzymatic and genetic analysis of G5α3N.4 binding showed a secondary structure with two stem-loop domains joined by single-stranded region contacting E7 in a clamp-like manner. The G5α3N.4 aptamer also produced specific complexes in HPV-positive cervical carcinoma cells. The affinity and specificity of G5α3N.4 binding domains for the HPV-16 E7 protein may be used for the detection of papillomavirus infection and cervical cancer.

Immortalization of oral keratinocytes by functional inactivation of the p53 and pRb pathways

A subgroup of head and neck squamous cell carcinomas (HNSCCs) contains high-risk human papillomavirus-type 16 (HPV16). The viral E6 and E7 oncoproteins inactivate the p53 and pRb proteins, respectively. We examined the causative effect of HPV16 E6 and E7 expression on the immortalization of normal oral keratinocytes (OKCs) and compared the resulting phenotype with alternative ways of p53- and pRb-pathway abrogation frequently found in HNSCCs without HPV. Primary OKCs were conditionally immortalized with temperature-sensitive SV40 large T-antigen and human telomerase, allowing these cells to return to their senescent primary state after temperature shift. HPV16 E6 and E7 were introduced to overcome senescence, determined with population doubling (PD) as read-out. For comparison, we downregulated p53 and p16 by short hairpin RNA genes and expressed mutant p53R(175)H and cyclinD1. Expression of HPV16 E6 caused an extended life span similar to expression of mutant p53R(175)H or p53 knockdown. Expression of mutant p53R(175)H seemed to cause additional activation of the hypoxia and WNT signaling pathways. HPV16 E7 expression had no direct effect on lifespan, similar to p16 knockdown or cyclinD1 expression. In combination with HPV16 E6 or other functional inactivations of p53, abrogation of the pRb-pathway by either HPV16 E7 or other manipulations caused an immortal phenotype. Our data show the causative role of HPV16 E6/E7 in early squamous carcinogenesis. Activity of each gene could be mimicked by other genetic events frequently found in HNSCC without HPV. This data provides the experimental proof of causal association of HPV in HNSCC carcinogenesis and further support the crucial role of the p53- and pRb-pathways.

Regulation of the human papillomavirus type 18 E6/E6AP ubiquitin ligase complex by the HECT domain-containing protein EDD.

Human papillomavirus (HPV) E6 oncoproteins target many cellular proteins for ubiquitin-mediated proteasomal degradation. In the case of p53, this is mediated principally by the E6AP ubiquitin ligase. Several studies have reported that E6 can target certain of its substrates in an apparently E6AP-independent fashion and that several of these substrates vary in the degree to which they are degraded by E6 at different stages of malignancy. To more fully understand the regulation of the E6AP/E6 proteolytic targeting complex, we performed a mass spectroscopic analysis of HPV type 18 (HPV-18) E6 protein complexes and identified the HECT domain-containing ubiquitin ligase EDD as a new HPV-18 E6 binding partner. We show that EDD can interact independently with both E6 and E6AP. Furthermore, EDD appears to regulate E6AP expression levels independently of E6, and loss of EDD stimulates the proteolytic activity of the E6/E6AP complex. Conversely, higher levels of EDD expression protect a number of substrates from E6-induced degradation, partly as a consequence of lower levels of E6 and E6AP expression. Intriguingly, reduction in EDD expression levels in HPV-18-positive HeLa cells enhances cell resistance to apoptotic and growth arrest stimuli. These studies suggest that changes in the levels of EDD expression during different stages of the viral life cycle or during malignancy could have a profound effect upon the ability of E6 to target various substrates for proteolytic degradation and thereby directly influence the development of HPV-induced malignancy.

Abstract CN01-03: Immunotherapy of established lesions caused by high-risk HPV

Abstract Therapeutic vaccination with a synthetic long peptide (SLP@) vaccine mediated the eradication of established human papillomavirus type 16 (HPV16)-positive tumors in mice and controlled wart growth and latent virus infection in rabbits persistently infected with cottontail rabbit papillomavirus. Subsequent phase I/II studies with an HPV16 SLP@ vaccine, consisting of 13 long peptides covering the HPV16 E6 and E7 antigens, in patients with advanced HPV16-positive cervical cancer, revealed that this vaccine was safe and highly immunogenic. The purpose of the current study was to test the clinical efficacy of this HPV16 SLP@ vaccine in HPV16-induced high-grade vulvar intraepithelial neoplasia (VIN3), a premalignant epithelial disorder, spontaneous regression of which occurs in less than 2% of patients and in which recurrence after standard treatment is high. In a phase 2 trial, 20 women with VIN3 were vaccinated three times sc in the limbs with a mix of the HPV16 E6 and E7 synthetic long peptides formulated in Montanide ISA-51. The endpoints were objective clinical responses, defined as reduction of at least 50% in lesion size (partial response) or complete regressions, and HPV16-specific T-cell responses, determined before and after vaccination. The vaccine was safe, as no side effects exceeding CTC grade 2 were observed. At 3 and 12 months after the last vaccination an objective response was observed in 12/20 (60%) and 15/19 (79%) patients respectively. Nine of them showed a complete and durable regression of the lesions at 12 months and at 24 months. The strength of the vaccine-induced HPV16-specific T-cell response was significantly higher in the group of patients with a complete regression of their lesions as compared to nonresponders. This study shows that in women with VIN3 objective clinical responses can be achieved by therapeutic vaccination with synthetic long peptides that is able to induce effective HVP16-specific T-cell responses. Literature: 1. Kenter GG, Welters MJ, Valentijn AR, Lowik MJ, Berends-van der Meer DM, Vloon AP, Essahsah F, Fathers LM, Drijfhout JW, Offringa R,Wafelman AR, Oostendorp J, Fleuren GJ, Burg van der SH, Melief CJ. Vaccination against Human Papillomavirus 16 oncoproteins for vulvar intraepithelial neoplasia. N Engl J Med. 2009 Nov 5;361(19):1838-47. 2. Melief CJ, van der Burg SH. Immunotherapy of established (pre)malignant disease by synthetic long peptide vaccines. Nat Rev Cancer. 2008 May;8(5):351-60. Citation Information: Cancer Prev Res 2010;3(12 Suppl):CN01-03.

A Systematic Analysis of Human Papillomavirus (HPV) E6 PDZ Substrates Identifies MAGI-1 as a Major Target of HPV Type 16 (HPV-16) and HPV-18 Whose Loss Accompanies Disruption of Tight Junctions

The E6 proteins from high-risk, cancer-causing types of human papillomavirus (HPV) are characterized by the presence of a PDZ (PSD95/Dlg/ZO-1) binding motif in their extreme carboxy termini, through which they interact with a number of cellular PDZ domain-containing substrates. In order to ascertain how many of these are degraded by E6 in vivo, we performed an extensive analysis of the effects of E6 ablation on the expression levels of a number of previously reported E6 PDZ substrates. Using HPV type 16 (HPV-16)-positive CaSKi cells and HPV-18-positive HeLa cells, we have found that MAGI-1 is a major degradation target of both HPV-16 and HPV-18 E6. In contrast, hDlg, hScrib, PTPN3, TIP2, FAP1, and PSD95 all exhibit various degrees of susceptibility to E6-induced degradation, and a high degree of HPV type specificity is observed for certain substrates. We also show that E6 preferentially targets MAGI-1 within the nucleus and at membrane sites. One of the direct consequences of MAGI-1 degradation is a loss of tight-junction integrity, as determined by mislocalization of the tight-junction protein ZO-1. Ablation of E6 expression restores tight junctions, and this restoration is dependent on the presence of MAGI-1. These results demonstrate that oncogenic HPV E6 proteins disrupt cellular tight junctions through the degradation of MAGI-1, and they provide further evidence of how the PDZ binding potential of E6 can contribute to HPV-induced malignancy.

Computer-aided analysis of structural properties and epitopes of Iranian HPV-16 E7 oncoprotein

Infection by human papillomavirus type 16 (HPV-16) is the cause of 50% or more of cervical cancers in women. The E7 oncoprotein of HPV-16 has long been known as a potent immortalizing and transforming agent. We used different servers like PseAAC, MHC_binding, MHC_II_binding and Expasy for the present computational prediction. The results for T cell epitopes showed that B1501, A0203, A0201, A0202, A6801 and DRB0405 alleles had lower IC50 than other alleles. We also predicted several peptides with the best binding affinities for alleles of the most frequent MHC class I and II alleles of the various ethnic groups living in the different region of Iran. Two peptides (26-35) and (44-52) were predicted as B-cell epitopes. According to this analysis 1 N-glycosylation site, 2 PKC sites, 4 CK2 sites and 3 disulfide sites were predicted. Our computational study predicted that B cell epitope 1 was Casein kinase II phosphorylated (site No. 31) and glycosylated (site No. 29). Putative MHC-I epitopes 3 and 5 and MHC-II epitopes 19, 21 and 26 were predicted to be casein kinase II phosphorylated. MHC-II epitopes 19 and 21 was predicted to be glycosylated. T cell epitopes 1, 13, 16 and 24 were demonstrated to be kinase C phosphorylated. The result of this analysis for Iranian HPV-16 E7 also indicated that 21.43%, 18.37% and 60.20% of the protein were in the α-helix, extended strand and random coil respectively.

Cytoplasmic Poly(A) Binding Proteins Regulate Telomerase Activity and Cell Growth in Human Papillomavirus Type 16 E6-Expressing Keratinocytes

The high-risk human papillomavirus (HPV) E6 and E7 oncoproteins are critical to the immortalization of keratinocytes. HPV type 16 (HPV16) E6 interacts with endogenous proteins to activate hTERT, the catalytic subunit of telomerase, thus avoiding cellular senescence signals. NFX1-123, the longer splice variant of NFX1, interacts with HPV16 E6, as well as cytoplasmic poly(A) binding proteins 1 and 4 (PABPC1 and PABPC4). HPV16 E6 affects hTERT expression posttranscriptionally through NFX1-123, as NFX1-123 interacts with hTERT mRNA and stabilizes it, leading to greater telomerase activity. The PAM2 motif of NFX1-123, with which it binds PABPCs, is required for the posttranscriptional regulation of hTERT by HPV16 E6 and NFX1-123. There is increasing evidence that RNA and DNA viruses utilize RNA-processing proteins, and specifically PABPCs, in the normal virus life cycle, and there is also evidence that RNA-processing proteins are perturbed in cancers. Here, we show that PABPCs are critical in hTERT regulation by HPV16 E6. Although the amount and cellular localization of PABPCs were largely unchanged in cervical cancer cell lines with or without HPV16 and in human foreskin keratinocytes (HFKs) with or without HPV16 E6, knockdown of PABPCs decreased hTERT mRNA and telomerase activity and overexpression of PABPC4 increased these in HPV16 E6-expressing HFKs. In contrast, knockdown of PABPCs in C33A cells had no effect on hTERT mRNA or telomerase activity. Additionally, overexpression of PABPC4 and hTERT led to greater growth of cultured HPV16 E6-expressing HFKs. This is the first evidence that PABPCs have a targeted role in hTERT regulation leading to a growth advantage in cells expressing HPV16 E6.

Variations of N-acetylation level of peptidoglycan do not influence persistence of Lactococcus lactis in the gastrointestinal tract

The food-grade Gram-positive bacterium, Lactococcus lactis, is recognized as a potential candidate to deliver proteins of medical interest by mucosal routes. The ability of carrier bacteria to persist and/or to lyse in the gastrointestinal tract needs to be considered to design optimal carrier strains to deliver proteins of interest at the mucosal level. Meyrand et al. (2007) have previously characterized in L. lactis, a peptidoglycan (PG) N-acetylglucosamine deacetylase (PgdA), which activity on PG influences bacterial sensitivity to lysozyme. Inactivation of pgdA gene in this bacterium, led to fully acetylated PG, resulting in a lysozyme-sensitive phenotype, whereas pgdA overexpression led to an increased degree of PG deacetylation, resulting in a lysozyme-resistant phenotype (Meyrand et al., 2007). In order to determine whether variations in L. lactis resistance to host lysozyme may influence its persistence in the GIT and its ability to deliver heterologous proteins in situ, we constructed L. lactis strains with different de-N-acetylation levels and producing a model antigen (the human papillomavirus type-16 E7 protein) and we compared the pharmacokinetics properties of these recombinant strains with that of a wild-type strain producing the same antigen in the GIT of mice. Our results show that there was no correlation between survival, at the ileum level, of bacteria intragastrically administered in mice and bacteria sensitivity or resistance to lysozyme. In addition, analysis of the E7-specific immune response evoked by the three strains after mucosal administration in mice suggest that neither lysozyme-sensitive nor lysozyme-resistant phenotype in L. lactis enhances significantly the potential of this bacterium as mucosal delivery live vector. In conclusion, our results suggest that either pgdA inactivation or pgdA overexpression in L. lactis leading to different levels of PG deacetylation does not confer any advantage in the persistence of this bacterium in the GIT and its ability to enhance host immune responses induced by delivered antigen in situ.