Human Pappilomavirus Genotype in Cervical Tissue of Patients with Cervical Intraepithelial Neoplasia (CIN) 1, CIN 2, and CIN 3

Objectives: to determine the genotype of HPV in patients with precancerous lesions of cervical tissue.Materials and Methods: An observational study with cross sectional study of patients paraffin block CIN1, CIN2, CIN3 was conducted in Dr Soetomo Hospital. HPV DNA was extracted from paraffin blocks, then performed PCR and genotyping of HPV. The sample consisted of 28 patients with cervical tissue paraffin blocks CIN1, CIN2 and CIN3. Patients aged between 26-74 years (standard deviation 10,12).Results: HPV genotypes that infect patients with CIN1 were HPV16 and 18, CIN2 were HPV16 and 52 and CIN3 were HPV16, 67, and combined infection HPV16/67 and HPV52/67. HPV genotypes in a single infection were 26/28 (HPV16, HPV18, HPV52 and HPV67), and multiple infections were 2/28 (HPV16/67 and HPV52/67).Conclusion: The most dominant HPV genotypes infect patients with precancerous lesions of the cervix were HPV16, HPV67, HPV52, and HPV18

Amino-functionalized poly(l-lactide) lamellar single crystals as a valuable substrate for delivery of HPV16-E7 tumor antigen in vaccine development

Background: Poly(L-lactide) (PLLA) is a biodegradable polymer currently used in many biomedical applications, including the production of resorbable surgical devices, porous scaffolds for tissue engineering, nanoparticles and microparticles for the controlled release of drugs or antigens. The surfaces of lamellar PLLA single crystals (PLLAsc) were provided with amino groups by reaction with a multifunctional amine and used to adsorb an Escherichia coli-produced human papillomavirus (HPV)16-E7 protein to evaluate its possible use in antigen delivery for vaccine development. Methods: PLLA single crystals were made to react with tetraethylenepentamine to obtain amino-functionalized PLLA single crystals (APLLAsc). Pristine and amino-functionalized PLLAsc showed a two-dimensional microsized and one-dimensional nanosized lamellar morphology, with a lateral dimension of about 15–20 µm, a thickness of about 12 nm, and a surface specific area of about 130 m2/g. Both particles were characterized and loaded with HPV16-E7 before being administered to C57BL/6 mice for immunogenicity studies. The E7-specific humoral-mediated and cell-mediated immune response as well as tumor protective immunity were analyzed in mice challenged with TC-1 cancer cells. Results: Pristine and amino-functionalized PLLAsc adsorbed similar amounts of E7 protein, but in protein-release experiments E7-PLLAsc released a higher amount of protein than E7-APLLAsc. When the complexes were dried for observation by scanning electron microscopy, both samples showed a compact layer, but E7-APLLAsc showed greater roughness than E7-PLLAsc. Immunization experiments in mice showed that E7-APLLAsc induced a stronger E7-specific immune response when compared with E7-PLLAsc. Immunoglobulin G isotyping and interferon gamma analysis suggested a mixed Th1/Th2 immune response in both E7-PLLAsc-immunized and E7-APLLAsc-immunized mice. However, only the mice receiving E7-APLLAsc were fully protected from TC-1 tumor growth after three doses of vaccine. Conclusion: Our results show that APLLA single crystals improve the immunogenicity of HPV16-E7 and indicate that E7-APLLAsc could be used for development of an HPV16 therapeutic vaccine against HPV16-related tumors

Evaluation of the management of Hr-HPV+/PapTest- women. Results at 1-year recall

With cervical cancer screening the choice of 1-year as a period of follow-up in positive high-risk HPV women without cytological lesions is still under discussion. We evaluated the management of these women and the role of HPV genotyping test. We did a cervical cancer screening study of women aged 35-64 with primary high-risk HPV test. Women positive for high-risk HPV with negative cytology were followed-up after 1 year. In this study we selected women with high-risk HPV+/PapTest- resulted high-risk HPV+ at recall and performed the PapTest and HPV genotyping test. The detection rate of squamous high grade (CIN2+) relative to the total screened cohort was 2.1‰, and it was 0.2‰ at the 1-year recall. The colposcopy performed in women referred at the 1-year recall accounted for 48.8% of the total (baseline + 1-year recall), and 84.3% of these women had no cytological lesions. The most frequent hr-HPV genotype detected was HPV16 and 66.7% of co-infections were due to HPV16 and HPV18. 54.5% of women presented a persistent infection at 1-year recall with the same HPV subtype, 50% of persistent infections was due to HPV16 and 16.7% of these were determined to be CIN2+ histological lesions. Our data show that it may be useful to extend the period of follow-up for women hr-HPV+/PapTest- so as to reduce the number of unnecessary colposcopies due to the transitory infections and that the genotyping test could help to identify the persistent infections in which HPV16 is involved

Human papillomavirus E2 regulates SRSF3 (SRp20) to promote capsid protein expression in infected differentiated keratinocytes

The human papillomavirus (HPV) life cycle is tightly linked to differentiation of the infected epithelial cell suggesting a sophisticated interplay between host cell metabolism and virus replication. Previously we demonstrated in differentiated keratinocytes in vitro and in vivo that HPV16 infection caused increased levels of the cellular SR splicing factors (SRSFs) SRSF1 (ASF/SF2), SRSF2 (SC35) and SRSF3 (SRp20). Moreover, the viral E2 transcription and replication factor that is expressed at high levels in differentiating keratinocytes could bind and control activity of the SRSF1 gene promoter. Here we reveal that E2 proteins of HPV16 and HPV31 control expression of SRSFs 1, 2 and 3 in a differentiation-dependent manner. E2 has the greatest trans-activation effect on expression of SRSF3. siRNA depletion experiments in two different models of the HPV16 life cycle (W12E and NIKS16) and one model of the HPV31 life cycle (CIN612-9E) revealed that only SRSF3 contributed significantly to regulation of late events in the virus life cycle. Increased levels of SRSF3 are required for L1 mRNA and capsid protein expression. Capsid protein expression was regulated specifically by SRSF3 and appeared independent of other SRSFs. Taken together these data suggest a significant role of the HPV E2 protein in regulating late events in the HPV life cycle through transcriptional regulation of SRSF3 expression. IMPORTANCE Human papillomavirus replication is accomplished in concert with differentiation of the infected epithelium. Virus capsid protein expression is confined to the upper epithelial layers so as to avoid immune detection. In this study we demonstrate that the viral E2 transcription factor activates the promoter of the cellular SRSF3 RNA processing factor. SRSF3 is required for expression of the E4̂L1 mRNA and so controls expression of the HPV L1 capsid protein. Thus we reveal a new dimension of virus-host interaction crucial for production of infectious virus. SRSF proteins are known drug targets. Therefore, this study provides an excellent basis for developing strategies to regulate capsid protein production in the infected epithelium and production of new virions

Identification of a Proliferation Gene Cluster Associated with HPV E6/E7 Expression Level and Viral DNA Load in Invasive Cervical Carcinoma

Specific HPV DNA sequences are associated with more than 90% of invasive carcinomas of the uterine cervix. Viral E6 and E7 oncogenes are key mediators in cell transformation by disrupting TP53 and RB pathways. To investigate molecular mechanisms involved in the progression of invasive cervical carcinoma, we performed a gene expression study on cases selected according to viral and clinical parameters. Using Coupled Two-Way Clustering and Sorting Points Into Neighbourhoods methods, we identified a Cervical Cancer Proliferation Cluster composed of 163 highly correlated transcripts, many of which corresponded to E2F pathway genes controlling cell proliferation, whereas no primary TP53 targets were present in this cluster. The average expression level of the genes of this cluster was higher in tumours with an early relapse than in tumours with a favourable course (P=0.026). Moreover, we found that E6/E7 mRNA expression level was positively correlated with the expression level of the cluster genes and with viral DNA load. These findings suggest that HPV E6/E7 expression level plays a key role in the progression of invasive carcinoma of the uterine cervix via the deregulation of cellular genes controlling tumour cell proliferation. HPV expression level may thus correspond to a biological marker useful for prognosis assessment and specific therapy of the disease

Evidence for cross-protection but not type-replacement over the 11 years after human papillomavirus vaccine introduction

Examination of cross-protection and type replacement after human papillomavirus (HPV) vaccine introduction is essential to guide vaccination recommendations and policies. The aims of this study were to examine trends in non-vaccine-type HPV: 1) genetically related to vaccine types (to assess for cross-protection) and 2) genetically unrelated to vaccine types (to assess for type replacement), among young women 13-26 years of age during the 11 years after HPV vaccine introduction. Participants were recruited from a hospital-based teen health center and a community health department for four cross-sectional surveillance studies between 2006 and 2017. Participants completed a survey that assessed sociodemographic characteristics and behaviors, and cervicovaginal swabs were collected and tested for 36 HPV genotypes. We determined changes in proportions of non-vaccine-type HPV prevalence and conducted logistic regression to determine the odds of infection across the surveillance studies, propensity-score adjusted to control for selection bias. Analyses were stratified by vaccination status. Among vaccinated women who received only the 4-valent vaccine (n = 1,540), the adjusted prevalence of HPV types genetically related to HPV16 decreased significantly by 45.8% (adjusted odds ratio [AOR] = 0.48, 95% confidence interval [CI] = 0.31-0.74) from 2006-2017, demonstrating evidence of cross-protection. The adjusted prevalence of HPV types genetically related to HPV18 did not change significantly (14.2% decrease, AOR = 0.83, 95% CI = 0.56-1.21). The adjusted prevalence of HPV types genetically unrelated to vaccine types did not change significantly (4.2% increase, AOR = 1.09, CI = 0.80-1.48), demonstrating no evidence of type replacement. Further studies are needed to monitor for cross-protection and possible type replacement after introduction of the 9-valent HPV vaccine

Frequent detection of high human papillomavirus DNA loads in oral potentially malignant disorders

Human papillomavirus (HPV) is estimated to be the cause of 40-80% of the squamous cell carcinoma of the oropharynx but only of a small fraction of the oral cavity cancers. The prevalence of oral HPV infection has significantly increased in the last decade, raising concerns about the HPV role in progression of oral potentially malignant disorders (OPMD) toward squamous cell carcinomas. We sought to study HPV infection in patients with oral lesions, and in control individuals, using non-invasive and site-specific oral brushing and sensitive molecular methods. HPV DNA positivity and viral loads were evaluated in relation to patient data and clinical diagnosis. We enrolled 116 individuals attending Dental Clinics: 62 patients with benign oral lesions (e.g. fibromas, papillomatosis, ulcers) or OPMD (e.g. lichen, leukoplakia) and 54 controls. Oral cells were collected with Cytobrush and HPV-DNA detected with quantitative real-time PCR (qPCR) for the more common high-risk (HR) and low-risk (LR) genotypes. HPV detection rate, percentage of HR HPVs and HPV-DNA loads (namely HPV16 and in particular, HPV18) were significantly higher in patients than in controls. Lichen planus cases had the highest HPV positive rate (75.0%), hairy leukoplakia the lowest (33.3%). This study detected unexpectedly high rates of HPV infection in cells of the oral mucosa. The elevated HR HPV loads found in OPMD suggest the effectiveness of qPCR in testing oral lesions. Prospective studies are needed to establish whether elevated viral loads represent a clinically useful marker of the risk of malignant progression

Keratinocyte differentiation-dependent human papillomavirus gene regulation

Human papillomaviruses (HPVs) cause diseases ranging from benign warts to invasive cancers. HPVs infect epithelial cells and their replication cycle is tightly linked with the differentiation process of the infected keratinocyte. The normal replication cycle involves an early and a late phase. The early phase encompasses viral entry and initial genome replication, stimulation of cell division and inhibition of apoptosis in the infected cell. Late events in the HPV life cycle include viral genome amplification, virion formation, and release into the environment from the surface of the epithelium. The main proteins required at the late stage of infection for viral genome amplification include E1, E2, E4 and E5. The late proteins L1 and L2 are structural proteins that form the viral capsid. Regulation of these late events involves both cellular and viral proteins. The late viral mRNAs are expressed from a specific late promoter but final late mRNA levels in the infected cell are controlled by splicing, polyadenylation, nuclear export and RNA stability. Viral late protein expression is also controlled at the level of translation. This review will discuss current knowledge of how HPV late gene expression is regulated

Evaluation of TypeSeq, a Novel High-Throughput, Low-Cost, Next-Generation Sequencing-Based Assay for Detection of 51 Human Papillomavirus Genotypes.

BackgroundHuman papillomaviruses (HPV) cause over 500 000 cervical cancers each year, most of which occur in low-resource settings. Human papillomavirus genotyping is important to study natural history and vaccine efficacy. We evaluated TypeSeq, a novel, next-generation, sequencing-based assay that detects 51 HPV genotypes, in 2 large international epidemiologic studies.MethodsTypeSeq was evaluated in 2804 cervical specimens from the Study to Understand Cervical Cancer Endpoints and Early Determinants (SUCCEED) and in 2357 specimens from the Costa Rica Vaccine Trial (CVT). Positive agreement and risks of precancer for individual genotypes were calculated for TypeSeq in comparison to Linear Array (SUCCEED). In CVT, positive agreement and vaccine efficacy were calculated for TypeSeq and SPF10-LiPA.ResultsWe observed high overall and positive agreement for most genotypes between TypeSeq and Linear Array in SUCCEED and SPF10-LiPA in CVT. There was no significant difference in risk of precancer between TypeSeq and Linear Array in SUCCEED or in estimates of vaccine efficacy between TypeSeq and SPF10-LiPA in CVT.ConclusionsThe agreement of TypeSeq with Linear Array and SPF10-LiPA, 2 well established standards for HPV genotyping, demonstrates its high accuracy. TypeSeq provides high-throughput, affordable HPV genotyping for world-wide studies of cervical precancer risk and of HPV vaccine efficacy