Human Papillomavirus Type 16 E7 Research Articles

DNA Vaccine Encoding HPV16 Oncogenes E6 and E7 Induces Potent Cell-mediated and Humoral Immunity Which Protects in Tumor Challenge and Drives E7-expressing Skin Graft Rejection.

We have previously shown that a novel DNA vaccine technology of codon optimization and the addition of ubiquitin sequences enhanced immunogenicity of a herpes simplex virus 2 polynucleotide vaccine in mice, and induced cell-mediated immunity when administered in humans at relatively low doses of naked DNA. We here show that a new polynucleotide vaccine using the same technology and encoding a fusion protein of the E6 and E7 oncogenes of high-risk human papillomavirus type 16 (HPV16) is immunogenic in mice. This vaccine induces long-lasting humoral and cell-mediated immunity and protects mice from establishment of HPV16-E7-expressing tumors. In addition, it suppresses growth of readily established tumors and shows enhanced efficacy when combined with immune checkpoint blockade targeted at PD-L1. This vaccine also facilitates rejection of HPV16-E7-expressing skin grafts that demonstrate epidermal hyperplasia with characteristics of cervical and vulvar intraepithelial neoplasia. Clinical studies evaluating the efficacy of this vaccine in patients with HPV16 premalignancies are planned.

Human papillomavirus type 16 E6 suppresses microRNA-23b expression in human cervical cancer cells through DNA methylation of the host gene C9orf3.

Oncogenic protein E6 of human papillomavirus type 16 (HPV-16) is believed to involve in the aberrant methylation in cervical cancer as it upregulates DNA methyltransferase 1 (DNMT1) through tumor suppressor p53. In addition, DNA demethylating agent induces the expression of one of the HPV-16 E6 regulated microRNAs (miRs), miR-23b, in human cervical carcinoma SiHa cells. Thus, the importance of DNA methylation and miR-23b in HPV-16 E6 associated cervical cancer development is investigated. In the present study, however, it is found that miR-23b is not embedded in any typical CpG island. Nevertheless, a functional CpG island is predicted in the promoter region of C9orf3, the host gene of miR-23b, and is validated by methylation-specific PCR and bisulfite genomic sequencing analyses. Besides, c-MET is confirmed to be a target gene of miR-23b. Silencing of HPV-16 E6 is found to increase the expression of miR-23b, decrease the expression of c-MET and thus induce the apoptosis of SiHa cells through the c-MET downstream signaling pathway. Taken together, the tumor suppressive miR-23b is epigenetically inactivated through its host gene C9orf3 and this is probably a critical pathway during HPV-16 E6 associated cervical cancer development.

Identification of the impact on T- and B- cell epitopes of human papillomavirus type-16 E6 and E7 variant in Southwest China

Cervical cancers almost are infected by human papillmavirus (HPV), encoding E6 and E7 oncoproteins which are regard as ideal targets on the mechanism of this disease and development of vaccines. HLA (human leukocyte antigen) participates in the local immune response to prevent tumor invasion and progression. But due to highly polymorphism of HLA, prediction shows its importance in this study. More effective immunoinformatics was used for predicting epitopes from HPV-16 E6 and E7, including T- and B-cell epitopes. Eight substitutions are detected. Specifically speaking, for HLA-I, HLA-A*33:03 (26), HLA-B*13:01 (14), HLA-C*03:02 (5) for E6 and HLA-A*02:01 (6), HLA-B*40:01 (5), HLA-C*03:04 (4) for E7 are most frequency. Epitope 41-48EVYDFAFR for HLA-A*33:03 (0.1) for E6 has best binding affinity, as well as HLA*02:01 and HLA-B*40:01 (0.2) for E7. The mutations of D25E and L83V of E6 and N29S of E7 produce new epitopes, and the percentile values change with them. For HLA-II, seventeen epitopes in the reference at percentile value from 0.22 to 4.76, while in variant from 0.22 to 4.96. For the B-cell epitopes, three most potent epitopes for E6 were listed, and N29S lead the growth of score from 0.81 to 0.83. In summary, E640-55REVYDFAFRDLCIVYR and E711-22YMLDLQPETTDL are the important regions, containing the majority of predicted epitopes. E6 72-83 for HLA-A*02:01 and E6 74-84 for HLA-B*15:02 maybe are the new direct for therapeutic vaccine aimed at L83V variants. HLA-DRB1*15:02 is better binder with T cell in our HLA class II.It is a systematic, detail recognition for T- and B-cell epitopes of HPV-16 E6 and E7 from Southwest China, which may be helpful to design vaccines specifically for women in Southwest China and testing methods specifically for this region. The results of our study may contribute to future researches on vaccines improvement, or screening methods for a particular population.

Abstract 5035A: Intratumoral HPV16-specific T-cells determine clinical outcome of HPV16+ oropharyngeal carcinomas

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer. While the number of HNSCC cases is decreasing, the number of oropharyngeal SCC (OSCC) is rising, especially in young adults. OSCC are increasingly (45-90%) caused by human papillomavirus type 16 (HPV16). Intriguingly, patients with HPV-induced OSCC respond better to therapy than HPV-negative OSCC. This is independent of nodal status, age, stage, tumor differentiation or gender. We hypothesized that the expression of the viral antigens E6 and E7 in HPV16+ OSCC equips the immune system with two strong tumor-specific antigens resulting in the development of a type 1 T-cell immune contexture supporting the better response of HPV+ OSCC to standard therapy. To test our hypothesis we studied 87 patients with OSCC. The tumors of these patients were tested for the presence of HPV by GP5+/6+ PCR and the expression of p16 by immunohistochemistry. Moreover, fresh tumor tissue was dispersed, cryopreserved and subsequently used for in-depth analyses regarding the composition of the leukocyte infiltrates using a large set of antibodies either by CyTOF or BD Fortessa. In addition, T-cell reactivity was tested directly ex vivo and after a short-term culture period. 57% of OSCC patients were HPV16+ and they showed the best overall survival (p<0.0001, HR 6.9). Whereas none of 37 HPV-negative patients displayed intratumoral HPV16 E6/E7-specific T-cell reactivity this was detected in 25 of 40 (63%) HPV16+ OSCC. Notably, for most patients these T-cells were already detectable directly ex vivo, indicative for their activity in vivo. The presence of intratumoral HPV16-specific T-cell reactivity for the response of patients to therapy was extremely important for a better clinical outcome (p = 0.0003, HR 23.9) as estimated by Kaplan-Meier curves and log-rank analysis. Division of HPV16+ patients on the basis of p16 expression revealed that p16+ patients had a much better clinical outcome (p<0.0001, HR 11.8). In line with the notion that overexpression of p16 is directly associated with expression of the HPV16 E6 and E7 oncogenes, a strong association between p16 expression and HPV16-specific TIL reactivity was found (p<0.0001). Mechanistically, we found that OSCC cell lines promoted the differentiation of monocytes to M2 macrophages. In vitro experiments showed that the presence of HPV-specific T-cell secreted cytokines prevented this and was even able to redifferentiate macrophages into better antigen-presenting cells, both phenotypically (including co-stimulatory molecules) and functionally (more IL-12 and less IL-10 production). In addition, these cytokines mediated a direct effect on the tumor cell proliferation and metabolism, thereby slowing down tumor growth as well as changing their impact on myeloid cells in the tumor microenvironment. In conclusion, HPV-specific T cells elicited by HPV+ OSCC are highly predictive for response to therapy. Citation Format: Marij J. Welters, Lilly-Ann van der Velden, Vanessa J. van Ham, Ilina Ehsan, Renske Goedemans, Saskia J. Santegoets, Sjoerd H. van der Burg. Intratumoral HPV16-specific T-cells determine clinical outcome of HPV16+ oropharyngeal carcinomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5035A.

Analysis of ROC: The value of HPV16 E6 protein in the diagnosis of early stage cervical carcinoma and precancerous lesions.

Cervical carcinoma is a multifactorial malignant tumor and diagnosis is therefore crucial. The aim of the present study was to examine the value of E6 oncoprotein, in human papillomavirus type 16 (HPV16), in the diagnosis of early stage cervical carcinoma and precancerous lesions. Receiver operating characteristic curve was used to analyze accuracy of diagnosis. A total of 124 patients infected with HPV16 were included in the study. The patients had an average age of 46.7±6.9 years and duration of disease of 10.5±3.4 months. To determine the expression level of HPV16 E6 the immunohistochemical Elivision method was performed. Proportion/horizon positive cells were used to count the cells, and pathologic diagnosis was employed for analysis of the results. The average follow-up time was 2.6±0.7 years. Sensitivity and specificity of diagnosing HPV16 E16 at 1 and 2 years, respectively, were calculated. The diagnostic rate of cervical carcinoma increased with time, and the positive expression of HPV16 E6 was also increased with the development of the disease. Differences among groups were statistically significant (P<0.05). Sensitivity, specificity and accuracy (AUC) of HPV16 E6 diagnosis improved with time, and the differences were statistically significant (P<0.05). Thus, HPV16 E6 oncoprotein can be used as an indicator with good sensitivity and specificity to diagnose early cervical carcinoma and precancerous lesions. The results therefore showed that accuracy increased with the development of the disease.

Construction and characterization of an attenuated recombinant Listeria monocytogenes vector vaccine delivering HPV16 E7

Listeria monocytogenes (L. monocytogenes, LM) is an excellent tumor vaccine vector. In this study, recombinant LM vaccine candidate expressing human papillomavirus type 16 (HPV16) E7 protein was constructed and its charactericts were determined. Through homologous recombination, E7 gene was cloned in frame with the LM4 Phly promoter-signal sequence, and introduced into the chromosome of LM4. The recombinant strain named LM4△hly::E7 with the plasmid-free and antibiotic-resistant gene-free was constructed. LM4△hly::E7 could express and secrete E7-LLO fusion protein; its size is 66 kDa and has immunological activity. Furthermore, LM4△hly::E7 could multiply in RAW264.7 macrophages by confocal laser scanning microscope. Additionally, LM4△hly::E7 could induce specific antibodies against E7 in immunized mice in ELISA. Also, the 50% lethal dose (LD₅₀) of LM4△hly::E7 strain was 3.863×10⁹ CFU (Colony-Forming Units) in C57BL/6 mice with intraperitoneal immunization, which was more attenuated than wild type LM4. Mice immunized with LM4△hly::E7 did not show obvious pathological change. These data show that LM4△hly::E7 expressing E7-LLO fusion protein has good safety, which may provide the materials for research of antitumor effect and would be a promising vaccine candidate for cervical cancer.

Human papillomavirus oncogenic E6 protein regulates human β-defensin 3 (hBD3) expression via the tumor suppressor protein p53.

Human β-defensin-3 (hBD3) is an epithelial cell-derived innate immune regulatory molecule overexpressed in oral dysplastic lesions and fosters a tumor-promoting microenvironment. Expression of hBD3 is induced by the epidermal growth factor receptor signaling pathway. Here we describe a novel pathway through which the high-risk human papillomavirus type-16 (HPV-16) oncoprotein E6 induces hBD3 expression in mucosal keratinocytes. Ablation of E6 by siRNA induces the tumor suppressor p53 and diminishes hBD3 in HPV-16 positive CaSki cervical cancer cells and UM-SCC-104 head and neck cancer cells. Malignant cells in HPV-16-associated oropharyngeal cancer overexpress hBD3. HPV-16 E6 induces hBD3 mRNA expression, peptide production and gene promoter activity in mucosal keratinocytes. Reduction of cellular levels of p53 stimulates hBD3 expression, while activation of p53 by doxorubicin inhibits its expression in primary oral keratinocytes and CaSki cells, suggesting that p53 represses hBD3 expression. A p53 binding site in the hBD3 gene promoter has been identified by using electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP). In addition, the p63 protein isoform ΔNp63α, but not TAp63, stimulated transactivation of the hBD3 gene and was co-expressed with hBD3 in head and neck cancer specimens. Therefore, high-risk HPV E6 oncoproteins may stimulate hBD3 expression in tumor cells to facilitate tumorigenesis of HPV-associated head and neck cancer.

HPV16 E6 Controls the Gap Junction Protein Cx43 in Cervical Tumour Cells.

Human papillomavirus type 16 (HPV16) causes a range of cancers including cervical and head and neck cancers. HPV E6 oncoprotein binds the cell polarity regulator hDlg (human homologue of Drosophila Discs Large). Previously we showed in vitro, and now in vivo, that hDlg also binds Connexin 43 (Cx43), a major component of gap junctions that mediate intercellular transfer of small molecules. In HPV16-positive non-tumour cervical epithelial cells (W12G) Cx43 localised to the plasma membrane, while in W12T tumour cells derived from these, it relocated with hDlg into the cytoplasm. We now provide evidence that E6 regulates this cytoplasmic pool of Cx43. E6 siRNA depletion in W12T cells resulted in restoration of Cx43 and hDlg trafficking to the cell membrane. In C33a HPV-negative cervical tumour cells expressing HPV16 or 18 E6, Cx43 was located primarily in the cytoplasm, but mutation of the 18E6 C-terminal hDlg binding motif resulted in redistribution of Cx43 to the membrane. The data indicate for the first time that increased cytoplasmic E6 levels associated with malignant progression alter Cx43 trafficking and recycling to the membrane and the E6/hDlg interaction may be involved. This suggests a novel E6-associated mechanism for changes in Cx43 trafficking in cervical tumour cells.

Replication-Competent Foamy Virus Vaccine Vectors as Novel Epitope Scaffolds for Immunotherapy.

The use of whole viruses as antigen scaffolds is a recent development in vaccination that improves immunogenicity without the need for additional adjuvants. Previous studies highlighted the potential of foamy viruses (FVs) in prophylactic vaccination and gene therapy. Replication-competent FVs can trigger immune signaling and integrate into the host genome, resulting in persistent antigen expression and a robust immune response. Here, we explored feline foamy virus (FFV) proteins as scaffolds for therapeutic B and T cell epitope delivery in vitro. Infection- and cancer-related B and T cell epitopes were grafted into FFV Gag, Env, or Bet by residue replacement, either at sites of high local sequence homology between the epitope and the host protein or in regions known to tolerate sequence alterations. Modified proviruses were evaluated in vitro for protein steady state levels, particle release, and virus titer in permissive cells. Modification of Gag and Env was mostly detrimental to their function. As anticipated, modification of Bet had no impact on virion release and affected virus titers of only some recombinants. Further evaluation of Bet as an epitope carrier was performed using T cell epitopes from the model antigen chicken ovalbumin (OVA), human tyrosinase-related protein 2 (TRP-2), and oncoprotein E7 of human papillomavirus type 16 (HPV16E7). Transfection of murine cells with constructs encoding Bet-epitope chimeric proteins led to efficient MHC-I-restricted epitope presentation as confirmed by interferon-gamma enzyme-linked immunospot assays using epitope-specific cytotoxic T lymphocyte (CTL) lines. FFV infection-mediated transduction of cells with epitope-carrying Bet also induced T-cell responses, albeit with reduced efficacy, in a process independent from the presence of free peptides. We show that primate FV Bet is also a promising T cell epitope carrier for clinical translation. The data demonstrate the utility of replication-competent and -attenuated FVs as antigen carriers in immunotherapy.

Human papillomavirus type 16 E7 oncoprotein upregulates the retinoic acid receptor-beta expression in cervical cancer cell lines and K14E7 transgenic mice.

Persistent infection with high-risk human papillomaviruses is the main etiological factor in cervical cancer (CC). The human papillomavirus type 16 (HPV16) E7 oncoprotein alters several cellular processes, regulating the expression of many genes in order to avoid cell cycle control. Retinoic acid receptor beta (RARB) blocks cell growth, inducing differentiation and apoptosis. This tumor suppressor gene is gradually silenced in late passages of foreskin keratinocytes immortalized with HPV16 and in various tumors, including CC, mainly by epigenetic modifications. We investigated the effect of E7 oncoprotein on RARB gene expression. We found that HPV16 E7 increases RARB mRNA and RAR-beta protein expression both in vitro and in the cervix of young K14E7 transgenic mice. In E7-expressing cells, RARB overexpression is further increased in the presence of the tumor suppressor p53 (TP53) R273C mutant. This effect does not change when either C33-A or E7-expressing C33-A cell line is treated with Trichostatin A, suggesting that E7 enhances RARB expression independently of histone deacetylases inhibition. These findings indicate that RARB overexpression is part of the early molecular events induced by the E7 oncoprotein.

Viral load and mRNA expression of HPV type 6 among cases with recurrent respiratory papillomatosis.

To determine viral load of human papillomavirus type 6 (HPV6), physical state of HPV6-DNA, and transcription level of HPV6 E7-mRNA in laryngeal papilloma and in adjacent healthy mucosa. Case series. A papilloma biopsy was collected from each of 25 adult patients with respiratory recurrent papillomatosis. From 14 of the 25 patients, we first collected a biopsy from healthy mucosa of the false vocal fold and another from the papilloma. Quantity of HPV6 and E7-mRNA was measured by polymerase chain reaction. For the papilloma, the median load of HPV6 was 41 copies/cell, and the lowest amount was 5.4 copies/cell. Human papillomavirus type 6 was detected in 50% (7/14) of the healthy mucosa, with a median of 1.1 copies/cell, and the highest amount was 6.6 copies/cell. Overall, viral load was higher in papilloma than in healthy mucosa (P < 0.05). The average HPV6 E2/E7-DNA ratio was 1.3, indicating an episomal state. Human papillomavirus type 6-mRNA was detected in all HPV6-DNA-positive samples. The transcription median ratio of HPV6-mRNA/HPV6-DNA was 1.5 in papilloma and 3.8 in healthy mucosa. The amount of HPV6-DNA was consistently higher in the papilloma than in healthy mucosa. The transcription level of HPV6 E7 mRNA was similar in the papilloma and in normal mucosa. We suggest that interfering with replication of HPV6 and suppression of HPV6 to fewer than five copies/cell may be curative.

Physical Status and Variant Analysis of Human Papillomavirus 16 in Women from Shanghai

Background/Aims: Human papillomavirus type 16 (HPV16) is the cause of more than half of all cases of cervical cancer. Genetic mutations in HPV16 and the integration of HPV16 DNA in the human genome are considered important genetic changes in cervical lesion progression. However, limited data concerning HPV16 lineages and physical integration status have been reported for Shanghai, China. The current study analyzed the genetic mutations in complete HPV16 genomes and the physical integration status of HPV16 DNA. Methods: A total of 30 samples of cervical exfoliated cells from patients with HPV16 infection were collected. The entire HPV16 genome was isolated, amplified by PCR and directly sequenced. The physical integration status was determined by 3′RACE nested PCR. Results: A total of 13 integration sites were identified, including 9 in common fragile sites and 1 not close to any fragile sites. Phylogenetic analysis identified two HPV lineages: the European (E) lineage and the East Asian (EA) lineage. Amino acid changes of D25E and N29S were the most common variations across the genome. The HPV16 early genes E1 and E7 and the late gene L1 tended to be highly conserved, whereas the early genes E2, E4 and E6 were more variable. Furthermore, 10 novel variations were identified in this study, which led to the 3 amino acid changes of S23I in E2 and E244K and T269I in E2/E4. Conclusion: Integrated HPV16 viruses were detected in all stages of cervical samples. Many variants in E2, E4, E7, and the long control region co-varied with E6 variations and helped to define the HPV16 lineages.

Tobacco smoke activates human papillomavirus 16 p97 promoter and cooperates with high-risk E6/E7 for oxidative DNA damage in lung cells.

We have previously shown a functional interaction between human papillomavirus type 16 (HPV-16) E6 and E7 oncoproteins and cigarette smoke condensate (CSC) in lung cells suggesting cooperation during carcinogenesis. The molecular mechanisms of such interaction, however, remain to be elucidated. Here we first present evidence showing that cigarette smoke condensate (CSC) has the ability to activate the HPV-16 p97 promoter by acting on the long control region (LCR) in lung epithelial cells. Interestingly, we observed that CSC-induced p97 promoter activation occurs in a dose-dependent manner in both tumor A-549 (lung adenocarcinoma), H-2170 (bronchial carcinoma), SiHa or Hela (cervical carcinoma) cells but not in non-tumor BEAS-2B (bronchial) or NL-20 (alveolar) lung cells unless they ectopically expressed the HPV-16 E6 and E7 oncogenes. In addition, we also observed a significant increase of primary DNA damage in tumor and non-tumor CSC-treated lung cells expressing HPV-16 E6 and E7 oncogenes suggesting a cooperative effect in this process, even though the contribution of E7 was significantly higher. Taken together, our results strongly suggest that tobacco smoke is able to induce the activation of the HPV-16 p97 promoter in cooperation with HPV-16 E6 and E7 oncogenes that, in turn, sensitize lung cells to tobacco smoke-induced DNA damage.

Human Papillomavirus 16 E6 Antibodies in Individuals without Diagnosed Cancer: A Pooled Analysis.

The increasing incidence of oropharyngeal cancer in many developed countries has been attributed to human papillomavirus type 16 (HPV16) infections. Recently, HPV16 E6 serology has been identified as a promising early marker for oropharyngeal cancer. Therefore, characterization of HPV16 E6 seropositivity among individuals without cancer is warranted. A total of 4,666 controls were pooled from several studies of cancer and HPV seropositivity, all tested within the same laboratory. HPV16 E6 seropositive controls were classified as having (i) moderate [mean fluorescent intensity (MFI) ≥ 484 and <1,000] or (ii) high seroreactivity (MFI ≥ 1,000). Associations of moderate and high HPV16 E6 seroreactivity with (i) demographic risk factors; and seropositivity for (ii) other HPV16 proteins (E1, E2, E4, E7, and L1), and (iii) E6 proteins from non-HPV16 types (HPV6, 11, 18, 31, 33, 45, and 52) were evaluated. Thirty-two (0.7%) HPV16 E6 seropositive controls were identified; 17 (0.4%) with moderate and 15 (0.3%) with high seroreactivity. High HPV16 E6 seroreactivity was associated with former smoking [odds ratio (OR), 5.5; 95% confidence interval (CI), 1.2-51.8], and seropositivity against HPV16 L1 (OR, 4.8; 95% CI, 1.3-15.4); E2 (OR, 7.7; 95% CI, 1.4-29.1); multiple HPV16 proteins (OR, 25.3; 95% CI, 2.6-119.6 for three HPV16 proteins beside E6) and HPV33 E6 (OR, 17.7; 95% CI, 1.9-81.8). No associations were observed with moderate HPV16 E6 seroreactivity. High HPV16 E6 seroreactivity is rare among individuals without diagnosed cancer and was not explained by demographic factors. Some HPV16 E6 seropositive individuals without diagnosed HPV-driven cancer, especially those with seropositivity against other HPV16 proteins, may harbor a biologically relevant HPV16 infection.

Human papillomavirus antibodies and future risk of anogenital cancer: a nested case-control study in the European prospective investigation into cancer and nutrition study.

Human papillomavirus (HPV) type 16 (HPV16) causes cancer at several anatomic sites. In the European Prospective Investigation Into Cancer and Nutrition study, HPV16 E6 seropositivity was present more than 10 years before oropharyngeal cancer diagnosis and was nearly absent in controls. The current study sought to evaluate the extent to which HPV16 E6 antibodies are present before diagnosis of anogenital cancers within the same cohort. Four hundred incident anogenital cancers (273 cervical, 24 anal, 67 vulvar, 12 vaginal, and 24 penile cancers) with prediagnostic blood samples (collected on average 3 and 8 years before diagnosis for cervix and noncervix cancers, respectively) and 718 matched controls were included. Plasma was analyzed for antibodies against HPV16 E6 and multiple other HPV proteins and genotypes and evaluated in relation to risk using unconditional logistic regression. HPV16 E6 seropositivity was present in 29.2% of individuals (seven of 24 individuals) who later developed anal cancer compared with 0.6% of controls (four of 718 controls) who remained cancer free (odds ratio [OR], 75.9; 95% CI, 17.9 to 321). HPV16 E6 seropositivity was less common for cancers of the cervix (3.3%), vagina (8.3%), vulva (1.5%), and penis (8.3%). No associations were seen for non-type 16 HPV E6 antibodies, apart from anti-HPV58 E6 and anal cancer (OR, 6.8; 95% CI, 1.4 to 33.1). HPV16 E6 seropositivity tended to increase in blood samples drawn closer in time to cancer diagnosis. HPV16 E6 seropositivity is relatively common before diagnosis of anal cancer but rare for other HPV-related anogenital cancers.

PDZRN3/LNX3 is a novel target of human papillomavirus type 16 (HPV-16) and HPV-18 E6.

High-risk human papillomavirus (HPV) E6 proteins have a C-terminal PDZ binding motif through which they bind, and target for proteasome-mediated degradation, a number of PDZ-containing cellular targets. Recent studies have suggested that the RING-containing ubiquitin-protein ligase PDZRN3 might also be an HPV E6 target. In this analysis, we show that HPV-16 and HPV-18 E6 can target PDZRN3 in a PDZ- and proteasome-dependent manner and provide a connection between the HPV life cycle and differentiation-related STAT signaling.

Human Papillomavirus E7 Oncoprotein Transgenic Skin Develops an Enhanced Inflammatory Response to 2,4-Dinitrochlorobenzene by an Arginase-1-Dependent Mechanism

We have shown that expression of Human Papillomavirus type 16 E7 (HPV16.E7) protein within epithelial cells, as occurs in HPV associated-premalignancy and cancers, results in local immune suppression, and a weak and ineffective immune response to E7 protein. However, a robust acute inflammatory stimulus can overcome this to enable immune elimination of HPV16.E7 transformed epithelial cells. 2,4-Dinitrochlorobenzene (DNCB) can elicit acute inflammation and has been shown to initiate the regression of HPV-associated genital warts. Although clinical use of DNCB is discouraged due to its mutagenic potential, understanding how DNCB induced acute inflammation alters local HPV16.E7 mediated-immune suppression might lead to better treatments. Here, we show that topical DNCB application to skin expressing HPV16.E7 as a transgene induces a hyperinflammatory response, not seen in non-transgenic control animals. The E7 associated-inflammatory response is characterized by enhanced expression of Th2 cytokines and increased infiltration of CD11b+Gr1intF4/80+Ly6ChiLy6Glow myeloid cells, producing arginase-1. Inhibition of arginase with an arginase specific inhibitor, N(omega)-hydroxy-nor-L-arginine, ameliorates the DNCB-induced inflammatory response. Our results demonstrate that HPV16.E7 protein enhances DNCB associated-production of arginase-1 by myeloid cells and consequent inflammatory cellular infiltration of skin.

Saliva and plasma quantitative polymerase chain reaction-based detection and surveillance of human papillomavirus-related head and neck cancer.

Human papillomavirus type 16 (HPV-16) is a major causative factor in oropharyngeal squamous cell carcinoma (OPSCC). The detection of primary OPSCC is often delayed owing to the challenging anatomy of the oropharynx. To investigate the feasibility of HPV-16 DNA detection in pretreatment and posttreatment plasma and saliva and its potential role as a marker of prognosis. This is a retrospective analysis of a prospectively collected cohort. Among a cohort of patients with oropharyngeal and unknown primary squamous cell carcinoma with known HPV-16 tumor status from the Johns Hopkins Medical Institutions and Greater Baltimore Medical Center (from 1999 through 2010), 93 patients were identified with a complete set of pretreatment and posttreatment plasma or saliva samples, of which 81 patients had HPV-16-positive tumors and 12 patients had HPV-16-negative tumors. Real-time quantitative polymerase chain reaction was used to detect HPV-16 E6 and E7 DNA in saliva and plasma samples. Main outcomes included sensitivity, specificity, negative predictive value of combined saliva and plasma pretreatment HPV-16 DNA status for detecting tumor HPV-16 status, as well as the association of posttreatment HPV DNA status with clinical outcomes, including recurrence-free survival and overall survival. The median follow-up time was 49 months (range, 0.9-181.0 months). The sensitivity, specificity, negative predictive value, and positive predictive value of combined saliva and plasma pretreatment HPV-16 DNA status for detecting tumor HPV-16 status were 76%, 100%, 42%, and 100%, respectively. The sensitivities of pretreatment saliva or plasma alone were 52.8% and 67.3%, respectively. In a multivariable analysis, positive posttreatment saliva HPV status was associated with higher risk of recurrence (hazard ratio [HR], 10.7; 95% CI, 2.36-48.50) (P = .002). Overall survival was reduced among those with posttreatment HPV-positive status in saliva (HR, 25.9; 95% CI, 3.23-208.00) (P = .002) and those with HPV-positive status in either saliva or plasma but not among patients with HPV-positive status in plasma alone. The combined saliva and plasma posttreatment HPV-16 DNA status was 90.7% specific and 69.5% sensitive in predicting recurrence within 3 years. Using a combination of pretreatment plasma and saliva can increase the sensitivity of pretreatment HPV-16 status as a tool for screening patients with HPV-16-positive OPSCC. In addition, analysis of HPV-16 DNA in saliva and plasma after primary treatment may allow for early detection of recurrence in patients with HPV-16-positive OPSCC.

Human papillomavirus 16-specific T-cell responses and spontaneous regression of anal high-grade squamous intraepithelial lesions.

Most anal cancers are attributable to persistent human papillomavirus type 16 (HPV-16) infection. The anal cancer precursor, high-grade squamous intraepithelial lesion (HSIL), frequently regresses spontaneously. We hypothesized that T-cell responses are associated with HSIL regression. In men who have sex with men undergoing anal cytology and high-resolution anoscopy, we measured responses to HPV-16 oncogenic proteins E6 and E7, using the CD25/CD134 assay for CD4(+) antigen-specific T cells and intracellular cytokine staining for CD4(+) and CD8(+) antigen-specific T cells. Of 134 participants (mean [SD] age, 51 [9.3] years; 31 [23.1%] infected with human immunodeficiency virus), 51 (38.1%) had HSIL. E6- and E7-specific CD4(+) T-cell responses were detected in 80 (59.7%) and 40 (29.9%) of the participants, respectively, and E6- and E7-specific CD8(+) T-cell responses were each detected in 25 (18.7%). HSIL was significantly associated with E7-specific CD8(+) T-cell responses (odds ratio, 4.09 [95% confidence interval, 1.55-10.77], P = .004), but not with any CD4(+) T-cell response (P ≥ .09). Twenty-six participants had HSIL a mean of 1 year before measurement of T-cell responses, and 6 (23%) of them were regressors. Five regressors (83%) had E6-specific CD4(+) T-cell responses vs 7 of 20 (35%) nonregressors (Pexact = .065). Systemic HPV-16 E6- and E7-specific T-cell responses were common in men who have sex with men. E6-specific CD4(+) T-cell responses may be associated with recent HSIL regression. NCT02007421.

Differences in the expression of human papillomavirus type 16 (HPV-16) E6 oncogene mRNA in SiHa cell line inoculated with CMV, HSV or ureaplasmas.

One of the factors associated with an increased risk of HPV-related malignant transformation may be bacterial and/or viral infections. The aim of our study was to examine whether the presence of infectious agents commonly detected in the genitourinary tract such as herpesviruses (HSV, CMV), and ureaplasmas (Ureaplasma urealyticum, Ureaplasma parvum) may lead to alterations in the expression of the HPV-16 E6 oncogene. Quantitative RT-PCR analysis was used to assess the level of HPV-16 E6 mRNA expression in SiHa cells. The presence of HSV-1 or HSV-2 in SiHa cells caused a 1.5-fold increase in HPV-16 E6 mRNA expression as compared with non-inoculated SiHa cells. Ureaplasma urealyticum presence but not Ureaplasma parvum stimulated the expression of HPV-16 E6 resulting in a nearly five-fold (4.8) up-regulated E6 mRNA level in SiHa cells. Our study is the first to suggest that infection of Ureaplasma urealyticum in an urogenital tract could increase the risk of cervical cancer by overexpression of the HPV E6 oncogene.