Better understanding of gender differences in the adoption of agricultural intensification strategies is crucial for designing effective policies to close the gender gap while sustainably enhancing farm productivity. We examine gender differences in adoption rates, likelihood and determinants of adopting strategy sets that enhance yields, protect crops, and restore soils in the West African Sahel, based on analysis of cereal production in Burkina Faso. Applying a multivariate probit model to a nationally representative household panel, we exploit the individual plot as unit of analysis and control for plot manager characteristics along with other covariates. Reflecting the socio-cultural context of farming combined with the economic attributes of inputs, we find that female managers of individual cereal fields are less likely than their male counterparts to adopt yield-enhancing and soil-restoring strategies, although no differential is apparent for yield-protecting strategies. More broadly, gender-disaggregated regressions demonstrate that adoption determinants differ by gender. Plot manager characteristics, including age, marital status, and access to credit or extension services do influence adoption decisions. Furthermore, household resources influence the probability of adopting intensification strategy sets differently by gender of the plot manager. Variables expressing the availability of household labor strongly influence the adoption of soil-restoring strategies by female plot managers. By contrast, household resources such as extent of livestock owned, value of non-farm income, and area planted to cotton affect the adoption choices of male plot managers. Rectifying the male bias in extension services along with improving access to credit, income, and equipment to female plot managers could contribute to sustainable agricultural intensification. Donors Developing agricultural technologies to improve women’s well-being poses a “much more difficult challenge” than merely “taking women into account” . Indeed,In conducting gender research, the selection of the appropriate unit of analysis matters. A large body of case studies conducted in Africa controls for the sex of the household head when estimating the determinants of technology adoption and productivity. In their seminal work, Similarly, Several recent studies have examined the determinants of adoption of multiple yield-enhancing, protective, and/or conserving strategies, especially in Eastern and Southern Africa , and to In this analysis, we examine three fundamental questions regarding gender and technology adoption in cereal production. We apply multivariate probit models to test for gender differentials in (1) adoption rates; (2) the likelihood of adoption; and (3) determinants of adoption in the farming context of the West African Sahel, using Burkina Faso as an example. We consider the adoption of yield-enhancing, yield-protecting, and soil restorative strategy sets on individually managed plots of maize, sorghum, and millet.Our analysis contributes to the literature on gender roles and technology adoption in several ways. First, we add to the ongoing discussion on the gender gap in technology adoption by exploring jointly the determinants of a wide range of intensification strategy sets in a different farming context- the West African Sahel. Second, reflecting the cultural specificity of gender roles and farm decision-making in this region, we control not only for the sex of the plot manager but whether or not the manager is the household head. Third, we use a nationally representative household panel to exploit the individual plot as a unit of analysis and control for additional plot manager characteristics along with other covariates.2Historical, ethnographic research e.g.,  and moreThe allocation of individual plots is associated with the status of household members in relation to the patriarch . For exaAnother important feature of the Burkinabe farming structure is the interplay between customary norms and formal tenure rights. In recent years, socio-cultural norms and incentives guiding resource use have evolved in response to food insecurity, spurred in part by the land fragmentation that accompanies high rates of population growth . Guirkin3Enquête Permanente Agricole (EPA)) collected by the General Research and Sectoral Statistics Department ) of the Ministry of Agriculture and Food Security (Ministère de l’Agriculture et de la Sécurité Alimentaire (MASA)) in Burkina Faso. The sampling frame for the EPA is based on the 2006 Population Census. The nationally representative sample includes 4,130 household farms in 826 villages across all 45 provinces. The EPA generates production, area and yield data for rainfed crops, serves as an early warning system for food insecurity, and also furnishes general information about livestock holdings, income and expenditures of rural households, and farm input use. In this article, we utilize data covering the 3-year period from 2009–10 through 2011–12, which are the last years for which fully cleaned data are available. The format of the EPA data reflects the socio-demographic structure of farm family decision-making in Burkina Faso, as described in Section The data for this analysis are drawn from the Continuous Farm Household Survey   .4We utilize a straightforward latent variable framework to model the decision of individuals  to adopt as a binary outcome. Consistent with the overall perspective of decision-making within a complex household in the face of imperfect or missing markets, we consider that individual choices are conditioned on household and market-related characteristics. Recognizing that even if markets were perfect and complete, differentiation in input use could result from plot-specific variable features of land and micro-climate, we also model choices as conditional on plot and agro-climatic characteristics. As explained in the introduction, consistent with current analyses of gender-differentiated adoption and productivity, we include plot manager characteristics other than gender as key covariates for hypothesis testing.i at time t, be:itX is a set of observable covariates, β is a vector of parameter estimates, itu is the normally distributed error term independent of itX, and ic are the time-invariant unobserved effects (ity) and is expressed as:It is expected that individual family members will adopt intensification strategy sets that maximize their perceived benefits. Let the perceived benefits derived from adopting an intensification strategy set by farmer  effects . Althougity is a limited dependent variable, which takes a value of 1 if the farmer i makes the observable decision of adopting any intensification strategy within a set at time t and zero otherwise. As specified, both ic and β are unknown parameters to be estimated by the probability model Prob(Given the binary nature of the dependent variable (to adopt or not), the possible interdependence across a set of intensification strategies, a multivariate probit model is used to examine the gender differentials in the determinants of adoption.The multivariate probit model can be seen as an extension of the probit model, since it allows estimating several probit models simultaneously, while allowing the error terms in those models to be correlated . Ignorinic) to be correlated with observed covariates in nonlinear models, through the projection of those effects on the time average . According to per se. Though a linear model is a useful starting point for estimating average partial effects, the model imposes the restriction that 0 ⩽ itβX + ic ⩽ 1 for all itX. The linear functional form for equation 5 cannot hold over a wide range of values of itX and ic, and may thus may provide a poor approximation , and organic fertilizer, which is recommended in combination with these to restore soil fertility, in terms of its physical, chemical, and biological properties . In contThe choice of explanatory variables draws from the concepts we outline above and a vast theoretical and empirical literature on the adoption of agricultural innovations in developing country agriculture, including reviews by In addition to gender, plot manager characteristics include age, education, marital status, and headship, as well as plot manager access to credit and extension services. The number of years since plot managers received any extension services is expected to be negatively correlated with technology adoption. Credit access is defined as whether or not the plot manager reported access to credit in the preceding year, regardless of whether credit was used in the survey season. Plot physical characteristics include size, location, and topography .We then include the covariates known to explain differences in input adoption among households, including wealth, tenure security, and availability of labor, which is a complementary input for all intensification strategy sets. Total landholding, livestock, non-farm income, and number of cotton hectares are used as proxies for wealth. The number of livestock owned also provides information regarding the availability of manure. Households growing cotton benefit not only from the cash it generates, but from better access to inputs on credit and extension services provided through the cotton ginneries . We expeMarket-related factors affect adoption incentives via transaction costs and endogenous prices. The share of households in the village who are members of a cereal association is included to express access to socio-political capital as exogenous to the decisions of individual plot managers. Variables measuring the number of agro-dealers and population density at the province level are included as proxies for market access.To account for potential interannual fluctuation in moisture availability for cereal crops, the coefficients of variation in total annual rainfall at the commune level over the last three years are included. Cereal dummy variables are also included to control for crop differences.6(a)(b)p-value 0.0000). Multivariate probit is preferred statistically to univariate probit regressions, indicating that the probability of adopting one set is interdependent on the decision of whether to adopt another set. The estimates of the correlation between the regression error terms are positive and significant, suggesting complementarities across the yield-enhancing, yield-protecting, and soil-restoring sets.The regression coefficients in Female plot managers are significantly less likely to adopt either the yield-enhancing or the soil-restoring strategy sets, after controlling for other covariates. This finding is consistent with the notion that within households, women have less access to these inputs. Fertilizer, which is lumpy and costly per unit, is often extended via formal programs and projects to the household head. To utilize soil restorative practices, plot managers need access to larger livestock, additional labor and equipment, which are typically managed by the head on behalf of the household since they are deployed across numerous plots and crops. By contrast, no gender differential is evident in the probability of adopting yield-protecting (damage control) inputs, which are divisible, scale-neutral, and widely available through agro-dealers .per se but rather reflects how the socio-cultural farming context combined with the economic attributes of technology affect adoption. We examine this in greater detail below, when testing for underlying differences in the determinants of adoption by men and women.Our findings are consistent with some previous studies regarding gender differences in fertilizer use . Ndiritu(c)vs. Chi-squared critical value of 112 with 75 d.o.f at 1% significance). In other words, the underlying process that explains adoption differs between male and female plot managers. The statistical results are supported by observable differences in statistically significant determinants between the two sets of regressions  regression for all plot managers to separate (unrestricted) regressions for males and females only using a modified Chow test that is appropriate for nonlinear regressions . Compariressions , Table 5Among the socio-demographic characteristics, age of the plot manager positively influences the probability of adopting the soil-restoring strategy set for both males and females. In this society, where elderly people are highly regarded , the staExtension services, which mostly target male farmers, statistically influence their decision to adopt modern inputs. The longer it has been since male plot managers received any extension services, the less likely they are to adopt the yield-enhancing and yield-protecting sets. This finding adds to the evidence that receiving advice from extension agents positively influences adoption of yield-enhancing inputs  and damaPlot characteristics, such as topography, size, and location from residence influence the adoption of intensification strategy sets. The probability of adopting the soil-restoring strategy set, for both female and male farmers, is lower on plots that are considered far from the residence, since distant plots require more time, labor, and energy in transport. The larger is the plot size, the higher is the probability of adopting modern inputs (yield-enhancing and yield-protecting sets) for both male and female plot managers. In contrast, the plot’s topography affects adoption of intensification strategy sets differently for male and female plot managers. Although men are as likely as women to manage plots on land with slopes, the probability of adopting yield-enhancing and soil-restoring sets on these plots is statistically significant for women only. On lowland farm plots, the yield-enhancing set is more likely to be adopted by women but less likely to be adopted by men.The probability of adopting intensification strategy sets on individual cereal plots is influenced by household characteristics, but the influence of these factors depends on the gender of the plot manager. In a patrilineal society, such as in Burkina Faso, women have less bargaining power than men, which limits their access to and control over household resources , which iIn contrast, factors related to labor availability more significantly affect the decision to adopt intensification strategy sets for female compared to male plot managers. These results reinforce the idea that women’s access to labor plays a crucial role in technology adoption . FurtherGender differences are apparent in the ways market factors affect the probability of adopting intensification strategy sets. Villages that have a large share of households belonging to a cereal association are more powerful in attracting resources and public support. By serving as platforms to share general knowledge and tips regarding agricultural practices, cereal associations positively influence technology adoption, especially for female plot managers. The number of agro-dealers in each province negatively influences adoption of the yield-enhancing and soil-restoring sets, but positively influences adoption of the yield-protecting set. This reflects the weaknesses of the Burkinabe private input supply sector, especially in regards to yield-enhancing inputs. The public sector has been highly involved in the marketing of fertilizer and improved seeds, notably in the cotton-cereal farming system, which has led to the displacement of private agro-dealers . These yThe coefficient of variation of annual rainfall at the commune level is positive and significant for two out of three intensification sets, regardless of whether the plot manager is male or female. This suggests that all farmers respond to increased variability in water supply in their commune by adopting yield-enhancing and soil-restoring sets. Our results are consistent with The choice of crops also influences adoption decisions, in similar ways for both male and female plot managers. Compared to sorghum, maize plots are more likely to benefit from the adoption of yield-enhancement and soil-restoring strategy sets by both male and female managers. This finding reflects the economic importance of maize as a cash crop in this region. In contrast, the adoption of yield-protection strategies is less likely on millet than on sorghum plots for either male or female managers. Millet, a subsistence crop, is more widely grown in drier regions, where pest and disease pressures may be lower.7In this article, we have tested gender differences in the rates, likelihood, and determinants of adoption of sustainable intensification strategies between male and female plot managers growing cereals in the West African Sahel. We contribute to the literature on the gender gap in technology adoption by controlling for the sex of the plot manager along with other plot manager characteristics . We also contribute to the literature on adoption of sustainable farming practices by exploring the use of numerous inputs grouped in terms of strategies , and their interrelationships, while controlling for major cereal crops .Overall, the soil-restoring strategy set is the most frequently adopted, followed by the yield-protecting and the yield-enhancing, regardless of whether the plot manager is male or female. Findings from the descriptive statistics also suggest that female plot managers are adopting any of the three intensification strategy sets at lower rates.More nuanced results emerge from the econometric analysis. The likelihood ratio test confirms the interrelatedness of intensification strategy sets and the appropriateness of the multivariate probit model as compared to independent probit models for predicting adoption. Results from the regression that includes a dummy for the sex of the plot manager show statistically significant differences in the probability of adopting the yield-enhancing and soil-restoring sets, after controlling for other plot manager characteristics and covariates. In contrast, sex of the plot manager does not statistically influence the probability of adopting the yield-protecting strategy set. This finding reflects how both the gendered, socio-cultural farming context and economic attributes of the technology affect incentives to adopt.Disaggregated regressions demonstrate that the determinants of adoption differ between male and female plot managers. Plot manager characteristics, including age, marital status, and access to credit or extension services do influence adoption decisions, but in different ways according to the sex of the plot manager. Access to extension is significant for male managers, who have been targeted by programs in the past—but not for female managers. Furthermore, household resources influence the probability of adopting intensification strategy sets differently by sex of the plot manager. Variables expressing the availability of household labor strongly influence the adoption of soil-restoring strategies by female plot managers. By contrast, household resources such as extent of livestock owned, value of non-farm income, and area planted to cotton more often affect the adoption choices of male plot managers.Our results provide insights on the influence of gender roles and economic attributes of technology on adoption decision that are relevant to the design and implementation of effective policies to sustainably increase production by farmers in Burkina Faso and similar areas of the West African Sahel. The interrelatedness of adoption strategies  confirms the policy importance of designing extension mechanisms and messages that encourage farmers to consider flexible combinations of techniques and practices so that they can benefit from agronomic and economic complementarities. The promotion of any single intensification strategy should take incentives for use of other sets, and current patterns of input use, into consideration.At the same time, the variation in input use confirms that farmers in these environments are not best approached with a fixed package in mind, but instead, with a recognition that due to constraints related to microclimate, labor, and cash, they will need the option of selecting from “menus” of practices and to suit their own conditions. Although plant breeders and agronomist must work to achieve complementarity by designing optimal packages of inputs, farmers’ optimal combinations will be as heterogeneous as the farm population and agro-ecology. The significance of plot characteristics in our models also supports this notion. Future research might explore the relative costs and benefits of research-and-extension designs that support this heterogeneity rather than blanket, uniform recommendations.Gender differentials in adoption rates for yield-enhancing and soil-restoring sets and among determinants of adoption confirm the need to collect data disaggregated at the plot level and design and promote policies that, while respecting socio-cultural norms, also respect opportunities and incentives for individuals within multigenerational, multi-family farms.The role of public extension services, for which funding support has declined in recent decades in favor of other types of information provision, nonetheless appears to be important in adoption decisions, especially in regards to modern inputs. Some changes are needed to redress the male bias in extension services, by including women as beneficiaries and covering topics on sustainable intensification, which takes into account women’s constraints to technology adoption. Credit access also remains crucial for inputs purchased with cash (yield-enhancing and yield-protecting sets), especially for women who have limited economic control within the household. Improving access to education, income, and equipment to women could contribute to increase their bargaining power and thereby, adoption of sustainable intensification strategies.Customary norms also appear to play key roles in adoption of strategies for sustainable intensification of agriculture. Marriage, in particular, appears as significant for young men, as compared to women plot managers, most of whom obtained usufruct rights upon marriage. Among men, major differences in social status are related to headship and marriage. Only a tiny minority of women in the sample are heads. Looking toward the next generation of farmers, and the transfer of rights and responsibilities, inter-generational differences among men within households may be as important to take into consideration as differences between men and women.


E. coli-derived concatenated, multitype L2-conserved epitopes of human papillomavirus (HPV) L2 protein might represent a less expensive and pan-type vaccine alternative (compared to type-specific HPV L1 virus-like particles), if stable protein expression and strong immunogenicity features could be met. Herein, three dual-type- (DT-) HPV L2 fusion peptides comprising the three head-to-tail tandem repeats (multimers) of either HPV 16 epitope “17-36” or “69-81” or one copy (monomer) of 11-88 fused to the same residues of HPV 18 were constructed and expressed in E. coli. SDS-PAGE and Western blot analyses indicated the proper expression and stability of the E. coli-derived DT peptides. Mice immunized by formulation of the purified DT peptides and Freund's adjuvant (CFA/IFA) raised neutralizing antibodies  which showed proper cross-reactivity to HPV types: 18, 16, 31, and 45 and efficiently neutralized HPV 18/16 pseudoviruses in vitro. Immunization studies in mice by formulation of the DT: 11-88 × 1 peptide with various adjuvants  indicated that Montanide adjuvants elicited the highest cross-reactive titers of NAbs and similar levels of IgG1 and IgG2a . The results implied development of low-cost E. coli-derived DT: 11-88 peptide formulated in human compatible ISA 720 adjuvant as a HPV vaccine. Human papillomavirus (HPV), a heterogeneous group of nonenveloped double-stranded DNA tumor viruses with around 8 kb genome encoding for early and late L1 and L2 capsids) proteins, infects the epithelia of the skin and mucosa in humans. HPV-induced diseases range from subclinical and self-limiting benign skin warts and mucosal papillomas (condylomas) to life-threatening dysplasias and carcinomas of the anogenital tract and oropharynx  capsids –5. Of noPrior studies indicated that neutralizing antibodies (NAbs) to capsid protein L1 confer sterilizing immunity against HPV infection. Although, NAbs are raised against both L1 and L2 , high titers of NAbs are raised against L1 since B cell epitopes of L1 are highly dominant compared to those of L2 . AccordiContrary to immune responses against strong conformational L1-VLP epitopes that are type-specific, the N-terminal of minor capsid protein L2 and amino acids 11-200  contain conserved, type-common (pan-type) but subdominant linear B cell epitopes. Thus, these epitopes are capable of inducing broadly cross-NAbs to highly divergent and heterogeneous HPV types, even in the isolated forms, but in much lower titers and potency . TherefoIn still another approach to develop low-cost HPV L2-based vaccines with improved immunogenicity by production in bacterial expression systems and multimeric, concatenated, and epitope-encoding peptides formulated with strong adjuvants was developed . It shouPrior studies indicated that application of three or more tandem repeats of the same epitope (amino acids 20-38) of L2 peptide in the context of linear protein induced more potent immune response in comparison with the same residues in monopeptide form . Since, E. coli host, synthesized  and subcloned into the 5′ NcoI and 3′ XhoI restriction sites of the pET-28a (+) expression vector (Addgene) in upstream of the C-terminal His-tag sequence. The three recombinant vectors corresponding to the abovementioned sequences are termed as the following: pET-28-HPV 17-36 × 3 , pET-28-HPV 69-81 × 3 , and pET-28-HPV 11-88 × 1 , respectively  of either HPV L2 RG-1 epitope “17-36 residues (17-36 × 3)” or “69-81 residues (69-81 × 3)” or one copy (monomer) of 11-88 amino acids (11-88 × 1) from HPV 16 (accession number: NC_001526) fused to the same regions, with the same patterns  of HPV 18 (accession number: NC_001357). All DNA sequences were codon optimized for expression in ectively .E. coli host, synthesized  and subcloned into the 5′ BamH I and 3′ HindIII restriction sites of the pET-28a (+) expression vector (Addgene). The recombinant vectors corresponding to the abovementioned sequences, encoding the four HPV L2 amino acids 11-200 peptides, are termed as the following: pET-HPV 16, pET-HPV 18, pET-HPV 31, and pET-HPV 45  induced by immunization of the dual-type fusion L2 peptides via ELISA-based neutralization assays, the coding sequences of L2 amino acids 11-200 from HPV types 16, 18, 31, and 45  were also codon optimized for expression in T-HPV 45 .The final recombinant constructs were confirmed by restriction analyses followed by agarose gel electrophoresis and DNA sequencing reactions. All molecular and cloning procedures were performed according to standard protocols .E. coli BL21 (Rosetta DE3) and induced for protein expression by IPTG induction (1 mM). The expressed proteins were further purified by Ni-NTA affinity chromatography according to the denaturing protocol of the manufacturer from the sonicated bacterial lysate . Subsequently, the recombinant polypeptides were renatured by dialysis against phosphate-buffered saline (PBS) and quantified using Bradford protein assay . The endotoxin level of the purified dual-type fusion peptides was further quantified by QCL-1000 chromogenic limulus amoebocyte lysate test (BioWhittaker) according to the manufacturer protocols. The recombinant protein was stored at −70°C until use.All three recombinant vectors encoding the dual-type fusion L2 peptides  and the four recombinant vectors encoding the L2 amino acids 11-200 from HPV types 16, 18, 31, and 45  were transformed into Expression of all the peptides was analyzed by 12% SDS-PAGE according to standard protocols . Specifid) mice, 4–6 weeks age were used for immunization. In the first immunization experiments, to compare the potency of the different peptides to elicit neutralizing antibodies, separate mice groups were immunized subcutaneously (s.c.) with 25 μg of each three dual-type fusion L2 peptides (in separate experiments), three times at two-week intervals. To this end, antigens were formulated in “complete Freund” adjuvant  for the priming dose and in “incomplete Freund” adjuvant  as booster immunizations in 100 μl total immunogen volume (at 1 : 1 ratio) for each immunization , or MF59 (at 1 : 1 ratio), or Montanide ISA 720 , or Montanide ISA 50V  which was pET-88-encoding antigen; 11-88 × 1 HPV 16 + 11-88 × 1 HPV 18 . To this end, 25 nization . ImmunizBlood samples were collected from all experimental groups through retroorbital bleeding two weeks after the final injection and sera were maintained in −20°C until use.7) were cultured 20 hour before transfection in 162 cm2 flasks containing Dulbecco's modified Eagle's medium (high glucose)  supplemented with 10 U/ml penicillin, 10 μg/ml streptomycin, and 10% FBS . Cells were cotransfected with 40 μg of a bicistronic L1 and L2 expression constructs (either HPV 16 L1/L2 (p16L1L2) or HPV 18 L1/L2 (p18L1L2); kindly provided by Professor Martin Muller, DKFZ, Germany) and 40 μg of a GFP expression packaging plasmid (pfwB) (Addgene number 37329) in two different reactions (for HPV 16 L1/L2 and HPV 18 L1/L2) using Lipofectamine 2000 transfection reagent . Media were replaced 6 h after transfection and cells were harvested 48 hours post transfection by centrifugation at 300g for 10 min and lysed by resuspension (108 cells/ml) in DBPS (Thermo Fisher Scientific) supplemented with 9.5 mM MgCl2, 0.5% Brij 58 , and 0.2% Benzonase followed by overnight incubation at 37°C to induce pseudovirion maturation. The next day, crude extract of cell lysates was chilled and subjected to salt extraction (adjusted to 0.8 M NaCl) and further incubated at 37°C for 1 h. Subsequently, cell lysates were clarified by spinning at 1500 ×g for 10 min at 4°C. Finally, supernatants were harvested and stored at −80°C until use.To assess the neutralizing capacity of the immunized mice sera, we first generated the HPV 18 and HPV 16 pseudoviruses (PsV) as described elsewhere  with minGeneration of HPV 16 L1/L2 and HPV 18 L1/L2 PsVs  was confirmed by transducing 293 TT cells using crude extract of cell lysates at 1 : 100 dilution in 24-well plates for each PsV and direct observation of GFP-expressing cells under fluorescence microscope  48 h after transduction. To measure the infectious titers of PsV, the 293 TT cells (1 × 105 cells/well) were transduced with serial dilutions of PsV preparations and 48 h post transduction, GFP-expressing cells were analysed by flow cytometry . Finally, the infectious titers were calculated as previously described for lentiviral vector titration , 41 and μl) of abovementioned preparations was placed on a 400-mesh Formvar/carbon-coated copper grid and after absorption, the grids were washed briefly and stained with 2% phosphotungstic acid for 2 min. Subsequently, the stain was removed, and the grid was allowed to air dry for 30 minutes. The microscopy was performed with transmission electron microscopy (TEM) (Zeiss 10A). Micrographs of random sections were taken at a magnification of 50,000x.For further visualization and confirmation of PsVs, extracted suspension  (1 : 20000) was added to each well and incubated for 1 hour at RT. After extensive washing steps, reactions were developed by adding 100 μl of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate , and color development was stopped by 2N sulfuric acid. Finally, the optical density of wells was measured at 450 nm by ELISA reader . Antibody (IgG) titers were expressed as the reciprocal of the highest dilution at which the OD 450 was greater than 2-fold in comparison with the same dilution at control sera.An in-house-developed ELISA was designed to assess the titer of the cross-reactive antibody (IgG) of mice immunized by DT: peptides to HPV L2 11-200 peptides of types 16, 18, 31, and 45 . To this end, ELISA 96-well plates (Nunc) were coated with 500 ng/well of recombinant HPV L2 amino acids 11-200 from HPV types 16, 18, 31, and 45 in separate wells (overnight) as described before . The coaAntibody IgG subclasses and isotypes  in the sera of mice immunized with different adjuvants formulations of the L2 11-88 fusion peptide were measured as described above using 1 : 4000 and 1 : 2000 dilution of sera and HRP-labeled goat anti-mouse conjugates of IgG subtypes , respectively.In all experiments, the cut-off value was taken as twice the mean of absorbance values of the negative control (unimmunized mice sera) and each sample was tested in triplicate.μg/ml streptomycin, and 10% FBS  in each well of a 96-well plates . The day after, pooled sera from each immunized group (5 mice per group) were serially diluted (at starting dilution 1 : 10) in culture media. Subsequently, 20 μl of diluted sera was mixed with 80 μl of diluted PsV (1 : 100), incubated on ice for 1 h and added to the preplated 293 TT cells. Two days after infection, the cells were trypsinized and GFP-expressing cells were analyzed by flow cytometry. Cells treated with the only diluted PsV were considered as negative control. Neutralization titers were determined as the highest sera dilution of each immunized group at which the HPV 16 and HPV 18 PsVs were neutralized (at least 50%) in comparison with cells infected with the diluted PsV without sera.The generated HPV 18/16 PsVs were used for performing the neutralization assay as previously described  with fewP values less than 0.05.Experimental data were analyzed using GraphPad Prism 6.0 . Significant differences among experimental groups were determined using one-way analysis of variance (ANOVA) and Bonferroni multiple comparison test. Significant differences between groups were set at NcoI/XhoI restriction enzymes followed by agarose gel electrophoresis resulted in two bands of 5350 bp corresponding to the body of digested pET-28a vector and 385 bp, 300 bp, or 500 bp for the fragment inserted into pET-17, pET-69, and pET-88, respectively  plasmids (Supplementary E. coli BL21 (Rosetta DE3) harboring the dual-type fusion L2 peptide-encoding pET-28a (+) vectors by IPTG resulted in the expression of recombinant protein with molecular weight (MW) of ~14 kDa, 11 kDa, and 17 kDa corresponding to expressed peptides of pET-17, pET-69, and pET-88, respectively  harboring the L2 11-200 peptides-encoding pET-28a (+) vectors by IPTG resulted in the expression of distinctive bands with various MVs of 38 kDa, 28 kDa, 38 kDa, and 36 kDa corresponding to expressed peptides from pET-HPV 16, pET-HPV 18, pET-HPV 31, and pET-HPV 45 vectors, respectively . Likewise, mice received the same mentioned peptides could elicit significant mean titers of 8 × 104, 3 × 104, and 26 × 103 against HPV 18  for DT: 17-36 × 3 peptide , followed by MF59 , while both Montanide adjuvants  elicited the highest and almost similar titers comparable to that of CFA/IFA (mean titer: 68 × 103)  .Results of IgG subtyping for different adjuvant formulations of the DT: 11-88 × 1 peptide indicated that in all groups , IgG1  was almoP values” for each antibody titer are provided in Figures In accordance with cross-reactivity results, evaluation of the induced antibodies by different adjuvant formulations of the DT: 11-88 × 1 peptide for neutralization of PsV 16/PsV 18 also indicated the higher neutralization titers for PsV 16 (mean titer of 50: 4800 and ISA 720: 4600) compared to PsV 18 (mean titer of 50: 3800 and ISA 720: 1600) which was highest for ISA 50 and 720  followed by CFA (mean titer: 2400 for PsV 16 and 2800 for PsV 18), MF59 , and alum , respectively Figures . The comE. coli host. These three peptides represented the most recognized immunogenic and conserved epitopes in the N-terminal of L2 peptide and encoded for three head-to-tail tandem repeats (multimers) of either HPV L2 RG-1 epitope “17-36 residues (17-36 × 3)” or “69-81 residues (69-81 × 3)” or one copy (monomer) of 11-88 amino acids (11-88 × 1) from HPV 16 fused to the same regions with the same patterns  of HPV 18. Immunization studies in mice indicated that L2 11-88 peptide formulated in the human compatible adjuvant “Montanide ISA 720” could induce high levels of cross-reactive antibodies towards HPV types 16, 18, 31, and 45 and neutralization potencies to HPV 16 and HPV 18 PsVs.In the present study, we demonstrated the stable expression and proper purification of three dual-type fusion HPV L2 peptides in E. coli cells harboring the L2 11-200 peptides-encoding pET-28a (+) vectors by IPTG resulted in the expression of distinctive bands with various MVs of 38 kDa, 28 kDa, 38 kDa, and 36 kDa corresponding to expressed peptides from pET-HPV 16, pET-HPV 18, pET-HPV 31, and pET-HPV 45 vectors, respectively  plasmids harboring the genes corresponding to the three DT L2 peptides resulted in the inserts, which in accordance with prior reports, were around 385 bp for of pET-36 (17-36 × 3 HPV 16 + 17-36 × 3 HPV 18) , 24, 300P < 0.05) than the other two peptides, but almost similar for both DT: 17-36 × 3 and DT: 69-81 × 3 peptides (P > 0.05). Although, to our knowledge, no prior study has addressed comparing of any monomeric/multimeric forms of all these three peptides together for immunization studies, but some reports on comparing either of the two pair of the peptides have provided results which are consistent with our conclusions. In an excellent study by Jagu et al. [Microscopic and flow cytometry analyses  indicateu et al. , immunizu et al. . In a cou et al.  demonstru et al. , which mu et al. , 52. In u et al.  also repu et al. . Of noteu et al. , 37–39 hu et al. , 43, 50.Adjuvants have a crucial role in subunit vaccine formulations to generate and enhance the weak immune responses induced by peptide antigens. Of note, antibody levels induced by L2 peptides are very weak compared to those induced by L1 VLP vaccines. Therefore, adjuvants have a critical role in formulation of L2 peptides as subunit vaccines to induce robust antibody responses. However, currently approved adjuvants for human use, like aluminum compounds, induce relatively weak responses and often require multiple immunizations to elicit protective antibody levels . In thisE. coli-derived, lipidated triple-repeat HPV 16RG-1 epitope fused with a FcgRI-specific single-chain antibody fragment (H22scFv) formulated with MF59 and poly I:C for raise of sufficient Abs [MF59 (Novartis), an oil-in-water adjuvant, was used in the first clinical trial of seasonal influenza virus vaccine around a quarter of century ago (in 1992) and is now licensed worldwide in 30 countries and has been used in a number of subunit vaccine formulation studies . As showient Abs . ResultsE. coli-derived HPV 16 L2 20-38 peptide with M720 adjuvant was also shown to be highly effective in induction of cross-NAbs against HPV types 16/33/58 [The Montanide (M720 and 50) adjuvants are relatively safe and have been studied in several therapeutic and prophylactic vaccine studies contain natural metabolized oils and a highly refined mannide that acts as a depot to slow the release of Ag. These adjuvants have been also targets of studies for formulation of cancer vaccines and improving Ag presentation to T cells. However, only M720 is known to be human compatible , 60. As 16/33/58 .ϒ and IL-4  are known to be secreted by Th1 and Th2 cells, respectively [Schistosoma mansoni cathepsin B [Although, the contribution of cellular responses might not be completely neglected, the crucial role of neutralizing antibodies to confer sterilizing immunity against HPV infection is well documented . Thus, iectively . Accordiectively –66. Thesectively , while Mectively . Consistectively , subunitectively , Leishmaectively , and Schhepsin B . The relhepsin B .Finally, it is noteworthy that although we did not compare our DT immunogens with currently available HPV L1-based vaccines (like Gardasil) or did not evaluate the cross-reactivity towards other important HPV types like HPV 58, but we consider the results of the prior studies , 20, 29.E. coli-derived DT peptides could raise efficient NAbs in mice, capable of cross-reacting with HPV types 16/18/31/45 and neutralizing HPV 16/18 PsVs, albeit with remarkably higher titers for DT: 11-88 × 1 peptide formulated in human compatible M720 adjuvant. Our results imply the possibility of development of a simple, low-cost preventive HPV vaccine based on this dual-type fusion L2 peptide with broad spectrum  for low-income countries.Taken together, to the best of our knowledge, this study presented the first report on construction of dual-type fusion L2 peptides (HPV 16/18) corresponding to the fused triplet epitopes of 17-36, 69-81, and 11-88 monomers and their proper/stable expression in bacterial system and comparing their potency for induction of NAbs. Our results indicated that all the three


Mental health conditions are prevalent among US veterans and pose a number of self-management and health care navigation challenges. Post-Traumatic Stress Disorder (PTSD) with comorbid chronic medical conditions (CMCs) is especially common, in both returning Iraq or Afghanistan and earlier war-era veterans. Patient-facing electronic health  technology may offer innovative strategies to support these individuals’ needs.This study was designed to identify the types of eHealth tools that veterans with PTSD and comorbid CMCs use, understand how they currently use eHealth technology to self-manage their unique health care needs, and identify new eHealth resources that veterans feel would empower them to better manage their health care.A total of 119 veterans with PTSD and at least one CMC who have used the electronic personal health record system of the US Department of Veterans Affairs (VA) responded to a mailed survey about their chronic conditions and preferences related to the use of technology. After the survey, 2 focus groups, stratified by sex, were conducted with a subgroup of patients to explore how veterans with PTSD and comorbid CMCs use eHealth technology to support their complex health care needs. Focus groups were transcribed verbatim and analyzed using standard content analysis methods for coding textual data, guided by the “Fit between Individual, Task, and Technology” framework.Survey respondents had a mean age of 64.0 (SD 12.0) years, 85.1% (97/114) were male, 72.4% (84/116) were white, and 63.1% (70/111) had an annual household income of < US $50,000. Mean score on a measure of eHealth literacy was 27.7 (SD 9.8). Of the respondents, 44.6% (50/112) used health-related technology 1 to 3 times per month and 21.4% (24/112) used technology less than once per month. Veterans reported using technology most often to search for health information , communicate with providers , and track medications . Five major themes emerged that describe how eHealth technology influences veterans with PTSD and comorbid CMCs: (1) interactions with social support, (2) condition management, (3) access to and communication with providers, (4) information access, and (5) coordination of care.The “Fit between Individual, Task, and Technology” model provided a useful framework to examine the clinical tasks that arose for veterans and their resourceful adoption of eHealth tools. This study suggests that veterans who use the Web are eager to incorporate eHealth technology into their care and self-management activities. Findings illustrate a number of ways in which the VA and eHealth technology developers can refine existing applications, develop new resources, and better promote tools that address challenges experienced by veterans with PTSD and comorbid CMCs. Foremost among the challenges facing the US Department of Veterans Affairs (VA) is providing care for the more than 40% of VA patients who are suffering from mental health disorders . The mosTo further complicate matters, PTSD often co-occurs with a number of chronic medical conditions (CMCs) ,16. VeteThe potential for electronic health  resources to better promote wellness in veterans with PTSD and comorbid CMCs depends on understanding patients’ health needs and preferences related to technology ,26. AmonTo date, however, very little research has examined eHealth use among veterans with PTSD. One study that surveyed veterans who attended a VA PTSD outpatient clinic  Given the multiple challenges that veterans with PTSD and comorbid CMCs face, it is vital to understand the ways in which the needs of these veterans interface with the types of eHealth resources available, as well as the health-related tasks veterans prefer and desire. The goals of this investigation were threefold: (1) to identify the types of eHealth tools used by veterans with PTSD and comorbid CMCs who use the Internet, (2) to understand how they currently utilize eHealth technology, and (3) to identify new eHealth resources that veterans feel would empower them to better manage their health care.We report on secondary data analyses from a sequential, mixed methods study comprising a survey followed by focus groups . In 2012For descriptive purposes, the survey asked patients to self-report their current health conditions using a list of 29 conditions that included “Post-Traumatic Stress Disorder.” Respondents could also write in additional conditions . BecauseThe 8-item eHEALS   was usedAmong the 119 individuals with PTSD and at least one CMC who completed the screening survey, 35 met eligibility criteria  for the focus groups . Using purposive sampling, we constructed 2 focus groups of patients with PTSD, stratified by sex to enable a forum for discussing potentially sensitive topics . Sampling considerations included patients’ experiences with PTSD and multimorbidity, their experience with technology, and their availability to participate in a scheduled group. Of the 35 eligible patients with PTSD, 10  participated in one of the focus groups.The 2 sex-stratified groups consisted of 7 men and 3 women. Written informed consent was obtained from each focus group participant, and each received a US $50 gift card for participation. The focus groups were moderated by a trained research specialist using a semistructured guide . The “FiDescriptive summary statistics were computed for demographic and technology variables. Focus groups were transcribed verbatim and analyzed using standard content analysis methods for coding textual data . As descUse of eHealth resources is presented in Five major themes describe how eHealth technology influences veterans with PTSD and comorbid CMCs, including their (1) interactions with social support, (2) condition management, (3) access to and communication with providers, (4) information access, and (5) coordination of care. Quotes from focus groups that represent each theme appear in an abbreviated version in Four subthemes emerged that describe how technology influences veterans’ interactions with their social support.Veterans with PTSD and comorbid CMCs reported using technology to overcome difficulties connecting socially with others in person or via the telephone. In particular, texting and social media were seen as useful for connecting with others without the stressors of social interaction, such as becoming “too emotional.” to meet so that the veterans can stay together and kind of help each other with the school issues.” In other words, technology can allow veterans to connect with peers and maintain supportive relationships when face-to-face support is not feasible.Several Veterans described using technology to facilitate mutual support within specific groups of veterans, for example women who attended a retreat together. Others stated that they were still seeking a means to connect with one another via social media. For example, a veteran who is attending a university was “trying to get our own place my therapist) in the middle of the night and she arranged for someone to come pick me up...If I hadn’t had that, you know, I probably wouldn’t have gone to (the) emergency room or called 9-1-1 or called one of those crisis lines where you have to talk to some stranger.”Veterans also reported using email and text messaging technology to give and receive support in times of crisis. Veterans indicated that they felt more comfortable using technology to connect with a peer or a professional who knew them, rather than someone they did not know, to help them cope with feelings of depression and suicidality. One veteran stated, “...when I got to my point where I was really at my lowest, you know, I called  is glued to my hand all the time and as soon as they got that PTSD app out...I loaded all my little pictures in there and my phone numbers and you can like send a text when you freak out and it will make a phone call (to the crisis line) for you.” Additionally, veterans used their digital devices to maintain a journal about the problems they encountered. One veteran opened a Web “blog” because he felt a website was a more secure location to write about his symptoms than in a book or on paper.Veterans described using technology to access Web-based tools to help them cope with their PTSD and comorbid CMCs. For example, some veterans used mobile apps, such as games, to distract themselves when their anxiety elevated. Others used apps that were specifically designed to manage physical and psychological symptomatology, such as anxiety and high blood pressure. For example, veterans identified the VA “PTSD Coach” app  as partiVeterans stated that their mobile phones, in particular, have become a source of grounding and security and thus function to reduce anxiety when they are in public. One veteran stated, “You know, holding a...smartphone or whatever, you know, like I just have a rock in my pocket or something that will calm me down or focusing on something in the room to like kind of calm my anxiety.” Others indicated that following maps or directions was very stressful for them because of memory and concentration difficulties, and so navigation apps were helpful to reduce their anxiety about navigating on the road.eVet”) as being helpful for renewing and keeping track of medications. One veteran stated, “I utilize Google mail appointment reminders and I set them all and so they email me and they can message on my phone to tell me to order my medication, to take my medication, to make an appointment, to go to an appointment, and then tell (me) things I have to check off.”Veterans described using computer programs, email, and mobile apps to keep track of a variety of health-related needs, including medical appointments and medication, as well as non–health-related tasks. Veterans identified the VA’s telephone system and Web portal  when they can. And sometimes the conversation (in office-based meetings) goes a little bit longer because you don’t always think about what you are going to say and it kind of drags on more than what it needs to.”Another theme focused on the convenience of engaging with providers via secure messaging. Veterans felt more connected to their providers and described that they and their providers could resolve problems more quickly using text messaging and email. One veteran stated, “Yeah, I think it’s faster for all of us, you know. It kind of frees up (providers’) time and they can answer (videoconferencing) would be easier.” Other veterans indicated that technology could be helpful in situations when they are unable to attend health care sessions at the VA facility because of anxiety and hypervigilance secondary to agoraphobia or military sexual assault.Similarly, veterans stated that they either used or desired access to home telehealth care services when in-office health care sessions were not feasible. A common theme was the importance of health care sessions provided via technology for veterans disabled by mental and/or physical symptoms. One veteran stated, “For me like sometimes my migraines are really bad and a trigger for me and my migraines is driving so Skyping , and be able to access it up here. So what I had to do is we had to sit there for two and a half hours and get him signed up in .” Veterans wished for greater access to and coordination of records across VA facilities to expedite access to health care regardless of location and to afford providers knowledge of veteran health care activities across the VA.This investigation used the FITT model  to identThe veterans in this study reported moderate levels of perceived knowledge, comfort, and skill at finding, evaluating, and applying electronic health information. Most had searched for health information, communicated with a provider via email, and tracked their medication, but fewer had used online health-related support groups, games, and mobile apps. The average eHEALS  score was comparable to scores reported by similarly aged nonveteran samples, including 283 baby boomers and older adults who use the Web   and 866 Our qualitative findings identified eHealth resources that empowered veterans to better manage their health care for their PTSD and comorbid CMCs. Findings suggest that health care systems should promote technology that addresses 5 themes: (1) interactions with social support, (2) condition management, (3) access to and communication with providers, (4) information access, and (5) coordination of care. Focus group themes aligned with our quantitative findings, which showed that the most common use for technology was to search for health information (consistent with the theme “information access”). Moreover, the second and third most common uses for technology were to communicate with providers and to track medications, respectively. These uses corroborated the focus group themes “access to and communication with providers” and “condition management.” As described below, qualitative findings went beyond quantitative results to more comprehensively define how veterans actually used technology to manage their health care needs and to prevent potentially injurious problems from occurring.Social support is a consistent correlate of positive outcomes in veterans with PTSD . HoweverVeterans who suffer from PTSD symptoms such as hypervigilance, negative mood, and flashbacks can experience high levels of stress when in public settings. As a result, veterans with PTSD and comorbid CMCs may avoid grocery stores, health care appointments, social gatherings, and other events ,50. In tIn addition to directly managing their symptoms, participants used electronic devices to increase communication with providers by mobile phone and by secure email messaging. Veterans felt that communication was more accurate and candid when delivered via digital modalities compared with face-to-face settings. Veterans also described feeling better able to report symptoms more accurately at the time they were occurring, which is consistent with some research on military populations ,52. AddiAnother modality embraced by the VA to increase access for veterans is clinical videoconferencing, which directly connects providers to veterans who are located in their homes or in another VA clinic ,54. VeteMoreover, whereas research supports the role of technology in increasing social support and reducing isolation , veteranVeterans used technology to access health information, but some indicated that they have become overwhelmed by the amount of resources available and, at times, by conflicting information. Materials designed for veterans with PTSD and comorbid CMCs should take into consideration the mild to moderate cognitive impairment that can be associated with their symptoms. There additionally appeared to be a desire for a trusted service that distinguishes high-quality consumer information from biased or lower-quality materials. Veterans need information presented in a format and at an educational level that engages them , and theFinally, veterans used technology to initiate treatment coordination between providers and, at times, across facilities. This care coordination was particularly important because study participants were often tasked with coordinating care for different health issues from multiple providers . VeteraneVet) in order to restrict the sample to veterans who have had Web exposure, which limits the generalizability of the results. Results are also limited by our survey response rate of 31.93% (479/1500). Survey respondents may be predisposed to view eHealth positively. We attempted to minimize this bias by offering veterans remuneration for their efforts filling out the survey. Additionally, participants in this study were primarily Caucasian veterans selected from a relatively computer-literate region in northern California. Their perspectives may be different from veterans who do not have access to or prefer not to use the Web, reside in other regions, or are of minority status. Finally, focus group members consisted of a small subgroup of veterans whose attitudes may not represent all veterans with PTSD and comorbid CMCs who use the Web. However, focus group members were similar to other study participants in that virtually all members of both groups had used and were comfortable using computers, the Web, and email but had less experience using online health-related support groups, games, and mobile apps. Additionally, eHealth literacy scores of veterans in the survey sample and focus group subsample were similar to those found in other samples of similarly aged adults who use the Web [The purpose of this research was to understand veterans’ experiences using eHealth technology to help manage their unique PTSD and comorbid CMC symptoms. Because our goal was to identify how veterans use eHealth, we purposefully selected from users of the VA's patient portal system (My Health  the Web .Our study suggests a number of opportunities to support veterans with PTSD and CMCs through eHealth technology. First, study participants expressed interest in increasing contact with peers through technology, for example, through social media groups for veterans in college or clinician-monitored chat rooms for group therapy patients to support one another between visits. Second, eHealth technology can provide symptom management support, for example, through mobile apps  or live In summary, the results of this investigation help establish that veterans with PTSD and CMCs who use the Web are eager to incorporate eHealth technology into their care and self-management activities. Furthermore, study findings suggest opportunities to augment the potential power of eHealth as an adjunct to care, particularly with regard to preventive care. The themes that emerged from this investigation help characterize approaches the VA and eHealth technology developers can take to refine existing resources and develop new tools to better serve veterans with PTSD and CMCs. Future research should evaluate whether such patient-centered endeavors facilitate the appropriate use of health care services and improve clinical outcomes.


The purpose of this study was to examine human papillomavirus (HPV) and HPV vaccine knowledge, attitudes, and uptake in college students and to identify factors associated with vaccination status utilizing the Precaution Adoption Process Model (PAPM). The sample included 383 undergraduates from a public university who participated in February and March 2015. Students were emailed an anonymous online survey assessing knowledge, attitudes, and perceptions related to HPV and HPV vaccination, as well as their stage in the PAPM regarding vaccination completion. Significantly more females (47.3%) than males (15.8%) were vaccinated. While most students had basic knowledge of HPV, they had low perceptions of their susceptibility to contract HPV. Most unvaccinated students were in the early stages of decision-making related to vaccination. Campus health centers have an opportunity to increase HPV vaccination rates. This study indicates that students need prompts from providers, as well as education regarding susceptibility to HPV. Human papillomavirus (HPV) is a highly prevalent sexually transmitted infection associated with increased cancer risks –2. An esLittle research has been conducted to understand how college-aged individuals begin transitioning to making their own health decisions as they become independent, and this knowledge gap extends to how they make decisions related to HPV vaccination specifically. Barriers identified by parents include cost and time, as the vaccine requires multiple visits to a health care provider to complete the series ,10. OtheThe national guidelines call for catch-up vaccination for young adults who were not vaccinated during adolescence . CollegeWeinstein’s Precaution Adoption Process Model  was deveIn an effort to address the need, this study sought to examine HPV vaccination awareness and uptake in male and female college students and to utilize the Precaution Adoption Process Model (PAPM) to assess how knowledgeable students are about HPV and HPV vaccines, evaluate concerns about vaccination, and identify factors associated with vaccination status.A cross-sectional online survey study design was utilized. We developed a survey to assess students’ knowledge, attitudes, and perceptions related to HPV and HPV vaccinations, as well as stage in the PAPM regarding vaccination completion. The survey included items developed in previous studies as well as additional items created for this study . Knowledt-tests (p < .05) were utilized to characterize the sample and to compare responses from female and male participants. A logistic regression model was developed to examine factors associated with vaccination status. Approval by the University of Mississippi Institutional Review Board was granted prior to the initiation of the study. Informed consent was provided by participants prior to survey completion.The survey was distributed via email to undergraduate students at a public university in the state of Mississippi and remained available for approximately one month. Analyses were conducted using SPSS (version 22.0). Descriptive statistics, chi-square tests, and independent The 383 participants were nearly evenly distributed across the four undergraduate classifications (freshmen through seniors), with a mean age of 21.01 years. The sample was 70.2% female and self-reported ethnicity as 76.8% White, 13.1% Black, 8.9% Asian, and 2.2% other, a distribution similar to that of the institution as a whole. The majority of the students (71.3%) indicated that they had a primary health care provider .p < .015). Significantly more females were at least aware of the HPV vaccine compared to males . The knowledge of HPV scale had a potential score range of 0 to 18. There were no significant differences on the knowledge scale between female and male respondents . Approximately 90% of respondents were aware that HPV is sexually transmitted, can be transmitted even when asymptomatic, and infects both men and women (p < .001). Provider recommendation rates differed as well, with 62.4% of females compared to 21.6% of males reporting that they had been offered the HPV vaccine by a doctor or nurse (p < .001).Responses indicated that most students had heard of HPV . Participants who were not vaccinated were asked to indicate their thoughts about getting vaccinated to determine which stage of the PAPM they were in at that point in time. The percent of participants in each stage indicated that most unvaccinated college students were in the earliest of the model’s stages as described in Table 3HPV vaccine uptake was reported by 47.3% of the females and 15.8% of the males (X2 = 21.78(4); p < .001). Unvaccinated participants were further asked how likely they were to get the HPV vaccine within the next six months. Vaccination intention was low, with 51.7% of females and 52.5% of males reporting that they were “very unlikely” or “unlikely” to get the vaccine within the next six months.The vast majority of males (90%) indicated that they had never seriously thought about getting the HPV vaccination, but a significant portion of females reported this as well (62.9%). Overall, females were further along in the stages compared to males  were predicted by this model described in The Advisory Committee on Immunization Practices (ACIP) recommends catch-up HPV vaccinations for young adults who were not vaccinated during adolescence . The resTo the best of our knowledge this is the first study to use the Precaution Adoption Process Model to evaluate where college students are in terms of decision making related to HPV vaccination. College campus health centers have a unique opportunity to significantly expand HPV vaccination coverage, thereby protecting the short and long-term health of this population. Understanding how to communicate to and engage students on this topic is essential to effectively develop interventions to motivate vaccination uptake.Limitations of this study include the use of a cross-sectional survey design and self-report measures. Additionally, the sample includes only those who self-selected to participate. However, the final sample was representative of the university’s population. This survey instrument combined items from previous investigations of HPV in college students and added additional constructs. This exploratory study did not validate this instrument. Further research is needed to investigate the reliability and validity of the assessment. This study is unique in utilizing the PAPM to examine college students’ intentions, knowledge, and attitudes related to HPV and HPV vaccination.There are suboptimal levels of HPV vaccine coverage in college students in the United States . ImportaS1 Fig(TIF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(XLSX)Click here for additional data file.


This research was accomplished to evaluate the IHC expression of p16 (ink4a) and CK17 in low grade cervical intraepithelial lesions (LSIL), high grade cervical intraepithelial lesions (HSIL) and invasive cervical carcinomas and to assess their correlation to HPV (16E6+18E6).The study included (127) formalin-fixed paraffin-embedded cervical biopsies; of which 22 cases were chronic cervicitis, 24 cases were LSIL, 28 cases were HSIL and 53 cases were invasive cervical carcinomas. Sections were immunohistochemically stained for p16 (ink4a), CK17 and HPV (16E6+18E6).P-value˂0.001). There was non-significant association between IHC expression of all makers with age of patients; types, grade and stage of cervical carcinomas . HPV (16E6+18E6) revealed a significantly positive correlation with p16 (ink4a)  and a non- significant correlation with CK17 ; in LSIL, HSIL and invasive carcinoma cases.The study established a highly significant increase in IHC of expression of p16 (ink4a), CK17 and HPV (16E6+18E6) from LSIL through HSIL to invasive carcinomas (p16 (ink4a) expression directly reflects infection with high risk HPV in cervical lesions and can add a significant diagnostic accuracy in the evaluation of CIN. CK 17 is a good marker of malignant transformation, with increasing in its expression according to the severity of cervical lesions; however, it is not related to HPV infection. Both markers are not related to prognostic variables of patients with cervical carcinoma. Cervical intraepithelial neoplasia (CIN) is a premalignant (dysplastic) lesion, which is marked with abnormal cell maturation and nuclear atypia . MajoritHR-HPV genotypes are essential for cervical cancer progression and its late precursor lesion, CIN-III. HPV type 16 is the most carcinogenic genotype, detected in about 60% of cervical cancers, followed by HPV18 found in nearly 15% of cervical cancers , 5. P16 (ink4a) is a cyclin dependent kinase inhibitor which coordinates transition from G1 to S phase of the cell cycle, acts as a tumor suppressor protein.Thus, thThe objective of this work was to study the expression of p16 (ink4a) and CK17 in LSIL, HSIL and invasive cervical carcinomas and to assess their correlation to HPV (16E6+18E6) expression in those lesions.This study enrolled 127 formalin fixed paraffin embedded cervical biopsies; of which 22 cases were chronic cervicitis, 24 cases were LSIL (CIN-I), 28 cases were HSIL  and 53 cases were invasive cervical carcinomas retrieved from the archival materials of two teaching hospitals for the period from January 2012 to October 2014. This study was permitted by study Institutional Review Board of College of Medicine /Al-Nahrain University, which funded this study.Twenty cases of LSIL were punch biopsies and 4 cases were cone biopsies. From cases of HSIL, 20 cases were punch biopsies, 6 cases were cone biopsies and 2 cases were total abdominal hysterectomy (TAH); for invasive cervical carcinomas 45 cases were TAH, 6 cases were punch biopsies and 2 cases were cone biopsy.Clinicopathological data  pathological stage of cervical carcinomas) were collected from patients’ reports. From each paraffin block, 4 representative (4 micrometer) sections were obtained, one section was stained with Hematoxylin and Eosin (H&E) and revised by a pathologist and the remaining three were subjected to immunohistochemical testing for : anti-HPV (16 E6+18 E6) antibody, clone (C1P5), anti-P16 (ink4a) antibody, clone (2D9A12) and anti-CK 17 antibody, clone (E3); all manufactured by Abcam. Interpretation of the results of immunohistochemical staining HPV (16E6+18E6):Brown nuclear staining or combined nuclear and cytoplasmic staining is considered positive reaction. Cytoplasmic staining only was considered as negative , 12. PosP16 (ink4a):Brown nuclear and cytoplasmic staining is considered positive. Positive control is astrocytoma. Scoring was semi-quantitative based on the following: The intensity scales: 0=no staining; 1=weak focally positive; 2=strong, focally positive or weak, diffusely positive and 3= strong, diffusely positive. Percentage of positive nuclear staining scales: 1=1-25%, 2=26-50%, 3=51-75%, and 4=76-100%. Final score ranges from 0, 2-7 .CK17Brown cytoplasmic staining is considered positive. Positive control is squamous cell carcinoma. Technical negative control was obtained by omission of primary antibody. The IHC expression of CK 17 was assessed semi-quantitatively by taking the percentage of stained cells. Cases showing <5 % positive cells were regarded as negative, and those having > 5% positive cells were considered positive. Scores were counted as the following: mild (+) when 5–30 % positive cells, moderate (++) when 31–60 % positive cells and strong (+++) when more than 60 % positive cells .Technical negative controls for all markers were prepared by exclusion of primary antibody.Statistical AnalysisStatistics was accomplished with SPSS V. 17  and Excel 2007 programs. Continuous variables were calculated as mean ± SEM (standard error of the mean), while categorical variables were expressed as numbers and percentages. Statistical relations between two categorical variables were tested using Chi-square or Fisher exact tests. Relations between categorical and continuous variables were tested using unpaired t-test and ANOVA. The correlations between various markers were tested using Spearman rank correlation. The clinicopathological data of LSIL, HSIL and carcinoma cases are summarized in HPV (16E6+18E6) immunohistochemical expressionP˂0.001). HPV (16E6+18E6) was expressed in 7 out of 24 LSIL cases (29.2%) while in HSIL, 13 cases out of 28 (46.4%) were positive for HPV (16E6+18E6) immunostaining (P˂0.001) (HPV (16E6+18E6) IHC was negative in all of the cases of chronic cervicitis, however, it was significantly increased in expression with rising severity of the lesions from LSIL to HSIL; and the highest expression was noted in carcinomas (staining . Positivstaining  .The difP˂0.001) .P=0.558) and (P=0.518), respectively. There was also non-significant association of HPV (16E6+18E6) expression with histopathological types (P=1), grade (P=1) and FIGO pathological stage of invasive cervical carcinomas (P=0.442)  in studied squamous intraepithelial lesion (SIL) and carcinoma cases, (P=0.442) .p16 (ink4a)immunohistochemical expressionP˂0.001),  from LSIL (37.5%) through HSIL (67.9%) to carcinomas (94.3%), (˂0.001), . None ofP˂0.001) (With reference to the score of p16 (ink4a) IHC expression; high scores(6-7) were noticed in 2 out of 24 LSIL cases (8.3%), 7 out of 28 HSIL cases 25%)  and 25 o%  and 25P˂0.001) .P=0.378) and (P=0.247), respectively. p16 (ink4a) expressions were not statistically different according to histopathological types, grade and FIGO pathological stage of invasive cervical carcinomas, (P=1), (P=0.570), (P=0.107), respectively (Results exposed non-significant association of p16 (ink4a) expression with age of patients in both SILs and invasive carcinomas, , )  through ˂0.001), .P˂0.001)  expression. In HSIL cases, CK17 reported strong expression in 7 cases (25%)  while CKP˂0.001) .P=0.913) and (P=0.948), respectively. There was also non- significant difference in CK17 IHC expression among different histopathological types (P=0.691), different grades (P=0.442) and pathological FIGO stage (P=0.631) of cervical carcinoma  and p16 (ink4a), CK17 IHC expressions in SIL and carcinoma casesP˂0.001). Nevertheless, CK17 showed non-significant with of HPV (16E6+18E6), (P=0.067)  expression significantly correlated with the expression of p16 (ink4a), (P=0.067) .P˂0.001). The correlation between HPV (16E6+18E6) and CK17 IHC expression was non-significant (P=0.775)  and IHC expression of p16 (ink4a) (P=0.775) .In Iraq, during the period 1976-1985, the cervical cancer ranked the sixth among the commonest 10 cancers in females , whereasAlthough this study is not a large epidemiological one that expresses the prevalence and incidence of HPV (16+18) according to different clinicopathological parameters of cervical lesions; but in general HPV (16E6+18E6) was significantly increased in its expression with mounting severity of the lesions from LSIL (29.2%) through HSIL (46.4%) to carcinomas (67.9%).  Activity of HPV infection is determined through early gene products’ expression to be active or latent or leads to malignant transformation .The E6 pIn an Iraqi study completed in Baghdad using ISH technique , HPV 1616 was reIn an Iranian meta-analysis study (including 20 studies) , the preIn an Indian works , and SowIn a retrospective international survey including 38 countries during the period 1949-2009, the prevalence of HPV (16+18) in invasive cervical cancer detected by PCR technique in Europe, North America (USA), central South America, Africa, Asia and Australia were 73%, 79%, 68%, 71%, 71% and 79%, in that order  in the current study is lower than other countries like Brazil, India, Turkey, Europe, USA and Australia and this is possibly due to difference in sample size, use of more sensitive methods for detecting HPV (16+18) like ISH and PCR compared to IHC method which was used in this study and may be attributable to the unique religious and cultural restrictions preventing women to have multiple sexual partners in Iraq, thus reducing unsafe sexual relationships therefore lowering the threat of HPV infection.This study reported non-significant connection of age and HPV (16E6+18E6) expression in studied SIL and carcinoma cases. This outcome is regular with other authors , 31. TheThe early age of the first sexual practice is unquestionably related to HPV infections . The immThe current research showed IHC expression of HPV (16E6+18E6) in 66.7% of adenocarcinomas, 66.7% of adenosquamous carcinomas and in 68.4% of squamous carcinomas with no significant difference (P=1). Those findings are in accordance to another Iraqi article , 30.There was non-significant difference in IHC expression of HPV (16E6+18E6) between the grade and pathological stage of studied cervical carcinoma cases in the present study. This result is accordance with the results obtained from other studies -36.P16 (ink4a) was also significantly increased in its expression from LSIL 37.5%) through HSIL (67.9%) to carcinomas (94.3%). This result is in agreement with several other studies .5% throu. In a stp16 overexpression could reveal the potential for cervical malignant transformation as it is augmented with higher CIN grade . In cervConcerning the age, the current work revealed no significant association between age and IHC expression of p16 (ink4a) in both studied SILs and carcinoma cases. This data is in harmony with other authors .On the subject of cervical carcinoma cases, the present study recorded non- significant difference in IHC expression of p16 (ink4a) among different histopathological types, grade and pathological stage of studied cervical carcinoma cases. These results were also observed by other researches , 40.The current work established that p16 (ink4a) is highly correlated with HPV (16E6+18E6) in SILs and cervical carcinoma cases, respectively. This result is akin to that obtained by other studies -49.P16 (ink4a) is a cell cycle inhibitor that binds to cdk4 and cdk6 that inhibits pRb phosphorylation and inactivation .The downThrough a negative feedback mechanism, E6 and E7 proteins can cause induce production of p16 (ink4a), as they bind to pRb and inactivate it . HPV-E7 CK 17 is present in cervical reserve cells (from which CIN is originated). Its expression in term of percentage and intensity is increase with increasing severity of CIN. In cervical carcinoma, keratin 17 was always detected, so the development of carcinoma from CIN-III is restricted to CK17 positive CIN-III , 10.This work set up positive expression of CK 17 in all cases of SIL and carcinomas, with significant augment in its expression with increasing severity of the lesions from LSIL (41.7%) through HSIL (71.4%) to cervical carcinomas (90.6%). This result is nearly analogous to that obtained earlier ,who founTaking age into consideration, this study reported non- significant association between age and IHC expression of CK17 in SILs and carcinomas, that is in tune with Ikeda et al., 2008 .The current paper showed non- significant difference in IHC expression of CK17 among different histopathological types, grade and pathological stage of cervical carcinoma that is in unity with other authors;  , and Ma.CK 17 was not correlated to IHC expression of HPV (16E6+18E6) in the studied SILs and carcinoma cases, that is in concord with other studies , 59. Misp16 (ink4a) expression directly reflects infection with high risk HPV in cervical lesions and can add a significant diagnostic accuracy in the evaluation of CIN. CK 17 is a good marker of malignant transformation, with increasing in its expression according to the severity of cervical lesions; however, it is not related to HPV infection. Both markers are not related to prognostic variables of patients with cervical carcinoma.


Human papillomavirus (HPV) infections and associated diseases remain a serious burden worldwide. It is now clear that HPV serves as the etiological factor and biologic carcinogen for HPV-associated lesions and cancers. Although preventative HPV vaccines are available, these vaccines do not induce strong therapeutic effects against established HPV infections and lesions. These concerns create a critical need for the development of therapeutic strategies, such as vaccines, to treat these existing infections and diseases.Unlike preventative vaccines, therapeutic vaccines aim to generate cell-mediated immunity. HPV oncoproteins E6 and E7 are responsible for the malignant progression of HPV-associated diseases and are consistently expressed in HPV-associated diseases and cancer lesions; therefore, they serve as ideal targets for the development of therapeutic HPV vaccines. In this review we revisit therapeutic HPV vaccines that utilize this knowledge to treat HPV-associated lesions and cancers, with a focus on the findings of recent therapeutic HPV vaccine clinical trials.Great progress has been made to develop and improve novel therapeutic HPV vaccines to treat existing HPV infections and diseases; however, there is still much work to be done. We believe that therapeutic HPV vaccines have the potential to become a widely available and successful therapy to treat HPV and HPV-associated diseases in the near future. Cervical cancer is the fourth most common cancer effecting women worldwide . Human pIdentifying HPV as the etiological factor for HPV-associated malignancies has created the opportunity to control those cancers through vaccination and other therapeutic strategies . VaccineMost sexually active women will be infected by HPV at some point in their life. For many women these infections remain asymptomatic and are cleared by the immune system. However, some women can develop persistent HPV infections, which may further develop into low or high-grade cervical intraepithelial neoplasia (CIN) and cervical carcinoma, or regress at any stage , 14. In Although prophylactic HPV vaccines have been a huge success and leap forward in the prevention of HPV infections and HPV-associated diseases, there is still a great HPV-associated disease burden worldwide. As such, there is an urgent need to develop treatments for the control and eradication of existing HPV infections and associated diseases. Our review will cover various therapeutic HPV vaccines in development for the treatment of HPV infections and HPV-associated diseases, including HPV-associated cancers. In addition, we will focus on the findings of latest clinical trials on therapeutic HPV vaccines.Several types of therapeutic vaccines have been developed and tested in preclinical and clinical trials, including live vector, protein or peptide, nucleic acid, and cell-based vaccines (for review see ). ImportThe following section discusses the characteristics of various therapeutic HPV vaccines being developed and tested. The section summarizes numerous recent clinical trials that have been implemented using various types of therapeutic HPV vaccines against HPV-associated lesions and malignancies. Table Live vector-based vaccines are often categorized as either bacterial or viral vectors depending on their vector platform. These vectors replicate within the body and facilitate the spread of the antigen , 16. LivListeria monocytogenes, Lactobacillus lactis, Lactobacillus plantarum, and Lactobacillus casei [Several bacterial vectors have been selected for the development of therapeutic HPV vaccines including us casei –26. Listus casei . Becauseus casei , 29.The first clinical use of a listeria-based therapeutic HPV vaccine was reported in 2009 . The vacL. casei that expresses a modified HPV16-E7 antigen, which is no longer carcinogenic [A recent study tested the efficacy and safety of oral administration of GLBL101c, a bacterial vector-based therapeutic HPV vaccine. The Phase I/IIa study involved 17 patients with HPV16+ CIN3 lesions . GLBL101inogenic . The bacSalmonella, Shigella, and Escherichia coli, can deliver plasmid encoding genes of interest to APCs. Previous studies have tested the use of Salmonella for the delivery of HPV16 E7 protein or E7 epitopes to elicit E7-specific responses [Other attenuated bacterial vectors can also be created through transformation with plasmids containing the genes of interest. For example, esponses .Several viral vectors have been examined to deliver HPV E6 and E7 antigens including adenoviruses, adeno-associated viruses, alphaviruses, lentivirus, and vaccinia viruses , 34–45.Vaccinia virus is an enveloped, double-stranded DNA virus belonging to the Poxvirus family. Vaccinia virus has a large genome, highly infectious nature, and low likelihood of unregulated integration of foreign DNA into its genome, making it a promising viral vector for vaccine delivery .Several Modified Vaccinia Ankara (MVA)-based therapeutic HPV vaccines have been created and tested in recent years. TG4001 is a suspension of MVATG8042 particles consisting of attenuated recombinant MVA including sequences encoding modified HPV16 E6/E7 and human IL-2. The safety and efficacy of this vaccine was evaluated in 21 patients with HPV16-related CIN2/3 lesions . Each paAnother MVA-based vaccine, MVA E2 was created to deliver E2 proteins into vaccinated hosts rather than E6 and E7 . The vacTA-HPV is a live recombinant vaccinia virus vaccine that encodes oncoproteins E6 and E7 of both HPV type 16 and HPV18. TA-HPV was first used in clinical trial in eight patients with advanced stage cervical cancer . In threAs mentioned earlier, one challenge to using live vector-based therapeutic HPV vaccines is the generation of antibacterial or antiviral immune responses and neutralizing antibodies after initial vaccine exposure. Consequently, this limits the efficacy of multiple vaccine administrations. This issue was partially addressed by a former study, which showed that cyclooxygenase 2 (COX-2) inhibitors may prevent the production of neutralizing antibodies to vaccinia virus .Peptide and protein that are derived from HPV antigens are processed by DCs and presented on either MHC class I or class II molecules to stimulate CD8+ or CD4+ T cell immune response , 57. FurAlthough peptide-based vaccines are safe, stable, and easy to produce they have poor immunogenicity and require lipids or other adjuvants, such as chemokines, cytokines, and Toll-like receptor (TLR) ligands, to enhance vaccine potency . These mHPV16 synthetic long-peptide vaccine (HPV16-SLP) and its therapeutic effects have been studied extensively in several clinical trials –61. HPV1n = 11) or third (n = 1) chemotherapy cycle. During CarboTaxol treatment lymphocyte count did not change; however, the number of circulating leukocytes increased significantly. Myeloid and lymphoid cell counts in CarboTaxol patients reached levels close to normalized levels of healthy donors. In addition, HPV16-SLP vaccination enhanced T cell response in patients, which remained the same in 11 patients after six cycles of chemotherapy. Significant regressions of tumors in patients were not observed, and 1 patient died 11 weeks after vaccination due to disease progression. However, vaccination was well tolerated and most adverse events observed were localized, injection site reactions. Additional clinical studies are being conducted to continue to evaluate the therapeutic potential of HPV-16 SLP vaccine. These includes a phase I/II trial in HIV+ male patient with HPV-16+ AIN2/3 (NCT01923116), a phase II trial in patients with HPV-16+ incurable solid tumors in combination with Nivolumab (NCT02426892), and a phase I/II trial in female patients with HPV-16+ advanced or recurrent cervical cancer (NCT02128126).Another study investigated whether HPV16-SLP vaccination combined with standard carboplatin and paclitaxel (CarboTaxol) chemotherapy could improve immunity in patients with cervical cancer . In addiPepCan is a therapeutic HPV vaccine consisting of four synthetic peptides covering HPV16 E6 and Candin, a novel adjuvant. The safety of PepCan was tested in a phase I clinical trial study in 31 patients with high grade squamous intraepithelial lesions (HSIL) . PepCan Additionally, a phase I dose escalation trial using therapeutic HPV-peptide based vaccine with adjuvant Montanide and GMCSF (GL-0810) has been tested in five patients with recurrent/metastatic (RM) squamous cell carcinoma of the head and neck (SCCHN) . GL-0810In addition to the vaccine candidates described above, a Phase I clinical trial has also been planned to evaluate the safety and therapeutic effect of PDS0101, a new therapeutic HPV vaccine candidate consist of Peptides from HPV-16 E6 and E7 as antigen and R-enantiomer of 1,2-dioleoyl-3-trimethylammonium-propane chloride as adjuvant, in female patients with high risk HPV infection or CIN1 (NCT02065973). Furthermore, a phase Ib/II trial has been planned to evaluate the therapeutic HPV vaccine DPX-E7, a HPV16-E7 aa11-19 nanomer peptide vaccine, in HLA-A*02 positive patients with incurable HPV16-related oropharyngeal, cervical, and anal cancer (NCT02865135).One benefit to using protein-based vaccines is that they contain all human leukocyte antigen (HLA) epitopes. This avoids the limitation of MHC restriction, which is a setback to using peptide-based vaccines . ProteinTherapeutic HVP vaccine TA-CIN is a subunit vaccine encompassing HPV16 E6E7L2 fusion protein . TA-CIN Bordetella pertussis CyaA expressed in E. coli. A phase I trial was conducted to examine the safety, tolerability, and immunogenicity of GTL001 in 47 women who had normal cytology but were positive for HPV16 or HPV18 infection. The participants were divided into four cohorts with two placebo sub-cohorts included within cohort three. Each patient was administered either 100ug or 600ug of GTL001 with imiquimod. Patients treated with GTL001 experienced injection site reactions including pain, swelling, induration, tenderness, and itching; however, the study demonstrated that vaccination with GTL001 was relatively safe. GTL001 induced a humoral response to CyaA in all subjects but anti-E7 antibodies were not induced. All patients showed similar immunogenicity and tolerability. Patients in cohort 4 (n = 9) who received 600ug GTL001 powder + imiquimod experienced the highest HPV16/18 clearance rate. However, in the recently press release by GENTICEL regarding their interim (18 months) results for the double-blinded, placebo controlled, phase II studies involving the use of GTL001 in 233 patients with HPV16/18+ normal or abnormal cytology  (NCT01957878), no significant difference in viral clearance was observed in HPV-16/18 positive patients receiving GTL001 vaccine versus placebo.GTL001 (ProCervix) is a therapeutic HPV protein-based vaccine that targets both HPV type 16 and 18 . GTL001 Additional clinical trials have been planned to test the potential of other therapeutic HPV protein vaccine candidates. One of such include the use of TVGV-1, a fusion protein of HPV-16 E7 antigen with ER targeting sequence, in a phase IIa trial with HPV induced cervical HSIL patients (NCT02576561).DNA vaccines have risen in popularity as an attractive and potentially effective approach for antigen-specific immunotherapy. DNA vaccines are safe, stable, easy to produce and can sustain antigen expression in cells for longer durations than RNA vaccines or protein vaccines. Furthermore, they do not produce neutralizing antibodies, which permits repeated vaccination . There iDNA vaccines are often administered via intramuscular (IM) injection; however, myocytes are usually the cells that uptake the DNA after IM injection (for review see ). Althouin vivo resulting in low immunogenicity. Consequently, several strategies have been developed to help overcome this hurdle (for review see [One limitation of DNA vaccines is that naked DNA is unable to amplify and spread from transfected cells to surrounding cells view see ).Therapeutic HPV DNA vaccines have undergone many clinical trials to evaluate the efficacy and safety of these vaccines. One such trial treated patients with HPV16-associated CIN2/3 lesions with heterologous prime-boost vaccination . The DNAA more recent clinical study was conducted to evaluate the safety, efficacy, and immunogenicity of pNGVL4a-CRT/E7(detox), a DNA plasmid vaccine . pNGVL4aA phase I clinical trial was conducted to evaluate the safety and efficacy of GX-188E, a therapeutic HPV DNA vaccine. The study was conducted in nine patients with high grade squamous intraepithelial lesions (HSIL/CIN3) . GX-188EAn additional clinical study tested the therapeutic effects of HPV DNA vaccine VGX-3100. VGX-3100 is a combination of two plasmids encoding optimized HPV16 and 18 E6 and E7 antigens , 84. VGXNaked RNA replicon vaccines can be derived from several RNA viruses including Sindbis virus, Venezuela Equine Encephalitis virus, and SFV , 87, 88.ex vivo have improved. DC-based HPV vaccines involve loading the DCs with HPV antigens ex vivo and delivering those DCs to the infected host [DCs play an important role in immune system regulation, and they are commonly identified as the most efficient professional APCs . DC-baseted host –98. One ted host ). Becausted host , 97, 100Due to the therapeutic potential demonstrated by DC-based vaccines in preclinical models, further clinical trials were developed to test DC-based vaccine efficacy in humans. One such study was conducted to evaluate safety, toxicity, and immunogenicity of a DC-based vaccine in ten patients with stage Ib or IIa cervical cancer . In thisAn additional phase I clinical trial investigated the toxicity and immunogenicity of a DC-based vaccine in 14 patients with HPV+ advanced, recurrent cervical cancer . PatientIn congruence with other vaccine types, DC-based vaccines have several limitations. DC-based vaccines are technically taxing, thus making them a poor choice for large-scale production. In addition, varying culturing techniques may lead to inconsistent vaccine quality and lack of standard criteria for vaccine evaluation. Lastly, the most effective administration route for DC-based vaccines has not yet been determined.ex vivo to express immune modulatory proteins, which can further enhance their immunogenicity in vivo. Cytokine genes IL-2, IL-12, and granulocyte macrophage colony stimulating factor (GMCSF) have been used to induce differentiation of naïve T cells into effector or helper T cells and to stimulate granulocyte production in HPV tumor cell-based vaccines in mice with HPV16 induced tumors [To create tumor cell-based vaccines, tumor cells are isolated and manipulated d tumors , 104. OnThe identification of high-risk HPV as the etiological factor for many diseases provides justification for the development of therapeutic HPV vaccines. The recent developments in the field as well as those discussed in this review have helped contribute to the foundational movement to eradicate HPV and HPV-associated diseases and malignancies. In this review we discussed the various methods of targeting HPV oncoproteins E6 and E7, which represent tumor-specific antigens and excellent targets for therapeutic HPV vaccines. Based on our own previous studies, and those conducted by other investigators in the field, we believe that the current therapeutic HPV vaccines mentioned in this review each possess advantages and limitations. Additional clinical studies are still necessary to further verify the antitumor efficacy of therapeutic HPV vaccines.With continued efforts to improve and develop therapeutic treatment strategies, we anticipate the continued success of therapeutic HPV vaccines over the next few years, and beyond. We believe that therapeutic HPV vaccines will become clinically available in the near future and be offered alongside other available therapies for the control of HPV-associated diseases.