Strong quantitative and functional antibody responses to the quadrivalent human papillomavirus (HPV) vaccine were reported in mid-adult aged men, but there are limited data on the avidity of the antibody response and the memory B-cell response following vaccination. Although circulating antibodies induced by vaccination are believed to be the main mediators of protection against infection, evaluation of avidity of antibodies and memory B cell responses are critical for a better understanding of the vaccine immunogenicity mechanisms. Both the modified enzyme-linked immunosorbent assay (ELISA) and the enzyme-linked immunosorbent spot (ELISpot) assay are tools to measure the humoral and cellular immune responses post vaccination to characterize vaccine immunogenicity. The avidity of HPV-16 and HPV-18 specific IgG in the serum of mid-adult aged men (N = 126) who received three quadrivalent HPV vaccine doses was examined using a modified ELISA. HPV-16 memory B-cell responses were assessed via ELISpot at month 0 (prior to vaccination) and 1-month post-dose three of the vaccine (month 7). The quadrivalent vaccine induced an increase in HPV-16 and HPV-18 antibody avidity at month 7. HPV-18 avidity levels moderately correlated with anti-HPV-18 antibody titers, but no association was observed for HPV-16 antibody titers and avidity levels. The HPV-16-specific memory B-cell response was induced following three vaccine doses, however, no association with anti-HPV-16 antibody avidity was observed. Three doses of quadrivalent HPV vaccine increased antibody affinity maturation for HPV-16/18 and increased the frequency of anti-HPV-16 memory B-cells in mid-adult aged men. HPV-16 and HPV-18 are the most common oncogenic HPV types, accounting for approximately 60% of oropharyngeal cancers, and approximately 90% of anal cancers ,3. WhileRoutine vaccination in mid-adult aged men could help reduce the incidence of oropharyngeal cancers, by preventing oral HPV infection. The quadrivalent HPV vaccine is safe and effective in reducing HPV-6/11/16/18 anogenital infections in young males ages 16–26 years ,8. FurthTo further evaluate the quality of the antibody response post vaccination among mid-adult aged men, antibody avidity and number of antigen specific memory B-cells were measured. Circulating antibodies against HPV induced by vaccination play an essential role in protection against repeated exposures and subsequent HPV infections. The antibody levels that are induced by vaccination are often much higher than that observed during natural HPV infection and more detailed analysis of this robust immune response will uncover key factors in long-term immune protection against HPV infection –12. The 2.2.1.2.1.1.www.clinicaltrials.gov, NCT01432574). A total of 145 men had specimens contributing to both month 0 and month 7 serum HPV antibody evaluations [Sera were collected at months 0 and 7 from 150 men, ages 27–45 who received quadrivalent HPV vaccine at 0, 2, and 6 months. This study was conducted in compliance with regulations by an ethical committee review and was monitored by the Moffitt Cancer Center Protocol Support Office. The study population consisted of mid-adult aged males from two clinics located in Cuernavaca, Mexico and Tampa, Florida  containing HPV 6/11/16/18 virus-like particles (VLP) was used to vaccinate men at month 0, months 2 and 6 in a volume of 0.5 mL by intramuscular administration in the deltoid or thigh muscle. At month 0 and month 7 serum and PBMC specimens were collected and stored frozen at ‒80 °C until tested.2.2.‒1 in 100 μl for at least 72 h at 4 °C. The plates were then washed three times with 350 μl of phosphate-buffered saline (PBS) containing 0.2% Tween 20 and blocked in 300 μl of PBS with 4% skim milk and 0.2% Tween-20 (blocking buffer) for 90 min at room temperature. The plates were washed three times. Serial dilutions of serum (starting dilution 1:100) in blocking buffer in 2-fold increments were added to the plate in a final volume of 100 μl. The plates were sealed and incubated at room temperature for 60 min on a plate shaker at 250 rpm. After washing the plate three times, peroxidase-labeled goat anti-human IgG  was added and the plates were sealed and incubated at room temperature for 60 min on a plate shaker at 250 rpm. Plates were then developed with tetramethylbenzidine substrate solution . After 25 min of incubation in the dark at room temperature, the reaction was stopped by the addition of 100 μl of 0.36 N H2SO4. The absorbance at 450 nm and 620 nm were measured with a microtiter plate reader . Antibody levels were expressed as ELISA units (EU mL‒1) which were interpolated from the standard curve. Antibody levels were then normalized for total IgG in each specimen and expressed as EU mg‒1. Both the HPV-16 and HPV-18 ELISA were calibrated with the World Health Organizations International Standards. The International Units (IU) conversion factor for anti-HPV-16 antibodies is 1 IU mL‒1 = 5.29 EU mL‒1, and for anti-HPV-18 antibodies is 1 IU mL‒1 = 6.81 EU mL‒1.Specimens were tested for anti-HPV-16 and HPV-18 IgG levels by the L1 VLP an enzyme-linked immunosorbent assay (ELISA), as previously performed . Flat-bo2.3.To approximate the binding strength of IgG antibodies to HPV, the avidity of antibodies was determined as previously described, using a modified ELISA-based method with the chaotropic agent guanidine hydrochloride  ,21. The 2.4.Frozen PBMC were thawed, washed and resuspended in assay media containing RPMI 1640 , 10% fetal bovine serum  and 1x penicillin–streptomy cin-glutamine (Gibco). Cell counts and viabilities were obtained with a MUSE Cell Analyzer (Millipore).6 viable cells per well were plated into 24-well tissue culture plates  in 2 mL stimulation media containing RPMI 1640 (Gibco), 10% fetal bovine serum (HyClone), 1x penicillin–streptomycin-glutamine (Gibco), 50 μM 2-mercaptoethanol (Sigma), 1 μM CpG ODN-2006 (Invitrogen), 1 μg mL‒1 Protein A from Staphylococcus aureus Cowan (Sigma), 1 μg mL‒1 pokeweed mitogen (Sigma). The stimulated PBMC were incubated in a 37 °C humidified 5% CO2 atmosphere for 6 days.Approximately, 1 × 102. After the two-hour incubation, the plates were washed six times with 0.05% Tween 20 (Sigma) in DPBS, followed by a 2-hour incubation at room temperature with a 1:500 dilution of biotinylated anti-IgG (Mabtech) in DPBS with 1% bovine serum albumin (Thermo Scientific) and 0.05% Tween 20. After incubation and four washes in DPBS to remove excess antibody, a 1:1000 dilution of streptavidin alkaline phosphatase (Mabtech) in DPBS with 1% bovine serum albumin (Thermo Scientific), was added to each well for 1 h at room temperature followed by 4 washes in DPBS. One hundred μl per well of BCIP/NBT substrate , was filtered and added for 7–10 min, resulting in the development of spots. The reaction was stopped by washing three times in distilled water. Plates were dried overnight, and the spots were visualized and counted using the ImmunoSpot Imaging Analyzer system (Cellular Technology Ltd.) and ImmunoSpot software v5.1. All wells were counted with set parameters and then each count was verified. The spot counts were adjusted to reflect the average number of spots per 1 × 106 cells and then the frequency of antigen-specific B-cells was calculated by dividing the number of antigen-specific spots by the estimated number of total B-cells. Pre- and Post-vaccination timepoints were run simultaneously for each participant and a positive and negative donor control were run with each batch.We adapted a previously established method for the HPV-16-specific memory B-cell ELISpot assay . The day2.5.t-test was used. Correlations between anti-HPV-specific IgG and anti-HPV-specific avidity indices were determined by Spearman correlation coefficients (ρ). Correlation between memory B-cell responses and avidity of anti-HPV-16 antibodies were determined by Spearman correlation coefficients (ρ). A p value of ≤ 0.05 was considered significant.Geometric mean avidity responses were measured, and their associated 95% confidence intervals were calculated using GraphPad Prism Version 8.4.3. Statistical significance was determined using either a Mann-Whitney test or a Wilcoxon Matched pairs signed rank test between time points. For determining statistical significance between two clinical sites an unpaired 3.3.1.All men that received three doses of the quadrivalent HPV vaccine developed HPV-16 and HPV-18 antibodies . The thrOf the 150 participants, 24 were seropositive for HPV-16 and/or HPV-18 antibodies on day 1, and thus, had a previous history of infection with HPV-16 and/or HPV-18 before vaccination. Twenty-three seropositive participants also had an increase in antibody avidity after receiving three doses of vaccine . There wIn this study, the participants seropositive for HPV-16 differed by location, with a higher prevalence of positivity (13.3%) at month 0 in Mexico compared to 3.9% positivity in the US . The par3.2.‒1) with avidity indices below the geometric mean and 63 individuals with avidity indices at or above the geometric mean with total anti-HPV-16 IgG levels below 500 EU per mg‒1. The concentration of circulating antibodies to HPV-16 does not appear to correlate with antibody affinity maturation for HPV-16. However, there was a moderate but significant correlation between anti-HPV-18 avidity levels and total HPV-18-specific IgG concentration .The avidity levels of anti-HPV-16 antibodies post vaccination at month 7 did not correlate with the total anti-HPV-16 IgG levels . There w3.3.HPV-16 specific memory B-cells were enumerated from PBMC using the ELISpot assay. The antigen-specific memory B-cells were calculated as a percentage of the total IgG positive memory B-cells. Up to 4.4 % of IgG positive B-cells were HPV-16-specific post vaccination (Median = 0.43% of IgG positive B-cells) . Antigen4.Tracking antibody affinity and memory B-cells were two independent parameters to evaluate the humoral and B cellular immune response to HPV vaccination. The avidity assay measured the binding strength of antibodies to HPV and thus, an important immunogenicity marker. The ELISpot provided quantification of memory B-cells that were antigen specific and was another independent measurement for vaccine immunogenicity and potency. Three doses of the quadrivalent vaccine elicited antigen-specific memory B-cells similar to levels seen with the anthrax vaccine . The freThree doses of the quadrivalent HPV vaccine provided both a high quality and high quantity antibody response against HPV in mid-adult aged men. Vaccines against influenza and hepatitis B virus also showed similar induction of antibody quantity and quality. An increase in the concentration and avidity of influenza-specific IgG was also detected after three doses of influenza vaccine . ProtectThis was the first study to evaluate the memory B cell response in males after three doses of the quadrivalent HPV vaccine, and its relationship with avidity. In women that received the quadrivalent HPV vaccine there remained a high level of protection against cervical cancer even though anti-HPV antibody levels eventually dropped below detection, emphasizing the importance for immune memory . Three d


Microplastic concentrations significantly differed between bait categories and companies, but microplastic characteristics did not vary. There was no correlation between microplastic contamination and the number of bait ingredients, but it was positively correlated with C:N ratio, indicating a higher contamination in baits with higher proportion of plant-based ingredients. We thus reveal that bait microplastics introduced accidentally during manufacturing and/or those originating from contaminated raw ingredients might be transferred into freshwaters. However, further studies are needed to quantify the relative importance of this cryptic source of contamination and how it influences microplastic levels in wild fish.High environmental microplastic pollution, and its largely unquantified impacts on organisms, are driving studies to assess their potential entry pathways into freshwaters. Recreational angling, where many anglers release manufactured baits into freshwater ecosystems, is a widespread activity with important socio-economic implications in Europe. It also represents a potential microplastic pathway into freshwaters that has yet to be quantified. Correspondingly, we analysed three different categories of industrially-produced baits  for their microplastic contamination (particles 700 µm to 5 mm). From 160 samples, 28 microplastics were identified in groundbait and boilies, with a mean concentration of 17.4 (± 48.1 SD) MP kg In freshwater, microplastics primarily originate from the fragmentation of larger plastic items3, and their prevalence in lakes5 and rivers7 can be high. Microplastics are consumed by aquatic organisms across trophic levels and taxa10, representing toxicological threats to individuals and subsequently affecting community composition and the functioning of freshwater ecosystems12. Microplastic characteristics, such as colour and shape, can modulate their consumption by aquatic organisms15, with their consumption being either direct  or indirect through the consumption of food resources contaminated with microplastics17. Identifying microplastic sources and their pathways into freshwater ecosystems is therefore important for reducing their potential impacts18.Microplastic pollution (plastic particles < 5 mm in size) represents a growing and ubiquitous threat to ecosystems19 and by up to 20% of populations in some European countries20. While angling is multifaceted in the way anglers capture a fish, most techniques release angling baits into the water to attract fish into a restricted spatial area in order to maximise the chance of fish capture21. Baits are introduced into freshwater ecosystems by anglers, either by hands and/or using devices such as a catapult. Anglers use, on average, 7.3 kg of baits per year22, with some specialised anglers using at least 200 kg of angling bait per year23. Evidence suggests that angling baits can represent an important food resource to wild fish, contributing to over half of the diet of fish in some ecosystems, and nearly 80% for some individuals24. This dietary contribution by bait tends to increase with fish size25, with larger specimens often being targeted by anglers more than smaller fish and they can be more vulnerable to capture26. Angling baits represent important trophic subsidies to freshwater ecosystems that can additionally contribute to eutrophication through the addition of phosphorous28.Angling is a widespread recreational activity practiced by more than 10% of the global population29, pet foods30 and canned fish for human consumption32. Therefore, angling baits could represent an unknown pathway of microplastic contamination within freshwater ecosystems that requires quantification, especially given their ubiquitous use in angling in many European countries28.Angling baits are usually purchased by anglers from commercial sources and can be categorised as ‘groundbaits’ , ‘boilies’  and ‘pellets’ . These angling baits differ in their composition but generally contain various flours  mixed with additional ingredients. Because commercially-available angling baits are primarily produced industrially, there is potential that they also contain substantial quantities of microplastics, either present in the raw materials or introduced during manufacture. Microplastics have already been reported in other industrially-produced and packaged wines34. Specifically, we tested the hypothesis that (1) the number of ingredients in angling baits was positively related to microplastic concentration, as it likely represents an increased diversity of the potential sources of contamination; and (2) the ingredients of the angling baits are a major determinant of their microplastic concentration, with animal-based baits containing more microplastic than plant-based baits.This study aimed to investigate the presence of microplastics within angling baits as a potential source of microplastic to freshwater pollution. The objectives were to firstly quantify the number, mass and characteristics  of microplastics within commercially-available, industrially-produced, angling baits , and to determine if contamination levels varied between bait categories and companies. This study also explored whether differences in microplastic number or characteristics could be related to the number of ingredients, and the origin of ingredients. The latter was assessed using the carbon to nitrogen (C:N) ratio to determine the relative amount of animal- versus plant-based ingredients Microplastics (700 µm–5 mm) were investigated within 16 commercially available angling baits products  that were purchased in France, with each bait replicated 10 times. Across the 160 analysed samples, a total of 86 particles were collected. Infrared spectroscopy analyses revealed that 39 particles were plastic, of which 28 were within the selected size range of microplastics, i.e. 700 µm to 5 mm Fig. . Therefop = 0.032) and companies . The microplastic concentration level ranged between 0 and 300 MP kg−1 .The mean occurrence of microplastics across all angling bait samples was 13.7 ± 17.01%, with occurrence varying between bait categories . There was a significant difference in the occurrence of microplastics between bait categories . There were a significantly higher concentration in groundbaits than in boilies .The range in MP concentration by mass across all samples was 0 to 232 mg kgp > 0.05), and microplastic size .The microplastics detected in groundbaits and boilies were almost exclusively comprised of fragments, with only one fibre detected Fig. e. PolyetThe bait packaging was composed of two polymers, polyethylene terephthalate and polyethylene   , the correlations between the number of ingredients reported on packages and microplastic concentrations were not significant  contaminated two out of three studied bait categories; groundbaits and boilies, with significant differences between categories and companies. Microplastics were mainly composed of polyethylene and artificial additives such as alkyd resins and paint additives, and were mainly white, red and blue. There was no correlation between the number of bait ingredients and their microplastic concentration and the C:N ratio of the baits was positively correlated with contamination level.26, whose diets may also depend heavily on angling baits25, suggests that larger fish might be most exposed to microplastics via this pathway. While several studies have already identified correlations between fish body size and microplastic loads in fish39, it is currently unknown whether this might be related to the consumption of contaminated baits.Given the incidence and number of microplastics per unit of bait mass, they could represent a significant source of microplastics to freshwater fish when fishing pressure is high. Once in the water, fish may consume the microplastics derived from angling baits either directly, i.e. microplastic released from bait, or indirectly through the ingestion of contaminated bait or other biota that have themselves consumed bait. The fact that anglers tend to target larger individualsCyprinus carpio) from several rivers43. Microplastic incidence and counts within cyprinid fishes have generally been high compared to other fish within the same system, ranging from 2.5 to 48 pieces per individual in the gastrointestinal tract43, with many of these studies implicating the benthic foraging habits of carp as a likely explanation. Fish feeding largely on baits, particularly large individuals, could reasonably achieve the levels of microplastic contamination observed in the wild, although it is acknowledged that identifying the sources of specific microplastics is difficult. While the potential effects of ingestion of contaminated baits have also yet to be determined, microplastic exposure has been shown to adversely affect C. carpio biochemistry, immunological activity, growth and oxidative pathways within the laboratory46.Species of the Cyprinidae family are the primary target for the angling baits investigated and microplastics have previously been detected in common carp 49. As such, it is difficult to draw inferences about microplastic contributions via angling baits by comparing polymer data alone.The diverse size range of microplastic particles detected in studies limits comparisons between the microplastic concentrations in the baits and those that are generally found in the biota50 that might gradually fragment over time. Various heating, milling and filtering processes during manufacturing may also alter, and fragment microplastic particles, potentially producing smaller particles falling outside the minimal threshold used in the present study52. This higher level of industrial manufacturing might explain the absence of larger particles in the pelletized angling baits. Considering that particle size is an important factor determining microplastic ingestion and impacts on organisms53, further investigations focusing on smaller fragment sizes (< 700 µm) are needed. That the microplastics identified in the baits were of a different composition to their packaging further suggests contamination existing within the raw ingredients and/or introduced during their manufacture, although further work is necessary to identify the exact stage(s) and source(s) of contamination.The absence of microplastics in the selected size range (700 µm–5 mm) in the pelletized angling baits suggests some key differences in their ingredients and/or manufacturing process compared with groundbait and boilies. Polyethylene microplastics and additives such as polyolefin and alkyd resins found in the angling baits are commonly present in machinery paints52, with often higher concentrations occurring in those with particular ingredients and/or manufacturing processes. Fishmeal was found to contain mostly fragments 100–1000 µm in size that were composed of polypropylene, polyethylene and polystyrene52, which are largely comparable to those particles found in our angling baits. Hanachi et al. (2019) additionally found higher microplastic concentrations in salmon and sardine than kilka-derived fishmeal, whereas, contrary to our first hypothesis, we found a positive correlation between bait C:N ratio and microplastic contamination, suggesting lower contamination in animal-based baits. Nevertheless, the similarity in microplastic features to those recovered from industrialized food29 suggests at least some procedural contamination from the manufacture process.Commercial fishmeal has previously been shown to contain microplastics28, but this does largely depend on the activity of anglers in a country and the amounts of baits they apply in their angling. Nevertheless, this estimate is comparable to the annual 0.15 tons of microplastics released through the use of winter de-icing salts applied to roads in some European countries54. At a larger scale, the microplastic contribution through angling baits can be considered minor compared to the top sources of European riverine microplastics  which may each contribute more than 0.3 kilotons of microplastic a year55. Nevertheless, angling bait-derived microplastics may make up a large proportion of local microplastic concentrations in particular locations or at particular times of the year when the baits are heavily used. Also, as expected for natural particles, microplastic concentration might increase with decreasing particle size56, and the total contamination by microplastics would probably be substantially higher, as only particles from 700 µm to 5 mm were considered here. Further investigations are needed to fully understand the potential contribution of this cryptic source of microplastic pollution compared to the global microplastic pollution in freshwater ecosystems. This contribution will likely be extremely variable among ecosystems and countries, depending on both the characteristics of the ecosystems  and of the fisheries (e.g. amount and type of bait used). We posit that in small lakes with intense fishing pressure targeting coarse fishes angling baits might represent an important source of microplastic pollution compared to other sources. Since angling baits are already known to contribute to freshwater eutrophication28, the additional release of microplastics from contaminated baits may represent another, co-occurring stressor to freshwater systems which requires further investigation. The awareness of hidden sources could contribute to the design of studies investigating consequences and impacts of microplastic to human and animal health.The estimates of the extent of cryptic microplastic emissions from angling baits were as high as 0.34 tons per year for a country, when considering 7.3 kg of groundbaits/angler/year and 3.3 million active anglers, as in GermanyThe ubiquitous presence of microplastic particles in the environment means that mitigating this angling source of contamination might have a negligible impact. Nevertheless, our results are important in highlighting a previously unknown source of microplastic loadings in freshwater fishes and thus have h elped identify a novel source and pathway. Such cryptic sources of microplastic contamination to freshwater ecosystems reveal the ubiquity of plastics within products used in daily hu man activities and, more specifically, on the relevance of angling activity in European countries in increasing the exposure of fish to these plastics. The manufacturing process of industrialized food, either for human or animal consumption, thus represents a potential source of microplastic contamination that has yet to be fully quantified and therefore further studies are encouraged in order to investigate the sources of these cryptic microplastics, and their fates and impacts in the environment.34. About 3 g of each angling bait, in triplicates, was oven-dried at 60 °C for 72 h before being ground (Retsch MM200) and analysed at the Cornell Isotope Laboratory  by isotope ratio mass spectrometry .We purchased some of the most popular, commercially-available, angling baits used in Europe, i.e. groundbaits, boilies and pellets, to target freshwater cyprinid fish. Angling baits were purchased in two angling shops and online from a popular angling website in France. In total, 16 different products were purchased , produced by 6 different companies, therefore including multiple bait categories from some companies. The products differed from each other by their commercial name or packaging and, in total, 27 commercial bags were purchased (Supplementary Table 2O2 (w/w) (30%)) solution  to digest organic matter. Then, samples were filtered through a 500-µm mesh size polyamide fabric  and washed with distilled water and ethanol (70% solution in water). The retained content in the Nitex was stored in polystyrene petri dishes. For each sample, two visual inspections of suspected plastic particles  to 5 mm) were performed by the same operator under a stereomicroscope (Leica MZ 75 and Nikon SMZ 800). Identified particles were then individually photographed using a binocular magnifier optical (Leica MZ16) equipped with a digital camera , with their size  measured using ImageJ v.1.8.057. Their colour was defined following previously described literature58. Particles were categorised as ‘fibres’  or ‘fragments’ (angular and solid or flexible), as adapted from Horton et al.59. Each particle was then weighed ; there were four particles of mass < 0.001 mg that were not included in calculations of mass concentration. The polymer composition of particles was assessed by attenuated-total-reflectance (ATR) Fourier-transformed infrared spectroscopy (FTIR) equipped with a diamond crystal . The crystal was cleaned with ethanol prior to the analysis of each particle and a background was collected at each set of four particles. The IR spectra was obtained with a resolution of 4 cm−1 and through the application of 32 scans over the wave-number range of 400 to 4000 cm−1. The ATR correction was applied to the spectra, which was then compared to available commercial libraries . A correlation factor of 0.6 was used as threshold to assign composition to the particle. When below this threshold, particles were considered as unidentified and removed from subsequent analyses. Identified particles were classified as microplastic with polymer category  following the Polymer Properties Database60 when possible, or as non-plastic , polyvinylester, polypropylene and polyacrylate. The size range was selected to optimise recovery during the stereomicroscope analyses and an unequivocal identification of particle composition63.Each sample consisted of 10 g of angling bait, with 10 replicates analysed for each angling bait product  to avoid external contamination. The original packaging of each angling bait was sampled and submitted to the same ATR-FTIR analysis to determine its polymer composition. A total of 10 replicates of silica powder , with around 10 g each, were used as blanks and submitted to the same entire process in the same sample batch. The microscopic inspection indicated the absence of suspicious particles in the size range of this study and contamination was therefore considered negligible.All procedures were performed under a fume-hood and metal- and/or glass-ware were used wherever possible. Nitrile gloves and cotton lab coats were always worn. The solvents, distilled water, ethanol and H−1) and as the mass of microplastics per unit of ground bait dry mass (mass concentration in mg kg−1). Fisher exact tests were first used to compare the occurrence of microplastics between angling bait categories and between companies. Generalized linear mixed-effects models (glmm) were used to test the difference in microplastic concentration (number and mass) between the categories of angling baits and between the companies (fixed effects) using angling bait product as a random factor and gamma distribution as family. Fisher exact tests were then used to compare the polymer composition and colour distribution of microplastics between angling bait categories. Spearman correlations tested the relationship between the microplastic concentration (number and mass) in angling baits , number of reported ingredients and C:N ratio  across all products (n = 16). All statistical analyses were performed using R v.4.0.264 and generalized linear mixed effects models were performed using the package lme464. Significant levels of generalized mixed effects models were obtained using the ‘Anova’ function in the car package66. Error reported around mean values are standard deviation.Microplastic occurrence in angling baits was calculated as the proportion between products containing microplastic and the total products available per category. Microplastic concentration in angling baits was calculated as the number of microplastics per unit of ground bait dry mass (number concentration in MP kgSupplementary Information.


Has the introduction of the vaccine for human papillomavirus (HPV) been associated with changes in infection rates among sexually active adolescent girls and young adult women?This cohort study assessed HPV infection rates of vaccinated adolescent and young adult women at a large adolescent-specific health center in New York City. Age-adjusted cervical HPV detection of vaccine-related types were lower year over year, while the prevalence of nonvaccine high-risk HPV types remained flat or higher.These results indicate the real-world effectiveness of the HPV vaccines in female youth in New York City; nevertheless, rates of some nonvaccine high-risk HPV types were higher. This cohort study of a single health center in New York City examines changes in human papillomavirus (HPV) infection rates among adolescent girls and young women following the introduction of a quadrivalent vaccine. Rates of human papillomavirus (HPV) infection have decreased since the introduction of HPV vaccines in populations with high vaccine uptake. Data are limited for adolescent and young adult populations in US metropolitan centers.To determine HPV infection rates in adolescent girls and young women aged 13 to 21 years in New York City following HPV vaccination.This cohort study of type-specific cervical HPV detection was conducted at a large adolescent-specific integrated health center in New York City between October 2007 and September 2019. Participants included an open cohort of adolescent girls and young adult women who received the HPV vaccine  over a 12-year period following HPV vaccination introduction. Data analysis was concluded September 2019.Calendar date and time since receipt of first vaccine dose.Temporal associations in age-adjusted postvaccine HPV rates.A total of 1453 participants, with a mean (SD) age at baseline of 18.2 (1.4) years, were included in the cohort . Approximately half (694 [47.8%] participants) were vaccinated prior to coitarche. Age-adjusted detection rates for quadrivalent vaccine types  and related types  decreased year over year, with the largest effect sizes observed among individuals who had been vaccinated before coitarche . By contrast, detection was higher year over year for nonvaccine high-risk cervical HPV types  and anal HPV types . The largest effect sizes were observed with nonvaccine types HPV-56 and HPV-68.Whereas lower detection rates of vaccine-related HPV types were observed since introduction of vaccines in female youth in New York City, rates of some nonvaccine high-risk HPV types were higher. Continued monitoring of high-risk HPV prevalence is warranted. This open cohort study (conducted between 2007 and 2019) assessed type-specific high-risk HPV detection rates in cervical and anal specimens of sexually active girls and women aged 13 to 21 years at enrollment in a longitudinal study conducted at a large adolescent-specific health center that provides integrated health services in New York City. Temporal associations in age-adjusted postvaccine HPV rates were evaluated in a large population of adolescent girls and young adult women in a metropolitan setting.33 Shortly after the approval of the first HPV vaccine in 2007, we initiated a prospective cohort study of sexually active female adolescents and young adults at the Mount Sinai Adolescent Health Center. Patients aged 13 to 21 years attending the clinic were invited to participate in a prospective study with repeated assessments every 6 months until age 26 years. Girls and women were eligible to participate if they had already engaged in sexual intercourse and intended to receive or had already received the Gardasil vaccine (Merck & Co). Girls and women who were pregnant at the time of recruitment were invited to return after their pregnancy. Written informed consent was collected from all participants prior to enrollment. The study was approved by the institutional review board at the Icahn School of Medicine at Mount Sinai. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.This was a longitudinal evaluation in an open cohort of adolescent girls and young adult women enrolled prospectively over a period of approximately 12 years.Study participants received a gynecological examination and completed a health history interview at each visit, including assessment of sexual behaviors, history of sexually transmitted infection, and immunizations. In addition, a self-report questionnaire was completed at each visit to assess lifetime and recent sexual activity and condom use. Vaccination status was confirmed using the New York City and state immunization registries.33 Briefly, cervical cells were obtained using a cytobrush placed in PreservCyt medium (Hologic) following the same procedure as for Pap smears. Anal cells were also collected in PreservCyt using a polyethylene terephthalate swab moistened in tap water. Specimens were stored at −20 °C immediately following collection.Specimen collection was performed at each 6-month visit by clinicians following previously described protocols.35 Briefly, samples were processed in a biosafety cabinet in a laboratory that was physically separate from where the PCR amplifications were performed to reduce the risk of contamination. DNA was purified from samples using DNA mini spin columns (Qiagen) according to the manufacturer’s protocols. Five microliters of purified DNA were then amplified in a 100-μl reaction using gold Taq and PCR with a mix of MY09/MY11-L1 consensus primers and a set of type-specific primers that amplify a 450-base pair HPV DNA fragment and improve the performance of the system. PC04/GH20 primers, which simultaneously amplify a 268-base pair cellular β-globin DNA fragment, were included as an internal control.35 Ten microliters of the PCR reaction mix were analyzed by gel electrophoresis to validate successful amplification.HPV infection outcomes were based on DNA of cervical and anal samples using a well-established polymerase chain reaction (PCR)–based protocol.27 as well as other low-risk types associated with anogenital warts.36 Hybridization signals were graded using a validated scale for signal intensity.37Samples were then analyzed for HPV DNA type by oligonucleotide allele hybridization. Seven microliter aliquots of the initial PCR reaction were applied to 10 replicate filters using a 96-well pipetting system. Filters were individually hybridized using biotinylated type-specific oligonucleotide probes for over 40 alpha HPV types known to infect the mucosal epithelia, including vaccine types and nonvaccine high-risk HPV types27; and (3) nonvaccine low-risk types identifiable by our PCR assay. Additional analyses looking at individual HPV types were also performed.To assess whether rates of HPV infection have changed since the introduction of the vaccine, we estimated the age-adjusted prevalence and 95% CIs by calendar date  and time since vaccination . We also assessed for possible breakthrough infections with vaccine types by restricting analyses to participants who initiated vaccination prior to coitarche . HPV types were grouped based on their relationship to the vaccine and oncogenic potential. Outcomes included prevalence and incidence of: (1) vaccine-related HPV types, representing the types affected by the quadrivalent vaccine  and the more recent 9-valent vaccine ; (2) nonvaccine high-risk HPV types  associated with increased risk of cancer40 with time since introduction of the vaccine as the primary independent variable. As age was a metameter of time, we used age as a within-patient time-varying covariate and year of cohort entry as a time-fixed covariate.We evaluated whether infection rates for HPV have changed since the administration of the vaccine by assessing longitudinally the probability of HPV detection over time among vaccinated participants while adjusting for changes in cohort characteristics over time. We used a multivariable logistic regression approach with generalized estimating equations for open cohorts,41 we adjusted for concurrent detection of vaccine types in the models looking at nonvaccine high-risk HPV types.Potential confounding factors were adjusted for in the multivariable models. Models for vaccine types were adjusted using number of vaccine doses received prior to the study visit (1 or 2 vs 3), age at coitarche (in years), age of first vaccine dose (in years), and number of recent partners in the past 6 months . Additional confounding factors and risk factors for cervical high-risk HPV adjusted for included consistency of condom use during recent sex  and history of a positive chlamydia test, the next most common sexually transmitted infection , which was updated at each visit to account for any new infections. For anal HPV, we adjusted for whether participants reported having anal sex in the prior 6 months (yes vs no) in addition to other variables that we fitted in the cervical HPV model. To address potential masking effects caused by differences in assay sensitivity to vaccine types over nonvaccine types reported with some PCR systems,P < .05 in 2-sided tests.We used a last value carry forward approach to impute missing data for the variables: number of recent partners in the past 6 months and history of chlamydia. As the proportion of missing information for other covariates was negligible, they were modeled under a missing completely at random assumption. The functional forms of variables and model goodness of fits were assessed using cumulative residuals. We assessed the correlations between model variables using variance inflation factors and empirical correlation coefficients, and centered age at 13 years (the minimum age of entry into the cohort) to reduce collinearity. The within-patient correlations over repeated measures were modeled using exchangeable structure based on quasi-likelihood information criterion, and robust standard errors were used for inference. All analyses were performed using SAS version 9.4 (SAS Institute). The threshold for significance was The study cohort included 1506 adolescent girls and young women enrolled between October 2007 and September 2019, with a mean (SD) age at baseline of 18.2 (1.4) years. Accrual occurred throughout the period of study with approximately half of the cohort enrolled before May 2012. Thirty-five participants did not complete a postvaccination visit, and HPV typing results were not available from any visit for 18 participants, leaving a final analytical cohort of 1453 vaccinated adolescent females , whereas age at vaccine initiation went from 18.6 (0.5) years in 2008 to 12.4 (0.5) years in 2019 (P < .001)  reported having 3 or more lifetime sexual partners at baseline. Mean (SD) age at first intercourse did not significantly change over time—from 15.2 (0.5) years in 2008 to 15.4 (0.7) in 2019  age at vaccine initiation of 13.0 (1.8) years and age at study entry of 17.9 (1.3) years. Participants in this subcohort were more likely to be enrolled after 2012 and were observed for a median of 2.0 years . Subcohort participants were also younger at time of entry, more likely to be vaccinated younger, had fewer lifetime sexual partners, were less likely to engage in anal sex, and were enrolled into the study sooner after becoming sexually active .P = .004) in the overall cohort, and from 8.8%  to 1.7%  in the subcohort vaccinated prior to coitarche (P = .03). Stratification on participants’ age at entry and recent number of sexual partners did not change the observed trends  between 2008 and 2010 to 4.7%  between 2017 and 2019 , we interpret this as insufficient to suggest type replacement at this time. Moreover, population evidence of cross-immunization protection against types genetically related to HPV-16, and to a lesser extent HPV-18, in vaccinated women has been reported by others.49As reported in other studies,52 Recent results, however, have found that the overall prevalence of HPV, including vaccine and nonvaccine types, has declined since the prevaccine era among vaccinated girls and women aged 14 to 19 years.53 By contrast, increases in nonvaccine high-risk HPV types have been reported in some countries with national school-based vaccine programs.48 These secular trends have important public health implications for prevention of cervical cancer using HPV screening in vaccinated populations. For example, HPV-68 represents a high-risk HPV type that has recently been reported to be increasing in some populations.54 Nevertheless, underlying mechanisms for the observed increased detection of specific types over time in a vaccinated cohort are not clear, as evidence for type replacement as a biological mechanism is lacking.One of the unique contributions that this study provides is the evaluation of a real-world example of the HPV infection rates following immunization in a population of adolescent girls and young adult women at a single health center in a large US city, reflecting strong evidence of vaccine effectiveness. Previous surveillance studies from the US have involved older women and populations with relatively low vaccine coverage.56 it is likely that the higher detection rates observed among recent enrollees may be a result of transient infections. Longer-term follow-up and a larger sample size would be required to assess more clinically relevant outcomes. It is also important to emphasize that while the prevalence of nonvaccine HPV may be increasing in our study cohort, the absolute risk and population attributable fractions of cancer for nonvaccine types are much lower than of vaccine types, which themselves have been declining.53 Moreover, population-wide reductions in HPV-associated diseases are already being seen.22Some limitations of our study require consideration. First, we did not measure for changes in persistent HPV infections or cytological end points over time. Assuming HPV clearance rates of 6 to 12 months, as has been reported for cervical and anal HPV,46 However, given the low prevalence of HPV-16  in this vaccinated cohort, we anticipated that the effect of masking would be minimized. Our regression analyses indicated masking was unlikely, as we did not see significantly higher prevalence rates over time for HPV-52 or HPV-58 with lower HPV-16 prevalence among vaccinated participants.Second, the elimination of HPV-16, which can produce a strong signal on L1-primer–based PCR assays, has been associated with higher detection of nonvaccine HPV types competing for the same primers, including HPV-52 and HPV-58.Lastly, because being sexually active was an inclusion criterion for participation in our study, there was likely significant HPV exposure in the study population before enrollment and potentially at time of vaccination. We attempted to correct for this by showing results restricted to participants who were vaccinated before coitarche. We also adjusted for other significant indicators of HPV exposure, including recent number of sexual partners and history of chlamydia. Nonetheless, we cannot confirm if detection of vaccine types after vaccination reflected breakthrough infections, or, as noted, simply detection of a prior (or transient) HPV infection.33 with now over 10 years of data in this cohort. However, our study findings suggest that postvaccination rates of nonvaccine high-risk HPV types may be greater in some higher-risk groups.5 As such, surveillance with HPV testing should be considered. While we did observe increases for some nonvaccine high-risk HPV types in this cohort, the clinical implications remain to be determined as the attributable risks of cancer associated with most nonvaccine high-risk HPV types remain low.31This cohort study provides real-world evidence of lower detection rates of vaccine-targeted HPV types following immunization of female youth in New York City,


INK4A (a surrogate marker for HPV-positivity) in the 8th TMN-classification has only prognostic, not therapeutic, intent. To avoid misclassification, highest precision in determining HPV-status is of utmost importance. Whenever possible, PCR-based methods, still referred to as the "gold standard”, should be used. New diagnostic antibodies represent some hope, e.g., to detect primaries and recurrences early. Prophylactic HPV vaccination should lead to a decline in HPV-driven HNSCC as well. This review discusses the above aspects in detail.Human papillomaviruses (HPV) cause a subset of head and neck cancers (HNSCC). HPV16 predominantly signs responsible for approximately 10% of all HNSCC and over 50% of tonsillar (T)SCCs. Prevalence rates depend on several factors, such as the geographical region where patients live, possibly due to different social and sexual habits. Smoking plays an important role, with non-smoking patients being mostly HPV-positive and smokers being mostly HPV-negative. This is of unparalleled clinical relevance, as the outcome of (non-smoking) HPV-positive patients is significantly better, albeit with standard and not with de-escalated therapies. The results of the first prospective de-escalation studies have dampened hopes that similar superior survival can be achieved with de-escalated therapy. In this context, it is important to note that the inclusion of p16 A considerable amount of benign and malignant diseases are caused by infections with human papillomaviruses (HPV) . The rolINK4A immunohistochemistry as the sole detection method for HPV infections in oropharyngeal carcinoma in the current TNM classification (AJCC/UICC) , there could be no correlation detected between HPV status (determined by HPV DNA and RNA detection plus p16INK4A IHC) of the investigated tonsillar tissues and sexual behavior of patients ) and the lowest rates in Brazil (11%). However, the alignment of various global HPV prevalence rates to official public governmental statistics on the burden of smokers in the population as performed above should not be conducted carelessly, since the proportion of smokers in the entire population might significantly differ from the proportion of smokers in the studies themselves. As mentioned, the official proportion of smokers in Germany and the USA has recently been estimated to be 24 and 14%, respectively, whereas in one of our own studies, the proportion of smokers was 50 and 70% in HPV-positive and -negative cases [The aspects of global HPV epidemiology are multifaceted and challenging. One of the reasons for the latter is the diversity of methodical approaches to detect HPV-positive, specifically HPV-driven cases as described in idelines , describve cases , respectve cases  reportedve cases . Meanwhive cases  and stilve cases . TherefoIn this review, it should be mentioned only briefly that, so far, the background for the described interaction between smoking habit and HPV status is not fully understood. However, based on our own results on more than 1000 patients (which add to the by-us investigated cases summed up in the introduction) and supported by two US-American studies, we postulated the following hypothesis, perhaps elucidating this interaction: smoking leads to increased SLPI and AnxA2 expression in mucosal tissues with significant SLPI excess. SLPI binds to AnxA2, which consecutively inhibits the binding of HPV, if present, to AnxA2. HPV binding to AnxA2 is crucial for successful HPV infection of mucosal cells. Conversely, in non-smokers with significantly higher levels of AnxA2 compared to SLPI, HPV can bind more readily to unoccupied—non-SLPI-bound—AnxA2, and successful infection of cells is likely. Our various studies on this topic are summarized in  and dataSmoking habit most likely influences HPV prevalence rates in combination with other factors of interest, such as, among others, alcohol uptake and sexual behavior, perhaps even in a synergistic way . The impAnother major aspect influencing the topic of epidemiology is the diversity of primary tumor sites in the head and neck region. So far in this article, we predominantly addressed head and neck squamous cell carcinomas (HNSCC) as a whole and partly addressed oropharyngeal (OP)SCC. Except for SCC of the nasopharynx, the various anatomical tumor sites all show a subgroup of HPV-positive and even HPV-driven cases according to the criteria addressed in Probably due to the significantly higher HPV prevalence rates described for TSCC and OPSCC with a supposedly higher clinical relevance, a clear trend has developed worldwide to examine almost exclusively oropharyngeal SCC instead of analyzing all tumor sites from the head and neck region for HPV infections. Considering the worldwide incidences of SCC in the various anatomical head and neck tumor sites, numbers of HPV-driven tumors of all non-tonsillar tumors reach approximately 50% of HPV-driven TSCC. The latter is based on the overall significantly higher incidence of non-tonsillar SCC in comparison to tonsillar SCC. Cramer and co-workers  estimateINK4A and perhaps p53 IHC, may be best suited to fulfill this requirement [As indicated in uirement . Indeed,uirement ,50,74,75Treatment options for HNSCCs are surgery alone or in combination with risk-adapted adjuvant radio(chemo)therapy (R(C)T) and primary R(C)T ,77. IndeINK4A overexpression. This statement seems to be challenging since in some countries of the Western World, there appears to be a paradigm to treat this tumor entity by primary RCT; this however, lacks clinical evidence based on prospective clinical trials.In 2018, we published a study on the survival of 126 TSCC cases which initially were all surgically resected and treated risk-adapted with or without adjuvant R(C)T . OverallIn agreement with studies by others , severalAnalyzing the association of smoking habit, co-morbidity, and achievement on planned R(C)T dosage in 643 HNSCC patients, we only recently confirmed the significant association between increased tobacco consumption and presence of co-morbidities (applying various co-morbidity indices as the CCI); however, we did not see a correlation between smoking and/or co-morbidities and an impaired achievement of R(C)T dosages ,82. MoreINK4A IHC, see In conclusion, future studies on HPV and survival in HNSCC should, along with substance abuse, additionally integrate the presence of co-morbidities with the latter being of higher relevance than smoking, gender, and age. Results of de-escalation studies, for instance, might be negatively biased by imprecisely applied detection methods  exclusively has prognostic intent and the application of de-escalated therapy, without any exception, should only be applied in clinical trials. Nevertheless, the detection of HPV infections continues to be of great importance: it should be clarified how exactly infection per se and transmission occur and which mechanisms and influences, e.g., immunological ones, finally lead to carcinogenesis. An intense and consequent comparison of characteristics of patients with HPV-driven or HPV-negative cancers may contribute to the identification of such mechanisms. For both intentions, novel detection methods with respect to, for instance, liquid biopsies might be useful in future. Furthermore, describing the molecular biological differences between active and inactive HPV infections in SCC may reveal deeper insights into HPV-related carcinogenesis. Knowledge of such mechanisms may eventually help to better understand the carcinogenesis of HPV-unrelated cancers, i.e., most likely tobacco- and alcohol-associated head and neck cancers, thus opening the chance to gain insights that possibly could benefit the large HPV-negative majority among HNSCC patients. Additionally, more data is needed to corroborate the need for global sufficient vaccination policies to finally overcome HPV-related diseases.A significant subset of HNSCCs is caused by an infection with HPV16. It is important to distinguish between tonsillar and non-tonsillar SCCs , as the HPV-attributable fraction  of these can be estimated to reach approx. 50 and 7%, respectively. With an estimated global overall number of HPV-driven cancers of 600,000 cases annually, the subgroup of HPV-driven HNSCC with approx. n = 52,000 represents 9% of all HPV-driven cancers . Patients with HPV-driven HNSCC show significantly superior survival rates, which specifically holds true for HPV-driven TSCC, with these patients showing a 10-year overall survival rate of 100% when (i) without smoking history and co-morbidity and (ii) when treated in a non-de-escalated treatment regimen. Prevalence rates as well as outcome differ, however, significantly between study populations and countries due to not-well-understood geographical differences and differences in study designs . Depending on whether or not patients have a smoking habit and are with or without co-morbidities, the subgroup of patients that will benefit from de-escalated treatment regimens in terms of reduced therapy-related morbidity appears rather small after all. Although the goal of lowering morbidity is important, it seems questionable whether de-escalation studies indeed are appropriate in light of today’s more gentle treatment modalities, including, for instance, intensity-modulated radiotherapy and in light of the excellent survival of patients with HPV-driven SCC when treated un-de-escalated. In this context, it again needs to be stressed that the literally down-grading of p16


Moreover, the role of [18F]PARPi as a broadly applicable imaging tool for head and neck carcinomas is investigated.  Human papillomavirus- (HPV-) associated oropharyngeal squamous cell carcinomas (OPSCCs) are clinically and pathologically distinct from HPV-negative tumors. Here, we explore whether HPV affects functional biomarkers, including γH2AX, RAD51, and PARP1 expression with immunoblotting and immunofluorescence. Effects of external beam ionizing radiation were investigated in vitro, and survival was investigated via colony-formation assay. [18F]PARPi uptake experiments were performed on HPV-negative and HPV-positive cell lines to quantify PARP1 expression. PARP1 IHC and γH2AX foci were quantified using patient-derived oropharyngeal tumor specimens.  HPV-positive and HPV-negative cell lines were used to evaluate the γH2AX expression in HPV-positive cell lines. Clonogenic assays confirm HPV-positive cell lines to be significantly more radiosensitive. PARP1 expression levels were similar, irrespective of HPV status. Consequently, [18F]PARPi uptake assays demonstrated that this tracer is internalized in cell lines independently from their HPV status.  Differences in DNA repair were detected, showing higher RAD51 and 18F]PARPi could serve as a general clinical tool for oropharyngeal cancer patients. The HPV status, often used clinically to stratify patients, did not affect PARP1 levels, suggesting that PARP imaging can be performed in both HPV-positive and HPV-negative patients. This study confirms that the PET imaging agent [ More than half a million patients were diagnosed with oral or oropharyngeal squamous cell carcinoma (SCC) in the United States in 2019 . Oral anThe treatment plan for a head and neck cancer patient depends on various factors such as location of the tumor, stage of the disease, and size of the lesion . MoreoveγH2AX nuclear foci, a DNA damage response marker PARPi) as an alternative imaging tool in head and neck carcinomas that can provide high-contrast images compared to [18F]fluoro-deoxy-D-glucose ([18F]FDG) FDG) .18F]FDG is a glucose-mimicking metabolic tracer; therefore, it is retained not only in tumors but also in some normal tissues with high metabolism [18F]PARPi is a DNA repair marker; therefore, it accumulates mostly in the nuclei of tumor cells, making it a good tracer for head and neck cancer imaging. PARP1 is a nuclear enzyme that plays an essential role in DNA damage repair. Several previous publications have demonstrated approaches to image PARP1 in vivo; see [18F]PARPi is derived from the scaffold of Olaparib, an FDA-approved PARP1 inhibitor widely used as a therapeutic agent in gynecological malignancies [18F]PARPi has been previously shown to bind specifically to tumor areas with a minimal off-target uptake PAt uptake , 25.18F]PARPi uptake in oral and oropharyngeal cancer. With the present study, we evaluated the diagnostic value of [18F]PARPi in oropharyngeal cancer which appears to be independent of a patient's HPV status. However, further in vivo and patient data is required to further interrogate this.As the HPV status affects DNA double-strand break repair, we aimed to address if the HPV status had an effect on PARP1 overexpression –30 and t2 humidified atmosphere. We cultured three HPV-positive  and three HPV-negative  cell lines. All media contain 10% (v/v) fetal bovine serum (FBS) and 1% penicillin and streptomycin. Both UPCI:SCC 090  and UPCI:SCC 154  cell lines were grown in MEM with 2 mM L-glutamine. The UPCI:SCC 152  was grown in MEM with 2 mM of L-glutamine, 50 μg/mL of Gentamicin, and 11.2 mL/L of NEAA. Cal 27  cells were cultured in DMEM and the FaDu  cells in MEM medium. The SCC 15  cell line was cultured in a 1 : 1 mixture of high glucose DMEM and F12 medium containing 1.2 g/L of sodium bicarbonate, 2.5 mM L-glutamine, 15 mM HEPES, and 0.5 mM of sodium pyruvate supplemented with 400 ng/mL of hydrocortisone 500 mL solution.All cell lines were grown in a monolayer culture at 37°C in a 5% COμL ice cold RIPA buffer with Triton X100 , one Mini Complete Protease Inhibitor Cocktail tablet , and one PhosSTOP tablet . Protein quantification was performed using bicinchoninic acid (BCA) protein assay . For the PARP1 and RAD51 expression, 20 μg of protein lysate was loaded on the gel. For γH2AX, 40 μg of total cellular lysate was loaded on the gel in order to be able to detect the specific signal. Also, the 0.45 μm pore size membrane was replaced with 0.20 μm nitrocellulose membrane  for γH2AX detection. The substitution was made because gamma-H2AX is a 17 kDa protein, smaller than all the other proteins detected in this study. During transfer processes, with the 0.45 μm pore size membrane, gamma-H2AX was eluted from the membrane resulting in no detection. Various methods were used to improve detection. The one that enabled detection was the replacement with the 0.20 μm pore size. Transfer time was lowered to 50 min because more extended periods led to the protein being eluted from the membrane. After optimization, 50 min was found to be the optimum time for the gamma-H2AX transfer process. Blocking was performed by 1% BSA in TBS-T solution. Proteins were detected using antibodies specific for PARP1 , γH2AX  RAD51 , β-actin , and Vinculin  with a corresponding horseradish peroxidase- (HRP-) conjugated secondary antibody , ab6789 (mouse), Abcam, USA). Loading control choice was based on the molecular size of the target protein, in order to not interfere with the protein of interest. When a membrane was probed for different proteins, the same loading control was used. Detection was performed using a chemiluminescent substrate . The bands were visualized using an automated blot processing machine  with a light-sensitive clear blue X-ray film  with 1 min exposure time.Cell pellets obtained from 80% confluent flasks were washed twice with PBS and lysed with 200 5 cells from all six cell lines were plated on a 6-well plate. On the following day, the plated cells were irradiated with 2 Gy γ-rays in a cesium irradiator . Before blocking, cells were washed with 300 μL of PBS. Using 3% paraformaldehyde (PFA) in a PBS solution, cells were fixed for 10 min at 2 different time points: 30 min and 24 h. Blocking was performed for at least 1 h in 300 μL PBS with 1% BSA, 0.1% Triton, and 1% Goat serum at room temperature. After blocking, cells were incubated with primary antibodies: γH2AX and RAD51 in 1% BSA for 24 h. After washing with PBS, cells were incubated with Alexa 488 goat anti-rabbit  and Alexa 594 goat anti-mouse  in 1% BSA for 1 h at room temperature in the dark. To mount the cover slip containing cells on the microscope slide, 20 μL of vector shield solution with DAPI  was used to counterstain the nuclei. Using an inverted confocal microscope , cells were imaged and the number of RAD51 and γH2AX foci present in the cells was quantified using an ImageJ plugin software. In order to detect foci in patient samples, 0.5 μg of γH2AX antibody and 0.1 μg, 0.2 μg, and 0.4 μg of RAD51 antibody were used. The patient data used in the manuscript was conducted under a protocol approved by the Institutional Review Board (IRB). No human approval was required for this study. IgG control was used to confirm specificity and control for false positive signals of the antibody. The slides were scanned  at 40x magnification. Adjacent sections were stained with hematoxylin and eosin (H&E). Regions of interest (ROIs) were drawn on the H&E slides (tumor areas) and transported using CaseViewer  to the consecutive slide, where immunofluorescence was carried out in order to analyze correct tumor areas. The images shown are representing one of the tumor areas in whole slide, and the mean number of foci includes foci count in all the tumor areas.4.0‐5.0 × 10In order to obtain a sufficient colony count, 800-3200 cells were seeded in 6-well plates in triplicates and exposed to known radiation doses. Cells were irradiated (IR) with 0, 2, 4, and 6 Gy using a cesium irradiator. Cells were allowed to grow for 21 days. After 21 days of incubation, the culture media were removed, and cells were stained in crystal violet (0.5%) in methanol for 2 min. Survival fractions were calculated based on the number of microscopic colonies where a colony is defined as >50 cells. The plating efficiency was calculated as previously reported . The meaImmunohistochemistry (IHC) was performed by the Memorial Sloan Kettering (MSK) Molecular Cytology Core Facility (MCCF) and as previously described by our group .18F]PARPi was tested in vitro (3 replicates) as previously described [18F]PARPi was synthesized as reported by our group PARPi were added to the cells. For blocking, cells were incubated with a 100-fold molar excess of Olaparib 1 h before adding [18F]PARPi. Media was removed, and cells were washed with PBS and lysed with 1 M sodium hydroxide (NaOH) after 1 h. The lysate was collected, and uptake was determined by measuring radioactivity on a Wizard2 automatic γ-counter .Uptake of [escribed . [18F]PAur group  and obtaP values were calculated using Student's unpaired t-test. Statistical significance was determined with alpha = 0.05, and the level of significance for each result was displayed as ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.Statistical analysis was performed using GraphPad Prism 8. Unless otherwise stated, data points represent mean values and error bars represent standard deviations of biological replicates. γH2AX (γH2AX expression in nonirradiated HPV-positive cell lines (4.06 ± 2.52) was higher in HPV-negative cell lines (0.78 ± 0.67) (γH2AX expression in irradiated cell lines decreased due to lower γH2AX levels in both HPV-positive (0.26 ± 0.34) and HPV-negative cell lines (0.61 ± 0.58).A panel of cell lines was tested for levels of PARP1, RAD51, and γH2AX . We obseγH2AX and RAD51 were used. The staining was performed on cells that were either nonirradiated or irradiated (30 min and 24 h postradiation)  at the 30 min postirradiation time point. Besides this, the mean number of the RAD51 focus count increased after irradiation and returned to baseline at 24 h. In HPV-positive cells, the mean number of gH2AX foci was closer to baseline, in both 30 min and 24 h postirradiation, compared to that of HPV-negative cells. Besides this, no difference was detected between pre and 30 min postirradiation in the RAD51 focus numbers. For the 24 h time point, instead of returning to baseline as HPV-negative cells did, the mean number of foci continued to show an increase.In order to visualize and quantify the DNA strand breaks pre- and postirradiation, antibodies against diation) . The mea) (γH2AX  and RAD5) (γH2AX  to detern = 3) and HPV-negative (n = 3) cell lines. Corroborating previously published literature PARPi, a PET imaging agent for head and neck cancers, which binds specifically to PARP. In order to confirm the correlation of the HPV status with cellular and molecular mechanisms underlying favorable prognosis, we investigated γH2AX, RAD51, and PARP1 protein expressions in HPV-positive and HPV-negative cell lines and oropharyngeal carcinoma patient samples.High-risk HPV subtypes, HPV-16 and HPV-18, encode two viral oncoproteins, E6 and E7, which play a role in disrupting normal host cell division by targeting p53 and the retinoblastoma protein (pRb), respectively . HPV-16 γH2AX  and RAD5γH2AX and RAD51 was detected. This reduction can be attributed to the intrinsic radiosensitivity of HPV-positive cell lines which was confirmed through clonogenic assays. HPV-positive cell lines displayed significant radiosensitivity at all dose levels which show a higher cell death and a higher probability of getting washed away during the washing steps in immunofluorescence microscopy and western blot. The inconsistency observed in RAD51 levels can be related to the divergent role of RAD51 where, on the one hand, the E7 oncoprotein increases the level of RAD51 protein PARPi imaging in oropharyngeal carcinomas.HPV correlation to single-strand DNA breaks was investigated through PARP expression. In immunoblotting, both HPV-negative and HPV-positive cell lines revealed an increasing trend in the expression of PARP1 in response to radiation. This observation aligns with the literature as radiation increases the formation of DNA breaks, and the expression of PARP1, being a DNA repair enzyme, increases , 36. PAR18F]PARPi as a potential clinical tool for imaging [18F]FDG PET/CT imaging due to its accumulation not only in the tumor area but also in normal cells [18F]PARPi is probably independent of HPV status. Through [18F]PARPi uptake assays, we demonstrated similar internalization in HPV-negative and HPV-positive cell lines. Since HPV-positive tumors have an entirely different prognostic profile compared to HPV-negative tumors, ensuring that [18F]PARPi uptake will not be biased towards either cohort was a necessary factor to explore. This study suggests that [18F]PARPi is a valuable imaging tool in head and neck tumors with probably no need to stratify patients based on their HPV status. However, new studies, encompassing larger cohort sizes, are necessary to more accurately access the tracer's capability of imaging both HPV-positive and HPV-negative patients. Since the current study was conducted solely in vitro, further in vivo and human studies are needed to interrogate the biological factors that may impact the uptake pattern and PARP expression.Our lab recently published a paper which demonstrated PARPi to image oropharyngeal tumors regardless of HPV status. Independence of HPV status and PARP allowed internalization of the tracer with no significant variability. Future experiments will focus on evaluating [18F]PARPi imaging in patient cohorts to determine its efficacy for imaging oropharyngeal squamous cell carcinoma with greater specificity.We have demonstrated the ability of [


Regular tests for human papillomavirus (HPV) and early treatment could represent an important strategy for preventing anal cancers among men who have sex with men (MSM). This study investigated facilitators and barriers to take up clinician-collected and self-collected HPV tests among Chinese MSM. This study was based on the baseline sample of a longitudinal study promoting HPV vaccination among 350 Chinese MSM who had never received an HPV vaccination. The baseline survey was conducted from August 2019 to April 2020. The prevalence of any HPV tests uptake in lifetime was 19.1%; 4.9% had HPV infection in anus, genital, oral cavity, and other places. Among the participants, 20% and 76.8% intended to take up self-financed and free clinician-collected HPV tests, and 76.8% intended to use free self-collected HPV tests. After adjusting for significant background characteristics, perceived risk of HPV infection, and perceived benefits, barriers, cue to action, and self-efficacy related to HPV tests in general and/or specific to self-collected HPV tests were associated with behavioral intention to take up free clinician-collected and/or self-collected HPV tests. Less than 20% of Chinese MSM reported HPV tests uptake. Modifying perceptions related to HPV tests may be useful to increase HPV tests coverage in this group. Men who have sex with men (MSM) are at high risk of human papillomavirus (HPV) infection . Meta-anHPV tests for MSM is most commonly done through anal cytology, which identifies patients with cellular changes in the epithelial cells that line the anal canal, and refers patients with abnormal results to high-resolution anoscopy and subsequent treatment of biopsy-confirmed anal intraepithelial neoplasia (AIN) . HPV DNAIn the literature, the prevalence of HPV tests uptake was low among MSM, ranging from 11–14.3% among MSM in general ,25 to 40Self-collected HPV tests refer to collecting samples by users instead of clinicians at home or other private places for HPV testing. Users can send the collected sample to designated clinics, who will examine the sample and inform them of the results . Self-coTo our knowledge, there was a dearth of studies investigating HPV testing behaviors or behavioral intention to receive clinician-collected or self-collected HPV tests among Chinese MSM. To address these gaps, this study investigated HPV testing behaviors and behavioral intention to take up free clinician-collected HPV tests and free self-collected HPV tests among a sample of Chinese MSM in Hong Kong. Potential associated factors were also investigated, which included socio-demographics, behavioral characteristics of the participants, perceptions related to HPV tests in general, and perceptions specific to self-collected HPV tests.Questions asked by this study was parts of the baseline survey of a longitudinal study promoting HPV vaccination among Chinese MSM, which was conducted from August 2019 to April 2020.Inclusion criteria are as follows: (1) Hong Kong Chinese-speaking males aged at least 18 years old, (2) self-reported having had oral or anal intercourse with at least one man in the last six months, and (3) willing to complete a follow-up telephone survey twelve months after the baseline survey. Those who had received HPV vaccination were excluded.Participants were recruited through multiple sources. Trained and experienced staff of a collaborative NGO providing HIV-related services worked as fieldworkers for recruitment. A recent mapping exercise was conducted by the government and identified 12 gay bars and 16 gay saunas in Hong Kong. Upon approval of the owners, field workers approach prospective MSM participants in these venues at different time slots during weekdays and weekends. They briefed prospective participants about study details and gave them an information sheet. The research team also conducted online outreach by posting information about the study periodically as discussion topics on two gay websites with highest traffic in Hong Kong. If prospective participants are interested in this study, they can contact the interviewers through private messaging or other means . Recruitment is supplemented by referrals made by peers and NGOs. Participants were guaranteed anonymity during the study, and they had the right to end participation in the study at any time. Their refusal or withdrawal from the study will not affect their access to any future services. Verbal instead of written informed consent was obtained due to maintaining anonymity, but the fieldworkers signed a form pledging that the participants had been fully informed about the study. Multiple forms of contact information were obtained in order to make an appointment to conduct the telephone interview. Upon completion of each survey, an HK $25 (US $3.2) supermarket or café coupon was mailed to participants as compensation for their time. Ethics approval was obtained from the Survey and Behavioral Research Ethics Committee of the Chinese University of Hong Kong (ref# KPF18HLF22).A panel that consisted of public health researchers, epidemiologists, health psychologists, physicians, and NGO workers was formed to develop the questionnaire. The questionnaire was tested among five MSM. Based on their feedback, the questionnaire was finalized by the panel.Information collected included socio-demographics , sexual orientation, lifestyles (smoking and drinking), HIV and STI status, utilization of HIV or STI prevention services, sexual behaviors with regular and non-regular male sex partners, male sex workers, and female sex partners in the last six months. A regular male sex partner (RP) was defined as a stable boyfriend, while a non-regular male sex partner (NRP) was defined as a man who was neither a RP nor a male sex worker. Participants also reported sexualized drug use (SDU) in the last six months. SDU is defined as the use of any of the following psychoactive substance before or during sexual intercourse: ketamine, methamphetamine, cocaine, cannabis, ecstasy, Dormicum/Halcion/Erimin 5/non-prescription hypnotic drugs, heroin, cough suppressant (not for curing cough), gamma-hydroxybutyric acid (GHB)/gamma-butyrolactone (GBL), 5-methocy-N, N-diisopropyltryptamine (Foxy), or mephedrone ,39.Participants were asked whether they had taken up any types of HPV tests in the lifetime and in the past year. Details related to the most recent episode of HPV test  were collected among MSM who had ever used HPV tests. Participants were briefed with the following statement: “Doctors can use an HPV test to identify HPV infection. Doctors can use a swab to collect samples from anus, genital, or oral cavity and examine whether you are infected with some types of HPV.” Then, they were asked about the likelihood of taking up free clinician-collected HPV tests . The chance of taking up self-collected free HPV tests was also measured after participants were briefed about the following: “You can also collect the sample by yourself at home or other private places for HPV testing. The commercial package includes a swab (such as a long and thin Q-tip), you can insert the swab about 1.5–2 inches into your anus to collect the sample. You can also use the swab to collect the sample from the surface of anus or penis. You may then put the swab in the specimen bag within the commercial package and send it to designated clinics, who will examine the sample and inform you of the results” . Behavioral intention to use clinician-collected and self-collected HPV tests was defined as the responses likely or very likely. Such definition has been used in previous studies .Three scales derived from the HBM were constructed. Perceived severity of HPV infection was measured by four items . The Perceived Severity Scale was formed by summing up individual scores (from 1 = strongly disagree to 5 = strongly agree). Higher score on the scale indicated perceived consequences of HPV infection to be more severe. The Cronbach’s α of the Perceived Severity Scale was 0.72; one factor was identified by exploratory factor analysis, explaining 54.9% of total variance.Two items (with response options ranging from 1 = strongly disagree to 5 = strongly agree) were used to measure the perceived benefit of HPV tests and perceived barriers to receive HPV tests. The Perceived Benefit Scale and Perceived Barriers Scale were formed by summing up individual item scores. The Cronbach’s α of these two scales was 0.71 and 0.75, respectively. Single factors were identified by exploratory factor analysis, explaining 77.9% and 57.4% of total variance.One item measured perceived susceptibility to HPV  with response options ranging from 1 = very low to 5 = very high. Two other single items assessed cue to action , and perceived self-efficacy  (response categories: from 1 = strongly disagree to 5 = strongly agree).Three items measured perceived benefits specific to self-collected HPV tests . The Perceived Benefit Specific to Self-collected HPV Tests Scale was constructed by summing up individual item scores (1 = strongly disagree to 5 = strongly agree). Higher score indicated perceived higher benefits of self-collected HPV tests. The Cronbach’s α of the Perceived Severity Scale was 0.67; one factor was identified by exploratory factor analysis, explaining 65.6% of total variance. Three other single items assessed perceived barrier , cue to action , and perceived self-efficacy  related to self-collected HPV tests (response categories: from 1 = strongly disagree to 5 = strongly agree).p < 0.05 in the univariate analysis, associations between independent variables of interest  and the dependent variables were then assessed by adjusted odds ratios (AOR). Each AOR was obtained by fitting a single logistic regression model, which involved one of the independent variables of interest and significant background variables. Principal component analysis with varimax rotation was used to perform exploratory factor analysis. SPSS version 21.0 was used for data analysis, with p values < 0.05 taken as statistically significant.Using behavioral intention to use free clinician-collected HPV tests and free self-collected HPV tests as the dependent variables, and background characteristics as independent variables, crude odds ratios (OR) were obtained by using logistic regression models. After adjustment for those variables with n = 176, 50.3%) and with a monthly personal income of HK $20,000 (US $2565) or higher . Most of the participants were currently single , had attained at least tertiary education , were employed full-time , and identified themselves as homosexuals . Among the participants, 1.7% (n = 6) self-reported as living with HIV, 23.1% (n = 81) reported a history of other STI and 23.1% (n = 81) utilized HIV or STI prevention services such as receiving free condoms, peer education or pamphlet, or attending seminars or workshops. In the past six months, 83.7% (n = 293), 48.6% (n = 170), and 9 (2.6%) had had anal intercourse with RP, NRP, and male sex workers, respectively. Moreover, 50.3% (n = 176) and 54.6% (n = 191) reported CAI with men and multiple male sex partnerships, respectively. In addition, 6 (1.7%) participants had sexual intercourse with female sex partners, and 3 (0.9%) of them reported condomless sex with such partners. The prevalence of SDU was 5.7% (n = 20)  completed the baseline survey. About half of the participants were 18 to 30 years old ((n = 20) .n = 67) and 9.7% (n = 34), respectively. Among 67 participants with experience of HPV tests, 25% (n = 17) were diagnosed as HPV infection in anus (n = 11), genital (n = 4), oral cavity (n = 2), and other place (n = 1). Their most recent episode of HPV tests were all clinician-collected HPV tests which mainly took place in NGOs  followed by public hospitals  and private clinics . Over half of them received free HPV tests, while others spent HK $350–7000 (US $44.9–897.4) (median: HK $700 or US $89.7) for their most recent episode of HPV tests. The prevalence of behavioral intention to receive self-financed (at HK $700 or US $89.7 per episode) and free clinician-collected HPV tests in the next year was 20% (n = 70) and 76.8% (n = 269), respectively. The prevalence of behavioral intention to take up self-collected HPV tests in the next year was 76.8% (n = 269)  .p < 0.001) and had anal intercourse with RP in the past six months  reported lower intention to use free clinician-collected HPV tests, while the history of other STI  was positively and significantly associated with this dependent variable , belief that significant others would suggest they take up HPV tests (cue to action) , and perceived self-efficacy to take up HPV tests  were associated with higher intention to take up free clinician-collected HPV tests. Perceived barriers to take up HPV tests were negatively and significantly associated with this dependent variable   .p = 0.02; reference group: self-reported as HIV negative) and had never tested for HIV  reported lower intention to do so. , while those with monthly personal income of HK $10,000–19,999 (US $1282–2564) , self-reported as HIV positive , and perceived benefits , cue to action , and self-efficacy  related to HPV tests were associated with higher intention to use self-collected HPV tests. A negative and significant association was found between perceived barriers to receive HPV tests and this dependent variable . Perceived benefits , cue to action  and perceived self-efficacy  specific to self-collected HPV tests were also significantly associated with this dependent variable.  ,25, lifeAbout 6% of the participants had never tested for HIV antibody. More attention should be given to this subgroup of MSM, as they reported lower intention to take up free clinician-collected HPV tests and self-collected HPV tests. Previous studies showed that many MSM who had never tested for HIV antibody engaged in high-risk behaviors and put themselves at a high risk for contracting HIV and HPV . As compThe findings provided some empirical insights for developing interventions promoting clinician-collected HPV tests. The HBM is a potentially useful framework to guide the development of future programs promoting clinician-collected HPV tests, as four out of its six constructs were significantly associated with the dependent variable in expected directions. A high proportion (78–89.7%) of participants perceived some benefits of HPV tests in early identification and treatment of HPV infection as well as anal cancer prevention. Those scored higher in the Perceived Benefit Scale were more likely to have behavioral intention to take up clinician-collected HPV tests. Future health promotion campaigns should strengthen such beliefs among local MSM. About 10% of the participants had concerns about being stigmatized by service providers and pain and discomfort caused by the tests. It is important to remove these concerns, as they were significantly associated with lower intention to take up clinician-collected HPV tests. Service providers should be made aware of such concerns among MSM. It may be useful to enhance their knowledge about MSM’s sub-culture and reduce stigmatization toward MSM; similar interventions were available . It is eEnhancing perceived benefits, providing strong cue to action, improving perceived self-efficacy, and reducing concerns related to stigmatization and testing procedures related to HPV tests in general are also useful strategies to promote self-collected HPV tests among MSM in Hong Kong. In addition, future interventions should increase perceived risk of contracting HPV, as it was associated with higher intention to use self-collected HPV tests. Similar to HIV self-testing , perceivThis was one of the first studies looking at behaviors and behavioral intention related to HPV tests among Chinese MSM. Although it had the strengths of being theory-based, it had several limitations. First, the participants were recruited by non-probabilistic sampling in the absence of sampling frame. The results may not be representative of MSM in Hong Kong. As compared to participants of a large-scale survey involving more than 4000 MSM conducted by the Hong Kong Department of Health, our participants were younger and reported a lower prevalence of HIV infection . Second,Chinese MSM in Hong Kong reported a low HPV tests uptake. There is a strong need for improvement. The prevalence of behavioral intention to receive free clinician-collected HPV tests and free self-collected HPV tests was equally high. Modifying perceptions related to HPV tests in general and those specific to self-collected HPV tests might be useful to promote HPV tests in future health promotion campaigns.


In 2014 and 2021, two nucleic-acid vaccine candidates named MAV E2 and VGX-3100 completed phase III clinical trials in Mexico and U.S., respectively, for patients with human papillomavirus (HPV)-related, high-grade squamous intraepithelial lesions (HSIL). These well-tolerated but still unlicensed vaccines encode distinct HPV antigens (E2 versus E6+E7) to elicit cell-mediated immune responses; their clinical efficacy, as measured by HSIL regression or cure, was modest when compared with placebo or surgery (conization), but both proved highly effective in clearing HPV infection, which should help further optimize strategies for enhancing vaccine immunogenicity, toward an ultimate goal of preventing malignancies in millions of patients who are living with persistent, oncogenic HPV infection but are not expected to benefit from current, prophylactic vaccines. The major roadblocks to a highly efficacious and practical product remain challenging and can be classified into five categories: (i) getting the vaccines into the right cells for efficient expression and presentation of HPV antigens (fusion proteins or epitopes); (ii) having adequate coverage of oncogenic HPV types, beyond the current focus on HPV-16 and -18; (iii) directing immune protection to various epithelial niches, especially anogenital mucosa and upper aerodigestive tract where HPV-transformed cells wreak havoc; (iv) establishing the time window and vaccination regimen, including dosage, interval and even combination therapy, for achieving maximum efficacy; and (v) validating therapeutic efficacy in patients with poor prognosis because of advanced, recurrent or non-resectable malignancies. Overall, the room for improvements is still large enough that continuing efforts for research and development will very likely extend into the next decade. Persistent infection with oncogenic types of human papillomavirus (HPV), including HPV-16, -18, -31, -33, -45, -52, -58 and close to 20 others, predisposes patients to cervical, penile, vulvar, vaginal, anal, and oropharyngeal malignancies ,8,9. GloThe roads to therapeutic, anti-HPV vaccines have seen a steady flow of traffic in the past two decades, as reflected by the large number of research articles directly related to this topic: a survey conducted in November 2021 revealed 73 PubMed papers in 1999–2004, 165 in 2005–2009, 120 in 2010–2014, and 228 in 2015–2021 . An initDespite all the premises and advantages of research and development (R&D) toward therapeutic, anti-HPV DNA vaccines , there aDNA vaccines encoding tumor-specific antigens are highly attractive because of their ability to induce potent, cell-mediated immunity  and oncology, investigators with diverse expertise often join force to find ways to carry the field forward across regional and international boundaries ,48,49,50Oncogenic HPV is well-known for its ability to evade host immune responses ,55,56, wPoor immunogenicity of two major oncoproteins, E6 and E7, has also proved to be challenging . To dateThe HLA class I (HLA-I) alleles known to recognize HPV CTL epitopes are also limited, with HLA-A*02 (mostly A*02:01) leading the way , and datTwo common tactics—codon optimization in vaccine design and electroporation in vaccine delivery—seem to be effective in enhancing the immunogenicity of HPV E6- and E7-based DNA vaccines , and theTo overcome limited choice of T-cell epitopes in the short E6 protein (158 amino acids) and E7 (105 amino acids), there is some success in the selective introduction of point mutations that enhance CTL responses . In the As in other drug development, preclinical evaluations are essential to the elucidation of pharmacodynamics and pharmacokinetics, as well as toxicity/safety profiles. For the preclinical evaluation of anti-HPV vaccines, protective immunity is typically determined in mice engrafted with a murine tumor cell line, TC-1, which expresses HPV-16-specific E6 and E8  or HPV-17 MVA E2 virus particles at weekly intervals. Overall, 1051 (89%) women showed complete elimination of intraepithelial lesions after treatment, and 81% women cleared oncogenic HPV [n = 20) group had total elimination of CIN2/3 without clearing HPV [Two promising DNA vaccine candidates against HSIL have gone through phase III clinical trials in Mexico and U.S., respectively . The firenic HPV . Among 1enic HPV . Howeverring HPV . The 11%ring HPV .N = 193), 23.7% of 131 patients in the vaccinated group responded (with HSIL regression and HPV clearance), while 11.3% of 62 patients in the placebo group did so at week 36. The rates of non-responders in both groups were quite high when compared with results for MVA E2 [p = 0.02). A concurrent phase III trial involving 198 participants (REVEAL 2) is expected to reach its primary endpoint in July 2022 [The second therapeutic candidate, VGX-3100, uses a mixture of two plasmids containing codon-optimized sequences corresponding to the E6 and E7 genes of HPV-16 and -18 . The four MVA E2 ,95, but uly 2022 . Of noteuly 2022  and vulvuly 2022 .In terms of mechanism of action, the BPV E2 antigen encoded by MVA E2 is intended to induce immune responses that are cross-protective against HPV—a practice similar to the one used for the highly successful smallpox vaccine. A clear advantage with MVA E2 is the potential activation of tissue-resident T-cells around the HSIL sites . Although little is known about the repertoire of conserved CTL epitopes shared between BPV and HPV E2 proteins , a recenAdverse effects associated with site-specific injection of MVA E2 vaccine ranged from headache and transient fever to abdominal and joint pains that were observed at various frequencies (6% to 69%), all of which were considered mild  ,95. For Overall, MVA E2 and VGX-3100 differ so starkly in their design, delivery system, administration sites, dose schedules and efficacy  that it Two other DNA vaccines against multiple HPV types (HPV-16 and -18) have completed phase I trials in South Korea (NCT01634503) and U.S. NCT00788164) . The res64 . The Four additional DNA vaccine candidates target HPV-16 only  in phase I or phase I/IIa trials, and their results were published between 2004 and 2021 ,112,113.Assuming that these candidates will successfully complete phase II and then advance to phase III trials, the timeline for completing these phases, as set by MVA E2 and VGX-3100 , ranges Therapeutic vaccine candidates that work well against HSIL  may not In the classic immunological toolbox, proper use of immune adjuvants improves the magnitude, breadth and durability of immune responses to protein-based vaccines , but theA new frontline in cancer immunotherapy focuses on the direct modulation of the tumor microenvironment ,125. In The success of new clinical trials may further depend on biomarkers of cancer invasiveness and prognosis, as patient enrollment and data analyses can be tweaked to maximize therapeutic outcomes ,137,138.The COVID-19 pandemic has brought a drastic change to the landscape of vaccine R&D  after twMuch effort and many resources are being redirected to these new, spot-lighted avenues, but one must first and foremost recognize that access to mRNA vaccines is still limited to a handful of nations where logistics with mRNA vaccine manufacturing, transport and storage are well within their reaches. Unless mRNA vaccines and the nanotechnology (lipid nanoparticle) behind them ,147,148 Following decades of heroic efforts, a steady progress in various R&D pipelines for developing therapeutic DNA vaccines against HPV-associated malignancies still falls short of delivering a final (licensed) product to advance patient care. Two leading candidates have shown modest efficacy in late-stage clinical trials, but they share no further similarity to allow any other consensus, even about the very basics of target immunogens and delivery systems. The lessons learned from these and other candidate vaccines with clinical data, along with analyses of HPV-specific TILs, point to multiple gaps and bottlenecks that remain challenging. As such, the field may require rounds of reiterative evaluation of alternative strategies for optimal results.