Two blinding corneal dystrophies, pediatric-onset congenital hereditary endothelial dystrophy (CHED) and some cases of late-onset Fuchs endothelial corneal dystrophy (FECD), are caused by SLC4A11 mutations. Three N-terminal SLC4A11 variants: v1, v2 and v3 are expressed in humans. We set out to determine which of these transcripts and what translated products, are present in corneal endothelium as these would be most relevant for CHED and FECD studies. Reverse transcription PCR (RT-PCR) and quantitative RT-PCR revealed only v2 and v3 mRNA in human cornea, but v2 was most abundant. Immunoblots probed with variant-specific antibodies revealed that v2 protein is about four times more abundant than v3 in human corneal endothelium. Bioinformatics and protein analysis using variant-specific antibodies revealed that second methionine in the open reading frame (M36) acts as translation initiation site on SLC4A11 v2 in human cornea. The v2 variants starting at M1 (v2-M1) and M36 (v2-M36) were indistinguishable in their cell surface trafficking and transport function (water flux). Structural homology models of v2-M36 and v3 suggest structural differences but their significance remains unclear. A combination of bioinformatics, RNA quantification and isoform-specific antibodies allows us to conclude that SLC4A11 variant 2 with start site M36 is predominant in corneal endothelium. Within the cornea are three major layers: outermost is the epithelium; the middle layer is the stroma, a dissolved proteoglycan and collagen layer, sparsely populated by keratocytes; innermost is the endothelium, a monolayer of endothelial cells, which rest on their basement membrane, the Descemet’s membrane (DM). Because of the stroma’s high solute concentration, there is a strong drive for osmotic fluid accumulation from the aqueous humor, across the endothelium. The endothelial layer’s principal role is to remove this fluid through a “pumping” mechanism, which reabsorbs fluid into the aqueous humor2.Cornea, the outer surface of the eye, serves as a physical barrier to protect the inner layers of the eye and contributes to refractive power in focusing the visual image1. FECD is responsible for 24% of corneal transplantation procedures in United States3. FECD is genetically heterogenous and caused by alterations in multiple genes including, SLC4A114, COL8A25, TCF86, AGBL17, LOXHD18, KANK49, LAMC19 and ATPB19, although trinucleotide repeat expansion in TCF410, is estimated to be responsible for about 70% of cases. Recessively-inherited, childhood-onset congenital hereditary endothelial dystrophy (CHED) arises only from SLC4A11 mutations16. Finally, SLC4A11 mutations also cause Harboyan syndrome (HS), characterized by childhood endothelial dystrophy and progressive sensorineuronal deafness17, although recently HS has been interpreted as a manifestation of CHED18.Defects in the endothelial “pump” give rise to stromal fluid accumulation, significantly thickening the layer. The edematous stroma blurs vision, significantly compromising visual acuity. Defects in the corneal endothelium give rise to posterior endothelial corneal dystrophies (ECD). Dominantly inherited, Fuchs endothelial corneal dystrophy (FECD) is most common, with a lifetime incidence of about 4% and disease onset typically over age 4020, Na+/OH− co-transport22, Na+-independent H+ (OH−) transport24, and NH3 transport26. Although plant SLC4A11 orthologs can transport borate27, the report that human SLC4A11 can28, has been disputed22. Most disease alleles of SLC4A11 result in protein misfolding and protein retention in the endoplasmic reticulum29, but some affect the protein’s transport function30.SLC4A11 is an integral membrane protein, with a 41 kDa cytoplasmic domain and a 57 kDa membrane domain. Consistent with an important role in the endothelial cell fluid “pump”, SLC4A11 facilitates transmembrane water movement23. Differences in the three sequence variants arise at the N-terminus within the first 20–60 amino acids. One RT-PCR analysis of RNA from corneal endothelium suggested that only splice form 3 was present23. Studies of recombinant SLC4A11 are ongoing, working to understand SLC4A11 function and ultimately to ameliorate corneal dystrophy symptoms associated with defective SLC4A11. To date, all earlier studies have explored the function of recombinant splice form 234.The genome database reveals three N-terminal variants of human SLC4A11: 918 amino acid splice form 1 (NCBI Reference Sequence: NP_001167561.1), 891 amino acid splice form 2 (NP_114423) and 875 amino acid splice form 3 (NP_001167560). These variants have also been respectively called SLC4A11-A, -B and –C39. SLC4A11 has a large N-terminal cytoplasmic domain, which has a role in SLC4A11 solute transport function31. The three N-terminal variants may thus affect the structure and/or function of the N-terminal cytoplasmic domain, with potential impact on transport activity. This underscores the need to identify the SLC4A11 isoform(s) present in human cornea. Accordingly, we analyzed intact human corneas and isolated corneal endothelium . We performed RT-PCR and real-time quantitative PCR of RNA from intact human cornea and isolated corneal endothelium. We developed antibodies able to detect variant 2 initiating translation at two possible start Methionine (Met) residues, and variant 3. We further characterized the cell surface trafficking, transport functions and structural differences of SLC4A11 variants.Definitively establishing the form of SLC4A11 protein in human cornea is critical to guide relevant future studies of the protein. Approximately 60 point mutations are spread throughout the two domains of SLC4A11To begin to examine what forms of SLC4A11 are expressed in human cornea, we performed bioinformatic analysis Fig. . Accordi40. We thus mapped the translational start efficiency of nucleotides surrounding human SLC4A11 variant 2 at M1 (v2-M1) and M36 (v2-M36), as well as the other mammalian sequences  was used to construct standard curves to enable comparisons of numbers of transcript copies present in each sample, for each SLC4A11 variant  downstream of M1 as a potential start site on SLC4A11 v2 as shown in Fig. 48. Data presented here indicate that for v2, M36, not M1, is the translational start site. The question thus arises: are these earlier reports valid? Do the properties of SLC4A11 differ depending on translational start site? We, therefore, compared the ability of these SLC4A11 variants to traffic to the cell surface and to facilitate transport function at the cell surface.Until now, SLC4A11 v2 with M1 start site has been the focus of studies . Immunoblots of cells used for water flux assays revealed similar levels of SLC4A11 expression for the two variants and as expected v2-M36 SLC4A11 migrated at a lower molecular weight that v2-M1 50 and Protein Homology/Analogy Recognition Engine V 2.0 (Phyre2)51. Homology models predicted through I-TASSER  and v3-SLC4A11  using the crystal structure of human erythrocyte Band 3 (SLC4A1) cytoplasmic domain (PDB: 1HYN) as the templateSER Fig.  had estiSER Fig. . The N-t52 with a cut-off score of 0.8. Threonine at position 5 (N-MAAATRRVFHLQPC-C) was predicted as a potential phosphorylation site for Protein Kinase C (PKC) with high score (0.887). PKC is crucial in corneal endothelial cell proliferation and cell cycle53. v3-SLC4A11 could be specifically phosphorylated at Thr5 as part of corneal endothelial cell proliferative regulation.Computational predictions of eukaryotic phosphorylation sites in the unique N-terminal region of v3 (M1-T19) were performed using NetPhos 3.1This study used bioinformatic, transcriptomic and protein level analyses to identify the SLC4A11 translational product in human cornea. Bioinformatic analysis predicted SLC4A11 v2 starting at Met36 to share the highest nucleotide and amino acid sequence conservation with SLC4A11 from other mammals. RT-PCR and qRT-PCR further revealed the presence of only v2 and v3 transcripts in human cornea and the endothelium, where variant 2 is the predominant transcript in the endothelium. Protein analysis showed the predominance of v2 protein in the endothelium when compared to v3. Further, antibodies targeting v2-M1 and v2-M36 SLC4A11 confirmed the presence of SLC4A11 v2-M36 in the human cornea. Homology models of the cytoplasmic domains of v2-M36 and v3 displayed substantial differences in the N-termini of both variants with a common c-terminal fold. No differences were, however, observed in the water flux function of these variants. Data here support that v2-SLC4A11, with translation start at the transcript’s second Met is the predominant SLC4A11 protein of human corneal endothelium, although the v3 variant is present at a significant level. No difference in cell surface trafficking or transport function could be detected arising from differences in the N-terminal region of SLC4A11.23, however, the source of the endothelial mRNA used was unclear and extraction method was not described. Additionally, the earlier study did not analyze the variants at the protein level.Using custom-designed antibodies, we found v2 to be four-times more abundant than v3 in the human corneal endothelium. We also found higher v2 abundance than v3 at the transcript level with negligible v1 in the cornea. Mass spectrometry, unfortunately, was unable to differentiate between v2 and v3 in the human cornea due to the absence of specific tryptic sites in the short unique N-terminal region of v3-SLC4A11 (data not shown). An earlier study found SLC4A11 v3 to be the predominant corneal endothelial SLC4A11 transcript31. Since we did not observe a difference of SLC4A11 water flux function between SLC4A11 v2 starting at M1 or M36, earlier functional studies of SLC4A11 v2-M1 are likely reliable. Our recent studies have used SLC4A11 v2-M36 after being identified as the relevant isoform to be studied in the cornea  but most studies of SLC4A11 variant 2 have used the protein starting at methionine 1 (v2-M1) . Q5 Site-directed mutagenesis kit was from New England Biolabs . Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), calf serum (CS), penicillin-streptomycin-glutamine (PSG), Chicken anti-mouse IgG conjugated with Alexa Fluor 594 and Chicken anti-rabbit Alexa Fluor 488 were from Life Technologies . Cell culture dishes were from Sarstedt . Complete Protease Inhibitor cocktail tablets were from Roche Applied Science . BCA Protein Assay Kit was from Pierce . High Capacity Immobilized Streptavidin Agarose Resin, SuperSignal West Femto Maximum Sensitivity Substrate, Taq DNA polymerase, CloneJET PCR cloning kit, phenyl methane sulfonyl fluoride (PMSF), glass coverslips and anti-GFP antibody were from Thermo Fisher Scientific . Poly-L-lysine was from Sigma-Aldrich . Immobilon-P PVDF membranes were from Millipore . Monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Santa Cruz Biotechnology . Monoclonal antibody against green fluorescent protein was from Thermo Fisher Scientific . Horseradish peroxidase-conjugated sheep anti-mouse IgG was from GE Healthcare Bio-Sciences Corp. . ECL chemiluminescent reagent was from Perkin Elmer Life Sciences . iQ SYBR green super mix was from Bio-Rad Laboratories . Trizol was from Life Technologies .34. This construct encodes “long” SLC4A11, starting at presumed start codon Met 1 (SLC4A11 v2-M1). A shortened version of pSKL1, removing the first 35 amino acids of the protein, resulting in an expression construct (pAMC1) encoding an 856 amino acid protein (SLC4A11 v2-M36), was created using the Q5® site directed mutagenesis kit. The primers used were 5′atctcgcagaatggatacttcg3′ and 5′gctagccagcttgggtct3′. gBlock gene fragment encoding the first 135 amino acids of SLC4A11 variant 1 with a BamHI site at the 5′ end was synthesized from Integrated DNA technologies and cloned blunt in pJET1.2 vector (pDM16) using the CloneJET PCR cloning kit. This was used as a positive control for variant 1 amplification as it contains the target sequence amplified by SLC4A11 variant 1 primers. SLC4A11 variant 3 with a c-terminal HA epitope tag was in pCDNA3.1 (pCML13). Integrity of all the clones was confirmed by DNA sequencing .Eukaryotic-expression construct for splicing variant 2 of human SLC4A11 encoding an 891 amino acid protein (NCBI Reference Sequence: NG_017072.1), N-terminally tagged with the HA epitope (pSKL1), was reported earlierSLC4A11 v2-M1 nucleotide sequences immediately upstream of the first and second possible translation initiation start codons (ATG) were compared to the human Kozak consensus sequence (GCCACC). To identify SLC4A11 genes in other organisms human SLC4A11 sequence was used as a BLAST query of other species genome databases available through Ensembl. Genomic DNA surrounding and inclusive of SLC4A11 coding sequence was exported and used to search for DNA sequences corresponding to the unique 297 bp, 184 bp and 147 bp regions of human SLC4A11 v1, 2, and 3, respectively. Genomic DNA was manually searched since many databases only contain predicted transcripts, not fully sequenced cDNAs. High sequence conservation allowed identification of the M36 codon readily. Sequences upstream of the M36 codon were carefully examined for the presence of an upstream M1 start codon corresponding to human v2-M1. Translated upstream sequences were aligned to human SLC4A11-v2-M1 amino acid sequences.Three polyclonal antibodies were raised in rabbits against synthetic peptides, corresponding to regions of human SLC4A11, by PrimmBiotech . Antibody “SLC4A11-v2-M1” corresponded to amino acids 11–26 of variant 2, starting at the presumed start Met1, CTQEVQGLVHGAGDLS. Antibody “SLC4A11-common” corresponded to amino acids 37–50 of variant 2, starting at M36, SQNGYFEDSSYYKC. Antibody “SLC4A11-v3” was generated against the N-terminal residues of the unique exon in SLC4A11 variant 3, MAAATRRVFHLQPC.2/air environment in complete DMEM  fetal bovine serum, 5% (v/v) calf serum, and 1% (v/v) penicillin-streptomycin-glutamine. All experiments involving transiently transfected cells were carried out 40 to 48 h post-transfection. cDNA encoding SLC4A11 variants and eGFP were transiently transfected, using the calcium phosphate method54.HEK293 cells were grown at 37 °C or 30 °C in 5% CO2. Lysates from intact corneas and endothelial layer were prepared separately. Briefly, intact corneas were frozen in liquid nitrogen and crushed using a mortar and pestle. The resulting tissue powder was solubilized in IP buffer (1% (v/v) IGEPAL CA-630, 5 mM EDTA, 150 mM NaCl, 0.5% (w/v) sodium deoxycholate, 10 mM Tris, pH 7.5), containing Complete Protease Inhibitor cocktail  and PMSF. For endothelial cell lysates, six corneas were micro-dissected and the endothelial layers were peeled off along with the Descemet’s membrane and pooled. Tissues were also lysed in IP buffer, containing Complete Protease Inhibitor cocktail. Both samples were incubated on ice for 20 min and were centrifuged at 13200 x g for 20 min at 4 °C and protein concentration was determined by BCA Assay55.Human cadaver corneas, obtained post-mortem with informed consent of next of the kin of deceased donors, were prepared for transplant by the Comprehensive Tissue Centre . Human corneas used in this study were deemed unsuitable for transplant and made available for research. Experiments using human corneas were approved by the University of Alberta Human Research Ethics Board (HREB) and experiments followed guidelines from HREB. For protein isolation, six corneal samples were pooled. Average age of donors was 54 years and average endothelial cell density was 2641 cells/mm56. Immunoblots were processed as described34. Mouse anti-GAPDH or mouse anti-GFP or Rabbit anti-SLC4A11-common or Rabbit anti-SLC4A11-v2-M1 or Rabbit anti-SLC4A11 v3 were used at 1:3000 or 1:3000 or 1:500 or 1:500 or 1:500 dilution in TBS-TM (5% skim milk powder in TBS-T: 0.1% (v/v) Tween-20, 0.15 M NaCl, 50 mM Tris-HCl, pH 7.5), respectively. After incubation with sheep anti-mouse or donkey anti-rabbit HRP conjugated secondary antibody at 1:5000 dilution, immunoblots were developed using Western Lightning™ Chemiluminescence Reagent Plus. Immunoblots containing corneal endothelial cell lysates incubated with Rabbit anti-SLC4A11-common or Rabbit anti-SLC4A11-variant 3 were developed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific). Immunoblots were visualized using ImageQuant LAS4000 . Quantitative densitometric analyses were performed, using ImageQuant TL 8.1 software.Cell lysates were prepared in 2X SDS-PAGE sample buffer (10% (v/v) glycerol, 2% (w/v) SDS, 0.5% (w/v) bromophenol blue, 75 mM Tris, pH 6.8), containing Complete Protease Inhibitor Cocktail (Roche). Lysates were made to 1% (v/v) 2-mercaptoethanol, heated for 5 min at 65 °C and insoluble material was removed by centrifugation at 16000 x g for 10 min. Samples were then resolved by SDS-PAGE on 7.5% (w/v) acrylamide gels33. Transfected cells were rinsed with 4 °C phosphate buffered saline (PBS) , washed with 4 °C Borate buffer  and labelled with Sulpho-NHS-SS-Biotin (0.5 mg/ml). After washing three times with 4 °C Quenching buffer , cells were solubilised in 500 µl of IPB buffer, containing Complete Protease Inhibitor. For each sample, half of the recovered supernatant was retained for later SDS-PAGE analysis . The remaining half of the recovered supernatants was combined with 100 µl of 50% suspension of High Capacity Streptavidin Agarose resin to precipitate the biotinylated proteins and the supernatant was collected . The T and U fractions of each sample were processed for SDS-PAGE analysis and immunoblotting as described above. Densitometry using GE image analysis Software and the formula (T-U)/T x 100% was used to calculate the percentage of biotinylated protein.Cell surface processing assays were performed as described earlier2. RNA was isolated from tissues using Trizol reagent (Life Technologies) according to the manufacturer’s instructions. The resulting RNA was then used as template with SuperScriptTM III Reverse Transcriptase (Invitrogen) (or without enzyme as a negative control) to generate cDNA according to manufacturer’s instructions. Resulting cDNA (100 ng) was used as template in PCR with Taq Polymerase along with the corresponding primers  in the similar reaction conditions as above. Fluorescence was recorded at the end of each elongation step. After 40 cycles, a melting curve was generated by slowly increasing the temperature (0.1 °C/s) from 54 °C to 95 °C, while the fluorescence was measured. The threshold cycle (CT) was calculated manually and kept consistent throughout. A standard curve was constructed for each variant by plotting the Log10 of the copies against the CT values. The CT for the samples were normalized for GAPDH and plotted in the standard curve to estimate the number of copies of each transcript per reaction. The values for No RT control were subtracted for each variant in each group.cDNA from total cornea and isolated endothelium were used with primer pairs for variant 1, 2, 3 and GAPDH  and washed with 1X PBS. During experiments, the chamber was perfused at 3.5 ml/min with isotonic MBSS buffer  and then with hypotonic (200 mOsm/kg) MBSS buffer, pH 7.4 (same composition as previous but lacking D-mannitol). The chamber was mounted on the stage of a Wave FX Spinning Disc Confocal Microscope , with a Yokogawa CSU10 scanning head. The microscope has a motorized XY stage with Piezo Focus Drive  and a live cell environment chamber , set to 24 °C for the duration of the experiment. Acquisition was performed with a Hamamatsu C9100-13 Digital Camera (EM-CCD) and a 20X objective during excitation with laser  at 491 nm. eGFP fluorescence, collected though a dichroic cube  at wavelengths 520–540 nm, was acquired at 1 point s−1 for 4–6 min. Quantitative image analysis was performed by selecting a region of interest for each HEK293 cell with Volocity 6.0 software . Following the switch to hypotonic medium, the rate of fluorescence change was determined from the initial 15 s of linear fluorescence change.HEK293 cells were grown on poly-L-lysine-coated 25 mm round glass coverslips and co-transfected with cytosolic enhanced green fluorescent protein (eGFP)  and, or pcDNA 3.1 (empty vector) or the indicated SLC4A11 plasmid constructs in a 1:8 molar ratioα-v3 × Detection ratio. So, v2 = Total SLC4A11 expression (detected with anti-common antibody) - v3 and thus v2 = Total SLC4A11 expression (detected with anti-common antibody) - (B.I.α-v3 × Detection ratio).HEK293 cells transfected with cDNA encoding SLC4A11 v2, v3 and empty vector were lysed as explained above. v2 lysate (10 µg protein), v3 lysates , vector control lysate (20 µg protein) and pooled human corneal endothelial cell lysate (20 µg protein) were processed on immunoblots in duplicates. Blots were probed with SLC4A11-common antibody and SLC4A11-v3 antibody, respectively followed by Donkey anti-rabbit secondary antibody and developed. GAPDH, used as a loading control, was detected with monoclonal antibody against GAPDH from Santa Cruz Biotechnology and secondary antibody was sheep anti-mouse . Band intensities (B.I.) were determined by densitometry. The ratio of v3 detection by anti-common and anti-v3 antibodies was determined 51 and Iterative Threading Assembly Refinement (I-TASSER)50. Crystal structure of human erythrocyte Band 3 (SLC4A1) cytoplasmic domain (PDB: 1HYN) was used as the template. Generated models were aligned in The PyMOL molecular graphics system v 2.0.7 using segments that were modelled with 100% confidence .Cytoplasmic domain sequence of SLC4A11 v2-M36 (M36-Y340) and SLC4A11 v3 (M1-Y359) were used to prepare their respective homology models using Protein Homology/Analogy Recognition Engine V 2.0 . Statistical analyses were performed using GraphPad Prism 7.0. (GraphPad software). Groups were compared with one-way and two-way ANOVA with Sidak-post tests, and two-tailed t-tests. p < 0.05 was considered significant.Supplementary Figures S1-S5 and Supplementary Tables S1 and S2


Human papillomavirus (HPV) vaccination and the implementation of primary HPV screening in the Netherlands will lead to a lower cervical disease burden. For evaluation and further improvement of prevention, it is important to estimate the magnitude and timing of health benefits of current and alternative vaccination strategies such as vaccination of boys or adults.We evaluated the impact of the current girls-only vaccination program and alternative strategies on cervical disease burden among the first four vaccinated five-year birth cohorts, given the context of primary HPV screening. We integrated the existing microsimulation models STDSIM (HPV transmission model) and MISCAN-Cervix . Alternative vaccination strategies include: improved vaccination uptake, including routine boys vaccination, and offering adult vaccination at sexual health clinics. Our models show that the current vaccination program is estimated to reduce cervical cancers and cancer deaths by about 35% compared to primary HPV screening in the absence of vaccination. The number needed to vaccinate (NNV) to gain 1 life year is 45. The most efficient alternative vaccination strategies are: 1) improving coverage of girls to 80% (NNV = 42); and 2) routine vaccination for girls and boys at 80% coverage , with cervical cancer mortality reductions estimated at 50% and 60% respectively.While the current program already substantially reduces cervical cancer incidence and mortality, prevention can be further improved by increasing vaccination uptake and extending vaccination to boys. As not all cervical cancer deaths will be prevented, screening participation should still be encouraged. Cervical cancer is the fourth most common cancer among women worldwide , with thMathematical modeling has been used to estimate HPV incidences and the associated cervical disease burden following vaccination, as well as to determine the most cost-effective cervical cancer screening strategy in the post-vaccination era –23. HoweWe developed an HPV transmission and cervical cancer screening modeling framework by integrating two established and comprehensive microsimulation models of HPV transmission (STDSIM) and cervical cancer development (MISCAN-Cervix). With this new modeling framework, we estimated the cervical disease burden for the first cohorts who have received HPV vaccination under the new cervical cancer screening program, while taking both the direct and indirect effects of HPV vaccination into account. In addition, we determined the change in cervical cancer burden under various foreseeable extensions of the current girls-only vaccination program.et al.[STDSIM is an established stochastic microsimulation model of the spread and control of sexually transmitted infections (STIs) –26. The et al..et al. [We have previously quantified STDSIM to the Netherlands to model the spread of HPV-16 and HPV-18. Briefly, we reproduced the Dutch population and its sexual network, based on demographic and sexual data. As an independent validation of the underlying sexual network, we simulated the transmission of chlamydia to validate the level of sexual risk behavior. The model was able to closely reproduce the observed chlamydia prevalence levels, reassuring us that the simulated sexual network is representative for the Dutch situation. We then introduced HPV-16 and HPV-18 in the population to estimate the transmission probabilities and acquired immunity dynamics necessary to reproduce the age-specific patterns in HPV-16 and HPV-18 prevalence. All details on the model structure, the parameter quantification, and model validation and underlying data for the Dutch setting, are described in detail by Matthijsse et al. MISCAN-Cervix simulates the individual life histories of a population of women, based on Dutch demographic and hysterectomy data. Women can acquire a high-risk HPV infection that either clears naturally or leads to the development of pre-invasive cervical lesions. These lesions regress or develop into invasive cervical cancer. Death can follow from cervical cancer or from other causes. Multiple infections can occur at the same time, which are independent of each other. Interventions such as hysterectomy and screening can affect these life histories. Pre-invasive stages and FIGO  1A cases can only be detected by screening, since they are assumed to be asymptomatic, whereas FIGO 1B or worse can also be clinically diagnosed .et al. [et al. [et al. [The model divides cervical disease into seven sequential stages: high-risk HPV infection, three pre-invasive stages  grade 1, 2, and 3), and three invasive stages . However, as bivalent vaccine efficacy estimates for boys are unavailable, efficacy for boys was assumed to be equal to the quadrivalent vaccine efficacy for boys  . Finallyet al. estimated that the effect of mailing self-sampling kits to all non-attendees of the Dutch cervical cancer screening program would generate an extra 5.2% attendance [We subsequently simulated the new cervical cancer screening program, in which primary HPV screening with reflex cytology and cytology triage after 6 months will be offered to women aged 30, 35, 40, 50, and 60 years. Additional HPV testing will be offered at age 45 and 55 for women who either have a positive HPV test or do not attend screening at ages 40 and 50, respectively. Women who attended screening at age 60 and tested positive for HPV will also be invited at age 65. We assumed that 10% of the population never attends screening and has a higher background risk than the 90% potential attenders . Attendatendance . Since ttendance .et al.[We used the same specificity and sensitivity of cytology for detecting precancerous lesions and invasive cancer for testing at least atypical squamous cells of undetermined significance (ASC-US) as in our previous study, i.e. a sensitivity of 40% for CIN 1, 50% or CIN 2, and 75% for CIN 3 and cervical cancer, and a specificity of 98% , 39. Foret al..STDSIM provides us with the estimated age-specific HPV-16 and HPV-18 incidence and vaccinated individuals over time. MISCAN-Cervix determines the most severe state of each woman  and the number of life years spent in each state see also . For theet al. [Huijsmans et al.  recentlyet al. . TherefoOur models predict that the HPV-16 and HPV-18 incidence reductions will lead to a 35% lifetime reduction in clinically detected cancers and cervical cancer deaths, and almost 40% in screen-detected cancers, compared to no vaccination . SomewhaUnder the current vaccination program, vaccinating 45 girls  will save one life year by preventing cervical cancer death, when compared to no vaccination . Most efThe sensitivity analyses in Using a comprehensive modeling framework, linking the established STDSIM and MISCAN-Cervix models, we have estimated the health impact of the current girls-only vaccination program and various alternative vaccination strategies under the new cervical cancer screening program in the Netherlands. HPV-16 and HPV-18 incidence reductions achieved under the current girls-only vaccination are predicted to lead to substantial reductions in cervical disease burden, with an estimated reduction of 35% in clinically detected cancers and cervical cancer deaths, and almost 40% in screen-detected cancers, compared to no vaccination. The NNV of the current girls-only program is 45. Largest health gains will be accomplished when full coverage can be achieved for routine vaccination of both girls and boys. For this strategy, the incremental NNV to gain one life year is 155, compared to the most efficient strategy of full coverage among girls (NNV = 42).et al., in which they concluded that the reduction of hazard in unvaccinated women increases with birth cohort year [The larger reductions in HPV incidence and corresponding age patterns for younger cohorts are attributable to higher levels of herd immunity. This was also demonstrated in the study of Bogaards ort year . For eveWe have estimated the reduction in cervical cancer due to HPV vaccination before, but in a crude way . In the Including also routine vaccination for boys with 80% coverage was predicted to prevent more deaths than reaching full coverage among girls (56% reduction versus 53%). However, including boys appears less efficient, due to a larger increase in number of vaccinations needed. We appreciate that full coverage of the vaccination program, either for only girls or both sexes, might be unrealistic. We therefore also explored the efficiency of the vaccination strategies when excluding the scenarios of full coverage . In thatEven though the models are quantified to data from the Netherlands, these results are generalizable to developed settings with a higher prevalence of HPV or cervical disease. While a higher prevalence of HPV-related cervical disease would lead to a higher absolute number of referrals and treatments, the relative reductions due to vaccination will not change substantially, as long as the proportion of cervical disease attributable to HPV-16 and -18 remains the same. This is supported by the sensitivity analysis with the higher HPV baseline prevalence , in which indeed absolute numbers differ from those in the base case analysis, yet the relative reductions due to vaccination are similar.Our study has four limitations that are noteworthy. First, in our model, we underestimated the proportion of CIN 2 attributable to HPV-16, and slightly overestimated the proportion of CIN 1 and CIN 3 attributable to HPV-18, compared to the observed proportions . These dWe conclude that, already for the first vaccinated birth cohorts, the current vaccination program will lead to substantial reductions in cervical cancer incidence and mortality in the Netherlands. Efficient strategies to further improve health gains are to increase vaccination uptake among girls, or to extend the target group to routine boys vaccination with increased vaccination uptake. As vaccination will not prevent all cervical cancer deaths, screening does not become obsolete and participation should continue to be encouraged in the post-vaccination era.S1 Text(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 FigThere are six disease pathways (types A through F) in MISCAN. All lesions start as either an HPV infection without CIN  or as a CIN 1 lesion without HPV infection (disease pathway E). Cleared/regressed denotes the absence of CIN and HPV infection; CIN 0 denotes the absence of CIN and cervical cancer. All cervical cancer states are HPV positive. The arrows between the states show which types of transitions can occur; the numbers refer to the duration distributions shown in (TIF)Click here for additional data file.S3 FigAnnual probability of acquiring a regressive HPV infection or CIN lesion (top graph) and annual probability of acquiring a progressive HPV infection (bottom graph).(TIF)Click here for additional data file.S4 FigCohort 1 is born between 1993–1997; cohort 2 between 1998–2002; cohort 3 between 2003–2007; and cohort 4 between 2008–2012.(TIF)Click here for additional data file.S1 TableLinear interpolation is used to determine the probability of having had a hysterectomy at intermediate ages.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TableAge-specific test results and histological diagnoses of cervical cancer screening in the Netherlands in years 2000–2007, for (A) women’s first lifetime screen and (B) subsequent screens. Data were obtained from the nationwide registry of histo- and cytolopathology in the Netherlands (PALGA).(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 TablePercentages in the table are estimated in the model calibration. Linear interpolation is used to determine the probabilities at intermediate ages.(DOCX)Click here for additional data file.S6 TableLinear interpolation is used to determine the probabilities at intermediate ages. Source: observed age-specific and stage-specific survival for the periods 1989–2002 and 2003–2009, obtained from the Dutch Cancer Registry.(DOCX)Click here for additional data file.S7 TableThe values in this table represent the percentages of cervical cancer deaths that occur within a given number of years after the moment of clinical diagnosis. It is assumed that no cervical cancer mortality occurs more than 10 years after clinical diagnosis. Source: observed age-specific and stage-specific survival for the periods 1989–2002 and 2003–2009, obtained from the Dutch Cancer Registry.(DOCX)Click here for additional data file.S8 Tableet al., who reported age-specific HPV prevalence in women aged 18–65 years, and Guan et al. who determined the distribution of HPV types in CIN and cervical cancer [The observed proportions are based on the studies of Coupé e ranges), 31. HPV(DOCX)Click here for additional data file.S9 TableIn the model, we assumed that 10% of women never attend screening at the general practitioner, and that 90% are potential attenders. Of the (DOCX)Click here for additional data file.


Many countries have initiated school-based human papillomavirus (HPV) vaccination programs. The real-life effectiveness of HPV vaccines has become increasingly evident, especially among girls vaccinated before HPV exposure in countries with high vaccine uptake. In 2009, Norway initiated a school-based HPV vaccination program for 12-year-old girls using the quadrivalent HPV vaccine (Gardasil®), which targets HPV6, 11, 16, and 18. Here, we aim to assess type-specific vaginal and oral HPV prevalence in vaccinated compared with unvaccinated girls in the first birth cohort eligible for school-based vaccination (born in 1997).® Brush and a FLOQSwab™, respectively. Sexual behavior was ascertained through a short questionnaire.This observational, cross-sectional study measured the HPV prevalence ratio (PR) between vaccinated and unvaccinated girls in Norway. Facebook advertisement was used to recruit participants and disseminate information about the study. Participants self-sampled vaginal and oral specimens using an EvalynAmong the 312 participants, 239 (76.6%) had received at least one dose of HPV vaccine prior to sexual debut. 39.1% of vaginal samples were positive for any HPV type, with similar prevalence among vaccinated and unvaccinated girls . For vaccine-targeted types there was some evidence of lower prevalence in the vaccinated (0.4%) compared to the unvaccinated (6.8%) group . This difference remained after adjusting for sexual behavior . Only four oral samples were positive for any HPV type, and all of these participants had received at least one dose of HPV vaccine at least 1 year before oral sexual debut.There is evidence of a lower prevalence of vaccine-targeted HPV types in the vagina of vaccinated girls from the first birth cohort eligible for school-based HPV vaccination in Norway; this was not the case when considering all HPV types or types not included in the quadrivalent HPV vaccine. Human papillomavirus (HPV) is a common sexually transmitted virus and infects a majority of women during their lifetime . The virFollowing the introduction of HPV vaccines, many countries initiated HPV vaccination programs . The reaIn 2009, Norway initiated a school-based HPV vaccination program, which vaccinated 12-year-old girls. From 2009 to autumn 2017, the quadrivalent vaccine was used; it was later replaced with the bivalent vaccine after a tender procurement process. Coverage has increased since the start of the school-based HPV vaccination program. Of those born in 2004, 83% received three vaccine doses in the 2016/2017 school year . In contA recent study from Norway demonstrated that the prevalence of vaccine-targeted HPV types in urine was 77%  lower in vaccinated compared with unvaccinated girls from the first birth cohort eligible for school-based vaccination in Norway . Howeverclinicaltrial.org (NCT02934724). The study included the first birth cohort eligible for the school-based HPV vaccination program, i.e., girls born in 1997 who were living in Norway in 2009, as they could provide the first sign of the effect of the vaccine on HPV prevalence.An observational, cross-sectional study design was chosen to measure the HPV prevalence ratio (PR) between vaccinated and unvaccinated girls in Norway. It is based on informed consent and was approved by the Regional Ethical Committee (#2014/2333). The study was registered nationally in HelseNorge.no  and in cPay per click (paying when someone clicks on the ads) was chosen as the payment method. The advertisements appeared in the Facebook newsfeed of girls in the target birth cohort, and those who clicked on the advertisements were directed to a web page with an overview of the study and an online registration form on which they could provide their name, birthdate, and home address, and opt to have the study kit delivered to the nearest post office instead of their home address. Information provided by the girls in the online form was later linked to the Population Registry for validation. Only girls with validated information  were sent a study kit. Invalid matches, including girls not born in 1997, received no further information. The study kit contained an informed consent form, study description, a questionnaire, a cervicovaginal sample kit, oral sample kit, and a return envelope. The sample kits contained the manufacturer’s sampling instructions (translated into Norwegian for study purposes). Girls who returned the study kits along with signed informed consent were included in the study.The recruitment period ran from September 2016 to February 2017, when girls in the target birth cohort were 18–20 years old. This ensured that the majority of the girls were sexually active and had likely been exposed to HPV. Facebook advertisements were used to recruit participants and to disseminate information about the study. We created advertisements according to guidelines that were approved by Facebook . Pay per® Brush, a device validated for HPV detection  [® solution . These tubes were vortexed three times for 15 seconds, and stored at +4–7°C overnight to let the cells detach from the brush. The suspension (400 μl) was used for DNA extraction using the automated nucleic acid extraction platform EasyMag . Oral samples were collected using a FLOQSwab™ (#552C) . The swab was transferred to 1 ml eNat buffer  and vortexed three times for 15 seconds, 20 minutes apart, in order to let the cells detach from the swab. 400 μl were used for DNA extraction using EasyMag. HPV detection and genotyping was performed using modified general primers-PCR followed by hybridization of type-specific oligonucleotide probes , which detect and genotype 37 HPV types [Cervicovaginal  samples were collected using an Evalynerlands) . At the PV types , 25.The objective of the study was to assess the effect of the school-based HPV vaccination program by comparing type-specific vaginal and oral HPV prevalence. Outcomes considered were prevalence of any HPV type; prevalence of individual vaccine-targeted HPV types ; prevalence of HPV16 or 18; and prevalence of HPV6, 11, 16, and 18 (vaccine-targeted types). The latter category was the main outcome due to the use of the quadrivalent vaccine in the school-based HPV vaccination program from 2009 to fall 2017. We also looked at the prevalence of high-risk HPV types  [Sexual behavior was ascertained through a short questionnaire. Two reviewers entered the responses into the study database to avoid misclassification. Using the national identification number extracted from the Population Registry, we retrieved the date and type of each dose of the complete vaccine regime from the Norwegian Immunization Registry SYSVAK. Norwegian Immunization Registry data were used, instead of self-reported vaccination status, to classify participants as vaccinated or unvaccinated (exposure classification). This was done as self-reported recollection of vaccination status may introduce recall bias . To be cThe study was not designed to estimate HPV prevalence in the general population, and therefore no specific efforts were made to recruit a nationally representative sample. However, the questionnaire allowed us in retrospect to assess if sexual behavior was similar to the general population of the same age.Sample size calculations for vaginal samples were based on the estimated HPV prevalence in 18-year-old Norwegian unvaccinated girls  and on r2) were used to test the association between sexual behavior and vaccination status. If one or more of the expected numbers was ≤5, the Fisher's exact test was used. Multivariable Poisson regression was used to estimate PRs adjusted for lifetime number of sexual partners, age at sexual debut, and time since last sexual intercourse. Missing values were ignored. All analyses were performed in Stata 15 .Chi square statistics  had received at least one dose of HPV vaccine at least 1 year prior to sexual debut, and were classified as vaccinated . The majThe most common low-risk HPV types detected in vaginal samples were HPV90 (7.4%), HPV42 (7.1%) and HPV89 (6.1%), while HPV51 (4.2%) was the most common high-risk type . No signOnly four oral samples were positive for any HPV type (1.3%), with a similar prevalence among vaccinated and unvaccinated girls . Each of these four participants had received at least one dose of HPV vaccine at least 1 year before oral sexual debut, and each was positive for a single HPV type . Two were positive for the same HPV type (HPV67 and 87) in their vaginal sample, while the other two were negative for the same HPV type in their corresponding vaginal sample.ad hoc analysis using a definition of participants as receiving either the quadrivalent or bivalent vaccine as vaccinated had negligible impact as only one participants moved from the unvaccinated to the vaccinated category , as the two participants who did not return their questionnaire  could be included. Among the participants who shifted from unvaccinated to vaccinated, one was HPV18-positive. The difference between vaccinated and unvaccinated girls with respect to vaccine types using the original definition of vaccinated remained evident when using the alternate definition of vaccinated and unadjusted prevalence ratios  . In addicategory . A similr trends .Using Facebook to recruit participants born in 1997, the first birth cohort that could benefit from the Norwegian school-based HPV vaccination program, we found a lower HPV prevalence among participants who received the quadrivalent vaccine compared to unvaccinated participants. This is in line with results from clinical trials , 29 and We observed a lower quadrivalent HPV vaccine type prevalence among vaccinated compared to unvaccinated women. In contrast, the prevalence of any type HPV and for high-risk HPV type was comparable between vaccinated and unvaccinated. This indicates that the change is vaccine specific. As the groups performed self-sampling in the same period, the reduction is not attributable to changes in sexual behavior over time. Another interesting finding was the very low overall prevalence of vaccine-targeted HPV types in vaginal samples . This is in line with a reduction in the prevalence of vaccine-targeted HPV types from urine samples from girls in birth cohorts who were not  and who were  eligible for school-based HPV vaccination . A reducOne major limitation of the present study is the small number of HPV-positive participants, which drastically reduced the power to observe differences in the prevalence of individual vaccine-targeted HPV types between vaccinated and unvaccinated participants. Our sample size estimates were based on the available HPV prevalence percentages from studies that turned out to be higher than the HPV prevalence among the participants recruited in this study , 28. ThiAt the end of the recruitment process, we experienced that very few girls showed interest to participate. We believe the current advertisements and recruiting strategy had reached its potential. Although, we were not aware of the overall low HPV prevalence at that time, in retrospect, the study could have benefited by including girls also born in 1998 in order to increase the sample size.Cervicovaginal self-sampling with the Evalyn brush has previously shown good concordance with general practitioner-collected samples –32; howePositivity for any HPV type was almost absent in oral samples (1.3%), even though less than 20% of participants were oral sexually naïve. Although it is not directly comparable, oral HPV prevalence among men and women aged 16–25 years in Scotland was 2.6% (95% CI: 0.1–13.2) . A studyPay per click in contrast to pay per impression (every time they show someone the advertisement) minimized the costs of the advertisement. In our study, Facebook recruiting allowed us to adjust the recruitment to target additional unvaccinated girls during the course of the study. Although it is a strength of the study that it recruited across the whole country, recruiting through Facebook also reduced the population that could be reached to those actively using that platform. However, at the end of 2017, 96% of girls aged 18–29 years in Norway had a Facebook account; 96% of those had an active account [Targeted advertising using social networking sites, like Facebook, is rapidly increasing to recruit research participants , 39. For account .A strength of the study is that we could adjust for differences in sexual behavior, based on the questionnaire. However, not surprisingly, the adjusted PR was only slightly adjusted, as no significant differences between the two groups were observed. This is in line with a large Nordic study, which showed that sexual behavior is not different in vaccinated compared to unvaccinated girls . The medWe conclude that there is evidence of a lower vaginal vaccine-targeted HPV type prevalence between vaccinated and unvaccinated girls in the first birth cohort of girls eligible for the school-based HPV vaccination program in Norway; this is not the case for the prevalence of any HPV type or non-vaccine-targeted HPV types.S1 FigShutterstock.com(A-B) Examples of advertisements used to target both vaccinated and unvaccinated. (C) Advertisement used to target only unvaccinated girls. Credits for images: (TIF)Click here for additional data file.S1 TableA participant is defined as vaccinated if she received at least one dose of quadrivalent HPV vaccine.(DOCX)Click here for additional data file.S2 TableA participant is defined as vaccinated if she received at least one dose of the bivalent or the quadrivalent HPV vaccine at least the calendar year before sexual debut.(DOCX)Click here for additional data file.S3 TableA participant is defined as vaccinated if she received at least three doses the quadrivalent HPV vaccine at least the calendar year before sexual debut.(DOCX)Click here for additional data file.S4 TableSTROBE Statement—Checklist of items that should be included in reports of cross-sectional studies.(DOCX)Click here for additional data file. 2 Jul 2019PONE-D-19-15806An observational study comparing HPV prevalence and type distribution between HPV-vaccinated and -unvaccinated girls after introduction of school-based HPV vaccination in NorwayPLOS ONEDear Dr Enerly,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.We would appreciate receiving your revised manuscript by Aug 16 2019 11:59PM. When you are ready to submit your revision, log on to If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsTo enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. 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Thank you for stating the following in the Competing Interests section:" We have read the journal's policy and the authors of this manuscript have the following competing interests: M.N. has received research grants from MSD/Merck through the affiliating institute.The other authors have declared that no competing interests exist.WHO-IARC: Where authors are identified as personnel of the International Agency for Research on Cancer / World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer / World Health Organization.Norwegian Immunization Registry SYSVAK  provided data, but is not responsible for the analyses and interpretation in this article."http://journals.plos.org/plosone/s/competing-interests).  If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” . The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: This is an interesting study, which confirms what has previously been seen in Norway with respect to impact of the HPV vaccine programme. Unfortunately, due to the small numbers of the females involved in the study, it is very difficult to see what more such a paper adds to the previously referenced studies from Norway and elsewhere. The utility of using Facebook to recruit women into providing samples and sexual behaviour data is of interest and it is that methodology that I think the authors could focus on more, as a potential for surveillance expansion. However, in its current format, I don't believe the data are particularly novel or representative of a population. The following may help to revise the manuscript.Questions/comments1. What has been the uptake of the HPV vaccine in Norway since the programme started? This is not included.2. Why did Norway move from 4v to 2v? This is not explained and at odds with most countries e.g. UK - the reverse occurred.3. Please expand on the Facebook recruitment strategy targeting the specific birth cohorts - this is interesting and will be of interest to other countries. Especially if samples are then provided via urine, which has been shown to have similar data with respect to HPV detection, when compared to clinically taken smears.4. Why are girls vaccinated with bivalent described as unvaccinated? This is not appropriate.5. Why do the authors change their vaccine status identification from registry to self-reported in the Methods? This is incongruous.6. Be careful using the term 'rate' with respect to vaccine uptake - they are only rates if a time component is included - otherwise percentage uptake is sufficient and preferable.7. The authors state that HPV 89 and 90 were most prevalent low-risk types - but no percentages included. Please include.8. I think it is important to state that while 'vaccine-specific' HPV has reduced, other HR-HPV has not - this suggests a vaccine effect rather than any change in sexual behaviour over time. The authors should develop this.Reviewer #2: Review Enerly et al.An observational study comparing HPV prevalence and type distribution between HPV-vaccinated and – unvaccinated girls after a school-based HPV vaccination in NorwayThe authors performed a cross sectional study of the HPV prevalence comparing vaccinated and non vaccinated girls in Norway. They used an Evalyn Brush and FLOQSwab device to retrieve material cellular material from the participants from either cervix and oral mucosa. They found a lower prevalence of HPV infections in the vaccinated girls compared to the unvaccinated. The could only ensure at least one vaccination of the participants, which was retrieved from the national epidemiology files.Major concerns:The authors present an approved study from the ethics commission and registered the study at the NCT files. However it is doubtful if the participants recruited via facebook are a representative group in this age group. In addition, one must doubt if all participants are able to handle the Evalyn device appropriate. How can the authors ensure that they do observe either false positive or false negative data. To my opinion, controlled studies that are published should ensure that participants see a physician who can ensure that the participant identify herself as the person that take part in the study and that the specimen is taken by an experienced physician under controlled circumstances.Furthermore, it is to my opinion critical that the authors can only ensure that the girls have received at least only one vaccine shot. Why isn’t it possible that they have all information about the full vaccination program. This is a major setback in the design of the study.Not surprising the authors have to exclude 210 girls because they did not send back the kit the consent was incomplete. This underscores the major disadvantage of the presented approach the authors have chosen.Reviewer #3: The sample size is small. But the authors have no commented on this. It is suggested to include justification for the small sample size, in the text. May be it can be included as limitation of the study.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  19 Aug 2019Response to reviewersReviewer #1Reviewer #1: This is an interesting study, which confirms what has previously been seen in Norway with respect to impact of the HPV vaccine programme. Unfortunately, due to the small numbers of the females involved in the study, it is very difficult to see what more such a paper adds to the previously referenced studies from Norway and elsewhere. The utility of using Facebook to recruit women into providing samples and sexual behaviour data is of interest and it is that methodology that I think the authors could focus on more, as a potential for surveillance expansion. However, in its current format, I don't believe the data are particularly novel or representative of a population. The following may help to revise the manuscript.1. What has been the uptake of the HPV vaccine in Norway since the programme started? This is not included.Answer: We have added this information to the Introduction along with two references.2. Why did Norway move from 4v to 2v? This is not explained and at odds with most countries e.g. UK - the reverse occurred. https://www.doffin.no/en/notice/details/2016-175572). The evaluation of the offers is not publicly available, but the evaluation weighted “Price 60% and Quality (Effect and safety) 40% (Part IV 2.1 in the link above). We have added a note to the Introduction about the tender.Answer: The change was done after a tender for procurement . Therefore we believe the data should be analysed using the definition defined upfront of the study. However, we have now included an ad hoc analysis. Adding bivalent vaccination to the definition moves only one participant from the unvaccinated category to the vaccinated category. This has negligible impact of the results. We have added this information to the results as a supplementary table.Answer: We certainly agree that treating bivalent vaccinated girls as unvaccinated is not appropriate per se. However, we did not foresee that Norway would change to the bivalent vaccine. When creating the analysis plan we choose the definition of vaccinated as “Women born in 1997, resident in Norway in 2009, vaccinated with the quadrivalent HPV vaccine”. This was used to describe the definition in the study in ClinicalT5. Why do the authors change their vaccine status identification from registry to self-reported in the Methods? This is incongruous.Answer: We used self-reported vaccination status only to optimize the recruitment process. We noticed that few participants reported to be vaccinated and therefore selectively recruited unvaccinated for a short period. During recruitment we did not have access to registry (sysvak) information. To clarify this use we have added a sentence in the method chapter.6. Be careful using the term 'rate' with respect to vaccine uptake - they are only rates if a time component is included - otherwise percentage uptake is sufficient and preferable.Answer: We have updated to percentage throughout the paper when appropriate. 7. The authors state that HPV 89 and 90 were most prevalent low-risk types - but no percentages included. Please include.Answer: Percentages are now included. HPV42 was missing and is now included too.8. I think it is important to state that while 'vaccine-specific' HPV has reduced, other HR-HPV has not - this suggests a vaccine effect rather than any change in sexual behaviour over time. The authors should develop this.Answer: We have added a sentence to the result to highlight this and we discuss it more thoroughly in the discussion. Reviewer #2: Review Enerly et al.An observational study comparing HPV prevalence and type distribution between HPV-vaccinated and – unvaccinated girls after a school-based HPV vaccination in NorwayThe authors performed a cross sectional study of the HPV prevalence comparing vaccinated and non vaccinated girls in Norway. They used an Evalyn Brush and FLOQSwab device to retrieve material cellular material from the participants from either cervix and oral mucosa. They found a lower prevalence of HPV infections in the vaccinated girls compared to the unvaccinated. The could only ensure at least one vaccination of the participants, which was retrieved from the national epidemiology files.Answer:Major concerns:The authors present an approved study from the ethics commission and registered the study at the NCT files. However it is doubtful if the participants recruited via facebook are a representative group in this age group. Answer: Although some similar sexual behaviour is seen comparing data from the questionnaire with data from other population-based studies of Norwegian women, we agree that there is likely a bias when recruiting through Facebook. We have therefore commented on this issue which we also touch upon in the last sentences of the method chapter “Human papillomavirus prevalence and sexual behaviour”. In addition, one must doubt if all participants are able to handle the Evalyn device appropriate. How can the authors ensure that they do observe either false positive or false negative data. To my opinion, controlled studies that are published should ensure that participants see a physician who can ensure that the participant identify herself as the person that take part in the study and that the specimen is taken by an experienced physician under controlled circumstances.Answer: We chose not to design the study to require the girls to see an experienced physician under controlled circumstances. Firstly, because the recruitment process would likely result in fewer participants and higher costs. Secondly, both vaccinated and unvaccinated were sampled the same way and reduced efficiency or contamination would likely be evenly distributed between the groups. However, we agree that we cannot ensure that girls sample correctly according to the instruction. Still, taken together we believe the study design is valid for answering the aims of the study.Furthermore, it is to my opinion critical that the authors can only ensure that the girls have received at least only one vaccine shot. Why isn’t it possible that they have all information about the full vaccination program. This is a major setback in the design of the study.Answer: Thanks for pointing it out that it was unclear as we in fact have the information. In the chapter “Human papillomavirus prevalence and sexual behavior” we have modified the sentence to clarify this. The distribution of the number of doses is listed in Table 1. An Ad hoc analysis using the definition of vaccinated as receiving all three doses at least the year before sexual debut was done and is added to the results and as a supplementary table.Not surprising the authors have to exclude 210 girls because they did not send back the kit the consent was incomplete. This underscores the major disadvantage of the presented approach the authors have chosen.Answer: Ideally, we wish that all girls that showed interest for the study would consent to the study after receiving the kits. However, that 60% consented and returned the kits we believe is still reasonable. The aim is not to measure the general HPV prevalence. Therefore we believe the return percentage is acceptable. The extra cost is of course a drawback. The two groups that finally consented to the study did show similar sexual behavior patterns and we believe therefore the 60% returns did not introduce biases between the groups that we could not adjust for in the analysis.Reviewer #3: The sample size is small. But the authors have no commented on this. It is suggested to include justification for the small sample size, in the text. May be it can be included as limitation of the study.Answer: We have expanded on this limitation in the discussion.AttachmentResponse to reviewers.docxSubmitted filename: Click here for additional data file. 25 Sep 2019An observational study comparing HPV prevalence and type distribution between HPV-vaccinated and -unvaccinated girls after introduction of school-based HPV vaccination in NorwayPONE-D-19-15806R1Dear Dr. Enerly,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Maria Lina TorneselloAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 2 Oct 2019PONE-D-19-15806R1 An observational study comparing HPV prevalence and type distribution between HPV-vaccinated and -unvaccinated girls after introduction of school-based HPV vaccination in Norway Dear Dr. Enerly:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. 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Papovaviridae because of their similarity in morphology and genome organization but are now classified in the separate families of Polyomaviridae and Papillomaviridae, respectively [Papillomaviridae that can cause human cancer mainly through the action of their viral oncoproteins E6 and E7. Epidemiological studies point to an etiological role of HR-HPV predominantly in cervical cancer, but also penile and anal cancers, and head and neck squamous carcinomas [Human polyomaviruses (HPyV) and papillomaviruses (HPV) were originally grouped in the family ectively ,2. Membeectively ,4. The rrcinomas ,6. ThereIn the Special Issue “Human Polyomaviruses and Papillomaviruses”, interesting findings on the role of the BKPyV agnoprotein in viral release, the biological importance of mutations in the promoter of HPyV9 variants, the epidemiology of HR-HPV in cervical and oropharyngeal cancer, the role of HPV in autophagy, and mechanisms of transformation are presented.N-ethylmaleimide-sensitive fusion attachment protein (α-SNAP) [So far, 14 HPyV have been isolated from different human samples , respect(α-SNAP) . This fi(α-SNAP) . In addi(α-SNAP) . They us(α-SNAP) . This stThe role of HPV in cervical cancer is well established, and there is increasing epidemiological evidence that HPV contributes to other malignancies. Four papers deal with different aspects of the molecular mechanisms by which HPV induces tumorigenesis ,14,15,16n = 12) contained HPV16 DNA. However, the genome copy number per tumor cell was low (0.001–2.5 copies/tumor cell). HPV genotypes 18, 31, 45, 52, and 58 were not found. Although the International Agency of Research of Cancer (IARC) declared that there is sufficient evidence to associate HPV 16 with oral cancers [n = 146) [n = 28; 34%). Although this study shows that co-infection with potential oncogenic viruses may play a role in the initiation and progression of these cancers, larger populations and the correlation with smoking and alcohol abuse must be further explored. Moreover, chronic inflammation with infiltrating EBV-positive blood cells should be considered when examining tumor tissues for the presence of EBV DNA.In this Special Issue, three epidemiological studies address the association of HPV with risk co-factors, namely, smoking, alcohol, and co-infection with other viruses ,21,22. A cancers , Gessner cancers . The eff cancers . Drop anTwo studies aimed at identifying possible biomarkers for HR-HPV-positive cancers. The group of Ramqvist compared the expression of cancer- and immune-related proteins in HPV-positive, HPV-negative tonsillar, and base of tongue squamous cell carcinomas and normal tissues . The expThis Special Issue offers an interesting perspective on the epidemiology of HR-HPV in different cancers, the mechanisms by which these viruses target host cell proteins to replicate their genome and to induce cancer, the role of co-factors such as smoking and co-infection, and possible novel therapeutic strategies. As for HPyV, this Special Issue provides novel information on the nuclear egress mechanism of BKPyV and opens the possibility of developing strategies to prevent viral release from infected cells. Moreover, studies with HPyV9 variants support previous observations that rearranged transcription control regions affect the biological properties of the virus, as previously shown for BKPyV and JCPyV.


Oral and oropharyngeal squamous cell carcinomas (OSCC and OPSCC) represent the majority of head and neck squamous cell carcinomas (HNSCC). Human papillomavirus (HPV) is an important etiologic factor together with Epstein-Barr virus (EBV). Little is known on the prevalence of major herpesviruses  in HNSCCs.INK4A protein was also investigated by immunohistochemistry (IHC).Fifty-one formalin-fixed paraffin-embedded (FFPE) tissue samples taken at surgery  were analyzed for 40 HPV genotypes (20 high-risk types), EBV, CMV, HHV-6, − 7 and − 8 by quantitative PCR. Expression of the HPV-induced p16P = 0.005). HPV infection was found in 25% of OPSCC and in none of the OSCC; conversely, higher prevalence of EBV was found in OSCC (72.7%). HPV and EBV co-infection was detected only in 4 (10%) OPSCC. CMV was detected in only two cases, whereas HHV-6, − 7 and − 8 resulted negative. The prevalence of HPV but no EBV was associated with the non-keratinizing SCC type (NKSCC) compared to the keratinizing SCC type (KSCC).In SCC, the prevalence of EBV was significantly higher compared to that of HPV (EBV 51% vs. HPV 19.5%; Single HPV or EBV positivity was higher in OSCC than in OPSCC. Other potentially oncogenic herpesvirus types were minimally or not represented. Head and neck squamous cell carcinomas (HNSCC) account for approximately 5% of all cancers and are a serious public health problem worldwide . HNSCC cINK4a a surrogate marker of oncogenic HPV infection. P16INK4a is a tumor suppressor protein that is downregulated in many tumor types [The Epstein-Barr virus (EBV) has long been related to nasopharyngeal carcinoma . A recenor types , but oveor types . Very liArchived formalin-fixed, paraffin-embedded (FFPE) tissue blocks were collected for 51 SCC cases. FFPE sections were obtained from the archive of the Pathology Department. Tissue specimens were obtained from patients with HNSCC  before initiation of radiotherapy, while samples of laryngeal HNSCC were taken at surgery. Eligible cases included newly diagnosed primary HNSCC. At the time of sampling, patients had not received antitumor therapy. The study was approved by the Ethics Committee of IRCCS San Martino IST . As archived tissue was used, no written consent was required.5 × 10 μm sections were removed from the blocks were deparaffinized with xylene and rehydrated through a graded series of distilled water-ethanol solutions. QIAamp DNA FFPE Tissue Kit  was used for DNA extraction. DNA was quantified with a NanoDrop 3300 fluorospectrometer using the RiboGreen dye diluted in TE buffer .Tissue sections were retrospectively analyzed for EBV and other HHV  by quantitative real-time PCR assays . HPV detINK4A monoclonal antibody  was used at 1:50 dilution. Cases were classified as either positive  or negative.Immunoperoxidase staining was done on 4 μm sections of FFPE tissue using the LSAB2 horseradish peroxidase system . Antigen retrieval was done by microwave heating for 10 min in 10 mM citrate buffer (pH 6.0). A p16INK4A positivity, clinical and demographic data using the Prism GraphPad software . Statistical significance was defined as p < 0.05.Statistical analysis was performed for virus positivity, p16The study included 51 patients, 40 with oropharyngeal cancer  and 11 with oral cancer . Demographic and clinical data were also recovered. Mean age was 65 years (standard deviation ±14 years) ranging from 43 to 94 years. Males (69%) with smoking (70%) and alcohol abuse (60%) problems were prevailing in the series. All cases resulted negative for HHV-6, − 7 and − 8. CMV was detected in only two cases (mobile tongue SCC and epiglottis SCC).INK4A.. Two patients had p16INK4Apositive and negative HPV PCR.Overall, of the 51 patients, 10 cases (19.6%) were positive for HPV (9 HPV-16 and 1 HPV-45) and 12 (23.5) for p16P = 0.05). Regarding the subsites of the head and neck region, HPV infection was found in 25% of OPSCC and in none of the OSCC. Conversely, EBV infection was found in 72.7% of OSCC and 45%.of OPSCC cancer cases .The prevalence of HPV and EBV, according to SCC primary site and hystotype and is presented in Table es Table  HPV and INK4A -positive. In contrast, only 1/15 KSCC cases was HPV positive and also p16INK4A -positive. The data indicate that NKSCC cases are more likey p16INK4A - and HPV- positive than KSCC . In Fig. INK4A-positive NK SCC at the base of tongue and in laryngeal. For EBV, although there was no difference in histopathological type, NKSCC cases were also more frequently EBV-positive compared to KSCC cases (data not shown).Of note, all 9 NKSCC cases HPV positive were also p16We explored the prevalence of HPV and potentially oncogenic herpesviruses (HHVs) in archival tissue of SCC cases from different anatomic sites. This is the first study from Italy reporting on the HPV and EBV co-infection in cancers.The overall HPV prevalence in HNSCC was low for OPSCC (25%) and absent in OSCC compared to the reported global prevalence of HPV in the same types of cancer  , 10, 18 Among the 12 HPV types defined as carcinogenic in cervical carcinoma by the International Agency for Research on Cancer , only type 16 appears to play an oncogenic role in OPSCC [The detection of HPV DNA alone is not sufficient to determine tumor causality. For cervical carcinoma and for oropharyngeal and laryngeal cancer the co-expression of several markers  is required to discriminate HPV-driven from non-HPV-driven cases .INK4a protein is considered a surrogate marker of HPV infection in SCC of the cervix and head and neck, as well as in HPV-associated anogenital cancer [INK4a appears as a reliable marker in tumors arising from the oropharynx, larynx, and oral cavity [INK4a, but lack HPV positivity.The p16l cancer . In HNSCl cavity , 22. A pIn our series, infection with high-risk HPV genotypes was not significantly associated with oral cancers. This is in line with what reported . In contINK4A positivity  compared to KSCC . Of interest is the frequent detection of HPV and EBV co-infection in oral and oropharyngeal cancer among Italian patients. HPV infection is considered a sexually-transmitted infection, while EBV is mostly transmitted through saliva [We classified SCC histotypes into two groups: nonkeratinizing and keratinizing SCC. In the investigated series, NKSCC was strongly associated with high-risk HPV genotypes and p16h saliva . Both agh saliva . Chemicah saliva  may be eh saliva .


Innate immune activation was characterized in human keratinocyte cell lines constitutively infected or not with HPV. ADAR1 knockdown induced an innate immune response through enhanced expression of RIG-I-like receptors (RLR) signaling cascade, over-production of type-I IFNs and pro-inflammatory cytokines. ADAR1 knockdown enhanced expression of HPV proteins, a process dependent on innate immune function as no A-to-I editing could be identified in HPV transcripts. A genetic association study was performed in a cohort of HPV/HIV infected individuals followed for a median of 6 years (range 0.1–24). We identified the low frequency haplotype AACCAT significantly associated with recurrent HPV dysplasia, suggesting a role of ADAR1 in the outcome of HPV infection in HIV+ individuals. In summary, our results suggest that ADAR1-mediated innate immune activation may influence HPV disease outcome, therefore indicating that modification of innate immune effectors regulated by ADAR1 could be a therapeutic strategy against HPV infection.Infection by human papillomavirus (HPV) alters the microenvironment of keratinocytes as a mechanism to evade the immune system. A-to-I editing by ADAR1 has been reported to regulate innate immunity in response to viral infections. Here, we evaluated the role of ADAR1 in HPV infection  In immunosuppressed individuals there is a higher rate of cervical and anal HPV infections2. HPV infection favors HIV acquisition, and HIV-infected individuals encompass a heavier burden of HPV-induced dysplasia and cancer due to progressive immune suppression and impaired cell-mediated immunity. Persistent HPV infections are strong determinants of anal high-grade squamous intraepithelial lesions (HSIL)3.HPV is a small non-enveloped double-stranded DNA virus that infects keratinocytes. Most HPV infections clear spontaneously, however, persistent HPV infection is strongly associated with risk of several cancers including cervical, laryngeal, esophageal and anal cancer4. Pattern recognition receptors (PRRs) include the RNA-binding helicase family of RIG-I-like receptors (RLR: RIG-I and MDA5), and the family of Toll-like receptors (TLR). In response to viral infections, produced type I interferons (IFN) activate the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway, which, in turn, induces the expression of additional interferon signaling genes (ISG) with direct antiviral properties5. HPV has developed mechanisms to evade the host surveillance by suppressing inflammatory immune pathways, by downregulating IFN response or through decreased expression of PRRs6.Innate immune responses are characterized by production of a complex network of cytokines that effectively activate cellular immune cells7. ADAR1 has been found to regulate innate immune responses and the IFN-signaling pathway10. Loss of ADAR1 function causes a severe autoimmune disease, as A-to-I editing is an intracellular mechanism that distinguishes cellular versus pathogen-derived nucleic acids11.ADAR1 is an adenosine deaminase that converts adenosines to inosines (A-to-I editing) in double stranded RNA (dsRNA). Inosine is recognized as guanosine by splicing and translational machineries, therefore A-to-I editing may lead to changes in coding and amino acid sequences12, including chronic myeloid leukemia13, hepatocellular carcinoma14, esophageal squamous cell carcinoma15 and cervical cancer progression16. A-to-I editing of dsRNA has been linked to the replication process of different viruses, being ADAR1 function associated to enhanced or reduced viral growth or persistence17. ADAR1 is a proviral factor In HIV-1 infection, i.e. ADAR1 expression is linked to enhanced viral replication19. Conversely, ADAR1 appears to prevent HCV replication22 and has been associated to HCV-associated liver fibrosis23. The role of ADAR1 on HPV infection and oncogenicity has not been explored.Importantly, there is strong evidence indicating that both catalytic and non-enzymatic functions of ADAR1 may participate in a number of malignant diseasesADAR1 haplotype was associated to recurrent relapse in HPV-associated dysplasia in a cohort of HPV/HIV co-infected subjects.Here, we have investigated the role of ADAR1 as a regulator of innate and antiviral immune function in HPV-infected cells and in a cohort of HPV/HIV coinfected individuals. We show that depletion of ADAR1 in keratinocytes alters RLR, IFN and ISG expression and leads to increased HPV expression in cell culture. Moreover, genetic association studies demonstrate that an Protein expression of innate immune effectors was first characterized in two keratinocyte cell lines SiHa HPV16+ and HaCaT HPV- ; IFNA7 (p = 0.0365); IFNB1 (p = 0.0006); IL12A (p = 0.0005); IL12B (p = 0.0252); IL15 (p = 0.0292); IL5 (p = 0.0011); IL6 (p = 0.0015); IL8 (p = 0.0025); LTA (p = 0.0004)) compared to siRNA-NT  were genotyped and tested for association to recurrence of HPV associated dysplasia (relapse in HPV associated dysplasia). Selected demographic and clinical information are summarized in Table To study the effect of ADAR1 in the development of HPV infection, a genetic association study was designed using a cohort of HPV/HIV coinfected individuals (n = 173). Six ADAR1 polymorphisms covering all known genetic variation in sim = 0.0064)  between groups of recurrent and non-recurrent HPV-associated dysplasia, indicating that ADAR1 was an independent factor from higher prevalence of oncogenic HPV types.To further explore whether ADAR1 may influence recurrence in HPV-induced dysplasia, estimation of haplotype frequencies were performed. Haplotype-based analysis represent a valuable resource for investigating the genetic basis of disease susceptibility, albeit the identification of the functional variant has proven challenging. When we estimated ADAR1 haplotypes to HPV-dysplasia recurrence, we found the low frequency haplotype AACCAT was significantly associated with recurrent HPV dysplasia (2% and 8% of non-recurrent and recurrent HPV dysplasia respectively) , we noticed that the associated haplotype only differs in one SNP from the most frequent ADAR1 haplotype32. SNP rs3766927 correlates with the expression of ADAR1, being the allele in the identified haplotype associated negatively with ADAR1 expression . Further investigation into the role of this haplotype would improve the significance of our findings. On the other hand, ADAR1 overexpression has been linked to better prognosis in individuals with oral squamous cells carcinoma33 and cervical cancer progression and angiogenesis16, providing further support to our data and pointing towards the involvement of epitranscriptomic modifications as determinants of HPV-associated pathogenesis. Importantly, loss of ADAR1 in tumors overcomes resistance to immune checkpoint blockade34, suggesting a potential benefit of immune checkpoint inhibitors in HPV infection.Upon closer inspection of the haplotype analysis through the Genotype - Tissue Expression (GTEx) Project  . HaCaT cells (HPV16 negative) were obtained from Eucellbank . The hepatoma cell line Huh7 cells and its derivative Huh7.5 expressing RFP-NLS-IPS and a constitutive mitochondrial marker, mito-EGFP (was a king.pngt of Dr. Charles M. Rice (The Rockefeller University). Cells were cultured in Dulbecco’s modified Eagle’s medium , supplemented with 10% heat-inactivated fetal calf serum , antibiotics  and maintained at 37 °C in a 5% CO2 incubator.HPV + SiHa cells35. Briefly, total of 50 pmol of the corresponding siRNA or mix of siRNA (25 pmol for each siRNA in double knockdown experiments) or 2 μg of Poly(I:C) was transfected in 1.25 105 of the corresponding cell line. RNA and protein lysates were collected 64 h post-transfection for siRNA and 16 h post-transfection for Poly(I:C). siRNAs used for transfection were ON-TARGETplus Non-targeting siRNA Pool (D-001810-10), human ADAR1 siRNA-SMARTpool (L-008630-00), human IFIH1 siRNA-SMARTpool (L-013041-00), siGENOME human DDX58 siRNA-SMARTpool (M-012511-01) -and siGENOME human ADAR1 siRNA-SMARTpool (M-008630-01) all from Dharmacon, Waltham, USA.Cells were transfected using Lipofectamine 2000  in 24-well plate following manufacturer instructions for RNAi transfections as described previouslyADAR1 Hs00241666, TaqMan DDX58 Hs01061436, TaqMan IFIH1 Hs01070321, TaqMan CXCL10 Hs00171042, TaqMan IFNB1A Hs01077958, TaqMan IRF7 Hs01014809, TaqMan ISG15 Hs01921425, HPV16-E1 Pa03453396) and previously described36HPV16-E7 primers and probe .Total RNA was extracted using the NucleoSpin RNA II kit , as recommended by the manufacturer. Reverse transcription was performed using the PrimeScript™ RT-PCR Kit  following manufacturer instructions. mRNA levels of all genes were measured by two-step quantitative RT-PCR and normalized to GAPDH mRNA expression using the DDCt method. Primers and DNA probes were TaqMan Gene expression assays from Life Technologies . mRNA relative levels of cytokine genes were measured by two-step quantitative RT-PCR and normalized to GAPDH mRNA expression by using the DDCt method.37. The following antibodies were used for immunoblotting: anti-rabbit and anti-mouse horseradish peroxidase-conjugated secondary antibodies ; anti-human Hsp90 , anti-ADAR1 ; anti-MDA5 ; anti-RIG-I ; anti-phosphoSTAT1 ; anti-phosphorylated IRF7 ; anti-GAPDH ; and anti-HPV16 E7 .Protein expression was evaluated by Western blot as described beforeNEIL1 transcripts were amplified as controls10, using the following primers: N1-TCOF 5′-TCCAGACCTGCTGGAGCTAT-3′, and N1-TCOR 5′-GGCCTTGGATTTCTTTTTG-3′. PCR product were then amplified using N1-TCIF 5′-CCCAAGGAAGTGGTCCAGTTGG-3′, and N1-TCIR 5′-CTGGAACCAGATGGTACGGCC-3′ for a nested amplification. PCR conditions were as follows 1 μL cDNA with thermocycler conditions of 98 °C for 30 s hot start, followed by 40 cycles of 98 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 7 min. The nested PCR conditions with 1 μL of input PCR products were 98 °C for 30 s hot start, followed by 30 cycles of 95 °C for 10 s, 55 °C for 30 s, 72 °C for 30 s, and 72 °C for 7 min final extension, and were run on a 2% agarose gel to confirm band size of approximately 150 bp.Total RNA was extracted and reversed transcribed as described above. Previously identified ADAR1 predicted modification sites in bioinfo.ut.ee/primer3-0.4.0/). Primer pairs used for sequencing HPV transcripts expressed in SiHa cells were as follows: HPV16 E6 5′-TTCATGTATAAAACTAAGGGCGTAA-3′, 5′-CAGCTGGGTTTCTCTACGTGT-3′ (546 bp); HPV16 E7 5′-CATGGAGATACACCTACATTGC-3′, 5′-CTGAGAACAGATGGGGCACA-3′ (285 bp); HPV16 E1_a 5′-TGATCCTGCAGGTACCAATG-3′, 5′-TGCTGCCTTTGCATTACTAGTTT-3′ (613 bp); HPV16 E1_b 5′-GGGGAGAGGGTGTTAGTGAA-3′, 5′-CACATTGTTGCACAATCCTT-3′ (700 bp); HPV16 E2_a 5′-AGATGATGGAGGTGATTGGA-3′, 5′-CACATTTAAACGTTGGCAAAGA-3′ (623 bp); HPV16 E2_b 5′-CTCAAGGACGTGGTCCAGAT-3′, 5′-AGTAGACATACTGGGTTATCTGAGG-3′ (590 bp). PCR conditions for HPV16 transcripts were 95 °C for 5 min hot start, followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, and 72 °C for 5 min final extension. PCR products were run on a 2% agarose gel and different transcript bands were purified using QIAquick PCR purification kit (QIAGEN).HPV primers were designed based on HPV16 genome sequence (GenBank accession number AF001599.1) using Primer3 software  as described by the manufacturer. Samples were sequenced in a ABI3730XL system (Applied Biosystems).NEIL1 transcripts was estimated calculating the ratio of edited peak height  for each sequence, and calculate the mean for the specific site for each independent experiment. Mean ratio of at least three independent experiments of siRNA-ADAR1 was then relativized to the mean ratio of at least three independent experiments of siRNA-NT.Relative ADAR1 editing efficiency in 39. Clinical characteristics of patients are shown in Table 173 HIV+ individuals were included in the study. This cohort is a single-center, prospective cohort of HIV-infected subjects annually assessed for HPV infection at anal, genital, and oral sites attending the Outpatient HIV Clinic of the Hospital Germans Trias i Pujol , as described elsewhereThe study was approved by the Ethics committee of Hospital Universitari Germans Trias i Pujol. Written informed consent was obtained from patients participating in the study. All methods were carried out in accordance with relevant guidelines and regulations and to the ethical principles suggested in the Declaration of Helsinki.40.Individuals in the study were those suspected for anal intraepitlelial dysplasia  or cervical high-grade squamous intraepithelial lesions  following a cytology. If lesions were visualized, during high resolution anoscopy (HRA) or cervical colposcopy, a directed biopsy was performed for histological analysis. All patients with biopsy-proven of anal intraepithelial neoplasia-2 or -3 were treated with infrared ablation or surgery. A surgical resection (conization) was proposed in case of HSIL diagnosed confirmed by histology (CIN2 or CIN3). Cytologic changes were classified according to the Bethesda System: normal (no cell changes), atypical squamous cells of uncertain significance (ASCUS) or low- or high-grade squamous intraepithelial lesions . Overall recurrent dysplasia was defined as detection of at least two biopsy proven AIN 2-3 or CIN 2-3 at any time point during follow-up at the treated location or at new site41. For HPV detection, the same samples were used to determine HPV genotype using the AnyplexTM II HPV28 real-time PCR  as described before41. HPV genotypes were classified as High  and low  oncogenic risk as described41.Anal samples were obtained introducing a cytobrush  3 cm into the anal canal and gently rotating it for 30 to 45 seconds. The cytobrush was introduced into and shaken in a 20 mL of PreservCyt/Thin solution  for 30 seconds and assessed by the PAP method. Cervical citology samples were obtained from the exocervix with an Avre spatula and from the endocervix with a cotton swab and was assessed by the Pap method38 using the QuickExtract™ DNA Extraction Solution 1.0 . Extracted DNA was used for each genotyping. Six ADAR1 SNPs  were selected based on linkage disequilibrium testing and cover all described variation in ADAR1 gene42. The variants were typed using TaqMan SNP genotyping assay  following manufacturer’s protocol. Reactions were analyzed on an ABI PRISM 7500 (Applied Biosystems) and allele calling was performed by AutoCaller Software v 1.1 (Applied Biosystems).Genomic DNA was extracted from anus or cervical samplesGenotyping data, minor allele frequency and Hardy-Weinberg equilibrium of selected SNPs are found in Supplementary Table Experimental data are presented as mean ± SD. Paired Student’s t test was used for comparison between two groups, using the GraphPad Prism software. P-values lower than 0.05 were considered significant.43 for recurrent HPV dysplasia adjusted by age and sex, number of months since the first proctologist evaluation, CD4 nadir, number and type of HPV genotypes. We consider four inheritance models , and the akaike information criterion (AIC) was considered for estimation of the relative value of statistical models. The level of significance was set up equal to 0.0083 (0.05/6 = 0.0083) based on Bonferroni correction for multiple comparisons. These analyses were carried out using the SNPassoc R library43. Haplotype estimation and association analysis for recurrent HPV dysplasia was performed using the haplo.stats R package. P-values were corrected by permutation after 10,000 permutations using the score test implemented in haplo.stats44. No formal sample size was calculated. The sample was defined as all HIV- infected with biopsy proven AIN2 or AIN-3, CIN2 or CIN3 confirmed by HRA or colposcopy.Genetic association analysis was computed using a logistic regression modelSupplementary information