High-risk human papillomavirus (HPV) is found in over 99% of cervical cancers. The aim of this study was to determine the prevalence of HPV in a population of women in Bobo-Dioulasso and to identify the high-risk types present in these women. From May to June, 2015, 181 women who came for consultation at the Souro Sanou University Hospital of Bobo-Dioulasso have been included in this study. Uterine endocervical swabs have been taken in these women. DNA obtained by extraction from the samples thus collected was used to determine the prevalence of high-risk human papillomavirus genotypes through real-time PCR. The age of the women ranged from 20 to 56 years with a mean of 35.3 ± 8.1 years. The prevalence of infection by high-risk HPV types was 25.4% (46/181). The most common high-risk HPV genotypes were HPV 39 (18.5%), HPV 52 (16.7%), HPV 18 (14.8%), and HPV 35 (13.0%). HPV 16 which is included in the HPV vaccines was not found in the population studied. This type of study which is the first one in Bobo-Dioulasso has showed a high prevalence of genotypes HPV 39, HPV 52, and HPV 35 which are not yet covered by a vaccine. The human papillomavirus (HPV) infection is one of the most common sexually transmitted infections in the world , 2. In mThe study was conducted at the Souro Sanou University Hospital of Bobo-Dioulasso, the second city of Burkina Faso. From May to June, 2015, 181 women who came for consultation in the Department of Gynecology, Obstetrics, and Reproductive Medicine (DGOMR) have been included in the study. Pregnant women or women who have undergone hysterectomy or menstruating women have been excluded. Each woman answered a questionnaire to provide information on their socioeconomic status and behavioral and sexual habits. Samples have been taken through endocervical swabbing of the uterus using a sterile cotton swab. The samples thus obtained have been immersed in a transport medium which was provided with DNA-Sorb-A kit  and kept at −20°C until the DNA extraction. Immediately after sampling, screening for precancerous lesions was done for the women by visual inspection with acetic acid and Lugol's iodine (VIA/VILI).This study has been approved by the Ethics Committee for Research in Health of Burkina Faso (Deliberation number 2014-9-110) and all the women have signed a consent form prior to participation.β-globin gene was used as internal control. The “HPV Genotypes 14 Real-TM Quant” made it possible to detect the following 14 high-risk HPV genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.The DNA extraction was made using DNA-Sorb-A kit . The genotyping of high-risk HPV was made by real-time PCR using the kit “HPV Genotypes 14 Real-TM Quant,” code V67-100FRT , and the Sacycler-96 Real-Time PCR . This genotyping is based on multiplex real-time amplification for each sample and the The PCR program used was as follows: 1 cycle of 95°C for 15 minutes; 5 cycles of 95°C for 5 seconds, 60°C for 20 seconds, 72°C for 15 seconds; and 40 cycles of 95°C for 5 seconds, 60°C for 30 seconds, and 72°C for 15 seconds.p < 0.05. Women under 30 years were considered as young women and these criteria were used in the study to group the women in age group: ≥30 years old versus <30 years old.Data were entered and analyzed using the SPSS software in its 20.0 version and Epi Info 7. The Chi-square test was used for comparisons with a significant difference for During the enrollment period in the Department of Gynecology, Obstetrics, and Reproductive Medicine (DGOMR), 181 women gave their consent to participate to this study. The main motivations for consultation were leucorrhea and pruritus , cervical cancer screening , abdominal-pelvic pain , dysmenorrhea , contraception needs , maternity desire , breast pain , and others .The sociodemographic, sexual, and behavioral characteristics of the study population are given in β-globin gene. The results of HPV research show that 25.4% (46/181) of the women were infected with high-risk HPV. The number of HPV genotypes per woman ranged from 1 to 3, and in considering multiple infections, we counted in total 54 genotypes among the 46 women infected with HPV. In this study, all tested samples were positive for p = 0.335).84.8% of the women infected with HPV were carriers of a single genotype. There was 15.2% (7/46) of multiple infections including 6 multiple infections with 2 genotypes and 1 multiple infection with 3 genotypes . This taIn This study shows the characteristics of the high-risk HPV infection in a general population of women in Bobo-Dioulasso. To the best of our knowledge, in Bobo-Dioulasso, it is the first study on a general population of women concerning the HPV. Indeed, the other studies conducted in this city have focused on a particular group of women who are sex workers , 13. HowThe prevalence of 25.4% of high-risk HPV obtained in this study is high compared to the worldwide prevalence of HPV infection estimated to be around 11-12% . SeveralHPV 39 (18.5%), HPV 52 (16.7%), HPV 18 (14.8%), and HPV 35 (13.0%) were the most common genotypes in this study. The frequency of the other genotypes found ranged from 7.4 to 1.8%. HPV 39 was not commonly found at the top of the most common genotypes in the literature. But it was part of the 3 most common genotypes in women with Atypical Squamous Cells of Undetermined Significance (ASCUS) . A high HPV 16 and HPV 18 are the high-risk genotypes included in the vaccines available to fight against cervical cancer. HPV 16 was not found in this study. Some studies had already shown a low prevalence of HPV 16 in some African countries compared to other high-risk HPV genotypes , 19, 26.In this study, the carrying of the high-risk HPV was not associated with risk factors such as the age at first sexual intercourse before the age of 16 , the useIn the present study, we did not investigate women's HIV status but it is known that HIV prevalence for the general population of Bobo-Dioulasso is 1.4% . This vaThe results of this study could certainly not be generalized to the entire population of Bobo-Dioulasso but they helped to know the HPV genotypes present in a population of women in this city. This is of real significance in the sense that the data obtained in a city other than Ouagadougou will be used to have a broader overview of the distribution of high-risk HPV genotypes in Burkina Faso.The most common genotypes in this study were HPV 39, HPV 52, HPV 18, and HPV 35. These results are consistent with those of other studies conducted in Burkina Faso, which showed that there was a predominance of high-risk HPV other than HPV 16 and HPV 18. The future vaccine trials should therefore consider these other high-risk genotypes in order to expand prevention through anti-HPV vaccination.


Recent literature data suggest that the high-risk human papillomaviruses (HR-HPVs) testing with several molecular techniques could be an alternative to cytology in the detection of cervical intraepithelial neoplasias of grade 2 or worse (CIN2+). However, any molecular techniques have its own limits and may give false negative results which must be clearly known before undertaking a primary HPV screening. This study aims to evaluate the performance of the high-risk HPV hybrid capture II detection kit (HCII) which is considered as a “gold standard technique” in a series of 100 women having proved both cytological lesions of atypical squamous cells-cannot exclude an HSIL (ASC-H) or high-grade squamous intraepithelial lesion (HSIL) and histological lesions of CIN2+. The clinical sensitivity of HCII in women with a cytological diagnosis of ASC-H/HSIL and a diagnosis of CIN2+ is high but not absolute and estimated at 96% . These data although they are infrequent must be clearly referred before to start an HPV primary screening of CIN2+ especially with HCII methodology. In Belgium, invasive cervical cancer is the 12th most common cancer type in women and about 600 new cases are diagnosed annually . In addin = 62) or ASC-H (n = 38) on cytology and CIN2+ lesions on biopsy (90 CIN 2/3 lesions and 10 invasive carcinomas/IC) were included in the study. All the patients  were living in either Wallonia or Brussels (“Belgian French Community”). For the cytology, as previously published, we used a thin-layer technology  from 20 mL vials that contained the collected cells  as we have previously described [This study was performed according to the rules of the Erasme Hospital's Ethics Committee . 100 consecutive patients with a proven HSIL  with CIN2/3 lesions on biopsy a positivity was observed in 86 cases . The results are summarized in 96 of the 100 (96%) of HSIL/ASC-H cytological lesions with a CIN2+ lesion on biopsy were HR-HPV-positive with HC2. From them, 10 cases with a histological diagnosis of invasive carcinoma (ISCC) were positive (100%). In ASC-H or HSIL  than cytology and appears as a future robust methodology for a large and rapid screening. Nevertheless, contrary to a conventional wisdom, we demonstrated that, in our population, there were high-grade or suggested high-grade cytological abnormalities with clearly proven CIN2+ lesions for which the research of viral HR HPVs sequences with HCII methodology was negative. These data, although they are infrequent, must be known and referred before to start an HPV primary screening of CIN2+ lesions especially with HCII methodology which is considered by many as a reference standard technology particularly in Belgium.


Persistent infection with the high-risk Human Papillomavirus type 16 (HPV 16) is the causative event for the development of cervical cancer and other malignant tumors of the anogenital tract and of the head and neck. Despite many attempts to develop therapeutic vaccines no candidate has entered late clinical trials. An interesting approach is a DNA based vaccine encompassing the nucleotide sequence of the E6 and E7 viral oncoproteins. Because both proteins are consistently expressed in HPV infected cells they represent excellent targets for immune therapy. Here we report the development of 8 DNA vaccine candidates consisting of differently rearranged HPV-16 E6 and E7 sequences within one molecule providing all naturally occurring epitopes but supposedly lacking transforming activity. The HPV sequences were fused to the J-domain and the SV40 enhancer in order to increase immune responses. We demonstrate that one out of the 8 vaccine candidates induces very strong cellular E6- and E7- specific cellular immune responses in mice and, as shown in regression experiments, efficiently controls growth of HPV 16 positive syngeneic tumors. This data demonstrates the potential of this vaccine candidate to control persistent HPV 16 infection that may lead to malignant disease. It also suggests that different sequence rearrangements influence the immunogenecity by an as yet unknown mechanism. Instead the viral E4 protein facilitates particle release from the desquamating cells by disrupting the intermediate filaments of the keratinocyte cytoskeleton. This strategy of low profile enables papillomaviruses to bypass the surveillance of the immune system and hence to persist for different periods of time within the affected epithelial site.Papillomaviruses comprise a large group of small DNA viruses with a very distinct biology. Being restricted to skin and mucosa without a viremic phase during virus replication, the pattern of their gene expression is tightly linked to the differentiation of the epithelium. The highest load of antigens appears in the keratinized upper cell layers where synthesis of the structural proteins and assembly of particles takes place. Virus maturation Persistence is a particular hallmark of the so-called high-risk human papillomaviruses (HPV) possibly since they replicate only in a few cells within a lesion HPV 16 and HPV 18 are the most important types for the development of cervical cancer and other malignant tumors of the anogenital tract and of the head and neck. A few years ago two vaccines against these viruses became commercially available. Clinical trials and the first reports after launching vaccination campaigns in countries such as Australia and the UK demonstrated highly efficient protection against persistent infection and precancerous lesions From the clinical trials, it also became evident that the current vaccines have no therapeutic activity, i.e. they are unable to eliminate existing infections An attractive alternative to application of proteins or peptides is genetic immunization either as naked DNA, by transfer through a viral vector or as a combination of both in a heterologous prime-boost scheme. For the use of the viral oncoproteins E6 and E7 as immunogen, we have developed the concept of gene shuffling to deprive them of their transforming yet to maintain all possible T cell epitopes Eight different versions of the HPV-16 E6/E7 shuffled genes were generated by assembly of synthetic oligonucleotides . E6 and HindIII and 3′-XbaI into the immunization vector pTHkan The artificial genes were synthesized and codon-optimized for the human system by GENEART  and cloned via 5′-RMA-S cellsC3 cells derived from embryonic mouse cells transfected with the complete HPV-16 genome 293tt cellsmusculustibialis anterior i.m.]).Ten to 12 days after vaccination, animals were sacrificed and spleens were isolated.Six-to-eight week old female C57BL/6 mice (owner bred) were kept under SPF isolation conditions and standard diet at the animal facilities of the German Cancer Research Center, Heidelberg, Germany. Agarose-gel verified plasmids  were injected. For CTL analysis, animals were immunized once  were used directly for Elispot-assays or co-cultured with 2×106 irradiated (100 Gy) RMA-S cells loaded either with E6 or E7 peptide in 25 cm2 culture flasks in αMEM  supplemented with 10% FCS, 0.1 mMβ-mercaptoethanol, 4 mM glutamine and antibiotics for 5–6 days at 37°C and 7.5% CO2 in a humidified incubator.Two×106 HPV-16 E7 expressing C3 cells musculustibialis anterior, as described above. Tumor sizes were measured with a caliper. Mice were sacrificed when the tumor size reached 400 mm2 or when tumors were bleeding. Tumor sizes of the mice within a group were calculated as arithmetic means with standard deviation (SD). All operations on live animals were performed under Isofluraneanaesthesia.C57BL/6 mice received 0.5×10All animal experiments were performed with approval by and in accordance with regulatory guidelines and standards set by the institutional review board at Regierungspraesidium, Karlsruhe, Germany (35-9185.81/G-48/06)IFN-γ Elispot assays were performed ex vivo as previously described 51Cr-release assays were performed after one in vitro restimulation of murine spleen cells. One×104 Na251CrO4labeled (0.05 mCi) target cells/well (unloaded RMA-S cells or RMA-S cells loaded either with E6 or E7 peptide) were incubated together with decreasing numbers of effector cells in 200 µl per well of a 96-well round bottom plate  for 4 h. Subsequently, 50 µl of supernatant was harvested from each well and the released radioactivity was measured in a Microbeta counter . Specific lysis was calculated according to the formula: percent specific lysis = [(cpm of the sample−spontaneous release)/]×100, where total release and spontaneous release are measured in counts per minute (cpm). Spontaneous chromium release was determined by using 51Cr-labeled target cells without effector cells, and total chromium release was determined by adding 2% Triton X-100 to lyse the labeled target cells.The Differences of means between experimental and control group were considered statistically significant when p was less than 0.05 by unpaired Students t-test.It was the aim of this study to generate a number of HPV 16 E6/E7 shuffled genes and to identify the construct with the highest immunogenicity for further development as a vaccine candidate. A total of 8 novel artificial HPV-16 E6 and E7 genes were generated as described in ex vivo, respectively. As positive controls, we included the shuffled E6 and E7 genes , immunogenicity of which had been determined earlier 4 splenocytes: 2±1 after E6 peptide stimulation and 2±2 after E7 peptide stimulation; vs control p: 0.0005) and E7-specific  IFN-gamma responses. The measured responses were also significantly higher compared to the animal group with the second strongest induction of the cytotoxic T lymphocytes .We determined the immunogenicity of the eight novel gene constructs providing all possible occurring CD8 epitopes of the E6 and E7 genes of HPV-16. First, we measured IFN-gamma and granzyme B secretion of splenocytes We obtained similar results in the granzyme B Elispot assays. Granzyme B is critical in the induction of apoptosis in target cells by cytotoxic T lymphocytes. Again, the HPV-16 E6E7 mixed shuffled construct induced the strongest activation of cytotoxic T lymphocytes  . The E6S51Cr-release assays after one round of in vitro restimulation of the splenocytes. Five to 6 days after in vitro restimulation, the splenocytes were co-incubated with 51Cr-labeled RMA-S target cells. Consistent with the Elispot assays, the splenocytes of the HPV-16 E6E7 mixed shuffled treated mice mediated the highest specific lysis of RMA-S cells loaded with E6- or E7-peptide, respectively   . The secin vitro assays.We concluded from the different experiments that the HPV-16 E6E7 mixed shuffled construct has the highest potential to induce a strong E6- and E7-specific cytotoxic T lymphocytes as measured in different in vitro data in an HPV-specific tumor model we performed tumor regression experiments using HPV-16 E6 and E7 positive C3 cells in C57/B6 mice 51Cr-release assay correlated with the tumor regression. Indeed, the injection of theHPV-16 E6E7 mixed shuffled vaccine provoked the most effective control of the tumor outgrowth. At the end of the experiment, the average tumor size in the control group (empty vector) was 209±77 mm2 but only 20±9 mm2 in the E6E7 mixed shuffled treated group (p<0.0001) (To verify the <0.0001) . AdditioCervical cancer is the second most common cancer in women worldwide resulting in about half a million newly diagnosed cases and 260,000 deaths every year Since the first description in the early 1990s, DNA vaccines proved to be a logical alternative to conventional protein-based vaccines. DNA vaccines stand out by their: 1) simple design and ease of production, 2) remarkable stability and long shelf-life, 3) considerable safety in clinical trials, 4) cost-effectiveness, and 5) ability to activate both arms of specific immunity in vitro and in vivo with no potential of oncogenicity In our previous work we choose another strategy, i.e. the generation of rearranged genes through disruption of the transformation active sites and reassembly of gene fragments in a random fashion. To preclude the loss of potential CTL epitopes as a result of gene shuffling, an appendix that includes the original sequences at the junctions was added at the end of the constructs. The potential of this technology was first evaluated using shuffled HPV 16 E7 gene (HPV 16 E7SH) in murine fibroblast cells and C57BL/6 mice. The results indicated that E7SH was able to induce CTL response against the authentic protein with complete loss of the transformation activity In the current study, we extended our strategy to combine E6 and E7 shuffled genes in a single construct aiming to provide the vaccine recipient with broader and more efficient immune repertoire. Eight different combinations of HPV 16 E6/E7 shuffled genes were designed with inclusion of 5′ Kozak sequence and codon optimization . Further51Cr-release assay and, most importantly, by in vivo tumor regression experiments. The aim of the study was the immunological characterization of newly developed E6 and E7-based therapeutic HPV-16 vaccine candidates rather than to investigate the mechanism(s) driving the different responses. Therefore, we can only speculate that changes in the nucleotide sequences may influence RNA abundance (either by synthesis or degradation) and therefore the amount of the encoded antigen. Indeed, it is well known that interactions between cis-regulatory sequences and different RNA-binding proteins as well as microRNAs influence RNA stability In a first round of experiments we analyzed E6 and E7-specific IFN-gamma and Granzyme B responses in Elispot assays of immunized animals. All vaccine candidates investigated were able to induce specific responses, but interestingly one out of the eight constructs (E6E7 Mixed Shuffled) displayed a significant stronger immunogenic potential. This finding could be reproduced in the functional Together, the outcome of this study clearly demonstrates that gene shuffling is a potential approach to increase the immunogenicity of antigens. This finding may generally influence the development of DNA- and protein-based vaccine in general.Materials and Methods S1Sequence of the eight different versions of the HPV 16 E6/E7 shuffled genes.(PDF)Click here for additional data file.


Oncoviruses cause tremendous global cancer burden. For several DNA tumor viruses, human genome integration is consistently associated with cancer development. However, genomic features associated with tumor viral integration are poorly understood. We sought to define genomic determinants for 1897 loci prone to hosting human papillomavirus (HPV), hepatitis B virus (HBV) or Merkel cell polyomavirus (MCPyV). These were compared to HIV, whose enzyme-mediated integration is well understood. A comprehensive catalog of integration sites was constructed from the literature and experimentally-determined HPV integration sites. Features were scored in eight categories  and compared to random loci. Random forest models determined loci classification and feature selection. HPV and HBV integrants were not fragile site associated. MCPyV preferred integration near sensory perception genes. Unique signatures of integration-associated predictive genomic features were detected. Importantly, repeats, actively-transcribed regions and histone modifications were common tumor viral integration signatures. Integration into the human genome is central to transposon mutagenesis, gene therapy and viral pathogenesis ,2,3,4. DNumerous studies have been conducted to determine preferential sites of DNA tumor virus integration, with differing conclusions. HPV and HBV integrations were previously detected at common fragile sites (CFSs), prone to breakage ,20,21,22Large-scale analyses of integration site selection have been conducted for transposons, gene therapy vectors and HIV ,2,3,4. Pn = 589) and for only those with precisely-mapped locations (n = 92). Viral integration site lengths are provided . A total of 589 HPV integration events from 436 cervical carcinomas (CESCs) and 59 HNCs were cataloged. Remaining integrations were from other carcinomas or cell lines. Several HPV types were represented, mostly HPV-16 (382) and HPV-18 (138) . Some anprovided . HBV intprovided . Thirty-provided . HIV intquencing . Viral iet al.  − Ct[input × 5.64]); IgG adjusted IP Ct value (∆∆Ct) = (∆Ct[IP] − ∆Ct[IgG]); fold enrichment above IgG = 2(−∆∆Ct).Real-time qPCR was performed using the Roche Lightcycler 480  using the following protocol: 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s, 60 °C (or 53 °C) for 1 min followed by signal acquisition. PCR reactions were assembled in a PCR hood using 1× SYBR Green PCR Master Mix (Roche Diagnostics), 300 nM primers and 2 μL of diluted template for a total reaction volume of 10 μL. Primers were chosen to approximate a ±500-bp window around the two HPV-16 integration sites at 13q22.1 in SiHa cells. The two integrated copies have identical breakpoints and are separated by a duplicated segment of the human genome . Primer


Oral and pharyngeal cancer (OPC) of multifactorial aetiology is a major health problem globally. Ranking first in all cancers, OPC poses a significant impact on the Sri Lankan male population. As Human Papillomavirus (HPV) high risk (HR) types are found to be significant risk factors for OPC globally, the current study was undertaken to examine the association between HR-HPV16 and 18 types with OPC in Sri Lanka.i.e. HPV, smoking, alcohol, betel quid, poor dentition, was established using Chi-square test. Logistic regression was used to calculate odds ratios (OR), adjusted for the influence of other risk factors.Serum samples of 78 OPC patients and 51 non-cancer controls were assayed for the presence of anti-HPV16 and anti-HPV18 IgG antibodies using in-house established Enzyme Linked Immunosorbent Assays (ELISAs). The association between OPC and its risk factors This prototype study in Sri Lanka showed a significant risk of 15 fold in developing OPC due to HPV16/18 seropositivity after removing variability due to other factors. Oncogenic HPV18 showed a higher rate of seropositivity being detected in 32% of OPC patients, and also in 2% of non-cancer control subjects. HR-HPV16 was detected in 23% of OPC patients and in 5.88% of controls. Moreover, seven OPC patients were detected with both anti-HPV16 and anti-HPV18 antibodies. According to the logistic regression models HPV18 seropositivity was associated with a 28 fold risk in developing OPC while that of HPV16 was associated with a 6 fold increase in risk for the development of OPC. A 5 fold risk of developing OPC was also pronounced among smokers while alcohol, betel and poor dentition was not significantly associated with OPC. Statistically significant differences with regard to age, gender, smoking, alcohol, betel use, poor dentition and site specificity of the tumour was not observed between HPV seropositive and seronegative OPC patients.Both in-house developed ELISAs detected significant proportions of HPV seropositives within the OPC study population suggestive of HPV as a strong risk factor for oral and pharyngeal carcinogenesis in Sri Lanka. Oral and pharyngeal cancer (OPC) is a significant component of the global burden of cancer, worldwide being the sixth most common cancer and the eighth most common cause of cancer death ,2. Sri LSeveral epidemiologic investigations carried out globally documented the correlation between consumption of tobacco, alcohol, and betel chewing as major risk factors for developing OPC with a dose-response relationship -7.In developing countries up to 23% of malignancies are caused by infectious agents, including HPV . HPV, inPresence of HPV DNA in a sample population of Sri Lankan patients with oral squamous cell carcinoma was previously reported. The positivity was detected by PCR and direct cycle sequencing and the highest HPV positivity was found in the buccal mucosa. The overall HPV prevalence of the study sample was 37.2% (N = 102) . Thus, aThe current study examined the associations between established risk factors of OPC, with special emphasis on HPV as a suspected major emerging risk factor determining oral and especially pharyngeal cancer in Sri Lanka. Sero-prevalence of HPV was detected with in-house developed indirect enzyme-linked immunosorbent assays (ELISAs) specific for HPV16 and HPV18, using virus-like particles (VLPs) produced for the L1 region of the HPV genome.Cases (N = 78) and controls (51) were comparable with regard to age and gender (P > 0.05). Males (94%) were more likely than females (6%) to be diagnosed with OPC. Age of cases ranged from 38 to 77 years. A higher proportion of the patients (66.7%) had only received primary education. From the 25 districts in Sri Lanka, residency of the cases covered ten.Among the cases, 33 (42%) had cancers of the oral cavity while 45 (58%) had pharyngeal cancers with sub sites including floor of the mouth, hard palate, tongue, retro molar buccal mucosa, soft palate, tonsil, cheek, maxilla antrum and oropharynx. Patients were not diagnosed for any other carcinomas previously with the exception of one case with a cancer in the sigmoid colon, and a majority (86%) had no family history of cancer.i.e. anti-HPV16/18 IgG antibodies, of the case and control subjects is shown in Figure Serologic response, Seropositivity to HPV18 L1 VLP was significantly different between cases (32%) and controls (1.96%). The seropositives pose a momentous 24-fold increase in risk for OPC when compared with seronegatives . Although, the wide confidence interval indicates an imprecise estimate due to the small sample size, the magnitude of risk estimate suggests a strong positive association between seropositivity to HPV18 and OPC. A significant difference (P = 0.019) was also detected in the seropositivity of HPV16 cases  compared with the controls , but with only a 4.8-fold risk in developing OPC with reference to the seronegatives . OPC patients seropositive for either HPV16 or HPV18 comprised 46% of the cases with a significant difference with the seropositivity of the control group (P = 0.00), and with a 13.71-fold risk of developing OPC with contrast to seronegatives  than those with anti HPV18 antibodies (0.177 to 0.536), indicative of a higher antibody response for HPV16 compared with HPV18. This was reiterated for OPC patients with co-infections of HPV16 and HPV18, where a majority presented with higher OD values for HPV16.Results of each indirect ELISA revealed 3 groups for anti-HPV IgG positivity Figure . Based oThe association of OPC with smoking, alcohol, betel quid chewing and poor dentition is shown in Table Influence of each risk factor for the development of OPC after adjusting for the influence of other significant risk factors using binary logistic regression models is shown in Table In the first model, after accounting for other factors  cases being seropositive for HPV16 was statistically significant with reference to the control subjects (P = 0.012) with a risk of 5.89-fold. A case subject being seropositive for HPV18 was statistically significant with reference to the control subjects (P = 0.003) after adjusting for smoking, alcohol consumption and betel quid chewing. The seropositives showed a marked increase of a 28-fold risk in developing OPC compared with the seronegatives (OR = 28.29) (model 2). The adjusted OR for both HPV16 and HPV18 was higher than the unadjusted OR suggesting that HPV16 and HPV18 unaided act as an important risk factor for OPC which can drive an oncogenic effect notwithstanding other risk factors. As per the third logistic regression model only HPV18 and smoking posed significant risks. Individuals seropositive for HPV18 were associated with a 20-fold risk of developing OPC after removing influence due to other factors. Smoking accounted for risk of 4.92 fold. Those who were seropositive with either high-risk HPV16 L1 or HPV18 L1 or both, showed a 15-fold risk of developing OPC relative to seronegative subjects for the two HPV types after controlling for other risk factors (model 4). Accordingly, smoking was also found to be significant (P = 0.004) with a 4.67-fold risk factor. Alcohol use and betel quid chewing were not statistically significant (P > 0.05) risk factors in developing OPC when exposure to HPV18 and/or HPV16 was present. Conversely, smoking was a significant risk factor in all models developed even after adjusting for HPV seropositivity.None of the established risk factors such as smoking, alcohol use, betel quid chewing, poor dentition nor age and gender (data not shown), were significantly different (P > 0.05) between the HPV16/18 seropositive and seronegative OPC patients.Of the OPC patients HPV seropositivity was detected in individuals ranging in age from 41-72 years of which a majority 38.8%) was within the 50-59 age group  and non-smokers (25%). Notably three of the seven patients co-infected with HPV16 and 18 types were non-smokers. The anti-HPV antibody magnitudes of these three cases were ‘high’ for HPV16 while high antibody magnitude for HPV18 was detected in a single patient.Patients with cancers of both the oral cavity and the pharynx were detected with anti-HPV16L1/18 L1 IgG antibodies. A higher seropositivity for anti-HPV antibodies were detected in patients with pharyngeal than those with oral cancer. None of the tumour characteristics  showed a significant difference (P > 0.05) with the HPV serologic status Table . Remarkard in the world. For pharyngeal cancers Sri Lanka ranks 4th in Asia and 11th in the world with an ASR/P of 7.2. Notably, both exceed the world ASR/P [Sri Lanka is facing a precarious health problem with oral and pharyngeal cancer (OPC) having the highest age standardized rate per 100,000 populations (ASR/P: 15.5) for oral cancer in Asia and ranking 3There is sufficient evidence for a causal role of tobacco, alcohol, betel quid chewing and poor oral hygiene with OPC in Sri Lanka -20, but The infection with HR-HPV typically lasts from 12-18 months and is eventually cleared by the immune system . CompareGlobal prevalence and type distribution of HPV in OPC remains unclear . The strNonetheless, this indicates a paradox with most of the prevailing records on HPV related OPC. A multicenter study conducted in nine countries evidenced that HPV16 unaided accounted for more than 90% of HPV-positive OPC, and that HPV18 was less of an important risk factor as this was detected infrequently in OPC patients . HoweverThe risk of malignancy and differential response towards OPC in individuals who are exposed to more than one type of HR-HPV is still unclear . HoweverThe aetiology of an infectious disease is dependent on the agent, host and environment . Howeverth decade of their life. The Sri Lankan situation may be different from that of other geographical regions having middle aged men more inclined to the risk, though the small sample size of the present study may pose a limitation on drawing presumptions.That HPV-seropositive OPC was not significantly associated with age of the patient in the current study, contradicts the postulate that younger age groups (30-50 years) are at a higher risk of developing OPC due to HPV . Since iStudies indicate that HPV plays a definite aetiologic role in cancers of the oropharynx and in a small group of cancers of the oral cavity ,15. The In general, at the base-line, tumor stage III/IV, poorly differentiated tumor grade and N2-3 nodal involvement show significantly increased risk in detecting anti-HPV antibodies . Due to The increasing prevalence rate of HPV in OPC highlights the change of life style in sexual behavior. The route of HPV infection in the oral and pharyngeal region is not firmly established . HoweverThe current study suggests four aetiologic categories of OPC in Sri Lanka: Low risk HPV positive tumors arising in non-smokers; moderate risk HPV positive tumors arising in former or current smokers; high risk HPV-negative tumors arising in current or former smokers; and the fourth category being HPV negative tumors arising in non-smokers. The fourth category may include heavy alcohol use, habit of betel chewing or any other variant associated with OPC.A major limitation of this research is the case-control design of the study which precludes establishment of a causal link between HR-HPV and OPC. As serological response to HPV is a surrogate marker of both current and past infection, it does not indicate whether infection was acquired prior to the development of OPC. An OPC patient could have acquired HPV infection few months prior to being diagnosed with OPC and hence had positive serology at the study enrollment, wherein HPV would not be ‘causally’ associated with OPC. Hence, a prospective longitudinal study should be undertaken to establish causal relationship between HPV and OPC.i.e analysis of HPV DNA in formalin fixed paraffin embedded cancer tissues and expression of its oncogenic genes E6 and E7, to support the role of HPV in the pathogenesis of oral and pharyngeal cancer, and epidemiologic evidence that will guide HPV related oral and pharyngeal cancer control and prevention programmes in Sri Lanka will be of great importance in the local and global scale.Interestingly, HPV positive tumors respond well to treatment showing a potential benefit of a more favorable outcome from therapy compared to HPV negative OPC . TherebyThis study was the first to follow established major risk factors with HR-HPV in both oral and pharyngeal carcinoma in Sri Lanka. Two in-house established ELISAs detected anti-HPV16 and anti-HPV18 IgG antibodies. The results showed a plausible association between HR-HPV and OPC with 46% of the case group being seropositive for HPV16/18; HPV18 and HPV16 were detected in 32% of the patients with a risk of 28 fold and in 23% with a 6 fold risk confounding other risk factors, respectively. Smoking was significantly associated with OPC posing a 5 fold risk.The case-control study received approval of the Ethics Review Committee, Faculty of Medicine, University of Colombo (EC12/29), Sri Lanka.Clinically diagnosed OPC cases were recruited from the National Cancer Institute, Sri Lanka between April-September 2012. Newly enrolled patients and those who received a single dose of chemotherapy or radiotherapy for OPC were the inclusion criteria. Exclusion criteria were severely ill patients and individuals who declined to participate. Written, informed consent was obtained prior to the interview and sample collection. Patients were interviewed using an approved questionnaire to accrue information on socio-demographics, use of tobacco, alcohol and betel, oral hygiene and sexual practices. Medical information of the patients was retrieved from their clinical records. Comparable to the enrolled cases (N = 78), 51 age and gender matched healthy individuals with no personal history of cancer were recruited as controls to the study.Variables on smoking history were ascertained by general discussion that assessed the number of cigarettes smoked per day, age at which an individual started to smoke, the number of years smoking was practiced and number of years since quitting. Similar information was obtained regarding lifetime consumption of alcohol and betel quid.A supplemental question included in the questionnaire, which inquired on sexual practices, was not pragmatic due to social and cultural barriers in Sri Lanka.A sample of blood (1 ml) was aseptically collected from the study subjects via intravenous bleeding. Separated serum samples were stored at -20°C until further use .IgG antibodies against HPV-specific proteins were assayed using two in-house established indirect enzyme-linked immunosorbent assays (ELISA).HPV virus-like particles (VLP) for HPV types 16 and 18 expressing L1 protein and H16.V5 and H18.J4 type-specific monoclonal antibodies  were useWells of microtiter plates  were coated with 100 μl of VLPs  in PBS (pH 7.4) and incubated overnight at 4°C. All subsequent incubation steps involved 37°C for 1 hour. Non-specific binding sites were blocked with milk buffer (5% w/v non-fat milk powder in PBS) and the plate was flick-washed with washing buffer (0.5% Tween20 in PBS). Human serum diluted in dilution buffer (5% w/v non-fat milk powder in washing buffer) at 1:100 was added to duplicate wells and following incubation, unbound reagents were removed by flick-washing. The bound antibodies were detected with alkaline phosphatase linked anti-human IgG antibody  and the reaction was visualized by the addition of PNPP . The reaction was arrested with 3 M NaOH and the optical density (OD) was measured at 415 nm in an automated ELISA plate reader .The negative control method  was usedStatistical analyses were performed using SPSS version 16 for Windows  software package. Comparisons of normally distributed variables of independent samples were performed using the t-test. When variable distribution was not normalized, the Mann-Whitney U test was used to compare variables between groups. The Chi-square test was used to compare categorical variables between groups . Binary logistic regression was used to model the relationships between presence of OPC and its predictor variables. Calculated odds ratio (OR) measured the risk of developing OPC. Wald statistic was used to determine parameter significance  in logistic regression analyses. The level of significance was set at P < 0.05.


Polistes paper wasps, caste remains flexible throughout adulthood, but there is evidence that nourishment inequalities can bias caste development with workers receiving limited nourishment compared to queens. Dominance and vibrational signaling are behaviors that have also been linked to caste differences in paper wasps, suggesting that a combination of nourishment and social factors may drive caste determination. To better understand the molecular basis of nutritional effects on caste determination, we used RNA-sequencing to investigate the gene expression changes in response to proteinaceous nourishment deprivation in Polistes metricus larvae.Social insects exhibit striking phenotypic plasticity in the form of distinct reproductive (queen) and non-reproductive (worker) castes, which are typically driven by differences in the environment during early development. Nutritional environment and nourishment during development has been shown to be broadly associated with caste determination across social insect taxa such as bees, wasps, and termites. In primitively social insects such as We identified 285 nourishment-responsive transcripts, many of which are related to lipid metabolism and oxidation-reduction activity. Via comparisons to previously identified caste-related genes, we found that nourishment restriction only partially biased wasp gene expression patterns toward worker caste-like traits, which supports the notion that nourishment, in conjunction with social environment, is a determinant of developmental caste bias. In addition, we conducted cross-species comparisons of nourishment-responsive genes, and uncovered largely lineage-specific gene expression changes, suggesting few shared nourishment-responsive genes across taxa.Overall, the results from this study highlight the complex and multifactorial nature of environmental effects on the gene expression patterns underlying plastic phenotypes.The online version of this article (doi:10.1186/s12864-015-1410-y) contains supplementary material, which is available to authorized users. Phenotypic plasticity provides an important adaptive mechanism by which morphology, physiology, and/or behavior can be adjusted to biotic and abiotic environmental factors including temperature, nutrition, population density, and predator presence . There aOne particular environmental factor, food availability, is especially important for caste polyphenism in social insects. Differential nourishment -10 and nPolistes are an informative group for understanding the evolution of eusociality and the origins of castes [Polistes¸ the quantity of larval nourishment changes according to seasonal changes in the adult-to-larva ratio [hexamerin 1 in gynes [Primitively eusocial taxa such as f castes ,32. Acrova ratio ,27. Firsva ratio . At thisva ratio ,33, whicva ratio ,35, greain gynes ,37, and in gynes . Experimin gynes ,39.Polistes, social factors, such as dominance behavior [Polistes.In addition to the established role of nourishment in caste differences in behavior ,31 and mbehavior -42 also behavior  propose behavior  that dirPolistes metricus. We had three main goals. First, we explored the transcriptional responses of wasp larvae to high and low nourishment levels during laboratory rearing using RNA-sequencing. Second, we tested the hypothesis that nourishment level relates to caste-related gene expression; specifically that low nourishment is associated with more worker-like gene expression patterns, and high nourishment is associated with more gyne-like expression patterns. To do this, we compared nourishment differential expression to a set of nearly 800 previously identified genes associated with caste development in field-reared P. metricus [In this study, we investigated the effect of experimental proteinaceous nourishment deprivation during larval development on caste-related gene expression in a primitively eusocial species, the paper wasp metricus , and we metricus  and dungmetricus . Our oveP. metricus; the transcriptome was based on both the sequence data described here in conjunction with additional samples described in a previous study [P. metricus differentially expressed transcripts (DETs) that differed between low and high nourishment treatments, using DESeq  were upregulated in low compared to high nourishment larvae . Thus, despite the fact that they had less food available, low nourishment led to a majority of genes having higher gene expression, and thus did not simply cause a general shutdown in transcription.To assess differential gene expression, we mapped reads to a previously assembled de novo transcriptome for us study . By comp ≤ 0.05, ), heretoPolistes wasps, submitted), and variable effects of nourishment level on physiology [One important feature of these results is the presence of outlier individuals, i.e. one individual low nourishment larva clustered with the high nourishment samples and a high nourishment larva clustered with the low nourishment samples Figure . The varysiology .P. metricus under starved vs. ad lib food conditions [It is important to validate RNA-Seq data using another method such as quantitative reverse transcription polymerase chain reaction (qRT-PCR). However, for this study, we had limited samples and quantities of RNA , so we cnditions . AlthougP. metricus brains reared under low and high nourishment conditions using qRT-PCR. We tested for a correlation (Spearman) in the log2 fold changes between the 24 candidate genes from Daugherty et al. [P. metricus transcriptome . For some genes, there is more than one best BLAST hit to the P. metricus transcriptome, so all transcripts were used for the correlation analysis. The gene PmTOR is absent from the P. metricus transcriptome, so this gene was removed from the analysis. Although none of the orthologous transcripts are significantly differentially expressed in the RNA-seq study, we nonetheless identified a significant positive correlation in log2 fold changes between these two studies , which provides support for our observed RNA-Seq results.Of 24 candidate genes, Daugherty et al.  identifiy et al.  and the y et al.  and the One reason we may not have observed statistically significant differential expression of these candidate genes in the RNA-Seq study, despite the strong correlation with the qRT-PCR data, is because of the limited statistical power. Differential expression calls are more stringent in this RNA-Seq study compared to the qRT-PCR analysis because familywise error correction is more severe due to the larger number of genes . Furthermore, the qRT-PCR study has a larger sample size (n = 47) compared to the RNA-Seq study (n = 8), which provides greater statistical power for detecting differential expression.P. metricus nourishment-responsive DETs compared to caste-related gene expression. A previous study [Next, we investigated how us study  comparedus study  show a pThere was a statistically significant but relatively small overlap (43 transcripts) between the nourishment-responsive and caste-related DETs (Chi-square with Yates’ correction: p < 0.0001). Of these shared DETs, the directionality of gene expression change was unexpected: eight (18.6%) were upregulated in low nourishment larvae compared to high nourishment larvae, whereas 38 (88.4%) were upregulated in worker-destined larvae relative to queen-destined larvae  were annotated with enzyme codes for the KEGG analysis with Blast2GO [Our previous study suggested gene expression similarity across studies is more pronounced on the level of pathways and gene functional categories, rather than specific genes or transcripts . TherefoBlast2GO   a microarray study of tissue-specific (fat body and muscle) gene expression derived from fruit fly larvae  and 2) aP. metricus nourishment-responsive transcripts with nourishment-responsive transcripts in D. melanogaster fat body and muscle tissues [When comparing  tissues , we idenP. metricus and D. melanogaster fat bodies  further supports common functional changes related to nourishment manipulation in both species. However, this signal is not observed in the comparison between P. metricus nourishment-enriched GO terms and D. melanogaster nourishment-enriched GO terms in the muscle tissue. There is only one common nourishment-enriched GO term , which is upregulated in the P. metricus low nourishment larvae but upregulated in the high nourishment samples for the D. melanogaster muscle tissue  between larvae raised experimentally on low vs. high nourishment, many of which are associated with lipid metabolism and oxidation-reduction activity. Most (73%) of the Polistes nourishment-responsive transcripts are upregulated in larvae with low nourishment, which is opposite to the pattern observed in other insects including fruit flies [In this study, we provide the first genome-wide transcriptional profiling of nourishment response during development in the genus it flies  and dungit flies . There wPolistes [P. metricus, we predicted that worker-destined larvae from natural nests in the field would have similar transcript expression patterns as experimental low nourishment larvae. With respect to directionality of overall transcript expression, this was the case — most DETs were upregulated in worker-destined larvae (92.1%) and in low nourishment larvae (72.9%). This work agrees with previous work in honey bees that also showed more genes with worker-biased expression in larvae, but more genes with queen-biased expression later in development  [Polistes  ,21. We aThere was also a significant overlap in nourishment-responsive and caste-related expression at the GO functional level, with terms related to metabolism, protein binding, and oxidation reduction activity Figures . On the Overall, our data suggest that nourishment level caused a partial shift in gene expression, with low nourishment individuals being more worker-like and high nourishment individuals being more gyne-like. However, it is pertinent to note that the nourishment manipulation may not have resulted in a strong nutritional stress; i.e., wasps may have compensated for low proteinaceous food availability by consuming more sugar. In a recent study examining adult wasps reared from the same nests as the larvae analyzed here, the nourishment manipulation caused only a partial shift in caste-related physiology . In thatPolistes wasps, submitted). Lab-reared wasps typically have higher lipid stores perhaps due to both overfeeding and inactivity [Polistes wasps, submitted). Two genes  known to exhibit caste-specific gene expression are perturbed due to lab rearing, but neither gene was differentially expressed in the current study. In addition, the low and high nourishment samples in our study were reared by single foundresses with unrestricted access to sucrose as opposed to the two feeding levels for caterpillars that they fed to larvae. Diets with a high carbohydrate to protein ratio can cause an increase in lipid levels in the insect fat body [Polistes metricus.One important caveat of our study is that the effects of the nourishment treatment on gene expression and physiology may be influenced by laboratory rearing, which may produce conflicting results compared to a natural field setting  that influence caste-related gene expression. These results support the notion that nourishment level during development can somewhat bias development into queen or worker caste as adults, but leave room for other factors, and thus underscore the complex and multifactorial nature of caste development.This study provides new data on nourishment-responsive gene expression in the context of caste development for P. metricus wasps in early March 2009 near Raleigh, NC, as they were exiting the attic of a house in which they had passed the winter in behavioral quiescence and reproductive diapause. Single wasps were placed in cages 30 cm in length, width, and height constructed of clear plastic with plastic screen on the top and two sides. An opening in the top was covered with a piece of hardboard 10 cm square with a nest from a previous season attached to the underside. Nests were trimmed to seven cells ca. 0.5 cm deep, and meconia  of the original nest occupants were removed. Each seven-cell nest therefore served as a “starter” nest on which the newly-captured wasps could initiate construction and then expand as their own. Construction paper was the pulp source for nest construction.We collected adult female 2 in the incandescent-only morning and evening periods and 225 micromoles/sec/m2 during the both-lights midday. Chamber temperature was 20°C at night and 30°C during full light, with a gradual ramp-up in the morning and ramp-down in the evening. Each day, we repositioned cages in a rotation pattern to distribute any light or temperature variation in the chamber across all colonies over the course of the experiment.Caged wasps were placed on wire racks in a growth chamber 1.2 m × 2.5 m × 2.1 m in the North Carolina State University Phytotron. The chamber contained incandescent lights on a 16 L/8D cycle; fluorescent lights came on 2 h following and went off 2 h before the incandescent lights. Light intensity was 21 micromoles/sec/mrd or early 4th-instar Manduca sexta larva ca. 2.0 cm in length. Low nourishment foundresses received one caterpillar every fourth day. After May 22, the feeding schedule for low nourishment foundresses was accelerated to one caterpillar every three days. In the high nourishment treatment, foundresses were provided with caterpillars ad lib, adjusted each day to one caterpillar above the number that had been consumed since the previous day’s provisioning. For this experiment, we collected the largest final instar larva in each nest immediately following spinning of a cocoon by the third larva in the nest to do so. Those three larvae were allowed to develop into adults that then were analyzed for developmental and physiological characteristics. These results were published separately [Each cage was provided with ad libitum sucrose (rock candy) and a water source (15 ml tube plugged with cotton). Both high and low nourishment groups had equal access to sucrose, but differed in the amount of proteinaceous food provided. Proteinaceous nourishment was provided in the form of late 3parately . For ourPolistes possess behaviorally distinct, not morphologically distinct castes, thus we were especially interested in developmental expression changes in larval brains. This led us to choose larval heads as the target tissue for our analysis. Whole heads were used rather than whole brains because of concerns about larval brain dissection quality and low RNA yields. We acknowledge that it is likely that the nourishment manipulation caused additional gene expression changes in other tissues we did not sample. However, we did not use whole bodies for two reasons: 1) our primary interest in brains and behavioral castes, and 2) the fact that brain gene expression differences in social insects can be subtle [e subtle , meaningde novo into a transcriptome . Raw sequence data has been deposited to the National Center for Biotechnology Information’s (NCBI) Short Read Archive . For a more details about the RNA-Sequencing and the transcriptome assembly, see [To preserve RNA during dissection, we removed the head region (indicated by dark coloration) of each individual larva, while kept on dry ice, using a sterilized razor blade. From these individual larval heads, we extracted total RNA using an RNeasy Mini Kit (Quigen), which was quality controlled using spectrophotometry (NanoDrop 2000) and a Bioanalyzer (Agilent). The High-Throughput Sequencing and Genotyping Unit of the W.M. Keck Center (University of Illinois at Urbana-Champaign) prepared 16 mRNA Seq libraries  derived from two experimental nourishment levels  and unmanipulated castes  using tbly, see .http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE61960). From these raw read counts, we identified differentially expressed transcripts  between ≤ 0.05,  download ≤ 0.05, . Raw reaDrosophila melanogaster sequences, we used Blast2GO (Version 2.6.5) [P. metricus transcriptomic sequences  to n 2.6.5)  to assigces (see ). Based ces (see ) betweenPolistes paper wasps, we compared molecular signatures of nourishment manipulation and caste determination [P. metricus transcripts by using a Chi-square test with Yates’ correction. For both the nourishment and caste datasets, we identified which KEGG pathways had at least one DET, and we then performed a Fisher exact test to determine whether there is a statistically significant number of shared KEGG pathways with DETs given the number of unique pathways with DETs per each dataset and all pathways without DETs. Finally, we assessed significance in the overlap between nourishment and caste GO enriched categories in relation to the total number of annotated, not enriched, GO terms and unique nourishment/caste enriched GO terms using a Fisher exact test.To assess the extent that differential nourishment biased gene expression patterns toward a particular caste phenotype in mination  at threeWe investigated an alternative approach (beyond comparisons of DET lists) to test for similarity in the transcript expression patterns associated with nourishment restriction and worker-caste development. In some sense, across the two studies we have a replicated experiment with respect to nourishment levels, which utilizes both the lab and field setting. As described in the introduction, worker-destined typically larvae receive more limited nourishment compared to queen-destined larvae ,27,30,33P. metricus nourishment manipulation dataset based on the following criteria: 1) the datasets compared late-stage subadult insects reared on two nourishment levels and 2) the studies used a transcriptome-wide approach, i.e. microarray or RNA-seq. We identified two studies that meet both of these criteria: one in fruit flies  between pairs of species. For the fruit fly comparison, we only used the wild-type fruit fly data, and we compared the paper wasp dataset to both tissue types in fruit flies (adipose and muscle). The GO terms listed in the publication of Teleman et al. [Based on our interest in whether nourishment differences induce similar molecular responses in transcript expression and biological processes, we reviewed the literature and searched data repositories (NCBI Gene Expression Omnibus and EMBL ArrayExpress) to identify the most directly comparable studies to the ogaster, ) and the Taurus, ). For thn et al.  were notn et al. ). For thhttp://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA242774). The transcript abundance data is available in the NCBI’s Gene Expression Omnibus repository . All other data sets supporting the results of this article are included within the article and its additional files.Raw sequence data has been deposited to the National Center for Biotechnology Information’s (NCBI) Short Read Archive (BioProject: PRJNA242774,


The prevalence of High-Risk Human papilloma virus (HR-HPV), a necessary cause of invasive cervical cancer (ICC) is relatively high in HIV infected women. Gaps exist in our knowledge of the optimal approaches for managing women who have HR-HPV with normal cervical cytology (NCC) particularly in settings of HIV infection.Between May 2012 and June 2013 we conducted a colposcopic assessment of HIV-infected women with prior (NCC) and known HR-HPV status to compare cervical abnormalities in women with and without HR-HPV. Colposcopic examinations were done at the Operation Stop Cervical Cancer (OSCC) unit of the Jos University Teaching Hospital (JUTH), Jos, Nigeria. Abnormal colposcopic finding (ACF) was defined as areas of aceto-white epithelium involving the squamo-coulumnar junction, areas of punctation, mosaic pattern or atypical vessels. We compared proportions of ACF as well as histologic grades of cervical intra-epithelial neoplasia (CIN) in women with or without HR-HPV. Statistical analysis was done on STATA.We conducted colposcopic examinations in 78 out of 89 (86.5%) eligible women. The mean age of the cohort was 32.4 years (SD ±4.6) with a median 32 years (IQR 29–36). After a mean follow up time of 20.1 months from the initial cervical pap cytology and HR-HPV testing, we found 12 of 78 (15.4%) women with ACF. The odds for an ACF was statistically higher [OR = 4.0 (95% CI: 1.1-14.7)] in women with HR-HPV compared to those without. Of the twelve women with ACF, subsequent histologic examination of colposcopically directed cervical biopsies confirmed CIN 1 in 4 cases (33.3%), CIN 2 in 1 case (8.3%), CIN 3 in 2 cases (16.7%), carcinoma-in-situ (CIS) in 2 cases (16.7%), and normal cervix in 3 (25.0%). Overall, the proportion of women detected with any grade of CIN was 11.5% (9/78) and 6.4% (5/78) were CIN 2 or greater lesion (CIN2+).HIV-infected women with NCC and HR-HPV had a four-fold higher likelihood for an ACF. The practice of early colposcopic examination of HIV-infected women with prior NCC and HR-HPV may increase early detection of higher grade CIN and CIS cancer stages in our setting. Cervical cancer was responsible for up to 25.4% of all new cancer cases in women in sub-Saharan Africa in 2012 .In developed countries of the world, well-organized cervical cancer screening programs using Pap smear cytology has led to significant declines in invasive cervical cancer. Such national programs do not exist in most developing countries like Nigeria. Studies have shown that women with HIV have an increase risk of having high grade squamous intraepithelial lesions (SILs) with higher rates of progression to invasive stages , 5. StudAdditionally, studies have shown that even in settings where cervical cancer screening is available, conventional Pap cytology is associated with high false negative and positive results  and someGaps exist in the knowledge of optimal management approaches for women with normal pap cervical cytology and a positive HR-HPV particularly in the setting of HIV infection. To assess the possible role of HR-HPV DNA testing in detecting underlying high-grade cervical lesions in women with prior NCC in the setting of HIV infection, we conducted a pilot study of HR-HPV DNA testing in women with NCC in a cohort of HIV infected women in Jos, Nigeria. The prevalence and epidemiologic factors associated with HR-HPV in our patient population have been previously reported . The curBetween May 2012 and June 2013, we enrolled a cohort of HIV-infected women with NCC at the Reproductive Health Unit (RHU) of APIN of the APIN/Harvard PEPFAR HIV Clinic, Jos University Teaching Hospital, (JUTH) Jos, Nigeria. The RHU was set up in 2008 and is staffed by two reproductive health nurses and a gynecologist to address issues related to contraception and cervical cancer screening.Routine care at JUTH includes CD4+ T cell  and viral load  measurement before antiretroviral therapy (ART), then approximately every 3 months during ART. Initiation of ART follows the Nigerian National Guidelines for HIV and AIDS treatment and care in adolescents and adults, 2007 . Other iA detailed questionnaire was administered to each participant to determine age, age at first coitus, parity, duration of HIV infection, ART history, previous abortions, history of contraception, previous Pap smear, smoking and alcohol consumption. Patient information not recalled by study participants such as duration of HIV disease, ART history etc. were extracted from their electronic database.During the first part of this study, this cohort of women had cervical samples obtained for HR-HPV diagnosis by Hybrid Capture 2 (HC2) signal amplification  unit of the department of Obstetrics and Gynecology, JUTH. The study participants were contacted through phone calls by the study nurse and a study examination visit was scheduled. In situations where the participants were not successfully contacted through phone calls , the researchers made efforts to track participants at their pharmacy drug pick-up visits where they received their regular antiretroviral drugs at the Adult HIV Treatment Clinic of JUTH. The colposcopic examinations were performed by a consultant gynecologist trained in VIA and colposcopic examination of the cervix. The gynecologist conducting the colposcopic examination was blinded to the HR-HPV status of the participant. Women who had detectable abnormalities were treated according to standard guidelines in our hospital. Abnormal colposcopic finding was defined as areas of aceto-white epithelium involving the squamo-coulumnar junction, areas of punctation, mosaic pattern or atypical vessels. Unsatisfactory colposcopic examination defined situations where the squamo-columnar junction was not clearly visualized. A tissue biopsy forceps was used to obtain cervical tissue as directed by the abnormal colposcopic area. Participants with normal colposcopic findings were reassured and booked for annual cervical pap cytology. The biopsy specimens were immediately fixed in formalin and transported to the histopathology laboratory for processing and histologic examination and interpretation by a trained pathologist who was blinded to the HR-HPV status of the specimen. The colposcopic findings were illustrated in a diagram and coded for subsequent entry into the study database.We used the Leisegang D-10625, Model1DS Ur Nr 55764, Colposcope, Berlin, Germany to examine the cervix. Each participant was placed in lithotomy position and an appropriate size Coscus speculum was introduced into the vagina to expose the cervix for examination. For each participant we first examined the vulva, vagina and the cervix before the application of 3% acetic acid solution. Prior to application of 3% acetic acid, the cervices seen to have excess cervical mucus were washed with 0.9% Normal saline to dislodge excess mucus discharge. Colposcopic abnormalities were described as normal, abnormal or unsatisfactory. Abnormal areas were biopsied using a punch cervical biopsy tissue forceps. The cervical tissues were processed in the histopathology laboratory. The Histopathologist was blinded to the HPV status of the participant and interpreted processed cervical samples either as Normal or CIN 1, CIN 2, CIN 3 or Carcinoma in Situ (CIS) with either well-differentiated, moderate differentiated or poorly differentiated squamous cell carcinoma or other variants if applicable.We created a study database that included routinely collected demographic parameters, CD4+ cell count , plasma HIV RNA , HC2 HR-HPV status, date of colposcopic examinations, colposcopic findings and histology results . To conduct statistical analyses, the database variables were coded for relevant statistical analysis on STATA. All analyses were performed using STATA version 11.0, College station, Texas, USA.Summary statistics were generated using two-way tables of association to compare baseline socio-demographic characteristics of study participants with normal versus abnormal colposcopic examination findings. The Pearson’s chi square and/or Fisher’s exact test was performed where applicable. We also created relevant indicator variables in order to run univariabe logistic regression analyses to determine odds ratio of having colposcopic abnormalities. In the multivariable logistic regression model the association between positive HR-HPV and abnormal colposcopic findings was adjusted for age category ≥30 years. Because of the relatively small numbers of participants in this pilot data, we estimated the proportions of histologic findings of the cervical biopsies in the study sample. All statistical tests were reported with corresponding 95% confidence interval and p-values. Probability estimates <0.05 were considered statistically significant.Institutional Review Board approval was obtained from the Harvard School of Public Health for identification and enrollment of HIV-infected women into care, treatment and other support services at APIN, JUTH. Secondary use of data approval was also granted by the Harvard School of Public Health to use CD4+ T cell count, viral load and other relevant patient data. The protocol for this study was approved by the JUTH Human Subject Ethics Committee.We studied 78 women out of 89 eligible participants (86.5%) who completed colposcopic examination Table . Of the Socio-demographic characteristics of the study cohort have been summarized in Table Colposcopic examinations were performed after a mean of 20.1 months from the initial cervical pap cytology and HR-HPV status evaluation. The mean follow up time between initial Pap cytology and subsequent colposcopic evaluation was comparable for women who were positive HR-HPV (20.5 months) and those negative HR-HPV (19.9 months); p-value 0.554. Colposcopy revealed 12 of 78 (15.4%) women with abnormal findings. The odds ratio (OR) of having an abnormal colposcopic examination finding was 4.0 (95% CI: 1.1-14.7) in women who were HR-HPV positive compared to HR-HPV negative; p-value 0.039 , CIN 2 in 1 case (8.3%), CIN 3 in 2 cases (16.7%), carcinoma-in-situ (CIS) in 2 cases (16.7%), and normal cervix in 3 cases (25.0%)  for age ≥30 years and an OR = 5.4  for an HR-HPV positive woman of having an abnormal colposcopic examination finding  HIV-infected women with prior NCC. Women with HR-HPV had a five-fold higher likelihood of an abnormal colposcopic description compare to those without after controlling for age. We also observed that women with positive HR-HPV had a higher proportion of CIN 2+ cervical abnormalities compare to women negative for HR-HPV. Overall, the proportion of any CIN lesion was 11.5% (9/78) and CIN2+ was 6.4% (5/78) in our cohort.In a cohort study reported from India , the aut3, high viral load  and those with a clinical evidence of HPV infection on visual inspection. A study from a Brazilian HIV-infected cohort, found a higher association of current cigarette smoking, CD4+ T cell count less than 350 cells/mm3, and HR-HPV with colposcopic/histopathologic diagnosis of CIN2+ lesions [Our pilot data showed that women who had a positive HR-HPV had a five-fold higher likelihood of an abnormal colposcopic examination after adjusting for age. Other factors such as CD4+ T cell count, HIV-1 viral load, previous abortions, and use of contraceptives were not significantly associated with having an abnormal colposcopic examination finding. A prior study in Jos during an era when accessibility and use of antiretroviral drugs was limited , found a lesions . Studies lesions . Our smaSubsequent colposcopically directed biopsy and histologic diagnosis a significant difference in the proportion of CIN 2+ lesions detected in HIV infected women who were HR-HPV positive compare to those HR-HPV negative. These findings have implications in the practice of cervical cancer screening in our setting. First, since colposcopy and directed biopsy with histologic examination is the gold-standard for the diagnosis of CIN/or invasive cancer; women with HR-HPV are more likely to have such colposcopic examination and directed biopsies thereby increasing the chances of diagnosing high-grade lesions and/or cancer. Results of HPV genotype distribution from meta- analysis data have shown that the HPV genotype distribution in low grades cervical lesions differs from that in cervical cancer , suggestOne alarming finding in our pilot study is the detection of 2 cases of CIS of the cervix following colposcopic examination and directed biopsies in women with prior NCC after only about 20 months of follow up. Indeed, studies have shown an increased hazard of rapid progression from CIN to ICC stages in women infected with HIV compared to HIV-uninfected controls . This fiThe major strength of this study lies in the fact that HR-HPV testing is not yet part of our screening for cervical cancer in Nigeria and this is first study examining its possible role in detecting high-grade cervical lesions particularly in HIV-infected women with NCC. We were also able to enroll 86% of the initial cohort and conducted colposcopic examinations irrespective of HR-HPV status. We must however, acknowledge the limitations of a relatively small sample size, which may have reduced the statistical power to control for potential confounding effects on detection of CIN2+ in our cohort. We also did not perform colposcopic examination at the time of initial Pap or HR-HPV determination and the abnormalities/CIN lesions detected at follow up colposcopy examinations may have existed at the time of initial Pap cytology examination.In all we detected 6.4% 5/78) CIN2 or greater in this small pilot cohort out of which 2 cases were CIS. Since HIV-infected women have a significant hazard of progression from CIN to ICC [/78 CIN2


Here, we demonstrate that electroporation-enhanced immunization with a rationally designed HPV DNA vaccine (GX-188E), preferentially targeting HPV antigens to dendritic cells, elicits a significant E6/E7-specific IFN-γ-producing T-cell response in all nine cervical intraepithelial neoplasia 3 (CIN3) patients. Importantly, eight out of nine patients exhibit an enhanced polyfunctional HPV-specific CD8 T-cell response as shown by an increase in cytolytic activity, proliferative capacity and secretion of effector molecules. Notably, seven out of nine patients display complete regression of their lesions and viral clearance within 36 weeks of follow up. GX-188E administration does not elicit serious vaccine-associated adverse events at all administered doses. These findings indicate that the magnitude of systemic polyfunctional CD8 T-cell response is the main contributing factor for histological, cytological and virological responses, providing valuable insights into the design of therapeutic vaccines for effectively treating persistent infections and cancers in humans. While several human papilloma virus (HPV) vaccines exist, a highly effective vaccine that mediates regression of HPV-induced tumours is lacking. Here the authors show that a therapeutic DNA vaccine-induced HPV-specific polyfunctional CD8 T cell in 7 out of 9 patients who all exhibited complete regression of lesions and viral clearance. Cervical cancer is one of the leading causes of cancer death in women worldwide15The pre-malignant cervical intraepithelial neoplasia 2 and 3 (CIN2/3), in particular those positive for HPV16, are considered as high-grade lesions that have approximately a 30% chance of developing into invasive cancerHPV E6 and E7 act as viral oncoproteins by binding and promoting degradation of tumour suppressor proteins, p53 and retinoblastoma (pRb), respectively11Induction of an HPV-specific T-cell response was observed with other vaccines, but without any meaningful clinical efficacy for the treatment of high-grade cervical lesions. One study found that subcutaneous immunization with an HPV16 E6/E7 synthetic long-peptide vaccine induced detectable level of HPV-specific interferon-γ (IFN-γ)-producing T-cell response in all five patients with high-grade cervical dysplasia, but without any reduction in HPV DNA and histological improvement of the cervical lesionsHere, we describe a newly developed HPV E6/E7 DNA therapeutic vaccine, designated GX-188, designed to facilitate the processing and presentation of HPV E6/E7 antigens by dendritic cells (DCs) through the co-expression of Fms-like tyrosine kinase-3 ligand (Flt3L). Vaccination with GX-188 by EP (GX-188E) elicits a significant E6/E7-specific T helper (Th) 1-polarized cellular immune response in all nine patients with CIN3. Importantly, GX-188E vaccination significantly induces polyfunctional HPV16-specific CD8 T-cell response in seven out of nine patients, which correlates with HPV clearance and complete resolution of high-grade cervical lesions on cytology and histology. Therefore, we offer an effective therapeutic vaccine strategy for eradicating persistent HPV infections and HPV-induced tumours, together with an important insight into the immune correlates to control persistent viral infection.GX-188 DNA vaccine is engineered to express E6 and E7 proteins of HPV16 and HPV18 fused to extracellular domain of Flt3L and the signal sequence of tissue plasminogen activator (tpa); the purpose for inclusion of Flt3L and tpa is to promote antigen presentation and trafficking of the fused protein to the secretory pathway, respectively . The act20According to the inclusion and exclusion criteria for this study, nine out of 11 screened patients with CIN3 were enroled . The scrA total of 49 adverse events (AEs) were recorded during all visits. Twenty-three AEs, including eczema, ecchymosis, vaginal itching, sleepiness, anorexia and dizziness were determined to be unrelated to the vaccination. Nineteen AEs including chills, injection site pain, swelling and hypoaesthesia, were recorded to be associated with GX-188E vaccination . AlthougSince it was reported that the administration of Flt3L protein could increase the frequency of white blood cells (WBCs)22To determine the immunological safety of our approach, we investigated whether the enhanced delivery of GX-188E generated anti-DNA antibodies, which are known to be associated with autoimmune disorders276 peripheral blood mononuclear cells (PBMCs). Interestingly, the response against the E6 antigen was more vigorous than against E7 as determined by the magnitude of response , during, at VT2 (2 weeks) and VT4 time points (8 weeks), and after, at VF1 (20 weeks) and VF2 time points (36 weeks), GX-188E vaccination. Relatively high pre-existing IFN-γ ELISPOT response was detected in one patient (A03), whereas the other eight patients displayed weak pre-existing HPV-specific cellular immunity before vaccination. On the basis of the criteria described in the Methods, all subjects exhibited a marked increase in the vaccine-induced E6- and E7-specific IFN-γ ELISPOT response compared with the background level before vaccination . Interes+ CD4 T-cell responses in all nine patients, while IFN-γ+ CD8 T-cell response was enhanced in eight out of nine patients. Thus, with the exception of patient A04, GX-188E vaccine elicited activation of both HPV16-specific CD4 and CD8 T cells.The establishment of memory T cells, normally starting to form about 4 weeks after immunization, is usually one of the indispensable factors for protective efficacy of a vaccine29Since persistent HPV infection impairs Th1 cellular response to HPV, leading to cervical cancer progression113132To determine whether GX-188E vaccination induced multiple aspects of HPV-specific CD8 T-cell functionality, we assessed the ability of CD8 T cells, on stimulation with a pool of HPV16 E6 and E7 peptides, to simultaneously express the following five different effector functions: cytokines IFN-γ, IL-2 and TNF-α, a chemokine MIP-1β and the cytotoxic activity as determined by expression of CD107a/b, which is exclusively found during degranulation of cytotoxic T cellsin vitro-expanded CD38+Ki67+ CD8 T cells. Although one patient (A01) displayed a relatively high pre-existing proliferative level prevaccination (VS), the rest of the patients demonstrated low levels of Ki67+CD38+ CD8 T cells  according to the histological evaluation of colposcopic-directed biopsy specimens  was found to be also co-infected with HPV18. At week 12 (VT4), four patients  and patient A05 showed clearance of HPV16 and HPV18 viruses, respectively , indicatIt is notable that the three patients  who cleared their lesions and HPV infection at the early time point (VT4) promptly displayed a relatively high magnitude of HPV-specific polyfunctional CD8 T-cell response . In addiin situ, and seven out of nine patients (78%) had complete regression of their high-grade lesion and clearance of HPV DNA, which are the highest response rate compared with the previous reports in clinical trials of HPV vaccines. Second, cytological, histological and virological evaluations yielded the same results in the responders, indicating induction of a complete response by GX-188E. Third, the normalization of lesions by GX-188E vaccination was maintained without recurrence of dysplasia during the entire study period of 36 weeks, indicating a sustained therapeutic efficacy of the vaccine. Finally, clinical and virological responses at the cervix were correlated with the magnitude of the polyfunctional CD8 T-cell response in the blood, suggesting that systemic HPV-specific CD8 T-cell responses might serve as a predictive biomarker for determining clinical outcomes of therapeutic vaccine in the setting of high-grade cervical lesions.In this study, we have demonstrated the efficacy of a novel HPV E6/E7 DNA therapeutic vaccine, designated GX-188, strategically designed to induce robust type 1 T cell-mediated immunity and to eradicate HPV infection-associated lesions. So far, several types of HPV therapeutic vaccines have been evaluated in patients diagnosed with CIN2/3 lesions using viral vectors13It has been reported that regression of CIN2/3 can occur spontaneously in some patients at a rate ranging from 11 to 30% over ≥1 year to 38% between 9 and 20 weeks after colposcopic biopsy3940+ lymphoid DCs464748431131The induction of strong HPV-specific Th1 and CD8 T-cell immunity by GX-188E vaccination leading to complete clearance of HPV DNA and high-grade cervical lesions observed in this study is likely due to the following reasons: codon optimization of HPV E6/E7 genes17The immunological and clinical outcomes after vaccination are generally variable and may be determined in part by host genetic factors. In the present study, GX-188E vaccine achieved complete response in seven out of nine patients (78%). Among seven responders, six patients carrying human leukocyte antigens (HLA)-A*02 exhibited high polyfunctional CD8 T-cell responses as well as complete regression of CIN3 . Among tin situ, probably providing more severe immunosuppressive microenvironment by which the effector function of vaccine-induced systemic CD8 T cells could be thwarted. It was reported that median cell density of stromal Foxp3+ regulatory T cells expressing IL-10 and indoleamine 2, 3-dioxygenase appear to increase significantly in the cervix with increasing pathology and cancerThe other non-responder patient (A09) exhibited a comparable magnitude of antigen-specific CD8 T-cell response with two effector functions, but had lower polyfunctional profile of CD8 T cells with three or more effector functions than responder patients, indicating that the polyfunctionality of vaccine-induced CD8 T cells may contribute to clinical outcomes. It is worth noting that the patient A09, who was infected with only HPV16, displayed a higher T-cell response to HPV18 than to HPV16 in IFN-γ ELISPOT assay. Thus, this biased response towards HPV18 in the patient A09 may have led to the impaired HPV16-specific polyfunctional CD8 T-cell response. Alternatively, it is possible that local immunity at cervix could be involved in the clearance of lesions. Recently, combined vaccination with a heterologous DNA prime-recombinant vaccinia vector-based boost regimen induced a tissue-localized T-cell response in most patients with CIN2/3, which was suggested to be informative for determining vaccine efficacyin situ, one patient (A03) who had a relatively high pre-existing HPV-specific cellular immune response completely cleared both HPV infection and the high-grade lesions on GX-188E vaccination. Thus, it would be of interest to investigate whether the pre-existing cellular immunity could be an indicator of the clinical outcome of therapeutic vaccine in CIN3 patients with carcinoma in situ. Most importantly, all six CIN3 patients with severe dysplasia showed eradication of HPV DNA and regression of their lesion following GX-188E DNA vaccination. Even though the number of patients (n=9) is too small to draw a definite conclusion, our findings may provide an important insight into the key requirements for a therapeutic vaccine to be effective against various persistent infections and chronic diseases. Considering that the current primary treatment option for CIN3 is surgical excision, which is often accompanied with several side effectsin situ.Among three CIN3 patients with carcinoma http://www.clinicaltrials.gov (NCT01634503)The clinical trial protocol was reviewed and approved by the Institutional Review Board at Cheil General Hospital and Women’s Healthcare Center (CGH-IRB-2012-35). This study was conducted in accordance with the ethical principles that had their origin in the current Declaration of Helsinki and was consistent with International Conference on Harmonization Good Clinical Practice (IHC GCP) and applicable regulatory requirements. All study participants gave written informed consent before undergoing screening for study eligibility and enrolment. This trial is listed at This phase 1 clinical study was conducted as an open label, single center, dose-escalation study at Cheil General Hospital & Women’s Healthcare Center, Seoul, Korea. The primary end point was to evaluate safety and tolerability in patients with CIN3. The secondary end points included systemic induction of HPV E6- and E7- specific T-cell immune responses measured by IFN-γ ELISPOT, and changes of involved lesions and HPV infection status at the uterine cervix. Women aged between 20 and 50 years with histologically and virologically proven HPV16- or HPV18-associated CIN3 were enroled in the study. The CIN3 was confirmed by colposcopy-directed biopsy and HPV16 or HPV18 positivity was determined by PCR. Subjects with hepatitis B virus, hepatitis C virus or human immunodeficiency virus infections, abnormal electrocardiography including arrhythmia, history of severe adverse drug events or severe allergic diseases were excluded. Females who were pregnant or planning to be pregnant were not recruited in the study. Vaccination consisted of a series of three vaccine injections administered intramuscularly to alternating deltoid muscles at weeks 0, 4 and 12. A standard 3+3 dose-escalation scheme was followed and dose levels of 1, 2 and 4 mg were tested. At the highest dose, 4 mg of GX-188E was split into 2+2 mg and injected to the left and right deltoids muscles. For the intramuscular injector, an EP device  was used to facilitate DNA uptake into cells.HLA typing was accomplished at Catholic Hematopoietic Stem Cell Bank, College of Medicine, The Catholic University of Korea, Seoul, Korea. Sequence-based typing (SBT) of HLA was performed by heterozygous amplification followed by sequencing of the complete exons 2, 3 of HLA-A and -B. For locus-specific amplification, primers were used in in-house method. After application by PCR, agarose gel electrophoresis of the PCR products was conducted to assess the quantity and quality. Cycle sequencing reactions using the ABI PRISM BigDye terminator kit  and the automated ABI377 DNA Sequencer  were performed. These data were analysed by using SBT analysis programme .E. coli DH5α under cGMP condition at Althea Technologies, Inc., San Diego, CA.The study vaccine, pGX-188, contains a plasmid DNA encoding E6 and E7 proteins of HPV serotypes 16 and 18 fused with tpa signal sequence and extracellular domain of Flt3L. Synthetic codon-optimized E6 or E7 genes were fragmented into two parts  with a small overlapping sequence (encoding 16 amino acids), and shuffled as shown in The assessments including colposcopy, histology, endocervical cytology and HPV genotyping test were conducted by local laboratory at the trial site. The assessments were performed in compliance with the standardized method or the internal protocol of Cheil General Hospital and Women’s Healthcare Center. Responses to treatment were evaluated using virology and histology results at weeks 20 and 36 post GX-188E vaccination.For histological evaluation, biopsy samples were taken during screening and two follow-up visits at weeks 20 and 36. Samples were fixed with 10% formaldehyde and 4–5 μm sections were stained with haematoxylin and eosin. Endocervical samples were collected using cytobrush  during colposcopic examination. This endocervical cytology test was also used in addition to histology for the assessment of GX-188E vaccination. Data from histological and cytological analyses were reviewed independently by at least two pathologists and results were confirmed after discussion with by conference of all pathologists and investigators.To evaluate virological response, HPV typing was performed to determine whether subjects were infected by either HPV16 and/or HPV18. Samples were collected from the cervix by using a swab-type device, and total DNAs were extracted using the AccuPrep Genomic DNA Extraction kit . HPV detection and genotyping was done by Multiplex-PCR system using the IVD CE marked Seeplex HPV4A ACE Screening kit  according to the manufacture’s protocol. The Seeplex HPV4A ACE Screening kit can identify HPV16, HPV18, other high-risk types , HPV6 and HPV11 types at the same time. PCR products were analysed using an automatic MultiNA instrument . HPV DNA genotyping was double checked in cervical cells using Cheil HPV DNA Chip with real time PCR to compensate the accuracy of HPV genotype as previously described2, and subsequently PBMCs (2 × 105 cells per well) were stimulated with 2 μg ml−1 of four different pools of HPV16 and HPV18 E6- or E7-derived peptides (20-mer with 10 amino acids overlapping) for 48 h. Phytohaemagglutinin and the medium only served as positive and negative controls, respectively. After stimulation, spots indicating IFN-γ-secreting cells were developed according to manufacturer’s instructions (BD Bioscience). The number of spots was analysed with an automated ImmunoSpot Analyzer (Cellular Technology Ltd.). The HPV-specific responses were calculated by subtracting the mean number of spots in the medium only control from the mean number of spots in experimental wells, which were expressed as SFUs per 106 PBMCsCryopreserved and thawed PBMCs were adapted with OpTmizer CTS medium (Life Technologies) for more than 6 h at 37 °C, 5% CO2. PBMCs were plated in duplicate and stimulated with a combined mixture of HPV16 E6 and E7 peptides in one pool (15-mer with eight amino acids overlapping) at a concentration 2 μg ml−1, α-CD3  or the medium alone (negative control) in the presence of 1 μg ml−1 of α-CD28  and α-CD49d  for 13 h. Secretion inhibitors  were added 90 min after initial stimulation. After stimulation, cells were washed with PBS for subsequent immunostaining and polychromatic flow cytometric analysis. Antibodies for staining cells were CD19-APCCy7 , CD4-PerCPCy5.5 , CD8-PECy7 , CD3-BV605 (Bright Violet 605) , CD3-BV500 , Live/dead-APCCy7 , MIP-1β-PE , IFN-γ-APC , TNF-α-BV421 , IL-2-BV711 , CD107a-FITC  and CD107b-FITC . Fluorescence-activated cell sorting analysis was accomplished by Fortessa flow cytomer (BD Bioscience), and the data were analysed using FlowJo software (Tree Star). Boolean gating was used to determine simultaneous cytokine production from CD8 T cells. Analysis of polyfunctionality was performed with SPICECryopreserved and thawed PBMCs were resuspended in OpTimizer CTS, and rested for more than 6 h at 37 °C, 5% CO5 per well) were resuspended in OpTimizer CTS, and rested for more than 6 h at 37 °C, 5% CO2, and subsequently PBMCs were plated in duplicate and were stimulated in RPMI 1,640 containing 10% fetal bovine serum, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin with a combined mixture of HPV16 E6 and E7 peptides in one pool (15-mer with eight amino acids overlapping) at a concentration of 2 μg ml−1 or the medium only as negative control in 96-well plates. Culture supernatants were harvested 48 h after the stimulation and cytokines were quantitated by Th1/Th2/Th17 cytometric bead array (CBA) kit (BD Biosciences). According to manufacturer’s instructions, the proposed detection limit was 2.5–5 pg ml−1  or 19 pg ml−1 (IL-17A), and the cutoff value was set at 5 pg ml−1 because the standard curve of each cytokine showed linearity starting at this concentration (Cryopreserved and thawed PBMCs (2 × 10ntration . Positiv6 cells per well) were adapted with OpTmizer CTS medium (Life technologies) for more than 6 h at 37 °C, 5% CO2. PBMCs were plated and stimulated with a combined mixture of HPV16 E6 and E7 peptides in one pool (15-mer with eight amino acids overlapping) at a concentration 2 μg ml−1 in RPMI 1,640 containing 10% fetal bovine serum, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin for 5 days. α-CD3 mAb and the medium alone served as positive and negative controls, respectively. After 3 days, cell cultures were replaced with 100 μl of fresh medium. At the end of culture, cells were washed with PBS for subsequent immunostaining and polychromatic flow cytometric analysis. The cells were stained with CD19-FITC , CD4-PerCPCy5.5, CD8-PECy7, CD38-BV421 , CD3-BV605, Ki-67-PE , and Live/Dead-APCCy7. Responses at least threefold greater than those of the medium control were considered to be positive. A post-analysed vaccine-induced response was defined as being at least threefold higher in the percentage of antigen-specific proliferating CD8 T cells than that at prevaccination.Cryopreserved and thawed PBMCs .Descriptive statistics of the safety, pharmacodynamics and pharmacokinetic outcomes was performed using SAS (V9.1) software. Standard and two-tailed paired Student’s T.J.K., H.-T.J., S.-Y.H., Y.-Y.H., Y.S.S., J.S.P. and Y.C.S. designed study; T.J.K., H.-T.J., H.G.Y., Y.B.S., S.R.H., C.-W.L., S.K., K.S.P., I.-H.L., K.-T.L. and M.S.J. performed experiments and analysed data; T.J.K., H.-T.J., S.-Y.H., Y.B.S., J.-W.W., J.P., C.D.S., Y.S.S., J.S.P. and Y.C.S. wrote the paper.How to cite this article: Kim, T. J. et al. Clearance of persistent HPV infection and cervical lesion by therapeutic DNA vaccine in CIN3 patients. Nat. Commun. 5:5317 doi: 10.1038/ncomms6317 (2014).Supplementary Figures 1-5, Supplementary Tables 1-4, Supplementary Methods and Supplementary References