Genital HPV Types Research Articles

Parents' Knowledge and Attitude towards HPV and HPV Vaccination in Poland.

HPV is one of the diseases of civilization that causes cervical cancer, among other diseases. For this reason, a vaccination program has been introduced worldwide for preadolescent, sexually inactive seronegative girls. However, the decision to vaccinate young girls must be made by the parents. In Poland, vaccinations are recommended but not financed by the government, which affects their choices, and there is insufficient knowledge of the diseases caused by genital HPV types. In addition, there are cultural, social, and even religious factors to be considered. Therefore, the aim of the study was to analyze the state of knowledge about HPV and HPV vaccines among parents. Two hundred and eighty-eight parents participated in the study, but only 180 of them declared that they had ever heard of HPV (62.5%). Therefore, only these parents completed the entire questionnaire consisting of 34 questions. The parents’ answers were analyzed with the Fisher’s and chi-squared tests. The study showed that parents’ knowledge of HPV and HPV vaccination in Poland is low (49.4% of correct answers). Parents’ attitudes were only influenced by knowledge and education and not by other parameters such as age, gender, place of residence, and the number of children. This study indicates that parents need to be educated about the threats of HPV and the possibilities of prophylactic vaccination.

Prevalence of human papillomavirus in the saliva of sexually active women with cervical intraepithelial neoplasias.

Background The main objective of this study was to estimate the prevalence of human papillomavirus-DNA (HPV-DNA) in the saliva of sexually active women with HPV-related cervical intraepithelial neoplasias (CIN) and compare the findings with a healthy control group. The secondary objectives were: 1) to determine the concordance between genital and oral HPV types in sexually active women with HPV-related CIN; 2) to analyze whether sexual habits influence the presence of HPV-related CIN; 3) to determine whether sexual habits influence the presence of oral HPV. Material and Methods Saliva samples were collected from 100 sexually active women, 50 with HPV-related CIN and 50 healthy subjects presenting normal cytology. PCR assay was used to detect HPV-DNA. Results The prevalence of oral HPV infection in saliva samples was 14% in women with HPV-related CIN, while in the healthy group it was 12%, without statistically significant difference (p =0.766). As for the concordance between genital and oral HPV types in women with HPV-related CIN, concordance was only observed for HPV-16, whereby among 22 women with genital HPV-16, only one (4.54%) also presented oral HPV-16. Regarding the possible influence of sexual habits on the presence of cervical pathology and presence of oral HPV, it was found that marital status, age at first intercourse, number of lifetime sexual partners, and condom use are related with the presence of cervical pathology (p<0.001; p=0.017; p=0.002; and p <0.001, respectively); condom use was also found to be related to the presence of oral HPV (p<0.001). Conclusions The prevalence of HPV-DNA in the saliva of sexually active women with HPV-related CIN is similar to healthy women. The concordance between genital and oral HPV types is low. Both the presence of cervical pathology and the presence of oral HPV are related to sexual habits. Wider sample size is required to confirm this results. Key words:Cervical intraepithelial neoplasias, HPV, saliva, cervix, cancer.

Incorporation of RG1 epitope concatemers into a self-adjuvanting Flagellin-L2 vaccine broaden durable protection against cutaneous challenge with diverse human papillomavirus genotypes.

Aim:To achieve durable and broad protection against human papillomaviruses by vaccination with multimers of minor capsid antigen L2 using self-adjuvanting fusions with the toll-like receptor-5 (TLR5) ligand bacterial flagellin (Fla) instead of co-formulation with alum.Methods:Fla fusions with L2 protective epitopes comprising residues 11–200, 11–88 and/or 17–38 of a single or multiple HPV types were produced in E. coli and their capacity to activate TLR5 signaling was assessed. Immunogenicity was evaluated serially following administration of 3 intramuscular doses of Fla-L2 multimer without exogenous adjuvant, followed by challenge 1, 3, 6 or 12 months later, and efficacy compared to vaccination with human doses of L1 VLP vaccines (Gardasil and Cervarix) or L2 multimer formulated in alum. Serum antibody responses were assessed by peptide ELISA, in vitro neutralization assays and passive transfer to naïve rabbits in which End-Point Protection Titers (EPPT) were determined using serial dilutions of pooled immune sera collected 1, 3, 6 or 12 months after completing active immunization. Efficacy was assessed by determining wart volume following concurrent challenge at different sites with HPV6/16/18/31/45/58 ‘quasivirions’ containing cottontail rabbit papillomavirus (CRPV) genomes.Results:Vaccination in the absence of exogenous adjuvant with Fla-HPV16 L2 11–200 fusion protein elicited durable protection against HPV16, but limited cross-protection against other HPV types. Peptide mapping data suggested the importance of the 17–38 aa region in conferring immunity. Indeed, addition of L2 residues 17–38 of HPV6/18/31/39/52 to a Fla-HPV16 L2 11–200 or 11–88 elicited broader protection via active or passive immunization, similar to that seen with vaccination with an alum-adjuvanted L2 multimer comprising the aa 11–88 peptides of five or eight genital HPV types.Conclusions:Vaccination with flagellin fused L2 multimers provided lasting (>1 year) immunity without the need for an exogenous adjuvant. Inclusion of the L2 amino acid 17–38 region in such multi-HPV type fusions expanded the spectrum of protection.

Sourcing of the WHO human papillomavirus type 18 international standards for HPV antibody levels

BackgroundHPV serology is important for studies of vaccine immunogenicity, but can not be performed in a comparable manner without international standardisation. ObjectivesTo find suitable candidate sera from naturally infected persons for use as International Standards (IS) for antibodies to high-risk HPVs, with priority for HPV-18. Study design946 healthy Thai women (median age 44, range 18–83) and 61 cervical cancer patients were screened using an HPV pseudovirion-Luminex assay to detect antibodies to genital (HPV-6,-11,-16,-18,-31,-33,-45,-52,-58,-68) and non-genital HPV types (HPV-5,-15,-32,-38 and -76). Suitable candidate sera should ideally be mono-specific (have reactivity against only one genital HPV) and have high antibody levels that are stable over time. ResultsSeroprevalences of HPV-16,-31,-52 and -58 were at least twice as high among cancer patients compared to healthy individuals. Thirteen healthy women who met the IS inclusion criteria in initial testing also consented to blood-bag donations. Donations from 2 women with high HPV-18 Ab titers were pooled to the HPV-18 candidate IS, later established as the WHO official IS for HPV antibodies. Sera that could potentially be used as candidate IS for other oncogenic HPVs have also been identified. ConclusionsIn the Thai population, seroepidemiology implicated HPV types HPV-16,-31,-52 and -58 as particularly associated with cervical cancer. A well characterized cohort study has allowed sourcing of materials for an IS for HPV-18 antibodies and could conceivably be used for IS for other HPV types as well.

Epidemiology of oral HPV in the oral mucosa in women without signs of oral disease from Yucatan, Mexico.

High-risk human papillomaviruses (HR-HPV) are considered necessary for the development of cervical cancer. Furthermore, there is no doubt that some types of oral squamous cell carcinoma are associated with HR-HPV. The epidemiology of oral HPV infections in healthy subjects remains unclear due to a lack of knowledge. The objective of this study was to investigate the epidemiology of human papillomavirus infections of the oral mucosa without pathology. A cross-sectional study was performed; samples from 390 women seeking prenatal care, Pap smears, family planning or gynecological diseases were studied. Oral cells were collected by direct swab sampling. Information regarding sociodemographic status, sexual behavior, infectious diseases, contraceptive history and tobacco and alcohol consumption were obtained through direct interviews. HPV and genotypes were detected by type-specific polymerase chain reaction. Our results revealed that 14% of the women studied had an oral HPV infection. Women ≤ 20 years of age had the highest HPV prevalence (24.5%). In total, seven genotypes were identified, including the high-risk genotypes 16, 18, 58 and 59 and the low-risk genotypes 6, 81 and 13, the latter of which is a type exclusive to oral mucosa. Sexual behavior was not associated with the presence of genital HPV types in the oral mucosa. Genital HPV types were present in the oral mucosa of women without associated clinical manifestations; however, sexual behavior was not associated with infection, and therefore others routes of transmission should be explored.

Genital HPV types in Australia

Genital HPV types in Australia

Detection of Human Papillomavirus infection in trichilemmomas and verrucae usingin situhybridization

It is hypothesized that trichilemmomas are 'burned out' verrucae. By performing in situ hybridization using HPV type-specific probes, we explored this concept. Verrucae vulgaris and plantaris were positive controls, and inverted follicular keratoses (IFKs) were negative controls. Additionally, all lesions were tested for HPV genital types (low and high risk). We analyzed 9 trichilemmomas, 20 verrucae vulgaris, 8 verrucae plana, 3 verrucae plantaris and 6 IFKs. All trichilemmomas were negative for HPV types 1, 2 and genital types. Conversely, 9/20 verrucae vulgaris, 2 verrucae plantaris, and 1 verruca plana were positive for HPV type 2. Among HPV-2 positive lesions, 2/2 verrucae plantaris and 1 verruca vulgaris (chin) were strongly positive for genital-type HPV. One verruca plana (shin) was positive for genital-type HPV only. All 6 IFKs were HPV negative. Using HPV type 1 and 2-specific probes and mixed genital-type probes, we were unable to detect HPV in trichilemmomas. This suggests that HPV-1, HPV-2 and low and high risk genital-type HPVs are not involved in the histogenesis of trichilemmoma. We also showed that genital HPV types could be present in non-genital verrucae.

Comparison of the performance of different HPV genotyping methods for detecting genital HPV types

Classification of high-risk HPV types for cervical cancer screening depends on epidemiological studies defining HPV type-specific risk. The genotyping tests that are used, are however, not uniform with regard to type-specific detection rates making comparisons between different studies difficult. To overcome the lack of a "gold standard" four tests were evaluated crosswise using 824 cervical smears pretested by HC2. The tests evaluated were the L1-PCR-based assays PGMY09/11 LBA, HPV DNA Chip and SPF LiPA and an E1 consensus PCR followed by cycle sequencing (E1-PCR). A subset of 265 samples was tested in addition with the GP5+/6+ reverse line blot assay. Differences were noted in the sensitivity and range for specific HPV types, e.g. with detection rates for HPV53 ranging from 2.3% to 11.6%. HPV16 was the most prevalent type detected by all tests except for the SPF-10 LiPa, which detected HPV31 more often. Kappa values calculated ranged from poor (k=0.20) to intermediate (k=0.54) for HPV positivity, but were higher for high-risk type positivity (k=0.31-0.61) and best for recognition of HPV16 (k=0.53-0.72). The analytical sensitivity of the tests ranged between 15% and 97% for individual types and specificity was highly dependent on which test system was used as "gold standard" for the analysis. The results of histology were used for calculation of clinical sensitivity and specificity. E1-PCR, PGMY09/11 LBA and SPF-10 LiPA had a high clinical sensitivity (>95%) for the detection of cervical intraepithelial neoplasia 2 or higher, whereas the HPV DNA Chip reached only 84.1%.

Homogeneous Amplification of Genital Human Alpha Papillomaviruses by PCR Using Novel Broad-Spectrum GP5+ and GP6+ Primers

Human papillomavirus (HPV) DNA detection and typing are important for diagnosis and management of HPV-associated diseases. One of the most commonly used PCR methods, GP5+/6+, shows weaknesses in amplifying certain types. To circumvent this limitation, we developed and validated broad-spectrum primers targeting the GP5+/6+ region. The addition of eight upstream and two downstream BSGP5+/6+ (BS) primers improved amplification of plasmids of 14 genital HPV types 10- to 1,000-fold versus GP5+/6+ PCR without altering sensitivity for the 10 others. For these 24 types, an analytic sensitivity of < or = 1,000 plasmid copies in the presence of 100 ng cellular DNA was obtained. Additionally, we integrated an internal beta-globin PCR into both HPV PCR systems, allowing simultaneous DNA quality control without affecting the sensitivity of HPV detection. Furthermore, we describe five additional low-risk HPV probes used in multiplex HPV genotyping (MPG) for simultaneous identification of all 15 high-risk, 3 putative high-risk, and 9 low-risk HPV genotypes. The performance of BSGP5+/6+ multiplexed with beta-globin primers was compared to that of standard GP5+/6+ with DNA from 1,112 cervical scrapings. There was 79% overall agreement (kappa = 0.816). BSGP5+/6+ was significantly more sensitive than GP5+/6+ for detection of HPV 30, 39, 42, 44, 51, 52, 53, 68, 73, and 82, detecting 212 additional HPV infections and increasing the proportion of multiple infections from 17.2 to 26.9% in cancer patients. In conclusion, BSGP5+/6+ multiplexed with beta-globin PCR provides an improvement in type-specific amplification sensitivity and homogeneity compared to GP5+/6+ and offers simultaneous internal control of DNA quality. BSGP5+/6+-MPG, therefore, is suitable for epidemiologic and also diagnostic applications.

Denaturing high-performance liquid chromatography for detecting and typing genital human papillomavirus.

Human papillomaviruses (HPVs) are important in the development of human cancers, including cervical and oral tumors. However, most existing methods for HPV typing cannot routinely distinguish among the more than 100 distinct types of HPV or the natural HPV intratypic variants that have also been documented. To address this problem, we developed a novel method, general primer-denaturing high-performance liquid chromatography (GP-dHPLC), for the detection and typing of genital HPV using an automated 96-well plate format. GP-dHPLC uses general primer PCR (GP-PCR) to amplify the viral DNA and then analyzes the GP-PCR products by denaturing high-performance liquid chromatography (dHPLC). A number of different primer pairs with homology to most known genital HPV types were tested, and the L1C1-L1C2M pair specific for the L1 region of the viral genome was chosen. A set of HPV standard control patterns, consisting of those for HPV types 16, 18, 31, 33, 39, 45, 51, 52, 56, 58, 59, 6, and 11, was established for genital HPV typing. One hundred eighty-six frozen and formalin-fixed cervical cancer tissue samples were analyzed for the presence of HPV and the HPV type by this method, and 95.8% of them were found to contain HPV DNA. GP-dHPLC accurately discriminated among HPV variants that differed by as little as one nucleotide. Several new variants of HPV types 16, 18, 39, 45, 52, and 59 were identified. Moreover, multiple HPV infections were detected in 26.6% of the samples. Our results indicate that HPV typing by GP-dHPLC permits discrimination of common genital HPV types, detection of multiple HPV infections, and identification of HPV variants in clinical samples.

Nongenital Human Papillomavirus Infections

Although genital HPV types produce a broad spectrum of disease, the nongenital types are a bit more predictive. Particularly in the immunocompromised patient, it appears as though when they become symptomatic they cause warts. These warts can be a particular problem with immunocompromised patients where the malignant potential can also be expressed. Additional understanding of the relationship between the papilloma viruses and cutaneous oncology is very important. There needs to be an application of seroepidemiologic techniques to understand better the epidemiology and further research on more effective and less painful therapies.

Identification of multiple genital HPV types and sequence variants by consensus and nested type-specific PCR coupled with cycle sequencing

Consensus and type-specific HPV primers were employed for PCR and cycle sequencing of genital HPVs in scrapings and colposcopically directed biopsies of the cervix from a cohort of 188 female sex workers. A total of 27 individuals tested positive for a broad spectrum of HPV types, including HPVs 6b, 16, 18, 31, 33, 34, 35, 45, 56 and 58, as well as a new HPV type, with seven individuals displaying dual infections. Good correlation between the results of individually paired samples was observed. A HPV 16 primer biotinylated at the 5' end was also used as a probe, which could successfully detect amplified products of HPV 16 but not other HPV types tested by an automated ELISA detection system. DNA sequence analysis revealed several HPV sequence variants that harbored mutations, especially in the E6 gene, many of which culminated in non-conservative amino acid substitutions in the transforming E6 oncoprotein. Such an approach of coupling PCR with cycle sequencing permits the determination of many known and even novel HPV types associated with varying degrees of risk to cervical carcinogenesis, and enables the identification of HPV sequence variants of putative biological and clinical significance, thus justifying its utility as an adjunct tool to complement cervical cytology and colposcopy. This study also emphasises the need for educational, interventional and behavioral modification to minimise HPV transmission, such as through consistent condom usage among sex workers.

Many different papillomaviruses have low transcriptional activity in spite of strong epithelial specific enhancers.

Transcription of the E6-E7 genes of human papillomavirus type 11 (HPV-11), HPV-16 and HPV-18 is specific to epithelial cells. This mechanism originates from synergism between different transcription factors such as AP-1, NFI and Sp1, which occur in many different cell types, but whose activity is biased in favour of epithelial cells. In this study, the transcriptional regulation of 14 different papillomavirus types in the absence of the viral E2 transcription factor was compared. Genital HPV types, including high-risk, low-risk and common wart-associated HPVs, were found to have strong epithelial specific enhancers, irrespective of mucosal or skin target cell and pathology. Skin specific non-genital HPVs, like HPV-1 and HPV-8, as well as bovine papillomavirus type 4 (BPV-4), had much lower enhancer activity. Contiguous genomic segments including the enhancer and the E6 promoter of genital as well as non-genital papillomaviruses generally had very low transcriptional activities, presumably due to silencers between enhancer and promoter sequences. This generalization applies to all cell types tested in spite of significant quantitative differences between the cervical carcinoma-derived cell line HeLa, the skin-derived cell line HaCat, undifferentiated and differentiated primary keratinocytes. The only enhancer with activity in fibroblasts was identified in BPV-1, apparently a reflection of the broader target cell specificity of this virus. The low transcriptional activity of papillomaviruses most likely reflects the low gene expression required during most or even all parts of the life-cycle of these viruses.

High risk genital papillomavirus infections are not spread vertically

The Medline-indexed literature on risk factors for HPV infection and HPV transmission is critically reviewed. Principles for assay validation and interpretation, reliability of different study designs and principles for interpretation of conflicting reports are discussed. The conclusions arrived at can be summarised as: (1) There is overwhelming epidemiological evidence that the only quantitatively important mode of transmission of infection with oncogenic genital HPV types is sexual. (2) There is also evidence that benign genital HPV types can be transmitted sexually, but the epidemiological data on the benign virus types are less extensive and less clear. (3) Perinatal HPV transmission is unequivocally demonstrated only for the rare disease juvenile respiratory papillomatosis. Copyright © 1999 John Wiley & Sons, Ltd.

High risk genital papillomavirus infections are not spread vertically.

The Medline-indexed literature on risk factors for HPV infection and HPV transmission is critically reviewed. Principles for assay validation and interpretation, reliability of different study designs and principles for interpretation of conflicting reports are discussed. The conclusions arrived at can be summarised as: (1) There is overwhelming epidemiological evidence that the only quantitatively important mode of transmission of infection with oncogenic genital HPV types is sexual. (2) There is also evidence that benign genital HPV types can be transmitted sexually, but the epidemiological data on the benign virus types are less extensive and less clear. (3) Perinatal HPV transmission is unequivocally demonstrated only for the rare disease juvenile respiratory papillomatosis.

In Vitro Infection and Type-Restricted Antibody-Mediated Neutralization of Authentic Human Papillomavirus Type 16

Human papillomavirus type 16 (HPV-16) is strongly associated with the development of cervical cancer. Studies of model systems with animal papillomaviruses have demonstrated the importance of neutralizing antibodies in preventing papillomavirus-associated disease. The assessment of neutralizing antibody responses against HPV-16, previously hampered by the lack of a viral source, was enabled by the recent propagation of an HPV-16 stock in xenografted severe combined immunodeficiency (SCID) mice. HPV-16 infection of an immortalized human keratinocyte cell line was demonstrated by detection of an HPV-16-specific spliced mRNA amplified by reverse transcriptase PCR. Infection was blocked by preincubation of the virus with antiserum generated against HPV-16 virus-like particles (VLPs) composed of the major capsid protein, L1. To examine potential cross-neutralizing activity among the different genital HPV types, rabbit antisera to L1 VLPs corresponding to HPV-6, -11, -18, -31, -33, -35, -39, and -45 were assayed for the ability to block the HPV-16 infection of cultured cells. Antiserum raised against HPV-33 L1 VLPs was the only heterologous antiserum which inhibited HPV-16 infection. Thus, a neutralization assay for HPV-16 may help to characterize the components required to compose a broadly efficacious genital HPV vaccine.

Identification of cis-regulatory elements in the upstream regulatory region of human papillomavirus type 59

Human papillomavirus type 59 (HPV-59) was cloned from a vulvar intraepithelial neoplasia and the complete nucleotide sequence was determined. This virus is closely related to HPV-18 and -39 (60% homology in nucleotide sequence) and is grouped with the genital HPV types. In the present paper, we demonstrate that the HPV-59 E2 transactivator represses its E6 promoter-mediated transcription. We have also analyzed cis-regulatory elements in the upstream regulatory region (URR) of HPV-59 using chloramphenicol acetyl transferase assays as well as electrophoresis mobility shift assays (EMSA). The results allow for a subdivision of the HPV-59 URR into three regions of activity: distal (nt 7149–7493), central (nt 7493–7742), and proximal (nt 7742–7748). In particular, the 250 bp (nt 7493–7742) of the central region plays an important role as a constitutive enhancer element for the maximal transcription of the E6 promoter. Our results suggest that the transcription factors AP1, Oct1, SP1 and unidentified factors bind to the HPV-59 E6 promoter region, whereas NF1, GRE and TFIID fail to bind despite the presence of putative binding sites in the DNA sequence.

CLINICAL MANIFESTATIONS AND NATURAL HISTORY OF GENITAL HUMAN PAPILLOMAVIRUS INFECTIONS

Over the past decade, the ever-increasing volume of evidence implicating HPV types in genital neoplasia has stimulated much research interest into all aspects of the biology of this interesting group of viruses. This research has led to the identification of growing heterogeneity of HPV types. It is not surprising, therefore, that the clinical profile of disease associated with genital HPV types is much broader than previously recognized. Knowledge of this clinical spectrum is mandatory to the understanding of the possible role of specific HPV types in human carcinogenesis.

In vitro generation and type-specific neutralization of a human papillomavirus type 16 virion pseudotype.

We report a system for generating infectious papillomaviruses in vitro that facilitates the analysis of papillomavirus assembly, infectivity, and serologic relatedness. Cultured hamster BPHE-1 cells harboring autonomously replicating bovine papillomavirus type 1 (BPV1) genomes were infected with recombinant Semliki Forest viruses that express the structural proteins of BPV1. When plated on C127 cells, extracts from cells expressing L1 and L2 together induced numerous transformed foci that could be specifically prevented by BPV neutralizing antibodies, demonstrating that BPV infection was responsible for the focal transformation. Extracts from BPHE-1 cells expressing L1 or L2 separately were not infectious. Although Semliki Forest virus-expressed L1 self-assembled into virus-like particles (VLPs), viral DNA was detected in particles only when L2 was coexpressed with L1, indicating that genome encapsidation requires L2. Expression of human papillomavirus type 16 (HPV16) L1 and L2 together in BPHE-1 cells also yielded infectious virus. These pseudotyped virions were neutralized by antiserum to HPV16 VLPs derived from European (114/K) or African (Z-1194) HPV16 variants but not by antisera to BPV VLPs, to a poorly assembling mutant HPV16 L1 protein, or to VLPs of closely related genital HPV types. Extracts from BPHE-1 cells coexpressing BPV L1 and HPV16 L2 or HPV16 L1 and BPV L2 were not infectious. We conclude that (i) mouse C127 cells express the cell surface receptor for HPV16 and are able to uncoat HPV16 capsids; (ii) if a papillomavirus DNA packaging signal exists, then it is conserved between the BPV and HPV16 genomes; (iii) functional L1-L2 interaction exhibits type specificity; and (iv) protection by HPV virus-like particle vaccines is likely to be type specific.

Detection of genital human papillomaviruses by polymerase chain reaction amplification with degenerate nested primers.

Degenerate oligonucleotide primers were designed for in vitro amplification by polymerase chain reaction (PCR) of a relatively well-conserved portion of the L1 capsid protein gene of genital human papillomaviruses. A specific 441bp fragment was amplified by PCR from genomic clones and clinical biopsy specimens containing DNA from HPV types 6, 11, 16, 18, 31, and 33, as well as from a number of other clinical specimens known to contain unclassified HPV isolates. As some HPV non-specific DNA was also often amplified, another set of degenerate primers was designed which amplified a 335 bp sequence contained within the 441 bp sequence. These nested primers could be used in a two-stage PCR reaction to obtain distinct HPV-specific DNA fragments suitable for direct sequencing. Two-stage PCR was used to demonstrate the presence of HPV DNA in 13 out of 16 biopsies, which had been classified histologically as cervical intraepithelial neoplasia (CIN), but which were negative for HPV on Southern blot hybridization. Seven of these amplification products were sequenced, and one proved to be a previously unsequenced HPV type. The results have important implications for the routine detection and typing of genital and other HPV types in clinical samples.