Seroprevalence of 34 human papillomavirus types in the German general population

The natural history of infections with many human papillomavirus (HPV) types is poorly understood. Here, we describe for the first time the age- and sex-dependent antibody prevalence for 29 cutaneous and five mucosal HPV types from 15 species within five phylogenetic genera (alpha, beta, gamma, mu, nu) in a general population. Sera from 1,797 German adults and children (758 males and 1,039 females) between 1 and 82 years (median 37 years) were analysed for antibodies to the major capsid protein L1 by Luminex-based multiplex serology. The first substantial HPV antibody reactions observed already in children and young adults are those to cutaneous types of the genera nu (HPV 41) and mu (HPV 1, 63). The antibody prevalence to mucosal high-risk types, most prominently HPV 16, was elevated after puberty in women but not in men and peaked between 25 and 34 years. Antibodies to beta and gamma papillomaviruses (PV) were rare in children and increased homogeneously with age, with prevalence peaks at 40 and 60 years in women and 50 and 70 years in men. Antibodies to cutaneous alpha PV showed a heterogeneous age distribution. In summary, these data suggest three major seroprevalence patterns for HPV of phylogenetically distinct genera: antibodies to mu and nu skin PV appear early in life, those to mucosal alpha PV in women after puberty, and antibodies to beta as well as to gamma skin PV accumulate later in life.     

Predominance of genomically defined A lineage of HPV16 over D lineage in Indian patients from eastern India with squamous cell carcinoma of the cervix in association with distinct oncogenic phenotypes

Human papillomavirus type-16 (HPV16) is classified into lineages, A, B, C and D and 10 sub-lineages portraying variable infectivity, persistence, and cytological outcomes, however, with geographical variations. Our objective was to delineate the distinctive features of lineages among cervical squamous cell carcinoma (SCC) in the eastern region of India. A total of 145 SCC cases and 24 non-malignant specimens, harboring episomal HPV16, were included. The presence of higher proportion of lineage A over D was observed among SCC cases (86.89% A1, 8.97% D1 and 4.14% D2), while only A1 sub-lineage viruses were found among control specimens. Among the A1 viruses, an association of variants in the E5 (Y44L, I65V), E6 (L83V) genes and LCR: C7577T with SCC, with combined Odd''s ratio (95% CI) of 20.5(4.61–91.25) was observed. Network analyses revealed the presence of 10 clades of lineage A viruses comprising of 64 HPV16 genomes harboring the risk alleles. High episomal HPV16 DNA copy numbers (adjusted p-value= 0.0271) and E7 mRNA expression (p-value=0.000017) predominated in SCC with lineage A, over D. Our study highlights the distinctive modalities of oncogenicity among different HPV16 lineages.     

Impact of Inhibitors and L2 Antibodies upon the Infectivity of Diverse Alpha and Beta Human Papillomavirus Types

The licensed human papillomavirus (HPV) vaccines elicit type-restricted immunity but do not target cutaneous HPV types of the beta genus that are associated with non-melanoma skin cancer in immune-compromised patients, and it is unclear if these diverse types share a common mechanism of infection. Residues 11-88 of minor capsid protein L2 contain cross-protective epitopes, and vaccination with concatamers of this region derived from as many as eight alpha HPV (L2 α11-88x8) is being developed as an alternative prophylactic vaccine with potentially broader efficacy. There is also interest in developing broadly protective topical microbicides, such as carrageenan or heparin that block HPV receptor interactions, or small molecule inhibitors of infection. Here we have examined several inhibitors of HPV infection and antisera to L2 α11-88x8 for their breadth of activity against infection by 34 HPV types from within both the alpha and beta families using pseudovirions (PsV) carrying a luciferase reporter as surrogates for native virus. We observed that both heparin and carrageenan prevented infection by mucosatropic HPV types, but surprisingly PsV of several epidermotropic alpha4 and beta HPV types exhibited increased infectivity especially at low inhibitor concentrations. Furin and γ-secretase inhibitors and L2 α11-88x8 antiserum blocked infection by all HPV PsV types tested. These findings suggest that the distinct tropism of mucosal and cutaneous HPV may reflect distinct cell surface receptor interactions, but a common uptake mechanism dependent upon furin and γ-secretase proteolytic activities. Carrageenan, which is being tested as a vaginal microbicide, broadly inhibited infection by the high-risk mucosatropic HPV PsV, but not most skin tropic alpha and beta HPV. Vaccination with an L2 multimer derived exclusively from alpha papillomavirus sequences induced antibodies that broadly neutralized PsV of all 34 HPVs from within both the alpha and beta families, suggesting each displays conserved L2 neutralizing epitopes.     

Comparative analysis of transforming properties of E6 and E7 from different beta human papillomavirus types

The cutaneous beta human papillomavirus (beta HPV) types appear to be involved in skin carcinogenesis. However, only a few beta HPVs have been investigated so far. Here, we compared the properties of E6 and E7 oncoproteins from six uncharacterized beta HPVs (14, 22, 23, 24, 36, 49). Only HPV49 E6 and E7 immortalized primary human keratinocytes and efficiently deregulated the p53 and pRb pathways. Furthermore, HPV49 E6, similarly to E6 from the oncogenic HPV16, promoted p53 degradation.     

Differential Expression of HPV16 L2 Gene in Cervical Cancers Harboring Episomal HPV16 Genomes: Influence of Synonymous and Non-Coding Region Variations

We tested the hypothesis that (i) synonymous variations within the coding regions, and (ii) variations within the non-coding regions of HPV, influence cervical cancer (CaCx) pathogenesis under the impact of intact HPV16 genomes. Whole genome sequence analysis of HPV16 isolates within 70 CaCx cases and 25 non-malignant samples revealed that synonymous variations were significantly higher within the E6 (p = 0.014), E5 (p = 0.001) and L2 (p = 0.0002) genes of HPV16 isolates within cases, compared to isolates within non-malignant samples. All of the 25 (100%) humanized codons identified within L2 ORF of the samples analyzed, were harbored by CaCx cases, while 8 out of 25 (32%) were harbored by HPV16 positive non-malignant samples (p = 3.87105E-07). L2 (mRNA and protein) expression was evident only among cases with episomal viral genomes and L2 mRNA expression correlated significantly with E2 gene copy numbers suggesting expression from all episomal genomes. Among such cases, Asian American (AA) isolates portrayed all of the humanized codons (100%; 4–6/sample) recorded within L2, which was significantly higher (p = 2.02E-7) compared to the European (E) isolates (22.8%; none or 1–2/sample). Additionally, majority of E variant isolates within cases (54/57; 94.7%) portrayed a variation (T4228C) within the short non-coding region (NCR2) between E5 and L2 genes, which portrays a weak promoter activity specific for L2 mRNA expression. This resulted in loss of 9 out of 14 miRNA binding sites (hsa-miR-548 family), despite the significant overexpression of miR548a-5p and miR548d-5p among such cases (28.64 and 36.25 folds, respectively), in comparison to HPV negative control samples. The findings exemplify the biological relevance of sequence variations in HPV16 genomes and highlight that episomal HPV16 in CaCx cases employ multiple mechanisms to sustain L2 expression, thereby justifying the potential role of L2 in such cancers, as opposed to those harboring viral integration.     

HPV-16 E2 contributes to induction of HPV-16 late gene expression by inhibiting early polyadenylation

We provide evidence that the human papillomavirus (HPV) E2 protein regulates HPV late gene expression. High levels of E2 caused a read-through at the early polyadenylation signal pAE into the late region of the HPV genome, thereby inducing expression of L1 and L2 mRNAs. This is a conserved property of E2 of both mucosal and cutaneous HPV types. Induction could be reversed by high levels of HPV-16 E1 protein, or by the polyadenylation factor CPSF30. HPV-16 E2 inhibited polyadenylation in vitro by preventing the assembly of the CPSF complex. Both the N-terminal and hinge domains of E2 were required for induction of HPV late gene expression in transfected cells as well as for inhibition of polyadenylation in vitro. Finally, overexpression of HPV-16 E2 induced late gene expression from a full-length genomic clone of HPV-16. We speculate that the accumulation of high levels of E2 during the viral life cycle, not only turns off the expression of the pro-mitotic viral E6 and E7 genes, but also induces the expression of the late HPV genes L1 and L2.     

A Humanized Mouse Model of HPV-Associated Pathology Driven by E7 Expression

Human papillomavirus (HPV) is the causative agent of human cervical cancer and has been associated with oropharyngeal squamous cell carcinoma development. Although prophylactic vaccines have been developed, there is a need to develop new targeted therapies for individuals affected with malignant infected lesions in these locations, which must be tested in appropriate models. Cutaneous beta HPV types appear to be involved in skin carcinogenesis. Virus oncogenicity is partly achieved by inactivation of retinoblastoma protein family members by the viral E7 gene. Here we show that the E7 protein of cutaneous beta HPV5 binds pRb and promotes its degradation. In addition, we described an in vivo model of HPV-associated disease in which artificial human skin prepared using primary keratinocytes engineered to express the E7 protein is engrafted onto nude mice. Expression of E7 in the transplants was stably maintained for up to 6 months, inducing the appearance of lesions that, in the case of HPV16 E7, histologically resembled human anogenital lesions caused by oncogenic HPVs. Moreover, it was confirmed through biomarker expression analysis via immunodetection and/or quantitative PCR from mRNA and miRNA that the 16E7-modified engrafted skin shares molecular features with human HPV-associated pretumoral and tumoral lesions. Finally, our findings indicate a decrease of the in vitro capacity of HPV5 E7 to reduce pRb levels in vivo, possibly explaining the phenotypical differences when compared with 16E7-grafts. Our model seems to be a valuable platform for basic research into HPV oncogenesis and preclinical testing of HPV-associated antitumor therapies.     

Organization of human papillomavirus productive cycle during neoplastic progression provides a basis for selection of diagnostic markers

The productive cycle of human papillomaviruses (HPVs) can be divided into discrete phases. Cell proliferation and episomal maintenance in the lower epithelial layers are followed by genome amplification and the expression of capsid proteins. These events, which occur in all productive infections, can be distinguished by using antibodies to viral gene products or to surrogate markers of their expression. Here we have compared precancerous lesions caused by HPV type 16 (HPV16) with lesions caused by HPV types that are not generally associated with human cancer. These include HPV2 and HPV11, which are related to HPV16 (supergroup A), as well as HPV1 and HPV65, which are evolutionarily divergent (supergroups E and B). HPV16-induced low-grade squamous intraepithelial lesions (CIN1) are productive infections which resemble those caused by other HPV types. During progression to cancer, however, the activation of late events is delayed, and the thickness of the proliferative compartment is progressively increased. In many HPV16-induced high-grade squamous intraepithelial lesions (CIN3), late events are restricted to small areas close to the epithelial surface. Such heterogeneity in the organization of the productive cycle was seen only in lesions caused by HPV16 and was not apparent when lesions caused by other HPV types were compared. By contrast, the order in which events in the productive cycle were initiated was invariant and did not depend on the infecting HPV type or the severity of disease. The distribution of viral gene products in the infected cervix depends on the extent to which the virus can complete its productive cycle, which in turn reflects the severity of cervical neoplasia. It appears from our work that the presence of such proteins in cells at the epithelial surface allows the severity of the underlying disease to be predicted and that markers of viral gene expression may improve cervical screening.     

Detection of Merkel cell polyomavirus in cervical squamous cell carcinomas and adenocarcinomas from Japanese patients

ABSTRACT: BACKGROUND: Merkel cell polyomavirus (MCPyV) was identified originally in Merkel cell carcinoma (MCC), a rare form of human skin neuroendocrine carcinoma. Evidence of MCPyV existence in other forms of malignancy such as cutaneous squamous cell carcinomas (SCCs) is growing. Cervical cancers became the focus of our interest in searching for potentially MCPyV-related tumors because: (i) the major histological type of cervical cancer is the SCC; (ii) the uterine cervix is a common site of neuroendocrine carcinomas histologically similar to MCCs; and (iii) MCPyV might be transmitted during sexual interaction as demonstrated for human papillomavirus (HPV). In this study, we aimed to clarify the possible presence of MCPyV in cervical SCCs from Japanese patients. Cervical adenocarcinomas (ACs) were also studied. RESULTS: Formalin-fixed paraffin-embedded tissue samples from 48 cervical SCCs and 16 cervical ACs were examined for the presence of the MCPyV genome by polymerase chain reaction (PCR) and sequencing analyses. PCR analysis revealed that 9/48 cervical SCCs (19 %) and 4/16 cervical ACs (25 %) were positive for MCPyV DNA. MCPyV-specific PCR products were sequenced to compare them with reference sequences. The nucleotide sequences in the MCPyV large-T (LT)-sequenced region were the same among MCPyV-positive cervical SCCs and AC. Conversely, in the MCPyV viral protein 1 (VP1)-sequenced, two cervical SCCs and three cervical ACs showed several nucleotide substitutions, of which three caused amino acid substitutions. These sequencing results suggested that three MCPyV variants of the VP1 were identified in our cases. Immunohistochemistry showed that the LT antigen was expressed in tumor cells in MCPyV-positive samples. Genotyping of human HPV in the MCPyV-positive samples revealed that infected HPVs were HPV types 16, 31 and 58 for SCCs and HPV types 16 and 18 for ACs. CONCLUSIONS: This study provides the first observation that MCPyV coexists in a subset of HPV-associated cervical cancers from Japanese patients. The prevalence of MCPyV in these lesions was close to that observed in the cutaneous SCCs. Further worldwide epidemiological surveys are warranted to determine the possible association of MCPyV with pathogenesis of cervical cancers.     

Protection of rabbits against challenge with rabbit papillomaviruses by immunization with the N terminus of human papillomavirus type 16 minor capsid antigen L2

Current L1 virus-like particle (VLP) vaccines provide type-restricted protection against a small subset of the human papillomavirus (HPV) genotypes associated with cervical cancer, necessitating continued cytologic screening of vaccinees. Cervical cancer is most problematic in countries that lack the resources for screening or highly multivalent HPV VLP vaccines, suggesting the need for a low-cost, broadly protective vaccinogen. Here, N-terminal L2 polypeptides comprising residues 1 to 88 or 11 to 200 derived from HPV16, bovine papillomavirus type 1 (BPV1), or cottontail rabbit papillomavirus (CRPV) were produced in bacteria. Rabbits were immunized with these N-terminal L2 polypeptides and concurrently challenged with CRPV and rabbit oral papillomavirus (ROPV). Vaccination with either N-terminal L2 polypeptides of CRPV effectively protected rabbits from CRPV challenge but not from papillomas induced by cutaneous challenge with CRPV genomic DNA. Furthermore, papillomas induced by CRPV genomic DNA deficient for L2 expression grew at the same rate as those induced by wild-type CRPV genomic DNA, further suggesting that the L2 polypeptide vaccines lack therapeutic activity. Neutralizing serum antibody titers of >15 correlated with protection (P < 0.001), a finding consistent with neutralizing antibody-mediated protection. Surprisingly, a remarkable degree of protection against heterologous papillomavirus types was observed after vaccination with N-terminal L2 polypeptides. Notably, vaccination with HPV16 L2 11-200 protected against cutaneous and mucosal challenge with CRPV and ROPV, respectively, papillomaviruses that are evolutionarily divergent from HPV16. Further, vaccination with HPV16 L2 11-200 generates broadly cross-neutralizing serum antibody, suggesting the potential of L2 as a second-generation preventive HPV vaccine antigen.     

The E6 and E7 proteins of the cutaneous human papillomavirus type 38 display transforming properties

Several studies have suggested the involvement of cutaneous human papillomaviruses (HPVs) in the development of nonmelanoma skin cancers. Here we have characterized the in vitro properties of E7 proteins of three cutaneous HPV types, 10, 20, and 38, which are frequently detected in skin specimens. We show that HPV38 E7 is able to inactivate the tumor suppressor pRb and induces loss of G(1)/S transition control, a key event in carcinogenesis. In contrast, HPV10 and HPV20 E7 proteins do not display these in vitro transforming activities. We also show that the two early proteins E6 and E7 of HPV38 are sufficient to corrupt the cell cycle and senescence programs in primary cells, inducing active and long-lasting proliferation of primary human keratinocytes, the natural host cells. Our study shows that E6 and E7 of this cutaneous HPV type have transforming activity in primary human cells, suggesting a role for HPV38 infection in skin carcinogenesis. In further support of such a role, we detected HPV38 DNA in approximately 50% of nonmelanoma skin cancers, but only in 10% of healthy skin specimens (P < 0.001).     

Differential regulation of cutaneous oncoprotein HPVE6 by wtp53, mutant p53R248W and ΔNp63α is HPV type dependent

UV exposure and p53 mutations are major factors in non-melanoma skin cancer, whereas a role for HPV infections has not been defined. Previous data demonstrated the wtp53-mediated degradation of cutaneous HPV20E6 by caspase-3. ΔNp63α and hot-spot mutant p53R248W conveyed a protective effect on HPV20E6 under these conditions. We demonstrate a differential regulation by wtp53 of the E6 genes of cutaneous types HPV4, HPV5, HPV7, HPV27, HPV38, HPV48, HPV60 and HPV77. Caspase- or proteasome-mediated down-regulation was HPV type dependent. Mutant p53R248W up-regulated expression of all these E6 proteins as did ΔNp63α except for HPV38E6 which was down-regulated by the latter. None of these cellular proteins affected HPV41E6 expression. Ectopic expression of both mutp53R248W and ΔNp63α in the normal NIKS keratinocyte cell line harbouring endogenous p53 and p63however led to a down-regulation of HPV20E6. We demonstrate that HPV20E6 expression in these cells is modulated by additional, yet unidentified, cellular protein(s), which are not necessarily involved in apoptosis or autophagy. We further demonstrate proliferation of HPV20E6-expressing keratinocytes. Levels of proteins involved in cell cycle control, cyclin-D1, cdk6 and p16(INK4a), phosphorylated pRB, as well as c-Jun and p-c-Jun, were all increased in these cells. HPV20E6 did not compete for the interaction between p16(INK4a) with cyclin-D1 or cdk6. Phosphorylation of pRB in the HPV20E6 expressing cells seems to be sufficient to override the cytokenetic block induced by the p16(INK4a)/pRB pathway. The present study demonstrates the diverse influence of p53 family members on individual cutaneous HPVE6 proteins. HPV20E6 expression also resulted in varying protein levels of factors involved in proliferation and differentiation.     

Heterogeneous Nuclear Ribonucleoprotein C Proteins Interact with the Human Papillomavirus Type 16 (HPV16) Early 3′-Untranslated Region and Alleviate Suppression of HPV16 Late L1 mRNA Splicing

In order to identify cellular factors that regulate human papillomavirus type 16 (HPV16) gene expression, cervical cancer cells permissive for HPV16 late gene expression were identified and characterized. These cells either contained a novel spliced variant of the L1 mRNAs that bypassed the suppressed HPV16 late, 5′-splice site SD3632; produced elevated levels of RNA-binding proteins SRSF1 (ASF/SF2), SRSF9 (SRp30c), and HuR that are known to regulate HPV16 late gene expression; or were shown by a gene expression array analysis to overexpress the RALYL RNA-binding protein of the heterogeneous nuclear ribonucleoprotein C (hnRNP C) family. Overexpression of RALYL or hnRNP C1 induced HPV16 late gene expression from HPV16 subgenomic plasmids and from episomal forms of the full-length HPV16 genome. This induction was dependent on the HPV16 early untranslated region. Binding of hnRNP C1 to the HPV16 early, untranslated region activated HPV16 late 5′-splice site SD3632 and resulted in production of HPV16 L1 mRNAs. Our results suggested that hnRNP C1 controls HPV16 late gene expression.     

Causal association between human papilloma virus infection and head and neck and esophageal squamous cell carcinoma

HPVs commonly cause proliferative lesions of squamous epithelium, and infection with certain HPV types carries a high risk of malignant transformation. We used molecular techniques to detect and type HPV in papillomas and carcinomas in the oral cavity and esophagus. DNA was extracted from 150 fresh or paraffin embedded biopsy specimens, and analyzed for HPV by PCR with 15 sets of consensus primers directed to conserved regions of L1 gene, three sets of HPV16E6 primers (specific for the HPV 16 prototype and L83V variant), and sets of primers specific for the E6 gene of other mucosa type HPVs including HPV 6, 11, 16, 18, 52, 58, 66 and 73. Overall, HPV sequences were detected in 61 of 150 specimens. HPV DNA sequences were detected in 16/32 specimens in the oropharyngeal region, in 13/36 specimens in larynx and 32/82 specimens in esophagus. Papillomas contained only the episomal form of HPV 16. In the esophagus, the most common type was HPV 73. In all specimens examined, HPV 6/11 (4/150), HPV 16 (23/150), HPV 35 (1/150), HPV 45 (1/150), HPV 54 (1/150), HPV 58 (1/150), HPV 61 (1/150), HPV 66 (1/150), HPV 68 (2/150), HPV 70 (3/150), HPV 72 (1/150), HPV 73 (16/150), double HPV infection (2/150), and unidentified HPV type (4/150) was detected. Interestingly, HPV was found in all verrucous carcinomas and in 18/22 basaloid squamous cell carcinomas. HPV16E6 T350G mutant were observed only in two of eight carcinomas. Using correspondence analysis, a segregation of specific virus types in specific clinico-pathologic lesions (verrucous carcinoma and basaloid squamous cell carcinoma) was proved. It was shown that the relative rates of the HPV positive tumors were significantly higher in women than in men. The synergic action of mucosal irritation and HPV infection may be necessary for the development of the papillomas and the specific types of carcinomas in the oral cavity and in the esophagus.     

Activation of cap-dependent translation by mucosal human papillomavirus E6 proteins is dependent on the integrity of the LXXLL binding motif

The human papillomavirus (HPV) type 16 (HPV16) E6 protein can stimulate mechanistic target of rapamycin complex 1 (mTORC1) signaling and cap-dependent translation through activation of the PDK1 and mTORC2 kinases. Here we report that HPV18 E6 also enhances cap-dependent translation. The integrity of LXXLL and PDZ protein binding domains is important for activation of cap-dependent translation by high-risk mucosal HPV E6 proteins. Consistent with this model, low-risk mucosal HPV6b and HPV11 E6 proteins, which do not contain a PDZ protein binding motif, also activate cap-dependent translation and mTORC1, albeit at a lower efficiency than high-risk HPV E6 proteins. In contrast, cutaneous HPV5 and HPV8 E6 proteins, which lack LXXLL and PDZ motif protein binding, do not enhance cap-dependent translation. Mutagenic analyses of low-risk HPV E6 proteins revealed that association with the LXXLL motif containing ubiquitin ligase E6AP (UBE3A) correlates with activation of cap-dependent translation. Hence, activation of mTORC1 and cap-dependent translation may be important for the viral life cycle in specific epithelial tissue types and contribute to cellular transformation in cooperation with other biological activities of high-risk HPV E6-containing proteins.     

Epithelium Expressing the E7 Oncoprotein of HPV16 Attracts Immune-Modulatory Dendritic Cells to the Skin and Suppresses Their Antigen-Processing Capacity

Antigen presenting cells (APCs) in skin can promote either antigen-specific effector functions or antigen tolerance, and thus determine clearance or persistence of cutaneous viral infections. Human papillomavirus (HPV) infections can persist in squamous epithelium in immunocompetent individuals, and some persisting HPV infections, particularly with HPV16, promote malignant epithelial transformation. Here, we investigate whether local expression of the HPV16 protein most associated with malignant transformation, HPV16-E7, affects the phenotype and function of APC subsets in the skin. We demonstrate an expanded population of Langerhans cells in HPV16-E7 transgenic skin with distinct cell surface markers which express immune-modulatory enzymes and cytokines not expressed by cells from non transgenic skin. Furthermore, HPV16-E7 transgene expression in keratinocytes attracts new APC subsets to the epidermis. In vivo migration and transport of antigen to the draining lymph node by these APCs is markedly enhanced in HPV16-E7 expressing skin, whereas antigen-processing, as measured by proteolytic cleavage of DQ-OVA and activation of T cells in vivo by APCs, is significantly impaired. These data suggest that local expression of HPV16-E7 in keratinocytes can contribute to persisting infection with this oncogenic virus, by altering the phenotype and function of local APCs.