In recent years, next generation sequencing (NGS) technology has been widely used for the discovery of novel human papillomavirus (HPV) genotypes, variant characterization and genotyping. Here, we compared the analytical performance of NGS with a commercial PCR-based assay (Anyplex II HPV28) in cervical samples of 744 women. Overall, HPV positivity was 50.2% by the Anyplex and 45.5% by the NGS. With the NGS, we detected 25 genotypes covered by Anyplex and 41 additional genotypes. Agreement between the two methods for HPV positivity was 80.8% (kappa = 0.616) and 84.8% (kappa = 0.652) for 28 HPV genotypes and 14 high-risk genotypes, respectively. We recovered and characterized 243 complete HPV genomes from 153 samples spanning 40 different genotypes. According to phylogenetic analysis and pairwise distance, we identified novel lineages and sublineages of four high-risk and 16 low-risk genotypes. In total, 17 novel lineages and 14 novel sublineages were proposed, including novel lineages of HPV45, HPV52, HPV66 and a novel sublineage of HPV59. Our study provides important genomic insights on HPV types and lineages, where few complete genomes were publicly available. Papillomaviridae family and are highly diverse. According to the International Committee on the Taxonomy of Viruses Study Group HPVs are classified in genera, species and types based on the L1 open reading frame (ORF), being the most conserved region of the HPV genome [L1 gene nucleotide sequence [Human papillomavirus (HPV) is one of the most common sexually transmitted infections and the principal cause of cervical cancer . HPVs beV genome . Differesequence . HPV typsequence ,3,4. Addsequence ,6.Recent studies indicate that lineages of HPV16, 31 and 58 genotypes have different carcinogenic properties ,10,11. FModern technological advances have resulted in the development of more than 250 HPV detection and genotyping methods . The AnyWhile PCR-based HPV assays detect by design only a limited number of targeted genotypes, next generation sequencing (NGS) may be an alternative approach for universal HPV detection and characterization ,23,24,25In this study, we aimed to investigate and characterize HPV diversity circulating in healthy young women attending vaccine surveillance study in Luxembourg using NGS-RCA technology. In addition, we compared the analytical performance of the Anyplex II HPV28 assay and NGS using RCA for HPV detection and genotyping. To the best of our knowledge, this is the first large scale study to present HPV diversity using RCA-NGS in healthy women and compare the analytical performance of a PCR based genotyping assay with NGS-RCA.A total of 744 cervical samples of women  participating in an HPV vaccination surveillance project were included in the study. More details on the study population and recruitment process have been published elsewhere . BrieflyDNA extraction from ThinPrep samples for sequencing and genotyping by the Anyplex II HPV28 was performed using the QIAamp DNA mini kit according to manufacturer’s instructions  . Prior tL1 gene. The Anyplex assay can simultaneously identify and distinguish of 14 carcinogenic or hr-HPV types , and 14 lr-HPV types  in two sets. The viral load is defined as high (+++) if a positive signal occurs before 31 polymerase chain reaction (PCR) cycles, as medium (++) if a positive signal occurs within 31 to 40 PCR cycles, and low if a positive signal occurs after 41 PCR cycles [Initially, HPV detection was performed by the Anyplex II HPV28, targeting the R cycles .L1 viral gene, as an internal positive control while water was used as a negative control. All reactions were performed according to the manufacturer’s instructions and the Seegene viewer software was used for data recording and interpretation [Real time PCR was performed in two multiplex reactions on the CFX96 real-time thermocycler . The human beta-globin housekeeping gene was amplified along with retation .Total DNA extracts were enriched using rolling-circle amplification (RCA) technology with the TempliPhi 100 kit according to manufacturer’s instructions  . LibrariIn order to assemble full HPV genome sequences, we followed two complementary approaches. For both approaches we performed an initial QC step, including visual inspection of FastQC report for each sample . The strThe first approach was to “blindly” assemble all reads from 744 samples (irrelevant of being HPV positive or negative) remaining after filtering against human, bacterial, plasmid and fungal reference sequences. The second approach was based on assemblies done on only dehumanized reads, which have been pre-mapped to all available HPV reference genome sequences. In both approaches mapping was performed with Bowtie2  and de nWe obtained 75 identical genomes by both methods, 86 not fully identical, 42 only with blind assembly and 43 only with bowtie assembly. We selected 75 identical genomes, while not identical genomes were compared and further processed to allow assembly selection. All assemblies were checked by remapping fastq reads to assembled genomes and by aligning to the respective reference genomes. Of the total 246 genomes assessed, three  were discarded from further analysis due to chimeric artefacts.https://pave.niaid.nih.gov/, accessed in November 2018) and one novel genotype detected in our laboratory. Accession numbers of reference genomes are provided in For HPV detection and genotyping, quality controlled and dehumanized fastq-files from (trimmed and filtered against human genome as described above) were mapped with Bowtie2 to a reference set of 318 HPV sequences downloaded from PaVE (Samples were considered positive if at least one concordantly mapping read pair was detected covering a minimum of 150 bp of the reference genome. To avoid possible mapping artefacts reads covering < 200 bp with the mutation rate > 0.03 (~4.5 variants on 150 bp read) were blasted against NCBI-based database (built on July 2019) and manually/visually checked. Artefacts were removed if the blast provided different results.https://pave.niaid.nih.gov/) [Complete HPV genomes obtained in this study n = 243) were assessed to investigate HPV variant distribution in healthy women in Luxembourg. Complete genomes were linearized according to the respective reference sequence available in PaVE (ih.gov/)  and aligih.gov/) . Each HP were assih.gov/) . For theih.gov/) . P-distaih.gov/) , whereasih.gov/) ,45. PlotStatistical analysis was performed using STATA 14 . We estimated HPV genotyping concordance by the percentage agreement and Cohen’s kappa (k). Cohen’s kappa values less than zero were classified as no agreement, 0.00–0.19 as poor, 0.20–0.39 as fair, 0.40–0.59 as moderate, 0.60–0.79 as good and 0.80–1.00 as excellent, as proposed in VALGENT ,47. AgreComplete genomes generated during the study have been submitted to the European Nucleotide Archive (ENA) repository and are available under accession numbers LR861810, LR861811, LR861836–LR861981, LR861983, LR861984, LR861986, LR861987, LR861989, LR861990, LR861992–LR862007, LR862010, LR862011, LR862014, LR862015, LR862017, LR862018, LR862020–LR862086 . SequencThe study was approved by the Comité National d’Ethique de Recherche (CNER # 201501/02 and 201909/01) and authorized by the Commission Nationale pour la Protection des Données (CNPD 288/2016).Overall, HPV positivity was 366/729 (50.2%) by Anyplex and 332/729 (45.5%) by NGS. From 28 genotypes covered by Anyplex, NGS detected 25  plus 41 additional genotypes in 171 (23.5%) samples, of which 43/171 (25.1%) were negative by Anyplex. Anyplex detected 146 genotypes in 97 samples negative by NGS. Overall, Anyplex detected 778 and NGS detected 888 type-specific infections, corresponding to an average of 1.1 and 1.2 genotypes per sample, respectively.p < 0.001)  of the samples with Anyplex and 204/332  with NGS. Anyplex viral load was significantly associated with the count of reads mapping to HPV by NGS (< 0.001) . The top< 0.001) A. The mo< 0.001) B. HPV67 < 0.001) C.General agreement for overall HPV positivity of 28 genotypes between the two methods was 80.8% (kappa = 0.616) (good concordance). When restricting the analysis to medium and high viral load results (++/+++), agreement was higher (86.3%) with a kappa value of 0.712 (good concordance). Agreement for 14 hr HPV positivity was 84.8% with kappa value of 0.65 (good concordance). Similarly, when restricting analysis to medium and high viral load (++/+++) agreement for 14 hr-HPV was slightly higher  (good concordance) . Type-spWe obtained 232 complete alpha HPV and 11 gamma HPV genomes from 153 samples  spanningOf a total 243 complete genomes, 49 (13 hr and 36 lr-HPV) were classified as belonging to 17 novel lineages and 14 novel sublineages of 20 HPV types including 4 hr and 16 lr-HPV genotypes . The remIn total, 118 complete hr-HPV genomes were assembled, spanning 24 existing lineages and 31 sublineages of 13 hr-HPV types . No compIn total, the 114 genomes obtained of 21 lr-HPV types clustered with 22 existing lineages and 20 sublineages . AccordiWe obtained 10 gamma-6 and one gamma-13 complete HPV genomes. In total, seven novel variants were identified including lineages A, B, C of HPV101 and lineages A, B of HPV108 and HPV226 . The difOur study showed a high diversity of human papillomavirus in women attending cervical cancer screening. We characterized 243 complete HPV genomes of 40 different genotypes and proposed the classification of novel lineages/sublineages for 4 hr-HPV and 16 lr-HPV genotypes. To the best of our knowledge, this is the first large scale study to present HPV diversity using unbiased NGS-RCA in healthy young women and comparing the analytical performance of a PCR-based genotyping assay with NGS-RCA.Our findings suggest that the PCR-based the Anyplex assay is superior to the NGS in terms of sensitivity since only 16% of genotypes with low viral load by the Anyplex were concordantly identified by NGS. This is not surprising considering that Anyplex detection is based on type-specific primers, whereas RCA is a random amplification of circular DNA. Nevertheless, NGS detected an additional 110 genotypes compared to the Anyplex PCR. Further, our study suggests that concordance between the two methods increases with higher viral loads. In a recent clinical evaluation of the Anyplex assay, the detection of cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) was associated with medium and high viral loads of the 14 high-risk types only, but not with low viral loads or low-risk genotypes . In our In total, we detected 66 different HPV genotypes including 90 lineages/sublineages, of which 27 were novel and not previously described. Although, the diversity of hr-HPV genotypes has been well studied ,13, we wSeveral NGS-based approaches have been trialed, including RNA-baits, PCR-based and untargeted metagenomics ,50,51,52Previous studies applied an RCA-based approach in immune-suppressed individuals, where high viral load would facilitate HPV detection and complete genome recovery. Pastrana et al. reported the discovery of 83 novel HPV types using RCA-NGS with the prior physical enrichment of viral DNA from immunodeficient patients . This meIn our study, we obtained 243 HPV complete genomes, which increases the number of publicly available complete HPV genomes on GenBank (~2283 complete genomes on April 2020) by approximately 10%. Our dataset provides important genomic insights on HPV variants where currently only a few complete genomes were available, such as HPV42, HPV59, HPV66, HPV67, HPV90, and HPV91.E6 oncogene, they may have a lower oncogenic potential [Interestingly, we also obtained 11 complete HPV genomes of gamma species, with a population prevalence of 3.7% in our population. Our findings are in line with the previous research which found gamma-6 species in cervical samples confirming their mucosal tropism ,56,57,58One of the limitations of our study was the absence of negative and positive control during the sequencing, in order to account for the cross contamination and index hopping. Therefore, our detection criteria might produce some false positive results. Nevertheless, only 3% of the samples were defined as HPV positive by NGS with at least one concordantly mapping read pair to the reference genome. Moreover, when considering more stringent detection criteria for HPV positivity by NGS (mapping coverage of 1000 bp), the agreement between methods did not change significantly . The NGSThe major advantage of the NGS method is that it requires no prior knowledge of which HPV genotypes are present in the samples. Overall, we were able to detect 51 different HPV genotypes including 26, which are not detectable by the Anyplex assay. As the majority of these HPV types belong to IARC Group 3 (not classifiable as to its carcinogenicity to humans) [


We found that microbial perturbations occurred in the early phase of hr-HPV infection. Lactobacillus and Sporolactobacillus were decreased, while bacteria related to bacterial vaginosis (BV), such as Gardnerella, Prevotella, Dialister, Slackia, Actinomyces, Porphyromonas, Peptoniphilus, Anaerococcus, Peptostreptococcus, Streptococcus, Ureaplasma, Megasphaera, and Mycoplasma were increased. Our results could offer insights into the correlations between hr-HPV and the vaginal microbiota in the early infection period, and provide indications that the predominance of some BV-associated bacteria during hr-HPV infection may increase the risk for cervical neoplasia.Persistent infection with the carcinogenic human papillomavirus (HPV) is a prerequisite for the progression of cervical lesions and cancer. A growing body of research has focused on the functional role of the vaginal microbiota in the persistence of HPV infection. Understanding the microbial composition and structure in women with high-risk (hr)-HPV infection may help reveal associations between the vaginal microbiota and HPV infection, and identify potential biomarkers. Our study investigated the vaginal microbial community in women with and without hr-HPV infection, by using  Chlamydia trachomatis . All participants provided written informed consent for the use and publication of their data prior to their enrollment into the study. The study protocol was approved by the ethics committee of the First Hospital of Jilin University, and the study was conducted in accordance with the principles of the Declaration of Helsinki.Three swab specimens were collected from each participant during a routine pelvic examination. The first specimen was collected from the cervix with a Cytobrush and used for cervical ThinPrep cytology testing . The second specimen was also collected from the cervix and used for hr-HPV DNA detection via the Hybrid Capture II (HC2) test , according to the manufacturer's guidelines. The HC2 test is a chemiluminescence-based assay that involves nucleic acid hybridization and signal amplification. This test can qualitatively detect 13 types of hr-HPV DNA, namely, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. The third specimen was collected from the mid-vagina, and was immediately frozen to−80°C and subsequently used for bacterial DNA extraction and sequencing.16S rRNA gene on the Illumina MiSeq PE300 sequencing platform . Amplification of the V3–V4 regions was performed with the following pair of universal primers, which contained a set of 8-nucleotide barcode sequences unique to each sample: forward, 338F (5′-ACTCCTACGGGAGGCAGCAG-3′); and reverse, 806R (5′-GACTACHVGGGTWTCTAAT-3′). The polymerase chain reaction (PCR) protocol was as follows: 5 min at 95°C, 25 cycles of 30 s at 95°C, 30 s at 55°C, and 30 s at 72°C, and final extension for 10 min at 72°C. PCR was performed in a 25-μL mixture containing 2.5 μL of 10 × Pyrobest buffer, 0.4 U Pyrobest DNA Polymerase , 2 μL of 2.5 mM dNTPs, 15 ng template DNA, and 1 μL of each primer (10 μM). All PCR assays were performed in triplicate. The resulting amplicons were subjected to 2% agarose gel electrophoresis, purified , and then quantified , according to the manufacturers' instructions. Finally, the purified amplicons were pooled in an equimolar ratio and paired-end sequenced (2 × 300) on the Illumina MiSeq platform.We extracted genomic DNA from a total of 60 cervical samples by using E.Z.N.A.® DNA extraction kits , according to the manufacturer's instructions. Next, we verified the quality of the extracted DNA by performing 1% agarose gel electrophoresis, and measured its concentration using NanoDrop spectrophotometry. The extracted DNA was then frozen to −20°C and stored until further analysis. We then performed high-throughput sequencing of the V3–V4 hypervariable regions of the bacterial version 1.2.1; http://qiime.org/) to extract high-quality sequences from the raw sequences obtained using MiSeq sequencing. Sequence selection was based on the length and quality of the sequences, tags, and primers. The set of unique sequences thus identified was categorized into operational taxonomic units  by using UCLUST . Using Usearch , we identified and removed chimeric sequences. Taxonomic identification of the remaining 16S rRNA gene sequences was performed using the Silva119 16S rRNA database and UCLUST software .We used QIIME , and co-occurrence network analysis was performed using the R packages “igraph” and “psych.” To identify significant differences in microbial composition, we performed linear discriminant analysis effect size  with the thresholds P < 0.05 and effect size > 2. Clusters of Orthologous Groups (COG) categories were assigned by performing BLAST searches to identify the closest matching sequence in the Search Tool for the Retrieval of Interacting Genes database. The raw reads were deposited into Sequence Read Archive (NCBI accession number: SRP212297).We evaluated the Chao1 and observed species indices, which estimate microbial richness, and the Shannon index, which measures species biodiversity , 25. TheP < 0.05 was considered to indicate statistically significant differences.Experimental comparisons were performed using analysis of variance and the Tukey honestly significant difference test. The results are presented as mean and standard error of the mean. Of the 60 women, 30 tested negative for hr-HPV infection, while the remaining 30 tested positive for hr-HPV infection. The average ages of HPV-positive and HPV-negative women were 36.70 ± 6.30 and 34.13 ± 6.43 years, respectively. All cytological tests were normal, and no CIN lesions were detected in any of the women from either group. The HPV-positive and HPV-negative groups did not differ in terms of age, parity, ethnicity, cigarette smoking, or use of hormonal contraceptives. The demographic characteristics of each group are presented in 16S rRNA gene sequencing. After MiSeq sequencing analysis, 2,874,102 sequences were identified from the 60 samples . In total, 138 OTUs were identified in the two study groups. The OTU rank abundance and rarefaction curves of all the samples indicated that the data sampling was sufficient, and the sequencing depth was adequate; the 16S rRNA gene sequences covered almost all microbial communities , Atopobium (2.1%), Gardnerella (0.72%), Prevotella (0.04%), and Mycoplasma (0.0001%) dominated the microbial community in the HPV-negative group, while in the HPV-positive group, Lactobacillus (86.34%), Gardnerella (6.42%), Prevotella (2.44%), Megasphaera (1.21%), Mycoplasma (0.80%), and Atopobium (0.37%) were the dominant microflora . In contrast, the abundances of Lactobacillus and Veillonella (Firmicutes) and Sneathia (Fusobacteria) were decreased in the HPV-positive group -8, IL-α, IL-β, and tumor necrosis factor (TNF)-α; both species were among the 6 most prevalent proinflammatory taxa in the vaginal microbiota in one South African study  is a common Gram-positive anaerobe that has not been reported to adhere to vaginal epithelial cells, but does usually adhere to epithelial cells at other body sites , Prevotella and Porphyromonas (Bacteroidetes), and Ureaplasma and Mycoplasma (Tenericutes) were consistent with the variations observed in the microbiota composition at the phylum level.In the present study, us study . Lactobabiofilms , 28. Thene study . A. vagiesponses –32. In aediators . Sneathitachment , 35. In an study . Gardner with BV , 37. G. an study . Peptoni with BV . Similardy sites . Mycopla with BV . Ureaplanfection , 42. TheMycoplasma (Mycoplasmataceae), Lactobacillales, Sporolactobacillaceae, and Sporolactobacillus (Sporolactobacillaceae) were candidate biomarkers for HPV infection. The rate of Mycoplasma infection is higher among women with high-grade squamous intraepithelial lesions (HSILs) than among women with low-grade squamous intraepithelial lesions (LSILs); however, in patients with cervical cancer, no Mycoplasma  infections were detected , cervical cancer, and the vaginal microbiota.Our study focused on analyzing the differences in the vaginal microbiota between HPV-negative women and HPV-positive women with normal cytology. We found that hr-HPV infection was associated with vaginal bacterial dysbiosis. Certain bacteria associated with BV showed increased abundance, while Lactobacillus and Sporolactobacillus decreased, while the abundance of BV-related pathogenic bacterial genera increased. By studying the early effects of hr-HPV infection on the vaginal microbiota, we provide candidate microbial markers to monitor persistent HPV infection and the development of cervical neoplasms.The present study demonstrated that vaginal microbial perturbations occurred in the early phase of hr-HPV infection when no CIN lesions had developed. The abundance of probiotics such as SRP212297, BioProject accession number-PRJNA551647).The datasets generated for this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: (NCBI Sequence Read Archive database and SRA accession number-The studies involving human participants were reviewed and approved by the ethics committee of the First Hospital of Jilin University. The patients/participants provided their written informed consent to participate in this study.All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Furthermore, each author certifies that this material or similar material has not been and will not be submitted to or published in any other publication before its appearance in the Frontiers in public health.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.


HPV (human papillomavirus) vaccinations have been introduced into the population of many countries through vaccination programs, although their acceptance varies from country to country, largely dependent on the state of knowledge about diseases caused by genital HPV types as well as cultural, social, and religious factors. The aim of the study was to analyze the state of knowledge about HPV and HPV vaccines among doctors during their specialization in gynecology and obstetrics, dermatology and venereology, and pediatrics. Another objective of the study was to analyze the impact of the state of knowledge about HPV vaccination on their attitude to primary prevention, i.e., vaccinations. A questionnaire was used to collect the data and 639 doctors took part in the study. The analysis was carried out mainly using descriptive statistical methods. In Poland, doctors’ knowledge about HPV is low, independent of gender, age, and subject of specialization. Doctors’ knowledge about the HPV vaccine is very low and independent of sex, age, and subject of specialization. However, doctors’ knowledge about HPV and the HPV vaccine influences the attitude to HPV vaccination and does not affect pro-active behaviors. HPV infection can be of two types: symptomatic and asymptomatic. Asymptomatic infections have been poorly studied. Approximately 15–20% of asymptomatic people can be diagnosed with features of HPV infection by the use of molecular tests [Benign HPV lesions are common warts, genital warts, and recurrent respiratory papillomatosis (RRP). Genital types of HPV may also predispose to the development of precancerous lesions in the anogenital area, i.e., CIN , VIN , VaIN , AIN , and PIN .Cancers proven to be related to HPV include cancer of the cervix, vulva, vagina, penis, anus, pharynx, and mouth.In 2008, Harald zur Hausen was awarded the Nobel Prize in Physiology or Medicine for his discoveries of the role of HPV in the pathogenesis of cervical cancer. Cervical cancer is the fourth most common cancer affecting women and the seventh most common in the general population (2012) [The incidence of cervical cancer in Poland has been falling in recent years. This success is attributed to the cervical cancer prevention program launched in 2006. This program is aimed at women between the ages of 25 and 59 who have not had a Pap smear test in the last three years. The incidence of cervical cancer fell from 11.5 per 100,000 women in 2005 to 10.3 per 100,000 women in 2010 [A large meta-analysis of 11 studies found HPV in 95% of cervical cancers. Oncogenic types  and probable oncogenic types  were selected [Every year in Poland, there are about 250 new cases of penile cancer, 250–300 new cases of rectal cancer, and about 500 new cases of vulvar cancer .Prophylaxis can be divided into primary prevention, secondary prevention, and tertiary prevention.Primary prophylaxis of HPV infections includes various types of procedures aimed at preventing the emergence and spread of infection. Primary prevention, in addition to vaccination, also includes educational activities aimed at eliminating or reducing the risk factors of HPV infection.A Pap smear is a screening test for the prevention of cervical cancer. In Poland, a national screening program for cervical cancer was introduced in 2006. This program covers preventive examinations for women 25–59 years of age who are offered smear tests by invitation as well as training doctors and midwives.A cervical smear test makes it possible to recognize pre-cancerous changes, as well as the duration of the menstrual cycle and the term of ovulation. A Pap smear is the first step in cervical cancer screening and further management depends on its result and follow-up tests.Despite invitations to preventive examinations, cytology is poorly accepted in Poland. Women are ashamed of taking this test or are afraid of taking it for fear of the results of the diagnosis. The Pap smear also has its limitations in the form of the subjectivity of the assessment of the material by the assessor and the experience of the doctor collecting the material.In Poland, colposcopy is not a screening test, but a test that verifies abnormal cytological results. This procedure, performed using a colposcope, makes it possible to indicate precisely the place from which—in the event of an abnormality—a biopsy should be taken. In Germany, colposcopy is a test that complements the Pap smear test. In Poland, it is more difficult to access due to the lack of support from the National Health Fund, the time-consuming procedure (compared to cytology), and the lack of qualified personnel. The varieties of colposcopy are penisoscopy, anoscopy, and vulvoscopy.HPV detection tests are additional tests supporting the management of neoplasia. In Poland, they are available, but they are rarely performed due to the lack of reimbursements from the National Health Fund. Many factors influence the level of inoculation and reception of the vaccine. The main ones include: lack of knowledge about HPV infections and vaccine, as well as motivational obstacles . Logistic barriers include: vaccination availability, the price of the vaccine, and the need to repeat vaccination (compliance). Vaccine myths—mistaken beliefs include sexual promiscuity, and negative information about the vaccine in the media .Doctors’ recommendations play key roles in the prevention of cervical cancer. A lack of recommendations from the NHP  and doctors is a well-researched topic in previous studies. People who received a positive recommendation from the NHP and/or their doctor were more likely to get vaccinated or vaccinate their children.It has also been previously studied that parents who had been strongly advised by healthcare providers to vaccinate their children had higher acceptance rates to immunization for their girls and boys and were more hesitant to postpone the immunization appointment and had fewer concerns about vaccines and were less likely to refuse vaccination .In previously conducted studies, the knowledge of health professionals varies depending on age , region Poland still remains high in the rankings for the incidence and mortality of cervical cancer. Despite this, there are no reliable studies defining the state of knowledge about HPV infections, primary and secondary prevention, and the impact of the state of knowledge about the attitude towards vaccination. This study, the first of its kind undertaken in Poland, undertook an analysis to determine the barriers to vaccination acceptance and factors to determine vaccination in our country.The aim of the work was to make an assessment of doctors’ knowledge about HPV infections, an assessment of doctors’ knowledge about HPV vaccines, and an assessment of the impact of doctors’ knowledge about HPV infection and HPV vaccines on their attitude to primary prevention, i.e., vaccinations.An observational cross-sectional descriptive study was carried out.All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Medical University of Warsaw.Data was collected using paper forms during obligatory study courses during residency. A researcher who monitored the conducted study and helped with technical problems was present to ensure that only residents were involved in the study.The survey was conducted in the largest cities in 2018 throughout Poland.No doctor refused to fill in the questionnaire, and those who did not complete it at a given course stated that they had completed one on a previous course. The study was completed when data from a minimum of 200 doctors of a given specialization were obtained.The analysis was conducted mainly with the use of descriptive statistics. The results are presented in the form of frequency tables and cross tables. Statistical inference was performed using the Chi-squared test or, in the case of low frequencies of the analyzed features, Fisher’s exact test.p = 0.05.The Fisher’s exact test is used in the case of samples that are too small—when the observed values calculated with the Chi-squared test are below 5. The calculations were performed using the statistics program R 3.5.1. All tests were performed at a significance level The responses of the doctors were presented depending on their age, sex, and size of the place of residence. Doctors’ knowledge about vaccination was also analyzed. The doctors’ responses to the questions related to their knowledge about vaccinations were classified as correct or incorrect, and each question was assigned a percentage. The attitudes of doctors towards the vaccination were then analyzed according to the results obtained.A goal of the study was to include as many participants as possible. At the initial stage, no formal calculation of sample size was carried out. The final number of 639 doctors ensured a precision  of 4 percentage points for assessing a trait whose true prevalence was 50% .The questionnaire survey  for doctThe survey consisted of both single-choice and multiple-choice questions. The age question was an open-ended question. The rest of the questions were closed questions. Nine questions related to knowledge about the HPV virus, eight related to the state of knowledge about the HPV vaccine. The rest of the questions were about the attitudes of doctors to vaccination and demographic data.The study included doctors during residency in pediatrics, gynecology, and obstetrics, and dermatology and venereology, participating in compulsory courses during their specialization studies. The questionnaires were completed by 639 doctors. A pooled analysis of doctors is presented in n = 362), doctors 30–40 years of age accounted for 34.4% (n = 199), and doctors 40–70 years of age accounted for 3.1% (n = 18). The largest group of doctors indicated town with over 500,000 inhabitants as their place of residence . A total of 204 doctors (33.7%) indicated town with 100,000 to 500,000 inhabitants as their place of residence, while 84 doctors (13.9%) indicated town with 20,000 to 100,000 inhabitants as their place of residence. Town up to 20,000 inhabitants was indicated by 25 doctors (4.1%). Inhabitants of villages accounted for 3% (n = 18).Altogether, 608 doctors answered the gender question, 492 women (80.9%) and 116 men (19.1%). Doctors 20–30 years of age accounted for 62.5% of the respondents (n = 203) of the subjects, in gynecology and obstetrics—32.1% (n = 205), in pediatrics—33.8% (n = 216). Almost all doctors  mentioned hospital as their place of employment. For most of them, it was the only place of employment—these doctors constituted 87.7% of the study group. Others combined employment in a hospital with employment in a clinic, private office, or other places of employment. Only 8 doctors (1.3%) did not work at a hospital. One of the respondents indicated three places of employment.Doctors specializing in dermatology and venereology constituted 31.8%  of doctors obtained a result of up to 50% of correct answers in the range of 51–60% (n = 219), 61–70% (n = 134), and 71–100% (n = 149). The highest number of doctors obtained a score between 51% and 60% of correct answers. If this were a test, then as many as 55.7% would not pass the test, assuming that the pass mark was a score of more than 61% of correct answers.Analyzing the second division, 21.4% , and other central nervous system disorders caused by thiomerosal, an ethylmercury compound used as a preservative in the vaccine”, and 79 doctors in total who believe that the vaccine may lead to “An anaphylactic reaction in children allergic to proteins connected with the cultivation of vaccine viruses in chicken embryos”. n = 20) directly said that they were not in favor of HPV vaccination, while 69 doctors indicated that they had no opinion. Among this group, more than half had a test result in the lowest quartile. Among doctors with a low-test result, 1.8% did not recommend HPV vaccination, and as many as 16.7% said that they had no opinion. In the group with the highest test result, 2% did not recommend HPV vaccination and 7.4% did not have an opinion.The state of knowledge about HPV infections in Poland has only been assessed sporadically within various groups . Doctors were never included in those studies. Questionnaires were usually used to examine the state of knowledge of the above groups, but the study groups were not always well defined. Our study showed that the knowledge of doctors about HPV in the study group is low, independent of sex, age, and specialization.The knowledge of doctors about the HPV vaccine in the study group is very low and independent of sex, age, and specialization. The state of doctors’ knowledge about HPV and the HPV vaccine has an impact on the attitude towards HPV vaccinations  and has no effect on pro-vaccine behaviors (encouraging or informing patients).In international studies in the United States, the United Kingdom, and Australia, which included 2409 people, the percentage of people who had heard of HPV was 61%. This knowledge was influenced by education, marital status, having a daughter 9–17 years of age, and previous vaccination. The overall knowledge about HPV has been assessed as low, which may have a negative impact in countries where HPV testing is a screening test . In othePrevious studies of the state of knowledge among health professionals have shown a high general knowledge about HPV, and the highest results are among doctors who are women . HoweverThe HPV vaccine is registered in Poland for young girls who are sexually inactive. Among doctors who took part in the study, pediatricians will most often have contact with this group. It is even more disturbing that the lowest test result in the field of knowledge about the HPV virus was obtained by doctors who specialize in pediatrics. A few studies conducted in the world on groups of pediatricians confirm their low level of knowledge about HPV .In the study conducted on a group of doctors, knowledge about the HPV vaccine was lower than the knowledge about HPV. Among the three study groups, the lowest score in terms of knowledge about the HPV vaccine was obtained by doctors who specialize in dermatology and venereology. Due to the fact that dermatologists and venereologists very often diagnose and treat patients with genital warts, pre-cancerous conditions of the external genital area, e.g., vulvar neoplasia, penile neoplasia, and peri-anal neoplasia. Training in dermatology and venereology also covers the prevention of HPV infections with the use of vaccines against these viruses. In the specialization program, knowledge of this subject is obligatory. Dermatologists should know the spectrum of protection of HPV infections depending on the type of vaccine. There are no other studies in this research field in the literature, apart from a single study that does not describe in detail the state of knowledge of dermatologists . In a stIn the results of this study, the state of doctors’ knowledge about the HPV virus and the HPV vaccine has an impact on the attitude towards HPV vaccinations , and it does not affect pro-vaccination behavior . Only one doctor among the study group cited the fear that the vaccine might induce sexual behavior in children as a reason for being anti-HPV (results not included).The study by Rosenthal et al. also noted that women who were strongly persuaded to vaccinate were four times more likely to get vaccinated than those who were poorly persuaded . DoctorsThere may be some possible limitations in this study. Our research takes into consideration only one aspect of the barriers towards vaccination, namely: doctors’ knowledge. Further investigations need to be conducted to include more anti-vaccine factors.Based on the research, the following conclusions were drawn. The knowledge of doctors about HPV in the study group is low, independent of sex, age, and specialization. The knowledge of doctors about the HPV vaccine in the study group is very low and independent of sex, age, and specialization. The state of doctors’ knowledge about the HPV virus and HPV vaccine has an impact on the attitude towards HPV vaccinations , and it does not affect pro-vaccination behavior .This research is the first attempt in Poland to identify the most important barriers to the effective implementation of the HPV vaccine and, therefore, to the prevention of diseases associated with this virus. The presented analysis may help in the implementation of HPV vaccination programs in Poland.


Self sampled HPV testing is a cervical cancer screening method . However, cytology in self-sampled specimen cannot be used as a triage test. Therefore, other methods for triage should be considered. CyclinA1 (CCNA1) promoter methylation has strong association with cervical precancerous and cancerous lesion. The objective of this study was to compare the diagnostic value of CCNA1 and self-sampled specimen for detecting high-grade cervical intraepithelial lesions or worse (CIN2+). A cross sectional study was conducted. Women with abnormal cytology or positive for high risk HPV (hrHPV) indicated for colposcopic examination were enrolled. Self-collected sampling for hrHPV DNA (SS-HPV) and CCNA1 were performed. hrHPV DNA testing was done by Cobas 4800 method. CCNA1 promoter methylation was detected by CCNA1 duplex methylation specific PCR. Histopathologic result as CIN2+ obtaining from colposcopic directed biopsy or excisional procedure was considered as positive a gold standard. The results of hrHPV and CCNA1 were reported as positive or negative. Sensitivity specificity, positive predictive value, and negative predictive value of SS-HPV and CCNA1 were calculated by comparing the results with the gold standard. Two hundreds and eighty women were recruited. High-grade cervical lesions and cervical cancer (CIN2+) were diagnosed in 21.8% (61 cases) of the patients. The most common type of hrHPV was non 16, 18 subtype, followed by HPV16 and 18. CCNA1 was positive in 13 patients out of whom, twelve were CIN2+. Sensitivity of CCNA1 was 19.7 % and its specificity and accuracy were 99.5% and 82.14%, respectively. The sensitivity of SS-HPV was 70.5%, and its specificity and accuracy were 39.2% and 43.3%, respectively. Due to high specificity and positive predictive value of CCNA1, it can be used as alarming sign of having high-grade cervical intraepithelial lesions, especially in patient who has positive hrHPV DNA test based on self-collected sampling. Cervical cancer is the third most common cancer in women and the seventh one among all cancers. GLOBOCAN 528,000 new cases per year in 2012. A large majority of cervical cancer diagnoses (about 87%) are made in less developed regions . Screening programs have been proved to decrease the numbers of cervical cancer-related death, especially in developed countries. Due to high sensitivity of the primary HPV testing, this test is going to be the first line screening for cervical cancer in several countries. However, the coverage of the screening test is one of the key strategies. The Ministry of Public Health (MOPH) of Thailand reported that only 25-38% of Thai women attended cervical cancer screening program. The obstacles against cervical cancer screening in Thai women are feeling of embarrassment, fear of pain, and feeling inconvenience. Therefore, self-sampled test may increase cancer screening coverage. Self-sampled HPV DNA testing devices are simple for analysis by laboratory technicians . The accuracy of self-sampled HPV test (SS-HPV) has been proved worldwide. Several studies have shown high concordance rate between clinician-sampling and self-sampling in HPV detection . The advantages of SS-HPV test were ease of use, higher accessibility, and induction of less painful . Although primary HPV testing for cervical screening has high sensitivity for the detection high-grade cervical lesion, the specificity and the positive predictive values are much lower than cytology . Not all HPV infected women have cervical lesions. Low specificity of HPV testing can lead us to unnecessary investigation and anxiety. According to ASCCP guideline, triaging scheme for positive hrHPV testing is required. Triaging of positive HPV test from physician- collected specimen can be performed by cytology. However, many researchers reported that the accuracy of self-sampled cytology test was significantly poorer than physician- collected test . For that reason, re-collection of specimen by the physician is recommended if triaging with cytology after positive self-sampled HPV test is required. Although triaging the positive HPV testing based on self-sampling with the same specimen is more convenience, the standard guideline for triaging with self-collected specimen is still inconclusive. Therefore, other options for triaging are required.DNA methylation of promotor region of certain genes is known to be a cause of carcinogenesis. Recently, a strong association between cyclinA1 (CCNA1) promoter methylation and high-grade cervical lesion was reported . Human CCNA1 gene has been mapped to chromosome 13q12.3-q13. CCNA1 may be an important tumor suppressor gene that plays a crucial part in head and neck carcinoma and cervical cancers. In a previous study, promoter methylation of CCNA1 gene was found in 45% of tumors but in none of normal tissues, suggesting the implication of CCNA1 gene methylation in carcinogenesis . However, data about CCNA1 promoter methylation test in self-sampled cervical cancer screening method are not available. The objective of this study was to evaluate the value or ability of self-sampled CCNA1 promoter methylation (SS-CCNA1) for the detection of high-grade cervical precancerous and cancerous lesions (≥ CIN2). The study was done after receiving approval form the Institutional Review Board, Faculty of Medicine, Chulalongkorn University (Protocol number 626/59). This cross-sectional study was conducted from March 1st 2017 to March 31th 2018. The sample size estimation was based on the sensitivity of CCNA1 promoter methylation for the detection of high-grade cervical lesions . With α error at 0.05 and β error at 0.1, at least 250 patients were required. Additional 30 patients were recruited due to probability of patients’ dropout. The patients who attended the Colposcopic Clinic and had abnormal Pap smear or hrHPV positive were recruited. Women who underwent hysterectomy or pelvic radiation, were diagnosed with cancer, were pregnant, and those who refused to do self-sampled test were excluded. Study interventionAll patients were informed about the study objectives and methods by the researchers and trained research assistant team. After that, the patients signed the informed consent voluntarily. Before performing colposcopy, the patients were asked to perform self-collected sampling without perineum preparation. The instruction was also handed to each patient as a leaflet. The self-sampling device contained a sterile 0.5x1cm soft brush and a transport media tube. Having understood the instruction clearly, the patients placed in a comfortable position (standing or sitting). A brush was inserted about 2 inches deep into their vaginas or until it was resisted then rotated 5 times. Next, the brush was taken out and put into the transport media tube. The specimens were sent to the Department of Microbiology, Faculty of Medicine, Chulalongkorn University within 48 hours. The transport media tubes were stored at the room temperature. High risk HPV DNA was detected by Cobas4800. Cobas 4800 HPV test is a molecular method based on real-time PCR (RT-PCR) with a fully automated system. Cobas4800 can detect HPV16, HPV18, and other 12 high-risk HPVs . The result of the test will be reported as positive for hrHPV type 16, 18 or non 16, 18 other high risk . Positive of any type of hrHPV would be considered as test positive for HPV . After that, CCNA1 promoter methylation test was performed on the specimens . CCNA1 promoter methylation was detected by two TaqMan™ probe real-time PCRs. The CCNA1 methylation set was composed of the forward primer reverse primer and probe. The Beta actin set primers were designed in the area of no CpG island to serve as internal control. The Beta actin set consisted of forward primer, reverse primer, and probe. Both PCR reactions were prepared in a volume of 20 µL containing 10 µL of 2X TaqMan GTXpress real-time PCR master mix, 0.4 µL of 10 µmoles of each primer as well as probes and 2 µL of bisulfite-treated DNA template; the remaining volume was adjusted by adding milliQ DNase-free sterile water. Real-time PCRs were performed in duplicates using Applied Biosystem® 7500 Real-Time PCR System . The PCR conditions were first denatured at 95 °C for 2 minutes then went after with 40 cycles as follows: denaturation at 95 °C 15 seconds, followed by annealing at 60 °C for 30 seconds. Negative control (dH20) and positive control ) were included in each PCR. A melting curve was generated to determine the specificity of the primers. Later, the threshold cycle (Ct) of the amplified methylation products was detected. The results of all samples must have Beta actin products as internal control. Data analysis reported positive if present of CCNA1 methylation product and reported negative if no CCNA1 methylation. The final diagnosis was achieved from histopathologic results considered as gold standard. Cervical tissue biopsy and/or endocervical curettage and/or endometrial sampling was performed by an experienced colposcopist who had passed the colposcopic training course and the 50 validated cases before performing colposcopic examination in this study. Those without significant colposcopic findings were also randomly biopsied at 6 and 12 o’clock positions. Histopathological diagnosis was reported according to WHO’s guidelines (2014) by gynecologic pathologists. The most severe histopathological result from cervical biopsies, conization, or hysterectomy was used as the gold standard.The statistical analysis was performed using SPSS version 22 . All information was analyzed as mean and percentage. Final histological results from colposcopic directed biopsy or excisional procedure were considered as gold standard. Histologic result included benign tissue was classified as low-grade cervical lesions (CIN 2-) and if included HPV effect or CIN1 was considered as negative in gold standard. High-grade cervical lesions (≥ CIN2) included CIN2, CIN3, and malignancy. CIN2+ was considered as positive in gold standard. CCNA1 and hrHPV testing results were reported as positive or negative. The diagnosis performance of self-sampled HPV test and self-sampled CCNA1 promotor methylation test for the detection of cervical precancerous and cancerous lesion (≥ CIN2) were calculated in respect to sensitivity, specificity, PPV, NPV, accuracy, likelihood ratio, and 95% confidence interval. The study was conducted from March 1st 2017 to March 31th 2018. Two hundred and eighty women were recruited from the Colposcopy Clinic, King Chulalongkorn Memorial Hospital. The mean age of the patients was 44.8 years (SD11.2). The majority of the patients were premenopause (68.2%).Cobas 4800 HPV testing was performed on all 280 patients. The percentage of hrHPV DNA detection and cytology results are shown in The diagnostic performance of CCNA1 promotor methylation and hrHPV DNA in self-sampled test for high-grade cervical lesion are presented in All of the CCNA1 positive cases also had positive HPV test, 6 of them were positive for non 16, 18 other high risk subtype; other 6 of them were positive for type 16 and one of them was positive for type 18. From 61 patients with high grade lesion, CCNA1 was positive in 12 ones. However, CCNA1 was negative in 218 patients out of 219 ones of negative gold standard , True negative value of CCNA1 was very high. From 13 CCNA1 positive patients, 10 were CIN2 or CIN3. The rest of them were squamous cell carcinoma, adenocarcinoma, and benign. This means that 12 out of 13 positive cases had significant lesions. The result of tests is shown in Primary hrHPV testing is becoming the first line screening for cervical cancer because of its high efficacy and cost effectiveness . Primary hrHPV testing specimen collection can be performed both by clinician and the patients. Self-sampling can reduce the barriers to cancer screening such as embarrassment, fear of pain, or fear of speculum examination . Self-sampling for cervical cancer screening has has enough accuracy and reliability for the detection of hrHPV. The performance of hrHPV DNA test in self-sampling devices was reported to be comparable with clinician-sampling . According to previous studies, sensitivity of self-sampled HPV test ranges between 84-100% . The sensitivity of self-sampled hrHPV for the detection of high-grade cervical lesion in this study was 70.49%, which was slightly lower than previous reports. HPV DNA testing has very high sensitivity but low specificity that can cause some problems such as overinvestigation and anxiety of the patients. Theoretically, not all women with HPV infection progress to cervical cancer. Approximately 70-80% of HPV infections will resolve spontaneously. Approximately 20% progress to precancerous lesions and only a few of them progress to cancerous lesion. Therefore, not all women with a positive hrHPV test develop lesions . Current recommended triage strategies for primary HPV screening include HPV partial genotyping with cervical cytology and alternatively host and viral methylation . However, self-samples are not suitable for cytology. Therefore, molecular testing is considered for a triage model. Recent evidence has demonstrated that methylation of cyclinA1 (CCNA1) gene may also considerably conduce to the process of tumorigenesis . It has been widely described in several studies that CCNA1 may be an important tumor suppressor gene. In a previous study, CCNA1 was proven to have high distinctive ability to differential benign or low-grade cervical intraepithelial lesion from high-grade intraepithelial lesion . This study found CCNA1 gene methylation positive 12 cases in high- grade lesion and positive one case in benign specimen, which was correlated with Yang et al.’s findings reporting CCNA1 can be positive 11.1 % in normal cervix, 25% in LSIL, 55.6% in HSIL and 80% in cervical cancer . Sensitivity of high-grade cervical lesion detection in self-sampled CCNA1 promoter methylation test in this study was quite low (19.67%), but its specificity was as high as 99.54% and its accuracy was 82.14%. The sensitivity obtained in our study was in contrast with the findings of a study in Chiangmai, a northern city in Thailand . They reported a high sensitivity and specificity for CCNA1 methylation in detection of high-grade cervical lesions, revealing sensitivity and specificity of 83% and 96.88%, respectively. We hypothesized that different methods of specimen collection may have an influence on the detection rate of CCNA1 methylation. Chujan et al., compared HPV testing and CCNA1 promoter methylation in terms of diagnostic performance in detection of high-grade cervical lesions based on cytology. However, the sensitivity and specificity of CCNA1 were compatible to our previous study that was performed on clinician-collected specimen . Based on the result of this study, CCNA1 positive was a strong marker for having high-grade intraepithelial lesions in the patient. This result was correlated with that of Kitkumthorn et al.’s study, reporting a strong association between CCNA1 promoter methylation and 3 . This study was performed to evaluate the role of CCNA1 in self-sampling method. This study had some limitations. Cost effectiveness of was not evaluated in this study, and long-term follow up of the patients especially those who had CCNA1 positive but their histological result was benign was not performed. In conclusion, we found that self-sampled HPV DNA testing had high sensitivity but low specificity that may cause women do unnecessary interventions such as colposcopic directed biopsy. Due to the low sensitivity of CCNA1 promoter methylation, it should not be used as screening method but its high specificity is valuable to be used as an alert sign of having high-grade lesion. Cytology cannot be used to triage the patients who have positive HPV in self-sampling method. However, CCNA1 methylation may be used as a triage test in SS-HPV positive test. Further studies are still required .


Squamous cell carcinoma of the head and neck (HNSCC) consist of two distinct biological entities. While the numbers of classical, tobacco-induced HNSCC are declining, tumors caused by human papillomavirus (HPV) infection are increasing in many countries. HPV-positive HNSCC mostly arise in the oropharynx and are characterized by an enhanced sensitivity towards radiotherapy and a favorable prognosis. To identify molecular differences between both entities on the protein level, we conducted a mass spectrometric comparison of eight HPV-positive and nine HPV-negative oropharyngeal tumors (OPSCC). Overall, we identified 2051 proteins, of which 31 were found to be differentially expressed. Seventeen of these can be assorted to three functional groups, namely DNA replication, nuclear architecture and cytoskeleton regulation, with the differences in the last group potentially reflecting an enhanced migratory and invasive capacity. Furthermore, a number of identified proteins have been described to directly impact on DNA double-strand break repair or radiation sensitivity , offering explanations for the differential prognosis. The unequal expression of three proteins  was confirmed by immunohistochemical staining using a tissue microarray containing 205 OPSCC samples. The expression levels of SLC3A2 and lamin B1 were found be of prognostic relevance in patients with HPV-positive and HPV-negative OPSCC, respectively. In recent decades, there has been an increase in the incidence of head and neck squamous cell carcinomas (HNSCC) with location in the oropharynx (OPSCC), whilst tumors arising from the larynx and hypopharynx have been declining. Sustained alcohol and tobacco consumption no longer represent the main risk factors for OPSCC in many countries as smoking cessation programs were implemented . The risTo identify biologically relevant differences in protein expression between HPV-positive and HPV-negative OPSCC in an open, unbiased experimental approach, we conducted a mass spectrometric comparison of the proteomes of HPV-positive and HPV-negative OPSCC tissue samples.INK4a antibody  and HPV status had been determined by genomic PCR using the MY09/11 primer set and subsequent sequencing of PCR products. Ten patients with HPV/p16-positive and 10 with HPV/p16-negative OPSCC with leftover formalin-fixed paraffin-embedded (FFPE) tumor tissue available from histopathological workup of the main tumor specimen were chosen for the mass spectrometric comparison. The tissue microarray (TMA) contains tumor samples from patients who had been diagnosed with a squamous cell carcinoma of the oropharynx and treated with curative intent at the University Medical Center Hamburg-Eppendorf between 1992 and 2013. The use of archived diagnostic leftover tissues and their analysis for research purposes as well as patient data analysis have been approved by local laws  and by the local ethics committee . The whole study has been carried out in compliance with the Helsinki Declaration.Patients treated for HNSCC at the Department of Otorhinolaryngology of the University Medical Center Hamburg-Eppendorf between August 2011 and March 2013 were reviewed for primary site and HPV/p16 status within our clinical cancer database. p16 status had been determined by immunohistochemistry using a mouse anti-p16The FFPE tissue blocks from HPV-positive and HPV-negative tumors were sliced with a microtome into sections of 6 µm thickness and transferred onto specimen slides. A total of 22 sections were cut from each tissue sample. The first and last specimens were stained with haematoxylin and eosin (HE) for microscopical assessment of the tumor. Areas of tumor growth were marked by a pathologist (T.S.C.) on both HE slides to provide guidance for the macrodissection of slides 2 to 21 in the next step.The tissue slices were washed subsequently by xylene  twice, 100% ethanol  twice, and once in 95%, 70% and 30% ethanol for 10 min for each washing step for deparaffinization. Then, tissues from tumor areas were transferred from the slides to reaction vials with a scalpel and incubated at 65 °C in antigen retrieval buffer ) for 4 h. Then, the tissues were washed twice with HPLC-grade water  and centrifuged at 12,000 rpm for 5 min.4HCO3 ) in HPLC-grade water were added to the reaction vials for reduction of cysteine residues, incubated for 10 min at 56 °C followed by centrifugation at 12,000 rpm for 5 min. Cysteine residues were alkylated with 300 mM iodoacetamide (Sigma-Aldrich Chemie) dissolved in 100 mM NH4HCO3. The samples were incubated in the dark at room temperature for 20 min, followed by centrifugation at 12,000 rpm for 5 min and removal of the supernatant. Finally, 200 µL sequencing grade modified trypsin  was dissolved to a final concentration of 0.1 µg/µL in HPLC-grade water and 1M ammonium bicarbonate of 100:4) was added to the tissue slices and incubated overnight at 37 °C. Finally, the samples were centrifuged in 12,000 rpm for 10 min and the supernatant was collected and transferred to new collection vials and dried with a vacuum concentrator.A quantity of 200 µL of 10 mM dithiothreitol ) dissolved in a 100 mM ammonium bicarbonate solution  and a C18-EMPORE-DISC . Single-use desalting columns were prepared by stamping out a 6 mm piece of the C18 disc and placing it into a gel loader tip, followed by washing with 100% acetonitrile . Then, 50 μL of Oligo R3 dissolved in 50% ACN were added on top of each column. The columns were conditioned by washing with 60 μL elution buffer (50% ACN in HPLC-grade water with 0.1% trifluoroacetic acid (TFA (Sigma-Aldrich)) and equilibrated with 60 μL wash buffer (0.1% TFA in HPLC-grade water). Then, the sample was dissolved in wash buffer and loaded onto the column, and the peptides were eluted with the elution buffer. The eluates were dried with a vacuum concentrator and stored frozen until mass spectrometric analysis was carried out.Dried samples were dissolved in a mixture of 2 µL 50% ACN and 18 µL 0.2% formic acid  in HPLC-grade water, then diluted 1:10 in 0.2% FA and centrifuged for 10 min at 15,000 rpm at 4 °C. The tryptic peptides were subjected to a reversed-phase nano-UPLC ) coupled via electrospray ionization (ESI) to an Orbitrap mass spectrometer (Orbitrap Fusion (Thermo Fisher Scientific)). Samples were injected onto a trapping column ) by an autosampler at 98% buffer A (0.1% FA in HPLC-water) and 2% buffer B (0.1% FA in ACN) and washed with 2% buffer B. The peptides were separated on a separation column ) by a gradient of 2% to 30% B in 90 min at a flow rate of 200 nL/min, followed by a gradient to 70% B in 10 min, another gradient to 90% B in 2 min and holding 90% B for 3 min before the column was equilibrated at 2% B for 15 min for the next run.The peptides eluting from the column were ionized with electrospray ionization in positive mode. The mass spectrometer was operating in data-dependent acquisition top-speed mode with the following parameters: MS1 scan range of 400–1500 Th, MS1 resolution of 120,000, AGC target of 4e5, maximum injection time of 50 ms. Ions with charge states 2–6 and a minimum intensity of 1e5 were selected for fragmentation, with a dynamic exclusion for 30 s for previously fragmented ions. Selected precursor ions were isolated with a width of 1.5 Th and fragmented at a HCD collision energy of 35%. MS2 spectra were acquired with the ion trap in rapid mode with a maximum injection time of 50 ms and an AGC target of 1e4.www.uniprot.org, 20,161 entries) and the internal contaminant database. The search parameters were set as follows: the precursor mass tolerance was set to 8 ppm, the fragment mass tolerance was set to 0.5 Da, and two missed cleavages were allowed for peptide identification; an FDR of 1% was given on the peptide and the protein level. Carbamidomethylation of cysteine residues was set as a fixed modification. Oxidation of methionine residues and acetylation of the protein N-terminus were set as variable modifications (five at most for each peptide). The MaxLFQ algorithm [Raw LC–MS data were processed with MaxQuant  . Peptidelgorithm  was usedTo compare the protein intensities between the HPV+ and HPV− groups, the quantitative protein data was loaded into Perseus 1.5.8.5 . First, R and the Bioconductor environment  were uset-test and regression analyses were performed using GraphPad Prism 6. Graphs depict mean values and standard error (SE) unless stated otherwise.The list of differentially expressed proteins was uploaded to the STRING database  to visuaTMA construction was described previously ,24. For R and the Bioconductor environment  were useTo characterize proteome differences, we conducted a mass spectrometric comparison of primary HPV-positive and HPV-negative OPSCC. The experimental workflow is outlined in p < 0.05; non-adjusted), the group means were at least different by a factor of 2 and corresponding observations had been made in a previous, comparable mass spectrometric study [The complete list of proteins including their respective intensity values in the individual tumors is presented in ic study .p-value and fold change in our and a similar study by Sepiashvili et al. [The random forest analysis identified a total of 24 proteins whose differential expression allowed separation of the HPV-positive and HPV-negative group in at least 5 of the 100 runs performed. A total of 15 proteins were concordantly identified through i et al. . With seCDKN2A gene) was exclusively detected in HPV-positive tumors. It was classified as differentially expressed by both definitions and among a group of six proteins identified in every run of the random forest analysis. Very similar patterns of  exclusive identification in HPV-positive tumors were observed for four other proteins: Nuclear pore membrane glycoprotein 210 (NUP210), heme-binding protein 2 (HEBP2 or SOUL), inositol polyphosphate 1-phosphatase (INPP1) and topoisomerase 2 beta (TOP2B)  B. Due toMore than half of the 27 proteins upregulated in HPV-positive OPSCC could be assigned to one of three distinct functional groups: 1. DNA replication, 2. Nuclear architecture and 3. Regulation of the cytoskeleton .This group includes five of the six minichromosome maintenance homolog proteins (MCM), which form the replicative helicase complex, a hexameric ring that separates the DNA double-strand preceding the replication fork. In fact, all detected MCM proteins showed higher expression levels in HPV-positive tumors .The MCM complex is critical for replication initiation as well as replication fork progression . A dissoLMNA gene through alternative splicing) was similar in both groups  is one of the most abundant structural components of the interphase nucleus and has been suggested to be a major constituent of the proposed nuclear scaffold termed the nuclear matrix . Nuclearh groups B. A subsh groups C.+ T cells [NUP210 is one of only two transmembrane proteins of the nuclear pore complex but is not necessarily required for either assembly or function and is lacking in some cell types ,46,47. I T cells ,50. WhetThis group comprises seven actin and microtubule organizing proteins . The cytFive proteins were selected for the validation of our findings by IHC staining of an HNSCC tissue micro array (TMA) containing 205 OPSCC samples with known p16 status . While Mp = 0.0678, one-sided t-test)  B.p = 0.036), despite a considerable association of SLC3A2 expression with lower N-stage. No such difference was observed in patients with p16-negative tumors (p = 0.05), but rather trended towards worse survival in those with p16-positive ones (p = 0.17)  for the few patients whose p16-positive tumors demonstrated the highest (3) SLC3A2 staining intensity (e tumors . A low l = 0.17) B. Intere = 0.17) . The undOur study represents the third mass spectrometric comparison of HPV-positive and –negative OPSCC. Slebos et al. performed repeated measurements of pooled tissue samples from 10 HPV-positive vs. 10 HPV-negative frozen tumors . A disadWithin our dataset, two factors clearly speak in favor of the accuracy and robustness of our measurements: i. the detection of p16 in seven of eight HPV-positive but in none of the HPV-negative samples B and ii.Regarding tumor cell biology, our results suggest differences between HPV-positive and HPV-negative OPSCC, especially in three functional groups: replication, nuclear architecture and cytoskeleton regulation. The expression of replication factors, especially the MCM-proteins, was congruently found to be upregulated in HPV-positive OPSCC in all three proteome studies as well as previous transcriptome analyses ,74,75,76The most relevant clinical features of HPV-positive OPSCC are the enhanced sensitivity towards treatment, especially radiotherapy ,13, and Regarding the enhanced radiation sensitivity of HPV-positive OPSCC, we had previously shown that HPV-positive HNSCC cells are characterized by a defect in the repair of radiation-induced DNA double-strand breaks , which wAnother interesting finding of our analysis is the enhanced expression of ubiquitin and of S-phase kinase-associated protein 1 (SKP1), an essential component of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex. Target specificity of the SCF complex is mediated by the specific member of the F-box protein family loaded. Ubiquitination mediates degradation via the 26S-proteasome but also the activity of a large number of proteins. Amongst other functions, SCF-complexes are critically involved in the regulation of the DNA damage response and double-strand break repair by mediating the degradation or triggering the activity of central factors, such as NBS1, Exo1, claspin, XRCC4 or CDC25A ,103,104.Our exploratory mass spectrometric study has several limitations, such as a limited number of tumors or the inclusion of tumor-associated stroma cells in the macrodissected tissue samples, which will to some extent, dilute the tumor-cell-specific signals. In addition, the detected peptides were not always sufficient to distinguish between isoforms or homologous proteins. For example, the three Rho proteins RhoA, RhoB and RhoC, which share 85% sequence identity , were idIn summary, our mass spectrometric comparison of HPV-positive and negative OPSCC tumors has identified protein level differences in replication, nuclear architecture and cytoskeletal regulation. We further identified a number of potential HPV surrogate markers and confirmed, respectively identified the negative prognostic value of strong expression of SLC3A2 and lamin B1 in HPV-positive and HPV-negative OPSCC, respectively. Finally, our data suggest several candidate proteins, which, based on altered expression in HPV-positive tumors, may contribute to the enhanced treatment sensitivity.


Is blue food desirable or disgusting? The answer, it would seem, is both, but it really depends on the food in which the color happens to be present. It turns out that the oft-cited aversive response to blue meat may not even have been scientifically validated, despite the fact that blue food coloring is often added to discombobulate diners. In the case of drinks, however, there has been a recent growth of successful new blue product launches in everything from beer to tea, and from wine to gin, arguing that coloring food products blue is more than simply a contemporary fad. In fact, the current interest in blue food coloring builds on the color's earlier appearance in everything from blue curacao to blue-raspberry candyfloss (cotton candy), and thereafter a number of soft drinks. Over the years, the combination of blue coloring with raspberry flavoring has also appeared in everything from bubble-gum to patriotic pop rocks (popping candy in The United States). Ultimately, it is the rarity of naturally-blue foods that is likely what makes this color so special. As such, blue food coloring can both work effectively to attract the visual attention of the shopper while, at the same time, being linked to a range of different flavors  depending on the food format in which it happens to appear. Note also that the basic descriptor “blue” covers a wide range of hues having a range of different associations, hence eliciting different reactions (be they positive or negative). While blue was once associated with artificiality, a growing number of natural blue food colorings have come onto the market in recent years thus perhaps changing the dominant associations that many consumers may have with this most unusual of food colors. Marketing, the article was subsequently republished 2 years thereafter in the fragrance industry newsletter, Dragoco Report, published by a German perfume manufacturer , I was very pleased eventually to track down what I believed to be the original citation for this anecdote, namely an article written by Wheatley . First pFast Food Nation  was apparently an editorial writer at the magazine in which the article first appeared. What is more, the text itself does not provide any statistics , and given her background and future career path (as a successful editor), it would seem unlikely that Wheatley herself conducted the blue steak experiment that she so famously reports. Rather, she would appear simply to have been recounting a story that she had heard, or read about, elsewhere. The main focus of Wheatley's article is to highlight the various ways in which packaging color can be used by marketers. That being said, the article also uncritically recounts the suggestion from Max Lüscher, a Professor of Psychology from Basle University  the effect that coloring foods has on our taste/flavor perception  cotton candy that had been a common feature of the fairground since at least the early 1950s, and possibly before . It really is all a matter of the form or substrate in which that food coloring appears. One can, I suppose, think of this as a version of Elliot's “color-in-context theory”  which may guide our flavor expectations, it is also worth noting that the visual appearance of food can take on a more symbolic meaning. This is perhaps especially clear in the case of the achromatic colors white and black, linked to purity/cleanliness and mourning, respectively .” Blue mashed potatoes may also have been served to children in the UK  to celebrate the end of the Second World War . Britain's first celebrity chef, Fanny Cradock, was also fond of presenting her mashed potatoes in vivid colors such as purple and blue on her TV shows from the 1950s onwards  examples of famous individuals intentionally coloring foods blue so as to deliberately disconcert their dinner guests  at the N = 29) with a soy analog bacon strip, an American-style cheese, as well as three other foods , that they had to rate in terms of flavor intensity/quality, aroma intensity/quality, and texture. The normally-colored bacon was perceived as having a more intense flavor than when abnormally colored bright blue instead. There was, however, no impact of blue food coloring on aroma intensity for either the bacon or cheese. That said, cooking and browning the bacon apparently reduced the color difference substantially. In a conference poster, Sakai  has been a feature of the fairground for a number of decades  operator, grape-purple and blue-raspberry.’In either 1970 or 1971, the blue raspberry ICEE took its place alongside red cherry as a signature flavor of the brand. It had an artificial raspberry flavor but was colored by FD&C Blue No. 1.”Hence, the blue-raspberry association would first appear to have been introduced into the marketplace in the format of candyfloss (cotton candy) at the fairground Park, . ThereafResearch conducted by my students and I a little over a decade ago, suggested that there were salient cross-cultural differences in the flavor expectations elicited by blue drinks  may have been primed in the Taiwanese participants in Shankar M. et al.'s  study byIn 2015, as one of the experiments conducted at The Science Museum in London, and also online as part of their “Cravings” exhibition more than 5,000 people from around the world were, once again, shown drinks of six different colors and asked to pick which looked the sweetest . According to the latter website, the color of the drink may just have been inspired by the azure color of the waters and sky surrounding this Caribbean island. Hence, in this case, the color association is, in some sense, symbolic, rather than based on a specific colored-flavored source object. The distinctive orange taste of this clear blue alcoholic drink is due to the Laraha oranges that are used to make it.However, going further back in time, the first commercial blue-colored food product was probably curacao . The product from the Dutch island of the same name was first made in 1896, achieved international recognition when a Dutch company Bols made a blue-colored version of curacao that became internationally recognized, as the color behind many a kitsch cocktail . In the first part of the fourteenth century in England, this was colored blue; by late fourteenth century, it was commonly yellow; and by the 1420s, it was an orange-red.” In the Medieval period, a blue color would therefore appear to be an unusual, desirable, and also temporary color in food.Over the course of history, blue had various different associations. For instance, according to Stewart , p. 58: Haslehurst's  kitchen Blue is a rarity among plants and animals.” She goes on to highlight how many of the things that appear blue to us do so only because of a trick of diffraction—the scattering of light—that is the case for bird feathers, sky, ice water, and butterfly wings. Consistent with such a view, according to a recent press article, no one has yet managed to grow a blue rose  may, in general be true, i.e., such foods are indeed rare, it is worth noting that rarity can actually act as a stimulant to desire – think only of truffle  was presumably once true it isn't obviously any more, given the rise in the number of natural blue food dyes – think pea flower, Blue Magik, iris flower, Jagua blue, etc.  Does not seem obviously true given that the blue-green moldy veining in blue cheese—think Blue Stilton, Stichelton, or Gorgonzola—are desirable attributes to many consumers. And, according to Nolan , moldy fTyrrells brand in UK).While chefs rarely introduce blue coloring into the foods they serve nowadays, it would not seem inconceivable that the contemporary consumer interest in unusually-colored produce has been sparked by the molecular/modernist chefs who have been deliberately miscoloring some of the foods they serve for years , could so easily have been otherwise. According to Gillian Kynoch, head of development and innovation Albert Bartlett's Purple Majesty potato variety, the reason why  we tend to think of potatoes as being white lies with Sir Walter Raleigh Carter, , who broCarrots originated in Afghanistan and Iran with purple, red, white, and yellow varieties of this root vegetable initially all being common. According to Carter , the oraCarrots of blueish nature  cabbage in Germany. According to a reviewer, in Northern Germany, this type (and treatment) of cabbage is called “Rotkraut” or “Rotkohl” (red kraut or red cabbage), whereas those living in the south, especially Bavarians, call it “Blaukraut.” It's always the same type of cabbage, and the color does not differ, yet those living in the North describe it (and thus presumably perceive it) as red, whereas those in the South perceive it as blue. The garnish is apparently very popular and liked across Germany, thus suggesting that taste (and familiarity) can sometimes beat the mere color property.Let me end though with the dishes of biochemist and self-proclaimed “mad scientist” Kurare Rose, . He has At the same time, there is also a sense in which people need repeated opportunities to appreciate more challenging material. Carbon and Leder  demonstrTo augment the 2020 reveal, Pantone included a twist of its own: As part of its marketing campaign, the company partnered with several brands to develop the smell, sound, taste, and texture of Classic Blue. The resulting package included a swatch of suede-like fabric from the Inside, a musk-and-sea-salt-scented candle, a blue, berry-flavored jelly, and a three-minute audio track titled ‘Vivid Nostalgiabeautiful aquamarine, flowy, kind of wavy color to it.”  its color of the year for 2020. According to Fixsen : “To augThe appetite-suppressing effects of blue in relation to food apparently extend beyond the food itself. Over the years, it has been suggested that everything from blue plates  capture our attention more effectively in a given context, and at the same time are less likely to have any well-established favor associations. A case can, I think, be made that the recent resurgence of blue foods is intimately linked to our digital consumption of food images.Ultimately, therefore, there is no single meaning of blue in food. The context, or food format, in which it appears is crucial to determining its meaning. Given the rarity of naturally-blue foods, this means that it is primarily added as a coloring and hence the link between color and flavor is essentially arbitrary (which is not the case for the majority of other food colors). Nevertheless, the majority of consumers nowadays would appear to associate this color in a drink or confectionary product with a sweet fruit flavor (e.g., Shankar M. U. et al., As for the blue steak story that we started with, that would appear to be nothing more than a much-publicized urban myth. As to why this particular story has proved so popular down through the decades, it might be argued that myths tend to stick when they “sound right,” when they fits our naïve expectations, and/or when we want them to be true (see also Tannenbaum, The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human papillomavirus virus (HPV) vaccines aim to provide durable protection and are ideal to study the association of cellular with humoral responses. We assessed the duration and characteristics of immune responses provided by the quadrivalent HPV (4vHPV) vaccine in healthy female adults with or without prior exposure with type 16 and 18 HPV. In a prospective cohort, vaccine naïve females received three doses of 4vHPV vaccine and were followed for two years to assess cellular  and humoral  and M4 ELISAs) responses. Frequencies of vaccine-specific CD4+ T cells correlated with antibody responses. Higher HPV antibody titers were found at all time points in participants previously exposed to HPV, except for anti-HPV-18 at Day 187 (one week post the third vaccination). Retrospective cohorts enrolled females who had previously received two or three 4vHPV doses and tested antibody titers by M4 ELISA and pseudovirion neutralization assay along with memory B cells (MBCs). Almost all women enrolled in a retrospective cohort with two prior doses and all women enrolled in a retrospective cohort with three prior doses had sustained antibody and memory responses. Our findings indicate that HPV vaccination induces a long-lasting, robust cellular and humoral immune responses. Human papillomaviruses (HPV) types 16 and 18 are the most important high-risk HPV types, causing approximately 70% of cervical cancers worldwide. The first two HPV vaccines developed, the bivalent AS04-adjuvanted HPV-16/18 vaccine  and the quadrivalent alum-adjuvanted HPV 6/11/16/18 vaccine  were licensed in 2006 for the prevention of cervical cancer and high-grade precursor lesions. In 2015, Gardasil 9 (9vHPV) targeting HPV 6, 11, 16, 18, 31, 33, 45, 52 and 58 was licensed; this is currently the only vaccine available in the US. When administered as three-dose schedules, these vaccines demonstrated high vaccine efficacy (>96%) against cervical cancer precursors in HPV-naïve individuals and induced high levels of type-specific antibodies that persist for over nine years .However, important questions have not been fully answered by earlier clinical trials, and immune correlates of protection have not been established. Vaccines that induce long-term protection are usually characterized by the generation of immune memory. Previous studies showed circulating HPV-16 and -18 specific memory B cells can be detected after vaccination with either 4vHPV or 2vHPV vaccines , but theIn this study, we report results from a prospective analysis of T and B cell responses following 4vHPV vaccination in HPV-naïve and HPV-exposed women and analyses of immune memory responses in two previously vaccinated cohorts (labeled as retrospective cohorts). Our overall goal was to provide further insights on the quality, magnitude, and duration of cellular and humoral responses induced by the HPV vaccine.ClinicalTrials.gov under identifier NCT01505049. The study was approved by the Emory University Institutional Review Board  and written informed consent was provided by subjects prior to study participationSubjects for all 3 cohorts were enrolled from April 2012 through May 2015 at the Hope Clinic of Emory Vaccine Center and Kaiser Permanente in Georgia, USA. In the prospective cohort, a total of 47 healthy women aged 18–26 years were enrolled and vaccinated with three doses of 4vHPV intramuscularly at months 0, 1 and 6. Participants were followed for two years. Results of prevaccination vaginal swabs for HPV DNA detection and serum ELISA antibody titers were used to categorize subjects’ prior exposure to HPV-16 or HPV-18 (assay methods detailed below). Blood for immunological testing was collected at baseline (Day 0), Day 67, Day 187, Day 365, Day 545 and Day 730 postenrollment. For the two retrospective cohorts, healthy females aged 18 to 30 years were enrolled, 78 who had previously received two 4vHPV vaccinations (Retro 2 cohort), and another 78 subjects who had previously received three 4vHPV vaccinations (Retro 3 cohort). Medical records were requested to confirm the number of doses of HPV and date of administration. Subjects from both retrospective cohorts attended only a single study visit during which blood samples were collected for antibody and memory B cell ELISpot assays. Subjects in the Retro 2 cohort also received a third dose of 4vHPV vaccine intramuscularly at enrollment as three doses were recommended for all vaccines at the time of the study. This study was registered at Peripheral blood mononuclear cells (PBMC) were separated from heparinized whole blood using a Ficoll-Hypaque gradient and stored in liquid nitrogen. Cryopreserved PBMCs were later thawed in a 37 °C water bath and washed. Before use, cells were counted and checked for viability by Trypan blue dye exclusion.Peptide pools for intracellular cytokine staining (ICS) and Carboxyfluorescein Succinimidyl Ester (CFSE) T cell proliferation assays were synthesized by JPT Peptide Technologies . These consisted of 15-mers with 11 amino acid overlaps spanning the L1 region of HPV16 HPV18 protein region of HPV-16 and-18 . The HPV-specific response was determined by intracellular cytokine  expression or CFSE dilution in CD4+ T cells upon ex vivo peptide stimulation. To define the percentage of vaccine-induced CD4+ T cell responses, Day 0 antigen (Ag)-specific responses from subjects who were shown to be seronegative and DNA-negative for HPV-16 and HPV-18 at baseline were used to establish a positive cut-off value corresponding to the 95th percentile of the Ag-specific responses in this HPV-negative population (HPV-naïve subjects). The positive responder was defined as subjects whose value was greater than the baseline value and with >180 HPV-16+ or >296 HPV-18+ CD4 T cells per million of total CD4 T cells.For analyses involving memory B cells and cytokine response, a threshold value was calculated for each antigen (HPV-16 and HPV-18). The threshold value was calculated by taking the mean plus 3 standard deviations of the response value at Day 0 (baseline) for all subjects who were ELISA HPV seronegative and vaginal swab DNA-negative at baseline.HPV16 and 18 L1/L2 viral-like particles (VLPs) were provided by the CDC for B cell enzyme-linked immunospot (ELISpot) assay. MBC response was reported as the number of HPV-16- or HPV-18-specific IgG-secreting B cells per million total IgG-secreting B cells. The associated 95% CI for the number of responders was calculated using a two-sided exact Clopper–Pearson method. A subject was classified as a responder in the prospective cohort if she had a postbaseline value greater than the baseline value and greater than the threshold value (negative cut-off) for the assay.Additional experimental methods for ICS, CFSE and MBC ELISpot assays are provided in Antibody responses to HPV-16 and -18 were measured using the multiplex M4ELISA as previously reported ,14. FurtHPV-16 and -18 pseudovirions were produced as previously described . The assSelf-collected vaginal swabs were placed in STM vials , extracted with MagNa Pure  and tested with RUO Linear Array HPV typing assay following previously reported methods .The absolute memory response and the percentage of subjects who were responders were summarized for HPV-16 and HPV-18. A subject was defined as a responder if her memory response was greater than the threshold value (negative cut-off). The minimum, maximum geometric mean frequency, geometric standard deviation and a 95% confidence interval were summarized for HPV-naïve and HPV-exposed groups for HPV-16 and HPV-18 in each cohort.A comparison of the absolute response was made using a two-sided, two-sample t-test. The difference in the absolute mean response was calculated on the log 10 scale and back-transformed for reporting. Both the geometric mean difference and two-sided 95% CI were reported. A comparison of the percentage of subjects who were responders was made using a two-sided Fisher’s Exact test. The difference in proportions and two-sided 95% CI for the difference was reported.Vaccine-induced CD4+ T cell responses to HPV-16 and -18 were assessed as total cytokine-expressing cells  at 7 days post-second and third vaccinations and compared with baseline levels. The magnitude of the correlation between CD4+ T cell responses and antibody titers at study time points was determined by a Spearman Rank Correlation Test.Additional statistical information is provided in We enrolled a total of 203 female subjects for the three studies, specifically 47 in the prospective cohort, 78 in the Retro 2 cohort, and 78 in the Retro 3 cohort . The medp = 0.0003) and 683 vs. 40 (p < 0.0001) for total cytokine-producing CD4+ T cells per 106 CD4+ T cells against HPV-16 and HPV-18, respectively. At Day 7 post-third vaccination (Day 187), magnitudes of HPV-specific CD4+ T cells increased relative to Day 67 for both HPV-16 (983), and HPV-18 (867), but were not statistically significant  at seven days post-second vaccination (Day 67) with geometric means (GM) of 718 vs. 66  when compared to naïve subjects. After vaccination, more CD4+ T cells expressed cytokines against HPV-16 and HPV-18 at each time point in HPV-exposed subjects compared with HPV-naïve subjects, but differences were only statistically significant for HPV-18 at Day 67 (Approximately 89% and 92% of subjects responded to HPV-16 at Day 67 (Day 7 post-second vaccination) and Day 187 (Day 7 post-third vaccination), respectively, and approximately 80% and 87% of subjects responded to HPV-18 at Day 67 and Day 187, respectively B.Antibody levels as measured by ELISA increased from baseline for all subjects and 100% of subjects were seropositive for HPV 16 and 18 following vaccination. Regardless of HPV type, geometric mean increased through Day 187, and remained measurable through Day 730 for both HPV types. .p < 0.001;  in either the HPV-naïve or exposed groups using the CFSE dilution assay . The CD4For HPV-16, the proportion of cytokine-producing HPV-16-specific CD4+ T cells at Days 67 and 187 significantly correlated with ELISA Ab titers at all time points . The corp < 0.0001) for the Retro 2 vs. Retro 3 cohorts; the medians were 475 days vs. 1608 days, respectively . ELISA geometric means for HPV-16 and -18 were lower for the Retro 2 cohort compared to Retro 3 . (The elapsed time from the last vaccinations differed significantly (ectively . ELISA aetro 3). . A similetro 3). . These cp = 0.10 and p = 0.053 for HPV-16 and HPV-18). After vaccination, although MBCs tended to be higher in those previously exposed, no statistically significant difference was observed (data not shown). For all subjects, whether exposed or naïve, 68%, 61%, and 46% responded for HPV-16 and 40%, 32%, and 26% responded for HPV-18 in the MBC assay at Days 365, 545, and 730, respectively  and CD4+ T cell immunity (by ICS and CFSE proliferation assays) in both HPV-naïve and HPV-exposed subjects. In addition, we evaluated the durability of humoral immunity up to seven years postvaccination in two retrospective cohorts.In the prospective cohort, HPV ELISA antibody levels after two doses were higher among HPV-exposed subjects compared to HPV-naïve subjects; these levels were further boosted by the third dose. Irrespective of HPV type, titers declined one year postdose one but remained detectable two years postvaccination, consistent with other reports ,19,20,21In our prospective cohort, higher baseline MBC magnitudes were detected in HPV-exposed subjects compared to HPV-naïve subjects, but these magnitudes were lower than those observed in both Retro 2 and Retro 3 cohorts, indicating that vaccination could induce more durable MBC response than natural exposure. A prior study showed that antibodies cloned from naturally elicited MBCs were generally non-neutralizing, whereas those isolated following vaccination were neutralizing . Our stu+ICOS+ Tfh1-like subset) with antibody and MBC responses using HPV vaccines as a successful model would be worth conducting on a larger scale [Although induction of antigen-specific memory B cells, a process in which CD4+ T cells are essential, is thought to be important for long-term vaccine-induced protection ,26, the er scale ,28 to beA literature search identified only a single published study of lymphocyte proliferation following HPV vaccination, with assays performed only one month post-second and third doses . In our A recent publication showed that in a high vaccine coverage setting, one dose had similar effectiveness as two or three doses in preventing high-grade disease cervical disease . We did The study sheds new light on the crucial role of the B and T cell responses and their importance in the duration of immunity after 4vHPV vaccination with no need for a booster per ACIP . The res