To date, the microbiota of the human penis has been studied mostly in connection with circumcision, HIV risk and female partner bacterial vaginosis (BV). These studies have shown that male circumcision reduces penile anaerobic bacteria, that greater abundance of penile anaerobic bacteria is correlated with increased cytokine levels and greater risk of HIV infection, and that the penile microbiota is an important harbour for BV-associated bacteria. While circumcision has been shown to significantly reduce the risk of acquiring human papillomavirus (HPV) infection, the relationship of the penile microbiota with HPV is still unknown. In this study, we examined the penile microbiota of HPV-infected men as well as the impact of HIV status.Corynebacterium and Prevotella were found to be the most abundant genera. Six distinct community state types (CSTs) were identified. CST-1, dominated by Corynebacterium, corresponded to less infections with high-risk HPV (HR-HPV) relative to CSTs 2–6. Men in CST-5 had greater relative abundances of Prevotella, Clostridiales, and Porphyromonas and a lower relative abundance of Corynebacterium. Moreover, they were significantly more likely to have HPV or HR-HPV infections than men in CST-1. Using a machine learning approach, we identified greater relative abundances of the anaerobic BV-associated bacteria  and lower relative abundance of Corynebacterium in HR-HPV-infected men compared to HR-HPV-uninfected men. No association was observed between HIV and CST, although the penile microbiota of HIV-infected men had greater relative abundances of Staphylococcus compared to HIV-uninfected men.The penile skin microbiota of 238 men from Cape Town (South Africa) were profiled using Illumina sequencing of the V3-V4 hypervariable regions of the 16S rRNA gene. Staphylococcus and BV-associated bacterial taxa , respectively. It is possible that these taxa could increase susceptibility to HIV and HR-HPV acquisition, in addition to creating conditions in which infections persist. Further longitudinal studies are required to establish causal relationships and to determine the extent of the effect.We found significant differences in the penile microbiota composition of men with and without HPV and HIV infections. HIV and HR-HPV infections were strongly associated with greater relative abundances of  To date over 40 human papillomavirus (HPV) genotypes have been shown to infect the anogenital tract, with 13 of these genotypes classified as oncogenic or high-risk HPV (HR-HPV) by the World Health Organisation , 3. The Prevotella, have been identified as a key risk factor for HIV acquisition in uncircumcised men [The best evidence to date for a potential role of the penile microbiome in sexually transmitted infections (STIs) and HIV acquisition have come from studies examining medical circumcision , 6. Maleised men , 14. Sevised men –22 have At present, there is no data on the impact of HIV infection on penile microbiota. Moreover, the role of the penile microbiota in HPV infection in men is mostly unknown. To fill this gap, we report here a study of penile skin microbiota in 238 men from the HPV Couples Cohort Study in South Africa [This cross-sectional study is a retrospective analysis of samples collected during a 2-year longitudinal HPV Couples Cohort Study as detailed elsewhere [The median age of the men was 36.0 years. All the men were sexually-active, with 54.6 and 37.0% of them being positive for HPV and HIV infections, respectively. Of the men positive for any HPV infection (low-risk (LR) and/or HR-HPV types), 28.5% (37/130) and 71.5% (93/130) were infected with single and multiple HPV types, respectively. HR-HPV infections were detected in 42.9% (102/238) of the men, with 20.6% (21/102) and 79.4% (81/102) infected with single and multiple HR-HPV types, respectively. All the circumcised men in our study were traditionally circumcised.Ae) bacteria require oxygen for respiration and growth, while anaerobic (An) bacteria do not [FAn) bacteria can survive in the presence or absence of oxygen, although growth activity in oxygen-free environment is usually slower [MAe) bacteria grow in the presence of oxygen but are sensitive to high oxygen concentrations [Corynebacteriaceae , Prevotellaceae , unclassified Clostridiales , Porphyromonadaceae , Staphylococcaceae , Bifidobacteriaceae , and Lactobacillaceae /FAn). Of the top 40 bacterial families, FAn bacteria were the most dominant (53.39%) followed by An bacteria (18.16%) and Ae (9.86%). The relative abundances of families with oxygen requirement categorised as An/FAn and MAe were 5.66% and, 3.98%, respectively. Families with unidentified oxygen requirement had an overall relative abundance of 7.33%. The relative abundances of families with MAe (0.18%) and MAe/Ae (0.26%) were very low.The top 40 most abundant bacterial families identified in the penile microbiota, together with their respective oxygen requirements, are summarised in Additional file a do not . Facultay slower . Microaetrations . The mosPseudomonadaceae and Oxalobacteraceae, that were not abundant in our study, were also included in the analysis as they have been shown to be positively correlated with each other in a previous penile microbiome study [To assess the potential relationships between the bacterial families in our study, we performed correlations between the most abundant bacterial families detected in the penile microbiota, using the Spearman’s rank correlation. Two families, me study .Veillonellaceae, Prevotellaceae, Porphyromonadaceae, unclassified Clostridiales, and Clostridiales Incertae Sedis XI. These bacterial families were positively correlated with one another, an indication of niche sharing and metabolic resource overlap. Generally, these bacterial families were negatively correlated with the eight bacterial families in Cluster-B, which included the most abundant family Corynebacteriaceae as well as Moraxellaceae, Flavobacteriaceae, Pseudomonadaceae, Oxalobacteraceae, Staphylococcaceae, Bifidobacteriaceae, and Lactobacillaceae, although family Clostridiales Incertae Sedis XI (in Cluster-A) was positively correlated with family Corynebacteriaceae (in Cluster-B). Negative interactions between families in Cluster-A and Cluster-B suggest that these bacteria may be competing against one another and may therefore co-exclude one another in the penile niche. The bacterial families in Cluster-B were positively correlated with one another, with Bifidobacteriaceae and Lactobacillaceae exhibiting the weakest positive interactions (Spearman r < 0.4) with the other families in this cluster. These two bacterial families clustered together following the first bifurcation of Cluster-B. Although Bifidobacteriaceae and Prevotellaceae were in different clusters , they were positively correlated with one another.The correlogram depicting the observed relationships between the bacterial families (positive and negative) is shown in Fig. Corynebacterium (47.12%), Prevotella (6.50%), unclassified Clostridiales (5.61%), Porphyromonas (4.85%), Staphylococcus (4.39%), Lactobacillus (3.81%), Gardnerella (3.78%), Chryseobacterium (2.44%), Acinetobacter (2.27%), and Negativicoccus (1.86%). Additional file A total of 650 genera were detected. Only 50 of the 650 genera had ≥0.08% relative abundance; they were distributed among 6 phyla. The most abundant genera were rium 47.1%, PrevotTo define community state types (CSTs), the penile samples were hierarchically clustered based on the microbiota composition. The clustering and heatmap of the relative abundance of the bacterial taxa across the 238 penile samples is shown in Fig. Corynebacterium was found to the most abundant genera in the penile microbiota  of the men, were characterised by diverse mixed populations of bacteria and lower relative abundances of Corynebacterium than CST-1. The most abundant genera (in decreasing relative abundances) in these CSTs were Corynebacterium, unclassified Clostridiales, and Porphyromonas in CST-2; Gardnerella and Corynebacterium in CST-3; Chryseobacterium, Corynebacterium, and Acinetobacter in CST-4; and Prevotella, unclassified Clostridiales, Corynebacterium, and Porphyromonas in CST-5. The remaining CST, CST-6, was the least prevalent CST and was dominated by Lactobacillus with very low relative abundance of Corynebacterium. In subsequent analyses, these CSTs were examined individually or in groups based on the dominance of Corynebacterium or the diversity of the microbial communities in the CSTs. The specific CST groupings were Corynebacterium-dominated (CST-1), non-Corynebacterium-dominated , low diversity (CST-1 and CST-6) and diverse communities .Table Corynebacterium-dominated CST-1 was significantly lower than that for CST 2, 3, 4, and 5 . Alpha diversity of CTS-6 was significantly lower than CSTs 2, 3, 4, and 5 . The alpha diversities of CST-1 and CST-6 were not significantly different (p = 0.213) and were the least diverse of the six CSTs  (p < 0.0001) : Simpson index: 0.5 (0.4–0.7) versus 0.3 (0.2–0.3), Dominance index: 0.5 (0.3–0.6) versus 0.7 (0.7–0.8), Shannon index: 1.3 (0.9–1.6) versus 1.9 (1.6–2.2), and Shannon Equitability index: 0.3 (0.2–0.4) versus 0.4 (0.4–0.5).The alpha diversity Fig. b of the R2 = 0.5569, p = 0.001).The beta diversities of the penile microbiota were also compared across CSTs using the weighted UniFrac distance metric. PCoA of the beta diversity of the penile microbiota confirmed that the six established CSTs were distinct and clustered according to the unique bacterial communities in each  versus non-Corynebacterium-dominated high-diversity microbiota (CSTs 2–5), using linear discriminant analysis (LDA). We found 86 features to be differentially abundant in CST-1 and CSTs 2–5 after the FDR-adjustment  occur in penile microbiota and that these taxa are more predominant in non-Corynebacterium-dominated microbiota than in Corynebacterium-dominated microbiota.Next, the differentially abundant bacterial taxa were determined in Corynebacterium-dominated penile microbiota (CST-1) were different from non-Corynebacterium-dominated penile microbiota (pooled CSTs 2–6), we assessed statistical associations of CSTs with participants’ metadata. The results of comparison of CST-1 versus CSTs 2–6 according to participants’ metadata are shown in Table Corynebacterium-dominated microbiota had lesser odds of being infected with HR-HPV  than men with non-Corynebacterium-dominated microbiota.To investigate whether the highly prevalent Corynebacterium-dominated microbiota (CST-1) had less HR-HPV (Table Corynebacterium-dominated microbiota (CSTs 2–6), we next compared the prevalence of HPV, HR-HPV, and HIV infections in CST-1 versus each of the other CSTs  .Since men with PV Table  compared0) Table . Men in p = 0.072).No statistical difference in HIV prevalence in the CSTs was observed, although a trend towards higher HIV prevalence in CST-5 compared to CST-1 was observed )  were obtained for HPV-negative (1.5 (1.1–1.9)) versus HPV-positive (1.6 (1.3–1.9)) groups  versus 1.5 (1.1–1.9), respectively (–2.0 versp = 0.050).The alpha diversities were not significantly different between men with and without HIV infection Fig. b, althoup = 0.037) and HIV-HPV+ men (Shannon: 1.6 (1.1–1.9); p = 0.045) , HIV-HPV- men (p = 0.828), and HIV-HPV+ men (p = 0.875). Similarly, alpha diversities did not differ between HIV-HPV+ men and HIV-HPV- men (p = 0.918). However, HIV-positive men with HR-HPV infections had significantly higher alpha diversity than HIV-negative men with HR-HPV .Further, we compared the diversity of the penile microbiota of men co-infected with both HIV and HPV (HIV + HPV+), HIV-positive men without HPV infection (HIV + HPV-), HIV-negative men with HPV infection (HIV-HPV+), and men without HIV and HPV co-infections (HIV-HPV-). The penile microbiota of HIV + HPV+ men had statistically higher alpha diversities (Shannon: 1.7 1.4–2.1) than HIV-HPV- men (Shannon: 1.6 (1.1–1.9); –2.1 than+ T-cell count and those with > 350 CD4+ T-cell count were not significantly different (Shannon: 1.6 (1.4–2.1) versus 1.7 (1.4–2.0), respectively; p = 0.873) . The beta diversity of the communities in HR-HPV-uninfected men was significantly different from that of HR-HPV-infected men . About 63.8% (81/127) of the men without HR-HPV infections had Corynebacterium-dominated penile microbiota that clustered together .The penile bacterial communities of men with and without HIV infection showed no distinct separation, indicating that HIV did not impact the clustering of these communities . However, the samples clustered according to HIV and HR-HPV co-infection status .Similarly, co-infection with HIV and HPV did not affect the clustering of the bacterial communities , thus suggesting no relationship of CD4+ T-cell count with community structure.No difference in beta diversities was observed in communities from HIV-positive men with ≤350 CD4Bacterial taxa that were differentially abundant in the penile microbiota of men with and without HR-HPV and HIV infections were analysed using Linear Discriminant Analysis (LDA) effect size (LEfSe) algorithm .p < 0.05) are shown in Fig. Campylobacteraceae, Prevotellaceae, and Veillonellaceae were found to be in greater relative abundances than in men without infection. The relative abundances of Prevotella, Peptoniphilus, and Dialister were found to be significantly greater in the penile microbiota of men with HR-HPV infection than uninfected men, after correction for multiple comparisons (q < 0.2). In uninfected men, the classified genera that were detected at greater relative abundances  included Corynebacterium, Micrococcus, Sanguibacter, and Brevibacterium. After adjustment for multiple comparisons, the relative abundances of the family Brevibacteriaceae and order Actinomycetales were significantly higher in uninfected men compared to HR-HPV-infected men.Biomarkers for HR-HPV were first assessed irrespective of HIV status. A total of 78 bacterial features for HR-HPV were detected at LDA > 2.0 or < − 2.0. The most significant of these biomarkers for HR-HPV infection  included Jonquetella, unclassified Clostridiales, unclassified Campylobacteraceae, Paenalcaligenes, Pimelobacter, unclassified Porphyromonadaceae, and Blastomonas  in uninfected men compared to HR-HPV-infected HIV-negative men after adjustment for multiple comparisons  bacterial features in the penile microbiota, which are presented in a histogram according to effect size  were found in men who were HIV-negative. Men with HIV infections had greater relative abundances of the genera Staphylococcus, Faecalibacterium, Strenotrophominas, Jonquetella, Ruminococcus, Roseburia, Pseudochrobactrum, and Lamia than men without HIV infections; although after correction for multiple testing the differences were not significant. On the other hand, the relative abundances of Propionibacterium, Nosocomiicoccus, and unclassified genera in the class Actinobacteria and order Actinomycetales, were greater in HIV-negative men than HIV-positive men. None of the predominant bacterial families in the penile microbiota  were found to differ significantly in relative abundances between HIV-infected and uninfected men. The relative abundances of the predominant penile bacterial families stratified by HIV infection status are shown in Additional file Corynebacteriaceae, Prevotellaceae, and unclassified Clostridiales were found to be the most common families regardless of HIV status. Each of these families occurred at relative abundances of between 5.2 and 49.7%.Bacterial taxa that were differentially abundant in the penile microbiota of HIV-positive and HIV-negative men were also evaluated using LEfSe Fig. . The anaize Fig. a and a cize Fig. b. TwentyFig. n = 0 were foIn this study, we examined the penile microbiota of 238 Black South African men. To date, this is the largest cross-sectional study using a culture-independent approach to detail the bacterial communities of the penile skin. Sampling of the penile skin microbiota was done by swabbing of the glans, coronal sulci (if present), and shaft of the penis. To date only one other study, by Zozaya and co-workers (2016) , has useCorynebacteriaceae, Prevotellaceae, unclassified Clostridiales, Porphyromonadaceae, and Staphylococcaceae. Facultative anaerobic (FAn) families (n = 7) were the most abundant families with a mean relative abundance over 50% of the penile microbiota, with the family Corynebacteriaceae contributing 47%. The next most abundant group was anaerobic (An) families (18.2%). High predominance of FAn and lower predominance of An families have previously been significantly associated with post-circumcision status [Corynebacteriaceae and Staphylococcaceae, both facultative anaerobes. The positive correlations highlight a possibility of cooperative interaction through metabolic resource overlap [Prevotellaceae and Porphyromonadaceae, as well as the unclassified Clostridiales. Negative correlations indicate the presence of competition for resources [Corynebacteriaceae and Staphylococcaceae might be the main competitors against the other three predominant families.The most common bacterial families in the penile microbiota were, in order of decreasing relative abundances, n status . Among t overlap . These tesources , 32 and Corynebacterium was the most prevalent (100%) and abundant (47.1%) genus. The relative abundance of Corynebacterium in our study is similar to that (45.9%) observed on the penile skin by Zozaya and colleagues (2016) [Corynebacterium was lower in uncircumcised men (31.7%) compared to circumcised men (55.2%). Thus, the relative abundance of Corynebacterium in our cohort of mostly traditionally circumcised men was intermediate to these published results and higher than uncircumcised men [Corynebacterium have also been reported in coronal sulci microbiota post-circumcision and in circumcised men [Corynebacterium in the coronal sulci microbiota of Ugandan men [Corynebacterium can also colonise the semen microbiota of men in appreciable abundances [We observed that s (2016) . Furthers (2016)  reportedised men . Greaterised men , 13. Forndan men . Apart fundances , 34.Corynebacterium-dominated penile microbiota (CST-1). Six of the 238 men (2.5%) had penile microbiota dominated by Lactobacillus (CST-6). Although Lactobacillus has been reported to inhabit the human penis [Lactobacillus in the vaginas of reproductive-age women [Lactobacillus has been reported to considerably increase in abundance in the penile microbiota following unprotected sexual exposure [Corynebacterium or Lactobacillus. The diverse CSTs, CSTs 2–5, had significantly lower relative abundances of Corynebacterium and Staphylococcus and greater relative abundances of several taxa, particularly the BV-associated bacteria compared to CST-1  with different bacterial diversities. Over 50% of the men had an penis , its orige women  and the exposure . The remCorynebacterium and Staphylococcus were abundant members of the coronal sulci microbiota, Streptococcus and Lactobacillus were the most abundant in the distal urethra of adolescent males [Corynebacterium and Staphylococcus, the predominant bacteria in CST-1, are abundant members of the superficial skin microbiota associated with moist sites [Lactobacillus are represented in CSTs 3–6 may therefore reflect sexual exchange of these taxa. Testing of this in future studies will require longitudinal sampling of the penile skin microbiota and surveillance of the microbiota in relation to incident sexual exposure.The distinct CSTs observed in the current study may relate to differences in penile moisture, sebum levels and oxygen availability. The penis provides several microenvironments that differ in levels of moisture, sebum and oxygen that may support distinct microbiota , 27, 36.st sites . Severalst sites , 39. OurCorynebacterium-dominated penile skin microbiota (CST-1) had less HR-HPV infections than those in CSTs 2–6. Among the diverse CSTs, CST-5 was characterised by a low relative abundance of Corynebacterium and greater relative abundances of Prevotella, unclassified Clostridiales and Porphyromonas; CST-5 was associated with greater numbers of HPV and HR-HPV infections when compared to CST-1. While no previous studies have been carried out to examine associations of penile microbiota with HPV, Corynebacterium is known to be abundant in post-circumcised penile swabs [Men with le swabs , 26; andle swabs , 40–42. le swabs . A randole swabs . The biole swabs .Prevotella, Peptoniphilus, Dialister, and unclassified Clostridiales  in the penile microbiota of men with HR-HPV infections compared to uninfected men. To our knowledge, no studies have investigated the associations between penile bacterial taxa and HPV infection. Of note, the BV-associated taxa Sneathia, Prevotella, Dialister, and Peptoniphilus have been identified in several vaginal microbiota studies in women [We used one of the machine learning approaches, LEfSe analysis, to identify differentially abundant bacteria in men according to HPV and HIV statuses. We found a significantly greater relative abundances of in women –47 as biActinomycetales compared to HIV-infected men. A greater number of men in CST-5 were HIV-infected when compared to CST-1, although the difference was not significant. However, HIV and HPV co-infected men had significantly more diverse penile microbiota than HIV-uninfected men with or without HPV infection. There are no published studies that have investigated the penile microbiome of HIV-infected men. A recent prospective study by Liu and colleagues (2017) [Prevotella, Dialister, Peptoniphilus, and Finegoldia at baseline than men who remained HIV-negative [Corynebacterium (mean: 12.9%) and was dominated by several BV-associated anaerobic bacteria and/or proinflammatory bacteria such as Prevotella (23.0%), unclassified Clostridiales (18.5%), and Porphyromonas (16.3%). In addition, CST-5 had other bacteria in appreciable relative abundances: Negativicoccus (4.8%), Dialister (2.6%), Finegoldia (2.2%), Sneathia (1.9%), Gardnerella (1.5%), and Fusobacterium (1.1%). BV-associated bacteria were found to be more prevalent and abundant in diverse CSTs; for example, CST-5 was less diverse than CST-1 (CST with Corynebacterium dominance) and CST-6 (CST with Lactobacillus dominance). Similar observation has also been reported in a penile microbiota study on a Ugandan cohort [We have observed no significant association between HIV status and penile microbiota CST, alpha diversity, or beta diversity. However, we found that men without HIV infection had significantly lower relative abundance of negative , 49. In n cohort . Penile Lactobacillus abundance in the penile microbiota following vaginal sex [An important consideration in studies examining the penile microbiota of sexually-active heterosexual men is the potential influence of regular exposure of the penile bacterial communities to the vaginal, oral and rectal microbiotas of their sexual partners. A recent case report, for example, found an increase in inal sex . Furtherinal sex . Differeinal sex .Corynebacteriaceae and genus Corynebacterium were the most abundant bacterial taxa in the penile microbiota of Black South African men. Six CSTs were established, with over 50% of the men having Corynebacterium-dominated penile microbiota (CST-1).This is the first study to characterise the penile microbiota of traditionally circumcised men and to examine the associations of these microbiota with HPV infection. We found that family Corynebacterium, and were mostly dominated by BV-associated bacteria (CSTs 2–6), were associated with prevalent HR-HPV infections in Black South African men. Specific penile bacteria  were identified as significantly enriched in the penile microbiota of HR-HPV-positive men compared to uninfected men. The role, if any, of these bacteria in HR-HPV prevalence, acquisition and clearance should be investigated.Penile microbiota that were not dominated by The present study included 282 randomly selected baseline specimens and their abstracted information from the parent study. Inclusion criteria were samples with baseline information on HIV status, positive human β-globin PCR results (a measure of sample integrity) and sufficient archived sample material for microbiota analyses. Dry Digene swabs  were used to swab the shaft, foreskin (if uncircumcised) and glans of the penis. These were placed in Digene Specimen Transport Medium  and frozen at − 80 °C until DNA extraction. Nucleic acids were isolated from the penile samples using the MagNA Pure Compact Nucleic Acid Isolation kit and the MagNA Pure Compact System . The Roche Linear Array HPV genotyping test  was used to detect 37 HPV types (13 HR and 24 LR types).The hypervariable region V3 and V4 of the 16S rRNA gene were amplified using the universal bacterial primers 319F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GACTACHVGGGTATCTAATCC-3′) . The 16Swww.arb-silva.de/), using the Needleman algorithm with kmer of 8 bp (nucleotide substring), − 2 gap opening and − 1 gap extension penalties. The kmer searching was used because it is faster and more reliable than blast and suffix tree template searching methods. Sequences with homopolymeric run longer than 12 bases were removed. The alignments were filtered to remove gaps as they do not have any genetic information. The sequences were further pre-clustered by relative abundance using a pseudo-single linkage algorithm to remove erroneous sequences with > 4 nucleotide mismatches. For additional filtering, singletons were also discarded. Potential chimeras were removed by a de novo method using the UCHIME algorithm [The qualities of the raw sequenced reads were assessed by FastQC v0.11.2 . The seqlgorithm .get.groups command. These sequences were then searched for errors against reference sequences of MOCK communities (“HMP_MOCK.v35.fasta”) using the seq.error command. Error rate was then calculated using the formula and expressed as a percentage:Non-chimeric sequences were assigned taxonomy using the Wang approach of 8 kmers implemented by RDP Classifier . The “trOTUs were defined at a 0.03 cut-off phylogenetic distance using the opticlust clustering algorithm. The quality (completeness) quality of sampling the bacterial communities was measured using Good’s coverage estimate . The covtaxonomy_mothur_abundance_silvaDB_v1.2.py). This was done at the phylum, family, and genus taxonomic ranks.The prevalence and relative abundances of the bacteria in the penile microbiota calculated in mothur was summarised at each taxonomic level using customised Python script  in RStudio v1.1.447 [Alpha and beta diversity analyses of the various metadata categories  were performed using a customised scripts . The overall oxygen requirement of each family was then assigned from the oxygen profiles of the genera in that family. For families with genera with different oxygen requirements were considered to have mixed aerotolerance profiles, e.g., Mae/Fan. For biochemically uncharacterised or taxonomically unclassified families the oxygen requirement was designated as “unidentified”. Finally, to examine the most to least common aerotolerance profile of the 40 most abundant families, we grouped all families with similar oxygen requirements together. For example, Flavobacteriaceae and Moraxellaceae were grouped together as “aerobic” while Veillonellaceae and Clostridiales Incertae Sedis XI were grouped as “anaerobic”. We then measured the overall prevalence of aerotolerance profiles of each of these grouped families.The aerotolerance (oxygen requirements) of the 40 most abundant families in the penile microbiota were accessed from published penile microbiome literature , 13 and Pseudomonadaceae and Oxalobacteraceae) that have been previously found to have a positive correlation by Price and colleagues (2010) [plotCorr function in the metagenomeSeq v1.12.1 [Positive and negative correlations between the counts of bacterial families in the penile microbiota were assessed using metagenomeSeq v1.12.1 , 66. The v1.12.1 , 66.p-value) was set at 0.05 while the threshold for discriminative features based on the logarithmic LDA score was set at 2.0. False discovery rate (FDR) was used to correct for multiple comparison testing and features with a q-value < 0.2 were considered to be significant. This was computed using MicrobiomeAnalyst [The Linear Discriminant Analysis (LDA) effect size (LEfSe) algorithm v1.0  was usedeAnalyst .R v3.2.2 (R Core Team 2016) and GraphPad Prism Software v6.01 were used to perform all statistical comparisons. Comparison of the HPV and CST groups with participant categorical and continuous metadata was computed by Fisher’s exact/Chi-square and Mann-Whitney unpaired nonparametric tests, respectively. A p-value of < 0.05 was used as the level of significance. Odds ratios (OR) with corresponding 95% confidence intervals (CI) were used to measure the magnitude of associations.2 value) that showed the extent of variation explained by the metadata category. A p-value of < 0.05 was used to measure the statistical significance.The alpha diversity indices of the various metadata categories  were compared using Mann-Whitney unpaired and Kruskal-Wallis nonparametric tests. For beta diversity comparisons, the Adonis nonparametric test with 999 permutations was used. The distance matrix was used to calculate the effect size  lines separate the genera of the different phyla.Additional file 3: Table S2. Differentially abundant genera in men with Corynebacterium-dominated versus diverse penile microbiota.Additional file 4: Figure S2. Alpha and beta diversity measures of penile microbiota. Comparison of the alpha diversity of penile microbiota grouped by a) human papillomavirus (HPV) infection status, and b) human immunodeficiency virus (HIV) and HPV co-infection status. In each plot, the box ranges from the first to the third quartile, with the median represented by the horizontal line. The whiskers extend to the smallest and largest non-outliers and outliers are represented by dots. Comparison of beta diversity (UniFrac distance) of the penile microbiota grouped by c) HPV infection status, and d) HIV and HPV co-infection status. The first two principal coordinate axes of variations and the percentage variation explained by each (Axis.1: 45.3% and Axis.2: 14.9%) are shown. Each solid point is a bacterial community.Additional file 5: Figure S3. Potential biomarkers for high-risk HPV (HR-HPV) infection by LEfSe in men without HIV infection. a) Histogram of differentially abundant taxa in penile microbiota of HIV-negative men with and without HR-HPV infections identified by LEfSe, and b) a six-level cladogram with a taxonomic hierarchical structure. Each coloured solid represents a taxon and its diameter is proportional to the taxon’s relative abundance. Blue and green solids represent statistically significant taxon ranks in HPV-positive and negative group, respectively. Only features with logarithmic LDA scores > 2.0 or < − 2.0 are shown. Asterisks indicate significantly differentially abundant taxa with q < 0.2 after FDR correction.Additional file 6. Metadata associated with all samples used in this study.Additional file 7. Unrarefied operational taxonomic unit table (with genus taxonomic assignments) used for this study.


Human papillomavirus (HPV) is a globally prevalent sexually-transmitted pathogen, responsible for most cases of cervical cancer. HPV vaccination rates remain suboptimal, partly due to the need for multiple doses, leading to a lack of compliance and incomplete protection. To address the drawbacks of current HPV vaccines, we used a scalable manufacturing process to prepare implantable polymer–protein blends for single-administration with sustained delivery. Peptide epitopes from HPV16 capsid protein L2 were conjugated to the virus-like particles derived from bacteriophage Qβ, to enhance their immunogenicity. The HPV-Qβ particles were then encapsulated into poly(lactic-co-glycolic acid) (PLGA) implants, using a benchtop melt-processing system. The implants facilitated the slow and sustained release of HPV-Qβ particles without the loss of nanoparticle integrity, during high temperature melt processing. Mice vaccinated with the implants generated IgG titers comparable to the traditional soluble injections and achieved protection in a pseudovirus neutralization assay. HPV-Qβ implants offer a new vaccination platform; because the melt-processing is so versatile, the technology offers the opportunity for massive upscale into any geometric form factor. Notably, microneedle patches would allow for self-administration in the absence of a healthcare professional, within the developing world. The Qβ technology is highly adaptable, allowing the production of vaccine candidates and their delivery devices for multiple strains or types of viruses. Human papillomavirus (HPV) is a globally-prevalent pathogen and the most common sexually-transmitted infection in the USA . There aDespite the protection offered by the HPV vaccine, only 53.7% of girls and 48.7% of boys in the US were vaccinated against HPV in 2019 . AnotherThe HPV capsid is composed of two proteins—the major capsid protein L1, which forms the virus structure, and the minor capsid protein L2, which is required for genome encapsidation and infection ,12,13. AChallenges remain to produce vaccine candidates that are stable without the cold chain, to be distributed worldwide. Further, the ideal candidate would be delivered using a vaccine delivery device, such as a microneedle, enabling self-administration in the absence of a healthcare professional. Toward this goal, we set out to develop a process to produce implants for sustained release of HPV vaccine candidates. Such delivery devices hold the potential to overcome the need for repeat dosing. Biodegradable polymers are ideal slow-release carriers for antigens, as sustained exposure to antigens over time leads to an improved immunological response ,22,23,24Here, we developed a single-dose HPV vaccine in which L2 peptide antigens from the high-risk HPV16 strain were displayed on the surface of VLPs derived from bacteriophage Qβ, a carrier used in several other vaccine candidates ,27. We cEscherichia coli cells (New England Biolabs) transformed with pET28CP containing the Qβ coat protein sequence were plated onto lysogeny broth (LB) agar (Thermo Fisher Scientific) containing 50 μg/mL kanamycin (Gold Biotechnology). The isolated colonies were picked into 100 mL of autoclaved LB medium plus kanamycin and cultured for 12 h at 37 °C to saturation. We added 50 mL of the culture to 1 L MagicMedia  and incubated at 37 °C for another 24 h, shaking at 300 rpm. The cell pellets were collected by centrifugation at 1500× g and frozen at −80 °C overnight. The pelleted cells were resuspended in 100 mL phosphate-buffered saline (PBS) on ice and lysed using a probe sonicator for 10 min. The lysate was centrifuged at 10,080× g, the supernatant was collected, and the Qβ particles were precipitated by adding 10% w/v PEG8000 (Thermo Fisher Scientific) at 4 °C for 12 h on a rotisserie. The precipitated fraction was pelleted by centrifugation at 10,080× g and dissolved in 40 mL PBS before extraction with a 1:1 v/v butanol/chloroform. The aqueous fraction was collected by centrifugation as above  and Qβ particles were purified on a 10–40% sucrose velocity gradient by ultracentrifugation at 96,281× g for 4.5 h. The light-scattering Qβ layer was collected and pelleted by ultracentrifugation at 160,326× g for 3 h. The purified Qβ particles were dissolved in PBS as a stock solution for later experiments.Bacteriophage Qβ VLPs were expressed and purified, as previously described . Chemica8 bifunctional linker containing an NHS group and a maleimide group (Thermo Fisher Scientific). In brief, 20 mg of Qβ was mixed with a 500-fold molar excess of SM(PEG)8 to Qβ in 0.5 mL PBS (pH 7.4), at room temperature for 1 h. The unreacted SM(PEG)8 was removed using a 100 kDa cut-off centrifuge filter at 3000× g for 15 min, and the recovered VLPs were washed with 0.5 mL PBS. The washed VLPs were resuspended in 0.5 mL PBS and reacted with a 500-fold molar excess of HPV L2 peptides at room temperature for 2 h. The unreacted peptides were removed by centrifugal filtration as above; the recovered HPV-Qβ vaccine candidates were washed twice in 0.5 mL deionized water, and then dialyzed against deionized water for 24 h. HPV peptides were conjugated to Qβ via an SM(PEG)2HPO4 and KH2PO4, pH 8.3), for 4 h at room temperature. The unreacted components were removed by centrifugal filtration as above, and the Cy5-Qβ particles were dialyzed against and then stored in deionized water.Bacteriophage Qβ (20 mg) was labeled with Cy5 through conjugation via a 500-fold molar excess of sulfo-Cy5 NHS ester (Lumiprobe) in 0.5 mL 0.1 M potassium phosphate buffer , using an FEI Quanta 600 scanning electron microscope coupled to a Bruker XFlash 6/60 EDX spectroscope. The EDX maps were prepared using the FEI AZtec software.The VLPs were characterized by fast protein liquid chromatography (FPLC), transmission electron microscopy (TEM), dynamic light scattering (DLS), and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). FPLC was performed using an AKTA-FPLC 900 system fitted with Superose 6 Increase 10/300 GL columns , using PBS (pH 7.4) as the mobile phase at a flow rate of 0.5 mL/min. TEM images were acquired on an FEI Tecnai Spirit G2 Bio TWIN transmission electron microscope. Samples were mounted on 400-mesh hexagonal copper grids and stained with 2% uranyl acetate. DLS was carried out on a Malvern Instruments Zetasizer Nano at 25 °C, in plastic disposal cuvettes. SDS-PAGE was performed on NuPAGE 12% Bis-Tris protein gels (Thermo Fisher Scientific) at 150 mV for 75 min. The gels were stained with Coomassie Brilliant Blue and images were acquired using the ProteinSimple FluorChem R imaging system. Qβ-loaded PLGA implants (~3 mg implant containing 300 µg VLPs) were placed in a 1.5-mL centrifuge tube and immersed in 500 µL PBS at 37 °C. At each time-point, 200-µL samples were removed and replaced with 200 µL fresh PBS. The quantity of released VLPs was determined by measuring the total protein concentration using a Pierce BCA assay kit (Thermo Fisher Scientific). All animal experiments were carried out in accordance with recommendations from the UCSD’s Institutional Animal Care and Use Committee . Female BALB/c mice aged 8 weeks  were anesthetized for the implantation process before shaving hair from the back and wiping the skin with Alcohol Prep pads. The FITC-PLGA implants loaded with Cy5-Qβ VLPs (~1 mg implant containing 100 µg VLPs) were introduced subcutaneously via an 18-gauge needle by pushing the implant out of the needle with a sterilized stainless-steel wire (0.51 mm diameter). The needle was withdrawn and the skin wiped with Alcohol Prep pads. Fluorescence images were acquired on an IVIS 200 imaging system at different time-points and were analyzed using Living Image v3.0. g for 20 min and were stored at 4 °C (short term) or −80 °C (long term).For vaccines in solution, female BALB/c mice aged 8 weeks were vaccinated subcutaneously by injection on days 0, 14, and 28, with blood collected 7 days after each vaccination. For the PLGA implants, mice were vaccinated by subcutaneous implantation, as described above, and blood was collected on days 7, 14, 21, 35, and 49. To compare the different HPV-Qβ conjugates, n = 5 mice were immunized three times with 30 µg of the specific HPV-Qβ conjugate (or the Qβ as a control). For the conjugates vs. mixture vs. peptide experiment, n = 6 mice were vaccinated with 30 µg of the specific HPV-Qβ conjugate, a mixture of 30 µg Qβ and 5 µg of the HPV peptide, or 5 µg of the HPV peptide alone. For the evaluation of single-dose vaccination using the loaded PLGA implants, the PLGA implants were subcutaneously introduced into n = 6 mice. The implants contained 100 µg HPV-Qβ, 500 µg HPV-Qβ, a mixture of 100 µg Qβ, and 20 µg HPV peptide, or 20 µg HPV peptide alone. To make a direct comparison among the three different vaccination techniques, three different groups of mice each comprising n = 6 mice were vaccinated subcutaneously with a single dose of 100 µg of the HPV-Qβ conjugate, three doses of 30 µg of the HPV-Qβ conjugate, and a single dose HPV-Qβ-loaded PLGA implants, loaded at 100 µg. Sera were separated from the collected blood through centrifugation at 2000× The abundance of antibodies against HPV peptides and Qβ particles in serum samples was determined by the enzyme-linked immunosorbent assay (ELISA). For the detection of α-HPV antibodies, 96-well Pierce maleimide-activated plates (Thermo Fisher Scientific) were coated with 100 µL HPV peptides (10 µg/mL) per well in coating buffer . After overnight incubation at 4 °C, the plates were washed three times with PBS containing 0.1% Tween-20 (PBST) and then blocked with 150 µL cysteine (10 µg/mL) per well for 1 h, at room temperature. After three washes in PBST, serial dilutions of serum in PBST containing 1% bovine serum albumin  were added and incubated at 37 °C for 1 h. After three washes in PBST, we added 100 µL of an alkaline phosphatase-labeled goat anti-mouse IgG secondary antibody (Thermo Fisher Scientific) diluted 1:2000 in PBST + 1% BSA for 30 min, at 37 °C. Finally, the plates were washed five times with PBST and developed with 100 µL of the 1-step PNP substrate (Thermo Fisher Scientific) for 30 min at 37 °C. The reaction was stopped by adding 100 µL of 1 M NaOH and the absorbance was read at 405 nm on a Tecan microplate reader. For the detection of α-Qβ antibodies, 96-well PolySorp plates (Thermo Fisher Scientific) were coated with 100 µL Qβ (10 µg/mL in PBS) and incubated at 4 °C overnight. After three washes in PBST, the plates were blocked with 150 µL PBST + 2% BSA at 37 °C for 1 h. The subsequent steps were as described for the detection of HPV peptides.The IgG subtype was also determined by ELISA. The procedure was similar to that described above for Qβ and HPV peptides, but the secondary antibody was replaced with goat anti-mouse antibodies specific for IgG2a, IgG2b, and IgG1 (diluted 1:2000), as part of the IgG subtype antibody kit . After three washes in PBST, we added an alkaline phosphatase-labeled rabbit anti-goat antibody (Sigma-Aldrich) diluted 1:3000, then washed five times in PBST. Finally, the plates were developed by adding the 1-step PNP substrate, as described above. For all ELISAs, the antibody titers were defined as the reciprocal serum dilution at which the absorbance exceeded the background value by >0.2.g for 6 h. Gradient fractions corresponding to the L1 band were collected and characterized by SDS-PAGE, agarose gel electrophoresis, and flow cytometry, as recommended [HPV16 pseudovirus encapsidating the reporter plasmid pfwB (Addgene #37329) encoding green fluorescent protein (GFP) was produced in 293 TT cells, as previously described . In brieommended . HPV16 pMCF10A cells were cultured in 96-well plates to produce the extracellular matrix (ECM), followed by the addition of the HPV pseudovirus (PsHPV), as previously described . The ECMt-test (GraphPad Prism software); * p < 0.05; ** p < 0.01; *** p < 0.005; ns, not significant p > 0.05. Values were expressed as means ± standard deviations. Sample sizes are stated in the figure legends.Comparisons of the differences between two different groups were performed using unpaired two-tailed student’s 17–31 is ideal for the development of an effective HPV vaccine because it is highly conserved among diverse HPV isolates [17–31 epitope, featuring alternative linkers (GPSL or GGSGGGSG) and orientations (epitope exposed at the N-terminus or C-terminus), as shown in The HPV16 epitope L2isolates ,16,17. Disolates , thus, w17–31 to Qβ was necessary to enhance its immunogenicity, because the injection of a mixture of unmodified Qβ and the free HPV peptide elicited antibodies against Qβ but not against HPV, and no antibodies against HPV were generated by the injection of the free HPV peptide alone  elicited the production of anti-HPV IgG in mice, but that the higher titers were achieved when the epitope was exposed at the C-terminus F. All fode alone . g of PLGA, to allow for flow at a reasonably low pressure and residence time in the extruder, to ensure complete melting of the polymer. This method can be easily adapted to other vaccine candidates but requires optimization for each candidate to determine temperature stability, candidate release rate, etc. To optimize the control of the implant parameters, we used a benchtop melt-processing system . To track the release of the particles in vivo, Qβ was labeled with Cy5 and the incorporation of the fluorophore was confirmed by FPLC and SDS-PAGE . FPLC anTo track the in vivo fate of the encapsulated VLPs and the polymeric implant, an FITC-PLGA matrix loaded with Cy5-Qβ particles was implanted subcutaneously into mice and fluorescence images were captured at different time-points A. This aTo assess the efficacy of the vaccine candidate delivered using the vaccine delivery device, we subcutaneously vaccinated the mice with PLGA implants loaded with HPV-Qβ at two different doses (100 µg and 500 µg). As controls, we vaccinated mice with PLGA implants loaded with a mixture of the HPV peptide and unmodified Qβ, or with the HPV peptide alone. Finally, we also subcutaneously immunized mice with three doses of free HPV-Qβ (30 µg per dose), as a conventional vaccination control. The immunization and bleeding schedule is summarized in We compared the anti-HPV IgG titers in serum samples taken from mice in each of the experimental and control groups B. The PLThe IgG subtype ratio following vaccination indicates the mechanism of the immune response. Mice vaccinated with the single-dose HPV-Qβ vaccine based on the PLGA implant, showed a similar HPV-specific IgG subtype ratio to mice in the conventional vaccination control group C. In botAdditionally, we aimed to evaluate to compare a single-dose soluble injection versus a single-dose biodegradable implant, in an effort to evaluate the necessity for a prime-boost regime with this vaccine candidate. Three groups were studied—(1) a single dose of biodegradable 100 μg HPV-Qβ loaded PLGA implant, (2) a single dose of subcutaneous injection of 100 μg of HPV-Qβ solution, and (3) three subcutaneous injections of 30 µg HPV-Qβ solution. The vaccination and bleeding schedule are shown in We find that in the case of slow release from the implant, the IgG antibody titer was slightly lower after 14 days and reached the same level as obtained by the other methods after 28 days. In all cases, the antibodies remained similar even after three months. Since the lifetime of HPV antibodies in mouse is found to be much longer (>90 days) than the release time of the vaccine cargo from the implant (about 20–25 days), a simple model of exponential loss of antibody in the mouse and an approximately linear release of vaccine cargo from the implant supports our observations (see Appendix S1). The model demonstrates that decay kinetics would dominate the concentration of antibodies in blood in the long run and would be essentially same for all methods of administration for this vaccine. Hence, we conclude that the mice vaccinated with the slow-release implants obtain, after a reasonable time, IgG titers comparable to that obtained by the traditional one single full dose or three-dose strategy. While this result might be disappointing, we would like to point out that subcutaneous injections are very expensive and require trained healthcare professionals, thus limiting its availability in developing and poor countries. Furthermore, we speculate that at a lower dose, the implant group might show some advantages of the single dose injection; and this will be tested in future work. If this holds true; this would reduce production costs, which would also be of benefit. Nevertheless, the implications of melt manufacturing of implantable devices has the potential to be immense. Microneedle patches can be manufactured through injection molding, which has the advantage of massive scalability. These devices show similar efficacy when self-administered, and given the known stability of Qβ, might eliminate cold-chain requirements. We anticipate that this technique would be of great value for vaccines that require a prime-boost dosing regimen. Additionally, several recent studies also point out the benefits of sustained release of antigen over bolus injection in terms of better antigen recognition features and enhancement of germinal center responses. ,34.In order to demonstrate the efficacy of the single-dose HPV-Qβ vaccine, serum antibodies from the vaccinated mice were tested for their ability to prevent the infection of pgsa-745 cells by HPV pseudovirus in an in vitro neutralization assay , as prevSubunit vaccine candidates were successfully formulated using conserved L2 peptide epitopes and the bacteriophage Qβ display nanotechnology. The HPV-Qβ were more immunogenic if the HPV peptides were exposed at the C-terminus rather than the N-terminus, and the best-performing VLP elicited a strong immune response in mice. The HPV-Qβ conjugate was encapsulated into PLGA implants through melt-extrusion, which can be controlled precisely using a new benchtop melt-processing system that recapitulates large-scale polymer manufacturing platforms. The immunogenicity of HPV-Qβ conjugate was retained during this process. The PLGA implants released the VLP cargo slowly, both in vitro and in vivo. Most importantly, the titer of IgG against the HPV peptide was similar in mice receiving a single-dose vaccination of HPV-Qβ via the PLGA implant and in those receiving traditional soluble vaccinations of HPV-Qβ. Furthermore, serum antibodies from mice vaccinated with the implant were able to block the infection of cells by HPV pseudovirus in an in vitro neutralization assay. Our results therefore demonstrate the feasibility and efficacy of a single-dose HPV vaccine based on a biodegradable polymer implant. This is of high importance for future development of self-administered vaccine devices made by polymer melt processing. It is also important to note that the high temperature stability of the HPV-Qβ vaccine candidate serves two functions—(1) it allows for traditional polymer manufacturing methods to scale-up fabrication of the implant, and (2) the vaccine and its implant would eliminate the need for the cold chain, thus enabling global distribution. The design concepts are highly adaptable and would be applicable to any infectious disease. The concepts described are especially appealing because they are scalable and fit within the traditional workflow of pharmaceutical production. Bacterial fermentation is a common method to generate therapeutic proteins and VLPs provide an especially high yielding platform with minimal optimization (Qβ evolved to be expressed in bacteria). Likewise, the device platform is common in the pharmaceutical industry, where hot melt extrusion is a common method to make pills and devices. The vaccine administration described herein mimics that of the clinically approved device, Nexplanon. Finally, since the VLP serves as a carrier, new epitopes can be quickly adapted to new diseases or strains.


BackgroundCharacterization of the sexual behaviours and lifestyle factors associated with human papillomavirus (HPV)-positive oral cavity and oropharyngeal squamous cell carcinoma (OPSCC) is crucial to optimal counselling. Our study aims to investigate the relationship between sexual behaviours, lifestyle factors and HPV-positive OPSCC in an Irish population.MethodsWe performed a case-control study of 60 patients with newly diagnosed HPV-positive and HPV-negative oral cavity and OPSCC.ResultsOral sexual activity was more common in the HPV-positive tumour subgroup; however, this association was insignificant on multivariate analysis. No association between age of onset of sexual activity, number of sexual partners or practicing anal sex and HPV-positivity was found. The HPV-positive tumour subgroup had significantly less tobacco use than their HPV-negative counterparts .ConclusionThe emergence of HPV-positive OPSCC means head and neck surgeons must adopt new roles as counsellors of sexually transmitted disease, in addition to their previous role of delivering a cancer diagnosis. Head and neck cancer is responsible for 3% of all invasive cancers diagnosed in Ireland each year . PatientOPSCC is broadly divided into two categories based on p16 expression, as represented by the 8th edition of the American Joint Committee on Cancer (AJCC) staging system: HPV-positive (p16 overexpression) and HPV-negative OPSCC . HPV-posThe recent upsurge in the incidence of OPSCC is speculated to be a consequence of the maturation of the “baby boom” generation, leading to a sexual revolution . Since tThe recognition of HPV-positive OPSCC as a sexually transmitted disease has led to a major shift in the traditional management of patients with head and neck cancer. The typical patient has evolved. Previously, patients were 60-year-old males with chronic exposure to alcohol or tobacco. Currently, patients are 50-year-old males without a significant smoking or alcohol history, and without other co-morbidities. This transformation has many implications for the diagnosing and treating clinician. Initial counselling now plays a pivotal role in a patient’s understanding of both their cancer diagnosis and its association with HPV. Previous studies have shown a diagnosis of HPV-positive OPSCC may lead to significant psychosocial issues concerning fidelity, and concern regarding the transmissibility of the virus to a patient’s partner . In addiDespite the recent surge of international data associating sexual behaviours, HPV-16 and OPSCC, a significant knowledge gap still exists in relation to the sexual behaviours of patients with OPSCC in an Irish population. Due to the increasing incidence of HPV-positive OPSCC, there is a growing importance in recognising this new demographic profile and characterizing the behaviours that put these patients at risk for developing the disease, so that we can counsel patients more effectively. In this study, we endeavor to build on the body of knowledge by comparing the sexual behaviours of patients diagnosed with squamous cell carcinomas of the oral cavity and the oropharynx in an Irish hospital setting.This retrospective case-control study was performed in St. James’s Hospital, Dublin, over a one-year period. Eligible cases included patients diagnosed with oral cavity and oropharyngeal squamous cell carcinomas. Histology was confirmed by histological analysis of formalin-fixed, paraffin-embedded biopsy specimens taken as a part of their standard treatment. The case group was identified using the p16 (p16INK4a) biomarker on immunohistochemistry as a surrogate for HPV-positivity, while the control group consisted of patients with p16 negativity. 30 patients were identified as eligible case subjects. Control group consisted of 30 unmatched patients with p16 negativity on immunohistochemistry. The study protocol was approved by St. James Hospital institutional review board and a written informed consent was obtained from all patients. All participants completed a structured interview at the Ear, Nose and Throat outpatients department during routine follow-up by the same interviewer. Reporting was in accordance with the recommended principles of the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement for case-control studies. Data were elicited on demographic characteristics, lifestyle factors , and sexual behaviours. Data regarding sexual behaviours included sexual orientation, age at first sexual intercourse, number of lifetime sexual partners, having ever participated in vaginal, oral-genital, oral-anal or anal sex, clinical history of sexually transmitted disease, regular correct condom use, and if their partner had a previous diagnosis of cervical dysplasia. Tobacco use data was measured by the mean number of cigarettes in pack years. Data on alcohol consumption was reported as the mean number of alcohol units consumed per week. Statistical analyses were performed using Stata software, version 12.1 . Case-control comparisons were made between the two groups. A series of univariate and multivariate analyses were performed. Multivariate analyses involved hierarchical logistic regression to identify associated factors. The multivariate models were used to calculate adjusted odds ratios, their 95% confidence intervals, and the corresponding p-values on covariates that revealed a statistically significant univariate p-value (p < 0.05).HPV-positive tumours were most commonly detected at the tonsils and the tongue base, accounting for 77% of the HPV-positive tumours. The most common sites in the HPV-negative group were the anterior tongue and tongue base, although location of HPV-negative tumours were more widely distributed compared to HPV-positive tumours. The tumour distribution of our cases and controls is shown in Table  The mean age of the participants interviewed was 59.5 years. The HPV-positive group was slightly younger overall, but this was not significant. The majority of the study population was male (76%), with marginally more males in the HPV-positive group than the HPV-negative group (40% v 36.7%). 78% of patients, from both groups, were in a relationship at the time of this study. Expectedly, a positive association was seen between tobacco use and the HPV-negative subgroup, with the mean smoking pack-year in this group being 56 pack-years . In contrast, the mean smoking pack-years for the HPV-positive group was 12.7 pack-years, including 15 participants who have never smoked. This association remained statistically significant after adjusting for confounding variables . The mean alcohol consumption per week was significantly higher in the HPV-negative cohort overall , but this did remain independently significant on multivariate regression analysis . In terms of sexual behaviour, there were no significant differences between age of onset of sexual activity  or whether the participant was sexually active or not at the time of diagnosis . Lifetime number of sexual partners was 17.5 in the HPV-positive cohort compared with 4.7 in the HPV-negative group. There was a significant difference in multivariate analysis ; however, this was not significant on univariate analysis , which may be the result of a dependent variable causing a suppression effect. All participants had performed vaginal intercourse and 55% reported being sexually active at the time of diagnosis.Table This is the first study investigating the differences in sexual behaviours in patients diagnosed with oral cavity and OPSCC in a representative sample of the Irish population. Our data elucidated a trend towards significance in patients engaging in oral sexual practices and the development of oral cavity and OPSCC, as seen in previous case-control studies . In addiAs expected, the majority of HPV-positive OPSCC were located in the tonsils and tongue base, which is likely due to the crypt epithelium present in lymphoid tissue which provides an entry point for HPV . AnotherIn our cohort, we found those who had engaged in oral sex were nearly twice as likely to have an HPV-related cancer (80% vs 43.3%). Despite this seemingly strong relationship, when factors such as tobacco use, alcohol consumption, age, and gender were controlled, having practiced oral sex failed to remain independently significant . It is plausible that the current study was underpowered to detect a positive association when adjusting for confounders. Other case-control studies demonstrated an exposure-response relationship between number of oral sexual partners and HPV-related OPSCC . In addiThe primary limitation of this study was a lack of power reducing the chance of detecting a significant result. Larger studies, such as the International Head and Neck Cancer Epidemiology (IHANCE) consortium, undertook a pooled analysis, including 5642 head and neck cancer cases and 6069 controls from several different countries . PositivThe aim of this study was to further characterize the sexual behaviours of patients with HPV-positive oral cavity and OPSCC in an Irish population, so that we could counsel them more effectively regarding their diagnosis. Despite a plethora of research internationally providing evidence of this association ,16,24, nThe development of an association between HPV and oral cavity and OPSCC has undoubtedly changed the landscape of head and neck oncology. Characterizing risk factors is an integral part of furthering our knowledge and obtaining the necessary information to adapt to this change. Head and neck surgeons must adopt a patient-centered role in counselling patients regarding sexually transmitted diseases, as well as the traditional role of diagnosing and treating cancer.


Oropharyngeal cancer (OPC) caused by human papillomavirus (HPV) is a rising global concern. Early lesions are small and are often located in difficult to access areas (such as the crypts of the tonsils or base of tongue). Unlike cervical cancer, there is no standard or routine screening program for HPV-driven OPC. HPV DNA from OPC tumors may shed directly into saliva, and this can be used as a biomarker for early diagnosis. In this study, we report the first-ever clinically occult OPC in an asymptomatic patient discovered through a saliva test. This case relied upon serial measurements of HPV-16 DNA in saliva, which fell to undetectable levels following low morbidity, curative treatment. The incidence of high-risk human papillomavirus HR-HPV−16,-18,-33) driven oropharyngeal cancer (OPC) is rapidly increasing in developed countries 6,-18,-33. It remaAn ongoing HPV-16 DNA prevalence study was approved by institutional ethics committees from the University of Queensland; Queensland University of Technology and the Royal Brisbane and Women's Hospital. A total of 665 cancer-free healthy individuals from Queensland Region, Australia between May 2016 and October 2017 were recruited. All participants gave written informed consent prior to sample collection.Six hundred and fifty cancer-free healthy individuals with sufficient amount of DNA were tested for oral HPV-16 DNA. Of these 3 have been identified to have persistent oral HPV-16 DNA infection. Following discussion with our ethics team we have approached these three participants and offered them consultation with an Ear, Nose, and Throat (ENT) surgeon. A 63-year-old caucasian male was assessed as part of this consultation process. He had consistently been HPV-16 DNA positive for a period of 36 months, with a steadily rising HPV-16 viral load in his salivary oral rinse samples . He was He is an ex-smoker, having quit 15 years ago, with a 45 pack year history of smoking. He drinks two standard drinks  per day. He is heterosexual, and his social history includes multiple oral sex partners in the past (>5), followed by a long term monogamous relationship. Initial clinical examination of the oropharynx including palpation and white light revealed no significant abnormalities. Both tonsils were irregular due to mucous retention cysts and there was slight tonsillar asymmetry (Left < Right) but no evidence of any malignant lesions. Narrow band imaging (NBI) showed some mild vascular changes at the left glosso-tonsillar sulcus. There were no palpable lymph nodes in the neck. An MRI examination of the oropharynx and neck demonstrated no occult lesions of the tonsils or the base of tongue and no cervical lymphadenopathy.He was offered continued surveillance, or a biopsy of the area of NBI change with bilateral tonsillectomy. The patient elected for bilateral tonsillectomy and biopsy of the base of tongue with NBI guidance under general anesthetic and informed consent was obtained. The surgical specimens were sent for histology and tissue HPV-16 DNA testing. The patient was discharged from hospital the same day. He had a routine postoperative course with a sore throat for 1 week and recovered fully. An ultrasound scan of his neck was performed 2 months post-surgery which showed no cervical lymphadenopathy. He is currently under routine oncological surveillance. The patient has a very high likelihood of cure with minimal morbidity from single modality treatment.Salivary oral rinse samples of this individual were collected at baseline, 6, 12, 36 month, and 2 weeks after his bilateral tonsillectomy using previously published method –10. Brie1-1 × 108 copies) of pHPV-16 plasmid DNA [American Type Culture Collection (ATCC)® 45113™].Total DNA was extracted from salivary oral rinse and tonsillar tissue samples using the QIAmp DNA Mini Kit  as per manufacturer's protocol. For detection of HPV-16 genotyping, the qPCR assay targeting the opening reading frame (ORF) region of HPV16 E6/7 was carried out with the QuantStudio™ 7 Flex Real-Time PCR System  as described previously , 12. ForH&E (Haemotoxylin and Eosin stains) staining on formalin-fixed paraffin-embedded (FFPE) slide was performed to investigate the cellular and tissue structure/morphology. HPV status was evaluated using CINtec® p16INK4a Histology Kit (E6H4 clone)  according to manufacturer's instructions. p16INK4a was considered positive by two independent pathologists when there was a strong, diffuse nuclear and cytoplasmic staining pattern in the majority (>70%) of tumor cells.Salivary oral rinse samples from this individual had been collected at baseline, 6, 12, and 36 month follow-up as well as 2 weeks after his bilateral tonsillectomy. HPV-16 DNA genotyping and viral loads in all samples were analyzed using an in-house developed qPCR assay. HPV-16 DNA viral load in saliva increased exponentially across the 36 month follow-up period  and subsequently declined to undetectable levels post-tonsillectomy .This individual was diagnosed as having a 2 mm squamous cell carcinoma (T1N0M0) in the left tonsil  using HaLong-term persistence of HPV-16 infection is likely to be a prerequisite for the development of malignancy , 14. WomThe impact of the pattern of salivary HPV persistence including changes in the absolute HPV viral DNA copies over time has never been investigated. The pattern of salivary HPV-DNA detection in this case demonstrates an exponential upward trend with the titer at first sample being 3.43 copies per 50 ng and the final titer before surgery of 1281.7 viral copies per 50 ng. This may represent progression of the lesion with subsequent shedding of increasing levels of HPV-16 DNA into the saliva. In future cases the presence of this pattern should be evaluated, as it may provide a critical marker for the progression of disease and hence a signal for intervention; indeed the pattern of viral copies in serial measurement may have more importance than the persistence itself.This case also has important implications with regards to the natural history of the disease. The left tonsil was strongly positive for HPV 16 DNA outside the region of malignancy and as anticipated was p16INK4a positive only within carcinoma. This implies that the malignancy is likely to have developed in a wider field of HPV infection with only a component undergoing malignant change. The existence of a precursor lesion to OPC has long been doubted and is cited as one of the obstacles to OPC screening  [HREC No: 2014000679 and 2014000862]; Queensland University of Technology [HREC No: 1400000617 and 1400000641]; and the Royal Brisbane and Women's Hospital (RBWH) [HREC/16/QRBW/447]. Written informed consent was obtained from this participant for publication of this case report.All authors have read and agree to the published version of the manuscript. KT and CP: conceptualization. All authors: methodology, validation, formal analysis, data curation, investigation, and writing—review and editing. KT, SV, and CP: writing—original draft preparation. CP: funding acquisition.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Many empirical studies have shown evidence of multiple health benefits provided by green and blue spaces. Despite the importance of these spaces, investigations are scarce in details for blue spaces rather than green. Moreover, most research has focused on developed regions. A limited number of studies on blue spaces can be found in China with a focus on the city level. Outcomes have been mixed due to varying research scales, methodologies, and definitions. This study relies on a national-level social survey to explore how the self-rated health (SRH) of senior individuals is associated with local green and blue space availability in urban and rural areas. Results indicate that the coverage ratio of overall green spaces and waterbodies around a resident’s home have marginal effects on SRH status in both urban and rural areas. In urban areas, living close to a park can is marginally beneficial for older people’s health. Regarding different types of blue spaces, the presence of a major river (within 0.3–0.5 km) or coastline (within 1 km and 1–5 km) in the vicinity of home negatively affects SRH among the elderly in urban areas. Close proximity to lakes and other types of waterbodies with a water surface larger than 6.25 ha did not significantly influence SRH. These findings not only evaluate general health impacts of green/blue space development on senior populations across the county but inform decision makers concerning the health-promoting qualities and features of different green/blue spaces to better accommodate an aging population in the era of urbanization. Green and blue spaces are critical components of humans’ living environment. Green space, in the form of parks, grasslands, forests, farmlands, and even backyard gardens, together with blue space, including lakes, rivers, and coasts, can promote people’s well-being in multiple ways. According to a number of literature reviews, there are several potential pathways for how different types of green/blue spaces benefit well-being ,2,3. GenCompared with the widely acknowledged health-promoting benefits of green spaces, studies concerning blue spaces are growing, yet remain scarce. Despite the recognition of the effects of blue space on people’s health, research on blue spaces has revealed largely mixed results due to varying scales, health indicators, definitions of blue space, and approaches to the measurement of blue spaces ,19,20. SResearch relies on place-based or people-based indicators for the availability of green/blue spaces around people . To measAs more than half of world’s population lives in urban areas, the negative impacts of urbanization, including environmental pollution, urban heat island effect, etc., are threatening increasing numbers of people . This isAccommodating the largest number of older people in the world, China is experiencing rapid population aging. In 2010, the number of senior people above the age of 60 was more than 177.6 million, accounting for 13.26% of the total population . MoreoveThis study examines how the availability of green and blue spaces is associated with general health among senior populations in China. To our best knowledge, this is the first attempt to examine green and blue spaces at the neighborhood level across China. Relying on a large-scale national survey, we examined how the self-rated health (SRH) of senior individuals is associated with the following factors: (1) green and blue space coverage within 1 km from a neighborhood, (2) closest distance to parks (only in urban areas), and (3) closest distance to different types of waterbodies.This study relies on a recently released national-scale social survey, the Chinese Social Survey (CSS). Launched by the Chinese Academy of Social Science in 2005, the CSS has been conducted every two years, relying on a multistage, probability-proportional-to-size sampling approach across the entire country . Each seWe used the 2011 wave as this was the only cohort that recorded the SRH of senior populations (age above 60 as a threshold). A one-item measure for SRH was asked for each participant above age 60: “How do you rate your current health conditions in general?” The answers are coded on a 4-point scale as “3” , “2” , “1” , and “0” (cannot take care of myself). The category “0” (cannot take care of myself) was eliminated as these responses came from an advanced aging population (above 80), who confront a higher risk of mortality and rarely visit or contact green or blue spaces daily . The totAnother important feature of the CSS 2011 wave was that it is the only version recording detailed local data such as the address of each respondent . Two trained research assistants manually searched each geolocation through Gaode Map  . The authors also double-checked all the returned geocodes on Gaode Map to ensure accuracy. Thus, the nearby availability of green and blue spaces and other location-specific environmental factors could be calculated, and the relationship between SRH and green or blue space availability could be analyzed.To measure the availability of green space, two indicators were used: overall green space coverage ratio and the closest linear distance to a park (in an urban area). Following a study at the same neighborhood scale, a green space coverage ratio was measured within a 1 km buffer from the centroid of neighborhoods as a proxy for total vegetation exposure around respondents’ homes . The greAdditionally, we calculated the closest linear distance to the entrance to a park, as this provides major accessible vegetated spaces for the public’s outdoor activities in urban areas. The park locations were also extracted from Gaode Map, which records the entrances in the form of “Points of Interest.” The closest distance to a park was only entered into regression analyses for urban areas as rural areas had no parks. It is admitted that the distances measured by a network-based approach can be more actual than a linear distance. However, the results calculated by the two types of measurement are often highly correlated ,53,54, a2 in the terrestrial land of China for the period 2000–2016 in an 8-day temporal and 250-m spatial resolution [Regarding blue space, we measured the coverage ratio of all types of water surfaces and the closest linear distance to different types of blue spaces. The data on water surfaces were derived from the Time Series of Inland Surface Water Dataset in China (ISWDC), which mapped inland waterbodies larger than 0.0625 kmsolution . We aggrsolution ,19,27,572 as control variables. The major road categories include national roads, provincial roads, and ordinary roads that are similar to the US highway [Individual-level socioeconomic characteristics were collected in CSS 2011 regarding the respondents’ age, marital status, ethnicity, insurance, lifestyle, education, household registration location (hukou in Chinese), occupation, income, and assets . The sur highway . Table 1To identify the association between senior people’s SRH and the availability of green and blue spaces, we used multiple multivariate regression in the form of ordinary least squares (OLS) regression. The OLS model is easier to interpret than the ordered probit model ,68,69. IThe regression was conducted separately on urban and rural areas, given widely varying geographical and socioeconomic features. Sensitivity tests were performed by (1) using different buffer distances  to calculate the green and blue space coverage ratio, and (2) categorizing continuous variables (distance to green and blue spaces) into dummies. The distances to parks, roads, and rivers were entered in logarithmic forms due to the detection of nonlinearity. Distances to lake and coast were excluded due to identified collinearities. We used distances of 300 m and 500 m as thresholds for creating dummies. A 300 m distance reflects a 5-min walking time for older people with or without mobility issues . A 500 mThe average SRH among senior populations above age 60 in China was 1.967 (S.D. = 0.706), with residents from urban areas having a slightly better value of 2.011 (S.D. = 0.685) than that reported by rural residents of 1.901 (S.D. = 0.733) . The higp value < 0.05). Having a major river (within 0.3–0.5 km) or coastline (within 1 km and 1–5 km) in the vicinity of an urban respondent’s home might negatively affect SRH , though such a trend was marginal in rural areas. Living near a coastline posed higher negative impacts on SRH of senior people than living close to a major river.2, tended to have higher SRH.Detailed regression results are shown in Using multiple indicators to measure the availability of green and blue space, this study examined how these were related to older people’s SRH status across China. Spatial-explicit data on green and blue spaces as well as other local environmental factors were obtained based on a nationwide social survey with detailed geolocation of respondents’ neighborhoods. Urban and rural areas were separately entered into regression estimations. Our results did not find strong evidence that living with a higher amount of green/blue spaces or within closer distance to green/blue spaces contributes to higher SRH among senior populations. A clear urban-rural difference was observed in that blue space tends to pose negative impacts in SRH in urban areas as nonsignificant outcomes were found in rural areas. Living close to certain types of waterbodies, such as rivers and coastlines, might be detrimental to older people’s health in urban areas. By offering a national observation in a developing region, this study enriches the current literature on public health and the environment, with a particular focus on green and blue spaces. The results provide reference information to assist decision-makers and urban planners to manage urbanization in an aging era.Our study identified both coverage of and distance to green space pose insignificant impacts on senior’s SRH. Despite the widely acknowledged positive impacts of green space on people’s general health, many studies returned mixed outcomes when investigating subpopulations, such as older people . For exaThe insignificant impacts of green space on people’s health might be due to the characteristics of the older population in our study, who are often less inclined to travel longer distances to use green spaces due to physical limitations and long-term illnesses ,18, and The coverage of water surfaces around neighborhoods as well as close distances to different types of waterbodies presents few positive impacts on older people’s SRH in both urban and rural areas. This result contrasts with previous observations in developed regions as studies showed in Canada , EnglandOne potential speculation on the minimal positive health effect of blue space is that the water-coverage ratio can hardly capture actual pathways showing how older people benefit from it. The actual quality and features of blue spaces might more substantially affect older people′s actual usages. Scholars have argued that senior populations are more concerned about safety , accessibility , or other public facilities , the lack of which might discount the attractiveness of a waterbody for them, and thus discourage their usage for health promoting activities ,39,84. AThe closest distances to various types of waterbody were not associated with the elderly’s SRH. Worse, living close to rivers or coastal areas negatively affected the reported general health statuses. Such negative impacts could be due to the poor or polluted water quality in rivers or coastlines, which might be prevalent in developing countries such as China . For exaOur study confronts several potential limitations regarding data and scale. Despite employing fine-resolution neighborhood locations for measuring green and blue spaces, the measurement might be less accurate than using exact residential addresses. A neighborhood often contains several residential quarters in China. The size of the neighborhood in urban areas is smaller than that in rural areas. Thus, urban areas might capture surrounding environmental characteristics such as green and blue coverage more accurately. Types of green and blue spaces might still be too coarse to reflect detailed features or qualities. Rivers, lakes, small waterbodies, and parks might present different qualities and features. For instance, other types of green spaces, such as pocket gardens or informal green spaces, might provide recreational services such as large parks do . HeavilyRelying on a recently released national-scale survey , this study examined fine-grained, neighborhood-level availability of green and blue spaces and the associations with the general health of senior people across China. We found that the coverage ratio of overall green spaces and waterbodies around a resident’s home had marginal effects on the SRH status of seniors in both urban and rural areas. Regarding different types of green and blue spaces, the presence of a major river (within 0.3–0.5 km) or coastline (within 1 km and 1–5 km) in the vicinity of home negatively affects SRH among the elderly in urban areas. Proximity to lakes or parks did not exert significant influences on SRH in older people. In urban area, older people living within 0.3 km from any types of waterbodies with surface measurements greater than 6.25 ha reported better SRH. As a pioneering study on nationwide public health effects of green and blue spaces, the empirical evidence expands current discourses on health outcomes influenced by local environmental features. In particular, this work adds to the less-explored literature on the impact of blue spaces on health particularly among senior populations, adding a national-level investigation from a developing country.


Collagen-induced arthritis (CIA) is the most widely used animal model of rheumatoid arthritis, but C57BL/6 strain is resistant to CIA because there is no collagen II peptide associated with H-2b. To establish a rheumatoid arthritis model in C57BL/6 mice, we immunized C57BL/6NJ (B6N) mice with human cartilage oligomeric matrix protein (COMP), which induced severe arthritis with high incidence, accompanied by a strong auto-antibody response. Native COMP was required, as denatured COMP lost its ability to induce arthritis in B6N mice. An immunodominant COMP peptide was identified as the key T cell epitope, with a perfect fit into the Ab class II peptide binding pocket. A critical amino acid in this peptide was found to be phenylalanine at position 95. Recombinant COMP mutated at position 95 (COMP_F95S) lost its ability to induce arthritis or a strong immune response in the B6N mice. In conclusion, A new model for RA has been established using C57BL/6 mice through immunization with COMP, which is dependent on a COMP specific peptide binding Ab, thus in similarity with CIA in Aq expressing strains.The most commonly used strains in experimental research, including genetically modified strains, are C57BL/6 mice. However, so far, no reliable model for rheumatoid arthritis is available, mainly due to the restriction by the MHC class II haplotype H-2 A new model of rheumatoid arthritis in C57BL/6 mice, the COMP induced arthritis (COMPIA), was established.The major T and B cell epitopes on COMP critically involved in the arthritis development were defined.Rheumatoid arthritis (RA) is a chronic inflammatory disease, which is characterized by leucocyte infiltration into synovial tissue and leads to cartilage and bone damage . It is aq beta gene (q and Ar developed arthritis whereas mice with the Ab (H-2b) haplotype, as in commonly used C57BL/6 strains, were resistant to CIA (b) (Collagen-induced arthritis (CIA) is the most widely used animal model of RA. The susceptibility to arthritis after immunization with heterologous type II collagen (CII) is strongly associated with MHC class II genes, more precisely the Aeta gene  due to beta gene . In a sct to CIA –12. Howe CIA (b) –15, in p CIA (b) , has the CIA (b) . One con CIA (b) .To establish a model for RA in C57BL/6 mice we turned our interest to other proteins that have earlier been shown to induce arthritis in mice, i.e. glucose-6-phosphoisomerase (GPI)  and cartq, H-2p, and also with transgenic human DR*0401  conditions and individually ventilated cages contained wood shavings and folded paper strips. The cages were kept in the same room with a climate-controlled environment in a 12-h light-dark cycle. All of the experiments were carried out when the mice were 9–12 weeks old. Experimental mice were divided randomly, blinded and age-matched. Clinical evaluation of arthritis was performed blindly and double-checked for each experiment. All experiments were approved by Stockholm regional ethics committee for animal research, Sweden (N35/16).Male C57BL/6NJ (denoted B6N) mice, which carry the H-2The COMP sequence was obtained from UniProtKB with accession number P49747. The construct contains the native leader sequence and the full-length COMP sequence and the C-terminal his-tag. The gene was synthesized at Eurofins with KpnI and XhoI restriction sites at the 5’ and 3’ ends. The synthesized gene was restriction enzyme digested using FastDigestTM enzymes (ThermoFisher Scientific). The digested DNA fragment was cloned into a mammalian expression vector pCEP4 (Life technologies) that was digested using the same restriction enzymes. After sequence verification, the recombinant plasmid was transfected into Expi393FTM cells (Life technologies) using the FectoPROTM DNA transfection reagent (Polyplus transfection). The supernatants were harvested 6 days post transfection. The recombinant protein was first captured using a 5 ml HisTrap Excel  affinity column followed by size exclusion chromatography on a HiLoad Superdex 200 pg column  equilibrated in PBS. The purified recombinant protein  were generated using the sequence of human COMP. Synthetic peptides containing interest sequences were synthesized by Biomatik Corporation .Synthetic peptides  were synthesized by Hangzhou Taijia Biotech Co., Ltd . Further details are given in Synthetic cyclic peptides were synthesized as previously described .For the expression of a single amino acid mutated COMP_F95S, two primers were designed with forward primer: 5’- AGGTTCCTGCTTTCCCGGAGTAGCG-3’ and reverse primer: 5’-GGTGCACAGTGCAGCAGAGGCC-3’. A Q5 site-directed mutagenesis kit  was used according to the manufacturer’s instruction. After transformation, several colonies were selected and the sequences verified. The plasmid with the right sequence was transfected into the Expi393FTM cells as described above.Mice were immunized intradermally (i.d.) at the base of the tail with 100 ug of native COMP emulsified 1:1 in Freund,s complete adjuvant  or in Freund,s incomplete adjuvant  containing Mycobacterium Tuberculosis H37Ra  in a total volume of 100 ul on day 0. All the mice were boosted on day 35 with 50 ug of native COMP in IFA in a total volume of 50 ul. Mice were double blind scored 3 times per week for the peripheral joint inflammation, starting 2 weeks after the first immunization. The evaluation of arthritis was conducted in a blinded manner according to an extended scoring protocol , using 0’s complete adjuvant  or CFA only (group 3) separately on day 0. On day 35, all the mice were boosted with or without COMP in IFA  in a total volume of 50ul . The evaluation of arthritis was conducted as described above.To evaluate the requirement of the native structure of COMP for the arthritis induction, mice were immunized with 100 ug of native COMP (group 1), denatured COMP (group 2) emulsified 1:1 in FreundTo prove the importance of phenylalanine at position 95 for the T cell epitope recognition and arthritis development, mice were immunized i.d. at the base of the tail with 100ug of COMP_F95S or native COMP emulsified 1:1 in Freund’s complete adjuvant  on day 0. On day 35, the mice were boosted with 50ug of COMP_F95S or native COMP in IFA  in a total volume of 50ul. The evaluation of arthritis was conducted as described above.1  and Goat Anti-Mouse IgG2b-HRP . After washing for 5 times, ExtrAvidin® -Peroxidase  was added and incubated for 45 mins. 5mg ABTS substrate  was diluted in 5 ml of freshly prepared buffer . After 20 min-incubation in the dark at 20, absorbance was measured at 405nm (OD405). Serum antibody against CII were analyzed by sandwich ELISA, as previously described  by ELISA using recombinant human COMP. Recombinant human COMP , PH 7.4) was precoated overnight at 4. Then the plates were blocked with 3% milk in PBS to decrease background disturbance. The plates were washed for 5 times with ELISA buffer (PBS with 0.1% Tween 20). The serum was diluted (*500) in PBS , and incubated for 2 h. Serum from COMP- induced arthritis mice were used as positive control. 50ul of secondary antibody was added and incubated for 1 h. The secondary antibodies were as following: Rat Anti-Mouse Kappa- Horseradish peroxidase (HRP) , Goat Anti-Mouse IgG(H+L) , Goat Anti-Mouse IgGescribed .In the first experiment, we performed epitope mapping of whole human COMP by ELISA to identify linear B cell epitopes with serum from COMPIA B6N model. Maxisorb plates were coated with COMP peptides or native COMP at a concentration of 5 ug/ml in PBS overnight at 4. The second day the plates were washed with PBST for 3 times and then blocked with 1% bovine serum albumin (BSA) in PBS. Different dilutions (1:100 to 1:2500) of mice serum (B6N mice immunized with COMP+CFA) were added to the wells and incubated for 1 h at room temperature. Then the plates were washed with PBST for 3 times. HRP-conjugated anti-mouse second antibody was added for further incubation of 1 h followed by the detection using ABTS-HRP system as previous description. Absorbance was measured at 405nm (OD405).6 cells per well for splenocytes, 0.5x106 cells per well for lymph node cells). The cells were stimulated with 50 ug/ml different peptides  in triplicates at 37for 24 h. Concanavalin A  used as a positive control. The plates were washed with PBS twice and a washing buffer (PBS containing 0.01% Tween 20) for 4 times followed by incubation with 50ul/well of biotinylated anti-IFN-γ  at room temperature for 2 h. After washing the plates with the washing buffer for 5 times, streptavidin-alkaline phosphatase  was added to each well and incubated for 45 min at room temperature. After washing with the washing buffer for 3 times and PBS for 3 times, cytokine spots were visualized using Sigma Fast BCIP/nitroblue tetrazolium (100 ul/well) and enumerated using an ImmunoScan ELISpot Analyzer (CTL Europe).The 96-well ELISpot plates  were prewetted with 70% EtOH and then precoated with anti-mouse IFN-γ capture antibody R46A2  overnight at 4. The second day the plates were washed with PBS for twice and then blocked with 1% BSA in PBS. Splenocytes or cells from draining lymph nodes were obtained from the immunized B6N mice and added to the plates  for decalcification, followed by dehydration and paraffin embedding. Tissue sections (7 μm) were then stained with hematoxylin and eosin (HE). HE-stained sections were blindly assessed in naive B6N mice or B6N mice immunized with native COMP.Mice were sacrificed at the end time point, and the hind paws were dissected and subsequently fixed in 10% buffer formalin solution. Fixed tissues were decalcified for 30 days in EDTA-solution  was constructed using the SWISS-MODEL (b with an PADI4 peptide (PDB code: 6MNM) was selected as a template due to high sequence similarity  was used as a basis, then several residues were modeled by FoldX program . To test the potential of COMP for the induction of arthritis, we immunized B6N mice with COMP emulsified in complete Freund, adjuvant  or in incomplete Freund’s adjuvant  containing Mycobacterium Tuberculosis strain H37Ra . Both groups immunized with COMP developed severe arthritis with high incidence . There was no difference between mice immunized with native COMP+CFA and mice immunized with native COMP + 0.5 mg/ml M.T.+IFA (p>0.05)  developed a prominent IgG anti-COMP autoantibody response, compared with naïve B6N mice . We selected 15 peptides  to repeat the experiment at different serum dilution (n=4), which confirmed the result . As expected, B6N mice immunized with CFA did not develop arthritis (p<0.05) (p<0.05). We conclude that antibodies to conformational epitopes are pathogenic and need for the induction of arthritis and that these antibodies are likely triggered by help from COMP specific T cell.To test whether the native conformational of COMP is recognized by antibodies developed after the immunization with both COMP preparations, we analyzed the sera at different time points during COMPIA (at days 35 and 88 post-immunization). Both native COMP immunized group and denatured COMP group developed an anti-COMP antibody response at both time points (*+ T cells was used as a read-out and ConA was used as a positive control. Spleen cells from mice immunized with COMP showed strong IFN-γ T cell recognition of peptide P9 (APGFCFPGVACIQTESGA)  were tested for their ability to activate T cells after immunization with human COMP. IFN-γ secretion by CD4b structure, COMP-P9 fits into the most regions of the Ab groove . B6N mice were immunized with COMP or COMP_F95S in CFA. As expected all mice immunized with COMP developed severe arthritis. Only 1 out of 8 B6N mice immunized with COMP_F95S developed arthritis, with very low scores  were clearly higher in mice immunized with native COMP group compared with those immunized with COMP_F95S group at both day 35 and at the endpoint  resulted in eight-time reduction of incidence of arthritis. Our experiments provide a conclusive biochemical evidence that the Ab molecule binds COMP-P9 leading to COMP-specific T cell response in B6N mice, that is a key step for induction of arthritis.Systemic delivery of nanoparticles coated with antigens (autoimmune-relative peptides) bound to MHC class II molecules trigger autoimmune reaction in different mouse models , which cb) mice through immunization with COMP, which is dependent on a COMP specific peptide, thus in similarity with collagen induced arthritis in H-2q (Aq) expressing strains.In summary, a new model for RA has been established using C57BL/6 .YZ was in charge of the animal experiments, methodology, software, formal analysis, resources, investigation, data collection, and wrote and edited the original draft. VU conducted the investigation and methodology. BX conducted the investigation and methodology. WC conducted the investigation. CG conducted the investigation, and provided the methodology and software. ZS conducted the investigation. RH conceptualized the study and subject design, wrote, reviewed, and edited the manuscript, provided the resources, and supervised the study. All authors contributed to the article and approved the submitted version.This work was supported by grants from the Swedish Research Council (2015-02662), Knut and Alice Wallenberg Foundation (KAM2015.0063), the Swedish Foundation for Strategic Research (RB13-0156) and Science and Technology Projects of Guangzhou (Grant No. 201707010365).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.