Correlation with HPV viral load and worsening cervical lesions had been reported, but its potential for triage after primary HPV screening has not been adequately explored, especially when combined with HPV-16/18 genotyping.To evaluate combinations of human papillomavirus (HPV) viral load and genotyping for HPV-16/18 as secondary screening strategies.The Shenzhen Cervical Cancer Screening Trial Ⅱ (SHENCCAST Ⅱ) database was re-analyzed to explore new screening algorithms using the results of Hybrid Capture 2 (HC2), Mass Array Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass spectrometry System (MALDI-TOF-MS) and the ThinPrep cytologic test (TCT) obtained by endocervical sampling.Compared with the recommended screening strategy of genotyping HPV-16/18 plus reflex to cytology, using viral load (10 RLU/CO as threshold) plus reflex to cytology resulted in less cytology but had a significantly higher sensitivity for cervical intraepithelial neoplasia 2+ (CIN2+)/CIN3+ without considerable changes in specificity and referral rates. Both of the strategy of using viral load ≥10 RLU/CO as cut-point for immediate colposcopy followed by triage genotyping HPV-16/18 for the other positive (≥1<10 RLU/CO) and the strategy of referring HPV-16/18 positives for immediate colposcopy followed by triage viral load (10 RLU/CO as threshold) for non-HPV-16/18 positives had comparable screening efficacy with algorithims that contain cytology.Primary HPV screening with triage of HPV-positive women by a combination of viral load and genotyping for HPV-16/18 provides good balance between sensitivity and specificity, the number of tests required, and referral rates. Today there is consensus that persistent infection with high risk types of the human papillomavirus (hrHPV) is the essential cause for the development of cervical cancer, 2. PrimIn 2014, the interim guidelines from the Society of Gynecologic Oncology and the American Society for Colposcopy and Cervical Pathology recommended applying HPV genotyping plus reflex cytology. This spThe objective of this study was to evaluate the feasibility of alternately using viral load and genotyping for HPV-16 and/or HPV-18 (HPV-16/18) with or without cytology for triage after primary endocervical hrHPV testing.The SHENCCAST Ⅱ trial, which was conducted in 7 sites in Guangdong Province, China from 2009 to 2010, enrolled more than 10000 women ages 25 to 59 years, non-pregnant, with no previous history of pelvic radiation, a total hysterectomy, or cervical cancer screening within the previous 3 years. All participants signed an informed consent document before enrollment. The study protocol was approved by the institutional review boards from the Peking University Shenzhen Hospital, Shenzhen, China and the Cleveland Clinic, Cleveland, Ohio, USA. All participants contributed a self-collected sample for 2 HPV assays  and a direct endocervical specimen for 3 HPV assays  plus cytology . All women with abnormal cytology  or those testing hrHPV positive by self-sampling or direct sampling were referred for colposcopy and biopsy–18. CervNine groups of algorithms using HPV viral load, 16/18 genotyping and cytology were evaluated.Group 1—primary cytology, consists of 2 subgroups  with referral for colposcopy all women ≥ASC-US or ≥LSIL.Group 2—primary HPV, consists of 2 subgroups , with referral for colposcopy all women testing positive: MALDI-TOF-MS any high risk sub-type (14 types) or HC2 ≥1 RLU/CO (13 pooled types).Group 3—primary HPV with secondary cytology, consists of 2 subgroups , with referral for colposcopy all women who are hrHPV positive (testing by HC2 or MALDI-TOF-MS) and ≥ASC-US. The women hrHPV positive with NILM are suggested 1yr F/U.Group 4—primary viral load, consists of 4 subgroups , with referral for colposcopy all women above the designated RLU/CO cut-points . The ones below cut-point are suggested 1yr F/U.Group 5—viral load plus reflex cytology, consists of 4 subgroups , with referral for colposcopy all women ≥10 RLU/CO , then the women ≥1 RLU/CO < chosen cut-point have reflex cytology, and the ones ≥ASC-US are referred. The women ≥1 RLU/CO < chosen cut-point with NILM are suggested 1 yr F/U.Group 6—primary HPV genotyping, with referral for colposcopy all women who are 16/18 positive. The women Non-16/18 positive are suggested 1yr F/U.Group 7—genotyping plus reflex cytology, with referral for colposcopy all women who are 16/18 positive, and the women Non-16/18 positive have reflex cytology, and the ones ≥ASC-US are referred. The women Non-16/18 positive with NILM are suggested 1 yr F/U.Group 8—viral load plus reflex genotyping, consists of 3 subgroups , with referral for colposcopy all women ≥10 RLU/CO , then the women ≥1 RLU/CO < chosen cut-point have reflex genotyping, and the ones 16/18 positive are referred.The women ≥1 RLU/CO < chosen cut-point with 16/18 negative are suggested 1 yr F/U.Group 9—genotyping plus reflex viral load, consists of 3 subgroups , with referral for colposcopy all womenwho are16/18 positive, then the women Non-16/18 positive have reflex viral load, and the ones ≥10 RLU/CO  are referred. The women Non-16/18 positive with < chosen cut-point are suggested 1 yr F/U.The specificities for different algorithms were compared using the McNemar test on the subset of patients with negative biopsy finding. Likewise, in the comparisons of the sensitivities, the McNemar exact test was used on the patients with CIN2+/3+ finding for the subset is small. Confidence intervals were exact binomial confidence intervals (not shown), The difference were considered significant when p values of was less than .05. Data analyses were performed using STATA 14.0 . The reverse of positive predictive value (PPV) was equal to the number of colposcopies needed to detect 1 case of CIN2+/3+.The mean age of the 8556 cases eligible for this analysis was 38.9 years. The prevalence of CIN2+/3+ were 2.72%(233/8556)/1.65%(141/8556); Cytological abnormalities of ≥ASC-US/LSIL/HSIL were found in 12.1%/4.8%/1.4% of the study population; The direct endocervical specimens showed a proportion of 13.67%/8.87%/7.61%/6.21%/5.20% for HPV viral load of ≥1/10/20/50/100 RLU/CO by HC2 assay. Using the MALDI-TOF-MS assay we found a positivity of 11.93% and 2.77% for all high-risk HPV types and 16/18 subtypes respectively.The primary cytology screening alone using ASC-US as threshold (Group 1a) showed a low sensitivity and a moderate specificity for CIN2+/3+ compared with primary HPV algorithm (Group 2) which had almost the highest sensitivity in all groups but referred many more people than others. Although Group 1b had a high specificity, the sensitivity was just 65.67% for CIN 2+ and 75.18% for CIN3+ .If HPV testing was the primary screening with secondary cytology, then only the HPV positive cases with cytology ≥ASC-US were referred (Group 3). This algorithm would reduce the referral rate to 6.60% and 6.11% (HC2 and MALDI), but results in a much lower sensitivity compared with the algorithm of primary HPV testing (Group 2) .Increasing the cut-point of the HC2 assay in Group 4 could improve the specificity and decrease the referral rate, but the cost as expected is a decrease in sensitivity. When 10 or 20 RLU/CO was used as the cut-point for viral load, the corresponding algorithms (Group 4a & 4b) had a comparable sensitivity for CIN2+/3+ using primary cytology (Group 1), but resulted in a significantly lower referral rate  .The algorithm of just referring HPV16 and/or 18 positives to colposcopy (Group 6) had the highest specificity, but the sensitivity for CIN2+ and CIN3+ were only 41.20% and 53.19% respectively .The algorithm viral load plus reflex cytology (Group 5a) used viral load 10 RLU/CO as the cut-point for immediate colposcopy and then triaged the other HPV positives (≥1<10 RLU/CO) using cytology. This model only added 4.80% cytology testing, but reached an sensitivity of 93.13%/96.45% and specificity of 92.32%/91.44% for CIN2+/3+, as well as referral rates (10.00%). By increasing the HPV positive cut-point all values changed; 10 RLU/CO seemed to be the most acceptable point to use .If cytology was eliminated and HPV-16/18 genotyping was used to triage primary HPV viral load screening directly (Group 8), a new balance between sensitivity and specificity could be reached. When viral load 10 RLU/CO was defined as the cut-point (Group 8a), no significant difference could be found on both sensitivity and specificity compared with Group 5a. Improving the cut-point could reduce the referral rate but again at the cost of decreased sensitivity .Group 7 used cytology as triage for Non-16/18 HPV positives. Though adding 9.16% more cytology testing, this algorithm could dramatically improve the sensitivity for CIN2+/3+  compared to primary HPV-16/18 genotyping alone (Group 6). However, compared to the algorithm of viral load followed by cytology (Group 5a), Group 7 was less sensitive, especially for CIN2+ , although it reduced the number of colposcopies .If when primary HPV genotyping was performed, and viral load level was used to triage the Non-16/18 positive cases (Group 9), a comparable result of sensitivity for CIN2+/3+ would be seen not only with Group 7  but also with Group 5a . Raising the cut-point for viral load (positive) would produce a new balance with decreasing sensitivity and an increasing specificity .Primary high-risk HPV screening is now widely accepted, but the switch from primary cytology to a primary HPV test has evolved over many years. The newer HPV genotyping technologies have added a new element to screening. HPV genotyping can be used for risk stratification in primary screening based on the correlation between different HPV subtype infections and cervical lesions, making it practical for combinations with an additional triage technology, such as cytology, to reach a balance between safety and test utilization appropriately.HC2 performs HPV testing and reports an HPV viral load surrogate simultaneously, and the viral load level is known to have positive association with the grade of cervical lesions–21. Our In view of the highest specificity of Group 6, we tried to use HPV-16/18 genotyping to triage the HPV positives with relatively low viral load instead of cytology in Group 5 (Group 8). When 10 RLU/CO was chosen as the cut-point, the sensitivity of such an algorithm (Group 8a) was significantly higher than the recommended algorithm, and was comparable with the algorithm viral load (10 RLU/CO as cut-point) plus reflex cytology (Group 5a). Because of increasing the cut-point of viral load (Group 4) could lead to a better specificity than cytology, we then tried to change cytology of the recommended algorithm (Group 7) to viral load detection to triage those Non-16/18 HPV positives (Group 9). Different RLU/CO values were analyzed, but only by using 10 RLU/CO as the cut-point (Group 9a), could such an algorithm produce a significantly higher sensitivity compared with Group 7.The prevalence of high-risk HPV types vary geographically. In parts of Asia we know that HPV-31/33/52/58 are reported as frequently as HPV-16 or 18 in pre-cancers–24. WhetIn Group 8 and Group 9, we alternately combined HPV viral load and HPV-16/18 genotyping and obtained a comparable secondary screening method without the recommended cytology. With this in mind, if the PCR based Cobas 4800 HPV test or similar assays had the capacity of outputting the data for viral load, a more efficient algorithm could easily be developed. If one HPV test could display the results of viral load and genotyping simultaneously, not only the cost could be reduced, but other triage methods could easily be combined.It is worth mentioning that, the difference to number of colposcopies needed to detect one CIN2+/CIN3+ was not considerably different (3.7/5.9 vs. 3.6/5.9) between Group 8a and Group 9a. However, between Group 4a and Group 6, it was 3.7/5.8 vs. 2.5/3.2, which reminded us the cutoff 10 RLU/CO might be unapplicable to triage all non-16/18 positives for its ruling out some type-specific ones which could cause high grade lesions in low viral load level. Therefore, grouping viral load according to different types and estimating their risks to reach CIN2+/3+ would refine the algorithms. Some studies reported P16/Ki-67 and methylation markers had better sensitivity and specificity than traditional cytology, 26, makAnother point of consideration in our current analysis was that all groups of algorithms contained the screening step of one year follow-up, but no data was available in the SHENCCAST Ⅱ trial data to assess the viability of this option. Therefore whether a strategy would perform the same over a lifetime in regions with repeated screening needs further research. However, it is important to recognize the great difficulty in achieving follow-up in many developing countries, especially in those remote areas, where the women might have the only one opportunity to receive screening.In conclusion, the current analysis of screening algorithms based on the SHENCCAST Ⅱ trial demonstrated that viral load appears to have an undeveloped potential for triage after primary HPV screening especially when combined with HPV-16/18 genotyping or cytology. Hopefully in the future the bio-informatic phase of the new PCR based assays can be designed to include viral load in their output.


Human papillomavirus (HPV), especially in immunosuppressed patients. However, the role of the mucosal types of HPV in SCC patients with normal immunity has not been extensively confirmed. The aim of this study was to investigate the association of some high-risk mucosal types of HPV with cutaneous SCC in an Iranian population. Squamous cell carcinoma (SCC) is the second most common non-melanoma skin cancer that may be caused by Sixty-five formalin-fixed, paraffin-embedded tissue specimens with a diagnosis of cutaneous SCC as the case group and sixty-five healthy skin specimens as the control group were included in our case-control study. Genomic DNA was extracted from tissue samples and then PCR was used for the detection of HPV genotypes by a commercial kit.P=0.028). Our data revealed that 6 out of 65 SCC samples (9.2%) were infected by high-risk mucosal types of HPV whereas none of the 65 control samples were infected by the mentioned HPVs. Statistical analysis showed a significant association between these types of HPV infection and SCC risk in our studied population (These findings suggested that some high-risk mucosal types of HPV are significant risk factors for cutaneous SCC. Human papillomavirus (HPV) infection (Squamous cell carcinoma (SCC) is the second most common skin malignancy after basal cell carcinoma (BCC), and its prevalence is increasing in many countries . SCC affnfection .papillomaviridae that has a circular double-stranded DNA with approximately 8kbp length  during the years 2011-2017 and SCC diagnosis was confirmed by the pathologist. To control the main confounding variable, subjects with immunosuppressive diseases, a history of transplantation, and immunodeficiency were excluded from the study. Also, other possible confounding variables such as age, sex, and location of the lesion were matched between healthy and patient groups.DNA ExtractionThe most suitable paraffin blocks were selected, then 10 cross-sectional cuts of each block with a thickness of 3-5 μm were prepared under sterile conditions by microtome. After transferring the samples to microtubes and deparaffinization, DNA extraction steps were performed according to the DNA-Sorb-C kit’s  instruction manual.Detection of HPV Genotypes After DNA extraction, 14 high-risk HPV genotypes were detected using an AmpliSens® HPV HCR genotype-titre-FRT PCR kit variant FRT-100 (AmpliSens®/Russia) within a DT-96 Real-Time PCR Cycler . The Beta-globin gene was used as an internal control to ensure proper DNA extraction. To carry out Real-time PCR, the entire volume of Taq F (60 μL) solution was initially mixed with 600 μL of PCR buffer B. Then 5μL of this mixture was added to 10 μL of four types of PCR mix F1 HPV . Of the four prepared solutions, 15 μL was added to each micro-tube containing DNA samples. Data analysis was performed using real-time PCR software by measuring the fluorescence signals obtained from 4 channels as follows: The DNA amplification signal for HPV types 58, 33, 39, 16 was identified in the FAM fluorescence channel. The DNA amplification signal for HPV types 52, 35, 45, 31 was detected in the JOE fluorescence channel. The DNA amplification signal for HPV types 66, 68, 59, 18 was identified in the ROX fluorescence channel. The DNA amplification signal for HPV types 51, 56 appeared in the Cy5 fluorescence channel. Finally, the internal control signal was detected by the Cy5 fluorescence channel in the microtubes containing PCR-mix F1 HPV 16, 18, 31/Glob and PCR-mix-F1 HPV 39, 45, 59/Glob.Statistical AnalysisData was collected and entered into SPSS version 19.0 . Independent t-test was used to analyze numerical data. Differences between the case and control groups regarding qualitative data, as well as genotype frequencies, were analyzed by χ2 test. P-values less than 0.05 were considered statistically significant.P<0.05).Of all patients with SCC, 53 (81.5%) and 12 (18.5%) individuals were respectively male and female, which indicates a higher incidence of SCC in men. In addition, the most common site of SCC lesions was observed in the head, neck, face, and hands, which are areas exposed to sunlight (87%). Out of 65 patients with SCC in this study, 6 (2.9%) were HPV-positive, while none of the controls were positive (0.0%). Statistical analysis showed significant differences between case and control groups regarding HPV infection , HPV 51 and 52 were observed in two SCC subjects but HPV 31 and 16 were observed in one SCC subject. Also, the most common HPV-positive sites of SCC lesions were the neck and face . Of all et al. (2013) found the DNA of HPV 18, 11, and 6 in 30% of malignant specimens of SCC while this level was reported to be 11% in controls that suggest an association between HPV infection and SCC risk  identified the DNA of HPV 18, 16, and 56 in 25.7% of malignant samples compared with 0.7% of benign ones  observed the DNA of HPV 16, 31, 35, and 51 in 59.7% of patients and 4.7% of controls (In this study, we investigated the presence of HPV high-risk types in patients with squamous cell carcinoma. Our study showed 9.23% of all patients with SCC were HPV-positive while none of the control group was HPV-positive. As seen, the incidence of HPV-infected cases in SCC patients was significantly higher than in healthy skin samples. According to previous studies, Shayanfar SCC risk . In a stAs mentioned, in some studies there was a significant association between HPV infection and SCC risk , 5. AlsoThere are some limitations in our study which should be noted. At first, we did not evaluate the cutaneous HPV types in our studied population. Also, we did not assess the high-risk HPV in benign skin lesions. In addition, we only employed the real-time PCR for HPV typing. However, proving the association between HPV and cutaneous SCC requires more experimental studies to determine the transcriptional activity of HPV in squamous cell carcinoma. Serological studies, HPV protein marker detection, and PCR being used simultaneously on SCC specimens, as well as studies on more patients in different geographical regions, can provide a better perspective of HPV’s role in SCC.


Point-of-care tests are needed for the screening of head and neck squamous cell carcinoma (HNSCC) and other malignancies. Luciferase immunoprecipitation systems (LIPS), employing light-emitting proteins, were used to examine serum antibodies against several cancer-associated targets in blood donor controls and subjects with colon cancer (CC) and HNSCC. The assessment of antibodies against the wild type p53 tumor antigen showed that approximately 25% of the CC and 20% of the HNSCC patients were seropositive. In addition, humoral responses against two p53 mutants, p53-R175H and p53-R273H, generally tracked the antibody responses seen against wild type p53. Analysis of antibodies against highly specific biomarkers of HPV-16-associated malignancy, E2, E6, and E7 oncoproteins, revealed no seropositivity in blood donors and CC patients. However, 45% (9/20) of the HNSCC patients showed E6 seropositivity, which overlapped all the detectable E2  and E7 seropositive subjects . Using neodymium magnets, ultrarapid LIPSTICKS testing of HPV-16 E6 antibodies in <60 s per HNSCC sample demonstrated almost the same diagnostic performance (40% sensitivity and 100% specificity) as LIPS testing in 2.5 h. While additional improvements and standardization are needed, these results highlight the possibility of using these approaches for the diagnosis of HPV-16-associated HNSCC. There is an interest in developing point-of-care tests in the oral health setting for the clinical detection of head and neck squamous cell carcinoma (HNSCC) . Based oWe and others have used a fluid phase immunoassay employing luciferase-tagged recombinant fusion proteins, called luciferase immunoprecipitation systems (LIPS), for diagnostic purposes . Owing tInformed written consent was obtained from all subjects in accordance with the human experimentation guidelines of the Department of Health and Human Services under IRB-approved protocols, and the studies were conducted according to the principles expressed in the Declaration of Helsinki. All human samples utilized in this report represented de-identified serum specimens with annotated data. Healthy volunteer (HV) blood donor controls were obtained from the NIH Blood Bank, Clinical Center, NIH, Bethesda, Maryland under NCT00001846 . The serum samples from colon cancer (CC), systemic lupus erythematosus (SLE), and HNSCC patients used in this report were obtained from Conversant Bio . Subjects provided written informed consent under the Western Institutional Review Board approved by Conversant Bio. All human serum samples from Conversant Bio were collected, processed, and distributed in full ethical and regulatory compliance with the sites from which they were collected. This includes independent ethical review, Institutional Review Board approval (where appropriate), independent regulatory review, and Conversant Bioethical review.n = 20) from the NIH Blood bank represented subjects with an average age of 57 years and were 80% male. For the subjects with HNSCC (n = 20), the average age of cancer diagnosis was 61 years and 75% were male. The clinical information on the CC (n = 20) was not available. The serum samples from SLE patients (n = 20) had an average age of 47.1 years and were 100% female.The HV controls (Renilla luciferase (Ruc) and p53 chimeric fusion protein were used [Previously described pREN2 constructs for ere used . Two knoere used . The E6 ere used , a highlEssentially as described, the LIPS assays were performed in approximately 2.5 h using a 96-well plate format at room temperature ,20,21. B® protein A/G magnetic beads, Waltham, MA, USA), diluted 1:5 in water are added. The reaction mix is tapped two times to disperse the magnetic beads and then 100 μL of buffer A is pipetted into the reaction mixture and the tube is immediately vortexed for 2 s. A 1/8 diameter neodymium magnetic stick  is then immersed into the tube containing the beads for 5 s to collect the immune complexes. The magnet is removed and dipped twice in wash buffer A. Lastly, the magnetic stick is placed in a tube, preloaded with 100 μL of the Nanoglow substrate (Promega) and the luminescent glow is measured with the tube luminometer with an integration time of 1 s.The LIPSTICKS technology  employedU tests were used to compare the antibody levels among groups and only statistically significant (p < 0.05) are shown in the figures. For calculations of sensitivity and specificity, cut-off limits for each antigen were derived from the mean value plus three standard deviations of the healthy blood donor controls [GraphPad Prism 6 software  was employed for data plotting and statistical analysis. Non-parametric Mann-Whitney controls .n = 20), and CC (n = 20) and HNSCC (n = 20). Testing an extract containing only Renilla luciferase (Ruc) as a control protein revealed low levels of antibodies in the HV, CC and HNSCC subjects  of the HNSCC as seropositive (data not shown). To further confirm this result, a different reporter, NanoLuc was employed to detect the HPV-16 E6 antibodies. As shown in p = 0.002).Due to the known high prevalence of HPV-associated HNSCC, antibodies against E2, E7, and E6 HPV-16 proteins were analyzed. LIPS analysis of HPV-16 E2 antibodies revealed low levels in the HV and CC patients that were similar to the buffer blanks A. LIPS ap = 0.03) than the value seen in the twenty HV controls (p < 0.0001) between the HPV-negative HNSCC and HPV-positive HNSCC cases (data not shown). These findings suggest that the E6 LIPSTICK test has the potential to diagnose HPV-16-associated HNSCC.Based on the substantial difference in antibody levels seen by LIPS between the HPV-negative and HPV-positive HNSCC samples, we sought to determine whether the rapid LIPSTICKS format, employing the NanoLuc-E6 fusion protein, could be used for detection of these antibodies in the HNSCC group D. The oncontrols D. Using Using the LIPS technology, antibody-based biomarkers against the human p53 tumor antigen and antibodies against the E2, E6 and E7 oncogenes of HPV were investigated. Approximately 25% of the colon cancer and 20% of the HNSCC patients harbored statistically significant autoantibodies against wild type p53 and is consistent with other published studies ,28. LIPSThere is also great interest in rapid point of care tests for infection, cancer, and autoimmunity . The mos


Cervical cancer is an important cause of death in women worldwide , 2. CancMethylation pattern of E6 promoter in low-risk HPV (type 11) and high-risk HPV (type 16 and 18) was examined by Bisulfite Sequencing PCR (BSP) method.Based on the results, methylation status of high-risk and low-risk HPV-E6 promoter is different. It was revealed that CpG dinucleotides were unmethylated in type 16 and 18 target sequences, whereas in HPV-E6 type 11 all of CpG dinucleotides were methylated except one of them.The result suggested that a significant correlation between methylation status and HPV-induced cervical carcinogenesis, and promoter of HPV-16 and 18 E6 has minimal methylation in comparison with low-risk HPV-11. Cervical cancer is the third leading cause of cancer deaths among women worldwide , 2. The High risk human papillomaviruses genome is detectable in more than 97 percent of cases . IntegraCervical cancer could be avoided by effective screening and management of pre-cancerous lesions, which can be successfully treated . The HPVMany studies indicated that E6 and E7 oncogenes of high risk HPV types in addition to other factors are responsible for immortalization of cells and tumor progression . GeneralHPV genome is a circular, double-stranded DNA which consists of three major regions: early, late, and a long control region (LCR). LCR contain origin of genome replication and several transcription factor binding sites that control viral genome transcription . E2 protAnother process that affects viral oncogene expression and tumor progression is methylation of CpG di-nucleotide in viral genome . CpG metStudies show that transient and persistent HPV infections have different methylation pattern. As mentioned, DNA methylation is one of the most important epigenetic mechanisms of oncogene expression. These epigenetic modifications involved in tumor progression and it somehow may be used for diagnostic purposes in the future .Bisulfite treatment of DNA is one of the DNA methylation assays. In this method cytosine residues on single stranded DNA convert to uracils but 5-methyl cytosine retains unaffected. Then methylated and unmethylated cytosines can be distinguished with methods such as methylation-specific PCR.Regarding the role and importance of papillomavirus E6 oncoprotein in cancer formation and possible correlation between DNA methylation in promoter region and gene expression, we investigated the differences in methylation patterns of high risk and low risk HPV target sequence. In this study methylation pattern of promoter of E6 in low-risk HPV (type 11) and high-risk HPV (type 16 and 18) was examined by Bisulfite Sequencing PCR (BSP) method.® CRL-1550™) and HeLa (ATCC® CCL-2) cell lines were obtained from a cell bank (Pasteur Institute of Iran). Confirmed positive clinical samples which included HPV-11, HPV-16, and HPV-18 obtained from Day General Hospital . The DNA of positive clinical samples and control cells were extracted using QIAamp® DNA Mini Kit .HeLa (with 10–50 copies of HPV 18) and CaSki (with 600 copies of HPV 16) cell lines were used as positive control for HPV-16 and HPV-18 respectively. Ca Ski  according to protocol.Methyl Primer Express v1.0 and Methprimer Softwares were employed for checking the methylation pattern of E6 promoter and designing primer sets . The chaE. coli competent cells were transformed with confirmed recombinant vectors in Luria-Bertani medium and the plasmids were extracted using intron commercial Kit (DNA_Spin™ Plasmid DNA Purification Kit). DNA concentrations were determined by measuring the optical density at 260 nm and the absence of the contaminating E. coli DNA or RNA was checked by agarose gel electrophoresis. The presence of the E6 promoter in the constructed vector (PTZ57R/E6 promoter) was determined using PCR . PCR product of HeLa and Ca Ski cell lines and PCR product of HPV-11, HPV-16, and HPV-18 were run on the gel agarose and observed band was consistent with the expected product length –4.After the results of PCR reaction were confirmed by gel electrophoresis, the PCR products of two types of 16 and 18 were cloned in the TA vector, with a positive result of the products of extracted plasmids . Then prAlthough Human papilloma virus (HPV) vaccines played a major role in cervical cancer prevention, but needs to be debated screening of patients . Pap smeEpigenetic regulation of gene expression including methylation of genome involved in different aspects is important. These epigenetic alterations are involved in main human diseases including cervical cancer progression . AlthougSeveral studies have documented about association between HPV methylome and HPV-related cancers. For example, methylation of HPV DNA especially in L1 gene in CIN2+ cases have different patterns compared to acute or cleared infections . It was DNA methylation is a stable characteristic which can be considered as a diagnostic marker and can be performed by using restriction enzyme based differential cleavages pattern of methylated DNA, sodium bisulfite conversion and sequencing and some newer methods, such as methylated DNA immunoprecipitation (Me-DIP) and deep sequencing technologies.Most of these studies investigated hrHPVs long control region (LCR) methylation pattern which contains E6 and E7 oncogenes promoter. Several studies have reported decreased methylation of LCR is accompanied by cancer progression in HPV16-positive CINs . Though,Since DNA methylation has been identified as a potential biomarker for cervical cancer , in thisThe present study shows differences between two types of low-risk and high-risk HPVs E6 gene promoter methylation patterns and thus between expression levels of E6 oncoprotein and carcinogenic power of these HPV types. HPV promoter methylation profile could be an easy and measurable biomarker to examination of potential carcinogenicity of high-risk HPVs. However, additional clinical sample testing in patients with different grades of CIN are needed to reach a better understanding of DNA methylation pattern as biomarker for different cancer stages.


Sequence variability in surface-antigenic sites of pathogenic proteins is an important obstacle in vaccine development. Over 200 distinct genomic sequences have been identified for human papillomavirus (HPV), of which more than 18 are associated with cervical cancer. Here, based on the high structural similarity of L1 surface loops within a group of phylogenetically close HPV types, we design a triple-type chimera of HPV33/58/52 using loop swapping. The chimeric VLPs elicit neutralization titers comparable with a mix of the three wild-type VLPs both in mice and non-human primates. This engineered region of the chimeric protein recapitulates the conformational contours of the antigenic surfaces of the parental-type proteins, offering a basis for this high immunity. Our stratagem is equally successful in developing other triplet-type chimeras , paving the way for the development of an improved HPV prophylactic vaccine against all carcinogenic HPV strains. This technique may also be extrapolated to other microbes. More than 18 human papillomaviruses (HPV) are associated with cervical cancer, and ideally vaccines should protect from all of them. Here, the authors engineer a triple-type, chimeric HPV vaccine, using loop swapping, that elicits robust neutralizing antibody titers in mice and non-human primates. Streptococcus pneumoniae vaccine1 and Human papillomavirus (HPV) prophylactic vaccine2. Yet, because pathogens, such as the influenza viruses and human immunodeficiency virus (HIV), have very high levels of antigenic variability, this approach is fraught with difficulties, as an increase in type coverage will dramatically enhance protein amount and adjuvant level per dose, as well as increase the manufacturing complexity and associated production costs. Studies that focus on designing immunogens capable of inducing a broader protection against multiple subtypes or variants require technical methods, such as computationally optimized broadly reactive antigen (COBRA)3, which uses the consensus sequence from multiple variants to increase the immunogenicity of the conserved epitopes that are shared between subtypes and targeted by broadly neutralizing antibodies among subtypes6. As yet, however, few studies have been successful, and there is thus an urgent need to identify or design antigens that can elicit antibodies with high and broad anti-virus potency.Vaccines are highly efficient weapons against infectious disease. However, multiple antigenic types or subtypes derived from the evolution of pathogenic microbes through sequence variation presents a serious obstacle in vaccine development. One way to tackle this variation is to include more antigenic variants into a single vaccine, as exemplified with the 7. To date, more than 200 distinct HPV genotypes have been identified, of which at least 18 belong to the “high-risk” group and are chiefly responsible for the development of cancer10. HPVs are non-enveloped, double-stranded DNA viruses comprising multiple copies of the major (L1) and minor (L2) capsid proteins. The native T = 7 HPV virion can be mimicked by an empty icosahedral shell, called a virus-like particle (VLP) which consists of 72 L1-only pentamers12. High-level neutralizing antibodies elicited by L1 VLPs can block HPV infection; however, protection against HPV infection is type-restricted, and there tends to be little cross-reactivity among HPV types15. Pre-clinical and clinical data show that vaccinated patients exhibit low titers of neutralizing antibodies against genetically related, non-vaccine HPV types in highly reactive immune sera, and these antibodies may drop below protective levels sooner than type-specific ones18. For these reasons, the current market-available prophylactic HPV vaccines: Cervarix19, Gardasil20 and Gardasil 9 are multivalent vaccines of the various HPV L1 proteins. For example, Gardasil 9 provides type-specific protection against infection from nine HPV types , the last seven of which are responsible for about 90% of cervical cancers.Oncogenic HPV infection is associated with several malignancies, including cervical and anogenital cancer21; however, it is possible that genetic mutation and recombination between different viruses may result in vaccine evasion; for example, a recombination event between HPV16 and HPV31 may result in a virus carrying HPV16 oncogenes but coding for HPV31 structural proteins21. Indeed, previous work shows that substitution of just a few residues on the FG loops of L1 proteins between HPV16 and HPV31 can create a new serotype, which would avoid the immunity conferred by both types22. Furthermore, it remains unclear whether widespread immunization with vaccines like Gardasil 9 would lead to an increase in infection rates from the other carcinogenic HPV types that are responsible for the remaining 10% of cervical cancer. Ideally, an even broader vaccine that could protect against these other types would help to avoid such concerns. However, the high dosage already required for Gardasil 9 (270 μg protein in a single dose) presents a significant challenge for the development of such a broad-acting vaccine through standard manufacturing practices.The evolutionary history of HPV indicates a slow rate of HPV mutation27 and structural analyses30 indicate that HPV type-specificity is largely dependent on five highly variable surface loops—BC, DE, EF, FG and HI—on the HPV capsid32. It stands to reason that a chimeric recombinant L1 capsid comprising the antigenic determinants of different HPV types may present an alternative strategy for the development of a cross-type or wide-spectrum HPV vaccine. Previous reports have observed that the recognition region  of type-specific neutralizing antibodies of one HPV type (type A) could be transferred to create a chimeric VLP of another type (type B)35. However, previous studies show that such a chimeric VLP of type B only generated low levels of neutralizing antibodies that could cross-react with type A34, and because of this poor response, there was little further interest in developing chimeric L1 VLPs based on such epitope grafting.Previous studies on HPV structures have shown that the HPV L1 monomer forms a canonical, eight-stranded β-barrel (BIDG-CHEF), and both biochemicalIn this study, we sought to develop a highly efficacious, cross-type vaccine candidate against the infection of two or more HPV types using an epitope grafting approach. We initially found that the L1 proteins of various HPV types shared an overall conserved structure in their core regions and even in their basic surface loop configurations, with phenotype variations between different HPV types able to be induced by minor structural movements of the surface loops. Our design is based on a rational understanding of the high structural conservation of L1 surface loops within phylogenetically close HPV types despite sequence variation; and we used this knowledge to select among hundreds of HPV variants to engineer chimeric particles. Here we characterized a chimeric HPV cross-type vaccine candidate with comparable efficacy to that of mixed wild-type (WT) VLPs. Our study confirms that the strict type specificity of HPV can be undermined by the introduction of a few residue substitutions between genetically close HPV types, introducing an appealing strategy for the design and development of wider-spectrum vaccines against HPV as well as other pathogenic microbes.36. To ascertain whether structural divergence drives HPV type specificity, we scrutinized the crystal structures of L1 pentamers of genetically close or distant HPV types  between the two most phylogenetically related HPV types—HPV33 and HPV58—and generated 10 chimeric L1 constructs: H33-58BC, H33-58DE, H33-58EF, H33-58FG, H33-58HI, H58-33BC, H58-33DE, H58-33EF, H58-33FG and H58-33HI Fig. . These m50) of the mAb panel to various chimeric HPV33/58 VLPs was determined by ELISA assay  and 80-fold  higher than that found in mice immunized with WT HPV33 VLPs , respectively , 94-fold , and 52-fold  higher titers against PsV33 than that elicited by WT HPV58 VLPs  and HPV58 VLPs . The neutralization titers of the anti-sera of these chimeras were measured and we found that most of the single-point mutants (except for H58-V87L) could elicit heterotypic neutralization antibodies  as compared with the H33-58HI control. H58-33BC-52DE and H58-33BC-52HI also showed high titers  as compared with the H58-33BC control , reaching comparable levels to those provided by a mix of the WT VLPs of HPV33, -52, and -58 , as before. All four mutants showed dose-dependent anti-HPV52 neutralization antibody responses Fig. . HoweverTo test whether this method could be applied to produce other triple HPV types, we attempted to graft immunodominant epitopes into a single L1 molecule within different groups. Four chimeras  were generated for Groups 2, -3, -5 and -6 Fig.  based onMacaca Fascicularis) with the lead HPV33/58/52 vaccine candidate to evaluate vaccine-induced immunity. Monkeys (n = 4 per group) were administered with 5 μg of the H58-33BC-52HI chimeric VLPs formulated with aluminum adjuvant at week 0 and 8. Neutralization titers were measured from antisera drawn at week 10. Immunization with WT HPV VLPs resulted in minor cross-neutralization of HPV33 and HPV58 in monkeys (titers ranging from 50 to 500); however, no cross-reactive antibodies to HPV52 were observed with HPV33 or HPV58 WT VLPs, and similarly, no cross-reactive antibodies to HPV33 or HPV58 were detectable after HPV52 VLP immunization   Fig. . This wo4. However, no immunogen to date has successfully elicited antibodies with high potent cross-type neutralizing ability, probably due to the limited information available about the conserved regions of natural antigens or the poor immunogenicity of the conserved areas, since such regions appear to be generally inaccessible in the pathogen’s native state. Here, we provide a rational approach for structure-based cross-type vaccine design: (1) Structure similarity (where the structure is available) and evolutionary distance analyses were performed to define type groups that could be incorporated into one chimera; (2) Chimeric loop-substituted mutants created between two most closely related types were initially generated to determine candidate(s) that would elicit an ideal double-type immunization response; constructs where swapping occurred between more structurally similar loops were prioritized, as only limited constructs could be generated and assayed for immunogenicity; (3) The remaining four loops of the double-type candidate(s) were then independently exchanged to the corresponding loop of a third genetically close type, and immunogenicity was evaluated to achieve the optimal triple-type chimeric VLPs; again, constructs where swapping had occurred on more structurally similar loops were prioritized. Although HPV sequence diversity is not as high as that of RNA viruses, such as the human immunodeficiency and influenza viruses, or single-stranded DNA viruses, with extensive structural information, the method provided here still could markedly benefit the development of a new generation of vaccines for a wide variety of pathogens.Increasing the immunity of conserved neutralizing epitopes offers a logical strategy for the design of broadly protective immunogens; this would be based on a molecular understanding of the interactions between isolated broadly neutralizing antibodies and protective antigenshttps://www.ncbi.nlm.nih.gov/protein/ANA05496]), HPV18 (AAQ92369 [https://www.ncbi.nlm.nih.gov/protein/AAQ92369]), HPV31 , HPV33 , HPV35 , HPV39 , HPV45 , HPV52 , HPV53 , HPV56 , HPV58 , HPV59 , HPV66 , HPV68  and HPV70  L1 genes were cloned into pTO-T7 vector. Mutations were constructed using Gibson assembly46, a potent reconstitution polymerase chain reaction (PCR) strategy. Briefly, two gene fragments from their templates were cloned by specific primers  and disrupted by sonication. The supernatant was purified by two chromatography procedures using SP sepharose  and CHT II resin . For VLP preparation, the purified L1 proteins were dialyzed with the above-mentioned buffer without reductant to allow VLP self-assembly; To prepare pentamers for crystallization, the Cys175 (as numbered according to the sequence of HPV16 L1) in L1 proteins was replaced with Ser and the mutated proteins were then subjected to limited trypsin digestion (mass ratio of trypsin vs. L1 protein was approximately 1:1000) at room temperature overnight and purified by Superdex 200.N-terminally truncated HPV16  PEG 3350; for HPV52 pentamer: 8% tasimate, 17.5% (w/v) PEG 3350; for HPV58-33BC pentamer: 0.2 M potassium thiocyanate, 23% (w/v) PEG 3350; for HPV58-33BC-52HI pentamer: 0.2 M calcium acetate, 18% (w/v) PEG 3350). Crystals were cryo-protected in their respective reservoir solutions supplemented with 30% glycerol, and flash-cooled at 100 K.Diffraction data from the HPV33, HPV52, HPV58-33BC, HPV58-33BC-52HI L1 pentamer crystals were collected at the Shanghai Synchrotron Radiation Facility beamline BL17U using a Quantum-315r CCD Area Detector. All datasets were indexed, integrated, scaled and merged using the HKL-2000 program package (http://www.hkl-xray.com). The structures were solved by molecular replacement with PHASER47. The crystal structure of HPV58 pentamer (PDB 5Y9E [http://dx.doi.org/10.2210/pdb5Y9E/pdb]) served as the search model. The interest models were built manually in Coot, refined by PHENIX48 and analyzed by MolProbity49 . Briefly, one round of rigid-body refinement was performed after molecular replacement. Afterwards, the resulting models were manually corrected and refined by COOT50. Coordinates and individual B factors of HPV52 and HPV58-33BC pentamers were refined in reciprocal space without non-crystallographic symmetry (NCS) restraints, whereas the coordinates and group B factors of HPV33 and HPV58-33BC-52HI pentamers were refined in reciprocal space with NCS restraints and secondary structure restraints to avoid overfitting. Data collection and structure refinement statistics are given in Table Purified HPV L1 pentamers of concentration at ~5 mg mL51 and the phylogenic tree was constructed by the neighbor-joining method using software MEGA 552 (http://www.megasoftware.net/). The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. The five additional types of HPV L1 protein sequences downloaded from GenBank  along with 15 types of sequences as mentioned above were used for phylogenetic analysis.Multiple sequence alignment of the L1 proteins from 20 different HPV types was conducted using Clustal Omega53. Briefly, samples were diluted with Laemmli sample buffer  SDS, 10% (w/v) glycerol, 100 mM dithiothreitol, and 0.001% (w/v) bromophenol blue) to a final protein concentration of 1 mg mL−1 or 0.2 mg ml−1. Samples were heated to 80 °C for 10 min, loaded into the wells of 10% acrylamide gels, electrophoresed, and stained with Coomassie Brilliant Blue.SDS-PAGE was performed using the Laemmli method with minor modificationsFor western blotting, resolved proteins were electrically transferred to nitrocellulose membranes for 55 min at 35 mA. Membranes were blocked with 5% skim milk, incubated with anti-HPV L1 linear mAb 4B3 , washed, and then incubated with goat anti-mouse alkaline phosphatase-conjugated antibody . Color was developed over 5 min using NBT/BCIP reagent .2SO4, and the absorbance read at 450 nm  using an automated ELISA reader . The median effective concentration  is defined as the antibody concentration for achieving 50% binding with the antigen. Data analysis was performed using GraphPad Prism .The wells of 96-well microplates were coated with wild type (WT) or chimeric VLPs (300 ng per well) and then blocked overnight at 4 °C. The wells were then incubated with two-fold serial dilutions of the antibody for 45 min at 37 °C. Antibody titers were detected using a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody , followed by 50 µL of 3, 3′, 5, 5′-Tetramethylbenzidine Liquid Substrate  per well for 15 min at 37 °C. The reaction was stopped by the addition of 50 μl of 2 M H280nm of 1 in a 1.2-cm light path. A total of 150 scans for samples were recorded and the rotor speed was set to 7,000 rpm for VLPs, and 30,000 r.p.m. for pentamers. The sedimentation coefficient was obtained using the c(s) method with the Sedfit software54, kindly provided by Dr. P. Schuck at the National Institutes of Health .The homogeneity and molecular mass of HPV L1 proteins were determined using sedimentation velocity (SV) experiments. SV was performed at 20 °C on a Beckman XL-A analytical ultracentrifuge, equipped with absorbance optics and an An60-Ti rotor . All the samples were diluted to an ODPurified WT or chimeric HPV L1 VLPs were chromatographed using a 1120 Compact LC HPLC system  separately, equipped with a TSK Gel PW5000xl 7.8 × 300 mm column ; columns were pre-equilibrated in phosphate buffer, pH 6.5 with 500 mM NaCl. The column flow rate and protein signal detection for the SEC analysis were 0.5 ml/min and 280 nm, respectively.−1) were measured at a heating rate of 1.5 °C min−1 using a scanning temperature that ranged from 10 °C to 90 °C. Software MicroCal Origin 7.0  was used to calculate the melting temperatures (Tm) based on the melting curves.DSC was performed on HPV L1 VLPs using a MicroCal VP-DSC instrument . Samples  absorbed with aluminum adjuvant for prime immunization. Mice also received two additional boost inoculations. Fusion was performed 2 weeks after the final immunization. The resulting hybridomas were screened using indirect HPV33 or HPV58 VLP-based ELISA and pseudovirus-based neutralization assays . Positive cells were cloned by limiting dilution at least three times until a single cell clone was attained. MAbs were prepared by injecting hybridoma cells into the peritoneal cavities of pristane-primed BALB/c mice; ascitic fluid was collected after 9–12 days, and the mAbs were purified by Protein A affinity chromatography. The purified anti-HPV33 mAbs  and anti-HPV58 mAbs  were diluted to 1.0 mg ml−1, followed by injection of 1 μg ml−1 of mAb 4E5 at 30 μl min−1 for 3.3 min, and then injections of serially diluted antigens at 30 μL min−1 for 3.3 min. Every measurement on the BIAcore 3000 biosensor was performed three times and the individual values were used to produce the mean affinity constant and standard deviation.CM-5 sensor chips were amine-coupled to a goat anti-mouse antibody Fc fragment (GAM-Fc) . One flow cell of a chip was coated with 15,000 RU (resonance units) of the GAM-Fc, whereas the other flow cell was left uncoated and blocked as a control. The affinity measurements of mAb 4E5 binding with HPV33, HPV58, HPV52, H58-33BC and H58-33BC-52HI pentamer, respectively, were initiated by passing HBS  over the sensor surface for 100 s at 30 μl min55. Briefly, plasmids carrying codon-optimized HPV L1 genes were individually co-transfected with an L2 expression plasmid and the marker plasmid into 293FT cells, which were purchased from Thermo Fisher  and had been authenticated by Short Tandem Repeat (STR) assay and single nucleotide polymorphism (SNP) genotyping, and tested for the absence of mycoplasma contamination. The cells were harvested 72 h after transfection, lysed with cell lysis buffer containing 0.5% Brij58 (Sigma-Aldrich), 0.2% benzonase , 0.2% PlasmidSafe ATP-Dependent DNase  and DPBS-Mg solution, and incubated at 37 °C for 24 h. Afterward, 5 M NaCl solution was added to the samples to extract the cell lysates. The TCID50 (tissue culture infective dose) of the supernatant was then measured to determine the titers of the PsVs, calculated according to the classical Reed–Muench method56. Maturation and purification of these samples followed the procedures described before59.The L1/L2 expression vector and pN31-EGFP used in the experiment were kindly provided by Dr. J. T. Schiller4 cells per well for 6 h. Sera were 2-fold diluted and PsVs were diluted to 2 × 105 TCID50/µL. Equal volumes (60 µl) of the PsV diluent and the serially diluted sera were mixed and incubated at 4 °C for 1 h. The negative control was prepared by mixing 60 µL of the PsV diluent with an equal volume of culture medium. Then, 100 µL of each mixture was added to designated wells and incubated at 37 °C for 72 h. The neutralization titers were calculated as the log10 of the highest sera dilution with a percentile of infection inhibition higher than 50%. Every sample was repeated at least three times, and the mean values and SD are reported.293FT cells were incubated at 37 °C in the wells of a 96-well plate at a density of 1.5 × 10Animal experiments in mice were approved by Xiamen University Laboratory Animal Center (XMULAC). The immunization and serum harvest of cynomolgus macaques were conducted at WuXi AppTec Co., Ltd. (Contract no. XMU-20160525A). The manipulation protocol was approved by WuXi Institutional Animal Care and Use Committee (IACUC). All procedures were conducted in accordance with animal ethics guidelines and approved protocols to minimize suffering during vaccination, blood collection, and euthanasia.To initially assess the immunogenicity of all chimeric VLPs, special pathogen-free (SPF) BALB/c mice (n = 4 or 5) were immunized intraperitoneally three times at an interval of 2 weeks  with chimeric or WT VLPs diluted in aluminum adjuvant (5 μg per dose). Serum samples were collected at week 8, and the neutralizing titers were analyzed by PBNA, as described above.To verify cross protection of H33-59HI and H58-59BC chimeric VLPs, WT VLPs and chimeric H33-58HI and H58-33BC VLPs (100 μg per dose), absorbed with Freund’s adjuvant, were used to immunize SPF BALB/c mice (n = 3) subcutaneously three times at an interval of 2 weeks . Serum samples were taken at week 8 after the first immunization to determine the neutralizing titers by PBNA.n = 5 or 6 per dose) at an interval of 2 weeks  with chimeric or WT VLPs diluted in aluminum adjuvant. Serum samples were collected at week 8 for PBNA.For dose-dependent response immunogenicity evaluations, SPF BALB/c mice were immunized intraperitoneally three times with three different doses  were vaccinated by intraperitoneal injection with a single dose  of each chimeric or WT VLP . The dose of each VLP in the mixed WT VLP group was equal to that of the same VLP in the single-type groups. Serum samples were taken 4 weeks after immunization, and the seroconversion at each dose was calculated. The ED50 was reported in units of micrograms of antigen per mouse, resulting in 50% seroconversion. The ED50 results were analyzed based on the dose-response curve using a Reed–Muench model60.For EDn = 4). The first group was inoculated with H58-33BC-52HI VLP (5 μg per dose); the other four groups were inoculated with HPV33 (5 μg per dose), HPV52 (5 μg per dose), HPV58 (5 μg per dose) VLPs, and the mixed VLPs of HPV33/52/58 , respectively. All five groups were immunized twice (months 0 and 2) by Intramuscular injection. Pre-immune serum samples of each monkey were collected individually, and serum samples were collected at 2-week intervals after immunization. The titers of HPV neutralizing antibodies in these sera were detected by PBNA. All data for the neutralization titers of per group analyzed by PBNA are plotted as the mean with SD.To assess the immunogenicity of H58-33BC-52HI VLPs in non-human primates, 20 cynomolgus macaques (Macaca Fascicularis)—with no detectable neutralizing antibodies against infection of HPV33, -52 or -58—were divided into five groups  with an electron dose of 25 e−/Å2 at 1 s. Particles were manually picked using the e2boxer.py program in EMAN2.163. After several rounds of reference-free 2D classifications using Relion2.064, good particles were sorted for further 3D reconstruction and refinement with AUTO3DEM65. Final map resolutions were determined by the gold standard FSC curve between two-half maps with a cutoff of 0.366. HPV58 pentamer crystal structure were manually fitted using the program Chimera, and further optimized into the corresponding maps using the “fit in map” command in Chimera67.Purified mAb 4E5 Fab fragments were incubated with HPV58, HPV58-33BC, or HPV58-33BC-52HI VLPs at 37 °C for 2 h . Small aliquots (3 μL) of mixtures were deposited onto glow discharged holey carbon Quantifoil Cu grids , blotted with filter paper for 6 s, and then vitrified by plunging into liquid ethane inside an FEI Mark IV Vitrobot at 100% humidity. CryoEM micrographs were collected at 300 kV with a FEI Tecnai F30 transmission electron microscope, equipped with Falcon II direct electron detector at a defocus level from 1.5 to 3.5 µm evaluated using Gctf68 , where the polar angles θ and ϕ represent latitude and longitude, respectively. The Fab density was projected as contour lines onto the sphere, which is presented as a stereographic diagram.The crystal structures of HPV33, HPV58-33BC, HPV58-33BC-52HI were fitted into the corresponding cryoEM density map using the tool “fit in map” in Chimera, and were then plotted onto stereographic spheres using RIVEMDescription of Additional Supplementary FilesSupplementary Data 1Supplementary InformationReporting SummaryPeer Review File


To assess the cost-effectiveness of HPV-based screening and management algorithms for HPV-positive women in phase 2 of the Cervical Cancer Prevention in El Salvador (CAPE) demonstration, relative to the status quo of Pap-based screening.Data from phase 2 of the CAPE demonstration (n=8000 women) were used to inform a mathematical model of HPV infection and cervical cancer. The model was used to project the lifetime health and economic outcomes of HPV testing every 5 years (age 30–65 years), with referral to colposcopy for HPV-positive women; HPV testing every 5 years (age 30-65 years), with immediate cryotherapy for eligible HPV-positive women; and Pap testing every 2 years (age 20–65 years), with referral to colposcopy for Pap-positive women.Despite slight decreases in the proportion of HPV-positive women who received treatment relative to phase 1, the health impact of screening in phase 2 remained stable, reducing cancer risk by 58.5%. As in phase 1, HPV testing followed by cryotherapy for eligible HPV-positive women remained the least costly and most effective strategy (US$490 per year of life saved).HPV-based screening followed by immediate cryotherapy in all eligible women would be very cost-effective in El Salvador. Yet conventional screening with Pap testing has faced challenges in El Salvador and other low-resource settings due to low population coverage, the need for frequent screening, and insufficient navigation to treatment for women who are screen positive.Cervical cancer is the leading cause of cancer death among women in El Salvador.2 Following a positive HPV test, WHO has endorsed management strategies involving either immediate treatment with cryotherapy, referral to colposcopy, or triage testing with visual inspection with acetic acid (VIA), in which only women who are both HPV positive and VIA positive are referred for treatment. Further data on the cost-effectiveness of these management strategies are needed.WHO recommends screening with HPV DNA testing where resources are available.3 Phase 2 scaled up HPV testing to 8000 women at eight health centers in order to compare the two management algorithms in a larger and more diverse screening population.4In 2012, the Cervical Cancer Prevention in El Salvador (CAPE) project was launched to assess the feasibility and cost-effectiveness of incorporating low-cost HPV testing into the national cervical cancer screening program. The CAPE project is a demonstration project in three phases, conducted by the Salvadoran Ministry of Health (MINSAL) with technical support from the non-profit organization Basic Health International. In phase 1—a pilot study of 2000 women aged 30–49 years screened at four health centers in the Paracentral region—women who tested positive for HPV received one of two management algorithms: colposcopy management , or screen and treat .In phase 1, more women received recommended follow-up in the screen and treat cohort than in the colposcopy management cohort, and screen and treat was found to be very cost-effective for management of HPV-positive women in El Salvador.7 as in phase 1. The model projects the lifetime health and economic outcomes associated with each screening strategy. As described in previous publications, individual girls enter the simulation model at age 9 years and transition between health states  each month until death. Monthly transition probabilities may vary by age, HPV type, duration of infection or lesion status, and prior HPV infection. Death from all causes can occur from any health state, and excess mortality from cervical cancer can occur after its onset, depending upon the stage of cancer. The model tracks disease progression and regression, screening and treatment events, and healthcare costs over the lifetime of each woman. These outcomes are then aggregated over the population and used for analysis.7The present study was conducted using an individual-based Monte Carlo simulation model of the natural history of HPV and cervical cancer,8 In brief, baseline parameter values were established for the natural history component of the model using longitudinal data for age- and type-specific HPV incidence, as well as type- specific and time-dependent rates of HPV clearance and progression.13 To reflect differences in HPV incidence and burden between settings, in addition to uncertainty in the degree of natural immunity following initial infection and in progression and regression of precancer, plausible ranges were set around these input parameter values. Repeated natural history model simulations selected a single random value from the range for each uncertain parameter to form a unique natural history input parameter set. A goodness-of-fit score was then computed for each unique set by summing the log-likelihood of model-projected outcomes to represent the quality of fit to epidemiologic data , including the age-specific prevalence of oncogenic HPV among women aged 30–49 years in phase 2 of the CAPE project,4 prevalence of HPV genotypes 16 and 18 in cervical cancer in South and Central America,14 and age-specific cervical cancer incidence in El Salvador.1 The 50 top good-fitting input parameter sets were selected for use in cost-effectiveness analysis, as a form of probabilistic sensitivity analysis . Results were reported as the mean and range of outcomes across these top 50 parameter sets.Details of the model parameterization and calibration process have been described elsewhere.As in the phase 1 cost-effectiveness analysis, Pap testing was compared with two HPV screening and management algorithms (colposcopy management and screen and treat) for women who tested positive for HPV. Following an initial screening visit at the clinic, women were scheduled to return for results. Women in the colposcopy management cohort who screened positive were then scheduled to receive colposcopy at the designated hospital, whereas women in the screen and treat cohort received visual assessment to determine eligibility for immediate cryotherapy (with ineligible women referred to colposcopy).4; and (3) compliance in the Pap strategy (which was not examined in the CAPE project) would be the same as in colposcopy management, as women were referred to colposcopy following a positive Pap result.It was assumed that (1) the initial screening populations were identical for each strategy; (2) the proportion of women who attended visits to receive results, cryotherapy, colposcopy, and treatment were based on phase 2 data from the relevant cohort (colposcopy management or screen and treat) and complied with recommended follow-up within 6 months 4; and (3HPV testing with provider collection of HPV specimens was assumed to take place every 5 years between ages 30 and 65 years (colposcopy management and screen and treat), while Pap testing with colposcopy management was assumed to take place every 2 years between ages 20 and 65 years .15 Cost data are presented in 5 For phase 2, all costs were updated from 2012 to 2014 US$ using gross domestic product (GDP) deflators;16 cost per cryotherapy was updated to reflect the cost per nitrous oxide tank refill and average number of patients treated per tank in phase 2; and the cost of fuel used to transport HPV and Pap specimens to the laboratory was added. Women's time spent traveling, waiting for, and receiving care was valued using national household income data; women's transportation costs to travel to healthcare facilities were estimated by in-country clinicians.17In accordance with guidelines for cost-effectiveness analysis, a societal perspective was applied, including costs irrespective of the payor.18Reported model outcomes include lifetime risk of cervical cancer, expected total lifetime cost per woman, and life expectancy. After discounting future costs and life-years at a rate of 3% per year, incremental cost-effectiveness ratios (ICERs) were calculated. ICERs represent the additional cost of a strategy divided by its additional benefit relative to the next most costly strategy after eliminating strategies that are either more costly and less effective, or have higher ICERs than more effective strategies . A “very cost-effective” intervention was defined as one with an ICER (in US$ per year of life saved [YLS]) less than El Salvador's per capita GDP of US$3990.Sensitivity analyses were conducted to examine the impact of alternative values for model inputs . AdditioEthics approval was not required owing to the retrospective design, as de-identified data were analyzed retrospectively as part of a previous analysis. All procedures conducted as part of CAPE were approved by the national ethics review board of El Salvador.In phase 2, HPV testing with screen and treat was the most effective screening strategy, predicted to reduce the absolute risk of cervical cancer by 58.5% . Pap witBecause screen and treat was both less costly and more effective than colposcopy management and Pap, it was the dominant strategy, with an ICER of US$490 per YLS, compared with no screening. Given El Salvador's per capita GDP of US$3990, screen and treat would be considered very cost-effective in El Salvador.Phase 2 results were robust across a wide range of sensitivity analyses . Screen In several sensitivity analyses, screen and treat no longer dominated Pap but still maintained the lowest ICER. When Pap test sensitivity to detect CIN2+ improved, screen and treat had an ICER of US$490/YLS, while Pap had an ICER of US$12 480. When eligibility for cryotherapy was reduced, the ICERs for screen and treat and Pap were US$570 per YLS and US$461 490 per YLS, respectively. When the effectiveness of cryotherapy was reduced, the ICERs for screen and treat and Pap were US$580 per YLS and US$348 470 per YLS, respectively. Though Pap was slightly more effective than screen and treat in these instances, the ICER for Pap was prohibitively expensive. Thus, screen and treat remained the most effective strategy with an ICER below per capita GDP in all sensitivity analyses.Results comparing phases 1 and 2—using the updated model, costs, and input parameters, except for phase-specific visit compliance indicators—are presented in In the present study, a microsimulation model of HPV infection and cervical cancer was updated to fit epidemiologic data from phase 2 of the CAPE project, in which HPV-based cervical cancer screening is being implemented in El Salvador's public sector. The cost-effectiveness of two management algorithms (colposcopy management and screen and treat) for HPV-positive women was estimated relative to Pap screening using phase 2 data for costs, visit compliance, and eligibility for cryotherapy. The present study found that HPV testing followed by immediate treatment with cryotherapy for all eligible women (screen and treat) every 5 years between ages 30 and 65 years would be very cost-effective in El Salvador, reducing the risk of cervical cancer by nearly 60% and costing US$490 per YLS. Across all sensitivity analyses, screen and treat was consistently the most effective screening strategy with an ICER below the cost-effectiveness benchmark of El Salvador's per capita GDP.The cost-effectiveness of screening strategies using phase 1 data on visit compliance to provide updated estimates for direct comparison was reanalyzed. Reassuringly, the slightly diminished compliance with recommended follow-up seen in phase 2 had little impact on the reduction in cancer risk or cost-effectiveness of screen and treat. The vast majority of eligible women in the screen and treat cohort received immediate cryotherapy in both phase 1 and phase 2; compliance with colposcopy and treatment was lower among women ineligible for immediate cryotherapy in phase 2. As scale-up of screen and treat continues, ensuring timely follow-up for ineligible women will remain a priority.5 The present analysis used an updated model with greater flexibility to adapt to settings with epidemiologic diversity, reflecting greater uncertainty in natural immunity and the progression and regression of precancer. The updated model fitted projected cervical cancer incidence in El Salvador well,1 while the previous model underestimated cancer incidence. This underestimation of cervical cancer incidence contributed to higher ICERs in the earlier analysis. In the present analysis of phase 1, Pap was associated with more life-years gained than screen and treat because it was assumed Pap started at age 20 years, while the previous analysis assumed all screening strategies started at age 30 years. In actuality, HPV-based strategies would likely be preceded by Pap testing for some women under 30 years, and followed by Pap testing in women over age 59 years, in accordance with national guidelines that were recently amended to reflect findings from CAPE.The phase 2 ICER for screen and treat in the present analysis (US$490 per YLS) was lower than the previously published phase 1 estimate (US$2040 per YLS) for several reasons.18 yet recent analyses suggest that using per capita GDP as a threshold for willingness to pay may displace interventions yielding substantial health benefits in favor of less effective interventions.24 Because the ICER for screen and treat is well below 50% of per capita GDP, it is likely that the strategy would remain “cost-effective” even at a lower threshold.Several limitations should be noted. First, programmatic cost data to reflect MINSAL's investment in information systems for creating a national screening registry were not available, nor were patient outreach costs included. Second, screening was evaluated for ages 30–65 years (HPV testing) and 20-65 years (Pap), but phase 2 only involved screening for ages 30-49 years. Thus, HPV prevalence data at older and younger ages was unavailable for model calibration, and the model may have under- or overestimated the benefits of screening outside the age range of 30–49 years. Model fit to cancer incidence data was also based on GLOBOCAN projections, because El Salvador does not have a national cancer registry. National guidelines regarding recommended screening ages and follow-up of treated women are being reconsidered; thus, the assumption in the present study that women receive a Pap test and colposcopy in the year following treatment may be in flux. Finally, there was lack of consensus around how to define a cost-effectiveness threshold. The WHO Commission on Macroeconomics and Health is often cited as the source of the per capita GDP benchmark,Phase 3 of the CAPE project, commenced in May 2015, is currently underway with the goal of screening 20 000 women per year with screen and treat to reach 80% of screening-eligible women within 5 years. As scale-up continues, health promotors will target underscreened women, potentially reaching women at greater risk of cervical cancer. Cryotherapy will not be available at all screening facilities and, in some cases, screen-positive women will be referred to receive treatment elsewhere. Continued follow-up of treated women in phases 1 and 2 will also provide data on cryotherapy effectiveness. As further data on implementation costs and health impact become available, cost-effectiveness estimates will continue to be updated to provide decision makers with information on one of the first national HPV-based screening programs in a lower-middle-income country.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.


European guidelines for cervical cancer screening now recommend the use of clinically validated assays for high-risk HPV-DNA sequences as primary test in women older than 30 years, performed in centralized laboratories, and run on systems providing automated solutions for all steps.We conducted a comparison study, according to the international guidelines, nested within the organized population-based cervical screening program, between the cobas 4800 and 6800 systems (Roche Diagnostics), to evaluate accuracy and reproducibility of HPV test results and laboratory workflow. In Italy implementation of HPV cervical screening is under way on a regional basis; in Veneto it started in June 2015, following a piloting phase; the assay in use in the three centralized laboratories is the cobas 4800 HPV test, run on the cobas 4800 system. Comparison of HPV results with a new version of the assay (cobas 6800/8800 HPV) run on the cobas 6800 system, and intra- and inter-reproducibility analyses have been conducted in samples collected in PreservCyt medium (Hologic) from women without and with a subsequent diagnosis of high-grade lesion.p = 0,0157 and p = 0,0056, respectively, at the recommended thresholds of 90 and 98%); kappa values of 0.967 and 0.969 were obtained for intra- and inter-laboratory reproducibility analyses in the 6800 system. The 6800 platform displayed several technological improvements over the 4800 system, with higher throughput and laboratory productivity, and lower operator’s hands-on time.Samples from women older than 30 years attending organized cervical cancer screening were used. Clinical sensitivity and specificity were evaluated on 60 cases and 925 controls, respectively; intra-laboratory reproducibility and inter-laboratory agreement by the 6800 system were evaluated on 593 and 460 specimens, respectively. Our results showed a very high agreement (> 98%) for overall qualitative results between the two systems; clinical sensitivity and specificity of the HPV assay run on 6800 were non-inferior to those of the HPV assay run on 4800 (The new cobas 6800/8800 HPV assay run on the 6800 instrument is suitable for use in large centralized laboratories included within population-based cervical cancer screening programs. DNA testing for HPV oncogenic types  as primaIn order to allow testing in large centralized laboratories and to ensure diagnostic accuracy, the assays must be run on systems providing automated solutions for both pre-analytical and analytical phases, starting from specimen primary tubes. Operator interaction, test throughput, workflow and system maintenance requirements are important determinants and substantial differences among some available automated systems have been shown ; for mosIn Italy, the Ministry of Health has introduced the implementation of HPV testing for cervical screening in the National Preventive Plan 2014–2018; cancer screenings are managed at regional level, and all regions are expected to comply by the year 2018. In the Veneto region, the use of DNA HPV testing in primary cervical screening was piloted in five organized programs from April 2009 to May 2015 –9, and fThe aim of our study is the evaluation of the cobas HPV assay performed on the new 6800 platform in comparison to the 4800 for use in cervical cancer screening.The study was nested within the organized population-based cervical cancer screening, and carried out in one of the three centralized HPV laboratories of the Veneto region , serving five programs. The HPV screening protocol is applied to women older than 30 years and includes HPV testing and cytology triage of HPV-positive samples, followed by immediate colposcopy in case of ASC-US  or more severe diagnosis, or HPV retesting at 1-yr recall in case of negative cytology . Women tThe study design aimed at evaluating clinical sensitivity and specificity and assay reproducibility by the cobas 6800 HPV assay in comparison to the cobas 4800 assay, according to the Meijer’s criteria , as wellThe instrumentation for the study (p 480 v2 and cobas 6800) was temporarily provided by Roche Diagnostics. The inter-laboratory analyses were performed at the Microbiology and Virology Unit of the General Hospital of Treviso, where a cobas 6800 system is in use for other assays, and the HPV dedicated software was implemented.All samples were previously tested by the cobas 4800 HPV assay, based on real-time polymerase chain reaction (PCR) technology, by use of the 4800 system, that provides full sample preparation (cobas × 480) and HPV detection ; the two instruments are physically separated and require the manual transfer of the reaction plate. The method detects 14 HPV types , provides individual HPV genotype results for HPV16 and HPV18, while detecting the other 12 types as a pool (other HR), and includes an internal quality control (beta-globin) for each sample . The primary vial is mixed and decapped/recapped by the p480 v1 instrument.Residual material of cervical samples collected in PreservCyt medium  from women older than 30 years, attending cervical cancer screening was used. Consecutive specimens were collected during February 1 through May 31, 2017 and during December 1, 2017 through February 15, 2018, and stored for no longer than 6 months before testing by 6800; selected samples were also included, in order to increase the number of cases (25 samples obtained from women with high-grade lesions and stored for up to 3 years at 4 °C before testing for the present study) and of HPV-positive samples . The 25 samples stored longer than 6 months were re-tested by cobas 4800 to verify amplificability and consistency of results; all samples gave a valid result  with overlapping qualitative results and Ct (cycle threshold) values, and were deemed suitable for inclusion in the study.For ethical reasons, all samples were anonymized before HPV analysis. The study has been approved by the local Ethical Committee (EC code 2017–07 plus EM 193/2017).Primary vials were initially processed by the p 480 v2 (with software 2.1.1) instrument, that performed spin-mixing, cap removal, barcode alignment and transfer of 2-ml sample aliquots to secondary vials, and recapping with the original primary vial cap.The secondary vials were then manually transferred to the cobas 6800 system, a fully automated unit that provides full sample preparation and HPV detection without further intervention by the operator.Both cobas HPV real-time PCR assays detect the same 14 types of HPV, use the same primers and probes, and provide partial genotyping for HPV16 and HPV18. The two assays do however differ in their Thermal Cycling (CT) profile , as well as in the elution sample volume amplified . Both HPV assays were performed according to the manufacturer’s instructions. Each plate, besides the 2 assay’s controls, contained 92 clinical specimens and 2 additional samples .2 (variation in Y explained by X/variation in Y) between the Ct values.Clinical sensitivity and clinical specificity were evaluated on samples from women with a histologically confirmed high-grade lesion (CIN2+) and on samples from women with no or low-grade lesions, respectively; the results with the 6800 assay were compared to those with the 4800 by a non-inferiority score test, according to the thresholds (relative sensitivity of at least 90% and relative specificity > 98%) recommended by Meijer et al. . The HPVThe cases’ group comprised 62 samples from women  with a histologically confirmed high-grade lesion  diagnosed either at baseline  or during follow-up (17 cases). The controls’ group comprised 925 women  with no or low-grade lesions. By the cobas 4800 HPV assay, all the cases and 67/925 (7.2%) controls were HPV-positive.A valid cobas 6800 HPV test result has been obtained for all but two samples ; 59/60 (98.4%) cases and 73/925 (7.9%) controls gave a positive result. A concordant result was recorded in 59/60 (98.4%) cases and in 915/925 (98.9%) controls. In Table Among the 60 women with a CIN2+ diagnosis and a cobas 6800 valid result, the HPV test was positive in all but one sample . Of the 10 discordant samples among the controls, 2 were 4800-positive/6800-negative and 8 were 4800-negative/6800-positive  showed complete concordance in 118 (95.2%) specimens and partial concordance in the remaining 6. The results of the partially discordant specimens are detailed in Table p = 0,0157 and p = 0,0056, respectively).Clinical sensitivity and specificity values of the cobas 6800 HPV assay were compared to those of the 4800 assay, according to the recommendations for the clinical validation of new HPV-DNA assays ; the scoThe intra-laboratory reproducibility of the 6800 assay was evaluated on 593 samples, 178 (30%) resulted HPV-positive at first testing. Overall qualitative agreement was recorded for 587/593 (99%), with a kappa value of 0.967 (95%CI 0.942–0.985). All 6 discordant samples  were negative on the second testing. Among the samples HPV-positive in both runs, type-specific agreement was observed in 169/172 (98.3%)  than those for 4800 vs 6800 comparison .Scatter plots of the Ct values of the HPV-positive samples are reported in Fig. In each plate, two control samples were included. The 10  internal quality controls (IQC), each tested 2–6 times, were always concordant but twice for the same HPV18-positive sample that on two-out-of-three repetitions was judged as negative; additional in-depth evaluation of its analytical data disclosed that for one of the two negative results the Ct for the HPV18 channel was very high and that the mean fluorescence intensity was very weak. The 5  external quality controls (EQC) were concordant in 3 HPV-positive cases and invalid in the other 2 . The 2 samples resulted invalid on two different runs with the 4800 assay, both gave a valid result on the 6800 assay.Comparison of the workflow by the two systems has shown higher laboratory operational performances of the 6800 over the 4800, and of the p 480 v2 in relation to the p 480 v1, respectively. The most important differences are summarized in Table The cobas HPV test detects 14 high-risk HPV types, is based on real-time PCR technology, has been clinically validated , 11 and Comparison of HPV type distribution among the samples positive by both systems, and on 6800 reproducibility assays, showed a very high complete concordance for type(s) assignment , in line with previous studies , 11, 13.HPV16 (and to a lesser extent HPV18) has been shown to have a stronger association than other high-risk types for CIN2+ and invasive cancer ; as a coAccording to the European and national guidelines, HPV testing for cervical cancer screening must be centralized in large reference laboratories and performed by using clinically validated assays run on automated systems. As a consequence, the degree of automation for all the steps of the process  and the laboratory productivity are essential components to assure high-quality results and high throughput. In our study, we have shown that the workflow and several instrumentation features are sensibly improved on the new platform, as already shown also for other assays . Among oOur data on the HPV assay performed in a cervical screening context on the new cobas platform composed by the p 480 v2 plus the 6800 instruments, have shown high consistency of results with the cobas 4800 system in use and high intra- and inter-reproducibility, together with higher performance in workflow and laboratory productivity, thus demonstrating its suitability for use in large centralized laboratories included within population-based cervical cancer screening programs.


Atopobium vaginae and Gardnerella vaginalis in patients with CIN3 lesions. We found no significant bacterial biomarkers associated with HPV infections. Fungal diversity was significantly higher in cervical samples with high-risk HPV and introitus samples of patients with Atypical Squamous Cells of Undetermined Significance (ASCUS). Fungal biomarker signatures for vagina and cervix include Sporidiobolaceae and Sacharomyces for ASCUS, and Malassezia for high-risk HPV infections. Our combined data suggests that specific cervicovaginal bacterial and fungal populations are related to the host epithelial microenvironment, and could play roles in cervical dysplasia.The human cervicovaginal microbiota resides at an interface between the host and the environment and may affect susceptibility to disease. Puerto Rican women have high human papillomavirus (HPV) infection and cervical cancer rates. We hypothesized that the population structure of the cervicovaginal bacterial and fungal biota changed with cervical squamous intraepithelial lesions and HPV infections. DNA was extracted from cervix, introitus, and anal sites of 62 patients attending high-risk San Juan clinics. The 16S rRNA V4 region and ITS-2 fungal regions were amplified and sequenced using Illumina technology. HPV genotyping was determined by reverse hybridization with the HPV SPF10-LiPA25 kit. HPV prevalence was 84% of which ∼44% subjects were infected with high-risk HPV, ∼35% were co-infected with as many as 9 HPV types and ∼5% were infected with exclusively low-risk HPV types. HPV diversity did not change with cervical dysplasia. Cervical bacteria were more diverse in patients with CIN3 pre-cancerous lesions. We found enrichment of  Cervical cancer, one of the most common cancers in women, causes an estimated 11,700 new cases in the United States each year, mostly in black and Hispanic women . The higHuman papillomavirus (HPV) is the etiologic agent for cervical dysplasia and carcinoma  with ∼50Lactobacillus spp.  we now know that 20% of all fatal cancers are microbially induced . The ectlus spp. .Gardenella vaginalis or Atopobium  with HPV infections and cervical neoplasia remains largely unexplored. The purpose of this study was to examine the association between cervical bacteria and fungi with HPV in varying degrees of CIN in women infected with high-risk (HR) and low-risk (LR) HPVs.We simultaneously examined the bacterial and fungal biota and genotyped HPV in a sample of Puerto Rican women attending urban San Juan clinics, to determine if their cervicovaginal microbial composition, distribution, and diversity could be related to cervical HPV infection, atypical cell changes, and neoplasia.Women coming for gynecology and colposcopy evaluation at the UPR and San Juan City clinics (San Juan Metropolitan area), who did not meet the exclusion criteria, were recruited to participate in this study. Exclusion criteria included: (1) antibiotics taken in the prior 2 months; (2) history of regular urinary incontinence; (3) treatment for or suspicion of prior toxic shock syndrome; (4) candidiasis; (5) active urinary tract infections; (6) active STD; and (7) vaginal irritation at the time of screening. We selected these exclusion criteria based on the indications from the Manual of Procedures of the HMP protocol .The study was approved by the Ethics Committees of the UPR-Medical Sciences Campus IRB (Protocol ref. 1050114/June 2014), San Juan City Hospital and the Inter American University of Puerto Rico IRB (Protocol ref. 1182327-2014). All subjects were informed  of the sampling procedure, risks and benefits of the study, gave written informed consent and signed HIPAA forms, in accordance with the Declaration of Helsinki. All staff involved with the project completed: CITI RCR, Social and Behavioral Research Best Practices for Clinical Research, HIPAA certifications and the NIH training on Protection of Human Subjects. Patients completed a metadata questionnaire with demographic characteristics , assessment of sexual risk , health history including antibiotic use, vitamins, and BMI.TM Specimen Collection Swabs , and placed in MoBio bead tubes with buffer  to collect specimens  with the ability to provide informed consent were enrolled in our study. All specimens  were collected using sterile Catch-Allpecimens . For theTM kit, following the manufacturer’s instructions with the following modifications: (1) the powerbead tubes were homogenized horizontally for 2 min at 2,000 rpm, using a PowerLyzerTM 24 Bench Top Bead-Based Homogenizer (MoBio), (2) we combined 100 μl of solution C2 and 100 uL of solution C3 and vortexed for 5 s for cell lysis and, (3) the elution solution was 100 ul sterile PCR water, that was warmed to 65°C, and to increase DNA yield, allowed to remain on the filter for 5 min before the final centrifugation step. The final amount of gDNA ranged from 5 to 40 ng/ul.Genomic DNA extractions were done using the MoBio PowerSoilWe used a highly sensitive short-polymerase chain reaction-fragment assay . This assay has been used for epidemiological and vaccination studies, due to high analytical sensitivity . Briefly1  using the universal bacterial primers: 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) as described in the Earth Microbiome Project1  using th1 .PRJNA429969.16S amplicons were sequenced with a MiSeq Reagent kit 2 bp × 250 bp (Illumina) at the Sequencing & Genotyping Facility of the University of Puerto Rico. The ITS-2 genes were sequenced with a Nextera XT Index Kit (Illumina) at the Louisiana State University (LSU) School of Medicine Genomics Core. We used both positive and negative controls for the library prep processing of 16S and ITS runs. 16S positive controls were mixed bacterial genomic DNA from Microbial Mock Community B  and the negative controls were from amplicon PCR and index PCRs without DNA templates. All the 16S and ITS raw reads were deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession Negative controls from the sequencing run were used to characterize contamination in downstream steps and monitor the quality of the sequencing runs as advised .uclust  . This ton qiimer  was used2.Fungal sequence analyses were done using the forward reads (R1) due to their higher accuracy. Primer-dimer reads were removed, as well as the sequences at the beginning of the R1 reads, using in-house scripts. Sequence reads were quality controlled using the QIIME 1.9.0 workflow . OTUs weIn previous studies, a significant problem in interpreting the significance of the fungal microbiota has been the relatively modest numbers of organisms in typical samples and the high representation of contaminating lineages in blank controls, reflecting admixture of fungal DNA from environmental sources during sample acquisition or DNA contamination in reagents . For impThe corrected abundance of each OTU was calculated by multiplying the post-PCR ITS DNA concentration (PicoGreen) provided by the sequencing facility, with the relative abundance of that OTU in the sample . Only geggplot2 package  binding the bacterial genus matrix with the fungal matrix, column-wise, that is in the combined matrix, each sample corresponds to one row, and each genus  corresponds to one column. On this new matrix the dice index was calculated using the  package , and end package . This me package , and end library .P-value in a PERMANOVA test is determined through permutations, and the test statistic is calculated directly from the distance matrix. Analyses of Variance tests, implemented in the aov function in P-values were significant after FDR correction (P < 0.05). Biomarker signatures were proposed only if P-values were significant after FDR correction (P < 0.05). The abundance of OTUs was tested to identify signatures significantly associated with the metadata categories (biomarkers). Here, non-parametric Wilcoxon rank sum tests were used, which have a lower false positive rate, are more robust to outliers, and do not require a normal distribution assumption   were HPV-positive and 10 (16.13%) were HPV-negative. Of the HPV-positive patients, 27 (51.9%), were infected with, exclusively high-risk HPV types, whereas 3 (5.76%), were infected with exclusively low-risk HPV types. Finally, a total of 22 (42.3%) patients were co-infected by both HPV types . We joined the group of women who exclusively had low-risk HPV types (n = 3), together with the HPV-negative women (n = 10), to form a low-risk HPV group (n = 13), comparing them with women who had high-risk types (n = 45)  and 31 subjects remained in the high risk group .Sneathia and Gardnerella. Anal samples were dominated by highly diverse taxa including Finegoldia, Peptinophilus, Prevotella bivia and Prevotella timonensis, among others . The structure of the bacterial community did not change significantly according to either HPV or cervical lesion . Although we found no bacterial biomarkers, we found an enrichment in the cervix of Lactobacillus kitasatonis and Tissierella praeacuta in high-risk HPV samples and Prevotella timonensis in CIN3 lesions compared to CIN 1 lesions . Introitus samples in women with ASCUS showed higher fungal diversity compared to NSIL and HGSIL (P = 0.04) . Sacharomyces and Sporidiobolaceae were significantly more dominant in introitus samples from patients with ASCUS compared to LGSIL . Sporidiobolaceae—the most-abundant fungi present in at least 70% of the samples  and low-risk HPV (Padj = 0.007)  Figure .L. iners, Candida, and other Lactobacillus species. Conversely, taxa with high co-occurrence included several species of Lactobacillus such as L. crispatus, L. jensenii, L. kitasatonis, and L. johnsonii  associated with diagnostic cervical lesions. Coinfections have been related to the number of sexual partners , Co-infeLactobacillus (V1–V2)  , we founiversity . L. inerobacilli , 2013. TL. kitasatonis and T. praeacuta (not significant) with high-risk HPV. Although we recognize our modest sample size that limits our ability to detect true biomarkers, a recent study with a smaller sample size reported a significant association of non- L. iners- Lactobacillus sp. in HPV positive samples, and also no significant differences in richness or diversity in HPV positive samples  glands in the lipid-rich prepuce and glans penis   . AhR recor (AhR) , thus itogenesis .Sporobolomyces, that has been associated with ulcerative colitis and Crohn’s disease (Sacharomyces and Cladosporium. These associations could reflect gut contamination arising from sexual practices, which may relate to vaginal microbiome changes (Sporidiobolaceae were significantly associated with low-risk HPV-infections and ASCUS in the introitus and cervix. Sporidiobolaceae are anamorphic yeasts, including  disease . Other a changes .C. albicans to mice after antibiotic treatment caused significant shifts to the gut microbiota (C. albicans are the most prevalent species in vulvovaginal candidiasis, with enhanced proteolytic activity and antigen modulation to penetrate the mucosal surface (Lactobacillus, Malassezia, and Candida have a low co-ocurrence. This suggests that either the reduction in Lactobacillus may result in an opportunity for fungi to colonize the cervicovaginal niche or, alternatively, these specific fungal infections create an environment that inhibits Lactobacilli growth. Our work, although on a small scale, illustrates the complex relationship between the mycobiome and the bacterial microbiota in the vagina and cervix during HPV infections.Fungi can colonize perturbed niches, as seen after the introduction of crobiota . Converscrobiota . C. albi surface . AlthougTo our knowledge, no other studies simultaneously assess HPV genotype diversity, bacterial and fungal communities associated with different histopathologic features. Despite the modest sample size, our data suggests that the structure of the cervicovaginal bacterial biota is correlated with CIN while the mycobiota is correlated with both high-risk HPV infections and CIN severity, in this reproductive-age Hispanic population. Characterizing the structural features of the cervicovaginal ecosystem may be an important step in integrating understanding of the biology of cervical neoplasia, to better develop therapeutic interventions that target the microbiota.FG-V conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents, materials, analysis tools, wrote the paper, prepared figures and/or tables, and was the responsible for funding. JR recruited the patients, acquired the samples and patient metadata and reviewed drafts of the paper. CZ helped analyze the data and reviewed drafts of the paper. DV-R and GO-M helped analyze data and reviewed drafts of the paper. FV-S helped in the patient recruitment process and reviewed drafts of the paper. MS-V and MM-F genotyped HPV and reviewed drafts of the paper. MG-C helped in the patient recruitment process and metadata acquisition, reviewed drafts of the paper. JW was responsible for Resphera Insight taxonomic assignments and reviewed drafts of the paper. KB, MD-B, and MB provided insights to the analyses, reviewed thoroughly drafts of the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Using predictive analytic modelling, the Veterans Affairs has identified Veterans considered to be High Need High Risk (HNHR) requiring increased support. This pilot study sent needs assessment questionnaires to 1112 HNHR Veterans to better understand gaps regarding technology use, access, physical function, and mobility. There were 341(30.7%) respondents: 270(80.4%) Non-Hispanic, 64(18.8%) Hispanic/Latino; 210(61.6%) White, 119(34.9%) Black/African Americans; and 310(90.4%) had ≥high school education. Average Barthel(ADL) score was 81.5±22.8 and Lawton(IADL) score was 5.8±2.2. Younger Veterans (age<70) were more likely able to use Internet ((117(65%) vs 74(46%)),(p≤0.01) and email (106(58.9%) vs 67(41.6%),( p≤0.01). They were also more likely enrolled in MyHealtheVet (87(48.3%) vs 58(36%),(p=0.043). Secure messaging was used by 62(34.3%) younger and 37(23%) older Veterans,(p=0.026). More higher functioning Veterans (140(55.1%)) used email than lower functioning (33(37.9%)),(p=0.018). Among higher functioning Veterans, 148(58.3%) were willing to use videoconference for care coordination and 116(45.7%) owned a smartphone or computer with camera for this; more than lower functioning Veterans (33(37.9%) and 28(32.2%)), (p≤0.01 for both). Less dependent Veterans preferred to be contacted via cellphone (88(62.4%)) or Internet (10(7.1%)) compared to the more dependent (96(48%) and 6(3%)) respectively (p=0.01). Only 71(44.1%) of older Veterans were willing to use videoconference (p≤0.01) and 54(33.5%) owned a smartphone or computer with camera,(p≤0.01). There are significant variations in technology use by age and ethnicity. However, although there are differences by functional ability, a significant number of disabled veterans are willing and able to use technology, and this may provide a way to address access barriers in this population.