Background. The high prevalence of HPV infection among Kazakhstani women and the absence of an HPV vaccination program are directly reflected in increasing rates of cervical cancer incidence and mortality. Kazakhstan made its first attempt at introducing the HPV vaccine in 2013, but was unsuccessful due to complications and low public acceptance. The attitudes of Kazakhstani women towards the vaccine were never measured. Therefore, this study aims to investigate the attitudes of women towards the HPV vaccine and determine factors associated with positive, negative, or neutral attitudes. Methods. A 29-item survey consisting of 21 demographic and contextual questions and 8 Likert-scale questions was distributed among women attending gynecological offices in four major cities of Kazakhstan from December 2021 until February 2022. Attitudes of women were measured based on their answers to the eight Likert-scale questions. Ordinal logistic regression was built to find associations between demographic characteristics and attitudes of women. Results. Two hundred thirty-three women were included in the final analysis. A total of 54% of women had positive attitudes towards the vaccine. The majority of women did not trust or had a neutral attitude towards the government, pharmaceutical industry, and traditional and alternative media. However, the trust of women was high in medical workers and scientific researchers. Women’s age, education, number of children, effect of the 2013 HPV program, and trust in alternative medicine were included in the ordinal logistic model. Women with a low level of education, a high number of children, who believe in alternative medicine, and who were affected by the failed 2013 vaccination program were less likely to have a positive attitude towards the vaccine. Conclusions. Contrary attitudes towards HPV vaccination exist among Kazakhstani women, with approximately half having positive and almost half having negative or neutral attitudes towards the vaccine. An informational campaign that takes into consideration women’s levels of trust in different agencies, as well as targets those who are the most uninformed, might help in a successful relaunch of the HPV vaccination program. However, more studies that cover a higher number of women are required. High-risk human papillomavirus (HR-HPV) infections cause a wide variety of benign and malignant conditions, including cervical cancer ,2,3,4. MThe association of HPV infection with anal, penile, head, and neck cancers is also well documented ,9,10. ThThe prevalence of HPV infection varies among countries worldwide, with the highest prevalence of HR-HPV infection among women in the developing regions of Southern and Eastern Asia  . A high p < 0.001) [The high prevalence of HR-HPV strains in Kazakhstan is contributing to increasing cervical cancer rates. Over the past 10 years 2009–2018), the crude rate of cervical cancer incidence in Kazakhstan has increased from 16.3 ± 0.4 to 19.5 ± 0.5 per 100,000 female population (< 0.001) , and eli, the cruIn 2013, the HPV vaccination campaign was introduced in Kazakhstan as a pilot program in four large regions . BivalenGiven the importance of the reintroduction of HPV immunization to prevent new cervical cancer cases, it is essential to understand what factors are associated with parents’ willingness to vaccinate their children. Studies have shown that attitudes towards HPV vaccination are associated with HPV vaccination uptake . MultiplAs one of the tasks announced by the World Health Organization (WHO) in the Global Strategy to Accelerate the Elimination of Cervical Cancer ,30 is toThis is a cross-sectional study, which was conducted from December 2021 until February 2022 in four major cities  representing the central, southern, western, and eastern regions of Kazakhstan. Women aged from 18 to 70 years old visiting gynecological offices were recruited for the study. A total of 399 women agreed to participate in the study, and 347 reported full demographic characteristics. Out of those 347 women, 321 (80.45%) fully answered the HPV vaccine attitude questions. Almost 27% were excluded due to incomplete answers to the contextual questions. In total, 233 women were included in the complete-case analysis .Two questionnaires were used in the study. The first collected data on demographic characteristics of women. The second questionnaire was adapted from the French Survey Questionnaire for the Determinants of HPV Vaccine Hesitancy (FSQD-HPVH) . The queIndependent variables were socio-economic and demographic characteristics such as age, ethnicity, level of education, city, family income, marital status, and number of children. According to age, women were categorized into four major groups: 18–27 years old, 28–33 years old, 34–43 years old, and 44 years old and older. Age of women was categorized according to the quartiles. Ethnicity was categorized into 2 groups: Kazakh and other ethnic groups. There were three levels of education: high school or below, college, and university. In January 2022, the average income in Kazakhstan was 269 149 tenge (USD ≈ 520) . Income In addition, contextual factors such as historical factors, policies, and mandates were used as independent variables. This section consisted of 6 Likert-scale questions. The major topics of the questions were the impact of COVID-19 vaccination on trust in HPV vaccine recommendations (COVID-19 effect), the 2013 HPV vaccination program in Kazakhstan (2013 HPV program), the relationship of HPV vaccination with sexual promiscuity, compulsory vaccine mandated by the government, freedom of vaccine choice for children, and alternative medicine strengthening body immune system . Statements about trust in different agencies were also used as independent variables. The Likert scale was used to measure trust in agencies and health systems such as the pharmaceutical industry, government, medical workers, scientific researchers, traditional media, and alternative media.The attitude of the participants was measured using 3 positive statements and 5 negative statement items on the Likert scale. Negative statements were reversed, and the mean attitude score of the participants was calculated. The attitude of the women was categorized as <3—negative attitude, 3—neutral attitude, and >3—positive attitude to separate women who have a positive or negative opinion about the vaccine from those who are hesitant. It is important to differentiate those who refuse vaccination from those who do not have an opinion and lack confidence in vaccination .p < 0.001) were performed before the factor analysis. All factors had a uniqueness lower than 0.6, except for the statement “Majority of my friends vaccinate their daughters from HPV”, which had a uniqueness of 0.81. The item was dropped from the attitude scale, as it was not relevant to the Kazakhstan context due to the HPV vaccine not being readily available in the country.Statistical analysis was performed using STATA 16 . CronbacDescriptive statistics consisting of mean values, standard deviations, and frequencies were obtained using univariable and bivariable analysis. Chi-square and Fisher’s exact tests with a significance value of <0.005 were used to analyze the relationships between categorical variables.Ordinal logistic regression was performed to explore factors associated with attitude towards HPV vaccination among women. Variables that showed significance in the bivariable analysis and were important as epidemiological factors were included in the final model. Among participants’ characteristics, variables such as age, education, and number of children were included in the final model. Although participants’ age was not a statistically significant factor, it was included in the final model due to its importance for the epidemiological picture. Two contextual factors such as distrust in the healthcare system due to complications of the HPV vaccination program in Kazakhstan in 2013 and trust in alternative medicine  were considered for the final model. The model assumptions were checked. The goodness of fit of the model was checked with Hosmer–Lemeshow, Pulkstenis–Robinson Chi-square, and deviance tests, as well as the Lipsitz likelihood-ratio test.The study was conducted in compliance with the Declaration of Helsinki and was approved by the Institutional Research Ethics Committee of Nazarbayev University (NU IREC) on 23 April 2019 (IREC Number: 146/4042019). Before inclusion in the study, all potential participants were informed about the aims, methods, risks, and benefits of the study. Verbal consent was received from participants after an explanation of the voluntary and anonymous nature of the study.The social and demographic characteristics of women are shown in The least trusted information sources among women were alternative media, the government, and the pharmaceutical industry. The most trusted were scientific researchers, closely followed by medical workers .Bivariable analysis between attitude towards HPV vaccination and patient characteristics and contextual factors is shown in The Chi-square test showed that all contextual factors were statistically significantly associated with the level of attitude towards HPV vaccination. Among women who had no prejudice that HPV vaccination encourages teenage girls to have sex, 65% had a positive attitude. Among those who disagreed with compulsory vaccination, a majority (54%) had a positive attitude to the HPV vaccine, and among those who agreed with compulsory vaccination, a majority (58%) had a positive attitude to the HPV vaccine. Similarly, a majority of women who agreed (52%) or disagreed (71%) with freedom of vaccine choice for their children were women with a positive attitude towards the HPV vaccine. The majority of women (73%) who disagree with the statement that alternative medicine strengthens the body’s defenses had a positive attitude towards the HPV vaccine.Women aged between 34 and 43 were 1.23 times more likely to have a positive  attitude towards HPV vaccination in comparison to women aged between 19 and 27 years, adjusting for other variables . Having Women who were neutral about their confidence in the healthcare system were 0.34 times the odds of a positive attitude towards HPV vaccination. Agreeing that alternative medicines strengthen the body’s defenses or being neutral about the statement decreased the odds of a positive attitude by 0.41-fold and by 0.48-fold compared with women who disagreed with the statement, adjusting for other variables .To date, there are no published studies investigating attitudes toward the HPV vaccine among Kazakhstani women. Thus, this is the first study that aimed to examine attitudes toward HPV vaccination and explore factors associated with a positive attitude toward HPV vaccination in Kazakhstani women. Since HR-HPV prevalence is high among women in Kazakhstan ,14,15,21Some historical contextual factors were taken into consideration in this study, such as previous HPV vaccination program complications/failure in Kazakhstan. The situation in Japan closely resembles the failure of the HPV vaccination program in Kazakhstan. Reports of side effects in mass media and prompt cancellation of the program had a long-lasting effect in Japan, causing the vaccine coverage to fall to less than 1% . In KazaAmong our respondents, those who were affected by the failed 2013 HPV vaccination program had a 79% lower likelihood of having a positive attitude towards the vaccine, adjusting for other variables. Overall, 16% of women indicated that the failure of the 2013 HPV vaccination program in Kazakhstan had affected their confidence in the healthcare system, and the majority of them (40%) had a negative attitude towards the HPV vaccine. Thus, an unsuccessful attempt to implement the HPV vaccination program in Kazakhstan in 2013 had a significant impact. This finding is similar to the study reporting data from Romania and France, where the initial HPV vaccination program coverage reported a poor response and required specific action plans to improve the situation ,39.Rapid introduction of the COVID-19 vaccine is another factor considered in this study, as it has been reported previously that the level of COVID-19 hesitancy is high in Kazakhstan ,41, and The ongoing debates around the COVID-19 vaccine, however, did not have the same effect. Among women who disagreed with having less confidence in HPV vaccination recommendations since the controversy over the COVID-19 vaccination, 69% still had a positive level of attitude towards HPV vaccination. Overall, 14% of women indicated that COVID-19 controversies have affected their trust in HPV vaccination, and their distribution among the attitude groups was even. This finding falls in line with previous studies that show a lack of hesitancy overlap between HPV and COVID-19 vaccines .Unfortunately, this study revealed a low trust of the respondents in the government  as the main policymaker in Kazakhstan and in pharmaceutical companies. The majority of the respondents (44%) did not trust the government; only 13% did, the rest remaining neutral. This evidence shows that the Ministry of Healthcare and other governmental agencies in Kazakhstan should reinforce the information campaign in trustworthy ways. Similarly, participants had low levels of trust in alternative media and mostly low or neutral levels of trust in traditional media. At the same time, the fact that in our study, researchers and healthcare professionals are the most trusted source of information about the vaccine gives hope and identifies directions that the Ministry of Healthcare can employ to increase HPV vaccine awareness and improve attitudes. Engaging and strengthening the relevant healthcare workforce in preparation for HPV vaccine introduction could be achieved through training/seminars, information about the evidence and necessity of HPV vaccination and cervical cancer, and communication training to address parents’ concerns and make recommendations. Our findings of trust are comparable with studies in European countries , where mistrust of health authorities was reported by 47% to 55% of the respondents . On the In this study, a positive attitude towards HPV vaccination was reported by 52% of the respondents. Education, place of residence, level of income, and number of children were the factors that were found to be significantly associated with the level of attitude towards HPV vaccination. Women with high income levels had the highest proportion of positive attitudes toward the HPV vaccine (62%). On the other hand, among women with high-school-level education and those who did not finish high school, 62% were neutral towards HPV vaccination. Thus, the level of education directly correlates with positive attitudes toward the HPV vaccine. Similar results were obtained in an Italian study, where 49.7% of the respondents had a positive attitude toward the HPV vaccine , and in However, having such a huge proportion of neutral respondents in our study indicates the direction of further actions. Informational interventions to increase awareness of the HPV vaccine and the advantages of being vaccinated should be developed, as a positive attitude toward the HPV vaccine is associated with a higher level of intention to receive the HPV vaccine ,50.Unfortunately, only 16% of mothers with four or more children had a positive attitude towards the HPV vaccine, and the majority (64%) had a neutral attitude. This is an important group of the population, who requires more information on the vaccine, as their attitude towards the vaccine has a direct impact on the HPV vaccination program coverage among the target group of 9–11 years old girls. The situation could be improved if relevant and effective informational programs are employed to explain the advantages of the HPV vaccine in HPV infection and cancer prevention, as has been demonstrated by a study among parents in Japan .As was confirmed by many studies, a significant reduction in the incidence of precancerous cervical lesions and cervical cancer was observed after the introduction of HPV vaccination programs ,52,53,54Study strengths and limitations. This was the first study to assess attitudes towards HPV vaccination among Kazakhstani women. Another strength of this study is covering the female population in the big cities of four Kazakhstani regions  and investigating the factors associated with attitudes towards HPV vaccination. However, since we approached only the population of big cities, but not these regions’ rural areas, this study cannot fully represent the whole country’s female population. Moreover, in this study, the proportions of participants with university-level and college-level education were 55% and 34%, respectively. These indicators could be different if we include participants from rural areas. These are the major limitations that we hope to overcome in our future studies to obtain a more precise picture in terms of participants’ residence and education. Another limitation of this study is its cross-sectional nature and resulting lack of causality. The study employed convenience sampling with self-reporting methods. This limits the representativeness of our findings due to selection and nonresponse biases. The descriptive nature of our statistical model, as well as the low number of respondents, prompts for expansion of the study beyond the pilot investigation. Moreover, in future studies, we should investigate parents’/mothers’ attitudes towards HPV vaccination further, as they are the main decision makers if the vaccination of the target group is planned.There are many issues related to cervical cancer prevention that require solving in Kazakhstan in the near future . AlthougThis study shows contrary attitudes toward HPV vaccination exist among Kazakhstani women, with approximately half of women having positive and almost half having negative or neutral attitudes towards the vaccine. We found that women’s age, education, number of children, confidence in the healthcare system, and belief in alternative medicine were associated with attitudes towards HPV vaccination. These factors, as well as high levels of trust towards medical and science workers, should be taken into consideration when planning context-specific health educational interventions to form positive attitudes towards HPV vaccination in Kazakhstani women. In addition, sharing accurate information regarding the safety, effectiveness, and benefits of HPV vaccination to prevent HPV infection and related diseases could potentially improve women’s attitudes. HPV educational interventions are warranted to successfully relaunch and include HPV vaccination in the national immunization program in Kazakhstan.


An estimated 15% of girls aged 9–14 years worldwide have been vaccinated against human papillomavirus (HPV) with the recommended two-dose or three-dose schedules. A one-dose HPV vaccine schedule would be simpler and cheaper to deliver. We report immunogenicity and safety results of different doses of two different HPV vaccines in Tanzanian girls.NCT02834637.In this open-label, randomised, phase 3, non-inferiority trial, we enrolled healthy schoolgirls aged 9–14 years from Government schools in Mwanza, Tanzania. Eligible participants were randomly assigned to receive one, two, or three doses of either the 2-valent vaccine  or the 9-valent vaccine . The primary outcome was HPV 16 specific or HPV 18 specific seropositivity following one dose compared with two or three doses of the same HPV vaccine 24 months after vaccination. Safety was assessed as solicited adverse events up to 30 days after each dose and unsolicited adverse events up to 24 months after vaccination or to last study visit. The primary outcome was done in the per-protocol population, and safety was analysed in the total vaccinated population. This study was registered in ClinicalTrials.gov, Between Feb 23, 2017, and Jan 6, 2018, we screened 1002 girls for eligibility. 72 girls were excluded. 930 girls were enrolled and randomly assigned to receive one dose of Cervarix (155 participants), two doses of Cervarix (155 participants), three doses of Cervarix (155 participants), one dose of Gardasil-9 (155 participants), two doses of Gardasil-9 (155 participants), or three doses of Gardasil-9 (155 participants). 922 participants received all scheduled doses within the defined window . Retention at 24 months was 918 (99%) of 930 participants. In the according-to-protocol cohort, at 24 months, 99% of participants who received one dose of either HPV vaccine were seropositive for HPV 16 IgG antibodies, compared with 100% of participants who received two doses, and 100% of participants who received three doses. This met the prespecified non-inferiority criteria. Anti-HPV 18 seropositivity at 24 months did not meet non-inferiority criteria for one dose compared to two doses or three doses for either vaccine, although more than 98% of girls in all groups had HPV 18 antibodies. 53 serious adverse events (SAEs) were experienced by 42 (4·5%) of 930 girls, the most common of which was hospital admission for malaria. One girl died of malaria. Number of events was similar between groups and no SAEs were considered related to vaccination.A single dose of the 2-valent or 9-valent HPV vaccine in girls aged 9–14 years induced robust immune responses up to 24 months, suggesting that this reduced dose regimen could be suitable for prevention of HPV infection among girls in the target age group for vaccination.UK Department for International Development/UK Medical Research Council/Wellcome Trust Joint Global Health Trials Scheme, The Bill & Melinda Gates Foundation, and the US National Cancer Institute.For the KiSwahili translation of the abstract see Supplementary Materials section. Cervical cancer results in more than 340 000 potentially preventable deaths annually, with most fatalities in low-income and middle-income countries.Evidence before this studySeveral authors of this work participated in a review to collate the evidence on single dose human papillomavirus (HPV) vaccination. This review identified the absence of evidence from randomised trials and highlighted that data from Africa were also limited. A 2019 systematic review published as part of this evidence review examined the effectiveness and immunogenicity of single dose HPV vaccination among participants who received their HPV vaccine through a clinical trial. Apart from one small randomised trial examining memory B-cell responses following single dose HPV vaccination, results came from observational studies nested within three large HPV vaccine trials  in which participants did not complete their allocated two-dose or three-dose schedules which resulted in single dose default groups followed up for immunogenicity and efficacy against HPV infection. We did an updated search of MEDLINE, Embase, Global Health Database, and Cochrane Central Register of Controlled Trials from Aug 1, 2018, to Dec 10, 2021, using the search terms “human papillomavirus AND vaccines AND (immunogenicity OR efficacy OR effectiveness) AND dosage”. From this search we identified two additional observational studies that extended the data from two of these studies and, in 2022, results were published from a randomised trial on the efficacy of single dose HPV vaccination in sexually active Kenyan women aged 15–20 years (KEN SHE study). The observational studies showed that frequency of HPV 16 and 18 incident and 12-month persistent infection and vaccine efficacy against infection endpoints was similar in women and girls who received a single dose of vaccine compared with those who received two or three doses. HPV 16 and 18 IgG antibody seropositivity was very high in all dose groups for vaccinated participants, although antibody mean concentrations were lower with one dose than with two or three doses, but remained stable over 11 and 9 years for all doses for two HPV vaccines. HPV infection endpoints were significantly lower in participants who received one vaccine dose compared to unvaccinated controls. The KEN SHE trial showed very high and non-inferior vaccine efficacy for one dose of the 2-valent and 9-valent vaccines compared with a control vaccine at 18 months after the first dose. Non-trials data include an observational cohort study of Ugandan girls who did not complete the 3-dose schedule of the 2-valent vaccine in a Government-administered HPV vaccination demonstration programme. Seroconversion was high for all doses. HPV 16 and 18 binding antibody responses were lower in girls who had received one compared with two or three doses but geometric mean concentrations for one dose recipients were not lower in these Ugandan girls compared with adult women who received one dose in the CVT and in whom efficacy had been demonstrated.Added value of this studyThis study is the first randomised clinical trial examining immune responses and safety of single dose HPV vaccine with either the 2-valent or 9-valent vaccine compared with two and three doses of the same vaccines in girls in the target age group of 9–14 years for vaccination. Antibody responses were comparable with those seen in the earlier observational studies, and were induced with both vaccines after one dose and increased after the second and third doses. Antibody geometric mean concentrations peaked at 1 month and then plateaued from month 7 for the single dose arm and peaked at month 7 then declined by month 24 for the two-dose and three-dose arms but stayed stable in the one-dose arms to 2 years. Single dose HPV 16 seropositivity at 24 months post dose was non-inferior to two and three doses and HPV 16 and HPV 18 avidity at month 24 did not differ by dose or vaccine. Both vaccines were well tolerated at all doses.Implications of all the available evidenceA single dose of either the 2-valent or 9-valent HPV vaccine was both immunogenic and safe, with high rates of seroconversion and antibody levels stable to 2 years after vaccination and antibody kinetics similar to those seen in other settings where single-dose efficacy has been demonstrated. Higher antibody levels observed with the 2-valent vaccine compared to the 9-valent vaccine are consistent with earlier studies that also found both vaccines to be highly efficacious. A single dose of HPV vaccine would very significantly simplify vaccine delivery and reduce costs of implementing national HPV vaccination programmes, in turn potentially increasing vaccine introductions and uptake in the regions that urgently need cervical cancer prevention.A single dose HPV vaccine would be simpler and cheaper to deliver than a multidose schedule but evidence is needed on the immunogenicity and efficacy of a single-dose schedule. Data from several observational studies in which some participants did not complete their allocated schedules suggest that a single dose of HPV vaccine provides efficacy against incident and persistent HPV 16 or 18 infection that is similar to efficacy with two or three doses.The first randomised trial to examine the efficacy of a single dose of HPV vaccine in sexually active Kenyan women aged 15–20 years (KEN SHE) reported that, at 18 months post vaccination, the incidence of persistent HPV 16 or HPV 18 infection was 0·17/100 woman-years with both the 2-valent and the 9-valent vaccines, compared with 6·83 per 100 woman-years in the meningococcal vaccine control group.We report the results of the DoRIS trial in Tanzania, the first randomised trial to examine immune responses after a single dose of HPV vaccine in the target age group for HPV vaccination.This open-label, randomised, phase 3, non-inferiority, immunobridging trial of two HPV vaccines was done in Mwanza, northwestern Tanzania (Dose Reduction Immunobridging and Safety Study of two HPV vaccines in Tanzanian girls [DoRIS]). The study was approved by the Tanzanian Medical Research Coordinating Committee and the ethics committee of the London School of Hygiene & Tropical Medicine. Regulatory approval was by the Tanzania Medicines and Medical Devices Authority.The trial protocol and procedures have been described previously.Participants were randomly allocated (1:1:1:1:1:1) to one of six arms comprising three different dose schedules of two different HPV vaccines , using random permuted block sizes of 12, 18, and 24. An independent statistician computer-generated the randomisation list. Sequentially numbered sealed opaque envelopes concealed the allocation from the study team and participants. Once allocated, participants and clinic staff were unmasked. Participants were not masked as we did not think immune responses would be affected by girls knowing their vaccine group, and one of the trial's secondary aims was the acceptability of reduced-dose schedules.We evaluated two prophylactic HPV virus-like particle (VLP) vaccines, both licensed by the US Food and Drug Administration and the European Medicines Agency. The 2-valent HPV vaccine  is an HPV 16 and HPV 18 VLP vaccine containing L1 major capsid proteins of HPV 16 and HPV 18 and a proprietary adjuvant system (ASO4) that is formulated with monophosphoryl-lipid A adsorbed to aluminium hydroxide. The 9-valent vaccine  targets 9 genotypes . The vaccine has an amorphous aluminium hydroxyphosphate sulfate adjuvant and each dose contains 60 μg of HPV 16 L1 protein and 40 μg of HPV 18 L1 protein. Both vaccines have excellent efficacy for preventing HPV 16 or HPV 18 associated grade 2 or grade 3 cervical intraepithelial neoplasia and HPV 16 or 18 associated adenocarcinoma in situ in women with no previous HPV 16 or 18 infection.At the screening visit, after informed consent, girls were screened for eligibility, including a medical history with clinical examination if warranted, HIV testing and counselling, and a urine pregnancy test. Girls were also asked to take a test of understanding (TOU) if aged 12 years or older to demonstrate appropriate understanding of the study. For girls younger than 12 years, a parent or guardian took the TOU. A screen failure was determined if the girl (or their parents) could not pass the TOU within three attempts.Girls who had passed the screening process were invited to an enrolment visit within 30 days of the screening visit, at which eligibility was reconfirmed and girls were randomly allocated to one of the six arms. Blood samples were collected for immunogenicity assays and a dried blood spot was made for malaria testing by PCR. Girls were asked to provide two nurse-assisted, self-administered vaginal swabs for baseline HPV DNA testing and genotyping with the Anyplex II HPV 28 detection assay  done at the Catalan Institute of Oncology, Barcelona. Participants were then randomly assigned and vaccinated according to their study arm and were asked to attend the clinic 1 month after vaccination.Subsequent vaccination visits were at 1 month after the first dose  or at 2 months after the first dose  and 6 months (girls enrolled in the two-dose and three-dose arms for either vaccine). At each vaccination visit, and at 6 months for the one-dose arms, we collected a dried blood spot for malaria testing. Participants were asked to attend the clinic 1 month after each vaccination visit for collection of information on adverse events (AEs). Whole blood samples of 15–20 mL (depending on participant's weight) were collected for immunological assays at 1, 7, 12 and 24 months after vaccination. Visit windows for vaccination and blood sampling visits were predefined in the protocol.All samples were processed at the Mwanza National Institute for Medical Research laboratory. HPV 16 and HPV 18 IgG concentrations were determined at the HPV Immunology Laboratory of the Frederick National Laboratory for Cancer Research in Maryland, USA, by use of an L1 VLP ELISA. This assay has previously been evaluated for monitoring antibody responses following single-dose HPV vaccination.The primary outcome was non-inferiority of HPV 16 and HPV 18 specific seropositivity following one dose of HPV vaccine compared with two or three doses of the same vaccine 24 months after vaccination. This corresponded to two overall analyses: one evaluating the reduced dose schedule of the 2-valent vaccine, and one evaluating the 9-valent vaccine.Vaccine immune responses were measured by the proportion of participants seroconverting to HPV 16 or 18, the GMC of HPV 16 and HPV 18 specific antibodies, HPV 16 and HPV 18 specific antibody avidity, and HPV 16 and HPV 18 specific memory B-cell responses.Secondary objectives are evaluation of HPV 16 and HPV 18 seropositivity and antibody GMC after one dose versus two or three doses at other timepoints up to 24 months post-vaccination; comparison of HPV 16 and HPV 18 antibody responses after two versus three doses; and evaluation of HPV 16 and HPV 18 antibody avidity.The trial had a coprimary immunobridging objective to demonstrate non-inferiority of HPV 16 and HPV 18 antibody GMC after one dose of vaccine compared with historical cohorts of women aged 10–25 years who received a single dose of HPV vaccine and in whom efficacy had been demonstrated; these results are reported in a companion publication.With 155 participants in each arm, assuming a 20% loss-to-follow up over 36 months, we expected to have 130 girls in each arm 24 months after vaccination. If the true proportion seroconverting is the same in each arm, with 130 girls per arm, the study would have more than 90% power to demonstrate that the lower limit of the 95% CI for the difference (one-dose schedule–comparison schedule) is above –5%, indicating that seropositivity with the one-dose schedule was not decreased by more than 5·0%. This was the same non-inferiority margin that was used in the trials leading to licensure of the two-dose regimen in girls younger than 15 years.1610 anti-HPV concentration and used a one-sided non-inferiority test at the 2·5% level.Our power calculations were also based on our coprimary objective of demonstrating non-inferiority of GMCs in the immunobridging analyses. If the true GMC ratio (one-dose schedule:comparison schedule) between arms is 1·0, with 130 subjects in each arm, we would have more than 90% power to demonstrate that the lower limit of the 95% CI for the ratio of GMCs is above 0·50, indicating that the one-dose schedule does not decrease HPV 16 or HPV 18 antibody GMC by more than 50%. This non-inferiority margin was based on pre-established standards from the US Food and Drug Administration that were used in other HPV vaccine bridging trials.In non-inferiority trials, intention-to-treat (ITT) analyses can increase the risk of falsely claiming non-inferiority, since these analyses often lead to smaller observed effects than if all participants had adhered to the protocol.Baseline characteristics were presented by arm. We tabulated the number and proportion of girls in each arm who were HPV 16 or 18 seropositive at each timepoint. For each vaccine type and HPV genotype, we calculated the difference (one-dose schedule–comparison schedule) in the proportion of girls who were seropositive, and estimated the 95% CI for the difference using the exact method of Chan and Zhang.For each vaccine and HPV genotype, there were two primary hypothesis tests of non-inferiority of seropositivity 24 months after vaccination: one dose versus two doses, and one dose versus three doses . Success was required for both tests to conclude non-inferiority; therefore, no adjustment for multiplicity was made to account for testing of multiple dose schedules. As a post-hoc sensitivity analysis, 97·5% CIs were calculated in accordance with the Bonferroni correction, to account for testing of multiple HPV genotypes.10-transformed HPV genotype-specific antibody concentrations; those below the assay cutoff were given a value of half the cutoff before log transformation. The arithmetic mean log10 antibody concentration and 95% CIs for each arm were calculated, assuming a normal distribution. Log10 antibody concentrations were compared by use of a linear mixed effect model with log10 concentration as the response, dose group, timepoint, and a dose group time interaction term as fixed effects, and participant as a random effect to account for correlation of repeated measurements within participant. Separate models were used for each vaccine type and HPV genotype. The difference in log10 concentrations (reduced dose schedule–comparison schedule) and its 95% CI at each timepoint was estimated from the mixed effect model; the GMC ratio and 95% CIs were obtained by back-transformation. We used a similar analysis to compare antibody avidity between dose regimens, with separate models for each vaccine type and HPV genotype, and fixed effects for dose regimen, timepoint, and dose regimen–time interaction. For the secondary objectives, multiple comparisons were taken into account when interpreting the findings but no formal adjustments were made for multiplicity. This study was registered with ClinicalTrials.gov, NCT02834637.For the analysis of antibody concentrations, we logThe funders of this study did not have any role in the study design, data collection and analysis, data interpretation, or writing of this report.Between Feb 23, 2017, and Jan 6, 2018, we screened 1002 girls for eligibility. 50 girls were excluded at screening for medical findings (n=20), no consent (n=15), HIV infection (n=7), not meeting vaccine deferral criteria (n=7), or failing the TOU (n=1). 22 girls were eligible at screening but did not attend the enrolment visit. 930 (93%) of 1002 girls were enrolled and randomly assigned to receive one dose of Cervarix, two doses of Cervarix, three doses of Cervarix, one dose of Gardasil-9, two doses of Gardasil-9, or three doses of Gardasil  of 930 participants. At 24 months, we included 856 (93%) of the 918 girls attending in the per-protocol analysis of anti-HPV 16 antibody responses, and 831 (91%) in the per-protocol analysis of anti-HPV 18. All but two participants were seropositive for HPV 16 IgG antibodies at 24 months (one participant in each of the one-dose arms was not HPV 16 seropositive). All but six participants were HPV 18 seropositive at 24 months . Non-inferiority of seroconversion of anti-HPV 16 antibodies at 24 months was met for one dose compared with two doses or three doses for both vaccines . Non-infvs 163 IU/mL for HPV16 and 107 IU/mL vs 50 IU/ml for HPV18, respectively), and non-inferiority was not met for either HPV genotype. Our immunogenicity results were similar among the total vaccinated cohort for both vaccines and both HPV genotypes  for HPV 16 and 10 IU/mL (95% CI 9–11) for HPV 18 . Among tenotypes .Table 3HAntibody GMCs among the two-dose and three-dose recipients peaked at 7 months and declined thereafter up until 24 months for both vaccines and both genotypes . HoweverBy contrast with antibody GMCs, there was no evidence of a difference between the one-dose, two-dose schedules and three-dose schedules in GM antibody avidity index for HPV 16 or HPV 18 of either vaccine  of 930 girls by 24 months . ClinicaThis is the first randomised controlled trial to assess immune responses and safety of a single-dose HPV vaccine compared with two-dose or three-dose regimens among girls in the target age group for these vaccines. This is also the first trial in sub-Saharan Africa to examine immune responses to the currently recommended two-dose regimen compared with the originally recommended three-dose regimen. Our study is very timely since a randomised controlled trial in sexually active Kenyan women and girls aged 15–20 years (KEN SHE) has demonstrated excellent (>97%) efficacy with a single dose of either the 2-valent or the 9-valent vaccine against incident persistent HPV 16 or 18 infections at 18 months post-vaccination.Consistent with previous studies, both vaccines in DoRIS were found to be well tolerated and no SAEs were considered related to vaccination. Malaria was the most common clinical event, which was not unexpected since malaria is endemic in the study area.Our serology data support observations from non-randomised studies that a single dose of HPV VLP vaccines can induce strong and sustained IgG antibody responses up to 2 years post-vaccination. HPV 16 and HPV 18 antibody GMCs reached a plateau after 7 months that was sustained to 24 months. Ongoing follow-up of this cohort will allow us to determine if these antibody concentrations remain stable over time, as observed following one dose of the 4-valent vaccine in India, where antibody levels have been stable for 4 years and efficacy has been demonstrated for 9 years, and the 2-valent vaccine in Costa Rica, where antibody levels have been stable for 11 years and efficacy has been demonstrated for 11 years.As has been shown in the CVT, PATRICIA, and India/IARC studies, antibody GMCs following one dose of HPV vaccine in the DoRIS trial were lower compared with GMCs after two or three doses.There is no known immune correlate of protection for L1 VLP HPV vaccines, but antibody responses are considered essential in the protection conferred by these vaccines. A single dose of a VLP HPV vaccine might be sufficient to protect against HPV infection and its sequelae for several reasons.Although we did not meet the non-inferiority criterion for anti-HPV 18 seropositivity at 24 months after a single dose of either vaccine, more than 98% of girls were anti-HPV 18 seropositive at that timepoint, and non-inferiority was met for anti-HPV 16 seropositivity. In the primary analyses, we did not adjust for multiplicity of testing. However, there remained good evidence of non-inferiority for HPV 16 when using a more stringent 97·5% CI in accordance with the Bonferroni correction.The two-dose vaccine schedule is being offered in many countries, including in sub-Saharan Africa, following the change in recommendation from three to two doses.B, AS01E, AS03, and AS04 compared with the Alum adjuvant.This is the first study in sub-Saharan Africa to measure HPV antibody avidity to any dose of HPV vaccine. Avidity is believed to reflect the degree of antibody affinity maturation and reflects how strongly the antibody binds to its target antigen. The India/IARC trial examined the HPV 16 antibody avidity index generated by the 4-valent vaccine in a subset of plasma samples by use of a modified HPV-L1 genotype-specific binding antibody assay.Our study found that vaccination with the 2-valent vaccine resulted in higher concentrations of HPV 16 and HPV 18 IgG antibodies compared with the 9-valent vaccine at any dose. These results are similar to other studies that compared the 2-valent and 4-valent vaccines.Study limitations include the sample size that did not allow us to evaluate efficacy of single dose HPV vaccination in this population. However, as has been done for other HPV vaccination studies and following recommendations arising from a WHO–IARC workshop in 2013, we have immunobridged our results to cohorts in which efficacy of a one-dose schedule has been demonstrated, and results are presented in the companion paper.Our study has several strengths. We enrolled girls in Tanzania which, like many countries in sub-Saharan Africa, bears a high burden of cervical cancer and associated mortality. The representativeness of the trial setting and study population will allow the results to be generalisable to other parts of the continent. The study population is also in the target age group for vaccination, allowing us to evaluate antibody responses to one and two doses over time as girls pass through puberty. The study had excellent retention at 2 years and nearly all participants were vaccinated according to protocol. Immunogenicity analyses for HPV 16 and HPV 18 immune responses were done in a laboratory with significant expertise in HPV serology that has participated in numerous studies of single-dose HPV vaccine responses.Our findings show that, in healthy African girls living in a malaria-endemic region, a single dose of the 2-valent or 9-valent HPV vaccines was well tolerated and resulted in high seropositivity rates and induced stable vaccine responses that persisted to 2 years. Antibody kinetics were similar to other studies in older females in other countries. Follow-up of the DoRIS cohort is continuing to provide data on durability and stability of single dose immune responses. New vaccine efficacy results for single dose in sexually active women are encouraging and efficacy data from observational studies are available up to 9–11 years. A single-dose regimen could encourage countries that have not yet included HPV vaccines in their national vaccination programmes to now introduce these vaccines. A single dose might also allow countries to do one-off activities to reach girls who missed HPV vaccination, including during the COVID-19 pandemic, and to focus on achieving high one-dose coverage rates which could in turn provide faster herd immunity to unvaccinated individuals. All these steps will contribute to the WHO cervical cancer elimination strategy targets for 2030.De-identified participant data presented in this manuscript can be made available after publication following written request to the London School of Hygiene & Tropical Medicine and the Mwanza Intervention Trials Unit, Tanzania. Requests must be accompanied by a defined analysis plan addressed to the corresponding author which will be reviewed by the Mwanza Intervention Trials Unit Data Sharing Committee and senior investigators at the London School of Hygiene & Tropical Medicine. Requesting researchers will be required to sign a Data Access Agreement if approval is given.DW-J reports a grant from GSK Biologicals in 2007 for a previous study on safety and immunogenicity of Cervarix in Tanzania unrelated to this submitted work. All other authors declare no competing interests.


PBMCs from previously cryopreserved venous samples were immunophenotyped by flow cytometry. Proportions of the CD4+ or CD8+ lymphocytes by their immunophenotype subsets were compared between HPV-positive and -negative mothers and their children. The mean rank distribution of CD8+ memory cells was significantly higher among mothers with persistent genital HPV16 infection. The median levels of both the antigen-presenting CD4+ cells and activated CD8+ cells were significantly lower in mothers with persistent oral HPV16 infection. When oral and genital HPV16-persistors were analyzed as a group, a marker of terminal effector cells was significantly increased as compared to HPV-negative women. Significantly higher levels of activated CD4+, CD8+ and circulating CD8+ memory cells were found among children whose mothers had persistent oral HPV16 infection. Persistent HPV16 infections are associated with changes in peripheral blood T-lymphocyte subsets. The mother’s persistent oral HPV16 infection possibly results in immune alterations in her offspring.Only few studies exist on the phenotype distribution of peripheral blood lymphocytes concerning persistent oral HPV infection. T-lymphocyte subsets were phenotyped in women who had persistent genital or oral HPV16 infection, using HPV-negative women as a reference group. A subset of 42 mothers and their children ( Human papillomavirus (HPV) can be acquired by nonsexual or sexual transmission ,3,4,5,6,The host’s immune system is presumed to be a fundamental determinant of the outcome of HPV infection . The ada+ T cells are of key importance. Previous studies have shown CD4+ T-cell-mediated immune response decreases in subjects who develop cancer precursor lesions or cancer due to a persistent HPV infection [+ T cells participate in initiating and maintaining the immune response, whereas CD8+ T-cell function as cytotoxic effectors. However, the role of CD8+ lymphocytes in HPV infection is less clear than that of CD4+ lymphocytes [regs) express CD4+ and CD25+ and because Tregs function in suppressing the immune system, these cells are believed to be involved in the progression of persistent HPV infections [+ T-cell function has been associated with chronic viral infections, due to the expression of several inhibitory receptors [Fortunately, most HPV-infected individuals can clear their infection by an efficient CMI response, in which CD4nfection ,16,17. Cphocytes . Regulatfections ,20. Compeceptors ,22.According to current understanding, the CMI system appears to play an important role in the progression or regression of an HPV infection. Immunophenotyping of peripheral blood T cells could cast further light on understanding the outcomes  of HPV infections even without any clinical HPV lesions. The prospective Finnish Family HPV Study (FFHPV) cohort was used to make comprehensive immunophenotyping of peripheral blood T-lymphocyte subsets in mothers with persistent HPV16 infection, accompanied by similar analyses of their children.The Finnish Family HPV (FFHPV) study is a longitudinal cohort study that was designed to clarify the dynamics of HPV infections within families. Since its onset in 1998, the study has been conducted jointly by the Department of Obstetrics and Gynecology, Turku University Hospital and the Institute of Dentistry, Faculty of Medicine, University of Turku. The original study protocol and its amendments were approved by the Research Ethics Committee of Turku University Hospital .Altogether, 329 pregnant women in their third trimester and their newborns were enrolled in the study between 1998 and 2001, based on written informed consent of all participants. The study design and the key characteristics of the participants have been detailed in a series of previous reports ,24,25. IGenital and oral scraping samples for HPV genotyping were taken with a cytobrush at the baseline and during the follow-up visits: at day 3  and at 1-, 2-, 6-, 12-, 24-, 36-month- and 6-year follow-up visits. The samples were fixed and immediately frozen and stored at −70 °C. HPV DNA was extracted by using the high salt method, as previously described ).Peripheral blood mononuclear cells (PBMCs) from previously cryopreserved venous samples of the mothers and their children were used. The participants were recalled for blood sampling, the mean age of the children being 12.2 years . A detai+CD69+ and CD8+CD69+; (2) Late-activated T cells: CD4+CD25+, CD8+CD25+, CD4+HLA-DR+, CD8+HLA-DR+, CD4+CD38+ and CD8+CD38+; (3) Memory T-cells: CD45RO+CD45RA−; (4) Naïve T cells: CD45RO−CD45RA+; (5) Central memory (CD45RO+CCR7+) and -naïve cells (CD45RA+CCR7+); (6) Effector-memory (CD45RO+CCR7-) and-naïve cells (CD45RA+CCR7-); (7) Resting memory cells and differentiated naïve cells: CD27+CD45RO+ and CD27+CD45RA+; (8) Differentiated memory cells and undifferentiated naïve cells: CD57+CD45RO+ and CD57+CD45RA+.T-cell subpopulations were defined by the presence or absence of different cell surface markers to (i) either early or late activated cells, (ii) memory or naïve cells, as well as (iii) differentiated or undifferentiated cells, closely following the protocol reported by Rodriguez et al. . The T-c+ and CD8+ T cells are reported as percentages of the total number of lymphocytes, the lymphocyte population being selected based on the forward scatter/side scatter pattern. The CD45RO, CD45RA, CD27 and CD57 markers are reported as percentages of CD3+CD4+ or CD3+CD8+ lymphocytes. HLA-DR and CD38 marker populations were estimated as a percentage of the CD3+CD4+ or CD3+CD8+lymphocytes. The CD57 subpopulation was estimated as a percentage of CD3+CD4+ or CD3+CD8+ lymphocytes that were CD27+CD45RA+ or CD27+CD45RO+.CD69 markers in CD4n = 28) was divided into four subgroups based on the mothers’ HPV infection status: Group (1) the first group included 10 mothers, who developed an incident CIN with persistent genital HPV16 infection during the follow-up and 10 children of these mothers; Group (2) the second group consisted of 7 mothers with persistent oral HPV16 infection and their 7 children. Persistent HPV16 infection  was defined by testing HPV16 positive at least in two (or more) consecutive follow-up visits. As to the reference groups, Group (3), consisted of 20 mothers who tested genital-HPV-negative during the follow-up and 8 children of these mothers; Group (4) consisted of 5 mothers who tested repeatedly oral-HPV-negative and their 3 children. To be eligible in these reference groups, the mother had to be always HPV-negative, with no HPV-positive genital or oral sample during the follow-up time.To be eligible for the present analysis, the mother had persistent HPV16 infection either in the genital or oral mucosa. As a reference group, the mothers who tested constantly HPV-negative during the follow-up time were selected as described earlier ,26. In a+ or CD8+ lymphocytes by their immunophenotype subsets were compared between the groups described above; first between the mothers, secondly between the children, and across the mother–child pairs. Bonferroni post hoc tests were used to control for multiple comparisons. Bonferroni uses t-tests to perform pairwise comparisons between group means but controls the overall error rate by setting the error rate for each test to the experiment-wise error rate divided by the total number of tests. Hence, the observed significance level is adjusted for the fact that multiple comparisons are being made. In null hypothesis testing by this post hoc test we used the same significance level  as the settings in options. All statistical analyses were performed by using SPSS statistical software (25.0). All tests were performed two-sided, and statistical significance was declared at the p-value < 0.05.In statistical analysis, the two HPV16-positive groups  were combined as the group of HPV16 carriers, to be compared with the combined group of always HPV-negatives. Between all the groups above, differences in the mothers’ mean age, follow-up time, gender of the children as well as oral HPV status of the children were assessed. The proportion of the CD4n = 28) included in the present analysis are shown in The baseline characteristics of the subgroups of the 42 mothers and their children  was seen in the mothers having persistent genital HPV16 infection (73.0%), while the lowest median proportion (47.0%) was detected in the mothers with persistent oral HPV infection (p = 0.019). In children, the distribution of CD3+ T cells was similar across the three comparison groups. No statistically significant differences were found in the distribution of the CD4+ (helper cells) and CD8+ (cytotoxic) T cells within the CD3+ T cells between HPV+ or HPV− mothers or the respective groups of children  was lower in the mothers with persistent oral HPV16 (p = 0.038). The same was true with the children: children of the mothers with persistent oral HPV16 infection had higher levels of CD38+HLADR+CD3+CD4+ cells; a marker of T-cell activation (p = 0.038).Among mothers, only one significant difference between HPV16 carriers and HPV-negative women was seen in the frequency of immunophenotypic subsets of CD3+CD8+ cells were compared , was significantly higher among the children of the mothers with persistent oral HPV16 infection as compared with the HPV-negative counterparts.When the immunophenotypic subsets of CD3compared , the levp-values that had been declared significant in + lymphocytes: Mothers persistent HPV16 infection vs. always HPV-negative, original p = 0.019, changed after BT to p = 0.084; (2) HLADR+CD3+CD8+: Mothers oral persistent HPV16 infection vs. always HPV-negative, original p = 0.038, changed after BT to p = 0.668; (3) CD38+HLADR+CD3+CD4+: Children of mothers with oral persistent HPV16 vs. always HPV-negative, original p = 0.038, changed after BT to p = 1.000. When BT was used in p-values that were declared significant underwent the following changes: (1) CD3+ lymphocytes: Mothers persistent genital HPV16 infection vs. always HPV-negative, original p = 0.019, changed after BT to p = 0.084; (2) HLADR+CD3+CD8+: Children of mothers with persistent oral HPV16 infection vs. always HPV-negative, original p = 0.006, changed after BT to p = 1.000; (3) CD38+HLADR+CD3+CD8+: Children of mothers with persistent oral HPV16 infection vs. always HPV-negative, original p = 0.008, changed after BT to p = 1.000; (4) CD38−HLADR+CD3+CD8+: Children of mothers with persistent oral HPV16 infection vs. always HPV-negative, original p = 0.018, changed after BT to p = 1.000; and (5) CD45RO+CD8+: Children of mothers with persistent oral HPV16 infection vs. always HPV-negative, original p = 0.033, changed after BT to p = 1.000.Supplemented statistical analysis was also further performed for the control of multiple comparisons by using the classical Bonferroni test (BT). When BT was used, the three + and CD8+ T-cell subsets stratified by the defined groups. This was conducted because the number of mothers defined by their HPV status was limited. Of the immunophenotypic subsets of CD3+CD4+ T cells, only the HLADR+CD3+CD4+ T-cell subset was significantly lower (p = 0.038) in mothers with persistent oral HPV16 infection than in HPV-negative mothers.+CD8+ T cells, only one statistically significant difference was found: CD45RO+CCR7−CD8+ cell population (a marker of memory cells), was significantly more abundant among mothers with persistent genital HPV16 infection than in HPV-negative women (p = 0.048). This difference to HPV-negative mothers remained significant also when the oral- and genital HPV16-positive mothers were pooled together, (p = 0.033).Of the immunophenotypic subsets of CD3+HLADR+CD3+CD8+ cells (a marker of activated T cells) were significantly lower (mean 3.67 ± SD 2.18) than in HPV-negative mothers . When the oral and genital HPV16 persisters were pooled, the CD45RA+CCR7−CD8+ cell population , was significantly increased in HPV16-positive mothers as compared with their HPV-negative mothers (p = 0.033).Among the mothers with persistent oral HPV16 infection, CD38+ and CD8+ T-cell subsets were recorded only between those in the oral  HPV16-persistor group and their control group  as seen in +CD3+CD4+ CD8+ cells  were significantly higher in children whose mothers had persistent oral HPV16 infection (p = 0.018). Similarly, a significant increase in the number of activated T cells was found both for CD4+ helper cells (CD38+HLADR+CD3+CD4+) (p = 0.038) and CD8+ suppressor cells (CD38+HLADR+CD3+CD8+) (p = 0.008). Additionally, the levels of HLADR+CD3+CD8+ cells , were significantly higher (p = 0.005) as were the levels of CD45RO+ CD8+ cells (p = 0.033), i.e., a population of circulating memory cells.Finally, in children, significant differences in the levels of CD4In the present study, we analyzed the distribution of T cells and their subpopulations in the peripheral blood of mothers with either persistent oral or genital HPV16 infection using constantly HPV-negative mothers as the reference group. Similar analyses were also performed in the children of these mothers because immunological recognition of HPV16 seems to occur in early childhood ,26,28,29+ lymphocytes and effector memory CD8+ T cells (CD45RO+CCR7−) in their blood as compared with always HPV-negative mothers. When all HPV16-positive mothers were pooled together, they had also higher levels of terminally differentiated (TEMRA) CD8+ cells (CD45RA+CCR7−), which are the most effective CD8+ cells in destroying tumor cells and virus-infected cells [+) and suppressor (CD8+) T cells among the CD3+ lymphocyte population.These analyses demonstrate that the mothers with persistent genital HPV16 infection had higher levels of CD3ed cells ,31,32. M+ T-cell population in the chronic carrier stage of the infection [+) and memory T-cell (CD45RO+) populations in peripheral blood in women with HPV infection [+CD27−CD8+ T cells in PBMCs were positively associated with HPV persistence whereas CD45RO+CD27+CD4+ T cells had a negative association [+/CD45RO+ expression in cervical intraepithelial lesions (CINs) might even be a prognostic biomarker because the expression is increasing in parallel with the increasing grade of CIN [As to other viruses, a longitudinal study on EBV-infected patients revealed differences in lytic versus latent epitope-specific composition of the CD8nfection ,34. Latenfection ,34. In lnfection ,35,36. Cociation . Howevere of CIN .+CD3+CD4+ and CD38+HLADR+CD3+CD8+ T cells as compared with their HPV-negative counterparts. HLA-DR is a histocompatibility antigen from the MHC II family and both CD4+ helper and CD8+ cytotoxic T cells bear HLA-DR molecules as important surface activation markers. Both HLA-DR and CD38 molecules are present on immature T- and B- lymphocytes and are re-expressed during an immune response. Antigen-presenting cells are essential for HLA-DR on CD3+CD4+ T cells. Co-expression of HLA-DR and CD38 is a key marker of CD8+ T-cell immune activation during several viral infections, e.g., influenza, Dengue, Ebola, and HIV-1 [We also found that mothers with persistent oral HPV16 infection had lower levels of peripheral HLADR+CD3+CD4+ T cells and persistent genital HPV infection [+ T cells, yet these women had also HIV infection [regs) [+ T cells can activate other T cells [+ T cells co-expressing of HLA-DR and CD38 accumulated over a prolonged period in HIV-infected lymphopenia patients. Several studies have also indicated that CD38+HLA−DR+CD8+ T cells could play a recovery role in activating immunity and eliminating the virus [Contradictory to our results, Rodrigues and coworkers reported a positive correlation between HLADRnfection . Additionfection . Importanfection ,42,43,44n [regs) . However T cells ,46. In lhe virus ,47.p = 0.500 or less in most instances where BT resulted in p = 1.000. Considering all the above, our results will need to be further replicated with larger study settings so that we can firmly confirm the findings of this study and the previous studies on this topic.Our contradictory results above compared to the study by Rodrigues and coworkers might also be due to their studies’ lack of correlation for multiple comparisons . Rodrigu+HLADR+CD3+CD4+, HLADR+CD3+CD8+, CD38+HLADR+CD3+CD8+ and CD38−HLADR+CD3+CD4+CD8+ T cells in peripheral blood, being significantly elevated as contrasted to their mothers with declined levels of these subsets. To the best of our knowledge, this is the first study immunophenotyping the peripheral blood lymphocyte subpopulations among mothers with persistent HPV16 infection and their children. Significant alterations in the distribution of lymphocyte subsets were found only in the children whose mothers had persistent oral HPV16 infection. Activated CD3+, CD4+ and CD8+ T cells expressing HLA-DR with or without CD38 were significantly more abundant in these children, compared with children of HPV-negative mothers, being a sign of activated immunity. Importantly, HPV-specific T-cell response was not analyzed in the present study. However, based on our previous data from the Finnish Family HPV Study, we know that the mother seems to be the main HP transmitter to her offspring via mouth [+ cells (CD45RO+) were expanded in these children. It has been suggested that persistent HPV infection could be associated with undifferentiated memory CD4+ T cells and other T-cell subsets that are incompetent in eliminating the viral pathogen [+ T cells expressing HLADR and CD3 were increased both in mothers with persistent oral HPV16 infection and their children (Interestingly, the children of the mothers with persistent oral HPV16 infection had entirely opposite changes in the proportions of CD38ia mouth ,20. In lpathogen ,16,49,50children .Despite its unique design, the present study has also limitations. First, the number of study subjects is limited, including only 42 mothers and 28 of their children. When stratified into cases and controls, this small number is the key limiting factor to reaching statistical power. In addition, the correlation for multiple comparisons needs to be considered as this might potentially help in obtaining false-positive results as discussed above. Similarly, the temporal relationship between HPV acquisition and collection of the PBMCs is not known. Therefore, the dynamics of adaptive immunity cannot be thoroughly investigated from these data. Finally, as previously affirmed , it is nIn general, a detailed dissection of the T-cell immunophenotypes and their relation to the known outcomes of HPV infections could ideally offer important predictive insights in clinical practice. Thus far, the number of studies assessing HPV immunity in relation to natural history is limited. As there is no reliable way to trace the time of the first HPV exposure, the studies on HPV immunology suffer from this inherent handicap. This study is, to our knowledge, the first to conduct T-cell immunophenotyping in mothers and their children in a longitudinal setting. Taken together, these data suggest that both genital and oral HPV16 infections in mothers are associated with alterations in the relative distribution of peripheral blood T-cell subsets. In children of these women, such alterations in T-cell subsets were only found when the mother had persistent oral HPV16 infection.


Dear Editor,Human papillomavirus (HPV), hepatitis B virus (HBV) and Epstein–Barr virus (EBV) are the three most oncogenic DNA viruses, contributing to 15 different types of cancer.n ≥ 5) in our dataset  non‐cancer HPV infection samples, 588 breakpoints in 38.0% (125/329) cervical precancer samples and 1597 breakpoints in 69.0% (158/227) cancer samples Figure . The tott Figure . Among tt Figure .Next, we found that the distribution of HPV integration strains and status in non‐cancer HPV infection, cervical precancer and cancer samples were different Figure . Specifip = .011; precancer vs. cancer, p < .0001; Wilcox test, False Discovery Rate corrected) and may serve as an early warning biomarker of carcinogenesis  from expanded integration datasets Table  and 5. PWe analysed the roles of SD‐EJ using computational simulation Figure  in 4341 n = 1 vs. n = .14, p = 1, Fisher's exact test) due to relatively small dataset  SD‐EJ pathway with SD‐EJ structures, followed by (ii) other alt‐EJ pathway with microhomologies overhangs and otherwise (iii) NHEJ pathway without the previous two signatures Figure . In 10‐bTogether, we report the largest genome‐wide landscape of HPV, HBV and EBV insertional mutageneses. We uncovered HPV, HBV and EBV to share the same common SD‐EJ integration mechanism. Based on our identified integration patterns and the biology features of three viruses, we proposed a new model of the integration process of HPV, HBV and EBV Figure , providiThis work was supported by the National Science and Technology Major Project of the Ministry of science and technology of China (Grant no. 2018ZX10301402); The National Natural Science Foundation of China (Grant no. 32171465 and 82102392); General Program of Natural Science Foundation of Guangdong Province of China (Grant no. 2021A1515012438); the National Postdoctoral Program for Innovative Talent (Grant no. BX20200398); the China Postdoctoral Science Foundation (Grant no. 2020M672995); Guangdong Basic and Applied Basic Research Foundation (Grant no. 2020A1515110170); the Major projects of Wuhan Municipal Health Commission (Grant no. WX19M02); the National Ten Thousand Plan‐Young Top Talents of China.The authors declare that they have no competing interests.Supplementary Note 1 The flow chart of VIPASupplementary Note 2 The performance of detecting virus integration sites in simulation dataSupplementary Note 3 The accuracy of indels calling at virus integration sites in simulation dataSupplementary Note 4 Study design and sample collectionSupplementary Note 5 Virus capture sequencingSupplementary Note 6 Statistical analysisFigure S1 The flow chart of VIPAFigure S2 The performance of detecting virus integration sites in simulation dataFigure S3 The sensitivities and specificities of indels calling at junction sites by VIPA in simulated dataFigure S4 The VIPA validation in cell line modelFigure S5 The IGV image of eight nanopore reads supporting HPV16 integration sites at chr19:55307406Figure S6 The Sanger sequencing results of all validated breakpoints in Ca Ski cell lineFigure S7 The Sanger sequencing results of all validated breakpoints in HepG2.2.15 cell lineFigure S8 The Sanger sequencing results of all validated breakpoints in Raji cell lineFigure S9 The MHs of human viral junctional sequences in other studies.Figure S10 The core algorithms of SD‐EJFigure S11 The display of integration events with MHs structures (10‐bp flanking regions) in three cell linesFigure S12 The statistics of integration events with SD‐EJ structures (10‐bp flanking regions) in three cell linesFigure S13 The schematic of simulation methodology used for comparisonClick here for additional data file.Table S1 The integration structures of Ca Ski validated by nanopore sequencingTable S2 The 24 recurrent integration genes of new HPV samplesTable S3 Dataset of HPV integration eventsTable S4 Dataset of HBV integration eventsTable S5 Dataset of EBV integration eventsTable S6 The common integration genes shared by HPV, HBV and EBVTable S7 The virus capture sequencing data source of dsDNA viruses (soft‐clip reads ≥3)Table S8 HPV Integration breakpoints per sample at each locusTable S9 HBV Integration breakpoints per sample at each locusTable S10 EBV Integration breakpoints per sample at each locusTable S11 HPV breakpoints confirmed by PCR amplification and Sanger sequencingTable S12 Characteristics of HPV infections women without cervical diseaseClick here for additional data file.


Clinacanthus nutans Lindau (C. nutans), a medicinal plant, has potent anti-inflammatory and antiviral effects. C. nutans cream is widely used in Thailand to treat the herpes simplex virus. We proposed that C. nutans might also induce CA clearance. There are no studies of C. nutans treatment of CA. This randomized controlled trial at Siriraj Hospital, Thailand, was conducted between January 2018 and December 2019. CA samples were obtained from 10 men with at least two CAs 1 centimeter apart. Each wart was randomized to a 4-week treatment with either C. nutans or podophyllin. The participants were 24 to 72 years old. Most HPV types were low-risk HPVs . Median CA clearance with podophyllin was a 97% CA clearance with podophyllin and 82% with C. nutans. C. nutans may be an alternative treatment for CA.Human papillomavirus (HPV) infection causes condyloma acuminata (CA). Podophyllin is the standard treatment.  Human papillomavirus (HPV) is the most common sexually transmitted infection worldwide . More thCA has a significant impact on the quality of life . Its trePodophyllin is a plant-derived compound that causes tissue necrosis by arresting cell division and mitosis. Podophyllin may be applied directly to CA once a week, and it should be washed off 4 hours after treatment. The purified form is preferred as it has a good safety profile .C. nutans) is another herb belonging to the Acanthaceae family and is used as a well-known herbal medicine. In Malaysia, Indonesia, Thailand, and China, the plant is used as an alternative medicine for insect bites, skin rashes, herpes simplex virus (HSV) and varicella-zoster virus infection, diabetes, and gout [C. nutans is also recommended as an antiviral agent against HSV and VZV [C. nutans has biological properties, such as antioxidant activity, antiviral activity, anti-inflammatory activity, and immunomodulatory effects [C. nutans is safe as in vivo study on the toxicity of C. nutans did not show any toxicity [Clinacanthus nutans Lindau  Thai men attending the Sexually Transmitted Disease Clinic at Siriraj Hospital; (2) 18 years or older; (3) willing to participate and give their written consent; (4) a diagnosis of anogenital warts by clinical manifestation; and (5) the presence of at least two CAs that were 1 centimeter apart. The exclusion criteria were as follows: patients (1) with HIV, (2) taking immunosuppressive drugs, or (3) with an autoimmune disease.Podophyllin was produced by Vidhyasom Co Ltd, Bangkok, Thailand. The concentration was 25% podophyllin which constitutes of podophyllum resin 25 g in compound tincture of benzoin 100 ml.C.nutans cream was produced by the Government Pharmaceutical Organization (GPO), Bangkok, Thailand, and the Thai Ministry of public health has approved C. nutans cream on the National list of essential medicine. The constitution was C. nutans extract powder 4.343 g in cream 100 g.β-globin gene as an internal control. The PCR, performed in a 100 μL reaction volume, was composed of a 50  μL linear array master mix and a 50 μL of DNA, as per standard protocol. After amplification, the biotinylated-PCR products were denatured immediately by adding 100 μL NaOH to each PCR tube. Hybridization of the denatured amplicons and genotyping were performed on the GT-Blot 48, as per the manufacturer's instruction. Only samples that give a positive signal for β-globin were considered for analysis. Using the linear array HPV reference guide, the strips were manually interpreted using the linear array HPV reference guide, by reading the individual types down the length of the strip. The 37 HPV types that were analysed for comprised 13 HR-HPV types  and 24 LR HPV types . Genital wart samples in one patient were randomized in a block of four to separate genital warts as A and B. Each wart was randomized for treatment with either C. nutans or podophyllin. The participants received C. nutans as a home remedy and were instructed to apply the cream 4 times daily. Follow-up appointments were scheduled for each week for 4 weeks. Wart sizes were measured at each of the 4 follow-up sessions.Before starting its trial, its protocol was approved by the Siriraj Institutional Review Board of the Faculty of Medicine Siriraj Hospital . Written informed consent was taken from the eligible patients, and questions were asked and clinically evaluated. Their medical history was taken, and a physical examination was performed. The lesion sites were the shaft of the penis, glans of the penis, neck of the penis, and glans of the penis. The participants were randomly assigned by a block of four. Specimens swabbed from lesions in the genital area were collected and kept in cryopreserved tubes at −80°C. HPV genotyping was performed by the linear array HPV genotyping test , based on a reverse hybridization of amplicons to immobilize membrane-bound probe. HPV DNA in the swabbing sample was detected by multiplex PCR targeted to amplify the conserved L1 region of the viral genome. The system involves coamplification of the C. nutans were compared using nonparametric Wilcoxon signed-rank tests and paired t-tests. Statistical significance was accepted for P values less than 0.05.Statistical analyses were carried out using PASW Statistics for Windows, version 18.0 . The mean, standard deviation, mode, median, maximum, and minimum were analyzed and reported as percentages. Pretreatment and posttreatment values for podophyllin and The ages of the 10 male participants ranged from 24 to 72 years, with a mean (SD) of 38.7 (16.2) years. All participants (100%) were HPV positive. There were both low- and high-risk HPV types. Mostly, the HPV types were HPV 11, followed by HPV 6. Approximately 50% of the patients were coinfected with multiple types of HPV, and 40% had multiple genital warts .There waC.nutans treatment, 6 of the 10 patients showed a more than 75% reduction, but only 1 patient completed the treatment with a 100% reduction  achieved a 100% reduction with podophyllin treatment. Regarding eduction .C. nutans treatment was an 82.5% CA reduction (C. nutans treatment (P < 0.001).The median total clearance for the podophyllin treatment was 97% CA reduction due to the CA clearance. However, the median total clearance for the eduction . There wCA is a benign disease caused by LR HPV. Our study showed that the most common types of HPV in CA were HPV 6 and 11. This result is consistent with the previous studies in Thailand, the United States, Brazil, Mexico, and China –20. We aC. nutans in clearing HPV-infected CA. We evaluated the efficacy of the 2 treatments using 10 patients.To our knowledge, this was the first controlled clinical trial to compare the efficacy of podophyllin and Treatments for CA include topical therapies applied by the patient or the clinician and cytotoxic or physically ablative therapy approaches. Recurrence rates have been reported to range from 25% to 67% . One-thiPodophyllum peltatum). Podophyllin is a cytotoxic agent that binds to the tubulin subunit of spindle microtubules and arrests mitosis in metaphase, eventually disrupting viral activity [Podophyllin is derived from the roots of the mayapple plant . The clearance rate of the podophyllin treatment was noticeably higher than that reported by an earlier study [C. nutans treatment group, the clearance rate was lower than that of the podophyllin group for all patients.This study evaluated the effects of treating HPV-infected CA with podophyllin  and 4% er study . As to tC. nutans treatment was lower than that of the podophyllin treatment. We suggest combining both to treat CA, for example, having patients apply C. nutans cream at home every day and visit their doctor weekly for podophyllin treatment.However, the efficacy of the C. nutans for the treatment of CA. Furthermore, the recommended application of C. nutans cream 4 times a day for an HPV infection may not be convenient or practical for some patients.Some limitations were in our study. First, as there was a small sample size, a larger cohort is needed to validate the optimal dosage of C. nutans may be beneficial for the treatment of CA. C. nutans may be used as an adjuvant treatment for CA.Our study found that