Objectives: The human papillomavirus (HPV) vaccination rate is generally low in China. This study aimed to assess the effect of web-based education on improving information-motivation-behavior skills related to HPV vaccination among Chinese female college students.Methods: From February to May 2020, female students were recruited from a university and divided into intervention and control groups. The intervention group received 7 days of HPV-related web-based education. Related information were collected using questionnaires in the baseline, 7 days, 1 month, and 3 months after the intervention. Chi-square test and repeated ANOVA were used to compare the differences between the two groups in the four surveys.Results: A total of 449 students (235 in the intervention and 214 in the control group) were included in the analysis. There were no statistical differences in demographic information between the two groups. Compared with the control group, students in the intervention group showed a richer knowledge and subjective norms of HPV vaccination (p < 0.05).Conclusion: The study provides preliminary support for a health intervention via web-based education in increasing HPV vaccination information among female college students. Cervical cancer is the fourth most common malignancy among women worldwide, with 604,127 new cases and 341,831 deaths in 2020 [It has been well acknowledged that persistent infection of high-risk human papillomavirus (HPV) is the primary cause of cervical cancer . FortunaEducation plays a crucial role in HPV vaccine awareness and acceptance in the general population , 15. StuA school-based, prospective, intervention study was conducted involving a 7-day web-based education intervention with a further 3-month follow-up. From February 2020 to May 2020, female first-year students were recruited online from a university in Zhengzhou, Henan Province through convenience sampling. An electronic version of the informed consent form was sent to voluntary participants. The inclusion criteria are as follows: 1) females 18 years of age and more; 2) first-year undergraduates; 3) no vaccination contraindications. The exclusion criteria are as follows: 1) males; 2) females under 18; 3) non-undergraduate first-year students; 4) medical students; 5) previous history of vaccination contraindications; 6) current pregnancy or breast feeding.Ethical approval for the study was obtained from the Institutional Review Board of the Chinese Center for Disease Control and Prevention on 24 October 2019. Before enrolment in the study, participants were well informed of the purpose, methods, expected risks, and benefits of this research. All participants voluntarily signed informed consent.The participants were divided on a class level to intervention and control groups after stratification by liberal arts and sciences, and were invited to join the corresponding DingTalk team. DingTalk is a free mobile office platform for communication and collaboration created by Alibaba. The students in the intervention group received 7 days of HPV-related web-based education, while the control group received just popular science education (not HPV-related). To maintain students’ interest in the control group, the non-HPV publicity materials were uploaded at the same time every day. The content of the HPV-related web-based education was developed under the guidance of IMB model after reviewing the related literature and conducting several rounds of panel discussions. It contains 2-day materials to popularize HPV knowledge, 2-day materials on situational stories to motivate participants to vaccinate themselves against HPV and 3-day materials on objective skills with self-decision making, self-efficacy, and objective conditions for making an appointment and receiving the HPV vaccine. The above intervention materials were designed as online readable texts or videos that could be easily accessed by the target population, see Information from participants was collected using a structured electronic questionnaire four times: at the study enrollment, after completing the 7 days of web-based education intervention, 1 month, and 3 months after the intervention. The content of the questionnaire includes sociodemographic information, participants’ information concerning HPV, motivation for vaccination, and behavioral skills regarding HPV vaccination.p < 0.001).For the information concerning HPV, there were 11 items, and the answers were “Agree,” “Disagree,” or “I do not know” , 22. EacThe online questionnaire was delivered through the DingTalk team. Participants in the corresponding team could complete the questionnaire anonymously and independently using DingTalk. The content of the questionnaire did not involve any personal information related to the participants. After the questionnaire was completed, the trained investigators checked and corrected the questionnaire logically.p < 0.05 (2-tailed test).Chi-square or Fisher’s exact test analysis was used to compare demographic information, and information concerning HPV at baseline and 3 months after intervention between the intervention, and control groups. Since the information, motivation and behavioral skills of each participant were repeatedly measured four times, ANOVA of repeated measurement data was used to evaluate the effect, and the resulting variables of subjects who lost the follow-up were analyzed according to the missing values. Statistical analysis was performed using SAS 9.4. Statistical significance was set at 455 students were recruited to the project, of which 6 students (1.3%) had HPV vaccination, and the remaining 449 unvaccinated students were included in the final analysis. In the baseline, there were 235 students in the intervention group and 214 students in the control group. In the follow-up survey, 93 students were lost, and only 356 students completed the fourth survey, including 189 in the intervention group and 167 in the control group .Most students were Han Ethnicity (98.9%) and lived in rural areas (56.6%). 63.0% of students had an average living expense of 1,000 to 2,000 Chinese Yuan (CNY) per month. Among the subjects, 77.1% of their parents were in the state of marriage, and 60.1% of them had junior high school education or below. About 52.6% and 51.0% of the students had heard of HPV-related diseases and the HPV vaccine, respectively. There were no statistical differences in demographic information between the intervention and control groups. More details are shown in There were 11 items about HPV-related information. At baseline, only 5 items had more than 50% accuracy. About 70% of the students knew that “HPV is related to the development of cervical cancer,” and more than 70% knew that “Regular cervical cancer screening is necessary after HPV vaccination” and that “The HPV vaccine does not protect against all types of cervical cancer.” More than 50% of the students knew that “HPV infection may result in oral cancer, condyloma acuminatum, and anal cancer.” Two items had less than 10% accuracy, including “HPV is almost asymptomatic” and “Most HPV infections will disappear on their own.” There were no statistical differences in all the 11 items between the intervention and control groups. More details are shown in p < 0.05). More details are shown in Three months after the intervention, the accuracy improved in both groups, and there were 7 items with more than 50% accuracy. Except for the item, “HPV is almost asymptomatic,” the accuracy of the other items in the intervention group was higher than in the control group . Statistically significant differences were observed between the groups in the scores of “information” and “subjective norms,” with higher scores in the intervention group (p < 0.05). Notably, there was an increase in the score of “perceived barriers” but a decrease in the score of “decision-making” in both groups within 3 months of intervention (p < 0.05). See Compared with the baseline, the mean score of “information,” “perceived susceptibility,” “subjective norms,” and “self-efficacy” increased significantly within 3 months after the intervention (all This study explored the effect of web-based education on improving college female students’ information-motivation-behavior skills related to HPV vaccination. The results showed that web-based education could significantly improve the HPV-related information in female college students. However, the effect on motivation and behavior skills related to HPV vaccination may require a longer follow-up. The results may provide a reference for the future promotion of HPV vaccination.via web-based education in increasing HPV vaccination information among female college students.The results of our study showed that Chinese female college students had a low understanding of HPV, indicating that although it has been 4 years since the HPV vaccine was approved in China , the awaIt is worth noting that after 7 days of web-based education, there were increases in students’ barriers to HPV vaccination, such as more doubts about the safety and effectiveness of the vaccine, more concerns about the side effects and pain of the vaccine. It is speculated that the reason may be that students pay more attention to the safety and effectiveness of vaccine after they have a certain understanding of it. So far, many studies have proven the safety and efficacy of the HPV vaccine –30, therThe study found a significant increase in HPV-related knowledge through web-based education, which was consistent with other study . HoweverThe results showed that students in the control group experienced increased HPV vaccination knowledge and perceived susceptibility but more perceived barriers and decreased decision-making in the post-intervention period. The reasons may be as follows: on the one hand, this study is not blind to the participants. Since the participants are students from the same school, they might discuss the contents of the survey with each other, making students in the control group also obtain some knowledge of HPV. On the other hand, the questionnaire in this study involved many HPV-related questions, and participants were surveyed repeatedly using this questionnaire four times. Thus, the students in the control group may inquire about HPV-related knowledge out of interest after finishing the questionnaire, which could improve their understanding of HPV-related information. However, such information is likely to be incomplete or negative, leading to a worse perception of HPV vaccination and decreased decision-making ability.There are several limitations of the study: first, the participants in our study were not blinded, and contamination may occur. Second, the study was carried out among female college students in a university in Zhengzhou, Henan Province. The HPV knowledge and vaccine receptivity may vary widely based on region, sociodemographic factors, and education level, so the findings may not be generalized to the entire Chinese female population. Third, the HPV-related educational intervention only lasted for 3 months, which rendered its impact on motivation, behavioral skills, and vaccination rate not obvious.In conclusion, our study found that web-based education could significantly improve the HPV-related information in female college students, but the effect on motivation and behavior skills related to HPV vaccination may require longer follow-up to be observed. Follow-up studies are needed to observe if an increased HPV-related knowledge might be translated into higher vaccination rates in China. The optimization of vaccination will need to be further enhanced to address the relevant barriers. The findings of our study could provide valuable information for future HPV vaccination policies during this critical time of the global acceleration towards the elimination of cervical cancer.


CRTC1/3-MAML2 gene fusions, but the additional molecular drivers remain largely uncharacterized. Here, we sought to resolve controversy around the role of human papillomavirus (HPV) as a potential driver of mucoepidermoid carcinoma. Bioinformatics analysis was performed on 48 MEC transcriptomes. Subsequent targeted capture DNA sequencing was used to annotate HPV content and integration status in the host genome. HPV of any type was only identified in 1/48 (2%) of the MEC transcriptomes analyzed. Importantly, the one HPV16+ tumor expressed high levels of p16, had high expression of HPV16 oncogenes E6 and E7, and displayed a complex integration pattern that included breakpoints into 13 host genes including PIK3AP1, HIPI, OLFM4,SIRT1, ARAP2, TMEM161B-AS1, and EPS15L1 as well as 9 non-genic regions. In this cohort, HPV is a rare driver of MEC but may have a substantial etiologic role in cases that harbor the virus. Genetic mechanisms of host genome integration are similar to those observed in other head and neck cancers.Mucoepidermoid Carcinomas (MEC) represent the most common malignancies of salivary glands. Approximately 50% of all MEC cases are known to harbor  CRTC1-MAML2 fusion gene is one of the most widely studied alterations in this disease )Given discrepant data around the prevalence of HPV in MEC, we sought to leverage our recently published MEC transcriptome data and advanced bioinformatics techniques to clarify the prevalence and status of HPV in MEC.CRTC1/3-MAML2 gene fusion status by RT-qPCR [A retrospective cohort of patients with MEC was previously identified from the University of Michigan pathology archive using an Institutional Review Board (IRB)-approved protocol for next generation sequencing of DNA and RNA (HUM00080561). However, patients were not consented for deposit of data in public databases. The cohort was previously typed for  RT-qPCR . As prev RT-qPCR . Total R RT-qPCR . Here, t RT-qPCR . A previImmunohistochemical staining was performed on the DAKO Autostainer  using Envision+ and diaminobenzadine (DAB) as the chromogen. De-paraffinized sections were labeled with the mouse p16 Ab-1 (DCS-50.1/47)  and a mouse Ab  was used as secondary antibody. Microwave epitope retrieval as specified was used prior to staining for all antibodies. Appropriate negative (no primary antibody) and positive controls (as listed) were stained in parallel with each set of slides studied. p16 immunostained slides were analyzed as previously described by our team [Formalin-fixed paraffin-embedded (FFPE) blocks were obtained for the single patient with HPV16+ MEC (as described below) and five additional patients without HPV reads by HPViewer, selected at random, for confirmatory NGS-based tumor analysis. Regions with >60% tumor content, as identified by our head and neck pathologist (J.B.M.), were identified for DNA isolation with the Qiagen Allprep DNA/RNA FFPE kit . Using the DNA Thruplex kit for library preparation (Takara Biosciences), targeted capture sequencing on DNA that passed our quality control standards was performed by the University of Michigan Advanced Genomics Core as previously described . We empl--max-bundle-frags parameter of cufflinks was adjusted from its default value of 1,000,000 to 100,000,000 to allow us to compute FPKM at loci with high depth of coverage. Additionally, we applied SurVirus [Quality of the sequencing reads was evaluated using FastQC v.0.11.5. The quality reports did not reveal any adapter contamination; therefore, it was not considered necessary to perform quality trimming. The reads were mapped to the hg19 reference genome following a two-step alignment workflow of STAR v2.5.3a. Next, samtools v1.2 was used to extract uniquely mapped reads and Cufflinks v2.2.1 was used to generate the FPKM data. The SurVirus  and SearSurVirus  to identThe first step in viral oncogene expression analysis was to build a reference genome of human and viral sequences. For this purpose, we used a modified version of the HPV16 genome, and its corresponding annotation file as described in . The humWe recently performed comprehensive transcriptome sequencing on 48 FFPE MEC tumors with a majority of tumors arising in the parotid. To now characterize the HPV content of tumors within this cohort, we analyzed the data using the HPViewer algorithm . This anCRTC1-MAML2 fusion [Clinically, MEC1 was a locally recurrent MEC of the anterior ethmoid sinuses in a 51-year-old male who underwent anterior sub-cranial resection with pathology showing high-grade MEC without perineural invasion and with negative margins. The patient had initially presented three years prior with a high grade MEC of the anterior ethmoid sinuses treated with subtotal resection followed by adjuvant radiation. He is now alive with no evidence of recurrent disease over 19 years out from salvage surgery. Our previous RT-PCR molecular sub-typing analysis of his tumor demonstrated the presence of a 2 fusion .To further validate our HPV annotation of this cohort, we performed PCR and Sanger sequencing of genomic DNA from MEC1, which confirmed the presence of HPV16 DNA  in this TMEM163, HIP1, and SIRT1 genes and reverse orientation insertions in the remaining ten integrations.  in our MEC cohort, with the single positive case of recurrent MEC of the anterior ethmoid sinus harboring transcriptionally active HPV16+ DNA with multiple complex integration events into various cancer-related genes. Concurrently, this tumor showed upregulation of p16 by IHC and altered expression of host genes affected by viral integration events. Our data raises several important points for the discussion of MEC tumor biology and the development of clinically useful, predictive, and prognostic biomarkers for this disease.The etiologic role and prognostic implications of HPV in head and neck malignancies besides squamous cell carcinoma of the oropharynx remains a contentious and much-debated topic in our field . ValidatCRTC1-MAML2 translocation. They utilized HPV E6/E7 RNA ISH to assay HPV status but failed to identify transcriptionally active virus in any of their samples, independent of CRTC1-MAML2 fusion status. Overexpression of p16 was not evaluated in their cohort. In a much smaller sample of six minor salivary gland MECs, Brunner et al. used HPV16/18 DNA ISH and p16 IHC to show the presence of HPV16/18 DNA in two (33.3%) MECs of the oral cavity [Our findings of rare HPV positivity in MEC are consistent with Bishop et al. . The autl cavity . InteresOur data conflicts most notably with that of Isayeva et al. in which the authors found a strikingly high prevalence of HPV16/18 DNA in 49/98 (50%) of their MEC cohort ,17,29. AHerein, we conclude that transcriptionally active HPV is a rare occurrence in MEC, independent of tumor subsite. Previous studies have failed to show a higher prevalence of HPV in MECs of anatomic subsites within lymphoid tissue of Waldeyer’s ring. Further, there seems to be no predilection for HPV positivity in mucosal subsites or minor versus major salivary glands. Our single HPV-positive tumor was a recurrent MEC of the anterior ethmoid sinuses. It is interesting to speculate that, although an infrequent event overall, MECs of sinonasal subsites may be more prone to harbor HPV as a molecular driver. However, we cannot definitively reach that conclusion with our data.Histologically, MECs are characterized by a variable, triphasic pattern of mucinous, intermediate, and epidermoid cells with histologic grade dependent on degree of nuclear atypia, mitoses, necrosis, perineural invasion, and cystic components . PreviouPIK3AP1, SIRT1, ARAP2, TMEM161B-AS1, and EPS15L1 as well as 9 non-genic regions. Interestingly, the PI3K pathway has been implicated in MECs, and alterations were identified in 52% of high-grade cases in a targeted sequencing analysis of one cohort, which was consistent with RNAseq analysis of 8 high-grade tumors showing a re-programming of PI3K signaling effectors [PIK3AP1 expression drives AKT phosphorylation in gastric and thyroid cancer models, suggesting an oncogenic role of this gene in other cancers [When MECs harbor transcriptionally active HPV however, the virus may significantly alter host gene expression by complex viral integration mechanisms. We showed that HPV integrated into 13 host genes, including ffectors . Previou cancers ,33. NotaSIRT1 is also of potential pathogenetic interest to MEC. SIRT1 has a complex and multi-faceted role in cancer that includes the regulation of TP53 as well as responses to DNA damage, metabolism stress, and inflammation [SIRT1 is a NAD+-dependent Class III histone deacetylase that has been shown to antagonize cellular senescence [CRTC2, and possibly CRTC1 as well [CRTC1-MAML2 expression in this tumor. Likewise, in the context of mutant p53, SIRT1 acts as a tumor suppressor [SIRT1 could act as a tumor suppressor in the context of HPV16+ MEC1, consistent with the relatively low expression of SIRT1 observed in this sample. To our knowledge, ARAP2, TMEM161B-AS1, and EPS15L1 have not previously been indicated as playing a role in MEC pathogenesis. As future genetic analyses of MEC tumors are published, it will be interesting to see if any of these genes disrupted by HPV integration are also altered by other genetic mechanisms, as the observation of multiple mechanisms of genetic disruption would support a critical role for the genes.Despite this notion, viral integration into ammation . SIRT1 inescence  and is a as well , suggestppressor . Given tCRTC1/3-MAML2 fusion negative cases. From a long-term perspective, while the prevalence of HPV-related cancers is increasing around the world, our data suggests that HPV-related MECs are likely to account for only a minor subset of this disease. Our data is consistent with recently published series in concluding that no particular MEC subsite is more prone to harbor transcriptionally active HPV. Indeed, while our data resolve fundamental questions about the role of HPV in this disease, it also supports a need for future research to help identify additional genetic drivers of MEC. Due to the rarity of transcriptionally active HPV in MEC, we cannot advocate for routine p16 or HPV DNA ISH testing in clinical practice.In conclusion, using our integrative sequencing analysis, we demonstrate that transcriptionally active HPV is a rare occurrence in MEC. However, when present, HPV can have a substantial role in altering the host genome, including through the direct integration and disruption of host genes. Our data suggest that alternative drivers other than HPV are much more frequently responsible for the pathogenesis of MEC, including


A new virus‐like particle based vaccine covering 14 types of high‐risk and disease‐inducing human papillomavirus (HPV) can offer better coverage against HPV‐induced diseases, particularly cervical cancers. However, the assessment of immunogenicity of the vaccine is an important task, representing not only its significant clinical characteristics, but also a major challenge, in terms of both the suitability of methods and the clinical sample testing throughput supporting clinical development. This work covers the development and evaluation of a method based on Luminex technology (a coded‐bead and flow‐cytometric approach) to assess the HPV‐type specific total immunoglobulin G (IgG). This method can evaluate the antibodies in sera post immunization against multiple types of HPV simultaneously , save time and cost, and improve test throughput with higher sensitivity and precision than the classical, plate‐based enzyme‐linked immunoassay and competitive Luminex‐based immunoassays. Using cynomolgus monkeys as model, we demonstrated the good correlation between the results from the pseudovirion‐based neutralization assay (PBNA), and the Luminex‐based total IgG assay, supporting that the latter method can be considered as a viable, dependable replacement method for the PBNA supporting immunogenicity evaluation of HPV vaccine in preclinical development and clinical investigation. It is well known that persistent infection of HPVs can first lead to cervical intraepithelial neoplasias with progressive grades and then to cervical cancer. In fact, 99.7% patients with cervical cancer could be detected with evidence of HPV infection.The majority of HPV types associated with human cervical cancers are from HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and likely 68.The discovery of HPV causing cervical cancers paved the road for vaccine development. Over the past decade or so (since 2006), virus‐like particle (VLP) based HPV vaccines had become the major product to prevent HPV infection and related diseases. Those VLPs are made by recombinant HPV L1 capsid proteins. The capsid structures of VLPs in the vaccines are highly similar to those of the natural virus particles so that they not only have the good immunogenicity to induce high titer of neutralization antibodies against individual HPV types but also offer long‐term protection against HPV‐induced diseases. With the absence of a viral genome, recombinant VLP‐based vaccines not only have proven efficacy but also have a good safety profile. Until June of 2021, there are 4 types of HPV vaccines which are all L1‐protein based VLP vaccines, approved for marketing, Gardasil® (approved in 2006),There are still many HPV vaccine candidates in various stages of the clinical development, including the one developed by Sinocelltech, Ltd. with 14 HPV types . This VLP‐based HPV vaccine has the potential to offer the best coverage against HPV‐induced diseases to date, with the potential cervical cancer protection rate worldwide as high as 95.4%.Since there is no adequate, relevant animal model for HPV infection due to viral species‐specificity, it is recommended by WHO that the pharmacodynamics properties of an L1‐VLP‐based vaccine should be assessed through immunogenicity studies in addition to other methods defined by an individual national regulatory authority.In this study, for the purpose of supporting the 14‐valent vaccine clinical studies, we developed a method based on the Luminex technology to assess the HPV‐type specific total IgG with the same mechanism as that of direct‐binding ELISA. Importantly, the Luminex‐based platform  can support the evaluation of the antibodies against multiple types of HPV‐VLP simultaneously, which has the advantages of saving time and improving test throughput, reliability, and data analysis. We have demonstrated with the nonhuman primate studies that the immunogenicity data obtained from the LTI high‐throughput method correlate well with those from the PBNA method, and the LTI method can be considered a useful and efficient supplement or replacement method for supporting both the preclinical and clinical immunogenicity assessment in the development of HPV vaccines with high valency.22.1Recombinant HPV VLP vaccine used in the preclinical study contained VLPs of HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 with 9 types overlapping with those in Gardasil 9® and the new 5 types .The 14‐valent recombinant vaccine is prepared from the purified individual VLPs of the individual major capsid (L1) proteins of 14 HPV types. First, the recombinant baculovirus for each HPV type can be acquired from the expansion in insect cells, after the construction of the recombinant plasmid containing the L1 gene of each type of HPV and transfection of baculovirus. The L1 proteins are produced by the expression and expansion of insect cells that have been infected with the baculovirus and self‐assembled into VLPs within the cells for each type. At the end of the expression, the insect cells were harvested and homogenized with the HPV VLPs isolated from the cell debris through centrifugation. The clarified supernatant for each HPV type was separately further purified through multiple steps of chromatography and filtration as well as virus inactivation and removal processes, leading to the aqueous drug substance bulk for each HPV type.To study and confirm the structural and quality properties of the individual drug substance to be used for the 14‐valent vaccine, dynamic light scattering (DLS) was used to assess the conformational integrity of each VLP type, which was a method to obtain the particle size information by measuring the fluctuation of the scattered light intensity of the particulate sample with time. The DLS analyses of the drug substance of the 14‐valent vaccine are carried out with the results showing that the protein particle size and its distribution of each batch of drug substance meet the quality standards (data not shown). Furthermore, the tungsten‐stained transmission electron microscope  was also used to assess the conformational integrity and particle morphology of each VLP type. The results showed that the VLP structure of each batch of the vaccine drug substance was fairly uniform with particle size about 60 nm in diameter. The structural features and protein particle size are consistent within the samples and among different production batches. The typical figures of electron microscope were shown in Figure To evaluate the type‐specific conformation of the VLPs used in this study, we have developed 14 type‐specific and neutralizing monoclonal antibodies to identify each type of VLPs. It is well known that type specific and neutralizing antibody targets conformational epitopes.50 of immunized serum by single type VLP was evaluated using 14 types of pseudovirus separately by the PBNA method. The data showed good specificity of VLPs, as shown in Table For VLP cross‐reactivity testing, EC2.2The individually expressed, separated, and purified VLP drug substances are absorbed on the preformed aluminum‐phosphate‐based adjuvant. Then, combining the adjuvant‐adsorbed VLPs of each HPV type in a predefined ratio to produce the final vaccine with formulation buffer containing histidine, sodium chloride, and polysorbate 80 post fill and finish process.2.3Cynomolgus monkeys were immunized via intramuscular injection with different doses and frequencies for various study objectives. In this study, we focused on the correlation between the two methods of immunogenicity assessment, namely the PBNA and LTI methods, and the data of both HPV type‐specific total IgG concentration and neutralizing antibody titer were collected, analyzed, and compared. The vaccination method and sampling time points are shown in Table 2.4The HPV PBNA, which was developed independently by Sinocelltech Ltd., was utilized in this study to investigate the immunogenicity of the candidate vaccine in cynomolgus monkeys. Specifically, the cotransfection of mammalian cells with two HPV capsid genes, L1 and L2, together with the green fluorescent protein (GFP) plasmid produced high infectious titers of pseudovirions, which presents the surface conformational epitopes of the capsid proteins similar to those of the native virions. As shown in Figure In PBNA, preimmunization and postimmunization sera were diluted serially and then incubated separately with each HPV type of pseudovirion to test the neutralizing‐antibody levels. Because it is difficult to cover all samples for all 14‐type HPV assays in one dilution range, a portion of samples were retested by a modified and more optimized dilution range. The mean of all test results for each HPV type was calculated in the final investigation.The immunogenicity investigation of the vaccine was based on the 14‐subtype HPV PBNA. Transfection of 293FT cells by the type‐specific HPV pseudovirion could be neutralized by neutralizing antibodies stimulated by vaccine immunization of cynomolgus monkeys. When 293FT was cultured with the pseudovirion‐sera mixture, the number of GFP‐positive cells would decrease with the concentration increase of the neutralizing antibodies in monkeys' sera. As a result, within certain limits, the relationship between the amount of neutralizing antibodies and the number of GFP‐positive cells showed a negative linear correlation. Thus, GFP cell counts could reflect the level of HPV neutralizing antibodies in animal serum in PBNA.g centrifugation for 5 min. 293FT cells were plated 4–6 h in advance in a 96‐well cell culture flat‐bottom plates at 15,000 cells/well in 100 μl cell culture medium and then cultured at 37℃, 5% CO2. Preimmunization and postimmunization sera were serially diluted with five dilutions from 400 to 2500 and then mixed separately with each diluted HPV type 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 pseudovrius individually. After incubation at 2–8℃ for 1 h, the 100 μl pseudovrius‐sera mixture was transferred into pre‐plated 293FT cells and incubated for 62–96 h. All samples were tested in duplicate wells, and meanwhile, pseudovirion only and medium only wells were set as positive and negative controls, respectively.Sera samples were inactivated at 56℃ for 30 min followed by a 10,0002.5The key technical core of the Luminex was to be able to address specific beads  by adjusting the ratio of two different fluorescent dyes to code multiple magnetic beads with different characteristics of fluorescence spectra. For the planned study, 14 kinds of commercial magnetic beads were covalently cross‐linked with an appropriate amount of specific high‐quality HPV VLPs, to obtain 14 individual beads covering 14 HPV types, i.e., one addressable bead per HPV type. The efficiency and specificity of each kind of VLP‐coupled magnetic beads were verified in the use of qualified specific anti‐HPV VLP monoclonal antibodies with high response to the target type and without cross‐interference with any other types. After quality was released, the VLP‐coupled magnetic beads were mixed and incubated with monkey serum; then, a phycoerythrin (PE)‐conjugated anti‐monkey specific secondary antibody was used as the detection reagent; finally, the complexes were detected by the Luminex 200 equipment. The fluorescence signal values  were related to the amount of serum total type‐specific IgG antibody bound with the individually immobilized HPV‐VLPs on the addressable beads. The mechanism of the Luminex‐based total IgG assay is schematically shown in Figure The anti‐HPV VLP serum obtained from cynomolgus monkeys immunized repeatedly with the investigational vaccine was chosen as the standard substance to achieve the quantitative determination of total IgG in serum. This is an ideal method for the efficacy or immunogenicity testing of vaccine candidates, and for this purpose, the comprehensive methodological validation of the Luminex method for detection of total specific IgG in the serum of cynomolgus monkey has been completed. Although there were no specific acceptance criteria for the parameters in the guidelines for method validation of vaccine immunogenicity evaluation, the validation conducted referred to the guidelines and recommendations for the bioanalytical method validation of ligand‐binding assays which were generally used to support pharmacokinetic assessments with more strict criteria, and the results indicated that the method was robust and accurate, showing good appearance in all the validated parameters, such as selectivity, linearity, precision, accuracy, and stability. The LTI methods were fully validated. Briefly, the intra‐batch accuracies (%RE) of QC samples of all HPV types were −5.82%–18.81%, while their inter‐batch accuracies were 2.50%–7.99%. Precision results showed that the intra‐batch precisions (%CV) of QC samples of all HPV types were 0.16%–17.22%, and the inter‐batch precisions of the same samples were 5.31%–11.70%. The stability samples from cynomolgus monkey standard serum were prepared and proved stable with the criteria of %RE within ±20% under different conditions, including a room temperature storage for 4 h, 2–8℃ storage for 24 h, 5 freeze‐thaw cycles, and −80℃ storage for 160 days. All results of the ISR  test showed good reproducibility, corresponding to the %Bias within ±30%.2.62.6.1The percentage of neutralization of pseudovirion infecting 293FT cells for a given sample of a specific HPV type is calculated as follows using HPV pseudovirion positive cell counts as below.No pseudovirion; and B = Preimmune sera − BackgroundNo pseudovirion.where A = Immune sera − Background50) achieving a 50% reduction of the maximal signal and were calculated with the Reed‐Muench method.The neutralization titers were defined as the effective concentration  as the standard. The reference serum and the Standard 05‐134 were diluted in gradient and evaluated in 3 runs of the PBNA assays. The data were calculated by the parallel lines method using CombiStats 5.0 Software to evaluate the potency or concentration of antibodies of each HPV type in the reference serum and then assigned relative potency accordingly. All potencies of 14 HPV types were expressed in mNU/ml , shown in Table 2.6.3p value are shown after the numeric analysis. If the significance/p value is less than or equal to 0.05, it proves that the correlation is significant, which means the data between PBNA and LTI are correlated. As for the correlation coefficient, the closer it is to 1, the higher extent is for the correlation between the two datasets.To compare the correlation of the data between PBNA and LTI methods, the Pearson correlation method was used in the analysis. In GraphPad Prism 8 software, the correlation coefficient (r) and correlation significance (two tailed)/33.1p value less than 0.0001, suggesting the presence of a clear correlation, particularly considering that the datasets are still limited in size.In Study #1, the monkeys were vaccinated with the low dose , but the immunogenicity data from the two methods were still clearly correlated. This correlation is significant even with the limited size and scope of the sampling (N = 36). The data were presented for the new 5 HPV types  that were not presented in Gardasil 9, and they were all shown with high correlation (at least 0.709), except HPV56 (r = 0.637), where the data spreading in neutralization concentration range was limited, but supporting the presence of correlation.In Figure p < 0.0001) , so that it is a broader measure of the immunogenicity resulting in increased sensitivity, given that the antigens (or VLPs) used in the assays are of high‐quality and representative. However, cLIA demonstrated by Merck is an assay to evaluate the limited neutralizing antibodies which has high specificity at the expense of reduced sensitivity and immunological coverage.There are several reported studies on the correlation analysis between PBNA and the other methods to evaluate protective antibodies in humans as mentioned above, but no such studies have been reported for cynomolgus monkeys covering upto 14 HPV types within one test. Here, we demonstrated the correlation between PBNA and LTI testing of postimmunization sera in the immunogenicity assessment in monkeys and our findings support the use of LTI assay in clinical settings, and, therefore, with the added advantage of the multiplex for the Luminex technology, it is possible to cover as many testing needs as desired, providing a truly suitable supplemental or replacement method for PBNA in both nonclinical and clinical pharmacodynamics studies. We have initiated the method validation work for human sera for the purpose of supporting Phase III efficacy study and immunological bridging studies of various age and ethnic groups in the future. Those works will be reported as well to further confirm the concepts reported here.In nonhuman primate studies, HPV VLPs have been tested and proved that repeated dosing with as low as 10 μg per dose was able to induce immune responses to reach a plateau.The data in Figure 5To support a new VLPs based 14‐valent HPV vaccine development, a new high throughput method (LTI) was developed, and its value and utility were confirmed through the comparative assessment of the immunogenicity in cynomolgus monkeys with the PBNA method. The results and analysis from the monkey studies confirm that LTI with higher throughput, sensitivity, and precision is a new dependable replacement analysis method for the PBNA supporting immunogenicity evaluation in clinical studies, especially for efficacy assessment of multivalent HPV vaccines with large study size and longer duration.Liangzhi Xie and Hongbin Qiu: conceived the study and provided overall guidance. Lei Bei and Xiao Zhang: wrote the original draft. Lei Bei, Xiao Zhang, Dan Meng, Shuman Gao, and Jilei Jia: conducted the studies and analysis on data. Chunxia Luo, Dandan Zhao, and Xuefeng Li: helped supervise the project. All authors contributed to revising the draft, providing final confirmation of the revised version, and being responsible for all aspects of the work.The authors declare no conflicts of interest.There is no experimental work with humans included in this article. All animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee (IACUC).Supplementary information.Click here for additional data file.Supplementary information.Click here for additional data file.Supplementary information.Click here for additional data file.Supplementary information.Click here for additional data file.


Tumour virology was born with the discovery by Peyton Rous in 1911 of a filterable agent in chicken cellular extracts that caused neoplasia in healthy chickens. Universally, 20% of all human cancers have a viral aetiology. Viruses are involved at various stages of the carcinogenesis pathway, depending on the viral pathogen, and likely require co-factors. Multiple risk factors have been associated with oesophageal and gastric malignancy, including carcinogenic pathogens. These viruses and bacteria include human papillomavirus (HPV) [oesophageal cancer], Epstein–Barr virus (EBV) [proximal stomach cancer], and Helicobacter pylori (HP) [non-cardia stomach cancer]. Viruses such as EBV have been firmly established as causal for up to 10% of gastric cancers. HPV is associated with 13 to 35% of oesophageal adenocarcinoma but its role is unclear in oesophageal squamous cell carcinomas. The causal relationship between hepatitis B (HBV), cytomegalovirus (CMV), HPV, and John Cunningham (JCV) and gastric neoplasia remains indeterminate and warrants further study. The expression of viral antigens by human tumours offers preventive and therapeutic potential (including vaccination) and has already been harnessed with vaccines for HPV and HBV. Future goals include viral protein-based immunotherapy and monoclonal antibodies for the treatment of some of the subset of EBV and HPV-induced gastro-esophageal cancers. It is estimated that between 15% and 20% of all human cancers worldwide are caused by infectious agents ,4. This Multiple risk factors have been associated with oesophageal and gastric malignancy, including carcinogenic pathogens. These viruses and bacteria include human papillomavirus (HPV) [oesophageal cancer], Epstein–Barr virus (EBV) [proximal stomach cancer], and Helicobacter pylori (HP) [non-cardia stomach cancer] ,9,10. ThIn this review, we discuss the published data linking viruses to oesophageal and gastric malignancy. We pay particular attention to the role of HPV and EBV in oesophageal and gastric cancers respectively. In addition, we discuss the role of lesser-known viruses e.g., John Cunningham Virus (JCV) and oesophageal and gastric tumours as well as the association between Hepatitis B and gastric neoplasia.Oesophageal cancer is the seventh most common cancer worldwide with a male preponderance (70%). There are an estimated 604,000 annual incident cases, and it is the sixth leading cause of cancer death with an estimated 544,000 deaths annually . The twoOSCC has a multifactorial aetiology depending on geographical location. In the West, excess alcohol and smoking is incriminated. Intriguingly, betel nut chewing in the Indian subcontinent and South-East Asia as well as drinking extremely hot tea in South America and Iran has been associated with OSCC. Other postulated risk factors include poverty, consuming pickled vegetables, and exposure to radiation ,17,18. IHPV is a non-enveloped DNA virus that belongs to the papillomaviridae family and has more than 150 genotypes. Although it demonstrates tropism for squamous epithelium, low copy numbers of HPV DNA have been shown to be integrated in the glandular epithelium of cervical adenocarcinoma, OAC, and its precursor lesion, Barrett’s dysplasia (BD) ,22,23. THPV-associated OSCC usually with genotypes 16 and 18 (0–78%) varies according to geographic location, study design, and detection methods employed ,32. IranAnother source for the anomaly in HPV prevalence rates in OSCC may relate to detection methods utilised to test for HPV. A systematic review conducted by Petrick et al. noted that HPV-OSCC prevalence varied depending on detection method; 7.6% for the Southern blot technique compared to 32.2% for HPV L1 serology. It should be noted that L1 is the major component of the HPV capsid and is indicative of cumulative exposure to HPV infection and does not necessarily denote an HPV-induced cancer. Nevertheless, the two most commonly used methods i.e., PCR and ISH, yielded similar HPV-OSCC rates . Other sAssessing transcriptional activity e.g., p16 or E6/E7 mRNA transcripts of HPV infection in OSCC, would be useful to determine if the virus has an aetiological role or otherwise in this malignancy. p16INK4A is widely considered a surrogate marker of hr-HPV infection in oropharyngeal cancers and cervical malignancy ,39,40.A systematic review revealed no significant correlation between HPV positive OSCC and p16 INK4A overexpression . A SwediIt is possible that the high p16 promoter methylation rate in OSCC may be responsible for the lack of p16 overexpression in HPV-positive cases .High antibody titers to the HPV16 L1 (late capsid protein) have been associated with cancers of the cervix ,45 and op = 0.023) and HPV6  has been reported [Significant associations between OSCC and antibodies to E6 for HPV16  in HPV-associated OSCC  or shownOverall, the role of HPV in OSCC is unclear. Well-designed, case-controlled studies using optimal viral detection methodology in appropriate tissue specimens whilst undertaking stringent steps to avoid contamination are required. Moreover, seeking viral transcriptional markers is essential in demonstrating a robust association (or otherwise) between HPV and OSCC.OAC is one of the fastest growing and deadliest cancers in the Western World ,56. TradRisk factors for OAC include obesity, smoking, long-standing gastro-esophageal reflux disease (GORD), family history of GORD, BO, or OAC, older age, male sex, white race, persistent human papillomavirus infection, and possibly reduction in H. pylori infection rates ,57,58. CSystematic reviews have reported HPV prevalence rates of between 13% and 35% in patients with OAC ,60. The Increasing hr-HPV viral load and integration status is associated with more severe disease in Barrett’s metaplasia–dysplasia–adenocarcinoma sequence . Whole e1 MCM, and Ki-67 in regard to survival, disease relapse/progression, metastatic spread, and disease-specific death [low, had a significantly improved DFS compared with HPV-/pRBhigh on univariable analysis  and also after adjusting for age, gender, BMI, ever smoked, excess alcohol, and resection margin . Similarly, HPV+/CD1low conferred a significantly favorable DFS  as did HPV+/MCM high  [low, conferring a significantly improved survival  [Rajendra et al. have reported that HPV-positive Barrett’s high-grade dysplasia and OAC have an improved survival compared with viral-negative osophageal tumours . They deic death . Patient = 0.02) . In rega = 0.04) .Negative association studies on HPV and BD/OAC have been reported, and the results may have been adversely affected by poor tissue classification, suboptimal testing methods, small sample sizes, racial and geographical variations, and the use of metaplastic tissue, which is not associated with the virus ,70,71,72EBV was the first human tumour virus identified in cells cultured from Burkitt’s lymphoma . It is aEBV primarily infects the epithelium of the oropharynx, then replicates and spreads to B cells establishing a latent infection that is responsible for many human malignancies . DependiBamHI W fragment and returned a total negative result [The first report of EBV DNA detection in OSCC was published in 1996 by Jenkins et al. . Using me result . Converse result . These re result . Hong ete result . The mose result .The contradictory data  is due to a combination of geographical, racial, and detection technique differences. Furthermore, it is unclear if strict measures were undertaken to prevent contamination. The use of old formalin-fixed tissue specimens can result in RNA degradation and hence a higher false-negative outcome with the use of ISH. Nevertheless, it seems a small minority of OSCC is associated with EBV.No association of EBV with OAC has been reported. In a study of 162 OAC, 92 cardia adenocarcinoma and 89 gastric adenocarcinoma, EBER transcripts were found in 0 (0%) of OAC, 3 of cardia adenocarcinoma (3.3%), and 8 of the gastric adenocarcinoma (8.9%) samples . In anotJohn Cunningham virus (JC) is a small DNA virus that causes progressive multifocal leukoencephalopathy; it is a rare, often fatal disease. Only one notable study exists in relation to the detection of JCV DNA in both oesophageal cancer tissue  and normal oesophageal epithelium . The detection of viral proteins i.e., JCV T antigen and agnoprotein, which signifies a productive infection was only found in carcinomatous tissue  and not benign oesophageal specimens (0%) . The autWu et al. assessed 164 OSCC surgical specimens from Shantou, China and found HSV DNA and protein of HSV I and II in 31.7% of moderately differentiated samples . AnotherConversely, Chang et al. reported a negative association of HSV and OSCC using IHC .Gastric cancer ranks as the fifth-highest malignancy by incidence in the world with just over a million new cases in 2020. It is also the fourth-deadliest tumour with 769,000 deaths worldwide . The higCardia cancers are generally associated with obesity and GORD ,101,102.Another classification of gastric cancers is based on the Cancer Genome Atlas Consortium (TCGA) molecular subtypes of (1) Epstein–Barr virus-positive tumours, (2) microsatellite unstable tumours, (3) genomically stable tumours, and (4) tumours with chromosomal instability .Epstein–Barr virus is associated with 8.4% of gastric cancers (predominantly adenocarcinomas). Proximal cancers i.e., cardia and corpus, were much more likely to be EBV positive  as compared with antral tumours (5.2%) . The TCGEBER-1 is the most used method to detect EBV infections in tissue specimens . It is pEBV entry into epithelial cells  involves multiple interactions between viral and host proteins, which is poorly understood . It is pMethylation of both viral and host DNA is important in the development and progression of EBV-induced gastric malignancy. Methylation of viral DNA controls the expression of both lytic and latent genes whereas methylation of host cell DNA inactivates the tumour suppressor genes . LMP2A ip = 0.002) [There have been contradictory studies on the role of HPV in GC. Both positive and negative association publications are abound ,129,130.= 0.002) .A systemic review/meta-analysis of nine studies that assessed 692 gastric cancer versus 664 controls found a pooled JCV prevalence of 35.6%. There was a significant correlation between this virus and gastric malignancy  . The preThe same meta-analysis investigated the role of HBV (hepatitis B virus) and 53, 396 stomach cancer patients as compared to 325, 458 controls. This revealed a pooled prevalence rate of 7.6% in gastric neoplasia. A significant association was detected between HBV and the risk of gastric malignancy  . LikewisThe role of viruses in carcinogenesis has been heavily debated for many years.The mere detection of viral DNA, RNA, or proteins is inadequate in demonstrating causality. Nevertheless, viruses such as EBV have been firmly established as causal for up to 10% of gastric cancers. The role of HPV in a significant minority of OAC is gaining momentum and acceptance given the weight of positive association studies ,138,139,


Over 17.4 million Americans have served in the U.S. armed forces. Although long-term mortality risk is reported to be higher among older veterans than nonveterans, research does differentiate whether there is variation by combat status. This study examined later-life mortality rates among nonveterans, noncombat veterans, and combat veterans. Data were from Wave 2 of the Midlife Development in the United States Survey . Participants included 3832 nonveterans, 584 noncombat veterans, and 217 combat veterans. Mortality rates did not differ when comparing nonveterans and noncombat veterans. Combat veterans, however, had a higher risk of mortality than did than nonveterans. Combat experience is a determinant of long-term mortality risk among veterans. Future studies should account for combat status when comparing health and mortality between veterans and nonveterans. Because of their heightened mortality risk, combat veterans should be provided with additional services during and after their time in the armed forces.


Intention to have daughter(s) receive HPV vaccination was assessed both before and right after reading the messages. A one-way ANOVA with Tukey’s or Games–Howell test was conducted. An intervention message targeting the fundamental motive of kin care and disease avoidance significantly increased intention of vaccination versus a control message . There was no significant difference between the two intervention groups. The evolutionary theoretical approach that focuses on fundamental human motives may have the potential to extend the communication strategy for HPV vaccination recommendations. Health professionals may be recommended to deliver messages that target the fundamental motive of kin care as well as messages about the susceptibility and severity of cervical cancer and vaccine efficacy . However, the present study only evaluated HPV vaccination intentions in Japanese mothers with daughters. Future studies should evaluate vaccination behavior in a wider range of subjects to confirm that the evolutionary theoretical approach promotes HPV vaccination.In recent years, researchers have discussed the introduction of an evolutionary perspective into public health and health behavior research. We aimed to examine the effects of messages that target the fundamental human motive of kin care on HPV vaccination recommendations among mothers with daughters, based on an evolutionary theoretical approach. This study consisted of a three-arm parallel-group single-blinded randomized controlled study. A web-based survey was conducted from 7 to 8 October 2021 in Japan. Mothers with daughters ( Cervical cancer is the fourth most common cancer in women, with over 570,000 new cases a year . CervicaThe situation is critical especially in Japan. Following the revision of the Japanese immunization law in April 2013, HPV vaccines were positioned in “Routine A” of the national immunization program . AlthougVarious models and theories have been used in studies of methods to encourage HPV vaccination, such as the health belief model, protection motivation theory, and theory of planned behavior ,9,10. ThIn the present study, we adopt another approach grounded in an evolutionary theoretical framework by focusing on fundamental human motives. In recent years, researchers have discussed the introduction of an evolutionary perspective into health behavior research ,14, and Because humans are a highly social species, they have faced and solved crucial social challenges to enhance their inclusive fitness. Evolutionary psychologists have assumed that these evolutionary challenges include self-protection (protecting oneself from enemies and predators), disease avoidance (avoiding infection and disease), affiliation , status (gaining and maintaining respect and prestige of their fellow members), mate acquisition (successfully attracting and acquiring a romantic partner), mate retention (fostering long-term mating bond with that person), and kin care (investing in and caring for family and kin) ,19,20. TAccording to the concept of domain specificity, which is one of the key features of modern evolutionary approaches ,22, a diPrevious studies have shown that vaccination recommendation messages have primarily communicated the causes of infectious diseases and the benefits of vaccines ,26,27. NContrarily, the fundamental motive of kin care is paramount in the minds of people with daughters, although messages targeting this fundamental motive  have rarely been used in communication to encourage vaccination to date ,26,30. THowever, to our knowledge, no study has focused on the fundamental human motives based on the evolutionary theoretical approach and examined their influence on health decision-making including HPV vaccination. The aim of this study is to examine the persuasive effects of messages that target the fundamental motive of kin care on HPV vaccination recommendations among participants with daughter(s), and to investigate the usefulness of developing messages to encourage HPV vaccination based on an evolutionary theoretical approach.The present study is based on the theoretical framework of evolutionary psychology and hypothesizes that a message that appeals to the fundamental human motive of kin care will increase mothers’ intentions to have their daughters vaccinated against HPV in order to protect their fertility. A key component of the intervention is an intervention message that appeals to the fundamental motive. It is assumed that the intervention message will increase the participants’ corresponding fundamental motive, and that the increased fundamental motive will influence their vaccination intentions.This study consisted of a three-arm parallel-group single-blinded randomized controlled study. The participants were randomly allocated 1:1:1 either to two intervention groups or a control group. A web-based survey was conducted from 7 to 8 October 2021 in Japan. Because mothers have a strong influence on their daughters’ HPV vaccination in Japan , mothersRecipients who were eligible and consented to participate were invited to complete a web-based survey. When participants consented to participate in the study via the website, they were blinded and randomly assigned to a group that received either an intervention message that targeted the fundamental motive of kin care, an intervention message that targeted the fundamental motive of disease avoidance, or a control message, using a computer-generated sequence included in the survey program.Participants responded to questions of outcome measures and sociodemographic information. Then, participants were invited to read the intervention or control messages. Immediately after reading the messages, participants responded to questions of outcome measures again, and to a question of a manipulation check. A total of 1089 participants were randomized and 969 participants completed the survey. Tens of yen worth of points that can be used as gift certificates or donations were given to all participants upon completion of the study by the survey company.The study was registered as a University Hospital Medical Information Network Clinical Trials Registry Clinical Trial  on 5 September 2021. The methods of the present study adhered to CONSORT guidelines . The proWe calculated the sample size during the study design phase. Because no study using an evolutionary theoretical approach examined the influence on health decision-making, based on a summary of effect sizes of social influence research in social psychology over a century , we estiParticipants in the intervention group that received a message targeting the fundamental motive of kin care read a short comment and viewed a short video about parenting, just prior to reading the intervention message. This comment and video were used to activate the fundamental motive of kin care.https://www.e-healthnet.mhlw.go.jp/, last accessed on 14 February 2022 ).In this study, in order to detect the effect of the message using an evolutionary approach on HPV vaccination by comparing the intervention and control groups, the control message had to be content that did not affect the outcome, i.e., content that was not related to HPV vaccination. Therefore, for a control message we obtained textual information about bruxism from the website of the Ministry of Health, Labour and Welfare ”.The primary outcome was intention to have daughter(s) receive the HPV vaccine. Participants responded to the following three questions on a scale of 1 to 6, ranging from “extremely unlikely” to “unlikely”, “a little unlikely”, “a little likely”, “likely”, and “extremely likely”: (1) If I could make an appointment right now to get the HPV vaccines, I would do it right now so that I could get my own daughter vaccinated. (2) Even if I am busy, I will make time to get my daughter the HPV vaccines. (3) Even if I am worried about the side effects of the HPV vaccines, I will get my daughter vaccinated. We adapted this measure from previous studies and modified it for the present study ,37.The secondary outcome was attitude toward HPV vaccination. Participants rated “having my daughter(s) receive the HPV vaccine” on a scale consisting of six 1–6 semantic differential items . We adapted this measure from previous studies and modified it for the present study ,39.Participants also responded to the following question on a scale of 1 to 6 as mentioned above, “Doing something for my daughter’s child (my grandchild) in the future is my greatest motive among my various motives”. This question served as a manipulation check to examine if the intervention message that targeted the fundamental motive of kin care activated this fundamental motive of the participants.All these questions were measured before and immediately after the participants read intervention or control messages. Mean scores (ranged from 1 to 6) were used for the analysis. Higher scores indicate greater intentions, more favorable attitudes, and greater motives. Additionally, all participants were asked for their sociodemographic information before they read intervention or control messages.p value of <0.05 was considered significant in all statistical tests. All statistical analyses were performed using IBM SPSS Statistics for Windows, Version 21.0 .Descriptive statistics were used to summarize participants’ sociodemographic information as percentages for categorical variables and as mean ± standard deviation for continuous variables. Cronbach’s α values were used to determine the internal reliability of the outcome measures. A one-way analysis of variance (ANOVA) was conducted with the absolute change in mean values before and after intervention and the mean values after intervention for each measure as the dependent variable and the group assignment as the independent variable. For multiple comparisons, Tukey’s test was conducted on significant main effects where appropriate. The Games–Howell test was performed when the assumption of homogeneity of variances was not satisfied. A Cronbach’s α for internal consistencies of questions were 0.919 in intention to have daughter(s) receive HPV vaccination and 0.935 in attitude toward having daughter(s) receive HPV vaccination.F = 54.281, p < 0.001, η2 = 0.101]. The Games–Howell post hoc test revealed significant differences between the kin care and the control groups , and the disease avoidance and the control groups . There was no significant difference between the two intervention groups. Regarding the mean values for intention of vaccination after the intervention, ANOVA and multiple comparisons revealed the same trend as the absolute change.F = 23.457, p < 0.001, η2 = 0.046]. The Games–Howell post hoc test revealed significant differences between the kin care and the control groups , and the disease avoidance and the control groups . There was no significant difference between the two intervention groups. Regarding the mean values for intention of vaccination after the intervention, ANOVA and multiple comparisons revealed the same trend as the absolute change.Regarding the absolute change in mean values for attitude toward vaccination before and after the intervention, ANOVA revealed a significant main effect of the group assignment . The motive of kin care increased the most in the intervention group that targeted the fundamental motive of kin care; the Games–Howell post hoc test revealed a marginally significant difference between the kin care and the disease avoidance groups  and revealed a significant difference between the kin care and the control groups .As We conducted interventions to examine the effectiveness of an HPV vaccine recommendation message that targeted the fundamental human motive of kin care of mothers with daughter(s), based on the evolutionary theoretical approach. Our hypothesis was supported by the study results: an HPV vaccination recommendation message that targeted the fundamental motive of kin care was as effective in encouraging HPV vaccination as a message that targeted the fundamental motive of disease avoidance. Our results indicate that previous strategies for communicating vaccination recommendations, which primarily used messages that targeted the fundamental motive of disease avoidance, was not the only optimal choice. It is recommended that health professionals add messages that target the fundamental motive of kin care to their repertoire of HPV vaccination recommendations.A message that targeted the fundamental motive of kin care significantly improved mothers’ attitudes toward HPV vaccination and significantly increased mothers’ intention to have their daughter(s) receive the HPV vaccine compared to a control message. Previous research and practice on behavior change, including vaccination recommendations, have been based mainly on cognitive behavioral models, and focused on cognitive beliefs such as perceived susceptibility and severity of infection ,10. PrevThis communication strategy targeting the fundamental motive of kin care may bring a new option to health professionals who experience complexity and difficulty in communicating about the HPV vaccine . First, A message that targeted the fundamental motive of disease avoidance also significantly improved mothers’ attitudes toward HPV vaccination and significantly increased mothers’ intention to have their daughter(s) receive the HPV vaccine compared to a control message. Vaccine recommendation messages to date have mainly targeted the fundamental motive of disease avoidance ,26,30; tThe present study found no significant difference in effectiveness between the two intervention messages; namely, a message that targeted the fundamental motive of kin care was as effective as a message that targeted the fundamental motive of disease avoidance to encourage mothers to have their daughter(s) receive the HPV vaccine. As mentioned earlier, we created the intervention disease avoidance message that mimics typical existing HPV vaccination recommendation messages in Japan. Therefore, the result indicates that the kin care message was as effective as existing messages. As noted above, using messages that target the fundamental motive of kin care will extend the communication strategy for HPV vaccination recommendations.Future studies should examine whether a combination message  is more effective than a kin care only message or a disease avoidance only message. Future studies should also examine the effect of messages that target the fundamental motive of kin care on encouraging health behavior other than HPV vaccination. For example, messages that target the fundamental motive of kin care may encourage women who wish for future childbirth and parenting to undergo cervical cancer screening. Additionally, there are other fundamental human motives than kin care, such as affiliation, status, mate acquisition, and mate retention. Future studies should examine the effectiveness of public health messages including vaccination recommendations that target these fundamental motives. It will be useful to conduct qualitative studies to get a deeper understanding of facilitators and barriers regarding HPV vaccination among parents in Japan in terms of evolutionary theoretical approach.The present study has several limitations. First, although participants were instructed to read messages carefully before reading the intervention or control message, it is unclear to what extent the participants perused the messages. Second, because the content of the control message was not associated with HPV vaccination, it cannot be determined that the intervention messages targeting the fundamental motives encouraged vaccination. For example, the results of this study may have just detected an effect of HPV vaccination information. However, we created the intervention disease avoidance message that mimics typical existing HPV vaccination recommendation messages in Japan. Therefore, the results indicate that the kin care message was as effective as existing messages, and they have important implications to extend strategies of HPV vaccination communication as mentioned earlier. Third, this study assessed vaccination intentions directly after message exposure. Our previous study showed that HPV vaccination intention 4 months after intervention using statistics, a patient’s and a mother’s narrative had decreased to a level that did not differ significantly from the level prior to intervention . Future This is the first study to examine the effectiveness of HPV vaccination recommendation messages by focusing on the fundamental human motive based on the evolutionary theoretical approach. We found that a message that targeted the fundamental human motive of kin care—which has been rarely used—was as effective in encouraging HPV vaccination among mothers with daughter(s) as a message that targeted the fundamental human motive of disease avoidance, which has been frequently used in cognitive behavioral models. This result indicates that the evolutionary theoretical approach that focuses on fundamental human motives has the potential to extend the communication strategy for HPV vaccination recommendations. Health professionals should deliver messages that target the fundamental motive of kin care, in addition to messages about susceptibility and severity of HPV infection and HPV vaccine efficacy, to encourage HPV vaccination among parents with daughter(s). Messages that target the fundamental motive of kin care are those such as, “Getting cervical cancer can prevent childbirth. To protect your daughter and your future grandchildren, get your daughter vaccinated against HPV”.


The authors aim to carry out an investigation on the impact of cervical oncogenic Human Papillomavirus (HPV) detection in pregnant adolescents, to clarify the prevalence and risk factors, considering the importance and lack of data on this topic in Brazil.A cross-sectional study was conducted with adolescents receiving prenatal care in a tertiary hospital in São Paulo, Brazil, with routine Pap smear and oncogenic HPV detection test in the uterine cervix. The authors sought to associate the results of these tests with demographic and obstetric variables.A total of 303 pregnant adolescents whose mean age was 15.30 ± 1.22 years comprised the study subjects. The frequency of high-risk HPV cervical detection was 50.50%. Multivariate analysis revealed that a large number of partners in their lifetime (OR = 1.27) and having a religion (OR = 2.05) were risk factors for cervical detection of oncogenic HPV, while schooling appeared as a protective factor (OR = 0.85). There was an association between this detection and colpocytological alterations, reaching almost 30% of patients, but without association with obstetric and neonatal outcomes.The prevalence found is one of the highest in Brazil and worldwide. A greater number of partners during their lifetime and having religion were detected as possible factors associated with cervical HPV detection. Detection of cervical HPV-DNA did not influence obstetric and neonatal outcomes. The findings of this study reinforce the need to implement educational measures capable of modifying the incidence of sexually transmitted infections in this population and capable of promoting adherence to HPV vaccination programs. More than 120 types of HPV have been identified and approximately half of them infect the genital tract. They can be divided into low-risk and high-risk viruses according to their ability to cause oncogenic cytological abnormalities in the uterine cervix. Among the high-risk oncogenic viruses, the authors would like to mention the following subtypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 67, 68, and 70.The cytological changes engendered by the Human Papillomavirus (HPV) were first reported in 1956.,,,,,The HPV-caused infections tend to clear within 9 to 15 months. Nevertheless, the infection may lie dormant in some women, and persist in a minority.,,The existence of evidence on a possible association between the gestational period and cervical HPV infection, as well as the existence of references informing about a higher rate of HPV isolation in young populations, motivated this study.This observational cross-sectional study was carried out in a tertiary teaching hospital, the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. The hospital is located in the city of São Paulo, the most populous metropolis in Brazil.An analysis was performed of the medical and laboratory data of all of the pregnant adolescents aged 10 through 18 years admitted to the outpatient clinic for the prenatal care of pregnancy in the adolescence of the Obstetric Clinic of the aforementioned hospital from January 01, 2010, to January 01, 2016. Excluded from the study were the pregnant adolescents who did not show up for prenatal care before the collection of vaginal smears, the adolescents with insufficient information in their medical records, or those with no access to it. Sociodemographic characteristics, clinical features, laboratory data, pathological history, obstetric history, and obstetric and neonatal outcomes were retrieved from the database of the sector. The adolescents’ gestational ages were estimated using the first day of the last menstruation or the first ultrasound exam during prenatal care.The collection of cervical cytology and the performance of hybrid capture for HPV-DNA in pregnant adolescents are part of the protocols of the hospital where the study was carried out, although official Brazilian guidelines do not recommend performing cytology before the age of 25 years.The data from 404 pregnant adolescents were analyzed. Four of the patients were excluded given the lack of access to their medical histories, and 97 of them were because they missed the Pap smear test, resulting in 303 cases for analysis.digene HC2 DNA Collection Device for detection of high-risk HPV. The search for HPV types was conducted with the digene HC2 High-Risk HPV DNA test using the Hybrid Capture® 2 (HC2) technology. The test is based on hybrid-capture strategies and the use of probes for detecting 13 viral subtypes of HPV .Investigation of HPV in the uterine cervix was usually performed during the first prenatal visit. The participants were subjected to a speculum examination for collecting cells from the ectocervix and the initial quarter of the endocervical canal with the aid of an Ayre spatula or a brush. The material that was gathered was transferred to a glass slide for an oncotic colpocytology (Pap smear) test and to a specific liquid medium from the A database was built with Microsoft Excel for Office 365 software. Statistical analyses were performed with the IBM SPSS Statistics for Windows 20.0  software.t-test. The medians of the nonparametric data were compared with the Mann-Whitney tests. The binary logistic regression was used to evaluate the associations between the variables predictive of an outcome with HPV present. The magnitude of the association was estimated by calculating the Odds Ratio (OR) and its respective 95% Confidence Interval (95% CI). Significant variables, those with a p-value lower than 0.15 (p < 0.15) in the univariate analysis and those of possible clinical importance were included in the multivariate regression model using the stepwise method. The goodness of fit of the logistic regression model was assessed through the Hosmer-Lemeshow test by seeking p values higher than 0.80 (p > 0.80). Associations with p values lower than 0.05 (p < 0.05) were deemed statistically significant.Qualitative data were reported as measures of absolute and relative frequency (percentage) and compared by means of the chi-square test or the Fisher exact test as appropriate. Quantitative data were displayed as measures of central tendency  and measures of dispersion (standard deviation). They were initially tested for normality of distribution with the Kolmogorov-Smirnov tests. Those with normal distribution had their means compared through the Student The Research Ethics Committee of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo approved the present study . An informed consent statement was waived because the project was a retrospective study with data retrieved from the patient's medical histories and there was no direct contact with the patients. All precautions were taken to protect the privacy of the research subjects and the confidentiality of personal information. The hospital's database identifies patients by numbers. Soon, the names of the women became anonymous to the researchers.Of the 303 pregnant adolescents included in the study, 153 (50.50%) tested positive for high-risk HPV, and 150 tested negative. An overall analysis of the group showed that the mean age of the participants was 15.30 ± 1.22 (range: 11‒18 years) and the average number of years of schooling was 9.08±1.79 (range: 4‒14 years). Most of the adolescents (83.50%) were married or cohabited or were single but had a partner. The partners’ mean age was 20.40 (range: 13‒47 years). A large proportion of the adolescents was Catholic (44.37%) or Evangelical (33.44%) whose monthly family income was less than 5 Brazilian minimum salaries, that is, less than USD 1500 per month (55%). Of those stating they were religious, only 42.21% (103/244) declared they actually practiced their religion.At the beginning of prenatal care, 10.56% reported the habit of alcohol consumption and another 10.56% declared they had the habit of smoking. Also, 7.92% stated they used or had used marijuana and 1.32% cocaine. Most adolescents had never been to a gynecologist before pregnancy (53.14%), and more than two-thirds were using some kind of contraceptive method (73.93%), mainly the male condom (45.36%) when they became pregnant. Adolescents aged over 14 years had a higher frequency of visits to the gynecologist before pregnancy than pregnant women aged less than or equal to 14 years . The mean sex frequency (per week) was 1.74 ± 1.80 and the mean time span of sexual activity (in months) prior to the pregnancy was 17.98 ± 13.14 (range: 1‒84). The average number of partners up to the time of the interview was 1.82 ± 1.64 (range: 1‒14), while the mean age of the patients at coitarche  was 13.86 ± 1.22 years (range: 9‒17 years).One adolescent (0.33%) presented a High-grade Squamous Intraepithelial Lesion (HSIL) on her Pap smear test, while 29 (9.57%) presented a Low-grade Squamous epithelial Lesion (LSIL) and 23 (7.59%) presented Atypical Squamous Cells of Undetermined Significance (ASC-US), totaling 17.49% of abnormal Pap smears.From the demographic perspective, a comparison between the subgroup of oncogenic HPV patients and those without HPV shows that the patients with viral detection had comparatively little schooling (p = 0.02) and were single  . There wOf the total of 303 adolescents evaluated in the study, only 289 had information about cervicovaginal cytology. Of these 289 pregnant women, 147 were diagnosed with oncogenic HPV and 142 were not diagnosed with oncogenic HPV. Of the adolescents diagnosed with high-risk HPV, 29.93% (44/147) presented cervical cytological alterations, while only 6.34% (9/142) of the adolescents without high-risk HPV presented cytological alterations in the cervix. The diagnosis of cervical oncogenic HPV was associated with both the ASC-US  and the LSIL  findings. Although there was only one HSIL case, this one case appeared to be related to the presence of oncogenic HPV .Table 3AWhen the authors evaluated the clinical characteristics of pregnant women, such as preconception weight and body mass index before pregnancy, we also did not observe a statistically significant difference between adolescents with HPV and those without a diagnosis of HPV . When thAs for obstetric outcomes, 55.21% of the overall group of pregnant adolescents underwent vaginal delivery without an instrument and 30.21% underwent a cesarean section. Interestingly, the oncogenic HPV detection did not interfere with the type of delivery nor with the neonatal result as shown in A multivariate analysis of the data was carried out to determine the risk factors independently associated with cervical detection of oncogenic HPV. Schooling (years of study) turned out to be an independent factor for protection against the cervical diagnosis of high-risk HPV . The number of sexual partners in the patient's lifetime  and having religion as opposed to not having one  were also shown to be independent risk factors. In the final model, adjustments were made for weekly frequency of sexual intercourse and marital status . This model proved to be highly adequate to predict the final event (p = 0.92 on the Hosmer and Lemeshow test). The other variables were not independent predictors of a diagnosis of cervical high-risk HPV .Table 5AThis study reported a high-risk oncogenic cervical HPV-DNA frequency of 50.50% among its 303 pregnant adolescents. In the literature, the prevalence of oncogenic HPV among pregnant women ranges from 5.80% to 51.70%.,,,,,,34The prevalence found in the present study does not seem to differ from the rate observed at the same clinic ten years previously, that of 51.70%, in a study with 60 pregnant adolescents.,Such a high prevalence of oncogenic HPV among pregnant adolescents prompts us to ponder the risk of not having them undergo a Pap smear as most recently recommended by the Brazilian Ministry of Health. The present policy is to limit the Pap smear to women aged 25 years or older in accordance with a few international protocols. The present study presents rates of colpocytological changes of 17.50%, with 10% of intraepithelial lesions, which are higher than that reported in most of the most recent studies of the Brazilian population, whose frequencies vary between 2.56% and 10.23%.,There is evidence that favors an earlier approach to cervical cancer screening and others that favor a later collection of oncotic colpocytology. An IARC  study showed that starting cervical cytopathological screening at age 25, rather than age 20, had a reduction of only 1% in the cumulative incidence of cervical cancer.Certainly, vaccination of preadolescents and adolescents against HPV is an important public health action, which should lead to lower rates of HPV-caused infection and cytopathological abnormalities in such patients. However, despite the fact that government vaccination in Brazil began in March 2014, the infection rates remain high when compared to previous data. In a study carried out with 152 pregnant adolescents in Brazil, not only was low adherence to the vaccination program found (only 44%), but also a remarkable ignorance of the ways to avoid being infected by HPV (66% did not know how HPV was transmitted).Furthermore, the detection of LSIL in the oncotic colpocytology of pregnant adolescents was strongly associated with the presence of oncogenic HPV  and of ASC-US . This result is in consonance with the current literature.No association was observed between the diagnosis of HSIL and the occurrence of high-risk cervical HPV. It was not statistically significant, though, most likely because there was only one case of HSIL among the 303 women assessed in the present study. As it is an infrequent event, a much larger cohort would be necessary to show a statistical connection between oncogenic HPV-caused infection and the presence of HSIL. As a matter of fact, such a link has already been well established in the literature. Another important piece of information is that the adolescent with cervical cytological results of HSIL had systemic lupus erythematosus and progressed to hypertensive pregnancy syndrome and delivery at 25 weeks gestation. Although the institutional protocol guides the performance of colposcopy in all pregnant women with a cytological diagnosis of the high-grade intraepithelial lesion, regardless of age, there was not enough time for further investigation of this adolescent. After delivery, there was loss of gynecological and obstetric follow-up of this patient.,Evaluation of the risk factors associated with the detection of oncogenic HPV-DNA revealed that less schooling, measured as fewer years of study, turned out to be an independent risk factor for the disease in pregnant adolescents. There is similar evidence in the literature.,,,,A large number of partners in an adolescent's lifetime was also identified as a predictor of cervical detection of oncogenic HPV. There is evidence in the literature to corroborate this finding.The practice of religion stood out as well as an independent risk factor for high-risk cervical HPV. There are no similar reports in the literature. This finding may be linked to the fact that women with religious beliefs show a behavioral pattern that differs from that of women with no such beliefs. However, one would imagine, at least in theory, that religion would encourage healthy behaviors and self-care, an expectation that runs counter to the findings of this research.One might say that adolescents with religious beliefs are at a greater disadvantage in terms of socioeconomic status. But this was not the case. These girls had a higher mean of school years  and higher rates of family income, which was over two minimum salaries . Besides, most of the adolescents who declared a belief in religion denied they actually practiced it (141/244 = 57.78%), These findings, therefore, add to the uncertainty about the factors which could explain the association between religious beliefs and cervical detection of high-risk oncogenic HPV-DNA.With regard to the pattern of sexual activity of young Christian women, Brooks et al. observed that the more religious a young woman is, the less likely she is to be sexually active; however, when initiating sexual intercourse, the probability of using condoms is lower.,,,,Additionally, being a single pregnant woman without a steady partner was also a risk factor in univariate analysis ; however, this was not confirmed by the multivariate analysis . This outcome may have resulted from the fact that patients without a stable relationship tend to have a pattern of sexual activity which is different from that which has been observed in adolescents with a steady partner.,,53 A meta-analysis of 36 studies concluded that cervical HPV infection was associated with preterm delivery , premature rupture of ovular membranes , with intrauterine growth restriction , with low birth weight  and with fetal death .None of the obstetric outcome variables turned out to be statistically significant when the groups were compared. An Israeli population-based retrospective cohort also found no association between cervical HPV infection and unfavorable obstetric outcomes, such as premature rupture of ovular membranes, small-for-gestational-age newborns, hypertensive pregnancy syndrome, or premature placental abruption.Since this was a hospital-based study, the study sample may not correspond exactly with the general community. Another point that may be considered a limitation is a fact that the hospital where this study was carried out assists primarily women of low socioeconomic status, thus preventing the results from being generalized to the overall population. As it was a retrospective study, there were significant data losses on some variables related to obstetric and neonatal outcomes, limiting the analysis of the results. The type of research carried out allows only raising hypotheses, requiring prospective and intervention studies to confirm the findings.However, the present findings confirm the relevance of the topic, indicating the need for educational measures to reduce the incidence of sexually transmitted infections in adolescents and to strengthen adherence to HPV vaccination programs.High-risk oncogenic cervical HPV-DNA was detected in more than half of the pregnant adolescents analyzed in the present study. Less schooling, a larger number of partners in their lifetime, and having a religion were shown to be independent risk factors for a cervical infection caused by high-risk HPV among adolescents. In the univariate analysis, being a pregnant adolescent without a steady partner turned out to be a risk factor for oncogenic cervical HPV. Obstetric outcomes, such as premature delivery, newborn weight at birth, APGAR score, number of days of hospitalization of the neonate, umbilical cord pH, or number of prenatal consultations, were not statistically different between the groups. However, studies with larger samples are needed to achieve more consistent results. The LSIL and the ASC-US colpocytological findings were strongly associated with high-risk cervical HPV. The LSIL, HSIL, and ASC-US rates among adolescents were, respectively, 9.57%, 0.33% and 7.59%, totaling 17.49% of cytological abnormalities. Such findings point to the need for additional qualitative studies to determine more precisely the type of behavior involved and the best way to address the risk associated with HPV-caused cervical infection during adolescence.Despite the high frequency of oncogenic HPV in the pregnant adolescent population, the literature shows a high tendency towards clearing cervical infections and a high rate of regression of cervical lesions. However, there is still a lack of information about the influence of the pregnancy period on the evolution of intraepithelial lesions in women under 20 years of age, requiring additional investigations. The present study also made it possible to identify adolescents at greater risk of acquiring sexually transmitted diseases, facilitating the orientation of possible health programs. Informational interventions to encourage vaccination can be presented as a solution to reduce the frequency of cervical infection and decrease the sexual transmission of oncogenic HPV in adolescents.The Research Ethics Committee of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo approved the present study .Authors accept the journal's rules for publication and assign their copyright to the publisher.The authors will make available the research data, according to personal request.The authors declare that they have not received any form of funding.The authors declare that they have no conflicts of interest for the subject of this publication. None of the authors received any benefits, financial or non-financial interests, directly or indirectly related to the work submitted for publication.