Evidence for Alteration of EZH2, BMI1, and KDM6A and Epigenetic Reprogramming in Human Papillomavirus Type 16 E6/E7-Expressing Keratinocytes

A number of epigenetic alterations occur in both the virus and host cellular genomes during human papillomavirus (HPV)-associated carcinogenesis, and investigations of such alterations, including changes in chromatin proteins and histone modifications, have the potential to lead to therapeutic epigenetic reversion. We report here that transformed HPV16 E6/E7-expressing primary human foreskin keratinocytes (HFKs) (E6/E7 cells) demonstrate increased expression of the PRC2 methyltransferase EZH2 at both the mRNA and protein levels but do not exhibit the expected increase in trimethylated H3K27 (H3K27me3) compared to normal keratinocytes. In contrast, these cells show a reduction in global H3K27me3 levels in vitro, as well as upregulation of the KDM6A demethylase. We further show for the first time that transformation with the HPV16 E6 and E7 oncogenes also results in an increase in phosphorylated EZH2 serine 21 (P-EZH2-Ser21), mediated by active Akt, and in a downregulation of the PRC1 protein BMI1 in these cells. High-grade squamous cervical intraepithelial lesions also showed a loss of H3K27me3 in the presence of increased expression of EZH2. Correlating with the loss of H3K27me3, E6/E7 cells exhibited derepression of specific EZH2-, KMD6A-, and BMI1-targeted HOX genes. These results suggest that the observed reduction in H3K27me3 may be due to a combination of reduced activities/levels of specific polycomb proteins and increases in demethylases. The dysregulation of multiple chromatin proteins resulting in the loss of global H3K27me3 and the transcriptional reprogramming in HPV16 E6/E7-infected cells could provide an epigenetic signature associated with risk and/or progression of HPV16-associated cancers, as well as the potential for epigenetic reversion in the future.

Human papillomavirus related head and neck cancer survival: A systematic review and meta-analysis

Human Papillomavirus (HPV) related oropharyngeal squamous cell carcinomas (OPSCCs) are reported to have improved prognosis and survival in comparison to other head and neck squamous cell cancers (HNSCCs). This systematic review and meta-analysis examines survival differences in HPV-positive HNSCC and OPSCC subtypes including tonsillar carcinoma in studies not previously investigated. Four electronic databases were searched from their inception till April 2011. A random effects meta-analysis was used to pool study estimates evaluating disease-specific (death from HNSCC), overall (all-cause mortality), progression-free and disease-free (recurrence free) survival outcomes in HPV-positive vs. HPV-negative HNSCCs. All statistical tests were two-sided. Forty-two studies were included. Patients with HPV-positive HNSCC had a 54% better overall survival compared to HPV-negative patients HR 0.46 (95% CI 0.37-0.57); the pooled HR for tonsillar cancer and OPSCC was 0.50 (95% CI 0.33-0.77) and HR 0.47 (95% CI 0.35-0.62) respectively. The pooled HR for disease specific survival was 0.28 (95% CI 0.19-0.40); similar effect sizes were found irrespective of the adjustment for confounders, HPV detection methods or study location. Both progression-free survival and disease-free survival were significantly improved in HPV-positive HNSCCs. HPV-positive HNSCCs and OPSCCs patients have a significantly lower disease specific mortality and are less likely to experience progression or recurrence of their cancer than HPV-negative patients; findings which have connotations for treatment selection in these patients.

Prevalence of human papillomavirus in women attending cervical screening in the UK and Ireland:New data from northern Ireland and a systematic review and meta-analysis

There is substantial international variation in human papillomavirus (HPV) prevalence; this study details the first report from Northern Ireland and additionally provides a systematic review and meta-analysis pooling the prevalence of high-risk (HR-HPV) subtypes among women with normal cytology in the UK and Ireland. Between February and December 2009, routine liquid based cytology (LBC) samples were collected for HPV detection (Roche Cobas® 4800 [PCR]) among unselected women attending for cervical cytology testing. Four electronic databases, including MEDLINE, were then searched from their inception till April 2011. A random effects meta-analysis was used to calculate a pooled HR-HPV prevalence and associated 95% confidence intervals (CI). 5,712 women, mean age 39 years (±SD 11.9 years; range 20-64 years), were included in the analysis, of which 5,068 (88.7%), 417 (7.3%) and 72 (1.3%) had normal, low, and high-grade cytological findings, respectively. Crude HR-HPV prevalence was 13.2% (95% CI, 12.7-13.7) among women with normal cytology and increased with cytological grade. In meta-analysis the pooled HR-HPV prevalence among those with normal cytology was 0.12 (95% CIs, 0.10-0.14; 21 studies) with the highest prevalence in younger women. HPV 16 and HPV 18 specific estimates were 0.03 (95% CI, 0.02-0.05) and 0.01 (95% CI, 0.01-0.02), respectively. The findings of this Northern Ireland study and meta-analysis verify the prevalent nature of HPV infection among younger women. Reporting of the type-specific prevalence of HPV infection is relevant for evaluating the impact of future HPV immunization initiatives, particularly against HR-HPV types other than HPV 16 and 18. J. Med. Virol. 85:295-308, 2013. © 2012 Wiley Periodicals, Inc. Copyright © 2012 Wiley Periodicals, Inc.

HPV prevalence and type-distribution in cervical cancer and premalignant lesions of the cervix: A population-based study from Northern Ireland

Assessment of Human papillomavirus (HPV) prevalence and genotype distribution is important for monitoring the impact of prophylactic HPV vaccination. This study aimed to demonstrate the HPV genotypes predominating in pre-malignant and cervical cancers in Northern Ireland (NI) before the vaccination campaign has effect. Formalin fixed paraffin embedded tissue blocks from 2,303 women aged 16-93 years throughout NI were collated between April 2011 and February 2013. HPV DNA was amplified by PCR and HPV genotyping undertaken using the Roche^® linear array detection kit. In total, 1,241 out of 1,830 eligible samples (68.0%) tested positive for HPV, with the majority of these [1,181/1,830 (64.5%)] having high-risk (HR) HPV infection; 37.4% were positive for HPV-16 (n=684) and 5.1% for HPV-18 (n=93). HPV type-specific prevalence was 48.1%, 65.9%, 81.3%, 92.2%, and 64.3% among cervical intraepithelial neoplasias (CIN) Grades I-III, squamous cell carcinomas (SCC) and adenocarcinoma (AC) cases, respectively. Most SCC cases (81.3%) had only one HPV genotype detected and almost a third (32.0%) of all cervical pathologies were HPV negative including 51.9% of CIN I (n=283), 34.1% CIN II (n=145), 18.7% of CIN III (n=146), 7.8% of SCC (n=5), and 35.7% of AC (n=5) cases. This study provides important baseline data for monitoring the effect of HPV vaccination in NI and for comparison with other UK regions. The coverage of other HR-HPV genotypes apart from 16 and 18, including HPV-45, 31, 39, and 52, and the potential for cross protection, should be considered when considering future polyvalent vaccines.

Human papillomavirus: a strong case for vaccinating boys

In the UK, human papilloma virus vaccination is restricted on the NHS to girls and only recently been recommended for men who have sex with men. The restriction is based largely on cost-effectiveness. In this article, Gillian Prue sets out the compelling case for vaccinating all boys. On page 10, Peter Baker describes HPV Action’s campaign for gender-neutral HPV vaccination.

Single-cell gene-expression profiling reveals qualitatively distinct CD8 T cells elicited by different gene-based vaccines.

CD8 T cells play a key role in mediating protective immunity against selected pathogens after vaccination. Understanding the mechanism of this protection is dependent upon definition of the heterogeneity and complexity of cellular immune responses generated by different vaccines. Here, we identify previously unrecognized subsets of CD8 T cells based upon analysis of gene-expression patterns within single cells and show that they are differentially induced by different vaccines. Three prime-boost vector combinations encoding HIV Env stimulated antigen-specific CD8 T-cell populations of similar magnitude, phenotype, and functionality. Remarkably, however, analysis of single-cell gene-expression profiles enabled discrimination of a majority of central memory (CM) and effector memory (EM) CD8 T cells elicited by the three vaccines. Subsets of T cells could be defined based on their expression of Eomes, Cxcr3, and Ccr7, or Klrk1, Klrg1, and Ccr5 in CM and EM cells, respectively. Of CM cells elicited by DNA prime-recombinant adenoviral (rAd) boost vectors, 67% were Eomes(-) Ccr7(+) Cxcr3(-), in contrast to only 7% and 2% stimulated by rAd5-rAd5 or rAd-LCMV, respectively. Of EM cells elicited by DNA-rAd, 74% were Klrk1(-) Klrg1(-)Ccr5(-) compared with only 26% and 20% for rAd5-rAd5 or rAd5-LCMV. Definition by single-cell gene profiling of specific CM and EM CD8 T-cell subsets that are differentially induced by different gene-based vaccines will facilitate the design and evaluation of vaccines, as well as enable our understanding of mechanisms of protective immunity.

HIV-1 vaccines and adaptive trial designs.

Developing a vaccine against the human immunodeficiency virus (HIV) poses an exceptional challenge. There are no documented cases of immune-mediated clearance of HIV from an infected individual, and no known correlates of immune protection. Although nonhuman primate models of lentivirus infection have provided valuable data about HIV pathogenesis, such models do not predict HIV vaccine efficacy in humans. The combined lack of a predictive animal model and undefined biomarkers of immune protection against HIV necessitate that vaccines to this pathogen be tested directly in clinical trials. Adaptive clinical trial designs can accelerate vaccine development by rapidly screening out poor vaccines while extending the evaluation of efficacious ones, improving the characterization of promising vaccine candidates and the identification of correlates of immune protection.

Nanocarriers for DNA Vaccines: Co-Delivery of TLR-9 and NLR-2 Ligands Leads to Synergistic Enhancement of Proinflammatory Cytokine Release

Adjuvants enhance immunogenicity of vaccines through either targeted antigen delivery or stimulation of immune receptors. Three cationic nanoparticle formulations were evaluated for their potential as carriers for a DNA vaccine, and muramyl dipeptide (MDP) as immunostimulatory agent, to induce and increase immunogenicity of Mycobacterium tuberculosis antigen encoding plasmid DNA (pDNA). The formulations included (1) trimethyl chitosan (TMC) nanoparticles, (2) a squalene-in-water nanoemulsion, and (3) a mineral oil-in-water nanoemulsion. The adjuvant effect of the pDNA-nanocomplexes was evaluated by serum antibody analysis in immunized mice. All three carriers display a strong adjuvant effect, however, only TMC nanoparticles were capable to bias immune responses towards Th1. pDNA naturally contains immunostimulatory unmethylated CpG motifs that are recognized by Toll-like receptor 9 (TLR-9). In mechanistic in vitro studies, activation of TLR-9 and the ability to enhance immunogenicity by simultaneously targeting TLR-9 and NOD-like receptor 2 (NLR-2) was determined by proinflammatory cytokine release in RAW264.7 macrophages. pDNA in combination with MDP was shown to significantly increase proinflammatory cytokine release in a synergistic manner, dependent on NLR-2 activation. In summary, novel pDNA-Ag85A loaded nanoparticle formulations, which induce antigen specific immune responses in mice were developed, taking advantage of the synergistic combinations of TLR and NLR agonists to increase the adjuvanticity of the carriers used.

Polymorphisme de la capside des papillomavirus appartenant à l’espèce 9 des alphapapillomavirus

Le gène L1 encode pour la protéine majeure de la capside des papillomavirus humains (VPH). L’information relative au polymorphisme de L1 pour les types autres que VPH- 16 est jusqu’ici limitée. Cet ouvrage explore le polymorphisme de L1 en comparant les séquences des types phylogénétiquement apparentés VPH-31, -33, -35 à VPH-16. Des spécimens génitaux recueillis de 732 femmes VIH-séropositives et 323 VIHséronégatives ont été criblés à le recherche d’ADN de VPH par PCR consensus au niveau du gène L1. Les échantillons positifs pour VPH-16 (n=74), -31 (n=74), -33 (n=37) et -35 (n=58) étaient analysés par PCR-séquençage pour la totalité du gène L1. Le nombre de nucléotides substitués pour L1 variait de 19 pour VPH-33 à 52 pour VPH-31. Le rapport du nombre de variantes sur le nombre d’isolats testés était plus élevé pour VPH-31 (56.4%, p=0.05) et VPH-35 (60.3%, p=0.04) comparativement à VPH-16 (40.5%), alors que ce ratio était inférieur pour VPH-33 mais sans différence statistiquement significative (24.3%, p=0.14). La distance entre les variantes était plus grande à l’intérieur des cinq boucles présumément exposées à la surface de la protéine L1 que dans la séquence à l’extérieur (p<0.01) Des variations synonymes étaient observées chez 1.7% (95% CI 1.1- 2.3) des nucléotides intra-boucles et 2.4% (95% CI 1.2-3.7) de ceux extra-boucles. Les variations non-synonymes étaient rencontrées pour 1.8% (95% CI 1.1-2.5) des nucléotides intra-boucles et 0.2% (95% CI 0-0.4) pour les nucléotides extra-boucles. Les ratios dN/dS étaient inférieurs à 1.0 pour les régions extra-boucles et encore davantage pour les régions intra-boucles. Ces résultats suggèrent que les séquences des régions hypervariables de L1 ont été sélectionnées positivement.

Identification et caractérisation d'un domaine de transactivation dans l’hélicase E1 des papillomavirus humains

Les papillomavirus sont des virus à ADN qui infectent la peau et les muqueuses. Ils causent des verrues et peuvent aussi mener au développement de cancers, dont le cancer du col de l’utérus. La réplication de leur génome nécessite deux protéines virales : l’hélicase E1 et le facteur de transcription E2, qui recrute E1 à l’origine de réplication virale. Pour faciliter l’étude de la réplication du génome viral, un essai quantitatif et à haut débit basé sur l’expression de la luciférase a été développé. Parallèlement, un domaine de transactivation a été identifié dans la région régulatrice N-terminale de la protéine E1. La caractérisation de ce domaine a montré que son intégrité est importante pour la réplication de l’ADN. Cette étude suggère que le domaine de transactivation de E1 est une région protéique intrinsèquement désordonnée qui permet la régulation de la réplication du génome viral par son interaction avec diverses protéines.

Charge virale intégrée du papillomavirus de type 16 dans la maladie anale préinvasive

L’histoire naturelle de l’infection anale par le virus du papillome de type 16 (VPH-16) est mal définie pour les hommes ayant des relations sexuelles avec d’autres hommes (HARSAHs) VIH-séropositifs. Le but de cette étude était d’évaluer l’association entre la charge épisomale et intégrée du VPH-16 et la progression de la néoplasie intraépithéliale anale (AIN). Les charges épisomales et intégrées du VPH-16 furent mesurées par PCR quantitatif en temps réel sur 665 spécimens anaux obtenus de 135 hommes VPH-16-positifs participant à l’étude prospective HIPVIRG (Human Immunodeficiency and Papilloma VIrus Research Group). Le grade de l’AIN fut déterminé sur des biopsies obtenues lors des anuscopies à haute résolution périodiques. L’intégration du VPH-16 fut confirmée par DIPS-PCR pour démontrer la présence de jonctions virales-cellulaires. La charge épisomale du VPH-16 [ratio de cote (OR) 1.5, intervalle de confiance (IC) à 95%=1.1–2.1], le nombre de types de VPH [OR 1.4 (IC 95%=1.1–1.8)] et le tabagisme actuel [OR 4.8 (IC 95%=1.3–18.6)], mais non la charge intégrée, furent associés aux lésions de haut-grade (AIN-2,3) après ajustement pour l’âge et le décompte des lymphocytes CD4. La charge épisomale du VPH-16 était le seul facteur prédictif de progression de l’AIN de bas-grade (AIN-1) vers l’AIN-2,3 [OR 8.0 (IC 95%=1.2–55.4)]. Les spécimens avec une charge épisomale du VPH-16 élevée étaient moins susceptibles de contenir de l’intégration [OR 0.5 (IC 95%=0.3–0.8)]. L’intégration du VPH-16 fut détectée en absence d’AIN, dans l’AIN-1 et dans l’AIN-2,3. L’analyse des jonctions virales-cellulaires ne permit pas d’identifier un site d’intégration spécifique.