Oncogenic HPVs Research Articles

Distribution of high-risk human papillomavirus in women with ASC-US or LSIL in Tunisian women: Place of HPV testing for women triage in Tunisia

The present study aimed to evaluate the distribution of oncogenic HPVs in Tunisian women diagnosed with ASC-US or LSIL in order to highlight the importance of HPV testing in the management of women with minor cytological lesions. The study involved 213 cervical samples from women aged from 18 to 82 years and diagnosed with ASC-US or LSIL. HPV detection and genotyping was performed by nested PCR followed by reverse Line Blotting. HPV DNA was identified in 161 cases (76.3%). Oncogenic HPV genotypes were detected in 53.1% of cases. The most frequent high-risk genotypes in this study were HPV16 (28.8%) followed by: HPV51 (9.6%), HPV18, HPV31 HPV56 (7.1%) and HPV45 (5.1%). Thus, 24 % of studied women were not infected by HPV and about 47% of infections are without oncogenic HPV. These results highlight the value of HPV testing in the decision algorithm of management of minor abnormalities lesions.

Human Papillomavirus 42 Drives Digital Papillary Adenocarcinoma and Elicits a Germ Cell-like Program Conserved in HPV-Positive Cancers.

We identify HPV42 as a uniform driver of DPA and add a new member to the short list of tumorigenic viruses in humans. We discover that all oncogenic HPVs evoke a germ cell-like transcriptional program with important implications for detecting, diagnosing, and treating all HPV-driven cancers. See related commentary by Starrett et al., p. 17. This article is highlighted in the In This Issue feature, p. 1.

Targeted Disruption of E6/p53 Binding Exerts Broad Activity and Synergism with Paclitaxel and Topotecan against HPV-Transformed Cancer Cells.

Simple SummaryThe identification of new specific anti-human papillomavirus (HPV) drugs is highly needed, as HPV-induced cancers still represent a significant medical issue. The aim of this study was to analyze in more detail the therapeutic potential of a compound, Cpd12, that acts by blocking the binding between HPV E6 oncoprotein and cellular tumor suppressor p53. We demonstrated that by blocking such an interaction, driven by highly conserved residues among oncogenic HPVs, Cpd12 exhibits broad activity against cervical cancer cell lines infected by different HPV genotypes and HPV-positive head-and-neck cancer cells. Interestingly, Cpd12 also showed the ability to inhibit cancer cell migration and to increase the activity of chemotherapeutic drugs such as taxanes and topoisomerase inhibitors. These findings improve the knowledge about the in vitro efficacy of Cpd12, paving the way to preclinical studies to develop new therapeutic strategies against HPV-induced tumors.High-risk human papillomaviruses (HR-HPV) are the etiological agents of almost all cervical cancer cases and a high percentage of head-and-neck malignancies. Although HPV vaccination can reduce cancer incidence, its coverage significantly differs among countries, and, therefore, in the next decades HPV-related tumors will not likely be eradicated worldwide. Thus, the need of specific treatments persists, since no anti-HPV drug is yet available. We recently discovered a small molecule (Cpd12) able to inhibit the E6-mediated degradation of p53 through the disruption of E6/p53 binding in HPV16- and HPV18-positive cervical cancer cells. By employing several biochemical and cellular assays, here we show that Cpd12 is also active against cervical cancer cells transformed by other HR-HPV strains, such as HPV68 and HPV45, and against a HPV16-transformed head-and-neck cancer cell line, suggesting the possibility to employ Cpd12 as a targeted drug against a broad range of HPV-induced cancers. In these cancer cell lines, the antitumoral mechanism of action of Cpd12 involves p53-dependent cell cycle arrest, a senescent response, and inhibition of cancer cell migration. Finally, we show that Cpd12 can strongly synergize with taxanes and topoisomerase inhibitors, encouraging the evaluation of Cpd12 in preclinical studies for the targeted treatment of HPV-related carcinomas.

Emerging Oncogenic Viruses in Head and Neck Cancers from Romanian Patients

(1) Background: Head and neck squamous cell carcinomas (HNSCCs) are some of the most frequent malignancies globally. Oncogenic viruses MCPyV, EBV and HPVs are recognized to be related to HNSCCs and skin cancers. There are no data from Romania regarding the involvement of herpes viruses and polyomaviruses in these types of cancer. We aim to evaluate the association of oncogenic viruses from Papillomaviridae, Herpesviridae, and Polyomaviridae families in HNSCCs and skin cancers. (2) Methods: A total of 26 fresh tumors (6/26 women) were tested for 67 viral agents using a multiplex PCR genotyping assay. (3) Results: A total of 23/26 (88.5%) samples were positive for one or more viruses. All the tested tumors were negative for any HPV (alpha or beta types). In total, we detected as positive samples: 16 (61.63%) EBV1, 12 (46.15%) HHV7, 8 (30.76%) MCV, 6 (23.07%) CMV and HHV6, 2 (7.69%) HHV8, 1 (3.8%) HPyV6 and EBV2. (4) Conclusions: We detected HPV-negative cases that are HPyV and HHV positive. In these fractions of HPV-negative HNSCCs cases, other oncogenic viruses may be involved, such as EBV1, MCV or CMV. Additional research is required for clarifying the natural history of these viruses in HNSCCs, as virus detection would have a decisive impact on diagnostic and decisional algorithms.

Frequent high-risk HPV co-infections excluding types 16 or 18 in cervical neoplasia in Guadeloupe

BackgroundCervical cancer is the fourth cancer worldwide. The Human Papilloma Virus is responsible for 99% of the cases but the distribution of its genotypes varies among populations. We aimed to identify HPV genotypes distribution in women with grade 2/3 cervical intraepithelial dysplasia or invasive cervical cancer in Guadeloupe, a French Caribbean territory with a population mainly of African descent.MethodsWe used paraffin-embedded tumors for viral DNA extraction from women diagnosed between 2014 and 2016 and identified by the population-based cancer registry. The HPV Genotyping was performed with the InnoLIPA HPV Genotyping Extra kit®.ResultsOverall, 213 samples out of the 321 eligible records were analyzed. The HPV status was positive for 94% of the cases. The five most common oncogenic HPV genotypes were HPV31 (47%), HPV33 (38%), HPV16 (32%), HPV44 (31%) and HPV26 (28%). HPV18 was found in only in 5% of the cases. Among the studied cases, 94% had multiple infections. More than 60% of single infections were HPV16-related, accounting for 35% of HPV16 infections.ConclusionsThese results show a different distribution of oncogenic HPVs in Guadeloupe with “31 > 33 > 16” and a high frequency of multiple infections. Despite a lower coverage, the nine-valent vaccine is nevertheless adequate.

Papillomavirus humains : dépistage et prévention

AbstractHuman papillomavirus (HPV) infections are among the most common sexually transmitted infections (STIs). Natural clearance is the main mode of evolution of these infections. Among the mucosal tropism HPVs, a distinction is made between low-risk oncogenic (LR) HPVs and high-risk oncogenic (HR) HPVs. When HR-HPVs persist, they can cause the development of precancerous and cancerous lesions at different anatomical sites (cervix, anus, vulva, vagina, penis and upper aerodigestive tract). After several decades of cervical cancer screening using cervical smears for cyto-pathological analysis, the detection of HR-HPV by molecular assay has been carried out as a first-line strategy since 2020. Several hundred tests are available on the market and the choice of a test meeting strict validation criteria is essential to the effectiveness of this screening. As no antiviral treatment is effective against these infections and condoms do not provide effective protection, prevention can only be envisaged using a vaccine targeting the 9 genotypes most frequently found in cancers and condylomas. Worldwide, countries with high vaccination coverage have already seen a decrease in HPV infections and lesions, from condylomas to cancers.

Oncogenic HPV promotes the expression of the long noncoding RNA lnc-FANCI-2 through E7 and YY1

Long noncoding RNAs (lncRNAs) play diverse roles in biological processes, but their expression profiles and functions in cervical carcinogenesis remain unknown. By RNA-sequencing (RNA-seq) analyses of 18 clinical specimens and selective validation by RT-qPCR analyses of 72 clinical samples, we provide evidence that, relative to normal cervical tissues, 194 lncRNAs are differentially regulated in high-risk (HR)-HPV infection along with cervical lesion progression. One such lncRNA, lnc-FANCI-2, is extensively characterized because it is expressed from a genomic locus adjacent to the FANCI gene encoding an important DNA repair factor. Both genes are up-regulated in HPV lesions and in in vitro model systems of HR-HPV18 infection. We observe a moderate reciprocal regulation of lnc-FANCI-2 and FANCI in cervical cancer CaSki cells. In these cells, lnc-FANCI-2 is transcribed from two alternative promoters, alternatively spliced, and polyadenylated at one of two alternative poly(A) sites. About 10 copies of lnc-FANCI-2 per cell are detected preferentially in the cytoplasm. Mechanistically, HR-HPVs, but not low-risk (LR)-HPV oncogenes induce lnc-FANCI-2 in primary and immortalized human keratinocytes. The induction is mediated primarily by E7, and to a lesser extent by E6, mostly independent of p53/E6AP and pRb/E2F. We show that YY1 interacts with an E7 CR3 core motif and transactivates the promoter of lnc-FANCI-2 by binding to two critical YY1-binding motifs. Moreover, HPV18 increases YY1 expression by reducing miR-29a, which targets the 3' untranslated region of YY1 mRNA. These data have provided insights into the mechanisms of how HR-HPV infections contribute to cervical carcinogenesis.

Assessing the Efficacy of Human Papillomavirus Disinfection and the Risk of Transmission from Clinical Lesions

<h3>Background</h3> HPV genomes can be detected on medical devices following hospital disinfectant procedures. Recent reports concluded that oncogenic HPVs derived from laboratory tissue-based models are not susceptible to commonly used high-level disinfectants, intensifying concerns that medical instruments may provide transmission of nosocomial HPVs infections. Therefore, we determined the infectious load of HPVs from clinical lesions and investigated the effectiveness of disinfectants on HPV virions derived from laboratory model systems. <h3>Methods</h3> Infectious HPV virions were isolated from cell culture, organotypic epithelial tissue cultures, and mouse xenografts. Clinical samples from recurrent respiratory papillomas (RRPs) and anogenital warts were obtained under IRB approval. The infectivity of HPV virion stocks was measured by the detection of spliced viral E1^E4 mRNAs in infected keratinocytes. Infections were validated by time-dependent detection of E1^E4 mRNAs, resistance to ribonuclease treatment and susceptibility to antibody-mediated neutralization. Our infectivity assay demonstrated a dynamic range of >3-5 log10. Suspension-based disinfection assays employed diluted ortho-phthalaldehyde (OPA) and 0.825% hypochlorite. <h3>Results</h3> In contrast to prior reports, we found that validated HPV virions obtained from a variety of sources were susceptible to a 2.5 to 4 log10 reduction in infectious titer when exposed as directed to OPA or hypochlorite. Some HPV virion stocks failed to meet the infectivity criteria of time-dependent detection of E1^E4 mRNAs, resistance to ribonuclease and susceptibility to antibody-mediated neutralization. Such unvalidated virus stocks are likely to produce spurious results and lead to confounding conclusions. Assessment of HPV infectious titers from clinical lesions suggests that compared to common warts, clinical RRP and anogenital warts have lower levels of virions present at apical surfaces. <h3>Conclusions</h3> We conclude that laboratory sourced HPVs are susceptible to disinfection by OPA and hypochlorite. As the levels of infectious virions recovered from HPV-induced lesions fall below those used in our assays, proper cleaning and disinfection processing is likely to be effective

Vaccination With Moderate Coverage Eradicates Oncogenic Human Papillomaviruses If a Gender-Neutral Strategy Is Applied.

BackgroundHuman papillomavirus (HPV) vaccination of girls with very high (>90%) coverage has the potential to eradicate oncogenic HPVs, but such high coverage is hard to achieve. However, the herd effect (HE) depends both on the HPV type and the vaccination strategy.MethodsWe randomized 33 Finnish communities into gender-neutral HPV16/18 vaccination, girls-only HPV16/18 vaccination, and hepatitis B virus vaccination arms. In 2007–2010, 11 662 of 20 513 of 40 852 of 39 420 resident boys/girls from 1992 to 1995 birth cohorts consented. In 2010–2014, cervicovaginal samples from vaccinated and unvaccinated girls at age 18.5 years were typed for HPV6/11/16/18/31/33/35/39/45/51/52/56/58/59/66/68. Vaccine efficacy for vaccinated girls, HE for unvaccinated girls, and the protective effectiveness (PE) for all girls were estimated. We extended the community-randomized trial results about vaccination strategy with mathematical modeling to assess HPV eradication.ResultsThe HE and PE estimates in the 1995 birth cohort for HPV18/31/33 were significant in the gender-neutral arm and 150% and 40% stronger than in the girls-only arm. Concordantly, HPV18/31/33 eradication was already predicted in adolescents/young adults in 20 years with 75% coverage of gender-neutral vaccination. With the 75% coverage, eventual HPV16 eradication was also predicted, but only with the gender-neutral strategy.ConclusionsGender-neutral vaccination is superior for eradication of oncogenic HPVs.

Repression of Human Papillomavirus Oncogene Expression under Hypoxia Is Mediated by PI3K/mTORC2/AKT Signaling.

Hypoxia is linked to therapeutic resistance and poor clinical prognosis for many tumor entities, including human papillomavirus (HPV)-positive cancers. Notably, HPV-positive cancer cells can induce a dormant state under hypoxia, characterized by a reversible growth arrest and strong repression of viral E6/E7 oncogene expression, which could contribute to therapy resistance, immune evasion and tumor recurrence. The present work aimed to gain mechanistic insights into the pathway(s) underlying HPV oncogene repression under hypoxia. We show that E6/E7 downregulation is mediated by hypoxia-induced stimulation of AKT signaling. Ablating AKT function in hypoxic HPV-positive cancer cells by using chemical inhibitors efficiently counteracts E6/E7 repression. Isoform-specific activation or downregulation of AKT1 and AKT2 reveals that both AKT isoforms contribute to hypoxic E6/E7 repression and act in a functionally redundant manner. Hypoxic AKT activation and consecutive E6/E7 repression is dependent on the activities of the canonical upstream AKT regulators phosphoinositide 3-kinase (PI3K) and mechanistic target of rapamycin (mTOR) complex 2 (mTORC2). Hypoxic downregulation of E6/E7 occurs, at least in part, at the transcriptional level. Modulation of E6/E7 expression by the PI3K/mTORC2/AKT cascade is hypoxia specific and not observed in normoxic HPV-positive cancer cells. Quantitative proteome analyses identify additional factors as candidates to be involved in hypoxia-induced activation of the PI3K/mTORC2/AKT signaling cascade and in the AKT-dependent repression of the E6/E7 oncogenes under hypoxia. Collectively, these data uncover a functional key role of the PI3K/mTORC2/AKT signaling cascade for viral oncogene repression in hypoxic HPV-positive cancer cells and provide new insights into the poorly understood cross talk between oncogenic HPVs and their host cells under hypoxia.IMPORTANCE Oncogenic HPV types are major human carcinogens. Under hypoxia, HPV-positive cancer cells can repress the viral E6/E7 oncogenes and induce a reversible growth arrest. This response could contribute to therapy resistance, immune evasion, and tumor recurrence upon reoxygenation. Here, we uncover evidence that HPV oncogene repression is mediated by hypoxia-induced activation of canonical PI3K/mTORC2/AKT signaling. AKT-dependent downregulation of E6/E7 is only observed under hypoxia and occurs, at least in part, at the transcriptional level. Quantitative proteome analyses identify additional factors as candidates to be involved in AKT-dependent E6/E7 repression and/or hypoxic PI3K/mTORC2/AKT activation. These results connect PI3K/mTORC2/AKT signaling with HPV oncogene regulation, providing new mechanistic insights into the cross talk between oncogenic HPVs and their host cells.

Vimentin Modulates Infectious Internalization of Human Papillomavirus 16 Pseudovirions.

ABSTRACTHuman papillomavirus (HPV) infection is the most common viral infection of the reproductive tract, with virtually all cases of cervical cancer being attributable to infection by oncogenic HPVs. However, the exact mechanism and receptors used by HPV to infect epithelial cells are controversial. The current entry model suggests that HPV initially attaches to heparan sulfate proteoglycans (HSPGs) at the cell surface, followed by conformational changes, cleavage by furin convertase, and subsequent transfer of the virus to an as-yet-unidentified high-affinity receptor. In line with this model, we established an in vitro infection system using the HSPG-deficient cell line pgsD677 together with HPV16 pseudovirions (HPV16-PsVs). While pgsD677 cells were nonpermissive for untreated HPV16-PsVs, furin cleavage of the particles led to a substantial increase in infection. Biochemical pulldown assays followed by mass spectrometry analysis showed that furin-precleaved HPV16-PsVs specifically interacted with surface-expressed vimentin on pgsD677 cells. We further demonstrated that both furin-precleaved and uncleaved HPV16-PsVs colocalized with surface-expressed vimentin on pgsD677, HeLa, HaCaT, and NIKS cells, while binding of incoming viral particles to soluble vimentin protein before infection led to a substantial decrease in viral uptake. Interestingly, decreasing cell surface vimentin by small interfering RNA (siRNA) knockdown in HeLa and NIKS cells significantly increased HPV16-PsV infectious internalization, while overexpression of vimentin had the opposite effect. The identification of vimentin as an HPV restriction factor enhances our understanding of the initial steps of HPV-host interaction and may lay the basis for the design of novel antiviral drugs preventing HPV internalization into epithelial cells.IMPORTANCE Despite HPV being a highly prevalent sexually transmitted virus causing significant disease burden worldwide, particularly cancer of the cervix, cell surface events preceding oncogenic HPV internalization are poorly understood. We herein describe the identification of surface-expressed vimentin as a novel molecule not previously implicated in the infectious internalization of HPV16. Contrary to our expectations, vimentin was found to act not as a receptor but rather as a restriction factor dampening the initial steps of HPV16 infection. These results importantly contribute to our current understanding of the molecular events during the infectious internalization of HPV16 and open a new direction in the development of alternative drugs to prevent HPV infection.

HPV-associated disease of the anal canal: A pathology primer

Anal cancer is a rare disease. Screening for it in high-risk populations is based on the model borrowed from cervical cancer screening with cytology and high-resolution anoscopy-guided biopsy. Squamous cell carcinoma of the anus is associated with oncogenic HPVs, particularly type 16. Squamous intraepithelial lesions (SIL) are graded on both anal cytology and histology using 2 tiers. Low-grade SIL is reflective of a productive HPV infection; high-grade SIL is the potential precancer. High-risk HPV testing may not be an efficient screening or triage tool given the high prevalence of the virus in populations targeted for anal cancer screening. However, genotyping for HPV 16 may prove useful. On biopsy, p16 immunostaining is used to adjudicate equivocal diagnoses on routine H&E tissue sections; a positive p16 immunostain supports a diagnosis of high-grade SIL.

Squamous intraepithelial lesions of the anal squamocolumnar junction: Histopathological classification and HPV genotyping

BackgroundHuman papillomavirus (HPV)-related anal cancer lesions are often found adjacent to the squamocolumnar junction (SCJ). We have assessed the histopathology and associated HPV genotypes in anal SCJ lesions in surgically excised anal warts in HIV-negative and –positive patients. MethodsHistopathology identified 47 squamous intraepithelial lesions (SILs) adjacent to the SCJ amongst a total of 145 cases of clinically diagnosed anal condylomata. The anal SCJ lesions were further analyzed with p16, CK7 and p63 immunohistochemistry and HPV genotyping. ResultsSixteen (16/47) of the excised anal wart lesions contained HSIL; Three were HSIL and exclusively associated with oncogenic HPVs. A further thirteen (13/47) were mixed lesions. Of these eight were HSILs with LSIL and six were HSILs with papillary immature metaplasia (PIM); Ten of the mixed lesions were associated with one or more oncogenic HPVs, while three cases were exclusively associated with HPV6. ConclusionsClinically diagnosed anal warts cannot be assumed to be limited to low-grade lesions as anal warts of the SCJ often show heterogeneous lesions, with coexistence of LSIL, PIM, and HSIL. Lesions showing PIM, however, may mimic HSIL, because they are hypercellular, but lack the nuclear atypia and conspicuous mitotic activity of HSIL; and are p16 negative.

Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements.

Human papillomavirus 18 (HPV18) is the second most common oncogenic HPV type associated with cervical, anogenital, and oropharyngeal cancers. Like other oncogenic HPVs, HPV18 encodes two major (one early and one late) polycistronic pre-mRNAs that are regulated by alternative RNA splicing to produce a repertoire of viral transcripts for the expression of individual viral genes. However, RNA cis-regulatory elements and trans-acting factors contributing to HPV18 alternative RNA splicing remain unknown. In this study, an exonic splicing enhancer (ESE) in the nucleotide (nt) 3520 to 3550 region in the HPV18 genome was identified and characterized for promotion of HPV18 929^3434 splicing and E1^E4 production through interaction with SRSF3, a host oncogenic splicing factor differentially expressed in epithelial cells and keratinocytes. Introduction of point mutations in the SRSF3-binding site or knockdown of SRSF3 expression in cells reduces 929^3434 splicing and E1^E4 production but activates other, minor 929^3465 and 929^3506 splicing. Knockdown of SRSF3 expression also enhances the expression of E2 and L1 mRNAs. An exonic splicing silencer (ESS) in the HPV18 nt 612 to 639 region was identified as being inhibitory to the 233^416 splicing of HPV18 E6E7 pre-mRNAs via binding to hnRNP A1, a well-characterized, abundantly and ubiquitously expressed RNA-binding protein. Introduction of point mutations into the hnRNP A1-binding site or knockdown of hnRNP A1 expression promoted 233^416 splicing and reduced E6 expression. These data provide the first evidence that the alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host trans-acting splicing factors. Expression of HPV18 genes is regulated by alternative RNA splicing of viral polycistronic pre-mRNAs to produce a repertoire of viral early and late transcripts. RNA cis elements and trans-acting factors contributing to HPV18 alternative RNA splicing have been discovered in this study for the first time. The identified ESS at the E7 open reading frame (ORF) prevents HPV18 233^416 splicing in the E6 ORF through interaction with a host splicing factor, hnRNP A1, and regulates E6 and E7 expression of the early E6E7 polycistronic pre-mRNA. The identified ESE at the E1^E4 ORF promotes HPV18 929^3434 splicing of both viral early and late pre-mRNAs and E1^E4 production through interaction with SRSF3. This study provides important observations on how alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host splicing factors and offers potential therapeutic targets to overcome HPV-related cancer.

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.

Molecular investigations of prevalence of human papillomavirus in Lithuania

HPV is one of the major risk factors of cervical cancer and precancerous changes. Infection with its oncogenic types determines a faster progress of pre-cancer changes. Women infected with oncogenic HPV types or multiple HPV infection must be under careful observation of a gynaecologist. The aim of this article is to identify infection of women who have various cytological changes in the cervix with the HPV and its genotypes by means of molecular methods. A total of 99 women took part in the investigation (the experimental group is 33 women, the control group is 66). Flat epithelium cells from the cervix were taken to determine HPV, and DNA was isolated from all samples. The isolated DNA was studied to determine general HPV, and positive samples were genotyped (HV6/11, HPV16, HPV18, HPV31, HPV33, HPV45 and HPV59) by multiplex PCR. Having carried out HPV tests, twice as high incidence rate of infection with HPV in the experimental group than that in the control group was established (40 and 21%, respectively). High cancer risk HPV genotypes were determined in 62% of women in the experimental group, whereas in the control group oncogenic HPVs of HPV16 and HPV31 genotypes were identified in 36% of women. Double HPV infection was diagnosed in 4 women in the experimental group, which can determine a faster progress of pre-cancer intra-epithelial lesions.

Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche.

In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential) is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution.

E6^E7, a novel splice isoform protein of human papillomavirus 16, stabilizes viral E6 and E7 oncoproteins via HSP90 and GRP78.

ABSTRACTTranscripts of human papillomavirus 16 (HPV16) E6 and E7 oncogenes undergo alternative RNA splicing to produce multiple splice isoforms. However, the importance of these splice isoforms is poorly understood. Here we report a critical role of E6^E7, a novel isoform containing the 41 N-terminal amino acid (aa) residues of E6 and the 38 C-terminal aa residues of E7, in the regulation of E6 and E7 stability. Through mass spectrometric analysis, we identified that HSP90 and GRP78, which are frequently upregulated in cervical cancer tissues, are two E6^E7-interacting proteins responsible for the stability and function of E6^E7, E6, and E7. Although GRP78 and HSP90 do not bind each other, GRP78, but not HSP90, interacts with E6 and E7. E6^E7 protein, in addition to self-binding, interacts with E6 and E7 in the presence of GRP78 and HSP90, leading to the stabilization of E6 and E7 by prolonging the half-life of each protein. Knocking down E6^E7 expression in HPV16-positive CaSki cells by a splice junction-specific small interfering RNA (siRNA) destabilizes E6 and E7 and prevents cell growth. The same is true for the cells with a GRP78 knockdown or in the presence of an HSP90 inhibitor. Moreover, mapping and alignment analyses for splicing elements in 36 alpha-HPVs (α-HPVs) suggest the possible expression of E6^E7 mostly by other oncogenic or possibly oncogenic α-HPVs (HPV18, -30, -31, -39, -42, -45, -56, -59, -70, and -73). HPV18 E6^E7 is detectable in HPV18-positive HeLa cells and HPV18-infected raft tissues. All together, our data indicate that viral E6^E7 and cellular GRP78 or HSP90 might be novel targets for cervical cancer therapy.

Efficacy of the HPV-16/18 vaccine: Final according to protocol results from the blinded phase of the randomized Costa Rica HPV-16/18 vaccine trial

BackgroundA community-based randomized trial was conducted in Costa Rica to evaluate the HPV-16/18 AS04-adjuvanted vaccine (NCT00128661). The primary objective was to evaluate efficacy of the vaccine to prevent cervical intraepithelial neoplasia 2 or more severe disease (CIN2+) associated with incident HPV-16/18 cervical infections. Secondary objectives were to evaluate efficacy against CIN2+ associated with incident cervical infection by any oncogenic HPVs and to evaluate duration of protection against incident cervical infection with HPV-16/18. Vaccine safety and immunogenicity over the 4-year follow-up were also evaluated. MethodsWe randomized (3727 HPV arm; 3739 control arm), vaccinated (HPV-16/18 or Hepatitis A) and followed (median 53.8 months) 7466 healthy women aged 18–25 years. 5312 women (2635 HPV arm; 2677 control arm) were included in the according to protocol analysis for efficacy. The full cohort was evaluated for safety. Immunogenicity was considered on a subset of 354 (HPV-16) and 379 (HPV-18) women. HPV type was assessed by PCR on cervical specimens. Immunogenicity was assessed using ELISA and inhibition enzyme immunoassays. Disease outcomes were histologically confirmed. Vaccine efficacy and 95% confidence intervals (95%CI) were computed. ResultsVaccine efficacy was 89.8% (95% CI: 39.5–99.5; N=11 events total) against HPV-16/18 associated CIN2+, 59.9% (95% CI: 20.7–80.8; N=39 events total) against CIN2+ associated with non-HPV-16/18 oncogenic HPVs and 61.4% (95% CI: 29.5–79.8; N=51 events total) against CIN2+ irrespective of HPV type. The vaccine had an acceptable safety profile and induced robust and long-lasting antibody responses. ConclusionsOur findings confirm the high efficacy and immunogenicity of the HPV-16/18 vaccine against incident HPV infections and cervical disease associated with HPV-16/18 and other oncogenic HPV types. These results will serve as a benchmark to which we can compare future findings from the ongoing extended follow-up of participants in the Costa Rica trial. Trial registration: Registered with clinicaltrials.gov: NCT00128661.

Human beta‐defensins‐1, ‐2, and ‐3 production by human amniotic epithelial cells in relation to human papillomavirus genotype (397.4)

HPV is common sexually transmitted infection. Based on the oncogenic potential, low‐risk and high‐risk HPV types were distinguished among approximately 40 HPV genotypes identified within the mucosa of the anogenital tract. Epithelial HBDs act as a potent protein adjuvants promoting cellular and humoral immune responses in viral infections. Unlike alpha‐defensins, HBDs are upregulated in HPV infections. We compared strenght of this response in respect to oncogenic (types 16 and 18) and non‐oncogenic (6 and 11) HPVs (groups I and II, respectively). After normal pregnancies (N = 96) HAEC were isolated from the amnions using trypsinization and divided into three groups (including negative controls; group III), according to the results of the vaginal smear real‐time PCR test for detection of HPV DNA. HAEC stimulated with lipopolysaccharide (1μg/ml) were cultured in vitro for 5 days. HBD‐1, HBD‐2, and HBD‐3 were quantified by ELISA in the culture media samples. Observed mean increases for HBDs were 5.7‐, 3.4‐, and 4.6‐fold, compared to the controls. The mean increases in HBDs concentrations were weaker in the oncogenic HPV infection, compared to the non‐oncogenic HPVs, and the differences between group I and II reached statistical significance (p &lt; 0.05) for HBD‐1 and HBD‐3. Thus, HPV‐related HBD overproduction depends on the virus genotype. Subdued response to oncogenic HPVs may be included in their pathomechanism.Grant Funding Source: Supported by WUM grant: 2M2‐W2‐13.