Thèses sur le sujet « HPV16 E7 »

To date, the success of cancer vaccines in human clinical trials has been limited. One of the reasons for this is the immunological tolerance to tumour antigens found in cancer patients. A novel DNA fusion vaccine design which links a pathogen-derived domain (DOM) of fragment C from tetanus toxin to a peptide epitope from a tumour antigen has been developed in our laboratory. The microbial sequence is able to activate a non-tolerised pool of helper T cells, providing T-cell help for immune induction against the linked tumour-specific sequence. The main aim of this project was to produce a therapeutic DNA vaccine against human papillomavirus (HPV)-associated cancers. A number of DNA fusion vaccines against the E7 antigen from HPV16 were constructed, including pDOM.E749-57, which encodes a well described H-2Db-binding epitope from E7 fused to the DOM sequence. CD8+ T-cell responses to the vaccines were demonstrated using flow cytometry and functional assays. Importantly, these responses were stronger than those induced by a published synthetic long peptide strategy. In vivo tumour challenge experiments showed that DNA vaccines had a protective and therapeutic effect. The vaccines were then tested in transgenic mice which develop spontaneous E7-expressing tumours in a setting of tolerance. DNA vaccine-mediated E7-specific CD8 + T-cell responses were successfully induced in these mice, together with a reduction in the mass of spontaneous tumours. This is the first demonstration of pDOM-epitope DNA vaccine-mediated therapy for spontaneous tumours and bodes well for translation into the clinic. One limiting factor for DNA vaccination in humans may be the delivery system. Electroporation (EP) is one approach which may overcome this. Therefore, a secondary aim of this project was to investigate the impact of EP on immune responses to DNA vaccination in more detail. EP proved essential for generating T-cell and antibody responses to the pDOM.E7 49-57 vaccine in sub-optimal conditions. This information will be crucial for the planning of therapeutic vaccination protocols in patients.

Thesis advisor: Junona Moroianu
Human papillomaviruses (HPVs) have been estimated to be the most common sexually transmitted infection in the United States. In addition to condyloma accuminata, infection of the squamous basal epithelium by high-risk HPVs, notably type 16 (HPV16), has been shown to be the primary etiological agent in the majority of cervical carcinomas. The E7 major transforming protein of HPV16, along with E6, has been linked to tumorigenesis and malignancy. While the E7 protein itself possesses no enzymatic activity, its ability to bind a number of nuclear and cytoplasmic targets subverts a variety of cellular regulatory complexes and facilitates viral replication. Previous studies in the Moroianu Lab have shown the HPV16 E7 oncoprotein to translocate across the nuclear pore complex (NPC) in a facilitated manner dependent on a non-canonical, c-terminal, nuclear localization signal (cNLS) for import, and a consensus leucine-rich nuclear export sequence (NES) for export (28). While the leucine-rich NES has been characterized, a full examination of the cNLS has yet to be performed. Here we present evidence that the karyopherin independent nuclear import mediated by the cNLS of 16E7 is dependent on its c-terminal Zn binding domain. Furthermore, we demonstrate that nuclear import is mediated by the direct interaction of a small patch of hydrophobic residues, 65LRLCV69, with the FG domain of the central FG-nucleoporin Nup62. In addition, we examined a potential regulatory mechanism of 16E7 nucleocytoplasmic translocation. Previous work has shown that a serine conserved in the high-risk HPVs at position 71 is phosphorylated by an unknown kinase. Here we present evidence that while phosphorylation of S71 is not required for either 16E7 nuclear localization or nuclear export in HeLa cells, mimicking phosphorylation of the S71 residue results in a statistically significant shift in the distribution of localization phenotypes of the resultant cell population toward a larger percentage exhibiting more nuclear localization. These data suggest that nucleocytoplasmic transport of 16E7 is, at least in part, a regulated process
Thesis (PhD) — Boston College, 2013
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

Human papillomavirus (HPV) is one of the most common sexually transmitted diseases in the world and has been associated with several human cancers, like cervical cancer. Thus, effective vaccination against Human papillomavirus represents an opportunity for the control of this cancer. The development of therapeutic Human papillomavirus vaccines is required to facilitate the control and eliminate on of a preexisting Human papillomavirus infection. In the last years, the expansion of efficient plasmid DNA purification processes has fostered therapeutics applications like gene therapy and DNA vaccination. Recently, the application of chromatographic operations based on affinity interactions between plasmid DNA or impurities with specific amino acids immobilized in stationary phases has demonstrated good results in the supercoiled plasmid DNA purification. Despite of selectivity achieved with these ligands, conventional matrices present limitations such as the low binding capacity and diffusivity for plasmid DNA samples. Owing to bottlenecks associated to conventional matrices, monolithic supports have emerged as interesting alternatives due to the versatility of their structural characteristics. The research work present in this thesis describes a new strategy that combines the selectivity of arginine as affinity ligand with the versatility of the epoxy-based monoliths to efficiently purify the supercoiled HPV-16 E6/E7 plasmid from other plasmid isoforms and Escherichia coli impurities present in clarified lysate. Additionally, breakthrough experiments were designed to compare the dynamic binding capacity of plasmid DNA to the conventional arginine-agarose matrix with the modified monolithic support. The dynamic binding capacity obtained for the arginine-epoxy monolith was significantly higher than the capacity achieved in the arginine conventional support. Quality control tests indicated that the plasmid sample resultant from the purification step presented a purity degree approximately 100% and an homogeneity higher than 97% of supercoiled isoform, with an extremely reduced level of impurities (RNA, proteins, genomic DNA and endotoxins). Overall, given that the plasmid DNA final product meets regulatory specifications, this combined support can be the key to obtain an adequate non-viral vaccine against a Human papillomavirus infection.
O Papiloma Vírus Humano é um vírus sexualmente transmissível que está relacionado com o desenvolvimento de vários cancros, como o cancro do colo do útero. Este tipo de cancro é a segunda maior causa de morte em mulheres, afetando mundialmente cerca de meio milhão de mulheres, das quais aproximadamente 274 mil morrem. A evidente associação que existe entre o Papiloma Vírus Humano e o cancro do colo do útero torna o Papiloma Vírus Humano um alvo interessante para o desenvolvimento de vacinas, no sentido de prevenir ou tratar o desenvolvimento do cancro. Atualmente, já são comercializadas duas vacinas preventivas, a Gardasil da Merck e a Cervarix da GlaxoSmithKline. Apesar de ambas mostrarem ser eficazes e seguras, possuem algumas limitações como elevado custo, não protegem contra todos os tipos de Papiloma Vírus Humano e trata-se de vacinas exclusivamente preventivas. Assim o desenvolvimento de vacinas terapêuticas pode ser uma estratégia promissora para colmatar estas falhas. A utilização do DNA plasmídico (pDNA) como uma vacina não viral tem-se tornado numa potencial estratégia terapêutica para prevenir ou tratar determinadas doenças de forma menos invasiva e segura comparando com os vetores virais. O mecanismo de actuação desta vacina baseia-se na expressão de proteínas antigénicas que desencadeiam uma resposta imunológica, evitando a progressão da doença . A preparação destas vacinas de DNA plasmídico requer o desenvolvimento de processos de produção e purificação que permitam obter grandes quantidades de plasmídeo na sua forma biologicamente ativa (isoforma superenrolada (sc)), cumprindo os requisitos das agências reguladoras no que diz respeito ao grau de pureza. Da ocorrência natural de complexos proteínas-DNA em sistemas biológicos sugeriu o desenvolvimento de uma estratégia de cromatografia de afinidade, utilizando matrizes convencionais de agarose com determinados aminoácidos imobilizados que reconhecem especificamente a isoforma superenrolada do DNA plasmídico. No entanto, as matrizes convencionais apresentam algumas limitações quando comparadas com os suportes monolíticos que possuem excelentes propriedades de transferência de massa e elevada capacidade de ligação para moléculas de grandes dimensões como o DNA plasmídico. Desta forma, o trabalho apresentado nesta tese consistiu na modificação de um monolito de epoxy por imobilização de aminoácidos de arginina, conjugando assim a selectividade deste ligando com a versatilidade do monolito de epoxy, tendo como finalidade purificar a isoforma sc do pDNA HPV-16 E6/E7. Numa fase inicial, foram realizados vários ensaios com amostras de DNA plasmídico pre-purificadas com o kit comercial, no sentido de avaliar e confirmar a presença dos ligandos de arginina, comparando o comportamento cromatográfico do monolito modificado com o mesmo suporte não modificado. Os resultados comprovaram que o monolito modificado reconhece especialmente o DNA plasmídico, permitindo a separação das isoformas superenrolada e circular aberta através de um gradiente por passos de NaCl. O mesmo não aconteceu com o monolito de epoxy não modificado, pois não ocorreu qualquer tipo de interacção do plasmídeo com o suporte. Posteriormente foi realizado um estudo de capacidade de ligação dinâmica para completar a caracterização do monolito com ligandos de arginina e comparar com a coluna convencional de arginina-argarose. Os resultados comprovaram que, para as mesmas condições de caudal e concentração de pDNA HPV-16 E6/E7, o monolito possui uma capacidade de ligação significativamente superior em comparação com a coluna convencional. Uma vez conseguida a separação das isoformas e a caracterização do monolito modificado com a arginina, a segunda fase do trabalho consistiu na purificação da isoforma sc do pDNA HPV-16 E6/E7 a partir de um lisado complexo de Escherichia coli. Os testes de controlo de qualidade revelaram que a amostra de sc de pDNA HPV-16 E6/E7, resultante da purificação com o monolito de epoxy modificado, apresentava um grau de pureza de aproximadamente 100% e uma homogeneidade superior a 97%. Para além disso, constituintes do hospedeiro como RNA e proteínas não foram detectados na amostra purificada e a quantidade de DNA genómico e endotoxinas estavam a baixo dos valores referenciados pelas agências reguladoras como a Food and Drug Delivery. Em suma, a combinação de ligandos de aminoácidos com suportes monolíticos pode ser uma solução promissora para obtenção de uma vacina não-viral baseada na isoforma sc do pDNA HPV-16 E6/E7, com o grau de pureza requerido para futuras aplicações terapêuticas contra a infecção por Papiloma Vírus Humano.

L'infection par le papilloma virus humain est associee au cancer du col uterin, la premiere cause de deces par cancer chez les femmes. L'oncoproteine e7 codee par le hpv-16 et responsable de la transformation des keratinocytes infectes, est exprimee a toutes les etapes, de la simple infection jusqu'au developpement du cancer. Dans la premiere partie du travail, nous demontrons que l'oncoproteine e7 exprimee par les cellules cancereuses du col uterin est relachee dans le milieu extracellulaire ou elle induit, in vitro, la surproduction par les apc de la cytokine immunosuppressive ifn- et inhibe la reponse immunitaire des cellules t. De plus, le e7 declenche la production, par les macrophages et les cellules dendritiques, des cytokines tnf-, il-1 et il-6, a effet angiogenique. La deuxieme partie du travail porte sur les effets de e7 sur les cellules endotheliales humaines issues de la microvascularisation du col uterin (crmven) et d'autres organes, afin d'en comparer les reponses. L'endothelium vasculaire joue un role central dans la dissemination des metastases. Nous demontrons que le e7 penetre dans les cellules endotheliales et se localise au niveau peri-nucleaire. De plus, l'incubation des cellules endotheliales avec e7 induit : 1) le remodelage du cytosquelette d'actine ; 2) l'expression des molecules d'adherence icam-1, vcam-1 et e-selectine, facteurs cle de la dissemination tumorale ; 3) la production des interleukines il-6 et il-8, facteurs de croissance des cellules tumorales et puissants facteurs angiogeniques. Les taux d'il-6 et d'il-8 apres l'incubation avec e7 sont significativement plus eleves dans les crmven que dans les cellules endotheliales d'autres organes, indice d'un effet organo-specifique de l'oncoproteine vis-a-vis de l'endothelium. Ainsi, nos resultats suggerent que le e7, presente dans le microenvironnement, pourrait jouer un role dans la progression tumorale, vu ses effets immunosuppressifs et l'activation selective des cellules endotheliales.

According to the American Cancer Society, it has been estimated that in 2019 alone, there will be approximately 53,000 new cases of oropharyngeal cancers. Oropharyngeal cancers are the largest subset of head and neck squamous cell carcinomas (HNSCCs), which are the sixth most common cancer across worldwide populations. They, along with other HNSCCs, fall under a category of cancers known as Human papillomavirus (HPV)-associated cancers, and it has been found that upwards of 70% of these cancers can be attributed to high-risk HPV infections. Specifically, the high-risk HPV gene, E7, plays a key role in relieving cell cycle repression by disrupting the DREAM complex via competitive binding with p130, driving the cell cycle and cell proliferation. In order to combat this interaction, a LIN52-S20C mutation was developed, in hopes of reducing E7 binding of p130 and stabilizing the DREAM complex. We utilized human cervical cell lines, immortalized keratinocytes, and mouse fibroblasts, all of which contained the HPV16 genome, as models to observe the effects of the LIN52-S20C mutation on HPV-mediated hijacking of the cell cycle. Not only were we able to replicate the increased proliferation and upregulated DREAM gene expression in infected cells, but we were also able to observe some reversal of these effects in many of our cell models through the expression of the LIN52-S20C variant. The findings of these studies have been promising and provide a basis for future works, and we hope that the effects of the LIN52-S20C mutation can be translated into studies in in vivo models.

O câncer cervical é o segundo câncer mais comum entre mulheres no mundo. A maioria dos casos (83%) ocorre em países em desenvolvimento, onde são encontrados em estágios relativamente avançados e, conseqüentemente, a sobrevida média é de cerca de 49% após cinco anos. Portanto, uma vacina eficaz contra as infecções pelo HPV pode levar ao controle do câncer do colo do útero. Apesar de prevenir, a vacina profilática não é acessível em função do alto custo, além de não eliminar o vírus em mulheres já infectadas pelo HPV. Assim, propusemos o desenvolvimento de uma vacina terapêutica eficaz utilizando duas abordagens: VLPs (virus-like particles) quiméricas, que poderiam apresentar propriedades profiláticas e terapêuticas, obtidas da fusão das proteína L1 e E7; proteínas quiméricas obtidas a partir da fusão de epítopos das proteínas E6 e E7 do HPV16, com e sem ubiquitina. Após subclonagens, com a obtenção dos vetores pPICHOLI-L1ΔCE71-50 e pPICHOLI-L1ΔCE743-77, partiu-se para a indução da expressão das VLPs quiméricas em Pichia pastoris, das quais não foram detectadas expressão protéica. Realizaram-se inúmeras modificações no protocolo de indução. Mesmo após essas alterações não foi detectada nenhuma expressão das fusões L1ΔCE71-50 ou L1ΔCE743-77. Como alternativa de uma vacina terapêutica, nos propusemos a expressar em E. coli proteínas sintéticas originadas da fusão entre epítopos das proteínas E6 e E7 do HPV16, com ou sem Ubiquitina, visando aumentar a apresentação de peptídeos via MHC de classe I de modo a estimular a eliminação de células infectadas com HPV16, evitando e regredindo o desenvolvimento dessas células cancerosas. Com a proteína E6E7 solúvel e purificada, realizou-se um ensaio de imunização. Nesse experimento, 20% dos animais imunizados com a proteína E6E7 não apresentaram desenvolvimento de tumor após a inoculação de células TC1. Assim isso nos leva a crer que com o aumento da concentração de proteína e utilização de adjuvantes seria possível aumentar o número de animais resistentes ao desenvolvimento do tumor. Em um segundo experimento de imunização, comparamos as proteínas E6E7 e E6E7Ub, em duas concentrações, 15 e 40 µg, e também com ou sem o adjuvante whole cell pertussis (WCP). Independentemente da concentração e presença ou ausência de WCP, os grupos imunizados com E6E7Ub apresentaram proteção contra o tumor entre 80% e 100% dos camundongos, enquanto os grupos imunizados com E6E7 apresentaram proteção entre 0% e 25%. Esses resultados são promissores, ainda que preliminares, indicando um potencial de uso da proteína E6E7Ub como imunógeno para vacina terapêutica contra o câncer cervical induzido por HPV16
Cervical cancer is the second most common cancer among women worldwide. Most cases (83%) occur in developing countries, where they are found in relatively advanced stages and, consequently, the median survival is about 49% after five years. Therefore, an effective vaccine against HPV infections can lead to control of cancer of the cervix. Although preventable, the prophylactic HPV vaccine is not accessible to all due to their high cost and in addition the vaccine does not eliminate the HPV in infected women. We have therefore proposed the development of effective therapeutic vaccines using two approaches: chimeric VLPs (virus-like particles), endowed with prophylactic and therapeutic properties, obtained from the fusion protein L1 and E7; chimeric proteins derived from the fusion of epitopes of proteins E6 and E7 of HPV16 with and without ubiquitin. After subcloning, we obtained the vectors pPICHOLI-L1ΔCE71-50 and L1 pPICHOLI- L1ΔCE743-77. After transformation of yeast Pichia pastoris with these constructions, the cells were induced, but it was not possible to detect any recombinant protein expression. As an alternative, we proposed the expression of synthetic proteins in E. coli derived from the fusion between epitopes of E6 and E7 proteins of HPV16 with or without Ubiquitin, in order to enhance the presentation of peptides through MHC class I to stimulate the elimination of HPV16-infected cells, preventing and regressing the development of cancer cells. Soluble E6E7 protein was purified and, 20% of the animals immunized with this protein did not develop tumor after inoculation of TC1 cells. In a second immunization experiment we compared the proteins E6E7 and E6E7Ub, in two concentrations, 15 and 40µg, with or without the adjuvant whole cell pertussis (WCP). Regardless of concentration and presence or absence of WCP, all the groups immunized with E6E7Ub showed protection against tumor between 80% and 100%, while the groups immunized with E6E7 showed protection from 0% to 25%. These results are promising and although preliminary, indicate the potential of E6E7Ub protein as an immunogen, for a therapeutic vaccine against cervical cancer induced by HPV16

Negli ultimi anni sono emerse evidenze che supportano un ruolo di HPV nella patogenesi del cancro al polmone ed è stata riscontrata la presenza e l’espressione, attraverso distinte linee di ricerca, degli oncogeni di HPV nei tumori polmonari supportando l’ipotesi che forme oncogeniche del virus possano agire come cofattori nel processo carcinogenico. Per chiarire il ruolo di HPV nello sviluppo del tumore del polmone abbiamo utilizzato la linea cellulare polmonare A549 come modello cellulare e, dopo infezione con costrutti esprimenti gli oncogeni E6, E7 ed E6/E7 di HPV16, con un approccio di tipo proteomico, abbiamo studiato i cambiamenti nel profilo di espressione proteica delle diverse linee cellulari infettate, rispetto alla controparte normale, associate alla presenza degli oncogeni. Dall’analisi dei replicati dei gels bidimensionali tra le cellule non infettate e infettate stabilmente con HPV16E6/E7, si sono trovate 17 proteine differenzialmente espresse di almeno 2 volte, la cui intensità media normalizzata fosse statisticamente significativa (p<0.05). L’identificazione delle proteine è stata effettuata con esperimenti di spettrometria di massa MALDI-TOF-MS e nLC-ESI-Q-TOF-MS/MS. Le possibili relazioni e associazioni funzionali tra le proteine espresse differenzialmente nelle linee infettate con gli oncogeni di HPV16 sono state valutate tramite il programma bioinformatico Ingenuity Pathway Analysis. Il risultato, derivante da tutte e tre le diverse condizioni di infezione, suggerisce un coinvolgimento funzionale di processi di inibizione dell’apoptosi e le proteine Annexin IV, Gp96, Hsp27 e Tumor protein-translationally controlled 1 identificate come regolatori principali della sopravvivenza cellulare e inibizione della morte cellulare programmata.
In recent years data have accumulated implicating the involvement of oncogenic HPVs in bronchial carcinogenesis and the presence and expression of oncogenic HPV transcripts in non-small cell lung cancers have been reported throughout distinct studies. Taken together these data seem to support the hypothesis that oncogenic HPVs could act as co-factor in lung carcinogenesis. To further understand the role of HPV in the development of lung cancer we employed the lung cell line A549 stably infected with HPV16E6, HPV16E7 and HPV16E6/E7 constructs to investigate by a proteomic approach the protein profile changes associated with the expression of these oncogenes. Replicated 2-DE gels from uninfected and stably HPV16E6/E7 infected A549 cells were compared for changes in protein profile. We identified 17 different polypeptides whose average normalized spot intensity was statistically significant (p<0.05) and differed by 2- fold. The protein identification was achieved by peptide mass fingerprinting by MALDI-TOF-MS and nLC-ESI-Q-TOF-MS/MS peptide ladder sequencing Relationships between differentially expressed proteins and the HPV-induced infection mechanism have been clustered by knowledge-base database functional association network analysis. The results, deriving from the networks obtained from all three different infection conditions, suggested the functional involvement of a cell death inhibition pathway with central nodes including Annexin IV, Gp96, Hsp27 and Tumor protein-translationally controlled 1 as major key proteins for cell viability and inhibition of apoptosis pathway.

O potencial oncogênico do papilomavírus humano (HPV) baseia-se na capacidade das oncoproteínas virais E6 e E7 alterarem o ciclo celular, levando à imortalização e malignidade das células. O importante papel das oncoproteínas na progressão tumoral e na interação com inúmeros alvos celulares tem relevância em estudos para o desenvolvimento de vacinas e terapias contra os cânceres associados ao HPV. Este estudo investigou a localização intracelular das oncoproteínas E6 e E7 de HPV16/18 e seus possíveis alvos celulares. Demonstrou a presença de E6 nuclear, citoplasmática e intramitocondrial, tanto em células naturalmente transformadas por HPV, como em células transfectadas com o oncogene E6 viral. E7 foi detectada no núcleo e citoplasma, porém nunca ocorreu E7 intramitocondrial. Confirmou a hipótese da presença intramitocondrial da oncoproteína viral E6 de HPV16/18 de alto risco. Dado inédito cuja relevância está relacionada com a aplicação clínica, no desenvolvimento de imunobiológicos e fármacos capazes de neutralizar a ação deste importante alvo terapêutico.
The oncogenic potential of HPV is based on the capacity of viral oncoproteins E6 and E7 to change cellular cycle leading to immortality and malignancy. The important role of oncoproteins in tumor progression and its interaction with numerous cellular targets have relevance in studies to the development of vaccines and therapies against HPV associated cancers. This study investigated intracellular localization of E6 and E7 HPV16/18 oncoproteins and its possible cellular targets. It showed the presence of E6 in the cellular nucleus, cytoplasm, and intramitochondrial in naturally HPV transformed cell, as well as in cells transfected with E6 viral oncogene. E7 was detected inside nucleus and cytoplasm, but E7 intramitochondrial did not occur. This study confirmed the hypothesis of the intramitochondrial presence of E6 viral oncoprotein from high risk HPV. This is an original data whose relevance is directly related to clinical application in the development of immunobiologicals and drugs, which are able to neutralize the action of this important therapeutic target.

Thesis advisor: Junona Moroianu
In this study we investigated the hydrophobic interactions between HPV11 E7 and the FG regions of Nup62N through transfection assays with EGFP-11E7 fusion plasmids in HeLa cells and binding assays with GST-Nup62N immobilized on Glutathione-Sepharose beads. We found that EGFP-11cE7 binds to Nup62N. This suggests a possible mechanism for the nuclear import of HPV11 E7 through direct hydrophobic interactions between its carboxy-terminus and the FG region of Nup62. The interaction between HPV11 E7 and CRM1 nuclear export receptor was also examined using similar methods. Binding between these proteins suggest that nuclear export of 11E7 is mediated by CRM1 binding to its leucine-rich nuclear export signal (NES)
Thesis (BS) — Boston College, 2013
Submitted to: Boston College. College of Arts and Sciences
Discipline: College Honors Program
Discipline: Biology

Du Jing.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2003.
Includes bibliographical references (leaves 70-89).
Abstracts in English and Chinese.
ABSTRACT --- p.I
ACKNOWLEDGMENTS --- p.IV
PUBLICATIONS --- p.V
LIST OF FIGURES --- p.VI
LIST OF TABLES --- p.VII
ABBREVIATIONS --- p.VIII
CONTENTS --- p.X
Chapter CHAPTER ONE: --- INTRODUCTION AND LITERATURE
Chapter 1.1 --- Laryngeal carcinoma and HPV --- p.1
Chapter 1.2 --- HPV --- p.2
Chapter 1.3 --- Human papillomavirus E6 protein --- p.6
Chapter 1.3.1 --- Transformation by HPV E6 --- p.7
Chapter 1.3.2 --- Inhibition of apoptosis by E6 --- p.8
Chapter 1.3.3 --- Alteration of gene transcription --- p.11
Chapter 1.3.4 --- E6 interation with other proteins --- p.12
Chapter 1.3.5 --- E6 as a therapeutic target --- p.14
Chapter 1.4 --- HPV E7 protein --- p.15
Chapter 1.4.1 --- Regulation of viral life cycle by HPV E7 --- p.16
Chapter 1.4.2 --- Degradation of retinoblastoma tumor suppressor by HPV E7 --- p.18
Chapter 1.4.3 --- Inhibition of p53 by HPV E7 --- p.22
Chapter 1.4.4 --- Interaction with other proteins by HPV E7 --- p.24
Chapter 1.5 --- Objective --- p.26
Chapter CHAPTER TWO: --- GENERAL MATERIALS AND METHODS --- p.28
Chapter 2.1 --- Materials --- p.28
Chapter 2.1.1 --- Materials for cDNA and RNA manipulation --- p.28
Chapter 2.1.2 --- Culture media and transfection reagents --- p.28
Chapter 2.1.3 --- Antibodies --- p.29
Chapter 2.1.4 --- Materials for protein manipulation --- p.29
Chapter 2.1.5 --- Kits --- p.30
Chapter 2.1.6 --- Instrumentation --- p.31
Chapter 2.2 --- Methods --- p.32
Chapter 2.2.1 --- Plasmid construction --- p.32
Chapter 2.2.1.1 --- DNA preparation --- p.34
Chapter 2.2.1.2 --- DNA ligation --- p.34
Chapter 2.2.1.3 --- Transformation of competent E. coli --- p.35
Chapter 2.2.2 --- Mini preparation --- p.35
Chapter 2.2.3 --- Clone selection and confirmation --- p.37
Chapter 2.2.4 --- Sequencing gel electrophoresis --- p.37
Chapter 2.2.5 --- Cell culture and cytokine treatment --- p.39
Chapter 2.2.6 --- Plasmid transfection --- p.39
Chapter 2.2.7 --- Confirming construction of stable cell lines by RT-PCR --- p.40
Chapter 2.2.7.1 --- Total cellular RNA extraction --- p.40
Chapter 2.2.7.2 --- First strand cDNA synthesis --- p.41
Chapter 2.2.7.3 --- Polymerase chain reaction (PCR) --- p.41
Chapter 2.2.8 --- Fluorescence microscopy and imaging --- p.43
Chapter 2.2.9 --- DNA fragmentation assay --- p.44
Chapter 2.2.10 --- Protein detection --- p.46
Chapter 2.2.10.1 --- Preparation of protein extract --- p.46
Chapter 2.2.10.2 --- SDS-PAGE electrophoresis and protein transfer --- p.47
Chapter 2.2.10.3 --- Immunoblotting analysis --- p.47
Chapter 2.2.11 --- Statistical analysis --- p.48
Chapter CHAPTER THREE: --- RESULTS --- p.49
Chapter 3.1 --- Plasmid construction --- p.49
Chapter 3.2 --- Expression of HPV16 viral oncogenes in transfected UMSCC12 --- p.51
Chapter 3.3 --- HPV16 E6 and E7 protect apoptosis induced by TNF-alpha and CHX --- p.53
Chapter 3.4 --- Detection of apoptosis with fluorescence staining --- p.55
Chapter 3.5 --- Regulation of the expression of apoptosis-associated proteins by E6 and E7 oncoproteins --- p.57
Chapter CHAPTER FOUR: --- DISCUSSION --- p.59
Chapter CHAPTER FIVE: --- CONCLUSION AND FUTURE PERSPECTIVE --- p.68
REFERENCES --- p.70
APPENDIX DNA SEQUENCING RESULTS --- p.90

IGFBP-3 expressing rekombinant vaccinia viruses used for tumor therapy Insulin-like growth factor-binding protein-3 (IGFBP-3) is a major regulator of endocrine effects of IGF and is capable to suppress the growth of variety of cancer. Several studies have shown that IGFBP-3 can induce the apoptosis of cancer cells via IGF-dependent and IGF-independent mechanisms. In our study, we have constructed recombinant vaccinia viruses (VACV) expressing IGFBP-3 under the control of the early H5 and synthetic early/late (E/L) promoter to investigate the potential effect on cancer growth in our cervical cancer model. We have shown that the expression of IGFBP-3 alone had no effect on tumor growth. On the other hand, the co-expression of IGFBP-3 enhanced the anti-cancer effect of immunization with the fusion protein SigE7LAMP, which gave rise to the anti-cancer immunity directed against HPV16 induced tumors. We have shown that the double-recombinant P13-SigE7LAMP-H5-IGFBP-3 can enhance the protective immune responses against MK16/ABC induced tumors. Furthermore, we have show that both double-recombinant viruses P13-SigE7LAMP-H5- IGFBP-3 and P13-SigE7LAMP-E/L-IGFBP-3 can increase the anti-cancer effect of SigE7LAMP expression in the therapy of TC-1 induced tumors. Key words: IGFBP-3, IGF, VACV, HPV16, E7 oncoprotein,...

Modulation of the tumor microenvironment represents a possible way to inhibit cancer growth and enhance anti-cancer immune responses. In the presented work we employ two strategies for tumor microenvironment modulation. Firstly, we have constructed rVACV co-expressing the tumor suppressor gene insulin-like growth factor-binding protein-3 (IGFBP- 3) and the fusion gene encoding the immunogen SigE7LAMP. The expression of IGFBP-3 was regulated either by the early vaccinia virus H5 promoter or by the synthetic early/late (E/L) promoter. We have shown that expression of IGFBP-3 regulated by the H5 promoter yielded higher amounts of IGFBP-3 protein when compared with the E/L promoter. Immunization with P13-SigE7LAMP-H5-IGFBP-3 was more effective in inhibiting the growth of TC-1 tumors in mice and elicited a higher T-cell response against VACV-encoded antigens than the control virus P13-SigE7LAMP-TK- . We found that high-level production of IGFBP-3 enhanced virus replication both in vitro and in vivo, resulting in profound antigen stimulation. Production of IGFBP-3 was associated with a higher adsorption rate of P13-SigE7LAMP-H5-IGFBP-3 to CV-1 cells when compared with P13-SigE7LAMP-TK- . We have identified two structural differences between the IMVs of the IGFBP-3 expressing virus P13-SigE7LAMP-H5-IGFBP-3...

A constante evolução da ciência tem permitido uma melhor partilha de conhecimentos na área da tecnologia do DNA recombinante, fornecendo um melhor conhecimento da informação contida nos genes e o impacto que alterações nesses genes poderão ter no organismo. A descodificação do genoma humano aliada ao progresso obtido no desenvolvimento de variados vetores de transporte de informação genética permitiu a evolução de terapias baseadas na entrega de genes terapêuticos, como a terapia génica e as vacinas de DNA. O desenvolvimento destas terapias trouxe uma nova esperança para o tratamento de certas patologias que, até então, permaneciam como intratáveis. Vetores biológicos e não biológicos têm evoluído largamente nos últimos anos, no entanto, a toxicidade demonstrada pela maioria dos vetores biológicos tem levado a um aumento de utilização de vetores não biológicos. O DNA plasmídico destaca-se entre os diversos vetores genéticos devido à simplicidade da sua produção, obtenção, baixo custo e ausência de toxicidade. As vantagens deste vetor têm levado a que a sua utilização como vacina de DNA tenha aumentado nos últimos anos, tornando-o o vetor de escolha na maioria dos estudos de investigação. As vacinas de DNA têm como modo de atuação a expressão de proteínas antigénicas com o objetivo de induzir uma resposta imunitária direcionada para essas mesmas proteínas, permitindo a prevenção e/ou tratamento de infeções virais e bacterianas. Torna-se imperativo o desenvolvimento de tecnologias que permitam a produção e purificação destes vetores, obtendo a maior percentagem de recuperação e pureza possíveis do plasmídeo na sua forma biologicamente ativa, a isoforma superenrolada (sc). A área da cromatografia tem progredido bastante no desenvolvimento de estratégias eficazes de purificação de plasmídeo, permitindo o aumento de produtividade e obtenção deste vetor e diminuindo eventuais custos associados à sua produção. O Vírus do Papiloma Humano (HPV) é um vírus sexualmente transmissível que se encontra atualmente associado ao desenvolvimento de massas tumorais devido à produção de duas proteínas oncogénicas, oncoproteínas E6 e E7, capazes de alterar o ciclo de proliferação celular e de provocar o crescimento anormal de células do organismo infetado. A tecnologia de vacinas de DNA apresenta-se assim como uma terapia promissora para infeções provocadas pelo HPV, através da indução de uma resposta imunitária contra as proteínas referidas. Recentemente, o nosso grupo de investigação conseguiu desenvolver de forma eficaz a produção e purificação da vacina de DNA sc HPV-16 E6/E7 através da utilização de um monolito modificado com ligandos de arginina, tirando partido dos princípios básicos da cromatografia de afinidade. Contudo, a recuperação do plasmídeo não foi a esperada, tendo sido apenas recuperado 39% da molécula alvo. O Desenho experimental é uma ferramenta estatística que, através da escolha correta dos fatores a serem avaliados, bem como os seus intervalos em estudo, permite a otimização de respostas de um sistema experimental. Deste modo, através do design experimental foi feita uma otimização ao sistema de purificação da vacina de DNA sc HPV-16 E6/E7 de modo a garantir um aumento de recuperação da molécula, mantendo o elevado nível de pureza. Com esse intuito, após uma avaliação inicial dos fatores e dos intervalos a serem usados, o design ‘Central Composite Face’ (CCF) foi utilizado para delinear um conjunto de experiências cromatográficas de modo a encontrar o ponto ótimo para a percentagem de recuperação do plasmídeo ser maximizada. A otimização foi bem-sucedida, permitindo a obtenção de uma percentagem de recuperação de cerca de 83%, mantendo-se a percentagem de 100% para a pureza. Após a otimização da estratégia de purificação, estudos de transfeção in vitro foram realizados de modo a avaliar a capacidade de transfeção celular e consequente expressão da proteína codificada pelo gene-alvo contido na vacina de DNA. Células CHO-1, isoladas a partir de tecido ovárico de rato chinês, foram cultivadas e transfetadas com a isoforma sc purificada através da estratégia otimizada com o monolito de arginina, bem como com a isoforma circular aberta (oc) e DNA plasmídico obtido através de um kit comercial, de modo a avaliar qual a melhor estratégia para transfeção. Através das técnicas de western blot e imunocitoquímica foi possível verificar que a entrada do pDNA nas células eucarióticas ocorreu com sucesso (processo de transfeção), observando-se um aumento significativo de expressão génica das proteínas E6 e E7 em comparação ao grupo de controlo (células não transfetadas). A avaliação da expressão génica da proteína E6 dos diferentes tipos de plasmídeos utilizados permitiu verificar que o aumento de expressão desta proteína foi mais significativo com a amostra de plasmídeo sc purificado pelo monolito de arginina, concluindo-se que de facto a isoforma sc induz uma maior eficiência de transfeção.
The infection by Human Papilloma Virus (HPV) is associated with the development of different tumours, in particular the cervical cancer. Oncoproteins E6 and E7, produced by this virus, are responsible for the disturbance of the cell cycle, through interaction with several proto-oncogenes, leading to uncontrolled proliferation of the infected host cells. Therefore, the development of a suitable therapy against HPV infection with these oncoproteins is a promising strategy. DNA vaccines arise as a potential therapeutic solution in cancer treatment, being able to trigger a strong immune response against the target antigen, normally expressed by the infected cells. The purification of supercoiled (sc) plasmid HPV16 E6/E7 DNA vaccine with the arginine monolith was recently developed by our research group. In spite of achieving 100% purity, only 39% of the target molecule was recovered. Experimental design is a new tool able to project several experiments, by evaluating and combining different factors, with the intent of improving and optimizing a given experiment. Through the use of Composite Central Face design and the choice of three factors to be evaluated, such as binding step, washing step and pH, different experiments were performed in order to achieve the optimal range for the sc HPV16 E6/E7 purity and recovery. The aim was successfully achieved with 83% of recovery and 100% of purity. Thereafter, transfection studies were performed in order to evaluate the plasmid DNA (pDNA) vaccine efficiency. Several plasmid samples obtained from different purification methods were tested: plasmid purified by a commercial kit, open circular isoform (oc) and sc isoform purified by our optimized strategy with the arginine monolith. After 72 hours of transfection, the expression of E6 protein in CHO-1 cells was evaluated through immunocytochemistry. Through immunofluorescence comparison, higher E6 protein expression was detected by sc pDNA, showing a significant increase, when compared to control group. On the other hand, pDNA purified with the commercial kit and oc pDNA had no significant immunofluorescence different in comparison with control group. These data suggest that the sc pDNA obtained by our optimized purification strategy is able to efficiently transfect cells and express the target proteins, encouraging us to proceed to in vivo studies in order to evaluate the immunogenicity of this DNA vaccine.