The Department of Veterans Affairs (VA) healthcare system routinely screens Veterans for food insecurity, housing instability, and intimate partner violence, but does not systematically screen for other health-related social needs (HRSNs).To (1) develop a process for systematically identifying and addressing Veterans’ HRSNs, (2) determine reported prevalence of HRSNs, and (3) assess the acceptability of HRSN screening among Veterans.“Assessing Circumstances and Offering Resources for Needs” (ACORN) is a Veteran-tailored HRSN screening and referral quality improvement initiative. Veterans were screened via electronic tablet for nine HRSNs  and provided geographically tailored resource guides for identified needs. Two-week follow-up interviews with a purposive sample of Veterans explored screening experiences.Convenience sample of Veterans presenting for primary care at a VA urban women’s health clinic and suburban community-based outpatient clinic (October 2019–May 2020).Primary outcomes included prevalence of HRSNs, Veteran-reported acceptability of screening, and use of resources guides. Data were analyzed using descriptive statistics, chi-square tests, and rapid qualitative analysis.n = 15), Veterans found screening acceptable and felt VA should continue screening. No Veterans interviewed had contacted recommended resources at two-week follow-up, although several planned to use resource guides in the future.Of 268 Veterans screened, 50% reported one or more HRSNs. Social isolation was endorsed most frequently (29%), followed by educational needs (19%), interpersonal violence (12%), housing instability (9%), and utility concerns (7%). One in five Veterans reported at least one form of material hardship. In follow-up interviews (In a VA HRSN screening and referral program, Veterans frequently reported HRSNs, felt screening was important, and thought VA should continue to screen for these needs. Screening for HRSNs is a critical step towards connecting patients with services, identifying gaps in service delivery, and informing future resource allocation.The online version contains supplementary material available at 10.1007/s11606-023-08181-9. To address this gap, our interprofessional team developed a Veteran-tailored HRSN screening and referral quality improvement initiative, “Assessing Circumstances and Offering Resources for Needs” (ACORN). ACORN aims to equip clinical teams with up-to-date information to better understand and address the social and economic factors impacting patients’ health.Veterans receiving care within the Department of Veterans Affairs (VA) healthcare system are routinely screened for housing instabilityWe evaluated ACORN at 2 VA outpatient primary care clinics. Specifically, we assessed (1) prevalence of identified HRSNs; (2) Veteran-reported acceptability of ACORN; and (3) use of geographically tailored resource guides.ACORN was implemented at 2 VA New England Healthcare System primary care clinics: an urban women’s health clinic and a suburban community-based outpatient clinic. The clinics were selected based on strong local leadership buy-in. This quality improvement initiative was determined by the VA Providence Healthcare System Institutional Review Board to not require regulatory review.An interprofessional Advisory Board including clinical leadership as well as staff physicians, psychologists, social workers, informaticists, and health services researchers met biweekly over 6 months to develop the ACORN initiative. VA, the country’s largest integrated healthcare system, has been a national leader in the integration of social care into medical care. Our team sought to develop a Veteran-tailored HRSN screening and referral initiative that could be efficiently scaled throughout VA.10 We created a tablet-based instrument that could be self-administered through a VA-developed electronic screening platform (eScreening).11 Veterans’ responses were then imported by nurses into a clinical note in the electronic health record (EHR) for clinical team review. Our team additionally developed geographically tailored resource guides with VA and non-VA resources to help connect Veterans with needed services.Though numerous HRSN screening tools exist, few have been validated and none are specifically designed for Veterans.1 and Centers for Medicare & Medicaid Services.2 The Advisory Board then determined which HRSNs could be addressed through VA, or other federal, state, or community resources, as our goal was to screen only for needs that could be addressed given existing services. Final ACORN domains included food, housing, utilities, transportation, education, employment, legal needs, interpersonal violence, and social isolation.We compiled candidate HRSN screening domains from expert bodies including the National Academy of Medicine15 To facilitate integration of ACORN into clinical workflows, we used existing VA screening questions for food insecurity8 and housing instability.7 For the remaining domains, we sourced questions used in other established screeners.19 For those domains without existing measures, or for which existing measures were not applicable for our population or setting, we adapted or developed new questions based on input from the Advisory Board and other subject matter experts. To ensure acceptability and clarity, we refined the screener based on cognitive testing with 10 men and 8 women Veterans. Veterans were recruited from the VA’s Compensated Work Therapy program20 (n = 11), which provides vocational rehabilitation services, and 7 Veterans who were also VA employees. The final screening instrument  codes linked to corresponding program websites. Information for designated VA contacts, often social workers, was also provided on each guide.To ensure clinical staff were equipped with information to address Veterans’ self-identified needs and that Veterans left their appointment with relevant resources, we developed geographically tailored resource guides corresponding to each domain in the ACORN screener.Prior to ACORN’s launch, each site identified a nurse champion with whom 2 team members  met weekly for 5 months before the launch. Our team worked alongside the champions and clinical and administrative staff to create site-specific standard operating procedures, encourage staff buy-in, and provide training on ACORN and eScreening. This collaborative approach allowed for tailoring to sites’ unique needs and staffing levels. Both sites identified nurses, who administer other clinical screenings during patient intake, as the most appropriate staff to review screening results with Veterans and provide follow-up support including distribution of resource guides. We also designated specific screening results warranting more intensive follow-up  and developed workflows to ensure referrals to appropriate VA clinical services. All screening and referral processes were detailed in an implementation guide.A convenience sample of Veterans presenting for care at each clinic was asked to complete the ACORN screener in the waiting room. Veterans were handed a tablet by an administrative clerk upon check-in. Positive responses were automatically flagged in eScreening to streamline identification of needs requiring follow-up. For non-urgent needs, nurses provided Veterans with corresponding resource guides. For urgent food, housing, and/or utility needs, interpersonal violence, or social isolation, resource guides were shared and nurses obtained Veteran consent to place a referral or facilitate a warm hand-off to social work and/or mental health.Clinical implementation started at both sites in October 2019. Once implemented, ACORN team members met weekly with nurse champions to discuss operational concerns and develop any needed workflow modifications. Given the rapid shift to telehealth at the start of the COVID-19 pandemic, our team worked with clinical informaticists to develop an ACORN EHR template that staff could administer during phone and video visits. The suburban clinic pilot ran through May 2020; the urban clinic stopped screening in March 2020 due to pandemic-related staffing limitations.22 We collapsed enrollment priority into 3 categories: Veterans receiving some percentage of service-connected disability compensation, Veterans with no service-connected disability compensation who are low-income, and those with no service-connected disability compensation who are not low-income (above the VA means test).In addition to HRSNs on the ACORN screener, demographic data including sex, age, race, ethnicity, marital status, and VA enrollment priority were obtained from the EHR. Enrollment priority refers to a determination of Veterans’ eligibility for and cost-share associated with VA health benefits, as well as service-connected disability compensation.n = 6) and suburban clinic (n = 9) were recruited for brief (~15–20 min) semi-structured telephone interviews 2 weeks after completing ACORN. Sampling was stratified across clinic sites by Veterans with zero identified needs (n = 7) or  1 or more needs (n = 8), with attempts to oversample women (n = 9) and Veterans from racial and ethnic minority groups . Ages of Veterans interviewed ranged from 35 to 74 years. Semi-structured interview guides were developed through an iterative process. Domains included Veterans’ reactions to being asked about HRSNs, screening acceptability, attitudes towards continued VA screening, preferences for screening administration, and motivators and barriers to use of resource guides. Interviews were conducted by project staff experienced in qualitative methods, audio-recorded, and transcribed verbatim.To understand Veterans’ experiences with ACORN, a purposive sample of Veterans from the urban clinic  and clinic site to identify and synthesize key trends.Two team members  independently analyzed interview transcripts using rapid qualitative analysis.p =  < 0.001), and more likely to identify as a racial and/or ethnic minority . Veterans at the urban clinic were also significantly more likely to be non-married/non-partnered , and low-income  , educational needs , interpersonal violence , housing instability , and utility concerns  . Across both clinics, social isolation was the greatest unmet need, which was endorsed by nearly one-third of Veterans. One in 5 participants reported at least some form of material hardship. Regardless of whether or not they reported HRSNs, Veterans felt screening was acceptable and important, and that VA should continue screening.27—have worsened globally since that time.29 We also found substantially higher rates of food insecurity and housing instability than has been previously reported within VA despite our using the same screening questions.31 These findings may be due, in part, to the fact that ACORN was Veteran self-administered whereas VA clinical reminders are staff-administered. Prior research has found patients prefer self-administered over staff-administered screening for HRSNs34, and that disclosure rates for food or financial insecurity and other sensitive topics such as exposure to violence and substance use are higher with self-administered screening.36 Rates of other HRSNs reported in ACORN were also higher than those found in other studies using VA EHR data such as ICD-10 codes37, which are known to be underutilized.40To our knowledge, this is the first study of a systematic HRSN screening and referral program among Veterans. The high rate of social isolation among those screened is particularly notable, as our data were collected primarily before the COVID-19 pandemic and reported social isolation and loneliness—which are associated with numerous adverse health outcomes among Veterans including increased risk for re-hospitalization and decreased well-being41 Previous research has further demonstrated that Veterans prefer tablet-based to paper-based screening, and that completion rates were significantly higher with tablet-based screening.11 Future work is needed to explore potential differences in Veteran and staff acceptability, perceived feasibility, and HRSN disclosure rates between Veteran self-administered and staff-administered screening.The ability to systematically screen for HRSNs and allow patients to self-administer screening is a strength of ACORN and has the potential to markedly improve the quality and accuracy of VA data. Self-administered, tablet-based screening allows for increased privacy, greater efficiency, and real-time review of responses by clinical staff.44 Notably, in our study and others, even patients who did not endorse unmet needs spoke to the importance of universal screening. Future work is needed to establish an evidence base for optimal screening frequency, and how frequency may differ for routine screening versus follow-up assessments once needs are identified or in high-risk populations where needs may be more prevalent.Our findings of high rates of acceptability of screening for HRSNs are consistent with prior work in non-VA healthcare settings.54 Among the subset of Veterans we interviewed, none who screened positive for HRSNs had used resource guides at 2-week follow-up, although several noted planning to use them in the future. Consistent with prior work, reasons Veterans did not use resources included not feeling they had a current need, competing priorities, and not remembering receiving the guides.52 Since this pilot, ACORN has expanded to over 15 VA facilities nationally across different clinical settings, and in collaboration with our operational partners, we have worked with local teams to support the development of site-specific workflows to ensure appropriate follow-up and referrals are provided.55 We have also established a Community of Practice to support and sustain implementation at both new and existing sites. Recognizing that optimal practices may vary based on patient preferences and needs, local resources, and staffing, there is likely not a one-size-fits-all approach to identifying and addressing HRSNs. Additional research is needed to better understand which patients may benefit from “low-touch” interventions such as tailored resource guides, and which patients need “higher-touch” interventions such as navigation or case management.Despite widespread support for healthcare-based social needs screening, previous work has found many patients screening positive for HRSNs decline assistance or do not follow-up with offered resources.Our findings should be interpreted within the context of several limitations. First, this initiative took place in 2 VA primary care clinics in New England, which may limit generalizability. Second, COVID-19 lockdowns led to a drop in screenings in the suburban clinic due to decreases in clinic volume and staffing, and screening in the urban clinic halted completely in March 2020. Future work should examine whether enabling Veterans to securely complete an electronic screener remotely, such as through a patient portal, may help scale HRSN screening efforts and response. However, it is important to note that digital health inequities  may prevent some Veterans from accessing these tools. Third, numbers and demographics of Veterans declining screening were not reliably collected by clinic staff and findings may have been susceptible to selection bias. Demographics of those screened, however, were generally similar to the broader patient population in each of the clinics with the exception that our sample was overall younger and more likely to be single. Fourth, Veteran-reported acceptability of ACORN was based on interviews with a subset of participants and may not be representative of all patients. We did, however, purposively sample Veterans who did and did not screen positive for HRSNs, and reached saturation within each stratum. Lastly, reported acceptability and importance of screening may have been subject to social desirability bias, although we tried to mitigate this bias by ensuring all respondents knew their responses would be anonymized and asking opened-ended questions in a non-judgmental way. Despite these limitations, our findings have important implications for our understanding of identifying and addressing HRSNs in healthcare settings.A self-administered electronic HRSN screening and referral program identified that Veterans have substantial HRSNs and want VA to screen for and address these needs. Although health systems cannot independently address upstream determinants of health, they can serve as leaders and advocates in the identification and management of patients’ HRSNs. Screening for HRSNs is a critical step towards connecting patients with needed services, identifying gaps in the current service delivery system, and informing future resource allocation.Supplementary file1 (PDF 250 KB)Below is the link to the electronic supplementary material.


Gardnerella spp. is closely related with this condition. Such bacteria are the major source of cervicovaginal sialidases, important for microbiota alterations. The sialidase-encoding gene nanH3 is account for their sialidase activity. Thus, a subset of 212 women positive for hrHPV at the first visit were included in the analysis of the current study aiming to compare the loads of nanH3 in cervicovaginal fluid (CFV) of women with persistent hrHPV infection and with those cleared the infection after a year. Participants were assigned to two study groups named “persistence”  or “clearance” , according to the HPV status upon enrollment and follow-up. Absolute quantification of nanH3 gene was performed using quantitative real-time PCR (qPCR). Persistence and clearance group did not show statistical difference in the load of nanH3 gene (p = 0.19). When considering the subset of women with HPV16, differences in number of copies of nanh3 gene was observed between the persistent (7.39E+08 copies/μL) and clearance group (2.85E+07 copies/μL) (p = 0.007). Therefore, baseline loads of nanH3 gene is increased in women that persist with cervical HPV16 infection after 12 months.Disturbed vaginal microbiota have a role in the persistence of high-oncogenic-risk human papillomavirus (hrHPV) and  Despite that, the majority of the cases of cervical HPV infection are cleared within 2 years5, being the immune responses important for clearance6.The cervical infection by human papillomavirus (HPV) is the most frequent sexually transmitted infection (STI) worldwide7, hormonal contraceptive use9, and parity10. In addition, the local cervical microenvironment, including the vaginal microbiota, may also influence the natural history of HPV infection11. Therefore, a Lactobacillus-deprived vaginal microbiota, as in bacterial vaginosis (BV), have been associated with persistent HPV infection and lesion progression14.Factors associated with developing cervical lesion and cancer include smokingLactobacillus and increased anaerobic and facultative anaerobic bacteria, of which, Gardnerella spp. that is present in nearly all cases16. Sialidase production is one of the most important virulence factors of Gardnerella spp.18. Among the deleterious effects of bacterial sialidases, it is the degradation of several protective factors of the vaginal mucosa and contribution to the exfoliation and detachment of vaginal epithelial cells19 facilitating bacterial adhesion to the epithelium and biofilm formation22, a condition already associated with persistence of BV23.Bacterial vaginosis is a polymicrobial dysbiosis, which occurs by the replacement of beneficial Gardnerella spp. and was thought to be the gene responsible for sialidase production24. However, the recent studies concluded that nanH2 and nanH3 account for the sialidase activity observed in cultured G. vaginalis25. Besides that, nanH2 is less prevalent in Gardnerella isolates and always detected when in the presence of nanH325.At first, the putative sialidase gene, nanH1 , was identified in Gardnerella was one single specie genus but now three other species in addition to G. vaginalis  were described26. Interestingly, sialidase genes nanH2 and nanH3 were only detected in G. piotii and in a subset of G.vaginalis isolates18.Until very recently, Gardnerella spp. in the cervicovaginal fluid of women between persistent hrHPV infection and those who cleared the infection after a 12-month period.Considering the importance of better understanding the relationship between the bacterial components of the vaginal microbiota and the outcome of hrHPV infection, the aim of this study was to compare the loads of the sialidase-encoding gene nanH3 of Table The most frequently genotype detected in the population  had normal microbiota, 21 (9.01%) intermediate and 80 (34.33%) had BV, detected by Nugent scoring. Data of Nugent evaluation were not available for five participants. Among BV positive women, 52/80 (65%) were nanH3 positive, while 28/80 (35%) were nanH3 negative. Thus, sensitivity and specificity of the test was 65%  and 75% (66–82%), respectively. Figure G. vaginalis27. Thus, it was hypothesized that gene encoding sialidase, frequently found in Gardnerella spp.18 could have a role in the outcome of HPV infection, being a useful marker for hrHPV persistence. Surprisingly, loads of nanH3 gene differed between clearance and persistence group in the presence of 5 most prevalent genotypes, however such difference remains only in the presence of HPV16.Recently our study group showed that women with persistent HPV16 and HPV18 infection have increased baseline loads of 28. Persistent rates in a large retrospective cohort study in China was 42.7% within 24 months29, while a multicentric study showed 54.1% of persistence rate over a year in American, Canadian and Brazilian women30. In relation to HPV16, the persistence rate was 52.1% after 12 months in Dutch cohort of young women31.This study showed high rates of HPV persistence, 58.49% for all hrHPV infection and 73.44% when considered only the HPV16. Such rates were similar to the rates showed in another study conducted in Brazil, with 61.8% of persistence rate32. However, other Brazilian study did not show differences of age when evaluating factors associated with clearance and persistence after 24 months28. The number of sex partners and the number of detected genotypes differed between the clearance and persistence group. Furthermore, one of five most prevalent HPV genotypes differed among the groups when considering their detection in single or mixed infection. In fact, a previous study showed that HPV genotypes differ in terms of the period for clearance, as women with mixed infections had a longer time to clear their infections compared to those with a single infection30. The increasing in number of sex partners could contribute to persistence since it increases the risk of acquiring new HPV genotypes.In relation to sociodemographic and behavioral characteristics of the study population, few variables differed between the groups. The median of age was higher in clearance group, agreeing with a study that showed that the highest HR-HPV persistence occurred in the 22–27 years old group, whereas clearance increased in women aged 28–33 yearsGardnerella spp. and persistent hrHPV infection, as well as progression of the cervical lesion34. Of the results of association between Gardnerella spp. with persistence and progression of hrHPV, Usik et al. proposed that in addition to role of Gardnerella spp. in the viral persistence, it also contributes for the disruption on vaginal microbiota , which also leads to the persistence and progression of HPV infection34.Studies on vaginal microbiota have demonstrated the association between Gardnerella produce sialidase18. Therefore, studying the presence of sialidase encoding gene of Gardnerella is important for paving the way for further studies focusing in the role of each individual Gardnerella sp. for HPV infection and persistence.As recently demonstrated, not all newly described species of Gardnerella allowing their uptake and catabolism and expose binding sites, facilitating bacterial adhesion to the epithelium and further biofilms formation35. Sialidases can still cleave mucin oligosaccharides, that decreased the cervicovaginal mucus viscosity, compromising the physical and biochemical barrier against pathogens36. Besides that, sialidases compromise the immune barrier by hydrolyzing immunoglobulin A37. In the present study, results on the quantification of nanH3 did not differ when included all hrHPV infection grouped, however loads of nanH3 were higher in the cervicovaginal fluid of women with HPV16 persistent.To the best of our knowledge, this is the first study to assess the relationship between hrHPV persistence infection and loads of nanH3 gene. It is known that sialidases hydrolyze the sialic acid of the host’s epithelial cells, provides a carbon source to 38, despite the mechanisms by it can influence in HPV infection remain unknow. It is known that Lactobacillus spp. produce several microbicidal factors, including lactic acid39, an important agent to acidify the vaginal environment and to control overgrowth of bacteria and preventing STIs41. The difference of nanh3 loads found in HPV16 infection is intriguing, are needed new approaches involving vaginal microbiota and HPV16, since this genotype is responsible for the most cases of high-grade intraepithelial lesions and invasive cervical carcinomas42.Studies have been reporting the association of altered vaginal microbiota with HPV infection43. In addition, cervical microbiome and cytokine profile showed notably different in all stages of the natural history of cervical cancer44. Thus, bacterial species may interact with immune signaling during HPV infection contributing to persistence and progression44. On the other hand, a consequence of HPV immune evasion is the imbalance in the vaginal microbiota due the reduced amino acid source sustaining the survival of Lactobacillus species45. Therefore, the association between BV and HPV seems to be bi-directional. In fact, a study showed that the stability of vaginal microbiome is necessary to maintenance of immune surveillance for HPV16 clearance46. In this sense, the differences in the load of nanH3 gene in the presence of HPV16 lead to hypothesize that the vaginal microenvironment would have a different modulation in the presence of different genotypes and the changes in the immune response might create a more permissive microenvironment for bacterial overgrowth.Human papillomaviruses modulate the host immune response by blocking immune-related gene expression and immune signaling pathways and different genotypes may act by different pathwaysGardnerella spp. is present in virtually all cases of BV, the most frequent dysbiosis of the vaginal microbiota47. In this study, an association between the nanH3 gene and BV was observed. Robinson et al. suggested the potential use of nanH3 as a molecular diagnostic marker of BV, such PCR test showing 80.95% sensitivity and 78.26% specificity compared with the Nugent score for BV diagnosis25. Although the association, in this study values of sensitivity and specificity were lower than the mentioned study. It is worth mentioning that studies differ in terms of prevalence of BV and number of study participants.37. Recently, our study group showed that sialidase activity in molecular-BV, assessed by V3–V4 16S rRNA sequencing, is associated with changes in bacterial components of the local microbiome as well as increased sialidase levels48. Indeed, sialidase activity in cervicovaginal fluid already was demonstrated in the presence of microscopy-detected BV50. In fact, sialidase activity could promote the growth or colonization of Prevotella, Bacteroides as well as G. vaginalis, vaginal bacterial sialidase producers52, being the production of sialidase an important step in the biofilm formation24. Such mechanism reinforces the role of Gardnerella spp. as the scaffold on vaginal mucosa for attachment of other bacterial species, which can start to form biofilm53, a condition already associated with refractory treatment of BV and persistence of BV-associated bacteria after treatment54.Besides the association between nanH3 gene and BV, the load of the gene was significantly increased in BV compared to normal microbiota. As previously discussed, sialidases have deleterious effects in the vaginal microenvironmentG. vaginalis sialidase-encoding gene in BV, since this condition seems have an important role in the persistence and progression of HPV infection. Thus, microbial products are promising tools for identifying women with increased risk for HPV persistence.Although the amount of sialidase-encoding nanH3 gene did not differ between clearance and persistence in the presence of all hrHPV infection, the loads are high in the persistent group and there is a difference among the groups when considering only HPV16 infection. A limitation of this analysis is due the low sample size when stratifying each genotype. Furthermore, such results contribute to better understanding the role of Between 2012 to 2014, 1635 reproductive-aged HIV-negative women screened for HPV infection in Botucatu, SP, Brazil. A sociodemographic, behavioral, gynecological history and clinical data were obtained during a face-to-face interview using a structured questionnaire and were entered into a Microsoft Excel spreadsheet . HPV positive women were then invited to a follow-up within 12 months. All participants were informed about the procedures of study and informed consent was obtained from all subjects as all reached the age of majority (18 years-old). All methods were carried out in accordance with relevant guidelines and regulations and this current study was reviewed and approved by the Ethics Committee of Botucatu Medical School  and the samples included in the present study belongs to the biorepository of principal study (Biorepository approved number: 3.140.843).15. Mid vaginal wall was utilized for Trichomonas vaginalis culture in Diamond’s medium55. Samples obtained with endocervical brushes were stored at −20 ºC until Chlamydia trachomatis and Neisseria gonorrhoeae detection, as described previously56 and for HPV detection and genotyping using the Linear Array HPV Genotyping kit . For cervicovaginal fluid sampling, 3 ml of sterile 0.9% NaCl was inserted into vaginal wall, homogenized with cervicovaginal secretion and recovered using a plastic sterile pipette. The cervicovaginal samples were stored at −20 ºC until genomic DNA extraction and G. vaginalis nanH3 gene absolute quantification.Same sampling procedures during physical examination were conducted by nurses/physicians at enrolment and at follow-up visit. After speculum insertion, vaginal content was obtained with swabs for smearing into microscopic glass slides for Nugent scoring, after gram stainingGenomic DNA extraction of cervicovaginal fluid was performed using the UltraClean Soil DNA Isolation kit  as recommended by the manufacturer. Extracted DNA was quantified using an Epoch spectrophotometer  and the quality of the extraction was confirmed by the 260/280 nm absorbance ratio.Gardnerella nanH3 gene, through polymerase chain reaction (PCR) products from clinical samples using the primers Gardnerella nanH3 F (5′-CAGTTCCAATGGAAGTGTGC-3′) and Gardnerella nanH3 R (5′-AGCATCTGGGAATGCTCTTG-3′) with 51 ºC of annealing temperature. Expected amplicon size was 322 bp for nanH3 gene, confirmed in 1.5% agaroses gel band25. Positive samples had the amplicon purified using Illustra GFX PCR DNA and Gel Band Purification Kit  and sequenced by Sanger method in ABI 3500  and confirmed by blast . The amplified products were used for the ligation step using the CloneJET PCR Cloning Kit , cloned into Escherichia coli DH5-α . Plasmid DNA was extracted using GeneJET Plasmid Miniprep Kit .A cloning step was performed to obtain the plasmid DNA with Gardnerella nanH3 F and Gardnerella nanH3 R under the following cycling conditions: 95 °C for 2 min, 25 cycles of denaturation at 95 °C for 5 s, annealing at 60 °C for 25 s, and an extension at 95 °C for 15 s followed by a dissociation stage: 95 °C for 15 s; 60 °C for 1 min and 95 °C for 15 min, according to the manufacturer's recommendations, performed in a StepOnePlus Real-Time System . Samples with a melting temperature value of 81 °C + / − 0.5 °C were considered positive for nanH3 gene. For constructing the standard curve, three plasmid dilutions  were utilized for sample cycle threshold interpolation. Loads of nanH3 gene were expressed as the number of copies per volume (µL) of cervicovaginal fluid.The absolute quantification of nanH3 gene was performed by quantitative real-time PCR (qPCR) using 2× qPCRBIO SyGreen Blue Mix Hi-ROX , primers Chlamydia trachomatis , Trichomonas vaginalis , Neisseria gonorrhoeae  and only low-risk HPV  were not included in the analysis. Five women were excluded of this study due conflicting data in the spreadsheet database. From that, 245 (14.96%) women were hrHPV positive , however, 12 samples were excluded due insufficient volume of cervicovaginal fluid for laboratorial analysis of the current study. Therefore, 233 cervicovaginal fluid samples were included for this study analysis. Participants were assigned at two groups according to the status of hrHPV infection at enrolment and at follow-up: ‘clearance group’ comprised participants who tested negative for any hrHPV at follow-up and in ‘persistence group’ were assigned participants positive for hrHPVs genotype at first visit and follow-up, matched with those detected at follow-up. For clearance group, 21 women who showed clearance of baseline genotype and had a new genotype detected at follow-up were not included.In the first visit, 544 (33.27%) women were positive for any HPV infection, of which 413 (25.26%) tested positive for hrHPV genotypes and were recruited for the follow-up visit after 12 months. Four hundred sixty-two (27.65%) women returned to the follow-up. For the present study, women positive for For sociodemographic, behavioral and clinical variables comparisons between the clearance and persistence groups, chi-squared or Fisher’s exact were utilized for categorical variables and Mann–Whitney tests was utilized for continuous variables. The loads and presence of nanH3 gene were compared between the study groups using, respectively, Mann–Whitney and chi-squared tests or Fisher’s exact test. All analyses considered a P-value < 0.05 to be statistically significant and were performed using GraphPad Prism software .Supplementary Table S1.


Considering the results of our study, we recommend including the high-risk genotypes HPV51, HPV68, HPV53 and HPV66 in future HPV vaccine formulations.Knowledge of human papillomavirus transmission from the genital tract to the oral mucosa remains unsatisfactory, with poor and often inconsistent literature results. The increase in HPV-associated oral malignancies prompts further analysis of the simultaneous detection of the virus in the two anatomical areas and on the identification of genotypes to be included in future vaccines. Therefore, in this retrospective study, we evaluated orogenital HPV concurrence, hrHPV, lrHPV and type-concordance in 337 samples, as well as the prevalence of the most common genotypes not included in HPV vaccines. Concurrence was found in 12.5% (31/248) of cases, hr-concordance in 61.3% (19/31) and lr-concordance in 12.9% (4/31). Finally, type-concordance was found in 32.3% (10/31) of concurrent infections. Regarding the identification of non-vaccine genotypes, the significantly prevalent genotypes in the anogenital area were HPV66 (12.6%,  The recent decades have witnessed major changes in the sexual behavior of both men and women, with the breaking of old taboos and rigid rules. There has been a lowering of the age of sexual debut, an increase in the number of sexual partners and general clearance of casual sex and formerly unacceptable sexual practices ,2.Recent statistical reports indicate how more than 80% of men and women engage in oral sex, making it the most common sexual practice along with vaginal sex ,4,5,6. PBoth viral and bacterial pathogens, such as human papillomavirus (HPV), Herpes simplex (HSV), Treponema pallidum and Chlamydia trachomatis, can be transmitted from the genital region to the oral cavity . While aAs stated by World Health Organization (WHO), HPV is the most prevalent sexually transmitted pathogen, with approximately 80% of sexually active women and men being infected at least once in their lifetime ,11. Basal keratinocytes of mucosal and cutaneous epithelia are the targets of the infection, which is usually asymptomatic. Failure to eradicate the virus in a space of 12–24 months exposes the patient to the risk of developing benign and malignant hyperproliferative lesions ,14. It is estimated that more than 500,000 cases of cancer are caused by HPV each year, making it the most common virus associated with cancer in humans. The first neoplasm linked to hrHPVs was cervical cancer, of which almost all cases (99.5%) have an HPV-induced etiopathogenesis ,15. LateThe recent rise of HPV-related head and neck cancers (HNSCC), specifically oropharyngeal squamous cell carcinoma (OPSCC) and oral squamous cell carcinomas (OSCC), has been linked to HPV transmission to the buccal mucosa ,18,19. IOn the other hand, lrHPV infections affecting the buccal mucosa can induce the development of benign oral papillary lesions, the most common of which are the following: condyloma acuminatum (CA), squamous papilloma (SP), verruca vulgaris (VV) and multifocal epithelial hyperplasia (MEH) . AnotherNevertheless, oral HPV infections may be present even in the absence of visible oral mucosal abnormalities and may remain undetected for various periods . Despite the well-known HPV tropism for both genital and oral sites and the rising concern about HPV-induced oral and oropharyngeal epithelial lesions, the exact route of transmission and the natural history of oral HPV infection are scarcely studied and still not fully understood .Orogenital contact has been reported as the primary route of oral HPV transmission, with the likelihood of infection appearing to increase with the number of sexual partners, with continued exposure to risk determined by, for example, an HPV-positive partner and being in a homosexual relationship . Some studies have suggested how autoinoculation, using the hands as intermediate contact and open-mouth kissing may also be a source of infection . The conflicting results in the literature regarding the prevalence of oral HPV infection and its association with genital infection may be due to the diversity of oral sampling procedures and HPV detection methods, as well as the different interpretations of “concurrence” and “concordance” data ,23,26.Several studies have been conducted on possible strategies to combat HPV infection ,28, howeTo date, three different vaccines have been approved by the Food and Drug Administration (FDA) and consequently by the European Medicines Agency (EMA): the bivalent Cervarix, against HPV16 and 18, the quadrivalent Gardasil, against HPV6, 11, 16 and 18, and the nonavalent Gardasil 9, against HPV6, 11, 16, 18, 31, 33, 45, 52 and 58. HPV vaccination campaigns, generally targeting women, have been shown to be an effective prevention strategy for cervical and anal cancer ,30,31,32Concerning oral infections, vaccine studies have shown encouraging but inconclusive results in preventing OPSCC and OSCC, with the search for evidence of efficacy complicated by the lack of specific premalignant lesions to use as surrogate endpoints ,34,35. Designing of HPV vaccines has been driven by the purpose of protecting against the genotypes most associated with cervical cancer, namely, HPV16 and HPV18, followed by HPV31, 33, 45, 52 and 58, and benign genital lesions, that is, HPV6 and 11. Recent studies describe how the same hrHPVs included in the vaccines cause 90–95% of HPV-related oropharyngeal cancers: an estimated 51–83% of HPV-positive patients had HPV16, while 0–20% had HPV31, 33, 45, 52 and 58, depending on the anatomical site ,37. However, changes in sexual practices, as described above, could lead to an increase in the prevalence of other genotypes in the future, both in the oral and in the genital region. This trend calls for the identification of other common genotypes not yet included in available vaccines so that their inclusion in future vaccines can eventually be considered.In light of the above, in this observational retrospective study, we evaluate the prevalence, concurrence and concordance of oral and genital HPV in a cohort of unvaccinated male and female patients. Specifically, we defined concurrence as the simultaneous detection of one or more HPV types in genital and oral samples from the same patient, whereas hrHPV-concordance, lrHPV-concordance and type-concordance were defined as the detection of hrHPVs, lrHPVs and the same genotypes in both sites, respectively. The identification of high frequencies of HPV types not included in the quadrivalent and nonavalent vaccines at both sites could provide important clues about the possibility of expanding the genotypes included in future vaccine formulations.The sample group consisted of 337 patients, 281 women and 56 men, referred for HPV testing to the Microbiology and Virology laboratory of the Department of Health Promotion, Mother and Child Care, Internal Medicine, and Medical Specialties (PROMISE) . Patients underwent HPV testing following the detection of cervical and penile HPV-related lesions, or because they had a close contact with a positive person . Cervical scraping for women and urethral and genital scraping for men were performed by gynecologists and urologists in private practice.® mouthwash , carefully reaching every part of the oral cavity and avoiding gargling. Before sampling, each patient was advised to avoid eating, drinking and using oral hygiene products. To investigate whether the suspected genital infection had spread to the oral cavity, both female and male patients provided an oral rinse. Sampling was performed at the Oral Medicine with Dentistry for Fragile Patients Unit of the Polyclinic “P. Giaccone” Hospital, Palermo, by rinsing the oral cavity for 30 s with 10 mL of Original Mint ScopeNo funding or financial compensation was provided to study participants.To isolate the cellular component, each sample was centrifuged at 1600 rpm for 10 min. The pellet was resuspended in 1–4 mL of phosphate-buffered saline, PBS  and then subjected to a second centrifugation at 13,000 rpm for 5 min. After discarding the supernatant, each pellet was stored at −20 °C or processed immediately.® HPV Genotyping Extra II , which identifies 23 genotypes classified as hrHPV or probable hrHPV  and 9 types classified as lrHPV . In this technique, a 65 bp fragment of the L1 gene, generated by an SPF10 PCR, is subjected to a reverse-dot blot hybridization assay. A volume of 200 μL of oral pellets resuspended in PBS was used to extract DNA using the QIAamp Mini Kit . HPV-DNA detection was carried out using INNOLiPAIn samples that were HPV positive but not genotyped, a nested PCR was performed consisting of a first amplification with PGMY09/PGMY11 primers followed by a second amplification with GP05+/GP06+ primers as described elsewhere . After dData are presented as numbers and percentages for categorical variables, while continuous data are expressed as the mean ± standard deviation (SD) unless otherwise specified. Particularly, medians with interquartile range (IQR) were used for variables with non-normal distribution.p-value < 0.05), residual analysis was performed using the Z-test. Test for normal distribution was performed using Shapiro–Wilk test. The t-test was used to test the differences between two means of unpaired data. Alternatively, the Mann–Whitney test was used when the distribution was not normal. Finally, all tests with p-value (p) < 0.05 were considered significant. Statistical analysis was performed using the Matrix Laboratory (MATLAB) analytical toolbox version 2008  for Windows at 32 bits.The chi-square test and Fisher’s exact test were performed to evaluate significant differences in proportions or percentages between the two groups. Fisher’s exact test was used when the chi-square test was not appropriate. Chi-square goodness of fit was used to assess significant differences between three or more modalities of a variable. If the chi-square goodness of fit was significant  females and 16.6% (56) males, with a mean age of 33.7 (10.2) years . p = 0.94), whereas a significant gender difference was observed at the oral site (p < 0.0001), i.e., we found a significant presence of HPV-positive men than HPV-positive women at the oral site.HPV-DNA was detected in 73.6% (248/337) of genital samples, and 10.1% (34/337) of oral samples. Specifically, 73.7% (207/281) and 7.1% (20/281) of women were positive in the genital and oral sites, respectively, whereas men showed a genital positivity rate of 73.2% (41/56) and an oral positivity rate of 25% (14/56). Notably, no significant difference was observed between women and men at the genital site  of cases in the genital area and 70.6% (24/34) in the oral cavity. Specifically, they were found in 87% (180/207) and 82.9% (34/41) of female and male genital specimens, respectively, and in 65% (13/20) and 78.6% (11/14) of female and male oral specimens, respectively.Single infections were the most common in the oral cavity, with a rate of 91.2% (31/34), while single and multiple infections were detected in 62.1% (154/248) and 37.9% (94/248) of genital specimens, respectively . For theConcurrence, namely, the simultaneous detection of one or more HPV types in genital and oral samples of the same patient, was found in 12.5% (31/248) of cases. In concurrent infections, hrHPV concordance, i.e., the detection of HR types in both anatomic sites, was detected in 61.3% (19/31) of patients, while lrHPV concordance was found in 12.9% of cases (4/31) .Type-concordance, i.e., the detection of the same HPV genotypes in both anatomic areas was found in 32.3% (10/31) of concurrent infection. Only three patients with a positive oral sample had a negative genital specimen, i.e., 3.4% (3/89), two men and one woman. The genotypes included in the quadrivalent and nonavalent vaccines were detected in 35.1% (87/248) and 61.7% (153/248) of genital samples and in 35.3% (12/34) and 44.1% (15/34) of oral samples .p < 0.0001), HPV53 , HPV51 , HPV42  and HPV68  in the genital area, while no significant differences were observed in the oral area for genotypes not included in the vaccines.p < 0.0001), HPV53 , HPV51 , HPV42  and HPV68 ; while in the men subgroup, we found HPV66 , HPV42 , HPV51 , HPV53 , HPV70  and HPV73 . Even in this case, at the oral site, no significant differences were observed between genotypes not included in the vaccines in either the male or female subgroups.p = 0.09), while significant differences were observed for sex , i.e., considering only subjects positive for HPV, we found a significantly higher HPV positivity at the oral site than at the genital site in men . As for HPV risk classification, we found a significantly higher frequency of patients with hrHPVs at the genital site than at the oral site   .The growth in unprotected oral sexual practices has made the transmission of STIs from the genital area to the oral mucosa a widespread event, which has been described for several pathogens. In particular, the transmission of HPV infection to the oral cavity is quite a hot topic, mainly because of the gradual increase in HPV-positive HNSCC observed in recent decades ,40,41. HHPV vaccines formulated on the basis of the most prevalent and disease-associated HPV genotypes are effective against genital HPV infection ,30,31,32Moreover, changes in sexual practices may result in the presence of other common genotypes, not yet included in the available vaccines, in both the oral cavity and in the genital area . The ideThe opportunity to analyze a cohort of men and women tested for oral and genital HPV in this study has allowed us to broaden our understanding of concurrent and concordant HPV infections, and, especially, to identify the most common HPV types that are not included in quadrivalent and nonavalent vaccines. This approach could represent a starting point for future vaccine design.Coherently with what has been described in the literature, HPV-positive genital samples were more numerous than HPV-positive oral specimens (73.6% vs. 10.1%) . Concerning oral infection, our findings fall within a wide range from 0% to 81% described in the literature. The lack of consistent data from one study to another may be explained by the diversity of oral sampling methods, HPV detection techniques and populations selected .p < 0.0001). In addition to these findings, The percentage of genital HPV positivity was equally high in the women and man subgroups, while the oral HPV prevalence was significantly higher in men compared to women . Additionally, oral samples were predominantly characterized by single infections (91.2% vs. 8.8%), unlike genital samples where multiple infections, although fewer than the single ones, accounted for a significant proportion of positive samples (37.9%).Some differences between oral and genital sites can be highlighted. First of all, although oncogenic genotypes were the most frequently detected in the two sites, in both women and men, their prevalence was significantly higher in genital samples than oral samples  that, even in this case, has already been reported in the literature . In termAlthough these results suggest an association between genital infection and the onset of oral infection, it should be noted that any consideration about concurrence and concordance must take into account the small numbers of the groups analyzed.The focus on non-vaccine HPVs detected in both areas allowed the high prevalence of certain genotypes to be identified. Specifically, the hrHPV genotypes HPV51, HPV68, HPV53 and HPV66 and the lrHPV genotype HPV42 were the most frequently detected genotypes not included in the commercially available vaccines in the genital samples of the whole cohort. A closer look at the two groups of female and male patients allowed the same genotypes to be identified as significant in the female samples; in males, however, statistical significance was lost for HPV68, while HPV70 and HPV73 were found to be significantly represented.Other studies carried out in different geographical areas, mainly in China, have shown similar results. HPV51 has been reported in several studies to be one of the most common circulating genotypes in women, similar to HPV53 and HPV68 ,55,56. ALooking at Italian data, Serretiello et al. recently identified HPV51 as one of the most common genotypes and HPV42 as the prevalent lrHPV in women . KiwerskThe lack of studies evaluating genital HPV prevalence exclusively in men does not allow a proper comparison with our results. Unfortunately, the same evaluation performed on oral samples did not reveal a significantly high prevalence of non-vaccine types.The classification of HPV as carcinogenic, probably carcinogenic or possibly carcinogenic by the International Agency for Research on Cancer (IARC) is based on the identification of strong epidemiological, biological and/or biochemical evidence. Focusing on the non-vaccine genotypes identified as significantly represented in this study, HPV51 is classified as “carcinogenic”, HPV68 as “probably carcinogenic”, while HPV53 and 66 are defined as “possibly carcinogenic to humans”. HPV51 has been shown to cause a small percentage, which varies greatly from region to region, of cervical cancers, but it has the ability to interfere with cellular tumor suppression mechanisms. The same can be said for HPV53 and HPV66, which are common in the general population but rarely found in cervical cancer samples. HPV73 has also been defined as suggestive of carcinogenicity .Our observations are limited by the small number of patients, especially males. The disproportion between male and female groups was due to the way patients accessed the Oral Medicine Unit with Dentistry for Fragile Patients. As oral specimen collection often followed genital examination, women undergoing regular gynecological examination and cervical HPV testing formed the majority of our study group.Nevertheless, our results, although preliminary, have potential public health implications. In fact, the identification of common non-vaccine genotypes, which have also been reported in other studies and whose carcinogenic potential is also described by IARC data, suggests the next steps in HPV vaccination strategies. Based on our results, it would be advisable to consider including the high-risk genotypes HPV51, HPV53 and HPV66 in future vaccine formulations, as they are significantly prevalent in both female and male genital samples, and to a lesser extent HPV68, due to its high prevalence in women.


Patients with viral infections are susceptible to osteoporosis. This cohort study investigated the correlation between human papillomavirus (HPV) infections and the risk of osteoporosis via 12,936 patients with new-onset HPV infections and propensity score-matched non-HPV controls enrolled in Taiwan. The primary endpoint was incident osteoporosis following HPV infections. Cox proportional hazards regression analysis and the Kaplan-Meier method was used to determine the effect of HPV infections on the risk of osteoporosis. Patients with HPV infections presented with a significantly high risk of osteoporosis  after adjusting for sex, age, comorbidities and co-medications. Subgroup analysis provided that populations at risk of HPV-associated osteoporosis were females , those aged between 60 and 80 years , and patients with long-term use of glucocorticoids . HPV-infected patients who did not receive treatments for HPV infections were at a greater risk  of osteoporosis, while the risk of osteoporosis in those who received treatments for HPV infections did not reach statistical significance . Patients with HPV infections presented with a high risk of subsequent osteoporosis. Treatments for HPV infections attenuated the risk of HPV-associated osteoporosis. Osteoporosis affects more than 200 million people and causes 8.9 million fractures annually , mainly As a trigger of the immune-mediated mechanisms that could affect bone quality, increasing studies have demonstrated the relationships between low bone mineral density and infections by viruses including human immunodeficiency virus (HIV) , hepatitAs some of the most prevalent yet less addressed viral infections in the field of research on osteoporosis, human papillomavirus (HPV) infections are the most commonly diagnosed sexually transmitted disease , with moHPV infections and other viral infections share several immune dysfunction pathogeneses that could contribute to the development of osteoporosis, which include a strongly elevated RANKL expression in the progression of HPV-associated cervical neoplasia . Bone reThe present population-based cohort study was performed using data from the National Health Insurance Research Database (NHIRD). The NHIRD contains registration files and original claim data, such as hospitalization records, diagnostic codes, records of outpatient visits, medication history, and personal information for over 99% of Taiwan’s population. All diagnostic records were validated by the Bureau of National Health Insurance (NHI) to ensure the accuracy of these data . In this2 laser operation, (4) chemosurgery for condyloma, (5) simple or complicated electro cauterization, (6) liquid nitrogen cryosurgery, (7) simple or complicated cryotherapy that involved CO2 freezing and liquid nitrogen. All participants were tracked until presence of osteoporosis, missing, death, or the end of the study, December 2013. A total of 12,936 subjects were included in the HPV group and 51,744 subjects were included in the non-HPV control group.In the present study, patients who had been clinically diagnosed with HPV infections between 2000 and 2013 were identified using International Classification of Disease Clinical Modification codes  ,27. OnlyAll subjects in both groups were tracked from the index date to the first osteoporosis event. The primary endpoint of this study was the occurrence of new-onset osteoporosis, which was diagnosed based on clinical history or spinal or hip bone mineral density (BMD) by dual-energy X-ray absorptiometry (DEXA) evaluation and the use of one of the following medications: denosumab, alendronate, risedronate, ibandronate, zoledronate, raloxifene, bazedoxifene, teriparatide, strontium ranelate . Patientp-value < 0.05 in the two-tailed test.The chi-square test was used to compare the distribution of age, sex, index date, and baseline co-morbidities between the two groups, including those with versus without a previous history of HPV infections. The mean age of onset was compared using Student’s t-test. The incidence rate of osteoporosis was estimated by dividing the amount of osteoporosis by follow-up person-years for HPV-infected cases and non-HPV controls. Multivariable Cox proportional hazards regression models were used to estimate the crude HRs (cHRs) and adjusted HRs (aHRs) for osteoporosis in patients with HPV infections, as compared to non-HPV controls. The covariates considered in the multivariable regression models included sex, age, index date, comorbidities, and co-medications. A multivariable Cox regression model adjusted for the covariates was used to reveal the effect of sex, age, and follow-up time on the incidence of osteoporosis. The HRs adjusted for covariates were calculated each subgroup, as stratified by sex, age, comorbidities, and co-medications. The cumulative incidence curve was obtained from the Kaplan-Meier method and examined by the log-rank test. All data analyses were performed using SAS . The statistical significance level was set at In this retrospective cohort study, 12,936 HPV-infected patients and 51,744 propensity score-matched non-HPV controls were included. Baseline characteristics are shown in p-value = 0.01) (The cumulative incidence of osteoporosis in patients with HPV was significantly higher than that in non-HPV controls (log-rank test  = 0.01) . The Cox = 0.01) . Consist = 0.01) .Findings in the subgroup analysis of Cox proportional hazards regression models provided that female patients with HPV infections , patients aged 60–70  or aged 70–80  with HPV infections, patients of the lowest  or the highest socioeconomic status  with HPV infections, and HPV-infected patients with long-term use of glucocorticoids  were predisposed to significantly great risk of HPV-associated osteoporosis . NotablyIn this 13-year nationwide population-based retrospective cohort study, there was a 32% increased risk of osteoporosis in patients with HPV infections, which was validated after adjusting for demographic variables, comorbidities, and co-medications. Patients who were women, aged between 60 and 80, of low and of high socioeconomic status, with long-term use of glucocorticoid, were susceptible to HPV-associated osteoporosis. Treatments for HPV infections lowered the risk of HPV-associated osteoporosis in patients with HPV infections.Studies have shown that virus infections, such as HIV, HBV, HCV, and herpes zoster infections, were independently associated with a higher risk of osteoporosis ,11. CollThe exact mechanism of how HPV infections undermined bone loss or osteoporosis has not been studied. However, highly expressed RANKL has been observed during the progression of HPV infection-associated cervical cancer, which was secreted by HPV-infected cells . The excIt was found in the present study that treatments for HPV infections attenuated the risk of HPV infections. This finding was in accordance with previous studies on benign lesions of anogenital warts , in whicThe major strength of the present study was the use of longitudinal data of large sample size and a long follow-up duration that was able to provide the temporal association between HPV infections and the risk of new-onset osteoporosis, and the effect of treatments for HPV infections on the attenuated risk of HPV-associated osteoporosis. Findings in the present cohort study were representative of the general population, and potential measurable confounders were balanced through propensity score matching ,59,60 foIn conclusion, patients with HPV infections were associated with a significantly greater risk of subsequent osteoporosis, especially in female patients, those aged between 60 and 80 years, and individuals with long-term use of glucocorticoids. Treatments for HPV attenuated the risk of HPV-associated osteoporosis. Prospective studies on the association between HPV genotypes and osteoporosis and studies of mechanistic approaches may shed light on HPV-associated bone loss and osteoporosis.


To achieve maximum efficacy, vaccines, such as subunit, recombinant, and conjugate vaccines, necessitate the incorporation of immunostimulators/adjuvants. Adjuvants play a vital role in bolstering and extending the strength of the immune response while also influencing its type. As antigen and adjuvant formulations become more intricate, it becomes imperative to establish a well-characterized and robust formulation to ensure consistent and reproducible outcomes in preclinical and clinical studies. In the present study, an HPV bivalent vaccine was developed using a BC02 adjuvant in conjunction with HPV 16 and 18 L1 VLP antigens produced from an E. coli expression system. The study involved evaluating the adjuvant formulation and in vivo immunogenicity in mice. Remarkably, a medium-dose of BCG-CpG-DNA combined with a low-dose of aluminum hydroxide substantially enhanced the immunogenicity of HPV16 and 18 VLPs, resulting in improved cellular and humoral immune responses. Human papillomaviruses (HPV) belong to the papillomaviridae family and are double-stranded DNA viruses that infect humans. There are over 100 identified types of HPV, categorized into cutaneous or mucosal types based on their tissue tropism . HPV infThe first HPV vaccine was licensed in 2006, and several bivalent, quadrivalent, and nonavalent vaccines have been approved since 2022 . Among t3), combined with a fragment of DNA from the Bacillus Calmette-Guerin genome that contains an unmethylated CpG motif. In the present study, we assessed the humoral and cellular immune responses in mice to E. coli-derived HPV16 and HPV18 L1 VLPs using different formulations of the BC02 compound adjuvant. The primary goal was to determine the most optimal compatibility dose of BC02 adjuvant with HPV16 and 18 VLPs and to evaluate the proprietary BC02 as a potential candidate adjuvant for the bivalent HPV vaccine.The BC02, also known as the BCG-CpG-DNA Compound Adjuvant System 02, is a compound adjuvant composed of an inorganic salt adjuvant, aluminum hydroxide (Al(OH)All animal-related procedures were conducted at the Institute for Laboratory Animal Resources of the National Institute of Food and Drug Control (NIFDC) under strict adherence to ethical considerations. The Institutional Animal Care and Use Committee of NIFDC granted approval for all laboratory animal experiments. The health of the mice used in the study was maintained under a specific-pathogen-free (SPF) condition, and they were housed in a constant environment with regulated temperature, humidity, and a 12 h light/dark cycle. Before immunization, the mice were given three days to acclimate to their surroundings. Throughout the study, the mice were provided with unrestricted access to food and water and received attentive care from specialized personnel.®) from InvivoGen  and procured the following materials from Gibco : phosphate-buffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM), RPMI-1640 medium, 100 × Penicillin-Streptomycin, and fetal bovine serum (FBS). Furthermore, IL-2 and IFN-γ ELISpot kits were supplied by Mabtech .In our laboratory, we developed and preserved the following biological components and reagents: BCG-CpG-DNA novel biological adjuvant, BCG-CpG-DNA compound adjuvants system 02 (BC02), HPV 16/18 L1 virus-like particles (VLPs), and human embryonic kidney cells . Additionally, we obtained aluminum hydroxide , or concanavalin A  for 48 h at 37 °C in a 5% CO2 atmosphere. The ELISpot kit was used to detect IFN-γ and IL-2-secreting cells, and positive spots were determined using a UV microanalyzer . The abundance of mouse splenocytes producing IL-2 and IFN-γ was measured using enzyme-linked immunospot (ELISpot) assays, following the kit’s instructions. Briefly, a single-cell suspension was prepared from the spleens of immunized mice at a concentration of 5.0 × 10g for 10 min at 4 °C. The purified pseudovirions were examined through 12% SDS-PAGE gels and titrated on HEK 293 cells to assess infectivity using SEAP analysis. The purified pseudovirions were subsequently pooled and stored at −80 °C until further use.HEK 293 cells were evenly distributed into a T-175 flask, followed by incubation for 16 h. Subsequently, they were transfected using Lipofectamine 2000 reagent . The L1 gene-containing plasmids were co-transfected into HEK 293 cells together with a reporter plasmid that encodes secreted alkaline phosphatase (pYSEAP). Following a 72 h incubation period, the cells were lysed and incubated overnight at 37 °C. The unrefined pseudovirions were dissolved by adding 5 M NaCl and then centrifuged at 1500× 5 cells/well) were pre-plated in a 96-well plate with 100 µL DMEM containing 1% penicillin-streptomycin and 10% FBS for 4–6 h. Purified HPV 16 or HPV 18 pseudovirions were subjected to a 2000-fold dilution. The diluted pseudovirion stock (60 µL/well) was added with 60 µL series diluted serum  in a 96-well non-treated plate and then incubated at 4 °C for 1 h. Subsequently, 100 µL of the pseudovirion-serum mixture was transferred onto the pre-plated cells, followed by incubation for 72 h at 37 °C under a 5% CO2 atmosphere. After that, 100 µL of supernatant was collected and centrifuged at 500× g for 5 min. The SEAP content in 15 µL of the supernatant was measured using the Great ESCAPE SEAP Chemiluminescence Kit . After adding the substrate for 20 min, each sample was analyzed using a 1420 multilabel counter (VICTOR3). Neutralization activity was determined by identifying the highest serum dilution that led to a >50% reduction in SEAP activity.HEK 293 cells  aluminum hydroxide (formulation A) or high-dose (50 µg) aluminum hydroxide (formulation B) compatible with BCG-CpG-DNA at a 2 week interval.(16-IFN-γ): p < 0.0001 and MA vs. A (16-IL-2): p = 0.0009; MA vs. B (16-IFN-γ): p < 0.0001 and MA vs. B (16-IL-2): p = 0.0227; MA vs. AgC (16-IFN-γ): p < 0.0001 and MA vs. AgC (16-IL-2): p = 0.0005). Similarly, HPV18-specific IFN-γ and IL-2 secretion were also remarkably increased in all groups receiving the adjuvants ((18-IFN-γ): p = 0.0073 and MA vs. A (18-IL-2): p = 0.0286; MA vs. B (18-IFN-γ): p = 0.0050; MA vs. AgC (18-IFN-γ): p = 0.0008; and MA vs. AgC (18-IL-2): p = 0.0014). Low-dose aluminum hydroxide and BCG-CpG-DNA appear to promote a Th1-type immune response to HPV types 16 and 18 L1 VLPs.The results showed that all mice receiving the adjuvants exhibited an obvious increase in HPV16-specific IFN-γ and IL-2 secretion A,C. Notadjuvants B,D. The (16), (18), Moreover, the addition of BC02 to HPV16 L1 VLP antigens stimulated the humoral response, leading to a 7.3-fold increase in antibody titer compared to antigen alone  were combined with the BCG-CpG-DNA adjuvant, a Th1-immune response was induced. However, further improvement is needed to increase the impact on antibody production. To address this, a third homologous booster immunization was given to the mice. Following the booster immunization, HPV16- and 18-specific IFN-γ and IL-2 secretion increased in response to all BC02-adjuvanted vaccines. However, there was a slight downward trend observed 4 weeks after the last immunization A,B. Coad3 adjuvant alone (LA vs. A (16-IFN-γ): p = 0.027; MA vs. A (16-IFN-γ): p = 0.001; HA vs. A (16-IFN-γ): p = 0.002; LA vs. A (18-IFN-γ): p = 0.005; MA vs. A (18-IFN-γ): p < 0.0001; HA vs. A (18-IFN-γ): p = 0.001, 3 adjuvant alone group (MA vs. A (16-IL-2): p = 0.0003; HA vs. A (16-IL-2): p = 0.006, 3 adjuvant alone group (MA vs. A (18-IL-2): p = 0.003, The above results demonstrated the success of combining low-dose aluminum hydroxide  with BCG-CpG-DNA as a novel formulation. This formulation was used to prepare HPV16 and 18 bivalent vaccines for further comparison of immune responses with different doses of BC02 compound adjuvant. The production of HPV16 and 18 L1 VLP-specific IFN-γ was dramatically higher in mice that received formulation A with low (LA), medium (MA), and high (HA) dose BCG-CpG-DNA adjuvant compared to those who received the Al(OH)3-adjuvanted vaccine 3-adjuvanted vaccine. Splenic IFN-γ and IL-2 secretion were measured at 1, 4, and 8 weeks after the final immunization and were found to be higher in all groups that received the BC02 compound adjuvant  compared to those who received the Al(OH)3 adjuvanted vaccine  vaccine A,B. ThisAdditionally, the strength and persistence of antibody responses were also enhanced in the LA, MA, and HA groups compared to the two-dose immunizations. Titers of anti-HPV16 and 18 pseudovirus-neutralizing antibodies were maintained for 8 weeks after the last immunization in the MA group C.The presence of HPV DNA and the virus-transforming protein in almost all cervical cancer cells highlights the potential of HPV protein to elicit an immune response during vaccine immunotherapy. HPV vaccines can be categorized as prophylactic or therapeutic. Prophylactic vaccines target HPV capsid protein L1 or L1/L2 chimerism and are commonly used to induce humoral immunity in individuals who have not been previously exposed to HPV. Therapeutic vaccines, on the other hand, target the HPV primary oncogenic factors, E6 or E7, and aim to induce cellular immunity in patients with pre-existing infections and associated pre-invasive lesions. Both prophylactic and therapeutic vaccines consist of two essential components: target antigens and adjuvants.E. coli was used as the source for the target antigen. Previous studies have shown that L1 capsids produced by the prokaryotic expression system can stimulate antibody production in animal models [E. coli, was successful in stimulating the production of anti-HPV 16 and anti-HPV 18 antibodies.HPV types 16 and 18 are recognized as the predominant HR HPVs responsible for cervical cancers and head and neck cancers. Globally, HPV16 accounts for 50% of HPV detections, while HPV18 accounts for 14% ,16,17. Al models ,25. The BCG-CpG-DNA activates the innate immune response via TLR9 receptor-mediated activation of both NF-κB and MAPK pathways [BCG-CpG-DNA with the inorganic salt, Al(OH)3. It stimulates both Th1 and Th2 immune responses to antigens derived from mycobacterium tuberculosis, toxoplasma gondii, or varicella-zoster virus [3 or BC02 compound adjuvant had elevated levels of IFN-γ and IL-2 compared to the naked antigen, but the BC02-based vaccine elicited a more robust Th1 cytokine response. These findings highlight the necessity of incorporating adjuvants in HPV 16 and 18 bivalent VLP vaccines and demonstrate that the cellular immunity response to BC02 vaccines is superior to that of Al(OH)3-adjuvanted vaccines. BCG-CpG-DNA was shown to be compatible with both low-dose  and high-dose  Al(OH)3, but the low-dose formulation had better performance in both 2- and 3-dose immunizations. This suggests that the binding of BCG-CpG-DNA to Al(OH)3 could reduce the amount of metal adjuvant, resulting in a stronger cellular immune response, particularly concerning HPV 16 antigen-specific IFN-γ levels, with improved safety. BCG-CpG-DNA, a novel biological adjuvant, possesses independent intellectual property rights and originates from the unmethylated CpG motif-containing DNA fragments from the Bacillus Calmette-Guerin genome. It demonstrates robust adjuvant properties in various species. Previous research has shown that pathways . Additiopathways . BC02, aer virus ,31,32,333 adjuvant or naked antigen in the two-shot immunization program. The optimal effect was achieved at 10 µg BCG-CpG-DNA, consistent with previous results. Although the anti-HPV16 and anti-HPV18 antibodies produced by the use of the BC02 adjuvant had lower titers than the PTC group in the two-dose immunization program, they showed persistent elevation after the addition of the third dose. This may be attributed to the mechanism by which Al(OH)3 adsorbs antigens, prolonging antigen release and action time. However, the BC02-adjuvanted bivalent HPV type 16 and 18 vaccines have not undergone additional validation in the mouse infection model, which represents a significant breakthrough for our future research.The study successfully optimized the compatible BCG-CpG-DNA dose in the compound adjuvant of the bivalent HPV 16 and 18 vaccines with formulation A by comparing it with the positive test control (PTC). The BC02-adjuvanted group induced higher HPV 16 and 18 antigen-specific IFN-γ and IL-2 levels compared to Al(OH)The current study investigated the BC02 compound adjuvant in combination with the HPV VLP vaccine. The cellular and humoral immunities were examined at different time points, and the optimal formulation of the bivalent HPV 16 and 18 L1 VLP vaccine was determined. BC02 emerges as a promising adjuvant for the domestic production of an independent, innovative HPV vaccine.


HPV+ patients had fewer positive biopsy cores  and a lower maximal tumor infiltration rate per core  compared to HPV- patients. However, when analyzing the whole prostate and the lymph nodes after RP, there were no significant differences in TNM stage, Gleason score or tumor volume between both groups. In a subgroup analysis of all high-risk HPV patients (n = 6), we found no significant differences in sociodemographic, clinical or histopathological parameters compared to HPV- or low-risk HPV+ patients. Conclusion: In our prospective study, we were not able to prove a clinically significant impact of HPV status on tumor characteristics in RP specimens. Most men with PCa had never heard of HPV, despite its proven causal association with other tumor entities.Introduction: The association between human papilloma virus (HPV) and the pathogenesis of prostate cancer (PCa) is still controversial. Existing studies often lack information about clinical risk factors, are limited by their retrospective design or only use a single detection method for HPV. Material and Methods: A total of 140 patients undergoing radical prostatectomy (RP) for PCa at the Department of Urology, Ludwig Maximilian University of Munich, Germany, were prospectively enrolled. Knowledge of HPV and sociodemographic parameters were assessed with questionnaires. The following methods were used for HPV detection: RP specimens were tested for HPV DNA by PCR. If HPV DNA was detected, an LCD-Array hybridization technique was used for HPV subtyping, and immunohistochemical staining for p16 was performed as a surrogate marker for HPV infection. Serological titers of HPV-16 L1 antibodies were measured using an HPV-16-specific immunoassay. Results: HPV DNA was detected in 9.3% (13/140) of RP specimens, with HPV-16 being the most predominantly detected subtype (5/13 = 39%). HPV-16 L1 antibody levels were below the limit of detection in 98% of patients (137/140). We found no significant difference between HPV PCR-positive (HPV+) and -negative (HPV-) patients in terms of HPV-16 antibody levels, history of HPV-associated diseases, level of education or marital status. Seventy-five percent of all PCa patients had never heard of HPV before. An acinar adenocarcinoma of the prostate was the most frequently detected histologic type in both HPV+ (100%) and HPV− (98%) patients ( Overall, 5.2% of the global cancer burden may be attributable to human papilloma viruses (HPV), with HPV-16 or -18 being the predominant subtypes , oropharp = 0.001) [In a meta-analysis of 46 studies with 4919 PCa patients from Yang et al., HPV was detected in PCa tissue in 19% of cases . Thirtee= 0.001) . Further= 0.001) . In deta= 0.001) .The association between HPV antibody levels and PCa is also controversial. In an analysis of men who donated serum samples in Finland, 165 PCa patients were matched with 165 control subjects. The presence of IgG antibodies against HPV-16 or -18 was associated with an increased risk of developing PCa  .There is an urgent need to clarify the association between HPV and PCa, as PCa is the second most frequent cancer in men and the fifth leading cause of death worldwide, constituting a huge burden for global health . FirstlyIn general, existing studies on HPV and PCa often lack information about clinical risk factors, are limited by their retrospective design, or only use a single detection method for HPV . TherefoA total of 140 patients with prostate cancer undergoing radical prostatectomy (RP) were prospectively enrolled after informed consent at the Department of Urology, Ludwig Maximilian University of Munich, Munich, Germany, between April 2021 and August 2021. The study was approved by the university ethics committee (project number: 20–1000).A patient-completed paper-based questionnaire was preoperatively distributed among all participants, evaluating sociodemographic factors and HPV-associated pre-existing conditions, such as genital/skin warts and anal, penile, or oropharyngeal carcinomas. The questions of the original questionnaire are shown in ®, Abviris Deutschland GmbH, Ahrensburg, Germany). The test was performed according to the manufacturer’s instructions. In short, 20 µL of patient serum was mixed with the provided HPV reagent, incubated for 5 min and pipetted onto a lateral flow test device. After 10 min, a coloured band appeared. Line intensities were read using a colourimetric reader  and converted to concentrations using a calibration function. Each sample was measured twice, and mean values were used for analysis. The limit of detection was calculated as the limit of blank +3 standard deviations (SD). The HPV-16 antibody level was then divided into three categories: below the limit of detection (LOD), detectable low (LOD—1000 ng/mL) and detectable high (>1000 ng/mL). After surgery, histopathological analysis was performed by board-certified pathologists at the Institute of Pathology, LMU Munich. All RP specimens were reviewed to confirm tumor type and stage, and Gleason grading was performed according to the 5th World Health Organization (WHO) classification of genitourinary tumors. DNA from all RP specimen was analysed using the VisionArray® HPV Chips assay . Briefly, a minimum of 15ng of DNA was amplified by polymerase chain reaction (PCR), using the CE-IVD VisionArray® HPV Primer Kit  together with AmpliTaq Gold DNA Polymerase . VisionArray® biotinylated HPV Primers target the L1 (late) region of Human HPV genomes resulting in amplicons with a length spanning 139–148 bp. PCR products were subsequently hybridized to HPV probes spotted on a glass chip covering the HPV types of: (i) low risk: 6, 11, 40, 42, 43, 44, 54, 55, 57, 61, 62, 72, 81CP8304, 83MM7, 84MM8, 90, 91; (ii) partial high risk: 26, 34, 53, 66, 67, 68a, 68b, 69, 70, 73, 82IS39, 82MM4; and (iii) high risk: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59. As a control of functional DNA as well as the PCR, reaction primers specific for a fragment of the human HLA-DQA1 gene (amplicon length: 222 bp) were also included by the vendor. Specifically bound biotinylated HPV-specific single-strand PCR products were detected by adding streptavidin–peroxidase conjugates in the presence of tetra-methyl-benzidine (TMB), resulting in a blue precipitate. For detection and analysis, HPV types were identified by employing VisionArray® Analyzer Software .Prior to surgery, serum was obtained from all patients and stored at −20 °C until further analysis. Serological detection of HPV-16 antibodies was performed using an HPV-16-specific, virus-like-particle (VLP)-based competitive immunoassay  staining for p16 was performed on formalin-fixed, paraffin-embedded (FFPE) tissues with representative tumor sections using an antibody against p16  on a Ventana Benchmark XT autostainer  according to the manufacturer’s protocol.t-test. All statistical tests were performed with R (v3.6.3) and DATAtab Statistics Calculator . A two-sided p-value < 0.05 was considered statistically significant.Baseline variables were calculated as mean and standard deviation (SD), as frequencies with percentages or as median and interquartile range (IQR). All parameters were assessed for normality with histograms and with the Shapiro-Wilk test and the corresponding comparisons were performed using a χ2 or two-sample A patient flowchart is shown in Patient age, education level and marital status are presented in p = 0.02). None of the patients were vaccinated against HPV, while 21% of patients reported that they were aware of the existence of an HPV vaccination. A total of 9.7% of the patients reported that they had suffered from skin warts before, and one patient (0.7%) reported genital warts. No other HPV-related diseases or cancers were reported. None of the patients reported the intake of systemic immunosuppressive drugs. There was one patient (0.7%) with an HIV infection treated with antiretroviral drugs. None of the patients met the criteria for hereditary prostate cancer; however, 22% of the patients (29/143) had a positive first-degree family history of PCa. There were no significant differences in terms of knowledge of HPV, history of HPV-related diseases or PCa family history between HPV+ and HPV- patients.The patients’ knowledge about HPV and history of HPV-related diseases are shown in p = 0.49). HPV+ patients had fewer positive biopsy cores  and a lower maximal tumor infiltration rate per core  than HPV- patients. In 90% of cases, PCa was detected in both lobes of the prostate, with no significant difference between groups . There was no significant difference between HPV+ and HPV- patients with regard to Gleason score (GS) distribution in prostate biopsies (p = 0.76), with GS 3 + 4 being the most frequently diagnosed GS in HPV+ and HPV- patients .Patient prostate biopsy results are shown in p = 0.35). RP was either performed by an open (73/140 = 52%) or robot-assisted (67/140 = 48%) approach. The median weight of the resected RP specimen was 59.8 g and median tumor volume of the RP specimen was 25%, with no statistically significant difference between HPV groups . The most common histological subtype was acinar adenocarcinoma (98% of cases). In two patients (1.4%), a small cell neuroendocrine carcinoma was found, and one patient (0.7%) was diagnosed with a ductal adenocarcinoma. Lymph nodes were removed in 116 of 140 patients (83%) and were positive in 11% (13/116). There were no statistically significant differences between HPV+ and HPV- patients in terms of histologic subtype (p = 0.86), GS (p = 0.086), local tumor stage  or local nodal stage .Characteristics of RP specimens are presented in HPV DNA was detected in 9.3% (13/140) of RP specimens. The results of the HPV subtyping are depicted in INK4a was performed on all HPV+ RP specimens (INK4a was found to be overexpressed (=active HPV infection) in two of the five HPV-16-positive patients (40%) and in two of three patients with a co-infection of HPV 6 and 11 (67%). In all other HPV+ patients, IHC for P16INK4a was negative.To discriminate between active and inactive HPV infection, IHC for P16pecimens . P16INK4HPV-16 L1 antibody levels are presented in p = 0.13). We further performed an analysis that only included high-risk HPV+ patients; but again, there was no statistically significant difference in HPV-16 L1 antibody levels between high-risk HPV+ and all other patients  after RP. In our study, HPV DNA was detected in 9.3%, which is lower in comparison to a meta-analysis from Yang et al. that included 46 studies with 4919 PCa patients and reported a 19% prevalence of HPV infection .HPV-16 L1 antibody levels were below the limit of detection in the vast majority of PCa patients (98%) in our study, and there was no association between detectable HPV-16 L1 antibody levels and HPV PCR status. A recent systematic review listed 15 serology studies of HPV in prostate cancer . The perWe found no significant differences in sociodemographic factors such as patient age, education level or marital status when comparing HPV+ and HPV- patients. Furthermore, there were no differences in terms of knowledge of HPV, history of HPV-related diseases or PCa family history between HPV+ and HVP- patients. Most PCa patients had never heard of HPV before, despite its proven causal association with several tumor entities . PatientOverall, 5.2% of the global cancer burden might be attributable to HPVs . It has To understand the complex interplay between viral and host factors that might be the reason for the incongruity of evidence and to find the best approach for prevention, early diagnosis and treatment, many efforts are still necessary . It has It should be noted that despite our extensive approach with prospectively collected data, there are several major limitations to the present study. First and foremost, although all patients were prospectively enrolled, we used a single-center setting without a control group. Second, only including patients who underwent RP in our department may have led to a selection bias, especially in low-risk PCa patients, who often choose active surveillance. Third, we only tested for HPV-16 L1 antibody levels, not for antibody titers of other HPV types. Fourth, the absence of HPV in RP specimens does not rule out HPV infections in the past. Fifth, expression levels of E7 might be a more specific surrogate marker for HPV than p16 expression levels.We were not able to prove a clinically significant impact of HPV status on tumor characteristics in RP specimens. Furthermore, HPV-16 L1 antibody levels were below the limit of detection in the vast majority of PCa patients (98%) in our study, and there was no association between detectable HPV-16 L1 antibody levels and HPV PCR status. Most men with PCa have never heard of HPV, despite its proven causal association with other tumor entities. Continued efforts should be made to raise awareness for HPV-related diseases and vaccination programs.