The most prevalent high-risk genotypes were HPV52 (4.16%), HPV16 (2.98%), HPV58 (2.15%), HPV53 (1.58%) and HPV68 (1.34%). HPV co-infection with up to 10 genotypes was reported for the first time. Our findings suggested a high burden of HPV infections among women in northern Guangdong. Establishing the prevalence and genotype distribution characteristics of HPV infections in the region can contribute to cervical cancer prevention through HPV vaccination.Globally, cervical cancer, whose etiologic factor is Human papillomavirus (HPV), is the third most common cancer among women. In cervical cancer screening, HPV testing is important. However, the prevalence of HPV in northern Guangdong Province has not been conclusively determined. A total of 100,994 women attending Yuebei People's Hospital Affiliated to Shantou University Medical College between 2012 and 2020 were recruited. HPV was tested by a polymerase chain reaction (PCR)-based hybridization gene chip assay. The prevalence of HPV among these women was established to be19.04%. Peak prevalence was observed in women aged 40–49 (7.29%). Besides, the prevalence of single-type HPV infection (14.46%) was significantly high, compared to multiple-type infection (4.58%) ( Therefore, HPV vaccination can significantly reduce the risk of cervical cancer3. HPV is a circular double-stranded DNA virus that mainly infects epithelial cells in the skin and mucosal cells. The International Agency for Research on Cancer (IARC) reports that there are more than 200 HPV genotypes, among which, about 40 genotypes infect the genitourinary system while 20 genotypes are associated with cancer development4. Most HPV infections have no obvious clinical symptoms, and the spontaneously resolve5. Based on their risk of causing cancer, HPVs are classified as high-risk HPV (HR-HPV) or low-risk HPV (LR-HPV). HR-HPV can cause cervical cancer, with HPV 16/18 exhibiting the highest risk of cancer development. It usually takes 10–20 years for HPV infection-driven lesions to progress to malignancy8.Globally, cervical cancer which can be caused by the major oncogenic types of HPV, is the third most common cancer in women10. The currently licensed HPV vaccines include bivalent vaccines (HPV16/18), quadrivalent vaccines (HPV16/18/6/11), andnine-valent vaccines (HPV16/18/6/11/31/33/45/52/58)12. Therefore, to select a suitable vaccine, it is important to understand local HPV incidences and subtype distribution.There are regional differences in HPV prevalence and genotype distribution, which explains the different types of vaccinesThis study aimed to investigate the prevalence and genotype distribution of HPV in northern Guangdong province, so as to provide a scientific basis for early screening strategies and vaccination for cervical cancer in the region.A total of 100,994 women attending the Physical Examination Center and Department of Gynecology of Yuebei People’s Hospital between January 1st, 2012 and December 31st, 2020 were enrolled in this study. The inclusion criteria were: (1) Aged 18–75 years; (2) All participants resided in northern Guangdong Province; (3) No history of sexual life or vaginal medication in the past one week; (4) Willingness to undergo an HPV test and participate in the present study, All participants signed informed consent. This research was approved by the Ethics Committees of Yuebei People’s Hospital . All methods were performed in accordance with relevant guidelines and standard operating procedures (SOPs).Gynecological practitioners collected the cervical specimens using cervical brush. First, the cervix was exposed using a vaginal dilator, secretions were wiped using a cotton swab, the cervical brush was extended into the cervix and rotated 4 to 5 times in a clockwise direction to obtain a sufficient amount of cervical epithelial cells. Then, the brush was placed in a vial containing the cell preservation solution . Cervical samples should be stored at room temperature for no more than 12 h and at 4 °C for no more than 7 days after collection. Specimens were sent to the department of clinical PCR laboratory in the hospital for testing.DNA from exfoliated cervical cells was extracted using a DNA extraction kit . Samples on the cervical brushes were eluted; the elution was transferred to a 1.5 ml centrifuge tube, centrifuged at 13,000 rpm for 10 min, and the supernatant discarded. To the cell pellet, 50 µl of the lysis buffer was added to suspend the pellet, incubated at 100 °C for 10 min, and centrifuged at 13,000 rpm for 10 min, to obtain DNA.HPV DNA PCR amplification and genotyping were performed using the HPV genotyping Kit  according to the manufacturer’s instructions. The test kit was approved by the National Medical Products Administration . The test included 23 type-specific oligonucleotides, designed to detect 17 HR-HPV genotypes  together with 6 LR-HPV genotypes . Amplification was performed using the BIOER Genetouch PCR amplification instrument. The amplification system consisted of 5 μl HPV-DNA and 20 μl reaction system. Reaction conditions were: 50 °C for 15 min, denaturation at 95 °C for 10 min, denaturation at 94 °C for 30 s, annealing at 42 °C for 90 s, elongation at 72 °C for 30 s, 40 cycles, elongation at 72 °C for 5 min, and storage at 4 °C. HPV genotyping was performed by hybridization in a YN-H16 thermostatic hybridization instrument. The final result was determined by directly observing color reactions on the chip. The negative control showed no color reaction at all sites except the blue dot on the internal control (IC) site. The positive control must be color reaction of the positive HPV genotype and IC site (shown in blue), and the genotype is read according to the site of the blue spot on the chip.p < 0.05.Data analysis was performed using SPSS22.0 software . Data were presented as mean ± standard deviation or frequency and percentage for numerical or discrete variables, respectively. The Chi-square test was used to determine significant differences between groups, and differences were considered statistically significant at This research was approved by the Ethics Committees of Yuebei People’s Hospital.p < 0.01; Table A total of 100,994 women visited the Yuebei People's Hospital and subjected to HPV DNA testing. The basic characteristics of study participants are shown in Table A total of 25,710 positive HPV genotypes were detected in samples from the 100,994 women . Twenty-three HPV genotypes were identified Table . The LR-p < 0.01). HPV co-infection with up to 10 subtypes was discovered for the first time . Single-type and HR-HPV infections were the most prevalent form. The most prevalent HR-HPV infections were HPV52, 16, 58, 53, 68, 51, 18, and 56 while LR-HPV infections were mainly HPV81 and 43. We also report, for the first time, HPV co-infection with up to 10 genotypes in two cases.13. According to a comprehensive meta-analysis involving over 1 million women, the global HPV prevalence is estimated to be 11.7%. Sub-Saharan Africa (24.0%), Eastern Europe (21.4%), and Latin America (16.1%) have the highest prevalence14. In Japan, the prevalence of HPV is 25%, with HPV52, 16, and 58 being the most common genotypes15. In Korea, the prevalence of HPV is 16.71%, with HPV 53, 58, and 52 being the most common genotypes16. A previous meta-analysis reported that HPV infection rate among women in mainland China is 19.0%, with HPV16, 52, and58 being the most common genotypes17. These types are also the most common types of CIN in China18. This contrasts with the situation in Western countries, where HPV16 and 18 infections are prevalent20.Studies have reported differences in HPV prevalence and genotype distribution in different countries24. Among the sampled regions, Haikou (31.94%), Beijing (21.06%), and Guangdong (20.02%) have the highest HPV incidences. Our results are consistent with HPV distribution characteristics previously reported in China27. The overall HPV infection rate is high and single-type as well as high-risk infections are more common. However, the top five genotypes are slightly different. For instance, the main genotypes in Meizhou28, were HPV 16, 52, 58, 18, and 81, while in northern Guangdong, they were HPV 52, 16, 58, 53 and 68. In short, the top three in most regions of China were HPV 52, 16, and 58.In China, there are regional differences in HPV prevalence and genotype distribution. Population-based screening revealed that the HPV infection rate in China ranges from 9.9% to 31.94%29.HPV screening and vaccination are the most effective measures for preventing cervical cancer. China's cervical cancer screening program adopts a combined method for HPV and cytology (TCT) screening, which can maximize screening Sensitivity and specificity to improve the detection rate. The program is led by the government, with the participation of social groups, medical institutions, third-party testing institutions, etc. It is completely free for the subjects, voluntary, and covers women aged 35–64. In short, cervical cancer screening has a long way to go, and various efforts are being made to explore solutions with Chinese characteristics. Most developed countries have adopted vaccination as their main preventive strategy. Therefore, if these prevention strategies can be promoted in developing countries, thousands of lives can be saved. Currently, the three available HPV vaccines are all prepared according to the existing epidemiological characteristics of HPV in Western countries. At the same time, there is controversy with regards the vaccination age. The American Cancer Society (ACS) recommends that HPV vaccination be performed at 9 to 12 years old to reduce cervical cancer incidences. However, ACS does not endorse the 2019 Advisory Committee on Immunization Practices recommendation for shared clinical decision for adults aged 27 to 45 years potential of vaccination30, and the vaccination situation is not optimistic. HPV vaccination is expensive, and uncertainties regarding the side effects of the vaccine are the main reason for reluctance. In this study, we found that the HPV infection rate remained stable without any downward trend from 2012 to 2020. Therefore, China needs to develop HPV vaccines that are suitable for the Chinese population, while putting into consideration the characteristics of vaccine genotypes and the age of vaccination, further localization, reducing costs, and including government free vaccination programs to reduce the incidences of HPV infections and cervical cancer.China approved the importation of bivalent vaccines in 2016. Nationally, the HPV vaccination rate for women is only 11.0%For the first time, we report on prevalence and subtype distribution of women HPV in northern Guangdong, which is of great significance for guiding the formulation of local vaccination and other prevention strategies. However, epidemiological investigation of HPV requires multi-center and long-term monitoring, especially evaluation of the effects after vaccination. Besides, persistent HR-HPV and multiple-type infections as well as cancer outcomes should be investigated further.


Discussion section, the word “Higher” was incorrectly written as “Lower”. The number “4" was incorrectly superscript applied in the sentence “T cells and memory CD4+ T cells might result in a better prognosis.”In the original article, there were several errors. The sentence “the risk score had the greatest weighing in this calculation” was repeated two times. In the Results, “Construction and Validation of a Nomogram for Predicting Prognosis of Patients With Bladder Cancer,” Paragraph 1:A correction has been made to “Consequently, 1- and 5-year OS can be estimated according to the total number of points; the risk score had the greatest weighing in this calculation.”Discussion, Paragraph 4:A correction has been made to PPFIBP2 and DENND2D are known to be associated with lower levels of tumor invasiveness and a better prognosis; our present findings were consistent with these earlier observations.”“Higher expression levels of Discussion, Paragraph 5:A correction has been made to + T cells and memory CD4+ T cells might result in a better prognosis;”“In triple-negative breast cancer, tumors with high levels of infiltrating CD8The authors apologize for these errors and state that these do not change the scientific conclusions of the article in any way. The original article has been updated.


Radiotherapy (RT) is a key player in the treatment of head and neck cancer (HNC). The RT response, however, is variable and influenced by multiple tumoral and tumor microenvironmental factors, such as human papillomavirus (HPV) infections and hypoxia. To investigate the biological mechanisms behind these variable responses, preclinical models are crucial. Up till now, 2D clonogenic and in vivo assays have remained the gold standard, although the popularity of 3D models is rising. In this study, we investigate the use of 3D spheroid models as a preclinical tool for radiobiological research by comparing the RT response of two HPV-positive and two HPV-negative HNC spheroid models to the RT response of their corresponding 2D and in vivo models. We demonstrate that HPV-positive spheroids keep their higher intrinsic radiosensitivity when compared to HPV-negative spheroids. A good correlation is found in the RT response between HPV-positive SCC154 and HPV-negative CAL27 spheroids and their respective xenografts. In addition, 3D spheroids are able to capture the heterogeneity of RT responses within HPV-positive and HPV-negative models. Moreover, we demonstrate the potential use of 3D spheroids in the study of the mechanisms underlying these RT responses in a spatial manner by whole-mount Ki-67 and pimonidazole staining. Overall, our results show that 3D spheroids are a promising model to assess the RT response in HNC. Head and neck cancer (HNC) is the sixth most common cancer and cause of cancer-related death worldwide, with more than 500,000 new cases and 380,000 deaths yearly . CurrentRadiotherapy (RT) is one of the main treatment options for locally advanced HNC. In recent decades, RT delivery and planning have underwent immense technological advancements, which have reformed RT to be an effective and sophisticated tool for cancer treatment ,2,6. AltClinical factors, such as tumor and patient characteristics and the inaccurate delivery of RT doses can explain some of the local recurrences. However, it is commonly observed that tumors with the same clinical characteristics treated with the same treatment protocol respond differently. These differential responses can be due to various tumoral  and tumor microenvironmental factors . Hence, a better understanding of the interplay between therapy response, molecular tumor biology and the surrounding tissue is of utmost importance and will lead to improved treatment responses ,9.Preclinical models resembling human tumors are essential to understand the biological mechanisms of cancer formation and therapy response. In the field of radiation biology, clonogenic radiation survival curves, in which the number of surviving clones are measured, are the gold standard to determine the relative radiosensitivity of cells or to determine the effect of drug treatments . This isHowever, these models lack the typical tumoral architecture, including cell–cell interactions and the tumor microenvironment (TME), making them poor representors of tumors and poor predictors of heterogenous treatment responses ,13,15. IIn HNC especially, preclinical studies show higher radiosensitivity for HPV-positive HNC when compared to HPV-negative HNC ,17. HoweTo assess whether we could recapitulate the differential RT sensitivity of HPV-positive and HPV-negative HNC cells in 3D, the growth of four HNC cell lines in 3D conditions was investigated. Although morphological differences were noted, all four HNC cell lines formed 3D spheroids in ULA plates A. FADU cGrowth curves were generated by measuring the mean area of the spheroids B. All ofTo determine whether 3D spheroids can be used for radiobiological studies, we assessed the RT response of 3D spheroids and compared it to the RT response determined by clonogenic survival assays.p = 0.049), and there was no reduction in the growth of the FADU spheroids compared to their respective non-RT-treated controls. After treatment with 8 Gy, the FADU spheroids showed an average reduction in area of 1.2-fold until day 19 post RT, whereas CAL27 showed an average reduction of 2.7-fold until day 28 (p = 0.019). These data indicate that the FADU spheroids showed a higher resistance to RT compared to the CAL27 spheroids.The RT response of 3D spheroids was assessed by growth curves with a follow-up time ranging from 4 to 8 weeks after treatment. The RT doses of 0, 4 and 8 Gy were chosen based on our previous experience with in vitro and in vivo models of HNC [p < 0.0001) compared to HPV-negative (FADU and CAL27) cell lines  compared to spheroids in the plateau phase, which mostly included HPV-negative spheroids (p = 0.33) difference in Ki-67 intensity was seen in RT-treated SCC154 spheroids compared to their untreated control spheroids. This corresponded with the exponential growth seen in the RT (4 Gy)-treated spheroids. It should be noted that we were not able to perform immunostaining on HPV-positive SCC154 and SCC47 spheroids treated with the RT dose of 8 Gy, since this dose resulted in the complete disintegration of the spheroids during the follow-up time.The proliferative capacity, measured as the mean Ki-67 staining intensity, of the spheroids was in accordance with the respective growth curves. Spheroids in the active growth phase at the end of the follow-up time, which mostly included HPV-positive spheroids, showed a significantly higher Ki-67 intensity  and hypoxia level of the spheroids at different time points during the observation period and not only at the end. This would have allowed us to assess the influence of RT and regrowth on the proliferative capacity of different HNC cell lines. Despite the limitations, this proof of concept study shows an agreement between differences in the RT responses of HPV-positive and HPV-negative 2D and 3D models, including in vivo xenografts. It clearly shows the advantages and opportunities of the use of 3D spheroid models in the field of radiobiology, and can form a solid base for further 3D model-based research.The HPV-positive HNC cell line SCC154 was purchased from the German collection of microorganisms and cell cultures . HPV-positive HNC cell line SCC47 was gifted by Dr T. Carey, University of Michigan. Both HPV-positive cell lines were cultured in Minimal Essential Medium  and supplemented with 10% fetal bovine serum , 1% L-glutamine  and 1% non-essential amino acids . HPV-negative HNC cell lines FADU and CAL-27 were gifted by Dr. Mechthild Krause, Technical University Dresden, and by Dr. A. Begg, the Netherlands Cancer Institute, respectively, and were cultured in Dulbecco’s Modified Eagle Medium , which was supplemented with 10% FBS and 1% sodium pyruvate . Cell lines were incubated at 37 °C and passaged via trypsinization. Cell lines were authenticated with short-tandem repeat profiling by the ATCC. All experiments were performed with mycoplasma-free cells.Cells were seeded at different densities: 555, 1110, 2220 and 13,320 cells per well for the HPV-positive cell lines and 580, 870, 1740 and 5800 cells for the HPV-negative cell lines. They were exposed to increasing RT doses of 0, 2, 4 and 6 Gy. RT was delivered via the Small Animal Radiation Research Platform . For the in vitro experiment, the following parameters were used: X-rays, 220 kV photons, 13 mA, a dose rate of 4 Gy/min, a source-to-skin distance (SSD) of 35 cm and a broad focus. Colonies were fixed after 2–3 weeks with 2.5% glutaraldehyde in PBS and stained with 0.4% crystal violet . Colonies containing 50 cells or more were counted with Gelcount . The plating efficiencies were determined by dividing the amount of counted colonies by the amount of seeded cells. The survival fractions were calculated via normalization to the plating efficiency of non-irradiated controls of the respective cell lines, as previously described .2 atmosphere at 37 °C. After 72 h of incubation, the medium was refreshed twice a week and cell growth was measured using an inverted widefield microscope . Spheroids were treated with single-dose RT of varying doses . HPV-negative spheroids were irradiated on day 7, whereas HPV-positive spheroids were irradiated on day 14 after seeding. These timepoints were determined based on the faster growth rate of HPV-negative spheroids.Spheroid formation was achieved by using ultra-low attachment (ULA) culture plates . HPV-positive spheroids were grown in MEM supplemented with 20% FBS, 1% L-glutamine and 1% non-essential amino acids, and HPV-negative spheroids were grown in DMEM supplemented with 20% FBS and 1% sodium pyruvate. The seeding densities were 5000, 4000 and 1000 cells per well for the HPV-positive (SCC154 and SCC47), CAL27 and FADU spheroids, respectively. Plates were centrifuged at 1300 RPM for 10 min at room temperature. Spheroids were allowed to rest for 72 h and were cultured in a humidified 5% CO6) or SCC154 cells (7.5 × 106) were subcutaneously injected in each flank of 6–7-week-old female nu/nu Naval Medical Research Institute (NMRI) mice . The mice were either treated with a vehicle or RT . RT was delivered via the Small Animal Radiation Research Platform . Dose calculations were performed with MuriPlan software  after cone beam CT. The dose was delivered using 220 kV photons, 13 mA and a 10 × 10 mm collimator.CAL27 cells (2 × 10Tumor volumes (V = π/6xd_1xd_2xd_3) were determined with caliper measurements. The body weight and welfare of the mice were monitored daily during treatment and three times per week during follow-up. The experiment was performed according to the Ethical Committee for Animal Experimentation of KU Leuven .After 35 (FADU) or 60  days in culture, spheroids were incubated with 20 μg/mL of pimonidazole  for 2 h at 37 °C, and the spheroids were fixed with 4% paraformaldehyde (PFA). After permeabilization with 0.1% Triton X-100  in 0.5% BSA-PBS, cells were stained with the mouse primary antibody against Ki-67  or the rabbit primary antibody against pimonidazole  for 24 h at 4 °C, followed by staining with goat anti-mouse 488 Alexa Fluor secondary antibody  or with goat anti-rabbit 647 Alexa Fluor secondary antibody , respectively, for 24 h at 4 °C. Nuclei were counterstained with DAPI . Immunofluorescence images were acquired with a confocal fluorescence microscope  and were quantified using Fiji (ImageJ).t-test or two-way ANOVA in Graphpad prism 6.01 . p-values < 0.05 were considered statistically significant.Statistical significance was tested by using the two-tailed


Accepting HPV vaccination is an essential factor affecting vaccine promotion among MSM. We aimed to explore the factors related to HPV vaccination intention among MSM and analyze the potential relationship between these factors. (2) Methods: We adopted a nonprobability sampling method to recruit MSM volunteers. Information collection included general demographics, personal behavioral characteristics, knowledge of HPV/vaccine attitudes, and risk threat perception. Bayesian networks (BNs) were used to analyze the data statistically. (3) Results: The BNs showed that perceived HPV risk and attitudes toward vaccine promotion were directly correlated factors, whereas knowledge of HPV/vaccines, a history of HIV testing, and the number of male sexual partners in the past 6 months were indirectly correlated factors. (4) Conclusions: The results of this study illustrate that MSM have a relatively high propensity to receive HPV vaccines. The proposal that strengthening the propagation of HPV and its relevant vaccines, encouraging MSM to undergo regular corresponding tests, and improving their risk perception of HPV infection can be raised to promote HPV vaccination among MSM. Papillomaviridae that causes squamous epithelial hyperplasia of the skin and is the most common sexually transmitted virus in the world [Helicobacter pylori [Human papillomavirus (HPV) is a spherical DNA virus of family he world . HPV infhe world ,3,4. It r pylori . Most HPr pylori . Sexual r pylori ,7. In wor pylori ,8. In cor pylori . In Chinr pylori .The most cost-effective way to prevent HPV infection is to be vaccinated against HPV . SeveralIn addition to national policy and vaccine availability, the willingness to receive the HPV vaccine is also an essential factor influencing the use of the vaccine among the MSM population. Therefore, a comprehensive analysis of the factors associated with HPV vaccination intention among the MSM population is vital to increase HPV vaccination coverage among the MSM population. Previous studies have used logistic regression based on independent conditions to explore factors associated with vaccination intention and have used odds ratios (ORs) to reflect the degree of association ,22,23. IThis study is a cross-sectional study. Nonprobability sampling was used to recruit MSM respondents from non-governmental organizations owing to the specificity of this population. Relevant data were collected by means of a web-based questionnaire distributed via WeChat, and respondents voluntarily chose to fill out the web-based questionnaire after understanding the purpose of the study and providing informed consent. The participants who completed the questionnaire were given a reward of 10 RMB. The study was approved by the Ethics Committee of Chongqing Medical University .A total of 1477 MSM were recruited, excluding 422 who took less than 2 min to fill in the form, 69 who were younger than 14 years or older than 55 years, and 17 who had missing or incorrect answers in the logic check. A total of 969 MSM were finally included in the study.The web-based questionnaire for this study consisted of three main sections focusing on the current status of HPV vaccination intention and its associated factors among the MSM population. The first section included general demographic characteristics, such as the age, education level, and monthly disposable income of the individual. The second section investigated the behavioral characteristics of the MSM population, such as the number of male and female partners in the last 6 months and whether they had been actively tested for human immunodeficiency virus (HIV) or HPV, etc. Finally, the third section investigated knowledge attitudes and perceived risk threat of HPV/vaccines in the MSM population, focusing on variables such as knowledge about HPV disease and vaccines, perceived HPV risk, and attitudes toward vaccine promotion. Twelve questions focused on knowledge of HPV disease and vaccines, in which a score of 10 or more indicated a high level of knowledge, and a score less than 10 indicated a medium or low level of knowledge.https://www.r-project.org accessed on 12 August 2022). Count data were analyzed descriptively using rates or composition ratios. The stats package in R software was used to analyze correlated factors. One-way logistic regression analysis was used for the initial screening of correlated factors, and the test level alpha was set at 0.05. The variables that were statistically significant in the one-way analysis were selected as the network nodes for BN construction, and the bnlearn package in R software was used to construct the BN model. The Tabu search algorithm was used for the structural learning of the BNs, and the great likelihood estimation method was used for the parameter learning of the BNs. Furthermore, GeNIe software  was used for BN model visualization and conditional probability inference.Statistical analysis was performed using R software .p < 0.05) in the one-way logistic regression analysis were used as the sample information, and BNs were used to construct a model of the factors associated with the willingness to be vaccinated against HPV in the MSM population, with 14 directed edges at 12 nodes. As shown in The statistically significant variables  are a high-risk group for HPV infection. In this study, 82.77% of MSM were willing to be vaccinated against HPV and had a high propensity to be vaccinated. Therefore, the authorities should consider including the MSM population in the national vaccination program. In addition, using a Bayesian network model, we identified HPV risk perception and vaccine promotion attitudes as direct correlates of HPV vaccination among MSM. Moreover, HPV/vaccine knowledge, HIV testing history, and the number of male sexual partners in the past 6 months are indirect correlates. Therefore, we should strengthen the promotion of knowledge about HPV and HPV vaccines, encourage MSM to take the initiative to be tested regularly for HIV/HPV, and increase the risk perception of HPV infection to aid the promotion of HPV vaccination among MSM.


Few studies have examined whether UK military veterans are at an increased risk of dementia. We explored the risk of dementia in Scottish military veterans aged up to 73 years in comparison with people who have never served.Retrospective cohort study of 78 000 veterans and 253 000 people with no record of service, matched for age, sex and area of residence, with up to 37 years follow-up, using Cox proportional hazard analysis to compare risk of dementia in veterans and non-veterans, overall and by subgroup.p = 0.879 (landmark age: 50 years), with no difference for men but increased risk in veteran women and Early Service Leavers. Post-traumatic stress disorder (PTSD) was associated with a higher risk of dementia in both veterans and non-veterans, although possibly to a lesser degree in veterans. A history of mood disorder was strongly associated with developing dementia, greater in veterans than in non-veterans, odds ratio 1.54, 95% CI 1.01–2.35, p = 0.045.Dementia was recorded in 0.2% of both veterans and non-veterans overall, Cox proportional hazard ratio 0.98, 95% confidence interval (CI) 0.82–1.19, There was no evidence to suggest that military service increased the risk of dementia, although this may change as the cohort ages. The well-documented association with PTSD shows no evidence of being stronger in veterans; by contrast, the association of mood disorder with dementia is much stronger in veterans. Healthcare providers should carefully assess the cognitive status of older veterans presenting with depressive illness in order to identify early dementia and ensure optimum management. The reduction in premature mortality in the latter half of the 21st century has paradoxically created a new challenge; more people than ever are living with dementia, creating a major burden on health and social care. The term ‘dementia’ covers a number of clinical entities of which the most common are Alzheimer's disease, diffuse Lewy body dementia, fronto-temporal dementia and vascular dementia  . Veterans were eligible for inclusion if they were born between 1 January 1945 and 31 December 1995 and were registered with National Health Service (NHS) Scotland both before and after service. The study follows on from the Scottish Veterans Health Study, using similar methodology which is described elsewhere  is provided by the Scottish Index of Multiple Deprivation (SIMD), which is based on 6505 data zones, derived from postcode of residence, with a mean population of 800. Deprivation status is calculated from information on income, employment, health, education (including skills and training), housing, crime, and access to services. The SIMD has been used to derive quintiles of SES for the Scottish population; ranging from 1 (most deprived) to 5 (least deprived)  as the censor time  veterans and 252 637 (100%) non-veterans were included in the analyses. There were 70 581 (90.3%) male veterans and 7573 (9.7%) female, in accordance with the gender balance of the armed forces. The mean period of follow-up was 32.5 years, and there were a total of 10.6 million person-years of follow-up among veterans and non-veterans combined. The oldest cohort members were 73 years of age at the end of follow-up.p = 0.879. The Nelson–Aalen cumulative hazard plot is shown in p = 0.481. There was an increased risk for veteran women which almost achieved statistical significance, HR 1.76, 95% CI 0.95–3.23, p = 0.070, although the number of cases was small.By the end of follow-up, 154 (0.20%) veterans had a record of a dementia diagnosis, compared with 490 (0.20%) non-veterans . Analysep = 0.040. Controlling for SES only slightly reduced the excess risk in ESL, HR 1.41, 95% CI 0.99–2.03, p = 0.059, but it was attenuated after adjusting for mood disorder, HR 1.31, 95% CI 0.92–1.87, p = 0.138. Analysis of HR by birth cohort showed no significant difference for any group  of the veterans with dementia had died, compared with 254 (51.8%) of the non-veterans. The difference was statistically significant, OR 0.81, 95% CI 0.66–1.00, p < 0.001. For veterans, the increase in risk was only threefold; 0.57% of those with a record of PTSD developed dementia compared with 0.19% of those without PTSD, OR 2.98, 95% CI 1.32–6.72, p = 0.006. Among those with a PTSD diagnosis, we found no evidence that veterans were at an increased risk of developing dementia compared with non-veterans, OR 0.73, 95% CI 0.28–1.88, p = 0.511. Testing for interaction was non-significant, p = 0.411. In all veteran cases, and the majority of non-veterans, the PTSD diagnosis preceded diagnosis of dementia, although in a small number of cases the interval between diagnoses was short, raising the possibility of the early symptoms of dementia having been misclassified as PTSD.A diagnosis of PTSD at any time increased the likelihood of developing dementia in both veterans and non-veterans, but to a greater extent in non-veterans. For non-veterans, there was a fourfold increase in risk. A total of 0.78% of those with a record of PTSD developed dementia, compared with 0.19% of those without PTSD, OR 4.13, 95% CI 2.47–6.89, p < 0.001. The increase in risk was lower in non-veterans; 0.88% of those with a history of mood disorder were diagnosed with dementia, compared with 0.17% of those without a record of mood disorder, OR 5.07, 95% CI 3.90–6.60, p < 0.001. Among those with a record of mood disorder, the risk of dementia was significantly higher in veterans compared with non-veterans, OR 1.54, 95% CI 1.01–2.35, p = 0.045. Testing for interaction closely approached statistical significance, p = 0.067.Mood disorder was also strongly associated with dementia, the diagnosis of mood disorder predating dementia, sometimes by many years, in the majority of cases. Of veterans who had a record of mood disorder at any time, 1.36% developed dementia, compared with 0.16% of those without mood disorder, OR 8.43, 95% CI 5.72–12.42, Despite the theoretical increased risk of dementia in veterans based on their known increased risks of cardiovascular disease .


Primary bladder tumors have a high degree of malignancy. To investigate the expression of human papillomavirus type 16 (HPV-16) in primary bladder tumors and the loss of cell differentiation and to explore the significance of HPV-16 detection, it is expected to be a disease. Treatment provides a theoretical basis. n = 28) and HPV unrelated group (n = 29). The general data of patients were collected, the expression of HPV-16 in bladder tissue samples was detected, and the correlation between pathological parameters and HPV-16 expression was analyzed.  Fifty-seven patients with primary bladder tumors admitted to our hospital from January 2019 to January 2022 were selected as the research subjects, and they were divided into HPV-related groups according to the human papillomavirus (HPV) infection status (P > 0.05); there was no significant difference in postoperative pathological expression (high expression and low expression) of patients (P > 0.05); there was no statistical difference in age and gender between HPV-related and HPV-unrelated groups (P > 0.05), HPV-related group and HPV-unrelated group compared daily regular drinking and smoking status, the difference was statistically significant (P < 0.05); HPV-16 expression was not correlated with tumor differentiation degree and age of patients (P > 0.05); the area under the curve (AUC) of HPV-16 for judging primary bladder tumor expression and cellular molecular deletion was 0.891, with a sensitivity of 83.94% and a specificity of 88.57%.  Among HPV subtypes, HPV 16 subtype accounted for the highest proportion, followed by HPV-18 and HPV-6 subtypes; there was no significant difference in tumor stage  between the HPV-related group and the HPV-unrelated group .  HPV-16 is an upper, expressed in primary bladder tumors and will participate in the differentiation and loss of cells, which can provide effective guidance and basis for the diagnosis of primary bladder tumors, which is an important factor for judging the pathological stage and prognosis of patients and can provide a theoretical reference for the formulation of therapeutic measures. The latest reported data show that bladder tumor is a highly malignant tumor. In China, the incidence of bladder cancer ranks first in malignant tumors of the urinary system, and transitional cell carcinoma (TCC) accounts for about 90% of the pathological types of bladder tumors. However, the understanding of the pathogenesis of primary bladder tumors is low, and exposure to ionizing radiation, aromatic amines, and cyclophosphamide are currently identified as risk factors . HPV maiBased on this, 57 patients with primary bladder tumors admitted to our hospital between January 2019 and January 2022 were opted as the study subjects to preliminarily investigate HPV-16 expression and analyze the relationship with patient prognosis, hoping to provide morphological evidence for disease healing, as reported below.n = 28); 7 females; 21 males; aged 27-64 years; mean age (61.46 ± 3.58) years; HPV subtypes: HPV-6 : 3, HPV-16 : 8, HPV-18 : 5, HPV-26 : 1, HPV-39 : 2, HPV-45 : 1, HPV-51 : 1, HPV-53 : 2, HPV-56 : 1, HPV-59 : 1, HPV-66 : 1, HPV-68 : 1, and HPV-82 : 1; tumor stage: 9, stage a: 10, and stage 2a: 9; postoperative pathology: upper expression in 17 and bottom expression in 11. In the HPV-unrelated cluster (n = 29), there were 8 females and 21 males, aged 27–64 years, with a mean age of (63.10 ± 3.29) years; tumor stage: 8 situations of stage 1, 10 situations of stage a, and 11 situations of stage 2a; postoperative pathology: 7 situations of upper expression and 21 situations of bottom expression. This study has been approved by the ethics department, and patients are aware of the study.Fifty-seven patients with primary bladder tumors admitted to our hospital from January 2019 to January 2022 were opted as the study subjects and divided into two clusters according to human papillomavirus (HPV) infection. HPV-related cluster (Inclusion criteria: (1) HPV positive; (2) older than 18 years old; (3) cervical biopsy or staining pathology diagnosis [iagnosis , diagnosExclusion criteria: (1) infectious diseases; (2) immune diseases; (3) combined with bladder cancer outside the tumor; (4) bottom compliance; (5) organ damage; (6) lactating or pregnant women.The specimens were opted from those who were pathologically diagnosed as primary bladder tumors. After the specimens were obtained, they were placed in EP tubes and then stored in -80°C refrigerator. Various data were collected from the patients, including age, gender, alcohol consumption, and smoking status.DNA sample extraction: centrifuge the cell suspension, extract DNA, and perform various operations according to the kit instructions, then uniformly dilute the DNA concentration to 100 ng/μL and store in -20°C equipment.Amplification PCR: PCR amplification is performed using primer nucleotide sequences.PCR product electrophoresis: agarose gel electrophoresis of PCR products, 10 μL of PCR products were subjected to agarose gel electrophoresis (1.0%) and photographed by Tianneng gel imaging system.Judgment result: if there is a bright band at the 217 bp position, and the prejudgment result is loaded at the same time, it is confirmed that the HPV-16 test is a positive result, and the product is analyzed by electrophoresis to verify the sequencing result.HPV subtype analysis of patients: including HPV-6, HPV-16, HPV-18, HPV-26, HPV-39, HPV-45, HPV-51, HPV-53, HPV-56, HPV-59, HPV-66, HPV-68, HPV-82 HPV subtypes.Tumor staging: count the number of situations of stage 1, stage a, and stage 2a, and calculate the proportion.Postoperative pathological expression: count the number of situations with upper expression and bottom expression, and calculate the proportion.General information: including age, gender, alcohol consumption , and smoking .Logistic regression analysis: the correlation between HPV-16 expression and the degree of tumor differentiation and age of patients.The value of HPV-16 in judging the expression of primary bladder tumor and the value of cell and molecular deletion: calculate the diagnostic sensitivity and specificity of HPV-16.Analysis of the prognosis of patients: count the number of situations of single tumor and multiple tumors, count the number of patients with tumor-free survival, recurrence, and death, and calculate the proportion.n(%), and the contrastion between clusters was performed by the χ2 test; the measurement data that obeyed the normal distribution was expressed as   between the HPV-related cluster and the HPV-unrelated cluster ( > 0.05) .P > 0.05)  of patients ( > 0.05) .P > 0.05). There was a notable variation between the HPV-related and HPV-unrelated clusters in daily regular drinking and smoking status (P < 0.05) (None notable variation in age and gender between the HPV-related cluster and the HPV-unrelated cluster ( < 0.05) .P > 0.05) (None correlation between the expression of HPV-16 and the degree of tumor differentiation and age of the patients ( > 0.05) .The area under the curve (AUC) of HPV-16 for judging primary bladder tumor expression and cellular molecular deletion was 0.891, with a sensitivity of 83.94% and a specificity of 88.57% .57 patients, including 11 patients with single tumor and 46 patients with multiple tumors, were followed-up for 3 years. The follow-up found that 37 patients survived tumor-free, 17 patients recurred, and 3 patients died .Males aged 40-70 are the main cluster of primary bladder tumors, with invasive growth and papillary growth as the main growth patterns, and the results are in good agreement with the results of most domestic studies . RegardiP > 0.05). The results of this study are consistent with other research results. Sex is not upper, which is related to the pathological composition and case conditions [Followed by HPV-18 and HPV-6 subtypes, no notable variation in tumor stage and pathological expression between the two clusters of patients, improving the comparability of the data between the two clusters , 12. In nditions , 14. Hownditions , 16. Thenditions , 18.Research on the expression of HPV-16 in primary bladder tumors and the analysis of the loss of cell differentiation can provide theoretical reference value for the judgment of disease status , 20. EveHPV-16 is an upper, expressed in primary bladder tumors and participates in cell differentiation and deletion. It may provide effective guidance and basis for the diagnosis of primary bladder tumors. It is an important factor in judging the pathological stage and prognosis of patients, and can be used for healing measures to provide theoretical reference.


This study investigated vaccine hesitancy in caregivers of girls in Grade 4 to 7 aged ≥9 years attending public schools in Sedibeng District. A cross-sectional survey using a self-administered questionnaire was conducted among caregivers of age-eligible girls attending all public schools in Sedibeng District with first dose HPV vaccination coverage of <70%. The questionnaire included demographics; HPV vaccination status of girls; reasons for not being vaccinated; and a 5-item tool measuring the determinants of vaccine hesitancy , using a 7-point Likert scale. Data were coded and captured on Microsoft Excel®. Except for collective responsibility which was reverse scored, the other 5C items  were captured as follows: 1 = strongly disagree, 2 = moderately disagree, 3 = slightly disagree, 4 = neutral, 5 = slightly agree, 6 = moderately agree and 7 = strongly agree. Descriptive and inferential statistical analyses were conducted using Epi InfoTM. Of the principals of all schools with <70% HPV vaccination coverage, 69.6% (32/46) gave permission. The response rate from caregivers of girls present on the day of data collection was 36.8% , with 67.1%  of respondents reporting that their daughters had received ≥1 dose of HPV vaccine. Only 63.1% (370/586) of respondents with unvaccinated daughters answered the question on reasons, with 49.2% (182/370) reporting reasons related to vaccine hesitancy. Statistically significant differences between caregivers of vaccinated and unvaccinated daughters were identified for four of the five determinants of vaccine hesitancy: confidence (vaccinated group higher), complacency (unvaccinated group higher), constraints (unvaccinated group higher) and collective responsibility (vaccinated group higher). This is the first South African study to (a) report results of the 5C scale, which was found to be very useful for predicting vaccination uptake; and (b) confirm that the relatively low HPV vaccination coverage in Sedibeng District is largely driven by reasons related to vaccine hesitancy.Girls aged ≥9 years attending South African public sector schools are provided with free human papillomavirus (HPV) vaccination, through a schools-based programme. HPV vaccine misinformation spread  While first dose HPV vaccination coverage in 2014 was relatively high at 86.6% (https://bit.ly/3nHAQXI).South Africa is home to <1% of the global population of women aged ≥15 years, yet bears 2.3% and 1.8% of global new cervical cancer cases and deaths from cervical cancer respectively . Also, Sedibeng District had the lowest HPV vaccination coverage in Gauteng Province in 2018 . This study aimed to investigate vaccine hesitancy in caregivers of girls in Grades 4 to 7 aged ≥9 years attending public schools in the Sedibeng District of Gauteng. Objectives included investigating reasons why girls had not been vaccinated, and measuring and comparing levels of determinants of vaccine hesitancy between caregivers of vaccinated and unvaccinated girls.https://municipalities.co.za/overview/114/sedibeng-district-municipality). In 2019 there were 156 public sector primary schools in the Sedibeng District, of which 46 had HPV vaccination first dose coverage below 70% in 2018 . All 46 schools were targeted by the survey, which was conducted from 26 August to 20 September 2019. This cross-sectional survey utilized a questionnaire which was self-administered by caregivers of girls in Grades 4 to 7 aged ≥9 years, attending the schools where permission had been granted by the school principals.The Sedibeng District is comprised of three local municipalities: Emfuleni, Lesedi and Midvaal Local Municipalities , the contact details of the researcher should further clarity be required, the aim and objectives of the survey, and ended with a consent statement. Thereafter there were sections on demographics of both caregivers and girls; the HPV vaccination status of the girls; a question on reasons for the girl not being vaccinated, with 15 selection options, the last option being “other (please specify)”; and a 5-item tool for measuring the determinants of vaccine hesitancy, based on the 5C model developed and proposed by Betsch et al. . ParticiWhen the WHO first defined vaccine hesitancy in 2014, they had built upon earlier work identifying the key drivers of vaccine hesitancy as confidence ; complacency (perception of low risk from vaccine-preventable disease [VPD]) and convenience , known as the “3Cs model” . Most vaIt has been argued that the term “vaccine hesitant” is a misnomer for a parent whose child is unvaccinated because of practical barriers to vaccination , 10. In The 5C model has subsequently become an important tool for measuring the determinants of vaccine hesitancy, and has been used for this purpose on various populations in different countries, including Hong Kong nurses ; USA patThe first author trained a team of four research assistants  to assist with (a) obtaining permission from school principals; (b) conducting information sessions with class teachers and learners; and (c) recording the numbers of surveys distributed to, and collected from each class at each school.Data collection commenced after obtaining (a) ethical clearance from the Sefako Makgatho University Research Ethics Committee (SMUREC/P/104/2019:PG), (b) permission from the Gauteng Department of Education and Sedibeng District Department of Education, and (c) permission from the individual school principals. Data were collected from 26 August to 20 September 2019. The questionnaire included a consent statement as well as the ethics clearance number. At each of the schools where the principal had given permission for data collection, a team member had sessions with the class teachers and female learners, where they explained the purpose of the survey, and the importance of giving the questionnaires to their caregivers, and returning them back to class.Self-administered questionnaires in the language of choice, were handed out in sealed envelopes to female learners in Grades 4 to 7 by a team member, who kept a record of the number of envelopes distributed per class in each school. The sealed envelope also contained an empty envelope in which to place the completed questionnaire for the learner to bring back to school. A sealed box with a posting slot for this purpose was placed in each classroom. Teachers were requested to remind the girls about returning the questionnaires daily, until the boxes were collected the following week by a team member, who counted and recorded the numbers of returned envelopes for each class.The raw data from the questionnaires were coded and captured on a Microsoft Excel® 2016  spreadsheet. The free text that respondents provided for the “other” reason option for not being vaccinated, was captured verbatim. Except for collective responsibility which was reverse scored, the 5C items were captured as follows: 1 = strongly disagree, 2 = moderately disagree, 3 = slightly disagree, 4 = neutral, 5 = slightly agree, 6 = moderately agree and 7 = strongly agree. Data were checked to ensure reliability of data entry. The checked spreadsheet was imported into Microsoft Access® 2016  for cleaning. Access® queries were run to identify records containing inconsistent data, and where these were found (e.g.: records where reasons for not being vaccinated were given for girls who were vaccinated), the relevant questionnaire was reviewed and any data capture errors were corrected.The questionnaire was pre-tested in a pilot study conducted in one of the primary schools in the Sedibeng District, which had >70% HPV vaccination coverage and thus was not included in the study. Pre-testing aimed to ascertain if the questions were well-comprehended and appropriate for eliciting the required information. Furthermore, piloting the study provided an overview of the actual study on a small scale, which assisted in making the necessary changes to the data collection instrument and the data collection process, prior to the commencement of the actual study. After the pilot study was conducted, necessary amendments and additions were made to the data collection instrument and process. The final data collection tool is available in t-test for measuring the statistical significance of the differences between the means of the 5C scores of caregivers of vaccinated and unvaccinated girls. P-values < 0.05 were considered statistically significant.Free text reasons were analyzed and coded by the first author, who created new codes for reasons that did not fit with the original list of reasons options. These codes were then checked by one of the research assistants, with discrepancies being checked by an independent researcher working within the over-arching project, who made the final decision and validated all the coding. A category for reasons related to vaccine hesitancy was created, based on the WHO definition of a “delay in acceptance or refusal of vaccination despite availability of vaccination services” . Data weThe pilot study was conducted on 67 volunteers. The maximum time taken to complete the questionnaire was 10 min. During piloting the principal advised that the majority of the learners spoke Afrikaans as their home language, thus the need for translating the data collection tool into Afrikaans was identified. No further changes were made to the data collection tool or data collection process.Of all schools requested to participate in the study, permission was denied by 30.4% (14/46). The reasons given by the 14 principals for denying permission included (a) the survey was being conducted at a very busy time in the school calendar ; (b) the perception that the majority of caregivers are opposed to HPV vaccination, thus the principal did not want to upset them ; (c) no reasons given . Thus permission to conduct the study was granted for 69.6% (32/46) of the schools. Of these 32 schools, all girls in Grades 4 to 7 present on the day of data collection  were handed questionnaires to take home for their caregivers to complete. In total, 63.9%  of questionnaires were returned. Of these 17.9%  were returned without any questions being answered, with 24.8% (137/553) of these being over-written with a statement indicating that the respondents do not give consent for HPV vaccination, i.e. these respondents apparently mistook the questionnaire for a consent form for HPV vaccination. Of the remaining questionnaires, the question on HPV vaccination status of the girls was answered by 1,784 respondents, 2 of whom had daughters too young to be vaccinated (i.e. <9 years old). Thus the final sample consisted of 1,782, giving a response rate of 36.8% .Data on age of caregiver was provided by 1,472 respondents, with the median age being 38.0 years and the mean being 40.5 years . The majority were aged between 30 and 49 years  and were the biological parents of the girls . The demographic characteristics of the respondents are detailed in Data on date of birth of girls was provided by 1,686 respondents, with the median age being 11.4 years and the mean being 11.5 years . Of the 96 girls for whom the respondents had not provided a date of birth, 66.7% (64/96) had received ≥1 dose of HPV vaccine, thus these girls must have been aged ≥9 years. None of the 32 respondents who did not provide a date of birth and whose daughters were not vaccinated, gave the reason “my daughter is too young.” It was thus assumed that all 32 girls without a date of birth were age-eligible for HPV vaccination.The majority of girls were African , with 16.0%  being white, 1.2%  being of mixed descent and 0.2%  being Asian. The majority  of girls were in Grades 4 and 5. The daughters of 67.1%  of respondents had received ≥1 dose of HPV vaccine. Of respondents whose daughters were vaccinated, 1,074 answered the question on number of doses received, and of these, 58.5%  reported that their daughter had received both HPV vaccine doses. Of respondents who answered the question on place where vaccination was received, 90.8%  reported that their daughters had been vaccinated at school. Caregivers of vaccinated girls were also asked a question on what grade their daughter was in, at the time she was vaccinated. Of those who answered this question, 80.4% (707/879) were vaccinated when they were in Grade 4; 13.4% (118/879) when they were in Grade 5; 1.5% (13/879) when they were in Grade 6; 0.8% (7/879) when they were in Grade 7; and 3.9% (34/879) were vaccinated in two separate grades.Respondents were allowed to select more than one reason for their daughter being unvaccinated. Of respondents whose daughters were unvaccinated, 63.1% (370/586) answered this question, and gave a total of 654 reasons . Of respOf the schools participating in the survey, 87.5% (28/32) were named by respondents who gave the reason “I am not aware of a HPV vaccination programme at school.” The number of girls at each school who were unvaccinated because of this reason, ranged from 1 to 19, with 25% (7/28) of the schools having ≥10 girls being unvaccinated because of this reason.confidence (higher in the vaccinated group), complacency (higher in the unvaccinated group), constraints (higher in the unvaccinated group) and collective responsibility (higher in the vaccinated group). There was very little difference between the mean scores of the two groups for calculation. See t-test p-values.Given that all South African public sector schools providing education to Grade 4 girls, are required by the DBE to participate in the two annual HPV vaccination schools-based campaigns, the relatively low permission rate of 69.6% obtained from school principals was unexpected. However, this permission rate was considerably higher than the 2.3% of South African private sector school principals who had allowed caregivers to participate in a similar survey that we had conducted online in 2018  was found to be 62.2%, which is higher than the official 2018 first dose coverage of 54.2% for Grade 4 girls attending the 32 schools participating in this study. This relatively large discrepancy may be explained by the lack of participation of schools managed by principals who were vaccine hesitant/vaccine deniers, and also the relatively large number of caregivers who either mistook the survey for an informed consent form for HPV vaccination, or used the opportunity to express their rejection of HPV vaccination.It is well-documented that vaccine hesitancy is a major driver of low HPV vaccination uptake globally , 8, 18. We had anticipated that reasons related to vaccine hesitancy would feature prominently in our study, since Sedibeng District was affected by anti-vaccination lobbying on social media during the HPV vaccination programme roll-out in 2014, suggesting that this community is susceptible to believing in social media misinformation. In essence, the reasons related to vaccine hesitancy reported by the respondents to our survey, were a reflection of most of the negative sentiments expressed by the public in response to an HPV vaccination-related Facebook post by the Western Cape Department of Health in 2019, six months prior to our survey . Our finConcerns about the safety of HPV vaccines (or perceived harms of HPV vaccines) has been identified as a major driver of HPV vaccine hesitancy in Europe  and the Since it has been reported that girls sometimes deliberately don't attend school on HPV vaccination day in order to avoid being vaccinated , we had The finding that almost 47% of respondents whose daughters were unvaccinated, were not aware that there was an HPV vaccination programme at their daughter's school, was highly concerning. While this reason for being unvaccinated was reported by respondents with daughters at 28 of the 32 schools, 7 schools were more affected than the others, having ≥10 girls being unvaccinated because of this reason. This may be an indication of gaps in the communication strategy being used for relaying information about the programme to caregivers. The ISHP strategy relies on the relevant class teachers distributing information packages to the girls in their class, who are expected to take these home and give them to their caregivers, and then return the signed informed consent forms to their class teachers before vaccination day , which aWe had opted to use the 5C scale for investigating vaccine hesitancy, based on the findings from a Western Cape study illustrating the suitability of this model for investigating vaccine hesitancy in South Africa . We founThis study has important limitations that must be considered when interpreting the results. First, while we endeavored to include all schools with <70% first dose HPV vaccination coverage, we were unsuccessful. Furthermore, at least 5 of the 14 schools that were not permitted to participate, were managed by principals who may have been either HPV vaccine hesitant, or HPV vaccine deniers. Three of these principals did not want to upset the majority of caregivers of girls attending their schools, who they described as being opposed to HPV vaccination. Since school principals are highly influential role models for their staff and the broader community, and the success of schools-based HPV vaccination programmes is significantly affected by the attitudes of school teachers , these eSecond, the relatively low response rate from caregivers has impacted negatively on the representativeness of this survey, by introducing volunteer bias. The direction of this bias may be explained by the relatively large proportion of questionnaires being returned with all questions being unanswered, including a relatively large number of caregivers using the opportunity to express their opposition to giving consent for their daughters to receive HPV vaccination. It is thus reasonable to suggest that volunteer bias may have resulted in an overestimation of HPV vaccination coverage, and an underestimation of vaccine hesitancy. The former assumption is supported by our finding that the first dose HPV vaccination coverage of Grade 5 girls, is considerably higher than the official coverage reported for 2018, the year that these girls were vaccinated.Finally, this study may have been subject to recall bias, especially for caregivers of girls who were in Grades 6 and 7, who had been offered HPV vaccination while in grade 4 in 2017 and 2016 respectively. These caregivers had reported a higher ≥1 dose HPV vaccine coverage by their daughters (72.4% and 71.6% respectively) than caregivers of girls in Grades 4 and 5 (64.7% and 62.7% respectively). However, the average official first dose HPV vaccine coverage for 2017 was 77.8%  compared to 72.4% reported by caregivers of Grade 6 girls in this study, while that for 2016 was 82.6% compared to 71.6% for Grade 7 girls in this study. This indicates that recall bias may have resulted in an underestimation of HPV vaccination coverage of older girls.This study is the first South African study to report results of the 5C scale, which was used to measure HPV vaccine hesitancy, with 4 of the 5 constructs found to be very useful for predicting vaccination uptake. It is also the first report confirming that the relatively low HPV vaccination coverage in Sedibeng District is largely driven by reasons related to vaccine hesitancy. What is clear from our results on reasons, is that the ISHP information packages are either not reaching all caregivers, or the language used in these information packages is not well-understood by all caregivers. This does not only pertain to the caregivers who reported being unaware of the HPV vaccination programme; it may also pertain to those reporting reasons related to vaccine hesitancy. Caregivers who do not fully understand the HPV vaccination information supplied by the ISHP may be less likely to allow their daughters to be vaccinated , and mayBest practice” and “worst practice” communication strategies used by schools in Sedibeng District need to be identified. This can be achieved in a relatively short space of time, since official coverage results are reported at school level, and it can safely be assumed that schools with very high coverage are using “best practice” communication strategies. While we have identified 7 schools where “worst practice” strategies are most likely being used, it is safer to assume that all 46 schools with low first dose HPV vaccination coverage may be using “worst practice” communication strategies. In addition, focus group discussions with caregivers at “worst practice” schools should be held in order to identify further specific concerns, information and communication gaps. These data will complement the results of this study, which should be used to update communication packages so that all relevant concerns are addressed. These focus group discussions should also be used to identify the communication strategies preferred by caregivers. Care must be taken to not only adopt the communication strategy preferred by the majority of caregivers, as none should be left uninformed. It could be that a combination of communication strategies will be required, in order to reach all caregivers.“Develop a standard operating procedure (SOP) for HPV vaccination communication, and mandate all public sector schools where HPV vaccination is offered, to follow this SOP. This SOP should be based on an analysis of the “best practice” and “worst practice” communication strategies, and educators  at all schools should receive training on how best to implement the SOP. While the focus of this training is capacity building of educators to promote HPV vaccination with confidence, it should also improve their role as advocates for public sector school health research and all health interventions offered through the ISHP.Conduct research to validate the 5C scale for South Africa. It is imperative to evaluate the success of the implementation of the “best practice” communication strategy intervention. This can easily be conducted by comparing pre- and post-intervention HPV vaccination coverage. However, comparing pre- and post-intervention vaccine hesitancy levels with a validated 5C tool will provide much-needed data that can be used to identify and address the drivers of vaccine hesitancy beyond the HPV vaccination programme. When conducting this future research, a concerted effort must be made to reach all caregivers and improve the response rate. The possibility that girls may not be taking ISHP information packages home to their caregivers as discussed above, may also have applied to the questionnaires for this survey, providing a further explanation for the low response rate. Although a much lower response rate was achieved in our online private sector survey .The South African Vaccination and Immunisation Centre, and the Network for Education and Support in Immunisation receive unrestricted educational grants from the vaccine industry. JM declares speaker/consultancy honoraria from the vaccine industry. FM is employed by the National Department of Health. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Circulating human papillomavirus DNA is a promising tool in the management of HPV-positive oropharyngeal cancer. The potential use of this tool will substantially change the way we treat, diagnose, and follow-up these patients.There has been a rising trend in HPV-induced head and neck cancers in the last several decades. This subgroup of squamous cell carcinoma is mostly located in the oropharynx and comprises a subset of patients who are typically younger and without the usual risk factors of smoking and alcohol use. As the prognosis of HPV-induced OPC is more favorable, there is a desire to properly select these patients for de-intensification protocols while identifying individuals who may suffer treatment failure. Here, we describe recent developments in circulating tumor HPV DNA as a marker of HPV-positive oropharyngeal cancer that can potentially be used as a diagnostic tool to stratify patients for de-escalation strategies and to survey for recurrence. Human papillomavirus (HPV)-induced oropharyngeal cancer (OPC) is a distinct entity with a rising incidence in the US and across the world . This suCirculating cell-free tumor DNA, also called liquid biopsy, is being used in several fields and can serve as a potential marker in cancers attributed to viruses ,7. SeverCirculating cell-free DNA (cfDNA) has been utilized in several fields of medicine since its advent in the mid-twentieth century. Cell-free DNA is fragmented DNA with an average size of 160–200 bp, and it is released into the blood and other body fluids through the apoptosis or necrosis of cells . It has The most common target for detecting ctDNA for HPV-OPC is the E6 or E7 open reading frame . One areIn addition to target variation, three different methods of assaying for ctDNA have been employed to date, most commonly quantitative PCR (qPCR), next-generation sequencing (NGS), and the more novel digital droplet PCR (ddPCR) ,21. AlthAn earlier stage at diagnosis correlates with improved survival in HPV-related oropharynx squamous cell carcinoma ,24. In tThe current gold standard for the diagnosis of HPV-OPC is tissue diagnosis on either the biopsy of the primary site or the fine needle aspiration of a metastatic lymph node, which is not without limitations. Clinical facilities in low-resource settings may not be well-suited to obtain, store, and process specimens for off-site histologic diagnosis. In addition, fine needle aspiration may yield insufficient cells for p16-immunohistochemistry, so additional biopsies may be necessary to obtain more tissue for determining p16 status. P16 status is critical to diagnostic work-up as it bears a strong prognostic indicator related to HPV-16 status . In thisTo date, the detection of ctDNA in HPV-OPC has been predicated on detecting circulating HPV DNA. This premise assumes that circulating HPV DNA is never observed in patients with either (1) Pnon-invasive HPV-associated lesions or (2) HPV infection without carcinogenesis. Indeed, Jeannot et al. detected no circulating HPV DNA in 18 patients with cervical intraepithelial neoplasia . HoweverFor diagnostic purposes, sensitivity is a measure of a screening tool’s efficacy. A high sensitivity ensures that few, if any, patients with HPV-OPC would test negative for ctDNA. For patients with a high pre-test probability of HPV-OPC in whom a false-negative ctDNA result is suspected, traditional tissue biopsy has a vital role.Siravegna et al. utilized ddPCR assays for the HPV E7 oncoprotein in HPV 16, 18, 33, 35, and 45 in 70 cases of HPV-OPC and 70 controls (HPV-negative (45) and non-cancer 25)) . With on) . With In a small study of 17 patients with HPV-related carcinoma of the cervix, Leung et al. tested the utility of a novel detection method called HPVseq, which utilizes 150 bp long “bait” probes to detect E6 and E7, as well as full-length HPV 16, 18, 33, 45, 31, 33, and 35. They reported a 100% sensitivity, as all patients had detectable ctDNA down to 0.03 copies/mL [No treatment modality for head and neck cancer is free of adverse effects. Surgery, radiation, and cytotoxic chemotherapeutics all bear permanent side effects such that the treatment of false positives is impermissible. Consequently, it is critical that diagnostic pathways incorporating ctDNA never produce a false positive.Ahn et al. performed qPCR using probes for HPV16 E6 and E7 and found a specificity of 100% in nine patients with HPV-negative tumors . Rettig Most methods for detecting ctDNA in HPV-OPC demonstrate sensitivity rates of 70–80%, although sensitivity can be improved depending on the used PCR technique. Increasing the number of targets  would likely increase sensitivity rates and diagnostic utility. However, increasing the number of targets may also introduce the added possibility of false positives and thereby decrease the specificity rate. As this area continues to expand, distinct screening and confirmation probe sets may be necessary.Pretreatment levels of circulating HPV DNA as a prognostic tool.As an early recurrence detection tool for follow-up after definitive treatment.As a tool to define the treatment strategy and implementation of deintensification therapies.Currently, surveillance is typically predicated on physical exams at regular intervals and post-treatment PET–CT scans. Unfortunately, even with state-of-the-art treatment, recurrence is not uncommon, reaching 26% (9–26%) in 5 years and 36% in 8 years ,38,39. CAlthough HPV-positivity is associated with a favorable prognosis, up to 35% of patients develop an aggressive disease . Currentp < 0.03). Furthermore, there was a significantly lower risk of tumor progression in patients with a significant increase in ctDNA two weeks into chemoradiation . Notably, smoking status did not predict freedom from persistence in this cohort.Emerging data suggest that ctHPV DNA bears promise as a prognostic tool; however, the timing of the ctDNA assessment and assay technique are critical. In a recent study by Cao et al. where HPV-OPC patients were all of the same stage (AJCC Stage III), both pretreatment ctDNA values and ctDNA two weeks into chemoradiation demonstrated prognostic value . Higher The timing of ctDNA measurement is important, as Dahlstrom et al. did not observe that patients without detectable pre-treatment ctDNA had a more favorable disease course than patients with detected ctDNA . One majThe importance of the assay technique was also highlighted in a study by Veyer et al. on 66 patients with HPV-OPC . Pre-treIn summary, higher levels of detectable ctDNA are associated with a worse prognosis. This finding appears most convincing when ddPCR is utilized instead of qPCR. As such, despite challenges, pre-treatment levels can potentially be considered prognostic for cases with classic HPV and oropharyngeal cancer with no or minimal other concurrent risk factors.Unlike HPV-negative head and neck cancer, HPV-OPC patients with recurrence can achieve high survival rates with salvage therapy. Surgery for locoregional recurrence has been shown to have two-year overall survival rate approaching 80%, and re-irradiation with either IMRT or protons can lead to a two-year overall survival rate of up to 70% . IncorpoSeveral studies have shown that detectable post-treatment ctDNA indicates recurrence and may precede imaging or exam findings . Cao et Rutkowski et al. explored the usage of post-treatment ctHPV DNA in 66 patients with HPV-OPC. An incomplete response  was associated with detectable ctDNA 12 weeks after treatment, as ctDNA was detected in 28% of patients with persistence and only in 4% of patients with a complete response. No patients showed negative ctDNA developed recurrence.Chera et al. studied 115 HPV-OPC patients treated with definitive CRT , and theRecently, the largest study of ctDNA for the detection of the occult occurrence of HPV-OPC was conducted by Berger et al. Digital droplet PCR was utilized as the assay technique. Of 1076 patients, post-treatment ctDNA was detected in 80 patients (7.4%). Only 2 of 80 patients with detectable ctDNA had no evidence of disease on the last follow-up. The remaining 98% either had recurrence at first follow-up or were subsequently found to have a biopsy-proven recurrence.In summary, the absence of detectable ctDNA after treatment for HPV-OPC portends a low risk of recurrence, and depending on the technique used, a positive assay for ctDNA appears to be associated with a more aggressive course. Larger prospective studies are needed to shed light on cases with several risk factors, potential HPV-negative second primaries, and the possibility of early intervention solely based on ctHPV DNA levels. In fact, several large prospective clinical trials are recruiting patients to answer these questions, with the targeted recruitment of several thousand participants, and results are expected in 2024–2025 .Several different strategies to de-escalate treatment are being studied in dozens of trials throughout the world. Such trials are attempting to reduce definitive radiotherapy dose, perform upfront surgery with modified adjuvant treatment, and/or use immunotherapy. Many of them face the challenge of selecting an appropriate patient population that will benefit from de-intensification while preserving oncologic outcomes. Potentially, ctHPV DNA can become such a marker. There are several ongoing clinical trials underway. The NCT05307939 trial being conducted at the Memorial Sloan Kettering Cancer Center uses ctHPV DNA levels to stratify patients for either observation or adjuvant radiation after surgery. If ctDNA is undetectable after transoral robotic surgery, the patient is stratified to a close observation group without any adjuvant therapy. The SIRS 2.0 trial uses a similar design but stratifies patients with undetectable levels of ctDNA into low and intermediate-risk groups based on final pathology (NCT05419089). The low-risk group receives close follow-up, and the intermediate-risk group receives de-intensified adjuvant RT. Investigators from the ReACT (NCT04900623) study set 40–50 Gy for the low-risk group and 50–60 Gy for the intermediate-risk group (risk group based on ctDNA levels).DART 2.0 (NCT05541016) is a study evaluating different de-intensification strategies using the ctDNA levels as a stratification variable. The low-risk group receives observation alone, while two other groups receive adjuvant radiation based on ctDNA levels.An emerging body of evidence suggests an evolving role for measuring ctDNA as an adjunct to standard care pathways for HPV-OPC. In the diagnostic setting, ctDNA  has a sensitivity in the 90–98% range and a specificity approaching 100%. On occasion, squamous cell carcinoma is diagnosed but with insufficient tissue for p16 staining (often on fine-needle aspiration samples). In such a situation, plasma ctDNA may help differentiate between HPV-negative and HPV-positive disease in lieu of repeating the biopsy. Moreover, ctDNA levels before treatment and in the initial phase of treatment have been shown to predict therapeutic responses. Furthermore, the detection of ctDNA after treatment has been shown to precede biopsy-proven recurrences by up to 4 months. Finally, ctDNA may have a role in cost-effectiveness. In a review, Haring et al. calculated the 2021 CMS costs for 10-year surveillance in OPSCC to be $17,381.68 . KowalchOngoing clinical trials are also exploring the role of ctDNA in selecting patients for de-intensification strategies. As new data emerge, the possibility of better care pathways heralds improved outcomes for patients with HPV-OPC.


CIGB-300 is a clinical-grade anti-Protein Kinase CK2 peptide, binding both its substrate’s phospho-acceptor site and the CK2α catalytic subunit. The cyclic p15 inhibitory domain of CIGB-300 was initially selected in a phage display library screen for its ability to bind the CK2 phospho-acceptor domain ofHPV-16 E7. However, the actual role of this targeting in CIGB-300 antitumoral mechanism remains unexplored. Here, we investigated the physical interaction of CIGB-300 with HPV-E7 and its impact on CK2-mediated phosphorylation. Hence, we studied the relevance of targeting E7 phosphorylation for the cytotoxic effect induced by CIGB-300. Finally, co-immunoprecipitation experiments followed by western blotting were performed to study the impact of the peptide on the E7–pRB interaction. Interestingly, we found a clear binding of CIGB-300 to the N terminal region of E7 proteins of the HPV-16 type. Accordingly, the in vivo physical interaction of the peptide with HPV-16 E7 reduced CK2-mediated phosphorylation of E7, as well as its binding to the tumor suppressor pRB. However, the targeting of E7 phosphorylation by CIGB-300 seemed to be dispensable for the induction of cell death in HPV-18 cervical cancer-derived C4-1 cells. These findings unveil novel molecular clues to the means by which CIGB-300 triggers cell death in cervical cancer cells. Cervical cancer is one of the most common causes of cancer-related death for women across the world. Human papillomavirus (HPV) is the causative agent of cervical cancer and a large number of other human malignancies ,2. AlthoHPV is a small non-enveloped DNA virus that infects keratinocytes in the differentiating epithelium of the skin and mucosae. The high-risk HPV-16 and HPV-18 subtypes are responsible for around 70% of all cervical cancer cases, whereas the low-risk types, including HPV-6 and HPV-11, cause benign genital warts (condylomas) . E7 targPhosphorylation is the major post-translational modification of E7 that affects some of these interactions. In particular, the presence of a CK2 phospho-acceptor site within the CR2 domain of E7 seems to enhance E7 interaction with different cellular target proteins, thereby increasing the ability of E7 to enhance cell proliferation and, potentially, malignant transformation ,12,13. RThe peptide CIGB-300 is a CK2 inhibitor with a dual mechanism; it binds to conserved phospho-acceptor sites on substrates, as well as directly targets the enzyme ,16,17. IFor the first time, we explored here the putative interaction of CIGB-300 with E7 oncoprotein in the cellular context, the relevance of the targeting, and its contribution to CIGB-300 cytotoxic effect. Our results demonstrate that the effect of CIGB-300 on CK2-mediated phosphorylation of E7 does not fully support its cytotoxic effect on cervical cancer. However, the interaction of the peptide with E7 impairs E7 ability to bind the pRB tumor suppressor. Altogether, the data presented here provide further molecular evidence as to the means by which CIGB-300 induces cell death in cervical cancer.TM), HeLa (CCL-2TM), SiHa (HTB-35TM), C4-1(CRL-1594TM) and HEK293 (CRL-1573TM) cell lines were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) , supplemented with 10% fetal bovine serum , penicillin–streptomycin (100 U/mL) and glutamine (300 μg/mL) .The CaSki  and was resuspended in dimethyl sulfoxide (DMSO) to obtain a 10 mM stock solution. The drugs were diluted directly into the growth media just prior to use.The plasmids expressing glutathione-S-transferasa (GST)-E7 from HPV subtypes 11, 16, and 18, GST alone, and the GST-fused N-terminal and C-terminal halves of HPV-16 E7 were described previously ,29.The plasmids pCMV:HPV-16 E7-FLAG-HA and pGEX:HPV-18E7 were a kind gifts from Karl Münger .2 (Solution A) was prepared and added dropwise to Solution B (2 × HBS), followed by 30 min of incubation at room temperature. The transfection mix was then added to the appropriate dish. Transfected cells were incubated at 37 °C for 48 h in a humidified CO2 incubator and then harvested for further analysis. Solutions A and B were prepared as described ). The cellular lysates were cleared by centrifugation, and 225 µL of total protein extract was incubated with biotin-tagged CIGB-300 (100 μM) or biotin-tagged scrambled peptide (10 mg/mL) for 2 h at 4 °C, then added to 20 µL of pre-equilibrated streptavidin-coated magnetic Sepharose beads  and incubated 1 h at 4 °C. The beads were then collected using a magnetic rack and extensively washed with cold RGMT. The streptavidin beads bound to CIGB-300-interacting proteins were resuspended directly in 2X SDS-PAGE sample buffer, resolved on a 15% SDS-PAGE gel and analyzed by western blot.The E7-CIGB-300 interaction was evaluated by in vitro/in vivo pull-down followed by western blot experiments. For the in vitro pull-down, the cells were seeded in 175 cm dishes and incubated to a confluency of 60–70%. Afterward, the cells were washed, collected, and lysed in lysis buffer RGMT  or PBS for 30 min at 37 °C in 5% CO2) in the presence of 10 nM ATP for 15 min at 30 °C. CIGB-300 was incubated with the GST-fusion protein on a rotating wheel for 1 h before the phosphorylation reaction, while CX-4945 was added before the enzyme. After extensive washing with a kinase wash buffer , the GST-fusion proteins were subjected to SDS-PAGE and western blot analysis using an anti-phospho-16-E7 antibody.For in vitro phosphorylation, purified GST-fusion proteins were incubated with CK2 enzyme  in 20 μL kinase buffer  and centrifuged at 14,000 rpm for 10 min. The supernatant was incubated with 30 μL of monoclonal anti-HA agarose beads  on a rotating wheel at 4 °C for 2 h. After incubation, the samples were washed with the lysis buffer. The immunoprecipitates were then run on SDS PAGE gels and analyzed by western blot.For immunoprecipitation, HEK293 cells were transfected with FLAG-HA-tagged pCMV HPV-16 E7 plasmid and empty vector. After 48 h, the cells were treated with 25 µM CX-4945 for 2 h and 200 µM CIGB-300 for 30 min at 37 °C. The cells were then harvested using a lysis buffer . The membranes were blocked in 5% non-fat milk powder dissolved in TBST . The membranes were then probed for different proteins using the appropriate primary antibodies, i.e., mouse monoclonal anti-HA , mouse monoclonal anti-HPV-16 E7 (1:200), mouse monoclonal anti-HPV-18 E7 (1:200) from Santa Cruz Biotechnology , mouse monoclonal anti-Rb (1:1000) , mouse monoclonal anti-α-tubulin, mouse monoclonal anti-HA-peroxidase (clone HA-7), and streptavidin–HRP (1:3000) . The HPV-16 E7 pS31/S32 peptide antibody generated by Eurogentec was described previously . Incubatp value below 0.05 was considered statistically significant.For the quantification of protein levels from the western blots, the band intensities were measured using Image J software. Differences between groups were determined using one-way ANOVA, followed by Dunnett’s multiple comparison test. The analyses were condicted using GraphPad Prism (v6.01) software . A We first investigated the putative physical interaction between the CIGB-300 peptide and the E7 viral protein from both high-risk HPV-16/-18 and low-risk HPV-11. We conducted in vitro pull-down experiments using biotinylated CIGB-300 and GST-fusion proteins. The peptide was incubated with GST-tagged HPV-11, -18, or -16 E7, or empty GST as negative control, followed by immunoblot analysis. The in vitro interaction was detected using an anti-streptavidin antibody recognizing the biotinylated peptide. The data in Having shown that CIGB-300 interacts with E7 in vitro, we wanted to assess the interaction in a relevant cellular context. Accordingly, we conducted in vivo pull-down assays using HEK293 cells transfected with constructs expressing FLAG-HA-tagged HPV-16 E7 or HPV-18 E7. The data shown in To investigate the effect of CIGB-300 on the CK2-mediated phosphorylation of E7, we conducted western blot analysis using GST-fusion proteins and total cell extracts derived from HEK293 cells overexpressing FLAG-HA-tagged HPV-16 E7. Using a specific anti-HPV-16 E7(S31/S32) antibody, we confirmed the in vitro inhibitory effect of CIGB-300 on E7 phosphorylation A. Accord50, of 200 µM, whereas the mutant clones showed a mean value of 259 µM; however, no significant sensitivity toward CIGB-300 cytotoxic effect was displayed. Our results indicate that the ability to target E7 phosphorylation is not an essential molecular event in the cell death induced by CIGB-300 in cervical cancer cells.Having demonstrated that CIGB-300 can inhibit CK2-mediated phosphorylation of the S31/S32 residues of E7, we explored the relevance of such inhibition for the cytotoxic effect of CIGB-300, using C4-1 cells expressing E7 mutated at the CK2 phospho-acceptor site. These cells were generated by a genome-editing approach in which the S32/S34 amino acid residues in E7 were changed to A32/A34, thereby impairing E7 susceptibility to phosphorylation . The impCIGB-300 was initially designed to bind the CK2 phospho-acceptor domain of HPV-16 E7 and, as we confirmed here, it was shown to bind preferentially to the N-terminal region of E7, near the pRB binding domain (Cys24). To determine whether the peptide disrupts the binding of HPV-16 E7 to pRB, we performed immunoprecipitation analysis of HEK293 cells transfected with constructs expressing FLAG-HA-tagged HPV-16 E7 or the empty vector as a negative control. We observed a clear decrease of the pRB signal in the HPV-16 E7 immunoprecipitated fraction after treatment with either the known CK2 inhibitor CX-4945 or CIGB-300 . This reIn addition to the standard regimen for cervical cancer therapy, treatments with new anticancer agents based on targeting the molecular pathways dysregulated in cervical cancer have emerged as strategies with great potential. Protein kinase CK2 has been shown to be involved in the regulation of cellular and viral proteins relevant for this malignancy ,33. For Although the development of CIGB-300 as a potential therapeutic arose from its ability to block HPV-16 E7 phosphorylation, its putative physical interaction with the viral oncoprotein in the cellular context remained to be confirmed. Here, we show for the first time, using in vitro pull-down assays, a clear physical interaction between HPV-16 E7 at a relevant therapeutic dose of CIGB-300. Importantly, that interaction was also confirmed for the E7 protein from the HPV-18 and HPV-11 types, which would support the clinical benefit of CIGB-300 in patients with HPV-18-positive tumors and those with low-risk HPV-infected lesions. To further evaluate the in vivo CIGB-300-E7 interaction between both molecules, we performed in vivo pull-down. We employed HEK293 as an epithelial cell model with high transfection efficiency that has been previously used for studying E6 and E7 interaction partners . Using H1 phase [The in vitro inhibition of HPV-16 E7 CK2-mediated phosphorylation by CIGB-300 has previously been documented ,41; here1 phase , it rema1 phase . To furtPrevious studies had indicated that CK2-mediated phosphorylation of E7 is required for pocket protein recognition . The besIn conclusion, we demonstrated for the first time that CIGB-300 targets E7 proteins from high- and low-risk HPV types. The effect of CIGB-300 on E7 phosphorylation does not appear to have a critical role in the CIGB-300-mediated cytotoxic effect in cervical cancer cells. However, the physical interaction of the peptide with E7 might affect HPV-16 E7 protein function by disrupting its interaction with pRB. Our study reveals novel molecular clues to the mechanism of action of CIGB-300 in cervical cancer.