O P. vivax é a segunda espécie mais prevalente causadora de malária no mundo. Medidas de controle ineficientes exigem o desenvolvimento de novas estratégias de prevenção, como vacinas, novas drogas e novos inseticidas. O objetivo geral do trabalho foi gerar uma formulação vacinal universal com proteínas e adenovírus recombinantes capazes de induzir anticorpos contra as diferentes formas alélicas da proteína circumsporozoíta (CSP) do P. vivax. As proteínas foram produzidas em E. coli e purificadas por cromatografia de afinidade e troca iônica. A obtenção destas proteínas nos permitiu testar qual seria a melhor formulação vacinal para a indução de anticorpos contra as três formas alélicas da proteína CSP de P. vivax (PvCSP). Anticorpos específicos reconheceram esporozoítas do P. vivax por imunofluorescência. Por fim testamos o uso de dois adenovírus recombinantes, um símio e um humano, deficientes em replicação, expressando as três regiões imunodominantes da proteína PvCSP em fusão. Estes foram capazes de induzir resposta imune específica contra as proteínas PvCSP sendo testados em esquema de prime-boost heterólogo, onde camundongos foram primados com os adenovírus e nas doses-reforço receberam a mistura com as três proteínas recombinantes. / The Plasmodium vivax is the second most prevalent species of malaria in the world. Inefficient measures of control used today demand the development of new strategies for prevention, as vaccines, new drugs and new insecticides. The central objective of this thesis was to generate a universal vaccine formulation with proteins and recombinant adenoviral vectors representing the different allelic forms of the circumsporozoite protein (CSP) of the P. vivax. The recombinant proteins were expressed in E. coli and purified. These proteins allowed us to test which would be the best vaccine formulation for the induction of antibodies against the three allelic forms of CSP. The specific antibodies also recognized P. vivax sporozoites by immunofluorescence. Finally we test the use of two recombinant adenoviral vectors, a simian and a human, both replication deficient, expressing a protein containing the repeat regions of the CSP in fusion. These adenoviral vectors induced specific immune response against CSP and were successfully used in an immunization regimen of heterologous prime and boost where in the first dose the mice received recombinant adenoviral vector and in the subsequent doses, the mixture with three recombinant proteins.

No description available.

Les adénovirus humains (HAdV) provoquent un large spectre de maladies cliniques chez les patients immunodéprimés et immunocompétents et sont également des outils polyvalents pour le transfert de gènes et la vaccination. L’immunité humorale acquise peut être en partie responsable des réactions indésirables envers les vecteurs AdV révélées dans plusieurs essais cliniques de vaccination. De plus, plusieurs protéines de l'hôte comme le facteur X de coagulation de la souris (FX) ou les immunoglobulines de type G se lient aux HAdV et exacerbent la réponse pro-inflammatoire. L’évaluation des risques précliniques se fait souvent chez la souris, même s’il existe plusieurs différences entre les humains et les souris dans l'interaction avec les HAdV. La liaison de FX aux HAdV active une réponse pro-inflammatoire chez la souris par l'intermédiaire des récepteurs Toll-like 4. Dans un autre scénario clinique pertinent, le complexe immun HAdV (IC-HAdV-C5) induit une activation plus forte de l’inflammasome dans les phagocytes humains que l’HAdV-C5 non complexé, mais par un mécanisme inconnu. Dans ce contexte, j’ai participé à deux études. Premièrement, nous avons étudié le rôle potentiel de FX et de TLR4 dans la réponse innée à l ‘HAdV-C5 en utilisant uniquement des cellules et des protéines d’origine humaine. Nous avons constaté qu'il n'y a pas d’activation de la voie de signalisation via TLR4 chez l’homme en présence de FX-HAdV. De plus, le FX n'a pas affecté la forte réponse immunitaire innée induite par IC-HAdV-C5 dans les phagocytes humains. Deuxièmement, nous avons abordé le mécanisme sous-jacent de l'inflammation induite par IC-HAdV-C5. Nous avons démontré que l‘IC-HAdV-C5 induit la formation de l’inflammasome dans les cellules dendritiques dérivées de monocytes et cela dépend de l’échappement endosomal dépendant de la protéine VI et de l'activation des récepteurs cytosoliques de l’inflammasome. Nos résultats nous aident à mieux comprendre les différences entre les tests précliniques réalisés chez la souris et les essais cliniques réalisés chez l'homme. De plus, cela nous permet de mieux comprendre comment l’immunité préexistante façonne la réponse immunitaire innée face aux HAdV, afin d’améliorer le traitement des maladies liées aux HAdV ainsi que l’efficacité des vecteurs HAdV. / Human adenoviruses (HAdV) cause a broad spectrum of clinical diseases in immunocompromised and –competent patients and are also versatile tools for gene transfer and vaccination. Pre-existing humoral immunity may be in part responsible for the adverse responses towards AdV vectors seen in several clinical vaccine trials. Furthermore, a variety of host proteins like mouse coagulation factor X (FX) or immunoglobulin G bind HAdV exacerbate the pro-inflammatory response. Pre-clinical risk assessment is often done in mice, albeit there are multiple differences between human and mice in the interaction with HAdV. The binding of FX to HAdV activates a pro-inflammatory response in mouse via Toll-like receptor 4. In another clinical relevant scenario, immune complexed-HAdV (IC-HAdV-C5) induces more inflammasome activation in human phagocytes than HAdV-C5 alone but by unknown mechanism. In this regard, I participated in two studies. First, we investigated a potential role of FX and TLR4 in the innate response to HAdV-C5 by using only human components. We found that there is no detectable FX-HAdV-TLR4 axis in human and FX did not affect the innate immune response elevated by IC-HAdV-C5 in human phagocytes.Second, we addressed the underlying mechanism of IC-HAdV-C5-induced inflammation. We found that IC-HAdV-C5 induces inflammasome formation in monocyte-derived dendritic cells and this is dependent on pVI-mediated endosomal escape and activation of cytosolic inflammasome sensors. Our findings help us to better understand the differences in preclinical testing in mice and clinical use in humans and how pre-existing immunity shapes the innate immune response to HAdV to improve treatment for HAdV diseases and HAdV vector effectiveness.

Master of Science / Department of Electrical and Computer Engineering / Masaaki Tamura / Steven Warren / Lung cancer mortality is the highest among all cancer–associated deaths. Despite early detection and treatment, prognosis of this disease remains poor. Therefore, development of new therapeutic agents and effective treatment procedures are urgently needed. Endogenous Angiotensin II (Ang II) type 2 receptor (AT[subscript]2), one of two isoforms of Ang II, has been shown to mediate apoptosis. Nanoparticle delivery systems make possible targeted drug delivery and controlled release of therapeutic molecules and genes. Thus, the aim of this study was to determine the anti-cancer effect of the over-expressed AT[subscript]2 gene on lung adenocarcinoma cells in vitro using adenoviral vector (Ad-) and nanoparticle (NP-) based gene delivery systems. This study showed that over-expression of Ad-AT[subscript]2 induced cancer cell-specific apoptosis in several human lung adenocarcinoma cell lines with minimal effect on normal lung epithelial cells. Ad-AT[subscript]2 significantly attenuated multiple human lung cancers' cell growth (A549 and H358) in vitro compared to the control viral vector, Ad-[Beta]-galactosidase (Ad-LacZ) when examined by direct cell count. The growth attenuation effect was detected as early as 24 hours after Ad-AT[subscript]2 transfection and lasted 12 days. Western Blot analysis revealed the activation of the caspase pathway. Examination for Annexin V by flow cytometry also confirmed activation of the apoptotic pathway via AT[subscript]2 over-expression. Similarly, AT[subscript]2 cDNA encapsulated poly(DL-lactide-co-glycolide) (PLGA) biodegradable nanoparticles (NPs) were shown to be effectively taken up into lung cancer cells. Surface conjugation of the angiotensin II peptide significantly stimulated uptake of the particles. This PLGA vector-dependent AT[subscript]2 transfection was effective in sustained gene expression and resultant cell death. These results indicate that the AT[subscript]2 over-expression effectively attenuated growth of lung adenocarcinoma cells through activation of intrinsic apoptosis. Since PLGA safety has been proven, whereas adenoviral vectors have several drawbacks in safety, the Ang II conjugated PLGA nanoparticles may be a better therapeutic gene delivery system. Therefore, it is concluded that the discovery of AT[subscript]2 DNA encapsulated PLGA conjugated with the Ang II peptide is a potentially useful tool for lung cancer gene therapy.

L’adénovirus (AdV) est un virus non enveloppé à ADN double brin qui entre dans la cellule par endocytose. Dans l’endosome un désassemblage partiel de la capside permet la libération d’une protéine interne de la capside, la protéine VI (PVI). Cette protéine code une hélice amphipathique qui va permettre la rupture de l’endosome. Des travaux antérieurs du laboratoire ont montré que le transport des particules virales vers le noyau nécessite la présence du motif conservé PPxY dans la PVI qui permet le recrutement d’ubiquitines ligases de la famille des Nedd4 (telles que Nedd4.1 et Nedd4.2). Il a précédemment été montré que la rupture des membranes induite lors d’infections bactériennes activait l’autophagie afin d’éliminer le pathogène intracellulaire via une dégradation lysosomale. Nos résultats démontrent que l’AdV induit également l’autophagie lors de son entrée dans la cellule. L’utilisation de différents AdV mutants nous a permis de démontrer que la rupture de l’endosome était responsable de l’induction de l’autophagie. De plus nos résultats montrent que le virus sauvage est capable d’éviter sa dégradation en contrôlant l’autophagie grâce au recrutement de la ligase Nedd4.2 via le motif PPxY de la PVI. Au contraire, un virus mutant dépourvu du motif PPxY et donc incapable de recruter la Nedd4.2 est séquestré dans les vésicules autophagiques puis dégradé par la fusion de ces vésicules avec les lysosomes. Ainsi le motif PPxY constitue un déterminant moléculaire permettant au virus de contourner les défenses cellulaires antivirales. / Adenoviruses (AdV) are linear ds-DNA containing, non-enveloped viruses that enter cells by receptor-mediated endocytosis. Once in the endosome it occurs a partial disassembly of the capsid allowing the releases of the membrane lytic capsid protein VI, which encodes an N-terminal amphipathic helix responsible for the endosome rupture. Our previous work showed that transport to the nucleus requires a conserved PPxY motif in PVI, which recruits ubiquitin ligases of the Nedd4 family (e.g. Nedd4.1 and 4.2). Previous work has shown that membrane damage induced by invasive bacteria elicits selective cellular autophagy to eliminate the pathogen via lysosomal degradation. In our current work we show that Adv also induce autophagy upon entry. Using a set of mutant AdV’s attenuated at each step of the membrane penetration process we show that indeed the membrane damage induced by the virus is causative for autophagy induction. Moreover the data show that wildtype AdV limit the level of autophagy induction and evade autophagic degradation by using a Nedd4.2 dependent process. In contrast, mutant viruses mutated for its PPxY and that fail to recruit Nedd4.2 are subject to autophagic degradation. Our data suggest that the presence of the PPxY motif in the virus subverts the autophagic process and thus identify the PPxY motif as an integral part of the virus to undermine cellular antiviral mechanism.

Adenovirus (Ad) is a widely studied DNA virus, but the nucleoprotein structure of the viral genome in the cell is poorly characterized. Our objective is to study Ad DNA-protein associations and how these affect the viral life cycle. Most of the viral DNA condensing protein, protein VII, is lost within a few hours of infection and this loss is independent of transcription. Cellular histones associate with the viral DNA after removal of protein VII, with a preferential deposition of H3.3. Micrococcal nuclease accessibility assays at 6 hpi showed laddering of the viral DNA, suggesting the genome is wrapped in physiologically spaced nucleosomes. Although viral DNA continues to associate with H3.3 at late times of infection, the overall level of association with histones is greatly reduced. Knockdown of the H3.3 chaperone HIRA had no effect on the viral life cycle suggesting that other H3.3 chaperones are involved. Our studies have begun to elucidate the nucleoprotein structure of Ad DNA in the infected cell nucleus.

No description available.

Les adénovirus (AdV), comme d'autres virus à réplication nucléaire, ont besoin d’arriver jusqu’aunoyau cellulaire afin de libérer leur génome. Pour ce faire, les particules des AdV contenant l’ADNviral sont transportées jusqu’au complexe du pore nucléaire (NPC), via le centre d’organisation desmicrotubules, par un mécanisme encore mal compris qui implique l’exportine cellulaire CRM1. Lacapside des AdV dépasse la taille limite d’entrée dans le noyau, et par conséquent, elle doit êtredésassemblée au niveau du NPC. Le mécanisme d’import de molécules d’ADN n’est pas un processusphysiologique. Pour cela, les AdV doivent détourner la machinerie cellulaire afin d’importer leurgénome dans le noyau. Le NPC est un complexe de protéines appelées nucléoporines. LaNup358/RanBP2, principal composant des filaments cytoplasmiques, sert de plateforme de liaison àdes karyopherines (e.g Importin-β, CRM1) et à la protéine GTPase Ran. Les karyopherinesreconnaissent des signaux spécifiques présents dans les cargos et facilitent leur transport d’unemanière très régulée dépendante de RanGTP. Nous avons constaté que l’import du génome AdV estmoins efficace en l’absence de Nup358. Dans ces conditions, nous avons observé que certaineskaryopherines deviennent limitantes pour l’import du génome viral, et identifié la région minimale deNup358 requise pour compenser ce défaut. D’autre part, nous avons confirmé l’implication de CRM1dans l’arrivé des particules virales au noyau et identifié un nouveau rôle de CRM1 dans ledésassemblage de la capside des AdV. Ces travaux contribuent à mieux connaître le mécanismed’entrée du génome AdV dans le noyau et donnent une idée de la façon dont les virus peuventcontourner la machinerie de transport cellulaire pour leur propre bénéfice. / Nuclear delivery of viral genomes is an essential step for nuclear replicating DNA viruses such asAdenovirus (AdV). AdV particles reach the nuclear pore complex (NPC) in the form of genomecontaining, partially disassembled capsids, through a poorly understood CRM1-dependent mechanism.These capsids exceed the NPC size limit and therefore, they must disassemble at the NPC to releasethe viral genome. Nuclear import of DNA cargos is not a physiological process. Consequently, AdVneed to divert the cellular transport machinery for nuclear genome delivery. The NPC is a multiproteincomplex consisting of nucleoporins (Nups). The Nup358/RanBP2 is the major component ofthe cytoplasmic filaments of the NPC and serves as binding platform for factors includingkaryopherins (i.e Importin-β, CRM1) and the small GTPase Ran. Selective transport of cargo throughthe NPC is mediated by karyopherins, which recognize specific signals within the cargos and facilitatetheir transport in a RanGTP-dependent regulated manner. We identified that Nup358-depleted cellsreduce nuclear import efficiency of the AdV genome. Indeed, we observed that karyopherins are ratelimitingfor AdV genome import under these conditions and we mapped the minimal region ofNup358 necessary to compensate the import defect. On the other hand, we could confirm therequirement of CRM1 in nuclear targeting of AdV capsids and identified and additional role inmediating AdV capsid disassembly. This work helps to understand the strategy used by AdV todeliver their genome and gives insight about how viruses hijack the cellular transport machinery fortheir own benefit.

The aim of the present study was to develop a dendritic cell (DC) vaccine encoding hepatitis C virus (HCV) multiple cytotoxic T lymphocyte (CTL) epitopes that can stimulate T cell responses in vitro, and can be used for immunization in vivo. DCs were infected with recombinant replication-defective adenoviruses (Ads) expressing 2 HCV sequences fused with green fluorescent protein (GFP) and FLAG tags. One sequence (sequence 1) contained the HCV CTL epitopes, NS4B 1793-1801 and P7 774-782, as well as the HCV Th epitope, NS3 1248-1261. A second sequence (sequence 2) was the positive epitope control which contained HCV core 35-44, core 132-140 and NS3 1248-1261. The efficiency of infection was detected by flow cytometry and the expression of HCV epitopes in the DCs was confirmed by RT-PCR and western blot analysis. Ad infection significantly enhanced DC maturation and interleukin (IL)-12p70 production, resulting in T cell proliferation and increased interferon-γ secretion. The CTLs stimulated by Ad-infected DCs specifically killed Huh7.5 human hepatoma cells. The recombinant Ad-expressing multiple CTL HCV epitopes effectively infected the DCs in vitro and promoted T cell antiviral immune responses, thereby laying the foundation for the development of anti-HCV DC vaccines.

The aim of the present study was to develop a dendritic cell (DC) vaccine encoding hepatitis C virus (HCV) multiple cytotoxic T lymphocyte (CTL) epitopes that can stimulate T cell responses in vitro, and can be used for immunization in vivo. DCs were infected with recombinant replication-defective adenoviruses (Ads) expressing 2 HCV sequences fused with green fluorescent protein (GFP) and FLAG tags. One sequence (sequence 1) contained the HCV CTL epitopes, NS4B 1793-1801 and P7 774-782, as well as the HCV Th epitope, NS3 1248-1261. A second sequence (sequence 2) was the positive epitope control which contained HCV core 35-44, core 132-140 and NS3 1248-1261. The efficiency of infection was detected by flow cytometry and the expression of HCV epitopes in the DCs was confirmed by RT-PCR and western blot analysis. Ad infection significantly enhanced DC maturation and interleukin (IL)-12p70 production, resulting in T cell proliferation and increased interferon-γ secretion. The CTLs stimulated by Ad-infected DCs specifically killed Huh7.5 human hepatoma cells. The recombinant Ad-expressing multiple CTL HCV epitopes effectively infected the DCs in vitro and promoted T cell antiviral immune responses, thereby laying the foundation for the development of anti-HCV DC vaccines.