Made available in DSpace on 2014-07-29T15:29:04Z (GMT). No. of bitstreams: 1 DISSERTACAO - HUGO DELLEON-1.pdf: 1681733 bytes, checksum: ec21194046431954674916776c620d9e (MD5) Previous issue date: 2009-05-27 / Although water is of vital importance for living beings, due to antropic action it becomes a way of dissemination of several microorganisms, which reach aquatic environments through the faeces of man and other animals and can cause several illnesses, especially for immunocompromised individuals. During routine environmental monitoring, coliform bacteria are normally used as a microbiological parameter of water quality, which does not evidence its contamination by viruses. Several researchers have proposed the detection of adenovirus (AdVs) by PCR as a molecular index to monitor other enteric viruses. AdVs are among the most persistent and ubiquitous enteric viruses present in water and associated with a variety of clinical manifestations. This study aimed to evaluate the quality of water collected from lakes and rivers in Goiânia as to the occurrence of AdVs. Water samples were collected monthly, from December 2007 to November 2008, at five different points in Goiânia (lakes of Bosque dos Buritis and Vaca Brava park, João Leite and Meia Ponte rivers downstream and upstream the municipal sewage treatment plant). The analyses were carried out at the Laboratório de Diagnóstico Genético e Molecular and Laboratório de Genética Molecular e Citogenética, Universidade Federal de Goiás. All the samples were filtered in a positively-charged nylon membrane followed by molecular detection using PCR and semi-nested PCR. Also, we performed physical-chemical and bacteriological tests to correlate these results with the occurrence of AdVs. Simultaneously, the Núcleo de Pesquisas em Agentes Emergentes e Re-emergentes carried out a meta-analysis to evaluate three methods of concentration of AdVs coupled to molecular detection in samples of untreated water. Since 29 out of the 54 water samples collected were positive for AdVs (39.2%), our results suggest the use of the methodology proposed in the present study for the detection of these pathogens in water. We observed statistically significant difference between nitrites, phosphates, fixed residues, total residues and the occurrence of AdVs, whereas no correlation was observed between fecal coliforms and AdVs. Furthermore, the occurrence of AdVs in the state of Goiás shows a seasonal trend. Based on the 33 studies selected for the meta-analysis, it was possible to get to the following interpretations: the most effective method to detect AdVs in samples from rivers or lakes was ultracentrifugation combined with nested-PCR; it is advisable to use a combination of microfiltration membrane and ultrafugation with the subsequent diagnosis using qPCR to detect AdVs in samples of treated and untreated sewage. This has been the first study carried out for the detection and monitoring of AdVs in water bodies in the Midwestern Region of Brazil and the present results may be useful to propose the eco-epidemiological profile of AdVs or even the routes of some neglected diseases, which points out the need to define a virus indicator / Embora a água seja de vital importância para os seres vivos, em decorrência da ação antrópica torna-se meio de disseminação de inúmeros microrganismos, que chegam aos ambientes aquáticos por meio das fezes do homem e de outros animais, podendo desencadear diversas doenças, sobretudo em indivíduos imunocomprometidos. Em rotina de monitoramento ambiental, normalmente são utilizadas bactérias do grupo dos coliformes fecais como parâmetro microbiológico de qualidade das águas, o que não evidencia sua contaminação por vírus. Alguns pesquisadores têm proposto a detecção de adenovírus (AdVs) por PCR como indexação no monitoramento de outros vírus entéricos. AdVs estão entre os mais persistentes e ubíquos vírus entéricos presentes em águas e associados com uma variedade de manifestações clínicas. Objetivou-se avaliar a qualidade da água de rios e lagos da cidade de Goiânia em relação à ocorrência de AdVs. Amostras de água foram coletadas mensalmente, entre dezembro de 2007 e novembro de 2008, em cinco diferentes pontos de Goiânia (lagos do Bosque dos Buritis e parque Vaca Brava e rios João Leite e Meia Ponte a jusante e montante da estação de tratamento de esgoto municipal). As análises laboratoriais foram realizadas pelo Laboratório de Diagnóstico Genético e Molecular e Laboratório de Genética Molecular e Citogenética, Universidade Federal de Goiás. Inicialmente, as amostras foram filtradas em membrana de nylon carregada positivamente e, em seguida, submetidas a detecção molecular por PCR e semi-nested PCR. Também foram realizados testes físico-químicos e bacteriológicos para correlacionar estes resultados com a ocorrência de AdVs. Concomitantemente, o Núcleo de Pesquisas em Agentes Emergentes e Re-emergentes conduziu uma metanálise avaliativa de três metodologias de concentração de AdVs acopladas à detecção molecular em amostras de águas não tratadas. Das 54 amostras coletas, 29 foram positivas para AdVs (39,2%), sugerindo o uso da metodologia proposta neste estudo para detectar estes patógenos em água. Foi observada diferença estatisticamente significativa entre nitritos, fosfatos, resíduos fixos, resíduos totais e a ocorrência de AdVs, mas não foi observada correlação entre coliformes fecais e AdVs. Além disso, a ocorrência de AdVs no estado de Goiás exibiu tendência à sazonalidade. Na metanálise, a partir dos 33 artigos selecionados foi possível fazer as seguintes interpretações: para detectar AdVs em amostras de rios ou lagos, o método mais eficiente foi a ultracentrifugação em combinação com nested-PCR; para a detecção de AdVs em amostras de esgoto tratado e não tratado é aconselhável utilizar uma combinação de microfiltração em membrana e ultrafiltração com subsequente diagnóstico por qPCR. Este foi o primeiro estudo a detectar e monitorar AdVs em cursos de água na Região Centro-Oeste do Brasil e os presentes resultados podem ser úteis para propor o perfil eco-epidemiológico dos AdVs ou até mesmo das rotas de algumas doenças que são negligenciadas, o que demonstra a necessidade de definir um indicador viral

Orientador: Rodrigo Pessoa Cavalcanti Lira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T10:10:15Z (GMT). No. of bitstreams: 1 Fulco_EnzoAugustoMedeiros_M.pdf: 625645 bytes, checksum: 871772c97dcfc7b49d6bc56d1a72c439 (MD5) Previous issue date: 2011 / Resumo: Introdução: Corticosteróides tópicos são utilizados comumente no tratamento da ceratoconjuntivite viral aguda. Tem sido sugerida a utilidade dos corticosteróides no tratamento sintomático da conjuntivite alívio dos sinais e/ou sintomas e prevenção dos infiltrados subepiteliais. Por outro lado, observou-se o relato dos possíveis efeitos colaterais, como o prolongamento da transmissão invitro do vírus e, no âmbito da medicina clínica, ensaios clínicos revelaram a eficácia duvidosa dos colírios de corticosteróides. O objetivo deste estudo foi comparar o uso dos corticosteróides tópicos, com quaisquer drogas usadas nos ensaios clínicos com o placebo. Objetivo: Avaliar se os corticosteróides tópicos são eficazes e seguros para o tratamento da ceratoconjuntivite adenoviral para melhorar os sintomas e evitar ou minimizar complicações relacionadas à doença. Desenho: Revisão sistemática. Métodos: Pesquisa documental na Cochrane Central Register of Controlled Trials (CENTRAL) (que contém o Cochrane Eyes and Vision Group Trials Register), MEDLINE, EMBASE, PubMed, nas listas de referência de relatórios de ensaio identificados e no o Science Citation Index. Foram incluídos ensaios clínicos aleatorizados comparando quaisquer apresentações de corticosteróides tópico com quaisquer outras formas de tratamentos da ceratoconjuntivite adenoviral aguda. Resultados: Foram incluídos ensaios clínicos randomizados onde os corticosteróides tópicos foram comparados com placebo no tratamento da ceratoconjuntivite adenoviral aguda. A busca digital inicial identificou quatro ensaios clínicos comparando corticosteroides e placebo no manejo da ceratoconjuntivite epidêmica somando 243 pacientes. Uma revisão sistemática foi realizada. Conclusão: Nenhum estudo mostrou melhora no alívio dos sinais ou sintomas. A prevenção dos infiltrados subepiteliais permanece controversa, mostrando mais comumente um adiamento na história natural da doença do que uma modificação nela. O uso destes colírios deve ser recomendado com cautela e novos ensaios clínicos são necessários para comprovar sua eficácia / Abstract: Introduction: Topical corticosteroids are commonly used in the treatment of acute viral keratoconjunctivitis. It has been suggested the usefulness of corticosteroids in symptomatic relief of the signs of conjunctivitis and / or symptoms and prevention of subepithelial infiltrates. On the other hand, we observed the reported possible side effects, such as the extension of transmission of the virus in vitro, and beside that, clinical trials showing the effectiveness of corticosteroids eye drops. The aim of this study was to compare the use of topical corticosteroids, with any drugs used in clinical trials with placebo. Objective: To assess whether topical corticosteroids are effective and safe for the treatment of adenoviral keratoconjunctivitis to improve symptoms and prevent or minimize complications related to the disease. Design: Systematic review. Methods: We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (which contains the Cochrane Eyes and Vision Group Trials Register), MEDLINE, EMBASE, PubMed and the reference lists of identified trial reports. We used the Science Citation Index to look for articles that cited the relevant studies. We included masked randomized controlled trials in which any form of topical corticosteroid treatment had been compared with placebo in the management of acute adenoviral ceroconjuctivitis. Results: Were included randomized controlled trials in which any form of topical corticosteroid treatment had been compared with placebo in the management of acute adenoviral keratoconjunctivitis. The initial digital search identified 4 clinical trials comparing corticosteroids and placebo in the management of Epidemic keratoconjunctivitis totaling 243 patients. A narrative review was conducted. Conclusion: No study has shown improvement in relief of the signs or symptoms. Prevention of subepithelial infiltrates remains controversial, most commonly showing a delay in the natural history of disease than a change in it. The use of eye drops should be recommended with caution and new clinical trials are needed to prove its effectiveness / Mestrado / Oftalmologia / Mestre em Ciências Médicas

Les pathogènes neurotropiques représentent une banque d’outils biologique afin de cibler spécifiquement le système nerveux central (SNC), pour son étude mais aussi dans l’optique d’une thérapie. Parmi eux, l’adénovirus canin de type 2 (CAV-2) est un vecteur prometteur pour cibler le SNC. CAR a été principalement étudié en tant que récepteur viral. Cependant, plusieurs études montrent que CAR est essentiel dans le développement du cœur ainsi que du système lymphatique. De manière intéressante, CAR est fortement exprimé pendant le développement du SNC, suggérant un rôle dans l’établissement des réseaux neuronaux. Dans ce travail, nous avons confirmé que CAR est lié aux mécanismes d’endocytoses et au trafic intracellulaire. L’endocytose de CAR est ligand dépendant. La partie intracellulaire de CAR régule son endocytose. Nos données suggèrent que CAR est l’unique récepteur pour CAV-2. Le présent travail de recherche montre aussi que CAR ne semble pas participer à la formation du SNC. En revanche, au niveau du SNC mature, CAR est impliqué dans la plasticité synaptique, dans la neurogénèse adulte et participe à l’homéostasie des synapses, mécanismes impliqués dans les processus mnésiques. / The coxsackievirus and adenovirus receptor (CAR) is a single-pass transmembrane protein belonging to the CTX subfamily of the immunoglobulin superfamily. CAR has been extensively studied as a viral receptor for coxsackie B viruses and some adenoviruses (AdVs). CAR is essential for the development of the cardiovascular and lymphatic system. Interestingly, CAR is highly expressed in the developing brain and has been hypothesized to regulate the establishment of the neuronal networks. In my PhD work, I showed that CAR can be link to the endocytic pathways and intracellular trafficking. CAR endocytosis is ligand-dependent and is regulated by CAR intracellular domain (ICD), suggesting strongly that CAR is most likely the unique receptor for canine adenovirus type 2 (CAV-2). Moreover, we demonstrated that CAR depletion in the developing brain did not significantly perturb brain development. In the healthy adult brain, CAR is relatively abundant and we demonstrated that CAR loss of function affected hippocampal plasticity, adult neurogenesis and synapse homeostasis, which affect cognition.

Les Adénovirus humains sont des virus non enveloppés se répliquant dans le noyau des cellules hôtes.Durant l’infection et après leur entrée par endocytose, les Adénovirus sont transportés au noyau pourinitier l’expression du génome viral. Dans l’endosome, les capsides virales subissent un désassemblagepartiel et libèrent le facteur viral lytique, la protéine VI (pVI). Au niveau de la membrane de l’endosome,cette protéine va alors induire sa rupture permettant ainsi le relargage des virions au sein du cytoplasmegrâce à son hélice amphipatique N-terminale. Par la suite, pVI est transportée vers des structuresnucléaires appelées PML nuclear bodies (PML-NB), associée une ubiquitine ligase cytoplasmique, laNedd4.2. Les PML-NB sont des complexes nucléaires multi-protéiques qui ont des propriétésantivirales. Celles-ci impliquent le recrutement de facteurs de transcription répressifs comme parexemple la protéine anti apoptotique Daxx ou encore le suppresseur de tumeur p53, impliqué dans larégulation du cycle cellulaire. Il a été montré que la protéine pVI en complexe avec Nedd4.2 induit larelocalisation de Daxx des PML-NB dans le cytoplasme, ce qui permet une expression efficace dugénome viral. Ainsi, l’inhibition fonctionnelle de Daxx par pVI suggère que cette protéine virale puisseaussi être impliquée dans la restriction de p53.Dans cette étude, nous avons montré que le nombre des modifications post-traductionnelles (PTM) dep53 augmentent en présence de pVI dans la cellule. De plus, les données obtenues montrent quel’expression de pVI affecte la transcription dépendante de p53 et que l’interaction avec Nedd4.2 n’estpas nécessaire pour inhiber les fonctions de p53. Pour étudier l’implication de pVI dans la modulationdu cycle cellulaire, nous avons créé une lignée cellulaire humaine exprimant cette protéine virale defaçon stable. La caractérisation de cette lignée a permis de mettre en évidence une prolifération cellulaireaccrue. Nos observations ont aussi montré une perte importante des PML-NB et une réduction desprotéines clés du cycle cellulaire p53 et pRb, un autre suppresseur de tumeur. Par des techniques demicro-injection et l’utilisation de l’inhibiteur MG132, nous avons observé que ces deux facteurscellulaires sont ciblés vers le protéasome et dégradés lors de la surexpression de pVI. L’étude desfonctions de cette protéine virale laisse penser que la protéine pVI présente un potentiel oncogéniquecar en effet, sa surexpression induit la dérégulation de l’homéostasie cellulaire et l’inhibition desuppresseurs de tumeur, comme p53 et pRb. / Human Adenovirus are non-enveloped viruses which replicate inside the host cell nucleus. Uponinfection and after receptor-mediated entry, they are transported towards the nucleus to initiate the viralgene expression. Viral capsids deliver from the endosome into the cytoplasm by partial disassembly andrelease inside the endosome mediated by viral lytic factor protein VI (pVI). pVI is targeted to themembrane via an amphipathic helix structure in the N-terminus of the viral protein. After membranerupture and capsid release, pVI is transported to sub-nuclear structures, so-called PML nuclear bodies(PML-NBs), together with the cytoplasmic ubiquitin ligase Nedd4.2. PML-NBs represent multiproteinaggregates in the host-cell nucleus with an antiviral capacity, as to several PML-associated repressivetranscription factors, such as the anti-apoptotic Daxx protein and the tumor suppressor p53 were reportedto localize at these foci. In addition, pVI-mediated displacement of Daxx from PML-NBs was shown tooccur in dependency of Nedd4.2 to support efficient viral gene expression. Therefore, we postulate thatbesides Daxx functional inhibition, pVI might also be involved in p53 restriction.Here, we show that p53 posttranslational modification (PTM) is increased when pVI protein is presentin the host-cell. Moreover, we obtained data that pVI expression severely impacts p53 inducedtransactivation of cellular transcription. Biochemical approaches indicate that pVI binding of theubiquitin ligase Nedd4.2 is no prerequisite for the capacity to inhibit p53 functions. In a next step toelucidate the role of pVI on cell cycle regulation, we generated a human cell line stably expressing theviral pVI protein. Our characterization analyses show significantly that these cells benefit from thepresence of pVI as we proved increased cell proliferation rates. We also observed an intense loss ofPML-NBs and reduced protein concentrations of cycle key regulators p53 and pRb. Usingmicroinjection and the inhibitor MG132 we were able to show that both cellular restriction factors weresequestered into the proteasomal degradation pathway of the cell. Evaluation of pVI functions temptedus to speculate, whether pVI might execute oncogenic potential upon overexpression, due toderegulation of host-cell homeostasis and inhibition of tumor suppressive determinants. / Humane Adenoviren (HAdV) sind unbehüllte Doppelstrang-DNA-Viren mit einem Proteinkapsid, diesich im Wirtzellkern replizieren. Der lytische Infektionsverlauf beginnt mit dem rezeptor-vermitteltenEintritt des Viruspartikels und dem gerichteten Transport des viralen Genoms zum Wirszellkern. Dasvirale Protein VI (pVI) ist nötig um den effizienten Austritt des bereits disassemblierten Viruspartikelsaus dem zellulären Endosom zu gewährleisten. Durch eine amphipathische Helix im N-terminalenProteinbereich interkaliert dieser lytische Faktor in die endosomale Membran und führt zum Aufbruchdes zellulären Organells. pVI wird anschließend an zelluläre Kernstrukturen, sogenannte PML nuclearbodies (PML-NBs) lokalisiert und komplexiert dort mit der zytoplasmatischen Ubiquitinligase Nedd4.2.PML-NBs stellen nukleäre Multiproteinkomplexe dar, die mittlerweile aufgrund ihrer antiviralenEigenschaften in den Mittelpunkt der virologischen Forschung gerückt sind. Diese zellulären Aggregatebestehen hauptsächlich aus repressiven Transkriptionsfaktoren, wie dem anti-apoptotischen DaxxProtein sowie dem Tumosupressor p53. In diesem Zusammenhang konnte bereits eine pVI-vermittelteRelokalisation des Daxx Proteins aus den PML-NBs gezeigt und als Vorraussetzung zur effizientenVirusgenexpression bestätigt werden. Es stellte sich im Rahmen der vorliegenden Arbeit die Frage, obneben der pVI-abhängigen Daxx Inhibition, auch p53 ein Zielprotein des viralen Capsidproteinsdarstellt.Unsere Arbeiten zeigen erstmals, dass nach der pVI Expression vermehrt posttranslationaleModifikationen am p53 Protein beobachtet werden. Weitere Befunde konnten außerdem einen Einflussvon pVI auf die p53-abhängige Transaktivierung zellulärer Promotoren beweisen. Mittelsbiochemischer Analysemethoden konnten wir zeigen, dass die Kooperation zwischen pVI und Nedd4.2keine Rolle bei der p53 Inhibition zu spielen scheint. Um im nächsten Schritt die Rolle von pVI imZellzyklus genau zu beleuchten, wurde zunächst ein zell-basiertes Modelsystem mit stabilerÜberexpression des viralen Faktors generiert, Anschließende phenotypische Analysen konnten zeigen,dass die Anwesenheit von pVI zur Steigerung der Zellproliferationsrate führt. Im Rahmen unsererUntersuchungen konnten wir auch einen signifikanten Verlust zellulärer PML-NBs beobachten sowieeine Reduktion der p53 und pRb Proteinkonzentration nachweisen. Mittels unter Verwendung vonMikroinjektion und dem Inhibitor MG132 war es uns möglich zu zeigen, dass pVI den proteasomalenProteinabbau der beiden Wirtszelldeterminaten p53 und pRb induziert. Deswegen kann man basierendauf den erhobenen Befunden zur pVI vermittelten Dysregulierung des zellulären Wachstums einonkogenens Potenzial des viralen Faktors annehmen.

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Replication defective adenoviruses have emerged as promising vectors for delivery of vaccine antigens. The development of new vaccine vectors has recently focused on serotypes t, which the human population is less exposed in order to circumvent pre-existing anti vector immunity. This thesis describes the construction and optimisation of ChAdOX1, a new vector based on chimpanzee adenovirus Y2S, which has recently been manufactured to clinical grade for a Phase 1 human trial. Comparative immunogenicity studies between vectors of different serotype were performed in mice, with careful consideration of the infectious titer of vector preparations, since this parameter can confound studies based solely on viral particle estimation. Aft intramuscular administration, HAdV-S (Human adenovirus C) based vectors elicited superior transgene product specific T cell and antibody responses compared to a selection of chimpanzee adenovirus vectors (from Human adenovirus EJ including ChAdOX1. The construction of ChAdOXl in a bacterial artificial chromosome (BA C), enabled precise, and flexible modification of the genome by recombmation mediated genetic engineering. (recombmeering). Reverse genetics was performed to identify vector determinants of immunogenicity. Chimeric ChAdOXl vectors were created by replacement of native virus associated RNA (VA-RNA) and fiber sequences with the corresponding sequences from HAdV-5 Using these chimeric vectors, the importance of innate immunity and vector transduction in determining vector immunogenicity was investigated. Though the mechanisms responsible ultimately remain unclear, superior transgene product specific immune responses with HAdV-5 correlated with higher levels of transgene expression in vivo after vector administration. The current study has conclusively demonstrated that neither VA-RNA sequences, nor the fiber protein, are responsible for differences in immunogenicity between vectors, contrary to hypotheses based on previous studies.

Early region 1A (E1A) protein is the first protein expressed following viral infection. E1A proteins initiate the cell cycle in infected cell by altering cellular gene expression and also activate expression of other viral genes enabling viral replication. The C-terminus of E1A is the least-characterized region of the protein, with few known binding partners, however DREF has been identified as a novel binding partner. My studies have determined that DREF directly binds to E1A and has a role in the virus life cycle. DREF is a restriction factor for virus growth and is a component of viral replication centres. DREF is SUMOylated and SUMOylation appears to affect localization to viral replication centres. DREF co-localizes with PML bodies and subcellular distribution of DREF is altered by the presence of E1A. This work provides a platform to study the role of DREF in uninfected cells, and in HAdV biology and its possible role in antiviral response. / October 2015

Defects within the DNA damage response (DDR) pathways are common in human malignancies. This is especially true in high-grade serous ovarian cancer (HGSOC) where defects within the Homologous Recombination (HR) pathway may be present in up to 50% of tumours. Oncolytic adenovirus is a potential novel therapy for human malignancies. These viruses infect malignant cells and multiply selectively within them causing cell death and release of mature virions. Here, I have investigated the role of the DDR in determining the efficacy of the E1A-CR2 deleted adenovirus type 5 (Ad5) vector, dl922-947, in ovarian cancer. I show that infection with dl922-947 stimulates a robust DDR within the host cell, which the virus manipulates in order to ensure optimal viral replication. In a panel of HGSOC cell lines, the extent of overreplication of genomic DNA and the degree of genomic damage following infection with dl922-947 was shown to correlate closely with viral efficacy. Functional HR, however, promoted viral DNA replication and augmented overall anti-cancer efficacy. Mechanistically, both BRCA2 and RAD51 localised to viral replication centres within the infected cell nucleus. RAD51 co-localisation was also demonstrated in cells with defective HR and occurred independently of BRCA2. In addition, a direct interaction was identified between RAD51 and adenovirus E2 DNA binding protein. Using functional assays of HR competence, I show that Ad5 infection does not alter cellular ability to repair DNA double-strand break damage via HR. These data suggest that oncolytic adenoviral therapy may be most clinically relevant in tumours with intact HR function. Using a high-throughput siRNA DNA repair screen, potential novel targets have been identified that can increase the efficacy of dl922-947 (for example: NONO) and also result in increased resistance (RPA). These results highlight the complex interplay between adenovirus and host cell. Further understanding of these pathways is vital to increase efficacy, develop biomarkers and improve patient selection into clinical trials for these therapies.

Prostate cancer is the most common non-skin cancer in America, and the most commonly diagnosed cancer among males. When metastatic, the disease can ultimately be incurable. Consequently, alternative strategies to current treatments are sought, especially in the area of immunotherapy. Vaccine immunotherapy using a specific antigen, such as prostate specific antigen (PSA) seeks to stimulate both the innate and adaptive immune system to destroy tumor cells in the body. PSA is an ideal target antigen given that it has a narrow distribution in tissues and is expressed in virtually all prostate cancer cases. An adenovirus encoding for PSA (Ad-PSA) can be used to deliver the genomic data encoding for PSA production and secretion to the target cell. This type of viral gene delivery system has already been shown to have the potential to stimulate anti-tumor activity. To enhance this activity and increase transfection efficiency, we proposed the combination of a viral system with a non-viral system, in the form of a cationic polymer such as poly(ethyl)enimine (PEI) or chitosan. Cationic polymers complex with the negatively charged adenovirus to form nanoparticles that can be used in gene delivery. Delivery in nanoparticle form can give enhanced uptake by the antigen-presenting cells necessary to initiate the targeted immune response. To further augment this response, previous research has shown that CpG sequences act as an adjuvant to enhance the efficacy of the Ad-PSA vaccines' tumor protection. CpG delivered in particulate form has also been shown to be more effective than delivery in solution. The objective of this proposal was to test the hypothesis that co-delivery of this targeted viral/non-viral gene delivery system will enhance tumor protection in a mouse model of prostate cancer. Using the OVA model antigen system, we found that the adenovirus encoding OVA (AdOVA), coupled with the polymer PEI, enhanced tumor protection in vivo compared to AdOVA alone. To move towards our therapeutic model, these experiments were repeated using chitosan as the cationic polymer carrier, delivering AdOVA, and incorporating CpG into some particles. In this set of experiments, we found that AdOVA + CpG gave the best tumor protection in challenge studies. AdOVA + chitosan + CpG showed a decrease in protective levels and numbers of antigen-specific immune cells. Further experiments focused on elucidating the mechanisms by which chitosan and CpG modulate the immune response. Using the therapeutic AdPSA model, chitosan was not found to enhance tumor protection or numbers of antigen-specific immune cells. Additional experiments found that this depression was not due to problems with viral infectivity or secretion due to chitosan complexation. A series of kinetics studies were performed which showed that peak levels of effector T cells were present 14 days later in AdPSA + CpG immunized mice than in AdPSA alone. This delayed effect may explain the increased levels of protection in AdPSA + CpG mice, and be useful in future vaccine design concerning the timing of peak response.