Arabidopsis thaliana ecotype Columbia and Nicotiana tabacum cv. Xanthi as potential sources for an inexpensive subunit vaccine.We have investigated the possibility and feasibility of producing the HPV-11 L1 major capsid protein in transgenic L1 gene with the C-terminal nuclear localization signal (NLS-) encoding region removed, and not with the full-length gene. The HPV-11 L1 NLS- gene was stably integrated and inherited through several generations of transgenic plants. Plant-derived HPV-11 L1 protein was capable of assembling into virus-like particles (VLPs), although resulting particles displayed a pleomorphic phenotype. Neutralising monoclonal antibodies binding both surface-linear and conformation-specific epitopes bound the A. thaliana-derived particles and – to a lesser degree – the N. tabacum-derived particles, suggesting that plant-derived and insect cell-derived VLPs displayed similar antigenic properties. Yields of up to 12 μg/g of HPV-11 L1 NLS- protein were harvested from transgenic A. thaliana plants, and 2 μg/g from N. tabacum plants – a significant increase over previous efforts. Immunization of New Zealand white rabbits with ~50 μg of plant-derived HPV-11 L1 NLS- protein induced an antibody response that predominantly recognized insect cell-produced HPV-11 L1 NLS- and not NLS+ VLPs. Evaluation of the same sera concluded that none of them were able to neutralise pseudovirion in vitro.Transformation of plants was only achieved with the HPV-11 L1 NLS- gene in two different plant species and increased yields of HPV-11 L1 protein by between 500 and 1000-fold compared to previous reports. Inoculation of rabbits with extracts from both plant types resulted in a weak immune response, and antisera neither reacted with native HPV-11 L1 VLPs, nor did they neutralise HPV-11 pseudovirion infectivity. This has important and potentially negative implications for the production of HPV-11 vaccines in plants.We expressed the wild-type HPV-11  Papillomaviridae. High-risk genital HPVs types 16, 18, 33 and 58 are the leading cause of cervical cancer [Papillomaviruses are small species- and tissue specific double-stranded DNA tumour viruses, classified in the taxonomic family l cancer -5.et al. [Given a HPV-11 prevalence rate of 5–12% in normal women -8, a seret al.  have fouWhile efforts to develop HPV vaccines have largely concentrated on the cervical cancer-causing HPV-16, HPV-11 has also received widespread attention. Extremely high healthcare costs are associated with management of non-cancerous HPV-6/11 disease, and inclusion of HPV-6 and 11 in a vaccine might be advantageous . Merck hA strategy for the large-scale production of inexpensive HPV vaccines is production in plants: this could be between 10 and 50 times cheaper than its production in fermentation systems. Delivery of these by the oral or "needle-free" route is ideally suited to the background setting of vaccination campaigns in many developing countries . We and Agrobacterium tumefaciens-mediated transformation, we genetically manipulated Arabidopsis thaliana and Nicotiana tabacum, to express the HPV-11 L1 gene. Comparative studies on the expression of the gene were performed on both expression systems. Products were characterised antigenically using monoclonal antibodies and used to immunise rabbits, in parallel with insect cell-produced antigens.This study aimed to develop a plant-derived L1 protein subunit vaccine against HPV-11 in a plant host from which it could be easily purified at reasonable yield, in order to formulate a potentially cheap vaccine that could help prevent HPV-11 infection in developing countries. Through A. thaliana and N. tabacum plants with the HPV-11 L1 and HPV-11 L1 NLS- genes, only plants containing the HPV-11 L1 NLS- gene could be regenerated. Attempts to introduce the full-length HPV-11 L1 gene failed in both plant systems. PCR screening of putative HPV-11 L1 NLS- transgenic A. thaliana first generation (T1) and N. tabacum regenerated generation (R0) plants, using primer pair 4 as listed in Table A. thaliana T1 generation and 4/15 N. tabacum R0 lines were positive for the gene.Despite several attempts to transform 1 plants and screening seeds on selective media. Random PCR screening of plant lines up to the T6 generation for A. thaliana and T3 generation for N. tabacum showed that the gene was stably integrated and successfully inherited  to HPV-6 ; HPV-11 (H11:B2 and H11:H3) and to HPV-16 (H16:D9) displaying specific and cross-reactive properties  also bound; however, binding efficiencies of these surface exposed linear epitope-recognizing and non-HPV-11-neutralising Mabs were significantly lower ; O.D.405nm comparisons allowed calculation of the total amount of L1 protein from transgenic A. thaliana and N. tabacum extracts for which the total plant weight and homogenisation buffer volume was known. Overall yields of L1 protein harvested from A. thaliana were calculated to range from between 3 and 12 μg/g, whereas N. tabacum plants yielded between 0.2 and 2.2 μg/g of fresh leaf material.The amount of L1 protein in transgenic plant extracts was measured by comparison to an ELISA-derived standard curve for known concentrations of insect cell-derived HPV-11 L1 NLSA. thaliana and N. tabacum protein extracts were immunotrapped onto grids using anti-HPV-11 L1 polyclonal antiserum and stained with 2% uranyl acetate  and N. tabacum  matched that of the positive control. In contrast, however protein from N. tabacum (B) was extensively proteolysed (~36 kDa) indicating that protease activities differ markedly between the 2 plant systems. HPV-11 L1 NLS- in insect cell lysate detected with H6:C6 (shown in C as Insect cell (IC)) was cleaved twice, resulting in 3 products: one of these is ~36 kDa in size. Non-infected insect cell lysate was used as negative control in C.Detection of HPV-11 L1 NLSA. thaliana and N. tabacum protein extracts. Serum collected on days 1, 15, 28, 42, 56 and 70 was analysed for anti-HPV-11 L1 reactive antibodies by ELISA and results are shown in Figure 3 generation transgenic A. thaliana-derived L1 protein extract calculated to contain between 21 μg and 43 μg.Eight New Zealand white rabbits were immunized with concentrated transgenic - VLPs . This lack of recognition of the HPV-11 L1 NLS+ VLPs is of great concern because these VLPs most closely resemble the actual virus: the implications of this will be discussed later.The most interesting result was obtained by evaluating serum reactivities in ELISA against insect cell-produced HPV-11 L1 NLS+ and NLS- VLPs by ELISA. The results shown in Figure - VLPs, and not with denatured protein from either type of VLPs. This was confirmed by western blots of the insect cell-derived NLS+ and NLS- protein. While very strong reactions were seen with baculovirus and Sf21 cell proteins in both prebleed and last bleed sera, neither L1 protein was detected (data not shown). This suggested that the predominant antibody response was most probably not targeted against any linear epitopes.To further investigate this, Day 56 antisera were tested against denatured and non-denatured insect cell-derived HPV-11 L1 NLS+ VLPs. Dilutions of the conformation-specific and neutralising MAb H11:H3 were the second positive control.All rabbit sera taken on days 1 and 56 were tested for their capacity to neutralise HPV-11 pseudovirions. Serum sample dilutions ranging from 1:50 to 1:12150 were incubated together with HPV-11 L1 pseudovirions before being assayed. One of two internal controls was serum from rabbit #19 (not previously shown), which was inoculated with insect cell-derived HPV-11 L1 NLS6, none of the post-inoculation day 56 sera were neutralising, even at the lowest dilution of 1:50.No specific neutralising capacity was observed for any prebleed serum. However, while rabbit #19 serum  neutralised the pseudovirion at the highest dilution tested (1:12150), and the known conformation-specific and neutralising MAb H11:H3 worked to a dilution of 1:10- VLPs are modified to some extent, yet still retain certain overall virion morphological features as seen by electron microscopy. Immunisation of animals, however, results in an antibody response that is not capable of recognizing or neutralising HPV-11 L1 NLS+ VLPs, the closest counterpart to the actual virus.Taken together, these serological results indicate that plant-derived HPV-11 L1 NLSL1 gene, as full-length and NLS- constructs, in transgenic A. thaliana and N. tabacum, with the overall aim of gaining some insight as to the feasibility of developing a plant-derived vaccine against HPV type 11. This is against a background of the release in 2006 of the GARDASIL™ yeast-derived quadrivalent vaccine against HPV types 6, 11, 16 and 18 by Merck: this vaccine has already shown 100% effectiveness in protecting from infection. However, the high cost of one course of this vaccine  means that it will not prevent the continuous spread of the virus-associated disease in the foreseeable future, because it will remain largely non-affordable to people in disadvantaged regions of the globe.The focus of this study was on the expression of a native HPV-11 A. thaliana and N. tabacum with the HPV-11 L1 gene missing the C-terminal nuclear localisation signal (NLS). This increases the number of plant systems used for the successful expression of HPV L1 proteins to include not only tobacco and potato, but now also Arabidopsis. Overall, our tobacco lines yielded ~2 μg of L1 protein per g of fresh leaf material, whereas up to 12 μg/g of L1 protein could be extracted from whole Arabidopsis plants. Our group has previously expressed unmodified CRPV L1 and HPV-16 L1 genes in transgenic tobacco to levels of 1 μg/g and 4 ng/g of plant tissue respectively [N. benthamiana to ~40 ng/g [et al. [L1 gene in tobacco at a level of ~0.5% of the total soluble protein content (TSP), and in potato to about 0.2% TSP or ~12 μg/g of tuber. Warzecha et al. [L1 NLS- gene in potato and reported extracting up to ~23 ng/g of fresh tuber. The HPV-16 L1 levels of expression have since been far surpassed by Maclean et al. [Here we report on the successful transformation of both ectively ,17, and ~40 ng/g . Biemelta et al.  expressen et al. ; this won et al. .in planta expression of the wild-type HPV-11 L1 gene presented great difficulties and that it was effectively impossible to transform either one of our host plants. Similar observations were made by Warzecha et al. in 2003 [L1 NLS+ gene, the number of putative transgenic plant lines remained low. The low number of confirmed HPV-11 L1 NLS- transgenic plant lines in this study reiterates the observation that integration and/or expression of the HPV-11 L1 gene (full-length vs truncated/wild-type vs codon-optimised) is generally not well tolerated in plants.We found that  in 2003  and despL1 gene, where expression of full-length L1 in either native  or human codon-optimised forms [L1 gene expressed better in plants than the plant codon-optimised or wild-type genes. Although HPV-11 and 16 L1 genes differ significantly, their L1 proteins are functionally very similar. Thus, the reasons for the difference in transformability remain obscure. It is also interesting in light of the HPV-16 results that our native HPV-11 L1 gene expressed so much better than the plant codon-optimised candidate of Warzecha et al. [L1 genes is best assessed empirically, rather than by assuming that gene optimisation according to plant codon usage will automatically improve expression.This stands in direct contrast to results obtained with the HPV-16 a et al. : this inL1 gene is not required for correct VLP assembly [in planta expression levels achieved with a spectrum of HPV L1 genes, we had decided to focus on maximizing the expression of the wild-type HPV-11 L1 NLS- gene in our two expression hosts.The NLS of the HPV-11 assembly . Resultiassembly . Therefoet al. [et al. [L1 mRNAs were somewhat degraded, was that such degradation was not detectable in our system.We were able to show successful transgene integration as well as stable inheritance in consecutive generations Figure  and 1B. et al.  and Warz [et al.  found byL1 NLS- gene via recombinant baculovirus in Sf21 cells resulted in a range of different sized particles that did not resemble the classical shape  using primer pairs 1 and 2 respectively (Table KRpavskpstapKRKRTKTKK) spanning aa 482 – 502. PCR was performed using standard cycling conditions, with an annealing temperature of 55°C. A silent mutation at position 257 (C to T) was found, otherwise this HPV-11 L1 sequence does not differ from the prototype HPV-11 L1 sequence (GenBank accession number M 14119).Full-length and truncated HPV-11 DNA fragments derived from the L1 and HPV-11 L1 NLS- ORFs were excised and cloned into pUC19 , and thereafter directionally cloned (EcoRI/HindIII) into plasmid pART7: this placed the L1 genes downstream of the CaMV35S promoter and upstream of the octopine synthase gene terminator (ocs 3'). This cassette was excised (NotI) and cloned into the A. tumefaciens binary vector pART27 [A. tumefaciens C58C1RifR competent cells were transformed with HPV-11 L1/HPV-11 L1 NLS- pART27 DNA using a freeze-thaw method [L1) and 4 (HPV-11 L1 NLS-) , transformed into Escherichia coli DH10BAC competent cells, and recombinant baculovirus was generated by transfection of the bacmid DNA into Sf21 cells . HPV-11 L1 protein was produced in Sf21 cells and virus-like particles (VLPs) were prepared as described [The full-length and NLSescribed .A. thaliana ecotype Columbia plants were transformed with A. tumefaciens constructs (HPV-11 L1/HPV-11 L1 NLS-) by a simplified transformation protocol [A. tumefaciens cultures containing either pART27 L1 or pART27 L1 NLS-. A. tumefaciens cultures were harvested after 2 days and resuspended to OD600 = 0.8 in phosphate buffered saline  containing 5% (w/v) sucrose and surfactant Silwet-77 at a concentration of 0.03% (v/v). Dipped plants were grown at 16/8 hr day/night photoperiod until seeds were harvested. To determine the transformation efficiency and to screen for transformed plants, dry seeds were sterilised in 10% bleach, washed 3 times in water and plated on plant nutrient sucrose medium (PNS) containing kanamycin (250 μg/ml). Germination was allowed to take place under a 16/8 hr photoperiod, at 25°C and 80% relative humidity. Seedlings were left on PNS media until complete development of a mature 4th leaf, before being transplanted to soil.protocol . Primaryet al. [N. tabacum plants. Sterilised leaf discs (±1 cm2) were dipped into a 48 hr A. tumefaciens culture carrying either the HPV-11 L1 or HPV-11 L1 NLS- construct. Squares were transferred to non-selective co-cultivation media [th leaves had grown to maturity.The transformation protocol of Horsch et al.  was usedon media  for 72 hA. thaliana and N. tabacum plants using the Dellaporta method [L1 and HPV-11 L1 NLS- genes using primer pairs 3 and 4 (Table Plant genomic DNA was extracted from putative transgenic and wild type a method  and screL1 NLS- mRNA was achieved by RT-PCR using the Access RT-PCR system (Promega). Primer pair 5 . Detection of the A. thaliana plants and leaves from N. tabacum were harvested and homogenised in 1:2 (w/v) cold 0.1 m phosphate/0.5 m NaCl ph 7.4 high salt buffer (HSP buffer). The homogenate was filtered through cheesecloth and centrifuged at 6000 × g for 10 min. 10% (w/v) polyethylene glycol (PEG mw 8000) was added to the supernatant before it was centrifuged at 6000 × g for 20 min. The pellet was resuspended in 1/10th original volume HSP buffer and centrifuged at 6000 × g for 20 min to remove further debris; the remaining supernatant was centrifuged at 100.000 × g for 180 min. The final pellet was resuspended in 1/10th of the previous volume of hsp buffer used.Entire - protein-containing extracts derived from A. thaliana and N. tabacum, together with non-transgenic plant protein extracts, were characterised by ELISA using a panel of Mabs to HPV-6, HPV-11 and HPV-16 [HPV-11 L1 NLSd HPV-16 , (Table 5% (v/v) trichloroacetic acid (TCA) was added to plant protein extracts before being centrifuged at 8000 × g for 20 min at 4°C. The pellet was washed twice in 100% acetone/20 mM HCl before the pellet was resuspended in 25 mM Tris, 2% SDS . Plant protein extracts were denatured at 100°C for 10 min in SDS-PAGE gel loading buffer, resolved on a 12% acrylamide gel and transferred onto nitrocellulose membrane by semi-dry electroblotting. L1 protein was detected with Mabs to HPV-6 L1  and then incubating grids with the secondary gold-labeled anti-mouse-conjugated antibody (1:100)  followed by staining with 2% uranyl acetate.A. thaliana and N. tabacum protein extracts (500 μl) were mixed 1:1 (v/v) with Freund's incomplete adjuvant and injected into New Zealand white rabbits (3 subcutaneous and 1 intramuscular site) on days 1, 28 and 56. Serum collected on days 1, 15, 28, 42, 56 and 70 was analysed by ELISA (1:20 dilution) against insect cell-derived HPV-11 L1 and HPV-11 L1 NLS- VLPs coated onto plates at a concentration of 0.4 μg/100 μl.Concentrated et al. [in vitro. Threefold dilutions of all pre- and post-inoculation sera ranging from 1:50 to 1:12,150 were prepared. Sera were incubated with HPV-11 pseudovirions and the resulting secreted alkaline phosphatase (SEAP) content was determined by application of the Great ESCAPE SEAP chemiluminescence kit (BD Clontech) according to the manufacturer's instructions.HPV-11 L1 pseudovirions were generated according to the protocol described by Pastrana et al.  with plaTOK participated in the design of the study, carried out most of the experimental work and drafted the manuscript. IIH participated in the design of the study, coordinated animal experiments and virus neutralisation assays and helped drafting and revising the manuscript. NDC supplied the monoclonal antibodies utilised in the study and participated in experimental design. EPR conceived the study, participated in its design and helped to revise the manuscript. All authors read and approved the final version.


Natural history models of human papillomavirus (HPV) infection and disease have been used in a number of policy evaluations of technologies to prevent and screen for HPV disease , sometimes with wide variation in values for epidemiologic and clinical inputs. The objectives of this study are to: (1) Provide an updated critical and systematic review of the evidence base to support epidemiologic and clinical modeling of key HPV disease-related parameters in the context of an HPV multi-type disease transmission model which we have applied within a U.S. population context; (2) Identify areas where additional studies are particularly needed.Consistent with our and other prior HPV natural history models, the literature review was confined to cervical disease and genital warts. Between October 2005 and January 2006, data were gathered from the published English language medical literature through a search of the PubMed database and references were examined from prior HPV natural history models and review papers. Study design and data quality from individual studies were compared and analyses meeting pre-defined criteria were selected.Published data meeting review eligibility criteria were most plentiful for natural history parameters relating to the progression and regression of cervical intraepithelial neoplasia (CIN) without HPV typing, and data concerning the natural history of HPV disease due to specific HPV types were often lacking. Epidemiologic evidence to support age-dependency in the risk of progression and regression of HPV disease was found to be weak, and an alternative hypothesis concerning the time-dependence of transition rates is explored. No data were found on the duration of immunity following HPV infection. In the area of clinical management, data were observed to be lacking on the proportion of clinically manifest anogenital warts that are treated and the proportion of cervical cancer cases that become symptomatic by stage.Knowledge of the natural history of HPV disease has been considerably enhanced over the past two decades, through the publication of an increasing number of relevant studies. However, considerable opportunity remains for advancing our understanding of HPV natural history and the quality of associated models, particularly with respect to examining HPV age- and type-specific outcomes, and acquired immunity following infection. It is estimated that genital human papillomavirus (HPV) infection is responsible for approximately 500,000 cervical cancer cases and 275,000 associated deaths worldwide each year ,2. HPV hIn recent years, a number of natural history models of HPV disease have been developed and used in policy evaluations of the cost-effectiveness of emergent technologies to prevent and screen for HPV-related disease, such as HPV vaccination -10, liquThe degree to which variation in a parameter value will influence model output and results will depend upon the particular parameter, model, intervention and output in question. Based on the observed variation in parameter estimates in existing HPV natural history models, we elected to conduct a critical and systematic review of the literature on the epidemiologic natural history and clinical outcomes of HPV disease, in developing an HPV multi-type disease transmission model which we have applied within a U.S. population context  Our findOverviews of the epidemiologic and clinical structure of our HPV multi-type model are presented in Figures Consistent with the model, our review is also confined to the epidemiologic natural history and clinical outcomes of cervical HPV disease and genital warts. Prior HPV natural history models to ours had generally examined only a single facet of HPV disease -9,15 andCertain clinical parameters  would be expected to vary by country. For instance, Pap testing coverage and hysterectomy rates may vary according to local guidelines, resources and cultural factors and cervical cancer mortality rates by stage in relation to the availability of specific treatments. In this review, parameters most likely to vary by country are described for the U.S. population, however data source selection issues may also be applicable to other settings. Where applicable, for further illustration, we additionally reference data sources used in adaptations of our model to selected other country settings. Finally, although often sharing many parameters in common, it is recognized that heterogeneity exists in the structure of different HPV natural history models. For instance, some models lump CIN 2 and CIN 3 into a single health state (CIN 2/3) ,21,22, wFor development of our model, between October 2005 and January 2006, data were gathered from the published English language medical literature through searches of the PubMed database as well as from published reports and statistics available on the internet and CD-ROM. References were also examined from prior HPV natural history models ,13,21-26A number of prior analyses have discussed methodological challenges in describing the natural history of HPV disease ,29-31, aThe multi-HPV type mode;   HPV types, which in turn were preferred over those with data for disease due to all HPV types (H-R and low-risk [L-R] combined). A similar hierarchy was employed for selecting studies in the modeling of HPV 6/11 infection.The combining of data into two groupings for types 6/11, and 16/18, respectively, was performed due to a general lack of published studies specifically describing the time to progression of individual incident HPV types in the development of disease at the time of our initial model design. The few available studies with stratification by HPV type combined reporting of results for HPV types 6/11 and 16/18 ,40,41. IThe model uses continuous mathematical functions to estimate the instantaneous rates of progression and regression of disease. In practice, however, all studies of HPV disease natural history collect observations at discrete time intervals, typically ranging from several months to several years apart. Given the relatively active progression and regression of some facets of HPV disease within a matter of months ,46, diffFor instance, suppose a woman developed an incident CIN 1 lesion just prior to baseline, progressed to CIN 2 at 12 months and regressed to negative for HPV infection at 24 months. A study with observations at only baseline and month 24 would conclude that this CIN 1 case regressed to normal, whereas a two-year study with a 12 month observation interval would observe progression to CIN 2, as well as subsequent regression. Given that many U.S. women undergo routine Pap screening as frequently as once a year , with thA decision was also made to pre-specify a uniform criterion across studies for the time point at which to assess event rates. Some HPV natural history studies report the regression or progression of CIN over a single year of total follow-up ,48. whilThere were two main reasons for choosing a 12 month horizon. First, for a number of natural history studies, HPV typing data for lesions observed on follow-up are unavailable, and as one looks over longer time horizons, it may become increasingly likely that apparent "progression" to higher grade HPV disease, or persistence, will be unrelated to the original HPV disease state of interest. For instance, a recent study found that among 16–27 year old U.S. women with incident HPV 6, 11, 16 and 18 infections who were observed to develop CIN during up to 36 months of follow-up, 50.4% had a lesion containing a different HPV type than that observed in the incident infection . While iWhere studies of HPV disease natural history with multiple years of follow-up are available, we will also compare the 12 month results across studies used in the base case to those observed through the mean or median follow-up time (if the mean is not reported). The choice of mean/median follow-up time is based on the observation that differential patient follow-up, as well as loss to follow-up, can render estimates derived from the longest follow-up times unstable. Unless otherwise noted, consistent with prior studies ,12,44, aWe considered the issue of whether to model HPV infection and disease natural history on an age-specific basis, or uniformly across age. Recent studies have failed to demonstrate a difference with age in the rate of clearance of HPV infection ,51, howerate = -[ln(1-p)]/t, where p is the risk or proportion and t is the follow-up time in years.Nearly all HPV natural history studies report a risk of progression or regression of disease, rather than a rate. Most models, however, use rates to express health state transitions. For ease of reference with respect to the original sources, we describe data selected from the literature in the form of a risk (proportion) in this review, and for our cost-effectiveness model . These rTable Only one prior study was found estimating the incidence of CIN 1 following HPV 16/18 infection . The 12 Although no additional studies of CIN 1 incidence were found in the literature specific to HPV 16/18 infection, Hoyer et al. ,54, follWhile some women with incident HPV 16/18 infections will be found on subsequent histology to have CIN 1 in natural history studies, there are also instances where the transition from HPV to CIN 1 to CIN 2 may occur before an initial histologic specimen is taken, and a CIN 1 diagnosis is never observed ,55,56. IThe 12-month risk of progression of incident HPV 16/18 infection to CIN 2 was estimated to be 5.8%, and to CIN 3 was estimated to be 3.5%, based on studies by Winer et al. and Insinga et al. ,43. In tThe Winer and Insinga studies also included data on incident HPV 16/18 infections through a mean follow-up of 38.8 and 21.9 months respectively, with respective cumulative risks of progression to CIN 2/3 during those time intervals of approximately 27.2% and 11.5%, or 9.4% and 6.5%, when annualized .The Winer study has a number of strengths for assessing HPV natural history with respect to CIN 2/3 development relative to other analyses ,53, inclNo studies were found describing the 12 month progression of HPV 16/18-positive CIN 1 to CIN 2. Three studies reported data on the 12 month progression of biopsy-confirmed CIN 1 to CIN 2 without regard to HPV type ,49. TwelSince type-specific data were not found in the literature, we instead examined data from Merck's F.U.T.U.R.E. I and II HPV vaccine phase III trials . Data weA limitation of the Merck trial-based analysis is that generally only women with continued cytologic abnormalities during the course of follow-up underwent repeat colposcopy and biopsy. Also, if the small number of women with persistent CIN 1 who were treated differed in their natural history from those with persistent CIN 1 who were untreated at the same follow-up time, the results could be affected. The finding that the 12-month risk of progression of CIN 1 due to HPV 16/18 (13.6%) was higher than that averaged from analyses reporting data for all CIN 1 cases (~7%) is consistent with the greater known risk of high-grade CIN and cancer due to these types ,64.No studies were found in the literature review documenting the 12-month progression of CIN 2 due to HPV 16/18 infection to CIN 3 or for any higher grade of cervical disease due to HPV 16/18 infection. For U.S. women in the placebo arms of F.U.T.U.R.E. I and II, it was estimated that 54% of initial CIN 2 biopsies (n = 37), that were not preceded by a higher grade of CIN or cancer, tested positive for HPV 16/18 infection. Thus, studies of disease regardless of HPV type status are likely to be more generalizable to HPV 16/18 disease natural history for CIN 2 than CIN 1.Two studies were identified reporting a 12 month risk of progression of biopsy confirmed CIN 2 to CIN 3 (severe dysplasia). In the first, by Kataja et al. (n = 70), women with CIN 2 on punch biopsy at baseline were followed up with Pap screening, colposcopy and biopsy at 6 month intervals, with treatment of disease if progression to carcinoma in situ (CIS) was observed on biopsy . The secThe mean follow-up time in the Kataja study was 3.75 years and in the De Aloysio study was 2.0 years. Progression to CIN 3 through the mean follow-up time in the Kataja study was ~23% and in the De Aloysio study was 33%. The corresponding estimated annual proportion progressing through the mean follow-up times were 7% and 18% respectively (mean 12%), similar to the estimates derived from the 12 month follow-up data. A limitation of both analyses in the context of the present review is that they did not report data specifically for HPV 16/18 disease and hence did not verify that CIN 3 cases observed over time were due to the same HPV types found in the CIN 2 lesions.The histologic diagnosis of CIN 3 may be sub-divided into the categories of severe dysplasia, and CIS, representing conversion of more than 2/3 but less than the full thickness of the cervical epithelium, and the full thickness of the epithelial layer, but without signs of invasion into the stroma, respectively ,67. ThisA study by Westergaard et al. analyzed data for 49 women with biopsy confirmed CIN 3 (severe dysplasia) . Women wProgression over time was reported through 21 months follow-up in the Westergaard study, with 1 woman treated for severe dysplasia during that period. However, as more women were treated following this time point  further data over time were not reported. Using data through 21 months from the Westergaard study, and 45 months from the Kataja study, yields an annual proportion progressing to CIS of 38% and 40% respectively, similar to the 12 month figures. Limitations of these analyses are similar to those described for progression from CIN 2 to CIN 3 (severe dysplasia).The literature review did not reveal a study meeting the pre-defined eligibility criteria documenting the 12 month progression of untreated CIS to localized invasive cervical cancer (LCC) [International Federation of Gynecology and Obstetrics (FIGO) Stage I]. Based on the limited data available, and calibration to population data for cervical cancer incidence, other cost-effectiveness models of HPV vaccination have assumed a very low rate of annual progression from CIN 2/3 to invasive cervical cancer -9. For iAs noted previously, prior cost-effectiveness analyses have modeled the progression of CIN 2/3 to LCC, rather than CIS to LCC and the existing literature is insufficient for precisely determining the risk of progression to LCC from either state. In the absence of literature-based data, we therefore undertook a strategy of calibrating the model to U.S. cervical cancer rates observed with ,69, and The decision to divide the CIS state into two compartments was motivated by a comparison of model output with observed data on cervical cancer incidence ,70. The average risk of progression over a 25 year period as that depicted in Figure For health states of shorter duration, with relatively high rates of progression and/or regression, such as for HPV infection and CIN (up to CIS), this formulation may reasonably describe natural history, as time in the health state will be relatively brief regardless of the distributional shape applied to the observed rates. However, for health states of relatively long duration, one may generate results in which individuals transition to another health state much more quickly than is consistent with the natural history of disease. Figure To gain insight into whether a distribution more similar to Figure If one were to utilize a distribution with a constant risk of progression, as depicted in Figure The division of the CIS state into two compartments was designed to approximate this latter distributional shape, with the relatively slower annual risk of progression from CIS1 to CIS2 (0.05), followed by a faster risk of progression from CIS2 to LCC (0.18), allowing for a delay in the time point of peak progression of CIN 3 to LCC (by 8–10 years) so as to better fit cervical cancer natural history data . Under tAs was the case for CIS, no studies providing natural history data on progression across cervical cancer stages were found that met the review eligibility criteria. For LCC to regional cervical cancer (RCC) [FIGO Stages II-III], we modeled an annual progression risk of 10%, and for RCC to distant cervical cancer (DCC) [FIGO Stage IV] a risk of 30%, within the ranges assumed in prior HPV natural history models ,9,22,23.The Insinga study described previously was the only analysis meeting review eligibility criteria found to describe the annual risk of clinically diagnosed CIN of any grade following HPV 6/11 infection . The HoyIn the Insinga analysis, from among 116 women with incident HPV 6/11 infections, 8.5% were diagnosed with biopsy-confirmed CIN 1 testing PCR-positive for HPV 6/11 infection within 12 months . ThroughIn the Insinga study, two woman with incident HPV 6/11 infections were observed to have a CIN 2 lesion testing PCR positive for HPV 6/11 infection within 12 months for a progression risk of 1.9% . No addiIn the F.U.T.U.R.E. study analyses described previously, there were 25 incident cases of CIN 1 testing PCR positive for HPV 6/11, none of which progressed to CIN 2 during the course of follow-up . The model transition probability for this parameter was therefore set to zero.In the Winer study described previously, anogenital warts were detected within 12 months in ~57% of women with incident HPV 6/11 infection . ThroughThe duration of HPV infection, in the model, refers to the persistence of type-specific HPV infection up to the point of progression to clinically detectable CIN, or clearance of HPV infection. Only two studies were identified reporting the mean duration of incident HPV 16/18 infections ,80. The From the Insinga study described previously , the meaNearly all previous HPV natural history models used in policy analyses have assumed that women infected with HPV have no protection against subsequent re-infection ,22,24,26The regression of CIN 1 lesions testing PCR positive for HPV 16/18 infection was examined in a study of placebo arm enrollees from the F.U.T.U.R.E. I and II trials as described previously. Women with incident CIN 1 lesions were presumed to have regressed if they had at least 2 consecutive negative cervical cytology or histology specimens following diagnosis. Within 12 months, 45.7% of incident HPV 16/18 positive CIN 1 cases (n = 64) were estimated to have regressed.A study by Sastre-Garau et al. reported that ~18% of prevalent CIN 1 lesions testing positive for HPV 16/18 infection (n = 28) regressed within 12 months . The intData beyond 12 months were not available from the trial analyses, but over a median follow-up of 24 months, the annual risk of regression in the Sastre-Garau study was approximately 19%. A limitation of both analyses is that cervical HPV typing results were not available during the follow-up periods, with the exception of instances in which histologic specimens were obtained. Thus it is possible that some women with evidence of persistent abnormal cytology/histology harbored disease due to other HPV types following the regression of their HPV 16/18 positive CIN 1 lesions, which may render the reported estimates of disease regression somewhat conservative. Several additional studies meeting review eligibility criteria estimated the risk of regression of CIN 1 without regard to HPV type ,49,57,85As no studies were identified reporting data for the regression of HPV 16/18-positive CIN 2 lesions, we utilized data from three studies reporting data across all biopsy-confirmed CIN 2 cases. The first two studies, by Kataja and De Aloysio, have been described previously and reported 12 month risks of regression of CIN 2 to HPV/normal of ~2% and 40% respectively ,65. In tThe mean or median follow-up time was not reported in the Matsumoto study, however, the mean annual risk of regression through the mean follow-up times in the Kataja and De Aloysio studies were 15% and 23% respectively ,65. LimiOf the three studies just described, only that of De Aloysio also reported on the regression of CIN 2 to CIN 1 over time. In addition to the group regressing to normal/HPV, another 13.3% of women with CIN 2 in that study regressed to CIN 1 within 12 months . This fiThe only study meeting the review eligibility criteria for the regression of CIN 3 (severe dysplasia) was that of Kataja . In thatThe Kataja study did not report data for the regression of severe dysplasia to CIN 2 or CIN 1. However, as was the case for CIN 2, in practice such regression is likely to occur . In the The study utilizing F.U.T.U.R.E. I and II trial data was the only analysis identified examining the regression of CIN 1 testing positive for HPV 6/11. The 12-month risk of regression was 55.2% .No studies were found documenting the 12 month risk of regression of anogenital warts in the absence of treatment. Although a number of clinical trials have reported wart clearance rates for patients receiving placebo, most have featured follow-up of 3 months or less, and included high proportions of patients with extensive, recalcitrant and previously treated warts ,87. OnlyHowever, in practice, individuals with the largest and most extensive warts may seek immediate treatment, and natural clearance rates may be even higher among those patients not seeking physician care. Considering those patients who are treated, the Winer study described previously, reported that 75% of women with incident warts undergoing treatment cleared the warts within 8.0 months . Data thNo studies were found estimating age- and stage-specific annual mortality rates among a representative sample of unscreened women with cervical cancer, as any such analysis would be fraught with ethical and methodological difficulties. As an approximation, data from the Surveillance Epidemiology and End Results Program (SEER) for 1997–2002 were used to estimate the 12 month excess risk of mortality due to cervical cancer, by age group and stage . Excess Table Age-specific hysterectomy rates were modeled based on 1994–1999 data reported from the U.S. National Hospital Discharge Survey . Other dlower than those estimated from nationally representative self-reported data for all women  [Nationally representative data on cervical cancer screening are based on self-reported behavior, which has been shown to over-estimate the actual receipt and recency of cervical cancer screening -101. Twoinsured) . We therinsured)  and to einsured) ,105.with a cervix using data reported in that analysis on the proportion of women in each age group estimated to have an intact cervix [The KPNW data were selected because they were derived from a large sample of women , provided a detailed breakdown of annual Pap screening utilization by age group, and distinguished between cervical and vaginal cytology and routine and non-routine Pap testing. The ovet cervix .Second, KPNW screening rates reflect the experiences of both screened (at least once in lifetime) and never screened women. Because we wished to model the never screened population separately, we elected to adjust the rates in the screened population to reflect the removal of never screened women from the denominator. Data on the proportion of women who have never undergone cervical cancer screening in the U.S. are only available from studies featuring self-reported data. As discussed previously, for a variety of reasons , the proOther data sources for cytology screening rates have been used in forthcoming adaptations of our model to country settings of the United Kingdom , Taiwan As most cervical screening in the U.S. is now conducted using liquid-based cytology (LBC)  we choseFor assessing the sensitivity of LBC for CIN 1, the only study observed to meet this criterion was conducted by Bigras et al. in a non-high-risk Swiss population  . In the The sensitivity of LBC for CIN 2 and CIN 3 were also estimated based on data from the Bigras study. Using data reported in their paper, a sensitivity of 61% was computed for CIN 2 and 55% for CIN 3, with an average sensitivity for CIN 2/3 combined of 59% . In a seFor assessing the specificity of LBC, we relaxed the criteria for study eligibility to include studies in which all women were referred for colposcopy, with a biopsy performed if an abnormality was suspected. Studies conducted exclusively or primarily among women with abnormal cytology results, or for which biopsy results for all grades of CIN (1–3) were not reported were excluded. The decision not to require that biopsy results be reported (or extrapolated) for all women, including those with negative cytology and HPV testing results, was based on the observation from the Bigras analysis that test specificity (98% using either criteria) is much less influenced by this requirement than overall sensitivity for CIN 1–3 (31% vs. 45%) . With a A meta-analysis by Mitchell et al. examined studies in which women with an abnormal Pap smear underwent both colposcopy and colposcopically directed biopsy, with results tabulated by grade of CIN and cancer . Nine stThe meta-analysis by Mitchell et al. also reported a mean specificity for colposcopy of 48%, and this figure was used in the model .It was assumed that women not diagnosed with asymptomatic cervical cancer through screening would be diagnosed if symptoms developed. The literature review did not identify any studies describing stage-specific symptom development in a representative cross-section of patients with cervical cancer. A difficulty in interpreting existing case series is that it is not possible to determine the true number of women within the population who harbor asymptomatic cervical cancer. In the absence of such data, we reviewed assumptions used in prior cost-effectiveness analyses ,9,21,22 For estimating the rate of eradication of CIN following treatment, we selected studies evaluating the performance of the commonly used LEEP procedure, subject to the following criteria: 1) Stratified reporting by CIN grade of the number of subjects with confirmed pre-treatment histology of CIN 1, 2 or 3; 2) Post-treatment follow-up of all women within 12 months with colposcopy and/or biopsy; 3) Definition of recurrent or residual disease as CIN 1 or more severe histology; 4) Stratified reporting of the number of subjects with recurrent/residual disease according to the grade of pre-treatment histology.Seven studies evaluating the treatment of CIN 1 met the review eligibility criteria, comprising 417 cases -124. A tFor cervical cancer, it was assumed that women surviving for more than 5 years following diagnosis were cured of disease . We therFor anogenital warts, current treatments often require multiple applications and often fail, requiring second and third-line therapies . Rather Women whose CIN is successfully removed and who do not develop recurrent CIN may still harbor HPV infection transmissible to others. To inform this model parameter, we searched for studies in which: 1) HPV testing was reported specifically for HPV types 16/18 or 6/11; 2) HPV DNA testing was performed on pre-treatment biopsies or treatment specimens of confirmed CIN histology; 3) Follow-up for all women and HPV testing occurred within 6 months post-treatment; 4) Colposcopy was performed on all women during follow-up to confirm the eradication of CIN lesions; 5) Persistent CIN disease during follow-up (to be excluded) was confirmed by histology. One previous study was identified which met these criteria. Cruickshank et al. studied women who were treated for CIN 3 via LEEP and laser ablation, had negative Pap smears and colposcopic assessments at 6 months, and remained free of disease thereafter for a minimum of 5 years . HPV tesOne study was identified through the literature review evaluating the PCR-based type-specific persistence of HPV infection following treatment for cervical cancer . Fen et The literature review identified two studies comparing the type-specific prevalence of HPV 6/11 infection prior to and following successful eradication of genital warts. The first, by Arany et al., evaluated 6 patients successfully treated via interferon therapy for HPV 6/11 positive genital warts and found 100% clearance of HPV 6/11 infection within normal skin at the wart site at 6–12 weeks follow-up  The secoNot all patients who harbor anogenital warts will be aware of their presence or seek physician care ,133. In Natural history models of HPV disease are increasingly being used to inform policy making for existing and emerging technologies ,21,24,44Given the breadth and complexity of HPV disease modeling, we have not comprehensively reviewed data to inform all possible model structures and permutations, or every potential limitation of existing studies to inform such models. Furthermore, although we attempted to be thorough in our review, it is also possible that our database and non-database search strategies may have missed some publications of relevance as we did not search all publicly available scientific literature databases . However, the present review has attempted to highlight available data sources and associated major strengths and limitations for informing parameters common to many existing models.Knowledge of the natural history of HPV disease has been considerably enhanced over the past two decades, through the publication of an increasing number of relevant studies. However, considerable opportunity remains for advancing our understanding of HPV natural history and the quality of associated models. For instance, more studies examining the type-specific progression and regression of HPV disease are needed, both for more accurate modeling of disease natural history and to inform policymaking for technologies such as vaccines that target specific HPV types ,14,15. AFor many disease areas, modeling is an iterative process. As one's understanding of disease natural history and clinical management are advanced, existing models can be adapted to incorporate new evidence. Comprehensive literature reviews for HPV disease and other health conditions can play an important role in assisting modelers and policymakers in critically evaluating the current evidence base and identifying areas for future study.AGC: atypical glandular cells; ALTS: ASCUS/LSIL triage study; ASCUS: atypical squamous cells of undetermined significance; CIN: cervical intraepithelial neoplasia; CIS: carcinomain situ; DCC: distant cervical cancer; FIGO: International Federation of Gynecology and Obstetrics; H-R: high-risk; HPV: human papillomavirus; HSIL: high-grade squamous intra-epithelial lesion; KPNW: Kaiser Permanente Northwest; L-R: low-risk; LBC: liquid-based cytology; LCC: localized invasive cervical cancer; LEEP: loop electrosurgical excision procedure; LSIL: low-grade squamous intra-epithelial lesion; PCR: polymerase chain reaction; RCC: regional cervical cancer; SEER: Surveillance Epidemiology and End Results Program.The authors are employed by, and may hold stock in, Merck & Co., Inc., which funded the article-processing charge for this study.RI conceived of the study and conducted the literature review, data extraction and analysis, and initial drafting of a manuscript. EE and ED assumed a primary role in defining the data parameters to be reviewed and assisted in the drafting of the manuscript and supplementary data analyses. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/119/prepub


Spinal cord-injured (SCI) patients experience poor health-related quality of life (HRQOL) and they usually report lower HRQOL than the general population or population subgroups in Iran and elsewhere. The aim of this study was to compare HRQOL between veterans and non-veterans with SCI in Iran.This was a cross-sectional study. HRQOL was measured using the 36-item Short Form Health Survey (SF-36). Thirty-nine male veterans and 63 non-veteran males with SCI were included in the study. Regression analyses were applied to determine the variables affecting physical and mental health-related quality of life among the patients.The male veterans had a lower HRQOL than the non-veterans with SCI. The differences were significant for all measures except for physical and social functioning. The greatest difference was observed for bodily pain (P = 0.001). The regression analysis results indicated that a longer time since injury was associated (P = 0.01) with better physical health-related quality of life (PCS), while being a veteran (P < 0.001) and having a spinal lesion in the cervical region (P = 0.001) were associated with poorer PCS. Older age (P < 0.001) and higher education (P = 0.01) were associated with better mental health-related quality of life (MCS), while being a veteran and having a spinal lesion in the cervical region (P = 0.02) were associated with poorer MCS.The study findings showed that veterans with SCI experienced lower HRQOL than their non-veteran counterparts. A qualitative study is recommended to evaluate why HRQOL was lower in veterans than in non-veterans with SCI although veterans had higher incomes as a result of their pensions and increased access to equipment, and medications. To improve quality of life in both veterans and non-veterans with spinal cord injuries, policy changes or implementation of new interventions may be essential so that veterans could receive additional support  and non-veterans could meet their basic needs. Spinal cord injury (SCI) causes several health problems that negatively affect not only the patient's physical condition but all aspects of their lives including their goals and communications, and more importantly their health-related quality of life -6. ActivOverall, the findings from several studies in the USA ,9, CanadDuring the 1980-1988 Iran-Iraq war, the human cost to Iran included more than 200,000 lives lost and more than 400,000 persons injured , of whomThis study has two aims: to assess quality of life in male spinal cord injured veterans; and to compare health-related quality of life between veterans and non-veterans with SCI. To the best of our knowledge this is the first paper from Iran that reports on the topic.This was a cross-sectional study of quality of life among a convenient sample of SCI veterans and non-veteran SCI patients in Tehran, Iran. Veterans were approached during a recreational trip and were asked if they wished to take part in the study. Thirty-nine out of 41 veterans agreed to be interviewed. Additional information was extracted from their case records kept in the Janbazan (Veterans) Medical and Engineering Research Center. Non-veterans were selected from a consecutive sample of patients attending a neurosurgery outpatient department in Imam Hospital, affiliated to Tehran University of Medical Sciences, during the study period. Eighty-three patients were approached and 63 agreed to take part. A face-to-face interview was arranged to collect data from both groups. Trained staff from the Janbazan (Veterans) Medical and Engineering Research Centre interviewed the veterans and trained staff from the Brain and Spinal Cord Injury Repair Research Center (BASIR) interviewed the non-veterans.Health-related quality of life was measured using the SF-36 questionnaire. The psychometric properties of the Iranian version of SF-36 are well documented . The queFor each individual, we also collected data on demographic factors, history since injury, level of injury, motor lesion, marital status at the time of injury and at the time of the study, and the number of children. It was thought that these variables might have significant positive or negative effects on health-related quality of life. For example, since patients might have suffered from infertility since their injury, this could affect their quality of life; hence we were interested in collecting data on the number of children.In univariate analysis, the chi-square test (or Fisher's exact test and Yate's correction where necessary) was used to compare categorical variables. To compare the SF-36 scores between veterans and non-veterans, Student's t-test was performed. We estimated regression models to examine the association between individual characteristics and HRQOL. Separate linear regression analyses were carried out for the PCS and MCS, which were treated as dependent variables. For this purpose, the distributions of the PCS and the MCS scores were examined. There was slight negative skewness but this was less than one in both cases, allowing us to using the test. Age, time since injury, being a veteran, educational level, having a spouse, and SCI in the cervical region were included in the model as independent variables. Variables that failed to represent significant P values (P < 0.05) were excluded from the model using a backward elimination procedure.The study received approval from the Sina Trauma and Surgery Research Center, affiliated to Tehran University of Medical Sciences, and the Janbazan (Veterans) Medical and Engineering Research Center ethics committee. All patients gave their informed consent.Thirty-nine veteran and 63 non-veteran spinal cord-injured patients were included in the study. All the patients were male. Table The SF-36 scores for veterans and non-veterans are shown in Table Finally, regression analyses were applied to determine the effects of variables associated with PCS and MCS. Longer time since injury (P = 0.01) was associated with a better PCS, while being a veteran (P < 0.001) and having a spinal lesion in the cervical region (P = 0.001) were associated with a poorer PCS Table . Older aThe study findings showed that male SCI veterans had lower HRQOL than male non-veteran SCI patients in Iran. Both the PCS and MCS and the SF-36 scores  were lower in veterans. Even comparison of the scores between veterans and the subset of non-veterans (those with complete motor lesions) yielded similar results. It is argued that complete motor lesions may lead to the occurrence of pressure ulcers and other complications by limiting the patient to bed or a wheelchair , so theyComparing role emotional (RE) in veterans and non-veterans revealed a significant difference between the two groups. It seems that male veterans with SCI had the more severe emotional disturbances in Iran. In contrast, two other published studies, the first from Iran concerning female veterans with SCI , and theIt has been shown that frequent mental distress and depressive symptoms are associated with poor self-reported health in individuals with spinal cord injury . In addiHigher expectation of life quality among veterans may be another explanation for the lower HRQOL among male veteran SCI patients. In fact, although SCI veterans enjoyed financial support and increased health care access, their higher expectations might have resulted in lower HRQOL than among the non-veteran SCI patients. To explain further, it should be noted that many Iranian veterans with SCI took part in the Iran-Iraq war voluntarily and intentionally, and expected that at the end of the war they would have an ideal society. They were ready to be injured or even to die to establish an ideal country. They self-sacrificed their health, but when they came back to their home towns after their injuries or 8 years of war, they were confronted with a society worse than they expected; a society with many broken values. Thus, their evaluation of their new life was now very different from that of a patient who, for instance, had suffered SCI following a road accident and consequently reported a lower quality of life. We believe this explanation is highly consistent with the quality of life definition offered by the World Health Organization: 'an individual's perception of his/her position in life in the context of the culture and value system in which he/she lives, and in relation to his/her goals, expectations, standards, and concerns' . There iWe used a well-known instrument (the SF-36) to measure quality of life in individuals with SCI. This is a generic measure, and it would seem much better to assess quality of life in this population by more specific questionnaires in addition to such instruments. For example, Luther et al. developed an 8-item SCI-specific physical functioning scale for veterans that showed strong psychometric properties . It is aThis study had several limitations. The major limitations were the small sample size and the fact that we did not collect data on the patients' chronic illnesses and common complications. There is evidence that chronic illnesses have a substantial effect on health-related quality of life for persons with SCI ,31,32. IThe study findings showed that veterans with SCI experienced lower HRQOL than their non-veteran counterparts. A qualitative study is recommended to evaluate why HRQOL was lower in veterans than in non-veterans with SCI although veterans had higher incomes as a result of their pensions and increased access to equipment, and medications. To improve quality of life in both veterans and non-veterans with spinal cord injuries, policy changes or implementation of new interventions may be essential so that veterans could receive additional support  and non-veterans could meet their basic needs.SCI: Spinal cord injury; HRQOL: Health related quality of life; PCS: Physical component summary; MCS: Mental component summary; SF-36: 36-item Short Form Health Survey questionnaire; RP: role limitation due to physical problems; RE: role limitation due to emotional problemsThe authors declare that they have no competing interests.SS contributed to the data analysis and writing the first draft. BSD wrote the proposal. AHT, MJ, and SMG confirmed proposal and collected the SF-36 data and took part in the study. AM helped as a consultant in all parts of the study, rechecked the data analysis and revised the final manuscript. VRM was the principal investigator and performed quality control on patients' case records, was responsible in all parts of the study, wrote draft and revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/10/6/prepub


Mutations in cartilage oligomeric matrix protein (COMP), a large extracellular glycoprotein expressed in musculoskeletal tissues, cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia. These mutations lead to massive intracellular retention of COMP, chondrocyte death and loss of growth plate chondrocytes that are necessary for linear growth. In contrast, COMP null mice have only minor growth plate abnormalities, normal growth and longevity. This suggests that reducing mutant and wild-type COMP expression in chondrocytes may prevent the toxic cellular phenotype causing the skeletal dysplasias. We tested this hypothesis using RNA interference to reduce steady state levels of COMP mRNA. A panel of shRNAs directed against COMP was tested. One shRNA (3B) reduced endogenous and recombinant COMP mRNA dramatically, regardless of expression levels. The activity of the shRNA against COMP mRNA was maintained for up to 10 weeks. We also demonstrate that this treatment reduced ER stress. Moreover, we show that reducing steady state levels of COMP mRNA alleviates intracellular retention of other extracellular matrix proteins associated with the pseudoachondroplasia cellular pathology. These findings are a proof of principle and the foundation for the development of a therapeutic intervention based on reduction of COMP expression. Cartilage oligomeric matrix protein (COMP), also known as thrombospondin 5 (TSP-5), is a low abundance glycoprotein that is found in the extracellular matrix (ECM) of cartilage, tendon, ligament and smooth muscle In contrast, COMP knock-out mice are normal in all growth parameters with only mild growth plate disturbances and flattening of the articular cartilage surface only found with exercise The challenges to using RNAi therapy in a clinical setting are significant and this barrier is particularly difficult with avascular tissues such as cartilage. Preventing or reducing PSACH cellular pathology will require long-term reduction of COMP expression because chondrocytes produce COMP throughout life In this study, recombinant lentiviruses encoding the elements necessary for expression of a COMP-specific shRNA were used to provide a continuous supply of shRNA for COMP-specific siRNA duplexes A and B) and protein (C and D) were markedly reduced by two shRNAs, 3A and 3B, that target different COMP type 3 calcium-repeat domain sequences , a resident rER protein, was associated with retained mutant COMP in the rER of PSACH growth plate chondrocytes *)  to attain high levels of COMP expression. As can been seen in We next tested the efficacy of shRNAs to reduce endogenous COMP mRNA levels in human chondrocytes. Chondrocytes containing shRNA integrants directed against COMP were grown in monolayer for one week. As shown in shown in left panel), in the presence of MT-COMP, MATN3, types IX and II collagens were co-retained. In comparison, shRNA 3B reduction of MT-COMP also reduced the retention of other ECM proteins retained ( right panel). This result demonstrates that reduction of intracellular MT-COMP by RNAi normalizes the export of the other matrix proteins preventing the premature protein interactions and intracellular matrix assembly. Moreover, expression of CRT was also reduced in cells expressing shRNA 3B. These findings are consistent with the COS-7 results shown in We have previously shown that MT-COMP expression induces the intracellular retention of ECM proteins and formation of intracellular matrix PSACH is a severe dwarfing condition caused by mutations in COMP that specifically affects growth plate chondrocytes. These mutations interfere with protein folding which initiates the ER stress response causing retention of COMP and other ECM proteins that assemble prematurely into ordered intracellular matrix in vitroMost of the mutations that cause PSACH are in the calcium-binding domain of COMP RNAi therapies are being tested in a variety of conditions including, cancer, macular degeneration, movement disorders, heart failure, storage diseases, prion disease, viral infection, chronic pain and addictive-induced behavior The PSACH and MED/EDM1 cellular pathology occurs for the first 15–18 years which coincides with human skeletal growth COMP is expressed at high levels during skeletal development and long bone growth and therefore it is important that RNAi therapy be capable of reducing high expression levels of COMP We also demonstrated in RCS cells that shRNA 3B efficiently reduced intracellular retention of MT-COMP, MATN3 and type IX collagen, thereby preventing the development of intracellular matrix that defines the PSACH and MED/EDM1 cellular phenotype. Moreover, in both COS-7 and RCS cells, shRNA 3B reduced the elevated CRT levels that result from the overexpression of COMP. Taken together, these results suggest that reducing mutant COMP mRNA levels may dampen the ER stress and matrix formation in the rER that is potentiated by the presence of mutant COMP.in vitro would be an effective treatment modality for the PSACH and MED/EDM1 chondrocyte pathology. This work provides a proof of principle for the potential development of treatment for this ER storage disorder. Delivery of RNAi therapy to avascular tissues, such as cartilage may be a challenge. In one approach, the delivery of siRNA molecules to bone metastatic tumors was mediated by their attachment to bovine pepsin treated type I collagen (atelocollagen) Overall, we show that suppression of COMP expression by RNAi 3A CCCAGAAGAACGACGACCAAA, 3B ATGCTTGTGACAGCGATCAAC, GA TGTGGGTTACACTGCCTTCAA and GB CAAACGTATTGGCAGGCGAAC.shRNA lentiviral particles that target human COMP were purchased from the MISSION® TRC-Hs 1.0 (Human) . MISSION shRNA clones were designed and developed by The RNAi Consortium (TRC) at the Broad Institute of MIT and Harvard University. Each shRNA is a hairpin with 21 base pair long stem and a 6 nucleotide loop. Expression of the shRNA is driven by the U6 promoter. The target region of each shRNA is shown in The bicistronic constructs were designed to express MT- or WT-COMP with a FLAG tag moiety (DYKDDDDK) at the carboxy terminus and GFP reporter gene under the control of the cytomegalovirus (CMV) promoter. The D469del mutation, used in this study, is the most common PSACH mutation accounting for approximately 30% of cases 5′CCAGTCACGTAGCGATAGCGGAGTG3′ For and 5′ATAGATCTAAGCTTGGTCGAGCTGATACTTCCCG 3′ Rev) and digested with Xho I and Bgl II. The resulting fragment was ligated into complementary sites in pShuttle  . Next, the WT- or MT-COMP-FLAG gene driven by the tetracycline responsive element (TRE) was inserted into pShuttle-CMV-rtTA to produce the final construct pShuttle-TRE-COMP-CMV-rtTA. All constructs were verified by sequencing. The AdEasy vector system kit  was again used as described above. Recombinant adenovirus were subsequently produced, isolated and quantified.Inducible WT- and MT-COMP expression cassettes were designed so that the level of COMP expression could be modulated by altering DOX concentration in the culture medium. Two subcloning steps were performed to generate the final vector pShuttle-TRE-COMP-CMV-rtTA. First, the CMV-rtTA cassette from pTet-On  was inserted into the pShuttle  using PCR  and RCS Lentivirus particles containing a shRNA were used to infect RCS and COS-7 cells at multiplicity of infection (MOI) of 0.1 to insure that single colonies of integrants could be isolated. Cells were cultured for three days in non-selective media and puromycin was then added to select stable integrants. Single colonies were moved to separate dishes and grown in standard culture media with puromycin for these experiments. Primary human costochondral chondrocytes were infected at MOI of 1 with shRNA lentiviruses and grown for five days in non-selective media and then in puromycin to select cell populations that contained an shRNA. All human primary chondrocytes in these experiments are from a population of shRNA integrants.COS-7 cells with and without an shRNA integrant were cultured until 70–80% confluent and infected with either the WT- or MT-COMP-GFP or DOX inducible WT- or MT-COMP-FLAG adenovirus  in DMEM with 2% v/v FBS. Cells were harvested 48 hrs later or at time points required for the experiments. RCS cells with and without a shRNA integrant were plated on to coverslips and 24 hrs later treated with 1 µg/ml hyaluronidase for 1 hour at 37°C and then infected with MT-COMP-FLAG (GFP) adenovirus (MOI 400–800) in OPTI-MEM media .Northern and Western analyses were used to assess mRNA and proteins in all experiments using standard techniques. Sample loading in Northern and Western analyses was normalized using Snu-6 and β-actin, respectively. Westerns were performed using anti-COMP rabbit polyclonal antibody  and detected using ECL Plus .v/v paraformaldehyde after 4 days and 100% methanol after 2 days, respectively, at 4°C for 20 min. All coverslips were incubated overnight at 4°C with the primary antibodies (1∶200). RCS cells and COS-7 cells were grown on coverslips and fixed using 4% RNA from chondrocytes with and without an shRNA integrant was collected for RT-PCR assays after one week in culture. Quantitative real-time RT-PCR (qRT-PCR) was performed utilizing the 7700 Sequence Detector . DNA contamination was removed using amplification grade DNAse  following manufacturer's instructions. Each assay was replicated three times and each sample was measured in triplicate including a control without reverse transcriptase. The final data were normalized to β-actin ×100 .


Human Papillomavirus (HPV) genotype distribution and co-infection occurrence was studied in cervical cytologic specimens from Murcia Region, (southeast Spain), to obtain information regarding the possible effect of the ongoing vaccination campaign against HPV16 and HPV18.A total of 458 cytologic specimens were obtained from two outpatient gynecological clinics. These included 288 normal benign (N/B) specimens, 56 atypical squamous cell of undetermined significance (ASC-US), 75 low-grade squamous intraepithelial lesions (LSIL) and 39 high-grade squamous intraepithelial lesions (HSIL). HPV genotyping was performed using PCR and tube array hybridization.The most frequent genotype found was HPV16 . Distribution of other genotypes was heavily dependent on the cytologic diagnoses. Co-infections were found in 15.3% of N/B, 10.7% of ASC-US, 48% of LSIL and 25.6% of HSIL cases (significantly different at p < 0.001). Strikingly, in N/B diagnoses, genotypes from A5 species were found as coinfecting in all cases. Genotypes from A7 or A9 species appeared in co-infections in 56.5% and 54% respectively whereas genotypes from A6 species appeared in 25.1% of cases.HPV vaccination might prevent 34.6% and 35.8% of LSIL and HSIL, respectively. Co-infection rate is dependent on both cytologic diagnosis and HPV genotype. Moreover, genotypes belonging to A5, A7 and A9 species are more often found as co-infections than genotype pertaining to A6 species. This suggests that phylogenetically related genotypes might have in common similar grades of dependency for cervical epithelium colonization. It is well established that human papilloma virus (HPV) is necessary to develop cervical cancer . There aIn order to prevent cervical carcinoma, two types of prophylactic vaccines have been developed: a bivalent vaccine against the high-risk HPV16 and 18 , and a tThe HPV genotypes found in different regions of the world vary both in type and relative incidence. There is also evidence of the prevalence of specific variants of defined genotypes in certain ethnic groups -17. In fThe Region of Murcia, located in the southeast of Spain, has a population of 1.4 millon and is currently divided into five administrative health areas. In the last ten years, a high influx of immigrants from northern Africa and Latin America has caused population increase of nearly ten percent. Regional health authorities have launched a campaign in 2008 to vaccinate all young females (aged 11 to 14 years old) against HPV16 and HPV18, thus making the Region of Murcia a good model system to study the effect of vaccination in HPV-related diseases.Four hundred and fifty eight cytologic consecutive specimens collected from November 2006 to April 2009 were included in this study. Cytologic specimens correspond to single patients (median age 38 (SD = 10.0); range 19 to 68) referred to the outpatient gynecological clinics of Hospital Universitario Santa María del Rosell (HUSMR) and Hospital Rafael Méndez (HRM) because of previous abnormal Pap smears. These two hospitals provide healthcare to a population of about 535.000 individuals (38.1% of population of Murcia Region). The incidence of cervical cancer in Murcia region is 9/100,000. Smears were stained with Papanicolaou stain. A second sample was taken by gynecologists either with cotton swab or with cytobrush and resuspended in 400 μl of PBS for immediate DNA extraction and HPV genotyping. Papanicolaou smears were reviewed by a pathologist trained in gynecologic pathology (SOR). The cytologic diagnoses of the cases included in this study were as follows: 288 normal or benign (N/B) cytologic diagnoses, 56 cases with atypical squamous cell of undetermined significance (ASC-US), 75 cases with low-grade squamous intraepithelial lesions (LSIL), 39 cases with high-grade squamous intraepithelial lesions (HSIL). Informed consent was not required for this study since the results presented here come from HPV genotyping routinely performed as an adjunct to Pap smears in pathology department. In any case all identifiers were deleted in order to protect patient confidentiality. The study was approved by the local ethical board.3 viral copies and diagnostic sensitivity and specificity for each genotype were 98.2% and 100%, respectively. A hybridization positive control containing biotin, a CFRT human gene internal control and HPV negative and positive controls were included in each run.DNA extraction was performed from all samples using Maxwell 16 Cell DNA Purification Kit  and Maxwell 16 automated DNA extraction station  according to the instruction manual. Average DNA concentration in these samples was 60 ng/μl. Genotyping was performed with Clart HPV 2 kit  ,23. AmplGenotypes were classified according to their cervical carcinoma associated risk as high, low and probable high-risk as reported by Muñoz et al . The mos2 was used to evaluate statistical significance between grade of lesion  and HPV genotypes found in the study.HPV genotype distribution analysis was performed using SPSS.14.0 . χHPV genotyping was successfully achieved in all samples. The percentage of samples positive for an HPV infection increased with the severity of the lesion, from N/B (49.7%) to LSIL (82.7%) and HSIL (87.2%) p < 0.0001)  of HPV positivity is within the range of positivity found in a Spanish study using linear arrays for genotyping in cytologic specimens from an outpatient gynecologic clinic (49.5%) [In the present study we have found an HPV detection rate of 49.7% in N/B cases, 53.6% in ASCUS, 82.7% in LSIL and 87.2% in HSIL. The N/B percentage is considerably higher than that found in other studies with Spanish general population . The rea (49.5%) . The perThere are some similarities in the HPV genotype distribution found in N/B cases from the cohort studied compared to that reported by Gomez-Roman et al. which genotyped cytologic specimens from a cohort of females from northern Spain attending an outpatient gynecologic clinic; most frequent genotypes encountered were HPV16, 53 and 58. In our study HPV53 was also found to be the second most common genotype and HPV58 the third most prevalent high-risk genotype. HPV53, although considered as low-risk  or as prHPV58 seems to be most frequently found than HPV18 in N/B cases. A similar finding is reported elsewhere in other regions of Spain ,34. ThisIn LSIL cases HPV51 and HPV53 were, along with HPV31 and 33, the most frequent genotypes detected after HPV16. Other studies from Spain and France have also reported a high prevalence of HPV51 and 53 in LSIL cases ,34.In our study the most common genotypes found in HSIL were HPV16, 45, 51, 33 and 31. HPV16, 51, 33 and 31 are amongst the five most frequent genotypes found in HSIL specimens in previous studies from Spain and France ,36. SurpAs seen in Figure In our work the presence of co-infection was observed in 15.3% of N/B cases, 10.7% of ASC-US, 48.0% of LSIL and 25.6% of HSIL. These differences in co-infection occurrence are statistically significant (p < 0.0001). Rousseau et al. also reported an increased frequency of co-infection from N/B cases (3%) to ASC-US (10%) and to LSIL (23%) and a decreased frequency from LSIL to HSIL (7%) [Our results suggest that LSIL may constitute a permissive state where the presence of certain genotypes as HPV16 may pave the way for other more co-infection-dependent genotypes, but as the lesion progresses only highly oncogenic types (i.e. HPV16) may persist whereas less oncogenic genotypes  are cleared off.The greater number of coinfecting genotypes associated with their lower frequency suggests that the acquisition of two genotypes do not favour subsequent HPV types acquisition. According to our data it seems that genotypes from certain species  appear as coinfecting more frequently than genotypes from other species (A6). This finding might indicate that there are different grades of dependency for each genotype or species to colonize cervical epithelium. The strength of the association and whether this probable dependency is intra or interspecies must be clarified in further studies. To the best of our knowledge this observation has not been previously reported. These results give support to the theory that the elimination of certain genotypes by vaccination may affect the distribution of other genotypes.Therefore, based on our results we hypothesize that the reason why HPV vaccine does not show cross-protection against HPV58 as shown in the PATRICIA study  may perhIn this study we show that coinfecting genotype distribution and its prevalence in different grade of lesions suggests probable genotype interplay in the cervical epithelium. Simultaneous synergy or competing relationship amongst them cannot be ruled out. Therefore the vaccination-driven elimination of one HPV type would affect the natural history of the rest of genotypes and subsequently that of cervical carcinoma and would reinforce the necessity of a constant survey of genotypes in any geographical area where the HPV vaccination campaign is being implemented.The authors declare that they have no competing interests.All authors state that the study sponsors (Fundación para la Formación e Investigación Sanitarias and Fundación CajaMurcia) have not been involved in either the study, design, the data collection, analysis and interpretation or the writing of the manuscript.PCZ and MEC designed experiments, SOR and MPG performed pathological diagnoses, ADP and JMB performed genotyping, FJOC performed statistical analysis. PCZ and MEC wrote grant applications and draft. Draft was corrected and approved by all authors.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/124/prepub


Infection with human papillomavirus (HPV) is a major risk factor for development of anal squamous cell carcinoma. Despite over 100 genotypes of the virus, HPV 16 and 18 are considered pathogenic as they are seen in the majority of cervical and anal cancers. We have employed a custom microarray to examine DNA for several HPV genotypes. We aimed to determine the accuracy of our microarray in anal cancer DNA for HPV genotypes compared to the DNA sequencing gold standard.We utilized a sensitive microarray platform to classify 37 types of mucosal HPVs including 14 known high-risk and 23 low-risk types based on cervical cancer data. We utilized DNA from pathologically confirmed cases of anal squamous cell carcinoma. All samples underwent microarray HPV genotyping and PCR analysis.HPV was detected in 18/20 (90%) anal cancers. HPV genotypes 16 and 18 were present in the majority of specimens, with HPV 16 being the most common. Eighty percent of anal cancers had at least two HPV types. Ten percent of cases (2/20) tested negative using our microarray; DNA sequencing confirmed the lack of presence of HPV DNA in these samples.Microarray technology is an accurate way to screen for various genotypes of HPV in anal cancer, with 100% correlation with genomic DNA detection of HPV. The majority of anal cancers in our study associated with pathogenic HPV 16 and/or 18. Other HPV genotypes are present simultaneously with HPV 16 and 18, and might contribute to its pathogenesis. Squamous cell cancer of the anus  is increasing in frequency in the general population in the United States, Europe and South America . There wUnder IRB approval, DNA was extracted from pathologically confirmed cases of anal cancer from two local hospitals: UC San Diego Medical Center and the Veteran's Administration Hospital in San Diego, California. A total of 20 patient samples were collected from these two cancer registries for use in this study. The average age of the patients was 51.5 (range 39-77) and 85% of patients were males. Staging was performed by computed tomography, and listed by AJCC classification in Table DNA tissue was extracted after manual or laser capture microscopy microdissection of pathologist-marked cancer areas from histological-confirmed slides of squamous cell cancer of the anus. This tissue was then deparaffinized in xylene, purified with absolute alcohol, and centrifuged at 15,000 rpm. The pellets were dried in a DNA SpeedVac (Eppendorf Vacufuge Concentrator), re-suspended in 100 μl Tris EDTA buffer, and incubated with 200 ug/ml proteinase K (Sigma Cat#P2308) at 55°C for 3 hours. The genomic DNA was purified with phenol/chloroform/isoamyl alcohol  and washed with alcohol. The resulting solute was re-suspended in TE buffer. Purified DNA was then subject to amplification for microarray analysis.2.DNA was also extracted from cell lines in which the HPV status was known. SiHa , HeLa , CaSki  and ME180 (HPV-68) were obtained from ATCC and grown in their ATCC-recommended media at 37°C in 5% COnd PCR. Subsequently, the labeled product was then coupled with Cy-3 NHS ester for array hybridization. This approach demonstrates the usefulness of this method for detection of the amplified viral amplicons when multiple viral types or genotypes might be present [We used a modification of the standard GP5+/GP6+ protocol . Modifie present ,10.A microarray platform was built to type 37 types of mucosal HPVs  genomic DNA was applied for titration experiment. The sensitivity with the HPVGP56 array is about 1-2 copies of HPV16 in the background of complex human genomic DNA. . We were able to unambiguously identify features representing HPV16 when only 1 or 2 copies of genome were present in the sample.We used SiHa  genomic DNA for analyzing the limit of detection to mimic the complexity of HPV16 in the presence of human genomic DNA. SiHa cells contain a single HPV-16 integrated into chromosome 13q . The humTo test the specificity of the array, 100 ng each of genomic DNA from SiHa , HeLa , Caski  and ME180 (HPV-68) were used for amplification, labeling and chip hybridization ,13. The After extracting the DNA for anal cancer specimens and hybridizing to the microarray, HPV was detected in 18/20 (90%) cases. All the samples were separately subject to PCR amplification and DNA sequencing analysis to confirm the presence or absence of HPV DNA in the cancer specimens and test the accuracy of the microarray platform. Genomic DNA from the anal cancer samples was used for GP5+/6+ primer PCR , a conseDNA sequencing confirmed a 100% correlation between the HPV microarray and DNA sequencing analysis  and HPV18 less frequently (10%) in anal carcinomas than in cervical carcinomas . HoweverThe analysis of HPV infection can be hindered by the lack of consistency in collection and detection methods, this is in part due to sample adequacy . The micIn summary, microarray technology is a novel and accurate way to screen for the various genotypes of HPV in anal cancer simultaneously, with a 100% correlation with genomic DNA detection of HPV in this cohort. The majority of anal cancers in our study is associated with HPV subtypes 16 and/or 18 and like cervical cancer, are the major genotypes associated with the cancer. Other HPV genotypes can be present in anal cancer and often occur simultaneously with genotypes 16 and 18, and might contribute to the progression of pathogenesis.The authors declare that they have no competing interests.Study concept and design: SR, Y-TL, JMC; acquisition of data: SR, Y-TL, LL, QL; analysis and interpretation of data: SR, Y-TL, LL, KM, QL, JMC; drafting of the manuscript: SR, JMC; critical revision of the manuscript for important intellectual content: SR, Y-TL, JMC; obtained funding: SR, Y-TL, JMC. All authors have read and approved the final manuscript.


HPV vaccine is an exciting promise of the preventive medicine. Although HPV-immunization programs still reveal a number of unanswered questions, they represent a novel opportunity for primary prevention against cervical cancer and other HPV-related pre-neoplastic and neoplastic diseases. It is reasonable that the short and long-term benefits of vaccination on cervical and vulvo-vaginal HPV-related pathology will emerge when assuring over time a clear and complete information to the community and harmonizing the prevention strategies. Indeed, HPV-vaccination programs will require an understanding of new paradigms of infection and cancer control, and thus will require a rationale integration with the currently operating screening systems. Much effort has been made in the last decades by scientists, epidemiologists, gynecologists, and other professionals including virologists, pathologists, and cytologists, in understanding the carcinogenetic pathways leading to cervical cancer. Consequently, a vast body of evidence has emerged indicating that the necessary cause, although not sufficient, for the development of cancer of the uterine cervix is the persistence of high-risk Human papillomavirus (HPV) genital infections -3. This Overall the disease burden attributable to HPV is significant, with more than 5% of all cancers worldwide attributed to such infections . Thus thvirus-like particles (VLPs) which are not infectious, nor oncogenic. Both vaccines protect against HPV types 16 and 18, that are together responsible for over 70% of the squamous and glandular cervical cancers [, but it is lower in women previously exposed to vaccine-targeted HPV types at the time of vaccination (modified-intention-to-treat) [Two HPV-vaccines are commercially available: the quadrivalent vaccine (GARDASIL™) and the bivalent vaccine (CERVARIX™), made by DNA recombinant techniques using expression systems based on Yeast and Baculovirus, respectively   nor prevThe quadrivalent vaccine has been approved by the US Food and Drug Administration (FDA) and the European Medical Evaluation Agency (EMEA) in 2006, and since then has received approval by other regulatory authorities in over 100 countries. The bivalent vaccine was approved in Australia and by the EMEA in 2007, and in over 60 countries, while is still awaiting FDA approval. The US-Advisory Committee on Immunization Practices (ACIP) and many scientific societies  recommended that the organized HPV vaccination programs target females 11 to 12 years of age, when the immunological response to vaccine is most effective and the HPV-seroprevalence is very low (i.e. ≤ 3% for HPV-16) . MoreoveSeparate studies are evaluating HPV vaccines in women over the age of 25. The rationale for vaccination of women over 25 years of age, is that, even though HPV infections tend to occur at relatively early ages and peak incidence rates occur between the ages of 15–25 for many oncogenic infections, the majority of women over 25 years of age have not been previously infected with HPV-16 and/or HPV-18 .In most European countries the national public health authorities, through their government advisory panels recommended the use of HPV-vaccines in national vaccination programs in order to maximize the public health benefit. In January 2007 the Italian Health Ministry has expressed an opinion on papillomavirus vaccination through its technical-scientific committee (Consiglio Superiore di Sanità) and stated to offer free vaccine doses to all Italian girls at 12 years old. In this context, vaccination should preferentially occur through organized programs in (multi)cohorts prior to sexual debut (pre HPV-exposition) or close to it. This setting represents the primary target population for HPV vaccination in Italy, and allows for better standardization, a more rigorous monitoring of vaccination, and is likely to benefit the community nation-wide. This is particularly true when organized vaccination is compared with the vaccination on an individual basis. Opportunistic vaccination is based on a different set of considerations than those used in nation-wide programs, and the ultimate goal is primarily to protect and provide benefit to an individual woman, sometimes irrespective of age. Moreover, vaccines should also be offered in catch-up programs at least to girls up to 16 years of age, which corresponds to the upper limit of the Italian obligatory school and will facilitate for high coverage.Many issues remain unsolved as we enter the era of HPV-vaccination against cervical cancer. Although vaccination has been the single most effective public health intervention to protect people against infectious diseases, it demands a capillary spread, a high acceptance among the population, an elevated coverage of the target population and the certainty of sustainable economic resources over time. Indeed, we are talking about the most expensive childhood vaccine proposed for a mass use, which may translate in the very near future (with the need for a maintenance of the screening programs) into higher health costs. Although religious or ethic reactions against vaccination in adolescents may be take into account , data concerning the parents acceptance are reassuring  and mothFurthermore, effective introduction of HPV-vaccines will require an understanding of new paradigms of infection and cancer control, and will require rationale integration with the currently operating screening systems. Indeed, at this moment the main challenges are how to properly combine the two major preventive tools, HPV vaccination and cervical screening programs, and how to optimize overall costs of such strategy.Cervical cancer screening has indeed showed to be highly effective as secondary prevention in most Western countries, although historically it was introduced without any randomized trials performed so far. Also in Italy due to organized, and also opportunistic, screening programs cervical cancer mortality significantly dropped from 8.6/100,000 in 1980 to 3.7/100,000 in 2002. Although it is well accepted that secondary prevention will have to continue in parallel to vaccination, because other high-risk HPV strains not prevented by vaccination may still cause cervical cancer, we can predict that screening protocols will change in the near future, for both vaccinated and non-vaccinated female populations. Due to the low sensitivity and low reproducibility of cervical cytology, screening strategies are already changing beyond the innovative wave of HPV-vaccination. We are moving from a prevention model based on cytology-colposcopy-histology to a biomolecular model based on virologic detection of HPV and its molecular interactions with the human host -29. The Some mathematical predictive models, within the setting of an organized cervical screening program, demonstrated that prophylactic HPV vaccination can reduce cervical cancer, CIN lesions and other genital HPV-related diseases ,31, indiAny changes in cervical cancer prevention policies (with or without vaccination) have to be addressed regarding all women at risk and, if not broadly accepted, could translate into higher disparities among the various socio-cultural layers of the female population. We should rather avoid increasing disparities among socio-economic groups, limiting the benefits of vaccine only to the higher-income subset of societies. This could be the scenario if vaccine uptake would be low in pre-teens, with a high-rate of opportunistic vaccine uptake among older girls (24–26 yrs old) already adhering to national screening recommendations. In this hypothetical scenario, despite high costs, we would not expect a significant decrease in cervical cancer mortality and morbidity.Another contemplated scenario concerns the false sense of safety produced by the vaccination. Some vaccinated women may perceive to be fully protected from cervical cancer and may be less likely to participate in screening programs. In the United States, it has been hypothesized that if 50% fewer vaccinated women participate in screening over a 5 year period following HPV vaccination  there woConsidering that full impact of vaccination on cervical cancer will take many decades to be revealed, our final recommendations about HPV-vaccination are concerning practical issues surrounding implementation of these vaccines in this moment:• Set-up cost-effective preventive programs combining vaccination and screening, based on the specific HPV-prevalence in the corresponding geographical area.• Intensify efforts to implement organized vaccination programs with high-coverage among HPV-naive girls.• Provide adequate HPV type specific surveillance to monitor any changes in HPV type distribution among the general population, to assess the impact of vaccination.• Evaluate cross-protection and HPV type-replacement.• Ensure adequate access to, and links between, vaccination-registries and screening-registries• Verify the vaccine efficacy in men and in high-risk subsets of patients Many clinical studies are still ongoing all-around the world to monitor the health impact of both HPV vaccines over the longer term and also to verify the economic impact for each country, concerning the local issues regarding the dynamic process of vaccination . It is reasonable  that the