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Anal Squamous Cell Carcinoma (SCCA) is a rare Human Papillomavirus type 16 (HPV16)-associated carcinoma whose pathogenesis is still poorly understood. Recent studies based on biopsy and Next Generation Sequencing (NGS) approaches have linked the viral episomal status to aggressive SCCA phenotypes, suggesting a potential role of the 16E5 oncoprotein in tumor development. Our previous findings indicated that 16E5 induces Fibroblast Growth Factor Receptor 2 (FGFR2) isoform switching, aberrant mesenchymal FGFR2c expression, Epithelial Mesenchymal Transition (EMT), and cell invasion in various in vitro human keratinocyte models, as well as in the in vivo context of cervical Low-grade Squamous Intraepithelial Lesions (LSILs). To further explore the role of 16E5 in epithelial carcinogenesis, this study aims to investigate the molecular profile in HPV-related anal lesions. The results showed a significant positive correlation between 16E5 and FGFR2c, as well as 16E5 or FGFR2c and key EMT-related transcription factors, particularly in the group of HPV16 positive anal samples not containing without high grade lesions. Additionally, by coupling the molecular analysis with an interactome investigation, we hypothesized a potential functional interplay between the Ca2+ channel Transient Receptor Potential Ankyrin 1 (TRPA1) and FGFR2c, mediated by 16E5 during the establishment of the oncogenic signaling. These findings will help to elucidate the actual relevance of 16E5 in the early progression of anal lesions and contribute to determine its potential as target for future preventive approaches for HPV16-positive SCCA. [ABSTRACT FROM AUTHOR]

Cervical cancer arises due to the progressive dysplasia of cervical epithelial cells, characterized pathologically as cervical intraepithelial neoplasia (CIN). Given the urgency for effective treatments against CIN, exploring novel pharmaceutical candidates is of paramount importance. Beta-Caryophyllene (BCP), derived from various plants, has shown promise in impeding the progression of various malignancies. However, its potential efficacy in CIN treatment remains unexplored. In this study, we investigate the impact of BCP on CIN progression and elucidate its mechanism of action. An immortalized cervical epithelial cell line (H8) infected with HPV16 was used as a model for CIN. The results revealed that BCP significantly inhibited the proliferation of H8 cells and induced apoptosis. Moreover, BCP demonstrated the ability to suppress autophagy in H8 cells. Mechanistically, our findings indicate that BCP exerts its effects by modulating the Wnt/β-catenin signaling pathway. Specifically, BCP was found to block this pathway, thereby impeding cell growth and autophagy in H8 cells. These results suggest that BCP holds promise as a therapeutic agent for CIN, potentially through its regulatory effects on the Wnt/β-catenin pathway. [ABSTRACT FROM AUTHOR]

Background: Human papillomavirus 16 (HPV16) is an oncogenic virus responsible for the majority of invasive cervical cancer cases worldwide. Due to genetic modifications, some variants are more oncogenic than others. We analysed the HPV16 phylogeny in HPV16-positive cervical Desoxyribonucleic Acid (DNA) samples collected from South African and Mozambican women to detect the circulating lineages. Methods: Polymerase chain reaction (PCR) amplification of the long control region (LCR) and 300 nucleotides of the E6 region was performed using HPV16-specific primers on HPV16-positive cervical samples collected in women from South Africa and Mozambique. HPV16 sequences were obtained through Next Generation Sequencing (NGS) methods. Geneious prime and MEGA 11 software were used to align the sequences to 16 HPV16 reference sequences, gathering the A, B, C, and D lineages and generating the phylogenetic tree. Single nucleotide polymorphisms (SNPs) in the LCR and E6 regions were analysed and the phylogenetic tree was generated using Geneious Prime software. Results: Fifty-eight sequences were analysed. Of these sequences, 79% (46/58) were from women who had abnormal cervical cytology. Fifteen SNPs in the LCR and eight in the E6 region were found to be the most common in all sequences. The phylogenetic analysis determined that 45% of the isolates belonged to the A1 sublineage (European variant), 34% belonged to the C1 sublineage (African 1 variant), 16% belonged to the B1 and B2 sublineage (African 2 variant), two isolates belonged to the D1–3 sublineages (Asian-American variant), and one to the North American variant. Conclusions: The African and European HPV16 variants were the most common circulating lineages in South African and Mozambican women. A high-grade squamous intraepithelial lesion (HSIL) was the most common cervical abnormality observed and linked to European and African lineages. These findings may contribute to understanding molecular HPV16 epidemiology in South Africa and Mozambique. [ABSTRACT FROM AUTHOR]

The E6 and E7 oncoproteins of high-risk types of human papillomavirus (HR-HPV) are crucial for the development of cervical cancer (CC). Small interfering RNAs (siRNAs) are explored as novel therapies that silence these oncogenes, but their clinical use is hampered by inefficient delivery systems. Modification (pegylation) with polyethylene glycol (PEG) of liposomal siRNA complexes (siRNA lipoplexes) may improve systemic stability. We studied the effect of siRNA targeting HPV16 E6, delivered via cationic liposomes (lipoplexes), on cellular processes in a cervical carcinoma cell line (CaSki) and its potential therapeutic use. Lipoplexes-PEG-HPV16 E6, composed of DOTAP, Chol, DOPE, and DSPE-PEG2000 were prepared. The results showed that pegylation (5% DSPE-PEG2000) provided stable siRNA protection, with a particle size of 86.42 ± 3.19 nm and a complexation efficiency of over 80%; the siRNA remained stable for 30 days. These lipoplexes significantly reduced HPV16 E6 protein levels and restored p53 protein expression, inhibiting carcinogenic processes such as proliferation by 25.74%, migration (95.7%), and cell invasion (97.8%) at concentrations of 20 nM, 200 nM, and 80 nM, respectively. In conclusion, cationic lipoplexes-PEG-HPV16 E6 show promise as siRNA carriers for silencing HPV16 E6 in CC. [ABSTRACT FROM AUTHOR]

Breast cancer risk factors include lifestyle, genetic–hormonal influences, and viral infections. Human papillomavirus (HPV), known primarily as the etiological agent of cervical cancer, also appears active in breast carcinogenesis, as evidenced in our study of 56 patients from northeastern Brazil. We assessed the clinical and sociodemographic characteristics, correlating them with various breast cancer tumor types. HPV detection involved amplifying the L1 region, with viral load measured using the E2/E6 ratio and viral activity indicated by E5 oncogene expression. Predominantly, patients over 56 years of age with healthy lifestyles showed a high incidence of invasive ductal carcinoma and triple-negative breast cancer. HPV was detected in 35.7% of cases, mostly HPV16, which is associated with high viral loads (80 copies per cell) and significant E5 expression. These results hint at a possible link between HPV and breast carcinogenesis, necessitating further studies to explore this association and the underlying viral mechanisms. [ABSTRACT FROM AUTHOR]

High risk human papillomavirus (HPV) infection is responsible for 99 % of cervical cancers and 5 % of all human cancers worldwide. HPV infection requires the viral genome (vDNA) to gain access to nuclei of basal keratinocytes of epithelium. After virion endocytosis, the minor capsid protein L2 dictates the subcellular retrograde trafficking and nuclear localization of the vDNA during mitosis. Prior work identified a cell-permeable peptide termed SNX1.3, derived from the BAR domain of sorting nexin 1 (SNX1), that potently blocks the retrograde and nuclear trafficking of EGFR in triple negative breast cancer cells. Given the importance of EGFR and retrograde trafficking pathways in HPV16 infection, we set forth to study the effects of SNX1.3 within this context. SNX1.3 inhibited HPV16 infection by both delaying virion endocytosis, as well as potently blocking virion retrograde trafficking and Golgi localization. SNX1.3 had no effect on cell proliferation, nor did it affect post-Golgi trafficking of HPV16. Looking more directly at L2 function, SNX1.3 was found to impair membrane spanning of the minor capsid protein. Future work will focus on mechanistic studies of SNX1.3 inhibition, and the role of EGFR signaling and SNX1-mediated endosomal tubulation, cargo sorting, and retrograde trafficking in HPV infection.

Aim: Aloysia citrodora has a long history of traditional use in treating various ailments. This study evaluated the in vivo chemopreventive efficacy and systemic toxicity of an extract of A. citrodora in a transgenic mouse model of HPV16 (human papillomavirus type 16)-induced cancer. Methods: The experiment involved six groups (n = 5): group 1 (G1, wild-type (WT), water), group 2 (G2, HPV, water), group 3 (G3, WT, 0.013 g/mL), group 4 (G4, HPV, 0.006 g/mL), group 5 (G5, HPV, 0.008 g/mL), and group 6 (G6, HPV, 0.013 g/mL). Throughout the assay, humane endpoints, body weight, food, and water consumption were recorded weekly. The internal organs and skin of the mice were collected for analysis after they were sacrificed. Toxicological parameters that were studied included hematological and biochemical blood markers, splenic and hepatic histology, and hepatic oxidative stress. Results: A. citrodora extract seems to reduce the incidence of dysplastic and in situ carcinoma skin lesions induced by HPV16 in this model, suggesting that dietary supplementation with concentrations of 0.008 g/mL and 0.013 g/mL may have beneficial chemopreventive effects. Conclusions: The extract did not induce any concentration-dependent toxicological effects on any of the parameters included in the study, indicating a favorable toxicological profile under these experimental conditions.

Abstract Head and neck cancers, particularly oropharyngeal cancers (OPC), have been increasingly associated with human papillomavirus (HPV) infections, specifically HPV16. The current methods for HPV16 detection primarily rely on p16 staining or PCR techniques. However, it is important to note the limitations of conventional PCR, as the presence of viral DNA does not always indicate an ongoing viral infection. Moreover, these tests heavily rely on the availability of tissue samples, which can present challenges in certain situations. In this study, we developed a RT-qPCR biplex approach to detect HPV16 oncogenes E6 and E7 RNA in saliva samples from OPC patients. Salivary supernatant was used as the liquid biopsy source. We successfully obtained RNA from salivary supernatant, preserving its integrity as indicated by the detection of several housekeeping genes. Our biplex approach accurately detected E6 and E7 RNA in HPV16-positive cell lines, tissues, and finally in OPC salivary samples. Importantly, the assay specifically targeted HPV16 and not HPV18. This biplexing technique allowed for reduced sample input without compromising specificity. In summary, our approach demonstrates the potential to detect viable HPV16 in saliva from OPC patients. Since the assay measures HPV16 RNA, it provides insights into the transcriptional activity of the virus. This could guide clinical decision-making and treatment planning for individuals with HPV-related OPC.

HPV16 is responsible for approximately 60% and 90% of global HPV–induced cervical and oropharyngeal cancers, respectively. HPV16 intratype variants have been identified by HPV genome sequencing and classified into four phylogenetic lineages (A–D). Our understanding of HPV16 variants mostly derives from epidemiological studies on cervical cancer (CC) in which HPV16 B, C, and D lineages (previously named "non-European" variants) were mainly associated with high-grade cervical lesions and cancer. Although a predominance of HPV16 lineage A (previously named "European variants") has been observed in head and neck squamous cell carcinoma (HNSCC), epidemiological and in vitro biological studies are still limited for this tumor site. Next Generation Sequencing (NGS) of the entire HPV genome has deepened our knowledge of the prevalence and distribution of HPV variants in CC and HNSCC. Research on cervical cancer has shown that certain HPV16 sublineages, such as D2, D3, A3, and A4, are associated with an increased risk of cervical cancer, and sublineages A4, D2, and D3 are linked to a higher risk of developing adenocarcinomas. Additionally, lineage C and sublineages D2 or D3 of HPV16 show an elevated risk of developing premalignant cervical lesions. However, it is still crucial to conduct large-scale studies on HPV16 variants in different HPV–related tumor sites to deeply evaluate their association with disease development and outcomes. This review discusses the current knowledge and updates on HPV16 phylogenetic variants distribution in HPV–driven anogenital and head and neck cancers. [ABSTRACT FROM AUTHOR]

Head and neck cancers (HNCs), primarily head and neck squamous cell carcinoma (HNSCC), are associated with high‐risk human papillomavirus (HR HPV), notably HPV16 and HPV18. HPV status guides treatment and predicts outcomes, with distinct molecular pathways in HPV‐driven HNSCC influencing survival rates. HNC incidence is rising globally, with regional variations reflecting diverse risk factors, including tobacco, alcohol, and HPV infection. Oropharyngeal cancers attributed to HPV have significantly increased, particularly in regions like the United States. The HPV16 genome, characterized by oncoproteins E6 and E7, disrupts crucial cell cycle regulators, including tumor protein p53 (TP53) and retinoblastoma (Rb), contributing to HNSCC pathogenesis. P16 immunohistochemistry (IHC) is a reliable surrogate marker for HPV16 positivity, while in situ hybridization and polymerase chain reaction (PCR) techniques, notably reverse transcription‐quantitative PCR (RT‐qPCR), offer sensitive HPV detection. Liquid‐based RT‐qPCR, especially in saliva, shows promise for noninvasive HPV detection, offering simplicity, cost‐effectiveness, and patient compliance. These molecular advancements enhance diagnostic accuracy, guide treatment decisions, and improve patient outcomes in HNC management. In conclusion, advances in HPV detection and molecular understanding have significant clinical management implications. Integrating these advancements into routine practice could ultimately improve patient outcomes. [ABSTRACT FROM AUTHOR]

Human papillomavirus type 16 (HPV16) is the most common cause of cervical cancer, but most infections are transient with lesions not progressing to cancer. There is a lack of specific biomarkers for early cancer risk stratification. This study aimed to explore the intrahost HPV16 genomic variation in longitudinal samples from HPV16‐infected women with different cervical lesion severity (normal, low‐grade, and high‐grade). The TaME‐seq deep sequencing protocol was used to generate whole genome HPV16 sequences of 102 samples collected over time from 40 individuals. Single nucleotide variants (SNVs) and intrahost SNVs (iSNVs) were identified in the viral genomes. A majority of individuals had a unique set of SNVs and these SNVs were stable over time. Overall, the number of iSNVs and APOBEC3‐induced iSNVs were significantly lower in high‐grade relative to normal and low‐grade samples. A significant increase in the number of APOBEC3‐induced iSNVs over time was observed for normal samples when compared to high‐grade. Our results indicates that the lower incidence of iSNVs and APOBEC3‐induced iSNVs in high‐grade lesions may have implications for novel biomarkers discoveries, potentially aiding early stratification of HPV‐induced cervical precancerous lesions. [ABSTRACT FROM AUTHOR]

Lemon is a fruit rich in antioxidant properties and has several health benefits, namely the reduction of skin edema and anticarcinogenic properties, which are due to its high content of bioactive compounds. Melatonin can improve and preserve the properties of lemon for longer and also has health benefits. The aim of this study was to evaluate the effects of oral administration of lemon juice after melatonin treatment on murinometric parameters of wild-type (WT) mice and transgenic mice carrying human papillomavirus (HPV). Two trials were performed for oral administration of the lemon extract compound: in drinking water and in diet. First of all, lemons were treated by immersion with melatonin at 10 mM. Then, lemons were squeezed, and the juice obtained was freeze-dried and stored to be subsequently added to drinking water or diet, according to the assay. Thus, mice were divided into eight groups in the drink assay (each with n = 5): group 1 (G1, WT, control), group 2 (G2, WT, 1 mL lemon), group 3 (G3, WT, 1.5 mL lemon), group 4 (G4, WT, 2 mL lemon), group 5 (G5, HPV16, control), group 6 (G6, HPV16, 1 mL lemon) group 7 (G6, HPV16, 1.5 mL lemon) and group 8 (G6, HPV16, 2 mL lemon). The diet assay was divided into four groups: group 1 (G1, WT, control), group 2 (G2, WT, 4 mL lemon), group 3 (G3, HPV16, control) and group 4 (G4, HPV16, 4 mL lemon). In the drink assay, the highest concentration of melatonin (308 ng/100 mL) was for groups 4 and 8, while in the food assay, there was only one concentration of melatonin (9.96 ng/g) for groups 2 and 4. Both trials lasted 30 days. During this time, body weight, food and water were recorded. Afterward, they were sacrificed, and samples were collected for different analyses. At the concentrations used, the lemon juice with melatonin had no adverse effects on the animals' health and showed a positive outcome in modifying weight gain and enhancing antioxidant activity in mice. Moreover, a reduction in the incidence of histological lesions was observed in treated animals. Further research is needed to better understand the effects of lemon extract on health and treatment outcomes in this animal model. [ABSTRACT FROM AUTHOR]

Head and neck cancers, particularly oropharyngeal cancers (OPC), have been increasingly associated with human papillomavirus (HPV) infections, specifically HPV16. The current methods for HPV16 detection primarily rely on p16 staining or PCR techniques. However, it is important to note the limitations of conventional PCR, as the presence of viral DNA does not always indicate an ongoing viral infection. Moreover, these tests heavily rely on the availability of tissue samples, which can present challenges in certain situations. In this study, we developed a RT-qPCR biplex approach to detect HPV16 oncogenes E6 and E7 RNA in saliva samples from OPC patients. Salivary supernatant was used as the liquid biopsy source. We successfully obtained RNA from salivary supernatant, preserving its integrity as indicated by the detection of several housekeeping genes. Our biplex approach accurately detected E6 and E7 RNA in HPV16-positive cell lines, tissues, and finally in OPC salivary samples. Importantly, the assay specifically targeted HPV16 and not HPV18. This biplexing technique allowed for reduced sample input without compromising specificity. In summary, our approach demonstrates the potential to detect viable HPV16 in saliva from OPC patients. Since the assay measures HPV16 RNA, it provides insights into the transcriptional activity of the virus. This could guide clinical decision-making and treatment planning for individuals with HPV-related OPC. [ABSTRACT FROM AUTHOR]

Little is known about the protection conferred by antibodies from natural human papillomavirus (HPV) infection. Our objective was to evaluate the association between HPV16 seroreactivity and HPV16 redetection, newly detected HPV infections, and loss of HPV DNA detection during follow‐up. We analyzed data from 2462 unvaccinated Brazilian women. HPV16 IgG and neutralizing antibodies at baseline were assessed by enzyme‐linked immunosorbent assay (n = 1975) and by the pseudovirus‐based papillomavirus neutralization assay (n = 487). HPV detection, genotyping, and viral load were assessed by PCR‐based methods. The associations were analyzed by Cox proportional hazards models. We observed a positive association between HPV16 IgG seroreactivity and redetection of HPV16 infections. Age‐adjusted hazard ratios (HR) with 95% confidence intervals (CI) ranged from 2.45 (1.04–5.74) to 5.10 (1.37–19.00). Positive associations were also observed between HPV16 IgG antibodies and (1) newly detected HPV infections by genotypes unrelated to HPV16 (age‐adjusted HR [95% CI] = 1.32 [1.08–1.2]) and (2) loss of detection of HPV infections by genotypes unrelated to HPV16 (age‐adjusted HR [95% CI] = 1.24 [1.03–1.50]). Naturally developed HPV16 antibodies do not prevent recurrent HPV infections. Overall HPV16 IgG and neutralizing antibodies seem to be serological markers for latent or past infections. [ABSTRACT FROM AUTHOR]

Human papillomaviruses (HPV), most commonly HPV16, are associated with a subset of head and neck squamous cell carcinoma (HNSCC) tumors, primarily oropharyngeal carcinomas, with integration of viral genomes into host chromosomes associated with worse survival outcomes. We analyzed TCGA data and found that HPV+ HNSCC expressed higher transcript levels of the bromodomain and extra terminal domain (BET) family of transcriptional coregulators. The role of BET protein-mediated transcription of viral-cellular genes in the viral-HNSCC genomes needs to be better understood. Using a combination of TAME-Seq, qRT-PCR, and immunoblot analyses, we show that BET inhibition downregulates E6 and E7 significantly, with heterogeneity in the downregulation of viral transcription across different HPV+ HNSCC cell lines. Chemical BET inhibition was phenocopied with the knockdown of BRD4, mirroring the downregulation of viral E6 and E7 expression. We found that BET inhibition directly downregulated c-Myc and E2F expression and induced CDKN1A (p21) expression, leading to a G1-cell cycle arrest with apoptotic activity. Overall, our studies demonstrate that BET inhibition regulates both E6 and E7 viral and key cellular cell cycle regulator E2F gene expression and cellular gene expression in HPV-associated HNSCC and highlight the potential of BET inhibitors as a therapeutic strategy for this disease while also underscoring the importance of considering the heterogeneity in cellular responses to BET inhibition.

Abstract Background Cervical cancer (CC) causes more than 311,000 deaths annually worldwide. The integration of human papillomavirus (HPV) is a crucial genetic event that contributes to cervical carcinogenesis. Despite HPV DNA integration is known to disrupt the genomic architecture of both the host and viral genomes in CC, the complexity of this process remains largely unexplored. Results In this study, we conducted whole-genome sequencing (WGS) at 55-65X coverage utilizing the PacBio long-read sequencing platform in SiHa and HeLa cells, followed by comprehensive analyses of the sequence data to elucidate the complexity of HPV integration. Firstly, our results demonstrated that PacBio long-read sequencing effectively identifies HPV integration breakpoints with comparable accuracy to targeted-capture Next-generation sequencing (NGS) methods. Secondly, we constructed detailed models of complex integrated genome structures that included both the HPV genome and nearby regions of the human genome by utilizing PacBio long-read WGS. Thirdly, our sequencing results revealed the occurrence of a wide variety of genome-wide structural variations (SVs) in SiHa and HeLa cells. Additionally, our analysis further revealed a potential correlation between changes in gene expression levels and SVs on chromosome 13 in the genome of SiHa cells. Conclusions Using PacBio long-read sequencing, we have successfully constructed complex models illustrating HPV integrated genome structures in SiHa and HeLa cells. This accomplishment serves as a compelling demonstration of the valuable capabilities of long-read sequencing in detecting and characterizing HPV genomic integration structures within human cells. Furthermore, these findings offer critical insights into the complex process of HPV16 and HPV18 integration and their potential contribution to the development of cervical cancer.

Human papillomaviruses (HPVs) are a major cause of cancer. While surgical intervention remains effective for a majority of HPV-caused cancers, the urgent need for medical treatments targeting HPV-infected cells persists. The pivotal early genes E6 and E7, which are under the control of the viral genome's long control region (LCR), play a crucial role in infection and HPV-induced oncogenesis, aswell as immune evasion. In this study, proteomic analysis of endosomes uncovered the cointernalization of ErbB2 receptor tyrosine kinase, also called HER2/neu, with HPV16 particles from the plasma membrane. Although ErbB2 overexpression has been associated with cervical cancer, its influence on HPV infection stages was previously unknown. Therefore, we investigated the role of ErbB2 in HPV infection, focusing on HPV16. Through siRNA-mediated knockdown and pharmacological inhibition studies, we found that HPV16 entry is independent of ErbB2. Instead, our signal transduction and promoter assays unveiled a concentration- and activationdependent regulatory role of ErbB2 on the HPV16 LCR by supporting viral promoter activity. We also found that ErbB2's nuclear localization signal was not essential for LCR activity, but rather the cellular ErbB2 protein level and activation status that were inhibited by tucatinib and CP-724714. These ErbB2-specific tyrosine kinase inhibitors as well as ErbB2 depletion significantly influenced the downstream Akt and ERK signaling pathways and LCR activity. Experiments encompassing lowrisk HPV11 and high-risk HPV18 LCRs uncovered, beyond HPV16, the importance of ErbB2 in the general regulation of the HPV early promoter. Expanding our investigation to directly assess the impact of ErbB2 on viral gene expression, quantitative analysis of E6 and E7 transcript levels in HPV16 and HPV18 transformed cell lines unveiled a noteworthy decrease in oncogene expression following ErbB2 depletion, concomitant with the downregulation of Akt and ERK signaling pathways. In light of these findings, we propose that ErbB2 holds promise as potential target for treating HPV infections and HPV-associated malignancies by silencing viral gene expression. [ABSTRACT FROM AUTHOR]

Recent evidence shows increased preterm birth risk with human papillomavirus-16 (HPV16) infection during pregnancy. This study aimed to measure the association between HPV16 viral load during pregnancy and preterm birth. We used data from participants in the HERITAGE study. The Linear Array assay was used for HPV DNA testing on vaginal samples collected during the first and third trimesters of pregnancy. The HPV16 viral load was measured with a real-time polymerase chain reaction. We used logistic regression to measure the associations between HPV16 viral load during pregnancy and preterm birth (defined as birth before 37 weeks of gestation). The adjusted odd ratios (aORs) and the 95% confidence intervals [CIs] were estimated with inverse probability treatment weighting of the propensity score. This study included 48 participants who tested positive for HPV16 during the first trimester of pregnancy. The aOR for the association between first-trimester HPV16 viral load (higher viral load categorized with a cutoff of 0.5 copy/cell) was 13.04 [95% CI: 1.58–107.57]). Similar associations were found using different cutoffs for the categorization of viral load during the first and third trimesters. Our findings suggest a strong association between a high HPV16 viral load during pregnancy and preterm birth, demonstrating a biological gradient that reinforces the biological plausibility of a causal association. [ABSTRACT FROM AUTHOR]

Human Papillomaviruses have been associated with the occurrence of cervical cancer, the fourth most common cancer that affects women globally, while 70% of cases are caused by infection with the high-risk types HPV16 and HPV18. The integration of these viruses' oncogenes E6 and E7 into the host's genome affects a multitude of cellular functions and alters the expression of molecules. The aim of this study was to investigate how these oncogenes contribute to the expression of immune system control molecules, using cell lines with integrated HPV16 genome, before and after knocking out E6 viral gene using the CRISPR/Cas9 system, delivered with a lentiviral vector. The molecules studied are the T-cell inactivating protein PD-L1, its transcription factor HIF-1a and the latter's negative regulator, miR-143. According to our results, in the E6 knock out (E6KO) cell lines an increased expression of miR-143 was recorded, while a decrease in the expression of HIF-1a and PD-L1 was exhibited. These findings indicate that E6 protein probably plays a significant role in enabling cervical cancer cells to evade the immune system, while we propose a molecular pathway in cervical cancer, where PD-L1's expression is regulated by E6 protein through a miR-143/HIF-1a axis. [ABSTRACT FROM AUTHOR]

Abstract The oncoprotein E6, a pivotal player in HPV-16-induced cancer, has long been the focus of extensive research. Building upon our previous study, we identified asarinin and thiazolo[3,2-a] benzimidazole-3(2H)-one-(2-fluorobenzylideno)-7,8-dimethyl (thiazolo) as potential potent anti-HPV-16 E6 oncoprotein agents. Utilizing the UniProt-derived E6 sequence, we employed I-TASSER to model the protein's three-dimensional structure. Subsequent molecular docking via AutoDock Vina, coupled with a 1,000 ps dynamic analysis under physiological parameters, revealed that both asarinin and thiazolo have a high chance of forming stable protein-ligand complexes with HPV-16 E6, displaying distinct molecular dynamic properties. Thiazolo exhibited superior stability in simulation, evident in ligand conformation and movement graphs, while asarinin excelled in terms of contact residues. Furthermore, SASA, hydrogen bond graphs, and the DCCM graph collectively underscore the comparable potential of both drugs as robust inhibitors of the HPV-16 E6 oncoprotein.

Background and Objective The current study aims to detect the frequency of human papillomavirus (HPV16) 16 from cervical samples in Dhi Qar Governorate / Iraq based on molecular analysis. Method Between 2017 and 2020, this survey was conducted on 93 adult females who had cervical cancer and 60 healthy people as controls. Pa- tients’ ages ranged from 32 to 78. DNA was collected for molecular analy- sis and treated to PCR for minor capsid protein L2 gene amplification and identification. Results According to PCR data, HPV16 was present in 60 (65%) of the 93 cervical cancer cases, but only 5 (8%) of the healthy control group were HPV16 positive. The current survey found the highest rates of high-risk HPV16 infections in 2019 (78%) and 2020 (69%) compared to the lowest rates in 2017 (47%) of infections. Additionally, age groups have an impact on the rate of HPV16 infection; according to the most recent findings, the infection rate among elderly women declined while it increased among young women. On the other hand, the distribution of HPV16 infections according to the stages of cervical cancer revealed that stage IV (70%) had the greatest infection rates, followed by stage III (68%) and stage II (60%). Conclusion For accurate HPV16 detection, PCR is a reputable tech- nique. In addition, viral infections have surged recently, especially in young women, and are unmistakably linked to the advancement of cervical cancer. In the Thi-Qar province of Iraq, HPV16 infections are significantly linked to cervical carcinoma among women.

Cervical cancer is a notable cause of mortality and morbidity among women of reproductive age. Human papillomavirus (HPV) is the leading cause of cervical cancer among women. Among 170 types of HPV; HPV-16 and -18 are responsible for cervical cancer. The overexpression of oncoproteins E6 and E7 are predominantly responsible for causing neoplasia. The presence of koilocytosis/koilocytotic atypia is the diagnostic point of HPV infection in pap smears. To identify the circulating types of HPV and determine the various risk factors associated with HPV infection, 100 vaginal biopsies or swabs were taken from patients suspected with cervical cancer, and qualitative and semi-quantitative real-time PCR were performed. PCR primers (GP5+/GP6+) based on a conserved region of the HPV-L1open reading frame(ORF) gene were used for the detection of HPV strains, while another set of primers was used for detecting the E6 gene (HPV-16) and E7 gene (HPV-18). The results showed an HPV infection rate of 23%. Furthermore, the prevalent genotype was found to be HPV-16 (73.91%), followed by HPV-18 (26.1%), while mixed infections of both HPV-16 and -18 accounted for 21.74%. In addition, an age of above 45 years, multiple pregnancies, low socioeconomic status, postmenopausal state, anemia, and early coitarche were significantly associated with HPV infection. These results provide the basis for the formulation of an appropriate strategy for disease monitoring to determine the frequency and distribution pattern of HPV infection.

Abstract Background Although the role of viral agents, such as human papillomavirus (e.g. HPV16, HPV18) in colorectal cancer (CRC) has been previously investigated, results remain inconclusive. Methods To further evaluate the involvement of oncogenic HPV types in CRC, 40 frozen neoplastic and 40 adjacent colonic tissues collected from Italian patients were analyzed by Luminex-based assays that detect a broad spectrum of HPV types, i.e. Alpha (n = 21), Beta (n = 46) and Gamma HPVs (n = 52). In addition, 125 frozen CRC samples and 70 surrounding mucosal tissues were collected from Czech patients and analyzed by broad spectrum PCR protocols: (i) FAP59/64, (ii) FAPM1 and (iii) CUT combined with Next Generation Sequencing (NGS). Results Using Luminex-basedassays, DNA from HPV16 was detected in 5% (2/40) CRC tissues from Italian patients. One HPV16 DNA-positive CRC case was subsequently confirmed positive for E6*I mRNA. Cutaneous beta HPV types were detected in 10% (4/40) adjacent tissues only, namely HPV111 (n = 3) and HPV120 (n = 1), while gamma HPV168 (n = 1) and HPV199 (n = 1) types were detected in adjacent and in tumor tissues, respectively. The NGS analysis of the CRC Czech samples identified HPV sequences from mucosal alpha-3 (HPV89), alpha-7 (HPV18, 39, 68 and 70) and alpha-10 species (HPV11), as well as cutaneous beta-1 (HPV20, 24, 93, 98, 105,124) beta-2 (HPV23), beta-3 (HPV49) and gamma-1 species (HPV205). Conclusions Our findings indicate that HPV types belonging to the mucosal alpha, and the ‘cutaneous’ beta and gamma genera can be detected in the colonic mucosal samples with a low prevalence rate and a low number of HPV reads by Luminex and NGS, respectively. However, additional studies are required to corroborate these findings.

Abstract Objective Similar to Human Papillomavirus (HPV) genotypes, different lineages of a genotype also have different carcinogenic capabilities. Studies have shown that specific genotype lineages of oncogenic HPV are associated with variable risks for the development of cervical intraepithelial neoplasia (CIN2/CIN3) and cervical cancer. The present study aimed to analyze the genetic diversity of the HPV16 genotype in women with CIN2/CIN3 and cervical cancer, from the northeast region of Brazil. Methods A cross-sectional multicenter study was conducted in the northeast region of Brazil, from 2014 to 2016. This study included 196 cases of HPV16 variants (59 and 137 cases of CIN2/CIN3 and cervical cancer, respectively). The difference of proportion test was used to compare patients with CIN2/CIN3 and cervical cancer, based on the prevalent HPV16 lineage (p < 0.05). Results According to the histopathological diagnosis, the percentage of lineage frequencies revealed a marginal difference in the prevalence of lineage A in CIN2/CIN3, compared with that in cervical cancer (p = 0.053). For lineage D, the proportion was higher in cancer cases (32.8%), than in CIN2/CIN3 cases (16.9%), with p = 0.023. Conclusion HPV16 lineage A was the most frequent lineage in both CIN2/CIN3 and cervical cancer samples, while lineage D was predominant in cervical cancer, suggesting a possible association between HPV16 lineage D and cervical cancer.

Background: Human papillomavirus 16 (HPV16) is an oncogenic virus responsible for the majority of invasive cervical cancer cases worldwide. Due to genetic modifications, some variants are more oncogenic than others. We analysed the HPV16 phylogeny in HPV16-positive cervical Desoxyribonucleic Acid (DNA) samples collected from South African and Mozambican women to detect the circulating lineages. Methods: Polymerase chain reaction (PCR) amplification of the long control region (LCR) and 300 nucleotides of the E6 region was performed using HPV16-specific primers on HPV16-positive cervical samples collected in women from South Africa and Mozambique. HPV16 sequences were obtained through Next Generation Sequencing (NGS) methods. Geneious prime and MEGA 11 software were used to align the sequences to 16 HPV16 reference sequences, gathering the A, B, C, and D lineages and generating the phylogenetic tree. Single nucleotide polymorphisms (SNPs) in the LCR and E6 regions were analysed and the phylogenetic tree was generated using Geneious Prime software. Results: Fifty-eight sequences were analysed. Of these sequences, 79% (46/58) were from women who had abnormal cervical cytology. Fifteen SNPs in the LCR and eight in the E6 region were found to be the most common in all sequences. The phylogenetic analysis determined that 45% of the isolates belonged to the A1 sublineage (European variant), 34% belonged to the C1 sublineage (African 1 variant), 16% belonged to the B1 and B2 sublineage (African 2 variant), two isolates belonged to the D1–3 sublineages (Asian-American variant), and one to the North American variant. Conclusions: The African and European HPV16 variants were the most common circulating lineages in South African and Mozambican women. A high-grade squamous intraepithelial lesion (HSIL) was the most common cervical abnormality observed and linked to European and African lineages. These findings may contribute to understanding molecular HPV16 epidemiology in South Africa and Mozambique.

Abstract Background There exists strong evidence that human papillomavirus (HPV) is associated with cervical cancer (CC). HPV E6 is a major oncogene whose sequence variations may be associated with the development of CC. There is not sufficient data on the distribution of HPV types in ThinPrep cytology specimens and HPV 16/18 E6 gene variations among CC patients in the southwest of Iran. This study was conducted to contribute to HPV screening and vaccination in Iran. Methods A total of 648 women screened for cervicitis, intraepithelial neoplasia or CC were included in the study. All participants underwent ThinPrep cytology testing, single-step HPV DNA detection and allele-specific reverse hybridization assays. Moreover, a total of 96 specimens previously tested positive for single infection with HPV16 or 18 were included for variant analysis. HPV16/18 lineages and sublineages were determined by PCR assays followed by sequencing the E6 gene and the construction of neighbor-joining phylogenetic trees. Results Overall, HPV DNA was detected in 62.19% of all the screened subjects. The detection rates of HPV DNA among individuals with normal, ASC-US, ASC-H, LSIL, and HSIL cervical cytology were 48.9%, 93.6%, 100%, 100%, and 100%, respectively. Low-risk HPVs were detected more frequently (46.9%) than high-risk (38.9%) and possible high-risk types (11.1%). Of 403 HPV-positive subjects, 172 (42.7%) had single HPV infections while the remaining 231 (57.3%) were infected with multiple types of HPV. Our results indicated a remarkable growth of high-risk HPV66 and 68 and low-risk HPV81 which have rarely been reported in Iran and HPV90 and 87 that are reported for the first time in the country. In addition, 3 lineages (A, D, and C) and 6 sublineages (A1, A2, A4, C1, D1, and D2) of HPV16, and one lineage and 4 sublineages (A1, A3, A4, and A5) of HPV18 were identified. The studied HPV16 and 18 variants mainly belonged to the D1 and A4 sublineages, respectively. Conclusion The present study suggests that the prevalence of HPV infection in women of all age groups with or without premalignant lesions in the southwestern Iran is high and the predominant HPV types in the southwest of Iran may differ from those detected in other parts of the country. This study also highlights the necessity of not only initiating HPV vaccination for the general population but also developing new vaccines that confer immunity against the prevalent HPV types in the area and national cervical screening programs using a combination of thinPrep cytology test and HPV detection assays in order to improve the accuracy of the screening.

Human papillomaviruses (HPVs) are a major cause of cancer. While surgical intervention remains effective for a majority of HPV-caused cancers, the urgent need for medical treatments targeting HPV-infected cells persists. The pivotal early genes E6 and E7, which are under the control of the viral genome’s long control region (LCR), play a crucial role in infection and HPV-induced oncogenesis, as well as immune evasion. In this study, proteomic analysis of endosomes uncovered the co-internalization of ErbB2 receptor tyrosine kinase, also called HER2/neu, with HPV16 particles from the plasma membrane. Although ErbB2 overexpression has been associated with cervical cancer, its influence on HPV infection stages was previously unknown. Therefore, we investigated the role of ErbB2 in HPV infection, focusing on HPV16. Through siRNA-mediated knockdown and pharmacological inhibition studies, we found that HPV16 entry is independent of ErbB2. Instead, our signal transduction and promoter assays unveiled a concentration- and activation-dependent regulatory role of ErbB2 on the HPV16 LCR by supporting viral promoter activity. We also found that ErbB2’s nuclear localization signal was not essential for LCR activity, but rather the cellular ErbB2 protein level and activation status that were inhibited by tucatinib and CP-724714. These ErbB2-specific tyrosine kinase inhibitors as well as ErbB2 depletion significantly influenced the downstream Akt and ERK signaling pathways and LCR activity. Experiments encompassing low-risk HPV11 and high-risk HPV18 LCRs uncovered, beyond HPV16, the importance of ErbB2 in the general regulation of the HPV early promoter. Expanding our investigation to directly assess the impact of ErbB2 on viral gene expression, quantitative analysis of E6 and E7 transcript levels in HPV16 and HPV18 transformed cell lines unveiled a noteworthy decrease in oncogene expression following ErbB2 depletion, concomitant with the downregulation of Akt and ERK signaling pathways. In light of these findings, we propose that ErbB2 holds promise as potential target for treating HPV infections and HPV-associated malignancies by silencing viral gene expression.

Cervical cancer is a notable cause of mortality and morbidity among women of reproductive age. Human papillomavirus (HPV) is the leading cause of cervical cancer among women. Among 170 types of HPV; HPV-16 and -18 are responsible for cervical cancer. The overexpression of oncoproteins E6 and E7 are predominantly responsible for causing neoplasia. The presence of koilocytosis/koilocytotic atypia is the diagnostic point of HPV infection in pap smears. To identify the circulating types of HPV and determine the various risk factors associated with HPV infection, 100 vaginal biopsies or swabs were taken from patients suspected with cervical cancer, and qualitative and semi-quantitative real- time PCR were performed. PCR primers (GP5+/GP6+) based on a conserved region of the HPV-L1open reading frame(ORF) gene were used for the detection of HPV strains, while another set of primers was used for detecting the E6 gene (HPV-16) and E7 gene (HPV-18). The results showed an HPV infection rate of 23%. Furthermore, the prevalent genotype was found to be HPV-16 (73.91%), followed by HPV-18 (26.1%), while mixed infections of both HPV-16 and -18 accounted for 21.74%. In addition, an age of above 45 years, multiple pregnancies, low socioeconomic status, postmenopausal state, anemia, and early coitarche were significantly associated with HPV infection. These results provide the basis for the formulation of an appropriate strategy for disease monitoring to determine the frequency and distribution pattern of HPV infection. [ABSTRACT FROM AUTHOR]

Background: Although the role of viral agents, such as human papillomavirus (e.g. HPV16, HPV18) in colorectal cancer (CRC) has been previously investigated, results remain inconclusive. Methods: To further evaluate the involvement of oncogenic HPV types in CRC, 40 frozen neoplastic and 40 adjacent colonic tissues collected from Italian patients were analyzed by Luminex-based assays that detect a broad spectrum of HPV types, i.e. Alpha (n = 21), Beta (n = 46) and Gamma HPVs (n = 52). In addition, 125 frozen CRC samples and 70 surrounding mucosal tissues were collected from Czech patients and analyzed by broad spectrum PCR protocols: (i) FAP59/64, (ii) FAPM1 and (iii) CUT combined with Next Generation Sequencing (NGS). Results: Using Luminex-basedassays, DNA from HPV16 was detected in 5% (2/40) CRC tissues from Italian patients. One HPV16 DNA-positive CRC case was subsequently confirmed positive for E6*I mRNA. Cutaneous beta HPV types were detected in 10% (4/40) adjacent tissues only, namely HPV111 (n = 3) and HPV120 (n = 1), while gamma HPV168 (n = 1) and HPV199 (n = 1) types were detected in adjacent and in tumor tissues, respectively. The NGS analysis of the CRC Czech samples identified HPV sequences from mucosal alpha-3 (HPV89), alpha-7 (HPV18, 39, 68 and 70) and alpha-10 species (HPV11), as well as cutaneous beta-1 (HPV20, 24, 93, 98, 105,124) beta-2 (HPV23), beta-3 (HPV49) and gamma-1 species (HPV205). Conclusions: Our findings indicate that HPV types belonging to the mucosal alpha, and the 'cutaneous' beta and gamma genera can be detected in the colonic mucosal samples with a low prevalence rate and a low number of HPV reads by Luminex and NGS, respectively. However, additional studies are required to corroborate these findings. [ABSTRACT FROM AUTHOR]

Persistent high-risk human papillomavirus (HPV) infection is a pivotal factor in the progression of cervical cancer. In recent years, an increasing interest has emerged in comprehending the influence of HPV on head and neck squamous cell carcinoma (HNSCC). Notably, it is well established that HPV-associated HNSCC show cases with distinct molecular and clinical attributes compared to HPV-negative cases. The present study delves into the epigenetic landscape of HPV16, specifically its L1 gene and untranslated region (UTR), through pyrosequencing, while the HPV16 DNA physical status was evaluated using E2/E6 ratio analysis in HPV16-positive HNSCC FFPE biopsies. Our findings reveal substantial methylation across six sites within the HPV16 L1 gene and seven sites in the UTR. Specifically, methylation percentages of two L1 CpG sites (7136, 7145) exhibit significant associations with tumor histological grade (p < 0.01), while proving concurrent methylation across multiple sites. The HPV16 DNA physical status was not correlated with the methylation of viral genome or tumor characteristics. This is the first study that examines epigenetic modifications and the HPV16 DNA physical status in Greek HNSCC patients. Our findings suggest an orchestrated epigenetic modulation among specific sites, impacting viral gene expression and intricate virus–host interactions. [ABSTRACT FROM AUTHOR]

Esophageal squamous cell carcinoma (ESCC) may be correlated with HPV infection, and the mechanism underlying the ESCC formation induced by HPV16 infection remains elusive. Here, we overexpressed HPV16 E6 and E7 and coordinated the overexpression of these two genes in EPC2 and ESCC cells. We found that E7 and coordinated expression of E6 and E7 promoted the proliferation of EPC2 cells, and upregulation of shh was responsible for cell proliferation since the use of vismodegib led to the failure of organoid formation. Meanwhile, overexpression of E6 and E7 in ESCC cells promoted cell proliferation, migration, and invasion in vitro. Importantly, E6 and E7 coordinately increased the capability of tumor growth in nude mice, while vismodegib slowed the growth of tumors in NCG mice. Moreover, a series of genes and proteins changed in cell lines after overexpression of the E6 and E7 genes, the potential biological processes and pathways were systematically analyzed using a bioinformatics assay. Together, these findings suggest that the activation of the hedgehog pathway induced by HPV16 infection may initially transform basal cells in the esophagus and promote following malignant processes in ESCC cells. The application of hedgehog inhibitors may represent a therapeutic avenue for ESCC treatment. [ABSTRACT FROM AUTHOR]

Epigenetic studies on the role of DNA-modifying enzymes in HNSCC tumorigenesis have focused on a single enzyme or a group of enzymes. To acquire a more comprehensive insight into the expression profile of methyltransferases and demethylases, in the present study, we examined the mRNA expression of the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B, the DNA demethylases TET1, TET2, TET3, and TDG, and the RNA methyltransferase TRDMT1 by RT-qPCR in paired tumor–normal tissue samples from HNSCC patients. We characterized their expression patterns in relation to regional lymph node metastasis, invasion, HPV16 infection, and CpG73 methylation. Here, we show that tumors with regional lymph node metastases (pN+) exhibited decreased expression of DNMT1, 3A and 3B, and TET1 and 3 compared to non-metastatic tumors (pN0), suggesting that metastasis requires a distinct expression profile of DNA methyltransferases/demethylases in solid tumors. Furthermore, we identified the effect of perivascular invasion and HPV16 on DNMT3B expression in HNSCC. Finally, the expression of TET2 and TDG was inversely correlated with the hypermethylation of CpG73, which has previously been associated with poorer survival in HNSCC. Our study further confirms the importance of DNA methyltransferases and demethylases as potential prognostic biomarkers as well as molecular therapeutic targets for HNSCC.

The E6 and E7 oncoproteins of high-risk types of human papillomavirus (HR-HPV) are crucial for the development of cervical cancer (CC). Small interfering RNAs (siRNAs) are explored as novel therapies that silence these oncogenes, but their clinical use is hampered by inefficient delivery systems. Modification (pegylation) with polyethylene glycol (PEG) of liposomal siRNA complexes (siRNA lipoplexes) may improve systemic stability. We studied the effect of siRNA targeting HPV16 E6, delivered via cationic liposomes (lipoplexes), on cellular processes in a cervical carcinoma cell line (CaSki) and its potential therapeutic use. Lipoplexes-PEG-HPV16 E6, composed of DOTAP, Chol, DOPE, and DSPE-PEG2000 were prepared. The results showed that pegylation (5% DSPE-PEG2000) provided stable siRNA protection, with a particle size of 86.42 ± 3.19 nm and a complexation efficiency of over 80%; the siRNA remained stable for 30 days. These lipoplexes significantly reduced HPV16 E6 protein levels and restored p53 protein expression, inhibiting carcinogenic processes such as proliferation by 25.74%, migration (95.7%), and cell invasion (97.8%) at concentrations of 20 nM, 200 nM, and 80 nM, respectively. In conclusion, cationic lipoplexes-PEG-HPV16 E6 show promise as siRNA carriers for silencing HPV16 E6 in CC.

Breast cancer risk factors include lifestyle, genetic–hormonal influences, and viral infections. Human papillomavirus (HPV), known primarily as the etiological agent of cervical cancer, also appears active in breast carcinogenesis, as evidenced in our study of 56 patients from northeastern Brazil. We assessed the clinical and sociodemographic characteristics, correlating them with various breast cancer tumor types. HPV detection involved amplifying the L1 region, with viral load measured using the E2/E6 ratio and viral activity indicated by E5 oncogene expression. Predominantly, patients over 56 years of age with healthy lifestyles showed a high incidence of invasive ductal carcinoma and triple-negative breast cancer. HPV was detected in 35.7% of cases, mostly HPV16, which is associated with high viral loads (80 copies per cell) and significant E5 expression. These results hint at a possible link between HPV and breast carcinogenesis, necessitating further studies to explore this association and the underlying viral mechanisms.

HPV16 is responsible for approximately 60% and 90% of global HPV–induced cervical and oropharyngeal cancers, respectively. HPV16 intratype variants have been identified by HPV genome sequencing and classified into four phylogenetic lineages (A–D). Our understanding of HPV16 variants mostly derives from epidemiological studies on cervical cancer (CC) in which HPV16 B, C, and D lineages (previously named “non-European” variants) were mainly associated with high-grade cervical lesions and cancer. Although a predominance of HPV16 lineage A (previously named “European variants”) has been observed in head and neck squamous cell carcinoma (HNSCC), epidemiological and in vitro biological studies are still limited for this tumor site. Next Generation Sequencing (NGS) of the entire HPV genome has deepened our knowledge of the prevalence and distribution of HPV variants in CC and HNSCC. Research on cervical cancer has shown that certain HPV16 sublineages, such as D2, D3, A3, and A4, are associated with an increased risk of cervical cancer, and sublineages A4, D2, and D3 are linked to a higher risk of developing adenocarcinomas. Additionally, lineage C and sublineages D2 or D3 of HPV16 show an elevated risk of developing premalignant cervical lesions. However, it is still crucial to conduct large-scale studies on HPV16 variants in different HPV–related tumor sites to deeply evaluate their association with disease development and outcomes. This review discusses the current knowledge and updates on HPV16 phylogenetic variants distribution in HPV–driven anogenital and head and neck cancers.

Lemon is a fruit rich in antioxidant properties and has several health benefits, namely the reduction of skin edema and anticarcinogenic properties, which are due to its high content of bioactive compounds. Melatonin can improve and preserve the properties of lemon for longer and also has health benefits. The aim of this study was to evaluate the effects of oral administration of lemon juice after melatonin treatment on murinometric parameters of wild-type (WT) mice and transgenic mice carrying human papillomavirus (HPV). Two trials were performed for oral administration of the lemon extract compound: in drinking water and in diet. First of all, lemons were treated by immersion with melatonin at 10 mM. Then, lemons were squeezed, and the juice obtained was freeze-dried and stored to be subsequently added to drinking water or diet, according to the assay. Thus, mice were divided into eight groups in the drink assay (each with n = 5): group 1 (G1, WT, control), group 2 (G2, WT, 1 mL lemon), group 3 (G3, WT, 1.5 mL lemon), group 4 (G4, WT, 2 mL lemon), group 5 (G5, HPV16, control), group 6 (G6, HPV16, 1 mL lemon) group 7 (G6, HPV16, 1.5 mL lemon) and group 8 (G6, HPV16, 2 mL lemon). The diet assay was divided into four groups: group 1 (G1, WT, control), group 2 (G2, WT, 4 mL lemon), group 3 (G3, HPV16, control) and group 4 (G4, HPV16, 4 mL lemon). In the drink assay, the highest concentration of melatonin (308 ng/100 mL) was for groups 4 and 8, while in the food assay, there was only one concentration of melatonin (9.96 ng/g) for groups 2 and 4. Both trials lasted 30 days. During this time, body weight, food and water were recorded. Afterward, they were sacrificed, and samples were collected for different analyses. At the concentrations used, the lemon juice with melatonin had no adverse effects on the animals’ health and showed a positive outcome in modifying weight gain and enhancing antioxidant activity in mice. Moreover, a reduction in the incidence of histological lesions was observed in treated animals. Further research is needed to better understand the effects of lemon extract on health and treatment outcomes in this animal model.

BackgroundThe integration of human papillomavirus (HPV) is closely related to the occurrence of cervical cancer. However, little is known about the complete state of HPV integration into the host genome.MethodsIn this study, three HPV-positive cell lines, HeLa, SiHa, and CaSki, were subjected to NANOPORE long-read sequencing to detect HPV integration. Analysis of viral integration patterns using independently developed software (HPV-TSD) yielded multiple complete integration patterns for the three HPV cell lines.ResultsWe found distinct differences between the integration patterns of HPV18 and HPV16. Furthermore, the integration characteristics of the viruses were significantly different, even though they all belonged to HPV16 integration. The HPV integration in the CaSki cells was relatively complex. The HPV18 integration status in HeLa cells was the dominant, whereas the percentage of integrated HPV 16 in SiHa and CaSki cells was significantly lower. In addition, the virus sequences in the HeLa cells were incomplete and existed in an integrated state. We also identified a large number of tandem repeats in HPV16 and HPV18 integration. Our study not only clarified the feasibility of high-throughput long-read sequencing in the study of HPV integration, but also explored a variety of HPV integration models, and confirmed that viral integration is an important form of HPV in cell lines.ConclusionElucidating HPV integration patterns will provide critical guidance for developing a detection algorithm for HPV integration, as well as the application of virus integration in clinical practice and drug research and development.

ABSTRACT Intramuscular vaccination of mice with the naked pBI-11 DNA plasmid targeting E6 and E7 of HPV16 and HPV18 via a conventional syringe and needle generates human papillomavirus (HPV) antigen-specific CD8+ T cell-mediated immune responses and therapeutic effects against the TC-1 tumor model. However, delivery of DNA vaccines by this method is less effective in patients, likely due to poor transduction of host tissues. Needle-free biojectors show great promise for DNA vaccination because of their simplicity of administration and high patient acceptability and also, we hypothesize, because of greater efficiency of cell transduction in host tissues. Here, we compared the kinetics of transgene expression from a plasmid DNA using intramuscular injection with a conventional needle administration to intradermal or intramuscular delivery with a customized Tropis biojector. Delivery using the customized Tropis biojector leads to enhanced transgene expression compared to intramuscular needle injection. In addition, we characterized the HPV antigen-specific CD8+ T cell-mediated immune responses and anti-tumor effects generated by pBI-11 DNA vaccination by each route of administration, as well as by split-dose multi-site injection. Intradermal, but not intramuscular, vaccination with pBI-11 DNA vaccine via customized Tropis biojector enhanced HPV antigen-specific CD8+ T cell-mediated immune responses over needle injection. Intradermal, but not intramuscular, vaccination via customized needle-free Tropis biojector elicited greater HPV antigen-specific CD8+ T cell-mediated immune responses as well as anti-tumor effects compared to intramuscular injection of pBI-11 with a needle. Good manufacturing practices (GMP) grade pBI-11 DNA vaccine delivered intradermally or intramuscularly via customized Tropis biojector was well tolerated by mice. IMPORTANCE Respectively, HPV16 and HPV18 cause 50% and 20% of cervical cancer cases globally. Viral proteins E6 and E7 are obligate drivers of oncogenic transformation. We recently developed a candidate therapeutic DNA vaccine, pBI-11, that targets HPV16 and HPV18 E6 and E7. Single-site intramuscular delivery of pBI-11 via a needle elicited therapeutic anti-tumor effects in mice and is now being tested in high-risk human papillomavirus+ head and neck cancer patients (NCT05799144). Needle-free biojectors such as the Tropis device show promise due to ease of administration, high patient acceptability, and the possibility of improved delivery. For example, vaccination of patients with the ZyCoV-D DNA vaccine using the Tropis device is effective against COVID19, well tolerated, and licensed. Here we show that split-dose, multi-site administration and intradermal delivery via the Tropis biojector increase the delivery of pBI-11 DNA vaccine, enhance HPV antigen-specific CD8+ T-cell responses, and improve anti-tumor therapeutic effects, suggesting its translational potential to treat HPV16/18 infection and disease.

Background/Aim: The most common genotypes of human papillomavirus (HPV) in patients with cervical cancer worldwide are HPV16 and HPV18. The persistence of these genotypes is associated with cervical cancer and detection, and HPV genotyping, particularly in women with abnormal Pap smears, has become a crucial tool for cervical cancer screening, diagnosis and management. We evaluated the overall prevalence of HPV in women with abnormal Pap smear cytology and also investigated age-specific HPV prevalence and HPV genotype distribution. Methods: We analyzed 716 cervical smear specimens in this retrospective cohort study. Cytological diagnoses of typical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesions (LSILs), and high-grade squamous intraepithelial lesions (HSILs) were made utilizing the Bethesda System. The Papanicolaou method was used for the staining of the Pap smears. The specimens were pre-screened for HPV DNA positivity using an HC2 assay (Qiagen, USA). After the prescreening, a Cobas 4800 HPV test system (Roche Diagnostics GmBH, Germany) was used to genotype the HPV-positive samples. Results: Of the 716 cervical smear samples, 520 (72.6%) were found to be HPV-negative. Among the HPV-positive samples, 106 (23.2%), 57 (28.8%) and 33 (53.2%) were identified from 456 ASCUS, 198 LSIL and 62 HSIL cases, respectively. These findings revealed a gradual decrease in HPV prevalence with increased cytological grade (P<0.05). For high-risk, low-risk and high-risk/low-risk HPV types, 76 (38.8%), 78 (39.8%) and 42 (21.4) were positive according to the HC2 assay, respectively (P<0.05) Only 117 of the 196 HPV-positive samples were found to be HPV-positive with the Cobas 4800 HPV test system. HPV16 was the most prevalent type detected by the Cobas 4800 HPV test: 55 out of 117 HPVpositive smear samples across all age groups (47%). HPV16 was significantly more frequently detected in the HSIL samples than HPV18 (P<0.05). The prevalence of HPV was the highest in women with ages between 29 and 38 (71/196, 36.22%) and declined with age. Conclusion: We found that HPV16 and HPV18 were the most prevalent genotypes of HPV in a cohort of Turkish women; HPV16 was most frequently detected in HSIL samples from women with ages between 29 and 38. We conclude that investigating the incidence of HPV16 and HPV18 genotypes will be important for implementing new programs and protocols to reduce the incidence of cervical cancer. These data may contribute to the development of preventive strategies to reduce the cervical cancer burden in Turkey. [ABSTRACT FROM AUTHOR]

Background: There exists strong evidence that human papillomavirus (HPV) is associated with cervical cancer (CC). HPV E6 is a major oncogene whose sequence variations may be associated with the development of CC. There is not sufficient data on the distribution of HPV types in ThinPrep cytology specimens and HPV 16/18 E6 gene variations among CC patients in the southwest of Iran. This study was conducted to contribute to HPV screening and vaccination in Iran. Methods: A total of 648 women screened for cervicitis, intraepithelial neoplasia or CC were included in the study. All participants underwent ThinPrep cytology testing, single-step HPV DNA detection and allele-specific reverse hybridization assays. Moreover, a total of 96 specimens previously tested positive for single infection with HPV16 or 18 were included for variant analysis. HPV16/18 lineages and sublineages were determined by PCR assays followed by sequencing the E6 gene and the construction of neighbor-joining phylogenetic trees. Results: Overall, HPV DNA was detected in 62.19% of all the screened subjects. The detection rates of HPV DNA among individuals with normal, ASC-US, ASC-H, LSIL, and HSIL cervical cytology were 48.9%, 93.6%, 100%, 100%, and 100%, respectively. Low-risk HPVs were detected more frequently (46.9%) than high-risk (38.9%) and possible high-risk types (11.1%). Of 403 HPV-positive subjects, 172 (42.7%) had single HPV infections while the remaining 231 (57.3%) were infected with multiple types of HPV. Our results indicated a remarkable growth of high-risk HPV66 and 68 and low-risk HPV81 which have rarely been reported in Iran and HPV90 and 87 that are reported for the first time in the country. In addition, 3 lineages (A, D, and C) and 6 sublineages (A1, A2, A4, C1, D1, and D2) of HPV16, and one lineage and 4 sublineages (A1, A3, A4, and A5) of HPV18 were identified. The studied HPV16 and 18 variants mainly belonged to the D1 and A4 sublineages, respectively. Conclusion: The present study suggests that the prevalence of HPV infection in women of all age groups with or without premalignant lesions in the southwestern Iran is high and the predominant HPV types in the southwest of Iran may differ from those detected in other parts of the country. This study also highlights the necessity of not only initiating HPV vaccination for the general population but also developing new vaccines that confer immunity against the prevalent HPV types in the area and national cervical screening programs using a combination of thinPrep cytology test and HPV detection assays in order to improve the accuracy of the screening. [ABSTRACT FROM AUTHOR]

Purpose: The mechanism of methylation of HPV CpG sites in the occurrence and prognosis of cervical carcinogenesis remains unclear. We investigated the effects of demethylation of the CpG sites of E2 and E6, essential genes of HPV16 integration, on cervical cancer cell expression, integration, and proliferation. Materials and Methods: HPV16-positive (Caski) cells were treated with different concentrations of the demethylation compound 5-aza-dc (0, 5, 10, 20 μmol/l) in vitro. After the intervention, the methylation statuses of HPV16 E2 and E6 were detected by TBS, the expression levels of E2 and E6 mRNA and protein were detected by real-time PCR and western blot, cell proliferation activity was detected by CCK8, and cell cycle and apoptosis were determined by FCM. GraphPad Prism version 8.4.2 and R version 4.2.3 were used for relevant data analyses. Results: The methylation levels of HPV16 E2 and E6 CpG sites decreased gradually with increasing 5-aza-dc intervention concentrations. With decreasing E2 and E6 methylation rates, E2 expression increased, the E2/E6 ratio increased, E6 expression decreased, and the growth inhibition rate of Caski cells increased. E2 and E6 expression were negatively and positively correlated with their degrees of methylation respectively, while the E2/E6 mRNA to protein ratio was negatively correlated with the methylation degrees of E2 and E6. Conclusion: Demethylation can be used as a prospective treatment to affect HPV expression and persistent infection, providing a new theoretical basis for the clinical treatment of viral infections. [ABSTRACT FROM AUTHOR]

Abstract Background High‐risk human papillomavirus (hrHPV) infection is a key factor that alters cervicovaginal microbiota patterns and causes cervical intraepithelial neoplasias (CINs) or even cervical cancer. Although local excisional treatment can clear hrHPV infection and restore the cervicovaginal microbiota, it is unclear which cervicovaginal microbiota represents recovery. Our objective was to describe the cervicovaginal microbiota before and after treatments and to assess the association between the microbiota and HPV persistence. Results A cohort of 91 participants was classified into four groups (healthy control women and HPV16‐infected women with CIN I, CIN II/III, and squamous cell carcinoma [SCC]). Endocervical swabs were collected 3 months prior to treatment and at 3 months post‐treatment for bacterial 16S rRNA gene pyrosequencing and for HPV DNA testing. There was an increase in the number of Lactobacillus bacterial species present after the clinical treatments, and the community state type (CST) profiles were shifted from dysbiotic CSTs II and IV to Lactobacillus‐dominated CSTs I and III. Specifically, the composition of Geobacter and Prevotella before treatment and Lactobacillus secaliphilus after treatment might have been related to CIN I, the composition of Burkholderia before treatment and Lactobacillus iners after treatment might have been related to CIN II/III, and the composition of Atopobium and Aerococcus before treatment and Bacilli after treatment might have been related to SCC. Further functional predictions revealed that the composition differences were linked to infectious disease‐ and cancer‐related genes. Conclusion Our study provides an illustration of the changes in CSTs and the cervicovaginal microbiota before and after HPV16 clearance in each disease state.

Abstract Background As a novel tumor suppressor mediator, activating transcription factor 3 (ATF3) has recently aroused an interest in its possible therapeutic applications in various cancers. In this study, we evaluated the effect of ATF3 overexpression on the cellular level of nuclear factor kappa B (NF-κB) in human papillomavirus (HPV)-infected Ca Ski cells. Further, we examined whether ATF3 could mediate cell cycle arrest and alter the apoptosis level of Ca Ski cells. Methods The biological behavior of Ca Ski cells was evaluated prior and subsequent to the overexpression of ATF3 by MTT assay, fluorescence microscopy, cell cycle and annexin V/PI flow cytometric analysis. The effect of ectopic ATF3 expression on the cellular level of NF-κB in HPV-positive cells was evaluated by western blotting assay. Results The overexpression of ATF3 in Ca Ski cells led to significant apoptosis and cell cycle arrest in the G1 phase. Western blotting assay revealed a discernible reduction of NF-κB p65 level in cervical cancer cells. Conclusion ATF3 acts as a tumor suppressor factor in HPV16-infected Ca Ski cells and exerts anti-cancer effects on HPV16-related cervical cancer cells potentially by hindering cell growth and inducing cell cycle arrest through the down-regulation of NF-κB. Our results suggest that ATF3 induction or NF-κB suppression may be useful targets for HPV16-related cervical cancer prevention and treatment.

Human papillomavirus 16 (HPV16) is considered to be strongly correlated with the development of cervical cancer. Among the 8 HPV16 genes, the E6 constitutes a remarkable marker to follow the evolutionary history and spatial phylodynamics of HPV16 in the Mediterranean basin. Thus, this work aims to decipher the major evolutionary events and crosstalks in the Mediterranean basin with a focus on Tunisian strains regarding the E6 oncogene. In this study, we first extracted the available and annotated Mediterranean strains of HPV16 E6 gene sequences (n = 155) from the NCBI nucleotide database. These sequences were aligned, edited, and used for the downstream phylogenetic analyses. Finally, a Bayesian Markov Chain Monte Carlo approach was applied to reconstruct the evolutionary history of HPV16 migration. Our results showed that the HPV circulating in Tunisia derived from a Croatian ancestor around the year 1987. This starting point spreads to most European countries to reach northern Africa through the Moroccan gateway in 2004. [ABSTRACT FROM AUTHOR]

Immunotherapies targeting truly tumor-specific targets focus on the expansion and activation of T cells against neoantigens or oncogenic viruses. One target is the human papilloma virus type 16 (HPV16), responsible for several anogenital cancers and oropharyngeal carcinomas. Spontaneous and vaccine-induced HPV-specific T cells have been associated with better clinical outcome. However, the epitopes and restriction elements to which these T cells respond remained elusive. To identify CD8+ T cell epitopes in cultures of tumor infiltrating lymphocytes, we here used multimers and/or a functional screening platform exploiting single HLA class I allele-engineered antigen presenting cells. This resulted in the detection of 20 CD8+ T cell responses to 11 different endogenously processed HLA-peptide combinations within 12 HPV16-induced tumors. Specific HLA-peptide combinations dominated the response in patients expressing these HLA alleles. T cell receptors (TCRs) reactive to seven different HLA class I-restricted peptides could be isolated and analysis revealed tumor reactivity for five of the six TCRs analyzed. The tumor reactive TCRs to these dominant HLA class I peptide combinations can potentially be used to engineer tumor-specific T cells for adoptive cell transfer approaches to treat HPV16-induced cancers. [ABSTRACT FROM AUTHOR]

Objective: To triage low-grade cervical smears (ASCUS/LSIL) by HPV 16/18 genotyping and dual staining with p16/Ki67 and to compare the sensitivity and specificity of these two triage methods for detection of high-grade cervical intraepithelial neoplasia (HGCIN). Methods: In this prospective cross-sectional study, we evaluated a total of 89 women with low-grade smears (54 ASCUS, 35 LSIL) recruited from a tertiary care hospital. All patients underwent colposcopy guided cervical biopsy. Histopathology was used as gold standard. All samples were subjected to HPV 16/18 genotyping (excluding 9) using DNA PCR and p16/Ki67 dual staining (excluding 4) using Roche® kit. We then compared the two triage methods to detect high-grade cervical lesions. Results: Overall, in all low-grade smears sensitivity, specificity and accuracy of HPV 16/18 genotyping, was found to be 66.7%, 77.1% and 76.2% respectively (p = 0.03). In low-grade smears sensitivity, specificity and accuracy of dual staining, was found to be 66.7%, 84.8% and 83.5% respectively (p = 0.01). Conclusions: Overall, in all low-grade smears the sensitivity of the two tests was comparable. However, dual staining had a higher specificity and accuracy than HPV 16/18 genotyping. It was concluded that both are effective triage methods but dual staining had a better performance than HPV 16/18 genotyping. [ABSTRACT FROM AUTHOR]

Human papillomavirus (HPV) vaccines based on HPV L1 virus-like particles (VLPs) are already licensed but not accessible worldwide. About 38.0 million people were living with HIV in 2020 and there is no HIV vaccine yet. Therefore, safe, effective, and affordable vaccines against both viruses are an urgent need. In this study, the HIV-1 P18I10 CTL peptide from the V3 loop of HIV-1 gp120 glycoprotein was inserted into the HPV16 L1 protein to construct chimeric HPV:HIV (L1:P18I10) VLPs. Instead of the traditional baculovirus expression vector/insect cell (BEVS/IC) system, we established an alternative mammalian 293F cell-based expression system using cost-effective polyethylenimine-mediated transfection for L1:P18I10 protein production. Compared with conventional ultracentrifugation, we optimized a novel chromatographic purification method which could significantly increase L1:P18I10 VLP recovery (~56%). Chimeric L1:P18I10 VLPs purified from both methods were capable of self-assembling to integral particles and shared similar biophysical and morphological properties. After BALB/c mice immunization with 293F cell-derived and chromatography-purified L1:P18I10 VLPs, almost the same titer of anti-L1 IgG (p = 0.6409) was observed as Gardasil anti-HPV vaccine-immunized mice. Significant titers of anti-P18I10 binding antibodies (p < 0.01%) and P18I10-specific IFN-γ secreting splenocytes (p = 0.0002) were detected in L1:P18I10 VLP-immunized mice in comparison with licensed Gardasil-9 HPV vaccine. Furthermore, we demonstrated that insertion of HIV-1 P18I10 peptide into HPV16 L1 capsid protein did not affect the induction in anti-L1 antibodies. All in all, we expected that the mammalian cell expression system and chromatographic purification methods could be time-saving, cost-effective, scalable platforms to engineer bivalent VLP-based vaccines against HPV and HIV-1 [ABSTRACT FROM AUTHOR]

Human papillomavirus (HPV) infection, especially HPV16, is one of the causative factors for the development of head and neck squamous cell (HNSC) carcinoma. HPV-positive and HPV-negative HNSC patients differ significantly in their molecular profiles and clinical features, so they should be evaluated differently depending on their HPV status. Given the tremendous variation in HNSC cancers depending on HPV, our goal in this study was to present biomarkers and treatment options tailored to the patient's HPV status. Gene expression levels of HPV16-positive and -negative patients were used as proxies, and the differential interactome algorithm was employed to identify the differential interacting proteins (DIPs). By assessing the prognostic capabilities and druggabilities of DIPs and their interacting partners (DIP-centered modules), we introduce eight modules as potential biomarkers specialized for either positive or negative phenotype. Finally, raloxifene was repositioned for the first time as a drug candidate for the treatment of HPV16-positive HNSC patients. • HPV16 is one of the causative factors for head and neck cancer development. • Patients have completely different characteristics according to their HPV status. • Prognosis or treatment of the patient should be different depending on HPV status. • Eight novel prognostic system biomarkers were determined. • Raloxifene was proposed for the treatment of HPV16-positive patients. [ABSTRACT FROM AUTHOR]