En utilisant une technique originale de détection et de quantification des HPV16 et 18 par PCR duplex, ce travail de thèse illustre la signification, l’intérêt et les limites de l’utilisation clinique de la mesure de la charge virale pour ces deux types d’hrHPV. Si nous n’avons pas démontré de réelle signification de la charge virale en HPV18, il n’en est pas de même pour l’HPV16 dont la charge virale augmente avec la sévérité des lésions constatées. Néanmoins, l’extrême variabilité des charges virales mesurées limite son utilisation en pratique clinique. Après un frottis cervico-utérin (FCU) anormal, une charge virale seuil en HPV16 à 3,0x106 copies par millions de cellules permet la prédiction optimale de la présence d’une CIN2+ (spécificité : 91 % et sensibilité : 58,2 %). Cette valeur seuil est particulièrement performante pour les patientes ayant un FCU de bas grade (spécificité : 96,4 % et sensibilité : 88 %). Si la charge virale en HPV16 et 18 ne semble pas être prédictive de la clairance virale chez les jeunes femmes de moins de 30 ans ayant un FCU normal, elle l’est chez les patientes HPV16 positives ayant une colposcopie normale malgré un FCU équivoque ou de bas grade (spécificité : 86,7 % et sensibilité : 85,7 %). / Using duplex PCR technique for the detection and quantification of HPV16 and 18, this work investigates the significance, value and limitations of the use of HPV16 and 18 viral load quantitation in routine clinical practice. Although HPV18 viral load was not found to be of any clinical relevance, HPV16 viral load was found to significantly increase with the severity of cervical lesions. However, the wide range of viral load observed strongly limitates its use in routine clinical practice. After an abnormal cervical cytology, a HPV16 viral load cut-off of 3.0x106 copies per million cells allows for the best prediction of CIN2+ (91% specificity and 58.2% sensitivity). Such cut-off is particularly efficient in case of low grade abnormal cytology (96.4% specificity and 88% sensitivity). Although HPV16 viral load does not appear to predict for HPV16 clearance in women under 30 with normal cytology, such prediction was observed among women with normal colposcopy following equivocal or low grade cytology (86.7% specificity and 85.7% sensitivity).

O câncer de colo do útero é a segunda principal causa de morte em mulheres por câncer, sendo causado principalmente pela infecção persistente por HPV. A principal forma atual de prevenção desse câncer é a realização de exames citológicos periódicos e a vacinação profilática disponibilizada recentemente pelo Ministério da Saúde. Entretanto, tais ações visam a prevenção da infecção por HPV ou a detecção de lesões, pois não há um tratamento específico contra infecções e lesões já estabelecidas. Dentre as proteínas expressas por HPV, L2 está presente no capsídeo viral e é bem conservada entre diversos tipos de HPV, enquanto E6 é uma proteína oncogênica capaz de induzir a transformação maligna das células. Este estudo visa o desenvolvimento de uma vacina profilática e terapêutica capaz de proteger contra a infecção por diversos tipos virais, assim como combater as células já modificadas por qualquer tipo de HPV. Assim, foi construído um vetor vacinal contendo peptídeos selecionados das proteínas L2 e E6 de HPV16 testadas em modelo murino, para avaliar sua eficiência como vacina de DNA ministrada antes ou após desafio com células tumorais. Os ensaios em modelo animal demonstraram que a vacina foi capaz de induzir a produção de anticorpos específicos anti-L2 e anti-E6, assim como induzir a produção de citocinas TNF e impedir o desenvolvimento tumoral de células HPV-positivas. Portanto, foi possível concluir que o vetor construído foi capaz de induzir uma resposta imune humoral e celular em camundongos, capaz de prevenir contra o HPV, assim como tratar os cânceres a ele associados. / Cervical cancer is the second most frequent cause of death in women due to cancer, mainly caused by persistent HPV infection. The primary prevention method of this cancer is through periodic cytological exams and prophylactic vaccination which has been recently made available by the Ministry of Health. However, such actions aim towards prevention of HPV infection or the detection of lesions, since there is no specific treatment against pre-existing infections and injuries. Among the proteins expressed by HPV, L2 is present in the viral capsid and is well conserved among several types of HPV, whereas E6 is an oncogenic protein, capable of inducing malignant mutations in cells. This study aims towards the development of a prophylactic and therapeutic vaccine, capable of protecting against infections caused by several viral types, as well as combating cells which have already been modified by any type of HPV. Thus, a vaccine vector, containing peptides selected from HPV16s L2 and E6 proteins, tested in the murine model, was constructed in order to evaluate its efficiency as a DNA vaccine, to be administered either before or after challenging with tumour cells. The animal model assays demonstrated that the vaccine was able to induce the production of anti-L2 and anti-E6 specific antibodies, as well as TNF cytokines, and to prevent tumour development in HPV-positive cells. Therefore, it was possible to conclude that the designed vector was indeed able to induce humoral and cellular immune responses in mice, capable of preventing against HPV, as well as of treating cancers associated with it.

INTRODUÇÃO: A epidermodisplasia verruciforme (EV) é uma rara genodermatose, associada ao beta-papilomavírus humano (Beta-HPV) e alto risco de desenvolvimento de câncer de pele. As claudinas são proteínas transmembranas expressas nos diversos epitélios e podem se alterar na carcinogênese. Para melhor compreensão do papel do Beta-HPV na carcinogênese cutânea, realizou-se um estudo da expressão das claudinas nos pacientes com e sem EV. MÉTODOS: Um painel de anticorpos anti-claudina -1, -2, -3, -4, -5, -7 e -11 foi empregado para analisar a expressão dessa proteína em 108 amostras de pele normal, 39 verrugas planas e 174 carcinomas espinocelulares cutâneo (CEC), obtidas de 33 pacientes com epidermodisplasia verruciforme (EV) e 112 indivíduos saudáveis (não EV- NEV). As amostras de CEC foram organizadas em 4 microarranjos teciduais. A análise estatística foi realizada por regressão logística para verificar as alterações do perfil de claudinas na carcinogênese e sua relação com idade, sexo e fotoexposição crônica nos doentes com EV e o grupo NEV. RESULTADOS: A expressão focal da claudina-1 apresentou associação com o CEC (p < 0,001) e a expressão não difusa (focal ou negativa) da claudina-2 esteve associado a verruga plana (p < 0,001), nos grupos EV e NEV. A expressão focal da claudina-3 apresentou associação com o CEC (p < 0,001), bem como a imunomarcação nuclear da claudina-3 (p < 0,001), em ambos os grupos. Adicionalmente, a chance da expressão nuclear da claudina-3 foi 53% menor nas áreas não fotoexpostas. A claudina-5 apresentou maiores porcentagens de expressão focal na pele normal, de expressão difusa no CEC e na verruga plana EV, e de expressão negativa na verruga plana NEV. O grupo EV apresentou menor chance de expressão focal e negativa (p < 0,001). Além disso, a negatividade da claudina-5 esteve associada a verruga plana (p < 0,001) e menor média de idade (p < 0,001). As claudinas -4, -7 e -11 apresentaram expressão difusa em quase todas as amostras estudadas, nos grupos EV e NEV. CONCLUSÕES: Verificou-se aumento progressivo da claudina-5 na carcinogênse cutânea e esse processo apresentou relação com a EV. Redução da expressão das claudinas -1 e -3 foi observada nos carcinomas cutâneos, e da claudina-2 nas verrugas planas, no entanto, não puderam ser relacionadas à infecção pelo beta-HPV. Idade e fotoexposição crônica foram fatores que influenciaram a expressão da claudina-5 e a expressão nuclear da claudina-3, respectivamente / BACKGROUND: Epidermodysplasia verruciformis (EV) is a rare genodermatosis, related to human beta-papillomavirus (Beta-HPV), with high risk for developing skin cancer. Claudins are transmembrane proteins expressed in epithelia and may be altered on carcinogenesis. Toward better understanding the role of Beta-HPV in cutaneous carcinogenesis, claudin expression study was performed in lesions of patients with and without EV. METHODS: A panel of anti-claudin antibodies was assembled to analyze claudins -1, -2, -3, -4, -5, -7 and -11 expression, by immunohistochemistry technique, in samples of 108 normal skin, 39 flat warts and 174 cutaneous squamous cell carcinomas (SCC), obtained from 33 patients with EV and 112 individuals without EV (not-EV - NEV). The SCC samples were organized in tissue microarrays. Statistical analysis was performed by logistic regression, aiming to verify changes in claudin profile in carcinogenesis and its relationship with age, sex and chronic sun exposure area, in patients with and without EV. RESULTS: Claudin-1 focal expression was associated with SCC (p < 0.001) and claudin-2 focal or negative expression with flat wart (p < 0.001), in EV and NEV groups. Claudin-3 focal expression was related with SCC (p < 0.001), as well as the nuclear immunostaining of claudin-3 (p < 0.001), in both groups. Additionally, chance of claudin-3 nuclear expression was 53% lower in not sun exposed areas. For claudin-5, focal expression was more common in normal skin, diffuse expression in SCC annd EV flat wart, and negative expression in NEV flat wart. The EV group showed lower chance of focal and negative expression (p < 0.001). In addition, the negativity of claudin-5 expression was associated with flat wart (p < 0.001) and lower mean age (p < 0.001). Claudins -4, -7 and -11 showed, in both groups, diffuse expression in almost all studied samples. CONCLUSIONS: Claudin-5 increasing expression was observed in cutaneous carcinogenesis and this process showed association with EV. Claudin-1 and -3 down expression were observed in cutaneous carcinomas, and claudin-2 in flat warts, however, they could not be related with Beta-HPV infection. Age and chronic sun exposure area were factors that influenced claudin-5 and claudin-3 nuclear expression, respectively

The latest model of tertiary structure of capsid protein of potato virus X (PVX CP) was used as a template to design new insertion sites suitable for the preparation of PVX-based antigen presentation system. Based on this model, seven insertion sites (A-G) located in putative surface loops were tested. As an antigen inserted into these sites was used 17 amino acids long epitope derived from human papillomavirus type 16 E7 oncoprotein (E7 epitope) fused with either 6xHis tag or StrepII tag in both possible orientations (6xHis-E7 and E7-6xHis, StrepII-E7 and E7-StrepII). Prior to plant expression, modified PVX CPs were expressed in Escherichia coli MC1061. The results showed that only PVX CP carrying StrepII-E7 or E7-StrepII in the insertion site A formed virus particles. The results from transient expression experiments with modified PVX CPs in Nicotiana benthamiana showed that only the insertion site A (located between 24th and 25th amino acid in the PVX CP) could tolerate all tested inserts. Importantly, viral particles were detected only in the presence of StrepII tag and their stability was affected by the insert orientation (StrepII-E7 vs. E7-StrepII) as only the viral particles presenting E7-StrepII could be purified. Besides the preparation of PVX-based antigen presentation system, an...

Les papillomavirus humains (HPV) sont des virus à ADN double brin qui infectent la peau et les muqueuses. Les infections par les HPV, bien que majoritairement asymptomatique, provoquent des défauts de prolifération cellulaire pouvant parfois générer des cancers. Selon leur pouvoir carcinogène, on distingue les HPV à bas risque oncogène (HPV-BR) provoquant des lésions bénignes, et les HPV à haut risque (HPV-HR) responsables de l’apparition de nombreux cancers ano-génitaux et de certains cancers des voies aéro-digestives supérieures. Parmi les HPV-HR, HPV16 est le plus prévalent. La carcinogenèse induite par les HPV-HR est corrélée à l’expression des protéines virales E6 et E7, qui dérégulent de nombreux processus cellulaires. L’expression des gènes viraux, réalisée par la machinerie de la cellule hôte, est finement régulée particulièrement au niveau posttranscriptionnel. En outre, l’épissage alternatif génère une vingtaine de transcrits viraux, permettant l’expression des protéines virales. L’épissage au sein de la région codante E6 permettant de former l’isoforme E6*I est présent uniquement chez les HPV-HR, mais pas chez les HPV-BR, ce qui suggère son implication dans la carcinogenèse induite par les HPV-HR. Toutefois, le rôle biologique de la protéine E6*I produite par les HPV-HR est encore controversé.Afin de mieux appréhender les mécanismes de la carcinogenèse induite par les HPV-HR, nous nous sommes intéressés à : (i) l’étude des fonctions biologiques de l’isoforme E6*I, et (ii) aux mécanismes impliqués dans la régulation de l’expression de E6 et E7.Pour appréhender le rôle biologique d’E6*I d’HPV16, nous avons utilisé le séquençage de l’ARN afin d’identifier des cibles dérégulées par son expression ectopique. L’expression des isoformes E6 et E6*I d’HPV16 dans des cellules HPV négatives dérégule des transcrits impliqués dans des processus biologiques relatifs à l’expression des gènes viraux, la carcinogenèse virale, la transduction du signal et la traduction. L’expression d’E6*I seule, dérégule des transcrits impliqués dans l’organisation de la matrice extracellulaire, des voies de signalisation et d’adhérence cellulaire. De façon intéressante, il a été montré que ces gènes dérégulés par l’expression d’E6*I sont communément affecté par le niveau intracellulaire de ROS (espèces réactives de l’oxygène). Cela corrobore le rôle d’E6*I dans l’augmentation de la production de ROS. Le stress oxydatif associé aux ROS pourrait favoriser l’intégration du génome viral à celui de la cellule hôte, caractéristique de carcinogenèse associée aux HPV-HR. En somme, E6*I pourrait avoir un rôle oncogénique indépendant de celui d’E6, et interviendrait dans la carcinogenèse associée aux HPV-HR.Nous avons aussi étudié le rôle du complexe de jonction des exons (EJC), dans la régulation posttranscriptionnelle de l’expression d’E6 et E7. L’EJC est un complexe multiprotéique déposé sur les ARNm via l’épissage influençant ainsi leur devenir. Nous avons montré qu’un facteur de l’EJC, eukaryotic initiation factor 4A3 (eIF4A3), se liait aux ARNm viraux. Par ailleurs, nous avons observé que les composants de l’EJC affectent, certes de différentes façons, l’expression d’E6 et E7. Enfin, nous avons aussi étudié l’effet du nonsense-mediated mRNA decay (NMD), un mécanisme lié à l’EJC, sur l’expression d’E6 et E7. Non seulement nos résultats suggèrent que le NMD inhibe l’expression d’E6 et E7, mais nous avons aussi observé que la protéine E6 d’HPV16 réduit l’activité du NMD. Cette inhibition permettrait à HPV16 d’avoir un contrôle sur ses transcrits mais d’affecter aussi des cibles cellulaires du NMD. Etant donné l’implication des gènes régulés par le NMD dans le maintien de l’homéostasie et l’adaptation cellulaires, il serait intéressant d’appréhender le rôle de cette nouvelle activité d’E6 dans la carcinogenèse associée aux HPV-HR. / Human papillomaviruses (HPV) are double strand DNA viruses that infect skin and mucosa. HPV infections, although mostly asymptomatic, cause cell proliferation defects that can sometimes give rise to cancer. According to their carcinogenic potential, we distinguish low-risk HPVs (lr-HPV) causing benign lesions, and high-risk HPV (hr-HPV) responsible for the appearance of numerous anogenital and some head and neck squamous-cell cancers. Among the hr-HPV, HPV16 is the most prevalent. Hr-HPV-induced carcinogenesis is correlated with the expression of the viral oncoproteins, E6 and E7, which deregulate many cellular processes. Viral gene expression, performed by the host cell machine, is finely regulated particularly at the post-transcriptional level. Besides, alternative splicing generates about twenty viral transcripts, leading to the expression of viral proteins. The splicing within the E6 open reading frame that generates an E6*I mRNA only in hr-HPV, but not in the lr-HPV, suggests its involvement in hr-HPV-induced carcinogenesis. However, the biological role of E6*I protein produced by HPV-HR is still controversial.In order to better understand the mechanisms of hr-HPV-induced carcinogenesis, we have interested in: (i) the study of the biological functions of the E6*I isoform, and (ii) the mechanisms involved in the regulation of E6 and E7 expression.To get insight the biological role of HPV16 E6*I, we used RNA sequencing to identify targets deregulated by its ectopic expression. Expression of HPV16 E6 and E6*I isoforms in negative HPV cells deregulate several transcripts involved in biological processes related to viral gene expression, viral carcinogenesis, signal transduction and translation. The expression of E6*I alone, deregulates transcripts involved in the organization of the extracellular matrix, signaling pathways and cell adhesion. Interestingly, it was shown that the genes deregulated by E6*I expression are commonly affected by the intracellular level of ROS (reactive oxygen species). These results support the role of E6*I in increasing ROS production. The ROS-associated oxidative stress could favor viral genome integration with that of the host cell, a characteristic of hr-induced carcinogenesis. In sum, E6*I may have an oncogenic role independent of E6, and intervene in the carcinogenesis associated with hr-HPV.We also studied the role of the exon junction complex (EJC) in the posttranscriptional regulation of E6 and E7 expression. EJC is a multiprotein complex deposited on mRNAs via splicing, thus influencing their fate. We have shown that a factor of EJC, eukaryotic initiation factor 4A3 (eIF4A3), binds to viral mRNAs. Moreover, we have observed that the components of the EJC affected, in different ways, the expression of E6 and E7. Finally, we also studied the effect of nonsense-mediated mRNA decay (NMD), a mechanism linked to the EJC, on the expression of E6 and E7. Our results suggest that not only NMD inhibits the expression of E6 and E7, but we have also observed that HPV16 E6 protein reduces NMD activity. This inhibition would allow HPV16 to have control over its transcripts but also to affect NMD cellular targets. Given the involvement of NMD-regulated genes in the maintenance of cellular homeostasis and adaptation, it would be interesting to understand the role of this new E6 activity in carcinogenesis associated with HPV-HR.

O carcinoma epidermóide de cabeça e pescoço (CECP) é um problema de saúde relevante no mundo, por sua prevalência e agressividade. Os papéis do tabaco e do álcool estão bem definidos na sua etiologia, mas uma minoria crescente dos pacientes acometidos pela doença não é tabagista ou etilista. A infecção pelo papilomavirus humano (HPV) parece ser responsável por parte desses casos. Sugere-se que o CECP que acomete pacientes não tabagistas e não etilistas (NTNE) tenha processos carcinogênicos e evolução clínica distintos daqueles de pacientes tabagistas e etilistas (TE). Os objetivos desse estudo foram verificar se os aspectos demográficos, clínicos e histopatológicos de pacientes com CECP são diferentes conforme os hábitos tabágico e etílico; verificar se os CECPs de pacientes NTNE e TE diferem em relação à positividade para HPV; e comparar a sobrevida específica pela doença e a expressão de marcadores biológicos em amostras tumorais e de mucosa normal do trato aerodigestório de pacientes NTNE e de pacientes TE. Para tanto, realizamos estudo transversal de pacientes com carcinomas epidermóides de cavidade oral (exceto lábio), orofaringe, laringe e hipofaringe, prospectivamente incluídos em um banco de tumores de CECP de 2001 a 2009, pelo grupo de pesquisa multi-institucional GENCAPO. Seus dados demográficos, clínicos e patológicos foram analisados conforme os hábitos de tabagismo e etilismo. Através de análise de pareamento, pacientes NTNE e TE foram comparados em relação à sobrevida, à positividade para o HPV no tumor por reação em cadeia da polimerase (PCR) e aos marcadores imunohistoquímicos p53, FHIT, Ki-67, VEGF, EGFR e p16 tanto em amostras tumorais como de epitélio não tumoral. Dos 1633 pacientes selecionados, 80 eram NTNE (4,9%), 1374 TE (84,1%), 140 tabagistas atuais ou no passado mas não etilistas (TNE:8,6%) e 39 apenas etilistas atuais ou no passado, mas não tabagistas (NTE:2,4%). O grupo de pacientes NTNE constituiu-se principalmente por mulheres, preferencialmente idosas, com tumores da cavidade oral, diferentemente de pacientes tabagistas e/ou etilistas (p<0,001). Considerando os diferentes padrões de hábitos, pacientes TE eram geralmente mais jovens (p<0,001), pacientes TNE apresentaram proporcionalmente mais tumores de laringe (p<0,001) e pacientes NTNE apresentaram menor número de parentes de primeiro grau tabagistas (p<0,001). Observou-se um provável efeito do álcool na ocorrência de metástases ganglionares (p<0,001), enquanto o tabaco pareceu relacionar-se a menor grau de diferenciação tumoral à histologia e a menores índices de massa corpórea (p<0,001). Detectou-se a presença de material genético do HPV em 32,8% dos tumores de pacientes NTNE. Desses tumores positivos para o HPV, metade apresentou também hiperexpressão pelo p16. Foi observado menor risco de óbito pela doença em pacientes NTNE quando apresentavam expressão tumoral intensa do p16 (p=0,011 / RR = 0,07; IC 0,01-0,55) e menor sobrevida se apresentavam marcação pelo FHIT em camada basal de margem não tumoral (p= 0,031). Ao comparar pacientes NTNE e TE, não se observaram diferenças na sobrevida específica pela doença ou na positividade para o HPV de forma independente de sexo, idade, sítio tumoral, grau de diferenciação, variante morfológica, estádio T e presença de metástases linfonodais. Pacientes NTNE apresentaram marcação nuclear pelo FHIT em camada basal menos frequentemente do que pacientes TE (p=0,021) / Head and neck squamous cell carcinoma (HNSCC) is a major health problem worldwide, due to its prevalence and aggressiveness. The role of tobacco and alcohol in its etiology is well established; however, a growing minority of patients with HNSCC neither smokes nor consumes alcohol. Human Papillomavirus (HPV) infection seems to be responsible for some of these cases. It is suggested that HNSCC affecting non smoking and non drinking (NSND) patients has different carcinogenesis and outcomes than those in smoking and drinking (SD) subjects. The objectives of this study were to test if demographic, clinical and pathological aspects of patients with HNSCC vary according to smoking and drinking habits; to test if HNSCC in NSND and SD patients differ in terms of HPV positivity; and to compare NSND and SD patients with HNSCC according to survival and biomarkers in tumor and mucosal samples of the aerodigestive tract. We conducted a cross-sectional study of patients with oral cavity (lips excluded), oropharynx, larynx and hypopharynx tumors prospectively included in a multi-institutional HNSCC tumor bank - GENCAPO, from January 2001 to February 2009. Demographic, clinical and pathological data were analyzed in regards of smoking and drinking habits. Using matched-pair analysis, we compared NSND and SD patients in relation to disease-free survival, HPV positivity through polymerase chain reaction (PCR) and Immunohistochemical staining of p53, FHIT, Ki-67, VEGF, EGFR and p16 biomarkers in tumor and mucosal samples. From 1633 patients, 80 were NSND (4.9%), 1374 SD (84.1%), 140 current or past smokers, but non drinkers (SND:8.6%) and 39 current or past drinkers, but non smokers (NSD:2.4%). NSND patients were most frequently women, remarkably elderly, with oral cavity cancers more commonly than the other groups (p<0.001).Comparing to the other groups, SD patients were younger (p<0.001); SND patients were more frequently affected by larynx tumors (p<0.001) and NSND patients had fewer smoking first degree relatives (p<0.001). We observed that alcohol may influence the presence on node metastasis (p<0.001) whereas tobacco may be related to less differentiated tumors and lower body mass indexes (p<0.001). We found HPV DNA in 32.8% of tumors of NSND subjects. Half of the HPV positive tumors were also positive for p16 staining. A lower risk of death from disease was observed among NSND patients with intense p16 staining (p=0.011 / RR = 0.07; CI 0.01-0.55) and lower survival rates in patients with positive nuclear staining for FHIT at the basal layer of mucosal epithelium (p=0.031). We found no differences in disease-free survival of NSND and SD patients in an independent manner of gender, age, tumor site, differentiation grade at histology, pathological variants, T stage and the presence of node metastasis. NSND patients presented less frequently with nuclear staining for FHIT at the basal layer of mucosal epithelium than SD patients (p=0.021)

O vínculo entre papilomavírus humano de alto risco e câncer cervical está bem estabelecido. Apesar da existência de vacinas profiláticas contra infecções pelos tipos mais comuns de HPV, para infecções e tumores causados por esses vírus as alternativas terapêuticas são restritas. Encontramos alguns motivos com homologias para proteínas do HPV de alto risco durante o imunoscreening de uma biblioteca de phage display com soros de participantes HPV-16-soropositivos da coorte Ludwig-McGill. Após enriquecimento das sequências, os bacteriófagos recombinantes foram purificados e amplificados para uso como imunógenos.Usando uma abordagem profilática, nós vacinamos experimentalmente camundongos imunocompetentes com um dos nossos bacteriófagos recombinantes, usando o bacteriófago sem inserto como controle. Estes camundongos foram então desafiados com células tumorais TC-1 (HPV-16 positivas), tendo-se avaliado as respostas imunes disparadas durante a progressão tumoral. Também usamos uma abordagem terapêutica, aonde os camundongos foram primeiro injetados com as células tumorais e imunizados com o bacteriófago após o estabelecimento do tumor. O crescimento tumoral foi monitorado e os tumores, baço e linfonodos foram avaliados quanto à quantidade e qualidade da resposta imunológica. Os testes de ELISA revelaram que todos os camundongos vacinados responderam à imunização com os diferentes bacteriófagos. O crescimento tumoral foi significativamente reduzido nas imunizações profiláticas e terapêuticas, embora a redução do tumor fosse mínima quando os camundongos foram tratados 9 dias após o enxerto. A redução no crescimento tumoral também se traduziu em uma sobrevivência significativamente maior para os camundongos imunizados. Estudos de infiltração celular não revelaram alterações em diversas sub-populações imunes, mas uma tendência de aumento de linfócitos T citotóxicos foi observada nos camundongos imunizados com PEP1 (bacteriófago contendo inserto). A importância deste aumento de CD8 na redução observada do crescimento tumoral foi confirmada utilizando camundongos CD8-knockout, onde a redução do crescimento tumoral previamente observada foi anulada. Foi observado um aumento de taxa CD8:CD4 nos camundongos imunizados e isto é uma indicação de ambiente tumoral citotóxico. Os ensaios de proliferação celular para testar a especificidade do antígeno dos linfócitos dos camundongos imunizados foram, no entanto, inconclusivos; da mesma forma, não pudemos alterar o padrão observado com o uso de adjuvante CpG. A utilidade da técnica de phage display também foi observada neste trabalho experimental. Trabalhos adicionais para entender o mecanismo de ação desses fagos recombinantes no controle do crescimento de tumores causados por HPV e seu potencial imuno-estimulador são necessários / The link between high-risk human papillomavirus and cervical cancer is well established. Despite the existence of prophylactic vaccines against infections by the most common types of HPV, therapeutic alternatives are limited for infections and tumors caused by these viruses. We found some homology motifs for high-risk HPV proteins during the immune-panning of a phage display library with sera from HPV-16- seropositive participants of the Ludwig-McGill cohort. After enrichment of the sequences, the recombinant bacteriophages were purified and amplified for use as immunogens. Using a prophylactic approach, we vaccinated experimentally immunocompetent mice with one of our recombinant bacteriophages using the insertless bacteriophage as a control. These mice were then challenged with TC-1 tumor cells (HPV-16 positive), and the immune responses triggered during tumor progression were evaluated. We also used a therapeutic approach where mice were first injected with tumor cells and immunized with the bacteriophage after tumor establishment. Tumor growth was monitored and tumors, spleen and lymph nodes were evaluated for the quantity and quality of the immune response. ELISA tests revealed that all vaccinated mice responded to immunization with the different bacteriophages. Tumor growth was significantly reduced in prophylactic and therapeutic immunizations, although tumor reduction was minimal when mice were treated 9 days after TC-1 cells grafting. The reduction in tumor growth also translated into a significantly greater survival for the immunized mice. Cell infiltration studies did not reveal changes in several immune subpopulations, but an upward trend in cytotoxic T lymphocytes was observed in mice immunized with PEP1 (insert-containing bacteriophage). The importance of this increase in CD8 in the observed reduction of tumor growth was confirmed using CD8-knockout mice, where the previously observed reduction of tumor growth was abolished. An increase in CD8:CD4 rate was observed in the immunized mice and this is an indication of a cytotoxic tumor environment. Cell proliferation assays to test the antigen specificity of lymphocytes from immunized mice were, however, inconclusive; likewise, we could not change the pattern observed with the use of CpG adjuvant. The usefulness of the phage display technique was also observed in this experimental work. Additional studies to understand the mechanism of action of these recombinant phages in the control of HPV tumor growth and its immunostimulatory potential are warranted

O desenvolvimento do câncer cervical está associado à infecção por alguns tipos de Papilomavírus Humano (HPV). Entre os mecanismos de carcinogênese associados ao HPV incluem-se alterações em moléculas que modulam a manutenção de componentes da matriz extracelular (MEC), como as metaloproteinases de matriz (MMP) e alguns de seus reguladores. A proteína RECK (reversion inducing cysteine rich protein with kazal motifs) apresenta função essencial na remodelação tecidual e na angiogênese fisiológica ou tumoral, através da regulação pós-transcricional da atividade de MMP-2, MMP-9 e MMP-14 (MT1-MMP). Resultados publicados previamente por nosso grupo apontam para a correlação entre a expressão da oncoproteína E7 de HPV16, a alta expressão e atividade de MMP-9 e a baixa expressão de seus reguladores, TIMP-2 e RECK. A expressão de RECK também é baixa em lesões do colo uterino de alto grau e em amostras de câncer cervical, quando comparadas a amostras de pacientes com cervicite. O presente estudo visa determinar o papel de RECK no processo de tumorigênese mediado por HPV. Para isto, estabelecemos linhagens derivadas de tumor de colo de útero (SiHa, SW756 e C33A) que superexpressam RECK a partir de transdução com lentivírus. Os efeitos da superexpressão de RECK sobre o potencial tumorigênico de SiHa, SW756 e C33A foram avaliados em modelos de estudo in vivo e in vitro. De maneira geral, a superexpressão de RECK foi associada com a capacidade reduzida de invasão em câmara de Matrigel® e de formação de colônia independente de ancoragem. Ainda, camundongos nude inoculados s.c. com células tumorais superexpressando RECK apresentaram atraso no estabelecimento e crescimento tumoral e sobrevida global estendida quando comparados aos controles. Ambos tumores derivados de SiHa RECK e SW756 RECK apresentaram redução na frequência de células tumorais e endoteliais, ao passo que mostrarm aumento no infiltrado inflamatório. Esta observação foi acompanhada de redução na população de neutrófilos e potenciais células mieloderivadas supressoras em tumores de ambas as linhagens. Em tempo, analisamos séries de dados de expressão de CIN e carcinomas cervicais do banco de dados GEO e verificamos que a hipermetilação do gene RECK e a inibição da expressão de mRNA de RECK são eventos precoces no desenvolvimento do câncer de colo de útero. Avaliamos que a baixa expressão de RECK foi associada a progressão de lesões CIN3+ e ao aumento de metástases em linfonodo pélvico em pacientes com câncer de colo de útero. Ademais, notamos que o tratamento com quimio radioterapia levou ao aumento dos níveis de mRNA de RECK em um outro grupo de pacientes com câncer de colo de útero. Concluímos que a superexpressão de RECK (i) reduz o potencial tumorigênico de linhagens celulares derivadas de colo de útero independente do status de infecção por HPV e que (ii) o seu efeito sobre as populações intratumorais se mostrou específico para as linhagens infectadas por HPV. Estes resultados apontam para uma possível interação entre as alterações no microambiente tumoral associadas ao HPV e a função de RECK. Finalmente, a regulação negativa da expressão de RECK é um evento precoce na história natural do câncer cervical. / Persistent infection with high-risk Human Papillomavirus (HPV) types is the main etiologic factor for the development of cervical cancer. The HPV carcinogenic mechanisms include alterations in extracellular matrix (ECM) components, as matrix metalloproteinases (MMP) and its regulators. The Reversion-inducing Cysteine-rich protein with Kazal motifs (RECK) plays a central role on tissue remodeling, tumor angiogenesis and exert inhibitory effects on the transcription, synthesis, activation and activity of MMP-2, MMP-9 e MMP-14 (MT1-MMP). As previously published by our group, it has been observed a correlation between the HPV16 E7 oncoprotein expression, the up-regulation of MMP-9 and the down-regulation of its inhibitors, RECK and tissue inhibitor of metalloproteinases 2 (TIMP-2). Also, RECK expression was downregulated in cervical intraepithelial neoplasias grades 2 and 3 (CIN2/3) and invasive carcinoma samples levels when compared with clinical samples of pacients diagnosed with cervicitis. The present study aims to determine the role of RECK in HPV mediated tumorigenesis. In order to do so, we generated cervical tumors derived cell lines (SiHa, SW756 e C33A) superexpressing RECK by lentiviral transduction followed by FACS. We assessed the effects of RECK superexpression in the tumorigenic potential of SiHa, SW756 e C33A using both in vitro and in vivo protocols. Overall, RECK superexpression is associated with reduced chamber invasion and reduced anchorage independent colony formation. Moreover, nude mice injected s.c. with RECK superexpressing tumor cells presented (i) delayed tumor establishment and (ii) increased overall survival, when compared with controls. Both SiHa and SW756 superexpressing RECK presented decreased frequency of tumor and endothelial cells, whilst showed increase in inflammatory infiltrate population. This observation was followed by a decrease in potential myeloid derived suppressor cell and neutrophil populations in both SiHa RECK and SW756 RECK tumors. Additionally, we observed hipermethylation and premature and consistent downregulation of RECK mRNA expression in CIN and cervical cancer expression datasets from GEO database. We observed that reduced RECK expression was associated with CIN3+ progression and increased pelvic lymph node metastasis in cervical cancer patients. Furthermore, we found that chemo radiotherapy treatment led to increased levels of mRNA in another set of cervical cancer patients. We conclude that RECK superexpression reduces the tumorigenic potential of cervical cancer derived cell lines regardless of HPV infection status. However, we found that the effect of RECK over the intratumoral cells populations is specific to HPV infected tumor cell lines. These results points to a possible interaction between HPV associated tumor microenvironment alterations and RECK. Finally, RECK downregulation is an early event in the natural history of cervical cancer.

Infecção pelo HPV é o principal fator de risco para câncer cervical. Os tumores apresentam microambiente complexo, células tumorais e inflamatórias integram sinais modulando a atividade de vias de sinalização. Caracterizamos o estado de ativação de três vias de sinalização importantes para a progressão tumoral. Com o aumento do grau da lesão, observamos: aumento da expressão de NF&#954;B e Akt fosforiladas no microambiente tumoral; redução de NF&#954;B fosforilada e aumento de STAT3 e Akt fosforiladas em leucócitos circulantes. Em modelos experimentais, a via de NF&#954;B também encontra-se inibida, principalmente em células apresentadoras de antígenos. Para testar se a modulação de vias de sinalização poderiam alterar as respostas a tumores, tratamos linfócitos B de pacientes com agonista de CD40, ativador de NF&#954;B, e observamos aumento do potencial de apresentação antigênica. Nossos resultados mostram o panorama do estado de ativação de importantes vias de sinalização para a progressão tumoral e trazem a possibilidade de uma ferramenta imunomoduladora. / Cervical cancers have HPV infection as a main factor. HPV tumors recruit leucocytes and change their phenotype as an evasion mechanism. We tried to understand how the tumor influences the immune cells behavior, analyzing signaling pathways important to tumor progression in cervical biopsy and PBMC from patients with high-grade lesions and cancer. We find an increase in NF&#954;B activation in immune cells from tumor microenvironment, but a decrease in PBMC. We could not find any difference in STAT3 signaling pathway in the tumor microenvironment, however, there was an activation increase in PBMC. Both tumor microenvironment immune cells and PBMC had an increase in Akt signaling pathway. To identify a possible strategy to reverse the tumor influence, we stimulated B lymphocytes from patients with sCD40L, achieving an increase in the numbers of CD80+CD86+ cells. Our results demonstrate a tumor effect over the immune cells, with an important systemic effect. However, the approach used to stimulate B lymphocytes from patients present us with a possible immunotherapy.

Les Papillomavirus Humains (HPV) sont responsables de près de 100% des cancers du col utérin. Récemment, ces HPV sont apparus comme étant aussi la cause de certaines tumeurs des voies aérodigestives supérieures, et particulièrement des carcinomes épidermoïdes de l’oropharynx. En France, la proportion des tumeurs oropharyngées HPV-induites est mal connue, notamment parce que le dépistage viral n’est pas recommandé. De plus, il est difficile d’évaluer la proportion de tumeurs HPV positives dans les tumorothèques car les échantillons tumoraux sont fixés dans du formol puis inclus en paraffine (FFIP), ce qui complexifie les techniques de détection. Nous avons, au cours de nos travaux, testé une méthode de détection des HPV à haut risque oncogène indiquée pour le traitement des frottis en phase liquide. Nous l’avons mise à l’épreuve sur des prélèvements FFIP et comparée à la technique de référence qu’est la PCR (Polymerase Chain Reaction) suivie d’une électrophorèse sur gel. Nos résultats indiquent que cette technique est applicable aux prélèvements tissulaires et apparaît même comme étant plus sensible. En France, deux tiers des patients atteints de tumeurs des VADS sont pris en charge à des stades tardifs. Ceci s’explique en partie par l’absence de dépistage organisé de ces cancers. Nous avons donc mené une étude prospective sur des patients atteints d’une tumeur des VADS afin de tester le frottis oral comme technique de dépistage des cancers mais également des infections par les HPV. Nos résultats indiquent que le frottis a une spécificité proche de celle de la biopsie (94,4%) pour le dépistage des cancers des VADS, mais une moindre sensibilité (66,7%). Cette étude nous a permis de mettre en évidence une tumeur HPV-induite dans 12,2% des cas. Parmi eux, nous avons détecté grâce à un frottis buccal (en zone saine) une infection par un HPV à haut risque oncogène dans 53,3% des cas. L’OMS a classé les HPV comme agents carcinogènes depuis 1995, et a établi que les patientes ayant développé un cancer du col utérin avaient un risque 6 fois plus élevé de développer une autre tumeur HPV-induite. Dans ce contexte, nous avons prévu une étude prospective multi-centrique visant à dépister une infection orale par un HPV oncogène chez des patientes porteuses d’une lésion pré-néoplasique ou néoplasique du col utérin. Le taux de co-infection des deux sites anatomiques est inconnu chez les femmes infectées au niveau génital. Dans la mesure où l’infection orale pourrait être à l’origine d’une seconde localisation tumorale, il semble important d’en connaître la proportion afin de proposer par la suite un suivi particulier aux populations « à risque ». Au-delà des traitements des cancers avérés se pose la question de la vaccination préventive, qui existe contre les HPV 16 et 18 dans la prévention des cancers du col utérin. Le type 16 étant retrouvé dans 90% des tumeurs épidermoïdes de l’oropharynx HPV-induites, l’extension des recommandations vaccinales apparaît comme une nouvelle question de santé publique / The Human Papillomavirus (HPV) are involved in almost 100% of cervical cancers. Recently, HPVs have been recognized as the cause of tumors of the upper aerodigestive tract, especially of squamous cell carcinoma of the oropharynx. In France, the proportion of oropharyngeal HPV-related tumors is unknown, partly because viral testing is not in guidelines. Moreover, assess the proportion of HPV-positive tumors in tumor banks is difficult because the tumor samples were fixed in formalin and embedded in paraffin (FFPE), which complicates detection techniques. We tested a high risk HPV detection method, indicated for liquid based pap smear, on FFPE samples. We compared this technique to the gold-standard : PCR (Polymerase Chain Reaction) followed by electrophoresis. Our results indicate that this technique is applicable to FFPE samples and even appears to be more sensitive. The majority of French patients (2/3) with head and neck consult with an advanced stage of disease. This is explained in part by the lack of organized screening of these cancers, contrary to breast, prostate, cervical, or colorectal cancers. But an early treatment is essential to increase the survival rate. We therefore conducted a prospective study on patients with head and neck tumors to test the oral brushing as screening cancer and HPV detection. We found tumor and/or dystrophic cells in 97.8% of patients with biopsy, and in 88.9% of patients by brushing. Compared with biopsy, our results suggested that smear has similar specificity for HPV detection in tumors (94.4%), but lower sensitivity (66.7%). This study has shown an HPV-related tumor in 12.2% of cases. Among them, we detected by brushing (in healthy area) an oral infection by high-risk HPV in 53.3% of cases. WHO has classified HPV as carcinogenic agents since 1995, and determined that patients who developed cervical cancer are six-times more likely to develop another HPV-related tumor. In this context, we have planned a multicenter prospective study to detect oral HPV infection in patients with a pre-neoplastic or neoplastic lesion of the cervix. Co-infection rate of the two anatomical sites is unknown in women infected with genital level. Insofar oral infection could be the cause of a second tumor location, it seems important to know how much women are co-infected to propose thereafter a special monitoring. The preventive vaccination, which exists against HPV 16 and 18 in the prevention of cervical cancer, is a future perspective. Because HPV 16 is found in 90% of HPV-related squamous cell carcinoma of the oropharynx, extending vaccine recommendations emerge as a new public health issue