HPV16 E7 Oncoprotein Research Articles

Virtual screening of antiviral peptides as novel blockers of human papillomavirus 16

Human papillomaviruses (HPVs) contribute to 5% of cancers, yet there is a lack of specific antiviral agents targeting HPV infection. Antiviral peptides (AVPs) present a promising alternative to conventional therapeutics. This study aims to explore the use of AVPs against the HPV16 E6 oncoprotein through virtual screening. The potential binding pocket of the E6 oncoprotein was determined, and using the antimicrobial CAMPR4 database 18 AVPs were shortlisted. These AVPs were then docked to the E6 oncoprotein using the HawkDock server, followed by dynamic simulation. Among the AVPs tested, AVP18, AVP10, and AVP7 demon­strated the highest inhibitory potential against the E6 oncoprotein. AVP18 exhibited more non-bonded contacts, hydrogen bonds, and electrostatic forces. Dynamics simulation confirmed the stability of the complexes formed by these top AVPs with E6. This research suggests that AVP7, AVP10, and AVP18 are promising lead candidates for blocking HPV16 by inhibiting the E6 oncoprotein. Keywords: antiviral peptides, docking, dynamics simulation, E6 oncoprotein, human papillomavirus

Human Papillomaviruses 16 and 18 E6 Oncoprotein Detection Test in Primary Oropharyngeal Carcinomas and Metastatic Lymph Nodes: A Cross-Sectional Study.

Accuracy in the diagnosis of HPV-associated oropharyngeal carcinoma (OPSCC) of a rapid, low-cost lateral flow immunochromatographic assay for detecting E6 oncoprotein of HPV-16 and HPV-18 was previously evaluated in a small pilot study. This cross-sectional study aimed to assess on a large case series the sensitivity and specificity of E6 oncoprotein as a diagnostic marker for HPV-associated carcinogenesis in OPSCC. 137 consecutive patients with histologically confirmed OPSCC were enrolled in two hospitals in Northeast Italy. HPV status was determined by PCR for HPV DNA and p16INK4a immunohistochemistry on primary tumor biopsies. An OPSCC was defined as HPV-associated when double positive for high-risk HPV-DNA and p16INK4a overexpression in primary lesion. Cytological samples from primary tumors and metastatic lymph nodes were obtained and tested for HPV16/18 E6 oncoproteins using the lateral flow immunochromatographic assay, which requires between 90 and 120 min to provide a result. Diagnostic performances were calculated as percentage with confidence intervals (CI). Of the 137 OPSCC cases, 68 (49.6%) were HPV-associated, testing positive for both high-risk HPV-DNA and p16INK4a, with HPV16 predominating (82.4%). An average waiting time of 22 days was observed to obtain the results of p16INK4a and HPV-DNA after primary lesions biopsy. In patients with HPV16/18-associated OPSCC, the HPV16/18 E6 oncoprotein was detected in 59 out of 60 cytological samples from the primary lesion (sensitivity: 98.3%; 95% CI: 91.1-100%) and in 45 out of 51 cytological samples from lymph node metastases (sensitivity: 88.2%; 95% CI: 76.1-95.6%). The E6 oncoprotein assay showed a specificity of 100% in both primary tumors and lymph node metastases. The low-cost lateral flow immunochromatographic assay for detecting HPV16/18 E6 oncoproteins confirmed high accuracy for identifying HPV-associated OPSCC, particularly in primary tumors, suggesting its potential as a valuable diagnostic tool in clinical practice. Its rapid diagnostic capability could significantly accelerate the process of treatment decision-making, enhancing the timely management of patients.

Potential Therapeutic Targets for the Treatment of HPV-Associated Malignancies

This review article aims to summarize broadly recent developments in the treatment of HPV-associated cancers, including cervical cancer and head and neck squamous cell carcinoma. Relatively new treatments targeting the key HPV E6 and E7 oncoproteins, including gene editing with TALENs and CRISPR/Cas9, are discussed. Given the increased immunogenicity of HPV-related diseases, other therapies such as PRR agonists, adoptive cell transfer, and tumor vaccines are reaching the clinical trial phase. Due to the mechanism, immunogenicity, and reversibility of HPV carcinogenesis, HPV-related cancers present unique targets for current and future therapies.

Comparative Molecular Docking of Apigenin and Luteolin versus Conventional Ligands for TP-53, pRb, APOBEC3H, and HPV-16 E6: Potential Clinical Applications in Preventing Gynecological Malignancies.

This study presents a comparative analysis of molecular docking data, focusing on the binding interactions of the natural compounds apigenin and luteolin with the proteins TP-53, pRb, and APOBEC, in comparison to conventional pharmacological ligands. Advanced bioinformatics techniques were employed to evaluate and contrast binding energies, showing that apigenin and luteolin demonstrate significantly higher affinities for TP-53, pRb, and APOBEC, with binding energies of -6.9 kcal/mol and -6.6 kcal/mol, respectively. These values suggest strong potential for therapeutic intervention against HPV-16. Conventional ligands, by comparison, exhibited lower affinities, with energies ranging from -4.5 to -5.5 kcal/mol. Additionally, protein-protein docking simulations were performed to assess the interaction between HPV-16 E6 oncoprotein and tumor suppressors TP-53 and pRb, which revealed high binding energies around -976.7 kcal/mol, indicative of their complex interaction. A conversion formula was applied to translate these protein-protein interaction energies to a comparable scale for non-protein interactions, further underscoring the superior binding potential of apigenin and luteolin. These findings highlight the therapeutic promise of these natural compounds in preventing HPV-16-induced oncogenesis, warranting further experimental validation for clinical applications.

Time to take HPV infection in colorectal cancer patients more seriously.

The association between viral infections and colorectal cancer (CRC) remains an enigma in cancer research. Certain types of Human Papillomaviruses (hr-HPVs), known for their oncogenic properties, have been observed in particular CRC biopsies, further adding to the enigma surrounding this association. This cross-sectional study was conducted on 40 confirmed cases of CRC adenocarcinoma. The presence and genotyping of HPV DNA in colorectal fresh tissue and urine samples was assessed using an HPV DNA hybridization kit. A subset of serum samples from both CRC cases and healthy volunteers was randomly chosen and subjected to western blot to investigate the presence of HPV16 E6/E7 oncoproteins carried by exosomes. It was observed that 26/40 HPV-positive CRC patients demonstrated 7 times more chance to develop colorectal cancer when compared to those 8/40 normal tissue (odds ratio [OR] = 7.4; confidence interval [CI] 95% = 0.483156-0.793718; p < 0.001). Of 26 HPV-positive CRC patients, 14 urine samples were also showed HPV DNA positivity (p = 0.013). High-risk HPV16 was the most prevalent genotype detected in both 24/40 tumor and 12/40 urine samples (p < 0.001). The tumor sample of a male was HPV45, while another male's urine sample was HPV31. A female CRC patient had HPV83 in tumor and HPV56 in urine. Here, was the first detection of HPV83 in a CRC patient. Notably among 20 randomly selected serum exosome samples, one serum sample concurrently tested positive for both HPV16 E6 and E7 oncoproteins, and one sample tested positive for HPV16 E7 oncoprotein. High risk HPV DNA detection in CRC urine samples supports non-invasive screening tools. Detection of HPV16 E6 and E7 oncoproteins in exosomes from serum samples shows potential for non-invasive diagnostics. HPV's potential role in CRC development is also underscored. HPV vaccination should be implemented in low- and middle-income countries to prevent cancer.

DNA Anchoring Strength Directly Correlates with Spherical Nucleic Acid-Based HPV E7 Cancer Vaccine Potency.

Vaccination for cancers arising from human papillomavirus (HPV) infection holds immense potential, yet clinical success has been elusive. Herein, we describe vaccination studies involving spherical nucleic acids (SNAs) incorporating a CpG adjuvant and a peptide antigen (E711-19) from the HPV-E7 oncoprotein. Administering the vaccine to humanized mice induced immunity-dependent on the oligonucleotide anchor chemistry (cholesterol vs (C12)9). SNAs containing a (C12)9-anchor enhanced IFN-γ production >200-fold, doubled memory CD8+ T-cell formation, and delivered more than twice the amount of oligonucleotide to lymph nodes in vivo compared to a simple admixture. Importantly, the analogous construct with a weaker cholesterol anchor performed similar to admix. Moreover, (C12)9-SNAs activated 50% more dendritic cells and generated T-cells cytotoxic toward an HPV+ cancer cell line, UM-SCC-104, with near 2-fold greater efficiency. These observations highlight the pivotal role of structural design, and specifically oligonucleotide anchoring strength (which correlates with overall construct stability), in developing efficacious therapeutic vaccines.

Quantification of HPV16 E7 Oncoproteins in Urine Specimens from Women with Cervical Intraepithelial Neoplasia.

We present the validity of using an ultrasensitive enzyme-linked immunosorbent assay (ELISA) for quantifying high-risk human papillomavirus (HPV) 16 E7 oncoproteins in urine specimens as a noninvasive method of analyzing the oncogenic activity of HPV. Some reports claim that the oncogenic activity of HPV is a more relevant clinical indicator than the presence of HPV DNA for estimating malignant potential. In the present study, urine containing HPV16 and related types were selected by uniplex E6/E7 polymerase chain reaction and classified according to the pathologic diagnosis of cervical intraepithelial neoplasia (CIN) in cervical biopsy specimens. Our ultrasensitive ELISA was able to detect attomole levels of HPV16 E7 oncoproteins, and it detected HPV16-positive SiHa cells at >500 cells/mL without detecting HPV18-positive cells. Our ELISA results showed E7 oncoproteins in 80% (4/5) of urine specimens from women with HPV16-positive CIN1, 71% (5/7) of urine specimens from CIN2 patients, and 38% (3/8) of urine specimens from CIN3 patients. Some urine specimens with undetectable E7 oncoproteins were thought to be negative for live HPV 16-positive cells or in an inactivated state of infection. These results provide the basis for assessing oncogenic activity by quantifying E7 oncoproteins in patient urine.

Field experience with the 8-HPV-type oncoprotein test for cervical cancer screening among HPV-positive women living with and without HIV in LMICs.

Overexpression of HPV-oncoproteins E6 and E7 is necessary for HPV-driven cervical carcinogenesis. Hence, these oncoproteins are promising disease-specific biomarkers. We assessed the technical and operational characteristics of the 8-HPV-type OncoE6/E7 Cervical Test in different laboratories using cervical samples from HPV-positive women living with (WLWH) and without HIV. The 8-HPV-type OncoE6/E7 Test (for short: "OncoE6/E7 test") was performed in 2833 HIV-negative women and 241 WLWH attending multicentric studies in Latin America (ESTAMPA study), and in Africa (CESTA study). Oncoprotein positivity were evaluated at each testing site, according to HIV status as well as type-specific agreement with HPV-DNA results. A feedback questionnaire was given to the operators performing the oncoprotein test to evaluate their impression and acceptability regarding the test. The OncoE6/E7 test revealed a high positivity rate heterogeneity across all testing sites (I2: 95.8%, p < .01) with significant lower positivity in WLWH compared to HIV-negative women (12% vs 25%, p < .01). A similar HPV-type distribution was found between HPV DNA genotyping and oncoprotein testing except for HPV31 and 33 (moderate agreement, k = 0.57). Twenty-one laboratory technicians were trained on oncoprotein testing. Despite operators' concerns about the time-consuming procedure and perceived need for moderate laboratory experience, they reported the OncoE6/E7 test as easy to perform and user-friendly for deployment in resource-limited settings. The high positivity rate variability found across studies and subjectivity in test outcome interpretation could potentially results in oncoprotein false positive/negative, and thus the need for further refinements before implementation of the oncoprotein testing in screen-triage-and-treat approaches is warranted.

Biochemical and toxicity evaluation of Retama sphaerocarpa extracts and in-silico investigation of phenolic compounds as potential inhibitors against HPV16 E6 oncoprotein

Cervical cancer is a type of cancer which affects the cervix cells. The conventional treatments for cervical cancer including surgery, chemotherapy, and radiotherapy are only effective in premature stages and less effective in late stages of this tumor. Therefore, the therapeutic strategies based on biologically active substances from plants are needed to develop for the treatment of cervical cancer. The aim of the present study was to assess in vivo toxicity, hematological and biochemical blood parameters in Wistar rats fed Retama sphaerocarpa aqueous leaf extract (RS-AE), as well as to perform in silico molecular docking studies and dynamic simulation of phenolic compounds against HPV16 oncoprotein E6 in order to identify potential inhibitors. RS-AE was found not to induce acute or sub-acute oral toxicity or significant alterations in hematological and biochemical blood parameters in Wistar rats. A total of 11 phenolic compounds were identified in RS-AE, including dihydrodaidzein glucuronide, chrysoperiol pentoside, genistin and vitexin, which turned out to have the highest binding affinity to HPV16 oncoprotein E6. Based on these results, these RS-AE phenolic compounds could be used as natural drugs against the HPV16 E6 oncoprotein.

HPV16 E7 modulates the cell surface expression of MET and CD109 via the AP2 complex

Multiple cellular pathways are affected by HPV E6 and E7 oncoproteins, including endocytic and cellular trafficking. HPV-16 E7 can target the adaptor protein (AP) complex, which contains proteins important during endocytosis transport. To further investigate the role of HPV E7 during this process, we analysed the expression of cell surface proteins in NIKS cells expressing HPV-16 E7. We show that different cell surface proteins are regulated by HPV-16 E7 via interaction with AP2. We observed that the expression of MET and CD109 membrane protein seems to be upregulated in cells expressing E7. Moreover, the interaction of MET and CD109 with AP2 proteins is disrupted by HPV-16 E7. In addition, in the absence of HPV-16 E7, there is a downregulation of the cell membrane expression of MET and CD109 in HPV-positive cell lines. These results expand our knowledge of the functions of E7 and open new potential cellular pathways affected by this oncoprotein.

Bioinformatics analysis of photoexcited natural flavonoid glycosides as the inhibitors for oropharyngeal HPV oncoproteins

The presence of oropharyngeal human papillomavirus (HPV)-18 E6 and E7 oncoproteins is highly significant in the progression of oropharyngeal cancer. Natural flavonoid compounds have potential as photosensitizers for light-activated antimicrobial therapy against HPV-associated oropharyngeal cancer. This study evaluated five natural flavonoid glycosides including Fisetin, Kaempferol, Morin, Myricetin, and Quercetin as photosensitizers against HPV-18 E6 and E7 oncoproteins using computational methods. After obtaining the amino acid sequences of HPV-18 E6 and E7, various tools were used to predict and verify their properties. The PubChem database was then examined to identify potential natural flavonoid glycosides, followed by predictions of their drug-likeness and ADMET properties. Subsequently, molecular docking was conducted to enhance the screening accuracy and to gain insights into the interactions between the natural compounds and the active sites of HPV-18 E6 and E7 oncoproteins. The protein structures of E6 and E7 were predicted and validated to be reliable. The results of molecular docking demonstrated that Kaempferol exhibited the highest binding affinity to both E6 and E7. All compounds satisfied Lipinski's rules of drug-likeness, except Myricetin. They showed high absorption, distribution volume and similar ADMET profiles with no toxicity. In summary, natural flavonoid glycosides, especially Kaempferol, show potential as photosensitizers for antimicrobial photodynamic therapy against HPV-associated oropharyngeal cancer through inhibition of E6 and E7 oncoproteins. These findings provide insights into the development of novel therapeutic strategies based on antimicrobial photodynamic therapy.

A novel method for colposcopic shunting in HPV-positive women: Quantitative detection of HPV E7 oncoprotein

The objective of the study was to evaluate the clinical application potential of quantitatively detecting human papillomavirus (HPV) E7 oncoprotein in HPV-positive women, with the goal of detecting potential high-grade cervical squamous intraepithelial lesions (HSIL) and cervical cancer improving the accuracy of colposcopic shunting in these patients.HPV-positive women (N = 611) were selected for quantitatively detecting HPV E7 protein levels by magnetic particle-based chemiluminescence immunoassay before colposcopy. Receiver operating characteristic (ROC) curve analysis was performed (n = 400) to determine diagnostic detection thresholds for HPV E7 oncoprotein. ThinPrep cytology test (TCT) and Aptima HPV E6/E7 mRNA analysis were also performed (n = 211). The diagnostic performance of these three diagnostic methods in detecting HSIL and cervical cancer was compared with the gold standard of pathological diagnosis. The area under the ROC curve was 0.724. The diagnostic detection threshold of HPV E7 oncoprotein was ≥10.88 ng/mL. The sensitivity (SEN), specificity (SPE), positive predictive value (PPV), negative predictive value (NPV), and Youden index of HPV E7 oncoprotein for the identification of HSIL and cervical cancer were 78.7 %, 77.9 %, 72.2 %, 83.3 %, and 56.6 %, respectively, which were higher than those of TCT and HPV E6/E7 mRNA.The results indicate that quantitative detection of HPV E7 oncoprotein can effectively shunt HPV-positive women and reduce unnecessary colposcopy and biopsy. It can detect potential HSIL and cervical cancer in a timely manner and prevent high-risk patients from missing diagnosis.

Oncoproteins E6 and E7 upregulate topoisomerase I to activate the cGAS-PD-L1 pathway in cervical cancer development.

Background: Cervical cancer (CC) stands as a significant health threat to women globally, with high-risk human papillomaviruses as major etiologic agents. The DNA damage repair (DDR) protein topoisomerase I (TOP1) has been linked to various cancers, yet its distinct roles and mechanisms in CC are not fully elucidated. Methods: We investigated TOP1 expression in cervical intraepithelial neoplasia (CIN) and CC tissues utilizing qRT-PCR and IHC, correlating findings with patient prognosis. Subsequent knockdown studies were performed in vitro and in vivo to evaluate the influence of TOP1 on tumor growth, DNA repair, and inflammatory responses. Results: TOP1 was highly expressed in CIN and CC, negatively correlating with patient prognosis. Inhibition of TOP1 impeded CC cell growth and disrupted DNA repair. TOP1 was shown to regulate tumor-promoting inflammation and programmed death-ligand 1 (PD-L1) production in a cGAS-dependent manner. HPV oncoproteins E6 and E7 upregulated TOP1 and activated the cGAS-PD-L1 pathway. Conclusions: TOP1 acts as a DNA repair mediator, promoting CC development and immune evasion. Targeting the TOP1-cGAS-PD-L1 axis could be a potential therapeutic strategy for CC.

In vitro activity of various Kenyan tea extracts against HeLa human cervical cancer cell line and molecular docking of tea catechins on high-risk HPV16E6 protein

Tea (Camellia sinensis) has been revealed to possess potential anticancer properties attributed to its catechin content. This study aimed to investigate the growth-inhibitory effects of various tea extracts on the HeLa cell line and molecular docking analysis of catechins on Human papillomavirus16 oncoprotein 6 (HPV16E6). Different catechins content in black, green, and purple tea were determined by using High-performance liquid chromatography (HPLC); molecular docking of four main catechins on HPV16E6 and in vitro antiproliferative activity and cell apoptosis assay were carried out. Molecular docking results suggested that the tea catechins have the potential to bind to the HPV16 E6 oncoprotein involved in cervical cancer development. The antiproliferative assay results indicated that purple tea had the highest inhibitory activity at both 24 h and 48 h, with IC50 values of 121.1 ± 3.25 μg/mL and 100.1 ± 3.76 μg/mL respectively, followed by green tea with 168.9 ± 12.59 μg/mL and 124.7 ± 3.53 μg/mL IC50 while black tea showed the least effectiveness with IC50 of 221 ± 19.35 μg/mL and 159.6 ± 2.33 μg/mL. DNA fragmentation, a characteristic of apoptosis, was observed in cells treated with the tea extracts. All three tea extracts demonstrated potential anti-cervical cancer activity bIy inhibiting cell proliferation and inducing apoptosis. The tea catechins showed promising interactions with the HPV16 E6 oncoprotein.

The E6 gene polymorphism of Human papillomavirus 16 in relation to the risk of cervical cancer in Tunisian women

Human papillomavirus type 16 (HPV-16) is the most prevalent HPV type worldwide and in Tunisia and the major carcinogenic HPV type found in cervical precancers and cancers. Previous studies have reported that genetic diversity of HPV16-E6 oncoprotein might be associated with cervical intraepithelial neoplasia progression. In this study we aimed to investigate the prevalence of HPV-16 E6 variants in precancerous lesions in Tunisian population to assess potential correlation with disease severity.Positive HPV cervical samples were obtained from the Laboratory of Anatomy Pathology of Pasteur Institute of Tunis. Cytological study was performed to identify cervical precancerous lesions. HPVs were typed using Reverse Line Hybridization. Only samples with HPV-16 single infection were selected for HP16-E6 genetic diversity investigation. HPV-16 E6 gene amplification was performed by PCR using specific primers and sequenced by Sanger Sequencing. The multiple alignment of generated sequences was performed using MEGAX software. Phylogenetic tree was constructed using Maximum Likehood method. The ternary complex of E6, E6AP and p53 core domain was used to perform in silico point mutations and thermodynamic calculations to assess stability and binding affinity. Genetic analysis of Tunisian E6-HPV16 sequences showed the presence of three lineages: European (A), African (C) and Asian American (D). Interestingly, the EUR variants were identified as the dominant lineage of HPV-16 and HPV-16 E6 350 G (L83V) was the most detected mutation in precancerous lesions. Modelling data showed that African variants induced the largest destabilizing effect on E6 structure and decreasing thereby in the affinity toward E6AP. Therefore, women infected with European variants are associated with low and high intraepithelial lesions.The findings give useful information for personalized decision algorithms of intra-epithelial cervical neoplasia in Tunisian women.

HPV16 E7 oncoprotein test as a triage strategy for HPV16-positive women in cervical cancer screening: long-term follow-up outcome.

Colposcopy is recommended once human papillomavirus (HPV)16/18 infection is detected. However, not all HPV16/18-positive women will necessarily develop cervical lesions. Therefore, this study aimed to investigate the application of quantitative HPV16 E7 oncoprotein detection as a cervical cancer screening method for more efficient screening while minimizing unnecessary colposcopy. E7 oncoprotein (HPV16) was quantitatively detected in cervical exfoliated cells of HPV16-positive women. The levels of HPV16 E7 oncoprotein in different degrees of cervical lesions were compared, and the optimal cut-off value for identifying HSIL+ was determined by receiver operating characteristic (ROC) curve analysis. With a pathological diagnosis as the gold standard, the sensitivity (SEN), specificity (SPE), positive predictive value (PPV), negative predictive value (NPV), and Kappa value were calculated to verify the diagnostic value of the method. Women diagnosed with low-grade squamous intraepithelial lesions (LSIL) and normal women were followed up for 5 years to evaluate the predictive value of HPV16 E7 protein for disease progression/persistent infection. The expression level of HPV16 E7 oncoprotein was positively correlated with the degree of the cervical lesion (r = 0.589, P < 0.01). The area under the ROC curve (AUC) was 0.817 (confidence interval: 0.729-0.904). The cut-off value of E7 oncoprotein for identifying HSIL+ was 8.68 ng/ml. The SEN, SPE, PPV, NPV, and Kappa values of HPV16 E7 oncoprotein for the identification of HSIL+ were 87.1%,70.0%, 87.1%, 70.0%, and 0.571, respectively, which were higher than those of ThinPrep cytology test (TCT). The SEN, SPE, PPV, and NPV of HPV16 E7 oncoprotein in predicting disease progression/persistent infection were 93.75%, 91.30%, 88.24%, and 95.45%, respectively. The quantitative detection of HPV 16 E7 oncoprotein can not only accurately screen cervical lesions but also achieve efficient colposcopy referral. Additionally, HPV16 E7 oncoprotein can accurately predict the progression of cervical lesions and persistent HPV infection.

Increased expression of HPV-E7 oncoprotein correlates with a reduced level of pRb proteins via high viral load in cervical cancer

Human Papillomavirus (HPV) is the most common cause of sexually transmitted diseases and causes a wide range of pathologies including cervical carcinoma. Integration of the HR-HPV DNA into the host genome plays a crucial role in cervical carcinoma. An alteration of the pRb pathways by the E7 proteins is one of the mechanisms that’s account for the transforming capacity of high-risk papillomavirus. For the proper understanding of the underline mechanism of the progression of the disease, the present study investigate the correlation of concentration of host pRb protein, viral E7 oncoprotein and viral load in early and advanced stages of cervical carcinoma. It was found that the viral load in early stages (stage I and II) was less (log10 transformed mean value 2.6 and 3.0) compared to advanced stages (stage III and IV) (Log10 transformed value 5.0 and 5.8) having high expression of HPV E7 onco-protein and reduced level of pRb protein, signifying the role of viral load and expression level of E7 oncoprotein in the progression of cervical cancer.

Expression of HPV-16 E6 and E7 oncoproteins alters Chlamydia trachomatis developmental cycle and induces increased levels of immune regulatory molecules.

Infection with Human Papillomavirus (HPV) is a recognized risk factor for Chlamydia trachomatis (CT) infection and vice versa. Coinfection of HPV and CT in women is a very common and usually asymptomatic finding that has been linked to increased risk of cervical cancer. It has been demonstrated that CT facilitates the entry of multiple high risk HPV genotypes, leading to damage of the mucosal barrier and interfering with immune responses and viral clearance, which ultimately favours viral persistence and malignant transformation. Although the facilitating effects elicited by CT infection on viral persistence have been reported, little is known about the consequences of HPV infection on CT development. Herein, we took advantage of a genetically modified human cervical cell line co-expressing HPV-16 major oncogenic proteins E6 and E7, as an experimental model allowing to investigate the possible effects that HPV infection would have on CT development. Our results show that CT infection of HPV-16 E6E7 expressing cells induced an upregulation of the expression of E6E7 oncoproteins and host cell inhibitory molecules PD-L1, HVEM and CD160. Additionally, smaller chlamydial inclusions and reduced infectious progeny generation was observed in E6E7 cells. Ultrastructural analysis showed that expression of E6 and E7 did not alter total bacterial counts within inclusions but resulted in increased numbers of reticulate bodies (RB) and decreased production of infectious elementary bodies (EB). Our results indicate that during CT and HPV coinfection, E6 and E7 oncoproteins impair RB to EB transition and infectious progeny generation. On the other hand, higher expression of immune inhibitory molecules and HPV-16 E6E7 are cooperatively enhanced in CT-infected cells, which would favour both oncogenesis and immunosuppression. Our findings pose important implications for clinical management of patients with HPV and CT coinfection, suggesting that screening for the mutual infection could represent an opportunity to intervene and prevent severe reproductive health outcomes, such as cervical cancer and infertility.

Novel competitive enzyme-linked immunosorbent assay for the detection of the high-risk Human Papillomavirus 18 E6 oncoprotein.

Cervical cancer represents a global concern with 604,000 new cases and 342,000 deaths reported annually, with the vast majority diagnosed in low income countries. Despite high-risk Human Papillomavirus (HR HPV)-induced cervical cancer has become highly preventable through prophylactic vaccines, screening programs are critical in the control of cervical carcinogenesis in populations with limited access to vaccination and in older generations of women who have already been exposed to HR HPV infection. The surge of HPV molecular tests has provided a more sensitive and accurate diagnostic alternative to cytology screening. Given that HPV DNA testing presents a low positive predicted value, leading to unnecessary treatment, the E6 oncoprotein from HR HPV types arises as a promising diagnostic marker for its overexpression in transformed HPV-positive cancer cells. For these reasons, this study aimed at obtaining monoclonal antibodies (mAbs) against the E6 oncoprotein of one of the most prevalent HR HPV types worldwide, HPV18, in order to develop a highly specific and sensitive indirect competitive ELISA (icELISA). The production of hybridomas secreting HPV18 E6 mAbs was carried out through a combined tolerization and immunization strategy, in order to avoid cross-reactivity with the E6 protein from low-risk HPV types 6 and 11. We selected the 7D2 hybridoma clone, which recognized HPV18 E6 and showed some cross-reactivity against the HR HPV45 E6 oncoprotein. The 7D2 mAb enabled the development of a sensitive, reliable and reproducible icELISA to detect and quantify small amounts of HPV18 E6 biomarker for cervical cancer progression. The present study establishes a valid 7D2-based icELISA that constitutes a promising bioanalytical method for the early detection and quantification of HPV18 E6 oncoprotein in cervical swab samples and cancer prevention.

HPV16 Impacts NHERF2 Expression in Oropharyngeal Cancers.

Infection with human papillomaviruses (HPVs), in particular with HPV type 16, is now considered to be a key risk factor for the development of a subset of oropharyngeal squamous cell carcinomas (OPSCC) that show different epidemiological, clinical, and prognostic characteristics from HPV-negative (HPV-) OPSCCs. So far, extensive research efforts aiming to distinguish these two distinct entities have not identified specific biomarkers, nor led to different therapies. Previous research has shown that HPV16 E6 oncoprotein binds NHERF2, inducing its proteasomal degradation, and consequently increasing cell proliferation; we therefore aimed to investigate how this might be reflected in human histological samples. We analyzed NHERF2 expression patterns in HPV16-positive (HPV16+) and HPV- OPSCC samples, to investigate any potential differences in NHERF2 pattern. Interestingly, we observed a statistically significant decrease in NHERF2 levels in HPV16+ and poorly differentiated HPV- OPSCCs, compared with healthy tissue. Furthermore, we observed a significant reduction in the percentage of NHERF2 immunoreactive cancer cells in HPV16+ tumors, compared with well and moderately differentiated HPV- OPSCCs, suggesting the importance of 16E6's targeting of NHERF2 in HPV-driven oncogenesis in the head and neck area.