Distinct Human Papillomavirus Research Articles

HPV-associated diseases

Nearly 200 distinct human papilloma viruses (HPVs) have now been recognized, and each is associated with a specific set of clinical lesions. They are associated with a spectrum of diseases, from benign verrucae vulgares and condylomata acuminata to the malignancies of the cervix, vulva, anus, and penis. Disease associated with HPV can be divided into skin and mucosal lesion of the genital and extragenital regions. The relationship between HPV and nonmelanoma skin cancer (NMSC) is important clinically, because NMSC is the most common form of malignancy among fair-skinned populations. HPVs have also been detected in skin tags, lichen sclerosus, seborrheic keratoses, actinic keratoses, epidermal cysts, psoriatic plaques, and plucked hairs, but cutaneous HPV can be found on healthy skin.

Human papillomavirus genotypes distribution in 175 invasive cervical cancer cases from Brazil

BackgroundInvasive cervical cancer is the second most common malignant tumor affecting Brazilian women. Knowledge on Human Papillomavirus (HPV) genotypes in invasive cervical cancer cases is crucial to guide the introduction and further evaluate the impact of new preventive strategies based on HPV. We aimed to provide updated comprehensive data about the HPV types’ distribution in patients with invasive cervical cancer.MethodsFresh tumor tissue samples of histologically confirmed invasive cervical cancer were collected from 175 women attending two cancer reference hospitals from São Paulo State: ICESP and Hospital de Câncer de Barretos. HPV detection and genotyping were performed by the Linear Array HPV Genotyping Test (Roche Molecular Diagnostics, Pleasanton,USA).Results170 out of 172 valid samples (99%) were HPV DNA positive. The most frequent types were HPV16 (77.6%), HPV18 (12.3%), HPV31 (8.8%), HPV33 (7.1%) and HPV35 (5.9%). Most infections (75%) were caused by individual HPV types. Women with adenocarcinoma were not younger than those with squamous cell carcinoma, as well, as women infected with HPV33 were older than those infected by other HPV types. Some differences between results obtained in the two hospitals were observed: higher overall prevalence of HPV16, absence of single infection by HPV31 and HPV45 was verified in HC-Barretos in comparison to ICESP patients.ConclusionsTo our knowledge, this is one of the largest studies made with fresh tumor tissues of invasive cervical cancer cases in Brazil. This study depicted a distinct HPV genotype distribution between two centers that may reflect the local epidemiology of HPV transmission among these populations. Due to the impact of these findings on cervical cancer preventive strategies, extension of this investigation to routine screening populations is warranted.

Revealing highly conserved regions in the E6 protein among distinct human papillomavirus types using comparative analysis of multiple sequence alignments

Several epidemiological and molecular studies have confirmed that cervical infection by certain human papillomaviruses (HPV) types is a precursor of the genesis of cervical neoplasia (Gravitt, 2011; Guzman-Olea et al., 2012). It is yet well elucidated that high risk genotypes lead to the development of cervical cancer and are also associated with other mucosal anogenital, head and neck tumors. The HPV genome is approximately 8 kb in length and is divided into three regions, the non-coding long control region (LCR, ~1 kb), and the protein coding early (E, ~4 kb) and late (L, ~3 kb) regions (Ganguly and Parihar, 2009). The viral genome encodes six early (E1, E2, E4, E5, E6 and E7) and two late (L1 and L2) proteins. HPV

Coexisting anal human papilloma virus infection in heterosexual women with cervical HPV infection

To evaluate the prevalence of coexisting anal human papillomavirus (HPV) infection and concordance of HPV types in women with cervical HPV infection and to investigate the possible predictors for anal HPV infection. Study group was composed of women referred with documented cervical HPV infection. All patients had undergone anal HPV DNA testing. One hundred and six patients presenting with cervical HPV infection were eligible for the study. Overall, 24 and 20 distinct HPV genotypes were detected from cervical and anal specimens, respectively. We observed a considerably high prevalence (51.9%) of coexisting anal HPV infection in our study group. A majority of the women who were found to have anal HPV infection were infected with oncogenic or probable oncogenic types (64.6%). There were 20.0 and 58.3% rate of total and partial concordance between the two sites, respectively. We found that no demographic parameter but history of anal intercourse was related with the risk of anal HPV infection. Women with cervical HPV infection have a considerable risk for coexisting anal HPV infection. Concordance of HPV types infecting the cervix and anal canal is relatively high. There is no strong predictor for anal HPV infection in this population.

Comparative analysis of virus–host interactomes with a mammalian high-throughput protein complementation assay based on Gaussia princeps luciferase

Comparative interactomics is a strategy for inferring potential interactions among orthologous proteins or “interologs”. Herein we focus, in contrast to standard homology-based inference, on the divergence of protein interaction profiles among closely related organisms, showing that the approach can correlate specific traits to phenotypic differences. As a model, this new comparative interactomic approach was applied at a large scale to human papillomaviruses (HPVs) proteins. The oncogenic potential of HPVs is mainly determined by the E6 and E7 early proteins. We have mapped and overlapped the virus–host protein interaction networks of E6 and E7 proteins from 11 distinct HPV genotypes, selected for their different tropisms and pathologies. We generated robust and comprehensive datasets by combining two orthogonal protein interaction assays: yeast two-hybrid (Y2H), and our recently described “high-throughput Gaussia princeps protein complementation assay” (HT-GPCA). HT-GPCA detects protein interaction by measuring the interaction-mediated reconstitution of activity of a split G. princeps luciferase. Hierarchical clustering of interaction profiles recapitulated HPV phylogeny and was used to correlate specific virus–host interaction profiles with pathological traits, reflecting the distinct carcinogenic potentials of different HPVs. This comparative interactomics constitutes a reliable and powerful strategy to decipher molecular relationships in virtually any combination of microorganism–host interactions.

Genome-wide methylation and expression differences in HPV(+) and HPV(-) squamous cell carcinoma cell lines are consistent with divergent mechanisms of carcinogenesis

Oncogenic human papillomaviruses (HPV) are associated with nearly all cervical cancers and are increasingly important in the etiology of oropharyngeal tumors. HPV-associated head and neck squamous cell carcinomas (HNSCC) have distinct risk profiles and appreciate a prognostic advantage compared to HPV-negative HNSCC. Promoter hypermethylation is widely recognized as a mechanism in the progression of HNSCC, but the extent to which this mechanism is consistent between HPV(+) and HPV(-) tumors is unknown. To investigate the epigenetic regulation of gene expression in HPV-induced and carcinogen-induced cancers, we examined genome-wide DNA methylation and gene expression in HPV(+) and HPV(-) SCC cell lines. We used two platforms: the Illumina Infinium Methylation BeadArray and tiling arrays, and confirmed illustrative examples with pyrosequencing and quantitative PCR. These analyses indicate that HPV(+) cell lines have higher DNA methylation in genic and LINE-1 regions than HPV(-) cell lines. Differentially methylated loci between HPV(+) and HPV(-) cell lines significantly correlated with HPV-typed HNSCC primary tumor DNA methylation levels. Novel findings include higher promoter methylation of polycomb repressive complex 2 target genes in HPV(+) cells compared to HPV(-) cells and increased expression of DNMT3A in HPV(+) cells. Additionally, CDKN2A and KRT8 were identified as interaction hubs among genes with higher methylation and lower expression in HPV(-) cells. Conversely, RUNX2, IRS-1 and CCNA1 were major hubs with higher methylation and lower expression in HPV(+) cells. Distinct HPV(+) and HPV(-) epigenetic profiles should provide clues to novel targets for development of individualized therapeutic strategies.

Multiple Human Papillomavirus Infections: The Exception or the Rule?

In this issue of the Journal of Infectious Diseases, Chaturvedi et al [1] report on a large epidemiological study of human papillomavirus (HPV) infection focusing on a topic of increasing interest in this field. HPV is recognized as a necessary cause of cervical cancer, but there are multiple oncogenic HPV types that may be found together in the same woman. What do such multiple infections mean, and how can they be analyzed in the context of epidemiological studies? In the earliest studies of HPV, multiple infections were seldom detected [2]. This is likely to be due to the characteristics of the earliest diagnostic tests. Some, such as Hybrid Capture 2 (HC2) (Qiagen, Gaithersburg, MD), do not distinguish individual HPV types, and the first polymerase chain reaction (PCR)–based assays included relatively few types other than HPV-16 and HPV18. In the past decade, however, PCRassays have become available that can detect broader ranges of oncogenic and nononcogenic HPV types (n . 40) and, most notably, have much higher analytical sensitivity (ie, ability to identify correctly HPV-positive women) [3, 4]. In a recent cross-wise comparison of 4 widely used PCR assays among women who had previously tested positive by HC2, the rate of detection of HPV infection ranged from 62.6% to 91.4%, and the proportion of multiple infections among all HPV-positive tests ranged from 24.8% to 52.6% [5]. This shows that there can be substantial disagreement even between high-quality PCR assays. The concordance between tests greatly increased, however, with increasing viral load and when the presence of cervical intra-epithelial neoplasia 2 or worse (CIN21) rather than HPV infection was set as an end point (clinical sensitivity) [5], [6]. There is no doubt that high clinical sensitivity and specificity are the most important features for an HPV test to help determine follow-up and treatment of HPV-positive women. Conversely, the use of a test with very high analytical sensitivity may be undesirable in cervical cancer screening programs, because it could lead to overdetection of infections that have no clinical consequences [6]. The shift toward highly sensitive PCR assays has, however, affected HPV findings in the most recent epidemiological studies in a way that cannot be ignored. Notably, (1) HPV infections are apparently more widespread than had previously been suggested,(2) coinfection with multiple types is very common in sexually active women and even more common than infection with single types among immunosuppressed human immunodeficiency virus (HIV)–infected women [7], and (3) the fraction of multiple HPV infections found in invasive cervical carcinomas has greatly increased during the past 2 decades, from 4.0% to 15.7% [8], despite the fact that this is a monoclonal disease that should have been caused by 1 HPV type only. The increasing frequency of detection of multiple HPV infections is, at present, complicating the estimate of the benefits of tests and vaccines including more or less broad ranges of HPV types [8], [3], [9], but it has also raised great interest in the biological implications of coinfections [10–14]. Much of the research on multiple HPV infections addresses 2 distinct questions. The first question considers the effects of infections with distinct HPV types on each other, either in terms of concurrent infections or sequential infections. The second question concerns the impact of multiple-type infections on the risk of future cervical disease, which, in screened populations, means CIN21. Both questions are addressed by Chaturvedi et al [1] in a study of 2478 HPV-positive young women, of whom 43.2% were infected with multiple HPV types. Received 29 October 2010; accepted 1 November 2010. Potential conflicts of interest: none reported. Reprints or correspondence: Silvia Franceschi, MD, International Agency for Research on Cancer, 150 cours Albert Thomas, 69372 Lyon cedex 08, France (franceschiS @iarc.fr). The Journal of Infectious Diseases 2011;203:891–3 The Author 2011. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com 1537-6613/2011/2037-0001$15.00 DOI: 10.1093/infdis/jiq146

HLA-Cw group 1 ligands for KIR increase susceptibility to invasive cervical cancer.

Inherited genetic polymorphisms within immune response genes have been shown to associate with risk of invasive cervical cancer (ICC) and its immediate precursor, cervical intraepithelial neoplasia grade 3. Here, we used the transmission/disequilibrium test to detect disease-liability alleles and investigate haplotype transmission of KIR and HLA class I polymorphisms in a large family-based population of women with cervical cancer and their biological parents (359 trios). The effect of distinct human papillomavirus types was also explored. HLA-Cw group 1 (HLA-Cw alleles with asparagine at position 80), which serves as ligand for certain killer immunoglobulin-like receptors (KIR), was significantly overtransmitted in women with ICC (P = 0.04), and particularly in the subgroup of women infected with high risk HPV16 or 18 subtypes (P = 0.008). These data support the involvement of the HLA-C locus in modulating the risk of cervical neoplasia perhaps through its function as ligands for KIR, but functional studies are essential to confirm this hypothesis.

Human papillomavirus (HPV) genotypes among HIV-infected and HIV-uninfected women in Mozambique

identified among the 141 HIV-infected women, of which 16 viral types were classified as cancer high-risk or probable high-risk viruses. The most common types identified were HPV types 58 (12.1%, p < 0.001), 16 (10.7%, p = 0.06), 61 (8.4%, p < 0.05), 53 (7.9%, p = 0.55) and 6 (7.6%, p = 0.41). Nineteen distinct HPV genotypes were identified among the 50 HIV-uninfected women. Ten types were of high-risk or probable high-risk. The most common types identified in HIV-uninfected subjects were HPV types 53 (10.4%), 6 (9.1%), 16 (9.1%), 18 (9.1%) and 66 (7.8%). Discussion HPV infection was mainly associated with HIV positive status. HPV multiple infections were high in the population studied, independently of HIV status. HPV Genotypes are different in the two groups. The HPV types identified are partially different from those more commonly identified in Western countries.

Human Papillomavirus Genotyping Using an Automated Film-Based Chip Array

The INFINITI HPV-QUAD assay is a commercially available genotyping platform for human papillomavirus (HPV) that uses multiplex PCR, followed by automated processing for primer extension, hybridization, and detection. The analytical performance of the HPV-QUAD assay was evaluated using liquid cervical cytology specimens, and the results were compared with those results obtained using the digene High-Risk HPV hc2 Test (HC2). The specimen types included Surepath and PreservCyt transport media, as well as residual SurePath and HC2 transport media from the HC2 assay. The overall concordance of positive and negative results following the resolution of indeterminate and intermediate results was 83% among the 197 specimens tested. HC2 positive (+) and HPV-QUAD negative (-) results were noted in 24 specimens that were shown by real-time PCR and sequence analysis to contain no HPV, HPV types that were cross-reactive in the HC2 assay, or low virus levels. Conversely, HC2 (-) and HPV-QUAD (+) results were noted in four specimens and were subsequently attributed to cross-contamination. The most common HPV types to be identified in this study were HPV16, HPV18, HPV52/58, and HPV39/56. We show that the HPV-QUAD assay is a user friendly, automated system for the identification of distinct HPV genotypes. Based on its analytical performance, future studies with this platform are warranted to assess its clinical utility for HPV detection and genotyping.

Distinct human papillomavirus type 16 methylomes in cervical cells at different stages of premalignancy

Human papillomavirus (HPV) gene expression is dramatically altered during cervical carcinogenesis. Because dysregulated genes frequently show abnormal patterns of DNA methylation, we hypothesized that comprehensive mapping of the HPV methylomes in cervical samples at different stages of progression would reveal patterns of clinical significance. To test this hypothesis, thirteen HPV16-positive samples were obtained from women undergoing routine cervical cancer screening. Complete methylation data were obtained for 98.7% of the HPV16 CpGs in all samples by bisulfite-sequencing. Most HPV16 CpGs were unmethylated or methylated in only one sample. The other CpGs were methylated at levels ranging from 11% to 100% of the HPV16 copies per sample. The results showed three major patterns and two variants of one pattern. The patterns showed minimal or no methylation (A), low level methylation in the E1 and E6 genes (B), and high level methylation at many CpGs in the E5/L2/L1 region (C). Generally, pattern A was associated with negative cytology, pattern B with low-grade lesions, and pattern C with high-grade lesions. The severity of the cervical lesions was then ranked by the HPV16 DNA methylation patterns and, independently, by the pathologic diagnoses. Statistical analysis of the two rating methods showed highly significant agreement. In conclusion, analysis of the HPV16 DNA methylomes in clinical samples of cervical cells led to the identification of distinct methylation patterns which, after validation in larger studies, could have potential utility as biomarkers of neoplastic cervical progression.

Difficulties in Estimating the Male-to-Female Sexual Transmissibility of Human Papillomavirus Infection

Estimates of transmissibility of infectious agents such as human papillomavirus (HPV) are important for developing preventive measures and determining the impact of such interventions. Based on the results of epidemiologic studies, HPV appears to be highly transmissible;1–8 however, empirical data on HPV transmissibility are lacking.9–11 We sought to design and conduct a study among couples to provide the first empirical estimate of male-to-female sexual transmissibility of HPV. However, we faced several difficulties in achieving this goal. Here we present the design and descriptive results of this study in brief and these difficulties in detail. Information on study population arose from a larger study described in detail elsewhere.5 Briefly, between December 2000 and November 2006, 244 women (age range: 18–22) were recruited to a longitudinal study of HPV infection through invitational letters mailed to 24,201 female University of Washington undergraduates and informational flyers at the student health clinic. Participants were eligible to enroll if they had never had vaginal intercourse with a male partner (i.e., virgin) or had first had intercourse with one male partner within the previous three months (i.e., peri-virgin). Consenting women were asked to refer their first male partner to the study and were given a letter of recruitment to forward to him. Every two weeks, women completed a Web-based diary designed to capture daily sexual behavior information such as date and number of acts of intercourse with a new partner.5, 12 Women were followed-up with gynecological examinations every four months. At each visit, self-collected vaginal swabs and clinician-collected cervical and vulvovaginal swabs were collected. Men were seen only for one visit. They underwent a clinician-administered interview about their medical and sexual history and a genital examination including specimen collection from the penis, urethra, and scrotal area, described in detail elsewhere.13 All samples were tested for HPV DNA by polymerase chain reaction (PCR)-based methods. Specimens testing positive by a generic probe were typed using a reverse line-blot assay (Roche Molecular Systems) for 37 distinct HPV types.14 PCR results of vaginal, cervical, and vulvovaginal swabs for women and penile, urethral, and scrotal swabs for men were combined to determine genital HPV status in each participant. Institutional Review Board of the University of Washington approved the study; informed consent was obtained from all participants. Male partners were enrolled for 33 women. Information on 19 couples was excluded due to the following reasons: one woman did not report any sexual activity in the diary; three men were not the first sex partner of their female partner; and 15 couples were not found to be infected with HPV. Of the remaining 14 couples, both partners were found to be infected with HPV in five couples and the male partner only was found to be infected in three couples. Descriptive results for these eight couples and for the other six couples in whom, unexpectedly, the female partner was found to be infected with HPV, while the male partner was not, are provided in Tables 1 and ​and22. Table 1 Characteristics of eight couples in whom at least one type of HPV was detected in the single visit of the first male partner Table 2 Characteristics of six couples in whom at least one type of HPV was detected in the female partner but not in the male partner Investigators who embark upon designing a methodologically sound study to find empirical estimates of male-to-female sexual transmissibility of HPV may face the following difficulties:

Role for Wee1 in Inhibition of G 2 -to-M Transition through the Cooperation of Distinct Human Papillomavirus Type 1 E4 Proteins

The infectious cycle of human papillomavirus type 1 (HPV1) is accompanied by abundant expression of the full-length E1;E4 protein (17-kDa) and smaller E4 polypeptides (16-, 11-, and 10-kDa) that arise by sequential loss of N-terminal E1;E4 sequences. HPV1 E4 inhibits G(2)-to-M transition of the cell cycle. Here, we show that HPV1 E4 proteins mediate inhibition of cell division by more than one mechanism. Cells arrested by coexpression of E1;E4 (E4-17K) and a truncated protein equivalent to the 16-kDa species (E4-16K) contain inactive cyclin B1-cdk1 complexes. Inactivation of cdk1 is through inhibitory Tyr(15) phosphorylation, with cells containing elevated levels of Wee1, the kinase responsible for inhibitory cdk1 phosphorylation. Consistent with these findings, overexpression of Wee1 enhanced the extent to which E4-17K/16K-expressing cells arrest in G(2), indicating that maintenance of Wee1 activity is necessary for inhibition of cell division induced by coexpression of the two E4 proteins. Moreover, we have determined that depletion of Wee1 by small interfering RNA (siRNA) alleviates the G(2) block imposed by E4-17K/16K. In contrast however, maintenance of Wee1 activity is not necessary for G(2)-to-M inhibition mediated by E4-16K alone, as overexpression or depletion of Wee1 does not influence the G(2) arrest function of E4-16K. Cells arrested by E4-16K expression contain low levels of active cyclin B1-cdk1 complexes. We hypothesize that differential expression of HPV1 E4 proteins during the viral life cycle determines the host cell cycle status. Different mechanisms of inhibition of G(2)-to-M transition reinforce the supposition that distinct E4 functions are important for HPV replication.

Detection of epidermodysplasia verruciformis-associated human papillomavirus DNA in nongenital seborrhoeic keratosis

DNA of epidermodysplasia verruciformis (EV)-associated human papillomaviruses (HPVs) has been widely detected in lesions of malignant skin tumours, benign tumours and other proliferative diseases of epithelial origin. To investigate the presence of EV-associated HPV DNA in nongenital seborrhoeic keratosis (SK) and to elucidate the prevalence of distinct HPV genotypes. We investigated HPV DNA in 55 nongenital SK biopsies, which were compared with 48 normal skin biopsies (healthy controls) using a nested polymerase chain reaction (PCR) using consensus primers CP65/CP70 and CP66/CP69. The positive PCR products were retracted and used to prepare recombination clones with T-vector. Distinct clones were analysed with endonucleases, and HPV genotypes were identified by direct sequencing. EV-associated HPV DNA was detected in 42 of 55 (76%) nongenital SK biopsies vs. only 13 of 48 (27%) healthy controls (chi2 = 22.087; P < 0.005). The prevalence was higher in patients with more than five lesions than in those with only one lesion (P < 0.05). Ten distinct HPV genotypes were detected in the nongenital SK biopsies: HPV 20, 23, 5, renal transplant recipient (RTR) X7, HPV 17, 37, 17b, RTRX4, RTRX4b and strain SK3. HPV 20 was found in 26 of 42 (62%) positive specimens, followed by HPV 23 (11 of 42, 26%) and HPV 5 (six of 42, 14%). Existence of multiple HPV genotypes was observed in 12 of 42 (29%) positive specimens. In healthy controls, five genotypes of EV-associated HPV (HPV 20, 23, 5, 17 and RTRX4) were detected, with the same predominant genotype of HPV 20 (five of 13, 38%). Several distinct HPV genotypes were found to coexist in four of 13 (31%) positive specimens. This study provides some evidence that EV-associated HPVs might play a part in the pathogenesis of nongenital SK.

High Prevalence of Cutaneous Human Papillomavirus DNA on the Top of Skin Tumors but not in “Stripped” Biopsies from the Same Tumors

Genomes of human papillomaviruses (HPV) are common in biopsies from non-melanoma skin cancers but are also found on healthy skin and it is possible that HPV positivity in tumor biopsies by PCR may merely reflect contamination of the lesion surface. To investigate this issue, 229 immunocompetent patients were tested for HPV DNA in swab samples collected on top of skin tumors and in biopsies of the same tumors, obtained after stripping with tape to remove superficial layers. HPV DNA was detected on top of 69% (159 of 229) of the lesions, and in 12% (28 of 229) of the stripped biopsies (p<0.001). The difference was seen for all four types of tumors studied. Seborrheic keratosis had 79% (34 of 43) HPV positivity on top of lesions versus 19% (eight of 43) in biopsies; actinic keratosis had 83% (38 of 46) HPV positivity on top versus 11% (five of 46) in biopsies; basal cell carcinoma had 63% (69 of 109) on top versus 8% (nine of 109) in biopsies and squamous cell carcinoma had 58% (18 of 31) on top versus 19% (six of 31) in biopsies. HPV DNA is common in superficial layers of lesions, but is not necessarily present throughout tumors.

Human papillomaviruses

Human papillomaviruses (HPV) infect exclusively the basal cells of the skin and of mucosal epithelia adjacent to the skin such as the mouth, the upper respiratory tract, the lower genital tract and the anal canal. HPV does not lead to a viremia. Basically there are three different types of HPV infection: Clinically visible lesions, subclinical HPV infections and latent HPV infections. Distinct HPV types induce morphologically and prognostically different clinical pictures. The most common HPV associated benign tumor of the skin is the common wart. Infections of the urogenitoanal tract with specific HPV-types are recognised as the most frequent sexually transmitted viral infections. So-called "high-risk" HPV-types (HPV16, 18 and others) are regarded by the world health organisation as important risk-factors for the development of genital cancer (mainly cervical cancer), anal cancer and upper respiratory tract cancer in both genders. Antiviral substances with a specific anti-HPV effect are so far unknown. Conventional therapies of benign skin warts and of mucosal warts are mainly nonspecific. They comprise tissue-destroying therapies such as electrocautery, cryotherapy and laser. In addition cytotoxic substances such as podophyllotoxin and systemic therapy with retinoids are in use. Systemically and topically administered immunotherapies represent a new approach for treatment. Both interferons and particularly the recently developed imiquimod, an interferon-alpha and cytokine-inductor lead to better results and are better tolerated then conventional therapies. HPV-specific vaccines have been developed in the last 5 years and will be used in future for prevention and treatment of benign and malignant HPV-associated tumors of the genitoanal tract in both sexes.

Hybrid papillomavirus L1 molecules assemble into virus-like particles that reconstitute conformational epitopes and induce neutralizing antibodies to distinct HPV types.

Human papillomavirus (HPV) hybrid virus-like particles (VLPs) were prepared using complementary regions of the major capsid L1 proteins of HPV-11 and -16. These hybrid L1 proteins were tested for assembly into VLPs, for presentation and mapping of conformational neutralizing epitopes, and as immunogens in rabbits and mice. Two small noncontiguous hypervariable regions of HPV-16 L1, when replaced into the HPV-11 L1 backbone, produced an assembly-positive hybrid L1 which was recognized by the type-specific, conformationally dependent HPV-16 neutralizing monoclonal antibody (N-MAb) H16.V5. Several new N-MAbs that were generated following immunization of mice with wild-type HPV-16 L1 VLPs also recognized this reconstructed VLP, demonstrating that these two hypervariable regions collectively constituted an immunodominant epitope. When a set of hybrid VLPs was tested as immunogens in rabbits, antibodies to both HPV-11 and -16 wild-type L1 VLPs were obtained. One of the hybrid VLPs containing hypervariable FG and HI loops of HPV-16 L1 replaced into an HPV-11 L1 background provoked neutralizing activity against both HPV-11 and HPV-16. In addition, conformationally dependent and type-specific MAbs to both HPV-11 and HPV-16 L1 VLP were obtained from mice immunized with hybrid L1 VLPs. These data indicated that hybrid L1 proteins can be constructed that retain VLP-assembly properties, retain type-specific conformational neutralizing epitopes, can map noncontiguous regions of L1 which constitute type-specific conformational neutralizing epitopes recognized by N-MAbs, and trigger polyclonal antibodies which can neutralize antigenically unrelated HPV types.

Degenerate and nested PCR: a highly sensitive and specific method for detection of human papillomavirus infection in cutaneous warts.

The role of human papillomavirus (HPV) in anogenital carcinogenesis is firmly established, but evidence that supports a similar role in skin remains speculative. Immunosuppressed renal transplant recipients have an increased incidence of viral warts and nonmelanoma skin cancer, and the presence of HPV DNA in these lesions, especially types associated with the condition epidermodysplasia verruciformis (EV), has led to suggestions that HPV may play a pathogenic role. However, differences in the specificities and sensitivities of techniques used to detect HPV in skin have led to wide discrepancies in the spectrum of HPV types reported. We describe a degenerate nested PCR technique with the capacity to detect a broad spectrum of cutaneous, mucosal, and EV HPV types. In a series of 51 warts from 23 renal transplant recipients, this method detected HPV DNA in all lesions, representing a significant improvement over many previously published studies. Cutaneous types were found in 84.3% of warts and EV types were found in 80.4% of warts, whereas mucosal types were detected in 27.4% of warts. In addition, the method allowed codetection of two or more distinct HPV types in 94.1% of lesions. In contrast, single HPV types were detected in all but 1 of 20 warts from 15 immunocompetent individuals. In summary, we have established a highly sensitive and comprehensive degenerate PCR methodology for detection and genotyping of HPV from the skin and have demonstrated a diverse spectrum of multiple HPV types in cutaneous warts from transplant recipients. Studies designed to assess the significance of these findings to cutaneous carcinogenesis are under way.

Association of rare human papillomavirus types with genital premalignant and malignant lesions.

Due to the limited number of reports concerning their association with particular dysplastic and neoplastic lesions, the oncogenic potential of so-called rare or novel human papillomavirus (HPV) types is still unclear. Cytologic smears or biopsy specimens from 538 patients were analyzed for dysplastic or neoplastic lesions and HPV infection. The HPV detection and typing system utilized allowed identification of all mucosal HPVs amplifiable by L1 polymerase chain reaction. Considering only patients infected with a single HPV type (n = 329), rare or novel HPVs (HPV-59, HPV-61, HPV-62, HPV-66, HPV-70, HPV-73, MM4, MM7, MM8, CP6108, and CP8304) were detected in 28% of normal specimens (n = 46), none of condylomatous lesions (n = 44), 12% of low-grade squamous intraepithelial lesions (SILs) (n = 42), 8% of high-grade SILs (n = 142), and 4% of cervical cancers (n = 54). Prevalence and oncogenic potential of distinct rare HPV types seems to be higher than previously assumed.

Detection of human papillomavirus in cervical intra-epithelial neoplasia, using in situ hybridization and various polymerase chain reaction techniques.

One hundred and forty-eight randomly chosen neutral-buffered formaldehyde-fixed cervical biopsies in which cervical intra-epithelial neoplasia (CIN) I-III had been diagnosed were tested for HPV (human papilloma virus) DNA by in situ hybridization (ISH) and polymerase chain reaction (PCR). For ISH, we utilized a biotinylated panprobe and type-specific, genomic probe sets. For PCR, we used the general primers GP5/GP6 and their recently described, elongated version GP5+/GP6+, and included the modification of hot-start PCR. Amplified DNA was detected by gel electrophoresis and slot blot hybridization. The positivity rate of ISH was 59% for all biopsies and 69%, 62% and 46% for CIN I, II and III, respectively. The sensitivity of GP5/GP6 was 74% with cold-start PCR and 78% with hot-start PCR. When GP5+/GP6+ was used, the sensitivity increased to 89% with cold-start PCR and to 95% with hot-start PCR. Based on the most sensitive PCR technique, HPV detection was 93%, 95% and 96% in CIN I, II and III, respectively. The number of HPV types decreased with the severity of the lesion, and HPV 16 was the predominant type. Multiple HPVs were rare and almost all HPV-positive cases could be typed. ISH and slot blot hybridization correlated well regarding HPV typing specificity. Our results confirm that distinct HPV types are present in a high proportion of cases of CIN. The sensitivity of ISH is lower than that of PCR. Furthermore, the modified general primers GP5+/GP6+ give a higher yield than GP5/GP6, while hot-start PCR increases sensitivity even further.