Advances in human papillomavirus detection and molecular understanding in head and neck cancers: Implications for clinical management.

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.

PAX1 methylation as a robust predictor: developing and validating a nomogram for assessing endocervical curettage (ECC) necessity in human papillomavirus16/18-positive women undergoing colposcopy.

OBJECTIVE: The major challenge in routine endocervical curettage (ECC) among Human Papillomavirus (HPV) 16/18-positive patients is that only a small fraction benefit. Nevertheless, current reported models often overestimate the validity and necessity of ECC, making it difficult to improve benefits for patients. This research hypothesized that assessing paired boxed gene 1 methylation levels (PAX1m) and clinical characteristics could enhance the predictive accuracy of detecting additional high-grade squamous intraepithelial lesions or worse (HSIL +) through ECC that were not identified by colposcopy-directed biopsy (CDB). METHODS: Data from 134 women with HPV16/18 positivity undergoing CDB and ECC between April 2018 and April 2022 were collected and analyzed. Quantitative methylation-specific polymerase chain reaction (qMSP) was utilized to measure PAX1m, expressed as ΔCp. Univariate and multivariate regression analyses were conducted to screen variables and select predictive factors. A nomogram was constructed using multivariate logistic regression to predict additional HSIL + detected by ECC. The discrimination, calibration, and clinical utility of the nomogram were evaluated using receiver operating characteristic curves (ROC) and the calibration plot. RESULTS: Age (odds ratio [OR], 5.654; 95% confidence interval [CI], 1.131-37.700), cytology (OR, 24.978; 95% CI, 3.085-540.236), and PAX1 methylation levels by grade (PAX1m grade) (OR, 7.801; 95% CI, 1.548-44.828) were independent predictive factors for additional detection of HSIL + by ECC. In HPV16/18-positive women, the likelihood of additional detection of HSIL + through ECC increased with the severity of cytological abnormalities, peaking at 43.8% for high-grade cytological lesions. Moreover, when cytological findings indicated low-grade lesions, PAX1 methylation levels were positively correlated with the additional detection of HSIL + by ECC (P value < 0.001). A nomogram prediction model was developed (area under curve (AUC) = 0.946; 95% CI, 0.901-0.991), demonstrating high sensitivity (90.9%) and specificity (90.5%) at the optimal cutoff point of 107. Calibration analysis confirmed the model's strong agreement between predicted and observed probabilities. CONCLUSION: The clinical nomogram presented promising predictive performance for the additional detection of HSIL + through ECC among women with HPV16/18 infection. PAX1 methylation level could serve as a valuable tool in guiding individualized clinical decisions regarding ECC for patients with HPV 16/18 infection, particularly in cases of low-grade cytological findings.

Prevalence of high-risk human papillomavirus in oral squamous cell carcinoma with or without chewing habits.

Oral cancer (OC) is the most common cancer in Pakistani males and the second most common in females. Major risk factors include peculiar chewing habits, human papillomavirus (HPV) infection and molecular pathways. However, less data is available for this avertible cancer regarding its association with high-risk HPV (HR-HPV) and chewing habits in this region. Therefore, this study was done to determine the prevalence of HR-HPV in oral squamous cell carcinoma (OSCC) and its correlation with p16 and chewing habits. Formalin-fixed paraffin-embedded (FFPE) biopsy specimens of 186 samples were tested for HR-HPV type 16/18 by PCR, followed by p16 immunostaining (IHC) in a subset of cases (n = 50). Appropriate statistical tests were applied to find the association between HR-HPV/p16 and peculiar chewing habits with significance criteria of p<0.05 with 95% CI. HR-HPV (type 16 &18) was present in seven out of 186 cases (3.8%). Of these seven cases, five were positive for HPV16, whereas two were positive for HPV16/18. The overall expression of p16 protein in 50 samples was 38% (n = 19), and among these 19-IHC positive samples, 26% were positive for HR-HPV DNA. No significant association was found between HR-HPV positivity and p16 and chewing habits (p>0.05). It was concluded that HR-HPV prevalence in OSCC was very low in our population, with no statistically significant correlation with p16 and chewing habits. These results suggest the role of HR-HPV as an independent risk factor in OSCC in the local setting.

Changes in intrahost genetic diversity according to lesion severity in longitudinal HPV16 samples.

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.

An Evaluation of High-Risk HPV in Squamous Cell Carcinomas of the Lip in a South African Cohort.

BACKGROUND: To determine the prevalence of HR-HPV in a series of lip SCC from South African patients, using currently accepted HPV-testing methodologies and to define the clinical and histomorphologic features of HPV-associated lip SCC. METHODS: Fifty SCC of lip and 50 control cases were tested for HR-HPV using p16 and HR-HPV DNA PCR. p16-equivocal/positive and HPV DNA PCR-positive SCC were further evaluated for the expression of HPV-16 and HPV-18 mRNA transcripts using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to confirm transcriptionally active HPV. RESULTS: p16 was positive in 22% (n = 11) and equivocal in 4% (n = 2) of the SCC. One p16-positive case showed positivity for both HPV-16 DNA and HPV-16 E6/E7 mRNA transcripts (HPV prevalence rate of 2%). The HPV-positive case was non-keratinizing and occurred in an 80-year-old female. The two p16-equivocal cases were HR-HPV DNA positive and mRNA PCR negative. p16 was found to have a positive predictive value of 9%. CONCLUSION: Findings from our cohort of lip SCC suggest that HR-HPV may have an insignificant role in the pathogenesis of SCC at this site. Due to its low ppv, p16 is insufficient to establish HR-HPV infection in SCC of the lip. The combination of p16 and DNA PCR appears to correlate with the presence of transcriptionally active virus. HPV E6/E7 mRNA detection is the gold standard for identifying HR-HPV. mRNA testing is not widely available in sub-Saharan Africa due to technical and financial constraints; however, the test appears to be of great value in p16-equivocal lip SCC.

Assessing the reduction of viral infectivity in HPV16/18-positive women after one, two, and three doses of Gardasil-9 (RIFT): Study protocol.

Human Papillomavirus (HPV) prophylactic vaccination has proven effective in preventing new infections, but it does not treat existing HPV infections or associated diseases. Hence, there is still an important reservoir of HPV in adults, as vaccination programs are mainly focused on young women. The primary objective of this non-randomized, open-label trial is to evaluate if a 3-dose regimen of Gardasil-9 in HPV16/18-positive women could reduce the infective capacity of their body fluids. We aim to assess if vaccine-induced antibodies could neutralize virions present in the mucosa, thus preventing the release of infective particles and HPV transmission to sexual partners. As our main endpoint, the E1^E4-HaCaT model will be used to assess the infectivity rate of cervical, anal and oral samples, obtained from women before and after vaccination. HPV DNA positivity, virion production, seroconversion, and the presence of antibodies in the exudates, will be evaluated to attribute infectivity reduction to vaccination. Our study will recruit two different cohorts (RIFT-HPV1 and RIFT-HPV2) of non-vaccinated adult women. RIFT-HPV1 will include subjects with an HPV16/18 positive cervical test and no apparent cervical lesions or cervical lesions eligible for conservative treatment. RIFT-HPV2 will include subjects with an HPV16/18 positive anal test and no apparent anal lesions or anal lesions eligible for conservative treatment, as well as women with an HPV16/18 positive cervical test and HPV-associated vulvar lesions. Subjects complying with inclusion criteria for both cohorts will be recruited to the main cohort, RIFT-HPV1. Three doses of Gardasil-9 will be administered intramuscularly at visit 1 (0 months), visit 2 (2 months) and visit 3 (6 months). Even though prophylactic HPV vaccines would not eliminate a pre-existing infection, our results will determine if HPV vaccination could be considered as a new complementary strategy to prevent HPV-associated diseases by reducing viral spread. Trial registration: https://clinicaltrials.gov/ct2/show/NCT05334706.

Prevailing of HPV-16 and 52 genotype in 2022-2023 in Sanandaj, Iran.

INTRODUCTION: Human papillomavirus (HPV) presents a potential threat to the onset of carcinogenesis in the cervix, anogenital regions, and oropharynx. HPV encompasses over 200 types, with at least 12 having the potential to cause cancer, impacting the majority of sexually active individuals. In this current research, we explore the occurrence and spread of HPV genotypes. MATERIAL AND METHODS: During this cross-sectional study conducted in Sanandaj, Iran from Feb 2022 to Aug 2023, diverse samples including oral, vaginal, and genital were collected from individuals referred to private laboratories in Sanandaj, Iran. After sample collection and DNA extraction (FAVORGEN, Taiwan), they were subjected to PCR and genotyping (MehrViru, Iran). The subsequent statistical analysis unveiled infection rates across different demographics and age groups. STATA (version 17) were used for statistical analysis. We examined infection rates across demographics using t-tests and Odds Ratio. RESULTS: Overall, 26% (249) out of 950 cases tested positive for HPV, with 69% of these classified as high-risk. Among the examined population, 98% (933) were female, and 2% (17) were male. Females aged 31-40 exhibited the highest percentage of HPV prevalence (115/460) in the study with the majority of positive cases belonging to HR genotypes. The overall most frequent genotypes identified were 6, 16, 52, 53, 51, 58, and 56. HPV-16 exhibited the highest frequency among HR genotypes, accounting for 42 (17%) occurrences, followed by HPV-52 with a frequency of 32 (13%). CONCLUSION: Our findings emphasize the significant prevalence of HPV among females, particularly in the 21-30 age group. The identification of high-risk genotypes, underscores the importance of targeted interventions for specific age cohorts. The age-stratified analysis highlights a consistent predominance of high-risk HPV across age groups, indicating the need for age-specific preventive measures. These results contribute valuable information for designing effective screening and vaccination strategies, to alleviate the impact of diseases associated with HPV.

Detection of free DNA based on metal-enhanced fluorescence triggered by CRISPR-Cas12a and colorimetric analysis.

The CRISPR-Cas system has been found to be extremely sensitive and there is an urgent demand to extend its potential in bioassays. Herein, we developed a novel nanobiosensor to detect the human papillomavirus 16 genes (HPV-16 DNA), which is triggered by CRISPR-Cas12a to amplify the fluorescence signal by metal-enhanced fluorescence (CAMEF). Along with the changing of the fluorescence signal, the aggregation of the substrate of MEF also leads to a change in the color of the mixture solution, enabling dual signal detection with the fluorescence and the naked eye. Furthermore, the designed CAMEF probe was verified to detect the HPV-16 DNA accurately and reliably in biological samples. Triggered by the CRISPR system, the designed CAMEF probe allows quantitative detection of the HPV-16 DNA in the wide range of 10-500 pM. Owing to the MEF, the fluorescence signal of the CAMEF probe was significantly amplified with the detection limit as low as 1 pM. Besides, we can determine the concentration of HPV-16 DNA simply by the naked eye, which also drastically reduces the possibility of false-positive signals. Theoretically, the target ssDNA could be any strand of DNA obtained by designing the crRNA sequence in the CRISPR-Cas system. We believe that the designed CAMEF sensor can present a reliable approach for the accurate detection of low amounts of target ssDNA in complex biological samples.

[Precancers of the oral mucosa: clinic, diagnostics]. / Predraki slizistoi obolochki rta: klinika i diagnostika.

OBJECTIVE: The aim of the study. Improving the efficiency of diagnosis and detailing the features of the clinic of «potentially malignant¼ diseases of the oral mucosa. MATERIALS AND METHODS: Clinical and laboratory examination of 124 patients of the department of oral mucosa diseases aged 35 to 80 years, among whom there were 75 women and 49 men, with diseases such as erythroplakia - 12 patients, verrucous leukoplakia - 52 patients, erosive form of leukoplakia - 35 patients, cheilitis Manganotti - 25 patients. Histological and immunohistochemical methods of investigation were used as diagnostics. To assess the proliferative activity of epithelial cells, the determination of the Ki-67 index was used. The synthesis of keratin 15 (K15) in epithelial layers was determined as a diagnostic criterion for the severity of neoplasia. The expression of human papillomavirus type 16 (HPV 16) antigens and p16INK4a protein in epithelial cells was studied, as well as the expression of p53 protein. RESULTS: A high prevalence of p53 mutations was observed in patients with erythroplakia. In leukoplakia, the expression of the Ki-67 protein was detected in the cell nuclei in both the basal and parabasal layers of the multilayer squamous epithelium, in 77% of cases, the expression of the p16INK4a protein in the epithelial nuclei with varying degrees of dysplastic changes was noted, and a positive reaction to HPV16 was also observed in the cell nuclei and cytoplasm of epithelial cells in the basal, parabasal and spiny epithelial layers. The appearance of K15 in the cytoplasm of cells above the basal layer with abrasive precancerous cheilitis was found in 48% of cases. CONCLUSION: To diagnose early manifestations of neoplastic processes in «potentially malignant¼ diseases of the oral mucosa, it is necessary to use both classical histological and immunohistochemical methods of investigation with various markers.

Exploring the Role of E6 and E7 Oncoproteins in Cervical Oncogenesis through MBD2/3-NuRD Complex Chromatin Remodeling.

BACKGROUND: Cervical cancer is among the highest-ranking types of cancer worldwide, with human papillomavirus (HPV) as the agent driving the malignant process. One aspect of the infection's evolution is given by epigenetic modifications, mainly DNA methylation and chromatin alteration. These processes are guided by several chromatin remodeling complexes, including NuRD. The purpose of this study was to evaluate the genome-wide binding patterns of the NuRD complex components (MBD2 and MBD3) in the presence of active HPV16 E6 and E7 oncogenes and to determine the potential of identified genes through an experimental model to differentiate between cervical precursor lesions, with the aim of establishing their utility as biomarkers. METHODS: The experimental model was built using the CaSki cell line and shRNA for E6 and E7 HPV16 silencing, ChIP-seq, qRT-PCR, and Western blot analyses. Selected genes' expression was also assessed in patients. RESULTS: Several genes have been identified to exhibit altered transcriptional activity due to the influence of HPV16 E6/E7 viral oncogenes acting through the MBD2/MBD3 NuRD complex, linking them to viral infection and cervical oncogenesis. CONCLUSIONS: The impacted genes primarily play roles in governing gene transcription, mRNA processing, and regulation of translation. Understanding these mechanisms offers valuable insights into the process of HPV-induced oncogenesis.

A human papillomavirus 16 E2-TopBP1 dependent SIRT1-p300 acetylation switch regulates mitotic viral and human protein levels and activates the DNA damage response.

An interaction between human papillomavirus 16 (HPV16) E2 and the cellular proteins TopBP1 and BRD4 is required for E2 plasmid segregation function. The E2-TopBP1 interaction promotes increased mitotic E2 protein levels in U2OS and N/Tert-1 cells, as well as in human foreskin keratinocytes immortalized by HPV16 (HFK + HPV16). SIRT1 deacetylation reduces E2 protein stability and here we demonstrate that increased E2 acetylation occurs during mitosis in a TopBP1 interacting-dependent manner, promoting E2 mitotic stabilization. p300 mediates E2 acetylation and acetylation is increased due to E2 switching off SIRT1 function during mitosis in a TopBP1 interacting-dependent manner, confirmed by increased p53 stability and acetylation on lysine 382, a known target for SIRT1 deacetylation. SIRT1 can complex with E2 in growing cells but is unable to do so during mitosis due to the E2-TopBP1 interaction; SIRT1 is also unable to complex with p53 in mitotic E2 wild-type cells but can complex with p53 outside of mitosis. E2 lysines 111 and 112 are highly conserved residues across all E2 proteins and we demonstrate that K111 hyper-acetylation occurs during mitosis, promoting E2 interaction with Topoisomerase 1 (Top1). We demonstrate that K112 ubiquitination promotes E2 proteasomal degradation during mitosis. E2-TopBP1 interaction promotes mitotic acetylation of CHK2, promoting phosphorylation and activation of the DNA damage response (DDR). The results present a new model in which the E2-TopBP1 complex inactivates SIRT1 during mitosis, and activates the DDR. This is a novel mechanism of HPV16 activation of the DDR, a requirement for the viral life cycle. IMPORTANCE: Human papillomaviruses (HPVs) are causative agents in around 5% of all human cancers. While there are prophylactic vaccines that will significantly alleviate HPV disease burden on future generations, there are currently no anti-viral strategies available for the treatment of HPV cancers. To generate such reagents, we must understand more about the HPV life cycle, and in particular about viral-host interactions. Here, we describe a novel mitotic complex generated by the HPV16 E2 protein interacting with the host protein TopBP1 that controls the function of the deacetylase SIRT1. The E2-TopBP1 interaction disrupts SIRT1 function during mitosis in order to enhance acetylation and stability of viral and host proteins. We also demonstrate that the E2-TopBP1 interaction activates the DDR. This novel complex is essential for the HPV16 life cycle and represents a novel anti-viral therapeutic target.

Exosomal EGFR and miR-381-3P Mediate HPV-16 E7 Oncoprotein-Induced Angiogenesis of Non-Small Cell Lung Cancer.

BACKGROUND: It has been demonstrated that exosomes derived from HPV-16 E7-over-expressiong non-small cell lung cancer (NSCLC) cells (E7 Exo) trigger increased levels of epidermal growth factor receptor (EGFR) and miR-381-3p. The purpose of this investigation was to examine the role of E7 Exo in NSCLC angiogenesis, and to analyze the contribution of exosomal EGFR and miR-381-3p to it. METHODS: The influence of E7 Exo on the proliferation and migration of human umbilical vein endothelial cells (HUVECs) was assessed using colony formation and transwell migration assays. Experiments on both cells and animal models were conducted to evaluate the angiogenic effect of E7 Exo treatment. The involvement of exosomal EGFR and miR-381-3p in NSCLC angiogenesis was further investigated through suppressing exosome release or EGFR activation, or by over-expressing miR-381-3p. RESULTS: Treatment with E7 Exo increased the proliferation, migration, and tube formation capacities of HUVECs, as well as angiogenesis in animal models. The suppression of exosome release or EGFR activation in NSCLC cells decreased the E7-induced enhancements in HUVEC migration and tube formation, and notably reduced vascular endothelial growth factor A (VEGFA) and Ang-1 levels. HUVECs that combined miR-381-3p mimic transfection and E7 Exo treatment exhibited a more significant tube-forming capacity than E7 Exo-treated HUVECs alone, but were reversed by the miR-381-3p inhibitor. CONCLUSION: The angiogenesis induced by HPV-16 E7 in NSCLC is mediated through exosomal EGFR and miR-381-3p.

Tobacco Smoke Condensate Induces Morphologic Changes in Human Papillomavirus-Positive Cervical Epithelial Cells Consistent with Epithelial to Mesenchymal Transition (EMT) with Activation of Receptor Tyrosine Kinases and Regulation of TGFB.

High-risk human papillomavirus (HR-HPV; HPV-16) and cigarette smoking are associated with cervical cancer (CC); however, the underlying mechanism(s) remain unclear. Additionally, the carcinogenic components of tobacco have been found in the cervical mucus of women smokers. Here, we determined the effects of cigarette smoke condensate (CSC; 3R4F) on human ectocervical cells (HPV-16 Ect/E6E7) exposed to CSC at various concentrations (10-6-100 µg/mL). We found CSC (10-3 or 10 µg/mL)-induced proliferation, enhanced migration, and histologic and electron microscopic changes consistent with EMT in ectocervical cells with a significant reduction in E-cadherin and an increase in the vimentin expression compared to controls at 72 h. There was increased phosphorylation of receptor tyrosine kinases (RTKs), including Eph receptors, FGFR, PDGFRA/B, and DDR2, with downstream Ras/MAPK/ERK1/2 activation and upregulation of common EMT-related genes, TGFB SNAI2, PDGFRB, and SMAD2. Our study demonstrated that CSC induces EMT in ectocervical cells with the upregulation of EMT-related genes, expression of protein biomarkers, and activation of RTKs that regulate TGFB expression, and other EMT-related genes. Understanding the molecular pathways and environmental factors that initiate EMT in ectocervical cells will help delineate molecular targets for intervention and define the role of EMT in the initiation and progression of cervical intraepithelial neoplasia and CC.

Concomitant human papillomavirus (HPV) vaccination and screening for elimination of HPV and cervical cancer.

HPV vaccination with concomitant HPV-based screening of young women has been proposed for faster cervical cancer elimination. We describe the baseline results of a population-based trial of this strategy to reduce the incidence of HPV. All 89,547 women born 1994-1999 and resident in the capital region of Sweden were personally invited to concomitant HPV vaccination and HPV screening with 26,125 women (29.2%) enrolled between 2021-05-03 and 2022-12-31. Baseline HPV genotyping of cervical samples from the study participants finds, compared to pre-vaccination prevalences, a strong decline of HPV16 and 18 in birth cohorts previously offered vaccination, some decline for cross-protected HPV types but no decline for HPV types not targeted by vaccines. Our dynamic transmission modelling predicts that the trial could reduce the incidence of high-risk HPV infections among the 1994-1998 cohorts by 62-64% in 3 years. Baseline results are prevalences of HPV infection, validated transmission model projections, and power estimates for evaluating HPV incidence reductions at follow-up (+/-0.1% with 99.9% confidence). In conclusion, concomitant HPV vaccination and HPV screening appears to be a realistic option for faster cervical cancer elimination. Clinicaltrials.gov identifier: NCT04910802; EudraCT number: 2020-001169-34.

Design and evaluation of a multi-epitope DNA vaccine against HPV16.

Cervical cancer, among the deadliest cancers affecting women globally, primarily arises from persistent infection with high-risk human papillomavirus (HPV). To effectively combat persistent infection and prevent the progression of precancerous lesions into malignancy, a therapeutic HPV vaccine is under development. This study utilized an immunoinformatics approach to predict epitopes of cytotoxic T lymphocytes (CTLs) and helper T lymphocytes (HTLs) using the E6 and E7 oncoproteins of the HPV16 strain as target antigens. Subsequently, through meticulous selection of T-cell epitopes and other necessary elements, a multi-epitope vaccine was constructed, exhibiting good immunogenic, physicochemical, and structural characteristics. Furthermore, in silico simulations showed that the vaccine not only interacted well with toll-like receptors (TLR2/TLR3/TLR4), but also induced a strong innate and adaptive immune response characterized by elevated Th1-type cytokines, such as interferon-gamma (IFN-γ) and interleukin-2 (IL2). Additionally, our study investigated the effects of different immunization intervals on immune responses, aiming to optimize a time-efficient immunization program. In animal model experiments, the vaccine exhibited robust immunogenic, therapeutic, and prophylactic effects. Administered thrice, it consistently induced the expansion of specific CD4 and CD8 T cells, resulting in substantial cytokines release and increased proliferation of memory T cell subsets in splenic cells. Overall, our findings support the potential of this multi-epitope vaccine in combating HPV16 infection and signify its candidacy for future HPV vaccine development.

Through the stringent selection of T-cell epitopes and other necessary elements, a novel multi-epitope vaccine targeting HPV 16 E6 and E7 oncoproteins was constructed using an immunoinformatics approach.The vaccine designed can induce both cellular and humoral immune responses, encompassing all the required immunogenic, physicochemical, and structural characteristics for an ideal vaccine design. Moreover, it offers decent worldwide coverage.In animal studies, the vaccine demonstrated strong immune responses, including expansion of CD4 and CD8 T cells, cytokine release, and enhanced memory T cell proliferation, resulting in long-term anti-tumor effects, inhibition of tumor growth, and prolonged survival in tumor-bearing mice.The immunological evaluation of the designed vaccine suggests its potential as a novel vaccine candidate against HPV 16.

Preliminary outcomes of the Cervical Cancer Screening Program of Northern Portugal: A snapshot.

BACKGROUND: Cervical cancer screening remains an essential preventive tool worldwide. First line high-risk Human Papillomavirus (HrHPV) genotyping became gold standard for cervical cancer screening, and has been adopted by several countries, including Portugal. Herein, we aimed to assess the early outcomes of the regional Cervical Cancer Screening Program of Northern Portugal. METHODS: The analysis of a representative set of cases evaluated during a one-month period (January 2020), with adequate follow-up was performed. Descriptive analysis was performed. RESULTS: Overall, 7278 samples were received, of which 15.2% were HrHPV positive, most of these disclosing a negative result in subsequent liquid-based cytology. Nearly half of the HrHPV-positive women were referred to colposcopy. Within this group, HPV16/18+ cases depicted the higher frequency of high-grade squamous intraepithelial lesion (HSIL) or worse, compared with abnormal cytology or persistent HrHPV infection. Among women with non-HPV16/18 HrHPV infection and negative cytology, which are eligible for repeat sampling in one year, 65% were re-tested. Importantly, nearly half of these cleared HrHPV infection. Furthermore, referral to colposcopy due to HPV16/18 infection and/or abnormal cytology results were associated with > 40% risk for HSIL or worse lesion. CONCLUSIONS: Our study confirmed the reliability and effectiveness of first line HrHPV genotyping in the Cervical Cancer Screening Program of Northern Portugal. Nonetheless, it also raised concerns about excessive referral to colposcopy, with the inherent human and financial costs. Thus, further improvement and optimization are key to ensure the sustainability of the program.

Naturally developed HPV16 antibodies and risk of newly detected cervical HPV infection outcomes.

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.

Biplex quantitative PCR to detect transcriptionally active human papillomavirus 16 from patient saliva.

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.

Cervical microbiota dysbiosis associated with high-risk Human Papillomavirus infection.

High-risk Human Papillomavirus (HR-HPV) genotypes, specifically HPV16 and HPV18, pose a significant risk for the development of cervical intraepithelial neoplasia and cervical cancer. In the multifaceted cervical microenvironment, consisting of immune cells and diverse microbiota, Lactobacillus emerges as a pivotal factor, wielding significant influence in both stabilizing and disrupting the microbiome of the reproductive tract. To analyze the distinction between the cervical microbiota and Lactobacillus-dominant/non-dominant status of HR-HPV and non-infected healthy women, sixty-nine cervical swab samples were analyzed, included 44 with HR-HPV infection and healthy controls. All samples were recruited from Human Papillomavirus-based cervical cancer screening program and subjected to 16s rRNA sequencing analysis. Alpha and beta diversity analyses reveal no significant differences in the cervical microbiota of HR-HPV-infected women, including 16 and 18 HPV genotypes, and those with squamous intraepithelial lesion (SIL), compared to a control group. In this study we identified significantly lower abundance of Lactobacillus mucosae in women with HR-HPV infection compared to the control group. Furthermore, changes in bacterial diversity were noted in Lactobacillus non-dominant (LND) samples compared to Lactobacillus-dominant (LD) in both HR-HPV-infected and control groups. LND samples in HR-HPV-infected women exhibited a cervical dysbiotic state, characterized by Lactobacillus deficiency. In turn, the LD HR-HPV group showed an overrepresentation of Lactobacillus helveticus. In summary, our study highlighted the distinctive roles of L. mucosae and L. helveticus in HR-HPV infections, signaling a need for further research to demonstrate potential clinical implications of cervical microbiota dysbiosis.

Analysis of related factors for pathological upgrading of cervical biopsy from CIN3 to cancer after conical resection.

BACKGROUND: To investigate related factors for postoperative pathological upgrading of cervical biopsy to cervical cancer (CC) in patients with cervical intraepithelial neoplasia (CIN)3 after conical resection. METHODS: This retrospective study collected data from patients diagnosed with CIN3 by cervical biopsies at the author's Hospital between January 2012 and December 2022. The primary outcome was the pathological results of patients after conical resection. The pathological findings were categorized into the pathological upgrading group if postoperative pathology indicated CC, while those with normal, inflammatory, or cervical precancerous lesions were classified into the pathological non-upgrading group. The factors associated with upgrading were identified using multivariable logistic regression analysis. RESULTS: Among 511 patients, there were 125 patients in the pathological upgrading group (24.46%). The patients in the upgrading group were younger (47.68 ± 9.46 vs. 52.11 ± 7.02, P < 0.001), showed a lower proportion of menopausal women (38.40% vs. 53.02%, P = 0.0111), a lower proportion of HSIL (40.00% vs. 57.77%, P = 0.001), a higher rate of HPV-16/18 positive (25.60% vs. 17.36%, P = 0.011), a higher rate of contact bleeding (54.40% vs. 21.50%, P < 0.001), lower HDL levels (1.31 ± 0.29 vs. 1.37 ± 0.34 mmol/L, P = 0.002), higher neutrophil counts (median, 3.50 vs. 3.10 × 109/L, P = 0.001), higher red blood cell counts (4.01 ± 0.43 vs. 3.97 ± 0.47 × 1012/L, P = 0.002), higher platelet counts (204.84 ± 61.24 vs. 187.06 ± 73.66 × 109/L, P = 0.012), and a smaller platelet volume (median, 11.50 vs. 11.90 fL, P = 0.002).The multivariable logistic regression analysis showed that age (OR = 0.90, 95% CI: 0.86-0.94, P < 0.001), menopausal (OR = 2.68, 95% CI: 1.38-5.22, P = 0.004), contact bleeding (OR = 4.80, 95% CI: 2.91-7.91, P < 0.001), and mean platelet volume (OR = 0.83, 95% CI: 0.69-0.99, P = 0.038) were independently associated with pathological upgrading from CIN3 to CC after conical resection. CONCLUSION: Age, menopausal, contact bleeding, and mean platelet volume are risk factors of pathological upgrading from CIN3 to CC after conical resection, which could help identify high risk and susceptible patients of pathological upgrading to CC.