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Hans Stevens
Leuven, BelgiumHans Stevens1, Elisabeth Heylen1, Roger Maes2, Matti Kiupel2, Annabel Rector1, Marc Van Ranst1. 1Laboratory of Clinical and Epidemiological Virology, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium; 2Diagnostic Center for Population and Animal Health, Michigan State University, East Lansing, MI, USA. Papillomatosis has been documented in a number of species from the suborder of the Feliformia, but all of them are found in species belonging to the Felidae family. The Crocuta crocuta papillomavirus type 1 is the first papillomavirus found in a species belonging to the Hyaenidae family of the Feliformia suborder.
A papillomatous lesion was removed from the mucosa of a wild spotted hyena (Crocuta crocuta) living in the Masai Mara Game Reserve, Kenya. Extracted DNA from this lesion was used as a template for multiply primed rolling-circle amplification (RCA) and degenerate primer PCR. This resulted in two small DNA fragments of 542 bp and 526 bp respectively in the L1 and E1 ORF. Long template PCR (LT-PCR) primers were selected and LT-PCR was performed on the RCA product. The two resulting fragments of approximately 3.5 kb and 6 kb were cloned and the complete genomic sequence was determined.
The Crocuta crocuta PV type 1 (CcrPV-1) genome contains 8344 basepairs and codes for 5 early open reading frames (ORF), E6, E7, E1, E2 and E4, and 2 late ORFs L2 and L1. The CcrPV-1 genome contains the normal noncoding region NCR1 between the end of L1 and the beginning of E6, but also a second noncoding region NCR2 between the end of the E2 ORF and the beginning of the L2 ORF, typical for the PVs of carnivores in the Lambdapapillomavirus genus. Phylogenetic analysis based on a concatenated nucleotide sequence alignment of the E1, E2, L2 and L1 ORFs of CcrPV-1 and 65 other PV types indicates that CcrPV-1 clusters with the other Lambdapapillomaviruses, between the PVs isolated from Felidae host species and the PVs found in Caniformia host species. This finding supports the co-evolution hypothesis, which states that PVs of closely related host species are closely related themselves. |
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Hans Stevens
Leuven, BelgiumHans Stevens1, Mark Bennett2, Elisabeth Heylen1, Philip Nicholls2, Marc Van Ranst1. 1Laboratory of Clinical and Epidemiological Virology, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium; 2School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, Australia. Papillomaviruses (PVs) have been found in numerous mammal species, all of which however were placental mammals. We report the first papillomavirus found in a marsupial host, the Bettongia penicillata papillomavirus type 1 (BpPV-1). BpPV-1 was found in a papillomatous lesion of a brush-tailed bettong (Bettongia penicillata) living in Western Australia. Total DNA was extracted from a frozen skin biopsy of this lesion and used as a template for multiply primed rolling-circle amplification (RCA). The RCA product was cut using the EcoRI, XbaI and HindIII restriction enzymes, subsequently cloned and the complete genomic sequence was determined. The BpPV-1 genome contains 7737 bp and codes for 5 early open reading frames (ORF), E6, E7, E1, E2 and E4, and 2 late ORFs, L2 and L1. Phylogenetic analyses of a concatenated alignment of the E1, E2, L2 and L1 ORF of BpPV-1 and 62 other PVs revealed that BpPV-1 clusters close to the root of the phylogenetic tree. Pairwise alignment revealed that the L1 and L2 ORFs are most similar to those of bandicoot papillomatosis carcinomatosis virus types 1 and 2 (BPCV) isolated from the marsupial hosts Perameles bougainville and Isoodon obesulus, respectively. These viruses have an early region similar to the early region of polyomaviruses and a late region similar to the papillomavirus late region. In a phylogenetic tree based on a concatenated alignment of the L2 and L1 ORF of BpPV-1, the 2 BPCVs and 62 other PVs, BpPV-1 also clusters together with BPCV-1 and BPCV-2. BpPV1 is the first fully sequenced PV found in a marsupial host. These sequence data provides support to the hypothesis that the BPCVs arose by genetic recombination between an ancient PV and an ancient polyomavirus.
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Kurt Tobler
Zurich, SwitzerlandChristian Lange1,2, Kurt Tobler2, Mathias Ackermann2, Claude Favrot1. 1Department of Dermatology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland; 2Institute of Virology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland. The number of described papillomavirus (PV) species and potential PV genera is steadily increasing. It seems that every host species might potentially harbour a large set of PVs, but the ensemble of PVs from each species appears to belong only to a few genera. This has been observed for more than hundred human, but also for ten bovine and recently for seven canine PVs. In most of the other host species too few PVs have yet been identified to consider this hypothesis.
We identified the DNA of a novel equine PV in aural plaques of a horse. The genome of a novel PV was amplified from a skin sample and was entirely cloned and sequenced. The novel equine PV genome contains the characteristic open reading frames (ORFs) E6, E7, E1, E2, L1 and L2, a large non coding region between the late and early region as well as a small non coding region between the early and the late region. The novel virus was consequently designated as equine PV3 (EcPV3).
The genomes of the three equine PVs were analysed and compared with each other and further PVs. Upon phylogenetic analyses the equine PVs group well together and appear in pylogenetic vicinity to some bovine PVs. Still the three equine PVs share less than 60% of nucleotide identities in L1, and may therefore be regarded as belonging to different clades. Pairwise alignment of the L1 amino acid sequences reveals that FdPV3 shares only 56.3% identities with EcPV1 and 54.9 with EcPV2. These findings may support an allocation of the equine PVs into some distinct clades, as seems to be the case in other species.
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Christian Lange
Zurich, SwitzerlandChristian Lange1,2, Kurt Tobler2, Jessica Gull3, Mathias Ackermann2, Elisabeth Vetsch1, Claude Favrot1. 1Department of Dermatology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland; 2Institute of Virology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland; 3Clinic for Zoo Animals, Exotic Pets and Wildlife, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland. In the past decades, papillomaviruses (PVs) infecting mammalian species have been discovered in a broad genetic diversity, forming a steadily growing number of genera. The more recent discovery of PVs infecting non-mammalian species extended the potential hosts to the whole clade of amniotes. Still only four whole genomes of PVs from non-mammalian hosts have been published as yet. We discovered PV DNA in pigmented papilloma-like lesions of a diamond python (Morelia spilota spilota). The genome of a novel PV was amplified from one of these lesions, was cloned and sequenced. It contains the characteristic open reading frames (ORFs) E6, E7, E1, E2, L1 and L2, a large non coding region between L1 and E6 as well as a small non coding region between E2 and L2. The size of the viral genome is, with only 7048 nucleotides, rather small, which is also the case in the two other published reptile PVs. A phylogenetic analysis based on the nucleotide sequence alignment of the L1 ORF placed the Morelia spilota papillomavirus1 (MsPV1) close to the root. Interestingly MsPV1 does not cluster with the other four PVs of sauropsids. Pairwise alignment of the L1 sequences on the amino acid level revealed only 42.9% and 42.5% identities with the described sea turtle PVs (CmPV1, CCPV1) as well as 43.9% and 43.6% identities with the two bird PVs (FcPV1, PePV1). Pair wise alignment with other L1 sequences revealed highest identities with the polar bear PV (UmPV1 - 56.5%) and two human PVs (HPV9 - 56%, HPV1 - 55.5%). These findings suggest that mammalian and sauropsid PVs may not be as genetically distinct in general as could be anticipated based to the previous findings.
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Ingo Nindl
Heidelberg, GermanyMarc Gottschling1, Eric Schulz2, Ignacio G. Bravo3, Rob Deaville4, Paul D. Jepson4, Marie-Françoise Van Bressem5, Eggert Stockfleth2, Ingo Nindl2,6. 1Department of Biology, Systematic Botany, Ludwig-Maximilians-Universität München, Munich, Germany; 2Clinic for Dermatology, Venerology und Allergology, Haut Tumor Centrum Charité (HTCC), Charité - Universitätsmedizin Berlin, Berlin, Germany; 3Centre for Public Health Research (CSISP), Valencia, Spain; 4Institute of Zoology, Regents Park, London, United Kingdom; 5Cetacean Conservation Medicine Group (CMED), CEPEC, Museo de Delfines, Pucusana, Peru; 6DKFZ - Charité, Viral Skin Carcinogenesis, Viral Transformation Mechanisms, German Cancer Research Center (DKFZ), Heidelberg, Germany. Currently known cetacean papillomaviruses (Omicronpapillomavirus) appear as chimeras. They exhibit genetic elements of viruses either with the shared tropism to mucosal tissue such as Alphapapillomavirus (i.e., early genes) or isolated from related host species such as Xipapillomavirus (i.e., late genes). We isolated and sequenced the complete genomes of four novel papillomavirus types and one variant from genital and oesophageal lesions of cetaceans, namely of Delphinus delphis (DdPV), Lagenorhynchus acutus (TtPV-3 variant) and Phocoena phocoena (PpPV-1, PpPV-2 and PpPV-4). Phylogenetic analyses were performed using Maximum Likelihood and Bayesian approaches. As inferred from early genes analyses, cetacean papillomaviruses, including the new types and variant, constituted a monophyletic group with (mucosotropic) Alphapapillomavirus. Analyzing late genes, they were the closest relatives of (ruminant) Xipapillomavirus, with the exception of PpPV-4. This was the unique cetacean papillomavirus that showed a consistent phylogenetic position close to the primarily mucosotropic clade. An explanation for incongruent tree topologies between early and late genes phylogenies are recombination events. PpPV-4 is the only known non-recombinant cetacean papillomavirus type and may represent a relative of a donor to chimeric Omicronpapillomavirus. Our study thus supports a complex evolutionary scenario with multiple driving forces for papillomavirus diversification, also including interspecies transmissions.
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Charles Wood
Winston-Salem, USACharles Wood1, Sarah Tannehill-Gregg2, Zigui Chen3, Koenraad van Doorslaer3, Mark Cline1, Robert Burk3,4. 1Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, NC, USA; 2Department of Drug Safety Evaluation, Bristol-Myers Squibb Research & Development, Mount Vernon, IN, USA; 3Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA; 4Departments of Pediatrics, Epidemiology & Population Health, and Obstetrics, Gynecology and Women's Health, Albert Einstein College of Medicine, Bronx, NY, USA. Betapapillomaviruses are a diverse genus of papillomaviruses (PVs) commonly associated with human skin papillomas and epidermal dysplasia. Few beta-PVs have been characterized in nonhuman species. This report describes a novel beta-PV, named Macaca fascicularis PV type 2 (MfPV-2), isolated from exophytic skin papillomas on the hands and feet of an adult male cynomolgus monkey (M. fascicularis). On histology the papillomas were composed of thickened epidermis with superficial foci of cytomegaly and cytoplasmic pallor. Nuclei exhibited moderate pleomorphism with frequent central clearing and peripheral chromatin. Positive immunostaining for PV antigen, p16, and the proliferation marker Ki67 was present multifocally within all layers of affected epidermal cells. Complete sequence identity (100%) was noted between PV genomes sequenced from hand and foot lesions. The MfPV-2 genome was 7632 bp in length and included putative open reading frames for E1, E2, E4, E6, E7, L1, and L2 genes, similar to other beta-PVs. The closest PV relatives to MfPV-2 are HPV115 (67.4% pairwise identity) and MfPV-1 (67.3% pairwise identity), which was also isolated from hand and foot papillomas in a cynomolgus macaque. Phylogenetic analysis placed MfPV-2 in a new species group (number 6) among the beta-PVs, distinct from MfPV-1. These findings provide further support for the idea that tissue tropism among ancient primate PVs developed prior to divergence of human and nonhuman primate PV types.
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Hao-Shun Huang
Madison, USAHao-Shun Huang1, Chris Buck2, Paul Lambert1. 1McArdle Laboratory for Cancer Research, Department of Cancer Biology, University of Wisconsin-Madison, Madison, WI, USA; 2Laboratory of Cellular Oncology, Tumor Virus Molecular Biology Section, National Cancer Institute, Bethesda, MD, USA. Human papillomaviruses (HPV) are small, nonenveloped double stranded DNA tumor viruses that induce various proliferative lesions upon infection of stratified squamous epithelia. Persistent infections by high-risk HPVs play a crucial role in the development of cervical cancer. HPVs also are the most common sexually transmitted pathogens. Recently, vaccines have become available for preventing infection by a subset of sexually transmitted HPVs. Microbicides that inhibit infection may provide alternative and economical means for preventing sexual transmission of HPVs. The recent development of protocols for producing useful amounts of HPV virions and HPV pseudovirions carrying reporter genes has provided opportunities to identify novel HPV microbicides. In a high throughput screen, drugs that inhibit the cellular protein Gamma secretase were identified as potential HPV microbicides. Secondary screens indicated that protein Gamma secretase inhibitors IX and X are highly effective at inhibiting infection of HPV pseudovirus infection both in human keratinocytes and in mouse cells, with IC50s in the picomloar to nanomolar range. Using a mouse model for in vivo infection of female reproductive tract, we observed that gamma secretase inhibitor IX could modestly inhibit HPV infection, to the same degree as its effectiveness in inhibiting gamma secretase activity in vivo. These studies indicate that Gamma secretase inhibitors may be effective microbicides for HPV.
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Anna-Lise Williamson
Cape Town, South AfricaDianne Marais1, Daniel Gawarecki2, Naomi Rutenberg2, Bruce Allan1, Khatija Ahmed3, Lydia Altini4, Nazira Cassim3, Felicity Gopolang 4, Margaret Hoffman 5, Anna-Lise Williamson1,6. 1Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa; 2Population Council, New York, NY, USA; 3Setshaba Research Centre, Soshangwe, Pretoria, South Africa; 4Centre for Infectious Disease Epidemiology and Research, University of Cape Town, Cape Town, South Africa ; 5Department of Public Health, Faculty of Health Sciences, University of Cape Town, Observatory, Cape Town, South Africa; 6National Health Laboratory Service, Groote Schuur Hospital, Observatory, Cape Town, South Africa.
Background: Carrageenan has been shown to inhibit Human papillomavirus (HPV) infection in vitro. A carrageenan containing vaginal microbicide, Carraguard developed by the Population Council was tested for its efficacy in prevention of genital HPV infection in South African women. This was a sub-study using data from the Population Council's randomised, double-blind, placebo-controlled trial which tested the efficacy of Carraguard to prevent HIV infection. Carraguard was not found to prevent HIV infection.
Methods: Participants in the study received Carraguard gel or placebo (methyl cellulose gel) plus condoms, and were instructed to use gel plus condoms during each act of vaginal intercourse. Oncogenic, high-risk, HPV (HR-HPV) detection was performed on study end cervical samples from 1723 women by Digene Hybrid Capture 2 analysis. Logistic regression analysis was used to assess the relative risk of contracting HR-HPV for individuals receiving Carraguard relative to individuals receiving placebo.
Results: End trial prevalence of HR-HPV was 23.2%. [95% CI(21.3, 25.2)]. In Carraguard users HR-HPV prevalence was 23.5% [95% CI (20.8, 26.3)] and 23.0% [95% CI (20.2, 25.8)] inplacebo users. Significant risk factors for HR-HPV infection were younger age, being single, an abnormal pap smear, multiple sexual partners (woman and primary partner) and promiscuous behaviour without the use of a condom. After adjusting for risk factors, Carraguard was found protective for HPV infection for women who inserted >80% of their returned applicators of test product with >30% confirmed covered sex acts. Within the latter category, Carraguard was almost 50% more efficacious than placebo. Carraguard users were 0.529 as likely to contract HR-HPV [95% CI (0.304, 0.920)] as women who used placebo (P=0.0176).
Conclusions: Carraguard was shown to be effective in protecting against vaginal transmission of HR-HPV. To our knowledge, this is the first report of successful protection against genital infection by a vaginal microbicide
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Christine White
Dublin, IrelandC.D. Spillane1,2, C. White1,2, K. Linder2, T. McEligott1, L. Kehoe1,2, O. Sheils1, C.M. Martin1,2, J.J. O'Leary1,2. 1Histopathology Department, Trinity College Dublin, Dublin, Ireland; 2Pathology Department, Coombe Women's Hospital, Dublin, Ireland. Background: Sulindac, a commonly used NSAID, has been investigated as a potential novel therapeutic for different forms of cancer, including colon and breast. A recent study looked at the molecular action of this drug on the HPV18 infected cervical cell line, HeLa, and demonstrated that not only could it induce apoptosis but also degrade one of the main oncoproteins, E7.
Design: This study aimed to validate the previous findings and to extend the analysis to other cervical carcinoma cell lines with differing origins, HPV status and viral DNA content. Three cervical cancer cell lines were examined, the adenocarcinoma derived HPV18 positive HeLa, the squamous cell carcinoma derived HPV16 positive SiHa and the HPV negative C33A.
Results: Sulindac had a time and dose dependent growth inhibitory effect on all three cell lines. However, the most potent response was observed in the HeLa cells, with the IC50 value approximately 200μM less than the other two cell lines. Analysis of the HeLa cells demonstrated that this activity occurred predominantly through the induction of apoptosis but additionally by cell cycle arrest. The previous findings that sulindac induced a post-transcriptional degradation of HPV18 viral oncogene E7 were validated. This decrease was dose dependent and appeared to correlate with an observed G1 arrest. However, in comparison with the previous findings these results were observed with significantly lower concentrations of sulindac, 115μM compared to 500μM. In addition, it was demonstrated that a decrease in COX activity may be partially responsible for the anti-proliferative activity of sulindac.
Conclusion: This data indicates that the antineoplastic activities of sulindac are multifaceted. Since most cancers progress through the action of multiple pathways, drugs that simultaneously block several pathways might be particularly effective as therapeutic agents. Therefore, these results suggest that NSAIDs may offer potential as novel therapeutics for cervical cancer.
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Jordan Meyers
Provo, United StatesJordan Meyers1, Eric Ryndock2, M.J. Conway2, Craig Meyers2, Richard Robison1. 1Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, USA; 2Department of Microbiology and Immunology, Pennsylvania State University, Hershey, PA, USA. Human papillomavirus (HPV) represents one of the most prevalent sexually transmitted infections in the United States. The susceptibility of HPV to disinfection has not been described due to the lack of a suitable infectivity assay as well as the inability to produce sufficient amounts of HPV particles. Using a qRT-PCR infectivity assay for high-risk HPV16, we present the first disinfectant susceptibility profile for HPV16, the most common high-risk HPV type. We tested particles from two different propagation methods: native organotypically-derived virions and synthetic 293TT-derived quasivirions. Common clinical disinfectants tested included: ethanol, isopropanol, hypochlorite, alkaline glutaraldehyde, ortho-phthalaldehyde, peroxyacetic acid (PAA- based disinfectant), and a triple phenolic. Of these disinfectants, only the hypochlorite and PAA-based disinfectants were able to produce greater than a three log reduction of infectious virus for both particle types. All disinfectants were tested at relevant concentrations and at a 45 minute contact time. These data suggest that HPV has a very unique susceptibility profile relative to other non-enveloped viruses such as poliovirus and adenoviruses. For two disinfectants, the quasivirions were more susceptible than the native virions. Both alcohols were able to reduce infectivity of quasivirions at a 70% concentration and surprisingly, were more effective at a 95% concentration. Both alcohols were ineffective against native virions, regardless of concentration. The results of these studies show that some disinfectants routinely used on medical instruments and devices may not be effective at inactivating native HPV, leaving open the possibility of iatrogenic transmission of HPV in a clinical setting. In addition, these data suggest that quasivirions do not have the same disinfectant resistance as more naturally-derived virions, suggesting that their use as surrogates in these types of studies may be problematic.
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Ana Montoya
México, MéxicoAna Gabriela Leija-Montoya1, Maria Luisa Benítez-Hess1, Julia D. Toscano-Garibay1, Luis M. Alvarez-Salas1. 1Laboratorio de Terapia Génica, Departamento de Genética y Biología Molecular, CINVESTAV, México, D.F., México . Cervical cancer is causally associated to persistent infection with high risk human papillomavirus (HPV), especially HPV-16. The HPV capsid is mainly composed of the L1 protein that can self-assemble in the absence of genetic material forming virus-like particles (VLPs) that are structurally and immunologically similar to the infectious virions. Aptamers are oligonucleotide ligands obtained by the iterative application of the SELEX method (Systematic Evolution of Ligands by EXponential enrichment) over combinatorial libraries and are capable of binding tightly and specifically to its targets. In this work, aptamers that specifically recognize HPV-16 virus-like particles (VLPs) were isolated from a combinatorial library of 415 variants using a modified SELEX method. Several aptamers were obtained after three cycles of negative selection and five cycles of positive selection against HPV-16 L1 VLPs. These aptamers were sequenced and divided into three groups according to sequence similitude. An aptamer representative of each group was probed for specificity against HPV-16 VLPs, the selection system and bovine albumin. Although all the probed aptamers efficiently bound the VLPs, the Sc5-3 aptamer showed the highest specificity. Later experiments using bacteria-produced HPV-16 L1 and GST-L1 chimeras resulted also in specific binding by Sc5-c3 aptamer, suggesting that recognition of L1 is not dependent in quaternary structure. Current experiments are aimed to establish the L1 domain associated to Sc5-c3 binding and cross-reactivity against other HPV types. Highly specific L1-recognizing aptamers have great potential as diagnostic tools and as antiviral agents to block HPV-16 infection.
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Tim De Schutter
Leuven, BelgiumTim De Schutter1, Dimitrios Topalis1, Graciela Andrei1, Robert Snoeck1. 1Laboratory of Virology, Rega Institute for Medical Research, K.U.Leuven, Belgium. Cidofovir (CDV, HPMPC [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine]) is an acyclic nucleoside phosphonate (ANP) with a broad spectrum anti-DNA virus activity. Both in vitro and in vivo data provide evidence of activity of cidofovir against HPV. Furthermore, successful case reports of off-label treatment with cidofovir support the potential use by either topical administration or local injection. Although it has previously been shown that cidofovir induces apoptosis in HPV-positive cell lines, the exact mechanism remains to be elucidated.
To study HPV in vitro, cells that harbour a part of the viral genome integrated in the cellular genome are frequently used. By subculturing SiHa cells (HPV-16 positive) in increasing concentrations of cidofovir, cells were selected for resistance. These resistant cells serve as an interesting tool to study the antiproliferative effect of cidofovir in HPV-positive cells.
In order to study the activity of cidofovir against HPV, gene expression profiling was performed in both cidofovir-treated and cidofovir-resistant SiHa cells by means of microarrays (HG-U133 Plus 2, Affymetrix) and Q-PCR. Different time points after the addition of the compound were tested, as well as both a low and a high passage of the resistant cells. All tested conditions, with their respective controls were performed in triplicates.
Genes with an altered expression, in comparison to their corresponding control, were found in all tested conditions. Different identified genes are related to essential functions, including cell death, cell cycle, cell-cell signaling and extracellular matrix. By using various software tools to analyze microarray data, it was possible to find several processes potentially involved in cidofovir-treatment and cidofovir-resistance in SiHa cells.
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Vincent Bonagura
Great Neck, USAEdward James1, Devoti James2,3, Rosenthal David2,3,4, Hatam Lynda2,3, Steinberg Bettie4,5, Abramson Allan3,5, Bonagura Vincent2,3,4. 1Benaroya Research Institute, Seattle, WA , USA; 2Feinstein Institute for Medical Research, North Shore-LIJ Health System, Manhasset, NY, USA ; 3Division of Allergy/Immunology, North Shore LIJ Health System, Manhasset, NY, USA ; 4Elmezzi Graduate School of Molecular Medicine, North Shore LIJ Health System, Manhasset, NY, USA ; 5Department of Otolaryngology, Long Island Jewish Medical Center, New Hyde Park, NY, USA. Objectives: The HLA-DRB1*0102, DRB1*0301 haplotype is associated with increased severity of RRP, a disease caused by HPV-6/11. TH1-like responses to E6/E2 proteins have been shown to protect against papillomavirus-induced disease in animals, and TH2 polarization may be related to RRP disease severity in DRB1*0102 and DRB1*0301 expressing patients with RRP.
Methods: Using tetramer guided epitope mapping, we identified the dominant immunogenic peptides within HPV-11 early proteins E6/E2, restricted by DRB1*0102 and/or DRB1*0301. Peptide binding, tetramer staining, and proliferation assays identified minimal epitopes within these peptides. The cytokine profile of sorted tetramer positive T-cell lines from RRP patients (n=6) and controls (n=10) was measured using a cytokine capture assay, to determine E2/E6-responsive T-cell polarization.
Results: Two distinct E6/E2 peptides (E6 113-132, E2 1-20) contained DRB1*0102 and DRB1*0301 restricted epitopes respectively. An additional peptide (E2 281-300) contained an epitope presented by both DRB1*0102 and DRB1*0301. Minimal epitopes within these peptides bound to recombinant DR protein, gave positive tetramer staining for sorted T-cell lines, and elicited T-cell proliferation in both RRP patients and HLA-matched healthy controls. While the magnitude of responses to these epitopes was similar in both groups, IFN-γ secretion was substantially lower in T-cell lines isolated from RRP patients and IL-13 was expressed by some tetramer-peptide binding T-clones.
Conclusions: CD4+ T-cells specific for E2/E6 epitopes are easily detected in RRP patients and healthy controls with DRB1*0102 and DRB1*0301 haplotypes. However, RRP patients exhibit HPV-specific, immune dysregulation, indicated by decreased IFN-γ, but increased IL-13 expression. Therefore, therapeutic vaccination or other interventions that repolarize T-cell responses and restore TH1-like cytokine responses to HPV proteins could improve outcomes for RRP patients. |
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Agata Kozar
Warsaw, PolandMaria Gołebiowska-Wawrzyniak1, Katarzyna Markiewicz1, Artur Jakimiuk2, Tomasz Maciejewski1, Agata Kozar1, Krzysztof Rytwinski1, Zbigniew Wawrzyniak3. 1Institute of Mother and Child, Warsaw, Poland; 2Central Clinical Hospital of Ministry of Interior, Warsaw, Poland; 3Medical University of Warsaw, Warsaw, Poland. Susceptibility to sexually transmitted infection with Human Papilloma virus is probably connected with disturbances in cellular immunity. Significantly more frequent HPV infections in women with impaired cellular immunity as well as intensification of papilloma lesions has been observed. Due to the increase frequency of infections it is essential to assess their coexistence.
Aim: Estimate value of the potential correlation between cellular immunodeficiency and the frequency of the HPV infection transmitted by genital tract in women.
Materials and Methods: The study group consisted of 141 potentially healthy women aged between 25 and 30. The distribution of peripheral blood T (CD3+, CD4+, CD8+), B (CD19+) lymphocytes and NK cells were studied by flow cytometry method. To identify HPV infections, DNA extracted from cervical squamous epithelial cells was tested by PCR.
Results: In the group of 141 women HPV infection was confirmed in 13 cases. During cytometric analyses we observed decreased number of lymphocytes: CD3+ in 9 cases, CD19+ in 19 cases, CD4+ in 8 cases, CD8+ in 5 cases, CD16+CD56+ in 29 cases.
Conclusions: No correlation between HPV infection and relevant deviation from the norm of the investigated immunological parameters has been observed.
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Diane Da Silva
Los Angeles, USADiane Da Silva1, Maaike Koopman1, Heike Brand1, Gregory McKee2, W. Martin Kast1. 1Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA, USA; 2Akela Pharma Inc, Austin, TX, USA. Background: The HPV family of viruses establishes persistent infections because it has evolved mechanisms that allow it to evade the human immune system. Studies from our laboratory have identified HPV-mediated suppression of antigen presentation by Langerhans cells(LC) as a key mechanism through which HPV evades immune surveillance. PolyICR is a stable TLR3 agonist that is a broad inducer of innate immunity and is being developed as a vaccine adjuvant and antitumor agent. An important feature of PolyICR is its ability to enhance dendritic cell expression of cell surface markers, cytokine production and functional activation of T cells.
Objectives: In this study, we determined whether PolyICR can overcome HPV-induced immune suppression by functionally activating LC in the presence of HPV16 and inducing activation of HPV16-specific T cells.
Methods: Human LC were analyzed for the expression of MHC and T-cell co-stimulatory molecules, production of Th1 inducing cytokines, in vitro migration, and activation of HPV16-specific T cells when LC were exposed to HPV16 VLP and subsequently to PolyICR.
Conclusions: PolyICR was able to activate LC that had been pre-exposed to HPV16 VLP such that expression of MHC, CD40, CD80, CD86, and CD83 were highly upregulated and LC secreted high amounts of Th1 and inflammatory cytokines and chemokines. Upregulation of the chemokine receptor CCR7 resulted in a significant increase in migration capacity. LC incubated with HPV16 VLP and treated with PolyICR induced an HPV16-specific CD8+ T cell response detected by interferon gamma Elispot and MHC tetramer analysis that was absent when LC were exposed to VLP alone. These data suggest that the TLR3 agonist PolyICR is a promising therapeutic molecule that can overcome HPV-induced immune suppression of LC and result in an LC capable of stimulating an anti-HPV T-cell mediated immune response.
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Chung Won Lee
SEOUL, REPUBLIC OF KOREAChung Won Lee1, Min Jong Song1, Keun Ho Lee1, Soo Young Hur1, Jong Sup Park1. 1Department of Obstetrics and Gynecology, School of Medicine, Catholic University, Seoul St. Mary's Hospital, Seoul, Korea. Objectives: The study aims to evaluate the effect of a human papillomavirus type 16 (HPV16) E7 synthetic peptides on the antigen-specific T-cell response in Carcinoma in situ (CIS) and cervical cancer patients.
Methods: We characterized the HPV-16 E7 specific T-cell epitopes using E7 overlapping peptide pools with peripheral blood lymphocytes obtained from normal healthy donors and HPV-16+ 5 CI patients and 3 invasive cervical carcinoma patients with informed consent. We then analyzed the difference in the HPV-16 E7-specific T-cell immune responses in patients during or after treatment of the lesion by ELISPOT assay.
Results & Conclusions: All of CIS patients were underwent loop electrosurgical excision procedure (LEEP) and all of cervical carcinoma patient were type III radical hysterectomy. Analysis of peripheral blood lymphocytes obtained from patients with HPV-16+ CIS and cervical carcinoma showed that the HPV-16+ E7 peptide pool 2-3 (aa 16-55) specific CD4+ T-cell immune response was significantly higher than other peptide pool. The HPV-16 E7 peptide specific T-cell immune response correlates with regression of established HPV16+ lesions and freedom from disease recurrence. Thus, this E7 epitope may be useful for the characterization of HPV-specific immune responses in patients infected with HPV-16 or immunized with HPV vaccines. |
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Paul Chan
Shatin, ChinaPaul Chan1, Shih-Jen Liu2, T.H. Cheung1, Winnie Yeo1, SM Ngai1, Jo Cheung1, Stephen Man3. 1The Chinese University of Hong Kong, Hong Kong; 2National Health Research Institute, Taiwan; 3Cardiff University School of Medicine, Cardiff, UK.
Background: A relatively high prevalence of HPV58 has been observed among invasive cervical cancers (ICC) from certain parts of the world, especially the eastern Asia, where a vaccine targeting specifically against HPV58 may be needed.
Subjects and Methods: T-cell responses to HPV58 L1, E6 and E7 peptides among Hong Kong Chinese women with transient infections, cervical intraepithelial neoplasia (CIN) grades 2 and 3, and invasive cervical cancers (ICC), were examined. Overlapping 9-mer peptides were screened using an in-vitro peptide binding assay, and followed by an interferon-γ enzyme-linked immunospot (IFN-γ ELISPOT) assay using peripheral blood mononuclear cells (PBMCs). Altogether, 91 women with HPV58 infection; including 32 transient infections, 16 CIN2, 15 CIN3, and 28 ICC, were examined.
Results: L1 peptides located at amino acid positions 296-304, 327-335, 101-109 and 469-477 showed positive IFN-γ ELISPOT results from 4 HLA A11 subjects, and the responses were mainly observed from women with transient infections. On the other hand, responses to E6 and E7 peptides were mainly observed from subjects with CIN2 or above. Two HLA A11-restricted E6 peptides (amino acid positions 64-72 and 94-102), and 3 HLA A11-restricted E7 peptides (positions 78-86, 74-82, and 88-96) showed positive responses. One HLA A2-restricted E6 peptide located at the position 99-107 had elicited positive responses from 2 CIN2 subjects. Another HLA A24-restricted L1 peptide located at position 468-476 was found to have positive responses from 2 CIN2 subjects.
Conclusions: T-cell responses were of low intensity and only detected in a small proportion of infected subjects across different grades of cervical lesion. The E6 and E7 epitopes identified in this study can be considered for further investigation for their role as therapeutic vaccines against HPV58-associated cervical neoplasia.
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Paul Chan
Shatin, ChinaPaul Chan1, Shih-Jen Liu2, Jo Cheung1, T.H. Cheung1, Winnie Yeo1, Stephen Man3. 1The Chinese University of Hong Kong, Hong Kong; 2National Health Research Institute, Taiwan; 3Cardiff University School of Medicine, Cardiff, UK. Background: HPV52 has been found in a relatively high prevalence among cervical neoplasia in East Asia. Vaccines target specifically at HPV52 may be required for this region. The E6 and E7 proteins encoded by HPV are prime targets for therapeutic vaccine development.
Subjects and Methods: 95 Hong Kong Chinese women with confirmed HPV52 infections; including 33 transient infections, 17 CIN 2, 15 CIN3, and 30 invasive cervical cancers had peripheral blood mononuclear cells collected to measure the T-cell responses using interferon-γ enzyme-linked immunospot (IFN-γ ELISPOT) assay.
Results: Epitopes located at amino acid positions 103-111, 332-340, 342-350 and 373-381 of the L1 protein; and at 27-35 and 86-94 of the E6 protein; and at 1-9 and 27-35 of the E7 protein, were found to elicit positive responses from HLA A11 individuals. A24-specific epitopes included 60-68 and 98-106 of the L1 protein, 42-50 and 59-67 of the E6 protein, and 24-32 of the E7 protein. One epitope (99-107) of the E6 protein showed positive responses for HLA A2 subjects.
Conclusions: The proportion of subjects showing detectable T-cell responses and the intensity of responses were low across all grades of cervical neoplasia suggesting that immune evasion mechanisms had set on early in the course of disease progression. T-cell responses against L1 were mainly confined to subjects who had recently cleared an HPV52 infection, whereas E6- and E7-specific responses were mainly observed from subjects who had developed cervical neoplasia. Further studies are warranted to evaluate the usefulness of these epitopes as therapeutic vaccine candidates.
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Paul Chan
Shatin, ChinaPaul Chan1, Jo Cheung1, Alfred Luk1, Wendy Ho1. 1Department of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China . Intoduction: HPV58 is more common in Asia and ranks the second or third among cervical cancers from Chinese populations.
Method: A total of 571 HPV58 samples, including 521 cervical scrapes and biopsies, and 50 penile scrapes collected form 15 countries were sequenced for the full length of E2, E4 and E5 genes. The phylogenetic relationship among different HPV58 variants was analysed by constructing maximum likelihood (ML) phylogenetic trees using PAUP* 4.0 b10.
Results: A total of 410 isolates were successfully sequenced for the E2 and E4 gene, and 421 isolates for the E5 gene. 76 variants of E2 were identified, but none was identical to the prototype. E2-WW01 and E2-WW02 were found in 43.9% and 16.8% of the specimens, respectively. Nucleotide positions 3445, 2935, 3685, 3571 and 2932 were the five most variable sites. The ML tree had a stem-like pattern with a main stem passing through variants E2_WW01, E2_WW39, E2_WW76, E2_WW02, and E2_WW07. 36 variants of E4 were identified, but none was identical to the prototype. E4-WW01 and E4-WW02 was found in 50.5% and 18.8% of specimens, respectively. Nucleotide positions 3445, 3571, 3596, 3411 and 3550 were the five most variable sites. No significant cluster was observed in the ML tree. 47 variants of E5 were identified. The prototype-like variant (E5_WW18) was found in only 1% of isolates. E5-WW01 and E5-WW02 was found in 48.2% and 22.1% of specimens, respectively. Nucleotide positions 3949, 3988, 3957, 4047 and 3930 were the five most variable sites. Only one significant cluster was observed in the ML tree, which only contained 2 variants.
Conclusion: Nucleotide sequence variations occur in the E2, E4 and E5 genes; but the variations are not sufficient for HPV58 lineage grouping. Clinical and epidemiological implication of these sequence variations remain to be established. |
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Satoko Kojima
Tokyo, JapanSatoko Kojima1, Kei Kawana1, Terufumi Yokoyama1, Kensuke Tomio1, Shiho Miura1, Katsuyuki Adachi1, Katsutoshi Oda1, Tomoyuki Fujii1, Shiro Kozuma1, Yuji Taketani1. 1Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, Tokyo, Japan. Introduction: Given that the cervical intraepithelial neoplasia (CIN) develops in the cervical mucosa followed by human papillomavirus (HPV) infection, the mucosal cytotoxic (type1) immunity against HPV is necessary for control of CIN. The aim of this study was to establish a method for measuring mucosal anti-HPV cellular immune response and to examine the relationship between the mucosal immunity and the CIN course. In this study, we focused on mucosal type1 immune responses to HPV16 E7 oncoprotein.
Patients and Methods: Cervical lymphocytes (105-6 cells) were obtained from cervix of 27 patients with CIN lesions by Cytobrash under written informed consent. Among them, 17 patients with CIN 2-3 were positive for HPV16, and 10 patients with CIN 1/2 were positive for other HPV ("Other HPV group"). Seventeen patients with CIN 2-3 were grouped according to their course; 8 patients who received conization after sampling due to progression were classified into "Cone group", and 9 who dispensed with conization after sampling due to stable diseases or regression to CIN1 into "Treatment-free (TF) group". Cervical lymphocytes were examined for ELISPOT assay to detect HPV16 E7-specific IFN-gamma producing cells. Numbers of E7-specific IFN-gamma producing cells (anti-E7 type1 immune cells) were compared between the three groups.
Result: FACS analysis revealed that 3.7-20% (median=6.0%) of the cervical lymphocytes expressed CD3 (CD4:CD8=6:4) whereas 0.1-0.5% expressed CD19. Number of anti-E7 type1 immune cells of TF, Cone and Other HPV groups was 63-126 (average=98.6), 0-70 (average= 36.0), and 0-71 (average=42.7) /104 cells, respectively. The cell number of TF group was significantly higher when compared to either Cone or Other HPV group (t test; p<0.005).
Conclusion: Patients who induced mucosal E7-specific type1 immune responses can control CIN2-3 lesions spontaneously. The number of anti-E7 type1 immune cells in the cervical mucosa was a promising-biomarker to determine a fate of CIN2-3 lesions.
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Joao Manuel Grisi Candeias
Botucatu, BrazilB. Maira Mösch1, Larissa Marcolino Doddi2, Jossimara Polettini2, Danielle Ferreira da Silva3, Joao Manuel Grisi Candeias3, Marcia Guimaraes da Silva2. 1Faculty of Earth and Life Sciences, Vrije Universiteit, Amsterdam, The Netherlands; 2Department of Pathology, Botucatu Medical School, São Paulo State University, Botucatu, Brazil; 3Department of Microbiology and Immunology, Botucatu Biosciences Institute, São Paulo State University, Botucatu, Brazil. Background: Current data shows that human papillomavirus (HPV) is the primary causal agent in the development of cervical cancer. Most HPV infections likely involve a balance of Th1 and Th2 type immune responses.
Objectives: To measure cervical levels of the interleukin (IL)-4, IL-6, IL-8, IL-10 and IL-12 from women with HSIL and from women in a control group.
Methods: A total of 40 women with a histopathological diagnosis of HSIL and 38 women without lesions but with high-risk HPV attended in Botucatu Medical School, and Amaral Carvalho Hospital, Jaú, SP. During the speculum exam cervical secretion was collected. The concentrations of the cytokines were determined using quantitative ELISA kits (R&D Systems). The research and genotyping assay of the HPV, in extracted secretions, was carried out on fragments of cervical biopsies obtained through a colposcopic examination of the cervix, using the L1 consensus PCR (GP5+/GP6+) and multiplex PCR.
Results: The median age of the control group was 33 years (22-60) and for the patients with HSIL, 37 years (19-70). Sixty patients (77.0%) were of Caucasian origin, 38.5% of the women were smokers and 23.0% reported lifetime monogamy. Of the women who were sexually active, the median of sexual intercourse was 1 in the control group and 2 in the HSIL group. In relation to oral contraceptives, 44.9% of all patients reported having used. DNA-HPV was detected in all cervical biopsy samples and the most common HPV types identified were, in order of decreasing prevalence, HPV-18, -33 and -16. No significant difference in local IL-4, IL-6, IL-8, IL-10 and IL-12 was found in patients with HSIL compared to the control group.
Conclusions: The production of cytokines in the cervicovaginal secretions is similar in patients with HSIL and patients without lesions contaminated with high-risk HPV. Financial support: FAPESP: 2008/58861-3
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Jozef Adam
Ko?ice, SlovakiaJozef Adam1, Monika Halánová2, Lýdia Čisláková2, Pavol Jarčuška3. 1Department of Gynaecology and Obstetrics, Faculty of Health Care, University of Presov, Presov, Slovakia; 2Department of Public Health, Medical Faculty, P.J. Šafárik University, Košice, Slovakia; 3Department of Infectology and Travel Medicine, Medical Faculty, P.J Šafárik University, Košice, Slovakia. Genital human papillomavirus (HPV) is one of the most common sexually transmitted pathogens. There are more than 40 HPV types that can infect the genital areas. Most people with HPV do not develop symptoms or health problems from it. In 90% of cases, the body's immune system clears HPV naturally within two years. But there is evidence that persistent HPV infection shows a close relationship to the cell-mediated immunity and is associated with T-cell immunodeficiency. A total 669 women (aged 20 to 60, sexually active, without a history of diseases that are related to the immune system) with suspected findings on the cervix were examined. The level of CD3+, CD4+, CD8+ lymphocytes and ratio of CD4+/CD8+ were determined by the direct immunofluorescence method. Of the total 669 women, 444 were HPV negative and 225 HPV positive. In 108 positive cases were detected HR-HPV, in 82 LH-HPV, and in 35 cases were detected both HR and LR HPV type. Comparison of the immunological parameters between HPV positive and HPV negative groups found statistically significant difference in average levels of complement components C3 (p<0.005) and C4 (p<0.001), the number of CD3+, CD4+, CD8+ ratio and CD4+/CD8+ (for all parameters p <0.001). When comparing the immunological parameters subgroup HR-HPV and LR-HPV group with HPV negative women, the difference was only in parameter C3 component of complement. Higher levels of C3 component of complement (p<0.01), higher number of CD3+ lymphocytes (p<0.005), lower percentage of CD4+ lymphocytes (p< 0.001), higher number of CD8+ lymphocytes (p<0.001) and lower CD4+/CD8+ ratio (p<0.01) were detected in the group of HR-HPV positive women in comparison with the group of LR-HPV positive women.
This study was supported by VEGA MŠ SR No. 1/0418/09. |
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Gloria Fernández-Tilapa
Chilpancingo, MéxicoGloria Fernández-Tilapa1, Hilda Jiménez-Wences1, Fabiola Padilla-Islas1, Dinorah Nashely Martínez-Carrillo1, Luz del Carmen Alarcón-Romero2, Amalia Vences-Velázquez3, Berenice Illades-Aguiar4. 1Laboratorio de Investigación Clínica, Unidad Académica de Ciencias Químico Biológicas, Universidad Autónoma de Guerrero, Chilpancingo, México; 2Laboratorio de Citopatología, Unidad Académica de Ciencias Químico Biológicas, Universidad Autónoma de Guerrero, Chilpancingo, México; 3Laboratorio de Inmunobiología y Diagnóstico Molecular, Unidad Académica de Ciencias Químico Biológicas, Universidad Autónoma de Guerrero, Chilpancingo, México; 4Laboratorio de Biomedicina Molecular, Unidad Académica de Ciencias Químico Biológicas, Universidad Autónoma de Guerrero, Chilpancingo, México.
Background: The host response to malignant tumors is a primary function in cellular immunity, modulated by Th1 cytokines. Objective: To determine RNA expression of IFN-γ, IL-12, IL-18 in premalignant lesions and cervical cancer associated with HPV infection.
Methods: Were analyzed 123 biopsies with diagnosis of low grade squamous intraepithelial lesion (LSIL), 40 with high grade squamous intraepithelial lesion (HSIL), 48 of cervical carcinoma (CC) and 66 cone fragments obtained from women with colposcopy findings of HPV infection and/or cervical lesion. Thirty-tree cervical scrapes from cytologically normal women (CN) were studied too. PCR was used for HPV screening with the MY09/MY11 and GP5+/GP6+ primers; RFLPs or sequenciation were used for typing and cytokines mRNA expression was detected by RT-PCR.
Results: The prevalence of HPV infection was 83% in LSIL, 77.5% HLSIL and 100% in CC (Table-1). The most common types viral were 16, 18, 58 and 31. IFN-γ mRNA was found in 27.6% of the CN samples, 3.4% of LSIL, 15% of HSIL and 39.4% of CC. The SIL and CC samples that expressed IFN-γ had high oncogenic risk HPV DNA; 48.5% of CC expressed IL-18 mRNA, and the level of transcripts was higher in CN than in CC; the lowest levels were found in SIL. Only 65% of IL-18 positive biopsies were HPV-positive of which 43% were HPV 16-positive. None of the samples with SIL or CC expressed the message of IL-12p35 and p40 subunits and only 25% of the CN samples had IL-12p35 mRNA. Only 48% of the samples were IFN-γ and IL-18 mRNA positives (Table-2).
Conclusions: In cervical tissues infected by HPV there is important dysregulation in IL-18, IL-12 and IFN-γ, which are key in the Th1 activation response and favoring persistent HPV infection and increasing the risk to develop premalignant lesions as well as cervical cancer.
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Wendy C.S. Ho
HKSAR, ChinaWendy C.S. Ho1, Jo LK Cheung1, Alfred CS Luk1, Paul KS Chan1. 1Department of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China. Introduction: HPV58, though rare worldwide, ranks the second or third among cervical cancers detected from Chinese populations. The E7 oncoprotein disrupts cell cycle regulation and is a key leading to the transformation of infected cells.
Method: A total of 571 HPV58-positive samples, including 521 cervical scrapes and biopsies, and 50 penile scrapes collected form 15 countries were analysed. The E7 gene was sequenced and aligned by CLUSTAL X to identify HPV58 E7 variants. The entropy of each variable nucleotide position was calculated. The phylogenetic relationship among different HPV58 variants was analysed by maximum likelihood (ML) trees generated by PAUP* 4.0 b10, and Bayesian phylogenetic trees constructed by MrBayes v3.1.2.
Results: A total of 441 isolates were successfully sequenced with 22 variants identified. The prototype-like variant being detected in 5.7% of isolates, was the forth most common variant found. E7-WW01 and E7-WW02 were found in 49.7% and 21.1% of the specimens, respectively. The second and forth most common variant, E7-WW02 and E-7-WW04, were found more commonly in Asia. Nucleotide positions 694, 760, 632, 793 and 801 were the five most variable sites, with an entropy value of 0.69, 0.57, 0.53, 0.40 and 0.39, respectively. All these variations involved amino acid changes. The 22 variants of E7 were divided into two main clusters in the ML tree, whereas three significant clusters were observed in the Bayesian tree.
Conclusion: Multiple nonsynonymous sequence variations occur in the E7 gene of HPV58. It is worthwhile to further study the oncogenic implication of these variants.
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Paciência Damião
Rio de Janeiro, BrasilPaciência Damião1, Michelle Silva1, Miguel Moreira2, Alcina Nicol1, Maria Emilia de Lima3, José Chiovo3, Claude Pirmez1. 1Instituto Oswaldo Cruz, Rio de Janeiro, Brazil,; 2National Institute of Cancer, Rio de Janeiro, Brazil; 3Principal Military Hospital, Luanda, Angola . Background and Objectives: Cervical cancer and the other HPV-associated cancers constitute a major public health worldwide. In Angola, this is the most frequent cancer affecting women between 15 and 44 years of age. So far, in a country with a high incidence of cervical cancer, no reports regarding the HPV genotypes prevalence in women are available. The purpose of this study was to identify HPV types in archival abnormal Pap smears from women attended at Luanda, Angola, Middle Africa. Material and Methods: A total of 55 archival Pap smears with cellular abnormalities from Luanda, Angola were examined. DNA was isolated from cytology slides, amplified through PCR using primers MY09/MY11 and GP5+/GP6+. The amplicons were sequenced using the DNA Sequencing Plataform (PDTIS-FIOCRUZ). The sequences were aligned and edited using the software MEGA 4.0 and type characterization of HPV strains.
Results: HPV DNA was detected by PCR in 81,3% (48/59) of archival Pap smears samples. HPV types were identified in 85,4% (41/48) of these cases, high-risk types such as HPV16, HPV18, HPV52, HPV66 and HPV58 being observed in 45,5% (28/41) and low-risk HPV6 in 31,7% (13/41) of the cases. Conclusions: The present data indicates the presence of high-risk HPV types commonly found in other parts of the world, such as HPV 16,18 , 33 52, 58, 66, and may contribute to increasing the knowledge of HPV epidemiology and designing adequate vaccine strategies to Middle Africa population.
Support: Principal Military Hospital, Luanda, Angola and IOC-FIOCRUZ, Brazil
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Sven Tiews
Soest, GermanySven Tiews1, Winfried Steinberg1, Wladimir Schneider1, Christoph Hanrath1. 1Dr. Steinberg und Partner, Laboratory for Cytopathology, Soest, Germany. Objectives: The purpose of this study was to determine the positive predictive value of HR-HPV testing for precancerous lesions of the cervix uteri. Therefore, we focused on the diagnostic accuracy of testing for one or more of the HPV types 16, 18 and 45 for all HR-HPV positive women. HR-HPV infections with subtypes 16, 18 and 45 have demonstrated a higher risk of developing cervical cancer [Bulk S, et al. Br J Cancer 2006; 94:171-5].
Methods: Between 2007 and 2008 a total of 586 women were recruited: a group of 477 women with a history of known cervical lesions and/or HPV infections (eligibility criterion: HR-HPVDNA positive test result with HC2T) and a group of 109 women who were examined as part of their routine cervical cancer screening. Baseline HR-HPV status was measured at enrolment with the FDA-approved Hybrid Capture® 2 HPVDNA Test and the HR-HPV 16/18/45 Probe Set Test (HC2T, PST; QIAGEN, Hilden, Germany). Cervical smears were classified according to the Second Munich Nomenclature (1989). The results were converted to the nearest equivalent in the Bethesda system. In general, study subjects were followed up semi-annually for a period of 11/2 years. The histopathological endpoint of CIN 2-3 lesion was used as a surrogate endpoint.
Results: To date data for 43.5 % of the risk group and for the complete control group were available. CIN 2-3 was confirmed in 77 HR-HPVDNA positive women. 85.7% of these lesions were positive for one or more of the HR-HPV types 16, 18 and 45 (PST+). 88.2% (60/68) of the histologically confirmed CIN 3 lesions and six out of nine (66.6%) CIN 2 lesions were positive PST+. Furthermore, all women with a histologically confirmed squamous cell carcinoma (n = 4) were PST+. Besides, three (50%) out of six detected CIN 1 lesions were PST+. Nonetheless, histology confirmed no malignancy in three cases. Two of them were PST+. Within the next few month study results will be updated.
Conclusion: These preliminary results demonstrate that starting cervical cancer screening at the age of 20 years remains important as seventeen (25%) of the 68 histologically verified CIN 3 lesions arose in women who were younger than 30 years. Our data suggest that adding an HR-HPV test that detects one or more of the HR-HPV types 16, 18 and 45 in conjunction with cytology could help to identify women with an underlying cervical lesion who have an elevated risk of developing severe cervical lesions. |
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Anja Kovanda
Ljubljana, SloveniaAnja Kovanda1, Boštjan J. Kocjan1, Katja Seme1, Mario Poljak1. 1Institute of Microbiology and Immunology, Faculty of Medicine, Ljubljana, Slovenia. More than a hundred HPV genotypes have been discovered so far, many being etiologically linked with particular mucosal or skin diseases. One of the most common HPV-associated conditions, verrucae vulgaris or common warts are caused by members of species 2 and 4 of genus Alpha-papillomavirus, and several species of Gama-, Mu- and Nu-papillomavirus genera.
In this report we describe the isolation and characterisation of a novel Alpha-papillomavirus HPV-125 from a hand wart of an immunocompetent individual. The full genomic DNA of this novel genotype was cloned using the overlapping PCR method. The sequencing of the full 7,809 bp genome showed HPV-125 to possess the classical genomic organisation of Alpha-papillomaviruses. The phylogenetic comparisons of the L1 gene with all other HPVs identified so far, showed HPV-125 to be a distinct genotype closely related to genotype HPV-3 (85.4% identity of the L1 gene) from species 2 of Alpha-papillomaviruses, a group etiologically linked with the development of cutaneous lesions. The amino acid sequences of the two proteins making up the viral envelope of HPV-125, L1 and L2, showed 90.9% and 90.3% identity with the L1 and L2 proteins of HPV-3. Unlike HPV-3, but similarly to other members of species 2, the genome of HPV-125 also contains a short ORF for putative E4 protein within the E2 gene, the expression of which needs to be further investigated.
The characterisation of a novel Alpha-papillomavirus genotype gives new insight into the phylogenetic diversity of cutaneous HPVs and provides helpful novel information for the potential development of future prophylactic or therapeutic vaccines against common warts and other cutaneous lesions. |
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Anja Kovanda
Ljubljana, SloveniaAnja Kovanda1, Bo?tjan J. Kocjan1, Katja Seme1, Mario Poljak1. 1Institute of Microbiology and Immunology, Faculty of Medicine, Ljubljana, Slovenia .
The knowledge of genomic diversity of human papillomaviruses is increasing rapidly with approximately 150 HPV genotypes being fully characterised. Most novel HPVs have been discovered within the Beta-papillomavirus group which are clinically important mostly for immunodeficient individuals. In this report we describe the isolation and characterisation of a novel Beta- papillomavirus HPV-151 from scrotal hair of an immunocompetent individual. As the small amount of HPV DNA in the original sample did not permit cloning, the genome of HPV-151 was first amplified using isothermal whole genome amplification and then cloned using the overlapping PCR method. The sequencing of the full 7,386 bp genome showed HPV-151 to possess the genomic organisation of Beta-papillomaviruses. The phylogenetic comparisons of the L1 gene with all other HPVs identified so far, showed HPV-151 to be a distinct genotype belonging to species 2 of Beta-papillomaviruses and to be most closely related to genotype HPV-22 (87.0% identity of the L1 gene). The amino acid sequences of HPV-151 L1 and L2 proteins showed 92.0% and 87.8% identity, respectively with the corresponding proteins of HPV-22. It is unclear why species 2 of Beta-papillomaviruses comprises such a large number of HPV genotypes, but the sheer diversity of this group points to its evolutionary success, with its underlying reasons meriting further research. In addition to improving our understanding of phylogenetic diversity of Beta-papillomaviruses the genomic characterisation of HPV-151 provides further information for the development of new diagnostic tests and potential future prophylactic or therapeutic vaccines directed against Beta-papillomaviruses.
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Anja Kovanda
Ljubljana, SloveniaAnja Kovanda1, Bo?tjan J. Kocjan1, Katja Seme1, Mario Poljak1. 1Institute of Microbiology and Immunology, Faculty of Medicine, Ljubljana, Slovenia . More than one hundred fifty HPV genotypes have been discovered so far, many being etiologically linked with particular mucosal or skin diseases. Although infection with Beta-papillomaviruses is mostly harmless in immunocompetent individuals, this group can cause both benign and malignant lesions in immunodeficient individuals. In this report we describe the isolation and characterisation of a novel Beta-papillomavirus HPV-150 from eyebrow hair of an immunocompetent individual. Because of minute amounts of viral DNA in the sample, the genome of HPV-150 was first amplified using isothermal whole genome amplification and then cloned using the overlapping PCR method.
The sequencing of the full 7,436 bp genome showed HPV-150 to possess the genomic organisation of Beta-papillomaviruses. The phylogenetic comparisons of the L1 gene with all other HPVs identified so far, showed that HPV-150 is a distinct genotype most closely related to genotype HPV-96 (79.6% identity of the L1 gene). HPV-96 is at present the only representative of species 5 of Beta-papillomaviruses which has been etiologically linked with the development of pre-malignant and malignant cutaneous lesions. The amino acid sequences of HPV-150 L1 and L2 proteins showed 88.1% and 86.4% identity, respectively with the corresponding proteins of HPV-96.
The genomic characterisation of HPV-150 provides helpful new information for the development of new diagnostic tests and potential future prophylactic or therapeutic vaccines directed against Beta-papillomaviruses. |
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Benjamin Smith
New York, United StatesBenjamin Smith1, Zigui Chen1, Koenraad van Doorslaer1, Mark Schiffman3, Robert Burk1,2. 1Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, USA; 2Department of Pediatrics, Division of Genetics, and Departments of Epidemiology and Population Health, Obstetrics, Gynecology and Woman's Health, and Albert Einstein Cancer Center, Albert Einstein College of Medicine, New York, USA; 3Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, DHHS, Bethesda, USA. Background: HPV DNA samples are now abundant. DNA sequencing, although progressively cheaper, easier and faster, is not yet at a level where we may feasibly obtain complete genome sequences (CGS) for all available samples. Analysis of HPV CGS information will allow insight into the genetic basis of viral pathogenicity.
Methods: A genotype imputation algorithm based on maximum partial matching was developed, as was an algorithm to assess informativeness for imputation across the entire genome. Genotype imputation for partially sequenced HPV16 DNA samples was performed using the imputation algorithm. The informativeness algorithm utilized a library of 62 previously sequenced complete genomes.
Results: Reconstruction of complete genotypes from partially sequenced HPV16 samples - URR and/or E6 regions - was performed. The low rates of recombination in HPV allowed >88% of total (ntotal=537) SNPs to be imputed accurately from these small fragments. Further analysis of complete HPV16 genomes identified regions, other than URR/E6, of higher informativeness that would lead to greater imputation accuracy, and thus may guide future sample processing.
Conclusions: The goals and challenges of genotype imputation and genome wide association studies in viral genetics in general, and HPV in particular, are substantially different from those of human and animal genetics where recombination is active. This study draws attention to the concept of lineage fixation in essentially non-recombinant DNA sequences, such as HPV, which allows genotype imputation based on small partial sequences, using computational methods, to supplement direct determination of CGS.
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Monica Molano
Bogotá, ColombiaOscar Buitrago1,3, Nicolas Morales1,3, Carolina Martín1,3, Antonio Huertas1, Pablo Moreno1, Teresa Martinez1, Monica Molano1. 1Grupo de Investigación en Biología del Cancer, Instituto Nacional de Cancerología, Bogotá, Colombia; 2Facultad de Medicina, Universidad Nacional de Colombia, Bogotá, Colombia; 3Grupo Investigación Epidemiológica, Instituto Nacional de Cancerología, Bogotá, Colombia.
Background: Human papillomavirus type 58 (HPV 58) is highly prevalent in women with normal cytology in the Colombian population. Also this type is highly prevalent in women with high grade squamous intraepithelial cervical lesions (HGSIL) and in women with invasive cervical cancer worldwide. There are few studies of E6 and E7/HPV 58 variants and some of them have shown an association of variants presence with a higher risk of HGSIL and cervical cancer. In Colombia there are no studies of E6 and E7/HPV58 variants in women with normal cytology.
Objectives: To identify E6 and E7/HPV58 variants in cervical scrapes of women with prevalent HPV 58 infections in the Bogotá cohort.
Methods: The E6 and E7 regions of 34 samples HPV 58 positives were amplified using the E6F1/E7R1 primers for the ORF E6 and E7P1/E7P2 primers for the ORF E7. E6 and E7/HPV 58 variants were detected using automated direct sequencing. The reference sequence of HPV 58 was used to compare sequences.
Results: 27/34 samples amplified the ORF E6 and E7 (79.4%). Twenty one of these samples have the T307/A694/G744/A761 variant (77.8%), three samples have the T307/T632/G744 /A760 variant (11.1%), one sample has the A169/T307/A599/A694/G744 /A761 variant (3.7%), one sample has the T307/A694/G744/A761/G763 variant (3.7%) and one sample has the T307/G744 variant (3.7%). None of the samples had the reference sequence of HPV58.
Conclusions: Five different variants were identified in the samples analyzed: 3 previously identified in Asian population and 2 new variants (A169/T307/A599/A694/G744/A761 variant and T307/A694/G744/A761/G763 variant). The follow up analysis of these variants will give important information about the role of these variants in the persistence of HPV 58 infection and in the development of cervical intraepithelial lesions.
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Ana Cid
OURENSE, SPAINSonia Perez1, Ana Cid2, Monica Pato4, M Teresa Saran3, Sonia Gonzalez1, M Carmen Alvarez2, M Gloria Esteban2, Julio Torres1, Isabel Lopez-Miragaya1, María Oña5. 1Microbiology Unit, Complejo Hospitalario Universitario Vigo, Vigo, Spain; 2Microbiology Unit, Complejo Universitario Ourense, Ourense, Spain; 3Gynecology Unit, Complejo Hospitalario Universitario Vigo, Vigo, Spain; 4Gynecology Unit, Complejo Hospitalario Ourense, Ourense, Spain; 5Microbiology Unit, Hospital Universitario Central de Asturias, Oviedo, Spain. Objective: To detect nucleotide polymorphisms within the LCR, E6 and L1 genes of HPV 16 in general population of Northwest Spain.
Materials and Methods: 2 protocols were carried out for HPV detection and genotyping in endocervical samples received from women attending a gynecological outpatient department during the year 2009: nested PCR MY09/11-GP5/6 (followed by DNA sequencing) in one Microbiology Unit and Amplicor HPVtest and Linear Array HPV Genotyping Test (Roche) in another one. HPV 16 was detected in 170 women. For identification of HPV 16 sequence variation, 761bp from the LCR-E6 region (Nt 7701-591) and 422bp from the L1 region (Nt 6599-7021) were amplified (Expand HiFi, Roche) as previously described (Xi LF, 2006. Wheeler, 1997) and sequenced (BigDye 1.1. Abi Prism 3100-Avant). All sequences from a sample were combined for the analysis. For the multiple alignment and the phylogenetic analysis the reference sequences European (E), Asian (As), African (Af1, Af2), Asian-American (AA) and NorthAmerican (NA) were included (Yamada, 1995).
Results: In 145 cases (average age 35.7 years) sequences of the three regions were available. The distribution of HPV 16 variants was: 131/145 (90.3%) E lineage and 14/145 (9.7%) non E variants. Of the 131 E lineage sequences, 66 (50.4%) grouped into the E-350G cluster (52 European Patron E-P, 4 E-C109G, 4 E-G131G, 2 E-C188G, 1 E-m), 61 (46.6%) into the E-350T cluster (all of them E-P) and 4 (3.1%) into the E-350G/350T one (E-P). Among the non E variants there were 7 AA, 4 Af2, 2 Af1 and 1 NA strains.
Conclusions: European lineage was the most prevalent HPV 16 variant. There was a homogeneous presence of E-350G and E-350T. No Asian Variant was found in the studied population. |
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R.C Stocco
São Paulo, BrazilP. Miranda M1,4, P. Felix P1,4, J.L. Lima Filho5, C. Carneiro M2, I. Silva DCG3, R. Carvalho F1, A. Lima A1,2, W. Beçak 1, R.C. Stocco1. 1Laboratório de Genética, Instituto Butantan, São Paulo,Brazil; 2Escola de Farmácia - UFOP, MG, Brazil; 3Laboratório de Tocoginecologia - UNIFESP, São Paulo, Brazil; 4Departamento de Morfologia - UNIFESP, São Paulo, Brazil; 5Laboratório de Imunopatologia Keizo Asami- UFPE, Recife, Brazil. We have analyzed the prevalence of human papillomavirus (HPV) types in 398 (18-65 years), sexually active women, attending routine gynecological evaluation in Ouro Preto, Minas Gerais, Brazil. In this region, bracken fern (Pteridium aquilinum) is included in human diet. Cytology analysis was done according to Bethesda, 2001. Endocervical samples were tested for HPV DNA with L1 primers MY09 and MY11, RFLP and sequencing. Univariate risk factor analysis was performed by chi-square test using SPSS software version 15.0.Table 1 and Figure 1 summarize the results. HPV was detected in 11% of all women (44, of which 32 with only one viral type). Twelve HPV types were found: the most frequent types were HPV16, HPV6, HPV61, HPV66 and HPV83, different from other regions in Brazil. The presence of high-risk HPV infection occurs more frequently in younger age, later sexual initiation, less children, single women, with many sex partners, urban residence and, surprisingly, it is not increased with bracken fern consumption. The analysis of codon 72 p53 polymorphism showed lower frequency of Proline homozygotes. Further studies mapping the distribution of HPV types worldwide and co-factors considering the characteristics of each population are necessary to direct future vaccine development.
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Ignacio G. Bravo
Valencia, SpainBeatriz Mengual Chulia1, Marc Gottschling3, Ignacio G. Bravo1,2. 1Genomics and Health, Center for Public Health Research (CSISP), Valencia, Spain; 2CIBER en Epidemiología y Salud Pública (CIBERESP), Barcelona, Spain; 3Department of Biology, Systematic Botany, Ludwig-Maximilians-Universität München, Munich, Germany. Despite the advances in basic and medical biology of PVs, our understanding on their origin and evolution is still based on notions and generalizations lacking in many cases a solid scientific foundation:
i) PV-host coevolution is commonly assumed and considered the primary, if not the only, driving force for PV diversification, despite the evidences for lack of global congruence between the phylogenies of PVs and their hosts;
ii) PVs are considered to be host species-specific, ignoring evidences for interspecies transmission and the possible role of arthropod vectors;
iii) potential for PV recombination is minimized, despite bioinformatic and experimental results against this view;
iv) PVs are considered epitheliotropic, although genomic and transcript material are present in a number of tissues;
v) knowledge about PV diversity is at a stage comparable to that of organismal taxonomy during Linnean times, 250 years ago;
vi) in the current L1-based ICTV PV taxonomy, genera are the highest-level category within Papillomaviridae, and all genera are considered to be biologically equivalent categories; this view ignores shared structural genome organisations across genera, and the strong support for the existence of monophyletic clusters of genera. As a consequence of this flat view on the PV landscape, alpha PVs (e.g. HPV16) and beta PVs (e.g. HPV38) are compared as if they were closely related, potentially misleading the research in virus-associated transforming mechanisms;
vii) host-based nomenclature (as in Human PV, Bovine PV or Canine PV) and tropism-based nomenclature (as in mucosal human PVs as opposed to cutaneous human PVs) generate the misleading impression that viruses infecting the same host or with a similar tropism are monophyletic.
We want to present an updated view on PV evolution in the last 150 millions years, and illustrate how the knowledge of PV phylogeny can help and guide our experimental research.
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Pierre Martin-Hirsch
Preston, UKPierre Martin-Hirsch1, Jemma Kelly1, Cara Martin2, Walter Prendiville2, John O'Leary2, Frank Martin1. 1Centre for Biophotonics, Lancaster Environment Centre, Lancaster University, Lancaster, UK; 2Department of Pathology, Coombe Women and Infants University Hospital, Dublin, Ireland. Human papillomavirus (HPV) is a sexually-transmitted infection associated with cervical cancer. Over 100 HPV types have been identified of which 13 are high-risk oncogenic. In unvaccinated women worldwide, the incidence of cervical cancer from HPV 16 and 18 will remain. Cervical cytology can be graded from normal (atypia-free) to low-grade (borderline nuclear abnormality/cervical intraepithelial neoplasia [CIN] 1) to high-grade (CIN2/3). Infrared (IR) spectroscopy is a non-destructive technique that allows the acquisition of a biochemical-cell fingerprint based on vibrational states of chemical bonds in the interrogated sample. Exfoliative cervical cytology specimens (n=147) were retrieved, graded by a cytologist and HPV-tested/genotyped using Hybrid Capture 2 and the Roche HPV Linear Array. Additionally, the spectral signatures cervical cell lines C33A, HeLa and SiHa were examined. After washing, cellular material was transferred to low-E glass slides and interrogated using attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy; 10 spectra were acquired per sample. Given the complex nature of a resultant dataset consisting of thousands of variables (wavenumbers), we used multivariate analysis for data reduction and information retrieval. Principal component analysis coupled with linear discriminant analysis (PCA-LDA) gave a visual representation of the data (scores plot) and, identification of the wavenumbers and consequent biochemical entities responsible for segregation (loadings plot). Immortalised cell lines were readily distinguishable from each other. It was difficult to segregate categories of cytology associated with specific HPV infection types. However, in low-grade cytology infected with high-risk oncogenic HPV 16 or 18, it was possible to segregate women based those aged 20 - 29 y and 30 - 39 y. This suggests a spectral phenotype in exfoliative cervical cytology associated with transient versus persistent HPV infection.
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Nicolas Wentzensen
Investigator, Hormonal and Reproductive Epidemiology Branch, National Cancer Institute, Division of Cancer epidemiology & Genetics
, USANicolas Wentzensen1, Ana Cecilia Rodriguez2, Raphael Viscidi3, Rolando Herrero2, Allan Hildesheim1, Jorge Morales2, Sholom Wacholder1, Mahboobeh Safaeian1, Robert Burk4, Mark Schiffman1. 1DCEG, NCI, NIH, Bethesda, MD, USA; 2Proyecto Epidemiológico Guanacaste, San Jose, Costa Rica; 3Johns Hopkins University, Baltimore, MD, USA; 4Albert Einstein College of Medicine, New York, NY, USA. Background: VLP-based HPV ELISA serology assays only partially predict immunity. A novel competitive Luminex Immunoassay (cLIA) has been developed by M. Esser and colleagues to specifically measure neutralizing antibodies against HPV types included in the quadrivalent vaccine.
Objective: We evaluated whether cLIA measured immunity against subsequent HPV6, 11, 16, and 18 infections in a subcohort of women nested in the Guanacaste Natural History Study.
Methods: We tested women who were HPV6, 11, 16, and 18 DNA negative at baseline and had a subsequent persistent infection or type-related CIN2+ with one of these 4 HPV types, and half of women with a subsequent transient infection (n=369 total). We also tested a sample of women from the cohort stratified by sexual activity and genotype status (n=498). The cLIA results are compared to previous VLP-based ELISA results for HPV16 (other types to be presented).
Results: The reproducibility for HPV16 cLIA testing was very good (R-square 0.95). Only 77/867 women were seropositive by HPV16 cLIA (including 0/69 virgins) compared to 224/867 by HPV16 VLP ELISA (p<0.001). ELISA but not cLIA seropositivity increased with number of previous sexual partners (p<0.001). HPV16 cLIA positivity was associated with protection against subsequent infections or HPV16-associated CIN2+ (OR 0.31, CI: 0.11-1.03). No women developing HPV16+ CIN2+ were cLIA positive. HPV16 ELISA positivity showed weaker protection (OR 0.63, CI: 0.42-1.04) that increased with strong seropositivity (OR 0.43, CI: 0.22-0.85). In paired, mutually-adjusted analyses, cLIA seropositivity was correlated with strong ELISA seropositivity; but cLIA positivity was the important predictor of immunity.
Conclusions: Neutralizing antibodies developing from the natural humoral immune response against HPV can be measured. Detection of neutralizing antibodies against HPV16 is associated with significant protection against subsequent infection and disease; however, the cLIA assay has low sensitivity to measure previous exposure.
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Zizipho Mbulawa
Cape Town, South AfricaZizipho Z. A. Mbulawa1, Dianne J. Marais1, Tim Waterboer4, Michael Pawlita4, David Cotzee2, Anna-Lise Williamson1,3. 1Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa; 2Centre of Infectious Disease, Epidemiology and Research, University of Cape Town, Cape Town, South Africa; 3National Health Laboratory Service, Groote Schuur Hospital, Observatory, Cape Town, South Africa; 4Infection and Cancer Program, German Cancer Research Center (DKFZ), Heidelberg, Germany. Background: This study examined human papillomavirus (HPV) serum antibodies in human immunodeficiency virus seropositive (HIV+) and seronegative (HIV-) women and men.
Methods: Sera from 265 HIV+ women, 171 HIV- women, 158 HIV+ men and 278 HIV- men were analysed for HPV antibodies (combined IgG, IgA and IgM) to the major capsid protein L1 of types 11, 16, 18, 31, 33, 35, 45, 52, 58 by Luminex-based multiplex serology. HPV genotyping in cervical and penile cells was performed by the Roche reverse linear array HPV genotyping assay.
Results: For women, HPV-11 (34%) seroprevalence was highest followed by HPV-31 (30%), HPV-16 (24%), HPV-18 (24%), HPV-45 (22%), HPV-35 (18%), HPV-52 (14%), HPV-58 (11%) and antibodies to HPV-33 (8%) were less prevalent. For men, HPV-11 (14%) seroprevalence was highest followed by HPV-31 (11%), HPV-35 (10%), HPV-16 (7%), HPV-52 (7%), HPV-58 (7%), HPV-18 (5%), HPV-45 (5%) and antibodies to HPV-33 (3%) were less prevalent. Overall, women had a 2-fold to 4-fold higher seroprevalence compared to men, and similar findings were observed when stratified by age. Women had a 2.7-fold higher seroprevalence of multiple (2-9) types compared to men (33%, 143/435 compared to 14%, 54/435, P<0.0001). HPV seroprevalence increased with increasing age in both women and men, however in women seroprevalence decreased in older women (41-67 years). More HIV- women and men had HPV antibodies to all types compared to HIV+ women and men respectively; however the difference was not significant. Presence of antibodies as well as antibody reactivity did not correlate with genital HPV DNA or HPV viral load, respectively.
Conclusion: More women had antibody responses than men, even when stratified according to age and HIV status. Multiple seroprevalence was more common in women. Serum antibodies correlated poorly with the presence of genital HPV DNA.
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Mirte Scherpenisse
Bilthoven, The NetherlandsMirte Scherpenisse1,2, Rutger Schepp1, Hein Boot1, Chris Meijer1,2, Guy Berbers1, Fiona Van Der Klis1. 1Laboratory for Infectious Diseases and Screening, National Institute for Public Health and the Environment, Bilthoven, The Netherlands; 2Department of Pathology, VU University Medical Centre, Amsterdam, The Netherlands. Background and Objectives: HPV infections are one of the most common sexually transmitted diseases. In March 2009 the Dutch government decided to routinely vaccinate all 12 year old girls against HPV-16 and HPV-18 with the bivalent HPV-vaccine (Cervarix, GSK). Data about the seroprevalence of high risk HPV types in the Dutch population are scarce. In 2006/2007 a large serum bank was established by means of a cross-sectional population-based study (ISRCTN 20164309). The serological antibody levels against 7 high risk HPV serotypes will be determined in this serum bank from the prevaccination era using a multiplex immuno assay (MIA) as developed by the vaccine producer. The MIA is validated for measurement of specific antibodies against HPV type 16, 18, 31, 33, 45, 52, and 58.
Methods: The cross-sectional serosurvey consists of samples from men and women between 0-79 years of age, n=6386 (1). The specific antibodies against seven high risk HPV types will be determined in this serum bank with a fluorescent bead-based multiplex immuno assay with virus like particles (VLPs) for the seven HPV serotypes coupled to the microspheres (luminex technology).
Results and Discussion: The antibody levels against the 7 high risk HPV serotypes obtained with the MIA will be presented. This study will contribute to gain insight into incidence of high risk HPV infections in the Dutch population. It is important to study the effects of the routine vaccination and the catch-up campaign started in 2009. Furthermore, possible shifts in the seroprevalence of the high risk HPV types can be distinguished by comparing the antibody levels in a serosurveillance study established in 1995/1996, with the data from the 2006/2007 study.
1. van der Klis et al, Neth. J. Med., 2009, 67:301-8 |
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Mark Esser
Wayne, USAKatie Matys1, Melissa Rose1, Lauren Ciminera1, Tina Green1, Jeff Gatz2, Paul Liberator1, Mark Esser1. 1PPD Vaccines and Biologics Lab, Wayne, PA, USA; 2Bioreclamation, Hicksville, NY, USA. Background: A Food and Drug Administration (FDA) or World Health Organization (WHO)/National Institute of Biological Standards and Controls (NIBSC) Human Papillomavirus (HPV) 6/11/16/18 human reference standard is not available. A 30L reference standard comprised of sera from women vaccinated with GARDASIL® was created to measure the antibody response to HPV 6, 11, 16 and 18 virus-like particles (VLPs).
Methods: A protocol was written and performed to collect serum and plasma from women within 6 months following vaccination with the HPV 6/11/16/18 VLP-containing vaccine, GARDASIL. The sera were screened in a competitive Luminex immunoassay (HPV-4 cLIA) for antibody titers to neutralizing epitopes on the VLPs and 31 individuals with high antibody titers were selected for plasma donation. The pooled reference serum was calibrated to an internal African Green monkey (AGM) reference standard and the WHO 05/134 type 16 reference standard. A set of 81 samples from GARDASIL vaccinees and non-vaccinees were used in the concordance analysis to determine the relative potency of the human reference standard in milli-Merck units/mL (mMU/mL).
Results: The provisional potency of the reference standard was determined to be 1274, 1198, 3544 and 686 mMU/mL for types 6, 11, 16 and 18, respectively and 276 IU/mL for type 16. The serostatus agreements using the two different reference standards were 95.5%, 97.5%, 99.4% and 93.2% for types, 6, 11, 16 and 18 respectively.
Conclusions: Since this reference standard is from women rather than African Green monkeys it is more qualitatively and quantitatively similar to sera from subjects enrolled in clinical trials and samples from sero-epidemiology studies. The results from this potency calibration suggest that this reference standard can be used by multiple laboratories to help harmonize results from vaccine clinical trials and sero-epidemiology surveys.
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Ligia Pinto
Frederick, USAJoseph Dauner1, Yuanji Pan1, Allan Hildesheim2, Clayton Harro3, Ligia Pinto1. 1HPV Immunology Laboratory, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD, USA; 2Division of Cancer Epidemiology and Genetics, NCI, NIH, Bethesda, MD, USA; 3Center for Immunization Research, Johns Hopkins University, Baltimore, MD, USA. Neutralizing antibodies generated by human papillomavirus (HPV) virus-like particle (VLP) vaccines are thought to be the primary correlate of protection against infection with HPV. However, the functional characteristics and the mechanisms involved in generating and maintaining these antibodies is not fully understood. The objective of this study was to analyze the relationship between HPV-specific memory B cells and the antibody response at months 2 and 7 after vaccination with a monovalent HPV-16 VLP vaccine in a cohort of 18-25 year old women. The memory B cell compartment was evaluated using an ELISPOT system and the antibody analysis included an antigen-specific ELISA, a pseudo-virus neutralization assay and a modified ELISA to measure avidity. The results show that all immunized patients studied (n = 19) had a detectable memory B cell frequency one month after the final dose was administered (median frequency = 0.20; IQR = 0.14-0.95). When memory B cell frequencies were correlated against measures of antibody response following vaccination, moderate correlations were observed between HPV-specific memory B cell frequency and anti-HPV IgG (Spearman ρ= 0.62, p = 0.0047) as well as neutralization titers (Spearman ρ= 0.76, p = 0.0002). Very weak correlation existed between memory B cell frequency and antibody avidity levels (Spearman ρ = 0.35, p = 0.14). These results provide a more comprehensive understanding of the kinetics and magnitude of the humoral response generated in response to a HPV VLP vaccine. The results suggest that while frequencies of HPV-specific memory B cells generated post vaccination are predictive of HPV-16 antibody levels generated by the vaccine, antibody avidity indices cannot be predicted from the frequency of memory B cells or the antibody titer levels. This, in turn, raises the need to better understand the role of avidity in long-term protection against infection following vaccination with the HPV vaccine. Funded by NCI Contract No. HHSN261200800001E.
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Ligia Pinto
Frederick, USAJoseph Dauner1, Yuanji Pan1, Allan Hildesheim2, Troy Kemp1, Carolina Porras3, Ligia Pinto1. 1HPV Immunology Laboratory, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD, USA; 2Division of Cancer Epidemiology and Genetics, NCI, NIH, Bethesda, MD, USA; 3Proyecto Epidemiológico Guanacaste, Fundación INCIENSA, San Jose, Costa Rica. Modified ELISAs estimate avidity through using various chaotropic agents and measuring the degree to which they disrupt the interaction between antibody and antigen. The theory behind the assay is the higher the avidity of an interaction the less susceptible it is to the effects of a chaotropic agent. The goal of this study was to develop a modified ELISA to measure the avidity of human papillomavirus (HPV)-specific antibodies raised after vaccination with HPV virus-like particles (VLP) and potentially in the context of natural infection. The HPV VLP used for coating antigens are complex, multimeric protein antigens and presumably also susceptible to the actions of the chaotrope. Therefore, a number of chaotropic agents, including urea, diethylamine, guanidine hydrochloride and ammonium thiocyanate were evaluated in the assay for their effectiveness in measuring avidity while not affecting the integrity of the plate-bound VLP. Guanidine hydrochloride (GuHCl) was selected as an ideal chaotropic reagent because it had a minimal effect on the integrity of the plate-bound VLP. The assay was then successfully applied to a subset of sera from Cervarix® recipients from the NCI Costa Rica trial (n = 50) collected at month 12 visit. Samples were diluted to give a target OD of 1.0 and a range of concentrations of guanidine 0.5 to 3.5 M was used. Overall, the assay was highly reproducible (overall CV = 3.6%) and captured a considerable range of antibody avidity indices (median = 2.78 M, IQR = 0.96-3.26 M). Therefore, a GuHCl-modified ELISA is an acceptable, low cost and high throughput method to measure avidity indices of HPV-specific antibodies that can be used within a clinical trial setting. Additional studies are warranted to address the potential role of antibody avidity in protection against infection. Funded by NCI Contract No. HHSN261200800001E.
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Marko Merikukka
Oulu, FinlandProscovia Bazanya Namujju1,2, Lea Hedman3, Klaus Hedman3, Cecily Banura4,5, Edward Katongole Mbidde2, Romano Byaruhanga5,6, Moses Muwanga7, Reinhard Kirnbauer8, Heljä-Marja Surcel1, Matti Lehtinen1. 1National Institute for Health and Welfare, Oulu, Finland; 2Uganda Virus Research Institute, Entebbe, Uganda; 3University of Helsinki, Helsinki, Finland; 4Makerere University, Kampala, Uganda; 5Karolinska Institute, Stockholm, Sweden; 6San Raphael of St. Francis Hospital Nsambya, Kampala, Uganda; 7Entebbe Hospital, Entebbe, Uganda; 8Medical University of Vienna, Vienna, Austria. Additional Author(s) & Affiliation(s): Marko Merikukka. National Institute for Health and Welfare, Oulu, Finland.
Background: Assessment of low avidity antibodies has been used for differential diagnosis of acute vs. recent infections with viral, bacterial and parasitic agents. The low-avidity antibodies may, however, persist for a longer period in some individuals. Objectives: We studied whether low avidity of human papillomavirus type (HPV16) IgG antibodies is associated with susceptibility to other HPV infections (indicated by corresponding seropositivity). Methods: A cross-sectional study was performed on HPV16 seropositive pregnant Finnish and Ugandan women. Three hundred and sixty-seven HPV16 seropositive women were randomly selected from four groups: A) HPV16 seropositives only, B) HPV16 and HPV6/11 or C) HPV31/33 or D) HPV18/45 seropositivity. We tested for avidity of HPV16 antibodies using HPV16 virus-like particles in a modified ELISA. Results: The overall prevalence of low avidity HPV16 antibodies among the HPV seropositive women was 18% (68/368). The proportions of low avidity HPV16 antibodies in the different groups were: A) 17% (28/160), B) 33.8% (23/68), C) 10.8% (9/84), and D) 14.3% (8/56). Women with the low-avidity HPV16 antibodies had an increased risk of being seropositive for low risk HPV types 6/11 (odds ratio, OR=2.4; 95% CI, 1.2-4.5). No increased risk of being seropositive for high-risk HPV types 18/31/33/45 was observed. Conclusions: Our findings suggest that there are differences in risk of being seropositive for low-risk and high- risk HPV types by low-avidity of HPV16 antibodies. Associated characteristics of host immunity could modify susceptibility to the different HPV types. |
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Joseph Carter
Seattle, USAJoseph Carter1, Mary Colangelo1, Shu-Kuang Lee2, Laura Sycuro2, Laura Koutsky2, Denise Galloway1,3. 1Program in Cancer Biology, Division of Human Biology and Public Health Science, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; 2Department of Epidemiology, University of Washington School of Public Health and Community Medicine, Seattle, WA, USA; 3Department of Microbiology, University of Washington, Seattle, WA, USA. Detection of antibodies to multiple HPVs in a single well using GST-L1 fusion proteins on a Luminex platform has not been well validated. This study investigates the association between HPV-type specific seropositivity and alpha HPV genital infection detected years earlier, and to assess antibody persistence.
Female university students (n=285) who had previously participated in a cohort study in which they were tested for HPV DNA at 4 month intervals over an average of 3.9 years were recalled for a follow-up study. The median elapsed time between HPV DNA detection in the first study and the initial follow-up visit was 7 years (range 3 to 12). At follow-up, antibodies to 16 genital HPV types were examined using a multiplex binding assay. Associations were confirmed if women in whom HPV DNA was detected in the 1st study were significantly more likely to be seropositive for that type at follow-up compared with women in whom HPV DNA was not detected.
Statistically significant associations between the type-specific detection of HPV DNA and antibodies were found for types 6, 16, 18, 33, 35, 39, 52, and 67. Antibodies to HPVs 11, 31, 51, 56, 59 and 68 were also detected more frequently among women in whom HPV DNA had been detected in the first study compared with women who consistently tested negative for that type but the associations did not achieve statistical significance. Only antibodies to HPVs 45 and 58 showed no relationship to previous DNA detection. Among women in whom HPV DNA had been detected there was considerable variation in the proportion of seropositive women.
To more fully address the persistence of antibodies, we are testing sera from the first study using the multiplex assay to 16 HPV types. The results will be compared with the long-term follow-up seroprevalence to assess persistence.
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Professor of Pediatrics and Director for Clinical Services, University of California
San Francisco, USAAnna-Barbara Moscicki1, Yifei Ma1, Sepideh Fahrat1, Julie Jay1, Janet Jonte1, Lynn Hanson1, Dien Vo1, Mark Scott1. 1Department of Pediatrics, School of Medicine, University of California, San Francisco, CA, USA.
Objective: Examine the correlation between anal and cervical immune responses to HPV in the same women.
Methods:40 women had samples obtained from the anus and cervix for cytokine expression using the Luminex platform. In 20 women, TLR expression was also examined using RT-PCR from both sites.
Results: 14 women were HPV negative (-) from both sites (NBS); 11 were positive (+) from both sites (PBS); and 15 were discordant (+/-). Levels of IL-6 were 40-fold higher in the cervix than in the anus and IL-8 levels were 16 fold higher. TLRs 2, 7, 8 and 9 had 17-70 fold higher expression in the anus than in the cervix. The overall correlation between sites for the cytokines was not strong for most of the cytokines. When we examined the correlation in women who were NBS, a moderate association was found for IL-8 and fair associations were found for IL-1α and IL-12 (Table 1). IL-6 had an inverse association. Among women who were PBS, TNF and MIP-1 α had strong associations and IL-6, IFN-α2 and IL-1α had moderate inverse associations. Strong correlations were found for TLRs 7, 8 and 9 in women NBS and inverse correlations were found in those PBS with higher levels found in the anal samples. TLRs 2, 3, 7, 8 and 9 in the anus and cervix were decreased in those with HR HPV compared to HPV negative samples. Correlation betweenTLR expression and the downstream cytokine, INF-α2, showed strong correlation in the anus (Table 2) but not cervix.
Conclusion: There appears to be inherent differences in anal vs. cervical mucosal immune responses to HPV. The higher levels of TLRs in anal samples may explain why HPV is more transient in the anus compared to the cervix.
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Zarina D'Costa
Dunedin, New ZealandZarina D'Costa1, Charles Matthews1, Merilyn Hibma1. 1Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand. Background: E-cadherin, a cell surface adhesion molecule, is needed for the retention of Langerhans cells (LC), the primary antigen presenting cells in the skin. We have previously reported that the E6 oncoprotein of HPV16 is able to cause a 50 % decrease in the levels of cell surface E-cadherin in vitro. This decrease is further enhanced when cells are co-transfected with both oncoproteins of the virus, E6 and E7. The objectives of this study were thus to investigate the potential transcriptional regulation of E-cadherin by HPV16 E6.
Methods: Quantitative RT-PCR was used to compare the levels of E-cadherin mRNA in E6 and control cells. A luciferase-based reporter assay was used to determine the activity of the wild type and deletion constructs of the E-cadherin promoter, in cells with and without E6. Spontaneously immortalized epithelial cells, HaCaTs, were transduced with retroviruses expressing HPV16 E6. Protein lysates were collected from these cells and stained for transcriptional repressors of E-cadherin via western blotting.
Results: The mRNA levels of E-cadherin were found to be 0.61 times less in cells expressing E6 compared to control cells. The measured luciferase activity showed that the E-cadherin promoter is less transcriptionally active in E6 expressing cells than control cells. Protein levels of Snail, the main transcriptional repressor of E-cadherin, were found to be elevated in cells expressing E6 relative to control cells.
Conclusion: HPV16 E6 is able to regulate the cell surface adhesion molecule E-cadherin at a transcriptional level. This manipulation of E-cadherin levels correlates with decreases in LC numbers in HPV16 infected skin, thus representing an immune evasion mechanism by HPV16.
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Merilyn Hibma
Dunedin, New ZealandCheng Mee Leong1, John Doorbar2, Ingo Nindl3, Han-Seung Yoon4, Merilyn Hibma1. 1Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand; 2National Institute for Medical Research, Mill Hill, London, UK; 3DKFZ-Charite Cooperation, Viral Skin Carcinogenesis, German Cancer Research Center, Heidelberg, Germany; 4Department of Pathology, University of Otago, Dunedin, New Zealand. E-cadherin is a cell adhesion molecule expressed predominantly on epithelial cells. Surface expression of E-cadherin is crucial for normal tissue organisation and morphogenesis and its loss is frequently associated with cancer. In the epidermis, E-cadherin has an additional role in Langerhans cell (LC) retention by mediating adhesion between keratinocytes and LC. We have previously reported that E-cadherin expression is frequently lost from the membrane of basal and parabasal keratinocytes in human papillomavirus type 16 (HPV16) infected cervical lesions. The aim of this study was to determine the degree of conservation of E-cadherin down-regulation among HPV types from other members of the α7 genus and from representative types from other genera. Lesions infected with α, β, γ or μ types (including only low-grade lesions from high-risk types), were examined for E-cadherin following immunofluorescence staining. In normal tissues (n=16), E-cadherin expression was membranous and strong. E-cadherin was cytoplasmic or heterogeneous (i.e. membranous staining was absent from the majority of cells) in 11 samples infected with high-risk α7 or α9 types. In cutaneous lesions (n=9) infected with low-risk types from α4, μ or γ genera, staining was heterogeneous or negative. Although the majority of α10-infected lesions (n=7) showed heterogeneous E-cadherin staining, two tissues exhibited membranous staining and significantly β-infected lesions had normal E-cadherin expression. The relationship between E-cadherin expression and LC was determined in all tissues following CD1a and E-cadherin double staining. In all tissues, LC were localised in areas where E-cadherin was expressed and were depleted from tissues with low E-cadherin expression. E-cadherin down-regulation by HPV therefore was not restricted to high-risk types or mucosa-infecting types but was also observed in low-risk and some cutaneous HPVs. There was a correlation between E-cadherin loss and LC depletion, supporting the hypothesis that repression of E-cadherin expression influences the immune microenvironment by mediating LC depletion. |
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Chamsai Pientong
Muang, ThailandChamsai Pientong1, Praphatson Malat1, Tipaya Ekalaksananan1, Bunkerd Kongyingyoes2, Bundit Chumworathayee3. 1Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; 2Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; 3Department of Obstetrics and Gynaecology,Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand. Low-grade cervical intraepithelial neoplasia (CIN I) lesions are almost always the result of human papillomavirus (HPV) infection. Spontaneous resolution of CIN I lesions is common, whereas persistent HPV infection is the risk of cervical cancer development. This study aimed to investigate the mRNA level of IFN-g, IL-10 and TNF-a in women with histology confirmed LSIL after cryosurgery and to determine the role of cryosurgery intervention in increased immune activation. At first visit, cervical cells and biopsies were collected from 100 women with LSIL within 3 months after histological diagnosis and detected for HPV DNA. The HPV positive cases were divided into cryosurgery (performed at second visit) and control groups. At third visit, cervical cells and biopsies were collected from both groups 6 months after cryosurgery, whereas only cervical cells were collected 12 months after cryosurgery. The HPV DNA was detected using PCR with GP5+/GP6+ primers and the mRNA level of IFN-g, IL-10 and TNF-a were determined in cervical tissues using real time PCR. The result showed that HPV DNA was found in 60 cases and the most common HPV was HPV16 (60%). Cryosurgery was performed in 29 cases within 4 weeks after the first visit and 31 cases were control group. At first visit, mRNA of IFN-g, IL-10 and TNF-a were expressed in 93%, 93% and 97% of 100 cases, respectively. The mRNA level of IFN-g, IL-10 and TNF-a were increased in both groups. However, cryosurgery did not differently activate mRNA expression from control when it was performed within 4 weeks after the first visit. Almost of HPV persistence detected 12 months after cryosurgery were found in cases with decreased mRNA level of IFN-g. These results suggested that, after biopsy or cryosurgery, inflammatory cytokines, especially, IFN-g were increased and correlated to HPV clearance. |
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David W. Rosenthal
Great Neck, USADavid W. Rosenthal1,2,3, James A. DeVoti2,3, Bettie M. Steinberg2,4, Alan Abramson4, Vincent R. Bonagura2,3. 1Elmezzi Graduate School of Molecular Medicine, North Shore-LIJ Health System, Manhasset, NY, USA; 2Feinstein Institute for Medical Research, North Shore-LIJ Health System, Manhasset, NY, USA; 3Division of Allergy/Immunology, North Shore-LIJ Health System, Great Neck, NY, USA; 4Department of Otolaryngology, North Shore-LIJ Health System, New Hyde Park, NY, USA. Objectives: RRP is characterized by recurrent benign tumors of the respiratory tract, caused by infection with human papillomavirus 6 and 11 (HPV). The immunologic mechanism(s) that govern disease variation remain unresolved, but we have previously shown an increased expression of TH2-like chemokines, (CCL17, CCL18, and CCL22). In a small pilot trial (n=4) we have shown clinical improvement in patient severity scores with administration of a COX2-inhibitor, celecoxib. We hypothesize that TH2-like chemokines play a critical role in the immune response of patients with RRP, and the pathogenesis of disease can be altered by COX2-inhibition.
Methods: Patients enrolled in a twelve month pilot trial of a COX2-inhibitor, celecoxib, were followed longitudinally during administration of celecoxib (n=3). Plasma samples were assayed by cytokine specific (CCL17, CCL18, CCL22) ELISA (DuoSet, R&D Systems) every 3 months during administration of celecoxib. Patients with RRP not enrolled in the pilot trial had multiple plasma samples assayed over a twelve month period as a control.
Results: CCL18 decreased in all three patients during celecoxib administration, while CCL17 and CCL22 did not change over twelve months. Patients not in the celecoxib pilot trial maintained similar chemokine expression levels in the plasma over 12 months.
Conclusions: These results support our previous studies that show RRP is a TH2-like disease, and suggests that the disease may be modified by alteration of the TH2-like chemokine CCL18. CCL18 has been shown to be altered by prostaglandin E2, the target for COX2 and CCL18 is produced by T-cells, dendritic cells, and alternately activated macrophages. This study examines the mechanism by which COX2 inhibition may alter the host's response to HPV.
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James A. Devoti
Manhasset, USAJames A. Devoti1, David W. Rosenthal1,2,3, Jennifer Diaz3, Alan Abramson4, Bettie M. Steinberg1,4, Vincent R. Bonagura1,3. 1Feinstein Institute for Medical Research, North Shore-LIJ Health Systems, Manhasset, NY, USA; 2Elmezzi Graduate School for Molecular Medicine, North Shore-LIJ Health Systems, Manhasset, NY, USA; 3Department of Pediatrics, Schneider Children's Hosptial, New Hyde Park, NY, USA; 4Department of Otolaryngology, Long Island Jewish Medical Center, New Hyde Park, NY, USA. Background: The transcriptional profile of papillomas is characterized by altered expression of immune response genes, and another group of genes that are frequently seen in cancers. Individual micro-RNAs (miRNAs) regulate a large number of target genes and more than one miRNA may regulate a specific gene, resulting in complex genetic regulation. To determine if miRNAs effect the altered transcriptome in RRP, we compared expression levels of a panel of 88 miRNAs from papilloma tissue, vs. unaffected, autologous laryngeal tissue.
Methods: Papilloma tissue (n=12) and unaffected, autologous laryngeal tissue (n=12) were obtained after obtaining informed consent. In order to enrich miRNA, small RNAs were separated by size exclusion from total RNA (>5μg each sample) that had been extracted from pairs of tissues. The small RNA directed cDNA synthesis, and the cDNA was then screened for individual miRNA expression levels using a commercially available expression profiling assay (MAH-001; SABiosciences).
Results: Four miRNAs were up-regulated, and 7 were down-regulated, utilizing a fold change greater than 4.0 in the data analysis. However only the upregulated miRNAs (miR-195, miR-21, miR-27a, and miR-26-b), which were from 5.7 to 8.5 fold increased, were also statistically significant (p= 0.009 to 0.001).
Conclusion: We have identified 4 miRNAs that may contribute to the altered transcriptional profile present in actively growing papillomas from patients with RRP. All of these miRNAs have been reported to also be upregulated in a variety of cancers and also in non-malignant diseases. We are currently confirming these findings in a larger set of archived RNA. This report is the first demonstration of altered miRNA levels in RRP. Understanding the pathways that are impacted by these regulatory molecules may provide new insight into the disease process, and could potentially identify rationale targets for therapeutic intervention.
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Alba Lucia Cómbita
Bogotá, ColombiaJosefa Antonia Rodríguez1, Constanza Gómez3, Martha Lucia Serrano1, María Mercedes Bravo1, Alba Lucia Cómbita1,2. 1Grupo de Investigación en Biología del Cáncer, Instituto Nacional de Cancerología, Bogotá, Colombia; 2Departamento de Microbiología, Facultad de Medicina, Universidad Nacional de Colombia, Bogotá, Colombia; 3Departamento de Patología, Instituto Nacional de Cancerología, Bogotá, Colombia. Although the infection with high risk HPV is an important risk factor in cervical cancer etiopathogenesis, there is increasing evidence suggesting that the ability to avoid immunosurveillance seems to be linked to transforming potential of HPV and to a rapid progression to cancer. A shift from a Th1 to Th2 cytokine profile and the subsequent expression of IL-10 has been associated with impairment of tumour immunosurveillance and progression of cervical cancer. In other cancer models, the IL-10 contribute to the impaired anti-tumour immune response by down-regulating HLA-I expression or by increasing HLA-G expression. To evaluate the contribution of these mechanisms in cervical cancer progression, we analyzed the association of HLA-I, HLA-G and IL-10 expression by immunohistochemistry in tumour cells from 63 patients: nine with CIN III, ten with invasive cervical cancer stage IBI-II, 20 with IIA-B and 24 with IIIB-IVB.
A down regulation of HLA-I expression was observed in 84,7% of patients. 39/59 (66.1%) displayed total loss of HLA-I expression, while 11/59 (18.6%) showed partial expression. HLA-G and IL-10 up regulation was observed in 16/58 (27,6%) and 27/58 (46,6%) of cases, respectively. Most of the HLA-G positive cases show concurrent up regulation of IL-10 cytokine 14/16 (87.5%) (Spearman's rho= 0.507, P=0.000). We found also a significant association between IL-10 expression and down regulation of HLA-I (P=0.028). Among patients that over expressed IL-10 cytokine, 13/27 (48,1%) had total loss of HLA-I expression, and 7/27 (25,9%) showed down regulation. Finally, we observed a higher HLA-G expression in patients with down regulation of HLA-I expression than those with normal HLA-I expression (X2 de Pearson, P=0.004). No significant association was observed between expression of these proteins and different stages of cancer.
Our results suggest that, in cervical cancer, the IL-10 expression could correlate cooperatively with up regulation of HLA-G expression and down regulation of HLA-I expression.
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Morgan Marks
Baltimore, USMorgan Marks1, Rafael Irizarry1, Yolanda Eby1, Philip E. Castle2, Justin Lessler1, Patti E. Gravitt1. 1Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA; 2US National Cancer Institute, NIH, DHHS, Bethesda, MD, USA. Ophthalmic sponges are used to collect undiluted cervical secretions for assessment of markers of genital tract immunity. Heterogeneity in absorbed and extracted sample volumes requires normalization in order to make valid inter-individual comparisons in marker levels. Issues with standard normalization procedures based on dilution factor calculation using sponge weight to estimate specimen volume prompted evaluation of alternative methods. We evaluated the validity of dilution factor adjustment across a range of secretion volumes as well as two alternative volume-independent methods for normalization. Sponges were collected from seventy eight (n=78) women enrolled in a study of the natural history of HPV in Perimenopause (HIP). Separately, we experimentally absorbed 5-200ul of PBS containing BSA onto sponges. Secretion volume was estimated by subtracting the weight of a representative dry sponge from a sponge after specimen collection. Total protein was extracted from each sponge in 300ul of PBS and quantified using a BCA assay. Cytokines measured using the Bio-Plex Human Cytokine 27-Plex Panel were normalized to 1) total protein; 2) dilution factor or 3) median cytokine concentration. The relationship of normalization factors and cytokine concentration pre- vs. post-normalization to specimen volume was assessed using Spearman rank correlation. Total protein and median cytokine concentration were positively correlated to specimen volume. Dilution factor was poorly correlated to specimen volume due to inaccuracies in measuring specimen weight at low volumes resulting in over-inflated or negative values. Total protein and median cytokine normalized markers were significantly correlated to each other. Individual cytokines were less likely to be correlated to specimen volume after normalization by total protein or median cytokine methods compared to dilution factor. Dilution factors based on specimen volume suffer from loss of precision and validity at low specimen volume. We present alternative methods that minimize the effects of sampling heterogeneity on cytokine measurement. Further validation of these methods is ongoing. |
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Hyoung Jin Kim
College of Pharmacy, Chung-Ang University
Seoul, KoreaHyoung Jin Kim1, So Young Kim1, Hong-Jin Kim1, John T. Schiller2. 1College of Pharmacy, Chung-Ang University, Seoul, Korea; 2Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, Maryland. Pseudovirus (PsV) has been used for human papillomavirus (HPV) study because native HPV do not efficiently replicate in the cell culture system. The Optiprep density gradient (OG) has been used for purifying the HPV PsV. In spite of advantage that OG provides recovering PsV with high infectious titer, its disadvantages such as requiring high priced equipment, 4 hours of centrifugation time and technical difficulty limit applicability of HPV PsV system. In this study, we introduced heparin chromatography (HC) and cation-exchange chromatography (CC) to develop a convenient purification system for HPV58 PsV, and the purification efficiencies between OG, HC and CC were evaluated. To simplify the column work of HC and CC, the columns were packed with 0.1 ml of resins, and samples were equilibrated with adding binding buffer instead of dialysis. These methods shorten the time for column working into 20 min. In addition, highly purified PsVs were obtained by two chromatographic methods. Almost all contaminants were removed as a flow-through in binding condition of 0.33 M NaCl and pH 7.0. The recovery ratios of infectious PsV titer by OG, HC and CC were 60 - 89%, 30 - 56% and 7 - 15%, respectively. Therefore, OG and HC showed superior recovery ratios while CC showed low recovery ratio. In the neutralizing antibody titration using PsVs purified by the three kinds of methods, the titers of mouse serum immunized with HPV58 PsV showed similar levels. These results indicate that HPV58 PsVs purified by HC and CC are useful for study of HPV type 58. Our two purification methods provide a simple and convenient system in obtaining highly purified PsV. In addition, two methods have considerable potential for scale-up to purify large amount of PsV. It is expected that our two purification methods could be applied to purifying different types of HPV PsV.
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Yuanming Shen
Hangzhou, ChinaYuanming Shen1, Yang Li1, Xing Xie1. 1Department of Gynecologic Oncology, School of Medicine, Zhejiang University, Hangzhou, China. Quantitative real-time RT-PCR (RT-qPCR), as a sensitive and reproducible quantization technique for detection of gene expression, is being used in microRNA (miRNA) expression research. Selection of optimal reference RNA targets for proper normalization is critical in miRNA RT-qPCR analysis, but it is still rather empirical. No optimal reference genes in miRNA RT-qPCR analysis have been identified in human cervical cancer up to date. In this study, we selected nine ubiquitous and stably expressed candidate reference RNA targets, including five (let-7a, miR-23a, miR-200c, miR-1979, and miR-26a) according to miRNA microarray analysis for 6 pairs of human normal and cancerous cervical tissues, and four (miR-103, miR-191, 5S rRNA, and U6 snRNA) used frequently in previous studies. All nine candidate genes were examined across 24 normal cervical tissues and 24 cervical carcinoma tissues to determine the most appropriate normalizer(s). The geNorm and NormFinder were employed for identifying the stability of gene expression and suitability for normalizors. Our results showed that all studied RNA targets reached a high expression stability with low M values(<1.5), five to nine Candidate miRNA normalizers [U6 snRNA (P = 0.39), 5S rRNA (P = 0.9), miR-23a(P = 0.5), miR-103(P = 0.12), and miR-191(P = 0.1) ] were equivalently expressed between the normal and tumor groups, while the expressions of the other four Candidate miRNA normalizers (miR-1979, let7a, miR-200C, and miR-26a ) were significantly increased in malignant samples (all P < 0.001). MiR-23a was identified as the most stable and reliable reference gene followed by miR-191 and miR-103. Our results suggested that miR-23a ,miR-191 and miR-103 are the optimal reference miRNAs that can be used for normalization in gene profiling studies of cervical cancer; of them, miR-23a is a novel miRNA normalizer.
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Helle Pedersen
Sonderborg, DenmarkHelle Pedersen,, Jalil Hariri1. 1Department of Pathology, Sonderborg Hospital, Sonderborg, Denmark. Objectives: The effect of storage on SurePathTM specimens as regard to DNA extraction is not clearly illustrated in the literature, although some companies recommend DNA extraction within a week of sample collection. The aim of this study is to test the effect of long time storage on DNA extraction from SurePathTM specimens.
Methods: The study included an old group of 98 leftover SurePathTM vials stored at room temperature for 2-8 weeks, and a new group of 97 leftover SurePathTM vials from the daily routine. DNA extraction was performed manually on all specimens using the PapilloCheckTM DNA extraction kit, and the SensoQuest LabcyclerTM for PCR. HPV genotyping was then made using PapilloCheckTM DNA-chip and CheckScanner.
Results: ASCUS+ was reported in 62 and 65 cases of the old and new groups respectively. The remaining specimens were WNL. Of these 40 (65%) and 46 (71%) samples harboured HR-HPV. The incidence of HR-HPV in the normal cases was on the other hand 7/36 (19%) and 15/32 (47%) in the old and the new groups respectively. The storage time of normal and ASCUS+ specimens in the old group was identical. The age range was comparable in normal and ASCUS+ cases in both groups.
Conclusions: It seems that 2-8 week of storage has no negative effect on HR-HPV testing in ASCUS+ cases. The significant difference of HR-HPV incidence in the normal cases is hardly due to failure in DNA extraction, because otherwise it would also have affected the ASCUS+ cases. All of the normal 36 cases in the old group, and 11 of the 32 normal cases in the new group were reported by cytotechnologists. The remaining 21 normal cases of the new group were reported by pathologists, which may explain the significant difference.
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Helle Pedersen
Sonderborg, DenmarkHelle Pedersen1, Gitte Sondergaard1, Jalil Hariri1. 1Department of Pathology, Sonderborg Hospital, Sonderborg, Denmark. Objectives: The PapilloCheckTM DNA testing is not validated for SurePathTM residual cell-pellet. The objective of this study was to compare the DNA extraction from SurePathTM vial and the corresponding cell-pellet for PapilloCheckTM HPV DNA-testing.
Methods: The study included 37 SurePathTM specimens reported as WNL and 62 specimens of ASCUS+. The DNA extraction from the cell-pellet as well as the vial from each case was performed manually using the PapilloCheckTM DNA extraction kit. The PCR was performed on SensoQuest LabcyclerTM. The PCR product was then hybridized to the PapilloCheckTM DNA-chip and scanned by CheckScannerTM.
Results: Three cases were HR-HPV positive in vials and negative in cell-pellet, while one was HR-HPV positive in cell-pellet and negative in vial. Another case was negative in vial and positive for LR-HPV in cell-pellet. HR-HPV was detected in 3 cases in the cell-pellet group, but the corresponding vials contained only LR-HPV. Otherwise there was full agreement regarding HR-HPV.
One of the 3 missed cases of the cell-pellet group was reported as HSIL, and the other two were reported as WNL. All of these 3 cases harboured HR-HPV in the corresponding vials. One of the 2 missed cases in the vial group was WNL and harboured HR-HPV in the cell-pellet, while the other was reported ASCUS but harboured LR-HPV in the cell-pellet. HSIL was reported in 10 cases. One of these was HR-HPV negative in cell-pellet as well as the vial, and 1 was HR-HPV negative in cell-pellet only. When both methods were considered, no HR-HPV was missed.
Conclusion: The study showed that the cell-pellet is feasible for DNA-testing by PapilloCheck,TM and there was no significant difference between the vial and cell-pellet groups regarding HR-HPV detecting. It also seems that the combination of both methods can be used in equivocal cases.
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Jalil Hariri
Sonderborg, DenmarkJalil Hariri1, Helle Pedersen1. 1Department of Pathology, Sonderborg Hospital, Sonderborg, Denmark. Introduction: The comparison of PapilloCheckTM HPV testing (PC-HPV) and immunocytochemistry (ICC) using HPV surrogate markers is to the best of our knowledge not sufficiently studied. The aim of this pilot study was to compare the reliability of PC-HPV and ICC to detect or predict HSIL.
Methods: PC-HPV and ICC using p16, ProExCTM, and Ki67 were performed on 37 normal and 62 ASCUS+ SurePathTM specimens. DNA extraction for PC-HPV was performed on the residual cell-pellet as well as left over material in the vials. The ICC was processed on BenchMark® XT Ventana/Roche. The ICC was interpreted positive when at least 2 of the markers were positive. Histology was available in 30 of the ASCUS+ cases.
Results: HSIL, LSIL, ASCUS, and ASC-H were found in 10 (16%), 14 (23%), 29 (47%) and 5 (8%) out of the 62 positive smears respectively. AIS and AGC were found in 1 and 2 cases respectively. HR-HPV was negative in 2 cases of the cell-pellet group; one of these was also negative in the vial group. Otherwise HR-HPV was detected in all of the HSIL cases in cytology as well as in histology. The ICC was negative in 2 histologically verified cases of HSIL. One of these was ASCUS in cytology while the other showed HSIL, which also was HR-HPV negative in the cell-pellet group.
Conclusion: The sensitivity of PC-HPV using vials, PC-HPV using cell-pellets and ICC to detect or predict HSIL was 91%, 82%, and 82% respectively. All 3 methods were thus comparable, although the sensitivity of PC-HPV using vials was slightly superior, but not significantly better. When all methods were used the sensitivity to detect HSIL was raised to 100%, but more efforts are needed to clarify this issue.
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Santina Castriciano
Brescia, ItalyDamien Jeantet1, Maria Bonhomme1, Faustine Meynier1, Arnalda Giambra2, Alice Squassina2, Ernestina Valagussa3, Paolo Libretti3, Maria Peroni4, Giuseppe Ciravolo4, Santina Castriciano2. 1Biomerieux, Grenoble, France; 2Copan Italia Spa, Brescia, Italy; 3Department of Cytology, Fleming Lab, Brescia, Italy; 4Department of Gynecology, Brescia University, Brescia, Italy . Objectives: NucliSENS sEasyQ® HPV v1.1 (bioMérieux) is a qualitative nucleic acid assay for the detection of E6/E7 HPV oncogene mRNA (HPV16, 18, 31, 33 and 45). The L-shape endo/esocervical flocked swab (LEC) is a device for Pap collection and CyMol is a cell and nucleic acid preservation medium (Copan Italia).
Study objectives were:
1) validate the performance of LEC and CyMol medium compared to the Cervex brush (CB) (Rovers) and PreservCyt solution (Hologic) for collection and preservation of specimens for HPV detection with the NucliSENS EasyQ® HPV 1.1 assay.
2) Evaluate stability of specimens in CyMol medium after 3 weeks at room temperature (RT) and 6 weeks at -20°C.
Methods: Six hundred clinical specimens were collected from voluntary women during a routine visit for Pap testing at 5 Fleming Lab sites in Brescia. Two clinical specimens (300 with CB in PreservCyt and 300 with LEC in CyMol) were collected from each patient in a randomized order and classified for PAP cytology status. Aliquots of all positive specimens in CyMol medium and the same number of negative specimens were used for 3 weeks RT and 6 weeks -20°C stability study. All specimens were extracted and tested with the NucliSENS® easyMAG® and EasyQ® HPV v1.1 assay.
Results: The results of the comparison study show a concordance of 100% between the specimens collected in CyMol and specimens collected in PreservCyt (Table 1). The invalid rates are comparable in the two transport media. Stability of specimens in CyMol stored at RT was demonstrated up to 3 weeks. The stability study after 6 weeks at -20°C is still in progress.
Conclusions: No statistical difference was found between the specimens collected with cervex brush in PreservCyt solution and specimens collected with L-shape endo/esocervical flocked swab in CyMol medium when tested with NucliSENS EasyQ® HPV v1.1.
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Yuanming Shen
Hangzhou, ChinaShen Yuanming1, Li Yang1. 1Women's Reproductive Health Laboratory of Zhejiang Province, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou , China. Quantitative real-time RT-PCR (RT-qPCR) is a sensitive and reproducible gene expression quantization technique which is now being used in microRNA (miRNA) expression research. As with mRNA analysis, selection of optimal reference RNA targets for proper normalization is a critical aspect of miRNA RT-qPCR data analysis. Unfortunately, the selection of reference RNA targets to normalize miRNA levels is still rather empirical and no suitable reference genes have been identified in human cervical cancer to date. In this study, first we filter five ubiquitous and stably expressed candidate reference RNA targets(let-7a, miR-23a, miR-200c, miR-1979,miR-26a) in 6 pairs of normal and malignant human cervical tissues using miRNA microarray Then the expression of these five miRNA genes and miR-103, miR-191 as well as two small nucleolar RNA genes (5S rRNA and U6 snRNA) that were already used in various studies, were examined across 24 normal cervical tissues and 24 cervical carcinoma tissues to determine the most appropriate normalizers .The expression stability and suitability of the 9 candidate reference RNA targets were validated employing geNorm and NormFinder. MiRNA23a and miRNA191 were demonstrated as the most stable reference genes and thus could be used as reference genes for normalization in gene profiling studies of cervical cancer, while the combination of two miRNAs (miRNA23a and mirRNA191) should be recommended as a much more reliable normalization strategy. This is the first study to identify reliable for analysis of miRNA by RQ-PCR in human cervical tissue.
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Sepehr N. Tabrizi
Parkville, AustraliaSepehr Tabrizi1,2,3, Nicole Austin1,3, Michael McCullough4, Gillian Phillips5, John Wark6, Rodney Petersen2, Suzanne Garland1,2,3. 1Department of Microbiology and Infectious Diseases, The Royal Women's Hospital, Parkville, Victoria, Australia; 2Department of Obstetrics and Gynaecology, University of Melbourne, The Royal Women's Hospital, Parkville, Victoria, Australia; 3Regional WHO HPV Reference Laboratory, The Royal Women's Hospital, Parkville, Victoria, Australia; 4Department of Dentistry, University of Melbourne, Parkville, Victoria, Australia; 5Victorian Cytology Service, Carlton, Victoria, Australia; 6Department of Medicine, University of Melbourne, Parkville, Victoria, Australia. Multivalent human papillomavirus (HPV) vaccines for cervical cancer prevention are now licensed in many countries. Efficacy of vaccine, in particular older women may require examination of archival samples to assess previous exposure to HPV. Papanicolaou (Pap) stained cervical smears are ideal for such assessment as most women would have had several such smears collected. In this study, efficiency of HPV detection and genotyping from Pap smears is compared to an ideal sample of endocervical brush collected in PreservCyt at the same time from 87 women undergoing treatment for high grade Pap abnormality. Smears were imaged prior to removal of cells from slides. Imaging enabled detection of all high grade abnormalities confirmed previously on the Pap slides. All Pap slides resulted in positive β-globin, indicating presence of recoverable amplifiable DNA. Over 87% concordance was achieved for detection of high risk HPV genotypes. Full genotyping concordance in 62% and partial in 26% of sample pairs was observed. In particular HPV 16 and 18 had very good agreement (κ=0.850 and 0.903 respectively). Only 1 Pap slide was positive where the PreservCyt had no detectable HPV demonstrating low false positive rates (1%). Overall this study confirms imaging and subsequent HPV detection and genotyping can offer a sensitive and relatively accurate HPV detection in archival cervical smears.
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Andrew Bailey
Manchester, UKAndrew Bailey1, Alexandra Sargent1, Joanna Nagorko1, Claire Gilham3, Henry Kitchener on behalf of the ARTISTIC Trial Study Group2. 1Clinical Virology, Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK; 2University Department of Obstetrics & Gynaecology, Manchester, UK; 3Institute of Cancer Research, London, UK.
Introduction: With the possible addition of high risk HPV (HR HPV) testing to cervical cancer screening programmes women who present with normal cytology but who are found to be HRHPV positive pose a particular dilemma. Confirmation of the presence of HR HPV in such samples could help in the clinical management of these women.
Objectives: To confirm the presence of high risk HPV (HR HPV) genotypes in cervical samples taken from cytology normal women who were found to be HR HPV positive using the Qiagen Hybrid Capture 2 (HC2) assay.
Methods: Stored archival samples of cervical cells obtained from approximately 2,500 women enrolled into the ARTISTIC trial are being examined during the course of this study. All women had normal cytology but were found to contain a HR HPV type by Qiagen HC2. Nucleic acid was extracted using either the Roche MagNAPure or the Biomerieux automated extraction systems. An aliquot of DNA was then amplified and genotyped using both the Roche Prototype Line Blot (LBA) and the Greiner-Bio-One HPV genotyping assays.
Results: Initial results obtained from testing 1281 HC2 positive/cytology normal samples showed that 57% contained a HR HPV genotype by Papillocheck compared to 63.8% which contained a HR HPV genotype when using LBA. The ability of either assay to confirm the presence of HR HPV genotypes was highly dependent on the RLU/cut off value of the HC2 assay.
Conclusions: The Roche prototype Line Blot assay appeared slightly more sensitive for the confirmation of HR HPV infections. These results indicate that approximately a third of women with normal cytology but who were found to be positive by HC2 positive do not have a HR HPV infection and, thus, could be managed more conservatively.
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Carina Eklund
Malmo, SwedenCarina Eklund1, Tiequn Zhou2, Joakim Dillner1,3. 1WHO HPV LabNet Global Reference Laboratory, Departments of Clinical and Medical Microbiology, Laboratory Medicine Skåne and Lund University, Malmö, Sweden; 2Quality, Safety and Standards Team, Immunizations, Vaccines and Biologicals Department, World Health Organization, Geneva, Switzerland; 3Departments of Laboratory Medicine, Medical Epidemiology & Biostatistics, Karolinska Institute, Stockholm, Sweden. Background: HPV serology is essential for both HPV vaccinology and epidemiology. VLP-ELISA correlates well with the neutralization assay but is easier to implement. Comparability and reliability of serological data requires international standardisation, particularly in terms of antibody units and cut-off levels, as well as international proficiency testing of serological laboratories.
Methods: The WHO HPV LabNet agreed on a common SOP for ELISA based on consensus from collaborative studies. The protocol was first tested by all LabNet members in terms of robustness, unambiguity and stability and a second version of the SOP established. VLPs were donated from 5 different sources and the most stable, sensitive and specific VLP preparation were selected for common use and distributed to WHO LabNet members as coated plates. A blinded challenge proficiency panel of 52 serum samples from PCR verified HPV-infected women and 11 virginal women were tested in parallel with the WHO HPV 16 International Standard serum to be able to report data in International Units (IU).
Results: Nine laboratories in 5 continents participated. Proficiency criteria for detection of HPV 16 antibodies were a sensitivity of at least 50% and a specificity of 100%. It was not possible to use a common cut-off in terms of IU (see example with data with a 4 IU cut-off in Table 1), but a mean value of the results from the control sera plus three standard deviations gave optimal performance (Table 1). Six of nine participating laboratories were proficient for HPV 16 antibody testing.
Conclusions: International HPV serology proficiency studies are feasible. Common SOPs, International Units and cut-off criteria have been established.
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Heather A. Cubie
Edinburgh, UKE.J. Fagan 1, C. Moore2, C. Jenkins1, A. Rossouw1, H.A. Cubie2, V.L.A. James1. 1Health Protection Agency, Centre for Infections, London, United Kingdom; 2Scottish HPV Reference Laboratory, Royal Infirmary of Edinburgh, Edinburgh, United Kingdom. Background: The United Kingdom National External Quality Assessment Service (UK NEQAS) distributes clinically relevant and educational specimens for external quality assessment (EQA).
Objectives: The aims were to assess the suitability of using liquid based cytology (LBC) samples for EQA of molecular methods and to review the methods used by participants to detect the presence of high risk (HR) human papillomavirus (HPV) genotypes.
Methods & Results: Three pilot distributions were dispatched between January 2008 and January 2009 with each distribution consisting of four different specimens. Performance was good with over 90% of participants reporting correctly on the presence or absence of high risk genotypes in all but one specimen where performance was 82.1%, due to the false detection of HR genotypes. This was a pooled specimen containing only low risk (LR) genotypes 61, 70 and 81. The most commonly used assay for the detection of the presence of HR HPV was digene Hybrid Capture II assay. In-house PCR assays were most commonly associated with incorrect results. The reporting of such assays to the EQA pilot decreased during the 13 months of participation.
Conclusions: The UK NEQAS scheme for Molecular Detection of HPV provides a standardized, homogeneous and characterized specimen. However, genotyping results reported by participants still showed variation. Robust EQA of HPV molecular screening programmes will be essential for monitoring the impact of the HPV vaccine. |
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William Seaman
Chapel Hill, USAWilliam Seaman1, Marion Couch1, Jennifer Webster-Cyriaque1. 1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, NC, USA. Integration of the oncogenic HPV genome into the host chromosome is a critical event in tumor initiation. Breakpoints in the viral genome occur in the E2 gene, resulting in disruption of E2 expression and dysregulation of the E6/ E7 viral oncogenes. Early detection of integration related E2 disruption is paramount in the treatment/management of oncogenic HPV-related lesions. We have developed a single tube multiplex assay capable of simultaneously detecting the HPV16 L1 gene as well as 3 different regions along the entire length of E2. This assay was used to determine the E2/L1 ratio in CaSki and SiHA cells. For CaSki DNA the E2:L1 copy number ratio for each E2 primer pair/probe was approximately one indicating that there is no detectable E2 disruption in these cells. Furthermore, our assay was capable of identifying the previously reported 3' E2 breakpoint in SiHA cells. We screened 65 oral cancers for the presence of HPV16 using qPCR and identified 10 oral cancers that were HPV16+. In one of these cancers, E2:L1 ratios of 1 were obtained using primer pair/probe combinations corresponding to the 5' end of E2 but no 3' E2 signal could be detected indicating that the E2 breakpoint occurred in the 3' end of the E2 gene and suggesting that there was complete integration of HPV16 genome. A low E2:L1 ratio (<0.75) was detected in 4 other oral cancer DNA samples suggesting that in these cancers there was a mixture of episomal and integrated HPV16 genomes. High E2/L1 ratios (>0.75) were detected in the other 5 cancers suggesting that high levels of HPV16 episomes were present possibly masking detection of integration events. This novel assay should allow for the detection and mapping of E2 disruption and allow for rapid screening of clinical samples for HPV16 integration events.
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William Seaman
Chapel Hill, USAWilliam Seaman1, Elizabeth Andrews2, Marion Couch1, Erna Kojic3, Susan Cu-Uvin3, Joel Palefsky4, Allison Deal1, Jennifer Webster-Cyriaque1. 1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, NC, USA; 2College of Dental Medicine, Western University of Health Sciences, Pomona, CA, USA; 3Department of Medicine, The Miriam Hospital, Brown University, Providence, RI, USA; 4Department of Medicine, University of California, San Francisco, San Francisco, USA. Human papillomaviruses (HPV) remains a serious world health problem due to their association with anogenital and oral cancers and warts. While over 100 HPV types have been identified, only a subset are associated with malignancy. HPV16 and 18 are the most common oncogenic types, while HPV6 and 11 are the most common types responsible for anogenital warts. These four types cause up to 90% of HPV-associated disease. While other quantitative PCR (qPCR) assays can be used to detect oncogenic HPV, there is no single tube assay that distinguishes the most frequent oncogenic types and the most common types found in warts. A qPCR assay was developed that allowed for detection and quantitation of these 4 HPV types. Type-specific primer pairs and TaqMan probes allowed single tube multiplex reactions to be performed. Each HPV type was detected over a range from 2 X 101 to 2 X 106 copies/reaction, providing a reliable method of quantitating type-specific HPV. A Sybr Green-based qPCR assay was developed that utilizes degenerate primers targeting the E1 region of all HPVs. DNA obtained from 253 specimens tissues and/or fluids from anogenital and oral compartments were analyzed using these qPCR assays. There was good agreement between the ability of the qPCR assays described here to identify HPV types in malignancies previously typed using standard methods. Comparisons to the gold standard detected a sensitivity of 89% (95% CI: 77% - 96%) and specificity of 90% (95%CI: 52% - 98%). These novel qPCR assays will allow rapid detection and quantitation of HPV to assess their role in viral pathogenesis.
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Luis Martins
Lisbon, PortugalLuis Martins1, Daniela Cochicho1, Mario Cunha1, Carmo Ornelas1. 1Portuguese Institute of Oncology of Lisbon, Clinical Pathology, Laboratory Virology, Lisbon, Portugal. The detection of HPV DNA in cervical specimens is usually made by PCR in the L1 region of the virus. The existing tests detect the presence of HPV, which is present in both transient infections, that resolve spontaneously, and in persistent infections. APTIMA HPV Assay (AHPV) detects E6/E7 mRNA of high-risk HPV types (14), using Transcription Mediated Amplification (TMA) technology. Detection of HPV mRNA may be more specific for detection of persistent infection which is more likely to progress to cervical dysplasia and cervical cancer.
Aim: Evaluation of the AHPV sensitivity and specificity, considering the endpoint for disease the detection of >=CIN1 and >=CIN2+.
Materials and Methods: To evaluate the clinical performance of AHPV assay, a total of 179 smears specimens - 15 negative, 25 ASCUS, 51 CIN1, 59 CIN2+ and 29 carcinomas - were analyzed by AHPV.
Results: The % of positive samples for ASCUS, CIN1, CIN2+ and Carcinoma were 50%, 67%, 91% and 96%, respectively. Considering the >=CIN2+ as the endpoint of disease, AHPV showed a clinical sensitivity and specificity of 84% and 51%. If we lower the endpoint of disease to >=CIN1, the sensitivity increases to 89.9% and the specificity lowers to 45.9%. These results are very similar to those published: 90.8% vs 84% and 56.2% vs 51%. The Positive and Negative Predictive Value (PPV and NPV) of AHPV is 62.7% and 77.01%, respectively, for >=CIN2+. If we consider the endpoint of disease as >=CIN1, the PPN becomes 76.3% and the NPV 70%. The PPV determined has higher value than those published (32.3%) and the NPV has a lower value.
Conclusions: This assay has a good clinical sensitivity and higher clinical specificity relative to the majority of the DNA tests. It's also a good marker of the viral activity of E6/E7 oncogenes, that can be associated with the progression to disease.
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Luis Martins
Lisbon, PortugalLuis Martins1, Daniela Cochicho1, Mario Cunha1, Carmo Ornelas1. 1Portuguese Institute of Oncology of Lisbon, Clinical Pathology, Laboratory Virology, Lisbon, Portugal. Background: Published studies indicate that HPV DNA testing for cervical intraepithelial neoplasia of grade 2 or 3 has a sensitivity of 94.6%, versus the 55.4% for the Pap testing. Regarding the specificity, the HPV DNA tests and Pap testing have, respectively, 94.1% and 96.8%. Generally, HPV DNA test have good sensitivity but poor specificity. Detection of HPV mRNA may improve specificity. Abbott Real Time High Risk HPV can detect 14 high risk HPV types, genotyping both HPV16 and HPV18; APTIMA HPV assay detects the oncogene E6/E7 viral activity from the same 14 high risk types and Papillocheck can detect 14+4 high risk types and 6 low risk types.
Objectives: To evaluate clinical Specificity, Sensitivity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) for >CIN2+ of the Abbott RealTime High Risk HPV (AbHPV), APTIMA HPV (AHPV) and Papillocheck (PC).
Methods: To evaluate clinical performance of AHPV, AbHPV and PC assays for the detection of CIN2+ lesions a total of 94 smears specimens - 12 negative, 24 ASCUS, 21 CIN1, 20 CIN2+ and 17 carcinomas - were analyzed by these tests.
Results: Overall agreement of AHPV, AbHPV and PC was 91.5% (86/94). Considering the endpoint as CIN2+, clinical sensitivity and specificity of AHPV was 97.2% and 50.9%, for AbHPV was 94.6% and 52.6% and for PC assay was 97.3% and 33.8%. All assays have high NPVs: 96.7%, 93.8% and 96%, respectively. The PC assay has a lower PPV than AHPV and AbHPV.
Conclusions: The clinical sensitivity for CIN2+ lesions is similar for all the tests (94.6-97.3%). Relatively to specificity, the AbHPV and AHPV have similar values and PC has the lower value. These values are in agreement by those published by other authors. All tests have a high sensitivity for detection CIN2+ lesions and can be used to redefine the screening protocols for cervical cancer. |
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Sonia Perez
Vigo, SpainSonia Perez1, Agar Araujo1, Ana Cid2, María José Lamas3, Jonathan Fernández1, Pura Garra1, Isabel López-Miragaya1, Julio Torres1, Santiago Melón4. 1Microbiology Unit, Complejo Hospitalario Universitario de Vigo, Vigo, Spain; 2Microbiology Unit, Complejo Hospitalario de Ourense, Ourense, Spain; 3Gynecology Unit, Complejo Hospitalario Universitario de Vigo, Vigo, Spain; 4Microbiology Unit, Hospital Universitario Central de Asturias, Oviedo, Spain. Background: it has been suggested that some Human papillomavirus (HPV) 16 E6 variants (for instance T350G) could be involved in viral persistence and progression of HPV infection. Detection of these variants by DNA sequencing is expensive and time consuming.
Objective: To evaluate a novel RT- PCR (Real Time PCR) assay for detection of HPV 16 350G Variant.
Materials and Methods: For identification of HPV 16 sequence variation, 761bp from the LCR-E6 region (Nt 7701-591) were amplified (Expand HiFi, Roche) as previously described (Xi LF, 2006), sequenced with the same primers (BigDye 1.1. Abi Prism 3100-Avant), and compared with NCBI database. For the T350G discrimination assay two different labeled probes were used: wild strain was detected in the VIC-filter and mutant strain in the FAM-filter (GTXpress Master Mix. 7500 Fast. Applied Biosystems). Primers and Taqman-MGB probes were designed targeting the conserved region between the different HPV 16 subtypes. Analytical performance was analyzed testing of 10-fold serial dilutions of the HPV 16 WHO International Standard (NIBSC 06/202) and DNA of HPV 16 LCR-E6 region from a wild strain, a 350G mutant strain, and mixes of both (350K). Clinical sensitivity and specificity were calculated by performing both RT-PCR and LCR-E6 sequencing in 121 endocervical samples positive for HPV16 in routine screening.
Results: For the T350G discrimination RT-PCR assay, the cut-off value was aprox 5.0x103 vp/ml for T350T, for T350G and for T350G detection in mixtures containing 20% of this mutant. The results of both RT-PCR and DNA sequencing were, respectively: 65/65 350G variant, 4/4 350K variant, 52/52 350T. In comparison with DNA sequencing, the novel assay sensitivity and specificity were 100% for the 350G Variant detection.
Conclusion: This novel real time genotyping assay is a rapid, sensitive and specific method for detection of HPV 16 E6-350G Variant. |
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Maria Gabriella Donà
Rome, ItalyMaria Gabriella Donà1, Maria Benevolo3, Fulvia Pimpinelli2, Mara Battista2, Livia Ronchetti3, Francesca Stivali2, Antonella Moscarelli2, Massimo Giuliani1, Aldo Di Carlo4, Amina Vocaturo3. 1UOC Dermatologia Infettiva, San Gallicano Dermatological Institute (IRCCS), Rome, Italy; 2Laboratorio di Patologia Clinica, San Gallicano Dermatological Institute (IRCCS), Rome, Italy; 3Pathology Department, Regina Elena Cancer Institute (IRCCS), Rome, Italy; 4Scientific Director, San Gallicano Dermatological Institute (IRCCS), Rome, Italy. Background: A plethora of manual and automated DNA extraction systems are used for the specimen preparation prior to HPV testing, not always in compliance with the guidelines of kit manufacturers.
Objectives: To investigate the influence of different DNA extraction methods on HPV typing by Linear Array® (LA, Roche) and InnoLiPa® (IL, Innogenetics).
Methods: Twenty samples, which tested positive by LA using the Roche-recommended AmpliLute® extraction, were selected. Two other methods were used for DNA extraction from the original PreservCyt® cervical samples: the QiAmp® Blood mini kit (Qiagen) and the NucliSens EasyMAG® automated system (bioMérieux). LA and IL were performed in parallel on the three different extracts.
Results: Type-specific concordance (i.e. same HPV types found in all the three extracts) was observed in 11/20 samples (55%) for LA and 15/20 (75%) for IL. When comparing the results obtained on the DNA extracted with the two manual methods, 5/20 (25%) and 4/20 (20%) cases were not concordant by LA and IL, respectively. When comparing LA results obtained on any of the manual extracts with those obtained after automated extraction, 7/20 samples (35%) did not show type-specific concordance in both cases. IL results were non concordant in 4/20 cases (20%) when comparing the AmpliLute® vs. the automated DNA extraction, while non concordant results were observed in 3/20 cases (15%) when comparing the Qiagen vs. the automated extraction. Lack of type-specific reproducibility in LA concerned 9 HPVs (2 LR and 7 possible HR or HR), mostly with medium/high inclusivity level (i.e. types the LA detects at higher concentration level).
Conclusions: HPV genotyping test performance is significantly affected by the DNA extraction method, although at lesser extent for IL. Use of consistent protocols for sample preparation is a priority to guarantee intra-assay and intra-laboratory reproducibility over time, particularly when monitoring transient vs. persistent HPV infections. |
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W.G.V. Quint
Voorburg, The NetherlandsD.T. Geraets 1, L.J. van Doorn 1, W.G.V. Quint 1, W.J.G. Melchers 2. 1DDL Diagnostic Laboratory, Voorburg, The Netherlands; 2Department of Medical Microbiology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands. Background: The Amplicor HPV test (Amplicor, Roche) permits the simultaneous detection of 13 HR-HPV types in a single reaction. However, to identify the specific HPV genotype, a separate PGMY-based amplification is required to perform the Linear Array HPV Genotyping Test (LA, Roche).
Objectives: The goal of the current study was to test whether the novel digene HPV Genotyping RH (reverse hybridization-based) and LQ Tests (xMAP-based) (QIAGEN, GP5+/6+ PCR-based identification of 18 classified HR-HPV genotypes) allow direct genotyping of Amplicor- and PGMY-based amplimers.
Methods: HPV-positive cervical scrapes for the current study were pre-selected based on genotyping results by a different method. A selection of these HPV-positive cervical samples was amplified and analyzed by Amplicor (n=258), while another panel of cervical specimens was PGMY-amplified and genotyped by LA (n=66). Both Amplicor- and PGMY-amplimers were additionally genotyped by the digene RH Test and LQ Tests.
Results: Amplicor-amplimers (n=258): 193/258 samples were Amplicor-positive, and these amplimers could be directly genotyped for 13 HR types by RH Test (183/193) and LQ Test (189/193). 65/258 samples remained Amplicor-negative, but contained one of 13 HR-HPV genotypes in 6/65 by RH Test and in 8/65 by the LQ Test. In addition, genotyping revealed untargeted types (HPV66 and 82) that were amplified but undetected by Amplicor.
PGMY-amplimers (n=66): Of 66 samples genotyped for presence of 18 HR types, 26 were positive by LA, 25 by RH Test, and 26 by LQ Test. RH and LQ Test demonstrated good agreement with LA for HPV genotyping.
Conclusions: The digene HPV Genotyping RH and LQ Tests can be directly applied on amplimers generated by the GP5+/6+, Amplicor, and PGMY systems. The use of a universal genotyping test could contribute to standardization of HPV testing for clinical practice. |
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W.G.V. Quint
Voorburg, The NetherlandsM.N.C. de Koning1, L.V. Khoe2, J.A.H. Eekhof3, M. Kamp1, J. Gussekloo3, J. ter Schegget1, J.N. Bouwes Bavinck2, W.G.V. Quint1. 1DDL Diagnostic Laboratory, Voorburg, The Netherlands; 2Department of Dermatology, Leiden University Medical Centre, Leiden, The Netherlands; 3Department of Public Health and Primary Care, Leiden University Medical Centre, Leiden, The Netherlands. A large number of HPV types, distributed over five papillomavirus genera, infect the human skin. HPV types belonging to the alpha, gamma and mu genus have been detected in cutaneous warts. Little is known about the natural history of these HPV types. Recently, a genotyping assay has been developed that was used in an intra-patient comparison of cutaneous wart-associated human papillomavirus types in 20 immunocompetent adults treated for persistent cutaneous warts. From each patient a swab of the forehead, wart and around the wart was tested. Additionally, scabs of the wart and deeper portions of the warts were analyzed.
All wart biopsies (n=20) were positive for a single HPV type. Fourteen were positive for HPV27, 3 for HPV57, 2 for HPV2, and 1 for HPV3. Scabs and swabs of the warts both confirmed 19 of the 20 typing results from the deeper wart portions. Swabs taken from around the warts and from the foreheads were less successful in confirming the HPV type present in deeper wart portions. These swabs also revealed 3 multiple infections.
There was an excellent HPV type agreement between swabs of warts and deeper wart portions validating the use of wart swabs for future studies of wart-associated HPV types. In this small study population HPV27 was highly prevalent. Whether this HPV type can be linked to persisting warts requiring treatment in adults needs further investigation.
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W.G.V. Quint
Voorburg, The NetherlandsD.T. Geraets1, D.A.M. Heideman2, M. Frank3, M.N.C. de Koning1, P.J.F. Snijders2, C.J.L.M. Meijer2, L.J. van Doorn 1, W.G.V. Quint1. 1DDL Diagnostic Laboratory, Voorburg, The Netherlands; 2Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands; 3QIAGEN Hamburg GmbH, Research & Development, Hamburg, Germany. Background: The GP5+/6+-based digene HPV Genotyping RH and LQ Tests were recently introduced for identification of 18 HPV genotypes and 2 subtypes (i.e., HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 (68a), 73, 82MM4 (82IS39)) in HC2-positive samples. Analytical validation data are presented here.
Methods: Assays were validated according to ICH guidelines for, among others, analytical sensitivity, analytical specificity, reproducibility, accuracy and robustness.
Results:
Analytical sensitivity
Formal Limit of Detection (LOD) for HPV16, 18, and 45 ranged between 4 and 23 copies/PCR. Analytical sensitivities for the remaining 15 types varied between 1 and 100,000 copies/PCR.
Analytical specificity
The assays did not cross-react with non-targeted types. In addition, no other pathogens commonly found in the female anogenital tract were reactive.
Reproducibility
Plasmids representing all targeted HPV types and 92 clinical samples were assayed in replicates on different days by different technicians with different kit lots. The results showed 100% identical genotypes in the plasmid panel and more than 95% reproducibility for clinical samples.
Accuracy
One hundred and eight clinical samples were tested in parallel by digene RH and LQ Tests and the homebrew GP5+/6+-PCR RLB system. Initial agreement was 91% and 93% with the RH and LQ Test, respectively. After discordant resolution the agreement was 98% for both assays.
Robustness
The assay results remained unaffected by deliberate variations in relevant method parameters, such as increased or decreased PCR temperature conditions, hybridization temperatures, and incubation times.
Interfering substances
The presence of blood and other potentially interfering substances had no effect on the assays.
Conclusions: The digene RH and LQ Tests show good analytical performance characteristics. Performance evaluation on clinical samples is ongoing. However, it is important to realize that assay efficacy is determined by the entire diagnostic algorithm comprising DNA isolation, PCR and amplimer analysis.
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Anuradha Rebbapragada
Toronto, CanadaAnuradha Rebbapragada1,2, Stephen Perusini1, Alessandro Di Prima1, Kumar Ramakrishna3, Irving Salit4, Vanessa Goleski5, Sam Ratnam6, Alberto Severini5,7. 1Ontario Agency for Health Protection and Promotion, Toronto, ON, Canada; 2Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada; 3Department of BioStatistics, Dalla Lana School of Public Health, University of Toronto, Toronto, ON, Canada; 4HIV Immunodeficiency Clinic, Toronto General Hospital, University Health Network, Toronto, ON, Canada; 5National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada; 6Newfoundland Public Health Laboratory, St. John's, NL, Canada; 7Department of Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada. Background: Identification of HPV types is required for epidemiologic measurements of vaccine efficacy and type-specific risk for cervical cancer. In order to identify the most suitable genotyping platform for population studies, we evaluated 5 genotyping platforms against a panel of 355 liquid based cytological (LBC) samples.
Methods: The testing panel of 355 samples consisted of N=171 cervical LBC from women who were referred to colposcopy for follow up and N=184 intra-anal LBC from HIV-positive men with clinical history of abnormal anal pathology. Nucleic acid was extracted from LBC by the AmpLilute kit (Roche), amplified as per individual assay protocol and detected with the Roche Linear Array (LA), INFINITI HPV 26-plex (INF), Seeplex HPV18 ASE (SG), Qiagen 16-plex (QIA), and an in-house Luminex (LU) assay. LA detection of HPV52 was confirmed by PCR.
Results: In order to overcome the lack of a "gold standard" when assessing test performance, we defined a HPV consensus result when 3 tests detected the same HPV type. Since only the 13 HR HPV types are commonly represented in all 5 tests, detailed comparative analysis was focused on HR HPV. The analytical sensitivity/specificity for HR HPV detection ranged from 10% to 100% for individual HPV types. Pair-wise test comparisons revealed that concordance varied by HPV type and kappa values ranged from 0.052 to 0.862 (poor to excellent). The tests also varied in their ability to detect co-infection with multiple HPV types. Infection with >3 HPV types was detected in 31.9%, 26.5% and 31.5% of samples by the LU, SG and LA assays and in 11% by the INF and QIA assays. Cost, throughput and labour varied between assays.
Conclusions: HPV genotyping assays vary in test performance and operational parameters. Our data will inform the decision framework for the laboratory component of HPV surveillance and vaccine evaluation. |
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Nicolas F. Schlecht
Newark, USAMahnaz Fatahzadeh1, Nicolas F. Schlecht2, Janae Ostoloza3, Anne Dunne3, Dannielle Botallico3, Sharod McKinney3, Robert D. Burk3. 1Department of Diagnostic Sciences, New Jersey Dental School, UMDNJ, Newark, NJ, USA; 2Department of Epidemiology & Population Health, Albert Einstein College of Medicine, Bronx, NY, USA ; 3Department of Pediatrics, Albert Einstein College of Medicine, Bronx, NY, USA . Background: Oral HPV has been implicated in the etiology of malignant oral disease. However, reported rates of oral HPV vary by methodologies to collect specimens and the sensitivity of assays used. We hypothesized that a specimen obtained upon arising in the morning would provide more exfoliated cells and a better substrate for HPV DNA detection.
Methods: We selected an HIV positive population attending an inner city oral health clinic with high expected prevalence of oral HPV infection. Oral rinse specimens were obtained from 52 consenting adults at two time points within the same day: samples were collected upon arising in the morning and approximately 8 hours later in clinic and tested for HPV DNA by MY09/11 and FAP PCR. Kappa statistics were used to assess agreement between paired samples.
Results: The detection rate of HPV DNA in oral rinse samples ranged from 53.8% for samples collected in the afternoon to 59.6% for morning samples. Sixteen (30.8%) individuals tested positive for high-risk HPV and 34 (65.4%) for low-risk types. There was good agreement in detection of HPV DNA between morning and afternoon samples (kappa=0.57, 95%CI: 0.35-0.79). This increased when restricting for high-risk types detected in either morning (28.9%) or afternoon (23.1%, kappa=0.75, 95%CI: 0.55-0.96). Distribution in number of concurrent types was also similar for morning and afternoon samples; 17 (54.8%) and 11 (39.3%) samples, respectively were positive for a single type, whereas 11 (35.5%) and 12 (42.9%) had multiple HPV.
Conclusion: Although HPV was detected more often in early morning oral rinse samples, detection rates on the same day (at home and in clinic) were similar. Self collected oral rinse sampling may provide a potential method for monitoring oral HPV infection.
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Rachel Winer
Seattle, USAQinghua Feng1, Viorica Popov1, Steve Cherne1, Rachel Winer4, Hector Zambrano2, Carlos Yerovi3, Stephen Hawes4, Laura Koutsky4, Nancy Kiviat1. 1Department of Pathology, School of Medicine, University of Washington, Seattle, WA, USA; 2Department of Dermatology, School of Medicine, Yale University, New Haven, CT, USA; 3Department of Obstetrics and Gynecology, School of Medicine, Catholic University of Santiago de Guayaquil, Guayaquil, Ecuador; 4Department of Epidemiology, School of Public Health, University of Washington, Seattle, WA, USA. We determined the feasibility of HPV detection in cervical exfoliated cells collected as dry swab samples. Both dry cervical swab and STM cervical swab samples were collected from 135 patients attending either colposcopy or women’s clinics in Guayaquil, Ecuador, who had a cytology diagnosis within 6 months. HPV was detected by dot blot hybridization and genotyped by the liquid bead microarray (LBMA) assay. Overall, 23.1% of dry samples were positive for any high-risk HPV types, and 24.6% of STM samples were positive for any high-risk HPV types. Of 125 paired samples, the type-specific high-risk HPV proportion positive agreement was 60.7% (kappa 0.69, 95% CI: 0.53-0.82). We found that dry samples were more likely to be insufficient for HPV testing than STM samples. Consistent with this observation, we showed that genomic DNA quantitated by β-actin gene was almost 20 times lower in dry samples than in STM samples when detected by the real time Taqman assay, while HPV DNA viral loads in dry samples were only 1.6 times lower than in matched STM samples. We concluded that cervical exfoliated cells could be collected as dry swab samples for HPV detection.
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Matthew A. Titmus
Stony Brook, United StatesMatthew A. Titmus1, Eli Hatchwell1, Kenneth R. Shroyer1. 1Department of Pathology, Stony Brook University, Stony Brook, NY, USA. Integration of the human papillomavirus episome into the host cellular genome is a key step in the progression from HPV infection to oncogenesis. In HPV-induced cancers this event has been correlated with constitutive expression of the HPV E6 and E7 oncoproteins. These oncoproteins negatively interfere with host p53 and pRb, respectively, resulting in an accelerated cell cycle and increased host genomic instability. It is widely assumed that this E6/E7 over-expression is due in the majority of cases to the simultaneous disruption of the E2 ORF, whose product acts to restrict E6/E7 expression in intact HPV.
The process of integration occurs in an undirected fashion and doesn't appear to follow any consistent pattern with regard to integration sites in either the host or viral DNA. This fact, and the observation that integrated HPV DNA appears to have an increased rate of mutation, limits the usefulness of PCR-based approaches for analysis of integration events. As a result, what little is currently known about HPV integration has been stitched together from a variety of studies employing a variety of experimental approaches which, by necessity, have generally been both low-resolution and reliant on a small number of samples.
The development of high-throughput sequencing has provided us with the means to overcome these long-standing limitations by not only making it possible to simultaneously produce thousands or millions of sequences, but to do so without a detailed knowledge of the target's flanking regions. To take advantage of this technology, our lab has developed a hybridization-based method to enriching genomic DNA recovered from archival formalin-fixed paraffin-embedded (FFPE) tissues for HPV-integrated sequences to a degree suitable for massively parallel sequencing. Using this approach, it is possible to sequence the regions surrounding the host:virus interfaces of a large number of HPV-integrated samples simultaneously.
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Matthew G. Hawkins
Cambridge, United KingdomMatthew George Hawkins1, Siolian L. R. Ball1, Katie Vaughan1, David M. Winder1, Jane C. Sterling1, Margaret A. Stanley1, Peter K. C. Goon1. 1Department of Pathology, University of Cambridge, Cambridge, United Kingdom. Human papillomaviruses (HPV) cause a variety of genital lesions, including anogenital cancers and Condylomata acuminata (anogenital warts). At present, approximately 40 anogenital HPV types have been identified. However, the overall contribution of each HPV type to anogenital wart aetiology has only recently been investigated. Current studies indicate that HPV6/11 cause ~90% of these lesions, with one-third present as a co-infection with other HPV types. However, HPV presence is identified using non-quantitative HPV DNA detection methods and HPV DNA presence alone does not necessarily indicate an active role in wart aetiology. Furthermore, detected virions may be present on the wart surface following their release from surrounding warts (i.e. surface contaminants) and thus would not contribute to wart aetiology. This phenomenon has not been addressed. Anogenital warts (n=23) were obtained from the department of GU/HIV medicine in Addenbrooke's Hospital, Cambridge, UK. Tissue samples were prepared for both DNA extraction and laser capture microdissection (LCM). HPV types were identified using the Roche Linear Array genotyping test. DNA isolated following LCM allowed the comparison of HPV types present within the wart (lower layers) and on the surface (upper layers), allowing incident HPV types to be excluded. HPV E6 mRNA levels were estimated following RNA extraction and RT-PCR from whole wart tissue. HPV type involvement in wart aetiology was determined following detection of HPV-specific E6 mRNA (figure 1), an indicator of transcriptional activity and thus a more accurate determination of HPV types contributing to wart aetiology. Surface contamination was evident in 19/23 tissue samples, 10 of which exhibited multiple contamination (figure 1). Exclusion of surface contaminants and detection of HPV E6 mRNA identified HPV types potentially involved in wart aetiology (figure 2), whilst highlighting caveats in current methodologies determining HPV prevalence in anogenital warts.
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Ramona Ursu
Iasi, RomaniaRamona Gabriela Ursu1, Luminita Smaranda Iancu1. 1University of Medicine and Pharmacy Gr. T. Popa, Iasi, Romania . Objectives: To estimate the HPV genotype prevalence in North East Romania, and to check the utility of HPV 16 viral load as a biomarker, for detecting women in early stages of HPV infection, before developing cervical cancer.
Methods: We tested cervical cell specimens collected from 84 women who signed the informed consent approved by the local Bioethical Committee. The age range of women tested for HPV was from 26 to 57 years, and the Pap test was performed according with Bethesda classification. HPV genotyping was performed with Linear Array HPV Genotyping (ROCHE DIAGNOSTICS®) and PCR reaction was realized with ABI 9700 Gold Plate System. The viral load of HPV 16 positive samples was quantified with Path-HPV16 Real-time PCR detection kit for Human Papillomavirus, MX3000P STRATAGENE instrument.
Results: Among the tested samples, 38 (46%) were negative for any HPV type, 15 (19%) patients presented multiple infections with oncogenic and non-oncogenic HPV types (16, 18, 26, 33, 35, 39, 42, 51, 54, 58, 52/33/35/58, 59, 61, CP6108, 62, 81), 10 (12%) were positive only with HPV 16, 5 (6%) for HPV 53, 2 (2%) were positive for HPV 31, 1 (1%) for HPV 52/33/35/58, 1 (1%) for HPV 68 and one (1%) for HPV 84 (fig. 1). Parameters of standard curve (efficiency, pipetting, reproducibility, slope) were between the accepted limits: Eff = 96,2%, slope = 3,416, Rsq = 0,994 (fig. 2) The viral load of tested HPV 16 positive samples was between 8,025e+003 - 4,549e+005 copies.
Conclusions: The epidemiological data provides information about distribution of HPV genotypes and may be important for determining the future impact of vaccines and potential changes in the country's epidemiological HPV profile. The prevalence of HPV 16 is similar to the results of others studies. High viral load of HPV 16 was associated with HSIL. We must continue our study on more cases, to have a broader image in our region.
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Nasuna Florence
Kampala, UgandaNasuna Florence1. 1Mulago Main Referral Hospital, Kampala, Uganda. Issues: The high prevalence of HIV/ HPV and TB co-infection is evident in sub-Saharan Africa. As antiretroviral use increases in this setting, it is imperative that we examine the impact of treatment intitiation where HIV/HPV and TB co-infection exist.
Description: We describe the case of a 51 year-old Ugandan woman with HIV/HPV / TB co-infection and a T-cell count of 18. The patient was diagnosed with extrapulmonary TB following aspiration of a supraclavicular abscess that decreased in size after intensive phase TB therapy. She initiated HAART thereafter. Twenty-nine days after antiretroviral treatment began she experienced recrudescence of symptoms with marked inflamation, local pain, fever and a fluctuant nodule at the prior site of infection. Aspirate of this revealed 3+ acid-fast bacilli on smear without evidence for other organisms. Culture results are pending.
Lessons learned: HAART restores the immune response to mycobacterium disease.
Recommendation: In this era where access to antiretroviral therapy in Africa is becoming more widely available, clinicians should be alert to the manisfestations of immune reconstitution syndromes. Particularly in settings where HIV/HPV/TB endemicity is high and where antiretroviral therapy is often initiated in advanced stages of HIV/HPV disease, providers ought to be vigilant for the development of acute mycobacterial symptoms and prepared to institute steroid therapy as warranted |
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Marta del Pino
Barcelona, SpainMarta del Pino1, Immaculada Alonso1, Jaume Ordi2, Lluís Puig-Tintoré1, Pere Fusté1, Joan Balasch1, Aureli Torné1. 1Department of Obstetrics and Gynecology, IDIBAPS-Hospital Clinic, University of Barcelona, Faculty of Medicine, Barcelona, Spain; 2Department of Pathology, IDIBAPS-Hospital Clinic, University of Barcelona Faculty of Medicine, Barcelona, Spain.
Background: Colposcopy examination of women with ASC-US or LSIL detects CIN2-3 in 5-20%; 10-15% of the rest of them develop CIN2-3 after 2 years follow-up. These facts justify that current protocols recommend colposcopy examination as an integral part of the follow-up of these women. Nevertheless, it is unknown whether colposcopy findings can provide any valuable information that might help to predict which patients will progress.
Objective: To compare the outcomes of women positive for HR-HPV with normal cytology, LSIL or ASC according to the colposcopy findings at initial evaluation.
Patients and methods: From January 2000 to July 2008, women referred to the Colposcopy Unit of the Hospital Clinic of Barcelona due to an abnormal cytology and/or HR-HPV infection who, after an exam including cytology, colposcopy (with colposcopically directed biopsy or endocervical curettage if indicated) and HPV detection, fulfilled following inclusion criteria were prospectively recruited: 1) positive HR-HPV testing, 2) negative, ASC or LSIL cytology. Follow-up controls were scheduled every 6 months. Colposcopy findings were recorded following the International Federation for Cervical Pathology and Colposcopy (Barcelona 2002) criteria. Patients were grouped according to their follow-up outcome: regression, defined as negative Pap, and negative HR-HPV detection; persistence, established on the basis of LSIL/CIN1 diagnosis and/or persistent HR-HPV infection; progression, defined as histological diagnosis of CIN2/3.
Results and conclusion: 465 women were recruited (mean age 33.6±10.4 years). Diagnoses at enrolment were: normal cytology in 92 women (19.8%), ASC in 67 (14.4%) and LSIL/CIN1 in 306 (65.8%). Table 1 shows colposcopy findings according to the initial diagnoses. No significant differences were found between groups (p=0.491). After a mean follow-up of 19.5 months (range 12.0-90.9), 161 (34.6%) women regressed, 261 (56.1%) showed a persistent lesion and 43 (9.3%) progressed. Table 2 shows the outcomes according to initial colposcopy findings (p=0.284). 21/174 (12.1%) of the women with an abnormal transformation zone (ATZ) involving <50% of the cervical surface, 2/38 (5.3%) of women with ATZ involving >50% and 1/4 (25.0%) of those showing only endocervical lesion progressed (p= 0.228). In conclusion, in women with HR-HPV infection and minimal or no cytological abnormalities neither colposcopy characteristics nor lesion surface area were related to progression risk.
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Robert Pickard
Columbus, USARobert Pickard1, Andrea Inman1, Weihong Xiao1, Tatevik Broutian1, Mike Koluder1, Xin He2, Maura Gillison1. 1Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA; 2Dept. of Epidemiology and Biostatistics, School of Public Health, University of Maryland, College Park, MD, USA . Background: Oral sexual behavior and marijuana smoking have been associated with HPV-positive oropharynx cancer. Despite dose-response relationships, residual confounding of the marijuana-tumor association by sexual behavior remains plausible.
Methods: A cross-sectional study of the association between sexual behavior, marijuana use, and prevalent oral HPV infection was performed among 1000 young adults age 18-30 at the Ohio State University, Columbus, Ohio. Behavioral data were collected by computer-assisted self-interview (CASI) and oral samples by Scope oral rinse and gargle (ORG). ORG DNA purified by QiaSymphony was evaluated for 37 HPV types by Roche Linear array. Logistic regression was used to model factors associated with infection.
Results: The study population was 54% female, 69% white, and median age was 21 years (IQR 19-23). Sexual behaviors and substance use were strongly associated with gender and race. Oral HPV infections were detected in 2.4% (95% CI:1.4–3.4), and half were high-risk infections. Factors associated with oral HPV infection in univariate analysis included black race, increasing age, sexual behavior (kissing, oral and vaginal sex) and frequency of marijuana use. Non-significant associations were observed with male gender, history of genital warts, young age at open-mouth kissing and intercourse, and infrequent alcohol use, but not measures of tobacco smoking or marijuana-joint sharing. In multivariable analysis, odds of infection were independently associated with black race (OR = 6.9; 95% CI: 2.3–21.0), five or more lifetime oral sex partners (OR = 5.7; 95% CI: 2.3–14.0), and frequent recent marijuana use (OR = 5.3; 95% CI: 1.3–20.1), after adjusting for gender and age.
Conclusions: Marijuana use is associated with oral HPV infection after adjustment for oral sexual behavior, consistent with a possible biological effect of cannabinoids on oral HPV natural history. Analysis of associations with incident and persistent infection is ongoing.
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Adrian Dana
North Wales, United StatesAdrian Dana1, Karyn Buchanan1, Mary Ann Goss1, Margaret Seminack1, Kristine Shields1, Scott Korn1, Richard Haupt1. 1Merck Research Laboratories, West Point, PA, USA. Objectives: The Pregnancy Registry for Gardasil (Merck) collects voluntary post-marketing reports of pregnancy exposures from health care providers or the public. This report covers the period from product licensure (June 2006) through May 31, 2009.
Methods: The registry is conducted in the US, Canada and France. Enrollment criteria include an identifiable health care provider and patient, with vaccine exposure occurring within 1 month prior to the date of onset of the last menstrual period (LMP) or during pregnancy. The primary outcomes of interest are birth defects, live births, fetal deaths, elective terminations, and spontaneous abortions. Outcomes from prospectively reported pregnancy exposures with estimated dates of delivery within the report period are used to calculate rates.
Results: 1897 women were enrolled in the registry with 1636 exposures reported prospectively (before the outcome of the pregnancy was known). Of the prospective reports with known outcome, there were 65 elective abortions, 64 spontaneous abortions, 2 ectopic pregnancies, 10 fetal deaths, and 968 live births, which included 6 sets of twins. Of 974 infants, 926 were normal infants with no defects, 21 had major and 26 had minor congenital anomalies. The overall rate of spontaneous abortion was 6.1 per 100 outcomes (95%CI: 4.7 to 8.0). The prevalence of congenital anomalies at birth was 2.4 cases per 100 live born infants (95%CI 1.5, 3.5). Of the 10 fetal deaths, 1 case included Turner's syndrome, 3 had attributable causes, and 3 had a history which might have contributed to the outcome.
Conclusions: Rates of spontaneous abortions and major birth defects were comparable to those reported in the literature (15% and 2.67%, respectively). Although the numbers of exposures are not sufficient to rule out a low risk, data collected in the pregnancy registry to date do not support a relationship between vaccine exposure and adverse fetal outcomes.
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Vincent R. Bonagura
Great Neck, USAVincent Bonagura1,2, Lynda Hatam1,2, James DeVoti1,2, David Rosenthal1,2, Allan Abramson1,3, Bettie Steinberg1,2,3. 1Feinstein Inst. Medical Res. NS-LIJ Health System, New York, NY, USA; 2Elmezzi Graduate School for Molecular Medicine, Feinstein Inst. Medical Res., NS-LIJ Health System, New York, NY, USA; 3Department of Otolaryngology, NS-LIJ Health System, New York, NY, USA. Introduction: We recently showed an altered frequency of killer cell immunoglobulin-like receptor (KIR) gene haplotypes in RRP. Specifically, KIR frequency haplotypes lacking 3 activating KIR genes, KIR3DS1, KIR2DS1, KIR2DS5, predicted disease severity. This suggested that these KIRs may be required to remove HPV-infected keratinocytes without HLA class I expression observed in papillomas. Reduced/absent HLA class I expression should prevent inhibitory KIR molecule-ligation on NK cells, and release NK inhibition of NK cytolysis, permitting activating KIR ligation by viral epitopes in/outside of the context of HLA class I molecules.
Methods: To determine if NK cells obtained from the blood of patients with RRP can cause cytolysis of target cells, we performed a CD107a mobilization assay using NK cells from 12 RRP patients and 13 controls. CD107a is a cell surface marker transiently expressed after cytolytic granule release, and correlates with cytokine secretion and NK cytolysis. PBMC were cultured 4hrs in media alone or with the HLA class I deficient cell line K562, at an effector:target ratio (E:T) of 5:1 in the presence of FITC-conjugated anti-CD107a and monensin. Following incubation, cells were stained with the amine-reactive live/dead fixable violet dead cell stain kit and CD3 APC, CD56 PE, CD14 and CD19 PerCP. Samples were analyzed by flow cytometry.
Results: The percentage of CD107a expression on CD3-56+ NK cells after subtraction of the spontaneous activity showed a significant reduction of CD107a expression on NK cells in patients with RRP, vs controls (p=0.044).
Conclusion: There is defective NK cellular innate responses in RRP, manifest as the failure of NK cells to be activated by target cells lacking HLA class I. Thus, papillomas containing keratinocytes lacking HLA class I are not likely to be cleared by NK cells in papillomas, thus adding cellular innate immune dysregulation to adaptive TH2-like/Treg polarization present in patients with RRP.
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Marilou Kiely
Québec, CanadaMarilou Kiely1, Philippe De Wals1, Chantal Sauvageau1, Ève Dubé1, Geneviève Deceuninck2. 1Institut national de santé publique du Québec, Québec, QC, Canada; 2Unité de recherche en santé publique, CHUQ, Québec, QC, Canada.
Background: Human papillomavirus (HPV) infections are the most common sexually transmitted infections (STIs) in North America. An HPV immunization program was established in Quebec in the fall 2008. Assessing the potential impact of vaccination on sexual behaviours and screening practices is needed for a comprehensive evaluation of the program. The aim of this study was to describe the knowledge and beliefs of women in Quebec regarding HPV infections as well as their sexual behaviours, cancer screening practices and their vaccination status against HPV. This survey represents the first step in a series of cross-sectional surveys aimed to identify trends.
Method: In spring 2009, 1347 women in Quebec of 24 years of age, randomly selected in the provincial health insurance database, participated in a cross-sectional descriptive survey conducted by mail questionnaire.
Results: The response rate was 56%. More than 85% of participants had heard of HPV, but less than half of them were able to correctly identify the mode of transmission of HPV. Over 80% of respondents knew what causes cervical cancer. 81% of surveyed women had heard about the HPV vaccine, but only 5% of participants had already received it. The average age at the 1st sexual intercourse was 16.5 years. Over 80% of participants have been screened for cervical cancer and average at initiation of screening was 17.5 years. Finally, over 55% of respondents said they were not satisfied with the information received about HPV and cervical cancer.
Conclusions: This study yielded data from a representative sample of 24-years-old women in Québec. Although most women surveyed had heard about HPV and the vaccine, gaps remained in their knowledge. In addition, the majority of respondents were not satisfied with the information received on HPV. A better way of transmitting information to women should be developed.
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Hester de Melker
Bilthoven, The NetherlandsJeanet Kemmeren1, Nicoline van der Maas1, Hester de Melker1. 1Centre for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven, The Netherlands.
Background: In March 2009, a catch-up campaign with HPV 16/18-vaccination for girls born between 1993 and 1996 was implemented in the Netherlands. Alongside implementation in the National Immunization Program monitoring of vaccine safety is performed.
Objectives: To study the reactogenicity of the bivalent HPV vaccine following administration of a first dose.
Methods: A questionnaire on local and systemic reactions was distributed by email one week after HPV vaccination to 5950 13-16-year-old girls vaccinated in the catch-up campaign. Response to the questionnaire was 68.7%. Within this descriptive study, the incidence, 95% confidence intervals, severity and duration of local reactions and systemic events were calculated.
Results: Local reactions were reported by 92.1% of the girls. In 1.6% these symptoms were severe. Pain at the injection site (83.7%) and reduced arm use (71.3%) were the most reported symptoms. Systemic events occurred in 91.7% and most often was myalgia (75.0%). However, 13.6% reported headache in the week before the vaccination. Fatigue and headache were reported by 33.9% and 30.0% of the vaccinated girls, respectively, and 0.7% had high fever (>= 39.5 ºC). 1.5% of the girls called for medical assistance.
Conclusion: In accordance with data from international studies, side effects after HPV 16/18-vaccination were common, but are in general mild and self-limited. The frequency of local reactions after HPV vaccination is quite comparable with the frequency of local reactions after dT-IPV vaccination at the age of nine years (92.1% vs. 83.4%, respectively), but the frequency of systemic events is substantially higher after HPV vaccination (91.7% vs. 33.4%, respectively). However, many of these symptoms also occur without vaccination and may be age-related; for example 13.6% of the girls reported headache in the week before vaccination.
Results will be used to inform the public and health care workers to increase confidence in the HPV vaccination program.
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Rosa Legood
London, CanadaRosa Legood1, Milena Farco2, Chris Roberts2, Henry Kitchener2. 1Health Service Research Unit, London School of Hygiene and Tropical Medicine, London, UK; 2University of Manchester, Manchester, UK. Background: Currently there are a range of future options for cervical cancer screening including primary HPV testing utilising hybrid capture II, cytology followed by HPV triage and further combinations based on type 16/18 testing. Future directions in research also need to consider the implications of strategies for the post-vaccination era. The objective of this modelling study was to utilise ARTISTIC trial data to assess the potential cost-effectiveness of alternative cervical cancer screening strategies to help identify likely optimal strategies going forward.
Methods: The setting of the ARTISTICtrial was Greater Manchester in the UK, and the study population were 24,000 women aged 20-64 who underwent primary cervical cancer screening. The trial cohort was randomised to combined screening with human papillomavirus (HPV) testing and liquid based cytology (LBC), or screening only with LBC (though HPV tests were performed and the results concealed). Genotyping data was also collected as part of the trial protocol.
In this study, clinical data from the first round of the trial were re-analysed and split by age group to assess the within trial colposcopy outcomes of alternative management strategies and matched with data on initial HPV types. A mathematical model was developed incorporating both trial data and further evidence synthesis, to predict management outcomes utilising alternative potential management strategies for both an unvaccinated and vaccinated cohort. Cost data has already been collected alongside the trial and further indicative prices of novel testing strategies were then obtained from the companies. The comparative cost per case detected of alternative management strategies over one screening round were then assessed in a stochastic framework.
Results: Results from the study will be presented at the meeting on the cost-effectiveness of alternative management strategies. These data will be utilised to inform on likely potential cost-effective options for cervical cancer screening. |
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Jerome L. Belinson
, Hui Du1, Bin Yang2, Xinfeng Qu3, Suzanne Belinson4, Robert Pretorius5, Gao Yang6, Chun Wang1, Jingjing Li6, Ruifang Wu1, Jerome L. Belinson4. 1Peking University Shenzhen Hospital Department of OB/Gyn, Shenzhen, PR China; 2Cleveland Clinic Department of Pathology, Cleveland, OH, USA; 3Royal Ladies Gynecology Clinic, Shenzhen, PR China; 4Preventive Oncology International Inc., Cleveland Heights, OH, USA; 5Kaiser Permanente, Fontana, CA, USA; 6Beijing Genomics Institute, Shenzhen, PR China. Objectives: The Shenzhen Cervical Cancer Screening Trial II (SHENCCAST II) is a 10,000 woman study examining self-sampling, multiple HPV technologies, cytology with computer assisted detection, and genotyping. We report here an evaluation of cytology and genotyping as secondary screens after primary HPV testing.
Methods: Women from 3 sites in rural Guangdong Province and city sites in Shenzhen, China were eligible if non-pregnant, 25-59 yrs of age, no screening ≥3 years, no hysterectomy, and no pelvic radiation. 10,000 women will be screened with self and direct HPV testing as well as ThinPrep cytology with computer assisted detection. The entire study population will be genotyped for 14 high-risk HPV types both on their self and direct samples using the high-throughput mass spectrometry based system MALDI-TOF. Positivity by any HPV test and/or cytology led to colposcopy where all patients were colposcoped and biopsied using the POI biopsy protocol of directed and random biopsies (minimum 5/patient).
Results and Conclusions: 4965 women are included in this interim analysis (full 10,000 will be presented). Mean age 38.8 years, >=ASCUS = 549/4965 (11.06%), >=LGSIL= 185/4965 (3.73%),>=HGSIL = 98/4965 (1.97%);>=CIN II = 148/4965 (2.98%), >=CIN III = 95/4965 (1.91%); HPV + rates for direct HC-II 725/4965 (14.60%), HPV + rates for self-sampling Maldi 791/4965 (15.93%). A clearly superior secondary screen is not identified. All options have strengths and major weaknesses depending on the clinical setting. |
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Jerome L. Belinson
, David Starks1, Lucybeth Nieves-Arriba1, Christine Enerson2, Suzanne Belinson3, Christine Booth4, Jennifer Brainard4, Andres Chiesa-Vottero4, Jerome L. Belinson3. 1Cleveland Clinic Department of Ob/Gyn, Cleveland, OH, USA; 2Prueba para La vida A.C, Morelia, Mexico; 3Preventive Oncology International Inc., Cleveland Heights, OH, USA; 4Department of Anatomical Pathology, Cleveland, OH, USA. Objective: To determine the effectiveness of triage VIA after primary HPV screening, for prevention of undertreament when performing cryotherapy prior to biopsy confirmation.
Methods: In the Mexican Cervical Cancer Screening Trial II (MECCS II) participants were women ages 30-50, non-pregnant, variable screening histories, and no prior hysterectomy or pelvic irradiation. All obtained a self-sample using the 4th generation POI/NIH brush. The sample was placed in PreservCyt for the high-risk HPV assays (HC-II and APTIMA). Then two direct cervical samples were obtained for cytology, HC-II, and APTIMA. Subjects positive on any test were recalled. At the 2nd visit, triage VIA was used to identify preinvasive disease too large for the cryo probe (>3 quadrant disease) or cancer. All patients were then colposcoped using the POI protocol of directed and random biopsies. All HC II positive subjects eligible by VIA triage were treated with cryotherapy. All patients treated with cryotherapy were requested to return in 6 months. At 6 months, a direct sample was obtained for cytology, HC-II, and APTIMA. All subjects who had abnormal biopsies prior to cryotherapy, or whose cervix appeared abnormal, were colposcoped and re-biopsied.
Results/Conclusions: 2512 women were included in the study with a mean age of 39 years. 300 received cryotherapy (297 with complete data). 81% received follow-up, 231 at 6 months and 10 by referral MD. 229/231 have complete 6 month data. Prior to cryotherapy 30/297 women (10%) had >=CIN 2 (15/30 ECC positive, 2/30 >3 quadrant disease). At follow up 5 women had > =CIN 2 (2/5 on ECC) and there were no cases of >= 3 quadrant disease. Aptima detected all five high grade lesions, cytology and HC-II each missed a CIN2. VIA triage does not evaluate the endocervix effectively, at 6 months 9/11(82%) with prior positive ECCs tested negative after cryotherapy. |
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Jerome L. Belinson
, Jerome L. Belinson1, Hui Du2, Bin Yang3, Suzanne Belinson1, Xinfeng Qu4, Phillip Castle5, Robert Pretorius6, Chun Wang2, Gao Yang7, Ruifang Wu2. 1Preventive Oncology International Inc., Cleveland Heights, OH, USA; 2Peking University Shenzhen Hospital Department of OB/Gyn, Shenzhen, PR China; 3Cleveland Clinic Department of Pathology, Cleveland, OH, USA; 4Royal Ladies Gynecology Clinic, Shenzhen, PR China; 5National Institutes of Health, Bethesda, MD, USA; 6Kaiser Permanente, Fontana, CA, USA; 7Beijing Genomics institute, Beijing, China. Objective: Compare the sensitivity and specificity for detection of ≥CIN 2 of two vaginal self-collection devices (POI/NIH brush vs. Qiagen brush) using HR-HPV assays Cervista, Maldi-TOF, and HC2.
Methods: 10,000 women age 25 to 59 years residing in Guangdong Province, China who were recruited for the Shenzhen Cervical Cancer Screening Trial II (SHENCCAST II). Women were randomly assigned to self-collect vaginal specimens with either the POI/NIH self-sampler or the Qiagen conical-shaped brush. All subjects had cervical specimens collected with the broom sampler by doctor directly and placed in PreservCyt for cytology and HPV testing. Cervical cytology specimens were prepared by the ThinPrep method. Vaginal self-collected specimens were tested for HR-HPV by Cervista and Maldi-TOF. The direct cervical specimens were tested for HR-HPV by HC2, Cervista, and Maldi-TOF assays. Women with HR-HPV in direct cervical or vaginal self-collected specimens or with cervical cytology of ASC-US or greater underwent colposcopy with our 5-biopsy protocol.
Results: This interim analysis includes the first 4,965 participating women. Their mean age was 38.8 years. 3% (148/4965) were diagnosed with ≥CIN 2. For direct cervical specimens HC 2 (sensitivity 94.6%), Cervista (90.5%) and MALDI-TOF (92.6%) were on similar ROC curves. Using Cervista for detection of HR-HPV, the sensitivity for ≥CIN 2 of the POI/NIH sampler (76.4%) was similar to that of the Qiagen brush (71.1%) p=0.58 . Using Maldi-TOF for detection of HR-HPV, the sensitivity for ≥CIN 2 of the POI/NIH sampler (95.8%) was similar to that of the Qiagen brush (88.2%) p=.16) and similar to that of direct cervical Maldi-TOF (92.6%) p>0.05.
Conclusion: For direct cervical specimens, areas under the ROC curves for Maldi-TOF, Cervista, and HC2 are similar; for vaginal self-collected specimens, the sensitivity and specificity for ≥CIN 2 of the POI/NIH sampler tested with Maldi-TOF is similar to that of the direct cervical specimens. |
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Jerome L. Belinson
, Ruifang Wu1, Hui Du1, Suzanne Belinson2, Bin Yang3, Xinfeng Qu4, Chun Wang2, Ruosong Wu4, Guoping Wang1, Gao Yang5, Jerome L. Belinson2. 1Peking University Shenzhen Hospital Department of OB/Gyn, Shenzhen, PR China; 2Preventive Oncology International Inc., Cleveland Heights, OH, USA; 3Cleveland Clinic Department of Pathology, Cleveland, OH, USA; 4Royal Ladies Gynecology Clinic, Shenzhen, PR China; 5Beijing Genomics institute, Beijing, China. Objectives: To test a new self sampling device, 2 new HPV platforms and a new computer assisted detection system in a population based screening trial.
Methods: 10,000 women were screened in Guangdong Province, China, if non-pregnant, 25-59 yrs of age, no screening < 3 years, no hysterectomy, and no pelvic radiation. Self-sampling was block randomized between the Qiagen brush and the new POI/NIH brush and tested with Cervista and MALDI-TOF for HR-HPV. Direct cervical sampling was placed in PreservCyt, from which Cytology, HC-II, Cervista, and MALDI-TOF were processed. All women with ≥ASCUS, and/or positive self or direct HR-HPV were asked to return for colposcopy and biopsies using the POI directed and random biopsy protocol.
Results: 4965 women are included at this time in this interim analysis, Mean age 38.8 years, ≥ASCUS = 549/4965 (11.06%), ≥HSIL = 98/4965 (1.97%); ≥CIN II = 148/4965 (2.98%), ≥CIN III = 95/4965 (1.91%); HPV + rates for direct tests HC-II 725/4965 (14.60%), Cervista 607/4965 (12.23%), Maldi 693/4965 (13.96%). HPV + rates for self-sampling: Cervista 901/4965 (18.15%), Maldi 791/4965 (15.93%). Cytology using the I2 imager had a sensitivity and specificity (≥ASCUS ) for ≥ CIN II of 89.9% and 91.4% respectively. The areas under the ROC curves for the direct HC-II, Cervista, and the Maldi were similar p=0.088. Using Cervista the sensitivity for ≥CIN II of the POI/NIH self-sampler was similar to the self-sampling Qiagen brush (76.4% vs.71.1% respectively, p=0.58). Using Maldi-TOF for detection of HR-HPV, the sensitivity for ≥CIN II of POI/NIH self-sampler was similar to self-sampling Qiagen brush (95.8% vs. 88.2% respectively, p=0.16) and similar to that of direct Maldi-TOF (92.6%) p>0.05.
Conclusions: The I2 imager functions as an excellent “cytotech” surrogate. All the HPV assays worked similarly well as direct tests, and the POI/NIH self-sampler with a PCR based assay equals their performance. |
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Hong-Jin Kim
College of Pharmacy, Chung-Ang University
Seoul, KoreaSu Jeung Lim1, Hyoung Jin Kim1, Hong-Jin Kim1. 1College of Pharmacy, Chung-Ang University, Seoul, Korea. In the virus-like particle (VLP) of human papillomavirus (HPV), conformational epitopes are an important factor affecting induction of neutralizing antibodies in the use of prophylactic vaccine. The structure of conformational epitope can be altered by binding with ligand. However, there has been no research investigating the effects of purification method in antigenicity and immunogenicity of VLP although the interaction between VLP and ligand on column resin is a potential factor affecting the structure of conformational epitope. Recently, we developed two one-step chromatographic methods for purifying the HPV VLP from Saccharomyces cerevisiae. One method uses heparin chromatography and the other, cation-exchange chromatography. In this study, we compared assembly characteristics and immune responses in mouse model between VLPs purified by the two methods above. As a result, it was confirmed that the VLPs purified by heparin chromatography (hHPV16 VLP) contained more unstable VLP than the VLP purified by cation-exchange chromatography (cHPV16 VLP). In addition, hHPV16 VLPs more strongly react with monoclonal antibody (Camvir-1) that reacts with linear epitope of HPV16 L1 than cHPV16 VLPs do. Therefore, two kinds of VLPs showed different assembly characteristics. In the study of mouse immunization, the hHPV16 VLP induced weak or no spleenocyte proliferation while the cHPV16 VLP effectively induced spleenocyte proliferation. In the humoral immune responses, the neutralizing antibody titer induced by cHPV16 VLP was three times higher than that induced by hHPV16 VLP while the total antibody titers against HPV16 VLP induced by two kinds of VLPs were similar. These immune response results indicate that the conformational change on the VLP by interaction between VLP and heparin on column resin results in alteration of immune responses. Therefore, we suggest that the selection of ligand in purification of HPV16 VLP is an important factor for determining the quality of VLP as a prophylactic vaccine.
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Sandra Giannini
Rixensart, BelgiumSandra Giannini1, Laurence Lockman1, Neil D. Christensen2, Sarah A. Brendle2, Karla K. Balogh2, Sally Mossman1. 1GlaxoSmithKline Biologicals, Rixensart, Belgium; 2Penn State University, College of Medicine, Hershey, PA, USA. Context: Clinical and preclinical data suggest that protective immunity to human papillomavirus (HPV) infection is afforded by high levels of HPV type-specific neutralizing antibodies. However, due to the fact that L1 conformational type-specific neutralizing antibodies play a major role in protection against papillomavirus, the cross-protective efficacy to HPV-31 and -45 suggested by the clinical trials with AS04-adjuvanted HPV-16/18 L1 VLP vaccine (Cervarix™, GSK) was unexpected.
Objective: To determine whether the rabbit HPV/CRPV quasivirion challenge model would be valid to assess the immune mechanism of cross protection induced by HPV L1 VLP vaccination.
Methods: New Zealand White rabbits received 3 intramuscular injections with either HPV-16/18 L1 VLPs (20/20 μg) adjuvanted with AS04 (50 μg MPL [3'-O-desacyl-4'-monophosphoryl lipid A] + 500 μg Al(OH)3), HPV-31/45 L1 VLPs (20/20 μg) adjuvanted with AS04, AS04 alone, or cottontail rabbit papillomavirus (CRPV) L1 VLPs (50 μg). Each rabbit was challenged 1 month post vaccination with different quasivirions. Serum samples were collected prior to challenge to assess antibody responses against HPV-16/18/31/45/58 L1 VLPs. Size and frequency of lesions were evaluated 2-8 weeks post challenge to assess protection.
Results: Both HPV-16/18 L1 VLPs/AS04 and HPV-31/45 L1 VLPs/AS04 vaccines induced high levels of specific and cross protection to HPV-16, -18, -31 and -45, but only partial protection to HPV-58. As expected, neither vaccine induced protection to CRPV. Homologous protection to HPV-16/18/31/45 was associated with high levels of ELISA and neutralizing antibodies. In contrast, heterologous protection was associated with relatively low cross-reactive ELISA titers and no or low levels of neutralizing titers.
Conclusions: Experimental findings are concordant with clinical studies demonstrating the potential for cross protection with HPV L1 VLPs following vaccination with HPV-16/18 or HPV-31/45 L1 VLPs formulated with AS04. The rabbit HPV/CRPV quasivirion challenge model will be used to help define the immune mechanism of cross protection. |
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Young Bong Kim
Seoul, South KoreaLee Hee-Jung1, Choi Jae-Yu1, Hur Yoon-Ki1, Oh Yu-Kyoung2, Kim Young Bong1. 1Department of Ainmal Biotechnology, Konkuk Univeristy, 1 Hwayang-dong, Gwangjin-gu, Seoul, South Korea; 2School of Pharmacy, Seoul National University, Gwanak-gu, Seoul, South Korea. Vaccination against the most common oncogenic human papillomavirus (HPV) types, HPV 16 and HPV 18, could prevent development of up to 70% of cervical cancers worldwide. We developed a novel DNA vaccine for HPV; a recombinant baculovirus bearing human endogenous retrovirus (HERV) envelope protein, which cannot replicate in mammals, was used as a nano carrier for HPV-L1 DNA vaccines (AcHERV-HP16L1, AcHERV-HP18L1). For in vivo test, mice were injected intramuscularly with 107 particles of the constructs, with two boosts at 2-week intervals. Compared with Cervarix®, or Gardasil® (1/20 of human dose), the AcHERV-HP16/18L1 immunized mice showed similar high levels of humoral immunity in IgG/IgA and elicited strong HPV16/18L1-specific CD8+ T cell immunity. Therefore, we suggest that AcHERV-HPV16L1 and AcHERV-HP18L1 vaccine can be further developed as a potential DNA vaccine which can provide both humoral and cell-mediated immune responses.
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Yu-Kyoung Oh
Seoul, South KoreaHee-Jeong Cho1, Taehoon Chun1, Young Bong Kim2, Yu-Kyoung Oh3. 1School of Life Sciences and Biotechnology, Korea University; 2Department of Animal Biotechnology, Konkuk University; 3School of Pharmacy, Seoul National University, Seoul, South Korea.
Needle-free nonparenteral vaccines offer a number of practical advantages, especially in developing countries. To address the effects of vaccine administration route, we tested mucosal and systemic immune responses against human papillomavirus 16 L1(HPV16L1) protein using intranasal, intravaginal, transdermal, sublingual (SL) and intramuscular routes. The SL route provided the most effective mucosal secretory IgA (sIgA) and serum IgG responses. After a 150 ug antigen dose via the SL route, saliva sIgA levels were 7.2- and 5.8-fold higher than those achieved via intravaginal and transdermal routes, respectively. Notably, SL administration even produced 4.6-fold higher levels of vaginal sIgA levels than did intravaginal delivery of 150 ug antigen. To enhance the immunogenicity of SL vaccines, we tested the adjuvanticity of nine molecules: three toll-like receptor agonists, three nucleotide-binding oligomerization-domain agonists, vitamin D3, poly-γ-glutamic acid and cholera toxin subunit B (CTB). Among the molecules tested, CTB provided the most enhanced mucosal sIgA and systemic IgG induction. SL-applied CTB enhanced the production of interleukin-4 and interferon-gamma from stimulated CD4+ T cells. Moreover, interferon-gamma–producing CD8+ T cell responses were increased 1.7-fold after co-treatment with SL CTB and HPV16L1. These results suggest the potential of the SL route for delivery of HPV16L1 vaccines using CTB as an adjuvant.
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Callie Bounds
Hershey, USACallie Bounds1,2, Jiafen Hu2, Nancy Cladel2, Karla Balogh2, Lynn Budgeon2,3, Neil Christensen1,2,3. 1Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, PA, USA; 2Jake Gittlen Research Foundation, Pennsylvania State University College of Medicine, Hershey, PA, USA; 3Department of Pathology, Pennsylvania State University College of Medicine, Hershey, PA, USA. The cottontail rabbit papilloma virus (CRPV) rabbit model has been widely employed to test the protective cell-mediated immunity generated to CRPV early viral proteins via intramuscular and gene gun mediated intracutaneous vaccination. With the advent of the HLA-A2.1 transgenic rabbit model system, new HLA-A2-restricted epitopes have been discovered and the epitope-specific cellular immune responses generated via intracutaneous gene gun mediated DNA vaccination have been assessed. Published reports from our laboratory indicate that gene gun delivery of DNA vaccines induce specific protective cellular immunity in the CRPV rabbit model and the HLA-A2.1 transgenic rabbit model. However, the parts necessary for this type of vaccination are costly and introduce variability into vaccination experiments as component composition can differ greatly by manufacturer. Furthermore, necessary gene-gun maintenance repairs are costly. For these reasons, we chose to find a more cost-effective and consistent means of DNA vaccination. DNA vaccination via tattooing has been employed in several animal model systems with great success and this technique allows for direct application of the DNA vaccine to the skin thus reducing the probability of inconsistency during vaccination. Vaccination of HLA-A2.1 transgenic rabbits with known HLA-A2-restricted CRPV E1 epitopes via DNA tattooing induced epitope-specific cellular immunity in the HLA-A2.1 transgenic rabbits and this immunity was completely protective as compared to week 7 papilloma clearance in gene gun vaccinated rabbits. Additionally, DNA vaccination of HLA-A2.1 transgenic rabbits with the HPV16/E7 82-90 vaccine using a tattoo gun or gene gun yielded similar levels of protection against an epitope-modified CRPV viral DNA genome. These data indicate that the tattoo gun is an alternative and useful means of DNA vaccine delivery.
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Callie Bounds
Hershey, USACallie Bounds1,2, Jiafen Hu2, Karla Balogh2, Neil Christensen1,2,3. 1Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, PA, USA; 2Jake Gittlen Research Foundation, Pennsylvania State University College of Medicine, Hershey, PA, USA; 3Department of Pathology, Pennsylvania State University College of Medicine, Hershey, PA, USA. Our laboratory employs both the HHDII mouse model and the HLA-A2.1 transgenic rabbit model to aid our pursuit of new HPV HLA-A2-restricted epitopes and to examine the epitope-specific immunity induced upon DNA vaccination and subsequent viral DNA challenge. We have recently employed a new technique of inserting HPV16 E7 HLA-A2-restricted epitopes into the E7 gene of the Cottontail Rabbit Papillomavirus (CRPV) genome. HLA-A2-restricted human papillomavirus type 16 (HPV16) E7 epitopes (amino acids 11-19 or modified 49-57*) that stimulated epitope-specific CTLs from spleen cells of HHDII mice after peptide vaccination were inserted into the CRPV genome. The newly created CRPV genomes were named CRPV/E7ins11-19 and CRPV/E7ins49-57*. The two epitope-modified genomes were assessed for viability and the HLA-A2.1 transgenic rabbits were utilized to study the epitope-specific cellular immune responses generated after gene gun mediated DNA vaccination and subsequent epitope-modified CRPV viral DNA challenge. Both epitope-modified genomes were capable of creating papillomas on the backs of NZW rabbits. These data indicate that the CRPV genome has plasticity and that the E7 gene is permissive for epitope-modification. HLA-A2.1 transgenic rabbits receiving the HPV16/E7 49-57* DNA vaccine were partially protected against challenge with the CRPV/E7ins49-57* genome as compared to control-vaccinated rabbits. Conversely, HLA-A2.1 transgenic rabbits receiving the HPV16/E7 11-19 DNA vaccine did not show increased protection against challenge with the CRPV/E7ins11-19 genome as compared to control-vaccinated rabbits. These studies demonstrate that epitope position and epitope sequence affect the stimulating capacity of individual epitopes.
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Callie Bounds
Hershey, USACallie Bounds1,2, Jiafen Hu2, Nancy Cladel2, Karla Balogh2, Neil Christensen1,2,3. 1Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, PA, USA; 2Jake Gittlen Research Foundation, Pennsylvania State University College of Medicine, Hershey, PA, USA; 3Department of Pathology, Pennsylvania State University College of Medicine, Hershey, PA, USA. The cottontail rabbit papillomavirus (CRPV) rabbit model has been used extensively to test the protective immunity elicited by VLP-based vaccines and the cell-mediated immunity generated to CRPV early viral proteins E1, E2, E6, E7, and E8. The newly established HLA-A2.1 transgenic rabbit model has proven useful for testing the immunogenicity of well-known HPV16 E7 A-2-restricted epitopes as well as computer-predicted CRPV E1 A2-restricted epitopes. In the current study, we compared the protective immunity induced to a preferred HPV16 E7 A2-restricted epitope, amino acids 82-90 (LLMGTLGIV), which has been relocated within the CRPV genome using two different methods. This epitope was inserted into the CRPV E7 gene creating the epitope-modified genome, CRPV/E7ins82-90. Additionally, the epitope was substituted into the CRPV L2 gene creating the epitope-modified genome, CRPV/L2sub82-90. Repositioning of the HPV16 E7 82-90 epitope within an early gene and a late gene of the CRPV viral DNA genome allowed us to examine and compare the ability of epitope-specific induced immunity to respond to an early expressed versus a late expressed target. Epitope repositioning within both the E7 gene as well as the L2 gene resulted in increased protection against epitope-modified CRPV viral DNA challenge of the vaccinated HLA-A2.1 transgenic rabbits as compared to control-vaccinated groups. Furthermore, the epitope-specific response was superior for the E7 epitope-target as compared to the L2 epitope-target. These data indicate that both early and late genes can be targeted by specific cellular immunity and point to increased cellular immunity to early rather than late genes of papillomaviruses. Furthermore, this study highlights the broad utility of the HLA-A2.1 transgenic rabbit model for testing numerous immunological facets involved in vaccine generated protective immunity.
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Sun-Woo Yoon
Daejeon, South KoreaSun-Woo Yoon1, Il-Han Lee2, Moon-Hee Sung2,3, Haryoung Poo1. 1Viral Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejon, Korea; 2Bioleaders Cooperation, Daejeon, Korea; 3Department of Nano- & Biochemistry, Kookmin University, Seoul, Korea. We developed a HPV mucosal vaccine candidate using the poly-γ-glutamic acid synthetase A (pgsA) protein from Bacillus subtilis Chungookjang to display human papillomavirus (HPV) type 16 L2 protein on the surface of Lactobacillus casei (L. casei). The papillomavirus minor capsid protein, L2 is a good candidate for prophylactic vaccine because of the cross-neutralizing activity of L2 specific antibodies. We confirmed the surface expression of L2 protein on L. casei by Western blot, flow cytometry and immunofluorescence assay. To check the L2-specific antibody production, the Balb/C mice were orally immunized with L. casei, L. casei-L2. Mice serum and vaginal wash samples were collected and then L2 specific IgG and IgA was measured by ELISA using recombinant HPV-16 L2 protein as a coating antigen. L2-specific serum IgG and mucosal IgA antibody productions were enhanced after oral administration and significantly enhanced after boosting. For cross-neutralizing activity, L. casei- or L. casei-L2-vaccinated serum samples were evaluated using a sensitive high-throughput neutralization assay based on HPV-16, HPV-18, HPV-58 and bovine papillomavirus 1 (BPV1) pseudoviruses carrying a secreted alkaline phosphatase (SEAP) reporter gene. The HPV-16 neutralizing activity of the serum from mice immunized with L. casei-L2 higher than those from L. casei. Notably, vaccination of mice with L. casei-L2 was cross-neutralized heterologous type of HPV-18 and HPV-58 pseudoviruses, but did not cross-neutralized type of BPV1 pseudoviruses. Additionally, oral administration with the L. casei-L2 was protected mice from HPV-16, HPV-18, and HPV-58 pseudovirions (encoding the red fluorescence protein reporter gene) infection at genital sites. These results collectively indicate that the oral administration of L2 displayed on Lactobacillus casei induces systemic immunity, mucosal immunity, and broad-spectrum neutralizing effects in mice.
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Subhashini Jagu
Elkridge, USASubhashini Jagu1, Sudha V Chivukula4, Kihyuck Kwak1, Richard B.S. Roden1,2,3. 1Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 2Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 3Department of Gynecology and Obstetrics, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 4Shantha Biotechnics, Hyderabad, India. Like L1 VLP vaccines, vaccination with minor capsid protein L2 protects animals from papillomavirus challenge by the induction of neutralizing antibodies. While L2 induces antibodies that cross-neutralize diverse papillomavirus types, L2-specific antibodies typically neutralize related types more effectively than less evolutionarily related types. Thus, to enhance cross-protection we designed L2 fusion proteins consisting of known cross-neutralizing epitopes of eight divergent HPV types 6, 16, 18, 31, 39, 51, 56, 73 (11-88x8) for expression in bacteria. Vaccination of mice with L2 11-88x8 polypeptide induced robust neutralization titers. However, 11-88x8 was insoluble in bacteria complicating the purification process. In silico analysis suggested a candidate transmembrane region at approximately residues 45-67 of L2 in each papillomavirus type examined. Since prior mapping studies had not identified a neutralizing epitope within this sequence, and to potentially enhance the solubility of the vaccine antigen, we generated a construct deleted for the candidate transmembrane region in each subunit (11-88x8DTm). To account for the possibility that deletion of the transmembrane regions eliminated critical T helper epitopes, we generated another construct in which a known T helper epitope, PADRE, was added (11-88x8DTm+PADRE). Groups of either Balb/c or C57BL6 mice were vaccinated three times with 11-88x8, 11-88x8DTm, or 11-88x8DTm+PADRE polypeptides formulated in alum+MPL, and their serum neutralizing antibody titers determined. Vaccination with 11-88x8 induced antibodies that broadly cross-neutralized oncogenic HPV types in both Balb/c and C57BL6 mice. While deletion of the transmembrane domains enhanced solubility of the antigen, the titers of cross-neutralizing antibodies were reduced in both strains of mice. Inclusion of the PADRE did not restore the levels of cross-neutralizing antibodies in C57BL6 mice, suggesting that elimination of T helper epitopes does not account for this effect. The findings suggest that the putative transmembrane region of L2 either contains unidentified cross-neutralizing epitopes, or contributes to the structure of adjacent cross-neutralizing epitopes.
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Kihyuck Kwak
Baltimore, United StatesKihyuck Kwak1, Subhashini Jagu1, John T Schiller2, Douglas R Lowy2, Sudha V Chivukula3, Richard BS Roden1. 1Department of Pathology, Johns Hopkins University, Baltimore, MD, USA; 2Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, MD, USA; 3Shantha Biotechnics, Hyderabad, India. At least fifteen oncogenic HPV types have been associated with cervical cancer. The HPV L2 minor capsid protein is a promising prophylactic antigen, as it has conserved neutralization epitopes that can be produced inexpensively in bacteria. If successful, it could overcome two limitations of the L1 VLP vaccines: their narrow type-restricted protection and their high manufacturing cost, which could be even higher for second generation L1 VLPs vaccines targeting additional HPV types. We have explored an HPV L2 multimer polypeptide (11-88x8) expression construct encompassing L2 residues 11-88 of eight genital HPV types to enhance and broaden the effectiveness of vaccination. The L2 multimer polypeptide was formulated with various adjuvants and used to vaccinate both rabbits and mice. To assess the breadth of the oncogenic HPV types that could be neutralized by immune sera from animals vaccinated with 11-88x8, we generated several new HPV pseudovirus types (33, 35, 39, 56, 59, 68, 73). After codon optimization, the new HPV pseudovirion types were produced containing a firefly luciferase reporter construct in 293TT cells. These new types were tested in the vaginal challenge model in BALB/c mice and in vitro with 293TT cells to validate their infectivity. Sera from rabbits vaccinated with 11-88x8 neutralized 11 types of pseudoviruses tested with robust titers but failed to detectably neutralize BPV4. Vaccination of mice with 11-88x8 in alum protected them from vaginal challenge with all of the HPV types tested, specifically HPV16, HPV59 and HPV68. These data suggest that an L2 multimer vaccine can be produced simply in bacteria and, when formulated in alum, can provide broad immunity in mice to vaginal challenge with diverse oncogenic HPV types, including those that were not used to derive the vaccine.
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Patricia Day
Bethesda, USAPatricia Day1, Rhonda Kines1, Cynthia Thompson1, Douglas Lowy1, John Schiller1. 1Laboratory of Cellular Oncology, NIH, Bethesda, MD USA. In vivo microscopic assessment of virion localization in the murine genital tract shows that capsids first attach to the basement membrane (BM), undergo a conformational change that allows furin cleavage of the N-terminus of L2, and subsequently transfer to the epithelial cell surface. Utilizing the murine cervicovaginal challenge model, we have investigated the mechanisms by which L1 VLP vaccine-induced antibodies interfered with this entry process. Following VLP vaccination, the resultant high titer neutralizing antibodies impede the infectious process at an early step, as antibody-bound capsids were prevented from BM interaction and instead bound to neutrophils and eventually became undetectable in the tissue. However, it is known that antibody titers wane over time in VLP-vaccinated women. Therefore, we have adapted the murine model to examine high and low titers, by passively transferring hyper-immune anti-HPV16 L1 VLP rabbit serum. Transfer of high amounts of serum resulted in complete protection against HPV16 pseudovirus challenge that was microscopically indistinguishable from that observed following VLP vaccination, confirming that the main protective activity of the vaccine is antibody mediated. By contrast, after passive transfer of the lowest amount of serum that was still highly protective, the capsids clearly interacted with the BM and were cleaved by furin. However, the capsids were subsequently lost from the BM and not transferred to the epithelium. Interestingly, L2 antibodies apparently neutralize infection by a means analogous to the second L1 mechanism (see Kines et al. abstract), suggesting that L2 vaccination might mimic L1 vaccination in protection from natural infection. We conclude that high levels of serum L1 antibodies neutralize by preventing BM binding while low levels, which may more closely reflect long-term plateau levels of antibodies in vaccinated women, neutralize at a post-furin cleavage step that prevents capsid interaction with the keratinocyte-specific receptor.
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Nicolas Cuburu
Washington, United StatesNicolas Cuburu1, Rhonda Kines1, Jeffrey Roberts1, John Nicewonger2, Christopher Buck1, Barney Graham2, Douglas Lowy1, John Schiller1. 1Laboratory of Cellular Oncology, National Cancer Institute, NIH, Bethesda, MD, USA; 2Viral Pathogenesis Laboratory, Vaccine Research Center, NIAID, NIH, Bethesda, MD, USA. Human papillomavirus pseudoviruses (HPV PsV) composed of the viral L1 and L2 proteins can encapsidate virtually any 6-8 kb circular plasmid. HPV PsV are attractive vehicles for genetic immunization, as high infectious titers can easily be generated in culture, and they transiently infect permeabilized genital epithelium. Therefore, we evaluated genital and systemic immune responses after HPV PsV genital immunization of mice, using respiratory syncytial virus M and M2 as model antigens (Ag). HPV PsV genital immunization induced a massive (5-fold) increase of total genital CD8+ T cells, which was attributable to the expansion of Ag-specific CD8+ T-cells. Genital CD8+ T-cell responses after HPV PsV genital immunization were far superior to those measured after HPV PsV intranasal or adenoviral vector Ad5 intramuscular immunization. In the most recent studies, Ag-specific genital tract CD8+ T-cells were predominantly detected within the epithelium, exhibited an effector-memory phenotype, and produced both IFN-γ and TNF-α. In the spleen, Ag-specific CD8+ T-cells exhibited either effector-memory or central-memory phenotype and were able to produce IFN-γ, TNF-α and IL-2. In addition to effector cytokine production, in vivo cytotoxic activity was detected in the genital tract and spleen. HPV PsV genital immunization elicited genital and spleen Ag-specific CD4+ T cell responses, and in vivo depletion of CD4+ T-cells abrogated the induction of Ag-specific CD8+ T-cell responses, suggesting a requirement for CD4+ T-helper cells. Importantly, Ag-specific CD8+ T-cell numbers in the genital tract declined only two-fold at 100 days post immunization. These data indicate that HPV immunization induces long lasting and functional CD8+ T-cell responses in the genital tract, which may have implications in the design of vaccines against sexually transmitted infections where CD8+ T-cells are desired for protection.
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Edward Rybicki
Cape Town, South AfricaMarieta Burger1, Gillian de Villiers1, Inga Hitzeroth1, Edward Rybicki1. 1Department of Molecular and Cell Biology, University of Cape Town, South Africa.
Background: Cervical cancer is caused by persistent infection with high-risk types of human papillomavirus (HPV), most notably types 16 and 18. Existing vaccines based on the L1 capsid protein are effective; however, second-generation vaccines preventing infection by other types of HPV or treating current infections are needed.
Objectives: The objective of this project was to produce chimaeric L1 prophylactic and/or therapeutic vaccine candidates in insect cells, and to test their immunogenicity in a mouse model.
Results: Eight different chimaeras were constructed, containing cross-neutralizing epitopes from the minor capsid protein L2 and/or proven immunotherapeutic epitopes from the non-structural protein E7. Three contained HPV-16 L2 peptides, one a bovine papillomavirus type 1 (BPV-1) L2 peptide, two had E7 peptides and two had both HPV-16 L2 and E7 peptides. All genes could be expressed in insect cells, but the L1/L2/E7 and L1/BPV L2 (1-88) chimaeras consistently expressed at lower levels. Six chimaeras were further tested for immunogenicity in mice. Anti-L2 antibodies could only be detected for L1/L2 (56-81) and L1/L2 (17-36). Anti-HPV-16 L1-binding antibody levels were highest for L1/L2 (56-81), while neutralizing antibody titres were high and at similar levels for three chimaeras. Neither was detected for L1/BPV L2 (1-88). No cross-neutralizing antibodies against HPV-45 pseudovirions were detected. Only the L1/L2/E7 chimaera elicited a cellular immune response in mice, but no significant prevention of tumour growth was seen.
Conclusions: Antisera elicited against the three L1/L2 chimaeras (excluding L1/BPV L2 (1-88)) neutralized the homologous, but not a heterologous papillomavirus type, even though antibodies against L2 were elicited by L1/L2 (56-81) and L1/L2 (17-36). However, the levels of anti-L2 antibodies may have been too low for neutralization. The L1/L2/E7 chimaera performed better than the L1/E7 chimaera with respect to cellular immunity. The chimaeras with HPV-16 L2 insertions, and the L1/L2/E7 chimaera, warrant further investigation as prophylactic and/or therapeutic vaccine candidates.
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Denise Nardelli-Haefliger
LAUSANNE, SwitzerlandMartine Bobst1, Stéphanie Longet1, John Schîller2, Patrice Jichlinski1, Denise Nardelli-Haefliger1. 1Dept. of Urology, CHUV and University of Lausanne, Lausanne, Switzerland; 2National Cancer Institute, NIH, Bethesda, MD, USA.
Background: The available prophylactic HPV vaccines provide up to 100% protection against HPV vaccine-type specific infections and associated lesions. It is inferred that protection is mediated by HPV type-specific neutralizing antibodies that transudate from serum to the ano-genital sites. However, the concentration of serum HPV-specific antibodies that is needed for protection is unclear. To gain insights into the quantitative relationship between serum antibody levels and protection from cervicovaginal infection, we are determining the amount/titer of passively transferred HPV-neutralizing antibodies that is necessary to prevent in vivo genital infection, using the mouse genital Pseudovirion (PsV) challenge model.
Methods: HPV16-neutralizing monoclonal antibody V5 will be i.p. transferred to naïve recipient mice before genital challenge with PsV16 encoding luciferase (luc). In vitro HPV16 neutralizing titers measured in serum using secreted alkaline phosphatase (SEAP) PsV 16 assay will be compared to those detected in cervico-vaginal secretions after i.p. transfer in mice, as well as to their ability to protect the recipient mice from in vivo PsV-16 - luc genital challenge.
Results: In vitro HPV16-neutralizing antibody titers peaked in both serum and cervico-vaginal secretions 24 hours after i.p. transfer of V5 antibodies into naïve recipient mice. In mice that were vaccinated with purified HPV16VLPs, we found that the mild detergent (4% nonxynol-9) treatment in the cervix/vagina, which is used to mimic local trauma during sexual intercourse before PsV genital infection, transiently increased the amount of VLP 16-specific IgGs in genital secretions by 20-fold. Ongoing experiment will determine the amount of serum V5 antibodies necessary to afford prevention of genital PsV16 luc infection.
Conclusions: If a correlation can be drawn between the serum HPV-neutralizing titers and the ability to prevent in vivo genital challenge in mice, this may help to establish a correlate of protection in human vaccines.
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Antoine Touzé
TOURS, FRANCEMaxime J.J. Fleury1, Antoine Touzé1, Pierre Coursaget1. 1University François Rabelais; Inserm U618 « Protéases et vectorisation pulmonaires » and IFR 136 « Agents Transmissibles et Infectiologie », Tours, France. Prophylactic vaccines against oncogenic HPV types 16 and 18 have been shown to have excellent safety and nearly 100% long-term efficacy in preventing persistent infections and HPV16- and HPV18-positive cervical pre-cancer lesions. Although it is obvious that HPVs are very stable viruses that evolve very slowly, investigation using Bayesian phylogenetic methods have demonstrated significant evidence of phylogenetic incongruence among the PV genes,indicating substantial recombination. Thus, the possibility of evolution under pressure from a vaccine or the selection of already existing serotypes cannot be eliminated. Improved understanding of the viral capsid sequences specific to a serotype would help to understand how papillomaviruses can escape after vaccination, due to immune selection. We analyzed the capacity of different HPV16 L1 FG loop protein mutants to self-assemble into VLPs, to transfer genes and to induce neutralizing antibodies. Most of the mutants had reduced reactivity to HPV16 neutralizing MAbs and lost their capacity to transfer genes. However, two chimeric HPV16/31 L1 FG loop substitution mutants retained the ability to transduce cells. Mice immunized with these two mutants evidenced low levels of neutralizing antibodies against HPV16 and HPV31, but had a strong neutralizing immune response to mutant pseudovirions. These findings suggest that a few point mutations within the FG loop are sufficient to generate a newserotype. Moreover, sera from mice immunized with HPV16 L1 VLP were not able to neutralize chimeric HPV16/31 L1 FG loop substitution mutant pseudovirions. These results suggest that a few point mutations or recombination within the FG loop of HPV16 and HPV31 might lead to the generation of a new serotype that can escape neutralization by anti-HPV16 antibodies.
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T-C Wu
Baltimore, USATae Heung Kang1, J-Y Chung2, Archana Monie1, Sara I Pai3, Chien-Fu Hung1,4, T-C Wu1,4,5,6. 1Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD, USA; 2Tissue Array Research Program, Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; 3Department of Otolaryngology/Head and Neck Surgery, Johns Hopkins Medical Institutions, Baltimore, MD, USA; 4Department of Oncology, Johns Hopkins Medical Institutions, Baltimore, MD, USA; 5Department of Obstetrics and Gynecology, Johns Hopkins Medical Institutions, Baltimore, MD, USA; 6Department of Molecular Microbiology and Immunology, Johns Hopkins Medical Institutions, Baltimore, MD, USA. Intramuscular administration of DNA vaccines can lead to the generation of antigen-specific immune responses through cross-priming mechanisms. We propose a strategy that is capable of leading to local inflammation and enhancing cross-priming, thus resulting in improved antigen-specific immune responses. Therefore, in this study, we evaluated the immunological responses elicited through electroporation-mediated intramuscular administration of a DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 E7 (CRT-E7) in combination with DNA expressing HLA-A2 as compared with CRT-E7 DNA vaccination alone. We found that the co-administration of a DNA vaccine in conjunction with a DNA encoding a xenogenic major histocompatibility complex (MHC) molecule could significantly enhance the E7-specific CD8+ T-cell immune responses and antitumor effects against an E7-expressing tumor, TC-1, in C57BL/6 tumor-bearing mice. Furthermore, a similar enhancement in E7-specific immune responses was observed by the co-administration of CRT-E7 DNA with DNA encoding other types of xenogenic MHC class-I molecules. This strategy was also applicable to another antigenic system, ovalbumin. Further characterization of the injection site revealed that the co-administration of HLA-A2 DNA led to a significant increase in the number of infiltrating CD8+ T lymphocytes and CD11b/c+ antigen-presenting cells. Furthermore, the E7-specific immune responses generated by intramuscular co-administration of CRT-E7 with HLA-A2 DNA were reduced in HLA-A2 transgenic mice. Thus, our data suggest that intramuscular co-administration of DNA encoding xenogenic MHC class-I can further improve the antigen-specific immune responses, as well as antitumor effects generated by DNA vaccines through enhancement of cross-priming mechanisms.
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Tae-Young Lee
Daejeon, South KoreaTae-Young Lee1, Il-Han Lee2, Moon-Hee Sung2,3, Haryoung Poo1. 1Viral Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejon, Korea; 2Bioleaders Corporation, Daejeon, Korea; 3Department of Bio & Nanochemistry, Kookmin University, Seoul, Korea. Human papillomavirus (HPV) subtype 16 is known as a high-risk type to induce the pathogenesis of cervical carcinomas. HPV E6 and E7 are two major viral oncogenic proteins that are consistently expressed in HPV infections and HPV-associated malignancies. Thus HPV16 E6 and E7 have been considered as target proteins of therapeutic vaccine for cervical cancer, especially given that local cell-mediated immunity (CMI) against the human papillomavirus type 16 E6 (HPV16 E6) protein is important for eradication of HPV16 E6-expressing cancer cells in the cervical mucosa. We express HPV16 E6 or E7 antigen on the surface of Lactobacillus by a novel display system using gamma-PGA synthetase, a surface anchoring protein originated from Bacillus subtilis, chungkookjang. Surface expression of HPV16 E6 or E7 antigens was confirmed by Western blot, flow cytometry, and immunofluorescence microscopy, and mice were orally inoculated with the L. casei-PgsA or L. casei-PgsA-E6, L. casei-PgsA-E7. E6 or E7 specific IgG and IgA production was observed in E6, E7- expressing L. casei immunized mice and significant increase of antibody production was observed after boosting. In order to check cell-mediated immunity, the lymphoproliferation and IFN-gamma ELISPOT assays were performed. L. casei expressing E6, E7 immunized group showed higher lymphoproliferation and IFN-? release than L. casei immunized control group. The antitumor effect of orally administered E6, E7 displayed on Lactobacillus against E6, E7 expressing tumor was also checked in C57BL/6 injected with E6, E7 expressing tumor cell, TC-1. In E6, E7 expressing L. casei immunized mice group, growth of tumor was reduced and tumor size was significantly smaller than L. casei immunized mice group, and L. casei expressing E6, E7 immunized mice also lived longer than control group.
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Oscar Peralta-Zaragoza
Cuernavaca, MéxicoOscar Peralta-Zaragoza1, Jonathan Salazar-Leon1, Andrea Contreras-Oliver1, Alma Falcon-Velasquez1, Elizabeth Meda-Monzon1, Guillermo Perales-Ortiz1, Victor Bermudez-Morales1, Vicente Madrid-Marina1. 1National Institute of Public Health, Cuernavaca, Morelos, México. Intoduction: HPV infection is the main etiologic agent in cervical cancer. HPV16 covers 50% of the cases and E6 and E7 oncoproteins have properties of cell transformation and immortalization cells. Prophylactic and therapeutic strategies had been developed against cervical cancer with limited results to patients. The targeted inhibition of E6 and E7 oncogenes in tumor cells infected with HPV may provide a rational approach towards the development of novel anticancer therapies.
Objective: Using HPV-transformed cells as a model system, we analyzed whether RNA interference (RNAi)-mediated E6 and E7 oncogenes silencing can be employed in order to overcome the carcinogenesis process.
Methods: For this aim, we generated two interfering RNA construct plasmids (psiRNAE6 and psiRNAE7) in a DNA-vector, specifically directed against HPV16 E6 and E7 oncogenes. SiHa cells (HPV16) were transfected with these plasmids to evaluate the effect on E6 and E7 oncogenes silencing by RT-PCR real time and end point and western blot.
Results: Our results indicate a selective reduced expression of mRNA E6 and E7 oncogenes in HPV16 transformed cells. In addition, we observed inhibition of HPV16 E6 and E7 oncoproteins expression as well as functional effects in cell proliferation, and an induction of p53 and pRb cellular protein expression.
Conclusions: Thus, these results suggest that HPV16 E6 and E7 oncogenes can be repressed by RNAi strategy and this approach may represent an alternative of treatment against the cervical cancer development.
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Yao-Ching Hung
Taichung, Y-C Hung1, L-M Chen1, Y-S Chen2, W-H Lin2, G-S Huang2. 1Department of Obstetrics and Gynecology, China Medical University and Hospital, Taichung, Taiwan, R.O.C,; 2Institute of Nanotechnology, National Chiao Tung University, Hsin-Chu, Taiwan, R.O.C.. Introduction and Objective: To elicit the size-dependent immune properties of gold nanoparticles (GNPs) for the application of drug (vaccine) carrier targeting cancer tissues, a synthetic peptide corresponding to Foot-and-mouth disease virus (FMDV) viral proteins was conjugated to GNPs ranging from 2-50 nm in diameter (2nm, 5nm, 8nm, 12nm, 17nm, 37nm, and 50nm). An extra cysteine was added to the C-terminus of the FMDV peptide (pFMDV), to ensure maximum conjugation.
Method(s): The pFMDV-GNP conjugates were injected into BALB/C mice. Immunization with pFMDV-keyhole limpet hemocyanin (KLH) conjugate was performed as the control. Blood was withdrawn from mice on weeks 4, 6, 8, and 10, and antibody titers against pFMDV and carriers were obtained. For pFMDV-GNP immunization, specific binding against peptide was detected in the sera of mice injected with 2nm, 5nm, 8nm, 12nm, and 17nm GNP conjugates.
Result(s) and Discussion: Maximum binding occurred with GNPs of sizes between 8nm to 17nm. The pFMDV-GNPs induced a 3-fold increase in antibody response compared to pFMDV-KLH. In particular, all sera exhibited undetectable binding against GNP, while antisera of pFMDV-KLH presented high levels of binding activity against KLH. The uptake of pFMDV-GNP in spleen was examined by ICP-MS and TEM.
Conclusion and Key Message: The amount of GNP accumulated in the spleen correlated to the immune response induced by pFMDV-GNP. In conclusion, we demonstrated the size-dependent immunogenic properties of pFMDV-GNP conjugates. GNPs ranging from 8nm to 17nm promotes the most intense immune response, thus should be avoided if used as drug (vaccine) carrier. GNPs of sizes outside of this zone will be potential drug (vaccine) carriers in the application of nanomedicine.
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Aldo Venuti
Rome, ItalySilvia Massa2, Francesca Paolini1, Laura Spanò3, Rosella Franconi2, Aldo Venuti1. 1Regina Elena Cancer Institute, Rome, Italy; 2 ENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Rome, Italy; 3University of L'Aquila, L'Aquila, Italy. A therapeutic cancer vaccine should trigger effector T-cells trafficking, overcome immunosuppression and generate acute inflammation at the tumour site. Such requirements could be fulfilled linking the tumor-specific antigen to molecules able to increase the immune 'visibility' of the target antigen. In a search of innovative immunological 'promoters' of weak antigens, creating the pro-inflammatory environment necessary for the clearance of cancer and with less clinical constraints than other experimental immune modifiers, we focused our study on plant proteins. Ribosome inactivating proteins (RIPs) are specific RNA N-glycosidase that arrest the cellular protein synthesis at the translocation step, killing the eukaryotic cells. RIPs toxicity has been already exploited for developing immunotoxins in 'passive' cancer immunotherapy. Nevertheless, some biological activities of RIPs, independent from the catalytic activity (i.e. generation of inflammation, anti-viral properties and cell death by apoptosis), considered detrimental for their former use as immunotoxins, represent, on the contrary, a unique advantage for a new application as cancer 'active' immunotherapy agents. Saporin, a stable protein expressed in different tissues of Saponaria officinalis plant, is a type I RIP with a weak aspecific toxicity. We decided to use a saporin non toxic mutant (SAPKQ) (unable to block protein synthesis) as a carrier for the E7GGG gene in the context of a DNA-based vaccine. Different fusions of HPV16 E7 with saporin were made. These fusions were transfected in HEK cell where they express high levels of E7 antigen. The fusion constructs were also injected in mice inducing E7-specific IgGs, CTLs and delayed-type hypersensitivity (Th1 cytokine-mediated inflammation). This specific immunological response was able to affect the growth of E7-expressing TC-1 cell tumours. Moreover this anticancer activity was confirmed in a new orthotopic mouse model of HPV-related head and neck cancer (AT84-E7 cells). Results from the intratumoral injection of these pro-inflammatory constructs will be reported.
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Chih-Ping Mao
Baltimore, USAChih-Ping Mao1, Chien-Fu Hung1,2, Tae Heung Kang1, Liangmei He1, Ya-Chea Tsai1, Chao-Yi Wu1, T-C Wu1,2,3,4. 1Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD, USA; 2Department of Oncology, Johns Hopkins Medical Institutions, Baltimore, MD, USA; 3Department of Obstetrics and Gynecology, Johns Hopkins Medical Institutions, Baltimore, MD, USA; 4Department of Molecular Microbiology and Immunology, Johns Hopkins Medical Institutions, Baltimore, MD, USA. DNA vaccines have recently emerged at the forefront of approaches to harness the immune system in the prevention and treatment of viral infections, as well as the prevention and treatment of cancers. However, these vaccines suffer from limited efficacy since they often fail to produce significant antigen-specific CD8+ T-cell responses. We report here a novel concept for DNA vaccine design that exploits the unique and powerful ability of viral fusogenic membrane glycoproteins (FMGs) to couple concentrated antigen transfer to dendritic cells (DCs) with local induction of the acute inflammatory response. Intramuscular administration into mice by electroporation technology of a plasmid containing the FMG gene from vesicular stomatitis virus (VSV-G)--together with DNA encoding the E7 protein of human papillomavirus type 16, a model cervical cancer antigen--elicited robust E7-specific CD8+ T-cell responses, as well as therapeutic control of E7-expressing tumors. This effect could potentially be mediated through the immunogenic form of cellular fusion and necrosis induced by VSV-G, which in a concerted fashion provokes leukocyte infiltration into the inoculation site, enhances cross-presentation of antigen to DCs, and stimulates them to mature efficiently. Thus, the incorporation of FMGs into DNA vaccines holds promise for the successful control of viral infections and cancers in the clinic.
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Shiwen Peng
Baltimore, USAShiwen Peng1, Archana Monie1, Tae Heung Kang1, Chien-Fu Hung1,2, Richard Roden1,2,4, T-C Wu1,2,3,4. 1Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD, USA; 2Department of Oncology, Johns Hopkins Medical Institutions, Baltimore, MD, USA; 3Department of Obstetrics and Gynecology, Johns Hopkins Medical Institutions, Baltimore, MD, USA; 4Department of Molecular Microbiology and Immunology, Johns Hopkins Medical Institutions, Baltimore, MD, USA. We have examined non-replicative human papillomavirus (HPV) pseudovirions as an approach in the delivery of naked DNA vaccines without safety concerns associated with live viral vectors. In the current study, we have generated HPV-16 pseudovirions encapsidating a DNA vaccine encoding the model antigen, ovalbumin (OVA) (HPV16-OVA pseudovirions). Vaccination with HPV16-OVA pseudovirions intramuscularly elicited significantly stronger OVA-specific CD8+ T cell immune responses compared to OVA DNA vaccination via gene gun in a dose-dependent manner. We showed that a single amino acid mutation in the L2 minor capsid protein that eliminates the infectivity of HPV16-OVA pseudovirion significantly decreased the antigen-specific CD8+ T cell responses in vaccinated mice. Furthermore, a subset of CD11c+ cells and B220+ cells in draining lymph nodes became labeled upon vaccination with FITC-labeled HPV16-OVA pseudovirions in injected mice. HPV pseudovirions were found to infect bone marrow-derived dendritic cells (BMDCs) in vitro. We also showed that pretreatment of HPV16-GFP pseudovirions with furin leads to enhanced HPV16-OVA pseudovirion infection of BMDCs and OVA antigen presentation. Our data suggest that DNA vaccines delivered using HPV pseudovirions represent an efficient delivery system that can potentially impact the field of DNA vaccine delivery.
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Shiwen Peng
Baltimore, USAShiwen Peng1,2, Sofia Lyford-Pike1, Chien-Fu Hung2,5, T.-C. Wu2,3,4,5, Sara Pai1. 1Department of Otolaryngology/Head and Neck Surgery, Johns Hopkins Medical Institutions, Baltimore, MD, USA; 2Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD, USA; 3Department of Obstetrics and Gynecology, Johns Hopkins Medical Institutions, Baltimore, MD, USA; 4Department of Molecular Microbiology and Immunology, Johns Hopkins Medical Institutions, Baltimore, MD, USA; 5Department of Oncology, Johns Hopkins Medical Institutions, Baltimore, MD, USA. Recurrent Respiratory Papillomatosis (RRP) is a common laryngeal neoplasm in children primarily caused by infection with human papillomavirus (HPV) types 6 and 11. Although a prophylactic vaccine has been developed that protects against viral infection, a therapeutic vaccine is needed for patients suffering from persistent disease. We have previously shown that a DNA vaccine encoding the HPV-6 and HPV-11 E6 and E7 genes generated robust E6-specific CD8+ T cell responses in C57BL/6 mice; however, no significant E7-specific T cell responses were observed. In order to enhance E7 CD8+ T cell responses, we generated a DNA vaccine encoding calreticulin (CRT) linked to HPV-6b E7. We found that C57BL/6 mice vaccinated with HPV-6b CRT/E7, but not E7 DNA alone, generated significant CD8+ T cell responses against the E7aa21-50 peptide. Further analysis demonstrated that these responses were specific to a H-2Db-restricted E7aa21-29 epitope. We also vaccinated HLA-A*0201 transgenic mice with the HPV6b CRT/E7 DNA vaccine and detected enhanced CD8+ T cell responses against the E7aa61-90 peptide, indicating a HLA-A*0201-restricted CD8+ T cell response. Further epitope analysis identified the HLA-A*0201-restricted E7aa82-90 as the recognized sequence. Taken together, our data demonstrate that a DNA vaccine encoding CRT linked to HPV-6b E7 can elicit strong H-2Db and HLA-A*0201-restricted HPV-6b E7-specific CD8+ T cell responses. Interestingly, HPV-6b and HPV-11 E7 share identical epitope sequences for both H-2Db-restricted E7aa21-29 and HLA-A*0201-restricted E7aa82-90 epitopes. The identification of these two CD8+ T cell epitopes can be used to evaluate the efficacy of E7-specific CD8+ T cell responses of both HPV-6 and -11. Furthermore, identification of the HLA-A*0201-restricted T cell epitope may be useful in monitoring HPV-specific immunologic responses in future vaccine clinical trials with RRP patients. |
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Rebecca Cerio
Bethesda, USARebecca Cerio1, Jeffrey Roberts1, Douglas Lowy1, John Schiller1. 1Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. HPV pseudoviruses composed of the HPV capsid proteins are promising candidates for gene therapy vectors due to their safety profile and ability to encapsidate tailor-made plasmid genomes. We previously demonstrated that HPV pseudoviruses infect most human tumor-derived cell lines in the NCI60 panel and also disrupted epithelium but are unable to infect, or even bind, normal tissue. These findings raise the possibility that pseudoviruses could effectively deliver cytotoxic genes to tumors in vivo, with little adsorption by or toxicity to normal cells. Here we report that HPV pseudoviruses also infect mouse epithelial tumors and human xenografts in vitro and in vivo. HPV pseudoviruses infected the murine WF-3 peritoneal and TC-1 lung epithelial tumor cell lines in vitro, as demonstrated by pseudovirus-delivered reporter expression. Furthermore, HPV-16-TK and HPV-45-TK (encapsidating thymidine kinase expression plasmids) pseudovirus infection followed by ganciclovir prodrug treatment killed WF-3 and TC-1 cells in a dose-dependent manner. After intraperitoneal growth, WF-3 tumors were permissive to pseudovirus infection, and HPV-16-TK followed by ganciclovir treatment was well-tolerated and increased median mouse survival time by 25% over HPV-16-TK treatment with no prodrug. These data suggest that HPV pseudovirus can act as an effective and specific gene therapy vector in vivo. To determine if susceptibility to pseudovirus infection was an artifact of in vitro passage, several human xenograft lines that had only been passaged in vivo were examined. Subcutaneous tumors of the MRI-H-207, MRI-H-258, and FM-108 human ovarian carcinoma lines were infected by HPV-16 and, to a lesser degree, by HPV-45 pseudovirus, with only minimal infection of normal subcutaneous tissue. HPV-16, but not HPV-45, pseudovirus also bound SWA-G human ovarian tumor xenograft cells in vitro. Thus, pseudovirus infection of human tumors may be HPV type- and tumor-specific. These results suggest that HPV pseudoviruses are potentially promising vectors for tumor-specific suicide gene therapy.
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Laetitia Carthagena
Paris, FranceLaetitia Carthagena1, Ken Y.C. Chow1, Émilie Brotin1, Youcef Ben Khalifa2, Sébastien Teissier2, Françoise Thierry2, Françoise Bachelerie1. 1INSERM U819, Unité Pathogénie Virale, Institut Pasteur, Paris, France; 2Papillomavirus Regulation and Cancer, Institute of Medical Biology, 8A Biochemical Grove, A*Star, 138648, Singapore. The CXCL12 chemokine directs migration of cells and controls angiogenesis by recruiting endothelial progenitor cells through the binding to the CXCR4 and CXCR7 receptors. CXCL12 is necessary during development for organogenesis and embryogenesis and, in adult life for tissue repair and leukocyte homeostasis. Those biological functions are critically involved in some pathological processes such as carcinogenesis and neo-vascularisation linked to tumor progression. Moreover, CXCL12 is up-regulated in numerous cancer cells and since pioneer works that linked its expression with the development of breast and prostate cancers, number studies have documented the role of the chemokine in human tumor pathogenesis. We reported that CXCL12 expression in the epidermis is correlated with human papillomavirus (HPV) infection. However, the role of the chemokine in the HPV life cycle remained unknown. Now we demonstrate that primary keratinocytes immortalized by high-risk type HPV up-regulate CXCL12 and its receptors in a manner dependent upon expression of the E6 and E7 oncogenes. Autocrine signaling set-in-motion by engagement of CXCL12 to its receptors controls motility and survival of immortalized keratinocytes. Moreover, binding of CXCL12 to CXCR7 induces the constitutive activation of the PI3K/Akt, which is involved in pro-survival pathways. Strikingly, expression of a CXCR4 mutant that mediates unregulated CXCL12-dependent signaling responsible for the rare combined WHIM immunodeficiency disorder featuring a high susceptibility to HPV infection, confers these cells with tumorigenic capacity. These results establish a pivotal role for the CXCL12-signaling axis in HPV-mediated cell transformation. We are now setting up organotypic "Raft" cultures with control and WHIM-derived cells in order to analyze the function of this axis in HPV replication. |
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Lyliana Barbosa
Pouso Alegre, BrazilLyliana Barbosa1,2, Ismael Silva1, José Correa3, Julisa Ribalta1. 1Department of Gynecology, Escola Paulista de Medicina, UNIFESP, Sao Paulo, SP, Brazil; 2Department of Gynecology, Faculdade de Ciências da Saúde, UNIVÁS, Pouso Alegre, MG, Brazil; 3Pathology Institute José Carlos Corrêa, Pouso Alegre, MG, Brazil. Objectives: To evaluate the expressions of survivin and telomerase in cervical samples from women with Human Papillomavirus induced lesions; to relate this findings with differents degrees of cervical intraepithelial neoplasia and invasive cervical carcinoma.
Methods: A hundred and five cervical samples were studied from women aged between 18 and 80 years old. They were distributed into five different groups: 20 patients without neoplasia, 24 women with low grade squamous intraepithelial lesions (CIN I), 20 patients with high grade squamous intraepithelial lesions (CIN II), 24 with high grade squamous intraepithelial lesions (CIN III) and 17 with invasive cervical carcinoma. The samples were analysed with PCR study for HPV, RT-PCR for quantitative telomerase detections and immunohistochemistry for survivin. The expression of survivin was described as negative (figure 2), weak, moderate and strong (figure 1 and 2). The quantitative telomerase detection was analysed by number of molecules per reaction.
Results: The strong expression of survivin was found in 10% of samples without neoplasia, 45,8% of CIN I, 70% of CIN III and 76,4% for invasive cervical cancer (p<0,0001). The telomerase quantitative analysis showed that HSIL and invasive cervical carcinomas presented higher levels of the enzyme, compared with LOSIL and non neoplastic samples. The quantitative analysis of telomerase showed and average of 0,007; 0,001; 0,003; 0,061 and 0,250 mol/reaction, respectively, for samples without neoplasia, CIN I, CIN II, CIN III and invasive cervical cancer (p<0,0001). There was a positive correlation between survivin and telomerase quantitative data, more significant in CIN III group.
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Eduardo Castañeda-Saucedo
Chilpancingo, MexicoMiguel A. Mendoza-Catalan1, Gema R. Cristobal-Mondragon1, Jesus Adame-Gomez1, Mirna A. Romero-Hernandez4, Laura Sierra-Lopez3, Jose Fco. Coppe3, Amalia Vences-Velazquez4, Luz del Carmen Alarcon-Romero2, Eduardo Castañeda-Saucedo1. 1Laboratorio de Biología Celular del Cáncer, UACQB-Universidad Autónoma de Guerrero, Chilpancingo, Guerrero, Mexico; 2Laboratorio de Investigación en Citopatología, UACQB-Universidad Autónoma de Guerrero, Chilpancingo, Guerrero, Mexico; 3Departamento de Patología, HGR Vicente Guerrero - IMSS, Chilpancingo, Guerrero, Mexico; 4Laboratorio de Inmunobiología y Diagnóstico Molecular, UACQB-Universidad Autónoma de Guerrero, Chilpancingo, Guerrero, Mexico. Cervical cancer is the second most common malignant neoplasia affecting woman worldwide. Infection with high-risk human papillomavirus (HR-HPV) is considered the main risk factor for developing cervical cancer and its precursor lesions. The HR-HPV oncoproteins E6 and E7 affect host cell function by binding to, and modifying the activity of a growing number of cellular proteins. Experimental evidences suggest that E6 and E7 may affect the function of GTPases of the Rho family. Rho GTPases are small signaling proteins involved in the regulation of crucial cellular functions such as cell-cell adhesion, proliferation, migration and invasion. Their activity is regulated by three groups of proteins: Guanine-nucleotide Exchange Factors (GEFs), GTPase activating proteins (GAPs), and GDP dissociation inhibitors (GDIs). expression of Rho GTPases and their regulatory proteins is altered in several cancers and precursor lesions. However, the expression pattern of these proteins in cervical pre-malignant lesions and cervical cancer has not been reported. The aim of this study is to evaluate the expression of the GTPases RhoA, Rac1 and Cdc42, and the GEFs Tiam1 and β-Pix in cervical pre-malignant lesions. Using immunohistochemistry, we studied 102 paraffin-embedded cervical biopsies: 20 normal cervical epithelium, 51 low-grade intraepithelial lesions (LSIL), and 31 high-grade intraepithelial lesions (HSIL). All five proteins are expressed in intermediate and superficial layers of normal cervical epithelium, but absent in basal cells. In LSIL and HSIL, expression of these proteins was found also in basal cells. Moreover, we found that Rac1 and Tiam1 are overexpressed in LSIL and HSIL, compared to normal epithelium, and that there is a statistically significant association between the overexpression of Rac1 and Tiam1 and the presence of LSIL (OR=4 and OR=22.3, respectively) and HSIL (OR=6 and OR=23.2, respectively). Our results suggest that overexpression of Rac1 and Tiam1 may play an important role during cervical carcinogenesis
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Claudio Villota
santiago, ChileClaudio Villota1,2,3,4, Enrique Boccardo5, Luisa Villa5, Luis Burzio1,2,3,4. 1Andes Biotechnologies, Santiago, Chile; 2Universidad Andrés Bello, Santiago, Chile; 3Millenium Institute of Fundamental and Applied Biology, Santiago, Chile; 4Fundación Ciencia para la Vida, Santiago, Chile; 5Ludwig Institute for Cancer Research, São Paulo, Brazil. Background: We have characterized a family of mitochondrial non-coding RNA whose members are differently expressed in tumor and pre-tumor cells derived from HPV infection when compared to their normal counterpart. HPV-derived pre-tumor cell lines express a transcript called SncmtRNA2, which is not detected in normal or tumor cells. Our results suggest that the SncmtRNA2 is a good marker for detection of pre-tumor cells during the development of cervix cancer. Previous results confirm that the SncmtRNA2 is expressed in early stages during cell transformation in cervix cancer development (CIN I-II).
Goal: The goal of the present study is to determine which HPV oncoprotein (E6 or E7) is responsible for inducing the expression of the SncmtRNA2 in HPV-inmortalized Human Foreskin Keratinocytes (HFK).
Methods: We immortalized HFK with a lentiviral vector coding for HPV16 E6, E7 and E6E7. The expression of the E6 and E7 mRNA was measured by reverse-transcriptase PCR (RT-PCR). The expression of the SncmtRNA2 was detected by in situ hybridization and RT-PCR using specific probes and primers.
Results: The SncmtRNA2 is expressed only in HFK expressing both viral oncoproteins (E6 and E7). Normal cells, or HFK expressing HPV16 E6 or E7 not express SncmtRNA2.
Discussion: Our results confirm that SncmtRNA2 is detected only in immortalized cells expressing HPV16 E6 and E7, results that agree with previous work described in the literature which indicates that the expression of both HPV16 E6 and E7 is necesary for keratinocyte immortalization. The detection of the SncmtRNA2 should be a important tool for future cervix cancer early detection. Further studies to characterize the molecular pathway of SncmtRNA2 induction by HPV16 E6E7 are warranted.
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Alcina F. Nicol
Rio de Janeiro, BrazilSergio Amaro Filho1, Andreia Pires2, Fabio Russomano3, Tristao Aparecida3, Cecilia Vianna A3, Gerard Nuovo4, Beatriz Grinstejen5, Cynthia Cunha5, Claude Pirmez1, Alcina F. Nicol1. 1Laboratory of Interdisciplinary Medical Research, LIPMED- IOC, Fiocruz, Rio de Janeiro, Brazil; 2Fonte Medicina Diagnostica Laboratory, Niterói, Rio de Janeiro, Brazil; 3Fernandes Figueira Institute, Fiocruz, Rio de Janeiro, Brazil; 4Comprehensive Cancer Center, Ohio State University Hospital, Columbus, Ohio, USA; 5IPEC, Fiocruz, Rio de Janeiro, Brazil. Background: The analyses of protein expression involved IN proliferation and tumor suppressor represents one of the most important studies to find possible indicator of carcinogenicity caused by HPV. Objectives: To analyze the expression of Ki-67, E6 and MCM-2 in invasive cervical cancer (ICC) samples and correlate this to high risk HPV type expression.
Materials & Methods: TMA blocks consisted of 88 ICC and 43 controls. The expression of Ki-67, HPV 16 E6 and MCM2 were analyzed by immunohistochemistry. In situ hybridization was employed to HPV DNA detection. The protein expression was quantified with ImagePro software. ANOVA and Bonferroni for multiple comparisons were used to analyze the data using R 2.10.0.
Results: Statistically significant increase (p<0.001) in the expression of Ki-67 was observed in the ICC samples (8.75% + cells/field; SE ± 0.61%) compared to controls (3.42% cells/field; SE ± 0.46%). No expression of E6 and MCM-2 were observed in controls compared with ICC samples, that showed 1.8% and 15.1% + cells/field, respectively. Three groups expressing different intensity and amount of HPV DNA by in situ hybridization were separated in stronger (? 7%), weaker (1-7%) and much weaker (? 7%) positivity and were correlated with E6, ki-67 and MCM-2 expression, Fig 1. Increased HPV copy number was correlated with Increasing expression to Ki-67 (p=0.033) and MCM-2 (p=0,001). The Bonferroni test revealed statistically significance difference between stronger and weaker HPV DNA expression in the Ki-67 (p=0.029) and MCM-2 (p=0.001) group.
Conclusions: Ki-67 and MCM-2 were highly expressed in the group where HPV DNA was strongly detected (p< 0.005), suggesting that Hr-HPV might be up-regulating the MCM-2 and Ki-67 expression, which could be favoring the development of cervical cancer. Further studies will be done in order to genotype the HPV-DNA to correlate them with these and others biomarkers which could be useful in diagnosis or represent a prognostic marker for cervical cancer.
Support: FAPERJ; IOC/Fiocruz, RJ-Brazil
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Mª. Clara Bicho
Lisbon, PortugalMª. Clara Bicho1, Alda Pereira da Silva1, Marcos Carvalho1, Andreia Matos1, Cláudia Marinho1, Rui Medeiros2, Manuel Bicho1. 1Laboratório de Genética e Centros de metabolismo e Endocrinologia e Cardiologia FML, Lisbon, Portugal; 2Instituto Português de Oncologia Francisco Gentil, Porto, Portugal. Introduction: Catechol-O-methyltransferase is an enzyme which catalyzes the metabolism of catecholestrogens harboring a Val/Met polymorphism at codon 158 (A/G) giving place to three phenotypes (HH, HL, LL) with decreasing activity. Its coenzyme, S-Adenosylmethionine, leads to homocystein (Hcy), which may be associated with cervix cancer. The aim of this work was to study the influence of COMT polymorphism on homocystein total plasma levels in women with cervix cancer compared to non pathologic controls.
Materials and Methods: A group of 96 women aged 19 to 77 years ( ? ±SD=41.4 ±12.1; median=39) with cervix cancer pathology (LSIL, HSIL; ICC), compared to a control group of 167 women aged 18 to 79 years ( SD=50.6 ±16.7; median=56). Genotype was determined by PCR-RFLP, and homocystein levels (nmol/L) by a commercial ELISA kit, in a total of 62 women.
Results: A significant difference was observed in homocystein levels between pathologic and control groups (N=32; SD=5.72 ±3.64 vs. N=30; SD=19.55± 20.06; p=0.001). There were differences on genotype frequencies (%) distribution between those pathologic, N=96: HH= 16.7, HL= 53.1 and LL= 30.2 vs. control, N=167: HH= 23.4, HL= 59.3; LL= 17.3; p=0.04, as well as in allele frequencies (pathologic H= 0.432; L= 0.568 vs. control H= 0.530; L= 0.470). Only in the control group were found differences in the homocystein levels accordingly to the genotype: control HH (8) = 18.62 ±20.73; HL (14) = 14.76± 13.05; LL (3) = 51.71± 33.56; p=0.016; pathologic HH (2) = 5.55 ±0.18; HL (19) = 5.15 ±3.98; LL (5) = 6.6 ±2.33; p=0.738.
Conclusions: There are differences in the genotype and allele distributions between healthy and cervix cancer pathologic women, which have higher LL genotype frequency, which presents the lowest activity; and consequently higher carcinogenic cathecol-estrogen concentrations. In pathologic women, low levels of homocystein weren't influenced by COMT polymorphism.
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Tsin-Wah Leung
Hong Kong , ChinaTsin-Wah Leung1, Stephanie S. Liu1, Annie N.Y. Cheung2, Hextan Y.S. Ngan1. 1Department of Obstetrics & Gynaecology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; 2Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China . Background: There are four E2 binding sites (E2BSs) at the LCR region of HPV16/18 genome. Binding of E2 protein to different E2BSs may exert either transcriptional activation/repression on E6 and E7 oncoprotein. Methylation status at the E2BSs may affect the relative binding of E2 protein to them.
Objectives of Study: To investigate the methylation status of E2BSs 1, 2 and 4 in HPV16-positive and/or HPV18-positive cervical cancer and to correlate this with clinical prognosis in cervical cancer patients.
Materials & Methods: DNA was extracted from paraffin sections of 52 cases of HPV16-positive and 24 cases of HPV18-positive cervical cancer. Methylation percentage of individual CpGs at E2BS 1, 2 and 4 were assessed by pyrosequencing. The average percentage of CpG methylation at each E2BS was calculated, compared and correlated with clinical parameters.
Results: Among the HPV16-positive cervical cancer, E2BS 1&2 were more methylated than E2BS 4 (14.2 ± 9.2% c.f. 10.0 ± 9.2%). Cases with heavier methylation at E2BS 1&2 relative to E2BS 4 had better overall survival (177.0 months vs 54.3 months, p=0.015) and disease-free survival (172.9 months vs 48.9 months, p=0.016). Among the HPV18-positive cervical cancer, E2BS 1&2 were more methylated than E2BS 4 (55.4 ± 25.3% c.f. 23.4 ± 20.8%). Radiosensitive HPV18-positive cervical cancer showed higher average CpG methylation at E2BS 1&2 compared with the radioresistant tumours (67.0 ± 16.1% vs 36.1 ± 26.3%, p = 0.048). Cases with hypermethylation at E2BS 1&2 had better disease-free survival (184.5 months vs 26.5 months, p=0.039).
Conclusion: Relative CpG hypermethylation at HPV16 and/or HPV18 E2BS 1 and 2 compared with E2BS 4 is associated with better patient survival in cervical cancer and is a potentially useful prognostic indicator for predicting patient survival and radiosensitivity.
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Deeksha Pandey
Manipal, IndiaDeeksha Pandey1, Peter Snijders2, Chris Meijer2, Renske Steenbergen2. 1Department of OBGYN, KMC, Manipal, India; 2Department of Molecular Biology, VUMC, Amsterdam, The Netherlands.
Background: Infection with hrHPV is a necessary, though insufficient, cause of cervical cancer. Progression of hrHPV-infected premalignant lesion to invasive cancer is driven by additional (epi)genetic events in the host cell genome.
Objective: We hypothesized that the rather low specificity of hrHPV testing may be improved by testing for (epi)genetic changes driving cervical carcinogenesis. Tumor suppressor gene silencing via promoter hypermethylation is an important event in cervical carcinogenesis, which can be detected in cervical scrapings.
Methods: We recently identified PCDH10 (pro-cadherin 10) as one of the progressively methylated genes in cervical oncogenesis. PCDH10 methylation was studied by methylation specific PCR (MSP) in cervical cancer cell lines and HPV transfected keratinocytes mimicking all stages of cervical carcinogenesis. Initial results were validated by quantitative MSP of 184 biopsy and 81 cytology samples.
Results: PCDH10 methylation was detected in 1/19 (5.3%), 4/35 (11.4%), 10/42 (23.8%), and 38/43 (88.3%) of normal, CIN1, CIN3 and squamous cell carcinoma samples, respectively (figure 1). PCDH10 methylation was also evident in 21/26 (80.7%) of adenocarcicnoma, but not in adenocarcinoma in situ samples (n=19). Cytology samples revealed PCDH10 methylation to be significantly more frequent in abnormal cytology samples of women who developed CIN3 lesions within 18 months compared to Pap 1 smears of women without cervical disease during 5 year follow-up. On drawing the ROC curve we could obtain cut off having high sensitivity up to 80% and specificity of 71%. Likewise as evident (figure 2) these cut offs can be adjusted to get the desired combination of sensitivity and specificity.
Conclusion: In conclusion, testing for PCDH10 promoter methylation may provide a valuable triage marker for risk assessment of hrHPV-positive women.
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Kazuyoshi Shigehara
Kanazawa, JapanKazuyoshi Shigehara1, Toshiyuki Sasagawa2, Syohei Kawaguchi1, Masayoshi Shimamura3, Takao Nakashima3, Kazufumi Nakashima3, Kazuhiro Sugimoto3, Atsushi Mizokami1, Eitetsu Koh1, Mikio Namiki1. 1Department of Integrative Cancer Therapy and Urology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan; 2Department of Reproductive and Perinatal Medicine, Kanazawa Medical University, Kahoku, Japan; 3Department of Urology, Ishikawa Prefectural Central Hospital, Kanazawa, Japan.
Objectives: We have recently found that human papillomavirus (HPV) infection in urothelial benign tumor named as the inverted papillloma. The causative role of HPV infection for bladder cancer was investigated.
Methods: HPV-DNA was tested in cellular DNA extracted from frozen samples of 92 cases of bladder carcinoma by modified GP5+/6+ PCR method, and the genotype was determined using a Hybri-Max HPV genotyping kit. In situ hybridization (ISH) of HPV genome and immunohistochemical analysis for p16, mcm-7 and HPV-L1 proteins were performed.
Results: DNA quality was confirmed by amplifying the β-globin by PCR. High-risk HPV types was detected in 17 (18%) of the cases with bladder cancer. HPV16 was most common type, followed by HPV18 and HPV33. Comparing the clinicopathological features between HPV-positive and negative cases, HPV was more frequently identified in younger age and lower grade (G1) cancer (p<0.05). Signals of high-risk HPV-DNA were observed in the nuclei of tumor cells of all HPV positive carcinomas. The p16 and mcm-7, which are surrogate markers for high-risk HPV-E7, were highly expressed in all HPV-positive cancer cells, whereas such expression was limited in HPV-negative ones. Their expression levels were significant higher in HPV-positive cases (p<0.05). HPV-L1, which is a marker for HPV propagation, was not observed in all of bladder cancer. All these findings suggest that HPV infection in bladder cancer is not accidental.
Conclusions: HPV may play an important etiological role for the development of low grade bladder cancer which is prevalently developed in younger age
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Elenae Vazquez Ulloa
MEXICO, MEXICOElenae Vazquez Ulloa1,2, Adriana Contreras Paredes1, Alejandro Aviles Salas1, Marcela Lizano Soberon1,2. 1Unit of Biomedical Research in Cancer, National Cancer Institute, Mexico; 2Biomedical Research Institute, National Autonomous University of Mexico, Mexico. Cervical cancer represents the second most common cancer among women worldwide. Persistent infection with high-risk human papillomavirus (HR-HPV) is the major risk factor in the development of cervical cancer. HR-HPVs E6 oncoprotein interacts with tumor suppressor p53 and promotes its degradation. Also E6 can bind to PDZ domain-containing proteins as hDlg, which is a scaffold protein since it localizes PTEN to the plasma membrane where it is functional as a lipid phosphatase. PTEN antagonizes the Akt pathway regulating cell survival and proliferation. Interestingly there is crosstalk between PTEN and p53, the last one binds to PTEN promoter and regulates its expression; on the other side PTEN can enhance p53 function. Our goal was to determine the expression of PTEN, hDlg and p53 during cervical cancer development. Expression levels of PTEN, hDlg and p53 were analyzed by immunohistochemistry in 48 cases of varying degrees of cervical intraepithelial neoplasia (CIN) and 105 cervical cancers. An over expressionof p53 and hDlg was found in cervical cancer; meanwhile hDlg is lost from cell membrane and accumulates significantly at the cytoplasm. In contrast, little or no PTEN was detected in invasive carcinomas and is lost from the cell nucleus. Our data suggest that PTEN is tightly regulated by p53 and hDlg in cervical carcinogenesis and, as in other types of cancers, loss of nuclear PTEN might be a promising biological marker. Work partially supported by CONACyT MEXICO. Grant: 69875
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Arkom Chaiwongkot
Heidelberg, GermanyArkom Chaiwongkot1,5, Chamsai Pientong1, Tipaya Ekalaksananan1, Bunkerd Kongyingyoes2, Pilaiwan Kleebkaow3, Bundit Chumworathayee3, Natcha Patarapadungkit4, Miriam Reuschenbach5, Svetlana Vinokurova5, Magnus von Knebel Doeboritz5. 1Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; 2Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; 3Department of Obstetrics and Gynaecology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; 4Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; 5Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, Heidelberg, Germany. Background: Upregulation of the E6-E7 oncogenes of high-risk human papillomaviruses (HR-HPVs) in basal squamous epithelial cells marks the shift from permissive to transforming HR-HPV-infections. It is commonly regarded as is the major transforming factor in the pathogenesis of cervical dysplasia and cancers. Viral integration into the host-cell genome and concomitant disruption of the E2-open reading frame has been hypothesized as potential mechanism resulting in overexpression of the E6-E7 oncogenes. In this study we further investigated chromosomal integration patterns of HR-HPV genomes during the preneoplastic progression of HPV-induced squamous epithelial lesions.
Methods: p16INK4a immunohistochemistry was used to identify lesions in the transforming mode of HPV gene expression and to distinguish them from lesions just in the productive viral gene expression mode. In p16INK4a positive lesions, the physical status of HR-HPV genomes was analyzed in 95 biopsies of squamous epithelial lesions induced by HPV types 16, 18, 31, 33, 45 and 58 using the amplification of papillomavirus oncogene transcripts (APOT) assay.
Results: None of the here analyzed p16INK4a negative samples displayed integrate derived transcripts, whereas they were all positive for episome derived transcripts. , None of the CIN 1 lesions displayed integrate derived transcripts that were however detected in 40% of HPV 16 and 33.3 % of HPV 58 but 100 % of HPV 18 and 45 associated carcinomas. In CIN 2/3 lesions, integrate derived transcripts were detected in 5% of HPV 16 and 16 % of 58 lesions.
Conclusions: These data further corroborate the hypothesis that the shift to the transforming mode of HPV-gene expression indicated by p16INK4a overexpression substantially precedes the emergence of squamous epithelial cell clones with chromosomally integrated viral genome copies in the progression cascade of HPV-induced preneoplastic lesions.
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Federico De Marco
Rome, ItalyElona Bucaj1,2, Ada Fiorini2, Kozeta Filipi3, Cesira Foppoli4, Carla Blarzino2, Maria Benevolo5, Federico De Marco1. 1Laboratory of Virology, "Regina Elena Institute for Cancer Research", Rome, Italy; 2Department of Biochemical Sciences, University "La Sapienza", Rome, Italy; 3Department of Epidemiology, Institute of Public Health, Tirana, Albania; 4CNR Institute of Molecular Biology and Pathology, Rome, Italy; 5Department of Pathology, "Regina Elena Institute for Cancer Research", Rome, Italy. Genital infection by HR-HPV is not per se sufficient to induce tumour development. To contribute to the identification of the role of Oxidative Stress (OS) in the neoplastic progression a study was conducted to evaluate the differences in protein expression and in protein oxidation among normal, dysplastic and neoplastic cervical tissues. Tissue specimens were obtained from patients affected by high grade dysplastic lesions and from patients with cervical cancer. Control tissues were obtained by women affected by non neoplastic cervical diseases (fibro-myoma). DNA extracted from tissue specimens (QIAGEN DNA extraction KIT) was used to analyse the presence of HPV infection, to evaluate the DNA viral load and the physical status of the viral genome. Tissues extracted proteins were separated in Bi-Dimensional Electrophoresis (2DE) and the analysis of differently expressed and oxidized proteins in the images acquired was performed using PD quest 2D software. Carbonyl adducts of proteins were determined by post-Western blot derivatisation of 2D nitrocellulose membrane with 2,4-dinitrophenyl hydrazine. DNP-protein adducts were then revealed by EIA detection with rabbit anti DNP specific antibody (Oxy-blot). Statistical analysis allowed the recognition of significantly modulated spots in dysplastic and neoplastic lesions in comparison with normal tissues. In addition a number of specific proteins were found to be selectively oxidized in pathological samples. Modulated and oxidised proteins were then identified by MALDI-tof Mass Spectrometry. Implication of proteins alteration in the malignant progression will be presented, discussed and correlated to virological status. Acknowledgements: Work partially supported by the Italian Ministry of Health and by the Italian Ministry of Foreign Affairs.
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Craig Woodworth
Potsdam, USAErik Vandermark1, Cynthia Schreiner1, Krysta DeLuca1, Rebecca Schilling1, Courtney Grayson1, Craig Woodworth1. 1Department of Biology, Clarkson University, Potsdam, NY, USA. Background: The NF-kB family of transcription factors mediates the host response to stress. NF-kB can stimulate cell growth and survival, and constitutive activation may contribute to malignant development. Several viruses have evolved mechanisms to alter NF-kB activation and this may serve a role in viral transformation.
Objectives: We examined whether HPV-16 E6 and E7 proteins alter NF-kB activity in epithelial cells cultured from human cervix.
Methods: Secondary cultures were infected with retroviruses encoding HPV-16 E6, E6 or E6/E7. As a control, cells were infected with retroviruses encoding the empty vector. NF-kB activity was determined by transfection with an NF-kB reporter gene and performing a dual luciferase assay.
Results: E6 and E7 acted differently. E6 significantly increased basal NF-kB activity and E7 significantly decreased activation. When cells were treated with TNF-alpha to strongly induce NF-kB, E6 enhanced and E7 inhibited TNF-induced activation. Co expression of E6/E7 generally reduced NF-kB activation, although results are variable. The p65 subunit of NF-kB was mainly localized in the cytoplasm, and E6/E7 slightly decreased both cytoplasmic and nuclear staining. The p50 subunit of NF-kB was mainly localized to the nucleus and E6/E7 did not alter expression or localization. To examine whether NF-kB contributed to immortalization, cells were infected with HPV-16 E6/E7 retroviruses and then stably transfected with either (1) the IkB-alpha dominant negative mutant, (2) the p65 subunit that increases activation, or (3) an empty vector as a negative control. Unexpectedly, the dominant negative IkB-alpha mutant stimulated colony formation and immortalization in several independent experiments. In contrast, over expression of p65 strongly inhibited colony formation and immortalization.
Conclusions: These results suggest that stable expression of HPV-16 E6/E7 causes variable reduction of NF-kB in cervical epithelial cells, and that reduction of NF-kB enhances growth and immortalization of human cervical cells in vitro. |
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Tipaya Ekalaksananan
Muang, ThailandTipaya Ekalaksananan1, Parichat Wongwarissara1, Chamsai Pientong1, Bunkerd Kongyingyoes2, Pilaiwan Kleebkaow3, Sumalee Siriaungkul4. 1Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; 2Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; 3Department of Obstetrics and Gynaecology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; 4Department of Pathology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand. Human papillomavirus type 16 Asian variant (HPV16 As) is found in most of Asian women diagnosed with cervical cancer. Transcription of HPV16 oncogenes, E6 and E7, is regulated by the long control region (LCR) containing several binding sites of both cellular and viral proteins. Nucleic acid sequence variation within the LCR may have an impact on oncogenic potential of the virus. The purposes of this study were to investigate the polymorphism and transcriptional activity of HPV16 Asian variant LCR in high grade squamous intraepithelial lesions and squamous cervical cancer of Thai women. The LCRs of HPV16 Asian variants were amplified, sequenced and compared with the reference and the previously reported sequences. The amplified LCRs of selected cases were analyzed for their transcriptional activities by insertion into the pGL3-Basic vector, a promoter-less luciferase reporter vector and comparison with vector of HPV16 LCR prototype. The results show nucleic acid sequence variation of 32 positions within the various LCRs and 11 positions mostly found at 3' end proximal to promoter have not been reported yet. Two cases containing nucleic acid sequence variation at position proximal to p97 promoter and HPV16 prototype were selected to analyze LCR transcriptional activity. Both of HPV16 As LCRs showed higher transcriptional activity than prototype. HPV16 As LCR with novel variations at nt position 46 T insertion with G, 61 T insertion with G and 28G insertion with A showed the highest transcriptional activity with 30 folds of the prototype while HPV16As LCR with previously reported variation had the activity 10 folds of the prototype. These results imply that HPV16 As, especially variant containing LCR nucleic acid sequence variation at 3' end proximal to p97 promoter, could lead to the overexpression of the HPV16 oncoproteins and contribute to the carcinogenesis of cervical cancer in Thai women.
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Berenice Illades-Aguiar
Chilpancingo, MéxicoBerenice Illades-Aguiar1, Mirna Vázquez-Villamar1, Ma. Elena Catarino-Chino1, Julio Ortiz-Ortiz1, Natividad Sales-Linares1, Javier Sánchez-Rendón1, Manuel Joaquín Romero-López1, Brenda Magali Lin-Li Lagunas-Bucio1, Eugenia Flores-Alfaro1, Marco Antonio Leyva-Vázquez1. 1Laboratorio de Biomedicina Molecular, Unidad Académica de Ciencias Químico Biológicas, Universidad Autónoma de Guerrero,Chilpancingo, México. Background: MGMT is a DNA repair gene, its expression decreases in various types of cancer. Hypermethylation of its promoter has been associated with the development of cervical cancer and its precursor lesions. Objectives: Determine the association between the MGMT -56 C>T polymorphism and the hypermethylation of its promoter in the development of LSIL, HSIL and cervical cancer.
Methods: Thirty cervical cancers, 21 with HSIL, 30 with LSIL and 31 with non-SIL samples from women from the State of Guerrero, Mexico were analyzed. The methylation state of the MGMT promoter was determined by methylation-specific PCR and the presence of the -56 C>T polymorphism by PCR and RFLPs (RsaI).
Results: MGMT was found to be hypermethylated in 47% of the samples and the frequency of the CT or TT genotype was 11% in hypermethylated and 17% in non-hypermethylated samples. An association was not found between hypermethylation and the -56 C>T polymorphism (OR=0.43, CI 0.13-1.4,p=0.17). The CT genotype was found in 27% of the cervical cancer, 19% of the HSIL, 7% of the LSIL and 3% of the non-IL samples. Meanwhile, the TT genotype was only found in 3% of the non-IL samples. An association was found between the presence of the CT or TTgenotype of the -56 C>T polymorphism and the development of HSIL (OR=6.2, CI0.7-52.1, p=0.09) and cervical cancer (OR=8.5, CI 1.2-62.6, p=0.035).
Conclusions: The CT or TT genotypes of the -56 C>T polymorphism are a risk factor for the development of HSIL and cervical cancer but not for the hypermethylation of the MGMT gene promoter.
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Berenice Illades-Aguiar
Chilpancingo, MéxicoBerenice Illades-Aguiar1, Irlanda Peralta-Arrieta1, Francisco Israel Torres-Rojas1, Luz del Carmen Alarcón-Romero1, Daniel Hernández-Sotelo1, Noelio Zamudio López1, Marco Antonio Leyva-Vázquez1. 1Laboratorio de Biomedicina Molecular y Laboratorio de Citopatología, Unidad Académica de Ciencias Químico Biológicas, Universidad Autónoma de Guerrero, Chilpancingo, México. Background: The increase in methylation of the CpG islands of gene promoters inhibits its expression.
Objective: Determine the methylation state of the promoters of the FHIT, MGMT, MLH1 and MSH2 genes in precancerous lesions and cervical cancer.
Methods: Exo-endocervical samples from women from the State of Guerrero, México were analyzed: ten of cervical cancer, 44 of LSIL, 34 of non-IL with HR-HPV and 24 of non-IL without HPV. The methylation state of the gene promoters was determined by PCR-SM. HPV detection was done by PCR-MY09/11 and/or GP5+/GP6+ and typification by RFLPs or sequencing.
Results: MGMT was found to be hypermethylated in 40% of cervical cancer, 50% of LSIL, 46% of non-IL with HR-HPV and 63% of non-IL without HPV. FHIT was hypermethylated in 60% of cervical cancer, 71% of LSIL, 60% of no-IL with HR-HPVand 42% of no-IL without HPV. MLH1 was hypermethylated in 10% of cervical cancer, 86% of LSIL, 67% of non-IL with HR-HPV and 58% of non-IL without HPV. MSH2 was not hypermethylated in cervical cancer, hypermethylated in 30% of LSIL, 42% of non-IL with HR-HPV and 33% with non-IL without HPV. A significant association was found between the hypermethylation of MSH2 and non-IL with HR-HPV (OR=2.86, CI 1.1-7.9), as well as between hypermethylation of MLH1 and LSIL (OR=9.69, CI 2.41-38.95). An association was found, although not significant, between the hypermethylation of the studied genes and cervical cancer precursor lesions.
Conclusions: Hypermethylation of the MGMT, FHIT, MLH1 and MSH2 genes is an early event in cervical carcinogenesis that remains throughout the progression of the tumor.
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Berenice Illades-Aguiar
Chilpancingo, MéxicoMaría Guadalupe Rivera-Trejo1, Berenice Illades-Aguiar1, Luz del Carmen Alarcón-Romero1, Daniel Hernández-Sotelo1, Eduardo Castañeda-Saucedo1, Marco Antonio Leyva-Vázquez1. 1Laboratorio de Biomedicina Molecular y Laboratorio de Citopatología, Unidad Académica de Ciencias Químico Biológicas, Universidad Autónoma de Guerrero, Chilpancingo, México. Background: DNMT3b over-expression increases the methylation of the promoters of repair and tumor suppressor genes associated with various types of cancer. It has been suggested that the -149C>T (C46359T) polymorphism of the DNMT3b promoter increases its expression.
Objective: Evaluate DNMT3b expression, its relationship with the -149C>Tpolymorphism and with methylation of the FHIT and MGMT promoters in women with precancerous lesions and HPV infection.
Methods: Twenty exo-endocervical samples of LSIL, 24 of non-IL with HPV and 5 of non-IL without HPV from women from the State of Guerrero, México were analyzed. HPV detection was done by PCR system (MY09/11 o GP5+/6+). Genotypification of the -149C>T polymorphism was done by PCR/RFLPs, DNMT3b expression by immunohistochemistry and methylation analysis of the promoters of the FHIT and MGMT genes by SM-PCR.
Results: DNMT3b was expressed in 85% of the LSIL, 21% of the non-IL with HPV and in 0% of the non-IL without HPV cases (p=<0.001). The frequency of the -149C>T SNP was: CT (50%), TT (42%) and CC (8%).There was no difference between the expression of DNMT3b and the -149C>T SNP (p=1.0). The frequency of the FHIT and MGMT promoter methylation was not significant between the LSIL, non-IL with HPV and non-IL without HPV cases (p=0.08 and p=0.79). When DNMT3b was expressed, FHIT was methylated in 77% of LSIL and 100% of non-IL with HPV; MGMT was methylated in 65% of LSIL and 40% of non-IL with HPV, but there was no significant difference between DNMT3b expression and methylation of the FHIT and MGMT promoters.
Conclusions: The increase in DNMT3b expression is more frequent as the lesion grade progresses, the -149C>T SNP does not influence the enzyme's expression level and the methylation of the FHIT and MGMTpromoters is not associated with the polymorphism.
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Dorothy J. Wiley
Los Angeles, U.S.A.Dorothy J. Wiley1, Emmanuel Masongsong1, Provaboti Barman1, Francois Coutlee2. 1School of Nursing, University of California, Los Angeles, Los Angeles, California; 2Laboratoire de virologie moléculaire, Centre de recherche du Centre hospitalier de l'Université de Montréal, Montréal, Québec, Canada.
Anal specimens derived from 83 HIV-HPV16 co-infected men were analyzed for cytosine methylation. HIV+ men ages 29-66 were recruited from the Montréal area; a single examiner performed anal liquid cytology, high-resolution anoscopy and biopsy, in addition to HPV genotyping using the Roche Linear Array Assay. HPV16 DNA was bisulfite modified, PCR amplified, cloned and sequenced; raw data were processed using the ClustalW alignment algorithm in BiQ Analyzer Software and imported into SAS 9.2 to generate heat maps and histograms. Between 8-10 clones were characterized for each specimen across 10 CpG sites within the 3'-L1 and 5'LCR region of HPV16 genomes. Data showed the average prevalence (standard deviation) of methylcytosines detected was 5% (0.03), 7% (0.03), and 4% (0.03) for seven NIL, 26 AIN1, and 50 AIN2 specimens tested, respectively. Mean and median methylation prevalence were closely approximated, and the highest methylation was observed at nucleotide 7091 for low- and high-grade anal intraepithelial neoplasias (LG-, HG-AIN), with minima at 7455 and 7561. Hypermethylation (>60%) in single CpG sites was observed more often in the 5' half of the target sequence. Also, multi-type oncogenic HPV infections (B=-0.15, p=0.03) and smoking (B=0.62, p=0.02) predicted the number of sites where clones showed >20% of methylcytosines, even after controlling for the effect of chronological age, HIV-load and grade of AIN disease. In a separate model, although the direction of the relationship was similar, the total number of HPV types detected did not significantly predict the number of sites with >20% methylation (B=-0.01, p=0.7). The inverse association between the number of oncogenic HPV infectants and methylation was unexpected but is consistent with data published by others.
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Koenraad Van Doorslaer
Bronx, USAKoenraad Van Doorslaer1, Robert D. Burk1. 1Albert Einsten College of Medicine, Bronx, NY, USA. Introduction: Most studies on the function of the papillomaviral oncogenes are based on the comparison of HPV16/18 and HPV6/11. These comparisons have proven valuable in understanding some functions of these proteins; however there are limitations in extrapolating these differences to actual carcinogenic risk. hTERT (human telomerase reverse transcriptase) levels are increased in most cervical squamous cell carcinomas, and it has recently been shown that HPV16 transcriptionally activates hTERT. In the present study, we used a luciferase reporter assay to assay whether E6 proteins derived from representative members of all human alpha-PV species-groups were able to activate the hTERT promoter.
Methods: U2OS cells, an osteosarcoma cell line that uses ALT to maintain its telomere length (telomerase negative), were transfected with 29 separate E6 proteins together with a plasmid carrying the hTERT promoter driving expression of a luciferase reporter. A Taqman RT-PCR method was developed to test whether activation of the hTERT promoter, as measured by an increase in luciferase signal, also resulted in increased hTERT mRNA levels.
Results & Discussion: All tested members of the alpha-9 species group (HPV16 and HPV16-related types) as well as HPV18 activated the hTERT > 3-fold compared to the empty vector control. These data indicate that activation of the hTERT promoter is associated with HPV type oncogenic risk (Fisher's exact test p<0.001) and suggest that maintenance of telomere length is an important feature of HPV carcinogenesis.
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Julio Velasco
Aviles, SpainJulio Velasco1,2, Cristina Perez2, Laia Alemany3, Silvia de Sanjose3. 1Pathology Department. Hospital San Agustín, San Agustín, Spain; 2University Institute of Oncology, Principality of Asturias (IUOPA), Asturias, Spain; 3Unit of Infections and Cancer, Catalan Institute of Oncology, IDIBELL, Barcelona, Spain.
Background: The oncological mechanism associated with HPV is one of the most stable of all known. Two common events are dysfunction of the regulatory proteins p53 and pRB for the viral proteins, E6 and E7 respectively. p16INK4a is an excellent indicator of the dysfunction of the pRB.The oncological mechanism associated with HPV is one of the most stable of all known.
Objectives: To improve the knowledge on the overexpression of the protein p16 INK4a in invasive cervical carcinoma (ICC), particularly: 1) if negativity of p16 INK4a is associated with some genotype 2) to analyze the relation between p16 INK4a positivity and LiPA results by age of the patient.
Methods: Two hundred and thirty four paraffin embedded tissue blocks of invasive cervical cancer samples diagnosed between 1998 and 2008 in all hospitals in Asturias were included. A) Tissue array preparation: Tissue slides were sectioned from paraffin-embedded blocks. Each tissue microarray contains 22 cervical squamous cell carcinomas or adenocarcinomas and its corresponding controls (their normal epithelial counterparts, vaginal or cervical mucosa of at least 15 mm away from the gross tumor border). Two hundred and thirty four cases were arrayed onto total of 11 slides. B) PCR method: The Human Papillomavirus detection and genotype was determined by SPF-10 broad-spectrum primers PCR subsequently followed by DEIA and genotyping by LiPA25 (version1: Labo Biomedical Products, Rijswijk, The Netherlands), C) Immunohistochemical method: the clone E6H4 TM to p16 INK4a human protein, IHC protocols were developed to stain tissue microarray.
Results: We observed negativity to the expression of p16 INK4a in 7 cases where the LIPA assay was positive (5 cases were HPV 16, 1 case was HPV 16/52 and 1 case HPV 31). In these cases, the difference between the detection of p16 INK4a and the positivity to HPV by LiPA was not statistically significant. The histology of these 7 cases was: 3 adenocarcinomas, 2 adenosquamous carcinomas and one classified as "other". The highest percentage of positive cases for both p16INK4a and LiPA was observed among women of 39 years old or younger, although differences were not of statistical significance.
Conclusions: 1) The pattern of expression of p16 INK4a is not associated with a particular HPV type although are data is based on limited number of cases. 2) The higher frequency of p16 negative cases in older women may be explained by the high prevalence of adenosquamous carcinomas and histologic types classified as "other, although it may be due to chance observation. 3) Both techniques have an excellent relationship and reinforce the hypothesis that HPV-related carcinogenesis is very stable.
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Jonathan Miller
Washington, USAJonathan Miller1, Xuefeng Liu1, Alison McBride2, Richard Schlegel1. 1Department of Pathology, Georgetown University Medical Center, Washington, DC, USA; 2Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA. The high-risk human papillomaviruses (HPV-16, -18) are critical etiologic agents in human malignancy, most importantly in cervical cancer. These oncogenic viruses encode the E6 and E7proteins that are uniformly retained and expressed in cervical cancers and required for maintenance of the tumorigenic phenotype. The E6 and E7 proteins were first identified as targeting the p53 and pRb tumor suppressor pathways in host cells, thereby leading to disruption of cell cycle controls. The simultaneous expression of E6 and E7 appears to be requisite for cervical cancer and indeed these two genes seem to have evolved both complementary and opposing functions that are necessary to prevent senescence and/or apoptosis. For example, while E7 stabilizes p53 protein, E6 degrades this tumor suppressor protein. Similarly, while E6 stabilizes RB protein, E7 inactivates and destabilizes it. In the current study, our microarray data demonstrated that E6 increased the expression of RB and ROCK1 mRNA in HFKs and this increased expression was confirmed by real time RT-PCR. The Rho/ROCK signaling pathways are complex and studies are often contradictory with regard to their effect on keratinocyte growth and differentiation. However, a relevant recent publication by the Hotchkins laboratory indicates that siRNA knockdown of ROCK1 stimulates terminal differentiation whereas knockdown of ROCK2 decreases terminal differentiation. General inhibition of both ROCKs induces cell proliferation and decreased differentiation. Indeed, the ability of ROCK inhibitors to mediate efficient keratinocyte immortalization has been recently reported. It is interesting that E6 and E7 appear to have opposing functions with respect to Rho/ROCK signaling, similar to the phenomenon seen with other HPV oncogene targets. E6 increases ROCK1 yet E7 has been reported to inhibit Rho A signaling. The yin-yang regulation of Rho/ROCKsignaling may be critical for fine tuning the growth and differentiation of keratinocytes as well as for regulating the viral replication cycle.
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Aleksandra Dakic
Washington, USAAleksandra Dakic1, Renxiang Chen1, Yuhai Dai1, Xuefeng Liu1, Richard Schlegel1. 1Department of Pathology, Georgetown University Medical Center, Washington, DC, USA. Cell immortalizationappears to be a critical event in the development of cancer and the oncogenichuman papillomaviruses (HPVs) encode two transforming proteins, E6 and E7, thatare sufficient and required for the efficient immortalization of primary humankeratinocytes. The E6 protein has multiple activities and interacting cellularproteins, including p53 and proteins containing PDZ domains. An early study showed that a dominantnegative p53 construct could substitute for E6 to immortalize keratinocytestogether with E7. Although theefficiency of this immortalization was not quantified, the results suggestedthat the p53 pathway was involved in cell immortalization. In the current study, we show that E6 mutantsdefective for p53 degradation (E6SAT8910, E6Y54D) are still able toinduce telomerase activity and immortalize cells with E7. In addition, E6 mutants lacking or mutated inthe PDZ motif retained their ability to induce p53 degradation and telomerase and to immortalize HFKs. Lastly, onemutant (E6Δ9-13), which was defective for both p53 degradation and telomeraseactivation, failed to immortalize keratinocytes with E7. Thus, E6-induced cell immortalizationcorrelates with its ability to induce telomerase and not with p53 or PDZ domainprotein interactions.
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Myeong-Kyun Shin
Madison, U.S.A.Myeong-Kyun Shin1, Julien Sage2, Henry Pitot1, Paul Lambert1. 1Cancer Biology Program, University of Wisconsin-Madison, Madison, WI, USA; 2Stanford University, Palo Alto, CA, USA. High-risk human papillomaviruses (HPVs) are associated with most of cervical cancers, other anogenital cancers, and a subset of head and neck squamous cell carcinomas (HNSCCs). Using transgenic mouse models, we previously found that HPV-16 E7 is the viral oncoprotein that most strongly contributes to the induction of cervical cancer in mice treated with exogenous estrogen (17 β-estradiol) and head and neck cancer in mice treated with 4-nitroquinoline 1-oxide (4-NQO) (Riley et al., 2003, Strati et al., 2007). However, tissue specific inactivation of the tumor suppressor pRb, a well known cellular target of HPV E7, was not sufficient to account fully for the oncogenic properties of E7 in cervical cancer (Balsitis et al., 2006) as well as HNSCC (Strati et al., 2007). In the current study, we are studying whether E7's ability to inactivate multiple pocket proteins, pRb, p107 and/or p130, is sufficient to explain E7's oncogenic potential in cervical cancer as well as HNSCC. Using combinations of conditional and germ-line knockout alleles of the genes encoding the pocket proteins, we observed that combinatorial loss of pRb and p130, or pRb and p107 lead to increased susceptibility of mice to cancers of the cervix and head and neck region in comparison to mice conditionally disrupted only for pRb. However, these double pocket protein-deficient mice still are not as susceptible to cancer as are E7 transgenic mice. Studies to investigate the cancer susceptibility of mice disrupted for all three pocket proteins are in progress.
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Christine How
Toronto, CanadaChristine How1,2, Angela Hui2, Anthony Fyles2, Fei-Fei Liu1,2. 1Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, ON, Canada; 2Ontario Cancer Institute/Princess Margaret Hospital, University Health Network, Toronto, ON, Canada. MicroRNAs (miRs) are small, non-coding RNAs that negatively regulate gene expression. Recent data indicates that miRs are aberrantly expressed in most human malignancies, including cervical cancer. To better understand how miRs mediate tumor progression in cervical cancer, dysregulated miRs were identified and functional studies were performed.
MiR expression was measured in 3 cervical cancer cell lines (SiHa, ME-180 and HT3) and 3 normal cervix tissues using a quantitative real-time PCR approach simultaneously examining 365 miRs along with 3 endogenous controls. Twenty-seven miRs were downregulated in all three cell lines compared to normal cervix tissues, including miR-196b. SiHa cells were transfected with miR-196b mimics to determine the cellular consequences of miR-196b overexpression, which resulted in reduced cell proliferation and clonogenicity in SiHa cells. Candidate miR-196b targets were elucidated using a tri-pronged approach combining in silico target prediction algorithms, clinical mRNA expression data, and in vitro global mRNA expression following miR-196b overexpression. Using this approach, HOXB7 was identified as a candidate target of miR-196b. To ascertain that miR-196b directly binds to the HOXB7 3' UTR, a luciferase reporter assay was performed. Reporter vectors were constructed containing either the HOXB7 3' UTR (pMIR-REPORT-HOXB7/UTR), or the HOXB7 3' UTR with a mutation in the predicted miR-196b binding site (pMIR-REPORT-HOXB7/mut). miR-196b overexpression resulted in significantly reduced luciferase activity in cells transfected with pMIR-HOXB7/UTR, but not in cells transfected with pMIR-HOXB7/mut, corroborating the direct and specific binding of miR-196b to the HOXB7 3' UTR. To determine the effect of knocking down HOXB7 expression, SiHa cells were transfected with siHOXB7, which recapitulated the reduced proliferation and clonogenicity observed following miR-196b overexpression.
The under-expression of miR-196b in human cervical cancer likely promotes tumour cell proliferation via increased activity of the HOXB7 transcription factor, which in turn, can upregulate several pro-survival pathways, ultimately leading to tumour progression. |
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Mariet Feltkamp
Leiden, The NetherlandsSiamaque Kazem1, Els Van Der Meijden1, Linda Struijk Struijk1, Frank De Gruijl2, Mariet Feltkamp1. 1Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands; 2Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands. Background: Betapapillomaviruses (betaPV) have been implicated in cutaneous squamous-cell carcinoma development. A mechanism of transformation could be the ability to inhibit UVB-induced apoptosis creating a pool of genetically unstable keratinocytes. In a previous study we have shown that among several betaPV types particularly HPV8 was able to impair apoptosis. As apoptosis and terminal keratinocyte differentiation share specific pathways, in this study we investigated the effect of betaPV E6/E7 on terminal keratinocyte differentiation.
Methods: HPV5, HPV8 and HPV38 E6/E7-expressing primary (PHK) and immortalized (N/TERT cells) keratinocytes were grown in organotypic skin (raft) cultures and compared with those expressing HPV16 E6/E7 or the empty retroviral vector.
Results: The HPV5 and HPV8 rafts grew thicker, contained ballooning cells and lacked the cornified layer, even after 18 days in culture. Ki67-staining revealed replication only in the basal layer, whereas the HPV16 rafts were Ki67-positive throughout. HPV38 resembled the empty vector control in every aspect. A marked accumulation of filaggrin and caspase 14-positive cells was observed in the HPV5 and HPV8 PHK rafts (Figure 1), in conjunction with loss of Akt1. This phenotype was also observed in PHK rafts expressing only E6, but not in those expressing only E7. In contrary, in the HPV8-expressing N/TERT rafts, filaggrin and caspase 14 expression was completely prevented (Figure 2). Western blot analysis confirmed the absence of profilaggrin in the HPV8 N/TERT cells, which was detectable in the HPV8 PHK rafts. In the latter, however, specific filaggrin intermediates were not detectable anymore.
Conclusions: HPV5 and HPV8 induced specific changes in keratinocyte outgrowth suggestive of late terminal differentiation arrest caused by E6. In the primary keratinocytes this arrest prevented filaggrin maturation which could explain the cornification defect. In the immortalized (p16ink4A-negative) N/TERT cells, HPV8 E6 expression reverted filaggrin expression, suggestive of dedifferentiation. The relevance of these findings to understand the role of betaPV in skin carcinogenesis will be discussed. |
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Eeva Auvinen
Helsinki HUS, FinlandDario Greco1,4, Niina Kivi2, Kui Qian1, Petri Auvinen1, Eeva Auvinen2,3. 1Institute of Biotechnology, University of Helsinki, Helsinki, Finland; 2Department of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland; 3Department of Virology, Helsinki University Hospital Laboratory, Helsinki, Finland; 4Present affiliation: Department of Obstetrics and Gynecology, Helsinki University Central Hospital, Helsinki, Finland. Tumor viruses have mechanisms to modulate crucial cellular signaling pathways to reprogram host cells and support their own life cycle, as well as to control host defense responses. We have earlier studied the effects of the human papillomavirus (HPV) type 16 E5 oncogene on the expression of cellular genes in a genome-wide cDNA microarray screen (Kivi et al., Oncogene 27:2532-41, 2008). Genes involved in cell motility, cell adhesion, cell proliferation, mitogenic signaling, differentiation, and antigen presentation were significantly altered due to E5 expression. Cell motility was shown to be enhanced in vivo in live cell imaging of E5 expressing cells.
In order to better understand the cellular pathways involved in the effects of E5 in cervical carcinogenesis, we have proceeded to perform a large time-scale analysis of E5 effects on cellular gene expression as well as on cellular microRNA (miRNA) expression in epithelial cells. MicroRNAs are small single-stranded regulatory RNA molecules which decrease the expression of their target genes by binding to the target mRNA and/or by inhibiting protein translation. MicroRNAs have been implicated in many chronic diseases, specifically in a number of cancers. Many viruses alter the expression of cellular microRNA expression, and several viruses, excluding papillomaviruses, have been shown to express their own microRNAs.
In this work, E5 expression was induced in HaCaT cells by dexamethasone and total RNA was at 0, 2, 4, 24, 48, 72 and 96 h after induction. Control cells without the E5 gene were treated in a similar manner. RNA was labeled and hybridized to Agilent human cDNA and miRNA microarrays in triplicates.
The expression of a set of cellular microRNAs was found significantly altered due to E5 expression. Microarray results were validated by TaqMan quantitative PCR assays and three microRNAs of interest were studied further. Using several prediction algorithms we searched for putative target genes of the selected microRNAs. The predicted microRNA target genes present among the genes that were significantly altered in expression array analysis were identified. We are currently carrying out bioinformatic and biological validation and analysis of the genes whose expression counter correlated with the expression of one or several of the selected microRNAs.
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Kazunori Nagasaka
Trieste, ItalyKazunori Nagasaka1, Lawrence Banks1. 1ICGEB, Trieste, Italy. High risk HPV E6 oncoproteins are characterized by having PDZ binding motifs at their extreme carboxy termini. Through these motifs they interact with a variety of cellular proteins that harbour PDZ domains. Whilst these interactions appear important for E6's ability to contribute to malignancy, the mechanism by which this occurs is unclear. Numerous cellular targets are reported to bind E6 through this motif, and these include the tumour suppressor proteins, hDlg and hScrib. The cell polarity regulator hScrib is a potential tumour suppressor whose loss is a frequent event in late stage cancer development. Little is yet known about the mode of action of hScrib, although recent reports suggest a role in the regulation of cell signaling. We now show that hScrib is itself a major regulator of the ERK signaling cascade. Loss of hScrib expression activates ERK and enhances nuclear ERK translocation. We also show that this is a result of direct interaction between hScrib and ERK and most likely involves recruitment of the protein phosphatase PP1c, by hScrib which subsequently de-phosphorylates ERK, thereby turning off the signaling pathway. Interestingly, hScrib is also phosphorylated by ERK and PKA in this complex, the consequences of which are enhanced susceptibility to HPV E6 induced degradation. These results show that HPV E6 can contribute to ERK activation through a variety of independent mechanisms.
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Tohru Kiyono
Chuo-ku, JapanMako Narisawa-Saito1, Yuki Yoshimatu1, Takashi Yugawa1, Shin-ichi Ohno1, Kei Haga1, Tohru Kiyono1. 1Virology Division, National Cancer Center Research Institute , Tokyo, Japan. Analyzing the cervical carcinogenesis model, we clarified that transduction of oncogenic HrasG12V (Hras) was sufficient for tumorigenic transformation of normal human cervical keratinocytes (HCKs) expressing human papillomavirus oncogenes E6 and E7. Subcutaneous transplantation of 200 HCKs carrying E6E7 and Hras resulted in the tumor formation within 2 months, indicating that this set of oncogenes readily confers tumor initiating property. Dissecting Ras signaling pathway, endogenous c-Myc stabilized by Hras was revealed to be one of the most important players in tumor formation. Unlike Hras, Akt1 with myristilation signal (myr-Akt1), constitutively active MEK1 (MEK1DD) or bcl2 did not cooperate with E6E7 to induce tumorigenicity in nude mice within 3 months. However, induced expression of c-Myc mutant (MycT58A), which is resistant to proteasomal degradation, conferred marked tumor forming abilities on myr-Akt- or MEK1DD-expressing HCK-E6E7 cells and less marked but still significant tumor forming ability on bcl2-expressing HCK-E6E7 cells. In contrast, induced expression of Myc inhibitor (Omomyc) to the levels which did not affect the growth in culture, markedly suppressed the tumor forming ability of HCKs with Hras and cervical cancer cell lines. Thus inhibiting Myc would be an ideal therapeutic strategy not only for the Myc altered cancers but also for the Ras activated ones.
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Yuki Yoshimatsu
Chuo-ku, JapanYuki Yoshimatsu1, Mako Narisawa-Saito1, Takashi Yugawa1, Shin-ichi Ohno1, Tohru Kiyono1. 1National Cancer Center Research Institute Virology Division, Tokyo, Japan . High-risk HPV E6 has been shown to interact with PDZ-domain-containing proteins through its carboxy-terminal motif and lead their degradation. It has been suggested that the PDZ domain-binding motif plays important roles in transformation of cultured cells, and hyperplasia and carcinogenesis of E6-transgenic mice. However, the biological effects of E6 oncoprotein especially through the PDZ domain-binding motif to the natural host cells have not been elucidated. We have examined the roles of the E6 protein in an in vitro model for cervical cancer, in which E6 and E7 together with activated Hras (HrasV12) can induce tumorigenic transformation of normal human cervical keratinocytes (HCK). Wild type (E6wt) or effector domain mutants of E6 (E6mts) were transduced to the HCK-E7- HrasV12 (HCK transduced with HPV16 E7 and HrasV12 with or without hTERT). HCK-E7-HrasV12-E6wt cells showed tumorigenic ability in nude mice. Then we determined the transforming ability of HCK-E7- HrasV12-E6mts with the cells carrying their wild type counterpart. A single amino acid deletion of the carboxy -terminal end of E6 (E6delta151) resulted in marked reduction of tumor size, whereas E6 (SAT) mutant which lacks the p53 degradation activity did not result in the significant reduction of tumorigenic ability. Then, we knocked down several PDZ-domain containing proteins in HCK-E7- HrasV12-E6delta151 cells to see whether knockdown of these proteins can restore the tumorigenic ability. Among them, single knockdown of Scribble or MAGI-1, both of which bind to E6wt but not E6delta151, significantly increased the tumorigenic ability, and the combinatorial knockdown of Scribble and MAGI-1 markedly restored the tumorigenic ability. These data indicate that E6 plays important roles in tumorigenic transformation of human cervical keratinocytes at least in part through targeting Scribble and MAGI-1.
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Yusuke Zushi
Tokyo, JapanYusuke Zushi1,2, Mako Narisawa-Saito1, Yuki Yoshimatsu1, Takashi Yugawa1, Nagayasu Egawa1, Shin-ichi Ohno1, Masatoshi Fujita1, Kazuma Noguchi2, Masahiro Urade2, Tohru Kiyono1. 1Virology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan; 2Department of Oral and Maxillofacial Surgery, Hyogo College of Medicine, Nishinomiya, Japan. Oral squamous cell carcinomas (OSCCs) are considered to arise from human oral keratinocytes. Like in cervical cancers, DNAs of oncogenic human papillomaviruses (HPVs) predominantly types 16 and 18 have been detected though less frequently (20-25%) in OSCCs. In accordance with the fact that E6 and E7 can inactivate p53 and pRb, respectively, mutations of p53 and inactivation of p16 are frequently observed in HPV-negative OSCCs. In addition to inactivation of the p53 and pRb pathways, other alterations such as overexpression of EGFR are often observed in both HPV-positive and -negative OSCCs. However, causal-relationship between accumulation of these abnormalities and multi-step carcinogenesis is not fully understood. To elucidate the carcinogenesis process, we newly established human tongue keratinocytes (HTK) cell lines derived from tongue mucosa with retroviral vectors expressing either HPV16 E6/E7 or a mutant CDK4 (CDK4R24C), cyclin D1 and human telomerase reverse transcriptase (hTERT). HTK-16E6E7 and HTK-CDK4R24C/cyclinD1/hTERT (termed HTK-K4DT) retaining most of the original characteristics of primary tongue keratinocytes showed no tumorigenicity in xeno-transplanted mice. Additional transduction of oncogenic Hras or erbB1 together with c-myc into the HTK-16E6E7 and HTK-K4DT expressing DNp53 (dominant negative form of p53) showed the anchorage-independent growth and subcutaneous tumor formation in nude mice. Orthotopic transplantation of HTK-K4DT-DNp53-HrasV12-c-myc-wt(wild type c-myc) and HTK-K4DT-DNp53-erbB1-c-mycT58A (mutant-c-myc) cells resulted in local growth of tumors in all the mice tested and some of them yielded regional metastases in 2-3 weeks. This experimental model should help to understand the carcinogenesis of OSCCs.
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Mario Castellanos
Brooklyn, USAMario Castellanos1, Vineet Punia2, Anita Szerszen1, Bette Lazzaro1, Adi Davidiv1, Mitchell Maiman1, Probal Banerjee2. 1Departments of Medicine, OB/GYN, and Pathology, Staten Island University Hospital,, Staten Island, NY, USA; 2Department of Biochemistry, College of Staten Island, Department of Chemistry, Staten Island, NY, USA.
Background: Pathogenesis of HPV to high grade cervical intraepithelial neoplasia (CIN) is well. In benign lesions, HPV-DNA is often packaged into virions. However, virion assembly stops during transformation. If cervical lesions are exposed to endonuclease digestion, the HPV viral capsid could alter nuclear susceptibility to DNA digestion.
Objectives: We propose that susceptibility to endonuclease digestion is a simple marker to identify which HPV lesions will progress.
Methods: Endonuclease resistance was examined in condyloma acuminata, CIN I-III and cervical carcinomas cases. From the paraffin-embedded tissue blocks sections were cut and placed in a bath containing DNASE I for DNA digestion. Undigested DNA was stained by a Feulgen process. H&E slides were used for comparison. DNA positivity was correlated to disease grade. In addition, Colposcopy Clinic records were examined to identify HPV-infected women. Two cohorts were formed: Progression groups - women that progressed to CIN II or higher (12-24 months) and Regression group - women on subsequent biopsies and HPV testing became negative. All biopsies stained with our method and results correlated to clinical outcomes.
Results: After endonuclease digestion, residual DNA was seen in 13/16 (81%) condylomas, and 12/15 (80%) CIN I cases. In contrast, 0/15 CIN II, 0/15 CIN III and 0/17 carcinomas had DNA staining. From the Colposcopy Clinic 24 HPV (+) women were identified with biopsies available over 12-24 month period. Ten of the 14 women (71%) that regressed had residual DNA staining, in contrast 7/10 (70%) that progressed were negative.
Conclusions: Susceptibility to endonuclease digestion identified 100% of high grade cases and cancers which had no residual DNA staining. In contrast, 80% of low grade lesions were positive. In addition, residual DNA positivity or negativity correlated with HPV regression or progression respectively. Endonuclease-resistant DNA may be simple marker to identify HPV lesions that are at risk for progression.
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William Bonnez
Associate Professor of Medicine,Department of Medicine, University of Rochester
School of Medicine and Dentistry; Infectious Diseases Division, University of Rochester Medical Center
Rochester, USAWilliam Bonnez1, Carrie DaRin1. 1University of Rochester School of Medicine and Dentistry, Rochester, NY, USA. Objective: The overexpression of cyclooxygenase-2 (COX-2) in many cancers and their precursors, has led to the successful demonstration in clinical trials that inhibitors of COX-2 could reduce the incidence of colonic adenomas and adenocarcinomas. COX-2 is also overexpressed in CIN and laryngeal papillomas, the later being predominantly caused by HPV-11. We wished to evaluate the antiproliferative efficacy of celecoxib, a COX-2 inhibitor, on HPV-11-induced human condylomas in the SCID mouse xenograft model.
Materials and Methods: Nine SCID mice (3 cages) were grafted under the skin of both flanks with a 4 mm diameter human skin implant infected with HPV-11. A different foreskin donor was used for each flank. After 1 week, each cage was randomized for the animals to be fed their regular diet supplemented with 0, 160, or 1,600 ppm of celecoxib for the following 11 weeks. At the end of the experiment the animals were sacrificed, and the grafts removed, measured, and processed for histology. The composite geometric diameter (cGMD) of the two grafts borne by each mouse and the presence of histologic markers of HPV infection were the endpoints. The experiment was repeated 4 times, thus involving 36 mice.
Results: Four mice died before the end of the experiment, one in each treatment group, and a fourth in the high dose group. 64 grafts were present in the remaining 32 mice.
Endpoints 0 ppm celecoxib 160 ppm celecoxib 1,600 ppm celecoxib p values
Graft size (cGMD), mean ± SD 3.65 ± 0.74 mm 3.55 ± 0.80 mm 3.06 ± 0.98 mm 0.16
Histology, HPV positive/total 16/19 (84%) 15/21 (71%) 14/20 (70%) 0.60
Conclusions: Celecoxib had a minimal and not statistically significant effect on the growth of HPV-11-infected condylomas. Its place in the management of laryngeal papillomas is unlikely.
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William Bonnez
Associate Professor of Medicine,Department of Medicine, University of Rochester
School of Medicine and Dentistry; Infectious Diseases Division, University of Rochester Medical Center
Rochester, USAWilliam Bonnez1, Carrie DaRin1, Galina Glazko1, Michelle E. Zanche1. 1University of Rochester School or Medicine and Dentistry, Rochester, NY, USA. Objectives: In a previous analysis of orthotopic human skin grafts from various donors experimentally infected by HPV-6 or -11 and implanted in severe combined immunodeficiency (SCID) mice, we had observed that the HPV-induced proliferation of these grafts was highly dependent on the foreskin donor. In order to evaluate if this donor effect was associated to a specific cellular gene expression profile we used the microarray technology. In addition, we also compared the gene expression profile of the grafts before HPV-infection and 12 weeks later.
Materials and Methods: Foreskin samples from 31 different donors were studied prior to HPV infection. Fifteen were chosen from donors whose HPV-infected graft showed the smallest proliferation, while 18 were from donors that showed the largest proliferation. Within these two groups, the "small" and the "large", we also chose a subset of paired grafts collected 12 weeks after their infection, 7 from the "small" group, and 8 from the "large" group. Total RNA from each sample was extracted, amplified, and hybridized to Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays.
Results: We found no significant gene over- or under- expression associated with HPV-induced graft proliferation. However, when comparing the gene expression profile before and after HPV-infection, especially in the grafts that showed the largest proliferation and demonstrated infection by histology, we found the over-expression of 105 genes involved in various aspects of cell physiology, and 16 under-regulated genes.
Conclusions: Given that differential gene expression did not account for the strength of graft proliferation after HPV infection, we speculate that allelic differences may account for differences in the magnitude of HPV-induced proliferation. HPV infection has a pleomorphic effect on cellular gene expression, consistent with complex interactions within the cellular machinery.
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Zhi-Ming Zheng
Head, Tumor Virus RNA Biology Section Senior Investigator, HIV and AIDS Malignancy Branch, National Cancer Institute, Center for Cancer Research
Bethesda, USARong Jia1, Lifang Zhang1, Mingfang Tao1, Xiaohong Wang1, Zhi-Ming Zheng1. 1HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. HPV16 E6 and E7 are expressed from a single bicistronic pre-mRNA which contains three exons and two introns. Splicing of the intron 1 from the E6 ORF is essential for E7 translation, whereas a bicistronic mRNA retaining the intron 1 produces only the E6 oncoprotein. Although majority of the intron 1 splicing takes place from nt 226 5' splice site (5' ss) to nt 409 3' splice site (3' ss) to generate E6*I, two minor 3' ss, nt 526 and nt 742 3' ss, are also used much less efficiently for alternative splicing to produce E6*II and E6^E7, respectively. The usage of all three 3' ss for HPV16 E6E7 RNA splicing is detectable in cervical cancer tissues and HPV16+ cell lines, CaSki, SiHa, and W12 subclones 20861 and 20863 cells, and can be reconstituted in in vitro splicing conditions. Although the HPV16 intron 1 in CaSki and SiHa cells has a few nucleotide variations from HPV16 reference strain, these natural occurring variations appear to have no effect on intron 1 splicing. In further characterization of the intron 1 in the E6 ORF, we have mapped a branch point sequence from the nt 409 3' ss and identified a polypyrimidine track that controls the nt 409 3' ss usage. Interestingly, we identified two additional 5' ss upstream of the nt 226 5' ss that had not been reported. The two novel 5' ss, positioned separately at nt 221 and nt 191 in the virus genome, remain silent in general, but were activated when the nt 226 5' ss was disrupted. Activation of the two novel 5' ss, with a frequency of 5:1 (nt 221: nt 191), was more dramatic in CV-1 cells than in HEK293 cells. This finding indicates a more complex RNA splicing regulation of HPV16 oncogene expression.
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Beatrice Orru
Dublin, IrelandBeatrice Orru1, Stefan Schwartz1,2, Fergus Ryan1. 1School of Biological Sciences, Dublin Institute of Technology, Dublin, Ireland; 2Dept. of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden. HPV-infected cells in high-grade cervical lesions are characterised by continued expressionof the early viral genes, and a complete shutdown of the late viral genes encoding the highly immunogenic viral structural proteins L1 and L2. We are speculating that inhibition of expression of the viral structural proteins L1 and L2 prevents detection and elimination of the infected cells by the host immune system. Therefore, it is of interest to delineate mechanisms that regulate late gene expression. Our aim is to identify cellular factors that can induce HPV-16 late gene expression. They could be targeted by small compounds that could act as antiviral drugs against HPV infection by prematurely inducing late gene expression and exposing the cell to the immune system of the host.
We generated a subgenomic HPV-16 plasmid named pBEL-CAT in which the L1 gene has been replaced with the chloramphenicol acetyltransferase (CAT) reporter gene preceded by the poliovirus IRES ( Figure 1). CAT is a surrogate for L1 and L2 expression and can be measured by CAT ELISA. pBEL-CAT produces 50,000 fold less CAT than a CMV-CAT plasmid upon transfection of HeLa cells as a result of efficient suppression of HPV-16 late genes in cervical cancer cells. Mutational inactivation of a splicing silencer in the HPV-16 L1 sequence induced CAT reporter gene expression 156 fold. We also show CAT expression can be induced by adenovirus E4orf4 protein (E4orf4), polypyrimidine tract binding protein (PTB) and arginine/serine-rich SRp30c protein (SRp30c) (Figure 2). Results revealed that induction of CAT was dependent on the levels of the E4orf4, PTB or SRp30c proteins.
These results demonstrate that this CAT reporter assay can be used to identify HPV-16 RNA elements that regulate late gene expression as well as molecules that can induce HPV-16 late gene expression.
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Sunghae Joo
Gen-Probe Inc
San Diego, USASunghae Joo1, Neurita Salva1, Tristan Nater1, Janel Dockter1. 1Gen-Probe Inc, San Diego, CA, USA.. Background: The APTIMA HPV (AHPV) Assay is a CE-marked multiplex screening assay that detects HPV E6/E7 mRNA from 14 high-risk (HR) types in PreservCyt liquid Pap specimens. We have recently developed the APTIMA HPV 16 18/45 Genotype (AHPV-GT) Assay, which specifically detects HPV types 16, 18 and 45 and discriminates type 16 from types 18 and 45. The AHPV-GT assay is intended for testing AHPV assay positive samples to determine if types 16, 18, or 45 are present.
Objectives: In this preliminary evaluation of assay performance, the analytical sensitivity and specificity of the AHPV-GT assay was determined, as well as a preliminary assessment of clinical performance for detection of HPV 16, 18, and 45 in liquid Pap specimens.
Methods: Analytical specificity was determined by testing in vitro transcripts (IVTs) for low-risk types (6, 11, 42, 43, 44, 53) and non-targeted HR types (31, 33, 35, 39, 51, 52, 56, 58, 59, 66, 68) at 1 million copies/reaction. The 95% detection limit was determined by testing serial dilutions of HPV 16, 18, and 45 IVT. The accuracy of the AHPV-GT assay for detecting HPV 16, 18 and 45 in AHPV assay positive clinical specimens (n=101) was also evaluated, using the Linear Array HPV DNA genotyping test (LA; Roche Molecular Diagnostics) as a reference.
Results: No cross-reactivity was observed with the low-risk and non-targeted HR IVTs. The predicted 95% detection limit was 100 copies/reaction for HPV 16, 18 and 45. Overall percent agreement with LA in AHPV Assay positive samples was 97% for type 16 and 93% for types 18/45.
Conclusions: The AHPV-GT assay is a sensitive and specific test for the detection of HPV types 16, 18 and 45 in PreservCyt liquid Pap specimens that are positive in the AHPV assay.
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Sung Soon Kim
Division of AIDS, Center for Immunology and Pathology, National Institute of Health
Seoul, KoreaDai-Ho Jang,, Jee Eun Rhee,, Sung Soon Kim,. 1Division of AIDS, Center for Immunology and Pathology, National Institute of Health, Seoul, Republic of Korea. Objectives: Previous studies have reported upon the association between Human papillomavirus 16 (HPV16) variants and increased risk of cervical intraepithelial neoplasia and invasive cervical cancer. In Korea, the incidence of the aspartic acid to glutamate at E6 amino acid 25 for HPV16 is predominant. The aim of this study is to establish in vitro cell line with stable E6 D25E and to investigate the role for cervical carcinogenesis of variant E6 gene through the expression of p53 and related cell cycle associated proteins p21, p27, cyclin A/E, MDM2, CDC2.
Methods: E6 D25E gene, amplified from HPV16 variant infected patient sample, was inserted into pLenti6.3/V5-TOPO lentiviral vector and the recombinant lentivirus was harvested from 293FT cells contransfected with the positive recombined plasmid and lentiviral packing materials. C33A cells were infected with the recombinant lentivirus and the cells with stable maintenance E6 gene were screened by blasticidin selection. The single clone was obtained by seeding the cells into 96-well plates with one cell per well. E6 expression in the cells was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoprecipitation. The expression of cell cycle proteins was detected with western-blot.
Results: By using lentiviral gene transfer technology, the E6 D25E gene was integrated into the chromosome of C33A cell. We selected 16 clones infected with the E6 D25E DNA from 40 clones and expression of the E6 D25E protein in the cell was confirmed by immunoprecipitaion. In the E6 D25E expressing cell line, tumor suppressor protein p53 levels were decreased. Additionally, the protein of the p53 regulated gene, p27, was down-regulated with similar response and cycline A/E levels were significantly increased. We did not observe consistent changes in protein levels of p21, CDC2 and MDM2.
Conclusion: The E6 D25E could differentially contribute to develop of cervical cancer by inducing of p53 and p27 degradation. These finding indicates that HPV16 E6 variant may be associated with G1/S phase cell cycle in these cervical carcinogenesis. |
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Markus Schmitt
Heidelberg, GermanyMarkus Schmitt,, Michael Pawlita,. 1Department of Genome Modifications and Carcinogenesis, Infection and Cancer Program, German Cancer Research Center (DKFZ), Heidelberg, Germany. Background: Infections with high-risk human papillomaviruses (HPV), mainly HPV type 16, can cause malignant transformation of the human cervical epithelium and cervical cancer (CxCa). Very little is known about the quantitative expression of all HPV16 transcripts in frequently used cervical cancer cell lines.
Methods: We have quantitatively analysed the viral transcriptome (10 spliced and 5 unspliced targets) in the HPV16-transformed cell lines SiHa, CaSki, MRI-H196, MRI-H186, HPK-IA (early and late passages) and C3. We used a nucleic acid sequence-based amplification (NASBA)-bead-based hybridisation assay quantifying spliced and unspliced HPV16 transcripts (Schmitt et al., 2009).
Results: The cell lines differed in their qualitative and quantitative expression of viral transcripts depending on the physical HPV genome status. In SiHa, transcripts containing the splice acceptor at nucleotide 3358 or downstream sequences were absent. In CaSki cells virtually all viral transcripts were present but with a reduced quantity of late transcripts. The tumorigenic phenotype of late passage HPK-IA cells appeared not to be mediated through changes in HPV expression. MRI-H186, HPK-IA and C3 cells expressed very high levels of full-length early transcripts ending at the early polyadenylation signal. MRI-H196 expressed L1 full-length (fl) but no E5 fl RNA suggesting the presence of integrated HPV16 genomes with a disrupted E2 region and rearranged L1 DNA sequence.
Conclusions: Quantitative expression changes of HPV16 transcript markers correlate with the physical state of the HPV genome in HPV16-positive cervical cancer cell lines.
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Christine Weyn
Antwerp, BelgiumChristine Weyn1, Joanne Rasschaert2, Jean-Marie Vanderwinden3, Yvon Englert1, Véronique Fontaine1. 1Research Laboratory on Human Reproduction and Unit of Microbiology, Institute of Pharmacy, Université Libre de Bruxelles, Brussels, Belgium; 2Department of Biological Chemistry and Nutrition, Faculty of Medicine, Université Libre de Bruxelles, Brussels, Belgium; 3Laboratory of Neurophysiology, Faculty of Medicine, Université Libre de Bruxelles, Brussels, Belgium. High-risk HPVs are associated with anogenital carcinomas and are found in > 99 % of cervical carcinomas. HPV could also infect the trophoblast, an epithelial cell from the placenta through which implantation and nutrient exchanges occur and could be responsible for some spontaneous abortions, although this should be further confirmed by additional experiments. Regulation of viral transcription at the LCR in trophoblastic cells has not been studied before.
This study compared the regulation of the HPV-16 LCR expression in different cell lines by steroid hormones and viral proteins. Since the HPV-16 LCR contains glucocorticoid and progesterone response elements (GRE/PRE), we determined the role of progesterone in viral transcription. RU-486, a competitive progesterone receptor antagonist, was shown to inhibit progesterone effects in trophoblastic cells. The effect on GRE/PRE of steroid hormones was confirmed by exogenous dexamethasone in these cells.
The role of the early viral proteins in HPV-16 transcription was determined using a plasmid expressing all the early viral proteins under the control of the entire LCR. This plasmid increased HPV-16 LCR transcriptional activity three- to fivefold in C33A and trophoblastic BeWo cells. Using various constructs harbouring mutated early regions, we observed that this induction is not due to E1 or E2, but to the other early viral proteins. In both BeWo and C33A cells, early viral proteins were shown to induce viral gene replication and as expected, absence of E1 or E2 expression abolished this effect. To elucidate the molecular mechanism responsible for the observed pHPV16early driven transcriptional activity, we determined a role for AP-1 transcription factors. To conclude, transcriptional control in trophoblastic and cervical cells can be conferred to, among other factors, progesterone and viral early proteins. This latter effect was not due to E1 or E2, but to the activity of E5, E6 and/or E7, probably via AP-1.
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Sandra Mazzoli
Firenze, ItalySandra Mazzoli1, Francesca Meacci1, Patrizia Addonisio1. 1STDs Centre, Santa Maria Annunziata Hospital, ASL 10, Firenze, Italy. HPV-DNA is prevalent in young fertile females and males populations where it is also a highly transient. Very few is known about the persistence of persistent/activated infections in healthy females and particularly in males. Commercial assays for simultaneous genotyping and detection of HPV-E6/E7-mRNA are now available. Recent papers have demonstrated that HPVE6/E7-mRNA can be more powerful than DNA in predicting the progression risk to cancer and in detecting a transforming HPV infection.
In 2009 we screened for HPV-DNA and subsequently for HPV-E6/E7-mRNA a sentinel population of 653 patients-(pts), 427 males and 226 females, presented to S.M. Annunziata STDs Centre, Florence, Italy. All were asymptomatic for HPV infection and did not present abnormal cytological findings. A total of 1,367 biological samples were analysed. Biological materials were analysed by INNO-Lipa/HPVGenotyping/Extra-Innogenetics, Italy. Nucleic Acids Extraction, was performed by EZ-1 BioRobot Workstation and DNA Tissue Kit, Qiagen. NucliSENS EasyQ®-HPV test and NucliSENS® easyMAG®, bioMerieux Inc., was used for HPV-E6/E7-mRNA.
HPV-DNA was positive in 33.2% of the patients: 217 pts., 93 females and 124 males: 54 pts, 24,8%, resulted positive for HPV-16, 18, 31, 33, 45. In 54 pts we evaluated HPVE6-E7mRNA: 28.5% (8/28) of the males and 50% (13/26) of the females were positive and demonstrated HR-HPVs activated towards a transforming HPV infection. 100% activated males viral strains were co-infections with other two or more than two HR-HPV genotypes. Among females HPV-16 were prevalently activated and as single infection. 5/8 mRNA-HPV16 positive women in six months follow-up developed high grade cervical lesions/cancer (62.5%).
In the present study we demonstrated that HPVE6/E7-mRNA activated oncogene transcripts are present also in asymptomatic populations: this represents the first report in males. HPVE6/E7-mRNA seems to be the best marker to select asymptomatic cancer progression risk cohorts for longitudinal long-term follow-up studies and the prevention of anogenital carcinomas both in males and females.
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Divya Patel
Ann Arbor, USADivya Patel1, Laura Rozek2, Justin Colacino2, Adrienne Van Zomeren-Dohm2, Mack Ruffin3, Elizabeth Unger4, David Swan4, Juanita Onyekwuluje4, Cecilia DeGraffinreid5, Electra Paskett6. 1Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI, USA; 2Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, MI, USA; 3Department of Family Medicine, University of Michigan, Ann Arbor, MI, USA; 4Division of Viral and Rickettsial Diseases, National Center for Zoonotic Vector-borne and Enteric Diseases, Centers for Disease Control and Prevention, GA, USA; 5Comprehensive Cancer Center, Ohio State University, Columbus, OH, USA; 6Division of Epidemiology, College of Public Health, Ohio State University, Columbus, OH, USA. Background: Aberrant methylation in promoter regions of biologically relevant genes in cervical cancer, and uneven cytosine-guanine dinucleotide (CpG) distribution and clustering of pairs within the enhancer region of HPV16 and HPV18 genomes, have been reported. We examined CpG island methylation in the HPV genome and promoter regions of candidate genes of women screened for cervical cancer in Appalachian Ohio.
Methods: Methylation was measured by bisulfite sequencing using Pyrosequencing technology (Qiagen) at 6 CpG sites in the E6 promoter region, 3 sites in the enhancer region, and 1 site in the L1 region of HPV16 in 88 HPV16-positive women. DNA methylation was measured at 6 CpG sites in the promoter region of Estrogen-Receptor α (ESR1) and 5 sites in an intronic region of Deleted in Colorectal Cancer (DCC) in the HPV16-positive women and 66 HPV16-negative women. Methylation levels were compared by cervical cytology and HPV16 status.
Results: No differences in mean HPV16 E6 promoter methylation in women with normal (2.5%) and abnormal (2.4%) cytology, or in the enhancer region in women with normal (11.1%) or abnormal (11.0%) cytology, were observed. Mean methylation at the site in the HPV16 L1 region was 25.2% (range: 0-96.9%) in women with normal cytology, and 23.3% (range: 0-54.5%) in women with abnormal cytology. Mean ESR1 promoter methylation did not differ by cytology status (normal, 6.7%; abnormal, 6.6%), nor did mean methylation of an intronic region of DCC (normal, 8.5%; abnormal, 10.6%). No samples with normal cytology had DCC methylation >16%, but 6 samples with abnormal cytology (all HPV16-positive) exceeded this value (range: 17.0-70.2%).
Conclusions: In this cross-sectional study, HPV genome CpG methylation was low except for the L1 region. ESR1 methylation was low regardless of HPV or cytology status. DCC methylation was highly variable but did not correlate with HPV or cytology status.
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Davit Bzhalava
Malmö, SwedenJohanna Ekström1, Davit Bzhalava1, Daniel Svenback2, Ola Forslund1, Joakim Dillner1. 1Dept. of Laboratory Medicine, Lund University, Malmö, Sweden; 2Department of Experimental Medical Science, Lund University, Lund, Sweden. There are at least 116 completely characterized human papillomavirus (HPV) types and putative new types are continuously found. Both squamous cell carcinoma of the skin (SCC) and other skin lesions commonly contain multiple infections with many cutaneous HPV types. The objective of this study was to identify as many HPV types as possible in possibly HPV-associated lesions such as SCCs, actinic keratosis (AKs) and keratoacantomas (KAs). Patient samples from 73 SCC/AK biopsies, 92 KA formalin fixed paraffin embedded (FFPE) tissue samples and 178 SCC/AK swab samples were pooled in three different pools (Table 1) and amplified using the general HPV primers FAP followed by high throughput sequencing. We obtained 1089 non-HPV, non human DNA reads and 380 HPV reads. The greatest amount of HPV-reads were found in the pool of SCC/AK swab samples, which are known to harbour a lot of HPV (Table 2). Fifty-nine reads, of varying length, from putative new types were identified (Table 2). Comparison between the three pools results in 27 unique putatively new types and 18 additional that could also be new types based on the finding that the sequences don't overlap. The biopsies taken from SCC and AK had previously been tested by 3 different labs using cloning and sequencing after PCR and a majority of the KAs had been tested for HPV using nested PCR. Although multiple types had already been identified in these samples in about 10,000 clones that had been sequenced previously by conventional methods, many additional putative new types were detected in the present study by high throughput sequencing. In conclusion, high throughput sequencing identifies sequences of many HPV types (both known and previously unknown) in several putatively HPV-associated lesions, implying that the diversity of HPV is even greater than revealed by conventional methods.
Pool number Sample
1 Fresh frozen biopsies from 37 SCC lesions and 36 AK lesions
2 FFPE biopsies from 92 keratoachantoma lesions
3 Swab samples from the top of the lesion from 86 SCCs and 92 AKs
Table 1: Description of the pooled samples.
Table 2: HPV-reads found in MID 1 to 3
Putative new types
Known Beta Gamma Unknown species*
SCC/AK biopsies 109 10 5 4
KA FFPE tissue samples 94 1 12 3
SCC/AK swab samples 118 9 10 5
*In this category we have placed putative HPV-types closest related to HPV101, 103 and 109, which have not yet been designated a species. |
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Xiaoze Li
Uppsala, swedenXiaoze Li1, Beatrice Orru2, Monika Somberg1, Fergus Ryan2, Stefan Schwartz1. 1Department of Medical Biochemistry and Microbiology, Faculty of Medicine, Uppsala University, Uppsala, Sweden; 2DIT,Dublin, Ireland. HPV-16 late gene expression is suppressed in cervical cancer cells. This may allow the HPV-16 infected cells to escape the immune system. Identification of factors that inhibit HPV-16 late gene expression may be important in order to understand the progression to cervical cancer. Two splice sites on the HPV-16 genome are used exclusively by the late L1 mRNA: SD3632 and SA5639. These splice sites are suppressed in proliferating cells, including cervical cancer cells. To identify cis-acting RNA elements that inhibit SD3632, we constructed subgenomic HPV-16 plasmids which contained SD3632 and SA5639, but none of the early splice sites. Plasmids were driven by a CMV promoter and contained CAT report gene as surrogate for L1. The plasmids started at nt position 3395bp, and contained 238 nucleotides upstream of SD3632, while other plasmids contained various deletions in this region. Plasmids were transfected into HeLa cells and CAT levels were monitored by CAT ELISA. Our results showed that deletion of all sequences between nucleotide positions 3395 and 3628 resulted in high production of CAT compared to plasmids containing this sequence. These results demonstrated that sequences between 3395bp and 3628bp suppressed SD3632. In order to map the inhibitory elements, 5'- and 3'-deletions were introduced between nucleotide positions 3395 and 3632.The results revealed that the smallest inhibitory element was located between 3587bp and 3628bp. Fine mapping by linker-scanning mutagenesis and point mutations identified the exact inhibitory element. Experiments are in progress to identify cellular factors that bind to the suppressor of late 5'-splice site SD3632.
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David Escarcega
Cuernavaca, MéxicoDavid Escarcega2, Fernando Ramos2, Jaime Berúmen1, Oscar Vazquez1, Ana María Espinosa1, Sarah Jenna3. 1Unidad de Medicina Genómica , Hospital General de México, México, D.F., México; 2Dirección de Posgrado e Investigación, Campus Cuernavaca, Tecnológico de Monterrey, Cuernavaca, México; 3CRC en Génomique Intégrative et Signalisation Cellulaire, PharmaQAM, Montreal, QC, Canada. Background: Human papillomavirus (HPV) genes reprogram several signaling pathways functions in host cells to achieve a cancerous behaviour in the cervix epithelium. Those pathways are not completely characterize neither clarified their dysfunctional effects. Heterogeneous efforts to create properly models are needed and should rely on a multidisciplinary approach.
Objectives: To achieve an extensive characterization of several singling pathways involved with Cervical Cancer.
Methods: First, we studied effects of the human papillomavirus (HPV) in epithelial cells using genome-wide single nucleotide polymorphisms and expression microarrays screenings. Secondly, we created a computational characterization of signaling pathways using a Petri net formalism. Finally, we integrated microarray data with modelled signaling pathways and implemented different data mining techniques to discover functional patterns.
Results: We found motifs of DNA alterations, correlated with sub or over expression levels, distributed in different signaling pathways. We propose a methodology to classify the key signaling pathways to each tumour by quantitative analysis. Furthermore, we created reliable models of signaling pathways related with the Cervical Cancer progression: Notch, MAPK, Hedgehog and Wnt pathways.
Conclusions: In this multidisciplinary work, our study provides a methodology to create integrative models and discover functional similarities between pre and transcriptional phenomena to clarify signaling pathways related with HPV disruption. |
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Ana Maria Espinosa
Mexico, MexicoAna Maria Espinosa1,2, Ana Alfaro1, Icela Palma2, Cynthia Serralde1, Ingrid Medina1, Eligia Juarez1, Luis Macias1, Edna Marquez4, Edgar Roman5, Jaime Berumen1,2. 1Genomic Medicine Unit, Hospital General de México (HGM), Mexico, D.F., Mexico; 2Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico, D.F., Mexico; 3Genetics Service, HGM, Mexico, D.F., Mexico; 4IIMAS, UNAM, Mexico, D.F., Mexico; 5Oncology Service, HGM, Mexico, D.F., Mexico . There are no procedures or molecular markers to screen cervical cancer that have, at the same time, high sensitivity and specificity. Comparative analysis of global gene expression between cervical cancer and normal cervical epithelial tissue may constitute the starting point for identification of potential markers for screening cervical cancer. The present study compares global gene expression between a sample of 43 cervical cancer positive for HPV16 and 12 control tissues using the Affymetrix HG-focus chip which explored ~8,600 genes. A total of 997 genes were expressed differently between the two groups, including genes involved in cell cycle, cell adhesion, DNA repair and apoptosis among others. Twenty-three (5 down- and 18 up-regulated) of the top ranked genes were validated by quantitative RT-PCR in the same samples analyzed by microarray, as well as in 22 cervical cancer positive for other HPVs and 13 additional controls. Based on these experiments, the proteins of 9 of the 10 better genes (all up regulated), that showed the higher sensitivity and specificity, which included 4 previously reported genes (MKI67, CDKN2A, CDC2 and PCNA) and 5 uncovered genes (PRC1, CCNB2, CDC20, CDKN3 and SYCP2), were selected to be explored by immunohistochemistry in 86 samples (20 controls, 20 CIN-I, 20 CIN2/3 and 26 invasive tumors) arranged in tissue micro arrays. The signal of four of those explored antigens did not correlate with the RT-PCR data (SYCP2, CDC20, MKI67 and CDC2). PRC1 showed signals mostly in invasive samples, CCNB2, CDKN3 and PCNA showed signals in all but control samples, whereas CDKN2A showed signals only in high grade and invasive neoplasias. The utility of these genes as markers for the screening program will be discussed. |
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Patricia Murphy
Durham, USAPatricia Murphy1, Dorian Henderson1, Melissa Adams1, Elizabeth Horlick1, Douglas Malinowski1. 1BD Diagnostics, Women's Health and Cancer, Durham, NC, USA. Objectives: Neoplastic transformation induced by HPV requires expression of the virally encoded oncogenes, E6 and E7. To determine whether E6/E7 mRNA expression correlates with cervical grade, we have analyzed the level of expression of the E6 and E7 mRNA (E6/E7) for five high risk HPV (HR-HPV) types using real time RT-PCR in a panel of cervical biopsy specimens.
Methods: DNA and RNA were extracted from 59 FFPE cervical biopsy tissues using a modified MasterPure protocol from Epicentre. The sample set consisted of 14 normal, 23 CIN III, and 22 SCC samples. HPV DNA genotyping was performed using real time PCR with primers/probes specific to genomic regions of 15 HR-HPV types. HPV16, 18, 31, 33, and 45 E6 and E7 mRNA detection was performed after reverse transcription using real time RT-PCR with different and non-overlapping primers/probes than those used for genotyping.
Results: HPV genotyping results indicated that 45/59 (76%) of specimens were positive for HR-HPV DNA and 14 were HR-HPV DNA negative, two of which were high grade disease. Among the 23 CIN III samples analyzed, 18 (78%) expressed E6/E7 for the five HR-HPV types analyzed; 4 of these negative samples were infected with HR-HPV types other than the 5 types analyzed, and 1 is currently being adjudicated. Among the 22 cancers analyzed, 20 (91%) were expressing E6/E7 for the five HR-HPV types, 1 sample was infected with a different virus and 1 sample is being adjudicated. None of the 14 normal samples expressed any detectable E6/ E7 mRNA.
Conclusions: In this research study, we have demonstrated that the majority of advanced cervical neoplasia samples express E6/E7 mRNA whereas normal samples do not. |
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Anastasia Yesnik
Bloomington, USAAnastasia Yesnik1, Christi Perkins1, Jennifer Im1, Molly Rodenbeck1, Darron Brown2, Peter Ortoleva1. 1Department of Chemistry, Indiana University, Bloomington, IN, USA; 2Department of Medicine, Indiana University School of Medicine, IN, USA.
Background: Altered gene regulation is a cardinal feature of HPV infection. The objective of this study was to identify changes within the transcriptional regulatory network (TRN) in human keratinocytes upon infection and subsequent integration of oncogenic HPV.
Methods: A cell line was created from human foreskin keratinocytes grown as an HPV 59-infected foreskin xenograft in an athymic mouse. Cells were passaged on feeder cells and RNA was extracted from uninfected foreskin keratinocytes (control cells), from HPV 59-infected cells at passage 11 (episomal HPV 59) and HPV 59-infected cells 94 (integrated HPV 59). Microarray analysis of the three RNA preparations was performed. For TRN reconstruction, a database of 5,946 genes and 15,883 experimentally validated gene-transcription factor (TF) interactions were integrated into a network reconstruction system (TRND). First, a large set of RNA expression profiles were used to establish the TRN of the "normal" epithelial cell. Comparing expression data taken at three stages (uninfected keratinocytes, early passage keratinocytes, and late passage keratinocytes) with the normal TRN, we identified network abnormalities underlying transition from infection to cancer. Second, RNA expression array profiles from 76 cervical cancers (since a limited number of male HPV-related cancer profiles) were used to establish a cervical cancer TRN. Statistical analysis validated the abnormalities discerned in the first analysis (normal epithelial cells) using findings from the second analysis (cervical cancer array profiles).
Results and Conclusions: Preliminary results suggest there are regulatory abnormalities in BASP1, CPVL, EZH2, KLF2, MAFK, MMP1, PCSK2, and PCYOX1. These were identified comparing groups of similarly regulated genes from the normal epithelial cell TRN to the three keratinocyte datasets, revealing genes which deviated from group trends. Using this information, gene ontology, and validation with the second analysis, we present suggestions for specific therapeutic targets and biomarkers.
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Damayanti Das
Kolkata, IndiaDamayanti Das1, Bornali Bhattacharjee2, Laikangbam Premi1, Sharmila Sengupta1. 1Human Genetics Unit, Indian Statistical Institute, Kolkata, India; 2University of Massachusetts Medical School, Department of Molecular Genetics and Microbiology, Worcester, MA, USA. Introduction: HPV16 is the major viral isolate identified in cervical cancer (CaCx) cases in India. Other than the significant role of viral E2 gene-disruption, viral DNA load is also likely to influence the CaCx risk.
Objectives: To investigate the role of HPV16 viral load and oncogene expression in cervical cancer pathogenesis.
Methods and Materials: A case-control study (152 CaCx cases and 87 controls, all HPV16 positive) was undertaken to estimate viral load and E7 and E4 expressions were studied by APOT assay combined with quantitative Real Time PCR (TAQMAN).
Results: Median HPV16 viral load (Mann-Whitney U test) was significantly higher among cases (2.99x107) compared to controls (1.92x104), irrespective of viral E2-status (p<0.001). Logistic regression of log viral load against disease status confirmed the finding both irrespective of E2-status [ORage-adjusted=1.261; 95% CI=1.161-1.369; p<0.001] and with E2-status as a co-variate of disease-risk [ORage-adjusted=1.333; 95% CI=1.207-1.472; p<0.001]. mRNA expression was studied on a subset of 40 cancer cases. APOT cum TAQMAN analysis identified 23 intact and 17 disrupted cases based on E7 expression in both intact and disrupted while E4 expression in only intact cases. The median viral load significantly varied (p=0.011; Mann-Whitney U test) between intact (2.55x107) and disrupted (9.96x105) cases. Both E7CT/β-actinCT and E2CT/β-actinCT were negatively correlated with viral load (p=0.016 and p=0.002, respectively) among intact cases. No association was found between viral load and E7CT/β-actinCT within disrupted cases. A significant difference (p<0.001; t-test) was observed in the mean E7CT between intact (6.69 ± 8.94) and disrupted (25.69 ± 14.31) cases, but no such difference was present in β-actin expression.
Conclusion: Thus association of high viral load with HPV16 positive cases, specifically those having intact E2, and higher E7 expression among E2-intact cases provide novel insights into disease pathogenesis.
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Laura Sichero
São Paulo, BrazilLaura Sichero1, João Simão Sobrinho1, Luisa Lina Villa1. 1Department of Virology, Ludwig Institute for Cancer Research, São Paulo, Brazil. The long control region (LCR) of human papillomavirus contains sequences important for the regulation of viral replication and transcription of early genes. Nucleotide variability among HPV molecular variants can be as high as 5% in the LCR. Some transcription factors (TFs) have been shown to bind to the LCR and modulate viral transcription. In order to identify additional TFs that might be involved in the regulation of E6/E7 expression, a high-throughput assay based on reverse transfection of 704 transfection ready cDNA plasmids covering all commonly know transcription factors was used. Briefly, the Transcription Factors GFC-Transfection Array (Origene, Madison, EUA) was tested for the regulation of a reporter plasmid containing the complete HPV-18 LCR driving the expression of luciferase. Three independent experiments were conducted in C33 cells. Of the 704 genes tested, twenty eight activated and thirty six inhibited HPV-18 LCR by more than 2 folds. TFs of each group were chosen for further validation. For this purpose, C33 cells were co-transfected with expression plasmids of the TFs under study in addition to recombinant vectors LCR-Luciferase of different variants of HPVs 16 and 18. Triplicates were carried out in five independent experiments. Most results obtained in the screening were confirmed in validation experiments. We observed some differences in activation or repression between prototypes and molecular variants of HPVs 16 and 18. Among the TFs identified which could potentially interfere with the regulation of HPV expression, only GATA3, p53 and myb have putative binding site within the LCR sequence as indicated using the TRANSFAC database. In conclusion, we identified several TFs not previously implicated in the regulation of HPV gene expression. Many of these factors have also been described to be mutated in cancer or are putative cancer biomarkers, and should be target for further studies.
Financial support: Fapesp 07/58590-7 |
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Mona Hansen
Lørenskog, NorwayMona Hansen1, Christine M. Jonassen1, G. Cecilie Alfsen2. 1Section for Genetechnology, Centre for laboratory medicine, Akershus University Hospital, Norway ; 2Department for Pathology, Centre for laboratory medicine, Akershus University Hospital, Norway. The aim of the study was to compare the differential gene expressions of HPV oncogenes (E6, E6* and E7) in lasermicrodissected areas of CIN1, CIN2, CIN3 and cervical cancer from cervical biopsies and cones from patients with proven HPV16 infection.
Samples from a total of 66 patients were evaluated. Liquid based cytology samples from 3 - 6 months prior to biopsy were positive for HPV 16 E6/E7 gene expression, proven with the PreTect HPV Proofer test from Norchip.
8 µm sections from formalin fixed and paraffin embedded samples (16 CIN1, 22 CIN2, 22 CIN3, 5 squamous cell carcinomas, and 1 adenocarcinoma) were dried overnight at 42°C and stained with hematoxylin and eosin, before lasermicrodissection of pathologic areas. RNA was extracted using the Stratagene Absolutely FFPE kit, with some minor adjustments from the protocol. cDNA-synthesis was performed using random hexamers and oligo(T) for priming. In order to increase the yield of mRNA expression, a pre-amlification step of 14 cycles was performed using the ABI PreAmp kit, prior to real-time amplification using ABI Gene Expression mastermix on a 7500 ABI BioPrism real-time PCR instrument. In order to normalize oncogene expression, two endogenous controls, ACTB and GAPDH were monitored.
Our data indicate that the level of HPV oncogene E6 expression is positively correlated to the histologic grade of dysplasia in cervical intraepithelial neoplasia.
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Lorenz Thomas
Heidelberg, GermanyLorenz Thomas1, Arkom Chaiwongkot1, Miriam Reuschenbach1, Friedrich Kommoss2, Dietmar Schmidt2, Magnus von Knebel Doeberitz1, Svetlana Vinokurova1. 1Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, Heidelberg, Germany; 2Institute of Pathology, A2, 2 Mannheim, Germany. Objectives: The life cycle of HPV is coupled to the cellular differentiation program of the squamous epithelial host cells. In the high-risk HPV types, the E6 and E7 oncogenes are expressed from a bicistronic mRNA initiated in the viral early promoter located just upstream of the E6 open reading frame (ORF). The early promoter is activated prior to viral genome replication. Activation of the differentiation dependent late promoter within the E7 ORF occurs in the more differentiated epithelial cell layers. Several additional minor promoters within the E6 ORF have been identified that may also play important roles during the viral life. Methylation of the viral genome may affect viral gene expression and promoter usage. We therefore aim to analyze the methylation pattern of HPV16 within E6 and E7 ORFs during epithelial differentiation.
Methods: The methylation pattern of the promoters located in E6 (p480, p542) and E7 (late p670 promoter) ORFs in epithelial cells with different degrees of differentiation was analyzed. DNA was isolated from micro-dissected basal, intermediate, and superficial cell layers of low-grade intraepithelial lesions and subjected for methylation analysis by bisulfite genomic sequencing technique.
Results: In low grade lesions, the E6 ORF promoter regions in the basal or intermediate cell layers contained unmethylated CpGs. In contrast, in the superficial cells most of the CpG dinucleotides were methylated including the potential E2 and SP1 binding sites. In contrast, the CpGs located within the late p670 promoter region were methylated in basal cells but showed less methylation in more differentiated cells.
Conclusions: These observations suggest that methylation of distinct CpG dinucleotides within the HPV 16 E6 and E7 ORFs is substantially changed during squamous epithelial differentiation of the host cells. Differentiation dependent changes of the HPV methylation correlate with the viral gene expression pattern in cells of the respective differentiation status.
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Alfredo Amador-Molina
Mexico, MexicoAlfredo Amador-Molina1,2, Alejandro Mohar2, Adela Carrillo-Garcia2, José Luis González-Montoya2, Marcela Lizano2. 1National Autonomus University of Mexico, UNAM, Mexico City, DF, Mexico; 2Unit of Biomedical Research in Cancer, National Cancer Institute of Mexico/IIBM, UNAM, Mexico City, DF, Mexico . Introduction: A persistent infection of HPV is the main etiologic factor of cervical cancer development. It has been proposed that HPV intra-type variations may rebound in differences in biological behavior. In Mexico, we have previously isolated three different HPV18 variants (AsAi, E, and Af). These isolates have been associated to specific histological types of cervical cancer harboring differences in relative prognosis. E1 viral protein plays a critical role in regulation of viral replication and viral load; this protein requires the E2 viral regulator to be stabilized on the long control region (LCR).
Objective: To determine whether variations in the LCR, E1 and E2 genes affect viral replication.
Methods: PCR products from E1 and E2 variant genes were obtained from tumors containing HPV18, and cloned in expression vectors. Transient transfections were done with E1, E2 and LCR in HEK-293 cell line, and expression was measured through RT-PCR, 72 hours post-transfection. Quantitative analyses of LCR plasmid replication were performed through real time-PCR.
Results: Individual analysis of each element showed that changes in the LCR and E1 gene had high impact on plasmid replication. In particular, LCR changes of European and African variants significantly decreased replication compared to the Asian-American variant. However, changes in the African E1 variant showed greater replication rate compared with other E1 variants. Nevertheless, when analyzing the three elements specific for each variant altogether, a higher replication rate for the African variant was found, compared to the other HPV18 variants.
Conclusions: Variations in HPV18 may rebound in viral replication. However, we suggest that viral load is not necessarily a risk factor for cervical cancer progression, as we find a higher replication rate for Af-HPV18, which has been associated with a better prognosis in HPV18 infections.
Partially supported by CONACYT grant 10003/60722 |
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Chun Fa Zhang
Clarksburg, USAShu Min Duan1, Gui Yu Wang2, Wei Fan Yang2, Chun Fa Zhang3. 1Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China; 2SR BioPharma Inc, Haikou, China; 3SR-BioExpress Inc, MD, USA. Background: High-risk human papillomaviruses (HPVs) are recognized cause for cervical cancer. Currently there is no effective therapy for cervical cancer and other HPV infections.
Objectives: We have investigated the antiviral activity of REBACIN, a plant-derived factor.
Methods: We examined the inhibitory effect of REBACIN on expression of E6 and E7 genes, the two oncogenes necessitated for HPV-induced malignancy. For this purpose, TC-1 and Hela cells in culture were used, which express E6/E7 genes of HPV16 and HPV18, respectively. We further examined the suppressive effect of REBACIN on the growth of tumor induced by HPV16 and HPV18 infection in a mouse model. TC-1 or Hela cells pre-treated with different doses of REBACIN were used to induce tumor in mouse followed by a 28-day regimen that includes three sequential subcutaneous injections of REBACIN at various doses. The isolated tumors were weighed and compared with the one without treatment. In addition, the activity of REBACIN against HSV-II virus-induced vaginitis was also examined.
Results: REBACIN effectively inhibited the E6 and E7 gene expression in a dose-dependent manner in both cell lines. At 0.25 µg/ml in the absence of cellular toxicity, the E6 and E7 gene expression were drastically repressed as assayed by RT-PCR (Fig.1.). In mouse, REBACIN reduced the weight of HPV16-, and HPV-18-induced tumor by up to 91.84% (Fig.2.) and 83.75%, respectively (p<0.05). Also, in a 6-day course of treatment of HSV-II-induced vaginitis in mouse, the efficacy of REBACIN reached 93.3% in contrast to 73.2% by Aciclovir as a positive control. The negative rate in REBACIN-treated group was 77% compared to 70% by Aciclovir.
Conclusion: REBACIN is a novel effective anti-HPV compound that is able to suppress E6 and E7 gene expression, and suppress tumors induced by HPV16 and HPV18 in vivo.
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Mohd Israr
Hershey, USAMohd Israr1, Danielle Mitchel1, Samina Alam1, Donald Dinello2, Joseph Kishel3, Craig Meyers1. 1Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, PA, USA; 2Oral Surgery Associates, Hershey, PA, USA; 3Department of Pharmacology, The Pennsylvania State University College of Medicine, Hershey, PA, USA. Human immunodeficiency virus positive patients taking antiretroviral drugs especially protease inhibitors have shown a significant increase in the development of oral complications including the emergence of HPV positive lesions. However, the effect of these drugs on the growth, and the life cycle of HPVs in oral epithelial tissues have not been widely studied. We have initiated studies to analyze the effect of the protease inhibitors Amprenavir and Lopinavir/Ritonavir on the growth of gingival epithelium using organotypic (raft) culture. Our results demonstrate that both Amprenavir and Lopinavir/Ritonavir severely inhibited the growth of gingival tissue when the drugs were present throughout the growth period. TEM observations revealed that tissue integrity of desmosomes was compromised in Lopinavir/Ritonavir treated gingival tissues. Further, Amprenavir and Lopinavir/Ritonavir treatments deregulated the proliferation and differentiation of gingival epithelium as determined from the expression patterns of cytokeratins 5, 14, 10, 6, PCNA and cyclin A.
HPV 16 continuously infected gingival and tonsil cell lines were developed. Gingival as well as tonsil tissues containing HPV16 were shown to produce infectious virus. Hematoxylin and Eosin staining was performed to examine the stratification and differentiation of these lines in raft culture. HPV infection was shown to alter the stratification and differentiation of gingival as well as tonsil epithelium. Further, our study showed that Amprenavir decreased infectivity of HPV16 in a concentration dependent manner. Experiments are underway to determine the effect of Lopinavir/Ritonavir on HPV16 infectivity.
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Tom Broker
Department of Biochemistry & Molecular Genetics, University of alabama at Birmingham
Birmingham, USAN. Sanjib Banerjee1, Hsu-Kun Wang1, Thomas R. Broker1, Louise T. Chow1. 1Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, USA. The productive program of human papillomaviruses occurs in differentiated squamous keratinocytes. We have previously shown that vegetative amplification of the HPV-18 genomic plasmid initiates in G2-arrested spinous cells in organotypic raft cultures of primary human keratinocytes (PHKs), as marked by their abundant cytoplasmic cyclin B1 (Wang et al., 2009. Genes Dev. 23: 181-194). We now demonstrate that the E7 protein, which induces S phase reentry in suprabasal cells, also induces cellular environments conducive for prolonged G2 phase. Western blots and indirect immunofluorescence assays were used to probe for host proteins known to control G2/M progression. E7 expression induced cytoplasmic cyclin B1 and cdc2 in the suprabasal cells. In addition, there were elevated Wee1 and Myt1 proteins that are responsible for inhibitory phosphorylation of cdc2 on Threonine 14 and Tyrosine 15. Indeed, the elevated cdc2 had inactivating phosphorylation on T14 or Y15, and possibly both. Moreover, in cells harboring cytoplasmic cyclin B1 or cdc2, there was an accumulation of elevated cdc25C phosphorylated on Serine 216, an inactive form unable to dephosphorylate and activate cdc2. Taken together, this study has revealed the mechanisms by which E7 induces prolonged G2 phase in the differentiated cells following S phase induction to enable viral DNA amplification. Similar results were observed in PHK raft cultures in which the fully productive life cycle of HPV-18 took place.
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Alma Mariana Fuentes
Unit of Biomedical Research in Cancer, National Cancer Institute and Biomedical Research Institute, National Autonomous University of Mexico
México, MéxicoAlma Mariana Fuentes1,2, Joaquin Manzo1,2, Adriana Contreras1, Marcela Lizano1,2. 1Unit of Biomedical Research in Cancer, National Cancer Institute, Mexico City, México; 2Biomedical Research Institute, National Autonomous University of Mexico, Mexico City, México. The most important risk factor for cervical cancer development is the persistent infection with human papillomavirus (HPV). In virtually all viral types analyzed, intratypic genomic variants can be detected. It is considered that HPV intratypic variations may influence its oncogenic potential. Through sequence analysis of HPV18 LCR three major phylogenetic branches, including European (E), African (Af) and Asian-Amerindian (AsAi), have been identified, the latter being the HPV18 prototype cloned.
E2 viral protein plays a critical role in the cycle of human papillomavirus because it has the ability to regulate both viral DNA replication and transcription of the E6 and E7 viral oncogenes. E2 is associated with induction of cell cycle arrest and apoptosis. Cell cycle arrest has been described in carcinoma cells specifically associated with HPV and it has been suggested that it occurs through transcriptional repression of E6 and E7 oncogenes. The transactivation and DNA binding domains of the E2 protein appear to be necessary for carrying out this cellular event. Little has been studied about the effect of E2 in apoptotic pathways. It has been linked with activation of caspases, specifically caspase 8, involving the participation of the extrinsic pathway in Saos-2 cells. However, events involving cellular effects on signal transduction are not yet elucidated.
Our aim is to determine which elements of the apoptosis signaling pathways are modulated by HPV18 E2 variant proteins. Cells negative for HPV (C33, HEK293, MCF7), as well as an HPV positive cell line (Hela), were transiently transfected with HPV18 E2 genes. Differences in proliferative rates were observed by crystal violet assays and flow cytometry. A decreased proliferative rate was observed for cells transfected with the African E2 variant gene, which also showed an increment in apoptosis, as demonstrated by TUNEL assay.
This work was partially financed by CONACYT Grant: I0003/60722 |
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Caroline Demeret
Paris, FranceManel Boulabiar1,2, Emna Ennaifer2, Med Samir Boubaker2, Michel Favre1, Caroline Demeret1. 1Genetics, Papillomavirus and Human Cancer Unit, Institut Pasteur, Paris, France; 2Human Papillomavirus Unit, Institut Pasteur, Tunis, Tunisia. NFkB signaling pathway is central in the control of many cell processes and of immune response to infection. Accordingly, many viral pathogens have evolved strategies to modulate the NFKB pathway to enhance viral replication, host cell survival, and evasion of immune responses. Persistent activation of the NFkB pathway probably contributes to oncogenic transformation by HPV, as it was found in high-grade squamous intraepithelial lesions and squamous cell carcinomas of the uterine cervix. Surprisingly, little is known about the regulation of NFkB pathway during the natural history of HPV infection. Here we show that expression of the E2 regulatory protein of mucosal and cutaneous HPV potentiates TNF-induced activation of NFkB in HaCaT keratinocytes. The E2 proteins from a series of 12 HPV genotypes are able to enhance TNF-induced NFkB activation, albeit to different extent, independently of HPV tropism and pathogenic potential. The N-terminal domain of E2 is responsible for this effect, while its transcriptional activation capacities are not involved. HPV16 and 18 E2 proteins have been shown to interact with Tax 1BP1, which is a negative regulator of NFkB signaling. Using a ''protein fragment complementation assay'' based on luciferase activity, we determined that all E2 but one (HPV-32 E2) efficiently interacted with Tax1BP1. However, Si-RNA mediated knock-down of Tax1BP1 did not relieve the enhancement of TNF-induced NFkB by E2, indicating the lack of correlation with E2 binding to Tax1-BP1. Since ubiquitination of signaling molecules, such as the TRAF proteins, is a common scheme in NFkB signaling, we are currently investigating whether the ubiquitination of NFkB signal transducers is affected in the presence of E2. Stimulation of TNF-induced NFkB activation by E2 unravels a novel aspect of the role of E2, which probably exploits the NFkB pathway to facilitate viral replication within the infected epithelium.
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Helena Faust
Malmö, SwedenHelena Faust1, Christopher Buck2, Diana Pastrana2, Joakim Dillner1, Johanna Ekström1. 1Department of Laboratory Medicine, Medical Microbiology, Malmö University Hospital, Lund University, Malmö, Sweden; 2Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, MD, USA.
Merkel cell polyomavirus (MCV) is associated with Merkel cell carcinoma (MCC). We previously reported a high throughput HPV serologic method based on pseudovirions bound to Luminex beads using heparin that retained native conformation of antigen and had improved sensitivity and specificity (Faust et al; Abstract 25th IPV).
We wished to investigate whether the same system could be used also for other tumor viruses like MCV. Serum antibodies to heparin-bound MCV pseudovirions and JCV virus like particles (VLP) were assayed using our multiplexed fluorescent heparin-coated bead-based assay. We tested 435 patients with squamous cell carcinoma (SCC), basal cell carcinoma (BCC), actinic keratosis (AK) or benign skin lesions. MCV DNA was assayed in the biopsies with real-time PCR. 18% of patients were MCV DNA positive and 67% of the study population was MCV seropositive. The sensitivity of the serology assay to detect patients positive for MCV DNA was high (97%) and seropositivity correlated strongly with DNA positivity (OR: 25.8 (95% CI=6.1-154.5). Antibodies to JCV were found in 66% of subjects but did not correlate with MCV DNA (OR: 1.28 (95% CI=0.73-2.24). The results obtained with pseudovirion-heparin-Luminex correlated well with an MCV neutralization assay. MCV DNA prevalence in biopsy or MCV seropositivity did not correlate with any of the studied diagnosis. In conclusion, a single-tube multiplexed heparin-Luminex can simultaneously detect antibodies against both HPVs and MCV.
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Helena Faust
Malmö, SwedenHelena Faust1, Joakim Dillner1. 1Department of Laboratory Medicine, Medical Microbiology, Malmö University Hospital, Lund University, Malmö, Sweden.
Prophylactic vaccines against Human Papillomavirus (HPV) based on Virus-Like Particles (VLPs) induce type-specific neutralizing antibodies. The major capsid protein L1 is highly conserved between HPV types, with a small number of hypervariable residues positioned in surface-exposed loops. To investigate the importance of these residues for the immunogenicity of HPV 16 and the L1 interaction with minor capsid protein L2, ten surface-exposed amino acid residues in 4 hypervariable loops of L1 were mutated. VLPs containing mutated or wild type L1 with or without L2 were produced in 293TT cells using pseudovirion expression vectors. The mutations reduced, but did not eliminate, the ability to bind neutralizing monoclonal antibodies and to induce neutralizing antibodies and cytotoxic T- lymphocyte responses. Cross-neutralization was not detected. The mutations in the L1 loops caused a strikingly decreased incorporation of L2 protein and a complete inability to encapsidate pseudogenomes, suggesting that intact L1 loops are important for the interaction of L1 with L2 in the HPV particle.
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Xuefeng Liu
Wahsington, USASawali Sudarshan1, Xuefeng Liu1, Richard Schlegel1. 1Department of Pathology, Georgetown University Medical School, Washington, DC, USA. We analyzed changes in cellular gene expression that were induced by the ER-resident human papillomavirus type 16 (HPV-16) E5 oncoprotein in primary human foreskin keratinocytes (HFKs). Our studies identified the downregulation of spliced XBP-1, a key player in the ER-stress response, as a biochemical marker of E5 expression. IRE1a, the endoribonuclease responsible for XBP-1 cleavage, was also downregulated. Furthermore, microarray analysis revealed that COX-2, another member of the ER-stress pathway, was downregulated. In contrast, low-risk HPV-6b E5 or a C-terminal 16E5 mutant (where conserved high-risk histidine and alanine residues were replaced with low-risk tyrosine and isoleucine residues) failed to alter the levels of COX-2 and XBP-1 mRNA. Since our preliminary data showed induction of XBP-1 splicing after E6 and E7 expression, we hypothesized that 16E5 might play a protective role against the host response to viral infection. Supporting this, we found that HFKs co-expressing E6, E7 and E5 had lower COX-2 levels than cells expressing E6 and E7 alone. COX-2 levels in E6/E7-transduced cells were also suppressed when compared to vector, suggesting that multiple pathways of COX-2 repression may exist. COX-2 downregulation by E5 can be overcome by thapsigargin or tunicamycin treatments, which initiate ER-stress via calcium fluxes and abnormal protein glycosylation respectively, making it unlikely that E5 is specifically tempering these pathways. Our data, therefore, suggest a potential role for E5 in repressing the cellular ER-stress response following HPV infection.
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Joaquin Manzo
Mexico city, MexicoJoaquin Manzo1,2, Alma M. Fuentes1,2, Jose F. Hernandez1, Adriana Contreras1, Marcela Lizano1,2. 1Unit of Biomedical Research in Cancer, National Cancer Institute, Mexico; 2Biomedical Research Institute, National Autonomous University of Mexico, Mexico.
Human cervical cancer is caused by high-risk types of human papillomavirus (HPV) such as HPV16 and HPV18, whose concurrent expression of E6 and E7 is a prerequisite for cancer development and maintaining malignant phenotypes.
E6 and E7 proteins from high risk HPV are considered the major oncogenic stimulus; these proteins bind a large range of cellular proteins, interfering with their functions, being p53 and pRb the most important.
The HPV E6 gene has the potential to encode a full-length protein as well as a truncated isoform (E6*I) by alternative splicing. Even when putative E6*I protein is suspected to be functionless, it has been demonstrated that E6*I can inhibit the p53 proteasome mediated degradation by binding to the E6 full-length protein. Nevertheless the E6*I function per se is not completely known, particularly concerning it’s activity on cellular proteins. Our work focuses on functions and interactions that E6*I exerts in the absence of full-length E6. In this work we show that HPV18 E6*I can inhibit cellular proliferation independently of its interaction with E6 full-length. Cresyl violet assays and flow citometry demonstrate that stably transfected MCF-7 cells and transiently transfected HaCaT cells carrying E6*I significantly decrease their proliferative rate at 48 hours post-transfection. We were also able to demonstrate by TUNEL assay at 24 and 48 hours post-transfection, that E6*I induce apoptosis in cells carrying this gene. Actually we are working on determining the cellular elements implicated on this effects.
Work partially supported by CONACyT MEXICO. Grant: 10003/60722
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Peter Hillemanns
Hannover, GermanyPeter Hillemanns1, Victoria Lange1, Ralf Hass1, Kristine Rose2, Jennifer Philips2,3, Marija Hillemanns4, Tom Evans3. 1Department of Obstetrics and Gynecology, Medical University Hannover, Hannover, Germany; 2Biomarker Development, Novartis Pharmaceuticals, East Hanover, NJ, USA; 33Translational Medicine/Infectious Diseases, Novartis Institutes for BioMedical Research, Inc., Cambridge, USA; 4Institute of Pathology, Medical University Hannover, Hannover, Germany. Persisting high-risk HPV infection may lead to cervical intraepithelial neoplasia (CIN) that can progress to invasive malignancy, whereby early viral oncogene products including the E6 protein play a major role in the pathogenesis of HPV-mediated cervix carcinoma development. This HPV E6 gene product is thought to mediate its cellular effects predominantly by interaction with the host factor E6-Associated Protein (E6-AP). An exploratory clinical investigation was performed in a cross-sectional population of women to evaluate E6-AP mRNA expression levels in different CIN stages (CIN 1 - CIN 3) and during the menstrual cycle. Thus, 38 premenopausal women between 18 and 45 years old were referred to the colposcopy clinic to undergo gynecologic examination. The recruited patients were evaluated for the following parameters, respectively:
- histopathology of cervical punch biopsies
- cytology processed using the ThinPrep® Pap test
- HPV DNA analysis by hybrid capture assay to discriminate low and high risk HPV status
- expression of the cyclin-dependent kinase inhibitor p16Ink4a by immunohistochemistry
- analysis of appropriate serum hormones by ELISA to discriminate the menstrual status
- expression of E6-AP mRNA levels in cervical smears and cervical biopsies by RT-PCR and comparison to cytology and histopathology
The results of the cytological and histopathological examinations of the tissue specimen corresponded with the p16Ink4a tissue expression. Moreover, the data demonstrated little if any influence of the menstrual cycle stage on E6-AP mRNA expression. Whereby E6-AP expression was detectable in all RNA samples isolated from PAP smears and cervical biopsies, E6-AP mRNA levels appear to be independent of the dysplasia stage. Furthermore, the normalized RNA levels from PAP smears were approximately 10-fold higher as compared to the appropriate biopsies. Together, these findings provide a favorable prerequisite for a specific molecular target in cervical cancer and other HPV-mediated neoplastic diseases using an E6-AP siRNA therapeutic approach. |
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Geneviève Morin
Montreal, CanadaGeneviève Morin1,2, Paola Di Lello2, Amélie Fradet-Turcotte1,2, James G. Omichinski2, Jacques Archambault1,2. 1Laboratory of Molecular Virology, Institut de Recherches Cliniques de Montréal (IRCM), Montreal, QC, Canada; 2Department of Biochemistry, Faculty of Medicine, University of Montreal, Montreal, QC, Canada . The E1 helicase is essential for replication of the papillomavirus genome. E1 binds to the viral origin where it assembles into hexamers that unwind DNA and interact with host factors at the replication fork. The origin-binding domain of E1, located in the middle of the protein, and its C-terminal helicase domain are necessary and sufficient to support DNA replication in vitro. In contrast, the N-terminal region (NTR) of E1 is only required in vivo, suggesting that it has a regulatory function. The first 40 amino acids of the E1 NTR interact with the cellular protein p80 to promote efficient viral DNA replication and maintenance of the genome in keratinocytes. In this study, we show that a region located immediately C-terminal of the p80-binding site has sequence similarity to the second p53 transactivation domain (TAD2). Accordingly, the E1 NTR from several HPV types could activate transcription in yeast when fused to the LexA DNA-binding domain. In all cases, E1 transactivation activity could be abrogated by mutation of three conserved hydrophobic amino acids that correspond to those important for the interaction of p53 TAD2 with its target proteins. As previously reported for p53, transactivation by E1 in yeast was found to require the SAGA histone acetyltransferase complex. Another important target of p53 is the Tfb1/p62 (yeast/human) subunit of the general transcription factor TFIIH. By NMR and ITC, we showed that the E1 TAD can also bind Tfb1/p62, on the same surface as p53 TAD2 and with a comparable affinity. Finally, we determined that mutation of the three residues in E1 that abrogate its transactivation activity also reduce by 50% its ability to support transient DNA replication in C33A cells. These results demonstrate the existence of a conserved TAD-like region in the E1 NTR that is required for efficient viral DNA replication.
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Junona Moroianu
Chestnut Hill, USAZachary Piccioli1, Anna Leszczynski1, Erin Hannah1, Courtney McKee1, Shahan Mamoor1, Patrick McManus1, Junona Moroianu1. 1Biology Department, Boston College, Chestnut Hill, MA, USA. In this study we investigated the nuclear import pathway for the low risk HPV11 E7 protein using both 1) transfection assays in HeLa cells with EGFP-fusion plasmids containing 11E7 wild type and its domains and 2) nuclear import assays in digitonin-permeabilized HeLa cells with GST-fusion proteins containing 11E7 and its domains. The EGFP-11E7, EGFP-11nE71-38 and EGFP-11cE739-98 localize mostly to the nucleus in HeLa cells. Both the GST-11E7 and GST-11cE739-98 proteins were imported into the nuclei of digitonin-permeabilized HeLa cells in the presence of only Ran-GDP and in the absence of nuclear import receptors. Moreover, the RanG19V-GTP mutant, deficient in GTP hydrolysis, could substitute for Ran-GDP in nuclear import of GST-11E7 and GST-11cE7. These data suggest that 11E7 enters the nucleus via a Ran-dependent pathway and independent of nuclear import receptors, and this pathway is mediated by a nuclear localization signal located in its C-terminal domain (cNLS). This cNLS contains the zinc-binding domain consisting of two copies of Cys-X-X-Cys motif separated by 29 amino acids. Mutagenesis of Cys residues in each Cys-X-X-Cys copy changed the localization of the resultant EGFP-11cE7 mutants to mostly cytoplasmic and inhibited the nuclear import of the GST-11cE7 mutants in digitonin-permeabilized cells. These data suggest that the zinc-binding domain is essential for the import activity of the cNLS and nuclear localization of 11cE7 and 11E7. Binding assays show that 11E7 and its cNLS interact with Phenyl-Sepharose in a similar manner as the nuclear import receptors, suggesting that 11E7 may enter the nucleus via direct interactions of its cNLS with FG-containing nucleoporins. In agreement with this model, Kap β1 and Kap β2 import receptors competed with 11E7's cNLS in nuclear import assays and inhibited its nuclear import. Ongoing experiments focus on determining other critical amino acids in the 11E7's cNLS required for nuclear import and analysis of its interactions with FG-containing nucleoporins.
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Ewa Krawczyk
Washington, USAFrank A. Suprynowicz1, Ewa Krawczyk1, Jess Hebert1, Vera Simic1, Christopher M. Kamonjoh1, Richard Schlegel1. 1Department of Pathology, Georgetown University Medical School, Washington DC, USA. It is widely thought that HPV-16 E5 enhances acute, ligand-dependent activation of the epidermal growth factor receptor (EGFR) by binding to the 16 kDa "c" subunit of the V-ATPase proton pump (16K), thereby inhibiting V-ATPase function necessary for endosome acidification and EGFR inactivation. Using an antibody we generated that recognizes endogenous (non-epitope tagged) 16K, we show that E5 associates with 16K only when both proteins are highly over-expressed. Nevertheless, the acidification of endosomes containing a pH-sensitive fluorescent EGF conjugate is inhibited in E5-expressing cells. E5 does not alter the cellular EGF binding capacity, number of EGFRs on the cell surface or initial endocytosis of pre-bound EGF; however, the subsequent fusion of EGF-containing endosomes with acidic cellular compartments is inhibited. This E5-dependent defect in endocytic membrane trafficking (transport and/or fusion) is not simply the result of disrupted actin, tubulin, vimentin or cytokeratin filaments.
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Yuki Inagawa
Chuoku, JapanYuki Inagawa1, Mako Narisawa-Saito1, Takashi Yugawa1, Yuki Kakuta-Yoshimatsu1, Nagayasu Egawa1, Shin-ichi Ohno1, Kei Haga1, Masatoshi Fujita1, Tohru Kiyono1. 1Virology Division, National Cancer Center Research Institute, Tokyo, Japan. The genital human papillomaviruses (HPVs) are classified as either high-risk (HR) or low-risk (LR) types according to the propensity for malignant progression of the lesions. The two viral proteins, E6 and E7, are consistently expressed in HPV-positive cervical cancers, and believed to be responsible for carcinogenesis. Although the oncogenic activities of E6 and E7 from HPV 16 and 18 have been well-documented, those of other HPV types are less characterized. Previous studies indicating that E6 and E7 expression alone is not sufficient to induce cervical cancer, and additional genetic alterations, namely Hras mutation, are required for the tumorigenic ability of the normal human cervical keratinocytes (HCKs). To compare the oncogenic functions of E6 and E7 proteins from genital HPVs, we transduced these E6 and E7 genes to HCKs bearing the doxycyclin-inducible Hras gene with activating mutation (Hras G12V).
Without HrasG12V induction, decreased levels of p53 were observed in HR-HPV E6 and E7 expressing cells. Up-regulation of the E2F-regulated gene products, MCM7, was evident in post-confluent cultures of HR-HPV E6 and E7 expressing cells. We also observed decreased levels of PDZ domain-containing proteins such as MAGI-1 in HR-HPV E6 expressing cells and enhancement of telomerase activity in HR-HPV E6 and E7 expressing cells. Furthermore, HR-, but not LR-HPV E6 and E7 expressing cells exhibited tumorigenicity with HrasG12V expression.
Taken together, our experimental system could evaluate the biological activities of HR- and LR-HPV E6 and E7, and the results suggest that most of the HR-HPV E6 and E7 proteins conserve the four biological activities; inactivation of the p53 and pRB pathway, enhancement of telomerase activity and degradation of PDZ-domain containing proteins such as MAGI-1. These functions could cooperatively play important roles in HPV-induced tumorigenesis. |
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Michelle A. Ozbun
Professor of Molecular Genetics and Microbiology, Molecular Genetics and Microbiology, University of New Mexico
Albuquerque, USAKelly J. Higgins1, Lanlan Wei1, Michelle A. Ozbun1. 1Department of Molecular Genetics and Microbiology, The University of New Mexico School of Medicine, Albuquerque, NM, USA. Human papillomavirus (HPV) infections have been postulated as causative agents of breast cancer. The data are contentious, but some studies show HPV DNA in significant percentages of breast cancer and mammillar samples. Although HPV16 E6 and E7 can immortalize normal human mammary epithelial cells (HMEC) in vitro, it has been unclear as to whether HPVs can naturally infect breast epithelium and thereby promote transformation. As HPVs demonstrate a strict tropism for differentiating squamous epithelium, we would not expect HPV infections of HMEC to be productive. Therefore, we set out to determine whether HPV virions could infect HMEC and how long viral gene expression and viral genomes could persist in HMEC. We find that HPV16 and HPV11 virions are capable of infecting HMEC lines, MCF10A and 76N+tert. HPV16 viral genomes persist and are expressed 9-14 population doublings. However, HPV11, the type found in a majority of breast cancers in some studies, was maintained in the HMECs for over 54 population doublings. We are in the process of investigating the specific mechanisms whereby HPV16 and HPV11 can infect HMEC lines and primary HMECs. An additional goal is to determine whether viral genomes can be persistently maintained in primary HMEC, either episomally or via integration, and if a growth advantage is bestowed upon such cells. We plan to determine if the infections induce signals and proliferative activities that might promote mammary epithelial cell transformation.
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Michelle A. Ozbun
Professor of Molecular Genetics and Microbiology, Molecular Genetics and Microbiology, University of New Mexico
Albuquerque, USASonya Flores1, Anastacia M. Maldonado1, Zurab Surviladze1, Michelle A. Ozbun1. 1Department of Molecular Genetics and Microbiology, The University of New Mexico School of Medicine, Albuquerque, NM, USA. Human papillomavirus (HPV) types 16 and 31 cause cervical cancers at different incidences and are differentially prevalent in the population. Our lab has shown that these two related oncogenic HPV types differentially interact with normal host human keratinocytes and utilize disparate early entry pathways. Both HPV types induce filopodia at the exposed cell membrane and both require endosomal acidification for uncoating. Yet, HPV31 infection is heparan sulfate proteoglycan (HSPG) independent whereas HPV16 relies on HSPG for infection, and viral entry kinetics are vastly different for the two viruses. Further, HPV31 uses a caveolin-1 and lipid raft dependent endocytosis route whereas HPV16 endocytosis is caveolae- and lipid raft independent (Smith et al. 07, JVI 81:992). Recent data from our lab suggests that the two viruses induce different signaling cascades at the keratinocyte plasma membrane. To determine the viral basis for these disparate cellular interactions, we are investigating capsid features that dictate the early entry events. HPV virions contain only two known viral proteins, the major capsid protein L1 that self assembles into capsid structures, and the minor capsid protein L2 that is essential for infection. Although L1 proteins are closely conserved between HPV16 and HPV31, we hypothesize that L1 dictates the main differences in attachment, signaling, entry kinetics, and infection routes used by these two HPV types. To examine these differences, we created chimeric pseudovirions (PsVs) composed of HPV16 L1 and HPV31 L2, and the counterpart PsVs made of HPV31 L1 and HPV16 L2. Our analyses in human keratinocytes consist of visual analyses of fluor-labeled virion binding and trafficking with confocal microscopy and infectivity assays for reporter gene expression, which indicate successful binding, entry, trafficking, uncoating, and transcription. We are in the process of testing binding, signaling, entry routes and kinetics. Results will be presented.
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Michelle A. Ozbun
Professor of Molecular Genetics and Microbiology, Molecular Genetics and Microbiology, University of New Mexico
Albuquerque, USAMichael P. Kivitz1, Agnieszka Dziduszko1, Michelle A. Ozbun1. 1Department of Molecular Genetics and Microbiology, The University of New Mexico School of Medicine, Albuquerque, NM, USA. Epithelial wounding and access to basal cells, thought to be the only permissive cells for early virus replication, are long held tenets of human papillomavirus (HPV) infection of keratinocytes; however, these have yet to be rigorously tested in human epithelium. Many HPV studies have used monolayer cell cultures, and while they have provided insights into HPV protein functions and some aspects of the viral life cycles, they fail to reproduce in vivo infection events. Some aspects of wounding can be investigated by scratch-wounding of monolayer epithelial cultures. Tissue-based models of infection, particularly organotypic or "raft" epithelial tissue culture systems, have some advantages over monolayer culture systems, including cells at various levels of differentiation in the epithelium and provision of 3-dimensional cellular interactions in the stratified tissue structure. Thus, organotypic epithelial cultures provide a fully permissive environment for generation of infectious virions as occurs in vivo. In this work our goal is to evaluate the contribution of wounding to infection, identify cells in relation to the wound site and in the stratified epithelium capable of binding and internalizing virions, and evaluate the cells able to express viral early genes. We began this work employing HPV16 pseudovirions (PsVs) carrying an EGFP reporter plasmid whereby the number of infected cells can be quantified by EGFP detection via FACS as well as visualization of infected cells within epithelial cell cultures by confocal microscopy. We find that more cells become HPV-infected in a wounded monolayer keratinocyte culture as compared to unwounded controls. We predict that cells at the wound edge are those more readily infected and are performing microscopy to test this idea. We plan to determine which early infection activities benefit from wounding and correlate this with the epithelial to mesenchymal transition at the wound edge. Further studies will employ raft tissues.
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Sharof Tugizov
San Francisco, USASharof Tugizov1,2, Rossana Herrera1, Rachna Sharma1, Piri Veluppillai2, Deborah Greenspan2, Joel Palefsky1,2. 1Department of Medicine, University of California, San Francisco, CA, USA; 2Department of Orofacial Sciences, University of California, San Francisco, CA, USA. Objective: The incidence of HPV-associated lesions is substantially higher in human immunodeficiency virus (HIV)-positive individuals than in HIV-negative individuals. HIV may potentiate HPV-related disease through attenuation of immune response to HPV; however, direct and/or indirect interactions between the two viruses may also facilitate HPV infection of mucosal epithelium. In this study we investigated the role of HIV tat in HPV infection of mucosal epithelium.
Methods: Oral biopsies from HIV-infected individuals were immunostained for HIV tat, immune cell markers and tight junction proteins using specific antibodies to tat, CD68, CD4, CD1a, occludin, ZO-1 and claudin-1. Polarized oral and cervical epithelial cells, and oral tissue explants were treated with HIV-1 tat and the cells and tissue explants were then exposed to HPV 16 pseudovirions (PsV) containing a reporter red fluorescent gene. Paracellular penetration of PsV across polarized cells and tissue explants were evaluated by detection of red fluorescence using confocal microscopy.
Results: Immunostaining analysis of oral mucosal epithelium of HIV-infected individuals showed that HIV tat was expressed in intracellular Langerhans cells, macrophages and CD4+ T lymphocytes. Analysis of integrity of oral mucosa epithelium of HIV-infected individuals showed that tight junction proteins occludin, ZO-1 and claudin-1 were disrupted. Treatment of polarized oral and cervical epithelial cells with wild-type but not mutant HIV tat disrupted epithelial tight junctions and activated integrin and mitogen activated protein kinase (MAPK) signaling. Tat-associated disruption of polarized epithelial cells potentiated paracellular passage of HPV 16 PsV from the apical compartment to the basolateral compartment. Treatment of oral epithelial explants with HIV tat also led to paracellular penetration of PsVs into mucosal epithelium and entry of PsV into basal/parabasal cells.
Conclusions: Our data indicate that HIV tat in the mucosal epithelial microenvironment disrupts epithelial tight junctions and may facilitate HPV penetration into basal epithelial cells where HPV infection is initiated.
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Michael Conway
Hershey, USAMichael Conway1, Linda Cruz1, Samina Alam1, Craig Meyers1. 1Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, PA, USA. Previous mutational analyses of in vitro-derived human papillomavirus (HPV) capsids have shown that certain cysteine residues are critical for proper capsid assembly, integrity, and maturation. Since previous studies utilized HPV capsids that were produced in monolayer culture, disulfide interactions linking L1 subassemblies have not been placed in the temporal context of stratified and differentiating human tissue - the natural host environment for HPV. Here, we extend upon our previous observation that HPV16 capsids become stabilized by utilizing a tissue-spanning redox gradient, by identifying the temporal roles of individual conserved cysteine residues. Specifically, at 10 and 20-days, C175S, C185S, C175,185S and C428S L1substitutions markedly increased the susceptibility of viral genomes to nucleases and/or they decreased encapsidation efficiency. A subpopulation of mutant virions that did protect their genomes remained as infectious as wild-type, although Optiprep gradient purification of all mutant virions led to dramatic loss of infectivity, suggesting that the mutations undermined capsid stability. Further, treatment of 8-day wild-type and mutant tissues with oxidized glutathione (GSSG) failed to enhance infectivity and endonuclease-resistant genomes in mutant tissues as compared to wild-type, suggesting that disulfide bonds are formed utilizing C175, C185, and C428 within suprabasal nuclei. Further research into the temporal disulfide bonding patterns of organotypic culture-derived HPV16 virions will be presented.
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Linda Cruz
Elizabethtown, USALinda Cruz1, Michael J. Conway1, Craig Meyers1. 1Pennsylvania State University, Elizabethtown, PA, USA. Studies using pseudovirions (PsV) have implicated the minor capsid protein L2 in early events of the viral life cycle including the entry of virions into the cell, escape of the viral genome from endosomes after viral uncoating, and bringing the viral genome into the nucleus. In HPV16 PsV, L2 is cleaved by a proprotein convertase, furin and/or PC5/6, at a consensus site in the N-terminal region during infection. We were interested in whether native virions (NV) produced under physiologically relevant conditions of differentiating host tissue would show the same dependence on cleavage by a cellular proprotein convertase to undergo a successful infection. Human foreskin keratinocytes infected with HPV16, HPV18, HPV31, and HPV45 were cultured to produce infectious virus. Surprisingly, infection of HaCat keratinocytes, Chinese hamster ovary cells, as well as primary foreskin keratinocytes with HPV16, was not dependent on cleavage by furin or PC5/6 as demonstrated by infection in the presence of a furin peptide inhibitor. In addition, infection with HPV16 produced from differentiated human cervical keratinocytes was insensitive to the furin inhibitor. HPV45 showed the same resistance to inhibition as HPV16. Interestingly, early studies suggest that the HPV16 NV L2 N-terminus may be cleaved during virion morphogenesis in the differentiating tissue. Further, this virus appears to be independent of primary binding to a heparan sulfate attachment receptor. However, infections with HPV18 and HPV31 suggest that the furin and/or PC5/6 enzyme is required during infection with these viruses.
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Craig Meyers
Professor of Microbiology and Immunology, The Huck Institutes Of The Life Sciences
Hershey, USASamina Alam1, Brian S. Bowser1, Michael J. Conway1, Mohd Israr1, Eric J. Ryndock1, Long Fu Xi2, Craig Meyers1. 1Department of Microbiology and Immunology, The Penn State College of Medicine, Hershey, PA, USA; 2Department of Pathology, School Medicine, Department of Epidemiology, School of Public Health, University of Washington, Seattle, WA, USA. Epidemiological studies suggest that Human Papillomavirus (HPV) infected women who smoke face an increased risk for developing cervical cancer. We have previously reported that exposure of HPV positive organotypic cultures to Benzo[a]pyrene (BaP), a major carcinogen in cigarette smoke, resulted in enhanced viral titers. Since BaP is known to deregulate multiple pathways of cellular proliferation, enhanced virion synthesis could result from carcinogen/host-cell interaction. Here, we report that BaP-mediated upregulation of virus synthesis is correlated to an altered balance between cell cycle specific cyclin dependent kinase (CDK) activity profile compared with controls. Specifically, BaP treatment increased accumulation of hyperphosphorylated pRb which coincided with increased cdc2/CDK1 kinase activity, but which further conflicted with the simultaneous upregulation of CDK inhibitors p16INK4 and p27KIP1, which normally mediate pRb hypophosphorylation. In contrast, p21WAF1 and p53 levels remained unchanged. Under these conditions, CDK6 and CDK2 kinase activities were decreased whereas CDK4 kinase activity remained unchanged. Addition of Purvalanol A, a specific inhibitor of CDK1 kinase, to BaP treated cultures, resulted in the production of non-infectious HPV31b particles. In contrast, infectivity of control virus was unaffected by Purvalanol A treatment. BaP targeting of CDK1 kinase occurred independently of HPV status since BaP treatment also increased CDK1 activity in tissues derived from primary keratinocytes. Our data indicates that HPV31b virions synthesized in the presence of BaP were dependent on BaP-mediated alteration in CDK1 kinase activity for maintaining their infectivity.
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Isao Murakami
Tokyo, JapanIsao Murakami1,2, Takamasa Takeuchi1, Iwao Kukimoto1, Tomomi Nakahara3, Rika Matsuo1, Takuma Fujii2, Daisuke Aoki2, Tadahito Kanda1. 1Pathogen Genomics Center, National Institute of Infectious Diseases; 2Department of Obstetrics and Gynecology, School of Medicine, Keio University, Minato, Tokyo, Japan; 3Virology Division, National Cancer Center Research Institute. Background: Nucleosome positioning affects gene expression and DNA replication. Nucleosome positioning on human papillomavirus (HPV) DNA during maintenance and propagation provides insight into the control of HPV life cycle.
Methods: We analyzed nucleosome positioning on episomal HPV DNA in cultured cells. W12 cells, which harbor episomal HPV16, and CIN612 cells, which harbor episomal HPV31, were cultured in proliferating or differentiating conditions. The cells were permeabilized by NP-40 and treated with micrococcal nuclease (MNase). Mononucleosomal DNA was gel-purified and the obtained MNase-resistant fraction was quantified by real-time PCR. PCR amplicons were set every 30bp of HPV genome spanning the long control region and E6 gene. Each amplicon was 140bp long including 30mer primers and the neighboring amplicons overlapped by 110bp.
Results: In proliferating W12 cells, nt.11-240 and nt.311-570 of HPV16 DNA were relatively MNase-resistant. In differentiating W12 cells, nt.11-150 and nt.191-330 of HPV16 DNA became more MNase-resistant and nt.41-240 became less MNase-resistant. In proliferating CIN612 cells, nt.11-270 and nt.431-570 of HPV31 DNA were relatively MNase-resistant. In differentiating CIN612 cells, nt.41-300 of HPV31 DNA became less MNase-resistant.
Conclusion: The MNase-resistant regions of the episomal HPV DNA, presumably in a less accessible conformation, changed according to host cell culture conditions. Some features were common to HPV16 and HPV31 and could be general among other HPV types. |
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Qian Wang
London, United KingdomQian Wang1, Deborah Jackson1, John Doorbar1. 1Division of Virology, National Institute for Medical Research, Mill Hill, London, United Kingdom. Recent work has suggested a role for E4 in genome amplification and life cycle completion. However, there appear to be differences between its role in different HPV types. In HPV 11, E4 loss has been reported not to compromise life cycle completion, while in HPV18 and 31, genome amplification was greatly reduced. Our previous work with HPV16 indicated differential effects depending on the nature of the E4 mutant expressed.
To clarify the role of E4 amongst the high-risk HPV types, we have looked in detail at E4 function in the context of the HPV16 and 18 genomes and the full productive life cycle. E4 loss does not abolish genome amplification or L1 production, but reduces the efficiency of life cycle completion. When raft cultures were propagated for 8 days, the E4 knock out (E4KO) mutants showed much reduced genome amplification and L1 expression. By 14 days, however, both genome amplification and L1 production were extensive even in the absence of E4. When taken together, an approximately 2-3 fold reduction in life cycle efficiency was noticed at all time points. Similar effects were noticed in methylcellulose experiments. It appears that the apparent differences between previous data may depend on which time points are examined in the differentiation system used.
The contribution of E4 to life cycle success may be related to its ability to bind E2 and/or to its association with mitochondria and the activation of certain caspases. In methylcellulose studies, the G2 arrest function of E4 appears to have a contributory role in genome amplification when analysed in the context of the whole genome. When E4 was reintroduced into HPV16 E4KO cells by transfection using an E4 expressing vector under the control of a K10 promoter, HPV16 genome amplification in methylcellulose was enhanced. These results suggest that E4 contributes to replication efficiency and life cycle completion rather than being essential for these events. Further work will establish whether low-risk E4 proteins also have this function. |
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Lietta Nicolaides
London, United KingdomLietta Nicolaides1, Ken Raj2, John Doorbar1. 1Division of Virology, National Institute for Medical Research, London, UK; 2Radiation Effects Department, CRCE, HPA, Oxfordshire, UK. The E6 protein from high-risk HPVs has previously been shown to be necessary for the persistence of viral episomes in cells but the mechanism for this remains unclear. E6 has many activities, one of which being its ability to bind to and degrade PDZ proteins, such as hScrib, via its C-terminal motif. In this work we have analysed the relationship between hScrib and HPV16 E6 by co-expressing the hScrib protein with either wild-type HPV16 E6 (16E6WT) or a mutant E6 protein which lacks the PDZ-binding motif (16E6PDZ). Interestingly, we observe that the levels of 16E6PDZ protein are considerably lower than those of 16E6WT. Analysis of RNA shows that this is not due to differences in transcription. In fact, it appears that the decreased level of 16E6PDZ protein results from it being more susceptible to proteasomal degradation than the 16E6WT protein. Furthermore, levels of 16E6WT protein are lower when hScrib is not co-expressed. This suggests that hScrib is able to stabilise the E6 protein and we hypothesised that this effect may play a role in the persistence of viral episomes. To study this, we constructed a mutant HPV16 genome that lacks the PDZ-binding motif of E6 (HPV16E6PDZ) and tested its ability to persist in NIKS cells compared to the wild-type HPV16 genome. We find that, whereas the wild-type genome does persist episomally, the HPV16E6PDZ genome does not. These results suggest a vital new role for the interaction between E6 and PDZ proteins in stabilising sufficient amounts of E6 protein to facilitate viral episome persistence.
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