Global Genomic Diversity of Human Papillomavirus 6 Based on 724 Isolates and 190 Complete Genome Sequences.

Human papillomavirus type 6 (HPV6) is the major etiological agent of anogenital warts and laryngeal papillomas and has been included in both the quadrivalent and nonavalent prophylactic HPV vaccines. This study investigated the global genomic diversity of HPV6, using 724 isolates and 190 complete genomes from six continents, and the association of HPV6 genomic variants with geographical location, anatomical site of infection/disease, and gender. Initially, a 2,800-bp E5a-E5b-L1-LCR fragment was sequenced from 492/530 (92.8%) HPV6-positive samples collected for this study. Among them, 130 exhibited at least one single nucleotide polymorphism (SNP), indel, or amino acid change in the E5a-E5b-L1-LCR fragment and were sequenced in full. A global alignment and maximum likelihood tree of 190 complete HPV6 genomes (130 fully sequenced in this study and 60 obtained from sequence repositories) revealed two variant lineages, A and B, and five B sublineages: B1, B2, B3, B4, and B5. HPV6 (sub)lineage-specific SNPs and a 960-bp representative region for whole-genome-based phylogenetic clustering within the L2 open reading frame were identified. Multivariate logistic regression analysis revealed that lineage B predominated globally. Sublineage B3 was more common in Africa and North and South America, and lineage A was more common in Asia. Sublineages B1 and B3 were associated with anogenital infections, indicating a potential lesion-specific predilection of some HPV6 sublineages. Females had higher odds for infection with sublineage B3 than males. In conclusion, a global HPV6 phylogenetic analysis revealed the existence of two variant lineages and five sublineages, showing some degree of ethnogeographic, gender, and/or disease predilection in their distribution. IMPORTANCE: This study established the largest database of globally circulating HPV6 genomic variants and contributed a total of 130 new, complete HPV6 genome sequences to available sequence repositories. Two HPV6 variant lineages and five sublineages were identified and showed some degree of association with geographical location, anatomical site of infection/disease, and/or gender. We additionally identified several HPV6 lineage- and sublineage-specific SNPs to facilitate the identification of HPV6 variants and determined a representative region within the L2 gene that is suitable for HPV6 whole-genome-based phylogenetic analysis. This study complements and significantly expands the current knowledge of HPV6 genetic diversity and forms a comprehensive basis for future epidemiological, evolutionary, functional, pathogenicity, vaccination, and molecular assay development studies.

A novel mucosal orthotopic murine model of human papillomavirus-associated genital cancers.

Cervical cancer results from infection with high-risk type human papillomaviruses (HPV). Therapeutic vaccines aiming at controlling existing genital HPV infections and associated lesions are usually tested in mice with HPV-expressing tumor cells subcutaneously implanted into their flank. However, effective vaccine-induced regression of these ectopic tumors strongly contrasts with the poor clinical results of these vaccines produced in patients with HPV-associated genital neoplasia. To assess HPV therapeutic vaccines in a more relevant setting, we have, here, established an orthotopic mouse model where tumors in the genital mucosa (GM) develop after an intravaginal instillation of HPV16 E6/E7-expressing tumor cells transduced with a luciferase-encoding lentiviral vector for in vivo imaging of tumor growth. Tumor take was 80-90% after nonoxynol-9 induced damage of the epithelium. Tumors remained localized in the genital tract, and histological analysis showed that most tumors grew within the squamous epithelium of the vaginal wall. Those tumors induced (i) E7-specific CD8 T cells restricted to the GM and draining lymph nodes, in agreement with their mucosal location and (ii) high Foxp3+ CD4+ infiltrates, similarly to those found in natural non-regressing HPV lesions. This novel genital HPV-tumor model by requiring GM homing of vaccine-induced immune responses able to overcome local immuno-suppression may be more representative of the situation occurring in patients upon therapeutic vaccination.

Validation of a Low-Cost Human Papillomavirus Genotyping Assay Based on PGMY PCR and Reverse Blotting Hybridization with Reusable Membranes.

The genotyping of human papillomaviruses (HPV) is essential for the surveillance of HPV vaccines. We describe and validate a low-cost PGMY-based PCR assay (PGMY-CHUV) for the genotyping of 31 HPV by reverse blotting hybridization (RBH). Genotype-specific detection limits were 50 to 500 genome equivalents per reaction. RBH was 100% specific and 98.61% sensitive using DNA sequencing as the gold standard (n = 1,024 samples). PGMY-CHUV was compared to the validated and commercially available linear array (Roche) on 200 samples. Both assays identified the same positive (n = 182) and negative samples (n = 18). Seventy-six percent of the positives were fully concordant after restricting the comparison to the 28 genotypes shared by both assays. At the genotypic level, agreement was 83% (285/344 genotype-sample combinations; κ of 0.987 for single infections and 0.853 for multiple infections). Fifty-seven of the 59 discordant cases were associated with multiple infections and with the weakest genotypes within each sample (P < 0.0001). PGMY-CHUV was significantly more sensitive for HPV56 (P = 0.0026) and could unambiguously identify HPV52 in mixed infections. PGMY-CHUV was reproducible on repeat testing (n = 275 samples; 392 genotype-sample combinations; κ of 0.933) involving different reagents lots and different technicians. Discordant results (n = 47) were significantly associated with the weakest genotypes in samples with multiple infections (P < 0.0001). Successful participation in proficiency testing also supported the robustness of this assay. The PGMY-CHUV reagent costs were estimated at $2.40 per sample using the least expensive yet proficient genotyping algorithm that also included quality control. This assay may be used in low-resource laboratories that have sufficient manpower and PCR expertise.

International collaborative proficiency study of Human Papillomavirus type 16 serology.

We performed an international proficiency study of Human Papillomavirus (HPV) type 16 serology. A common methodology for serology based on virus-like particle (VLP) ELISA was used by 10 laboratories in 6 continents. The laboratories used the same VLP reference reagent, which was selected as the most stable, sensitive and specific VLP preparation out of VLPs donated from 5 different sources. A blinded proficiency panel consisting of 52 serum samples from women with PCR-verified HPV 16-infection, 11 control serum samples from virginal women and the WHO HPV 16 International Standard (IS) serum were distributed. The mean plus 3 standard deviations of the negative control serum samples was the most generally useful "cut-off" criterion for distinguishing positive and negative samples. Using sensitivity of at least 50% and a specificity of 100% as proficiency criteria, 6/10 laboratories were proficient. In conclusion, an international Standard Operating Procedure for HPV serology, an international reporting system in International Units (IU) and a common "cut-off" criterion have been evaluated in an international HPV serology proficiency study.

Afatinib versus placebo as adjuvant therapy after chemoradiation in a double-blind, phase III study (LUX-Head & Neck 2) in patients with primary unresected, clinically intermediate-to-high-risk head and neck cancer: study protocol for a randomized controlled trial.

BACKGROUND: Over 50% of patients with head and neck squamous cell carcinoma (HNSCC) present with locoregionally advanced disease. Those at intermediate-to-high risk of recurrence after definitive therapy exhibit advanced disease based on tumour size or lymph node involvement, non-oropharynx primary sites, human papillomavirus (HPV)-negative oropharyngeal cancer, or HPV-positive oropharynx cancer with smoking history (>10-pack-years). Non-surgical approaches include concurrent chemoradiotherapy, induction chemotherapy followed by definitive radiotherapy or chemoradiotherapy, or radiotherapy alone. Following locoregional therapies (including surgical salvage of residual cervical nodes), no standard intervention exists. Overexpression of epidermal growth factor receptor (EGFR), an ErbB family member, is associated with poor prognosis in HNSCC. EGFR-targeted cetuximab is the only targeted therapy that impacts overall survival and is approved for HNSCC in the USA or Europe. However, resistance often occurs, and new approaches, such as targeting multiple ErbB family members, may be required. Afatinib, an irreversible ErbB family blocker, demonstrated antiproliferative activity in preclinical models and comparable clinical efficacy with cetuximab in a randomized phase II trial in recurrent or metastatic HNSCC. LUX-Head & Neck 2, a phase III study, will assess adjuvant afatinib versus placebo following chemoradiotherapy in primary unresected locoregionally advanced intermediate-to-high-risk HNSCC. METHODS/DESIGN: Patients with primary unresected locoregionally advanced HNSCC, in good clinical condition with unfavourable risk of recurrence, and no evidence of disease after chemoradiotherapy will be randomized 2:1 to oral once-daily afatinib (40 mg starting dose) or placebo. As HPV status will not be determined for eligibility, unfavourable risk is defined as non-oropharynx primary site or oropharynx cancer in patients with a smoking history (>10 pack-years). Treatment will continue for 18 months or until recurrence or unacceptable adverse events occur. The primary endpoint measure is duration of disease-free survival; secondary endpoint measures are disease-free survival rate at 2 years, overall survival, health-related quality of life and safety. DISCUSSION: Given the unmet need in the adjuvant treatment of intermediate-to-high-risk HNSCC patients, it is expected that LUX-Head & Neck 2 will provide new insights into treatment in this setting and might demonstrate the ability of afatinib to significantly improve disease-free survival, compared with placebo. TRIAL REGISTRATION: ClinicalTrials.gov NCT01345669.

Human papilloma virus type and recurrence rate after surgical clearance of anal condylomata acuminata.

BACKGROUND: Anal condylomata acuminata (ACA) are caused by human papilloma virus (HPV) infection which is transmitted by close physical and sexual contact. The result of surgical treatment of ACA has an overall success rate of 71% to 93%, with a recurrence rate between 4% and 29%. The aim of this study was to assess a possible association between HPV type and ACA recurrence after surgical treatment. METHODS: We performed a retrospective analysis of 140 consecutive patients who underwent surgery for ACA from January 1990 to December 2005 at our tertiary University Hospital. We confirmed ACA by histopathological analysis and determined the HPV typing using the polymerase chain reaction. Patients gave consent for HPV testing and completed a questionnaire. We looked at the association of ACA, HPV typing, and HIV disease. We used chi, the Monte Carlo simulation, and Wilcoxon tests for statistical analysis. RESULTS: Among the 140 patients (123 M/17 F), HPV 6 and 11 were the most frequently encountered viruses (51% and 28%, respectively). Recurrence occurred in 35 (25%) patients. HPV 11 was present in 19 (41%) of these recurrences, which is statistically significant, when compared with other HPVs. There was no significant difference between recurrence rates in the 33 (24%) HIV-positive and the HIV-negative patients. CONCLUSIONS: HPV 11 is associated with higher recurrence rate of ACA. This makes routine clinical HPV typing questionable. Follow-up is required to identify recurrence and to treat it early, especially if HPV 11 has been identified.

Aerosol immunization with NYVAC and MVA vectored vaccines is safe, simple, and immunogenic.

Each year, approximately five million people die worldwide from putatively vaccine-preventable mucosally transmitted diseases. With respect to mass vaccination campaigns, one strategy to cope with this formidable challenge is aerosol vaccine delivery, which offers potential safety, logistical, and cost-saving advantages over traditional vaccination routes. Additionally, aerosol vaccination may elicit pivotal mucosal immune responses that could contain or eliminate mucosally transmitted pathogens in a preventative or therapeutic vaccine context. In this current preclinical non-human primate investigation, we demonstrate the feasibility of aerosol vaccination with the recombinant poxvirus-based vaccine vectors NYVAC and MVA. Real-time in vivo scintigraphy experiments with radiolabeled, aerosol-administered NYVAC-C (Clade C, HIV-1 vaccine) and MVA-HPV vaccines revealed consistent mucosal delivery to the respiratory tract. Furthermore, aerosol delivery of the vaccines was safe, inducing no vaccine-associated pathology, in particular in the brain and lungs, and was immunogenic. Administration of a DNA-C/NYVAC-C prime/boost regime resulted in both systemic and anal-genital HIV-specific immune responses that were still detectable 5 months after immunization. Thus, aerosol vaccination with NYVAC and MVA vectored vaccines constitutes a tool for large-scale vaccine efforts against mucosally transmitted pathogens.

Induction of human papillomavirus oncogene-specific CD8 T-cell effector responses in the genital mucosa of vaccinated mice.

Cervical cancer, the second leading cause of cancer mortality in women worldwide, results from infection with a subset of human papillomaviruses (HPV), HPV-16 being the most prevalent type. The available prophylactic vaccines are an effective strategy to prevent this cancer in the long term. However, they only target 70-80% of all cervical cancers and cannot control existing HPV infections and associated lesions. Therapeutic vaccines are thus necessary for women who cannot benefit from prophylactic vaccination. Induction of protective immune responses in the genital mucosa (GM) may be crucial for efficacy of HPV therapeutic vaccines. We report here that mice that received a single subcutaneous (s.c.) vaccination of an adjuvanted long synthetic HPV16 E7(1-98) polypeptide showed induction of 100% tumor protection against s.c. TC-1 tumors and that tumor regression was mainly provided by CD8 T cells. In vivo cytotoxic assay revealed high E7-specific cytolytic T lymphocytes activity in spleen and in genital draining lymph nodes (LN), and E7-specific CD8 T cells could be detected in GM by tetramer staining. More importantly, high-avidity E7-specific INF-gamma secreting CD8 T cells were induced not only in blood, spleen and LN but also in GM of vaccinated mice, thus providing evidence that a parenteral vaccination may be sufficient to provide regression of genital tumors. In addition, there was no correlation between the responses measured in blood with those measured in GM, highlighting the necessity and relevance to determine the immune responses in the mucosa where HPV-tumors reside.

Place du frottis cytologique à l'adolescence [Role of the PAP smear in adolescence]

The publication of the 2006 directives concerning the management of PAP smears has incited us to update our internal protocols at the CHUV, which are described in this article. A new addition to these directives is the specific management of adolescent PAP smears, who present both a high HPV carriage rate and increased incidence of cytological abnormalities with a favorable outcome in most cases. Our goal is to avoid over-treating dysplasias in this type of patient in order to avoid long-term complications. Emphasis is placed on the first gynecological consultation where a listening ear and clear and targeted information remain essential in the proper management of a young patient.

Immunobiology of human papillomavirus infection and vaccination - implications for second generation vaccines.

Prophylactic human papillomavirus (HPV) L1 virus like particle (VLP) vaccines have been shown, in large clinical trials, to be very immunogenic, well-tolerated and highly efficacious against genital disease caused by the vaccine HPV types. However these vaccines, at the present, protect against only two of the 15 oncogenic genital HPV types, they are expensive, delivered by intramuscular injection and require a cold chain. The challenges are to develop cheap, thermo-stable vaccines that can be delivered by non-injectable methods that provide long term (decades) protection at mucosal surfaces to most, if not all, oncogenic HPV types that is as good as the current VLP vaccines. Current approaches include L1 capsomers, L2 protein and peptides, delivery via recombinant L1 bacterial and viral vectors and large-scale VLP production in plants. Rational design and successful development of such vaccines will be based on an understanding of the immune response, and particularly the 'cross talk' between the innate and adaptive responses. This will be central in the development of adjuvants and vaccine formulations that induce the response to provide effective protection.

Squamous-cell carcinoma arising in a non-irradiated child with recurrent respiratory papillomatosis.

We describe a patient with recurrent respiratory papillomatosis (RRP) associated with human papilloma virus (HPV), who developed a fatal squamous cell carcinoma of the lung. At the age of 1 year he presented with hoarseness, dyspnoea and inspiratory stridor but the diagnosis of RRP was made only 1 year later. At the age of 4 years he was tracheostomized because of upper airway obstruction. In spite of multiple surgical excisions and topic treatment with 5-fluorouracil the papillomata extended to the lung parenchyma. At the age of 16 years he developed a squamous-cell carcinoma of the lung and died 4 months later. Transformation to pulmonary carcinoma is a rare complication in non-irradiated patients with lung papillomatosis. We found only 11 similar cases in the literature.

A murine genital-challenge model is a sensitive measure of protective antibodies against human papillomavirus infection.

The available virus-like particle (VLP)-based prophylactic vaccines against specific human papillomavirus (HPV) types afford close to 100% protection against the type-associated lesions and disease. Based on papillomavirus animal models, it is likely that protection against genital lesions in humans is mediated by HPV type-restricted neutralizing antibodies that transudate or exudate at the sites of genital infection. However, a correlate of protection was not established in the clinical trials because few disease cases occurred, and true incident infection could not be reliably distinguished from the emergence or reactivation of prevalent infection. In addition, the current assays for measuring vaccine-induced antibodies, even the gold standard HPV pseudovirion (PsV) in vitro neutralization assay, may not be sensitive enough to measure the minimum level of antibodies needed for protection. Here, we characterize the recently developed model of genital challenge with HPV PsV and determine the minimal amounts of VLP-induced neutralizing antibodies that can afford protection from genital infection in vivo after transfer into recipient mice. Our data show that serum antibody levels >100-fold lower than those detectable by in vitro PsV neutralization assays are sufficient to confer protection against an HPV PsV genital infection in this model. The results clearly demonstrate that, remarkably, the in vivo assay is substantially more sensitive than in vitro PsV neutralization and thus may be better suited for studies to establish correlates of protection.

Clinical follow-up of women infected with human papillomavirus-16, either alone or with other human papillomavirus types : identification of different risk groups

Rapport de synthèse Objectifs : Évaluer l'impact clinique de femmes infectées par de multiples papillomavirus human (HPV) à haut risque dont le HPV 16 en comparaison de l'évolution de femmes infectées par du HPV 16 seul. Méthode : 169 femmes ont été classifiées en trois groupes, dépendant de leur profile HPV: HPV-16 seul, HPV-16 et un HPV de type bas risque, HPV-16 et un autre HPV à haut risque. Le HPV-DNA des frottis cervicaux a été analysé par polymerase chain reaction (PCR) et reverse line blot hybridization (RLBH). Toutes les femmes ont été suivies à la consultation de colposcopie pour une durée de 24 mois ou plus. La prise en charge s'est faite selon les recommandations de Bethesda. Résultats : Les femmes infectées par du HPV 16 et un autre HPV à haut risque n'ont présenté aucun changement voire une progression de leur dysplasie en comparaison des femmes des autres groupes (RR: 1.39; 95%CI: 1.07 à 1.82; p value: 0.02 à 6 mois; RR: 2.10; 95%CI: 1.46 à 3.02; p value: <0.001 à 12 mois; RR: 1.82; 95%CI: 1.21 à 2.72; p value: 0.004 à 24 mois). Conclusions : Les femmes présentant une co-infection par du HPV 16 ainsi qu'un autre HPV de haut risque voient leur risque d'évolution défavorable augmenter.

Clinical and molecular characteristics of HNSCC patients with brain metastases: a retrospective study.

Among the metastasis patterns of head and neck squamous cell carcinoma (HNSCC), intracranial spread is a rare but dreaded event. To date only very few cases have been reported and clinical and molecular data are sparse. We screened our archives for HNSCC patients from 1992 to 2005 who were diagnosed with brain metastases (BM). For retrospective analysis, all clinico-pathological data including disease-free survival (DFS), local progression-free survival (LPFS), and overall survival (OS) were compiled. Additionally, we assessed the mutational status of the TP53 gene and the prevalence of HPV serotypes by PCR and Sanger sequencing. Immunohistochemistry was applied to detect p16INK4A expression levels as surrogate marker for HPV infection. The prevalence rate of BM in our cohort comprising 193 patients with advanced HNSCC was 5.7 %. Of 11 patients with BM, 3 were female and 9 were male. Seven of the primary tumors were of oropharyngeal origin (OPSCC). LPFS of the cohort was 11.8 months, DFS was 12.1 months and OS was 36.0 months. After the diagnosis of BM, survival was 10.5 months. Five tumors showed a mutation in the TP53 gene, while five of the seven OPSCC tumors had a positive HPV status displaying infection with serotype 16 in all cases. Compared with patients who harbored TP53wt/HPV-positive tumors, patients with TP53 mutations showed a poor prognosis. Compared with the whole cohort, the interval between diagnosis of the primary and the detection of BM was prolonged in the HPV-infected OPSCC subgroup (26.4 vs. 45.6 months). The prognosis of HNSCC patients with BM is poor. In our cohort, most tumors were OPSCC with the majority being HPV positive. Our study points toward a putatively unusual metastatic behavior of HPV-positive OPSCC.