INK4A and p53 expression between treatment naive and recurrent anal cancer patients. HPV was genotyped via AmpliSens® HPV HCR-genotype-titre-FRT kit. HPV16 VL was determined via quantitative polymerase chain reaction-based in-house test. p16INK4A and p53 expression was tested via immunohistochemistry. The cohort comprised 13 treatment-naive and 17 recurrent anal SCC patients. High-risk HPV was detected in 87% of cases, and HPV16 (73%) was the predominant genotype. The rate of HPV positivity was higher among women and nonsmokers, and majority of HPV-positive cases were also p16INK4A-positive. All p53-negative tumors were HPV16-positive. The most predominant p53 staining pattern in the HPV-positive group was scattered type, whereas it was diffuse type in the HPV-negative group. The HPV16 VL was higher in the treatment-naive group. Further, in the treatment-naive group, cases with scattered staining pattern of p53 had higher HPV16 VL than cases with diffuse staining pattern. The opposite result was noted in the recurrent cancer group. Moreover, p16-positive cases with scattered p53 staining pattern in the treatment naive group had higher HPV16 VL than their counterparts in the recurrent cancer group. In conclusion, the HPV VL, as is the association between VL and p16INK4A /p53, is in an inversed trend in treatment naive and recurrent cancer patients, highlighting the importance of HPV VL measurement in anal SCC.Human papilloma virus (HPV) is highly frequent among patients with anal squamous cell carcinoma, but the viral load (VL) differs between patients. This study aimed to compare the rate of HPV positivity, HPV16VL, p16 Anal squamous cell carcinoma (ASCC) is rare, accounting for only 0.5% of new cancer cases and a lifetime risk of only 0.2%. However, its incidence during the last 10 years has increased by 2.2% annually [CDKN2A and TP53 gene mutations. Further, PIK3CA alterations, although more frequent in HPV-positive cases, are also highly common in the HPV-negative cohort.It is already known that HPV-positive and HPV-negative cancers are separate entities with respect to their molecular landscape and clinical behavior. HPV-positive tumors better respond to radiotherapy or chemoradiotherapy than their HPV-negative counterparts . These dDespite the high complete response rate 80%) in anal cancer patients, some remain resistant to chemoradiotherapy. To establish the molecular alterations behind these differences, a comparative genomic analysis of treatment-naive and recurrent tumor was performed . Unexpec0% in anaINK4A based on the finding that only a combination of HPV/p16INK4A-positivity corresponds to transcriptionally active HPV [This study aimed to investigate whether the HPV16 viral load (HPV16 VL) can discriminate between treatment-naive and recurrent tumors. Additionally, we aimed to analyze the correlations between HPV VL and the expression of viral E6/E7 proteins, as well as cellular p53, p16tive HPV . To the Because studies on HPV16 VL do not have an established cut-off value, their results cannot be directly compared . Thus, aThe study was performed using formalin-fixed, paraffin-embedded (FFPE) tissue samples obtained during diagnostic or therapeutic procedures from patients with ASCC. FFPE blocks were obtained from the archives of the Pathology Department of Maria Sklodowska-Curie National Research Institute of Oncology, Gliwice Branch. Patients were treated according to diagnosis and current disease status between 2006 and 2016. Data on age, sex, and tumor/nodal classification were collected from medical records. Smoking status was self-reported during admission.x, Gdańsk, Poland). Depending on the tumor area, 3–10 consecutive tissue slices (5 μm) were used for DNA isolation. Macrodissection was performed, if necessary, to enrich tumor samples with cancer cells. For the quantitative polymerase chain reaction (PCR), 10 μL eluate containing 50 ng DNA (measured via NanoDrop) was used. HPV was genotyped using AmpliSens® HPV HCR-genotype-titre-FRT kit , which enables detection and quantification of 14 high-risk HPV (hrHPV) strains . Positivity was defined as HPV Ct values below 30. To quantify HPV DNA viral load (copies/genome) in tissue samples we used real time PCR based on TaqMan technology. Amplification of TERT (human telomerase reverse transcriptase) was used as a marker of the total amount of genomic DNA presented in samples. The oligonucleotides (probe and primers) for HPV and TERT gene have been synthesized by Genomed S.A . Each measurement consisted 2 standard curves (genomic DNA and plasmid construct with HPV genome), negative control and a sample. All PCR reactions were performed using the Bio-Rad CFX96 qPCR instrument . Copies of TERT were converted to genome and viral load of HPV was expressed as the number of copies per genome.HPV DNA was isolated from FFPE tumor samples using the commercially available GeneMATRIX Tissue DNA Purification Kit  was employed prior to antibody incubation. ImmPRESS™ Universal Antibody, Polymer Detection Kit; Peroxidase  with DAB as a chromogen was used for antibody detection. The following antibodies were employed: mouse monoclonal HPV16/HPV18 E6 Antibody (C1P5) ; mouse monoclonal HPV Type 16 E7 Antibody (8C9) ; monoclonal mouse anti-human p53 protein (DO-7) ; and CINtec® p16 Histology . Antibodies used for viral E6 and E7 immunohistochemical staining showed weak or very weak signal. Despite testing various conditions of immunodetection, high background staining was constantly observed. This precluded a reliable evaluation of immunoreaction, and therefore the results of E6 and E7 staining were excluded from further analysis. Positive p16INK4A expression was defined as strong, diffuse nuclear, and cytoplasmic staining in ≥70% of cancer cells. This cut-off is consistent with previously published criteria developed by Singhi and Westra [INK4A negativity was defined as absence of or faintly diffuse immunoreaction. p53 immunoreaction was assessed according to the staining pattern and number of positive cancer cells as follows: negative, 0% of cells are positive; scattered, less than 60% of cells are positive; and diffuse, more than 60% of cells are positive. p53 staining patterns were assessed according to the method proposed by Ando et al. [The expression of viral E6 and E7, and p53 and p16d Westra . By conto et al. . Brieflyo et al. .10 transformed to achieve normal distribution. Two cases with coinfection of HPV16 and other hrHPV genotypes were excluded from the analysis. Continuous variables were analyzed via nonparametric Mann–Whitney U test. All statistical analyses were performed using Statistica software ver. 13.1 , and p < 0.050 was considered significant.Chi-square test was used to evaluate the association between categorical variables. HPV16 VL was logThe cohort comprised 30 patients; of these, 13 were treatment-naive and 17 had recurrent ASCC. All patients in our cohort did not have distant metastases (M0), while 12 patients had lymph node metastases . hrHPV was detected in 26 cases (87%). HPV16 was found in 22 cases (73%), while other HPV subtypes  were detected in 7 cases. Coinfection of HPV16 and other hrHPV subtypes was found in 2 cases. The incidence of hrHPV-positive cases was similar between the treatment-naive group and the recurrent cancer group. Three of the four hrHPV-negative cases belonged to the recurrent cancer group. The clinicodemographic data of the treatment naive and recurrent ASCC groups are presented in Table 10 transformed HPV16 VL ranged from 0.628 to 3.77 with median 2.12. In 2 cases co-infection of HPV16 (VL = 2.13 and 2.67) with HPV45 (one case) and HPV52 (one case) was found, both in trace amounts. In 3 HPV16-negative cases, the log10 transformed HPV31 VL, HPV33 VL and HPV39 VL were 1.71, 1.73 and 1.89, respectively. In 2 other HPV16-negative cases, trace amounts of HPV33 DNA and HPV45 DNA were found. Comparison between HPV16 and other types did not show statistically significant differences in VL levels (p = 0.819). However, it was observed that VL values of the other types were within the lower quartile of HPV16 VL.The logHPV16 VL was higher in the treatment-naive group than that in the recurrent cancer group, but the difference was not statistically significant. Associations between clinical and molecular data with respect to HPV16 positivity/VL in the two groups are shown in Tables p = 0.09). The correlation between smoking and HPV16 was similar in both groups. However, a comparison of HPV16 VL in these groups showed marked differences. In the treatment-naive group, nonsmokers had significantly higher HPV16 VL than smokers (p = 0.03). Meanwhile, the opposite finding was found in the recurrent cancer group, that is, smokers had higher HPV16 VL than nonsmokers.Analysis according to smoking status showed that the rate of HPV16-positivity was higher in nonsmokers than that in smokers . The rate of lymph node involvement was also higher in HPV-positive, advanced tumors than their HPV-negative counterparts (p = 0.08). Meanwhile, the correlation between HPV positivity and lymph node status was not significantly different between advanced-stage and early-stage tumors. Advanced tumors (T3/T4) also had higher HPV16 VL than T1/T2 tumors, but the difference was not statistically significant.Advanced-stage tumors (T3/T4) were more frequently HPV positive than early stage tumors (T1/T2) (p = 0.05). With respect to the correlation between HPV16 VL and p53 pattern, cases with scattered staining pattern in the treatment-naive group had higher HPV16 VL than cases with diffuse staining. By contrast, cases with diffuse staining pattern had higher HPV16 VL than those with scattered staining pattern in the recurrent cancer group . There was no correlation between nodal status and p53 expression in both groups.INK4A positive. Women were more frequently p16 positive than men in both groups, but the difference did not reach statistical significance (p = 0.08). However, although p16INK4A was significantly correlated with HPV16 (p = 0.007) .p16-positive cases had higher HPV16 VL than p16-negative cases, without statistical significance. Further, p16-positive cases with above median HPV16 VL were predominant in the treatment-naive group, whereas p16-positive cases with below median HPV16 VL were predominant in the recurrent cancer group (p = 0.05). This trend was observed in both groups. p16-positive cases with scattered p53 staining pattern in the treatment-naive group had significantly higher HPV16 VL than their counterparts in the recurrent cancer group (p = 0.07).In total, 19 cases were p16+/HPV+ Table . ScatterStudies have shown the importance of HPV VL testing for stratifying HPV-positive patients. High VL was found to be a favorable prognostic factor in various HPV-related cancers, including cervical cancer , anal caIn our study, we directly compared HPV16 VL in treatment-naive and recurrent ASCC. We found that HPV16 VL was higher in treatment-naive than in recurrent cancer, but the difference was not statistically significant. As we were not able to find any study comparing HPV16 VL between treatment naive and recurrent anal cancer, we referred to a study on primary and recurrent HPV-dependent vulvar cancer . There wOther well established factors influencing HPV VL level are sex and smoking. The association between sex hormones and HPV is best documented in cervical diseases. Epidemiological studies have shown that oral contraceptives and multiple pregnancies are risk factors for cervical cancer . FurtherAnother factor associated with HPV VL is smoking. Smoking among HPV-positive women has been shown to be among the precipitating factors increasing the risk of cervical cancer. The chemical components of tobacco and its metabolites have been found in the cervical mucus of active and passive smokers . MoreoveIn anal cancer no association between HPV16 VL and smoking was reported . By contTP53 mutational status was not analyzed. However, in the current study, scattered staining pattern was predominant in the HPV-positive cohort, while it was diffuse staining pattern in the HPV-negative cohort. This observation indirectly confirms the findings reported by Meulendijks et al. [Of interest is the relationship between p53 staining pattern and HPV. In this study, we analyzed p53 staining pattern following the classification described by Ando et al. , who reps et al.  that theINK4A/p53, further characterization of samples not only with respect to HPV positivity, but also to VL will provide novel information on the mechanisms of cancer recurrence.Considering that HPV16 VL is a valuable prognostic marker in HPV-dependent SCC cancers, its measurement should be standardized. Moreover, given that treatment naive and recurrent cancer showed opposite/inversed characteristics, including the association between viral load and p16


Cervical cancer (CC) remains a leading cause of gynaecological cancer-related mortality with infection by human papilloma virus (HPV) being the most important risk factor. We analysed the association between different viral integration signatures, clinical parameters and outcome in pre-treated CCs.Different integration signatures were identified using HPV double capture followed by next-generation sequencing (NGS) in 272 CC patients from the BioRAIDs study [NCT02428842]. Correlations between HPV integration signatures and clinical, biological and molecular features were assessed.p < 0.0001). We identified >300 different HPV-chromosomal junctions (inter- or intra-genic). The most frequent integration site in CC was in MACROD2 gene followed by MIPOL1/TTC6 and TP63. HPV integration signatures were not associated with histological subtype, FIGO staging, treatment or PFS. HPVs were more frequently episomal in PIK3CA mutated tumours (p = 0.023). Viral integration type was dependent on HPV genotype (p < 0.0001); HPV18 and HPV45 being always integrated. High HPV copy number was associated with longer PFS (p = 0.011).Episomal HPV was much less frequent in CC as compared to anal carcinoma (MACROD2; involved in impaired PARP1 activity and chromosome instability.This is to our knowledge the first study assessing the prognostic value of HPV integration in a prospectively annotated CC cohort, which detects a hotspot of HPV integration at  Both patterns may be present jointly .5 It is thought that the longer half-life of integrated viral transcripts compared to half-life of episomal transcripts favours cellular immortalisation and transformation into cancer cells while also providing a selective growth advantage.6 Most often, the integration of HPV DNA leads to a breakpoint in the E2 gene, resulting in de-repression of the E6 and E7 viral oncogenes. When the virus remains episomal, expression of E6 and E7 proteins may result from leaky expression or epigenetics dysregulation. E6 and E7 proteins impact the function of p53 and pRb proteins, allowing squamous cell tumorigenesis.6Cervical cancer (CC) remains a leading cause of gynaecological cancer-related mortality worldwide and constitutes the second most common malignancy in women.7 is the most widely accepted but not experimentally reconstituted. HPV DNA integration into the human genome triggers various genetic alterations, such as oncogenes amplification, tumour suppressor gene inactivation, inter- or intra- chromosomal rearrangements as well as genetic instability.8 Genes localised near the integration sites of viral genomes can experience changes in RNA and protein expression levels, leading to over- or under-expression. In 2015, whole-genome sequencing and high-throughput viral integration methods identified as many as 3667 HPV integration breakpoints in cervical neoplastic lesions. Frequent integration sites have been reported in genes relevant to the neoplastic process, such as the MYC oncogene.9 Loss of function (LOF) in the RAD51B tumour suppressor gene following HPV DNA insertion was reported to affect the DNA repair pathway and genomic instability in CC.10Several mechanisms of integration have been reported in the literature; the “looping” model of HPV integration following DNA replication and recombination (resulting in DNA concatemers)11 In 2016, Holmes et al. developed a Capture HPV method to identify five different HPV signatures in 72 CC. The first two signatures contain two hybrid chromosomal–HPV junctions which are co-linear (2 Junctions Colinear “2J-COL”) or non-linear (2 Junctions Non-Linear “2J-NL”) depending on their relative orientations. It reflects two modes of viral integration, associated with chromosomal deletion or amplification events, respectively. The third and fourth signatures exhibit several hybrid junctions either clustered in one chromosomal region (Multiple Junctions Clustered “MJ-CL”) or scattered at distinct loci (Multiple Junctions Scattered “MJ-SC”) while the fifth signature consists of episomal forms of HPV (EPI).12HPV DNA integration occurs as a single copy or in multiple repeats (in tandem or dispersed).On the assumption that HPV integration types/signatures/pattern might predict clinical outcomes, we analysed the association between the different viral integration signatures, clinical and pathological parameters and outcome in the large cohort of 272 HPV-positive CC patients enrolled in the prospective BioRAIDs study [NCT02428842].www.raids-fp7.eu) prospective CC BioRAIDs study [NCT02428842]. The clinical protocol together with tumour sampling procedures, quality control of samples and treatment in 18 European centres (seven European countries) as well as study results have been previously published.15Patients included in this study were enrolled in the EU-funded RAIDs Network  according to the manufacturers’ protocol. For DNA isolation, one to five 10 μm tissue sections were cut depending on the size of the tumour biopsy. DNA was isolated using the automated Tissue Preparation System .PIK3CA gene had been previously carried out on all tumour samples.15 In summary, paired-end whole-exome sequencing was performed on a HiSeq2500 platform, with an Agilent SureSelectXT Human. The sequencing was performed to reach an average depth of coverage of at least 80× per sample. Dedicated filtering strategies were applied to somatic variants depending on their functional impact per gene category: oncogene or tumour suppressor gene or uncharacterised. For oncogenes as PIK3CA, hotspot missense mutations known in the COSMIC database were considered. Among the 87 PIK3CA mutations, three patients had an H1047R mutation (exon 20) and 84 patients had a E452K/E545K mutation (exon 9).A mutational analysis of the The DNA libraries were prepared using 500 ng of genomic DNA (extracted from frozen tissue), starting with ultra-sonication (Covaris) to produce double-strand DNA fragments of approximately 280 bp. End-Repair and A-tailing were applied to facilitate ligation of the adapters, containing unique barcodes for each sample, specific to the Illumina technology for amplification and sequencing. KAPA Hyper Prep kit was used, according to the manufacturer’s instructions.The double capture method was carried out using the SeqCap EZ Rapid Library Small Target Capture method, developed by Roche, which is adapted to capture small DNA targets. The DNA libraries were multiplexed (by 12) and hybridised for 16 h with the biotinylated HPV oligonucleotide probes, recognising all HPV genotypes. The DNA sequences were then captured by streptavidin beads and amplified by PCR. We performed a double capture (i.e. two rounds of hybridisation and capture) to improve the efficiency and specificity. Post-capture libraries were sequenced using Illumina MiSeq system , in paired-end 150, with 24 samples multiplexed on a V2 micro flow-cell.KLK3) in order to normalise the starting amount and quality of genomic DNA. Similar results were obtained with other endogenous diploid controls .The HPV copy number shows the abundance of the target relative to the endogenous control  quality controls and cleaning of raw sequencing Illumina data, (ii) HPV genotyping, and (iii) the detection of the HPV insertion sites within the human genome. Nf-VIF is implemented through the Nextflow workflow management system, ensuring a high portability, reproducibility, and scalability  was calculated as a single measure to discriminate efficacy. Survival curves were estimated by the Kaplan−Meier method, and compared using the log-rank test. For all statistical tests, significance level was defined as p < 0.05.The correlations between HPV integration signatures and clinical, biological and molecular features were analysed using chi-square tests, chi-square tests with Yates’ correction or Fisher’s exact tests, as appropriate. Progression-free survival (PFS) was defined as the time interval from the date of CC diagnosis to progression. Survival data were censored on the date of last follow-up. To visualise the efficacy of a molecular marker  to discriminate two populations (patients who progressed) in the absence of an arbitrary cut-off value, data were summarised in an ROC curve.PIK3CA mutational status) and outcome of the 272 HPV-positive CC patients from the BioRAIDs European study are presented in Table Clinical, histological, biological  followed by HPV18  and HPV45  , no HPV-chromosomal breakpoint was observed and the viral genome persisted in an episomal form (EPI). Five HPV integration patterns were observed: 2J-COL , 2J-NL , 2J , MJ-CL , MJ-SC . The BioRAIDs CC series differed significantly from that of HPV-positive anal carcinoma (p < 0.0001)  were more often represented in CC. The results were similar in HPV16-positive cancers that represent the majority of the subtypes in both cervical and anal cancers  , TP63 (n = 5), and several others such as ERBB2 (two sites); KLF12, and RAD51B with a single site  , HPV integration signatures were not associated with histological subtype, with FIGO stage/lymph nodes (presently FIGO stage 3), or treatment assignation but they were associated with HPV genotype status (p < 0.0001). HPV18 and HPV45 genotypes were always integrated (most frequently as 2J). Multiple (MJ) viral integration signatures were predominant in HPV16-positive samples  as compared to other HPV genotypes  (Table p < 0.0001).The distribution of HPV integration signatures according to clinical, biological and pathological characteristics is presented in Table s p = 0.03, HPV inThere was no significant correlation between the HPV integration signatures  and the PFS   did not have a significantly poorer outcome but the numbers are insufficient to draw any conclusions .The most frequent integration site was in the KLK3. The optimal cut-off was four (as determined in the “Methods” section). Patients were classified into low  vs. high HPV copy number . HPV16-positive patients consistently had a higher HPV copy number  (p < 0.0001) as compared to patients with other HPV subtypes  (Table p < 0.0001). Furthermore, patients with a low HPV copy number showed poor outcome in comparison to patients with a high HPV copy number (p = 0.011) , followed by MIPOL1/TTC6 (n = 5) and TP63 (n = 5). Interestingly, our data identified a new CC-related recurrent integration site in the MACROD2 (mono-ADP-ribosylhydrolase) gene. Non-coding and structural mutations/variations in the germline MACROD2 gene have been associated with psychiatric disorders, obesity and cancer predisposition.20 Deletions in the MACROD2 gene are frequent in colorectal cancer22 and are reported to alter DNA repair and sensitivity to DNA damage and consequently impact colorectal tumorigenesis.23 Neither RNA expression nor functional studies support a tumour suppressor role of MACROD2 gene. This gene spans more than 2 Mb and constitutes a common fragile site contributing to increased genomic instability.25 Our results report intronic integration sites in the MACROD2 gene yet there is still lack of evidence concerning the functional consequence of these intronic integrations within MACROD2. Functional analyses are not straightforward due to the high rate of splicing in MACROD2 and the important number of alternative transcripts (coding and non-coding) of variable size. MACROD2 deletions and haploinsufficiency were linked to impaired PARP1 activity and chromosomal instability in colorectal cancer26 and in liver cancer,27 suggesting a tumour suppressing function of this gene. Importantly, the present study identifies HPV integration as a new molecular pattern of MACROD2 alteration likely causing loss of function, but the seven patients in our cohort with HPV integration in the MACROD2 gene are presently insufficient to discern a meaningful impact on CC evolution, albeit responsible for genomic instability.In this CC patient population from the prospective BioRAIDs study, we were able to identify >300 HPV-chromosomal (inter-genic or intra-genic) junctions; the MYC, TMEM49, FANCC and RAD51B genes30 as well as in the following: POU5F1B, FHIT, KLF12, KLF5, HMGA2, LRP1B, LEPREL1, DLG2 and SEMA3D. Slightly less common integration sites were reported in the following genes: AGTR2, DMD, CDH7, DCC, HS3ST4, CPNE8, C9orf85, MSX2 and CADM2.9 Several of these previously reported integration sites into genes such as FHIT, KLF12, RAD51B were detected in a single or in two patients of the present CC cohort. HPV integration in MIPOL1/TTC6 and TP63 genes were reported in five patients each. Concordant with our results, Parfenov et al. reported in a head and neck squamous cell carcinomas a rearrangement between chromosomes 3 and 13 close to the HPV integration site in a non-coding region but involved in a region of chromosome 3 where TP63 genes are located.31 P63 plays a key role in epidermal keratinocyte proliferation and differentiation and is a master regulator of gene expression pattern and epigenetic landscape that define epidermal fate.32TP63-driven enhancer reprogramming promotes aggressive tumour phenotypes in primary pancreatic ductal adenocarcinomas.33 HPV integration in TP63 genes was recently reported in HPV-positive vulvar cancer patients.34 In another HPV-positive head and neck squamous cell carcinoma study, HPV sites of integration into MIPOL1/TTC6 were identified in more than one tumour sample. The integration of HPV into the ERBB2 gene site was observed in two patients in association with ERBB2 amplifications, in concordance with previous reports.10Previously, frequent integrations in other SCCs were reported in the 17Twelve percent of CC patients did not display any HPV integration, while 43% had double junctions and 49% multiple junctions’ signatures. The distribution of HPV signatures in our CC cohort differed from that previously described in HPV-positive anal squamous cell carcinoma with a lower rate of episomal HPV as compared to anal cancer (45%).PIK3CA than those harbouring episomal HPV, confirming previously reported data.12 Similar results were also observed when considering only HPV16 patients (data not shown). This is in accordance with the literature where HPV integration is reported to provide a selective growth advantage of cancer cells.6 CC patients with a high HPV copy number had significantly better PFS, as compared to patients with low HPV copy number. These results are consistent with other reports in the literature.36No significant association was observed between HPV integration signatures and treatment type, histological subtype or FIGO staging. MJ viral integration signatures were predominant in HPV16-positive samples and tumours with viral integration (2J or MJ) had less frequent activating mutations in PIK3CA were significantly associated with high HPV copy number and with the episomal form of HPV. The analysis of outcome based on PIK3CA alone did not show an association with poor outcome. In a prior analysis of the BioRAIDS dataset, the association of PIK3CA with epigenetic alterations was associated with a shorter PFS.15In conclusion, while HPV integration is thought to be a random event, our results point out that some hotspots may impact cancer evolution. This would need analyses in larger aggregated datasets. The episomal form of HPV was less frequent in cervical carcinoma as compared to another genital carcinoma  and its presence was significantly associated with high HPV copy number, suggesting a decrease of viral replication upon integration. Mutations in MACROD2 gene, known to be implicated in impaired PARP1 activity and chromosome instability.To our knowledge, this is the first study assessing the prognostic value of HPV integration in a prospectively annotated patient cohort and reporting an HPV integration at the Supplementary file


Diagnosed in more than 90% of cervical cancers, the fourth deadliest cancer in women, human papillomavirus (HPV) is currently the most common pathogen responsible for female cancers. Moreover, HPV infection is associated with many other diseases, including cutaneous and anogenital warts, and genital and upper aerodigestive tract cancers. The incidence and prevalence of these pathologies vary considerably depending on factors including HPV genotype, regional conditions, the study population, and the anatomical site sampled. Recently, features of the cervicovaginal microbiota are found to be associated with the incidence of HPV-related diseases, presenting a novel approach to identify high-risk women through both blood and cervical samples. Overall, the HPV repartition data show that HPV infection and related diseases are more prevalent in developing countries. Moreover, the available  vaccines based on virus-like particles, despite their proven effectiveness and safety, present some limitations in terms of system development cost, transport cold chain, and oncogenic HPV variants. In addition, vaccination programs face some challenges, leading to a considerable burden of HPV infection and related diseases. Therefore, even though the new  vaccine seems promising, next-generation vaccines as well as awareness programs associated with HPV vaccination and budget reinforcements for immunization are needed. Papillomaviridae family, HPV is a small, double-stranded DNA virus classified into two categories: low-risk HPVs (LR-HPVs) responsible for anogenital and cutaneous warts, and high-risk HPVs (HR-HPVs) responsible for oropharyngeal  cancers and anogenital cancers, including cervical, anal, vulvar, vaginal, and penile cancers (Human papillomavirus (HPV) is the most common sexually transmissible infection (STI) in the world, with a high negative impact on individual social life. In their lifetime, sexually active women and men will be infected at least once , without cancers –6. Cervi cancers , is an H cancers . Overall cancers . The thrEpidemiological surveillance of HPV infection and related diseases represents a crucial topic for monitoring and evaluation of the three currently available antiviral prophylactic vaccines , as well as their acceptance all over the world. Moreover, knowledge of HPV infection characteristics and evolution at the molecular level allows for a better understanding of the actual distribution of the burden of HPV-related diseases and their impact. This would in turn inform new therapeutic strategies for the development of next-generation antiviral vaccines, to overcome the shortcomings of current prophylactic regimen, including high costs, limited antiviral protection spectrum, and immunization management. In this review, we summarize the current prevalence distribution of HPV infection and related diseases worldwide and discuss the involvement of factors, specifically the genetic and molecular characteristics of the antigenic HPV-L1 protein, and reveal microbial factors associated with the natural history of HPV. We also present the actual infection progression worldwide with an emphasis on its evaluation, specifically the limitations of current HPV vaccines, highlighting the improvements to be made to reduce the burden of this disease.HPV infection has reached a considerable proportion worldwide, particularly among women, in whom it is the primary cause of cancer , thus maAmong women with normal cervical cytology (NCC), earlier reports show 10.4%  and 11.7The intra-continent distribution of HPV prevalence fluctuates significantly, allowing worldwide regional repartition.Regarding the geographical world regions , the HPVIn men, the global prevalence rate of genital HPV infection is almost similar to that in women (3.5–45% vs. 2–44%) , 19; theTaken together, these data lead to the conclusion that the most blatant HPV incidence and prevalence are recorded in the low- and middle-income regions in both genders, healthy and unhealthy . In factDue to their competent immunity, and their sexual and financial stability, some women generally eliminate the infection very quickly, explaining the decrease in HPV prevalence after 25 years of age. The low rates of persistence, recrudescence, or reinfection, observed in older women, are generally caused by the multi-sexual partnership due to social and financial instability (poverty) and lack of education , 30.Second, within only 3 years (from 2007 to 2010), the overall prevalence of HPV infection in women with NCC increased by about 1% , with the same trend as those described in the different sub-continents mentioned earlier . These dIn men, the high prevalence of HPV, which is invariable with age, could be explained by their sexual activity preferentially with young girls under 25 who are highly infected , and by Cutaneous warts (CWs) are mainly caused by the genera β and γ (HPV4 and 65), and rarely by the genera α  and μ (HPV1) from low-risk oncogenic genital HPVs (LR-HPV), called cutaneous HPVs , 35. A rThe CW prevalence does not exceed 33% in healthy subjects and tends to be higher in males than in females , 40, 45.Mainly caused by the low prevalence <1%,  of HPV6 In children, the prevalence of AGW is low, almost zero. In contrast, several new studies revealed a net increase in the AGW prevalence in both adults and children, leading to the hypothesis of sexual abuse in children  because In the general adult population, the incidence  and prevalence (0.13–0.56%) of AGW are similar in women and men, with peaks observed between 20 and 29 years of age . This maRegionally, SSA appears to have slightly higher AGW incidence and prevalence rates , especiaEach year, around 569,847 new cases of cervical cancer (CC) are diagnosed . In 2018, it was responsible for 3.3% of deaths due to cancers by causing nearly 311,365 deaths, with more than 85% in developing countries . This deSince the 1980s, HPV infection (HR-HPV) has been associated with CC , 6. It iIn summary, aside from anal, vulvar, and oropharyngeal cancers, over 90% of anogenital cancers, especially CCs, are associated with HPV  and are Chlamydia tracomatis, and Propionibacterium acnes have significantly been identified in prostate cancer (PC) tissues. The viral load and HPV prevalence in the same tissues are also low , also low , 67. Howalso low , 68.Likewise, several studies debate the implication of HPV in the occurrence of breast cancer (BC) , 70. HowIn summary, the presence of HR-HPVs in an organ or tissue susceptible to a tumor (other than those with a direct link with HPV) implies that HR-HPVs would be involved in cell immortalization, and therefore in the progression to malignant states in the tissue or organ involved. Thus, the presence of HPV in mammary or prostatic tissues should not be considered a carcinogenesis etiological factor until proven otherwise but rather as a risk factor for aggravation of already established benign states toward metastatic states , 70, 72.Data based on HPV genotypic epidemiology and the presence of disease clusters detailed herein would reinforce the implication of HR-HPVs as an aggravating factor, but not as an etiological factor. The prevalence and incidence of PC and BC are higher in European and North American regions/populations. Yet, in developing countries, including Latin America and the Caribbean, and SSA, the death rate due to these cancers, as for CC, is the highest , 66, 73.Moreover, co-infections with other pathogens , 73 servTherefore, death rates related to such diseases can be reduced as HPV infection is a preventable STI.Although the HPV infection-associated diseases are the same according to viral types  around the world, there is nevertheless a variability of HPV genotypic prevalence by geographical region. Knowledge of the genotypic prevalence in a given continental region will facilitate policy-making for strengthening vaccination programs in these specific regions or, to a lesser extent, develop a new generation of broad-spectrum vaccines. Strengthening vaccination programs or choosing the vaccine under the influence of the most prevalent genotypes will help reduce the health and socioeconomic burdens related to HPV infection .Some HPV genotypes are present all over the world. Indeed, several studies have shown that the most common types are HR-HPVs, with genotypes 16, 18, 59, 45, 31, 33, 52, 58, 35, 39, 51, 56, and 53 mainly found in descending order of prevalence. Among the LR-HPVs, the most common are HPV 6 and 11 types, which are responsible for almost all GWs. However, their prevalence differs by region  7, 11, , 46, 75.Briefly, unlike the HPV global distribution described earlier, HPV 16 and 18, detected in 71% of ICC, are less prevalent in developing regions  12), wh, wh12), Genotypes 6 and 11 are the most common LR-HPVs in America and more or less in Europe, while in Asia and Africa, they are not very common. For instance, as shown in Finally, there is no positive correlation between the distribution of the regional prevalence of HPV genotype and the burden of the associated diseases. Rather, this HPV-related burden is associated with molecular and genetic characteristics of HPV, revealing a regional variability within HPV, which we describe hereafter.Papillomaviridae family. The 72 capsomeres covering the virus are repetitions of pentameric monomers composed of five identical L1 proteins, anchoring one L2 protein . On. OnPapil protein , and car protein .The ~55 kDa major capsid protein (L1), encoded by a 1.7-kb ORF  when they have a homology >60%; to the same genus and the same species  when they have a homology > 70%; to the same genus, the same species, and the same sub-type when they have a homology between 90 and 98%; and are different variants when they have <2% homology difference , 95.Further, based on comparative genomic criteria, the existence of a geographical genotypic variability within the HPV variants has been described, referred as “inter-population or intra-type variability or intratypic molecular variants.” Indeed, comparative studies of genetic sequences of LCRs and ORFs encoding E1, E2, E5, and/or E6 proteins and, more specifically, the L1 protein showed that in the same geographical region, variants from the same HPV strain have a very low amount of divergence , whereas this divergence between variants from two distinct regions is higher. The existence of these natural intratypic molecular variants had been demonstrated years ago from comparisons of the LCR sequences of HPV-16, proving to be specific or more widespread in certain parts of the world . Based oMost studies describing the HPV infection cycle were conducted on a few viral strains, including HPV 16, 18, and 31 . HoweverThe virus entry into undifferentiated epithelial cells depends on specific molecular interactions involving the viral antigens and host receptors. In fact, structural studies of L1 revealed the presence of four heparan-sulfate proteoglycan (HSPG)–specific binding sites, rich in lysine (K)  requiredHPV replication depends on epithelial cell differentiation. Indeed, by infecting undifferentiated basal cells, the virus guarantees its multiplication and persistence. Consistently, by dedifferentiating, the basal cells at the same time ensure viral protein synthesis sequentially, as presented in Therefore, the L1 protein is the most important in the infection process . Owing to the presence of multiple surface epitopes, it possesses the ability to boost immunity against HPV by producing a high amount of specific and effective antibodies that recognize HPV in the physiological medium. Although the modalities of HPV infection are the same in general, as seen previously, the pattern of gene expression remains different, with differences in virulence  due to vIn a normal cell cycle, basal epithelial cells divide asymmetrically to renew the basal layer on the one hand and the cutaneous or mucosal epithelium on the other hand. Thus, the number of divisions during cell differentiation remains limited to the formation of the apical epithelium. In contrast, HR-HPV infection leads to the maintenance of this cell division, which is the long-term origin of the HPV-related diseases presented previously. At the molecular level, it is the hyper-expression of the E6 and E7 proteins, which leads to the maintenance of cell division. Indeed, studies have shown that the overexpression of E6 and E7 oncoproteins occurs as a result of the viral genome integration into the host genome, causing a loss of expression of some viral genome parts, including E2, that negatively regulates p97 –114. In primo-infection.Clinically, after a primary infection, the manifestations of infection are not perceptible early; this makes acute HPV infection very poorly documented. The anti-HPV antibody kinetics have not yet been determined because the prodromes associated with HPV acute infection occur very late after infection; however, studies are underway . MoreoveAfter the establishment of infection, replication is maintained by the E6 and E7 proteins, and HPV L1 protein plays only a minor role limited to the repeated binding with the neighboring cells. The modification of the natural history in terms of the infection rate reduction would be achieved by reducing the infection spread over other cells or by focusing mainly on the naïve population (children), who are not or LR-HPV carriers.Chlamydia) infections have been associated with HPV infection progression. Furthermore, it has been revealed that a significant correlation exists between the vaginal and cervix microbiota and the natural history of HPV infection  and bacterial , along with Mobiluncus sp., Prevotella sp., Mycoplasma hominis, and Atopobium vaginae, marked the persistence of HR-HPV infections and precancerous lesions  and the number of colposcopies performed after a primary HPV screening  were greater than those achieved after a cervical cytology screening   , 138. In = 1934)  as the p = 1934) , 147. HoFurthermore, as stated in section Cervicovaginal Microbiome and Precancerous Lesion Occurrence RISKS, it is now known that the CVM is implicated in the natural history of HPV infection, and the monitoring of bacterial variation in the CVM is now a novel and effective measure in the follow-up diagnosis of HPV-infected women and could help to identify women at risk of developing cancer. However, the causality effect needs to be determined to know whether the disruption of CVM is caused by HPV infection or the presence of a disrupted CVM is a risk factor for HPV infection and related complications. This knowledge could help in the prevention of HPV infection and related diseases.Despite all these control strategies, particularly vaccination, there are still high rates of morbidity due to HPV and related diseases in certain regions of the world because of challenges encountered. The main challenges are two-fold: (1) immunization coverage is not complete, or the programs are not well established/followed; (2) vaccinations are usually given very late—or not—accepted. Regarding the first case, in certain regions, vaccine coverage assessment studies showed that the vaccination programs are not adequately completed in many patients who received at least one (1) dose , 149, maIn many countries, ignorance of the susceptibility to HPV infection and associated disorders, and the lack of confidence in vaccination programs are some of the major challenges leading to a refusal of parents to vaccinate their children , 155. SeFinally, it is worthy of note that the high burden of HPV is also associated with the antigenic protection limits of current vaccines. Among the 17 HR-HPVs , these vaccines can only protect against 7 , offering 89.6% protection . FurtherWhile HPV is widely spread, the related infection and its incidence as well as the burden of the related diseases differ considerably by and within regions according to several factors, including both extrinsic  and intrinsic  factors . HPV is http://hdr.undp.org/en/content/table-1-human-development-index-and-its-components-1). Most of these countries are characterized by poor quality of life, particularly with limited access to basic needs such as healthy food, drinking water, decent housing, good hygiene, and medical care  increases the burden associated with HPV infection and HPV-associated diseases in developing countries. More interestingly, it has recently been shown that the CVM plays a very important role in the progression of infection toward the development of precancerous lesions, and therefore in the natural history of HPV infection. High variability in the cervicovaginal microbial population associated with depletion of  lesions , 131. It lesions , lead toountries , thus juFrom an HPV molecular genetics perspective, this uneven distribution of HPV-associated burden across the world is a rigorous and rational explanation. The major HPV viral capsid proteins (L1 protein) are the most important in the early stages of infection and the reinfection process during viral persistence, and are therefore mainly responsible for the activation of the immune response . They arThis variability within HPV variants called “inter-population or intra-type variability or intratypic molecular variants” described earlier has been characterized particularly from the L1 protein, especially within the hypervariable surface loops  containing the antigenic epitopes responsible for the production of antibodies  104, 16. This exThe current preventive strategies, which include diagnostics and vaccination programs with the current HPV vaccines  and, in particular, the difficulties linked to their implementation in certain regions, is another aspect that explains the uneven distribution of HPV-associated diseases, specifically found to be higher in less developed regions. Developed regions, including Australia, Europe, and America, where at least two of the three current HPV vaccines have been introduced, have reported a decrease in the incidence of HPV-related HPV infections, along with an absence of adverse effects , 140–143G. vaginalis and depletion of Lactobacillus spp., is involved in the occurrence of CINs; thus, it is a risk factor supporting the high HPV burden in developing countries. Moreover, the limited action of the current vaccine against the African intratypic variants prevalent in these regions explains the observed HPV burden because the current vaccines are not developed from these specific L1 variants, but general (or Caucasians) strains.HPV is a common sexually transmitted infection worldwide, which disrupts normal social life and has lethal consequences. Apart from a few exceptions, the burden of HPV infection and related diseases remains high in developing countries, and factors that explain these high rates include poor living conditions, co-infections with other pathogens, poor healthcare facilities, and high cost of vaccines. The high CVM variability caused by multiple genital infections, associated with high abundance of Therefore, even though the current vaccines have demonstrated satisfactory efficacy and safety, and even if the future expectations for the 9-valent vaccine seem satisfactory, the need to develop broad-spectrum vaccines targeting other HR-HPV variants and next-generation vaccines targeting other HPV proteins remains existential. Furthermore, the adoption of one- or two-dose vaccination programs is highly desirable, as it will reduce the cost and allow broad coverage , 153. AwAK conceived the presented idea, extracted the data, wrote the original draft, and formatted the article for submission. BL, HM, and AZ reviewed and edited the original version of the article. G-AB extensively reviewed, edited, and formatted the article for submission. YZ provided resources and technical assistance in data extraction, critically reviewed, and edited the article. TJ conceptualized the main idea, provided resources in data extraction and financial assistance during the whole study, and supervised the whole paper. All the authors read and approved the final version of the article for publication.G-AB was employed by the company Sinomedica Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A total of 188 out of 272 (69.1%) preadolescents attending five out eighteen schools involved in the project, who had not previously received the HPV vaccine, were vaccinated. During past years, education campaigns on HPV were mainly dedicated to adult women, excluding teenagers and omitting young males. It should therefore be of primary importance that school-based education and vaccination programmes be standardized.In recent years, vaccination coverage rates against Human Papilloma Virus (HPV) in Europe have shown a decreasing trend and remain below the required standard. The present study aims to assess knowledge and attitudes regarding HPV infection and vaccination among a representative sample of preadolescents of Palermo, Italy. A survey was carried out throughout two questionnaires, before and after carrying out an educational intervention scheduled during school hours. A total of 1702 students attending first-grade secondary schools of the province of Palermo were enrolled (response rate 68.9%). Students attending third classes , being of higher socioeconomic status , who had previously received information about sexually transmitted diseases (STDs) at home  or at school  and who had ever heard in the past about HPV  showed a significantly higher baseline level of knowledge regarding HPV. Willingness to receive HPV vaccination, in a 10-point Likert scale, significantly increased between the pre-  and post- (9.01 SD ± 1.52) intervention questionnaires ( The carcinogenic role of the Human Papilloma Virus (HPV) in the development of malignant tumors  is widely recognized .Several factors contribute to an increase in the risk of HPV infection, such as the number of previous and current sexual partners, the age of sexual debut, smoking attitudes, and congenital or acquired immunosuppression states .Due to its infectious etiology, the main primary preventive strategy for HPV infection is represented by vaccination .In several European countries, including Italy, HPV vaccination has been offered actively and free of charge to female subjects from 12 years old since 2008. Afterwards, the offer of a HPV vaccination was also extended to male preadolescents (from the age of 12 years old) since 2015 . HPV vacNevertheless, in recent years, vaccination coverage rates against HPV in numerous European countries have shown a decreasing trend among preadolescent females, and remain below the required standard . In ItalMoreover, there is a significant gap between females and males who received at least one dose and those who completed the vaccination schedule (two or three doses), imposing a careful evaluation of both organizational obstacles and reasons that determine the failure of vaccination schedule completion ,10.Several factors associated with poor knowledge and attitudes regarding HPV vaccination, such as the low education level of parents, not participating in school seminars on HPV, low risk-perception of the disease, and a limited knowledge of the effectiveness and safety of the HPV vaccination have been previously documented ,12.Objectives of the present study were to assess the baseline level of knowledge and attitudes regarding HPV infection and vaccination and the willingness to be vaccinated against HPV in a representative sample of preadolescents of both sexes attending secondary schools of Palermo; and after a formative intervention scheduled during school hours, to re-evaluate the knowledge on and willingness to undergo a HPV vaccination.A multicenter project coordinated by the Department of Health Sciences of the University of Genoa was conducted in four Italian Regions  with the main objective being to evaluate the offer of vaccination, coverage rates, and determinants associated with HPV vaccination uptake or refusal in different age classes and target groups ,14,15.Sicily is a southern Italian region with about five million inhabitants, and is fourth for demographic density in Italy . Sicily In particular, the province of Palermo and the corresponding LHA, accounting for 1,252,588 inhabitants residing in 82 municipalities, is the most populous in the region .A pre–post interventional study evaluating knowledge and determinants associated with HPV vaccination in primary target cohorts  was conducted in association with formative interventions in a sample of schools of the LHA of Palermo by healthcare professionals  of the Department of Health Promotion, Maternal and Infant Care, Internal Medicine and Excellence Specialties of the University of Palermo, Italy, during two consecutive school years (2017/2018 and 2018/2019) in 18 first-grade secondary schools (accounting for 2469 students) located in Palermo.The schools were sampled through cluster sampling based on urban location and were divided into three levels (from A: less deprived to C: more deprived), according to the deprivation index of the neighborhood or municipality in which they arose.The deprivation index was calculated by using an algorithm including the average household income, the percentage of adults aged 25 with a high school diploma or university degree, and the percentage of employed people of the neighborhood or municipality of the first-grade secondary school .In relation to the main outcome of the study  and to the population of preadolescents aged 11–14 years in the province of Palermo , 1492 preadolescents should be enrolled, so that the sample could be considered representative  .An informative note on the objectives and purposes of the study was provided to all parents of the students attending the schools involved in the study, and a consent/dissent form was completed in order to agree to the participation to the survey and to the educational interventions on sexually transmitted diseases (STDs) and HPV vaccination.During the 2018/2019 school year, with the logistical support of the Palermo LHA that provided material and human resources, the on-site HPV vaccination was contextually offered after the intervention in five schools in a dedicated and fully equipped caravan of the LHA, parked in the courtyards inside the schools.A survey was carried out throughout two (pre- and post-intervention) questionnaires, both administered in paper form, before and after carrying out an educational intervention on STDs, HPV infection, and preventive strategies.The two questionnaires were borrowed from previous experience and tested for its comprehensibility and reproducibility on a sample of 102 preadolescents attending three first-grade secondary schools of Palermo.The pre-intervention questionnaire included 15 items and was divided into two main sections: (1) a section included questions regarding socio-demographic characteristics and attitudes about STDs, particularly HPV; (2) a section aimed at investigating knowledge on HPV-related diseases and vaccination strategies, with five “sentinel” questions investigating HPV infection and vaccination .A knowledge score, created for the study and tested in the analysis of the responses obtained by the first 102 students enrolled, that assigned one point for every correct response to the “sentinel” questions was calculated for every respondent. Specifically, students with three or more correct responses were assigned to the “adequate knowledge” group.Moreover, a previously tested deprivation index was assigned to each participant in accordance with socio-demographic characteristics of students . In partFrom the sum of the obtained scores (from 0 to a maximum of 11), a range of values was defined, assigning to each student a deprived (from 0 to 5) or not deprived (from 6 to 11) status.Furthermore, a post-interventional questionnaire included four items assessed, through a Likert scale , knowledge about HPV infection or HPV vaccination before the seminars.Finally, a 10-point Likert scale question on how much the participant was willing to receive the HPV vaccine was included both in the pre- and post-intervention questionnaires, with the aim to evaluate the effectiveness of the intervention.The educational interventions for preadolescents included a frontal lesson of about 20 min, conducted by medical doctors and researchers of the Department of Health Promotion, Maternal and Infant Care, Internal Medicine and Excellence Specialties of the University of Palermo.In supporting the intervention, a set of slides relating to STD prevention with a particular focus on HPV infection, related diseases, and vaccination was presented in plenary sessions to all students for which the consent form signed by parents was obtained.The personal data and the results of the pre- and post-interventional questionnaires were collected in accordance with the rules of good clinical practice, and each questionnaire was anonymous.In order to verify that each student completed both questionnaires and that the pre- and post-educational intervention willingness to HPV vaccination might be compared, all participants were provided two unique anonymous codes that were used at the beginning of both questionnaires.The study protocol was authorized and approved by the Regional Ethics Committee of the Liguria Region (Protocol number 162 REG 2017 or 11 April 2017) and by the Ethical Committee Palermo 1 (Protocol number 6/2017 of 5 June 2017).Data collected through written questionnaires were entered into a database created with EpiInfo 3.5.4 . All the data were analyzed using the statistical software package Stata/MP 12.1 .Absolute and relative frequencies were calculated for the categorical  variables. The differences in the categorical variables for hesitancy and refusal and between before and after the intervention were analyzed using chi-squared tests (Mantel–Haenszel) and the McNemar test, respectively. Correct responses to at least three of the five “sentinel” questions investigating HPV infection and vaccination knowledge was considered as a dependent variable (good knowledge) in the uni/multivariate analysis conducted with principal variables examined in the pre-intervention questionnaire.p-value ≤ 0.20 at the univariate analysis, to guarantee a more conservative approach, were included in the two multivariate backward stepwise logistic regression models carried out. The crude odds ratio (crude OR) and the adjusted OR (adj-OR) with 95% confidence intervals (CIs) were calculated in the logistic regression models. A p-value ≤ 0.05 was considered significant throughout the study.Variables found to have a statistical association with a A total of 1702 students attending first-grade secondary schools of the province of Palermo were enrolled (response rate 68.9%), of which 52% were female. About half of the preadolescents (47.9%) attended third classes, and 50.4% belonged to schools of an intermediate level (B) of neighborhood socioeconomic index, followed by students belonging to schools of high (A = 25.7%) and low levels (C = 24%). A large majority of respondents (98.1%) were Italian, and 64.2% attended schools in the Municipality of Palermo. Finally, regarding the deprivation index, 67.1% of enrolled students reported a deprived socio-economic status .A large majority of students had already attended educative lessons about STDs at school (66.5%), while only 32.4% of respondents previously had information about STDs at home, and 21.7% with at least one physician. Moreover, 71.3% of the sample had previously heard about the HPV infection or vaccine and 88% knew the correct way of HPV transmission  . Almost In n = 97 students did not participate in the pre–post interventional studies). Specifically, about 85% of students participating to the formative intervention and responding to the post-intervention questionnaire provided a correct response to the questions.In p < 0.001; data not shown in figure).Willingness to receive HPV vaccination, in a 10-point Likert scale, among students enrolled in the study significantly increased between the pre-  and post- (9.01 SD ±1.52) intervention questionnaires  preadolescents that participated in the formative interventions and that had not previously received at least one dose of HPV vaccination were vaccinated against HPV after the participation in the formative interventions, in collaboration with medical officers of the LHA of Palermo and after the collection of consent forms signed by parents (data not shown).In Italy, only a few studies evaluating the main reasons associated with low HPV vaccination knowledge and attitudes were conducted during recent years ,19.Among preadolescents and their parents, one of the main reasons was represented by the role of the family paediatricians or the general practitioners in recommending the vaccination to parents ,18.Results of the present study, on the other hand, demonstrate that better attitudes and knowledge regarding HPV infection and vaccination are attributable to the formative role of families and schools. Students that had previously received information about STDs at home or at school showed statistically significantly higher knowledge scores on HPV than others.Formative intervention conducted at school was strongly associated with HPV vaccination knowledge and uptake and, for instance, female young adults who did not participate in school seminars on HPV demonstrated lower vaccination uptake rates in previous studies ,20,21.A systematic review also confirmed that the best strategies to increase attitudes regarding HPV vaccination were both parental education (through brochures and information sheets or invitation letters) and adolescents’ education  .This could explain data obtained among students of the Palermo province, in which better knowledge on HPV infection and vaccination were observed among students with a low deprivation index. In general, minor attention given to informative and educative messages and a better response to invitation letters sent by LHAs were shown by most deprived subjects in Sicily ,24.In order to improve vaccination awareness and adherence among deprived social classes, school seminars and vaccination offer could probably be considered effective interventions aimed at improving the adherence to HPV vaccination .According to results obtained in the present study, satisfying data were reported in the sentinel questions regarding HPV infection and vaccination knowledge of the post-intervention questionnaire. The willingness to receive HPV vaccination also showed a statistically significant 6% increase.Usually, information and recommendations provided by healthcare professionals can be considered one of the strongest predictive factors by increasing the chances of adherence to HPV vaccination . HoweverDuring past years, education campaigns on HPV were mainly dedicated to adult women, excluding teenagers, and systematically omitted young males .In the study sample, an increased willingness to receive HPV vaccination was observed among female and male students attending schools of the Palermo municipality. Generally, these findings could be attributed to major attention of these students given to personal risk perception of contracting the infection  or better school education due to the lower number of students in classrooms, which is more frequent in the schools of the Palermo municipality ,30.Finally, parental awareness represented one of the main factors influencing the acceptance of the HPV vaccine among preadolescents, and was associated with the quantity and quality of information provided on HPV, also at school .It should therefore be of primary importance that medical and sanitary teams support and carry out school-based education and vaccination programmes about teenagers’ sexuality, STDs, and preventive strategies, such as HPV vaccination.In particular, students attending schools in more deprived areas or students with poor socio-economic conditions are least informed about STDs or the opportunity to protect themselves with vaccination. For these reasons, the school environment should represent an optimal place for sharing correct information and to improve knowledge and attitudes on STDs prevention, regardless of personal or school deprivation index .As a matter of fact, during the study duration, 188 preadolescent students of five schools (69.1% of the susceptible sample) not already immunized against HPV were vaccinated, demonstrating the high efficacy of school-based formative intervention and a vaccination offer.Some limitations could have affected the present study. First, a selection bias should be considered, since greater participation of students whose parents were more prone to vaccine compliance could not be excluded. Secondly, the study was based on self-reported information of a representative sample of preadolescents—although self-reporting can be a risk for inaccuracy, it was recognized as a cost-effective and feasible method for gathering data from large population samples, also among secondary school students . FinallyHPV vaccination represents a clear example of under-use of a practice with a very high scientific value . In ItalThe large-scale organization of school-based educational interventions on STDs, HPV-related diseases and preventive strategies should probably be standardized and extended in order to improve awareness and willingness of students on the importance of HPV vaccination.


E6, E7, and L1 genes of HPV18, to assess the prevalence of HPV18 variants in Korea and to explore the relationship between HPV18 genetic variants and the risk for cervical cancer.High-risk human papillomavirus (HPV) infection is an essential factor for the development of cervical cancer. HPV18 is the second most common carcinogenic HPV type following HPV16, but the lineages of HPV18 have been less well studied than those of HPV 16. The purpose of this study was to analyze the nucleotide variants in the A total of 170 DNA samples from HPV18-positive cervical specimens were collected from women admitted to a secondary referral hospital located in Seoul. Among them, the lineages of the 97 samples could be successfully determined by historical nomenclature.All the studied HPV 18 variants were lineage A. Sublineages A1 and A4 comprised 91.7% (89/97) and 1.0% (1/97), respectively. Sublineages other than A1 or A4 comprised 7.2% (7/97). We identified 15 new nucleotide substitutions among 44 nucleotide substitutions: C158T, T317G, T443G, A560G, A5467G, A5560C, A5678C, A6155G, G6462A, T6650G, G6701A, T6809C, A6823G, T6941C and T6953C. Among them, 6 substitutions at positions 317, 443, 5467, 5560, 6462, and 6823 resulted in amino acid changes . The pathologic results were classified as normal in 25.8% (25/97) of the women, atypical squamous cells of undermined significance (ASCUS) in 7.2% (7/97), cervical intraepithelial neoplasia (CIN) 1 in 36.1% (35/97), CIN2/3 in 19.6% (18/97), and carcinoma in 12.4% (12/97). There was no significant association between the HPV18 sublineages and the severity of pathologic lesion or the disease progression.This study is the first to analyze the distribution of HPV18 variants in Korean and to associate the results with pathologic findings. Although the HPV18 variants had no significant effect on the degree and progression of the disease, the newly discovered nonsynonymous mutation in L1 might serve as a database to determine vaccine efficacy in Korean women. Cervical cancer is the fourth most common cancer among all malignancies in females worldwide and the seventh most common cancer in Korea. According to the World Health Organization’s (WHO’s) GLOBOCAN project in 2018, 569,847 new cases occur and 311,365 people die annually due to cervical cancer worldwide. In Korea, cervical cancer is the seventh most common cancer, with the development of 3348 new cases and 1029 deaths reported annually [E1, E2, E4, E5, E6, and E7), late genes (L1 and L2) and a long control region (LCR) [E6 and E7 are major oncogenes that are highly expressed in tumors and are related to cellular immortalization, malignant transformation, and carcinogenesis. Based on these roles, proteins E6 and E7 are generally regarded as ideal targets for the development of therapeutic HPV vaccines [HPV is a small double-stranded DNA virus with an 8-kb genome containing early expressed genes , Asian-Amerindian (AA) or African (AFR) lineages according to E6-E7, L1, and/or LCR sequences [Over 200 HPV types have been identified based on equences –14. Thisequences  that canequences , 17. In HPV genetic variants have been reported to differ in their viral assembly, pathogenic potential, and host immune response depending on geographic and ethnic features , 17–20; From 2010 to 2017, 7992 women admitted to the Seoul National University Boramae Medical Center were tested for cervical HPV genotype more than once. Among them, 3926 (3926/7992 = 49.1%) were positive for HPV, and 170 (170/3926 = 4.3%) were positive for type 18 and negative for other types. HPV detection and typing were performed using a liquid bead microarray, namely, the GeneFinder HPV PCR Kit .E6, E7, and L1 genes were performed using type-specific primers, which are shown in Table E7 sequencing had a high success rate (165/170), so the sequencing was completed with only 1 primer set . However, E6 and L1 sequencing had low success rates, so we attempted 3 trials each (Table Amplification and sequencing of HPV18 http://web.expasy.org/translate/) for the determination of amino acid changes. PSIPRED v.4.0 (http://bioinf.cs.ucl.ac.uk/psipred/) was used for secondary structure prediction, as it provides a simple and accurate secondary structure prediction method.Nucleotide sequences were translated by the translate-tool of ExPASy (http://megasoftware.net) [All sequence data were assembled against the HPV18 reference strain, GenBank sequence NC_001357, using Sequencher version 5.2.3 . Twenty-eight other sequences were downloaded from GenBank, as in the latest Taizhou area study [are.net) , 23.Based on cytological and histological evaluations of fresh specimens, the cervical lesions were graded according to their severity as follows: normal, atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), cervical intraepithelial neoplasia grade 1, 2 or 3  and cervical cancer. The histological diagnosis of each case was reviewed by an experienced pathologist who was unaware of the HPV testing results.Mann-Whitney, Fisher exact and linear by linear association tests were used for comparisons between AA and E lineages. Variables affecting cervical cancer risk were analyzed by a logistic regression model. All statistical analyses were carried out with SPSS, version 22.0 .E6, 165 (165/170 = 97.1%) E7, 74 (74/170 = 43.5%) entire L1 and 90 (90/170 = 52.9%) partial L1 sequences from HPV18 isolates were successfully amplified by PCR and sequenced. In total, only 97 samples (E6-E7-L1 sequences) could be classified as lineages and sublineages according to E6-E7 sequence-based historical nomenclature rules of previous studies [Ninety-seven (97/170 = 57.1%)  studies .E6-E7-L1 sequences (BRM01 ~ 36 variants). In total, we found 44 nucleotide substitutions. Among them, 29 nucleotide substitutions were already reported: T104C, A171G, T173G, C287G, A482C, T485C, C549A, C554T, C751T, C860T, C5478T, A5497G, G5609A, T5619A, C5701G, C5875A, C5920T, A6059G, T6131G, T6146G, A6401G, A6430C, A6441C, C6460G, C6625G, C6842G, A6970G, and G7032A [E6; H13R, H44P, A345T, and N465S in L1, respectively). In addition, all the 15 newly identified sequences in this study were submitted to GenBank under the accession numbers MK813921-MK813935  and/or cervical biopsies  after HPV genotyping within 3 months. The cervical pathologic results were classified as normal in 25 women (25/97 = 25.8%); ASCUS in 7 (7/97 = 7.2%); LSIL/CIN 1 in 35 (35/97 = 36.1%); HSIL/CIN2 and 3 in 18 (18/97 = 18.6%); and carcinoma in 12 (12/97 = 12.4%). There was no statistically significant association between HPV18 lineage and cervical lesion severity  . There wP = .773)  Table .From 2010 to 2017, almost half (49.1%) of requested cervical HPV genotyping tests were positive for any type of HPV in a secondary referral hospital in Seoul, Korea. Among the positive results, the frequency of HPV 18 single positive was 4.2%. The frequency of the present study was comparable to other reports in Korea  –33.E6 and G5503A, C5701G, C6460G, C6625G, and C6842G in L1, were found in all HPV18 variants. These 6 substitutions were also found in all HPV18 isolates in southeastern and northeastern China [The distributions of HPV variants differ among geographic origins, evolutionary dynamics, and pathogenicity. In our population, 6 substitutions, namely, C287G in rn China , 18, whirn China . These fWe previously discovered that the HPV 16 variants were composed of A1 ~ 3, A4, C, and D sublineages  in Korea . Upon coPrevious studies reported that the risk of developing high-grade CIN is significantly increased with the non-European variants , 34. OneIn the aspect of public health, many countries have implemented national policies of HPV vaccination. In Korea, HPV vaccination has been free for 12-year-old girls since 2016, and the government intends to expand the vaccination targets. Three prophylactic vaccines have received licensure from Korea Food and Drug Administration: the AS04-adjuvanted bivalent (HPV16/18) vaccine , which was licensed in 2008; the aluminum hydroxyphosphate sulfate (AAHS) adjuvant quadrivalent (HPV6/11/16/18) vaccine , which was licensed in 2007; and the AAHS adjuvant 9-valent (HPV6/11/16/18/31/33/45/52/58) vaccine , which was licensed in 2016. These prophylactic HPV vaccines are composed of L1 proteins of multiple HPV combinations [E6-E7-L1 sequences over the past 20 years does not have significantly reduced reliability compared to WGS. Thus far, many studies have been selectively conducted on the oncogenic proteins E6 and E7, and the major capsid protein L1 plays an important role in the prophylactic vaccine, as our study suggests.With the development of next-generation sequencing (NGS), the whole-genome sequencing (WGS) of 8 kb HPV became easier, making the analysis of lineages and single-nucleotide polymorphisms (SNPs) relatively faster and more accurate. However, WGS analysis pipelines in microbiological fields have not yet been established systematically, and it is difficult to analyze multiple samples with limited resources. In addition, although WGS identifies more variants and contributes to the construction of more accurate phylogenetic trees than partial sequencing, the data composed of In summary, this research identified 15 new nucleotide substitutions and is the first to analyze the distribution of HPV18 variants in Korean women and to match the results to pathologic results. Data regarding the geographic/ethnic HPV18 genetic diversity distributions may be helpful for designing diagnostic probes, correlating epidemiologic cervical cancer risks, and analyzing the efficacy of HPV vaccines for targeted populations in Korea.


Human papillomavirus (HPV) infection affects both men and women and causes a wide range of diseases from benign lesions, such as genital warts, to more serious diseases such as anogenital and oropharyngeal cancers. HPV has evolved effective strategies to evade the host immune system and while antibodies are important, our understanding of other host immune factors involved in protection, particularly at the mucosal surface, is poorly understood. Prophylactic HPV vaccines are highly effective in preventing vaccine genotype-related-HPV infections and their associated diseases. While these vaccines do not clear existing lesions, there are several promising therapeutic vaccines targeting HPV-associated lesions that are undergoing clinical trials. A number of important questions related to HPV infection and vaccination remain unanswered. This includes identification of novel immunological markers of protection  against oncogenic and non-oncogenic HPV types, as well as understanding the critical immune responses required for optimal immune protection in high-risk groups who are particularly vulnerable to HPV infection and associated diseases.This collection of articles under this Research Topic focuses on current concepts and state of the art knowledge on the immunology of HPV infection and vaccines.Zhou et al. provides an overview of the current landscape of HPV vaccines, including important aspects such as immunogenicity, cross-protection and potential adverse reactions. They refer to the global call for action on the elimination of cervical cancer by WHO and highlight the current strategies needed to improve vaccine coverage in both high-income and low-income settings. Importantly, using China as an example where HPV disease burden has increased over the last 20 years, the recent licensure of prophylactic HPV vaccines is a fundamental step that will go a long way to reducing the prevalence of HPV-associated diseases among the Chinese population in future years.The review article by Pattyn et al. describe the benefits and accuracy of first-void urine for the measurement of both HPV DNA, as well as antibodies. This sample represents a non-invasive approach to measuring HPV DNA and particularly antibodies that are present at the cervicovaginal surface where HPV typically infects. They suggest that while there are limitations with this type of approach, HPV-specific antibodies can be detected with moderate to good correlations with serum antibodies, offering a promising alternative for monitoring vaccination programs post-implementation. Indeed, simpler approaches to measurement of HPV antibodies are needed as the ‘gold-standard’ pseudovirus (PsV)-based neutralization assays are labor intensive and require a high level of technical expertise while the virus-like particle (VLP)-based assays require specialized reagents and equipment which can be difficult to source. The manuscript by Toh et al. describes the use of a PsV ELISA to measure HPV-specific antibodies following vaccination, showing that the production of PsV compared with VLP is simpler and provided excellent correlations with the neutralization assay, offering an alternative method for HPV antibody measurement.Monitoring the effectiveness of HPV vaccines during epidemiological studies and clinical trials relies on accurate measurement of both HPV DNA, as well as HPV-specific neutralizing antibodies. While these are routinely assessed using swabs and blood, respectively, novel approaches such as urine have also been investigated. In their Perspective article, Zhang et al. reported that in mice, CD40 activation accelerated vaccine-induced HPV16 tumor-specific CD8+ Tscm (stem-like memory) formation which were more potent and longer-lived that other HPV-specific CD8+ T cells. Importantly, there was a higher proportion of Tscm cells in patients who were HPV16+ but tumor negative compared with HPV16+ cancer patients. This offers new insights into cancer immunotherapy strategies aimed at targeting these cells to improve patient outcomes. In another paper by Zottnick et al., they highlight the advantages of newly developed orthotopic models of HPV cancer using a humanized mouse system. Such an approach would overcome the limitations of existing preclinical mouse models and allow novel HPV therapeutic vaccination strategies to be examined for their ability to induce human immune responses at the site of infection and hopefully improve translation to the clinic.Understanding the immunological basis of protection against HPV infection is also critical in the context of developing and harnessing therapeutic strategies to improve survival. To this end, Skeate et al. demonstrate using in vitro models that the theta-defensins from rhesus macaques have unique properties in terms of inhibiting HPV infection through preventing binding to specific cell surface receptor complexes on target epithelial cells. The development of low-cost antiviral strategies that prevents infection and transmission of high-risk HPV types would be an important component in the overall goal to reduce HPV infection and their associated diseases globally.Novel antiviral compounds that can disrupt HPV infection are also an attractive area of research. In their manuscript, This Research Topic provides an up-to-date summary of the latest research and insights into the immunology of HPV infection and vaccination that will ultimately provide the basis for improved strategies to reduce HPV-associated diseases in the future.PVL drafted the manuscript. All authors contributed to the article and approved the submitted version.PVL is supported by an Australian National Health and Medical Research Council Career Development Fellowship Level 2 (GNT1146198). IHF is supported by an Australian National Health and Medical Research Council Investigator Grant (GNT1173927). SMG is supported by an Australian National Health and Medical Research Council Investigator Grant (GNT1197951).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human papillomaviruses (HPVs) depend on the cellular RNA-processing machineries including alternative RNA splicing and polyadenylation to coordinate HPV gene expression. HPV RNA processing is controlled by cis-regulatory RNA elements and trans-regulatory factors since the HPV splice sites are suboptimal. The definition of HPV exons and introns may differ between individual HPV mRNA species and is complicated by the fact that many HPV protein-coding sequences overlap. The formation of HPV ribonucleoproteins consisting of HPV pre-mRNAs and multiple cellular RNA-binding proteins may result in the different outcomes of HPV gene expression, which contributes to the HPV life cycle progression and HPV-associated cancer development. In this review, we summarize the regulation of HPV16 gene expression at the level of RNA processing with focus on the interactions between HPV16 pre-mRNAs and cellular RNA-binding factors. Human Paillomaviruses (HPVs) are small DNA viruses that infect cutaneous or mucosal epithelial keratinocytes ,2. HPVs Control of HPV gene expression is of paramount importance in the viral life cycle as well as in the rare event of carcinogenesis. Although transcriptional regulation of HPV gene expression plays a major role during the life cycle, HPV has relatively few promoters ,10, whicHere, we review the regulation of HPV mRNA splicing and polyadenylation with focus on individual HPV splice sites and polyadenylation signals and the cellular and viral factors that control these sites. In addition, we discuss the connection of these HPV RNA-processing events with transcription, RNA modifications, epigenetics, and the DNA damage machinery. Multiple previous reviews have covered RNA processing in human as well as bovine papillomavirus ,15,16,17HPV is a small DNA virus with an approximately 8 kb circular, double-stranded DNA molecule as a genome ,19 Figu. The genHPV pre-mRNAs are synthesized by cellular RNA polymerase II (Pol II). Transcription initiation is followed by RNA processing that includes 5′-cap attachment, alternative RNA splicing, and polyadenylation. Translation of HPV mRNAs is conducted by the cellular translation machinery in a cap-dependent manner. Since the majority of the HPV pre-mRNAs/mRNAs are structurally polycistronic ,21 Figu, it is iHPV life cycle progression is coupled to host epithelial cell differentiation . Since HIn transition to the late stage of the HPV life cycle, the expression levels of E1 and E2 increase, enabling vegetative amplification of the HPV DNA in the differentiated epithelial cell layers . The veghttps://pave.niaid.nih.gov/#home). We have listed some of the alternatively spliced mRNAs that are key to the discussion in this review  the strength of splice sites or polyadenylation (polyA) signals; (2) the presence of cis-acting regulatory RNA sequences on pre-mRNAs; and (3) the expression levels of various trans-acting factors, e.g., RBPs ,46,47. SGU(A/G)AGU, whereas the splice acceptor is (C/U)nX(C/U)AG_(A/G) (the most conserved nucleotides are in bold face) [GUA) are highly consistent with consensus sequence (The HPV16 genome encodes at least four splice donors (SD or 5′-splice sites)  and seven splice acceptors (SA or 3′-splice sites)  . The consequence .Among HPV16 splice donors, SD3632 appears to conform better to the consensus splice donor than the remaining HPV16 splice sites. However, HPV16 splice donor SD3632 is strongly suppressed and exclusively used for HPV16 late mRNA splicing, suggesting that this splice site is silenced by cis-acting regulatory RNA elements and/or trans-acting factors. Regarding HPV16 splice acceptors, the sequences flanking the cleavage position of the splice sites are diverse and contribute to the strength of each splice site . The conIn conclusion, all HPV16 splice sites are suboptimal and are regulated to respond to cellular differentiation during the course of the HPV life cycle. Unscheduled alterations of HPV splice site efficiency will invariably affect the balanced usage of other HPV splice sites, thereby distorting the relative levels of more than one HPV16 mRNA. Thus, it is of paramount importance to strictly control the usage of all HPV splice sites during the entire HPV life cycle.HPV16 splice sites SD226, SA409, SA742, SD880, SD1302, SA2582, SA2709, and SA3358 are all used to generate alternatively spliced HPV16 early mRNAs, while SD880, SD1302, SA2582, SA2709, SA3358, SD3632, and SA5639 generate HPV16 late mRNAs . A numbeHPV16 splice sites SD226, SA409, SA526, and SA742 are utilized for alternative splicing of mRNAs spanning the E6 and E7 coding region, with the possible exception of SD226 that may be alternatively spliced to downstream 3′-splice sites SA2709 and SA3358 in addition to SA409, SA526, and SA742 . mRNAs tThe control of these splice sites by cis-acting sequences and trans-acting factors is currently under investigation, and hitherto it has been reported that 226^409 splicing in HPV16 and the corresponding splicing event in HPV 18 (233^416) are negatively regulated by hnRNP A1 that binds to exonic splicing silencers located in the E7-coding region ,57 Figu. This suHPV16 splice acceptor SA3358 is the most commonly used 3′-splice site on the HPV16 genome and is efficiently used during both early and late stages of the HPV16 life cycle. Despite these properties, it conforms poorly to the consensus 3′-splice site . The splSince HPV16 splice sites SD3632 and SA5639 are exclusively involved in HPV16 late L1 mRNA splicing, they are conditionally inactive during the early stage of the HPV16 life cycle and active during the late stage. Even then, only partial activation of L1 mRNA splicing is allowed as complete activation would inhibit L2 mRNA production. Inhibitory RNA elements that suppress HPV16 late splice sites SD3632 and SA5639 have been identified ,63,64. SSplicing silencers at HPV16 3′-splice site SA5639 were mapped to a region downstream of SA5639 in the L1-coding region and were shown to consist of AU-rich sequences that interact with hnRNP A1 ,66 Figu. The regIn addition to the HPV16 splice sites discussed previously, HPV16 polyadenylation signals are also controlled by regulatory RNA elements ,16. The Upstream sequences of HPV16 pAE also positively control the activity of the pAE via binding to polyadenylation factor Fip1 at uridine-rich sequences in the HPV16 early UTR  as well The HPV16 late polyA signal, pAL, is strongly suppressed by negative RNA elements located in the late UTR and that interact with U1snRNP and CUGBP ,74,75,76Regarding the HPV16 5′-UTR region, cellular RNA-binding proteins interacting with this region have not been identified to the best of our knowledge. However, cis-acting regions in the LCR interact with trans-acting transcription factors or DNA replication factors ,79 that Although it is clearly important to understand how production of each HPV mRNA is regulated, it is also important to understand what each HPV mRNA produces and how—in particular in the light of the ability of HPVs to produce so many alternatively spliced and polycistronic mRNAs. However, translation of HPV16 mRNAs has been investigated only for E6/E7, E5, and E2 mRNAs ,83,84,85A number of reports suggest that splicing is dramatically affected by changes in transcription . It has Epigenetic changes of the HPV16 chromatin have been described . More thThe activation of the HPV early promoter which is located in the HPV LCR is regulated by a number of cellular transcriptional factors, such as Sp1, AP1, TEF1, NF1, Oct1, YY1, and CDP  and by HmRNA export is a critical step of gene expression and includes mRNA transport through the interchromatin space, docking at and export through the nuclear pore complex (NPC), and release of the mRNA into the cytoplasm which are modulated by assembly of pre-mRNP complexes and acquisition of export receptors. There are two major export receptors described, NXF1 and CRM1 . The maiThere is no direct evidence linking particular RBPs to HPV mRNA export. However, it has been shown that HuR binds specifically to the HPV1 late UTR and to the HPV16 late UTR, suggesting that HuR could potentially enhance HPV late gene expression by promoting nuclear export of late mRNA ,136,137,mRNA stability or half-life can be regulated by sequence-specific RBPs that bind to regulatory RNA elements and modulate the interaction of the mRNA with the cellular RNA degradation machinery. mRNA decay rate is transcript-specific ,145 and The stability of many labile mRNAs is frequently controlled by cis-acting sequences called AREs (AU-rich elements) that are generally located in the mRNA 3′-UTRs. A number of ARE-binding proteins associate with AREs and recruit components of the core degradation machinery, e.g., hnRNP D, TTP, BRF1, TIA-1, TIAR, and KSRP ,155,156.mRNA instability element has been identified in the L1 and L2 ORFs of high-risk HPV16, but not in the L1 and L2 ORFs of low-risk HPV1 . These RRecent studies demonstrated that RNA methylation—primarily N6-methyladenosine—has an important biological function in the regulation of different cellular processes such as metabolism, embryonic development, and stem cell self-renewal in an RNA-processing-dependent manner . N6-methExpression levels and RNA-binding activity of the RBPs are strictly controlled during tissue development, cell differentiation, and other major cellular events. Many diseases, including cancer, have been linked to defects or dysregulation of RBP expression or function ,171,172.ELAVL2, GRB7, KHSRP, and PTBP1) in HPV-infected cervical tissue samples and keratinocytes [Expression levels of RBPs can be modulated during the HPV life cycle in response either to epithelial cell differentiation or to the HPV infection itself. It was originally observed that HPV infection altered the expression levels of a variety of RNA-binding proteins in HPV-infected cells in vivo as well as in HPV-positive precancerous cervical lesions and cervical cancer . Recentlinocytes . Among Hinocytes  as well inocytes ,177, caninocytes , telomerinocytes , cell cyinocytes  and to tinocytes . HPV16 Einocytes ,183, CBPinocytes ,185, IRFinocytes , c-Jun [inocytes , ATF-2 [inocytes , or c-Myinocytes ,189 or vinocytes ,191. In inocytes  and SR pinocytes ,175,192 Functional dysregulation of RBPs is caused by abnormal post-translational modifications (PTM) in many tumors. Abnormal PTM of RNPs originates from defects in one or multiple signaling pathways that cause changes in phosphorylation, acetylation, methylation, and/or ubiquitination of proteins . PTM canIn recent years, it has become increasingly clear that the cellular DNA damage response machinery plays a major role in the HPV life cycle. HPV infections activate the cellular DNA damage response (DDR) , and HPVIt has previously been shown that changes in PTM of cellular RNA-binding proteins affect HPV16 RNA processing  Figure . More spIn addition to the Akt signaling pathway, it has been demonstrated that the ERK signaling pathway affects HPV16 alternative splicing of HPV16 E6 mRNAs via ERK-responding RBPs, Brm and Sam68  Figure . Since EIt has been demonstrated that the expression of HPV late transcripts occurred in suprabasal cells of differentiated epithelium in organotypic raft cultures ,207,208 It has been shown that E2 inhibits HPV early polyadenylation . This alIt is apparent that regulation of HPV RNA processing by HPV–ribonucleoprotein interactions is critical for the control of HPV gene expression, in particular for the temporal production of all alternatively spliced and polyadenylated HPV mRNAs during the entire life cycle, including the switch from the early to the late HPV gene expression program. A number of cis- and trans-acting factors that regulate HPV16 RNA processing have been identified, but additional cis- and trans-acting factors are clearly required for the control of the entire HPV16 gene expression program. To understand the full network of cis- and trans-acting factors that give rise to the HPV transcriptome, a complete identification of these regulatory circuits is required. In addition, many questions remain to be addressed: is HPV pre-mRNA splicing affected by RNA polymerase II kinetics including epigenetic changes on the HPV genome, are RNA modifications contributing to HPV RNA processing, is RNA nuclear export or RNA stability contributing to regulation of HPV gene expression, which PTMs on RBPs affect HPV RNA processing and how are they controlled by intracellular response and/or extracellular stimuli. It is also of importance to perform comparative studies on different HPV types, including cutaneous HPVs, low-risk HPVs as well as all high-risk types, since it could potentially lead to a better understanding of the contribution of regulation of HPV RNA processing to pathogenesis and cancer development. Recent global transcriptomic studies and techniques such as CLIP-seq will help to reveal how RNA processing regulates HPV gene expression. This knowledge may contribute to future development of diagnostic tools for detection of HPV infection as well as for the development of novel drugs to treat HPV infections.