The human CIB1-EVER1-EVER2 complex governs keratinocyte-intrinsic immunity to ß-papillomaviruses.

Patients with epidermodysplasia verruciformis (EV) and biallelic null mutations of TMC6 (encoding EVER1) or TMC8 (EVER2) are selectively prone to disseminated skin lesions due to keratinocyte-tropic human ß-papillomaviruses (ß-HPVs), which lack E5 and E8. We describe EV patients homozygous for null mutations of the CIB1 gene encoding calcium- and integrin-binding protein-1 (CIB1). CIB1 is strongly expressed in the skin and cultured keratinocytes of controls but not in those of patients. CIB1 forms a complex with EVER1 and EVER2, and CIB1 proteins are not expressed in EVER1- or EVER2-deficient cells. The known functions of EVER1 and EVER2 in human keratinocytes are not dependent on CIB1, and CIB1 deficiency does not impair keratinocyte adhesion or migration. In keratinocytes, the CIB1 protein interacts with the HPV E5 and E8 proteins encoded by α-HPV16 and γ-HPV4, respectively, suggesting that this protein acts as a restriction factor against HPVs. Collectively, these findings suggest that the disruption of CIB1-EVER1-EVER2-dependent keratinocyte-intrinsic immunity underlies the selective susceptibility to ß-HPVs of EV patients.

Cutaneous HPV and alpha-mucosal 9-valent HPV sero-status associations.

Seroepidemiology of human papillomaviruses (HPV) among men is poorly understood. We examined the association between seropositivity to cutaneous HPV and 9-valent HPV (9vHPV) types. Six hundred men were randomly selected from the HPV Infection in Men (HIM) Study. Archived serum specimens were tested for antibodies against 9vHPV types [low-risk (6/11) and high-risk (16/18/31/33/45/52/58)], and 14 cutaneous types, including ß-types 5/8/12/14/17/22/23/24/38/47, α-type-27, γ-type-4, µ-type-1, and ν-type-41, using a GST L1-based multiplex serology assay. Risk factor data were collected through questionnaires. Logistic regression was used to evaluate associations between mucosal and cutaneous HPV types. Approximately 21% of men were positive for ≥ 1 cutaneous HPV type, and ≥ 1 nine-valent HPV vaccine type at the same time. Men who were seropositive for any-cutaneous HPV were nearly twice as likely to be seropositive for 9vHPV (adjusted odds ratio (AOR) = 1.97, 95% confidence interval (CI): 1.30-2.99), high-risk (AOR = 1.83; 95% CI: 1.04-3.20), low-risk (AOR = 1.92; 95% CI: 1.16-3.18), and four-valent, 4vHPV, (AOR = 2.01; 95% CI: 1.25-3.21). Type-specific cutaneous HPV seropositivity (types: 8/14/17/23/38/27/4/1) was also positively associated with seropositivity to 9vHPV, high-risk, and low-risk categories. These data indicate that exposure to cutaneous HPV and 9vHPV types is common. Future longitudinal studies are needed to assess the temporality of these associations.

Association between betapapillomavirus seropositivity and keratinocyte carcinoma-prospects for prophylactic vaccination?

In this issue, Genders et al. (2015) demonstrate in a cohort of organ transplant recipients that betapapillomavirus seropositivity around transplantation significantly increases the risk of developing keratinocyte carcinomas. These results further substantiate an etiologic role of betapapillomaviruses in skin cancer and raise prospects of anti-viral immunotherapy.

The presence of betapapillomavirus antibodies around transplantation predicts the development of keratinocyte carcinoma in organ transplant recipients: a cohort study.

Organ transplant recipients (OTRs) have an increased risk of developing keratinocyte carcinomas (KCs). The aim of this study was to correlate infection with human papillomaviruses (HPVs) belonging to the beta genus (Beta-papillomavirus (Beta-PV)) at transplantation with later development of KCs. In a cohort study, sera collected between 1 year before and 1 year after transplantation of OTRs transplanted between 1990 and 2006 were tested for antibody responses against the L1 capsid antigen of Beta-PV and other HPV genera (Gamma-, Mu-, Nu-, and Alpha-PV) using multiplex serology. The OTRs were followed for a maximum of 22 years. Cox regression models with KC, squamous cell carcinoma (SCC), and basal cell carcinoma (BCC) as outcome variables were used. Out of 445 OTRs, 60 had developed KC: 14 developed only SCC, 24 only BCC, and 22 both types of KC. The time-dependent hazard ratio (HR) to develop either or both types of KC, adjusted for age, sex, and transplanted organ, in tested Beta-PV-seropositive OTR around the time of transplantation compared with Beta-PV-seronegative OTR was 2.9 (95% confidence interval (CI) 1.3-6.4). The HR for SCC was 2.9 (95% CI 0.99-8.5) and for BCC it was 3.1 (95% CI 1.2-8.0). There was also an association between Mu-PV seropositivity and KC, but there were no significant associations between other HPV genera tested and KC. A positive seroresponse for Beta-PV around transplantation significantly predicted the development of KC in OTRs up to 22 years later, providing additional evidence that infection with Beta-PV has a role in KC carcinogenesis.

Human Beta-papillomavirus infection and keratinocyte carcinomas.

Although the role of oncogenic human Alpha-papillomaviruses (HPVs) in the development of mucosal carcinomas at different body sites (eg cervix, anus, oropharynx) is fully recognized, a role for HPV in keratinocyte carcinomas (KCs; basal and squamous cell carcinomas) of the skin is not yet clear. KCs are the most common cancers in Caucasians, with the major risk factor being ultraviolet (UV) light exposure. A possible role for Beta-HPV types (BetaPV) in the development of KC was suggested several decades ago, supported by a number of epidemiological studies. Our current review summarizes the recent molecular and histopathological evidence in support of a causal association between BetaPV and the development of KC, and outlines the suspected synergistic effect of viral gene expression with UV radiation and immune suppression. Further insights into the molecular pathways and protein interactions used by BetaPV and the host cell is likely to extend our understanding of the role of BetaPV in KC.

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.

Tumor prevention in HPV8 transgenic mice by HPV8-E6 DNA vaccination.

The genus beta human papillomavirus 8 (HPV8) is involved in the development of cutaneous squamous cell carcinomas (SCCs) in individuals with epidermodysplasia verruciformis. Immunosuppressed transplant recipients are prone to harbor particularly high betapapillomavirus DNA loads, which may contribute to their highly increased risk of SCC. Tumor induction in HPV8 transgenic mice correlates with increased expression of viral oncogenes E6 and E2. In an attempt to prevent skin tumor development, we evaluated an HPV8-E6-DNA vaccine, which was able to stimulate a detectable HPV8-E6-specific cell-mediated immune response in 8/15 immunized mice. When skin of HPV8 transgenic mice was grafted onto non-transgenic littermates, the grafted HPV8 transgenic tissue was not rejected and papillomas started to grow within 14 days all over the transplant of 9/9 non-vaccinated and 7/15 not successfully vaccinated mice. In contrast, no papillomas developed in 6/8 successfully vaccinated mice. In the other two of these eight mice, a large ulcerative lesion developed within the initial papilloma growth or papilloma development was highly delayed. As the vaccine completely or partially prevented papilloma development without rejecting the transplanted HPV8 positive skin, the immune system appears to attack only keratinocytes with increased levels of E6 protein, which would give rise to papillomas.

Human papillomavirus type 8 interferes with a novel C/EBPß-mediated mechanism of keratinocyte CCL20 chemokine expression and Langerhans cell migration.

Infection with genus beta human papillomaviruses (HPV) is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and 8 in patients with epidermodysplasia verruciformis (EV), a genetic skin disease. So far, it has been unknown how these viruses overcome cutaneous immune control allowing their persistence in lesional epidermis of these patients. Here we demonstrate that Langerhans cells, essential for skin immunosurveillance, are strongly reduced in HPV8-positive lesional epidermis from EV patients. Interestingly, the same lesions were largely devoid of the important Langerhans cells chemoattractant protein CCL20. Applying bioinformatic tools, chromatin immunoprecipitation assays and functional studies we identified the differentiation-associated transcription factor CCAAT/enhancer binding protein ß (C/EBPß) as a critical regulator of CCL20 gene expression in normal human keratinocytes. The physiological relevance of this finding is supported by our in vivo studies showing that the expression patterns of CCL20 and nuclear C/EBPß converge spatially in the most differentiated layers of human epidermis. Our analyses further identified C/EBPß as a novel target of the HPV8 E7 oncoprotein, which co-localizes with C/EBPß in the nucleus, co-precipitates with it and interferes with its binding to the CCL20 promoter in vivo. As a consequence, the HPV8 E7 but not E6 oncoprotein suppressed C/EBPß-inducible and constitutive CCL20 gene expression as well as Langerhans cell migration. In conclusion, our study unraveled a novel molecular mechanism central to cutaneous host defense. Interference of the HPV8 E7 oncoprotein with this regulatory pathway allows the virus to disrupt the immune barrier, a major prerequisite for its epithelial persistence and procarcinogenic activity.

NF-κB signalling is attenuated by the E7 protein from cutaneous human papillomaviruses.

The high-risk Alpha-types of human papillomavirus (HPV) are the causative agent of cervical cancer, which is the second major cause of death among women worldwide. Recent investigations have shown that E7 from the Alpha-papillomavirus HPV-16 interacts with IKKα and IKKß of the IKK complex in the NF-κB pathway leading to an attenuation of the activity. There is a possible link between development of non-melanoma skin cancer and cutaneous Beta-papillomavirus but if these HPV types attenuate the NF-κB pathway is unclear. Seven different E7 proteins, representing four out of the five different species of the Beta genus (HPV-20, -37, -38, -92, -93 and -96) and one from the Gamma genus (HPV-4) were investigated for potential modulation of the NF-κB pathway in U2OS cells. Our results demonstrate that E7 from all the cutaneous HPV types were capable of inhibiting the NF-κB activity as well as E7 from HPV-16. In addition, E7 proteins from the cutaneous HPV types demonstrated interaction with IKKα but not with IKKß. The deregulation of the NF-κB pathway by cutaneous HPVs might contribute to the pathogenesis of non-melanoma skin cancers and its precursors.

A case-control study of betapapillomavirus infection and cutaneous squamous cell carcinoma in organ transplant recipients.

We examined the association between betapapillomavirus (betaPV) infection and cutaneous squamous cell carcinoma (SCC) in organ transplant recipients. A total of 210 organ transplant recipients with previous SCC and 394 controls without skin cancer were included. The presence of 25 betaPV types in plucked eyebrow hairs was determined using a human papillomavirus (HPV) DNA genotyping assay, and antibodies for the 15 most prevalent betaPV types were detected using multiplex serology. We used multivariate logistic regression models to estimate associations between various measures of betaPV infection and SCC. BetaPV DNA was highly prevalent (>94%) with multiple types frequently detected in both groups. We found a significant association between SCC and the concordant detection of both antibodies and DNA for at least one betaPV type (adjusted OR 1.6; 95% CI 1.1;2.5). A borderline-significant association with SCC was found for HPV36 (adjusted OR 2.4; CI 1.0;5.4), with similar associations for HPV5, HPV9 and HPV24. These data provide further evidence of an association between betaPV infection and SCC in organ transplant recipients. Confirmation of a betaPV profile predictive of risk for SCC may pave the way for clinically relevant pretransplant HPV screening and the development of preventive and therapeutic HPV vaccination.

The Association between cutaneous squamous cell carcinoma and betapapillomavirus seropositivity: a cohort study.

BACKGROUND: It is currently unclear whether betapapillomaviruses (betaPV) play a role in the etiology of cutaneous squamous cell carcinoma (SCC). We investigated the association between betaPV antibodies and subsequent SCC in a population-based cohort study. METHODS: Serum samples were collected in 1992 and/or 1996 from 1,311 participants of the community-based Nambour Skin Cancer Study. These were tested for the presence of L1 antibodies against 21 different betaPV types. Histologically diagnosed SCCs were ascertained through three full-body skin examinations and linkage with the local pathology laboratories. We used age- and sex-adjusted Cox proportional hazards models to analyze the relationship between betaPV antibodies and SCC occurrence from 1992 until 2007. RESULTS: SCC was newly diagnosed in 150 people. No associations were found between the presence of any betaPV L1 antibodies and the occurrence of SCC (HR = 1.0), and stratification by sex, skin color, and sunburn propensity did not affect these results. However, among people who were less than 50 years old in 1992, the presence of betaPV antibodies was associated with a two-fold increased risk of SCC. There was no significant association between antibodies to any individual betaPV type examined and the later development of SCC. CONCLUSIONS: Whether betaPV infection of the skin, and indirectly betaPV antibodies, are involved in the oncogenic process in the general population remains unclear, and this longitudinal study provides only limited support. IMPACT: This study emphasizes the need for additional longitudinal studies of HPV (human papilloma virus) and SCC, to avoid the possibility of reverse causality in case--control studies.

Marked differences in Betapapillomavirus DNA and antibody prevalence between patients with psoriasis and those with atopic dermatitis.

BACKGROUND: Recent studies revealed that Betapapillomavirus (betaPV) infections are highly prevalent. Skin diseases such as psoriasis, characterized by keratinocyte hyperproliferation, and atopic dermatitis (AD), dominated by cutaneous inflammation, might have an impact on viral life cycle and immune response induction. OBJECTIVES: To investigate whether betaPV infection is different in psoriasis and AD. METHODS: Twenty-seven patients with psoriasis and 17 with AD were included for betaPV genotyping using eyebrow hairs, and for seroresponse determination. RESULTS: BetaPV DNA was found significantly more often in patients with psoriasis than in those with AD (100% vs. 81%, P=0·022) and the mean number of betaPV types was higher (4·8 vs. 2·1 types, P=0·002). In contrast, the seroprevalence in patients with AD was significantly higher compared with that in patients with psoriasis (88% vs. 56%, P=0·023). Type-specific concordance of serological response to the betaPV type detected in eyebrow hairs was 27% in patients with psoriasis and 47% in those with AD (P=0·019). CONCLUSIONS: We speculate that the condition of the skin and the immunological state of the patients have an important impact on the life cycle of betaPV.

Multicenter study of the association between betapapillomavirus infection and cutaneous squamous cell carcinoma.

Human papillomaviruses (betaPV) from the beta genus cannot be classified according to their oncogenicity due to a paucity of information. This study evaluates the association between betaPV infection and cutaneous squamous cell carcinoma in conjunction with measures of UV exposure and susceptibility. We performed case-control studies in the Netherlands, Italy, and Australia, countries with profoundly different UV exposures. The presence of 25 betaPV types in eyebrow hair follicles was determined using a highly sensitive HPV DNA genotyping assay, and antibodies for the 15 most prevalent betaPV types in a total of 689 squamous cell carcinoma cases and 845 controls were detected using multiplex serology. Multivariate logistic regression models were used for case-control comparisons and interaction analyses. BetaPV DNA was detected in eyebrow hairs of more than 90% of all participants. The presence of betaPV DNA was associated with an increased risk of squamous cell carcinoma in the Netherlands (OR = 2.8; 95% CI 1.3-5.8) and Italy (OR = 1.7; 95% CI 0.79-3.6), but not in Australia (OR = 0.91; 95% CI 0.53-1.6). Seropositivity for betaPV in controls ranged between 52% and 67%. A positive antibody response against 4 or more betaPV types was associated with squamous cell carcinoma in Australia (OR = 2.2; 95% CI 1.4-3.3), the Netherlands (OR = 2.0; 95% CI 1.2-3.4) and fair-skinned Italians (OR = 1.6, 95% CI 0.94- 2.7). The association between UV susceptibility and squamous cell carcinoma was stronger in betaPV-seropositive people. These combined data support the hypothesis that betaPV may play a role in the development of cutaneous squamous cell carcinoma.

Lack of association between the presence and persistence of betapapillomavirus DNA in eyebrow hairs and betapapillomavirus L1 antibodies in serum.

Betapapillomavirus (betaPV) DNA and seroresponses are highly prevalent in the general population and both are frequently used as infection markers in epidemiological studies to elucidate an association with cutaneous squamous cell carcinoma (SCC). Little is known about the natural history of betaPV infection and the aspects of infection that drive antibody responses. To investigate the relationship between these markers, this study assessed whether the presence or persistence of betaPV DNA in eyebrow hairs and L1 antibodies of the same betaPV type co-occurred more frequently than would be expected by chance in both a cross-sectional assessment and a longitudinal study. betaPV DNA in plucked eyebrow hairs and L1 antibodies in serum were measured in 416 participants of the Australian community-based Nambour Skin Cancer Study in 1996. Similar data were available for a subset of 148 participants in 2003. Observed co-occurrence of betaPV DNA and antibodies was compared with expected values based on prevalence. A case-wise concordance index was used to calculate the overall concordance of betaPV DNA and antibodies of the same type. No significant associations were found between the presence or persistence of betaPV DNA and antibody responses. The age and sex of the host did not influence the association, and nor did SCC status or a history of sunburns. It was concluded that betaPV antibody responses in adults are not primarily driven by betaPV infection as measured in eyebrow hairs. Other factors, such as viral load, may play a more pivotal role in the induction of detectable seroresponses.

An analysis of clustering of betapapillomavirus antibodies.

Betapapillomaviruses (betaPVs) may contribute to the aetiology of cutaneous squamous cell carcinoma. However, no high-risk types have yet been identified, possibly because the high frequency of co-infection prevents a straightforward analysis of the independent effects of individual viruses. This study aimed to determine whether specific virus types were more likely to co-occur than others, thereby reducing the number of parameters needed in statistical models. Antibody data were analysed from controls who participated in case-control studies in The Netherlands, Italy and Australia and from participants in the German Nutrition Survey. Cluster analysis and two ordination techniques were used to identify patterns. Evidence of clustering was found only according to the number of viruses to which antibodies were detected. The lack of clustering of specific viral types identified suggests that if there are betaPV types that are independently related to skin carcinogenesis, they are unlikely to be identified using standard epidemiological methods.

High beta-HPV DNA loads and strong seroreactivity are present in epidermodysplasia verruciformis.

Epidermodysplasia verruciformis (EV) is a rare disease, characterized by cutaneous warts and associated with a strong predisposition to beta-genus human papillomavirus (HPV). Earlier studies reported high copy numbers of HPV-DNA in nearly all skin tumors from EV patients, but neither HPV replication status in non-lesional skin nor anti-HPV seroreactivity in these patients have been reported yet. We therefore performed a comprehensive viral load analysis for the more common beta-HPV types on skin samples and plucked eyebrow hairs from four EV patients treated at our dermatology department. The results clearly demonstrate that they carry a multiplicity (up to eighteen types) of beta-HPV genotypes in both skin sites. Worthy of note, a high intrapatient concordance for specific types between hair bulbs and skin biopsies was observed and the same beta-PV profile was maintained over time. Viral load analysis revealed a load range between less than one HPV-DNA copy per 100 cells to more than 400 HPV-DNA copies per cell in both eyebrow hairs and skin proliferative lesions. Evaluation of seroreactivity to beta-HPV types in the four EV patients revealed that antibodies against the 16 beta-HPV were significantly more prevalent and showed higher titers than in the controls.

Serological relationship between cutaneous human papillomavirus types 5, 8 and 92.

Evidence of a possible association of cutaneous human papillomavirus (HPV) types, especially members of the genus Betapapillomavirus, and the development of non-melanoma skin cancer (NMSC) is accumulating. Vaccination with virus-like particles (VLPs) consisting of self-assembled L1, the major capsid protein, has been introduced to control anogenital HPV infection. This study examined the serological relationship between betapapillomavirus (beta-PV) types 5 and 8 and the new type HPV-92, which has recently been isolated from a basal cell carcinoma containing a high number of viral genomes. Following expression by recombinant baculoviruses, the L1 protein of HPV-92 self-assembled into VLPs that elicited high-titre antibodies after immunization, similar to VLPs from HPV-5 and -8. Haemagglutination inhibition (HAI) assays were used as a surrogate method for the detection of virion-neutralizing antibodies, which correlates with protection from infection. Antisera raised against HPV-5 and -8 VLPs displayed HAI activity not only against the homologous type, but also against heterologous HPV types 5, 8 and 92, whereas HAI activity of antisera against HPV-92 VLP was restricted to the homologous type. The results of neutralization assays using HPV-5 pseudovirions were consistent with those from HAI assays. Cross-neutralizing immune responses by VLP vaccination against heterologous HPV types may provide broader protection against the multiplicity of HPV types detected in NMSC. If a close link to HPV infection can be conclusively established, these results may provide a basis for further evaluation of VLPs of beta-PVs as candidates for a prophylactic skin-type HPV vaccine, aimed at reducing the incidence of NMSC.

Alpha- and betapapillomavirus E6/E7 genes differentially modulate pro-inflammatory gene expression.

Keratinocytes, the target cell of human papillomavirus (HPV) infection, can produce numerous cytokines and pro-inflammatory molecules which are important for the generation of an effective immune response. How this biological response, which involves the tumor stroma, is affected by the HPV oncoproteins within the epithelial cell itself is not clear. Here it is shown that oncoproteins of different HPV genotypes (alpha- versus beta-HPV genus) alter the expression of pro-inflammatory molecules in early passage primary human keratinocytes and the immortalized cell line HaCaT. HPV5 E6/E7 oncoproteins significantly induced interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) expression. By contrast, the same molecules were down-regulated or not modulated in HPV16 E6/E7 transduced keratinocytes. Interestingly, HPV38 oncoproteins expression resulted in a lower induction of pro-inflammatory molecules, resembling the behavior displayed by the mucosal carcinogenic HPV16. Finally, inducible nitric oxide synthase (iNOS) expression levels and nitric oxide (NO) production were induced at similar levels by all the HPV genotypes tested. These results further emphasize the different biological activities among HPV genotypes, and offer new insights into HPV-associated skin diseases.