Variations in HPV LCR/E6/E7 have been shown to be associated with the viral persistence and cervical cancer development. So far, there are few reports about the polymorphisms of the HPV-58 LCR/E6/E7 sequences in Southwest China. This study aims to characterize the gene polymorphisms of the HPV-58 LCR/E6/E7 sequences in women of Southwest China, and assess the effects of variations on the immune recognition of viral E6 and E7 antigens.Twelve LCR/E6/E7 of the HPV-58 isolates were amplified and sequenced. A neighbor-joining phylogenetic tree was constructed by MEGA 7.0, followed by the secondary structure prediction of the related proteins using PSIPRED v3.3. The selection pressure acting on the HPV-58 E6 and E7 coding regions was estimated by Bayes empirical Bayes analysis of PAML 4.8. Meanwhile, the MHC class-I and II binding peptides were predicted by the ProPred-I server and ProPred server. The transcription factor binding sites in the HPV-58 LCR were analyzed using the JASPAR database.+ and CD8+ T-cells. In the LCR, C7265G and C7266T were the most variable sites and were the potential binding sites for the transcription factor SOX10.Twenty nine SNPs  were identified at 27 nucleotide sites across the HPV-58 LCR/E6/E7. From the most variable to the least variable, the nucleotide variations were LCR > E7 > E6. The combinations of all the SNPs resulted in 11 unique sequences, which were clustered into the A lineage . An insertion (TGTCAGTTTCCT) was found between the nucleotide sites 7280 and 7281 in 2 variants, and a deletion (TTTAT) was found between 7429 and 7433 in 1 variant. The most common non-synonymous substitution V77A in the E7 was observed in the sequences encoding the α-helix. 63G in the E7 was determined to be the only one positively selected site in the HPV-58 E6/E7 sequences. Six non-synonymous amino acid substitutions  were affecting multiple putative epitopes for both CD4These results provide an insight into the intrinsic geographical relatedness and biological differences of the HPV-58 variants, and contribute to further research on the HPV-58 epidemiology, carcinogenesis, and therapeutic vaccine development.The online version of this article (10.1186/s12985-018-0986-7) contains supplementary material, which is available to authorized users. Human papillomavirus type 58 (HPV-58) is one of the high-risk members of the papillomavirus family, which are considered to be associated with cervical cancer . Its preThe high-risk HPV oncoproteins E6 and E7 can disturb the transcriptional activity of the tumor suppressor protein p53 and the retinoblastoma tumor suppressor gene product pRB, respectively, and induce their proteolytic degradation. E7 can also interact with p53. They are thought to perturb the cell cycle to provide a cellular milieu to favor the replication of the HPV genome DNA and the production of viruses , YY1 9]9], and sIn recent years, there have been many relevant reports about the E6, E7 and LCR of HPV-16. However, studies of the HPV-58 sequences originating in Southwest China are few, especially for the LCR. It is necessary to further research the nucleotide polymorphisms within the HPV-58 LCR, E6, and E7. Besides, the effects of the variations on the immune system’s ability of recognition of the viral E6 and E7 antigens were little reported and should be evaluated. In this paper, the single nucleotide polymorphisms (SNPs) in the HPV-58 LCR, E6, and E7 in Southwest China, were identified and the phylogeny was described to determine the circulating lineages. To assess the possible association of polymorphisms in the HPV-58 E6, E7 and LCR with the virus infection, replication and propagation, the amino acid changes in the HPV-58 E6 and E7 which affect multiple putative epitopes for both CD4+ and CD8+ T-cells were described, and the binding sites of the transcription factors in the LCR were analyzed. Our results could provide basic data for further studies on the HPV-58 epidemiology, prevention, and therapeutic vaccine development.Each participant was informed of the study aims for obtaining informed consent. This study was approved by the Institutional Ethics Committee of Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College (No. 2009ZC187M), and was carried out in line with the Helsinki Declaration.Two thousand one hundred forty six cervical scrapings of gynecological outpatients aging from 22 to 59 were collected from the First Affiliated Hospital of Kunming Medical University in Southwest China in 2015. The routine cytology and HC2 testing were performed. Then each sample was stored in a 1.5 mL microtube at − 80 °C for further study.The DNA of cervical scraped cells was extracted according to the previous publications , 16. TheThe nested PCR assay was performed for HPV typing. Firstly, the primer pair MY09 (CGTCCAAAAGGAAACTGAGC) /MY11 (GCACAGGGACATAACAATGG) was used to amplify the extracted DNA. The 20 μL PCR reaction volume contained 1 μL of the extracted DNA, 10 μL 2× Power Taq PCR MasterMix (BioTeke), 2 μM MY09 primer and 2 μM MY11 primer. The reaction condition was 94 °C for 5 min, followed by 38 cycles of 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min, and then 72 °C for 10 min. Secondly, the primer pair GP5+ (TTTGTTACTGTGGTAGATACTAC) /GP6+ (GAAAAATAAACTGTAAATCATATTC) was used as previously described , 18. 5 μAccording to the HPV-58 prototype D90400.1, two partially overlapping fragments of the HPV-58 LCR were amplified by using two pairs of primers to obtain the entire LCR. Primers used for the amplification of the complete nucleotide sequence of the HPV-58 E6 and E7 were described previously . All theThe E6, E7 and LCR fragments were amplified in 50 μL PCR reaction volumes containing 2 μL of extracted DNA, 25 μL 2× Power Taq PCR MasterMix (BioTeke), 0.4 μM forward primer and 0.4 μM reverse primer. The PCR condition was: 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 55 °C for 45 s, 72 °C for 50 s, and then 72 °C for 10 min. The positive fragments were subjected to the bi-directional DNA sequencing  for further analysis. The sequences obtained in this study were submitted to the NCBI GenBank database and got the accession number: MF741321 to MF741331.To identify the SNPs in the LCR, E6 and E7, the studied HPV-58 LCR/E6/E7 sequences were aligned to the HPV-58 prototype sequence (GenBank: D90400.1) by MEGA (version 7.0)  after sehttp://bioinf.cs.ucl.ac.uk/psipred/) using the default parameters [The secondary structures of the HPV-58 E6 and E7 proteins were predicted by PSIPRED v3.3 (rameters .The neighbor-joining phylogenetic tree based on the HPV-58 LCR/E6/E7 was constructed by MEGA software (version 7.0) using the Kimura 2-parameter model. The number of bootstrap replications was set at 1000. To construct the phylogenetic branches, the following reference sequences were used: D90400.1 (A1), HQ537752 (A2), HQ537758 (A3), HQ537762 (B1), HQ537764 (B2), HQ537774 (C), HQ537768 (D1) and HQ537770 (D2) [To estimate the selection pressure acting on the HPV-58 E6 and E7 coding regions, the non-synonymous and synonymous nucleotide divergence were calculated by CODEML in PAML 4.8. Using the Bayes empirical Bayes (BEB) analysis, the sites with the posterior probability > 95% were identified as the positively selected sites –25.http://www.imtech.res.in/raghava/propred1/) and ProPred server (http://www.imtech.res.in/raghava/propred/) were used to predict the major histocompatibility complex (MHC) class-I binding peptides [The ProPred-I server  [To analyze the transcription factor binding sites in the HPV-58 LCR, the JASPAR database (eg.net/)  was usedAmong the 2146 collected outpatient samples in Southwest China, 248 samples  were HPV positive . Followi12 entire LCR/E6/E7 sequences were obtained and further analyzed. The small copy number of HPV in some women might be a possible explanation for not all sequences successfully amplified. When aligned to the HPV-58 reference sequences (GenBank: D90400.1), a total of 29 single SNPs were identified in 27 nucleotide sites across the LCR, E6 and E7. The combinations of all these SNPs made for 11 unique LCR/E6/E7 sequences (variant IDs: 1 to 11), of which the variant 2 was represented in 2 samples . C7265G, C7266T and A7793G were the most variable sites and were identified in 27% of the variants (3/11). In addition, an insertion (TGTCAGTTTCCT) was found between the nucleotide sites 7280 and 7281 in 2 variants , and a deletion (TTTAT) was found between the nucleotide sites 7429 and 7433 in 1 variant (variant ID: 2). In the E6, 3 SNPs were detected . C307T and A388C were identified in 36% variants (4/11). C321T and A388C resulted in the amino acid changes including S71F and K93 N, respectively. No non-synonymous variations were observed in the E6 sequences encoding the α-helix or the β-sheet. In the E7, 6 SNPs were detected , of which T744G was identified in 64% variants (7/11), and 5 SNPs resulted in amino acid changes including T20I, G41R, G63S, G63D and V77A. The non-synonymous variation V77A was detected in the E7 sequences encoding the α-helix. There was no insertion or deletion detected in either E6 or E7 sequences.A neighbor-joining phylogenetic tree based on the HPV-58 LCR/E6/E7 was inferred from 11 obtained unique variants and 8 reference sequences. As shown in Table The selective pressure analysis results by the Bayes empirical Bayes (BEB) analysis of PAML 4.8 were summarized in Table To evaluate the impacts of the HPV-58 E6 and E7 sequence polymorphisms on the immune recognition of these antigens, the binding peptides specific for both MHC class-I and class-II were predicted by the ProPred-I and ProPred servers, respectively. The results showed that the non-synonymous substitutions within the HPV-58 E6 and E7 were potentially affecting the multiple epitopes. The results were summarized in Table The potential binding sites for the cellular transcription factors in the HPV-58 LCR variants were investigated by the online tool JASPAR. The results indicated the affected transcription factor binding sites due to some of the nucleotide variations spanning nucleotides 7140 to 109. The variations at the nucleotide sites 7265 and 7266 were suggested to be the binding sites for the transcription factor SOX10, and 7304 and 7714 for SOX9. In addition, the nucleotide sites 7172, 7540, 7556, 7749, 7755, 7793, 7807, and 24 potentially affected the binding sites for the transcription factors FOXA1, SRY, ELK4, FOS, FOXP3, ESR2, CEBPA, and HOXC11, respectively.A specific geographic and population research on the characterization of each HPV type is important to learn the HPV prevalence, find out the biological evidences of carcinogenesis, and help improving the strategies of vaccine development. So far, the HPV family is comprised of more than 170 different types . They caIn the HPV-58 genome, each region differs in the sequence diversity. The HPV-58 LCR variants from Yunnan, Southwest China have not been reported yet. Here, the variation of the HPV-58 LCR was showed in a higher ratio than those of the E6 and E7 in Yunnan, Southwest China. From the most variable to the least variable, the nucleotide variations were LCR > E7 > E6 found in 2.3%, 2.0% and 0.7%, respectively, of the studied variants. Additionally, the most common substitutions in the HPV-58 LCR were C7265G, C7266T and A7793G. And the most common non-synonymous substitutions in the HPV-58 E6 and E7 were A388C (K35 N) and T803C (V77A), respectively. V77A in the E7 was observed in the sequences encoding the α-helix. Whether this substitution changed the secondary structure of the E7 protein and then affected the potential ability of the E7 to cause cervical cancer needs to be further studied. Besides, 63G in the HPV-58 E7 coding sequences was determined to be a positively selected site, indicating an adaptive molecular sequence evolution.The HPV variants co-evolve with the specific populations and their prevalence varies across the geographic regions. In this study, phylogenetic analysis based on the HPV-58 LCR/E6/E7 revealed a high prevalence of the A lineage, among which the A1 sublineage with a proportion of 64% accounted for the majority of HPV-58 variants obtained. No variant belonged to the lineages B, C, or D. Nevertheless, the B lineage was found in Sichuan  and HenaThough phylogenetically related, the HPV-58 variants can differ in pathogenicity. The occurrence of C632T and G760A in the HPV-58 E7, which were identified in our study and resulted in the amino acid changes including T20I and G63S, respectively, has been implied to be linked to an increased risk for cervical cancer . The amiThe HPV LCR has been shown to contain kinds of the binding sites for both viral and cellular factors and regulate the replication of HPV and the transcriptional activity of the E6 and E7 through the interactions between the regulatory protein complex and the viral DNA , 13, 36.This study provides a useful data for understanding the phylogenetic relatedness of the HPV-58 variants, the possible influence of non-synonymous substitutions in the HPV-58 E6/E7 on the T-cell responses, and the impacts of the HPV-58 LCR variations on the bindings of the cellular transcription factors. Further studies of the HPV-58 variant genomes from a large population of the asymptomatic carriers and cervical cancer patients are required to establish the carcinogenesis of HPV-58 and to make improvements to the immunotherapeutic approaches and vaccine development strategies.Additional file 1:Table S1. The PCR primers for validating the variations of HPV-58 LCR, E6 and E7. (DOC 33 kb)


Approximately 200 human papillomaviruses (HPVs) infect human epithelial cells, of which the alpha and beta types have been the most extensively studied. Alpha HPV types mainly infect mucosal epithelia and a small group of these causes over 600,000 cancers per year worldwide at various anatomical sites, especially anogenital and head-and-neck cancers. Of these the most important is cervical cancer, which is the leading cause of cancer-related death in women in many parts of the world. Beta HPV types infect cutaneous epithelia and may contribute towards the initiation of non-melanoma skin cancers. HPVs encode two oncoproteins, E6 and E7, which are directly responsible for the development of HPV-induced carcinogenesis. They do this cooperatively by targeting diverse cellular pathways involved in the regulation of cell cycle control, of apoptosis and of cell polarity control networks. In this review, the biological consequences of papillomavirus targeting of various cellular substrates at diverse anatomical sites in the development of HPV-induced malignancies are highlighted. Persistent infection with the human papillomaviruses (HPVs) is the main risk factor in the development of numerous human malignancies at various anatomical sites ,2. Of thβ-HPVs commonly infect cutaneous epithelia. Originally they were found to be present in skin warts and in cutaneous squamous cell carcinomas (CSCC) of patients with the rare disease epidermodysplasia verruciformis (EV) . In EV pAlpha-HPV types infect mucosal epithelia and, as demonstrated in Much information about the viral life cycle has been obtained from the organotypic raft culture model system, which reconstructs the full productive life cycle of HPVs in vitro . In thisThere have been few successful attempts to study β-HPVs life cycles; although some limited information has been obtained from organotypic raft culture studies that have shown certain similarities between LR α types and β types. Like HPV-11, HPV-8 episomal genomes are not well maintained in tissue culture and tend to be lost over time. In addition, it appears that HPV-8 viral genomes do not increase the cellular growth rate, which is why the complete HPV-8 life cycle has not been reproduced in this in vitro system ,25. ThisThe normal productive viral life cycle of the HR α-types is a highly regulated and coordinated process. However, in some instances, mostly during persistent infection, the viral DNA is randomly integrated into the host genome, leading to cellular immortalization and eventually to malignant progression. The outcome of this process is the collapse of the viral replicative capacity: most of the viral genes are lost while the two major viral oncogenes, E6 and E7, remain uncontrollably expressed, driving cellular immortalization, further progressing towards cellular transformation, and ultimately resulting in cancer development ,32. InteThe HPV E6 protein is approximately 150 amino acids long and has two zinc fingers formed by two pairs of CXXC motifs ,47 FiguA. These On its own, E6 cannot target p53 for proteasomal degradation, rather, it hijacks a cellular E3 ubiquitin ligase UBE3A/E6AP (E6-associated protein), binding through E6’s LXXLL motif, and the stable E6/E6AP complex then labels p53 for degradation in a proteasome-dependent manner ,62,63,64Although the major anti-apoptotic activity of E6 is its inactivation of p53, it also has a number of other cellular pro-apoptotic targets, including Bak. Bak is highly expressed in the upper epithelial layers, indicating that it could play a role in terminal differentiation . After UIn addition to their association with pro-apoptotic cellular proteins, HR E6 oncoproteins are also involved in the deregulation of the cellular DNA replication machinery. Normal somatic cells end their life by entering senescence, which results from the progressive shortening of telomere DNA over successive rounds of replication, and most normal cells do not exhibit telomerase activity . HoweverPSD95/Dlg/ZO-1)-binding motif (x-T/S-x-L/V [All HR α-HPV E6 proteins have a class I PDZ (/S-x-L/V ) at thei/S-x-L/V  and PDZ /S-x-L/V . PDZ dom/S-x-L/V . The two/S-x-L/V ,45. Seve/S-x-L/V ,90 and t/S-x-L/V . The PBM/S-x-L/V ,93.The specificity of the PDZ interaction is an important factor in E6-PDZ target recognition, and different E6 proteins interact preferentially with different PDZ domain-containing proteins ,8,45,94.Interestingly, the HR PBM can also be post-translationally regulated. It contains a protein kinase A (PKA) phospho-acceptor site, and its phosphorylation by either PKA or AKT blocks PDZ binding and instead allows E6 to interact with 14-3-3ζ , a membeHPV E7 is a 98 amino acid protein that has a C-terminal zinc-binding domain, whose structural integrity is critical for E7 activity ,106. TheE7 is post-transcriptionally regulated by the proteasome and by phosphorylation. It interacts with the Skp-Cullin-F box (SCF) ubiquitin ligase complex, which leads to increased ubiquitination of the protein . It has The CD1 domain comprises the first 20 amino acids of E7 and is critical for E7’s ability to induce S-phase progression and cellular transformation ,113,114 Amino acid residues from 20 to 38 constitute the CD2 region of E7 B. This cpRb has a critical role in cell cycle regulation, controlling the G1 to S phase transition. Under normal conditions, pRb is unphosphorylated in early G1 and is progressively phosphorylated towards S phase. Unphosphorylated pRb interacts with the E2F transcriptional factors and acts as a transcriptional repressor of promoters containing E2F sites . E2F famIn addition to the pRb interaction, E7 also binds to the other pocket proteins p107 and p130. These play important roles in the regulation of cellular proliferation, differentiation and apoptosis, through their interactions with different molecules . They inAmino acid residues 38–98 constitute the C-terminal CD3 region B, which Centrosomes are the major microtubule organizing centers in the majority of animal and human cells . Under nBoth E6 and E7 oncoproteins have been shown to independently cause centrosomal abnormalities when stably expressed in cell culture and in transgenic mouse models ,148. HowThe most extensively investigated site of HPV-induced malignancy is the anogenital region, where almost all cancers arise at the cervical or anal transformation zones ,155, wheRecently, HR HPV infections have also been associated with a number of head-and-neck cancers (HNSCC), the majority occurring in the oral cavity and the tonsillar crypts ,164,165.About 80% of cervical cancers are caused by HPV-16 and HPV-18 and many E6 and E7 cellular targets are common to both virus types, with any differences lying only in the degree of interaction. In contrast, virtually nothing is known about their cellular substrates in the oropharynx, which may be very different owing to the tissue complexity and the surrounding environment. Biochemical and mechanistic studies of the viral oncogenes and their interacting partners in oropharyngeal cells are needed to clarify potential differences both in the productive life cycle of the virus and in virus-induced malignancy.The E6 and E7 oncoproteins of the β-HPV types share some common interacting partners and affect similar cellular pathways as the HR α-HPV oncoproteins, but there are also some significant differences.Unlike HR α-types, the majority of the β-type E6 proteins do not target p53 for proteasome-mediated degradation . Howeverβ-type E6 proteins interact very strongly with MAML1, through a binding motif similar to the LXXLL motif used by α-type E6s to bind E6AP ,173. MAMLike α-HPV types, β-types also interfere with the expression of p53-regulated genes by associating with the transcriptional co-activators CBP and p300. The E6s from HPV-5, HPV-8 and HPV-38 all interact with p300 ,177, andThe E6 proteins of β-HPV types 5, 20, 22 and 38 also associate with and activate hTERT in an E6AP-dependent manner, albeit to a lesser extent than HPV-16 E6, but the E6 proteins from other β-types show a significantly lower ability to activate telomerase . FurtherLike the α-type E6 proteins, the β-HPV E6s can abolish p53-dependent and -independent apoptosis, which is induced in cutaneous sites in response to UV-induced DNA damage . β-typesCellular interacting partners of β-HPV E7 proteins have not been as well-characterized as those of the α-type HR E7s. However, they also contain an intact LXCXE binding motif and interact with the pocket proteins pRB, p107 and p130 ,185, butIn addition to its role in regulating cell cycle progression, HPV-38 E7 was shown to induce changes in p53-regulated gene expression ,187,188.Only two β-HPV types, HPV-8 and HPV-38, have actually been shown to promote tumorigenesis in transgenic mouse models ,191. WheThe environment of viral infection of the β-HPV types is significantly different from the environment where HR α-types infect their host cells. Different β-HPV types infect and cause lesions in various different types of skin, commonly in proximity to hair follicles or nails, as well as between the digits and at the eccrine and apocrine sweat apparatus , all of It is becoming clear that whilst there are some intriguing similarities between alpha and beta HPV types, there are also many major differences, and this is most likely a reflection of their differing sites of replication. In addition, it is also apparent that the same virus type, for example HPV16, might well display different properties depending upon the site of infection. Certainly its cancer-causing activity appears to be much higher in tissues with a transformation zone, such as the cervix, anus or nail-bed, than in those without. Thus, whilst we are gaining a greater understanding of how the viral oncoproteins function, there is still much to learn about their respective activities in different tissue types and environmental settings.


Rabies is a fatal disease, claiming the lives of around 59,000 people annually worldwide. It is considered a neglected and underreported disease leading to inadequate support from governments. Apart from dog vaccination and proper animal bite management, an integral part of a successful rabies control program is community education. The Philippine government conducts an extensive nationwide annual World Rabies Day (WRD) celebration as part of its community education.Strong inter-sectoral collaboration at the national level is a key factor for the success of WRD, capitalizing on the partners’ strengths to mobilize various sectors. Strategies include the National WRD Celebration and releasing national government memorandums. An invitation letter campaign was initiated, encouraging stakeholders to register their activities. Banners were given as an incentive for those who registered. Mass and social media were also utilized to promote WRD.Registered WRD events held in the Philippines rose from 10 events in 2012, to 37 events in 2013, to 66 events in 2014 and 76 events in 2015. The individual activities involved veterinary services and information, communication, and education (IEC) activities. Nine unique WRD IEC activities are highlighted in this paper. Promotion of WRD through social media was also utilized in recent years. More news items were published online than those printed in newspapers and aired on television.The campaign’s success underlines the value of a national government-led program. The national rabies program sets the agenda for priority activities including the WRD campaign. Its capacity to allocate funds for the program also denotes stability which is beneficial for local program implementers. Different segments of society were tapped through various strategies. The campaign’s flexibility allowed for a large range of activities and presented opportunities for expanding partnerships and integration with others interventions for its sustainability. With appropriate tools and government support, the extensive WRD campaign in the Philippines can be replicated in other countries. The strategies discussed prove that since different localities celebrate WRD in their own way, other countries can also organize activities adapted to their culture and contribute to the global campaign against rabies. Canine-mediated rabies is a fatal viral disease that continues to kill approximately 59,000 people annually on a global scale . It is mThe World Health Organization (WHO) categorizes rabies among the ‘neglected zoonotic diseases.’ This refers to a group of diseases that are insufficiently addressed by governments and the international community . In mostFurther proof of the neglected disease status of rabies is that many developing countries still lack the proper facilities and personnel to conduct rabies surveillance and diagnosis . This leTragically, it is the communities living in the poorest regions in developing countries that suffer the brunt of rabies. It remains endemic in most parts of Asia and Africa where over 90 % of human rabies deaths occur at present. In rabies-endemic Southeast Asian countries , about 608 million people are at risk of rabies infection and from 2009 to 2013, rabies was reported to have been the cause of death of approximately 3475 people and 7617 animals [In the Philippines, rabies remains a public health concern and is the most fatal infectious disease. At least one-third of these deaths occur in children aged 15 years old and below . Data shThe World Rabies Day (WRD) campaign was created by the Global Alliance for Rabies Control (GARC) in 2007 in partnership with WHO, World Organisation for Animal Health (OIE), United Nations Food and Agriculture Organization (FAO), the Centers for Disease Control and Prevention (CDC), and the Pan-American Health Organization (PAHO) as a response to the call to raise global awareness and mobilize resources for human rabies prevention and animal rabies control. It is celebrated annually every September 28 and is the largest unifying initiative focused specifically on the prevention of human and animal rabies [The goal of the WRD campaign is to increase and sustain global awareness and encourage different sectors to get involved and organize activities that would cascade life-saving information on rabies, animal bite prevention and management, and responsible pet ownership to communities. It also provides a platform to urge governments to support the implementation of national rabies prevention programs , 8. WRD GARC serves as the overall coordinator of the campaign including the monitoring and promotion of WRD activities as well as collaborating with partner organizations and governments to extend the campaign’s reach . The SouThe WRD celebration in the Philippines is anchored on Republic Act No. 9482 also called the Anti-Rabies Act of 2007 . This acIt is the goal of the NRPCP to eliminate rabies and declare the Philippines as a rabies-free country by the year 2020. To achieve this, the Program implements the following key components based on its Manual of Operations—Post Exposure Prophylaxis and Pre-Exposure Prophylaxis; health promotion; dog vaccination; dog population management, a central database system; and responsible pet ownership .Among the strategies adopted by the NRPCP under its health promotion component is the annual WRD celebration in the country. This case study will discuss the strategies of the extensive WRD campaign in the Philippines as a component of its national rabies prevention and control program. It will include data from 2012 to 2015 as documented by GARC.The Philippines, through its national and local government units, has celebrated WRD since its launch in 2007. A crucial factor in the effective implementation of the WRD campaign is the strong collaboration among the various sectors involved. Figure The NRPCC holds several meetings months in advance to prepare for WRD. Information regarding the celebration is then cascaded to the sub-national offices of government committee members. In the case of DILG, information is disseminated to the LGUs from the provincial to the municipal level. This has proven to be effective in encouraging participation of the LGUs as the Philippines implements a decentralized government. This implies that LGUs have the capacity to allocate resources for their rabies control programs including the celebration of WRD in their respective localities through their veterinary and health offices . Other mAs a multi-sectoral committee, the NRPCC espouses a ‘One Health’ approach in controlling and eliminating rabies in the country. The concept of One Health is not new and has been constantly explored and re-discovered over the course of history. While there is no universally accepted definition of One Health, it is fundamentally rooted in the belief that the well-being of humans, animals, and the environment are all interdependent .The One Health approach has gradually gained momentum over the past decades. As the world continuously strives for convergence and globalization, it also brings upon threats and issues that shape the landscape of human health, animal health, and ecosystem health . One HeaIn the context of neglected zoonotic diseases such as rabies, the need for a One Health approach is crucial for disease control and prevention as it entails the collaboration of various stakeholders. While they may use different approaches to control and prevent the disease, these efforts are effectively integrated through the One Health approach . The WRDA key feature of the WRD campaign is its flexibility. Provided with a global theme, participants have the opportunity to organize a diverse range of WRD activities that are tailor-made for their country. Fundamental messages on rabies prevention and control can be translated into activities based on their cultural relevance . For theThe highlight of the National WRD Celebration is the Declaration of Rabies-Free Zones. Of the seven rabies-endemic countries in Southeast Asia, the Philippines is among only three  that have an official internal process for declaring rabies freedom in parts of the country . This inFor DA-BAI, WRD is celebrated concurrently with the Animal Welfare Week. This joint celebration highlights the importance of promoting animal welfare as part of rabies elimination. It also seeks to increase public awareness of the importance of vaccinating dogs against rabies .NRPCC member-agencies such as DepEd and DILG have also been equally supportive of the WRD celebration. In 2008, DepEd released a Memorandum Circular enjoining all DepEd units and offices as well as public and private elementary and secondary schools to organize educational and social activities to strengthen rabies prevention education and disseminate information on responsible ownership, immunization, and vaccination of pets . A similAnother strategy which was valuable in promoting the WRD celebration was the invitation letter campaign initiated by GARC in close coordination with the NRPCC. Letters were sent to the DOH and DA-BAI sub-national offices, veterinary schools, LGUs, and other government agencies nationwide encouraging them to conduct and register their WRD activities on the GARC website. The letter campaign has evolved through the years, adapting to the advances in technology. From sending letters only through fax in 2012 and 2013, invitations were instead sent to event organizers mainly through email in 2014 and 2015. Online registration of events makes it easy for organizers to add their events to the GARC website themselves. Promoting WRD using social media, particularly through Facebook, was especially helpful in getting the public involved. A “text brigade” or the mass sending of short message service (SMS) or text messages was also initiated by PCMVLP for its members.As support for local WRD activities, banner tarpaulins (designed in consultation with the NRPCC) were distributed as an incentive for those who registered their activities on the GARC WRD web platform. In 2015, the DOH National Center for Health Promotion led the creation of a standard banner design. Event feedback was obtained by contacting event organizers to thank them for their participation in WRD. A brief update of the activity was also requested along with consent to post of their photos on GARC’s social media accounts. This mechanism allowed GARC to document the outcomes of the activities including the estimated number of people reached by these initiatives.The WRD celebration was also publicized through mass media. Numerous media outlets nationwide covered the WRD activities held all over the country. News items, press releases, online posts, and video clips, among others released before and after WRD, were monitored and collated.For the past 4 years (2012 to 2015), the Philippines has registered one of the largest number of WRD events globally, as documented through the GARC WRD platform. ‘Events’ generally refer to a set of activities conducted by a WRD event organizer  and organizers may carry out several individual ‘activities’. Figure Events held in the Philippines accounted for 8 % of the global total in 2012, rising to 25 % in 2014 and 2015 as seen in Fig. It should be noted though that not all those who were listed were given WRD banners see Fig. . The orgAs the letter campaign was aimed at the LGUs, most of the event organizers, not surprisingly came from this sector. For 2015, the large number of LGUs that registered can also be attributed to the Memorandum Circular from DILG that were sent out. In some cases, the sub-national offices assisted the LGUs in their WRD activities.As part of the national WRD celebration, local governments throughout the country conducted activities such as mass dog vaccination and information, education, and communication (IEC) campaigns to coincide with the launch of WRD activities each year. While there is a national directive for LGUs and agencies to conduct activities in celebration of WRD, only a portion of the estimated total number of these activities was documented through the invitation letter campaign. Table While there were more IEC activities conducted over the years (since it covered a wider range of activities), the veterinary services were an equally significant element of the WRD celebration in the Philippines. The greater part of the activities in this category involved mass dog vaccination which was generally accompanied by lectures and seminars educating the community on the impacts of rabies and the importance of having their dogs and cats vaccinated against the disease. LGU veterinarians usually provide these services for free during WRD to promote their rabies control and elimination programs and encourage more people to avail of the services. For the mass dog vaccination, staff from the Provincial and City Veterinary Office or Municipal Agriculture Office either went from house to house or set up vaccination stations in public areas  to ensure that the majority of the dogs and cats in the locality were vaccinated.The organizers have shown great imagination and effort in conducting the IEC campaign with activities ranging from fun runs and dog shows, to various school-based and community-based competitions, and the use of mass media, among others. Based on organizers’ feedback, it is estimated that more than 100,000 Filipinos were reached and educated on rabies prevention and control through the various community and school-based IEC activities held as part of WRD. For the purpose of this paper, we have chosen to highlight nine innovative WRD IEC activities conducted in the Philippines over the years. Table Various communication channels were also utilized to further promote the observance of WRD to a wider audience. Table In 2012 and 2014, print advertisements see Fig.  were plaPromotion through radio and television was also essential in involving more people in the campaign. Several major television networks covered the national WRD celebration. Other networks invited members of the NRPCC on to their shows to talk about WRD, rabies, animal bite prevention and control, and responsible pet ownership .While the contributions of print media and television cannot be disregarded, placing an advertisement in the primary segments of newspapers was costly. Hence, social media has been increasingly utilized to promote WRD to the general public over the years since most newspaper outlets and television stations also have online portals and Facebook pages. Filipinos are among the most active internet users in Southeast Asia, spending an average of five hours daily using the internet. There are also 47 million active Facebook users in the country as of 2015 which makes it a more practical and cost-effective platform to promote the campaign and reinforce key messages on rabies control and prevention .In 2014, a digital advertisement was placed on the Philippine Star’s website which ran on its leaderboard on five pages  for 5 days that extended after WRD. The advertisement was linked to GARC’s website to provide more information on the campaign. Online articles about WRD posted by different websites were also monitored and collated. These include articles from newspaper, radio, and television online portals, official websites of LGUs and national government agencies including the Philippine Information Agency, and personal web blogs, to cite a few. The NRPCC promoted individual WRD activities on their official Facebook page as well .Rabies continues to be a largely underreported and neglected disease in many developing countries. The majority of rabies cases remain unreported hence there is a lack of accurate data reflecting the grave impacts of the disease. As a result, rabies remains under the radar of most government policies and programs and this is considered one of the challenges in controlling canine-mediated rabies in rabies-endemic areas .The Philippines is among the eight Southeast Asian countries that have committees designated to specifically address and prioritize the prevention and control of rabies . The sucThe NRPCC’s inter-sectoral approach has also been an essential factor in encouraging the participation of the public in the WRD celebration. Each sector is appropriately represented through its members, which allows the NRPCC to maximize the strengths of each agency and its networks. Notable examples are the Memorandum Circulars released by DILG and DepEd to tap local policy-makers and students, respectively to celebrate WRD. It also highlights the importance of a close partnership between the public and private sector since each agency and organization has something to offer to make the campaign a success.While campaign organizers have already made great strides in the celebration of WRD, there are still opportunities to further improve it. Its annual celebration can open outlets for other interventions and for the integration of other diseases. For instance, the Provincial Veterinary Office of Ilocos Norte plans to include other diseases such as dengue in their annual Provincial Rabies Quiz Bee. On the other hand, the Marikina City Veterinary Office was able to tackle two community issues (i.e. conserving the environment and rabies control and prevention) through their Trash-to-Art contest for students. Inter-sectoral networks can also be expanded to include other partners. Since WRD is celebrated alongside the Animal Welfare Week, different Animal Welfare groups can be involved in the implementation of the campaign, sharing the same advocacy effort.Based on the results of the invitation letter campaign, which was the main method used to gather information, there is reason to believe that the number of WRD activities that took place in the country far exceed those that were registered. The strategies mentioned in this review each possess strong and weak points. The letter campaign in particular was valuable in promoting WRD to different stakeholders. The LGUs responded positively to the campaign particularly on the provision of banners. To sustain their active participation in the campaign including the registration of their WRD events, a potential way forward would be to capitalize on the strength of social media. With the fast-paced advancements in technology and the growing number of social media users in the country, the possibility of registering WRD activities through Facebook can be explored. For a more thorough analysis of the impacts of the activities, the feedback mechanism can also include interviews and surveys for the organizers.In addition to WRD, the Philippines also celebrates Rabies Awareness Month annually every March as mandated by a Presidential Executive Order. Given that WRD is a 1-day event, the government can use this month to keep up the momentum in raising the public’s awareness and strengthening rabies control and prevention efforts nationwide.The activities mentioned here are just a small number of the overall picture of the WRD celebration in the Philippines but we believe that the wide range of activities discussed in this paper more than adequately demonstrates the adaptability of the campaign. With a global theme as a guide, organizers are given the opportunity to think outside the box and create innovative activities for different segments of society. The WRD campaign also recognizes and intensifies the existing rabies control programs and activities of the partners. This is especially true for local rabies program implementers who showcase their regular dog vaccination and IEC activities by providing free services during the WRD celebration. These activities can likewise be replicated by other countries and adapted based on their culture and traditions.In countries such as the Philippines where rabies is still endemic, the WRD celebration presents an opportunity to educate and raise the awareness of national and local government staff as well as communities on how fatal rabies is, and call for action for the elimination of this deadly disease.The World Rabies Day campaign in the Philippines owes its success to the enthusiasm and active involvement of the public and private sectors in the country. It clearly demonstrates that an extensive campaign can be achieved through multi-sectoral support and participation.The strategies discussed also demonstrate that different localities were able to celebrate WRD in their own unique way. Therefore, other countries can also organize their own WRD activities, no matter how big or small, and contribute to the global campaign against rabies.


We report here that HPV infection associates with a 2.5-fold increase in HIV acquisition risk in this population (95% CI: 1.2–5.3). Among 48 vaginal cytokines profiled, cytokines associated with HPV infection overlap substantially with cytokines associated with HIV risk, but are distinct from those observed in HPV negative women. Although our data do not establish a causative link between HPV status and the risk of HIV, we suggest that increasing HPV vaccination coverage may carry an additional benefit of reducing the risk of contracting HIV infection, particularly in regions with high HPV prevalence.Human papillomavirus (HPV) infection correlates with higher rates of HIV acquisition, but the underlying biological mechanisms are unclear. Here we study associations between HPV and HIV acquisition and relate these to vaginal cytokine profiles in an observational cohort of women at high risk of HIV infection  are separately associated with increased risk of HIV acquisition. Here the authors longitudinally profile 48 cervicovaginal cytokines and HPV status in a large observational HIV high-risk cohort, and show the same cytokines associate with HPV infection and HIV risk. Almost all cases of cervical cancer are caused by human papillomaviruses (HPV)3. These DNA viruses preferentially infect basal epithelial cells, and their effective evasion of host immunity can ultimately result in the formation of intraepithelial lesions and cervical cancer. Over 40 HPV types are known to infect the human genital tract, with 12–20 classified as being oncogenic or “high risk” for causing cervical cancer6.Cervical cancer disproportionately affects women in southern Africa, with double and triple, respectively, the incidence and mortality rates that are observed worldwide7. These types have been included in each of the three HPV vaccines showing high efficacy in preventing HPV infection11. Because the risk of HPV infection is known to be very high following sexual debut12, efforts are underway to implement these vaccines in young women worldwide, as the degree of protection is much higher in HPV uninfected individuals.Two vaccine-preventable HPV types, HPV-16 and -18, contribute to over 70% of cervical cancer cases13, but also human immunodeficiency virus (HIV) infection16. In addition to its role as a major factor in the development of cervical and several anogenital cancers, HPV has also been associated with increased risk of HIV acquisition18. Multiple HPV strains can coinfect the same individual, thus life history of HPV infection can include simultaneous persistence, clearance (non-persistence), and/or acquisition of different HPV strains. Increased HIV infection risk in sub-Saharan Africa has been associated with prevalent HPV infection, infection with either oncogenic or non-oncogenic HPV types, infection with non-oncogenic HPV types, and concurrent infection with multiple HPV types21. Clearance of HPV infection occurs relatively quickly, with about half of infections clearing by 6 months; and more slowly over the next few years, with an average persistence of ~10 months22. In two studies that assessed HPV persistence and HIV risk empirically in sub-Saharan Africa, non-persistence was associated with increased HIV risk21, suggesting that active immune responses to HPV might underlie the observed HPV–HIV associations.Young women in sub-Saharan Africa bear a disproportionate burden of not only HPV24. However, the stimuli responsible for the elevated cytokine concentrations remain unclear. Here we hypothesized that the female genital immune environment may differ according to HPV infection status; a concept that, if confirmed, would shed light on possible mechanisms for the reported associations between HPV infection and HIV risk. It is biologically plausible for cervical HPV infection, or other potentially correlated changes in the female genital tract to impact HIV risk, since sexual transmission of HIV is primarily mediated through interaction of the virus with genital cellular targets for infection, in the same mucosa where HPV replicates. It is therefore reasonable that immune cell recruitment to eliminate or contain HPV infection in the genital epithelium may promote an immune environment that favors HIV infection. A precedent for this is supported by the requirement of an inflammatory response, particularly those mediated by T and NK cells, to effectively facilitate HPV-associated genital wart or CIN1 regression30. These activated NK cell correlates of HPV clearance are also biomarkers of increased HIV risk in CAPRISA 004 participants31; the T cells are relevant to HIV infection as preferred targets of infection (CD4+ T cells expressing the CCR5 co-receptor for HIV entry i.e. CD4+CCR5+ T cells)34. Given that inflammatory cytokines in cervicovaginal lavage (CVL) fluid are strong surrogates of both absolute numbers of endocervical HIV target cells and impaired barrier function35, we measure here multiple cytokines relevant to cell recruitment (chemokines), regulation, barrier growth and repair, and mediation of adaptive and pro-inflammatory immune responses, and correlate these to HPV status over time.Previous work by our group has demonstrated that elevated mucosal cytokines in the reproductive tract of HIV-uninfected women are strong predictors of subsequent HIV infection outcome36, we characterize the epidemiology and mucosal immunology of HPV–HIV relationships in this setting. Specifically, we leverage the existing data and specimens collected as part of the CAPRISA 004 effectiveness study of tenofovir gel, carried out in high-risk women from rural and urban KwaZulu-Natal37. These data provide insights into the epidemiology of HIV and warrant investigation on whether HPV vaccines can decrease the risk of HIV infection.Given the high HIV incidence that has been observed in KwaZulu-Natal, particularly in young women aged 15–24 yearsp < 0.001; Wilcoxon rank sum test), marital status , living away from the regular sex partner , and baseline infection with Chlamydia trachomatis .HPV DNA was detected in 73.8% (95% CI 70.7–76.9%) of participants at baseline , 51 (9.8%), and 35 (9.4%), and the non-oncogenic viruses HPV 84 (11.7%) and 62 (9.6%). Approximately one-quarter of the cohort (24.3%) was infected by a single HPV type. The proportions of participants infected by 1–2, 3–5, >5 HPV types were 42.1%, 25.4%, and 6.3%, respectively, suggesting that multiple-type HPV infections are relatively common in this population.About half of participants were infected with an oncogenic HPV type , and baseline coverage of vaccine types was 16.8%, 22.0%, and 41.3% for the Cervarixp = 0.015; Cox proportional hazards regression). Infection with oncogenic HPV was associated with a 2.8-fold increased HIV risk , during which 66 HIV infections occurred ; Table 38. Similarly, the term “acquired” here refers to the absence of DNA for an HPV type at one visit but the detection of it at the next consecutive visit, acknowledging that this definition may reflect new or re-infection, or reactivation39. Clearance of any HPV type was observed in most participants , clearing any HPV type , and for persistence of any HPV type ; Category 2, women who only cleared HPV ; Category 3, women who only acquired HPV ; and Category 4, women with no changes to the HPV types detected at both visits. The remaining categories captured women who were HPV DNA positive at both visits, with combinations of HPV acquisition, clearance and/or persistence. These women were classified as Category 5, women who both cleared and retained some HPV types; Category 6, women who both cleared and acquired some HPV types; Category 7, women who persisted and acquired some HPV types; and Category 8, women with evidence of clearing, acquiring, and persisting with some HPV types  were elevated in the genital tract of HPV positive women relative to HPV negative women , acquisition (N = 404) or persistence (N = 247) were observed, respectively, relative to remaining HPV negative , both cleared and acquired (Category 6), and where it was both acquired and persisted  to examine unsupervised multivariate clustering of cytokines by HPV group Fig. . Women w41, the data underscore the importance of further investigation to fully understand the complex immunological environment that may mediate the increased risk of HIV associated with HPV infection.This study confirmed the epidemiological link between HPV and increased risk of HIV infection, and tested the hypothesis that the female genital tract cytokine milieu would differ by HPV status. The data presented are in support of this hypothesis, particularly in women who clear their HPV infection. Since clearance was most consistently associated with increased HIV risk, and with the broadest cytokine response, including all cytokines previously associated with HIV risk in this cohort42, but markedly higher than the 20.8% observed in Cape Town43, where the HIV prevalence is the lowest in South Africa. The 2.5-fold increase in HIV infection observed for women with prevalent HPV in our study is in line with the HPV–HIV infection risk estimates published in the meta-analyses, which showed an approximate 2-fold effect18. Similarly, the estimates in our associations between HIV risk and HPV clearance and persistence resemble those observed in another study in sub-Saharan Africa that reported increased HIV risk when any HPV types were cleared, but no association when only type-specific persistence occurred19. This study is the first to investigate the impact of tenofovir microbicide gel on HPV infection in a clinical trial setting. Here, contrary to the observed impact on preventing HIV and HSV-2 infections44, the use of topical tenofovir microbicide gel was not associated with the prevention of HPV infection, nor its persistence or clearance in this region.Approximately three-quarters of women in this HIV prevention trial of high-risk women were HPV-infected; this suggests a high overlap between burdens of both HPV and HIV infections in KwaZulu-Natal, South Africa. The prevalence of oncogenic HPV infections was 52%, somewhat lower than the 70% observed in a small female sex worker cohort in KwaZulu-Natal45 demonstrate a link between increased HIV risk and oncogenic HPV. These oncogenic types were predominantly represented by IARC class 1 HPV types and included several specific associations with vaccine-preventable HPV types. Considering the association between increased risk of HIV acquisition and infection by vaccine-preventable HPV types, these data underscore the importance of protecting young women from HPV infection through vaccination and cross-protection46 against the broadest number of oncogenic HPV strains. The data also suggest the potential for increased HIV risk if infected by non-vaccine types, indicating that the risk to HIV transmission may not be related to the oncogenicity of the HPV alone, but to general immune control of HPV. This concept emphasizes the importance of larger studies of HIV incidence in regions with differential HPV vaccine uptake to comprehensively determine the excess HIV risk attributed to infection with vaccine-preventable versus non-preventable types. Our finding that infection with any of the 9 vaccine-preventable HPV types was associated with raised concentrations of several genital cytokines associated with HIV risk suggests that any HIV protection provided by HPV vaccination may be biologically mediated. Considering the observed overlap in cytokine profiles associated with HIV risk and with HPV clearance, persistence and acquisition, additional studies will be required to determine whether the ability of HPV vaccination to prevent HIV infection may need to be bolstered through complementary efforts to address any immunological effects associated with infection by non-vaccine HPV types.In addition, we and others50), but on associations between current HPV infection and female genital cytokines, including cytokine biomarkers of HIV risk. Our data suggest that prevalent HPV infection is associated with increased concentrations of several pro-inflammatory, chemotactic, regulatory, growth-related, and adaptive response-associated cytokines. While our study agrees with the increased concentrations of certain chemokines associated with prevalent HPV infection49, our measurement of a broad and substantial panel of cytokines in a large clinical cohort allows a more robust and expanded assessment of cytokine associations with prevalent HPV. Importantly, many of the most prominent HPV-associated cytokines have previously been associated with HIV risk in this cohort and in other studies of African women51. Multivariable modeling showed heavy overlap in cytokine profile between mutually exclusive HPV categories, but reasonably strong delineation between HPV categories compared to those who remained HPV negative during the study. Considering the relationship between genital cytokines and HIV risk, these data highlight the importance of including HPV status in risk scoring profiles and emphasizes the benefit of prospective HPV measurements for informing potential HIV infection risk.This study focused not only on genital immune environments associated with subsequent HPV clearance, persistence or acquisition 30 were elevated in women with a history of HPV infection compared to HPV negative controls; more often significantly in categories featuring HPV clearance. However, while the association between HPV and increased genital cytokine concentrations is strong, the data cannot assign specific mechanisms of HPV-mediated HIV transmission, nor can it confirm a causal relationship between HPV-associated cytokines and HIV risk. Further studies are warranted to assess cellular and other mechanism(s) for the relationship between these viruses. Defining cellular sources of HPV-associated cytokines may also contribute to the design of targeted interventions to control the inflammation and reduce HIV risk.HPV clearance occurred frequently, was associated with increased HIV risk, and was the common thread that best captured increased cytokine concentrations compared to those who remained HPV negative. Of the eight categories, clearance alone was associated with the highest number of significant differences in single cytokine concentrations relative to controls. The contribution of transient HPV infections to the genital cytokine environment is therefore highly relevant and should likely be considered in biomedical efforts to reduce HIV infection. HPV infection can be resolved spontaneously, or through innate and adaptive immune responses. A potential mechanism for the relationship between inflammation and HIV risk is cytokine-mediated recruitment of HIV target cells to the genital tract. The strong association between HPV infection and genital cytokine responses suggests a role for cellular immunity in efforts to control HPV infection. Here, several cytokines, including beta chemokines critical for attracting CCR552, this study is limited by the potential for misclassification of HPV categories between visits, which may have contributed to the similar cytokine patterns observed between categories. The misclassification of HPV categories also extends to the difficulty in determining whether acquisition events represent reactivation or new infection, whether cleared infections refer to latent or eliminated infection, and the challenges of understanding the immunological profiles associated with each. Considering that acute HIV infection is demonstrated to increase the number of HPV types detected39, the survival analysis is limited by this potential for overestimation of the HIV incidence associated with multiple-type infection, or with clearance, persistence, or acquisition of HPV. Although the CAPRISA 004 trial included frequent HIV testing and rigorous retrospective viral load confirmations37, HPV genotyping data were available for only baseline and study exit visits of most seroconverters, and longitudinal HPV classification would have been based on genotyping conducted after HIV infection. Additionally, the difficulty in timing the changes in HPV status in the context of HIV infection, we were unable to determine the degree of HPV–HIV co-infection occurring from the same infected partner. Cytokine data were, however, available for the majority of visits for each participant. Considering the established relationship between HIV infection and immune dysregulation, our linear mixed models only included cytokine data collected from confirmed HIV-negative samples with complementary HPV genotyping data for that visit, and therefore reflects the genital mucosal immune milieu associated with a current HPV status in HIV-negative women. Since cytokine data were available for confirmed HIV-negative specimens, we can only speculate on the immune profile associated with HIV infection that could promote an HPV status. Our data does, however, support the hypothesis that immune profiles observed in HPV infection may contribute in some way to the established link between HPV infection and HIV risk.Since HPV can be rapidly cleared, maintained and acquiredAnother limitation of the study is that only a subset of clinically relevant HPV types was assessed in this study, meaning that some HPV negative women could have been infected by unmeasured types. However, had this been of significant consequence, the difference between cytokine concentrations and HIV risk in any HPV category relative to HPV negative women should not have been as strong. Data on cervical cytology were not documented within the CAPRISA 004 trial. However, considering the population was HIV uninfected and mainly in their twenties, we would expect very little high grade abnormal cytology. However, given that this was a microbicide safety trial, extensive vaginal observation data were available, and these data do not support any widespread clinically obvious abnormal cytology likely to confound the analysis presented. Lastly, this study was also limited in the number of participants with both HPV and vaginal microbiome data, and the relationship between HPV and the vaginal microbiome in this context is not well understood.The strength of this study lies in its combination of very strong epidemiological data and mucosal immune assessments in a large prospective cohort, one in which HPV and HIV rates are extremely high. In summary, although not a definitive mechanism, these data provide some insight into how HPV might increase HIV risk and highlight the importance of further studies to validate and expand on the concepts raised. The data suggest, among others, an urgency in understanding and controlling the immune predictors of HPV status and multiple-type infection, in both HIV infected and uninfected women; in determining the cellular or other factors associated with the cytokines observed in the different HPV status categories; and in understanding the immune impact of HPV infection in vaccinated individuals and its relation to HIV risk. These data emphasize the importance of HPV vaccination since oncogenic HPV types are linked to HIV infection. The study assessed genital inflammation and HPV in specimens representing the mucosal surface over which sexual transmission of both viruses takes place, and demonstrated a difference in genital cytokines by HPV status over time, which informs possible mechanisms to explain the complex epidemiological relationship between HPV and increased HIV risk. While these data do not rule out possible unmeasured explanations for the increased HIV risk associated with HPV infection, they do provide compelling biological evidence for elevated immune responses in HPV positive women, consistent with increased HIV infection risk. It is important to highlight the high prevalence of HPV (74%), and that the broad coverage of vaccine types observed in this study supports the notion that the current roll-out of HPV vaccination in South African schools could have a strong cancer prevention benefit. It is also important to note the frequent changes in HPV status detected in this cohort of young South African women (>50% acquired or cleared HPV infection) and each HPV category’s link with HIV-associated cytokines. Combined with large effect estimates for increased HIV risk, the population attributable fraction of HIV acquisition due to HPV infection is substantial in this region with hyperendemic HIV prevalence. Therefore, in addition to preventing cervical cancer, further development of more effective ways to prevent and/or treat HPV infection, or modify its immunological effects, could have major HIV prevention benefits.37. The enrollment criteria and characteristics of the population have been described53, and details of those excluded can be found in Fig. 24, and cervicovaginal aspirates were used for measurements of Tenofovir concentrations by ultraperformance liquid chromatograph-mass spectrometry54. No assessments of genital epithelial and immune cell phenotype and function were possible as cervical cell sampling was not conducted during the trial.The CAPRISA 004 study was a Phase IIb clinical trial to evaluate the safety and efficacy of tenofovir 1% gel applied vaginally to prevent HIV infection. This trial was completed in 2010 and demonstrated both safety and an effectiveness of 39% in preventing HIV in the intent-to-treat analysisCVL specimens were collected by applying 3 ml of sterile saline to the ectocervix and aspirating the liquid from the posterior fornix. CVL specimens were transported on ice to the CAPRISA laboratory within 6 h, where samples were centrifuged and the CVL pellet and supernatant fractions were separated and each were stored in −85 °C for future use.DNA was extracted from CVL pellets using an automated MagNA pure instrument  according to the manufacturer’s instructions. HPV DNA was amplified by PCR using the Roche Linear Array® HPV Genotyping kit  according to manufacturer instructions. Briefly, a pool of biotinylated primers was added to CVL pellet DNA to amplify HPV by defining capture sequences of nucleotides within the polymorphic L1 region of the HPV genome. This assay distinguishes between 37 HPV genotypes, including those considered a significant risk factor for progression to cervical cancer (oncogenic HPV types). The HPV genotypes detected in this study include HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, IS39, and CP6108; with CP6108 denoting a subgroup of HPV type 89, IS39 a subgroup of HPV type 82, HPV type 55 a subgroup of type 44, and HPV type 64 a subgroup of type 34. Further, the assay contains a cross-reactive probe hybridizing with HPV genotypes 33, 35, 52 and 58 from the Alpha 9 genus, and single probes for each type to aid in the determination of these similar HPV types. Additional primer pairs were included to amplify the human β-globin gene, thereby providing an internal control for the sample adequacy of the DNA extraction and amplification process.56. Herpes simplex virus types 1 and 2, Treponema pallidum and Haemophilus ducreyi and C. trachomatis lymphogranuloma venereum (LGV) strains were detected by real-time multiplex PCR , while Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma genitalium, were detected using in-house optimized real-time multiplex PCR tests 56.Serology was performed for herpes simplex virus type 2 (HSV-2) using the HSV-2 gG2 enzyme-linked immunosorbent assay . DNA was extracted from vulvovaginal swabs [Xtractor Gene (Corbett Robotics Pty Ltd.)] to detect the presence of other current sexually transmitted infections54. The intraday and interday assay accuracy was 96–107% and 92–106%, respectively; both with a precision of Tenofovir concentrations were measured in cervicovaginal fluid by ultraperformance liquid chromatograph-mass spectrometry24. Here we report the measurements of 48 cytokines, including several pro-inflammatory, hematopoietic, regulatory, adaptive, and/or growth-related cytokines: interleukin (IL)-1β, IL-1Rα, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-12p40, IL-16, IL-18, IL-1A, IL-2RA, IL-3, IL-13, IL-15, IL-17, basic fibroblast growth factor (FGF), cutaneous T-cell attracting chemokine (CTACK), eotaxin, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM–CSF), growth regulated (GRO)-α, hepatocyte growth factor (HGF), interferon (IFN)-γ, IFN-α2, interferon gamma-induced protein (IP)−10, leukemia inhibitory factor (LIF), monocyte chemotactic protein (MCP)−1, MCP-3, macrophage colony-stimulating factor (M-CSF), monokine induced by gamma-Interferon (MIG), macrophage migration inhibitory factor (MIF), macrophage inflammatory protein (MIP)–1α, MIP-1β, nerve growth factor (NGF)-β, platelet derived growth factor (PDGF)-ββ, regulated upon activation normal T cell expressed and presumably secreted (RANTES), stem cell factor (SCF), stem Cell Growth Factor (SCGF)-β, stromal derived factor (SDF)−1α, tumor necrosis factor (TNF)–α, TNF-β, TNF-related apoptosis inducing ligand (TRAIL), and vascular endothelial growth factor (VEGF). Analyte concentrations were measured using Bio-Plex Pro Human Cytokine kits and a Bio-Plex Array Reader (Bio-Rad Laboratories) as previously described24. The sensitivity of these kits ranged between 0.2 and 45.2 pg/ml for each of the 48 cytokines measured. Data were collected using Bio-Plex Manager software (version 6), and a 5 PL regression formula was used to calculate sample concentrations from the standard curves. Cytokine levels below the lower limit of detection of the assay were reported as the midpoint between the lowest concentration measured for each cytokine and zero. All specimens were plated undiluted, and all laboratory assays were conducted blinded. Duplicates were included within plates to assess intra-plate variability (n = 20 duplicate wells per plate), and replicates were placed across separate plates to assess inter-plate variability (n = 32 replicate wells across plates). Cytokine assessments were repeated if correlation coefficients of associations between replicates were below Spearman rho = 0.8, and if significant differences in magnitudes among replicates were observed. Individual cytokines undetectable in >40% of specimens were assessed as categorical values. To minimize variability in longitudinal analyses all specimens from the same patient were placed in the same run.The concentrations of cytokines were previously measured in undiluted CVL fluid of CAPRISA 004 participants using Luminex multiplex assays4 and correspond with several meta-analyses’ definitions21: HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. The remainder of the 37 types investigated are defined as low risk for cervical cancer. Several baseline HPV infection categories were defined in order to assess the relationship between HIV acquisition and HPV DNA detection , multitype HPV infection, oncogenicity, and with vaccine type HPV infection. Considering the high number of multitype HPV infections, the potential for co-infection is acknowledged, particularly within the latter two groups. For example, while women in the non-Gardasil®9 HPV types group were infected exclusively with non-Gardasil®9 HPV types, women in the Gardasil®9 group could be infected exclusively with Gardasil®9 HPV types (27%), or co-infected with vaccine and non-Gardasil®9 HPV types (73%).Oncogenic HPV investigated here were defined as Group 1, 2 A carcinogens by the International Agency for ResearchThree overlapping HPV classifications were defined as follows: women who (i) “acquired any” HPV types, irrespective of whether they concurrently cleared and/or had persistent HPV infections; (ii) who “Cleared any” HPV types, irrespective of whether they concurrently acquired and/or had persistent HPV infections; or (iii) “Persisted any” HPV types, irrespective of whether they concurrently acquired and/or cleared any HPV infections between any two study visits Fig. . Eight mn = 494), STIs were less prevalent (24%) than HPV, suggesting that concurrent STI is unlikely to fully explain the HPV-associated cytokine differences. Sensitivity analyses controlling for laboratory STI data were carried out in the “acquired any” HPV category , presence of STI symptoms, HSV-2 status, frequency of condom use, marital status, and whether women were living with regular partners. Multivariable linear regression models were used to analyze the effect of baseline HPV status on the levels of each cytokine, controlling for false discovery rate (FDR) using the Benjamin–Hochberg test. Multivariable linear mixed models with a random effect of the individual participant were used to analyze the effect of HPV strata on the levels of each cytokine, controlling for FDR using the Benjamin–Hochberg test. Autoregressive of order 1 (AR(1)) structure was used to account for correlation between measurements from the same participant. This structure was well suited for our data since we have evenly spaced cytokines measurements. The log-rank test was used to test for trend in HIV IRs across ordinal variables. Ninety-five percent confidence intervals for HIV IRs were calculated based on the Poisson distribution. Associations of HIV incidence were determined by modeling time to HIV infection with multivariable Cox proportional hazards regression models, where time spent in the study was calculated from randomization to the estimated date of HIV infection or the date of withdrawal from the study. Due to the fact that cytokines are correlated, we used PCA to model the relationship between HPV status and genital cytokine concentrations observed at the study end visit of all HIV- study participants. The aim was to identify patterns of cytokine expression for different HPV categories while using information from all 48 cytokines together. All analyses were performed using SAS version 9.4 . Two-sided P-values less than 0.05 were considered statistically significant.Further information on experimental design and reagents is available in the Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


Ogg1-/-) animals display increased propensity to age-induced and diet-induced metabolic diseases, including insulin resistance and fatty liver. Since the intestinal microbiome is increasingly understood to play a role in modulating host metabolic responses, we examined gut microbial composition in Ogg1-/- mice subjected to different nutritional challenges. Interestingly, Ogg1-/- mice had a markedly altered intestinal microbiome under both control-fed and hypercaloric diet conditions. Several microbial species that were increased in Ogg1-/- animals were associated with increased energy harvest, consistent with their propensity to high-fat diet induced weight gain. In addition, several pro-inflammatory microbes were increased in Ogg1-/- mice. Consistent with this observation, Ogg1-/- mice were significantly more sensitive to intestinal inflammation induced by acute exposure to dextran sulfate sodium. Taken together, these data indicate that in addition to their proclivity to obesity and metabolic disease, Ogg1-/- mice are prone to colonic inflammation. Further, these data point to alterations in the intestinal microbiome as potential mediators of the metabolic and intestinal inflammatory response in Ogg1-/- mice.OGG1-deficient ( Oxidative stress can result from endogenous and exogenous generation of reactive oxygen species (ROS) in response to environmental and dietary factors. Induction of oxidative stress has been implicated in the onset and progression of a number of pathologies, including metabolic syndrome and chronic inflammation. ROS exert their effects by altering the redox status of the cell and by reacting with and damaging cellular constituents. One of the important targets of ROS-induced damage is DNA, which is subject to oxidative lesions that must be repaired to maintain genomic stability –3. OxidaThe gastrointestinal tract is colonized by a large number of microorganisms, including bacteria, viruses, archaea, fungi, and protozoa. These microorganisms are collectively referred to as the gut microbiome and have now been demonstrated to serve a variety of functions, including energy harvest, xenobiotic metabolism, vitamin production, and immune function. Accordingly, aberrant intestinal microbial colonization, or intestinal dysbiosis, has been implicated in numerous pathologies, including the development of obesity –36. FurtGiven our prior observations of increased propensity to obesity in OGG1-deficient mice, we sought to determine if OGG1 status, in the context of a regular low-fat diet or a hypercaloric diet, impacts intestinal microbial composition and whether any observed changes are associated with disease risk.Ogg1-/- mice has been previously described . No. No54]. weighted  and weigweighted , with soCoprococcus, Dorea, and Oscillospira, in addition to several unidentified Clostridia genera  was also positively correlated to body fat and body weight, while a taxon from the order RF39.f (Tenericutes phylum) was negatively correlated with both  ≤ 0.01, n = 24) . As antia genera . The Firith both .Ogg1-/- mice, we were struck by the observation that several of the changes in gut microbes in Ogg1-/- mice have been reported to play a role in intestinal inflammation and colitis. For instance, increased levels of Deferribacteres, Mucispirillum; and Proteobacteria, particularly Sutterella have been shown to be associated with active colitis [Ogg1-/- mice, we asked the question of whether Ogg1-/- mice would have increased sensitivity to an acute inflammatory challenge. To induce an acute inflammatory condition in the colon, WT and Ogg1-/- mice were challenged with 3% DSS in 5% sucrose in water for 5 days. Mice were euthanized either at the end of the exposure or after being transferred to regular water for 5 additional days. In contrast to wild-type controls, by the end of the 10 day protocol, Ogg1-/- mice experienced considerably greater weight loss (Ogg1-/- mice were twice as severely affected (~2-fold), confirming a much more severe inflammatory response in the Ogg1-deficient mice -1β levels were upregulated by DSS treatment in WT and Ogg1-/- mice, but this difference reached statistical significance only in the latter genotype  and Bacteroidetes (B) phyla represented nearly 100% of microbial diversity in feces from chow-fed WT mice, these phyla only contributed to 88% of the bacterial population in chow-fed Ogg1-/- mice. Contributing to this lower overall F+B % in the Ogg1-/- group, there was a statistically significant decrease in the Firmicutes (p = 0.036), and a significant reduction in the F:B ratio in Ogg1-/- mice (0.30 in WT-chow vs. 0.20 in Ogg1-/- chow). Thus, although reduced F:B ratios have been associated with improved metabolic phenotypes in some reports [Ogg1-/- mice were in phyla  unique to this genotype . This bacterial group, found only in the Ogg1-/- animals, is known to degrade cellulose and xylans and has been associated with increased energy harvest and fecal short-chain fatty acids (SCFAs) [Ogg1-/- mice and their altered microbiomes.One of the most striking findings from these studies was the significantly increased alpha diversity in fecal samples from -/- mice , Fig 3.  reports , 78, 79, reports –83. Integenotype , Table 1 (SCFAs) , providiOgg1-/- mice. For instance, we observed significant increases in the relative abundance of Prevotellacaea and TM7 groups. Elinav et al. [Lactobacillus in Nlrp6-/- mice, in another model where modulation of the host genotype alone resulted in dramatic alterations in the intestinal microbial environment. NLRP6 is a sensor of both endogenous and exogenous stress and is involved in the production of IL-18, which is critical for the maintenance of the intestinal epithelial cell barrier. NLRP6-/- mice exhibit reduced IL-18 levels in intestinal epithelial cells and, interestingly, like Ogg1-/- mice were highly susceptible to DSS-induced colitis. The correlation between colonic inflammation and changes in pro-inflammatory taxa such as Prevotella and TM7 in the Ogg1-/- intestines suggests that local changes in the intestine may mediate this pro-inflammatory phenotype.With regard to colonic inflammation and associated changes in the gut microbiome, several notable alterations were observed in v et al.  reportedAllobaculum, Rikenella, Bacteroides, Parabacteriodes, Odoribacteria, and an unidentified group of the order Bacteriodales) were also present in significant numbers only in Ogg1-/- gut microbiomes. This was also true for the Firmicutes genus Dorea, Proteobacteria taxa Sutterella, and Helicobacteraceae (other), Deferribacteres (Mucillispirillum) and an unassigned taxon. In addition to being positively correlated with body weight and body fat in this study, increases in Dorea have been correlated with the onset of nonalcoholic fatty liver and its progression to nonalcoholic steatohepatitis in pediatric patients [Ogg1-/- mice being associated with increased inflammatory status, Mucispirillum, an intestinal commensal normally associated with the gut epithelium and found to be increased in Ogg1-/- animals, has been shown to activate T cell–dependent immunoglobulin A (IgA) production and to increase in abundance in the presence of inflammation [Ogg1-/- gut microbiomes and > 5% in Ogg1-/- HFD), these microorganisms are of note as they have been reported to increase in the gut microbiomes of mouse models exhibiting DSS-induced or spontaneous chronic colitis in comparison to wild-type controls [Ogg1-/- mice and were further exacerbated in HFD-fed animals. This baseline increase in inflammatory species is consistent with our observations of increased underlying inflammatory histological changes in Ogg1-/- mice, even prior to DSS administration , AlkB homology (Alkbh)2 or Alkbh3 were all greatly sensitized to the development of severe ulcerative colitis, following DSS treatments [Aag-/- mice that were challenged with an acute DSS regimen accumulated significantly greater quantities of DNA base damage caused by reactive oxygen and nitrogen species, and showed severe colonic damage [Aag, Alkbh2, and Alkbh3 knockout animals, thus demonstrating the essential role of the initiation of BER in colonic inflammation. This investigation also demonstrated that shifting from an acute to a chronic DSS challenge coupled with a single dose of azoxymethane resulted in a significantly increased colonic tumor burden [Ogg1-/- mice that were exposed to conditions of chronic DSS treatment [Mutyh-/- mice was attributed to a role for MUTYH in mounting an inflammatory response [Similar to eatments , 95. Aagc damage . In addir burden . Increasreatment . This streatment . These rreatment –102. Theresponse . Any potOgg1-/- mice. If so, our data would again suggest that transcriptional regulation by OGG1 occurs in a tissue-specific manner, with OGG1 binding promoting inflammation in the enterocyte while being anti-inflammatory in the lung. Alternatively, it is plausible that upon acute induction of severe DNA damage across the genome, as might occur during acute colitis, channeling and sequestration of OGG1 to its DNA repair functionality may result in its reduced availability as a transcriptional regulator.While the best described role for OGG1 is in excision of oxidized guanine bases through the base-excision DNA repair pathway, it should be noted that additional functions for OGG1 have been described. For instance, binding of OGG1 8-oxoG upstream in promoter regions of pro-inflammatory genes has been shown to upregulate their expression in lung cells , 103. ThOgg1-/- mice. For instance, our observation of significantly elevated IL-1β Click here for additional data file.S1 FigRarefaction curves generated from the Goods coverage of OTUs (index > 0.99) across samples.(EPS)Click here for additional data file.S2 FigResults are from the analysis of sequences recovered from the fecal samples as described in Methods.(EPS)Click here for additional data file.


Globally, breast cancer is the most frequent cancer among women. Studies reported an increased risk of breast cancer among women with prior cervical dysplasia. This study aimed to describe the prevalence of human papillomavirus (HPV) in breast cancer and explore if women with prior cervical neoplasia carry an increased risk of HPV-positive breast cancer compared to women without.10 PCR-DEIA-LiPA25 and an in-house semi-Q-PCR assay.This case–control study identified 193 Danish women diagnosed with breast cancer (1998–2012) at Aarhus University Hospital or Copenhagen University Hospital Herlev. Cases were 93 women with cervical intraepithelial neoplasia grade 3 or worse (CIN3+) prior to breast cancer. Controls were 100 women without prior cervical dysplasia. HPV testing and genotyping were done using SPFp = 0.61 and 1.08 vs. 0.00%, p = 0.48). HPV prevalence in CIN3+ was 94.62% (95% CI 0.88–0.98). Concordance between the assays was 98.60%.Overall HPV prevalence in breast cancer for the assays was 1.55% (95% CI 0.32–4.48) and 0.52% (95% CI 0.01–2.85). There was no difference in HPV prevalence between cases and controls (2.15 vs. 1.00%, HPV prevalence in breast cancer is very low suggesting no etiological correlation between HPV and breast cancer. Human papillomavirus (HPV) has been established as the leading cause of cervical cancer , and theBreast cancer accounts for 25% of cancer cases and 15% of cancer-related deaths among women worldwide . As a reWe conducted a hospital-based case–control study in Denmark, where all patients have access to the health care system at no cost. Upon birth or immigration, each Danish citizen is assigned a CPR-number, which is a unique code that reflects the person’s age, sex, and the date of birth. Estimates in this study are based upon women who were diagnosed with breast cancer during 1998–2012 at Aarhus University Hospital and Copenhagen University Hospital Herlev.The Danish Pathology Data Bank (DPDB) is a national databank storing results on all patho-anatomical tests conducted in Denmark, and it used the Systematized Nomenclature of Medicine (SNOMED) as nomenclature and classification system.in situ, squamous cell carcinoma in situ, squamous cell carcinoma, or adenocarcinoma).The study population was identified through two SNOMED searches in the DPDB Figure . A complWomen in the control group were eligible if they had a record of at least two normal cervical cytology results within 5 years of their breast cancer diagnosis. Women were excluded if they had a previous record of cervical dysplasia or cervical cancer. Overall, 100 women with a diagnosis of breast cancer were included in the control group , the case was excluded. A total of 93 women with a history of both CIN3+ and breast cancer were included in the case group  containing CIN3+ tissue and breast cancer tissue were collected at the participating pathology departments during May through November 2016. All blocks were sectioned at the Department of Pathology, Aarhus University Hospital. The sandwich technique was applied to ensure histopathological confirmation of tumor tissue in the sections flanking the sections subjected to HPV analysis. First, a 3-μm-thick section was cut for hematoxylin and eosin staining (HE). Second, four to eight 10-μm-thick sections were cut and subsequently macro-dissected to ensure that only the neoplastic area was dissected off the slide and collected in a sterile tube. To avoid contamination between specimens, gloves were changed before cutting each block, the knife was changed before cutting each tissue sample, and the microtome, tweezers, and brush were carefully cleaned with 1% sodium dodecyl sulfate and 99% ethanol before and after cutting every block. Furthermore, no paraffin block containing cervical tissue was cut on the microtome during the process of breast tissue sectioning. Finally, after collecting the tissue into tubes, a 3-μm-thick section was cut for HE staining. As quality control, both positive and negative controls were included and analyzed on the same terms as the CIN3+ and breast cancer samples. Negative controls were used to ensure no contamination of HPV from the persons performing the procedures, and they consisted of two components; tubes with sections from an FFPE block containing only pure paraffin  and tubes with purified material from cytolomegavirus embedded in paraffin. DNA extraction was performed at the Department of Pathology, Aarhus University Hospital using the QIAsymphony DSP DNA Mini Kit, version 1 .10 PCR-DEIA-LiPA25 assay  (SPF10LiPA25), which used the SPF10 primer set to amplify a 65 base pair (bp) region in the L1 open-reading frame. After PCR, HPV-positive samples were distinguished from HPV-negative using the DNA enzyme immunoassay (DEIA). HPV genotyping of HPV-positive samples were subsequently performed with a reverse hybridization technique (LiPA25) that allowed the detection of 25 HR and LR HPV genotypes   were chosen for blinded analyses of the HPV genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59.In Herlev, analyses were carried out using a semi-quantitative PCR assay (semi-Q-PCR) based on Taqman probes , 23. ThiAll analyses were conducted according to the manufacturer’s instructions.10LiPA25 and semi-Q-PCR, and Cohen’s kappa statistic was used to determine the rate of agreement  and 100 controls   with a mean time from CIN3+ diagnosis to breast cancer diagnosis of 4.61 years (SD 95% CI 3.78–5.45) . Most CIN3+ cases were CIN3  or squamous cell carcinoma in situ  (Table There was no difference in mean age (±SD) at breast cancer diagnosis between cases [51.82 years (±11.48)] and controls [50.35 years (± 8.49)] and in both groups, ductal carcinoma was by far the most frequent breast cancer diagnosis . In the case group, mean age at the time of CIN3+ diagnosis was 47.20 years (±11.95). The vast majority of cases had been diagnosed with CIN3+ prior to their breast cancer diagnosis  . In the case group, two breast cancer samples were positive for HPV according to SPF10LiPA25 (2.15%); one was HPV 16 positive and one was HPV 56 positive. Both women had a CIN3+ specimen that was positive for HPV 58. The semi-Q-PCR assay found one HPV 16-positive breast cancer sample in the case group (1.08%), and this was the same sample, that was HPV 16 positive with SPF10LiPA25. In the control group, one breast cancer sample was positive for HPV 16 (1.00%) according to SPF10LiPA25, and none tested positive for HPV (0.00%) according to the semi-Q-PCR.Overall prevalence of HPV in breast cancer was 0.52% (95% CI 0.32–4.48) when using semi-Q-PCR and 1.55% (95% CI 0.01–2.85) when using SPF10LiPA25 and the semi-Q-PCR analyses , and HPV 16 was the most commonly detected genotype (45.16 vs. 44.09%) followed by HPV 31 (11.96 vs. 11.96%)  in both the SPF10LiPA25 analyses were HPV-negative in 195  samples and HPV-positive in 91  samples, whereas the semi-Q-PCR analyses were HPV-negative in 197  samples and HPV-positive in 89  samples. Among the 92 cervical and breast samples that tested positive for HPV with one of the assays, SPF10LiPA25 detected one HPV genotype in 87 (94.57%) samples, two types in four (4.35%) samples, and no HPV in one (1.09%) sample. Using the semi-Q-PCR, one type was detected in 85 (92.39%) of these 92 samples, two types in four (4.35%) samples, and no HPV in one (1.09%) sample  were concordant and four 1.40%) were discordant  in CIN3+. All samples were tested using two highly sensitive PCR-based HPV assays, and the agreement rate between them was high with an overall concordance of 98.60%.10LiPA25) and reported no HPV-positive breast cancer cases among 76 cases tested. The difference in HPV prevalence across studies may also be explained by false positive results, where contamination is a crucial point. Studies have shown that HPV can be detected in up to 18% of samples obtained from fomites in an gynecological outpatient clinic  in addition to invasive breast cancer cases, whereas others included benign tumors. However, if HPV is hypothesized to play a role in breast cancer carcinogenesis, it seems reasonable to assume that DCIS cases as well as benign tumors would, at least to some extent, turn out to be HPV-positive with rising prevalence rates with increasing severity of the disease similar to what is observed in precancers and cancers of the cervix. The prevalence of HPV is known to be positively correlated with the severity of the cervical disease  were chosen for blinded analysis of the HPV genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 using the semi-Q-PCR. We, therefore, cannot rule out an underestimation of the overall prevalence and the prevalence of multi-infections when using the semi-Q-PCR. This may also have biased the concordance rate between the two assays toward a higher agreement rate. Second, we cannot exclude that selection bias may have occurred. We chose not to include cases considered at HR of being a BRCA-mutated breast cancer  as we hypothesized that these cancers occur due to somatic mutations and not as a result of an HPV infection. However, we cannot know if the HPV prevalence would have been higher had we included these cancers as well. Unlike many other countries where population-based registries do not exist, we were able to retrieve the screening history of the entire study population at the individual level making this case–control study of high quality. However, we cannot preclude that some women in the control group may have had dysplasia prior to the establishment of the DPDB  although most pathology departments have transferred data. Nevertheless, because the overall HPV prevalence in breast cancer was very low and only a minority of women would have been eligible for screening before 1998, we find this less likely to affect our results.This study has some limitations that must be addressed. First, all samples were analyzed by SPFThis study was approved by the Danish Data Protection Agency (case citation 1-16-02-111-16) and has formal ethics approval by the Region Committees on Health Research Ethics (case citation 1-10-72-55-16). It was furthermore ensured than none of the study subjects were registered in Tissue Use Register, which would imply that they have decided that their tissue cannot be used for research.All authors contributed with designing of the study. SB, JB, TS, and SL completed the first draft of the manuscript, and MLS, AH, EH, SGJ, and EB supervised on the content and on the laboratory procedures at the pathological departments. SGJ and HK performed laboratory procedures, and SGJ and EH interpreted the Q-PCR results. MLS performed statistical analyses, which were subsequently verified by the remaining authors. All authors participated in editing the manuscript.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.