Papillomaviruses are small DNA viruses that are associated with the induction of epithelial tumors. HPV is an important infectious agent causing almost 100 % of cervical tumors but it can also cause tumors in other anogenital and head and neck locations in both men and women. Active HPV infection induces changes in miRNA expression that contribute to the tumor formation and progression. It is already known that papillomaviruses do not encode their own viral miRNAs but they affect the expression of cellular miRNAs. In my thesis I have in selected epithelial tumors (vulva, cervix, anus and tonsils) determined their etiology and analyzed the presence of miRNAs in tissues by next generation sequencing. From these data I determined the expression profiles of deregulated miRNAs in tumors relation to healthy tissues of corresponding location. Even though, sufficient number of samples was analyzed, it was not possible to detect HPV-core miRNA common to all analyzed HPV-induced tumors due to the absence of statistically relevant differentially expressed miRNAs in HPV positive vulvar tumors. Among the tumors of the other sites I found an overlap in three miRNAs. One of these miRNAs (miR-139-5p) and another one (miR-9-5p) which I have selected based on the study of other published data, were used for...

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La dynamique de l’infection par le virus du papillome humain (VPH) chez l’enfant et les facteurs de risques associés à la transmission périnatale sont peu connus. À partir des 422 femmes enceintes infectées par le VPH dans la cohorte HERITAGE, le but était de décrire le risque de transmission périnatale, de récurrence et de persistance chez l’enfant et de mesurer l’association entre la charge virale du VPH durant la grossesse et la transmission périnatale. Des tests VPH ont été effectués sur des prélèvements vaginaux et placentaires collectés chez les femmes, et sur des prélèvements conjonctivaux, oraux, pharyngés et génitaux collectés chez les enfants aux 3-6 mois de la naissance jusqu’à 2 ans. Des analyses descriptives et des modèles de régression logistique ajustés ont été utilisés. La transmission périnatale du VPH a été de 7,3% (IC95%: 5,0%-10,4%), mais peu d’infections récurrentes et persistantes ont été observées chez l’enfant. La charge virale durant la grossesse a été fortement associée au risque de transmission périnatale (ratio de cotes ajusté = 7,01; IC95%:1,89-25,98) pour les femmes ayant une charge virale moyenne aux deux trimestres au-dessus d’une copie/cellule comparativement à celles en dessous de ce seuil. Cette étude suggère que la détection du VPH chez l’enfant n’est pas rare quoique le risque de persistance et de récurrence reste faible. La charge virale durant la grossesse a été fortement associée à la transmission périnatale. Cette étude permet de mieux comprendre la dynamique du VPH chez l’enfant et les facteurs de risque associés à la transmission périnatale. / The dynamics of human papillomavirus (HPV) infection in children and the risk factors associated with perinatal transmission remain poorly understood. From the 422 HPV-positive pregnant women in the HERITAGE cohort, the aim was to describe the risk of perinatal transmission, recurrence and persistence of HPV in children and to measure the association between viral load during pregnancy and perinatal transmission. HPV testing was done on vaginal samples collected during pregnancy, on placental samples and on conjunctival, oral, pharyngeal and genital samples collected in children every 3-6 months from birth to 2 years of age. Descriptive analyses and adjusted logistic regression models were used. Perinatal transmission was 7.3% (95%CI: 5.0%-10.4%) but few cases of recurrent and persistent infections were observed among children. HPV viral load during pregnancy was strongly associated with perinatal transmission (adjusted odd ratio= 7.01; 95%CI: 1.89-25.98), for women with a mean viral load measured at both trimesters of at least one copy per cell of HPV (compared to those with less than one copy per cell). This study suggests that the detection of HPV in children is not rare, although the risk of persistence and recurrence during childhood remains low. Viral load during pregnancy is strongly associated with perinatal transmission. This study provides a more comprehensive insight into the dynamics of HPV infection in children, and the risk factors associated with perinatal transmission.

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The Human Papillomavirus (HPV) is a virus of the Papillomaviridae family and its genome consists of DNA. It is the main cause of viral sexual transmission. Cervical cancer or uterine colon cancer is the second most common occurrence among women worldwide. Recent studies have proven that some types of HPV are mainly responsible for the development of this type of cancer. In accordance to its oncogenic activity this viral group was divided in low and high risk types. Brazil does not have a representative amount of data related to the prevalence of HPV infection. The incidence data of the virus is obtained through the analysis of carriers of the invasive carcinoma of uterine colon, cervical intraepithelial neoplasias, and others types of infections associated. Molecular assays have been showing throughout the years an excellent sensitivity and specificity in the detection of the HPV s DNA. Nowadays, in situ hybridization techniques are being used as method of choice for the detection of the viral DNA. In many studies, the Polymerase Chain Reaction (PCR) with the application of the primers MY09 and MY11 have shown efficiency in viral tracking, consequently, the development of molecular diagnostics allow monitoring of the disease, decreasing mortality rates caused by cancer of the cervix. The DNA sequencing is an efficient methodology used for the molecular characterization of the viral types, which allows the accomplishment of the alignment for similarity of the sequences obtained through others that were already identified and deposited in the GenBank. The current study s purpose was to identify the different types of HPV and the presence of co-infections through PCR and sequencing of a hyper-variable region of the L1 gene. The studied sample consisted of 515 female volunteers, which 111 (21.55%) presented the incidence of the HPV DNA. Within the acquired specimens, 50% were considered high oncogenic risk (the major incidence was types 16 and 58) and 7.21% had multiple infection. The determination of the base sequencing of the L1 gene is essential to the identification of the viral type. However, the sequencing of the complete gene and both DNA chains become financially unviable for the majority of the specialized laboratories. A practical approach applied in this study was to determine the sequencing of a specific region of the L1 gene with 450pb, where the identification of the HPV became possible by following this methodology. The techniques used showed the necessity of the application of a more sensible and specific viral diagnosis, which contributes to the viral infection control and to the reduction of the mortality rate caused by cervical cancer. / Fundação de Amparo a Pesquisa do Estado de Alagoas / O Papilomavírus Humano (HPV) é um vírus da família Papillomaviridae e possui o seu genoma constituído de DNA. É o causador da transmissão sexual viral de maior frequência. O câncer cervical ou câncer de colo de útero possui a segunda maior ocorrência nas mulheres de todo o mundo. Estudos recentes têm comprovado que alguns tipos de HPV são os principais responsáveis pelo desenvolvimento deste tipo de câncer. De acordo com a sua atividade na carninogênese esse grupo viral foi dividido em tipos de baixo e de alto risco oncogênico. O Brasil ainda não possui uma quantidade representativa de dados relacionados à prevalência de infecção por HPV, os dados de incidência do vírus são obtidos através da análise de portadores de carcinoma invasivo de colo uterino, neoplasias intraepiteliais cervicais e outros tipos de infecções associadas. Ensaios moleculares vêm mostrando ao longo dos anos uma alta sensibilidade e especificidade na detecção do DNA do HPV. Atualmente são utilizadas técnicas de hibridização como método de escolha para a detecção do DNA viral. Em muitos estudos a Reação em Cadeia da Polimerase (PCR) com a aplicação dos primers MY09 e MY11 demonstrou eficiência no rastreamento viral, consequentemente, o desenvolvimento desse diagnóstico molecular possibilitaria o monitoramento da doença, diminuindo as taxas de mortalidade causada pelo câncer de colo do útero. O sequenciamento de DNA é uma metodologia eficiente para a caracterização molecular dos tipos virais, permitindo a realização do alinhamento por similaridade das sequencias obtidas com outras já identificadas e depositadas no GenBank. O presente trabalho teve como propósito identificar os diferentes tipos de HPV e a presença de co-infecções, através de PCR e sequenciamento de uma região hipervariável do gene L1. A amostra estudada foi composta de 515 voluntárias das quais 111 (21,55%) apresentaram a presença do DNA do HPV. Entre os espécimes encontrados 50% são considerados de alto risco oncogênico (maior incidência dos tipos 16 e 58) e 7,21% possuíam infecção múltipla. A determinação das sequencias de base do gene L1 é fundamental para a identificação do tipo viral. Entretanto o sequenciamento do gene completo e em ambos os sentidos da cadeia de DNA se tornam inviáveis financeiramente para a maioria dos laboratórios especializados. Uma abordagem prática aplicada no estudo foi determinar a sequencia de uma região específica do gene L1 contendo 450pb, sendo possível a identificação do HPV seguindo essa metodologia. As técnicas utilizadas mostraram a necessidade da aplicação de um diagnóstico viral mais sensível e específico, contribuindo para o controle da infecção viral e para a diminuição da incidência e da mortalidade causadas pelo câncer cervical.

5 Abstract Even though prophylactic vaccine against human papillomavirus (HPV) is currently licensed, infections by the virus continue to be the major health problem mainly in developing countries. Considerable effort is being devoted to preparation of therapeutic vaccine and to decrease of the production costs of current vaccine. Viral proteins such as the E7 oncoprotein and the L2 capsid protein from HPV type 16 are promising targets for the development of the experimental anti-HPV vaccine. The aim of our work was optimization of expression of mutagenized E7 oncoprotein (E7ggg) fused to the C-terminus of Tobacco mosaic virus (TMV) coat protein (CP) or Potato virus X (PVX) CP in viral vectors derived from these plant viruses. The impact of linkers connecting CP and E7ggg fusion partners on expression and stability of fusion proteins was examined. The fusion proteins were first expressed in Escherichia coli (E. coli) MC1061 to assess the characteristics of the recombinant protein prior to their transient expression in both non-transgenic or transgenic Nicotiana benthamiana (N. benthamiana). We have obtained the high level expression in E. coli, but most of the expressed proteins based on TMV CP remained in insoluble inclusion bodies. To increase the ratio of soluble protein various molecular...

Contexte : La littérature suggère que le virus du papillome humain (VPH) puisse être transmis verticalement. Or, le mécanisme exact de transmission verticale demeure inconnu et les données ne permettent pas de savoir dans quelle mesure la transmission verticale est affectée par le mode d’accouchement. L’objectif de l’étude était de mesurer l’association entre le mode d’accouchement et la détection d’ADN du VPH chez les bébés. Méthode : Nous avons utilisé les données de 1052 femmes enceintes de la cohorte HERITAGE. Des échantillons vaginaux auto-collectés ont été obtenus chez les mères durant la grossesse, et des échantillons des muqueuses de la bouche, la gorge, les yeux et de la région anogénitale ont été collectés chez les bébés à la naissance et à 3 mois. Nous avons inclus les 282 femmes ayant eu un test VPH positif au premier et troisième trimestre de grossesse. Tous les échantillons ont été analysés pour la détection d’ADN du VPH par la méthode de réaction de polymérase en chaîne (PCR) avec le test Linear ArrayMC. Les informations sur l’accouchement ont été collectées dans les dossiers médicaux. L’association entre le mode d’accouchement et la transmission verticale du VPH a été mesurée par régressions logistiques. Résultats : La probabilité de transmission verticale du VPH a été de 8,9% (25/282), soit 3,7% (3/81) pour les césariennes et 10,9% (22/201) pour les accouchements vaginaux. Chez 21 des 25 enfants positifs au VPH (84%), il y avait au moins un génotype concordant avec leur mère, et tous sont nés par accouchement vaginal. Une augmentation significative du risque de transmission verticale du VPH a été observée pour l’accouchement vaginal, en comparaison avec la césarienne (OR ajusté: 3,63, intervalles de confiance à 95% (IC 95%): 1,03-12,82). Nous n’avons pas observé d’association significative entre la césarienne suivant la rupture des membranes et le risque de transmission, lorsque comparé avec la césarienne avec membranes intactes (OR ajusté : 1,31, IC 95% : 0,10-17,76). Il n’y a pas eu d’association entre la durée écoulée entre la rupture des membranes et la naissance (en heures continues) et le risque de transmission verticale (OR : 1,00, IC 95% : 0,97-1,02). Conclusion : L’accouchement par césarienne a été associé à un risque significativement plus faible de transmission du VPH chez les bébés. La transmission verticale du VPH surviendrait principalement lors du passage dans le canal vaginal car très peu d’enfants nés par césarienne ont été infectés au VPH. Puisque la rupture des membranes avant la césarienne et la durée entre la rupture des membranes et la naissance n’ont pas été associées à un risque de transmission du VPH plus élevé, nos résultats suggèrent que la transmission par infection ascendante après rupture des membranes est probablement rare. / Background: The literature suggests that human papillomavirus (HPV) can be transmitted vertically. However, the exact mechanism of vertical transmission remains unknown and the data do not allow us to know to what extent vertical transmission is affected by the mode of delivery. The aim of the study was to measure the association between mode of delivery and the detection of HPV DNA in infants. Method: We used data from 1052 pregnant women from the HERITAGE cohort. Self-collected vaginal samples were obtained from mothers during pregnancy, and specimens from the mucous membranes of the mouth, throat, eyes and anogenital region were collected from infants at birth and at 3 months. We included 282 women who had both positive HPV tests in the first and third trimester of pregnancy. All samples were analyzed for detection of HPV DNA by the polymerase chain reaction (PCR) method with the Linear ArrayTM assay. Information about the delivery was collected from medical records. The association between the mode of delivery and HPV detection in infants was measured using logistic regressions. Results: The probability of transmission of HPV was 8.9% (25/282); 3.7% (3/81) for caesarean sections and 10.9% (22/201) for vaginal deliveries. In 21 of 25 HPV positive infants (84%), there was at least one genotype concordant with their mother, and all were born vaginally. A significant increase in the risk of transmission of HPV was observed for vaginal delivery, compared to caesarean section (adjusted OR: 3.63, 95% confidence intervals (95% CI): 1.03-12.82). We found no significant increase in the risk of HPV transmission for caesarean section following rupture of membranes, compared to caesarean section with intact membranes (adjusted OR: 1.31, 95% CI: 0.10-17.76). There was no association between the time between rupture of membranes and birth (in continuous hours) and the risk of vertical transmission (OR: 1.00, 95% CI: 0.97-1.02). Conclusion: Caesarean delivery is associated with a significantly lower risk of HPV vertical transmission. Vertical transmission is thought to occur mainly during passage through the vaginal canal, because very few infants born by caesarean section have been infected with HPV. Since rupture of membranes before caesarean section and the time between ruptured membranes and birth have not been associated with a higher risk of HPV transmission, our results suggest that transmission by ascending infection after rupture of membranes is unlikely.

Detection of p16 through immunohistochemistry (IHC) is the standard for determining the HPV status of the tumor according the TNM eighth edition released in 2017 and has become crucial for determining the HPV status of oropharyngeal squamous cell carcinomas (OPSCC) with direct impact on staging and prognostication. In recent years, detection of HPV DNA in mouthwashes has been proposed as a noninvasive alternative, both for OPSCCs and for other head and neck squamous cell carcinomas (HNSCCs). However, the prospect of using the mouthwashes to monitor the response to therapy is unclear. To evaluate the effect of curative therapy on the detection of HPV DNA, we performed a prospective study comparing the detection frequency of high-risk HPV DNA (HR-HPV-DNA) in pre- and post-therapy mouthwashes. We collected 137 mouthwashes from 88 pathologically confirmed HNSCC patients for DNA isolation and HPV genotyping with the Inno- LiPA assay. We show that HPV DNA in pretherapeutic mouthwashes can detect HPV-driven HNSCCs with a sensitivity of 50.0% and specificity of 85.4%, alongside a high negative predictive value of 79.5% and an accuracy of 74.5%. Furthermore, we observed a notable decrease in the detection frequency of HR-HPV-DNA after successful treatment (pre-therapy 50.0% (9/18) versus post-therapy 9.7% (3/28)). However, the comparatively low sensitivity regarding detection of HPV-driven OPSCC argues against its use in clinical routine.

L’accouchement prématuré a été récemment associé à la persistance du virus du papillome humain de type 16 (VPH-16) en grossesse. Il demeure toutefois difficile de savoir si cette association est causale et d’en expliquer les mécanismes biologiques potentiels. Afin de mieux caractériser cette association, nous avons étudié l'association entre la charge virale du VPH-16 en grossesse et l’accouchement prématuré. Les données de 48 femmes enceintes qui étaient positives pour le VPH-16 dans la cohorte HERITAGE ont été analysées avec un modèle de régression logistique, où la confusion a été ajustée avec scores de propension et pondération par l’inverse de probabilité de traitement. La charge virale du VPH, mesurée avec test PCR en nombre de copies/cellule au 1er et 3ième trimestre de grossesse, a été analysée en continue et dichotomisée à l’aide de différents seuils (0,5, 1,0 et 2,0). La charge virale (en continue) au 1er trimestre de grossesse a été associée à l’accouchement prématuré avec un OR ajusté (aOR) de 1,13 [IC 95% 1,03-1,25]. Le aOR pour la charge virale catégorisée avec seuil de 1,0 copie/cellule au 1er trimestre était de 15,03 [IC 95 % 1,75- 129,26]. Les analyses avec des seuils différents et au 3ième trimestre de grossesse ont données des résultats similaires quoique les ORs n’étaient pas toujours statistiquement significatifs. Nos résultats suggèrent une forte association entre la charge virale du VPH-16 et l’accouchement prématuré. Cette étude contribue à une meilleure compréhension des mécanismes tout en supportant la causalité. / Preterm birth has recently been associated with the persistence of human papillomavirus 16 (HPV-16) during pregnancy. However, it remains difficult to determine whether this association is causal and to explain its potential biological mechanisms. To better characterize this association, we investigated the association between HPV-16 viral load during pregnancy and preterm birth. Data from 48 pregnant women positive with HPV-16 infection from the HERITAGE cohort were analyzed using a logistic regression model, where confounders were adjusted with propensity scores and inverse probability treatment weighting. HPV viral load, measured with a PCR test as copy numbers/cell during the 1st and 3rd trimester of pregnancy was analyzed continuously, and categorized using different cutoffs (0.5, 1.0 and 2.0). Continuous viral load at 1st trimester of pregnancy was associated with preterm birth with an adjusted OR (aOR) of 1.13 [95% CI: 1.03-1.25]. The aOR viral load categorized with cutoff 1 copy/cell at 1st trimester was 15.03 [1.75-129.26]. Analyses with different cutoffs in 3rd trimester of pregnancy gave similar results although the ORs were not always statistically significant. Our results suggest a strong association between HPV-16 viral load and preterm birth. This study contributes to a better understanding of the mechanisms and provides an additional argument on causality.