Seroprevalence of Human Papillomavirus 6, 11, 16, and 18 in Young Primiparous Women in Sao Paulo, Brazil

Introduction: Data on epidemiology of HPV infection are needed for the development of human papillomavirus (HPV) vaccine recommendations, especially in countries where HPV vaccination is not yet included in public vaccination programs. The aim of this study was to determine the prevalence of serum antibodies to HPV types 6, 11, 16, and 18 and associated factors among young women after birth of the first child. Methods: This cross-sectional study was carried out in a large public maternity hospital in Sao Paulo, Brazil. Three hundred one women aged 15 to 24 years who gave birth to their first child were recruited between 43 and 60 days after delivery. Seroprevalence was performed using a type-specific enzyme-linked immunosorbent assay based on HPV Late protein 1 viruslike particles. The association of seroreactivity with these 4 HPV types with selected demographic and behavioral factors was assessed by Generalized Linear Model analysis. Results: Fifty-eight (19.3%) women (95% confidence interval, 15.0%-24.2%) had antibodies to any of the 4 viruslike particles tested. The overall seroprevalence rates of the HPV types were: HPV16, 9.0%; HPV18, 7.0%; and HPV 6+11, 7.7%, which are targeted by the HPV prophylactic vaccines. In the multivariate analysis, only age (inversely, P = 0.044 for trend) and previous sexually transmitted disease (P = 0.008) were 2 factors independently associated with HPV seropositivity. Conclusions: These data offer additional information on the epidemiology of HPV in a group of young Brazilian women after first delivery and contribute to establish a baseline of HPV seroprevalence against which post-HPV vaccine era seroprevalence can be compared.

Expression and characterization of HPV-16 L1 capsid protein in Pichia pastoris

Human papillomaviruses (HPVs) are responsible for the most common human sexually transmitted viral infections. Infection with high-risk HPVs, particularly HPV16, is associated with the development of cervical cancer. The papillomavirus L1 major capsid protein, the basis of the currently marketed vaccines, self-assembles into virus-like particles (VLPs). Here, we describe the expression, purification and characterization of recombinant HPV16 L1 produced by a methylotrophic yeast. A codon-optimized HPV16 L1 gene was cloned into a non-integrative expression vector under the regulation of a methanol-inducible promoter and used to transform competent Pichia pastoris cells. Purification of L1 protein from yeast extracts was performed using heparin-sepharose chromatography, followed by a disassembly/reassembly step. VLPs could be assembled from the purified L1 protein, as demonstrated by electron microscopy. The display of conformational epitopes on the VLPs surface was confirmed by hemagglutination and hemagglutination inhibition assays and by immuno-electron microscopy. This study has implications for the development of an alternative platform for the production of a papillomavirus vaccine that could be provided by public health programs, especially in resource-poor areas, where there is a great demand for low-cost vaccines.

Human Papillomavirus (HPV) Type Distribution in Females with Abnormal Cervical Cytology. A Correlation with Histological Study

The aim of this study was to determine human papillomavirus (HPV) types distribution in cervical preneoplasic lesions in a Southern Spanish population and their relationship between HPV type and grade of histopathological abnormality. Finally, 232 cervical samples from 135 women with previous cytological abnormalities were included in this study. Colposcopy studies and biopsies were performed. Haematoxylin-eosin stained slides were observed and detection of HPV DNA in cervical swabs was carried out with use of a polymerase chain reaction and microarrays technology. The relationship between the presence of HPV infection and diagnostic variables was evaluated. HPV 16 was the most common type followed by HPV 58, 51, 33 and 31. However, the two HPV types targeted in the prophylactic vaccines such as HPV type 16 and 18 were detected in only 37 (21.2%) and 2 (1.1%) cases respectively. Thirty-three (18.9%) of samples were infected with multiple types, the majority of them with two types. In addition, during the follow-up of patients many changes in type distribution were observed. Several studies will be necessary in order to evaluate the HPV type distribution for therapeutically and prophylactic purposes such as vaccine treatment. Also, because of the differences obtained depending of use of various DNA technologies, the performance of some comparative studies of the different methods from detection of HPV would be advisable in a high population of patients and with the most homogeneous conditions possible.

Adult-type rhabdomyosarcoma: analysis of 57 cases with clinicopathologic description, identification of 3 morphologic patterns and prognosis.

Adult-type rhabdomyosarcoma (RMS) has been classically defined as a pleomorphic sarcoma with desmin expression occurring in adult patients. To reevaluate this entity, we analyzed a series of 57 cases using immunohistochemistry for desmin, myogenin, alpha smooth muscle actin, h-caldesmon, pankeratin AE1/AE3, epithelial membrane antigen (EMA), S100 protein, CD34, MDM2, and CDK4. In this series, there were 36 men and 21 women aged from 22 to 87 years (median: 59). Tumors were mainly located in the lower limbs (27 cases), trunk wall (15 cases), and upper limbs (10 cases). Most tumors were deeply located (51/54) with a size from 1 to 30 cm (median: 8 cm). Cases were classified in 3 histologic categories: spindle cell RMS (25 cases), pleomorphic RMS (16 cases), and mixed type (16 cases). Forty-one tumors were grade 3 and 16 grade 2. Immunohistochemistry showed that every case was positive for desmin and myogenin. Alpha smooth muscle actin was positive in 21%, pankeratin AE1/AE3 in 20%, and CD34 in 13.2%. Treatment modalities and follow-up were available in 46 cases. Median follow-up was 60.9 months. Eight patients developed a local recurrence and 16 a distant metastasis with a 5-year overall survival rate of 52.6% and a 5-year metastasis-free survival of 62.9%. The only predictive factor for metastasis was histologic grade. In conclusion, adult-type RMS is a rare sarcoma occurring mainly in the extremities and trunk wall with 2 main histologic patterns, spindle cell, and pleomorphic patterns, which represent the end of the spectrum of a single entity.

Identification of Novel Cellular Transcription Factors that Regulate Early Promoters of Human Papillomavirus Types 18 and 16

Background. The long control region (LCR) of human papillomavirus (HPV) regulates early gene transcription by interaction with several viral and cellular transcription factors (TFs). Methods. To identify novel TFs that could influence early expression of HPV type 18 (HPV-18) and HPV type 16 (HPV-16), a high-throughput transfection array was used. Results. Among the 704 TFs tested, 28 activated and 36 inhibited the LCR of HPV-18 by more than 2-fold. For validation, C33 cells were cotransfected with increasing amounts of selected TF expression plasmids in addition to LCR-luciferase vectors of different molecular variants of HPV-18 and HPV-16. Among the TFs identified, only GATA3, FOXA1, and MYC have putative binding sites within the LCR sequence, as indicated using the TRANSFAC database. Furthermore, we demonstrated FOXA1 and MYC in vivo binding to the LCR of both HPV types using chromatin immunoprecipitation assay. Conclusions. We identified new TFs implicated in the regulation of the LCR of HPV-18 and HPV-16. Many of these factors are mutated in cancer or are putative cancer biomarkers and could potentially be involved in the regulation of HPV early gene expression.

Recombinant Kunjin virus replicon vaccines induce protective T-cell immunity against human papillomavirus 16 E7-expressing tumour

The persistence of the E7 oncoprotein in transformed cells in human papillomavirus (HPV)-associated cervical cancer provides a tumour-specific antigen to which immunotherapeutic strategies may be directed. Self-replicating RNA (replicon) vaccine vectors derived from the flavivirus Kunjin (KUN) have recently been reported to induce T-cell immunity. Here, we report that inclusion of a CTL epitope of HPV16 E7 protein into a polyepitope encoded by a KUN vector induced E7-directed T-cell responses and protected mice against challenge with an E7-expressing epithelial tumour. We found replicon RNA packaged into virus-like particles to be more effective than naked replicon RNA or plasmid DNA constructed to allow replicon RNA transcription in vivo. Protective immunity was induced although the E7 CTL epitope was subdominant in the context of other CTL epitopes in the polyepitope. The results demonstrate the efficacy of the KUN replicon vector system for inducing protective immunity directed towards a virally encoded human tumour-specific antigen, and for inducing multi-epitopic CTL responses. (C) 2004 Elsevier Inc. All rights reserved.

A polytope DNA vaccine elicits multiple effector and memory CTL responses and protects against human papillomavirus 16 E7-expressing tumour

Vaccine-induced CD8 T cells directed to tumourspecific antigens are recognised as important components of protective and therapeutic immunity against tumours. Where tumour antigens have pathogenic potential or where immunogenic epitopes are lost from tumours, development of subunit vaccines consisting of multiple individual epitopes is an attractive alternative to immunising with whole tumour antigen. In the present study we investigate the efficacy of two DNA-based multiepitope('polytope') vaccines containing murine (H-2(b)) and human (HLA-A* 0201)-restricted epitopes of the E7 oncoprotein of human papillomavirus type 16, in eliciting tumour-protective cytotoxic T-lymphocyte (CTL) responses. We show that the first of these polytopes elicited powerful effector CTL responses ( measured by IFN-gamma ELISpot) and long-lived memory CTL responses ( measured by functional CTL assay and tetramers) in immunised mice. The responses could be boosted by immunisation with a recombinant vaccinia virus expressing the polytope. Responses induced by immunisation with polytope DNA alone partially protected against infection with recombinant vaccinia virus expressing the polytope. Complete protection was afforded against challenge with an E7-expressing tumour, and reduced growth of nascent tumours was observed. A second polytope differing in the exact composition and order of CTL epitopes, and lacking an inserted endoplasmic reticulum targeting sequence and T-helper epitope, induced much poorer CTL responses and failed to protect against tumour challenge. These observations indicate the validity of a DNA polytope vaccine approach to human papillomavirus E7 - associated carcinoma, and underscore the importance of design in polytope vaccine construction.

Serologic response to human papillomavirus 16 among Australian women with high-grade cervical intraepithelial neoplasia

This study evaluated the detection of human papillomavirus (HPV) 16 antibody in HPV 16-associated cervical intraepithelial neoplasia (CIN) in Australian women. Seroreactivity to HPV 16 L1 virus-like particles was assessed in patients with CIN 2 (n = 169) and CIN 3 (n = 229) lesions previously tested for the presence of HPV DNA. Seropositivity was significantly commoner in women with HPV 16 DNA-positive lesions (98/184) than in women with no HPV DNA in the lesion (15/47) or with HPV of types other than 16 in the lesion (43/167) (P = 0.0004). In addition, seropositivity was observed in 33% (55/169) of women with CIN 2 and 46% (106/229) of women with CIN 3, in keeping with the lower fraction of CIN 2 (57/169) than CIN 3 (127/229) biopsies positive for HPV 16 DNA. HPV 16 seropositivity is most common in women with HPV 16-associated CIN, but many patients with HPV-associated CIN 3 are seronegative, and HPV 16 seropositivity is common in women with CIN associated with other HPV types. Overall, HPV 16 serology is a poor predictor of presence of HPV 16-associated CIN 3 in patient population studied.

Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment

Background: Persistent infection by high risk HPV types (e.g. HPV-16, -18, -31, and -45) is the main risk factor for development of cervical intraepithelial neoplasia and cervical cancer. Tumor necrosis factor (TNF) is a key mediator of epithelial cell inflammatory response and exerts a potent cytostatic effect on normal or HPV16, but not on HPV18 immortalized keratinocytes. Moreover, several cervical carcinoma-derived cell lines are resistant to TNF anti-proliferative effect suggesting that the acquisition of TNF-resistance may constitute an important step in HPV-mediated carcinogenesis. In the present study, we compared the gene expression profiles of normal and HPV16 or 18 immortalized human keratinocytes before and after treatment with TNF for 3 or 60 hours. Methods: In this study, we determined the transcriptional changes 3 and 60 hours after TNF treatment of normal, HPV16 and HPV18 immortalized keratinocytes by microarray analysis. The expression pattern of two genes observed by microarray was confirmed by Northern Blot. NF-kappa B activation was also determined by electrophoretic mobility shift assay (EMSA) using specific oligonucleotides and nuclear protein extracts. Results: We observed the differential expression of a common set of genes in two TNF-sensitive cell lines that differs from those modulated in TNF-resistant ones. This information was used to define genes whose differential expression could be associated with the differential response to TNF, such as: KLK7 (kallikrein 7), SOD2 (superoxide dismutase 2), 100P (S100 calcium binding protein P), PI3 (protease inhibitor 3, skin-derived), CSTA (cystatin A), RARRES1 (retinoic acid receptor responder 1), and LXN (latexin). The differential expression of the KLK7 and SOD2 transcripts was confirmed by Northern blot. Moreover, we observed that SOD2 expression correlates with the differential NF-kappa B activation exhibited by TNF-sensitive and TNF-resistant cells. Conclusion: This is the first in depth analysis of the differential effect of TNF on normal and HPV16 or HPV18 immortalized keratinocytes. Our findings may be useful for the identification of genes involved in TNF resistance acquisition and candidate genes which deregulated expression may be associated with cervical disease establishment and/or progression.

Mice expressing the E7 oncogene of HPV16 in epithelium show central tolerance, and evidence of peripheral anergising tolerance, to E7-encoded cytotoxic T-lymphocyte epitopes

In order to derive mice which expressed both the E7 open reading frame transgene of human papillomavirus type 16 in skin and MHC class 1 restriction elements for several E7-encoded cytotoxic T-lymphocyte (CTL) epitopes, K14.HPV16E7 mice which express E7 in basal keratinocytes were crossed to the F1 generation with A2.1 K-b transgenic mice which express the MHC binding cleft domains of human HLA A*0201, and murine H-2(b). F1 mice (denoted K14E7xA2.1) expressed E7 in the thymus at least as early as 2-5 days before birth. Immunisation of FVBxA2.1 control mice (transgenic for HLA A*0201 and H-2(b) but not for E7), with two HLA A*0201-restricted epitopes of E7 and one H-2(b)-restricted CTL epitope of E7, gave strong primary CTL responses recognising epitope-pulsed or constitutively E7-expressing syngeneic target cells. In contrast, in immunised K14E7xA2.1 mice, the CTL responses to the H-2(b) epitope and one of the HLA A*0201 CTL epitopes were strongly down-regulated, and to the other HLA A*0201 epitope, completely abolished, as demonstrated by percentage specific killing by bulk splenocyte cultures in cyrotoxicity assays, and by CTL precursor frequency analysis, In thymus-transplanted bone marrow radiation chimeras in which the immune system of K14E7xA2.1 mice was replaced by a FVBxA2.1 immune system, specific immunisation did not result in reemergence of strong E7-directed CTL responses. In agreement with these in vitro findings, specific immunisation failed to significantly alter the course of E7-associated tumour development in K14E7xA2.1 mice. These data are consistent with a model of central deletional CTL tolerance to E7-encoded epitopes recognised in the context of two distinct MHC class 1 restriction elements, and with the possibility of peripheral T-cell anergy maintained by expression of E7 in the skin. (C) 1998 Academic Press.

Split tolerance to a viral antigen expressed in thymic epithelium and keratinocytes

When expressed as a transgene from the keratin 14 (K14) promoter in an MHC class II-deficient mouse, I-Ab expressed in thymic cortical epithelium promotes positive but not negative selection of I-Ab-restricted CD4(+) T cells (Laufer, T. M. et al., Nature 1996. 383:81-85). Transgenic mice expressing the E7 protein of human papilloma virus 16 from the K14 promoter were studied to determine the consequence of expression of a cytoplasmic/nuclear protein from the K14 promoter. K14E7-transgenic mice express E7 in the thymus and skin without evidence for autoimmunity to E7. Repeated immunization of FVB(H-2(q)) or F1(C57BV6JxFVB) mice with E7 elicited similar antibody responses to the defined B cell epitopes of E7 in K14E7-transgenic and non-transgenic animals. In contrast, for each genetic background, a single immunization with E7 elicited demonstrable T cell proliferative responses to the major promiscuous T helper epitope of E7 in the transgenic but not the non-transgenic animals. Further,E7-immunized non-transgenic F1 (FVBxC57BL/6J) animals developed strong E7-specific cytotoxic T lymphocyte (CTL) responses and were protected against challenge with E7(+) tumors, whereas similarly immunized K14E7-transgenic animals had a markedly reduced CTL response to E7 and no E7-specific tumor protection was observed, although the antibody and CTL response to ovalbumin was normal. Expression of E7 protein as a transgene from the K14 promoter in the skin and thymus thus induces E7-specific tolerance in the cytotoxic T effector repertoire, together with expansion of the E7-specific T helper repertoire. These findings demonstrate that limited tissue distribution of an autoantigen may result in split tolerance to that autoantigen.

Simplifying the molecular mechanisms of human papillomavirus

The human papillomaviruses (HPVs) are associated with several human epithelial diseases. These diseases are confined to cutaneous and mucosal epithelia and comprise papillomas (warts) and benign or malignant neoplasms. Globally, infection by HPVs presents a considerable health problem given that at any one time approximately 10% of the population may have warts of one form or another. Of more serious concern is the prevalence of HPV-associated cervical carcinoma. It is estimated that 500,000 new cases of cervical neoplasia are diagnosed per year (primarily squamous carcinomas). Thus, HPV-associated cancer represents one of the most common cancers afflicting women and is one of the three most common causes of cancer death among women globally.(15) Although some genotypes of human papillomaviruses are clearly associated with the development of cancer (in particular, HPVs 16 and 18) these viruses share significant structural and functional similarity to the nononcogenic genotypes, and one of the puzzles of HPV biology is why essentially similar viruses vary so widely in their oncogenic potential.

Potential strategies utilised by papillomavirus to evade host immunity

The co-evolution of papillomaviruses (PV) and their mammalian hosts has produced mechanisms by which PV might avoid specific and non-specific host immune responses. Low level expression of PV proteins in infected basal epithelial cells, together with an absence of inflammation and of virus-induced cell lysis, restricts the opportunity for effective PV protein presentation to immunocytes by dendritic cells. Additionally, PV early proteins, by a range of mechanisms, may restrict the efficacy of antigen presentation by these cells. Should an immune response be induced to PV antigens, resting keratinocytes (KC) appear resistant to interferon-gamma-enhanced mechanisms of cytotoxic T-lymphocyte (CTL)-mediated lysis, and expression of PV antigens by resting KC can tolerise PV-specific CTL. Thus, KC, in the absence of inflammation, may represent an immunologically privileged site for PV infection. Together, these mechanisms play a parr in allowing persistence of PV-induced proliferative skin lesions for months to years, even in immunocompetent hosts.

Immune responses induced by BCG recombinant for human papillomavirus L1 and E7 proteins

Recombinant bacille Calmette-Guerin (BCG) based vaccine delivery systems could potentially share the safety and effectiveness of BCG. We therefore prepared recombinant BCG vaccines which expressed the L1 late protein of the human papillomavirus (HPV) 6b or the E7 early protein of the HPV 16. The two recombinants were evaluated as immunogens in C57BL/6J and BALB/c mice, and compared with a conventional protein/adjuvant system using E7 or L1 mixed with Quil-A adjuvant. rBCG6bL1 and rBCG16E7 primed specific immune responses, represented by DTH, T-proliferation and antibody, and rBCG16E7 induced cytotoxic immune response to E7 protein. The magnitude of the observed responses were less than those elicited by protein/adjuvant vaccine. As recombinant BCG vaccines expressing HPV6bL1 or HPV16E7 persist at low levels in the immunised host, they may be beneficial to prime or retain memory responses to antigens, but are unlikely to be useful as a single component vaccine strategy. (C) 2000 Elsevier science Ltd. All rights reserved.

The human papillomavirus E7 protein is able to inhibit the antiviral and anti-growth functions of interferon-alpha

It is now well recognized that cervical cancer is caused by infection with certain human papillomavirus (HPV) subtypes and while interferon-alpha (IFN-alpha) is used to treat HPV-infected lesions, HPV appears to have developed a means to avoid the effects of IFN-alpha. Clinically, resistance appears to be associated with the expression of the E7 oncoprotein. Here we investigated the effects of expression in cells of the E7 protein from high- and low-risk papillomavirus subtypes on a range of responses to IFN-alpha. 2fTGH, a cell line dependent on IFN-alpha for growth in selection medium, grew significantly less well in the presence of E7, and the antiproliferative effects of IFN-alpha upon epithelial cells was lost upon E7 expression. The antiviral effects of IFN-alpha were abrogated in E7-expressing cells. Loss of response to IFN-alpha was found to occur in both high- and low-risk papillomaviruses. Finally, deletion of amino acids 21-24 of HPV type 16 E7 protein partially reversed repression. We conclude that E7 inhibits the functional effects of IFN-alpha and that this property is shared by all HPV subtypes tested. (C) 2000 Academic Press.