Human Papillomavirus Gene Research Articles

Low PD-L1 expression, MAP2K2 alterations, and enriched HPV gene signatures characterize brain metastases in head and neck squamous cell carcinoma

BackgroundBrain metastasis (BM) is a rare but severe complication of head and neck squamous cell carcinoma (HNSCC), with limited knowledge of molecular characteristics and immunogenicity.MethodsWe analyzed 61 cases of HNSCC-BM from three academic institutions (n = 24) and Foundation Medicine Inc (FMI, n = 37). A subset of cases underwent next-generation sequencing, multiple immunofluorescence, and proximity ligation sequencing. Gene enrichment analysis compared alterations in FMI BM samples (n = 37) with local samples (n = 4082).ResultsDemographics included: median age of 59 years, 75% male, 55% current/former smokers, 75% oropharyngeal primary, and 67% human papillomavirus (HPV) +. ATM (54%), KMT2A (54%), PTEN (46%), RB1 (46%), and TP53 (46%) were frequently altered in BM samples from academic centers (62% HPV/p16+). Structural rearrangements ranged from 9 to 90 variants by proximity ligation sequencing. BMs had low densities of CD8+, PD-1+, PD-L1+, and FOXP3 + cells, and 92% had PD-L1 combined positive scores < 1%. CDKN2A (40.5%), TP53 (37.8%), and PIK3CA (27.0%) alterations were common in the FMI BMs (51% HPV+). MAP2K2 alterations and HPV + signature were enriched in FMI BMs compared to local tumors (11.8% vs. 6.4%, P = 0.005 and 51.25% vs. 26.11%, P = 0.001 respectively), and pathogenic TSC1 inactivating mutations were enriched in local tumors (67.3% vs. 37.8%, P = 0.008). Median overall survival from BM diagnosis was 9 months (range 0–27).ConclusionsHNSCC patients with BM frequently have oropharyngeal primary sites and are HPV+. Common molecular alterations in BM samples, including targetable PIK3CA and ATM, were identified. MAP2K2 alterations were enriched and densities of immune cells were low, highlighting potential targets for further research and immunotherapy considerations.

Abstract 6082: Rapid on-site nucleic acid detection using CRISPR and digital signal processing for portable and integrated cervical cancer screening in low resource settings

Abstract Addressing the global disparity in cancer care necessitates the development of rapid and affordable nucleic acid (NA) testing technologies. This need is particularly critical for cervical cancer. Molecular detection of human papillomavirus (HPV) has emerged as a highly accurate screening method, surpassing traditional Pap smears. However, implementing this transition in low- and middle-income countries has been challenging due to the high costs and centralized facilities required for current NA tests. Here, we present CreDiT (CRISPR Enhanced Digital Testing), an advanced diagnostic system for rapid, on-site NA detection. CreDiT integrates two major technical breakthroughs: i) a one-pot CRISPR strategy that simultaneously amplifies both target NAs and analytical signals, and ii) a robust fluorescent detection method based on digital communication (encoding/decoding) technology. These innovations enhance CreDiT's practical utility, offering a rapid assay (&amp;lt;35 minutes) that integrates NA extraction and detection in a single streamlined workflow. Furthermore, CreDiT's straightforward probe design enables easy incorporation of new NA targets, while its compact device provides robust signal detection. We adapted CreDiT for point-of-care HPV screening by designing probes for high-risk HPV genes (HPV16, HPV18, HPV45, HPV31, HPV33, HPV58) and oncoprotein mRNAs (E6, E7, p16INK4a) and developing a portable CreDiT device capable of processing 12 samples. CreDiT demonstrated sensitive detection of cell-derived HPV DNA targets down to single copies and accurately identified HPV types in every clinical cervical brushing specimen (n = 121) we tested. This technology has the potential to facilitate prompt and reliable triaging of high-risk HPV, overcoming pathology bottlenecks and circumventing geographical and socioeconomic barriers to effective cervical cancer screening in resource-limited regions. This work has spearheaded recent screening research efforts in Uganda and Ghana. Citation Format: Chang Yeol Lee, Hyunho Kim, Hanna Lee, Thomas Randall, Amy Ly, Hakho Lee, Cesar M. Castro. Rapid on-site nucleic acid detection using CRISPR and digital signal processing for portable and integrated cervical cancer screening in low resource settings [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6082.

Enhancing Cervical Cancer Screening: Review of p16/Ki-67 Dual Staining as a Promising Triage Strategy.

Cervical cancer, primarily caused by high-risk human papillomavirus (HR-HPV) types 16 and 18, is a major global health concern. Persistent HR-HPV infection can progress from reversible precancerous lesions to invasive cervical cancer, which is driven by the oncogenic activity of human papillomavirus (HPV) genes, particularly E6 and E7. Traditional screening methods, including cytology and HPV testing, have limited sensitivity and specificity. This review explores the application of p16/Ki-67 dual-staining cytology for cervical cancer screening. This advanced immunocytochemical method allows for simultaneously detecting p16 and Ki-67 proteins within cervical epithelial cells, offering a more specific approach for triaging HPV-positive women. Dual staining and traditional methods are compared, demonstrating their high sensitivity and negative predictive value but low specificity. The increased sensitivity of dual staining results in higher detection rates of CIN2+ lesions, which is crucial for preventing cervical cancer progression. However, its low specificity may lead to increased false-positive results and unnecessary biopsies. The implications of integrating dual staining into contemporary screening strategies, particularly considering the evolving landscape of HPV vaccination and changes in HPV genotype prevalence, are also discussed. New guidelines and further research are necessary to elucidate the long-term effects of integrating dual staining into screening protocols.

The human papillomavirus late life cycle and links to keratinocyte differentiation.

Regulation of human papillomavirus (HPV) gene expression is tightly linked to differentiation of the keratinocytes the virus infects. HPV late gene expression is confined to the cells in the upper layers of the epithelium where the virus capsid proteins are synthesized. As these proteins are highly immunogenic, and the upper epithelium is an immune-privileged site, this spatial restriction aids immune evasion. Many decades of work have contributed to the current understanding of how this restriction occurs at a molecular level. This review will examine what is known about late gene expression in HPV-infected lesions and will dissect the intricacies of late gene regulation. Future directions for novel antiviral approaches will be highlighted.

RNA elements that controlhuman papillomavirus mRNA splicing-targets for therapy?

Human papillomaviruses (HPVs) cause more than 4.5% of all cancer in the world and more than half of these cases are attributed to human papillomavirus type 16 (HPV16). Prophylactic vaccines are available but antiviral drugs are not. Novel targets for therapy are urgently needed. Alternative RNA splicing is extensively used by HPVs to express all their genes and HPV16 is no exception. This process must function to perfection since mis-splicing could perturb the HPV gene expression program by altering mRNA levels or by generating dysfunctional mRNAs. Cis-acting RNA elements on the viral mRNAs and their cognate cellular trans-acting factors control papillomavirus RNA splicing. The precise but delicate nature of the splicing process renders splicing sensitive to interference. As such, papillomavirus RNA splicing is a potential target for therapy. Here we summarize our current understanding of cis-acting HPV16 RNA elements that control HPV16 mRNA splicing via cellular proteins and discuss how they may be exploited as targets for therapy to papillomavirus infections and cancer.

Epigenetic Peculiarity of Alphapapillomavirus Genus Based on the CpG Dinucleotides’ Island Variability

Background: Methylation plays a crucial role in genome regulation, serving as an essential epigenetic mechanism. CpG islands, which are regions of DNA rich in CpG dinucleotides, are ubiquitous epigenetic regulatory features that can modulate the functional dynamics of genes, thereby influencing the pathophysiology of pathogenic organisms within a host. Objectives: In this study, we aimed to investigate the distribution of CpG islands on Papillomavirus genomes and assess their potential impact on the neoplastic progression of different Alphapapillomavirus genera within host cells. Methods: We conducted an analysis of dinucleotide frequencies in DNA sequences from various Alphapapillomavirus genera. The sequences for our analysis were sourced from the specialized HPV (human papillomavirus) database (pave.niaid.nih.gov). Following a comprehensive comparative examination of entire genomes from different HPV species, we specifically focused on 63 genomes belonging to the Alphapapillomavirus genus to identify internal CpG island profiles. These investigations were conducted using online bioinformatics software (DBCAT), and statistical analysis was performed using GraphPad Prism v. 8.0.1. Results: Our preliminary findings revealed that 76.1% of the viruses in our study had fewer than 3 CpG islands but multiple CpG sites, while fewer than 25% of the viruses possessed at least 3 CpG islands. High-risk viruses were identified in 68.42% of genotypes with fewer than 3 CpG islands, whereas low-risk genotypes were observed in 15.7% of cases. Notably, some oncogenic viruses lacked CpG islands entirely, and this pattern was also observed in viruses with a single CpG island, occurring in 50% of cases. Based on the absence of CpG islands, genotypes such as HPV 67, HPV 97, and HPV 34 could potentially be classified as carcinogenic. Additionally, the distribution of CpG islands across HPV genes did not appear to be random. Genes like L2 and E2 contained a CpG island in 50% of cases, whereas oncogenes E6 and E7 consistently had a CpG island in 100% of cases in high-risk genotypes. Conclusions: Our findings suggest that the loss of 1 or more CpG islands could potentially transform an HPV genotype into a high-risk genotype. This process may be accelerated in the presence of host-related risk factors. Further research is required to validate whether CpG island distribution can aid in identifying oncogenic viruses.

Reconnoitering correlation between human papillomavirus infection-induced vaginal microecological abnormality and squamous intraepithelial lesion (SIL) progression

ObjectiveThis study aims to investigate the relationship between abnormal vaginal microecology and human papillomavirus (HPV) infection, as well as the squamous intraepithelial lesions (SIL) progression.MethodsA total of 383 patients diagnosed with HPV infection in our hospital between March 2017 and February 2022 were selected as the experimental group. In addition, several volunteers (n = 898) who underwent physical examination during the same period were randomly selected as the control group. Subsequently, we conducted several investigations, such as HPV detection and gene typing, examined vaginal microecological imbalances, and performed cytological examinations to analyze the correlation between microecological changes, different types of HPV infection, and SIL progression.ResultsHPV detection primarily included single and high-risk types of HPV infections. Moreover, significant disparities in the vaginal microecological environment between patients with persistent HPV infection and the control group, as well as patients with low-grade and high-grade SIL (LSIL and HSIL), were observed. The regression analysis revealed a correlation between LSIL and microflora density, diversity, bacteriological vaginosis (BV), vulvovaginal candidiasis (VVC), trichomonas vaginalis (TV), sialidase, as well as Lactobacillus. In addition, we identified an association between HSIL and pH, flora density, diversity, BV, VVC, candida vaginitis (CV), leukocyte esterase, catalase, and Lactobacillus levels.ConclusionThese findings revealed a significant association between abnormal vaginal microecology and both HPV infection and the SIL progression.

Human Papilloma Virus Infection and Gene Subtypes Analysis in Women Undergoing Physical Examinations: A 2015-2020 Study in Wenzhou, China.

Persistent infection with high-risk human papillomavirus (HPV) is closely related to cervical cancer. The epidemiologic characteristics of cervical HPV have regional differences. Therefore, it is necessary to develop the most favorable policies according to the actual situation of each region to prevent and reduce the prevalence of cervical cancer. This retrospective cross-sectional study investigated the prevalence, gene subtypes, and temporal trends of HPV in women undergoing physical examination in Wenzhou, to provide a decision-making basis for further prevention and control of HPV. A total of 31131 cervical exfoliated cell specimens obtained from physical examinations in Wenzhou, a coastal city of China, from 2015 to 2020 were collected. The age distribution was analyzed using the chi-squared test, and the time change trend was analyzed using the Mann-Kendall trend test. On this basis, the distribution characteristics of the HPV subtypes were analyzed. The total prevalence rate was 9.55%, and the prevalence rate in different age groups ranged from 7.77% to 14.16%. The prevalence rate in different years was 8.84%-11.83%. The prevalence rate was bimodal; it was highest in the group 25years old, followed by the group >61years old. The top five high-risk gene subtypes were HPV52, HPV58, HPV53, HPV16, and HPV39, whereas the low-risk subtypes were HPV61, HPV81, HPV44, HPV43, and HPV55. Of all the positive samples, 76.03% were infected with a high-risk subtype. Most female HPV patients in Wenzhou are infected with high-risk gene subtypes. Therefore, physical examination and screening for HPV should be further strengthened, and the corresponding vaccination policy should focus on high-risk gene subtypes.

Human papillomavirus type 16 E6 promotes cervical cancer proliferation by upregulating transketolase enzymatic activity through the activation of protein kinase B.

Over 99% of precancerous cervical lesions are associated with human papillomavirus (HPV) infection, with HPV types 16 and 18 (especially type 16) found in over 70% of cervical cancer cases globally. E6, a critical HPV gene, triggers malignant proliferation by degrading p53; however, this mechanism alone cannot fully explain the oncogenic effects of HPV16 E6. Therefore, we aimed to investigate new targets of HPV oncogenic mechanisms. Our results revealed significant changes in nonoxidative pentose phosphate pathway (PPP) metabolites in HPV16-positive cells. However, the role of nonoxidative PPP in HPV-associated cell transformation and tumor development remained unexplored. In this study, we investigated the impact and mechanisms of HPV16 E6 on cervical cancer proliferation using the HPV-negative cervical cancer cell line (C33A). HPV16 E6 was found to promote cervical cancer cell proliferation both in vitro and in vivo, activating the nonoxidative PPP. Transketolase (TKT), a key enzyme in the nonoxidative PPP, is highly expressed in cervical cancer tissues and associated with poor prognosis. HPV16 E6 promotes cervical cancer cell proliferation by upregulating TKT activity through the activation of AKT. In addition, oxythiamine (OT), a TKT inhibitor, hindered tumor growth, with enhanced effects when combined with cisplatin (DDP). In conclusion, HPV16 E6 promotes cervical cancer proliferation by upregulating TKT activity through the activation of AKT. OT demonstrates the potential to inhibit HPV16-positive cervical cancer growth, and when combined with DDP, could further enhance the tumor-suppressive effect of DDP.

The tumor microenvironment state associates with response to HPV therapeutic vaccination in patients with respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP) is a rare, debilitating neoplastic disorder caused by chronic infection with human papillomavirus (HPV) type 6 or 11 and characterized by growth of papillomas in the upper aerodigestive tract. There is no approved medical therapy, and patients require repeated debulking procedures to maintain voice and airway function. PRGN-2012 is a gorilla adenovirus immune-therapeutic capable of enhancing HPV 6/11-specific T cell immunity. This first-in-human, phase 1 study (NCT04724980) of adjuvant PRGN-2012 treatment in adult patients with severe, aggressive RRP demonstrates the overall safety and clinically meaningful benefit observed with PRGN-2012, with a 50% complete response rate in patients treated at the highest dose. Responders demonstrate greater expansion of peripheral HPV-specific T cells compared with nonresponders. Additional correlative studies identify an association between reduced baseline papilloma HPV gene expression, greater interferon responses and expression of CXCL9 and CXCL10, and greater papilloma T cell infiltration in responders. Conversely, nonresponders were characterized by greater HPV and CXCL8 gene expression, increased neutrophilic cell infiltration, and reduced T cell papilloma infiltration. These results suggest that papilloma HPV gene expression may regulate interferon signaling and chemokine expression profiles within the tumor microenvironment that cooperate to govern clinical response to therapeutic HPV vaccination in patients with respiratory papillomatosis.

Introduction of E-Score: A gene expression score to characterize immunogenic versus oncogenic HPV expression in HNSCC.

6057 Background: Human Papillomavirus (HPV) integration in the human genome occurs in a majority of HPV(+) head and neck squamous cell carcinomas (HNSCC), with direct and indirect effects on host gene expression and tumorigenesis. Although previous studies showed that HPV integration in HNSCC is overrepresented in genes often somatically mutated in head and neck, lung, and urogenital cancers, reports are mixed for whether patients with HPV integration have worse clinical outcomes. These disparate results may be due to differences in how integration is defined. Integration can often lead to partial loss of the viral genome (e.g. HPV genes E1, E2, or E5) with retention of the oncogenes E6 and E7, which led to a low E2/E7 expression ratio being used as a biomarker for integration. Conversely, others have used the direct detection of DNA or RNA HPV-human breakpoints to identify tumors harboring integrated HPV. HPV integration and the retention or deletion of HPV genes can dramatically change the expression of HPV genes, and we hypothesize it also affects the host immune response. Hypothesis: Loss of non-oncogenic HPV genes relative to E6 and E7 levels reduces immunogenicity, leading to weakened immune cell infiltration and worse clinical outcomes. Methods: Bulk RNA sequencing data from HPV(+) HNSCC tumors (n = 136) was used to characterize the differences in retention or loss of HPV genes and calculate the E-Score as the sum of the logCPM of HPV genes E1, E2, E4, and E5 relative to E7. Cell type deconvolution was performed using ComBat to correct for batch effects, and CIBERSORTx to quantify cell type proportions within the tumor samples. Kaplan-Meier estimates of recurrence and overall survival (OS) were calculated, and Cox proportional hazards tests with covariates were used to test for association with recurrence and OS. To test the correlation between immune cell type inferred proportions and E-Score, T-Tests were performed between samples with high and low E-scores. Results: Low E-Score was significantly associated with recurrence (p = 0.012) but not with OS (p = 0.39) in the multivariate model accounting for age, tumor stage (AJCC8), smoking status, drinking status, BMI, marital status, and molecular tumor subtype (defined as IMU or KRT). For integration positive samples (n = 66), higher E-Score, suggesting lower rates of HPV gene loss, was correlated with higher percentages of dendritic cells (p = 0.044), macrophages (p = 0.021), and CD8+ T Cells (p = 0.024), but not CD4+ T cells (p = 0.14). This significance did not hold when looking at integration negative samples. Conclusions: These results suggest that HPV+ HNSCC patients with a loss of E1, E2, E4 and E5 tend to have less immune cell infiltration and are more likely to have recurrence. We conclude that E-Score is a potential prognostic biomarker of recurrence in HPV+ HNSCC and potential predictive biomarker for immunotherapy.

Abstract LB350: Novel mouse model to study HPV associated pathologies

Abstract Papillomaviruses are species specific, non-enveloped, double-stranded DNA viruses that have an ~8kb circular genome enclosed by a 55 nm icosahedral capsid. Human papillomaviruses (HPV) are associated with both benign and malignant epithelial lesions. High-risk HPV subtypes are the causative agents of cervical and the majority of oropharyngeal squamous cell carcinomas. Persistent HPV infection is required for HPV-associated tumorigenesis; however, cellular factors needed to support the viral persistence and HPV genes expression are not completely understood. Several transgenic mouse models, developed in last decades, helped tremendously to study HPV-induced carcinogenesis. However, the absence of HPV non-coding upstream regulatory region and late coding region, as well as targeted early genes expression driven by an artificial promoter - features that are shared by all existent HPV transgenic mice - limit our ability to investigate HPV-associated malignant transformation and the role of host factors in this process. Therefore, we generated HPV16 transgenic mouse by using CRISPR/Cas9-mediated genome editing to insert the full-length HPV16 genome into the Rosa26 locus. Two founder mice, with single HPV copy and with tandem HPV integration, were used to establish mouse colonies. Interestingly, regardless of HPV copy number, ~30% of the HPV knock-in mice exhibited early death during the first 2 weeks after birth, while survived pups were significantly smaller than the same age wild-type mice, and had severe hair loss. Intriguingly, the growth retardant and hair loss phenotype in the HPV16 knock-in mice disappeared in ~4 weeks, most likely indicating that viral genes compromise specific stages of mouse development. We evaluated the expression of HPV genes during development, in different mouse organs, and in established fibroblasts and epithelial cell cultures. Our newly developed HPV16 knock-in mouse model provides viral gene expression from natural viral promoters offering an essential basis to explore the role of cellular factors in HPV-driven pathologies. Citation Format: Xue Li, Travis Schrank, Aditi Kothari, Hina Rehmani, Jason Tasoulas, Sulgi Kim, Wendell Yarbrough, Natalia Isaeva. Novel mouse model to study HPV associated pathologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB350.

Abstract 1212: Horizontal transfer of functional human papillomavirus genes in head and neck squamous cell carcinoma

Abstract Human papillomavirus (HPV) infections are currently associated with more than 70% of oropharyngeal squamous cell carcinomas (OPSCCs). HPV-positive OPSCCs display a distinct biologic behavior and treatment response and thus are now considered a distinct entity than HPV-negative OPSCCs. However, the underlying process of HPV-related OPSCC carcinogenesis is not entirely clear. HPV as a driver of cervical carcinogenesis is well-characterized with identification of a canonical mechanism dependent on HPV DNA integration, loss of HPV E2 gene expression, and increased expression of HPV E6 and E7 oncogenes with no active infection present in malignant lesions. However, whole genome sequencing found no HPV DNA integration in ~25% of HPV-associated OPSCC, suggesting an alternative mechanism of HPV carcinogenesis. Expression of all HPV genes, including self-assembling capsid proteins, in a substantial portion of HPV+ OPSCC, led us to hypothesize that tumors with episomal HPV are capable of transferring HPV genes to surrounding cells and that HPV gene transfer contributes to carcinogenesis in this subset of OPSCC. Our data support this hypothesis, showing that growth media from HPV+ OPSCC cell lines, as well as isolates from HPV+ tumors or nodal metastases, effectively transferred functional HPV genes to cells lacking HPV. The HPV DNA Transferring Agent (HDTA) derived from HPV+ OPSCC contained HPV L1 capsid protein and intact HPV genome protected from DNAase degradation. Initial characterization revealed that anti-L1 antibody blocked gene transfer and that exposure of cells to the HDTA initiated a robust innate immune response in recipient cells. Negative staining of HDTA isolate from metastatic lymph nodes revealed many pleomorphic, electron-dense particles with diameter ranging between 20 and 80nM, with approximately 40% of particles being between 50 and 60nM, consistent with the size of HPV. We propose that development and/or maintenance of OPSCCs depend on continuous production of HDTA capable of transferring HPV genes into neighboring cells. This paradigm of tumorigenesis/maintenance in OPSCC is most likely to apply to cancers lacking HPV integration, but may also apply to tumors with integration, since recent data suggested that integrated HPV may excise from the genome to recreate a transient episomal like structure, even containing cellular DNA. Our findings offer novel insights into oropharyngeal carcinogenesis and suggest targeting HDTA as a new therapeutic strategy for HPV-positive OPSCCs. Citation Format: Jason Tasoulas, Wendell Gray Yarbrough, Natalia Issaeva. Horizontal transfer of functional human papillomavirus genes in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1212.

Direct Virus Gene Detection: A CRISPR/dCas9-Mediated Surface-Enhanced Raman Scattering Strategy with Enzyme-Catalyzed Signal Amplification.

The aberrant growth of cervical cells caused by the infection of human papillomavirus (HPV) may cause cervical cancer. In order to effectively prevent the occurrence of cervical cancer and for better follow-up treatment after surgery, a rapid and reliable detection method of HPV DNA is essential. Here, a surface-enhanced Raman scattering (SERS) detection method was developed based on the clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 technique and the enzyme catalysis amplification reaction, which achieved a simple and rapid detection of low-content HPV genes. The CRISPR/dCas9/sgRNA complex was anchored above a magnetic bead, which can precisely capture target DNA sequences, exhibiting high selectivity for HPV genes. When the biotinylated target DNAs exist, they can bridge a streptavidin-modified horse radish peroxidase (HRP) to the magnetic bead, producing an HRP-decorated conjugate. This conjugate allows an HRP-catalyzed reaction for its substrate (3,3',5,5'-tetramethylbenzidine, TMB). Gold nanostars with a silica shell exhibiting the lightning rod effect of SERS were employed to measure the SERS spectra of the oxidative product of TMB. Enzyme catalysis and SERS co-contribute to the SERS signal output, ensuring a high detection sensitivity. This method is a proof of concept for detecting HPV DNAs in a complex system. The current method can be applied to other target DNAs simply by changing the sgRNA sequence. Many superiors portend that the CRISPR/dCas9-based SERS method is promising for further clinical application.

The Role of Methylation of Host and/or Human Papillomavirus (HPV) DNA in Management of Cervical Intraepithelial Neoplasia Grade 2 (CIN2) Lesions.

Cervical intraepithelial neoplasia grade 2 (CIN2) is an intermediate stage between CIN 1, which is a low-grade lesion, and CIN3, which is the immediate precursor of cervical cancer (CC). Traditionally, CIN2 was regarded as a high-grade lesion and was treated with conization or ablative methods. In recent years, there has been a shift in the management of younger patients, who are now more often being managed conservatively due to frequent spontaneous CIN2 regression and possible adverse effects of treatment on future pregnancies. Because the risk of progression to CC still exists with conservative management, a personalized approach is needed to identify patients with a higher probability of progression. In this regard, research has focused on the role of host and human papillomavirus (HPV) gene methylation. This systematic review summarizes the current knowledge regarding conservative CIN2 management focusing on the main methylation markers and its implementation in conservative CIN2 management, and it describes major ongoing longitudinal studies on the subject. The review showed that DNA methylation is an accurate predictor of disease progression and a valid triage tool for HPV-positive women, with CIN2 performing better than triage cytology. Because virtually all CCs are methylation-positive, methylation-negative women at baseline have an extremely low risk of CC.

Relationship between vaginal microecological changes and oncogene E6/E7 and high-risk human papillomavirus infection

High-risk (HR)-human papillomavirus (HPV) is the leading cause of precancerous cervical lesions in patients with chronic untreated infection. We investigated the relationships among several vaginal microbiological alterations, oncogene E6/E7 expression, and HR-HPV. A total of 1327 women who underwent HPV screening, vaginal microecology determination, and fluid-based thin-layer cytological test were enrolled and classified into the HPV-negative group, the low-risk (LR)-HPV-positive group, and the HR-HPV-positive group. The status of cervical HPV infection, vaginal microecology, and E6/E7 mRNA expression were examined sequentially. The effect of HR-HPV infection on cervical cancer (CC) was meticulously assessed, and associations between HR-HPV infection and vaginal microecology and E6/E7 mRNA were identified. In total 548/1327 patients were HPV positive, including LR-HPV infection (N = 132) and HR-HPV infection (N = 416). Patients in the HR-HPV positive group revealed higher detection rates of bacterial vaginosis (BV), trichomonal vaginitis (TV), and vulvovaginal candidiasis (VVC) relative to the HPV negative group. A higher E6/E7 mRNA expression was identified in HR-HPV patients compared to LR-HPV patients. BV and E6/E7 mRNA were classified as independent risk factors for HR-HPV infection. Patients with HR-HPV infection were more susceptible to CC development. Overall, BV and E6/E7 mRNA expression were identified as independent risk factors for HR-HPV infection. IMPACT STATEMENT What is already known on this subject? Through literature review, we found that vaginal ecological changes increase the risk of HPV infection, and HPV persistent infection is an important risk factor for cervical precancerous lesions and cervical cancer. In addition, HPV gene E6/E7 is expressed in HPV-positive cervical cancer cells, which is related to cell malignant transformation and even tumorigenesis. What do the results of this study add? This study further revealed that bacterial vaginosis (BV) and E6/E7 mRNA were independently correlated with HR-HPV infection, and HR-HPV infection increased the risk of cervical cancer. What are the implications of these findings for clinical practice and/or further research? E6/E7 mRNA detection may be used as a new auxiliary diagnostic index for HR-HPV infection. In addition, this study provides a reference for whether the restoration of vaginal microecological balance in patients with BV undergoing clinical treatment is conducive to HR-HPV regression, and provides theoretical support for the prevention and control of cervical cancer microecological approach and the occurrence and development of cervical cancer.

A novel photoelectrochemical array platform for ultrasensitive multiplex detection and subtype identification of HPV genes

The rapid, low-cost and user-friendly methods for nucleic acid detection is urgently needed. Here we developed a miniaturized and convenient photoelectrochemical biosensor array (PEBA) platform for multiplexing and simultaneous detection of nucleic acid. The array system containing nine sensing units was integrated on one piece of conductive glass by laser etching and screen printing. Moreover, human papillomavirus (HPV), the main cause of cervical cancer, was selected as the model marker to evaluate the applicability of the fabricated PEBA. The proposed PEBA for HPV genotyping involved the TiO2@Au nanoparticles (NPs) as the photoelectrochemical (PEC) material and CdS quantum dots (CdS QDs)-labeled DNA hairpin probe anchored on the TiO2@Au NPs as the recognition element. With the addition of HPV target, the probe undergoes a significant conformational change and forces CdS QDs away from TiO2@Au, resulting in decreased PEC signals. The established array platform coupled with nucleic acid amplification exhibited high sensitivity as low as 0.1 copies/μL and a linear range of 0.6–600 copies/μL for nine HPV genotyping. Method evaluation with 40 clinical samples including 20 HPV-positive and 20 negative samples, gave a 97.5% concordance result in comparison with commercial kits. The genotyping results obtained by the PEBA reveal that HPV52, HPV18, and HPV11 are the most prevalent genotypes in positive samples, which is in good concordance with the official report concerning the trend of HPV prevalence among women with cervical lesions in Shenzhen. The designed PEBA platform has potential applications in extensive fields like biomedicine analysis and clinical healthcare diagnosis.

B-cell Epitope Mapping of Capsid L1 from Human Papillomavirus to Development Cervical Cancer Vaccine Through In Silico Study

Human papillomavirus (HPV) is a virus that plays an important role in the occurrence of cervical cancer. The HPV gene is composed of two parts: early and late gene. The L1 protein has a conserved region composed of cysteine ​​and lysine residues, both of which have involved in the binding process between virions and host receptors. Previous research has shown that vaccines can be developed based on epitopes that have conserved areas. This study is important to identify conserved protein sequences in L1 of HPV capsid, predict epitope mapping of B cells and antigenicity in the conserved region of L1 HPV capsid, as well as the similarity of amino acid residues of epitope composers with surface receptors of human body cells. The conserved areas were identified in L1 HPV as a potential epitope of B cells based on epitope mapping analysis of positions 23-46 and 97-119 with EGRGQPLGGSGHPNDDE DRDKQ and RHNGGPGPSGSSQFNKPYWAQGN peptides and each had a peptide length of 22-mer and 23-mer. The 97-119 epitope has a high antigenicity score and the similarity of the low amino acid residue sequence to the cell surface receptor of the human body, the 23-mer RHNGGPGPSGSSQFNKPYWAQGN peptide can be used as a reference for the development of cervical cancer prevention vaccine.

Biomolecular Condensation of the Human Papillomavirus E2 Master Regulator with p53: Implications in Viral Replication

p53 exerts its tumour suppressor activity by modulating hundreds of genes and it can also repress viral replication. Such is the case of human papillomavirus (HPV) through targeting the E2 master regulator, but the biochemical mechanism is not known. We show that the C-terminal DNA binding domain of HPV16 E2 protein (E2C) triggers heterotypic condensation with p53 at a precise 2/1 E2C/p53 stoichiometry at the onset for demixing, yielding large regular spherical droplets that increase in size with E2C concentration. Interestingly, transfection experiments show that E2 co-localizes with p53 in the nucleus with a grainy pattern, and recruits p53 to chromatin-associated foci, a function independent of the DNA binding capacity of p53 as judged by a DNA binding impaired mutant. Depending on the length, DNA can either completely dissolve or reshape heterotypic droplets into irregular condensates containing p53, E2C, and DNA, and reminiscent of that observed linked to chromatin. We propose that p53 is a scaffold for condensation in line with its structural and functional features, in particular as a promiscuous hub that binds multiple cellular proteins. E2 appears as both client and modulator, likely based on its homodimeric DNA binding nature. Our results, in line with the known role of condensation in eukaryotic gene enhancement and silencing, point at biomolecular condensation of E2 with p53 as a means to modulate HPV gene function, strictly dependent on host cell replication and transcription machinery.

Development of an HPV Genotype Detection Platform Based on Aggregation-Induced Emission (AIE) and Flow-Through Hybridization Technologies.

Genetic mutations can cause life-threatening diseases such as cancers and sickle cell anemia. Gene detection is thus of importance for disease-risk prediction or early diagnosis and treatment. Apart from genetic defects, gene detection techniques can also be applied to gene-related diseases with high risk to human health such as human papillomavirus (HPV) infection. HPV infection has been strongly linked to cervical cancer. To achieve a high-throughput HPV gene detection platform, the flow-through hybridization system appears to be one of the commercialized diagnostic techniques for this purpose. The flow-through hybridization technique is based on a vacuum-guided flow of DNA fragments which is continuously directed toward the oligoprobes that are immobilized on the testing membrane. However, the conventional colorimetric method and signal read-out approach suffers a problem of low sensitivity. On the contrary, fluorescence approaches allow more sensitive detection and broad sensing ranges. In this work, a fluorescent dye HCAP, which possesses aggregation-induced emission (AIE) properties and is responsive to alkaline phosphatase, was developed and applied to the flow-through hybridization platform to achieve HPV genome diagnosis of clinical samples. Also, an automatic membrane reader was constructed based on the AIE-based diagnosis platform which can identify the diagnostic result of patient DNA with a total concordance rate of 100% in the clinical trial.