Virus-like particle-based vaccines for high-risk human papillomaviruses (HPVs) appear to have great promise; however, cell culture-derived vaccines will probably be very expensive. The optimization of expression of different codon-optimized versions of the HPV-16 L1 capsid protein gene in plants has been explored by means of transient expression from a novel suite of Agrobacterium tumefaciens binary expression vectors, which allow targeting of recombinant protein to the cytoplasm, endoplasmic reticulum (ER) or chloroplasts. A gene resynthesized to reflect human codon usage expresses better than the native gene, which expresses better than a plant-optimized gene. Moreover, chloroplast localization allows significantly higher levels of accumulation of L1 protein than does cytoplasmic localization, whilst ER retention was least successful. High levels of L1 (.17 % total soluble protein) could be produced via transient expression: the protein assembled into higher-order structures visible by electron microscopy, and a concentrated extract was highly immunogenic in mice after subcutaneous injection and elicited high-titre neutralizing antibodies. Transgenic tobacco plants expressing a human codon-optimized gene linked to a chloroplast-targeting signal expressed L1 at levels up to 11 % of the total soluble protein. These are the highest levels of HPV L1 expression reported for plants: these results, and the excellent immunogenicity of the product, significantly improve the prospects of making a conventional HPV vaccine by this means.

BackgroundHIV-infected women are at increased risk for developing cervical cancer. Women living in resource-limited countries are especially at risk due to poor access to cervical cancer screening and treatment. We evaluated three cervical cancer screening methods to detect cervical intraepithelial neoplasia grade 2 and above (CIN 2+) in HIV-infected women in South Africa; Pap smear, visual inspection with 5% acetic acid (VIA) and human papillomavirus detection (HPV).MethodsHIV-infected women aged 18–65 were recruited in Johannesburg. A cross-sectional study evaluating three screening methods for the detection of the histologically-defined gold standard CIN-2 + was performed. Women were screened for cervical abnormalities with the Digene HC2 assay (HPV), Pap smear and VIA. VIA was performed by clinic nurses, digital photographs taken and then later reviewed by specialist physicians. The sensitivity, specificity and predictive valves for CIN-2 + were calculated using maximum likelihood estimators.Results1,202 HIV-infected women participated, with a median age of 38 years and CD4 counts of 394 cells/mm3. One third of women had a high grade lesion on cytology. VIA and HPV were positive in 45% and 61% of women respectively. Estimated sensitivity/specificity for HPV, Pap smear and VIA for CIN 2+ was 92%/51.4%, 75.8%/83.4% and 65.4/68.5% (nurse reading), respectively. Sensitivities were similar, and specificities appeared significantly lower for the HPV test, cytology and VIA among women with CD4 counts ≤200 cells/mm3 as compared to CD4 counts >350 cells/mm3.ConclusionsAlthough HPV was the most sensitive screening method for detecting CIN 2+, it was less specific than conventional cytology and VIA with digital imaging review. Screening programs may need to be individualized in context of the resources and capacity in each area.

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Among the more than one hundred formally described human papillomavirus (HPV) types, 18 are referred to as high-risk HPV types due to their association with anogenital cancer. Despite pathogenic similarities, these types form three remotely related taxonomic groups. One of these groups is called HPV species 9 and is formed by HPV-16, the most common and best-studied type, together with HPV-31, -33, -35, -52, -58, and -67. Previous worldwide comparisons of HPV-16 samples showed about 2% nucleotide diversity between isolates, which were subsequently termed variants. The distribution of divergent variants has been found to correlate frequently with the geographic origin and the ethnicity of the infected patients and led to the concept of unique African, European, Asian, and Native American HPV-16 variants. In the current study, we address the question of whether geography and ethnicity also correlate with sequence variations found for HPV-31, -35, -52, and -58. This was done by sequencing the long control region in samples derived from Europe, Asia, and Africa, and from immigrant populations in North and South America. We observed maximal divergence between any two variants within each of these four HPV types ranging from 1.8 to 3.6% based on nucleotide exchanges and, occasionally, on insertions and deletions. Similar to the case with HPV-16, these mutations are not random but indicate a relationship between the variants in form of phylogenetic trees. An interesting example is presented by a 16-bp insert in select variants of HPV-35, which appears to have given rise to additional variants by nucleotide exchanges within the insert. All trees showed distinct phylogenetic topologies, ranging from dichotomic branching in the case of HPV-31 to star phylogenies of the other three types. No clear similarities between these types or between these types and HPV-16 exist. While variant branches in some types were specific for Europe, Africa, or East Asia, none of the four trees reflected human evolution and spread to the extent illustrated by HPV-16. One possible explanation is that the rare HPV types that we studied spread and thereby diversified more slowly than the more abundant HPV-16 and may have established much of today's variant diversity already before the worldwide spread of humans 100,000 years ago. Most variants had prototypic amino acid sequences within the E6 oncoprotein and a segment of the L1 capsid protein. Some had one, two, or three amino acid substitutions in these regions, which might indicate biological and pathogenic diversity between the variants of each HPV type.The phylogeny-based taxonomy of papillomaviruses (PVs) places these viruses into a separate family which is further divided into genera and species (11). On lower taxonomic levels, PVs are classified as types, subtypes, and variants. Basic and clinical research normally addresses PVs on these lower three levels of taxonomy. More than one hundred different human papillomavirus (HPV) types have been formally described (1, 9, 11). Eighteen HPV t...

Background: Cervical cancer and infection with human immunodeficiency virus (HIV) are both major public health problems in South Africa. The aim of this study was to determine the risk of cervical pre-cancer and cancer among HIV positive women in South Africa.

Human papillomavirus-like particles (HPV VLPs) have shown considerable promise as a parenteral vaccine for the prevention of cervical cancer and its precursor lesions. Parenteral vaccines are expensive to produce and deliver, however, and therefore are not optimal for use in resource-poor settings, where most cervical HPV disease occurs. Transgenic plants expressing recombinant vaccine immunogens offer an attractive and potentially inexpensive alternative to vaccination by injection. For example, edible plants can be grown locally and can be distributed easily without special training or equipment. To assess the feasibility of an HPV VLP-based edible vaccine, in this study we synthesized a plant codon-optimized version of the HPV type 11 (HPV11) L1 major capsid protein coding sequence and introduced it into tobacco and potato. We show that full-length L1 protein is expressed and localized in plant cell nuclei and that expression of L1 in plants is enhanced by removal of the carboxy-terminal nuclear localization signal sequence. We also show that plant-expressed L1 self-assembles into VLPs with immunological properties comparable to those of native HPV virions. Importantly, ingestion of transgenic L1 potato was associated with activation of an anti-VLP immune response in mice that was qualitatively similar to that induced by VLP parenteral administration, and this response was enhanced significantly by subsequent oral boosting with purified insect cell-derived VLPs. Thus, papillomavirus L1 protein can be expressed in transgenic plants to form immunologically functional VLPs, and ingestion of such material can activate potentially protective humoral immune responses.Infection with specific human papillomavirus (HPV) genotypes is a necessary factor in the development of cervical cancer and its precursor lesions (7, 43). Multiple sexually transmitted "high-risk" genotypes (e.g., types 16,18,31,33,35, 45, 51, 52, 58, and 59) have been associated with malignant disease, while other sexually transmitted types (notably 6 and 11) primarily cause only benign disease (e.g., anogenital warts) (20). Recombinant virus-like particles (VLPs) are promising immunogens for controlling HPV-associated diseases because they induce high-titer neutralizing antibody responses that have been associated with protection from infectious challenge (27, 36). Results from phase I studies in human subjects indicate that VLPs are safe, well-tolerated, and immunogenic when administered parenterally (12,16). Encouraging data from a recent phase II study suggest that VLPs may be highly efficacious for HPV prophylaxis (18).Vaccination for disease prevention is a major success of modern medicine; however, the full potential of this strategy to reduce human suffering has not yet been realized. For example, several so-called "vaccine-preventable" diseases continue to affect the lives of millions of individuals who live mostly in economically disadvantaged regions. With few exceptions, recommended immunizations rely on parenteral administration, and in...

ObjectiveTo examine the association between CD4 counts, HPV infection and the risk of cervical neoplasia among HIV-seropositive women.MethodsA cross-sectional observational study was conducted among 1,010 HIV-seropositive women using cytology-based Pap smears. HPV DNA testing using Linear Array genotyping assay (Roche) was carried out in a subset of 191 patients. Multivariable-adjusted prevalence ratios (mPR) and 95% confidence intervals (CIs) were estimated with log-binomial regression.ResultsAmong 1,010 HIV-seropositive women, the prevalence of AGC/ASCUS, LSIL and HSIL or greater was 8.3, 23.5 and 18.0%, respectively. The risk of cervical lesions was higher with CD4 < 200 cells/mm3 vs. CD4 levels > 500/mm3. HPV types 16 (41.7%) and HPV 56 (22.2%) were the most common types in HSIL cases. Women with CD4 levels < 200/mm3 had a higher prevalence of HPV types 16 (p < 0.01) and 66 (p = 0.04). No statistical relationship between cervical lesions and HAART use was found.ConclusionThe burden of HPV infection and HSIL was high and correlated with HIV-induced immunosuppression. HPV 16 was the most common type in HSIL and increased in prevalence with greater immune suppression. Prophylactic HPV 16 vaccination could prevent approximately 40% of HSIL cases. Strengthening screening programs is imperative in this population.

SummaryCervical cytobrush sampling is a relatively non-invasive method for obtaining mucosal cells from the female genital tract. To define mucosal immune cells sampled by cervical cytobrushing and to validate this approach for local immunity studies, we investigated the impact of human immunodeficiency virus (HIV) status and inflammation on the yield and composition of cervical cytobrush specimens. Cervical cytobrush samples were obtained from 89 chronically HIV-infected and 46 HIV-negative women. The HIV-infected women had significantly higher yields of CD3 + , CD45 + , CD19 + , CD14 + , Langerin + and CD24 + cells than the uninfected women. While cytobrush-derived T cells from uninfected women were predominantly CD4 + (4Á2 CD4 : 1 CD8), CD8 + T cells were predominant in HIV-infected women (0Á6 CD4 : 1 CD8). The majority of CD4 + and CD8 + T cells from HIV-infected and uninfected women were of the effector memory (CD45RA ) CCR7 ) CD27 ) ) phenotype. HIV-infected women had significantly elevated levels of interleukin (IL)-1b, IL-6 and IL-8 in cervical supernatants compared with uninfected women. We observed a significant positive correlation between T-cell counts and IL-1b, tumour necrosis factor (TNF)-a and IL-12 concentrations. Neutrophil counts correlated significantly with cervical concentrations of IL-1b, TNF-a, IL-8, IL-6 and IL-10. Antigen-presenting cell numbers correlated significantly with TNF-a and IL-12 concentrations. HIV-infected women on antiretroviral therapy had similar levels of cervical lymphocyte infiltration and inflammation to women naïve to therapy. In conclusion, we suggest that inflammation at the cervix and HIV infection are likely to be key determinants in the absolute number of mucosal immune cells recovered by cervical cytobrushing.